Hemangiomas are the most common head and neck tumors in the pediatric age group. Despite their frequency, little is known about the factors controlling their growth and regulation. We have identified two transcription factors CTCF (CCTCCC binding factor) and BORIS (Brother of the regulator of imprinted sites) that play a major role in the regulation of hemangioma growth, acting through IGF2, a critical growth factor in pediatric tumors. MicroRNAs are recently discovered molecules that are known to regulate a host of physiological functions including growth and development of tumors. We have additionally identified two microRNAs, MicroRNA23a (mir23a) and MicroRNA23b (mir23b) that are very strongly correlated to the expression of the transcription factors CTCF and BORIS, thus influencing the growth of hemangiomas. We therefore propose: 1. To formally demonstrate the regulation of CTCF and BORIS by mir 23a and 23b in an in vitro model 2. To create a transgenic mouse model of hemangiomas by over expressing microRNA 23b, using an endothelial cell specific promoter Tie-2 3. To propose a new method of molecular classification of hemangiomas based on microRNA profiles through a microarray analysis

Classic and atypical Spitz nevi are uncommonly occurring melanocytic lesions with an incidence of 7 in 100,000. They most commonly present in children and adolescents but may also occur in adults. The clinical and histological features of melanoma can be similar to both the classic and atypical Spitz nevi. Spitzoid melanoma is the variant of melanoma that shares clinical features with spitz nevi. There is no single discriminating factor between Spitz nevi and melanomas as every trait of Spitz nevi has been described in melanoma. While spitz nevi most commonly occur in children, a population where melanoma is quite rare, it is estimated that 40% of children who do develop melanoma are originally misdiagnosed. As melanoma is on the differential of every Spitz nevus, differentiating between the two is incredibly important. Over treatment of Spitz Nevi can result in significant morbidity while under treatment of melanoma can result in premature death. Our hypothesis is that the subcellular localization of Gp100, Nedd9 and Gab2 will be consistently altered in spitzoid melanoma when compared to benign spitz nevi.

Keloids represent aggressive and prolonged fibroblast responses to cutaneous trauma with significant disfigurement, and in many instances functional impairment. Many treatments have been employed with unacceptably high rates of recurrence. Recent advances in studies of systemic markers of disease and the role of circulating progenitor cells in other inflammatory conditions have demonstrated a common pathway for regional trafficking of fibroblasts that could be inhibited in order to prevent aberrant postoperative wound healing and keloid recurrence. Our goal is to determine if patients who form keloids demonstrate a higher proportion of circulating fibroblast precursors in peripheral blood samples, and if keloid specimens contain a significant concentration of surface markers responsible for local trafficking of this precursor cell. This phenomenon has been demonstrated in similar patient populations within our collaborating laboratory at UV A (R. Strieter). We hypothesize that peripheral blood samples taken before, during and after elective keloid excision will demonstrate a higher concentration of circulating fibroblast progenitor cells (CD451184), compared to case controls without keloids undergoing minor elective surgery. We anticipate that keloid specimens will demonstrate significant expression of fibroblast trafficking ligand CXCLI2, and hypoxic inducing factor (HIF-I), a marker of hypoxic insult present in states of prolonged inflammation and angiogenesis. The ability to detect the local upregulation of this pathway, and to inhibit its aggressive function will allow for proper peri-operative counseling before elective surgery and provide a novel, specific treatment to help decrease keloid recurrence.

Worldwide, 38 hand allografts have been performed 28 patients, with survival times from 2 to 9 years. More recently two separate French groups and a Chinese group have performed partial face transplants. All of these transplants are dependent on chronic immunosuppresion, with its attendant side-effects including malignancy and opportunistic infection. In addition, all of these transplants have demonstrated episodes of rejection of the skin that required treatment with steroids, monocolonal antibodies or an increase in the overall level of immunosuppression. Our long-term goal is to devise practical methods for inducing immunologic tolerance that would allow for the long term survival of these complex tissue allografts without the need for chronic immunosuppression. This would significantly improve the risk-benefit ratio and allow for the more widespread use of composite tissue allotransplant (CTA) in the reconstruction of lost limbs and severe facial deformities. One method of inducing tolerance to an allograft is to establish a state of mixed chimerism. In a mixed chimera, the donor's immune system has become tolerant not only of the alloantigens expressed on host tissues but also those on hematopoietic and immune cells. In turn, the recipient's cells are tolerant of the alloantigens on the surface of the donor's hematopoietic cells. We have a pre-clinical large animal that will address the application of mixed chimerism to a clinically relevant application to the use of CTA. Aim I. Determine if tolerance to vascularized myocutaneous allografts can be achieved when tissue transplantation is performed at the same time as the establishment of mixed chimerism. Aim 2: Examine the role of graft infiltrating cells in the establishment of immune tolerance to composite tissue allografts in the canine model of mixed chimerism, with a specific focus on the presence of T -regulatory cells, their expression of FoxP3, and the analysis of the expression of chemokines and cytokines after transplantation.

This proposed study is to define the neurovascular anatomy and specific technical issues for the harvest of functional composite flaps of the eyelid, nose, ear and upper and lower lips, as single, vascularized facial subunits. The facial subunits, which include the specialized tissues that makes up the ear, eyelid, nose, cheek, and upper and lower lips serves to define topographic areas with characteristic skin qualities, outlines, and shape. The subunit approach to facial reconstruction has been a useful principle to follow for concealing scars, maintaining skin qualities, and restoring symmetry. While microvascular free flaps can often provide tissue replacement for large defects, it has been less successful in restoring complex, specialized tissues such as the upper and lower lip. Therefore, the reconstruction of facial subunits that have lost tissues with specialized functions, such as the eyelid, continues to be an unsolved challenge. The ability to reconstruct these specialized tissues with the exact subunit from cadaveric sources would revolutionize the field of reconstructive surgery. Trauma, especially bums, to the face often leads to the loss of critical specialized structures such as the eyelids, lips, ears and nose .. Composite tissue transplantation of specialized subunits would allow for the transfer of tissues that could closely match the dimensions, texture, thickness, and function of any given facial defect. A near-normal facial appearance could be achieved in a single stage without any donor-site morbidity. The recent clinical transplants in France and China highlight the possible future use of these specialized transplants. The French patient had lost the upper and lower lips as well as much of the nose. These complicated structures were reconstructed in a single stage with no donor site issues. While there may be patients that require transplantation of the entire face, the majority of patients that will benefit from this CTA will need a segmental composite transplant. The goal of this study is to define the anatomic and technical issues surrounding these transplants. Preliminary studies that we have conducted show that it may be possible to raise a functional composite eyelid flap based on the transverse facial arterial system. Further study is needed to confirm these initial observations and show that such a flap can be harvested on a consistent basis.

The identification of a new molecular target to prevent radiation induced skin fibrosis and injury may lead to an increased understanding of the fibrosis mechanisms occurring after ionizing radiation therapy and the development of novel therapeutics against this morbidity. We propose that matrix metalloproteinases (specifically, MMP-2 and MMP-9) provide such a target. MMPs have been shown to increase in radiation-induced dermal injury and are integral to the regulation of the TGF-b fibrotic pathway. We hypothesize that knockout of this gene will therefore result in radioprotection.

The purpose of this study is to evaluate the potential for adipose-derived stem cells (ASCs) to enhance interface zone fibrovascular incorporation, vascularity and regenerative potential at the acellular dermal matrix (ADM) -fascia interface of ventral hernia repairs. The following are the goals of the study: 1. Compare scaffold pre-seeding to direct injection of ASCs into the fascia-ADM interface. 2. Quantify the fascia-ADM interface zone engraftment of ASCs over the first two weeks post-operatively. 3. Determine the phenotype of engrafted stem cells at the fascia-ADM interface zone. 4. Evaluate the functional capacity of ASCs in the setting of ventral hernia repair. Clinical problems in this setting include technical challenges in the reduction and repair of complex chronic hernias, difficulty in achieving complete reconstruction of abdominal wall elements, and significant rates of recurrence. The potential to improve current materials-based repairs with a stem cell-based, tissue engineered approach could provide a superior reconstruction at the tissue and cellular level, yielding a reduced rate of recurrence. Our hypothesis is that ASCs will enhance vascular support, tissue regeneration, and bioprosthetic incorporation at the graft-abdominal wall interface.

The need for deformable elastic cartilage to reconstruct ear deformities, including microtia, has not been met by tissue engineering strategies. The most significant hurdle to generating deformable cartilage is the inability to control elastin production. In this project, we will utilize growth factor and nanofiber scaffold strategies to improve elastin production in umbilical cord derived mesenchymal stem cells. Scaffolding will consist of nanofibers, which are submicron fibrils, on which augmented cell growth and differentiation has been demonstrated. We will compare chondrogenesis on nanofiber mats and within nanowebs, the latter of which has a greater 3-dimensionality, and ability to interact with and influence cells. In order to test deformability, we will use microindentation techniques. For all biochemical and biomechanical parameters, we plan to compare human auricular cartilage to our tissue engineered cartilage. The potential clinical impact of generating a deformable tissue engineered elastic cartilage is significant. This cartilage will be a valuable adjunct in the reconstruction of patients with microtia and other ear abnormalities. Broader applications are also possible in patients with nasal deformities.

Tissue engineering of bony constructs presents a promising reconstructive option for large bony defects. While the use of bone allograft as a matrix has the advantage of immediate tensile strength, the porosity of polymer matrices is advantageous for cellular ingrowth and nutrient diffusion. The formation of a vascular network is a key factor for the success of larger or less porous bony constructs. Adipose tissue-derived stem cells (ASCs) which can be isolated with minimal donor site morbidity have the ability to differentiate into both osteoblasts and endothelial cells by culture in specific media. In addition, endothelial cells have the potential to populate a matrix and form capillary networks. Since critical-sized rat calvarial defects are a well-established model for testing the osteoregenerative capacity of bone substitutes, this model will be used to evaluate the osteoinductive properties of ASC derived osteoblasts and the effect of ASC derived endothelial cells on those osteoinductive properties in both polymer and bone allograft scaffolds. Specifically, this project seeks to evaluate the following areas in the development of bony constructs from ASCs. (1) Evaluate the ability of ASC derived endothelial cells to populate and form capillary networks in both polymer and calvarial allograft scaffolds. (2) Create bony constructs with viable osteoblasts and a capillary network by co-culturing ASC derived osteoblasts and endothelial cells in polymer and calvarial allograft scaffolds. (3) Compare osteoregenerative capacity of bony constructs containing ASC derived osteoblasts and endothelial cells, ASC derived osteoblasts alone, and matrix/allograft alone.

Composite tissue allotransplantation (CT A) represents a novel surgical tool in the reconstructive surgeon's armamentarium. It provides a true replacement of "like for like" and does not involve any donor site morbidity. However, long term CTA survival requires chronic immunosuppression, which is associated with complications such as drug and metabolic toxicities, opportunistic infections and malignancies. To overcome the risk-benefit ratio of CTA as routine surgical reconstructive option, limiting chronic immunosuppression and preventing acute rejection is absolutely vital. The potential efficacy of bone marrow (BM) transplantation for tolerance induction is well established. Use of donor BM transplantation to induce tolerance to CTA represents an attractive option. However, its full potential has not yet been realized due to the need for recipient myeloablative preconditioning, risk of graft versus host disease (GVHD), which limits the amount of BM transplantable and risk of sensitization. Specific Aim: To use massive doses of donor BM to induce tolerance to CTA and assess the mechanism without causing any side effects. Hypothesis: The concomitant use of massive doses of donor BM in presence of co-stimulatory blockade without any recipient preconditioning along with short-ten11 rapamycin will induce robust tolerance to CTA without risks of GVHD, sensitization or chronic immunosuppression.

Delayed or failed wound healing is a serious problem acutely, but with long term repercussions. Chronic wounds are the cause of suffering to thousands of patients and require an immense utilization of health care. In the bum unit, patients who heal slowly are the same ones who later struggle with scars, contractures, leading to impaired function and return to normal life. Active MMP-9 has been found at pathologically higher levels in chronic wounds when compared to a normal wound or normal skin. We believe that excess MMP-9 causes wounds to be unable to close. In the proposed project, we seek to prove our hypothesis that unbridled overexpression ofMMP-9 in keratinocytes slows the healing of wounds. Further, we then evaluate the ability of MMP-9 inhibitors to restore the slowed wound healing into a normal trajectory.

In the project described here, the effect of local immunosuppression in the skin as the component representing the primary target for rejection in composite tissue allotransplantation is addressed. In contrast to previous attempts, the investigators aim for inhibition of lymphocyte infiltration and not for inactivation of previously infiltrated lymphocytes while avoiding an immunosuppressive "overkill" and interference with the known tolerogeneic effect resulting from the continuous antigen presentation to the recipient of a composite tissue allograft. For this purpose, four inhibitors of lymphocyte migration (anti-CD11a-Ab, anti-ICAM-1-Ab, Efomycine M and Bb15-42) have been selected and will be studied for their efficacy in prevention of rejection in experimental rat models for vascularized skin and limb transplantation. All of these compounds act on lymphocyte adhesion, rolling or transmigration, Inhibition of lymphocyte migration into the skin shall ultimately allow for profound reduction or avoidance of systemically applied long term immunosuppression in composite tissue transplantation involving the skin. Inhibition of lymphocyte trafficking will be combined with a steroid free immunosuppressive protocol using a depletional polyclonal antibody (ALG) followed by very low dose monotherapy with Tacrolimus. This approach provides a clinically oriented protocol for immunosuppression with minimal toxicity in hand transplantation and any other type of CTA that includes the skin. In preliminary experiments, Bb15-42 was tested in a Brown-Norway to Lewis rat hind-limb allotransplant model. Untreated controls were compared with a group receiving Bb15-42 (5mg/kg) and sub-therapeutic doses of Tacrolimus (0.1 mg/kg). Treatment with Bb15-42 together and sub-therapeutic doses of Tacrolimus significantly prolonged limb allograft survival from 7 +/- 0 days to 17.25 +/-2.872 days (p=0.0056). Although hand transplantation is of major current interest for reconstruction of a variety of defects, there is an urgent need for minimizing toxicity of the required immunosuppressive treatment. The protocol described here shall ultimately allow for clinical application of hand transplantation thus avoiding the severe side effects associated with high dose long-term systemic immunosuppression. This would allow for the wide spread application of composite tissue allotransplantation as a therapeutic option for a great variety of disabling and disfiguring injuries.

The purpose of this experiment is to evaluate the potential of non-cross-linked Porcine Acellular Dermal Matrix (P ADM; Strattice, Lifecell Corporation, Branchburg, NJ) for abdominal wall reconstruction as compared to Human Acellular Dermal Matrix (HADM; AlloDerm, Lifecell Corporation, Branchburg, NJ). We hypothesize that PADM will have similar characteristics to HADM in respect to its ability to integrate into the native tissue with few adhesions. The specific aim is to compare these two biomaterials in respect to their integration into the native tissue. This comparison will be made through evaluating cellular and vascular density, strength of the biomaterial and the biomaterial-musculofascial edge interface, and the area, grade and types of adhesions formed. Abdominal adhesions are a worldwide health problem which can cause a multitude of pathologies including bowel obstruction, female infertility, enterocutaneous fistulas and bowel perforation. Lysis of adhesion surgeries are frequently required. HADM has been used with advantages, however, they have high costs and bulge potential. PADM would be advantageous over HADM in that it may be more readily available; there can be more stringent quality control; and costs may be decreased. Identifying a prosthetic mesh with greater strength and integration and reduced abdominal adhesions would have a great clinical impact by reducing complication rates and reducing hospital cost.

Composite tissue allotransplantation (CTA) presents a viable option for reconstructing difficult defects resulting from traumatic injury, congenital deformities, and surgical extirpation, CTA offers the advantage of providing an intact, preserved graft without donor site morbidity. A hand transplant is superior to a prosthetic only, in that recovery of sensation and precise neuromuscular function. Similarly, a face transplant is simply a mask if there is no ability for facial expression, sensation, and oral consumption. The overall goal of the proposed research is to improve sensory recovery after CTA through targeting the somatosensory cortex. Central nervous system modulations are key to optimizing sensory recovery after CTA. At the laboratory of W.P. Andrew Lee at the University of Pittsburgh in the Starzl Institute for Transplantation laboratory, preliminary experiments focused on defining changes in somatosensory cortical mapping and neuronal response properties after face transplantation in a rat model. Using electrophysiology, it was found that there n as cortical reintegration of sensory inputs, and thus reinnervation, after CTA. However, there are substantial changes in the pattern of sensory reinnervation in gene mapping from the periphery to the somatosensory cortex. The focus of the current research is therefore to investigate therapeutic interventions to enhance cortical functional restitution after CTA. Therapeutic strategies that mitigate central nervous system remodeling after CTA ultimately will help to improve functional recovery. Cytokines that mediate peripheral and central nervous system modulations that occur after injury may provide means to improve reinnervation and cortical reintegration after CTA. One such cytokine is Erythropoietin (Epo), a naturally occurring cytokine that initiates erythropoiesis. In addition to its hematopoietic effects, it has shown tissue protective activity in both the central nervous system (CNS) and peripheral nervous system (PNS) in vivo. Carbamylated Erythropoietin (Cepo), a synthetic polypeptide represents the prototype of a designer cytokine for therapeutic intervention in central and peripheral nervous system injury without erythropoietin side effects. The specific aim of the proposed study is to investigate the potential for the novel therapy, Cepo, to mitigate central nervous system changes that occur after composite tissue allotransplantation.

The US Surgeon General has reported that diseases of the craniofacial region are among the most common health problems affecting the general population. Devastating head and neck cancer (HNC) single-handedly impose a significant biomedical burden by accounting for 8000 deaths and 35,720 new cases each year in the U.S. alone (Jemal). Most of these patients will necessitate multimodality therapy with surgery, radiation and chemotherapy. Although radiotherapy has significantly improved survival, it drastically impairs bone healing and adjacent normal tissues leading to significant morbidity and thus precluding the use of mandibular Distraction Osteteogenesis (DO) as a viable reconstructive option for HNC. Distraction Osteogenesis, or the creation of new bone by gradual separation of two osteogenic fronts, generates an anatomical and functional replacement of deficient tissue from local substrate. The central hypothesis to be tested in this proposal is that the deleterious effect of radiation on bone formation can be mitigated to allow functional restoration and successful regeneration of the mandible. To test this hypothesis we will utilize our unique and reliable rodent model for DO and the specific metrics of diminished bone quality within the regenerate of irradiated distracted mandibles. We will employ tissue-engineering strategies (human adipocyte derived stem cells transplant or fat grafting) alone and in combination with a pharmacologic agent (Simvastatin), to assuage the adverse impact of radiation induced injury on new bone formation and healing in order to optimize reconstruction and repair. The long-term goal of this proposal is to provide fundamental information that can be translated from the bench to the bedside to lead to improved treatment modalities to this severely compromised patient population.

The effects of Botulinum toxin treatment on facial musculature have previously relied upon subjective, qualitative patient- and physician-reported outcomes. We seek to apply a novel quantitative metric in a prospective observational study. Digital Image Speckle Correlation (DISC) analysis is a non-invasive, sensitive technique that quantifies facial muscle recruitment by detecting subtle deformations of the overlying skin. A photo is taken of a patient volunteer with the face at rest and during a slight facial movement, and the software calculates the force of contraction. DISC quantifies the initial paralysis of each individual muscle group and monitors their return of function over time. Botulinum toxin injections will be dosed to cosmetic effect in the forehead, glabellar, and crow's feet areas. Photos will be taken of patient volunteers immediately before and after treatment using a head stabilizer that standardizes position and distance from the camera, with their faces at rest and then raising their eyebrows, frowning, and blinking. This series will be repeated at one week, two weeks, four weeks, and monthly to six months follow-up. DISC software will then be used to calculate the contractile force of the facial muscles initially and at each subsequent follow-up visit, comparing to the baseline to determine the percentage change over time. We will corroborate our quantitative analysis with patient-reported data using the validated FLO-11Questionnaire and SPA Measure, as well as SGA and FWS scored by plastic surgeons. DISC is a sensitive, non-invasive measure of facial muscle dynamics that provides a novel approach to quantitative, prospective outcomes data to Botulinum toxin treatment. It has the potential to enhance clinical judgment in tailoring treatment to individual patients. Additional applications of this technology include monitoring recovery following nerve repair, or quantifying prospective outcomes data for facial transplant recipients.

Severe peripheral nerve injuries often leave patients with longstanding pain, paralysis and numbness. Longer distances to the target end organs produce worse outcomes. We have recently found that a novel strategy of side-to-side nerve grafting leads to improved regeneration: conducting very small numbers of fibers from an intact donor nerve to an adjacent injured recipient nerve results in enhanced regeneration of the recipient nerve. Very few axons are required to enter the recipient denervated nerve stump to exert the protective effect, suggesting that soluble chemical mediators are likely to play a role. The overarching hypothesis of this proposal is that donor axons sustain the growth permissive state of Schwann cells (SC) in the denervated stump. We will assess this hypothesis by testing the following sub-hypotheses: 1. The protective effect of side-to-side nerve grafts increases with the number of grafts, and that bridging axons regenerate both proximally and distally within the recipient injured nerve. We will use our novel transgenic rats whose axons fluoresce to examine whether the axons regenerate both proximally and distally within the recipient injured nerve. We will also examine using retrograde labeling of neurons the relationship between number of grafts and the magnitude of the protective effect. 2. Locally produced soluble factors from regenerating axons promote SC differentiation and/or proliferation, thus mediating protection of chronically denervated stumps. We will examine whether leading candidate molecules produced by neurons including calcitonin gene related peptide and neuregulin are the prime mediators of protection by blocking their actions with siRNA in vivo. This discovery will likely alter the paradigm by which surgeons will reconstruct devastating proximal injuries. Clinical use of the technique will be optimized by detailed knowledge of its mechanism.

Objective evaluation of surgical education and core competencies is rapidly gaining importance in residency training for numerous reasons: patient safety, reduced resident work hours, mandatory Accreditation Council for Graduate Medical Education (ACGME) requirements regarding skills lab training, and specialty-specific competencies. Although over 40 microsurgical models have been published, no consensus currently exists regarding the best method for skills assessment. With the increased national focus on objective evaluation within a surgical skills laboratory, it is important to provide plastic surgery training programs and residents with an assessment tool that accurately reflects trainees' progress and facilitates constructive feedback. Purpose: We propose to develop and publish an objective tool for evaluating microsurgical skills in a laboratory setting. This project represents a collaborative effort by members of the Association for Reconstructive Microsurgery to create a valid, reliable, and openly available assessment tool measuring microsurgical proficiency. Methods: We will finalize our evaluation form based on input from national microsurgery experts, ensuring face, construct, and content validity. Ten PGY 1-6 residents will be recorded performing a 1mm rat femoral artery anastomosis. Surgeries will be evaluated by 10 microsurgery experts using our assessment tool. Statistical analyses will include internal consistency, inter- and intra-rater variability, and reliability. Multivariant analysis of individual components will eliminate institutional bias and non-predictive elements while reducing redundancies. Significance: A robust assessment tool that accurately reflects microsurgical proficiency is a necessary response to national changes in surgical education. Once validated, we will make the tool freely available, creating an unprecedented opportunity for the inter-institutional conduct of multi-center surgical education studies.

Our group has developed a new way to regenerate skin after major burns and other trauma. The basis for this leap forward is that we have developed technology to significantly expand the surface area of skin available for regeneration compared to conventional skin grafting technology. Creating a device to mince skin into small skin micrografts we are able to release migratory cells into the healing milieu. Using this controlled mincing technology a split thickness skin graft can be reliably expanded over one hundred fold in the operating room within minutes from harvest. In pilot wound experiments, we created micrografts (0.8 x 0.8 mm) consisting of epidermis and dermis and transplanted these micrografts into full thickness wounds with an expansion ratio of over 1 to 100 of micrograft to total wound surface area. In these pilot studies 100 % epidermal healing was achieved within 14 days in healthy, as well as diabetic pigs, compared to 62 % and 49 % respectively in healthy and diabetic controls, proving that the regenerative capacity of the skin graft far exceeds what is harnessed in current clinical practice. We have also found that in moist environments the orientation of the micrograft, dermal side up or down, intriguingly is not dependent upon the initial orientation of the skin micrograft within the wound bed. In this proposed research project, we plan to build upon this technology platform by determining the most favorable size of a micrograft to optimize the skin micrograft's regenerative capacity. Moreover, we want to apply this optimized micrograft size to determine the maximum possible expansion ratio of micrograft to total wounded surface area needed to regenerate a full-thickness wound. Early clinical results have confirmed the utility of this method in the treatment of large burn injuries and other complex wound scenarios.

Upper extremity amputations occur with a relatively high frequency, are disabling, debilitating, and have a high cost to the health care system. One solution to this difficult problem is composite tissue allotransplantation (CTA). Composite tissue allografts (hand transplants) have been performed in multiple centers worldwide. Allograft survival is high, with functional outcomes superior to prosthetic devices. Despite this success, composite tissue transplantation has not reached widespread clinical use because recipients require lifelong high-dose multi-drug immunosuppression to prevent graft rejection. These regimens carry a high risk for metabolic, infectious, and neoplastic side effects that have limited the widespread adoption of CTA. In light of these challenges, we propose a model of targeted immunomodulation based on experience from solid organ transplantation performed with the "Pittsburgh Protocol". This protocol utilizes non-ablative induction with alemtuzumab, followed by donor bone marrow cell infusion and low-dose immunosuppression using a single drug, tacrolimus. In human solid organ transplant recipients treated with this protocol, immunosuppression was reduced to minimal levels or even withdrawn completely. To translate this novel concept to composite tissue transplantation, our laboratory has developed a preclinical model of heterotopic hindlimb transplantation in Yucatan miniature swine. We were able to successfully translate the Pittsburgh Protocol to this preclinical model, but have not had complete success with long term tolerance; muscle and bone are accepted indefinitely, but skin eventually rejects. In an effort to further minimize systemic immunosuppression and maximize allograft survival we propose to use a novel technique, costimulatory blockade with custom-designed fusion proteins to achieve operational tolerance. The goal of this proposal will be to demonstrate tolerance in a preclinical model of composite tissue allotransplantation.

Composite tissue allotransplantation helps improve the lives of patients with mutilating injuries of the hand and face. It's use is limited due to the need for systemic immunosuppression. Currently, clinical protocols using bone marrow cell infusion have allowed a reduction in the amount of immunosuppression these patients need to take. Future advances in cell based therapies will require a deeper understanding of the immunomodulatory affects of bone marrow derived cells. The current proposal will extend our understanding by comparing the use of bone marrow derived stem cells from donor, recipient, and third-party sources. We will study their ability to delay rejection of a hind limb transplant in rats as well as study their direct effect on a mixed lymphocyte reaction essay. These results will help us design new cell-based therapies in the future, help make CTA more available to patients, and extend the donor pool of mesenchymal stem cells for these patients.

Specific Aims: To study conditions which may favorably alter tendon biomechanics. Our preliminary data has shown that release of the A1 pulley at the time of tendon repair decreases forces across tendons, compared to repaired tendons without alteration of the pulley system. Techniques performed at the time of tendon repair (including A1 pulley release) may decrease the forces across tendons in the postoperative period, allowing more aggressive rehabilitation protocols, and decreasing rupture rates. Research Methods: Cadaveric hands with the distal forearm intact will be obtained, flexor tendons will be dissected in the forearm, the hands will placed in a tensile testing machine, force excursion curves will be generated for various testing conditions. Force excursion data is then used to calculate work of flexion, which is an extremely sensitive method of quantifying all forces that resist tendon gliding. Testing conditions will include but not be limited to, 1. uninjured tendon with A1 pulley intact, 2. uninjured tendon with A1 pulley divided, 3. profundus tendons lacerated and repaired with A1 pulley intact, 4. profundus & superficialis tendons injured and repaired with A1 pulley intact, 5. injured & repaired tendons (including both profundus and superficialis) with partial release of A1 pulley, injured and repaired tendons (including both profundus and superficialis) with complete release of A1 pulley. Loads will be applied to the tendons to simulate passive & active motion protocols. The clinical impact of this study is significant because release of the A1 pulley at the time of tendon repair would be a simple, low cost, maneuver which can be employed by hand surgeons to improve outcomes and avoid rupture postoperatively.

As researchers identified the pluripotentiality of a cell line within adipose tissue known as preadipocytes, these cells came to be known as adipose-derived stem cells (ADSCs). Ease of ADSC harvest, compared to bone marrow aspiration, gave way to research studies looking to utilize ADSCs in a variety of tissue regenerative applications. Plastic surgeons have learned to refine the process of harvesting fat grafts, and to use these grafts for soft tissue augmentation of the face, breast, etc. While contour restoration remains the primary objective of fat grafting, anecdotal reports of skin rejuvenation and scar softening overlying grafted fat are frequently noted. Many of these reports suggest such effects result from the impact of ADSCs on overlying skin. Objective evidence of ADSC contribution to epithelial and dermal cell lineages, however, is lacking. Despite this, researchers have been able to identify ADSC's role in neovascularization, through both angiogenesis and vasculogenesis. While these secondary roles are notable, they do not specifically demonstrate how fat grafts improve skin composition. The purpose of this study is to histologically describe how ADSCs contribute to skin cell lineages, after being grafted into a subdermal plane. To our knowledge, no study has attempted to identify the differentiation of ADSCs towards skin cell lineages after subdermal implantation. Identification of donor fluorescent ADSC cells, as differentiated skin cells in recipient nude mice, aims to validate claims of ADSC's contributions to skin rejuvenation. Objective data, demonstrating cellular contribution of fat grafts to overlying skin, may validate the potential use of sub-dermal fat grafts for management of sclerotic skin conditions (i.e. scleroderma). Validation of future studies directed towards scleroderma "skin rejuvenation", through fat grafting, offers a novel treatment strategy that may significantly alter how these patients are managed.

Achieving hemostasis during surgery is vital to a successful outcome. In cosmetic surgery even small amounts of extravasated blood can lead to increased bruising, pain and excessive swelling producing sUboptimal outcomes. Currently available topical hemostatic products such as Tisseal TM are limited in their applicability as a result of their significant cost and potential to transmit infectious disease or cause allergic reaction. Clearly, the development of a safe, effective, and inexpensive topical hemostatic agent would be a significant clinical advance and improve cosmetic surgical outcomes. Furthermore this work promises significant benefit to many areas beyond cosmetic surgery, including reconstruction, surgical oncology, and trauma. The objective of this study is to assess the efficacy of a novel injectable hydrogel based on polyethylene glycol and a polycarbonate of dihydroxyacetone (PEG-pDHA), in achieving hemostasis when applied topically in a clinically relevant rat model of hemorrhage due to traumatic liver injury. This compound is manufactured from dihydroxyacetone (DHA), which occurs naturally in the human body as part of the glycolytic pathway. Our preliminary studies characterizing this polymer demonstrate that PEG-pDHA may have bioadhesive properties. This material is non-inflammatory and non-immunogenic as shown in previous in vivo animal studies and has a superior safety and biocompatibility profile compared to some commonly used topical hemostatic agents. We hypothesize that PEG-pDHA gel is equally or more effective as a topical hemostatic agent than currently available hemostatic products.

Our long term efforts are concentrated on facilitating growth and signal transduction through a neuroprosthetic "living interface" which will provide fine motor control and sensory feedback to patients with artificial limbs. The interface is located where the peripheral nerve stump exchanges ionic signals with wiring for electronic prosthetic controllers. In our preliminary studies we found that highly electroconductive polymer- PEDOT (poly 3,4-ethylenedioxythiophene) supports electrical signal transmission through a 2 cm nerve gap in situ. We believe PEDOT can play a major signal processing role if it is in contact with a surviving nerve stump. In this study we examine the influence of PEDOT on peripheral nerve regeneration and the end organ reinnervation process. We form a 1.5 cm gap in the rat peroneal nerve and reconstruct this nerve gap with decellular nerve which is polymerized with PEDOT. Compound muscle action potentials are recorded at monthly intervals during the postoperative period until POD #90. During recovery, the nerve fibers are expected to regenerate through the decellular nerve material. At POD #90, muscle contractile tests are utilized to determine the degree of functional recovery following the nerve grafting procedures. Qualitative and quantitative characteristics of regenerating nerve fibers in the presence of PEDOT are revealed by both light and scanning electronic microscopic analysis of the constructs taken from the reconstruction site. With this study we will demonstrate the effect of PEDOT on regenerating nerve conductivity, morphology, and end organ function. Results of this work will contribute to furthering our understanding about peripheral nerve health in the presence of conductive polymers used in conjuction with biomaterials. Our next step is to test PEDOT as a component of the "living interface" with an animal amputation model. With favorable results, it is possible that the study of a "living interface" may reach clinical trial.

The prolongation of survival afforded by radiotherapy in Head and Neck Cancer (HNC) patient management continues to be paralleled with the costly incidence of bone related pathologies. Despite our efforts, the incidence of osteoradionecrosis (ORN), late pathologic fractures, and non-unions in this setting remains largely unchanged. It is estimated that 5 to 10 percent of patients undergoing high dose radiotherapy for HNC will develop ORN and its debilitating complications. It is commonly accepted that the pathologic effects of radiation on bone formation and healing are mediated through the mechanisms of vascular damage, direct cellular depletion and diminished function of the existing cells responsible for the generation and maintenance of osteogenesis. The global hypothesis of this proposal is that the mechanisms of vascular damage and diminution in cellular number and function can be prevented or reversed with therapeutic manipulations to allow for prophylaxis of ORN and radiotherapy induced non-unions. To investigate this hypothesis, we propose the utility of Deferoxamine—an angiogenic factor, and Amifostine—a cellular radioprotective agent. The effects of these agents will be explored in our unique murine model of radiotherapy-induced non-union after mandibular fracture repair. Our methods will consist of the irradiation of four groups of Sprague Dawley rats divided as follows: No therapy, Amifostine (AMF) pre-treatment, Deferoxamine (DFO) therapy, and a combination of AMF and DFO. All groups will undergo mandibular fracture with external fixator repair and will be allowed to complete a 40-day consolidation period according to our established protocol. Upon completion, detailed clinical and gross assessments will be utilized for the development of ORN. Micro-Computed Tomography (µCT) and Histology will be utilized to analyze bone quality, cellularity and mineral production.

The incidence of contracture formation in burns is ~ 40%. There are no effective treatments to prevent contractures. Contractures continuously shrink over time as a result of cell contractility. Cell contractility is caused by actomyosin force generation. There are three forms of actin: β-cytoplasmic (β-actin), γ-cytoplasmic (γ-actin), and α-smooth muscle actin (α-SMA). αSMA is the characteristic form of actin present in contractile myofibroblasts. But the three actin isoforms may substitute for each other to cause contracture formation. We will test the hypothesis that β-actin and γ-actin can substitute for αSMA to promote scar contracture and fibroblast contractility. The long-term objective is to develop a drug to prevent and treat contracture formation. Specific Aim 1 To characterize the specific role of αSMA within the inflammatory, proliferative, and remodeling phases of repair that cause scarring in vivo. Wound models representative of all phases of wound healing will be performed in wild-type, αSMA KO, and heterozygous mice for αSMA. The wounds will be comprehensively analyzed using methods aimed at the unique charactistics of each phase. These will include gross appearance, rate of contraction, histology, and immunohistochemistry. qRT-PCR will be used to quantify the actin isoforms. And wound breaking strength will also be used to assess the remodeling phase. Specific Aim 2. To determine how α-sma, β-actin, and γ-actin expression alter fibroblast contractility. This Specific Aim will transfect α-sma KO fibroblasts with β-actin and γ-actin siRNA to determine whether β-actin, and γ-actin can substitute for α-sma to produce the same contractile forces. Contractile forces will be measured using fibroblast populated collagen lattice contraction, and fibroblast migration assays.

Traumatic neuroma-in-continuity (NIC) result in profound neurological deficits and its management poses the most challenging problem to peripheral nerve surgeons today. Clinically prototypical NIC is encountered in brachial plexus injuries, with close to 1/20 motorcycle and snowmobile accident victims sustaining brachial plexus injuries. Early diagnosis of NIC remains remains a challenge and delayed repair strategies are employed with and often limited success.To our knowledge no rodent or other animal model has yet been refined or validated to reproduce NIC injuries by employing physical forces akin to those responsible for the majority of injuries encountered clinically. The novel traumatic neuroma-in-continuity injury model in rodent sciatic nerves, developed in our laboratory requires further characterization. Employing a combination of traction and intense compression forces, we were able to consistently reproduce the histological characteristics of NIC. We compared the behavioral effects of our sciatic nerve NIC injury model, to the established simple sciatic crush injury and demonstrated significantly worse functional recovery in the NIC group. A larger study is required to properly compare our NIC model also with nerve transection injuries in order to characterize relative axonal misdirection and functional deficits. Retrograde motor neuron labeling from distal sciatic nerve branches will enable us to quantify and compare misdirection of motor axons. Serial skilled locomotion tasks up to 8 weeks post injury will allow us to quantify and compare functional deficits between study groups. A validated simple small-animal model for NIC would be invaluable to make this elusive and devastating injury amenable to basic science research. Our relatively simple and accessible model, once further validated, would be a useful tool to investigate and develop new diagnostic techniques and intervention strategies to help improve our patient outcomes.

Despite the fact that early and intermediate functional outcomes after composite tissue allotransplantation (CTA) are highly encouraging rejection and the need for high-dose multi-drug immunosuppressive treatment continues to be the bane of CTA preventing wider clinical application. However, a better understanding of the mechanisms and identifying surrogate markers of rejection or acceptance in CTA by exploring novel methods to diagnose skin rejection could fulfill the important clinical need to enable reduction of systemic immunosuppression in CTA. This study therefore proposes to establish a non-invasive method for immune monitoring in a murine CTA model, based on novel commercially available dermal tape stripping technologies. While such methods have been used primarily in dermatology to assess skin inflammation, and other skin-related pathologies, measurement of inflammatory cytokines profiles captured in these tapes by means of Luminex might have great potential for extension of this technology into development of a clinically relevant non-invasive immune monitoring tool for acute rejection in CTA. Samples will be collected using a rat hind-limb transplantation model from both transplantated and native legs by tape stripping (Sebutape & D-Squame) and conventional skin punch biopsy. Cytokine expression profiles (protein and mRNA) will be analyzed using Luminex assays and compared between tape stripping and biopsy methods. Tissue samples will be also stained for the presence of T-lymphocytes (CD4, CD8, Foxp3), B-lymphocytes (CD20), macrophages (CD68), and other cell types that become of interest as a result of the Luminex data analysis. Selection of specific stains will consider Luminex and histology analysis results to assist in gaining the maximum mechanistic information possible. Predictive computational models for rejection, developed on existing preliminary data, will be calibrated to the data gathered by tape stripping then assessed for predictive preci

The broad goal of this project is to measure treatment outcomes in adolescent patients with benign breast disorders such as macromastia, gynecomastia, breast asymmetry, and tuberous breast. These disorders are common in adolescent males and females and are a significant source of morbidity and psychological distress. There are limited outcome data on adolescents with benign breast disorders and no prospective studies have been conducted in this population. As a result, there are no evidence-based treatment guidelines for pediatricians and plastic surgeons. The described study is the first of its kind to prospectively study long-term functional and psychosocial outcomes following treatment of male and female adolescents with benign breast disorders. Benign breast disorder patients enrolled in the study will complete validated questionnaires regarding self-esteem, quality of life, and eating attitudes prior to and at several time points following surgical treatment. Females with macromastia will complete an additional survey on breast symptoms. A control group of healthy male and female adolescents will also complete the surveys at similar timepoints. The control group will complete an additional survey at baseline to assess breast and/or chest symptoms. This study will provide new insight into these common problems in adolescents and will provide the foundation for studying long-term outcomes in this population. This study will ultimately contribute to the development of evidence-based treatment algorithms to optimize patient care.

This study aims to investigate the efficacy of autologous fat grafting for treatment of scleroderma induced skin changes in a mouse model. To date, no cure exists for scleroderma, with a quoted figure of 55% survival at 10 years for the diffuse cutaneous form. Skin changes are characterized by diffuse fibrosis and thickened dermis with profound inflammation. Recent studies have shown that injected adipose tissue has both a volumetric effect and a regenerative effect on dystrophic skin, which has been postulated due to be due to the presence of multipotent mesenchymal stem cells which promote local vascularization and healing. Our hypothesis is therefore that autologous fat grafting will have a beneficial effect on cutaneous manifestations of scleroderma through probable mechanisms which include: a)Neovascularisation and reversal of skin fibrosis through local paracrine signaling by injected adipose cells b)Reversal of inflammatory changes through local paracrine signaling by injected adipose cells c)Downactivation of aberrant SSC fibroblasts and abnormal signaling pathways such as the TGF-B/Smad-CTGF axis We aim to investigate this question through a well-established mouse model of scleroderma, created through serial subcutaneous injections of bleomycin. Autologous fat grafting will be performed, and assays performed which aim at detecting the following: a)Local skin changes: decreased skin thickness, collagen content, tissue fibrosis b)Reversal of typical SSC skin changes: manifestations of inflammation and dermal fibrosis c)Detection of alterations in signaling in the TGF-B/Smad-CTGF axis d)Survival of transplanted fat graft This project has clinical relevance to plastic surgery because fat grafting has potential to result in both a tactile and visual change in skin quality for scleroderma patients, and would be particularly relevant in areas such as the face and hands, which are areas of high aesthetic concern, marking the patient with stigmata.

Dupuytren's Contracture, or Dupuytren's disease, is an inherited condition of the hand that causes one or more fingers to curl permanently onto the palm. Surgery to remove the contracted tissues to restore hand function fails to prevent recurrence of this disease in at least 3 out of every 10 patients. Our research group has identified a novel and previously unrecognized molecular pathway by which cells in the palm are induced to grow abnormally and become contractile, the insulin-like growth factor pathway. This pathway is also active in several different types of cancer, and cancer researchers are currently trialing inhibitors of this pathway as potential therapies. Our research findings suggest that inhibitors of the insulin-like growth factor pathway may be beneficial to patients suffering from Dupuytren's disease. This project will test that possibility by isolating cells from the hands of patients undergoing surgery for Dupuytren's disease and growing these in conditions that promote growth and tissue contraction, in the presence or absence of insulin-like growth factor inhibitor. If successful, this project will identify a new approach that, in combination with hand surgery, may prevent recurrence of these debilitating contractures.

Composite tissue allotransplantation (CTA) is dramatically expanding the capabilities of reconstructive plastic surgeons. Pediatric craniofacial patients offer unique reconstructive challenges as the surgeon must account for growth and development in designing immunosuppressive regimens and operative plans. We endeavor to employ our institution's novel immunomodulatory monotherapy in the pediatric setting in an effort to minimize immunosuppression-associated morbidities thus rendering the risk/benefit ratio of CTA favorable in this population. An understanding must be developed of a craniofacial transplant's potential to facilitate normal craniofacial growth in the pediatric recipient. The Specific Aim of this study is to demonstrate that immunomodulatory monotherapy and craniofacial transplantation is compatible with normal somatic development and craniofacial growth in skeletally immature recipients. We hypothesize that allogeneic Le Fort II transplantation in a juvenile rat model using immunomodulatory monotherapy will result in normal somatic development and craniofacial growth. We will begin by assessing the effect of immunomodulatory monotherapy on juvenile rat somatic growth and craniofacial development in comparison to control data. Next, craniofacial growth will be compared between controls and juvenile rats undergoing syngeneic Le Fort II CTA; as this group will not require immunomodulation, it will serve as a surgical control. Finally, the combined effects of allogeneic Le Fort II CTA and immunomodulatory monotherapy on craniofacial growth will be assessed by comparing juvenile rats undergoing this procedure to controls. The potential impact of this work on Plastic Surgery is twofold. First, it would provide early data for the suitability of immunomodulatory monotherapy in the pediatric setting. Second, it would serve as proof of concept that craniofacial transplantation is compatible with normal craniofacial growth and development.

The fabrication of a bioreactor capable of generating compressive forces and active on-board feedback control to induce a chondrogenic phenotype in an animal model with autologous tissue has not previously been attempted. We plan to create a bioreactor that allows a trackable, non-destructive assessment of engineered tissue in vivo. This proposal also lays the groundwork for studying multiple other parameters that are of interest in generating cartilage (including fibrillin and elastin production, oxygen tension, and calcium concentration). In sum, this project will be part of a larger vision of making significant advances in the fields of tissue engineering, bioreactor construction, and the generation of clinically useful, flexible cartilage initially using a small animal model, and transitioning to a large animal model to complete preclinical trials in anticipation of clinical use.

Breast cancer affects one in eight women. Patients may undergo a variety of surgical options including local excision, partial mastectomy or complete mastectomy. Reconstruction varies depending on the surgical defect. A growing number of plastic surgeons are using fat injection techniques to restore post-mastectomy defects. In any given fat harvest site (abdomen, flank, buttock, etc.) there is an ample supply of adipocytes and a small percentage of adipocyte derived stem cells (ADSC) that can be purified from lipoaspirates. ADSCs have potent angiogenic and regenerative properties capable of treating numerous medical and surgical problems. However from a safety standpoint, it is not known whether these beneficial properties of ADSCs for reconstruction could have detrimental effects on any remaining breast cancer cells post-resection, or if ADSCs could promote transformation of normal breast epithelium. The purpose of this study is to investigate the safety of using ADSCs when exposed to breast cancer cells. We hypothesize that adipocyte stem cells influence the growth and metastasis of breast cancer whereas primary lipoaspirates will not affect tumor progression. Aim 1: in vitro biochemical analysis of ADSCs versus lipoaspirate co-culture with breast cancer cells measuring cancer cell proliferation, migration, invasion, and pro-angiogenesis ability. Aim 2: in vivo analysis of ADSCs versus lipoaspirate on breast cancer xenograft growth and metastasis in immunodeficient mice. The completion of this study will generate the critical mass of data needed to initiate larger, extramural funded studies for the timely surgical topic of fat injection procedures and the potential impact on cancer. This completely novel study is one that could only be implemented by a unique interdisciplinary research team of a reconstructive surgeon (Dr. Chiu), cancer biologist (Dr. Rowan) with participation of stem cell biologists (Drs. Bunnell and Gimble).

While two-stage tissue expander/implant (TE/I) technique is an excellent reconstructive option in well-selected mastectomy patients, the primary drawback, however, is that it requires two separate procedures under general anesthesia and multiple office visits for expander inflation to create the breast mound. While the effect of acellular dermis on various clinical outcomes in the setting of two-stage TE/I reconstruction is currently investigated by a multi-center randomized controlled trial led by McCarthy, the use of acellular dermal matrix in one-stage prosthetic breast reconstruction has been reported only by a few small case series. Thus, in order to rigorously evaluate the use of acellular dermal matrix in single-stage reconstructions, we propose a multi-center, randomized controlled trial designed to study patient satisfaction and quality of life (QOL) associated with the use of acellular dermis in one-stage, implant reconstruction compared to the traditional, two-stage, TE/I reconstruction. By eliminating the intermediate process of tissue expansion and converting the conventional two-stage procedure to one-stage with the use of human acellular dermis, we hypothesize thatpatient satisfaction and quality of life will be significantly improved following skin-sparing mastectomy. In addition, other important implications for successful single-stage immediate breast reconstruction include: 1. Decreasing the surgical wait-list for primary cancer cases; 2. Increasing the efficiency within the clinic; 3. Expediting the patient's return to productivity; 4. Easing the overburdened hospital OR usage; and 5. Potentially offloading the financially strained healthcare system by eliminating the cost of tissue expanders, serial expansion with clinic visits, and a second OR under general anesthesia. To facilitate valid post-hoc comparisons between our outcome measures and those used by McCarthy et al, we have intentionally set up our research design (participant eligibility, treatment plan, surgical technique using the acellular dermis, timing of postoperative evaluations) to mirror their protocol. This will allows us to directly compare the intervention effect on QOL and patient satisfaction scores between the two studies. The conclusion generated by comparing the confidence interval of effect size, therefore, would help answer the question of whether patient-satisfaction is improved by the use of acellular dermal matrix alone or its ability to convert a two-stage reconstruction to one-stage. Therefore, our proposed study follows the natural progression of scientific research with a multi-center RCT on the safety and patient-perceived benefits derived from the use of acellular dermis in the novel setting of one-stage implant reconstruction.

Breast reconstruction using the free muscle-sparing transversus abdominus myocutaneous (TRAM) or deep inferior epigastric perforator (DIEP) flap are common methods for restoring mastectomy defects for breast cancer patients. Despite its increasing popularity and safety, the abdominal donor site remains a major source of postoperative pain. Adequate pain control is important as it has been shown to reduce serious medical complications, in-hospital death, shortens hospital stay, and lowers health-care costs. The current postoperative pain relief protocol consists primarily of a patient-controlled anesthesia (PCA) device delivering intravenous opioids. Opioids can cause numerous side-effects such as sedation, headache, nausea, vomiting, breathing difficulties, bladder and bowel dysfunction. A promising approach to provide postoperative pain control of the abdominal incision is the newly developed transversus abdominis plane (TAP) peripheral nerve block. Although the TAP block has been found to be an effective pain-relief following major abdominal surgeries, its use has never been studied for breast reconstruction using abdominal tissue. Therefore, we propose this study to rigorously test the effectiveness of a TAP catheter delivering continuous local anesthetic in reducing postoperative abdominal pain following abdominal tissue breast reconstruction. This study has significant potential implications in improving both clinical care and health outcomes in patients undergoing microsurgical abdominally-based breast reconstruction.

Repair of calvarial defects in children is difficult. Autologous bone is limited and alloplastic substances are not advocated because they can become unstable or inhibit cranial growth. Although split cranial bone is the preferred material for inlay cranioplasty in children, it cannot be harvested in patients less than six years of age because of the lack of a diploic space. Particulate bone graft, consisting of small corticocancellous bone pieces harvested with a hand-driven brace and bit, can be obtained from the cranium without a diploic space. We previously have shown that this material heals full-thickness cranial defects experimentally and clinically when placed over normal dura (primary cranioplasty). However, the ability of particulate graft to heal inlay defects with scarred dura (secondary cranioplasty) has not been studied. Particulate graft may be less effective when used over an injured area (e.g., infection, prior failed cranioplasty), compared to being applied over normal dura. The specific aim for this project is: Aim: To determine whether particulate bone graft heals secondary inlay cranioplasty defects. Using a leporid model, critical-size calvarial defects will be created and treated with (1) no implant for 32 weeks, (2) particulate bone for 32 weeks, (3) no implant for 16 weeks followed by particulate bone over the scarred site for 16 weeks. Animals will undergo CT to determine ossification. The results of this study will be immediately translatable to patient care. If particulate graft can heal defects with fibrotic periosteum and dura, it would become the first-line technique for secondary cranioplasty in young children.

Defects requiring craniofacial reconstruction, especially in children, are often complicated by a limited supply of bone. Tissue engineering is a potentially valuable strategy in repairing these deformities. Some of the most promising results have come in the form of BMP-2-generated endochondral bone. However, most calvarial bone is intramembranous in origin. BMP-2-based successes in cranial bone generation are therefore essentially creating ectopic bone at an orthotopic site, and it is not certain that this endochondral bone is ideally suited to respond to local environmental cues. The central hypothesis of this study is that by leveraging emerging bioprinting technologies to deposit precise doses and patterns of proteins known to play a key role in intramembranous ossification, we can enhance existing molecular osteoinductive pathways for calvarial defect repair. Specific Aim 1 is to compare the efficacy of different doses of TGF-B1 and TGF-B2 bio-printed on acellular dermal matrix (ADM) in generating intramembranous bone to repair calvarial defects. We will achieve Specific Aim 1 by testing the osteoinductive capacity of different doses of TGF-B1 and TGF-B2 bioprinted on ADM in a rabbit calvarial defect model. Specific Aim 2 is to evaluate the utility of printing FGF in combination with the ideal dose of whichever TGF-B isoform proves most osteoinductive in Specific Aim 1. We will achieve Specific Aim 2 by printing variable doses of FGF in combination with the ideal dose of the most osteoinductive TGFB isoform identified in Specific Aim 1 in an effort to produce an intramembranous osteoinductive synergy between the selected TGF-B isoform and FGF. The potential impact of this work is the development of a tissue engineering strategy that enhances local calvarial osteoinductive pathways to physiologically heal bone defects in the craniofacial skeleton. Calvarial reconstruction would no-longer be complicated by donor site morbidity and tissue shortages.

Tissue expansion and implants are widely used for breast reconstruction after mastectomy; however, clinical application has been greatly limited by adjuvant radiotherapy. Progressive damage of the overlying skin not only leads to low expansion efficiency, but also poor aesthetics and increased complications. Identifying a therapy that may be tissue protective or enhance tissue regeneration during expansion in spite of radiotherapy may decrease the poor sequelae encountered by these patients during breast reconstruction. Interest in adipose-derived mesenchymal stem cells (ADSC) in promoting tissue regeneration is currently under investigation. However, its effect on skin regeneration during tissue expansion in the setting of a radiated field has yet to be determined. We hypothesize that ADSC transplantation will decrease tissue damage and enhance the efficiency of tissue expansion in animals treated with radiotherapy. To test this hypothesis, a rat model will be used, wherein tissue expansion will be undertaken in the setting of irradiation. ADSCs will be transplanted into the experimental group after radiation. The effects on tissue regeneration will be measured by evaluating ADSC differentiation into various regenerative skin components and quantifying ADSC paracrine secretion of growth hormones.

The goal of this project is to investigate the function of the sirtuin genes in aging human primary adipose tissue, to determine the epigenetic modifications leading to adipose senescence, and to identify specific epigenetic targets for stem-cell based regenerative medicine interventions. While adipose tissue plays a central role in metabolism, caloric restriction pathways, and is the largest known reservoir of adult stem cells, the epigenetic factors resulting in human adipose senescence are poorly understood. This work aims to broaden our understanding of normal tissue aging and advance the treatment and prevention of age-related pathologies. Specifically, the proposed work will: 1) validate a functional role for the sirtuin genes in human adipose aging; 2) establish critical sirtuin modifiers and target proteins that are specific to human adipose aging by sirtuin ChIP-seq analyses; 3) determine whether positive and negative sirtuin regulators can recapitulate youthful or aged phenotypes in human adipose-derived stem cells. Understanding the mechanisms of sirtuin gene activity in human aging through the study of primary human adipose tissue as proposed by this work will expand our understanding of human cellular and tissue aging, ultimately leading to advances in stem-cell based regenerative medicine and reconstructive surgery therapies.

Obesity is one of the most important known preventable causes of cancer. Emerging evidence suggests that cytokines secreted by adipose tissue may be critical drivers of ER-breast cancer. One key adipose-derived cytokine that is increased in obese women is interleukin-6 (IL6). IL6 induces epithelial-to-mesenchymal transition (EMT) and has been recently shown to promote stem cell turnover. Activation of IL6-signaling predicts aggressive disease and a poor outcome in ER- breast cancer. This project will consist of 3 concise aims. In aim 1, I will test for activation of IL6-network signaling in pre-cancer (atypia) and invasive cancer from women who are normal weight, overweight, and obese. To accomplish this I will perform microdissection and IL-6 signaling profiling on tissue samples obtained from a cohort of 150 high-risk premenopausal women. In aim 2, I will investigate whether increased mammary adipose IL6 production results in activated epithelial IL6-network signaling in obese women with pre-cancer and invasive cancer. To accomplish this I will test whether levels of adipose IL6 from patients with pre-cancer and invasive cancer correlate with BMI. I will then compare adipose IL6 levels with epithelial expression of IL6 network proteins. In aim 3, I will perform a pilot trial to determine whether: 1) supervised exercise or 2) bariatric weight loss surgery or 3) body contouring plastic surgical procedures decreases adipose generated IL6 and epithelial IL6 network signaling. As a future investigator, I hope to study the interaction of adipose tissue and breast cancer initiation to identify modes of prevention. This study will have a direct impact on the way in which plastic surgeons view the interaction between adipose tissue and breast epithelium. Both in terms of the current practice of injecting fat into breast epithelium for augmentation or reconstructive purposes as well as the current practice of adipose reducing surgery.

There has been a strong national push to improve health care quality. However plastic surgery care has not been systematically reviewed. This project plans to look at factors impacting the quality of plastic surgery by assessing what hospital characteristics are associated with better outcomes. We will study three index plastic surgical procedures: cleft palate repair, autologous breast reconstruction, and digit replantation. These procedures represent a broad spectrum of the plastic surgery field and also require a hospital stay. Our analysis will assess for associations between hospital factors and the following quality indicators: mortality, length of stay (LOS), and inpatient complications rates. We will utilize data from the health care utilization project (HCUP) and the American Hospital Association (AHA) to run models looking at what hospital factors are associated with our quality indicators for each index procedure from a national perspective. Our secondary aim is to examine if volume is associated with shorter LOS, lower mortality, and lower complications for our index procedures. Hospital volume has been found to be associated with better quality for several surgical procedures leading to the implementation of high volume centers. Yet this is not true for every surgical technique. We will use our HCUP data to compile a dataset of our index procedures. We will then use the AHA ID number to calculate annual volume for each index procedure for each hospital and assess if high volume hospitals are associated with shorter LOS or lower complication rate.

With an estimated 230,480 newly diagnosed invasive cancer cases and 57,650 in-situ cases in 2011, breast cancer is the number one cancer diagnosed in women. Since breast conservation surgery (BCS) with radiation, mastectomy (M), and post-mastectomy breast reconstruction (BR) have similar recurrence rates and survival, evaluating health related quality of life (HRQOL) outcomes and patient satisfaction in women who have had these procedures is necessary to facilitate the surgical decision making process and to maximize short and long-term health of those facing a breast cancer diagnosis. With the increase in the rate of prophylactic mastectomies and the persistent 25% of patients choosing mastectomy, a large proportion of women are candidates for BR. However, rates of BR remain low at a mere 8% of those undergoing breast cancer surgery. This may be related to insufficient knowledge of the value of BR on short and long term outcomes; particularly compared to the other procedures. Current studies about this are limited by small patient numbers, few institutions, and the use of surveys that are not specific to breast cancer patients or their procedures. What is needed are patient-reported outcomes which are collected using targeted items and scales specific to the surgical groups. We propose a study of breast cancer survivors enrolled in the Susan Love/Avon Army of Women foundation which consists of 360,758 women interested in accelerating breast cancer research; 29% of whom are survivors. We aim to evaluate and compare HRQOL and patient satisfaction in those who had BCS, M, and BR and to evaluate the effect of time on these outcomes. The long-term goal of this study is to improve the treatment of patients by providing the evidence needed to guide surgical decision making and influence disparities in breast cancer care. As quality metrics and equal access to care become increasingly central to healthcare reform, such data will be crucial for physicians and patients.

Craniosynostosis is premature fusion of the cranial sutures. The cause is unknown, and while it is associated with numerous syndromes, most cases are sporadic. We possess an ideal model to investigate its causes and treatments, a colony of rabbits with familial, non-syndromic coronal craniosynostosis. Tissue interactions within sutures maintain their patency. Elucidating the gene expression of suture tissues will help us to understand these interactions and why sutures fuse. This will aid in designing tissue engineering strategies to treat craniosynostosis. Tools to study this were previously lacking. Cell culture, immunohistochemistry, and in situ hybridization have been used but have limitations. Laser capture microdissection (LCM) isolates small, homogenous cell populations from tissues. Gene expression in these samples is quantified using real time PCR (rtPCR). This allows exact localization of gene expression and accurate quantification for comparison between normal and synostotic tissues. As craniosyostosis is a condition of abnormal bone formation, we expect craniosynostotic rabbit tissues to exhibit a more mature osteoblast phenotype. To test this, we will obtain coronal sutures from wild-type and synostotic rabbits. Samples of dura, periosteum, osteogenic fronts, and suture contents will be isolated by LCM. We will perform qPCR using probes for genetic markers of osteoblast differentiation. We will determine relative expression levels of each gene with qPCR and compare the tissue samples of wild-type and synostotic rabbits to assess their degree of osteoblast maturation. Many signaling factors have been implicated for their role in synostosis. To investigate the role these play in our rabbits we will do qPCR on wild-type and coronal suture LCM samples with primers for these genes, and demonstrate if their expression is different in the synostotic rabbits. Any differences observed will offer insight into the processes causing premature fusion.

The repair of injured cutaneous tissue is a fundamental biological sequence essential to the continuity of life. However, the potential for dysregulation and overexuberance leading to hypertrophic scar is a serious clinical problem with a lack of good therapeutic options. Hypertrophic scarring can result in severe disfigurement, life-threatening restriction of motion, and disabling pain. Research in the field of facial scarring has been limited to the effect of incision location, and to the efficacy of delayed treatment on already formed hypertrophic scars. However, tension during early scar formation is an important contributor to hypertrophic scarring, and the tension in the face is compounded by the action of the muscles of facial expression. Only recently have targeted interventions been applied, including the use of botulinum toxin to decrease the tension created by facial musculature adjacent to incision sites. In spite of advances in our understanding of the acute mechanisms of wound repair, the role of tension on the molecular signals that culminate in pathologic scar formation remain mostly unknown. The exciting new field of “mechanotransduction” tries to elucidate the process by which cells sense mechanical forces and convert these forces to biochemical signals. This process has been difficult to study because of the absence of hypertrophic scarring in most animals, and the lack of applicability of in vitro systems. We propose a novel animal model for testing the effect of cyclic tension on cutaneous scarring. We aim to show that we can produce not only stretching of the scar, but also an increase in scar height through the use of a novel cyclic tension application device. We believe that our research will lead to an improved mechanistic understanding of the role of cyclic tension in surgical scars. Further, we believe that this understanding will provide a foundation for new interventions, both operative and medical, to decrease facial scarring.

Critical bony defects are those that can't heal spontaneously due to their large size. They arise from trauma, both military and civilian, from non-union following a fracture or osteotomy, or may be secondary to ablative surgery for cancer. These defects may be repaired using vascularized autograft harvested from elsewhere in the body. However, there may be inadequate bone available, and autograft harvest carries significant donor site morbidity. The primary alternative material available to reconstructive surgeons is processed cadaveric bone, or allograft. Allograft is particularly useful for repair of large, load-bearing defects; however it is limited by a lack of vascularity. Without a blood supply allograft bone fails to grow or remodel in vivo, leading to weakening of the graft and ultimately fracture, with failure rates near 50% after 10 years. Laboratory-based bone tissue engineering strategies have made great strides but have not yet been successful for clinical applications due to an inability to produce adequate volumes of bone and to become integrated into the host's vascular network. Our goal is to engineer large volumes of vascularized bone, ultimately providing a new standard for repair of critical bone defects. Our preliminary data suggest that this is possible. We previously applied basic principles of bone tissue engineering to create large volume, well-vascularized bone in vivo, using allograft as a scaffold. This process is called allograft revitalization. This proposal will investigate the mechanism of allograft revitalization using our established hemi-mandible porcine model, and adapt our model for repair of critical porcine tibial defects. The ability of revitalized allograft to repair a critical defect will be directly compared to the current clinical standards, allograft and autograft. Repair of critical defects using allograft revitalization represents a potentially dramatic improvement over these modalities for reconstructive surgeons.

Carcinoma of the skin is the most common type of cancer diagnosed in the United States, with the most deadly type being malignant melanoma. The incidence of this disease has continued to rise for the last three decades, with an estimated 70,000 individuals expected to receive a new diagnosis of malignant melanoma in 2011 (ACS). The American Cancer Society predicts that 9,000 Americans will die from this disease this year alone. Despite the emergence of multiple new therapies and ongoing clinical trials, and a focus on education and skin cancer prevention, the death rate in patients over 50 years old continues to rise. Squalamine, a recently discovered aminosterol, has been shown to be effective as both an antibiotic and anti-viral as well as maintaining antineoplastic properties. This intrinsic antineoplastic effect of squalamine has been studied in the literature for its clinical capacity to reduce small cell lung cancer progression. Here, we suggest an additional role of squalamine in inducing the destruction of malignant melanoma via induction of apoptosis and/or necrosis of melanoma cells through angiogenic inhibition. It is with these funds, we offer a potential novel therapy using squalamine as an injectant adjuvant for the treatment of cutaneous melanoma.

In 2009, over 86,000 women underwent breast reconstruction. Tissue expander/implant based reconstructions were the most frequent method with approximately 57,000 operations performed last year. Despite a continued increase in the amount of procedures performed yearly, breast reconstruction still has a high overall complication rate, reported to be as high as 53%, with incidence of infection reaching 20%. Surgeons commonly place closed-suction drains and prescribe prophylactic antibiotics until drains are removed, which may be several weeks. This contrasts current CDC recommendations of stopping antibiotics at a maximum of 24 hours after clean elective surgery. Currently, there are no prospective randomized controlled trials demonstrating that long-term antibiotic prophylaxis improves outcomes for breast reconstruction patients. We plan to determine whether there is a difference in complications in tissue-expander based immediate breast reconstruction between patients who receive 24 hours of postoperative antibiotics versus patients who receive prophylactic antibiotics until drain removal. We hypothesize that there is no significant difference in infection rates between these patients. Patients will be enrolled in a randomized controlled trial in which they will be placed into two study arms based on antibiotic duration, with operative and postoperative antibiotic and drain care protocols standardized. Our primary outcome is surgical site infection as defined by the CDC. The study endpoint is one year post-op or second stage implant exchange procedure. Unnecessary prolonged antibiotic administration presents additional risks to our patients including systemic side effects, increased costs, super-infection, and growth of resistant organisms. This study will provide evidence-based guidelines for postoperative antibiotic prophylaxis in breast reconstruction.

Peripheral nerve injuries and diseases are a major cause of chronic pain conditions and muscle hyperactivity. The pathology is usually due to aberrant signal transmission along sensory or motor nerve fibers. Better control over peripheral nerve signal transmission can lead to better management of peripheral nerve injuries, chronic pain and muscle hyperactivity and thereby significantly improve public health. The recent birth of the field of optogenetics has opened up the possibility of controlling nerve signal transmission using light. This project aims to use optogenetic technology to understand how to reversibly block neural signals traveling from the central nervous system to the peripheral nervous system and vice versa. This understanding will establish the necessary building blocks that will facilitate the development of an implantable optical nerve cuff device. This device could potentially allow one to reversibly block nerve conduction with light alleviating pain and muscle hyperactivity. Preliminary EMG data from our lab demonstrates that we can reversibly stimulate or block peripheral nerve signal transmission using specific wavelengths of light. The project has a high likelihood of success because it represents a strong partnership among the fields of neuroscience, biomedical engineering and plastic surgery. The key personnel involved offer expertise in the areas of neural signal processing, microfabrication, peripheral nerve anatomy, physiology and surgery. A successful outcome could provide plastic surgeons with an accurate and precise tool to reversibly block peripheral nerve signal transmission. This tool will help promote a better understanding of nerve disease processes, chronic pain and muscle hyperactivity and therefore significantly impact public health and advance the field of peripheral nerve surgery.

Peripheral nerve injuries are estimated to occur in 5% of patients with traumatic injury presenting to level 1 trauma centers and have a limited ability for natural recovery. The ideal surgical repair of a peripheral nerve injury is the end- to-end coaptation of the proximal and distal ends to produce a tension free repair. Following peripheral nerve injury and reconstruction, a major factor contributing to limited functional recovery is the inability of regenerating motor neurons to accurately find their denervated muscle targets. We have shown that constitutive overexpression of glial derived neurotrophic factor (GDNF) in skeletal muscle (i.e. the periphery) increases peripheral nerve regeneration and overexpression of GDNF in the central nervous system actually reduces regeneration. We have also demonstrated that controlled release of GDNF at the site of injury can enhance functional recovery. Despite these observations, others have demonstrated that GNDF does not increase the number of regenerating motor neurons immediately after nerve injury. Instead it is proposed that GDNF only stimulates increased axonal sprouting. Given these observations, we hypothesize that GDNF enhances functional recovery in rodent models of peripheral nerve injury by increasing the efficiency with which regenerating axons reinnervate motor end plates. To evaluate this hypothesis we will evaluate the relationship between muscle reinnervation efficiency and function recovery in multiple clinically relevant nerve injury paradigms (Aim 1). We will then use this information to evaluate the ability of increased GDNF levels in the denervated muscle to augment reinnervation efficiency and functional recovery following peripheral nerve injury (Aim2). The completion of this proposal will lead to the study of new clinically relevant treatments that target the denervated muscle to enhance functional recovery following peripheral nerve injury.

The broad objective of this project is to investigate a novel drug delivery method to improve peripheral nerve regeneration. Despite improved surgical techniques, recovery is usually incomplete after nerve injury in the peripheral nervous system (PNS). Delays in treatment further compromise recovery because the neurons' capability to regenerate axons diminishes when the axons have not yet connected to their end-organs (“chronic axotomy”). This decline is in part due to reduced neurotrophic factor availability within the neurons and from the progressively denervated Schwann cells in the growth pathway of the distal nerve stump1. Reduced availability becomes even more critical during placement of grafts to bridge nerve gaps, as is commonly required in human nerve repair. Unfortunately nerve grafts or conduits have made little impact on clinical outcomes despite reducing donor site morbidity. Exogenous sources of neurotrophic factors, including brain derived neurotrophic factor (BDNF) and glial cell line derived neurotrophic factor (GDNF) promote neuron survival and axon regeneration under conditions where endogenous supplies decline with time. However, these factors have not yet been included in clinically available grafts and conduits. Our OBJECTIVE is to develop a clinically applicable biodegradable delivery system of polymer microspheres (MSs) releasing neurotrophic factors to enhance PNS regeneration. The delivery of BDNF and GDNF via microspheres will be tested in a rat animal model of nerve injury to determine their capacity to promote axon regeneration and functional recovery after nerve resuture. To this end, both histological assays and muscle functional analysis (evoked contractile muscle force) will be evaluated in the animals following experimental treatment. Our long-term goal is to promote nerve regeneration and functional recovery in patients who suffer PNS injuries.

Our research is aimed at improving breast cancer treatment and reconstruction. Specifically we are studying the use of fat grafting in re-contouring the breast after cancer resection. Fat grafting for reconstruction is currently in widespread use. This is despite unpredictable results that vary widely among patients. The recent discovery of stem cells in fat, so called adipose-derived stem cells (ADSC), has provided a potential explanation for the varying degrees of fat retention. The objectives of our research project are to investigate factors influencing the survival of grafted fat. This will be accomplished in two parts. First, we will characterize the effect of increasing patient age on ultimate fat graft survival. We will collect demographic data and information regarding previous cancer chemotherapy treatment on patients undergoing fat grafting. Using a 3D camera, patients will undergo volumetric measurements prior to and post-surgery at selected time points (one, three and six-months postoperative). Each patient population will be assessed for a correlation with graft retention. Second, we will categorize patients by percent of graft survival (e.g., >75%, 50-75%, <50%) and assess for a correlation in number of ADSC. We will quantify pre- and post-surgery volumes and group patients by percent of fat graft retention. These groups will be compared by mean number of ADSC. We believe there is a correlation between fat graft retention and fraction of cells capable of differentiation present in each person. Quantifying stem cells will be performed using laboratory methods including cell counts and flow cytometry. Since cellular fraction of stem cells likely decreases with age, we believe increasing patient age is inversely proportional to the amount of fat graft remaining as long-term viable tissue. By defining which patients benefit most from fat grafting, clinicians will be able to properly select patients for this procedure.

Critical size bony defects of the mandible remain a complex surgical challenge for plastic and reconstructive surgeons. Although vascularized bone flaps can provide enough bone to repair such defects, the associated donor site morbidity remains a challenging problem which surgeons have not been able to circumvent. The possibility of creating vascularized bone flaps with ‘endogenous tissue engineering' techniques has the potential to provide large sized bone flaps with minimal donor site morbidity. Tissue transformation, performed by treating skeletal muscle flaps with osteogenic factors, has the potential to transform muscle flaps into custom shaped vascularized bone flaps for reconstruction of critical size mandible defects in vivo. The conversion of “expendable” skeletal muscles into bony flaps is a way to circumvent the morbidity associated with the harvest of autologous vascularized bone flaps for mandible reconstruction. However, this technology has not been further sophisticated and the characteristics of the bone produced have not been fully analyzed. We hypothesize that skeletal muscle can be transformed into high quality mandible-shaped vascularized bone flaps in vivo with the use of osteogenic factors and bone growth enhancement drugs. The possibility of creating a mandible-shaped vascularized bone flap from autologous muscle flaps in vivo with FDA approved osteogenic factors (BMP-2) and vascularity enhancement drugs (Deferoxamine) can potentially revolutionize the source of bone flaps for reconstruction of critical size mandible defects.

Thumb carpometacarpal (CMC) arthritis is a disease of aging occurring in the 5th to 7th decade of life. It is symptomatic in up to 20% of people over the age of 50 years and results in significant physical disability and deterioration of hand function. The age group affected by thumb CMC arthritis represents the fastest growing segment of the US population projected to double current estimates to 112 million by 2050. Healthcare resource utilization for thumb CMC arthritis is likely to increase significantly. Clinicians have several options when considering non-surgical or surgical treatment for thumb CMC arthritis and review of published literature suggests that there are variations in the patterns of treatment. Accounts published in literature are however limited to the experience of contributing physicians and may not reflect how variations are manifest at the population level. We propose conducting a population level study to evaluate the variations in treatment of thumb CMC arthritis nationally. We hypothesize that patient factors such as co-morbidity, age, gender and health system factors, such as health coverage plan type and health provider type, play a role in determining variations in patterns of non-surgical and surgical treatment at the national level. We will use a national claims database to accomplish our 3 study aims: (1) To evaluate variations in utilization of surgical and non-surgical treatments. (2) Describe trends in the choice between 8 surgical techniques used to treat thumb CMC arthritis and (3) Perform a cost-analysis of surgical and non-surgical treatment for thumb CMC arthritis. Our study will establish the groundwork for cost-utility studies comparing different techniques of surgical treatment as current evidence does not support the superiority of any particular technique. Furthermore, we believe this will provide impetus for high level evidence studies to establish evidence-based approach for treatment of thumb CMC arthritis.

The skin is directly exposed to the environment and thus vulnerable to ultraviolet (UV) radiation from the sun. Long term exposure to UV radiation has deleterious effects in human skin, including sunburn, cancer, immune suppression and premature aging. Sunburn and immune suppression occur acutely, whereas skin cancer and photoaging result from accumulated damage caused by chronic, repeated exposures. Skin cancer frequently occurs in skin that is photoaged. Photoaged skin is characterized by thickening, rough texture, coarse wrinkles, and mottled pigmentation. With the increase in life expectancy and the emphasis that the American culture places on beauty, the "aging face" is a rapidly growing field. Consequently, the resulting facial skin changes can be detrimental to both one's health and self-esteem. The molecular mechanisms of photoaging secondary to UV irradiation implicate that c-Jun, part of the transcription factor activator protein 1 (AP-1), is the common mediator following injury. Previous studies have characterized c-Jun up-regulation following UV radiation resulting in degradation of collagen and an increase in matrix metalloproteinases (MMPs), culminating in the loss of elasticity that is the phenotypic hallmark of photoaging. Previous work in our laboratory has demonstrated topical short interfering RNA (siRNA) can be used for local gene silencing of cutaneous wounds, which has since been adapted to achieve transcutaneous silencing of intact skin. We will test the hypothesis that transcutaneous inhibition of c-Jun production via topical application of siRNA will reverse UV irradiation-induced skin injury and improve skin elasticity. The first aim is to develop a model of UV irradiation injury to the dorsal skin that shows reliable up-regulation of c-Jun and AP-1 as well as collagen degradation. This work will be performed in a mouse model and will characterize fibrosis by histology, immunohistochemistry, Western blot, and RT-PCR. The second aim is to demonstrate siRNA blockade of c-Jun production in vitro. Dermal fibroblast explants will be used to determine optimal siRNA delivery vehicles and doses, as well as the time course of the effect. Our third aim is to demonstrate in the mouse model that after injury, topically applied siRNA blocks local and not systemic c-Jun production, and that the result is alteration of the skin phenotype. Skin will be evaluated by the techniques mentioned above. In addition, mechanical testing (tensiometry) will be employed to measure elasticity and tensile strength of siRNA treated and control skin.

Cleft lip and palate malformation is one of the most common birth defects, where improved understanding of its genetic basis is critical to advance its treatment. Palate formation involves complex morphogenetic events which include neural crest cell migration and cell-cell signaling. Wnt signaling is critical for neural crest cell (NCC) migration and facial morphogenesis in vertebrates. The zebrafish is a powerful vertebrate model that can be used to study the genetic basis and mechanisms that pattern the early craniofacial skeleton. We have demonstrated that knockdown of wnt9a in zebrafish results in loss of ethmoid plate and failure of lower jaw structures to form. To investigate the role of wnt9a signaling on NCC migration, we have generated sox10::kaede transgenic lines. Endogenous sox10 gene expression is restricted to NCCs, while kaede, a fluorescent protein allows for cell specific labeling. Using this model, fate mapping analysis of NCC migration can be performed following wnt9a gene disruption to elucidate its role in palatogenesis and lower jaw development. Our second aim is to dissect Wnt regulatory pathway in zebrafish craniofacial development. We have recently shown expression patterns of wnt9a in zebrafish to be localized to the pharyngeal arch, oropharyngeal epithelium that circumscribes the ethmoid plate, and ectodermal cells superficial to the lower jaw structures. Wnt signaling can be modulated by antagonists including secreted Frizzled Related Proteins (sFRP). frzb is one gene which encodes a secreted protein with homology in the ligand binding domain to Wnt receptor Frizzled, but lacks the domain encoding the seven transmembrane segments. Furthermore, frzb has been shown to have similar spatial and temporal expression profile similar to that of wnt9a. To elucidate the key molecular pathway regulating differentiation of NCCs into the palate and jaw structures mRNA overexpression as well as morpholino disruption of frzb will be performed.

A major focus in the field of craniofacial surgery is the development of tissue-engineered bone with biomimetic functionality allowing for its translation to the clinical setting. One of the greatest goals is achieving complete bone regeneration even on a large-scale defect. One challenge is the difficulty associated with successfully engrafting a bioactive construct with significant metabolic, circulatory, oxygenic, and nutritional needs into a potentially hostile growth environment. Our approach to this challenge is engineering a “smart scaffold” that incorporates the osteoconductive properties of an apatite-coated scaffold, the osteogenic potential of huma mesenchymal stem cells (HMSCs), and the angiogenic effects of vascular endothelial growth factor (VEGF) to effectively create an in situ regenerative bioreactor with self-sustaining qualities ensuring cell survival and prolonged tissue regeneration. By engineering HMSCs that overexpress VEGF, hypoxia at the defect site can be mitigated and the chemotactic effects of VEGF can support the homing and attachment of HMSCs to the scaffold, boosting its osteogenic potential.

Tissue defects from trauma, tumor resection or congenital malformations require soft tissue repair. Standard care includes free tissue flap transfer, or prosthetic components such as silicone or saline implants. Adipose tissue retention during autologous fat transfer has been one of the major challenges in plastic and reconstructive surgery. One strategy involves the controlled delivery of adipogenic factors, such as insulin and dexamethasone, within the fat graft. This project outlines the novel design and assessment of encapsulated insulin and dexamethasone in poly(lactic-co-glycolic acid), (PLGA) microspheres mixed with lipoaspirate and their adipogenesis effect in vivo, using a combined drug therapy approach. We hypothesize that the slow release of combined insulin and dexamethasone can enhance adipogenesis and angiogenesis, thus retaining the fat graft volume.We expect that the combination of two FDA-approved adipogenic factors will significantly enhance the retention of lipoaspirate during fat grafting, and will be easily translated into clinical trials. Insulin/dexamethasone-loaded PLGA microspheres (Ins/Dex MS) will be prepared using double emulsion/solvent extraction technique. The bioactivity of the drugs will be assessed by mixing the microspheres with human lipoaspirate and injecting subcutaneously into an athymic mouse model. Animals will be divided in 10 groups, 5 animals per group. The first group will contain the highest dose of insulin/dexamethasone MS followed by decrease dose in MS with the last group containing only lipoaspirate with empty microspheres. Samples will be analyzed grossly and histologically after 5 weeks in vivo. We expect to demonstrate that the controlled delivery of adipogenic factors such as insulin and/or dexamethasone via polymer microspheres to significantly affect tissue formation and vascularization. This represents a clinically relevant method of stimulating fat retention in tissue engineering therapies.

The majority of chronic wounds occur in people over age 60, significantly impacting their quality of life. Many studies support that an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) results in degradation of the extracellular matrix and impaired wound healing. Induction of MMP expression by reactive oxygen species (ROS) has been reported to be mediated by mitogen-activated protein kinases (MAPKs) in vitro, but the interaction between ROS and MMPs in vivo has not been explored. Using a young rat ischemic wound model, we found both high ROS production and MMP expression in the ischemic wound, which correlated with high levels of phosphorylated MAPKs. Based on these preliminary results, we hypothesized that the ROS/MAPK/MMPs/Apoptosis pathway plays a key role in chronic wounds in the elderly. To test this hypothesis we have modified the previously utilized random ischemic flap wound to one with axial blood flow. This allows us to selectively and accurately deliver MAPK siRNA to the ischemic wound tissue via the axial artery. Using this novel animal model in aged rats, combined with special inhibitors delivered locally, we will elucidate whether the following three imbalances exist in the aged ischemic wound: 1) imbalance between removal and production of ROS; 2) imbalance between MMPs and TIMPs; and 3) imbalance between cell proliferation and apoptosis. The use of MAPK siRNA will then allow us to determine the relationship between those imbalances and MAPK signaling. This research is intended to delineate the cellular and molecular mechanisms that result in chronic wounds so that rational therapies can be developed to improve healing in the elderly.

Injuries to the upper extremities that involve tendons and ligaments are very common. Many of these injuries, including for example a sprained wrist, heal with no special treatment. However, injuries that cause the tendon or ligament insertion to tear off its bony attachment often needs surgical treatment. A good result after surgical reconstruction of this insertion part is however hard to accomplish since it typically heals as a fibrous scar which looses its former strong properties. New ruptures and poor function may be the post operative result. We hypothesize that we can create a stronger healing if we can replace the old injured insertion point with a "spare part" construct, based on the patients own cells. This is called tissue-engineering. New tissue, in this case tendon and its insertion into bone, are developed by culturing multipotent stem cells on a scaffold that is used as a skeleton for the cells. To test our hypothesis, and to optimize the conditions for our cells to populate these constructs by adding growth factors, we will work in an animal model of tendon insertion healing in the rat foot. The ultimate, long term goal with this project is a translation of these techniques to the production of human tendon/ligament - bone insertion constructs that can be clinically used in reconstruction of extremity injuries.

A literature review identified a number of cases of an aggressive form of T-cell Anaplastic non Hodgkins Lymphoma associated with breast implants. We know of up to 40 cases reported in the literature, by word of mouth and a few by reasonably reliable hearsay. Despite the very malignant nature seen microscopically and in the laboratory data, the tumor rarely spreads and the patients do well after removal, chemotherapy and/or radiation. All but one are tumor free to date. The implant (both saline and silicone) brand and style when known were all textured by a "lost salt" method. that leaves tiny loops of silicone for fixation. These tumors are very rare (a possible 40 out of many thousands) so far and fortunately seem to be curable. Our research plan is to accumulate all the information available including as many paraffin blocks as possible. The data will be evaluated epidemiologically to determine risk and the tissue will be evaluated microscopically and by a battery of immunological tests to confirm ALCL. These include: TLBR-1 cells from culture and tissue sections of TLBR-1 tumors grown in SCID for immunohistochemical studies. Cytospin preparations of TLBR-1 cells fixed with 2% parafolmaldehyde (Polysciences, Warrington, Pa) Wright-Gemsa and hematoxilin and eosin stains,, cytospin and paraffin tissue slides will be stained with monoclonal antibodies against human ALK (ALK1 clone, DAKO, Glostrup, Denmark), EMA (clone E29, Cell Marque Corp., Rocklin, Ca), CD30 (clone Ber-H2, DAKO), PAX-5 (clone24, Cell Marque Corp.), and pancytokeratins (clone AE1/3, DAKO) among others. We have received one sample of freshly excised capsule which is growing robustly in tissue culture demonstrating unique characteristics suggesting that this represents a new manifestation of lymphoma.

Since the development of the Michigan Hand Outcomes Questionnaire (MHQ) in 1998, this questionnaire has become the dominate outcomes tool in hand surgery in the US and around the world. Over 100 presentations and publications have used the MHQ as the outcomes tool in research for a variety of upper extremity problems. However, its application for assessment of plastic surgery practices with regard to hand surgery procedures is uncertain. Because the comprehensiveness of the 37-item MHQ can be time consuming for routine evaluation of outcomes of medical practices, there is a need for a brief questionnaire that can be administered to patients as part of the routine intake history examination. A brief questionnaire can be used to track outcomes of a practice but also to potentially satisfy the part 4 module of the Maintenance of Certification (MOC) for performance in practice using patient surveys. In this project, we will use existing prospective data sets in applying the MHQ for item reduction to develop a reliable and valid brief-MHQ that follows the standard protocol for this process. The newly created brief MHQ will be field tested on over 300 consecutive patients with upper extremity disorders in order to test this questionnaire for validity and responsiveness to clinical change and more importantly, applicability for routine administration to track patients' outcomes.

Background: Metacarpophalangeal (MCP) joint deformities caused by rheumatoid arthritis can be treated using silicone metacarpophalangeal joint arthroplasty (SMPA). There is no consensus whether this surgical procedure is effective. The purpose of the study was to prospectively compare outcomes for a surgical and a non-surgical cohort of rheumatoid arthritis patients. Methods: The prospective cohort trial was conducted from January 2004 to May 2008 at 3 referral centers in the US and England. Over a 3 year period, 70 surgical and 93 nonsurgical patients were recruited. All patients had severe ulnar drift and/or extensor lag of the fingers at the MCP joints. The patients all had one year follow-up evaluations. Patients could elect to undergo SMPA and medical therapy or medical therapy alone. Outcomes included the Michigan Hand Outcomes Questionnaire (MHQ), Arthritis Impact Measurement Scales (AIMS2), grip/pinch strength, Jebson-Taylor test and ulnar deviation and extensor lag measurements at the MCP joints. Results: There was no difference in the mean age for the surgical group (60) when compared to the non-surgical group (62). There was also no significant difference in race, education, and income between the two groups. At one year follow-up time, the mean overall MHQ score showed significant improvement in the surgical group, but no change in the non-surgical group, despite worse MHQ function at baseline in the surgical group. Ulnar deviation and extensor lag improved significantly in the surgical group, but the mean AIMS2 scores and grip/pinch strength showed no significant improvement. Discussion: This clinical trial demonstrated marked and significant improvement for RA patients with poor baseline functioning treated with SMPA. The non-surgical group had better MHQ scores at baseline and their function did not deteriorate during the one year follow-up interval.

Hand transplantation is the next frontier in plastic surgery. To date, 46 hand transplantations have been performed around the world, with 5 in the US. The promising long-term results of the US procedures have several centers contemplating starting hand transplantation programs. In an era of fiscal constraints on the healthcare system, new innovations will be scrutinized not only on their quality of life enhancements, but also on their costs. Despite the diverging ethical opinions regarding hand transplantation, a formal decision analysis has never been performed. In addition, the economic impact of this operation has never been addressed. The specific aims of this study are: (1) to perform a decision analysis, using various perspectives (plastic surgeons, hand therapists and patients) to assess the utility of hand transplantation, prosthesis and amputation, (2) to compare the utilities assigned by surgeons, hand therapists and patients, and (3) to conduct a cost-utility analysis of comparing hand transplantation and prosthesis.

Fat grafting has become an increasingly popular adjunct to aesthetic and reconstructive breast surgery. This projects aims to apply 3D imaging technology to assess the efficacy of fat grafting to the breast as well as stability of post-operative results. Numerous factors that have yet to be established will be determined with particular focus on migration of the autologous fat transfer.

The research objective is to translate previous work on flexor tendon tissue engineering in the rabbit model to human cases. We have established that acellularized tendon provides a viable scaffold. Now, we hope to enhance recellularization by increasing intratendinous cell penetration. Specific goals are to: 1) Compare the microarchitectural properties of sonicated acellularized human flexor tendon. 2) Compare recellularization rates of sonicated versus unsonicated acellular tendon. 3) Compare the mechanical properties of sonicated-reseeded tendons with controls. The eventual aim is to allow translation to select human cases by using tissue-engineered tendon constructs for tendon reconstruction in mutilating hand injuries. Human flexor tendons will be harvested and preserved. Acellularization is performed with SDS, Triton x-100, and freeze-thaw cycles to minimize antigenicity. Tendons will be divided and immersed in PBS. Pulsed sonication is performed at power settings ranging from 90W to 465W for a total sonication time of 1min. Primary cultures of dermal fibroblasts and adipoderived stem cells will be expanded in culture and seeded at a density of 2x106 cells/cc. Seeding efficacy will be determined by H&E microscopy, cytostaining, and quantitative analysis of collagen I & III. A human tissue bioreactor providing uniaxial tendon strain will subject tendons to a stretch force 5N over a 5 day course at 1cycle/min in alternating 1 hour periods of loading and rest. The specimens will undergo tensile testing to compare ultimate tensile stress and elastic modulus. With these techniques, surgeons could remove patient cells and allow cells to proliferate in culture while the patient is stabilized. Banked cadaver allograft tendons can be acellularized and seeded with the patient's cells. When extremity reconstruction is undertaken, large amounts of biocompatible tendon would be available. This would create a tissue bank of biocompatible allograft tendons.

Adipose tissue is emerging as a valuable and practical source of therapeutically useful cells. Emerging data suggests that fresh uncultured SVF cells can enhance the vascularization, " take" and volume maintenance of fat grafts. The real-time, intraoperative use of these uncultured cells provides certain regulatory advantages, and this may explain in part the growing application of this cell-based therapy on a global scale. However, the SVF (defined herein as freshly isolated, uncultured cells) represents a relatively crude, highly heterogeneous collection of numerous cell types, nearly half of which are leukocytes with undefined activity and benefit. In order to establish standardized methods for reproducible and predictable clinical effect, it would be useful to separate and enrich specific subpopulations of cells from within the crude SVF and to do so in a cheap, practical, efficient and effective manner within the operating room setting. Of the various methods available for cell separation/enrichment, microfluidic technology offers a unique approach to achieving the stated objectives. We propose to design and validate a versatile microfluidic system that can selectively enrich for specific cell populations from freshly isolated adipose SVF in the point-of-care setting. In Specific Aim 1, we will define protocols for the effective microfluidic handling of fresh SVF cells and design specifications for a cell enrichment microchip to process SVF. In Specific Aim 2, we will test and validate our microchip design with fresh human SVF samples. This first-of-its-kind work will establish the feasibility of using MEMS microfabricated silicon chip technology to improve the accuracy and reliability of adipose-derived cell enrichment, leading to improved therapeutic outcomes for cell-based therapies and decreased medical costs by significantly reducing failed and inconsistent grafts/treatments.

Limited availability and morbidity associated with suitable bone grafts has driven tissue engineering approaches to osseous repair. We plan to create a cellular based construct that will form structurally and functionally normal bone that is clinically applicable by studying the osteogenic potential of genetically manipulated human adipose derived stem cells (ASCs) within a 3-dimensional culture environment. Ideally, osteoprogenitor cells would be autologous, easily harvested, and would maintain strong osteogenic potential in patients of all ages. Although ASCs are abundant and easy to obtain/process, their potential for osteogenesis decreases with increasing donor age. It has been shown that ASCs can be reprogrammed by four virally transduced genes, Oct3/4, Sox2, Klf4, and c-Myc, to express embryonic stem cell (ESC) characteristics. By attaining an ESC-like state, induced ASCs (iASCs) may be able to not only increase bone formation but also maintain this response despite increasing donor age. We plan to test our hypothesis by examining differences in proliferation and osteogenic potential between iASCs and control ASCs from the same donor. ASCs will be harvested from multiple donors allowing us to study the effect of increasing donor age. Lentiviral vectors containing the defined factors noted above and a lacZ marker will be used to transduce ASCs into iASCs which will be compared against appropriate controls. Population doubling time and DNA assays will be used to assess proliferative potential. Osteogenic differentiation will be determined by quantifying expression of osteogenic markers using RT-PCR, alkaline phosphatase assays, and Von Kossa mineralization. All experiments will be performed in both 2-D (PLGA film) and 3-D (PLGA Scaffold) cultures. This work may provide further advance toward the creation of a clinically applicable bone graft substitute that can aid the plastic surgeon in the repair of bony defects.

Scar contracture is estimated to affect more than 40 million people worldwide. There are no effective treatments to prevent contractures. The long-term objective of this research is to develop a drug to prevent scar contracture formation. Contractures develop gradually, and as they progress the scar matrix becomes increasingly stiff. Contractures are believed to be caused by fibroblast contractility, which is stimulated by Rho kinase (ROCK). It is hypothesized that ROCK activation causes scar contracture formation and that increasing matrix stiffness acts synergistically with TGF-beta to promote ROCK activation through Rho A. Two Specific Aims are proposed in this innovative proposal to test the hypothesis. Successful completion of the Specific Aims will further the understanding of the pathogenesis of scar contracture and identify a drug to prevent contracture formation. Specific Aim 1. To determine how increasing tensile strain works synergistically with TGF-beta to promote ROCK activation. In the heart, ROCK is stimulated by increasing mechanical strain and in the lung ROCK is activated by TGF-beta stimulation of Rho A. The effect of increasing mechanical strain and TGF-beta stimulation on ROCK induced fibroblast contractility in the skin is unknown. This Specific Aim will use polyacrylamide gels of varying stiffnesses (soft (1kPa), and medium (20kPa)) with glass slides as a positive control (>100kPa), to test how changes in matrix rigidity act synergistically with TGF-beta to alter ROCK mediated fibroblast contractility through Rho A. Specific Aim 2. To determine the efficacy of ROCK inhibition in contracture prevention. The FDA approved drug, fasudil, has been shown to prevent lung, liver, and kidney fibrosis. Fasudil will be used to test whether ROCK inhibition prevents rat wound contraction. Osmotic pumps will be subcutaneously implanted to continuously deliver systemic fasudil. The effects of fasudil on wound contraction will be quantified by planimetry.

Repair of calvarial defects in children is difficult because the supply of autologous bone is limited, and alloplastic substances are not advocated because they inhibit cranial growth. Although split cranial bone is the preferred material to correct cranial defects in children, it cannot be harvested in children less than 6 years of age. In order to overcome the limited supply of autologous bone graft in young children, bone-dust produced by high-speed drilling and particulate bone obtained with a hand-driven brace and bit have been used to fill cranial defects. The purpose of this project is to test the hypothesis that autologous cranial particulate bone graft is superior to bone-dust for inlay cranioplasty because it is osteoconductive, osteoinductive, and osteogenic. The specific aims of this project are: 1. To compare the ossification, osseointegration and vascularization of inlay autologous cranial particulate bone and bone-dust. 2. To determine the osteogenic, osteoinductive and osteoconductive properties of cranial particulate bone and bone-dust.

It is estimated that 50-60% of all extremity injuries in the US military are complicated by delayed heterotopic ossification (HO). Although not fatal, the considerable morbidity associated with HO includes pain, inability to fit amputation prosthesis properly, and a grotesque appearance to the extremity that prevents proper psycho/social rehabilitation. The estimated 50,000-battlefield injuries secondary to Operation Iraqi and Enduring Freedom has increased awareness of this entity beyond orthopedic surgeons who have traditional treated HO. There is evidence that muscle derived progenitor cells (MDC)s have osteogenic potential and are the source of the structurally sound mature endochondrial bone formed in HO. We seek to establish a murine translational HO model to harness MDC mediated osteogenesis. Although the initial goals of my research include the understanding and inhibition of the processes of HO, the information that will be gained in pursuit of these goals should be directly applicable to strategies designed to enhance or induce bone regeneration. If we understand how to stop bone from forming, we may better understand how to initiate bone formation. Our laboratory has established a technique for isolating MDCs from murine muscle. We have also fabricated 3D cell cultures and gathered preliminary data that suggests the osteogenic potential of these MDCs. In this study, we hypothesize that MDCs isolated from murine muscle will display in vitro osteoegenic differentiation in response to osteoinductive factors. We expect cyclical motion and inflammatory mediators (both being intrinsic to the HO process) to further enhance the in vitro osteogenic differentiation of MDCs. Finally, we hypothesize that osteoinduced MDCs will promote in vivo bone growth when implanted into heterotopic locations or into large orthotopic defects in syngeneic animals.

Vascularized composite allotransplantation (VCA) is a promising reconstructive option for select patients, but need for chronic immunosuppression limits more widespread use of this therapy. Studies support the induction of donor-recipient hematopoietic chimerism as a way to achieve allograft tolerance. Bone-marrow transplantation is a widely used approach, but AMD3100 (Plerixafor), a CXCR4 antagonist that mobilizes hematopoietic stem and progenitor cells (HSPCs) in vivo, is a potential alternative. AMD3100 has prolonged survival of pancreatic islet-cell and liver grafts in animal transplant models. By harnessing the immunomodulatory properties of HSPCs, administration of AMD3100 has also led to clinical improvement in autoimmune disorders. Here, we will examine the mechanism and effect of AMD3100 on graft survival in a murine VCA model. CXCR4 antagonism has not been studied in VCA, which presents unique immunological barriers. Further, the co-transplantation of bone marrow, unique to VCA grafts, makes AMD3100 an especially promising approach for improving graft survival, as the transplanted host has both donor and recipient sources of stem cells. We will study AMD3100's ability to induce tolerance or decrease maintenance immunosuppression doses. We will also measure mobilization of HSPCs and assess levels of peripheral hematopoietic chimerism. To further explore AMD3100's mechanism and the relative effects of donor- and recipient-derived cells on graft survival, we will perform transplants with myeloablated recipients and donors, respectively. CXCR4 inhibition is promising in VCA, where protocols involving transfusion of donor stem cells have shown benefit. If shown to promote graft survival, AMD3100 could be a valuable alternative to the costly, complex harvest and transfusion of bone marrow cells. Given the growing clinical reality of VCA and the FDA-approved status of AMD3100, our study results could translate rapidly to the clinical arena.

The relative paucity of autologous bone available for use in the reconstruction of complex osseous defects and the potential morbidity associated with autologous bone graft harvesting has driven the search for an engineered bone graft substitute (BGS) for years. The long-term goal of this project is to generate a clinically useful BGS. In this study, we plan to specifically address that goal by testing the usefulness of an alternative osteoinductive molecule: oxysterol. Oxysterols are naturally occurring cholesterol oxidation products that have been shown to induce the expression of markers of osteogenic differentiation. We have previously shown that treatment of multi-lineage progenitor cells (MLPC) cells with oxysterols results induces osteogenic markers and mineralization. However, little is known about the molecular mechanisms that govern the osteogenic activity of oxysterols. The specific aims of this project are to further investigate these mechanisms and to test the osteogenic activity of oxysterols in vivo. In particular, we plan to (1) study the effects of oxysterols on osteogenesis in human MLPCs, (2) identify signal transduction pathways utilized by oxysterols in their stimulation of osteogenesis, and (3) test the efficacy of oxysterols in healing critical-sized rabbit cranial defects. We will culture MLPCs and expose them to various oxysterol isomers and look at the expression of markers of osteogenesis and at mineralization. We will also study the effect of oxysterols on the MAP Kinase, Hedgehog, and Wnt signaling pathways. Finally, we will use an oxysterol-based BGS construct to heal a critical-sized rabbit cranial defect. This work may help provide a clinically available product to be used in the reconstruction of bone defects associated with congenital anomalies, craniofacial or extremity trauma, or tumor extirpation, which would be of great use to plastic surgeons who manage these types of problems.

Worldwide there are currently 38 patients and 54 hand allografts that have been performed, with survival times from 2 to 9 years. More recently there have been 8 partial face transplants performed. All of these transplants are dependent on chronic immunosuppression, with their attendant side-effects including malignancy and opportunistic infection. In addition, all of these transplants have demonstrated episodes of rejection of the skin that required treatment with steroids, monocolonal antibodies or an increase in the overall level of immunosuppression. Our long-term goal is to devise practical methods for inducing immunologic tolerance that would allow for the long term survival of these complex tissue allografts without the need for chronic immunosuppression. This would significantly improve the risk-benefit ratio and allow for the more widespread use of composite tissue allotransplant (CTA) in the reconstruction of lost limbs and severe facial deformities. One method of inducing tolerance to an allograft is to establish a state of mixed chimerism. In a mixed chimera, the donor's immune system has become tolerant not only of the alloantigens expressed on host tissues but also those on hematopoietic and immune cells. In turn, the recipient's cells are tolerant of the alloantigens on the surface of the donor's hematopoietic cells. We have a pre-clinical large animal that will address the application of mixed chimerism to a clinically relevant application to the use of CTA. Aim 1: Determine if tolerance to vascularized myocutaneous allografts can be achieved across a single haplotype mismatched after simultaneous tissue and hematopoietic stem cell transplantation. Aim 2: Examine the role of graft infiltrating cells in the establishment of immune tolerance to composite tissue allografts in the canine model of mixed chimerism, with a specific focus on the presence of T-regulatory cells, their expression of FoxP3, and the analysis of the expression of chemokines and cytokines after transplantation.

Finger replantation was first successfully accomplished more than 40 years ago. Since that time, this procedure has become commonplace in hand and reconstructive surgery. The surgeon must weigh many characteristics of the patient and the severed digit to determine whether replantation can be accomplished with reasonable anticipated outcomes. Some situations arise in which replantation is only marginally indicated. Success is measured not only in survival, but also function of the replanted digit. The specific aim of this project is to perform a cost-utility analysis to assess various types of digit replantation procedures and to compare replantation surgery to revision amputation, specifically considering differences in mechanism of injury, level of amputation, and the age of the patient. First we will perform a systematic review of outcomes of finger replantation and revision amputation to determine rates of complications and anticipated impairment. Second, we will design a decision-analysis model based on methodology proposed by The Panel on Cost Effectiveness in Health and Medicine to assess the societal preferences from groups of informed subjects for the various surgical options. The data from the systematic review and decision analysis will be used to conduct a cost-utility analysis of the various treatment options. Cost will include direct medical costs and indirect costs of time away from work. Utility is determined by preferences for health states from different perspectives associated with each treatment. The utilities will be translated to the common measure of quality adjusted life years (QALYs). The data obtained from this study will help guide surgeons in the difficult process of treating patients with digit amputations in situations where replantation is not clearly indicated. Because of rising healthcare costs and lack of access to replantation surgery in the United States, it is critical that evidence-based data are presented to guide resource allocation and encourage use of treatments that improve patient outcomes. More economic analysis is needed in Plastic Surgery to define the value of our treatment for the nation.

Repair of large nerve injuries relies upon reconstruction from autologous sources. These repairs are limited by the amount of available autograft and donor morbidity. Nerve allograft transplantation provides a temporary scaffold for host nerve regeneration and allows for the repair of nerve injuries. They have the advantages of requiring temporary immunosuppresion, decreased donor morbidity, and are abundant in supply. We seek to characterize the immunological mechanisms involved in nerve allograft rejection, and the effects of costimulatory blockade and cold preservation on allograft survival. The immune response, is triggered by two distinct extracellular signals. The first, occurs with antigen(Ag) presentation on major histocompatibility complexes (MHC) to T-Cell receptors (TCR). The second, occurs with co-stimulatory molecule binding to specific TCRs. The first signal, antigen presentation, can occur via the direct or indirect pathway. In the direct pathway, donor Antigen Presenting Cells (APCs) present Ag on donor MHCs to TCRs. In the indirect pathway, recipent APC present Ag on recipient MHCs to TCRs. The second signal, co-stimulatory molecules bind to TCRs and regulate the activation and expansion of alloreactive T-cells. Two well established methods to modulate the immune response include cold preservation and co-stimulatory blockade. Cold preservation of nerve allograft has been shown to reduce antigenicity through decreased expression of donor MHCs. Co-stimulatory blockade has been shown to blunt the activation and expansion of alloreactive T-cells and lead to longer allograft survival. We will isolate the direct and indirect pathways using MHC deficient mice. Nerve allograft will be used to repair sciatic nerve defects. Using return of muscle function, muscle reinnervation, and histologic axon growth as end points, we can assess the pathway of dependency, and the effects of co-stimulatory blockade and cold preservation in nerve allotransplantation.

Hemangioma is the most common tumor of infancy, affecting approximately 10% of all children. This benign vascular tumor is composed of rapidly dividing endothelial cells which progress through three phases; proliferating, involuting, and involuted. The purpose of our study is to further delineate the genetic and cellular differences between hemangiomas in the proliferative and involuted phases. These differences will help to elucidate the mechanisms that cause a particular hemangioma to involute and ultimately guide the development of novel therapies with greater specificity and less toxicity. Patients (6mon.-13yrs) whose lesions require surgical excision will be considered for our study. We estimate obtaining 25 samples per year, with a final goal of 50 tissue samples (2 yrs. duration). After adequate counseling (by the PI or a co-investigator) and full disclosure, an informed consent (see attached) will be obtained. Small pieces of fresh hemangioma tissue (some in the proliferative and some in the involutional phases), as well as adjacent normal tissue (free surgical margin), will be obtained from intra-operative specimens. The specimens will be brought immediately to our laboratory facility and appropriately prepared for tissue culture as an in vitro human model of hemangioma. Any extra tissue specimen will be securely stored, indefinitely, in a -80 degree freezer. Using this model we will utilize a variety of techniques including histochemistry, immunohistochemistry, quantitative assays of angiogenesis, reverse transcriptase-polymerase chain reaction analysis, and gene micro-array analysis to uncover genetic and cellular differences between proliferative and involuting hemangiomas. We will also be testing novel therapies, using this in vitro model, to assess their potential to induce a potential to induce a proliferative hemangioma to involute or regress, with the ultimate goal to find a treatment with greater specificity and limited toxicity.

The field of tissue engineering has seen tremendous exponential growth in recent years. The initial concept of tissue engineering as defined by Langer and Vacanti was "an interdisciplinary scientific field that combines the principles of life sciences and engineering toward the development of biologic substitutes that will serve to restore, maintain, or improve tissue function". This concept relied on the combined use of donorderived cells, a biodegradable scaffold and possibly a bioreactor to engineer a cellscaffold construct with potential clinical application as a tissue or organ substitute. In the special context of hand surgery, a number of tissues are of interest for engineering components of the upper extremity. These include nerve, bone, tendon, skin, vessel, cartilage and composite tissue. While much progress has been made in the field, a number of limitations have prevented widespread clinical application of tissue engineering aside from a number of skin substitutes which have been approved for use by the Food and Drug Administration (FDA) One major limitation has been the issue of donor site morbidity from cell harvest. This is a major issue whether cells are derived from end organs or from more multipotent sources such as bone marrow derived mesenchymal stem cells. While groups have explored the use of embryonic stem cells (ESCs) for use in musculoskeletal tissue engineering6, the use of ESCs is fraught with ethical and moral issues. An alternative cellular approach to tissue engineering would be to induce cell migration into an implanted scaffold through creation of a biomimetic environment by selective constant release of cytokines. We would term this approach "cell guidance". The use of cells and scaffold materials to replace diseased or injured tissue, per the traditional paradigm of tissue engineering, was also found to be ineffective for purposes of nerve tissue engineering, with a consensus emerging that it will ultimately require the coordinated presentation of multiple permissive signals to be incorporated into tissue engineered biomaterial platforms for regrowth of tissues. Efforts in this area to recreate the complex neural microenvironment have been termed "axon guidance". Another problem that has emerged with translation of traditional tissue engineering techniques has been inadequate vascularization, and hence perfusion of engineered constructs. In the hand, this will potentially pose a major problem in engineering of bone and composite tissue. Inherent limitations with osseous tissue only forming on the surface of a polymeric scaffold were noted early on, and continue to limit engineering of large vascularized bone constructs10. By patterning biomimetic vascular channels in a polymer scaffold, induced vascularization of constructs would potentially allow fabrication of much larger constructs than that currently possible following the traditional tissue engineering paradigm. This approach has been termed "vascular guidance". Here, we describe recent progress in "Guidance" strategies, which aim at manipulating the microenvironment around cells and scaffolds to achieve a biomimetic milieu. These strategies offer an alternative and novel approach to overcoming limitations inherent to the traditional paradigm of tissue engineering.

An ideal cell source has not been described for bone and cartilage tissue engineering, with clinical translation limited by problems such as donor site morbidity inherent in harvest of such cell sources as end-organ cells and MSCs; as well as unpredictable quality of tissue formed in the cell-scaffold construct. Use of embryonic stem cells (ESCs), while promising, is fraught with ethical and moral issues. A potential alternative is offered by a technique which we have termed "cell guidance"1. This technique obviates the requirement for a donor cell source, by inducing homing of desired cells into cytokine-impregnated scaffolds to induce tissue formation. The concept of "cell guidance" relies on the SDF-1- CXCR4 axis, which is a pivotal regulator of trafficking of various stem cells in the human body4. It has been shown previously that mesenchymal stem cells (MSCs) expressing CXCR4 migrate in response to a SDF-1 stimulus5. In a previous study1, we were able to demonstrate site-directed homing of cells into a poly-e-caprolactone (PCL) scaffold implanted in a subcutaneous murine model, with resultant tissue ingrowth and angiogenesis.. In this study, we hypothesize that the use of SDF-1 as a cytokine to induce homing of stem cells, in combination with a differentiation cytokine, delivered through a cytokine microdelivery system, will be sufficient to induce osteogenesis and chondrogenesis in the absence of any implanted cells, within a PGA mesh scaffold. We will perform animal studies designed to test this hypothesis, and analyse for cell proliferation and differentiation using quantitative assays, immunohistochemistry and histology. This proposed study serves as both a "proof of principle" experiment, building on previous published data1, and also attempts to build a foundation for future head-to-head comparative studies against established cell sources in bone and cartilage tissue engineering, such as end-organ cells and mesenchymal stem cells (MSCs).

Despite extensive progress in scar research, hand surgeons still rely primarily on early active motion hand therapy protocols to combat morbidity of reduced function after flexor tendon repair. To date, studies have not generated a clinically available product, applied intraoperatively at the time of flexor tendon repair, to ensure reduction in peritendinous adhesions and smooth tendon glide. Various growth factors have been implicated in the scar process. Research focused on enhancing or inhibiting single factors has demonstrated that the central mechanism for scar control is unknown. A universal regulator, through which all fibrotic pathways must pass to elicit scar formation following tendon injury, and repair, has not been discovered. Early growth response factor-1 (EGR-1), a transcription factor associated with synthesis of a variety of key profibrotic growth factors and expression of extracellular matrix genes, holds promise as that primary mechanism of control. The purpose of this study is to develop a scar model, useful for quantification of the tendon scar process, and to identify EGR-1 in the healing tendon wound bed. To our knowledge, no study has addressed EGR-1's presence or absence at flexor tendon repair sites. By application of sodium morrhuate, a sclerosing agent, to an established rabbit flexor tendon repair model, a novel model of tendon scar formation is hypothesized to develop. Sodium morrhuate infiltration into rabbit tendons has been previously shown to increase tendon diameter and cellularity. Application of EGR-1 antibody to tendon harvested at various time points along the inflammatory and early proliferative phases of healing will, in theory, identify this factor in the wound matrix. Future research focused on modulation of EGR-1 expression could lead to novel treatments for preventing adhesion formation following flexor tendon repair. Applicability of these potential treatments spans all medical disciplines interested in scar manipulation.

Cutaneous wound healing is a complex process that involves the orchestration of multiple different cell types to produce a functional biological patch. In the case of chronic wounds, factors such as local tissue hypoxia and ischemia-reperfusion injury (IRI) combine to impede the usual progression of these events and indefinitely forestall the healing process. Despite the advent of bioengineered skin products and recombinant growth factor therapies for the treatment of problem wounds such as venous stasis ulcers, pressure sores, and diabetic ulcers, many chronic wounds have proven refractory to these methods and continue to pose a great burden to the health care system in terms of both cost and time. We propose that mesenchymal stem cells (MSCs) represent a novel therapeutic approach for the treatment of chronic wounds based on their ability to function as intelligent bioactive agents of repair. Early reports have demonstrated that these cells are capable of fully integrating into the wound environment and, via either direct cellular differentiation or the context-dependent elaboration of signaling molecules, can not only enhance wound healing but also promote the development of more regenerative phenotypes. In our experiments, we aim to test the ability of adipose-derived stem cells (ASCs), a specific type of MSCs that are readily harvested from adipose tissue stores, to rescue healing deficits in two rabbit models of chronic wounds. These models include a previously established ischemic rabbit ear wound model as well as a novel rabbit ear wound model that employs a reversible arterial clamp for the induction of ischemia-reperfusion injury (IRI). The influence of ASCs on basic wound healing parameters will be rigorously compared with that of fibroblast controls for all studies and the fates of transplanted cells will then be analyzed with a strategy for GFP labeling.

Background: Management of end stage rheumatoid wrist disease remains controversial. Total wrist fusion (TWF) provides reliable pain relief and stability and is the most commonly applied management strategy. Total wrist arthroplasty (TWA) is a motion-preserving alternative that is gaining popularity. The purpose of this study is to perform a cost - utility analysis comparing TWA to TWF for the rheumatoid wrist Methods: A time trade-off utility survey was developed to investigate patient and physician preferences for the potential outcomes of TWA and TWF. The study sample consisted of rheumatoid patients (N=49) recruited as part of an ongoing NIH study and a national random sample of hand surgeons and rheumatologists (N=-109). A decision tree was created using utility values derived from the survey, and the expected quality-adjusted-life-years (QALYs) for each procedure were determined. Using the societal perspective, we derive the costs based on the Medicare fee schedules for the CPT codes associated with TWA and TWF and their potential complications. Costs per QALY were calculated and compared. Results: Patients and physicians both showed a preference for operative management over nonoperative management. Application of cost data indicated that the incremental cost per additional QALY gained for TWA over non-operative management was $841 - $960 and the incremental cost per QALY gained over TWF was $458 - $1,820, which is substantially less than the national standard of $50,000/QALY deemed acceptable for adoption. Conclusion: In the absence of rigorous outcome data, a cost-utility analysis is useful to guide treatment decisions. TWA and TWF are both extremely cost-effective procedures. This study incorporated patient and physician utilities to demonstrate that TWA has a small incremental cost over the TWF procedure, and TWA is worthy of adoption based on this economic model.

Hemangiomas are the most common tumors of infancy predominantly affecting the head and neck. They are vascular tumor comprised of endothelial cells. However, little is known about their origin. Nearly all lesions turn into a fibrofatty residuum, suggesting a default pathway that converts them into fat. We wish to propose a novel hypothesis that hemangiomas are stem cell tumors comprised of pericytes derived from the neural crest. Based on this premise we will investigate a novel drug treatment that forces hemangiomas into the default adipocyte pathway. In an animal model, pericytes were precursors to adipocytes.(Tang et al. 2008) We hypothesize that markers for pericytes will be present in hemangioma tissue. Delta like kinase(dlk) is a critical inhibitory factor that controls the maturation of preadipocytes to adipocytes and is a strong marker of pericytes. To show the link between pericytes and hemangiomas, we will infect hemangioma cultures with a dlk silencing gene with the hypothesis that silencing dlk will cause the hemangioma to turn to fat. We will also analyze microRNAs that regulate adipocyte formation, mi195, mi299 and mi143. Dupin et al. have shown that pericytes as well as a significant component of the soft tissue of the head and neck region are derived from the neural crest. We hypothesize that hemangioma tissue will demonstrate neural crest markers accounting for its predilection to the head and neck region as well as stem cell markers given these cells' phenotypic malleabilty. In order to demonstrate the presence of pericyte, neural crest, and stem cell markers we will selectively excise tissue with laser capture microdissection followed by RNA extraction and qRTPCR for selected markers. Finally, hemangioma tissue will be placed subcutaneously in mice and treated with thiazolidionediene(TZD). TZD acts on dlk and PPARgamma, both involved in adipogenesis. This will add to the understanding of pathogenesis and treatment of hemangiomas.

Patient reported outcomes (PRO's) that are relevant to and valid for patients who have sustained injuries to the upper extremity are needed to characterize the effects of these injuries, and to allow standardization of future research of the upper extremity. The purpose of this project is to develop an upper extremity specific PRO instrument for patients who have sustained injury to the hand or arm. The instrument developed will be valuable in determining the effects of upper extremity conditions on individual patients and society as a whole. The proposed project will be conducted in two parts. The first portion will be the assembly of an expert panel. This panel will assimilate and organize multiple survey items regarding function of the upper extremity. These items will come from the NIH Patient-Reported Outcomes Measurement Information System (PROMIS) physical function bank as well as existing upper extremity surveys such as the Disabilities of the Arm, Shoulder and Hand (DASH) and the Michigan Hand Questionnaire (MHQ). These items will be combined and distilled to approximately 40 items that are felt to be pertinent and applicable to patients with upper extremity injury. The second portion of the project will be cognitive interviews with patients who have sustained upper extremity injury. During these interviews, each patient will be administered each item generated by the expert panel, and feedback regarding clarity and answerability of the items will be elucidated. Based on patient feedback, the items will be further revised to then generate an item bank suitable for large-scale administration. This qualitative project will have potential benefit for plastic surgeons, especially those practicing hand surgery. This will be the first step in standardizing outcomes studies for upper extremity conditions. Further, this will allow the development of patient reported outcome tools that entail less respondent burden.

Although much research has investigated the cellular and molecular mechanisms underlying both the physiologic and pathologic mechanisms of suture fusion, the critical events remain to be fully elucidated. Many studies have alluded to the role of various growth factors, as well as bone morphogenic proteins as critical cellular mediators in suture fusion, however little research has focused on the role of osteoimmunology, or the intricate crosstalk between immunologic cells and osteoblasts and osteoclasts. The role of osteoimmunology in skeletal homeostasis is well investigated and reveals that the key mediators, consisting of T lymphocytes, antigen presenting cells, as well as osteoblasts and osteoclasts, interact through the Tumor Necrosis Factor, TNF, superfamily signaling cascade involving TNF-related Activation-induced Cytokine, TRANCE (RANK-L), its receptor RANK, and downstream signaling adaptor proteins, TNF receptor-associated proteins (TRAFs). Absence of these mediators have been shown to lead to osteopetrosis, excessive bone formation, while over expression leads to osteoporosis, excessive bone loss in murine models. (Kim, 2000; Naito, 1999) The purpose of this proposal is to assess the role of osteoimmunology as it relates to suture patency and fusion. We hypothesize that the immune system plays a role in the development of the craniofacial skeleton and that an over-stimulation of this interaction locally at a suture results in premature fusion of the suture and retardation of skeletal growth.

Similar to how flight simulators are used to train pilots, computer-based surgical simulators provide a milieu to train physicians in a safe environment. Past technical limitations were prohibitive in creating robust 3D simulation software, but recent advancements in processing power and graphic cards now allow real-time rendering and interaction with complex, highly detailed anatomic models. The benefits of surgical simulation have been well documented in the literature and are an integral component of surgical training in most surgical disciplines. As true innovators in the fields of medicine and surgery, we should utilize technology to further advance this unique specialty. The construction of a 3D virtual reality library of plastic surgery simulations would be a progressive advancement and its impact on plastic surgery has the potential to be revolutionary. An interactive surgical simulator that will engage the surgeon, ask questions, test competency and provide feedback has the potential to quantify surgical knowledge. Internet connectivity allows for rapid deployment of surgical modules, networked testing formats, data aggregation, comparative analysis and guided tutorials. Combined with the approval of a surgical society, the Association of Academic Chairman of Plastic Surgery, this platform has the potential to set measurable quantitative surgical standards for residency training. Although the benefits of simulation training have been reviewed in the literature, we want to validate our model by conducting a proof of concept study prior to developing additional simulators. We will extend this initial pilot to a multi-center project to determine the efficacy of simulator training in plastic surgery education. We anticipate this collaborative effort involving experts from outstanding institutions will pioneer the integration of surgical simulation in training and provide the groundwork for standardized curriculum development.

This project aims to understand the role of Notch signaling in the regulation of the lifecycle of infantile hemangioma (IH) from proliferation through involution. IHs are the most common benign tumor of infancy, yet its pathogenesis is still poorly understood, and there is no FDA-approved therapy for the treatment of IHs. Most His develop in isolation and never require intervention. However, problematic hemangiomas can cause major morbidity including amblyopia, permanent disfiguring scarring, and even mortality. In addition, in PHACE syndrome, a large, segmental IH is associated with major abnormal vascular architecture, and affected infants have missing blood vessels and circulation to the brain, increasing their risk of strokes. Notch signaling plays a pivotal role in cell fate determination and cellular differentiation during embryogenesis. Notch genes have been shown to be expressed in IHs. Since there are no viable animal models for the study of IHs, this project will use isolated hemangioma stem cells (HemSCs) and hemangioma endothelial cells (HemECs) to study Notch signaling. Using silencing RNA (siRNA) technology, expression of specific Notch genes will be inhibited in order to study the function of these isolated HemSCs and HemECs. Other methods include using commercially available pan-Notch inhibitors such as gamma-secretase, as well as specifically designed antibodies produced in our laboratory. Cellular function such as proliferation, migration, and endothelial network formation will be studied. In particular, preliminary results in our laboratory has shown that human umbilical venous endothelial cells (HUVECs), when co-cultured with HemSCs, results in a more complex vascular pattern, with thicker tubular structures, and increased branching points. Understanding specific regulators of IHs will help in the design of a specific treatment for affected infants. Moreover, it will provide insight into vasculogenesis--the formation of blood vessels from stem cells. This knowledge will have broad applications, including the design of vascularized, tissue-engineered products.

Biofilm covering of prosthetics has resulted in the loss of multiple implanted devices used in orthopaedic procedures such as knee replacements or hip replacements. Biofilm, an aggregate of microorganisms which then produce a protective matrix of DNA, protein and polysaccharides, adheres to the prosthesis and provides protection to the bacteria from immune system defenses as well as antimicrobials. The eradication of these biofilms from implanted hardware has been a topic of research. When implants are coated in these biofilms, they result in an infection in the surrounding tissue cause joint infections thus resulting in an increased morbidity, multiple surgeries for washouts and a dramatic increase in health care spending. By eradicating these biofilms, we believe the rate of infection resulting from bioprosthesis will decrease. We believe that there is a device currently available that can penetrate and remove biofilm on prosthetic material without damaging the device. This device, the VersaJet uses hydrodissection to lift the biofilm off the surface without damaging the underlining materials. We propose to test the product in an in-vitro model using titanium disks.

Microsurgery requires a unique set of skills that takes time to develop. Increased efficiency and overall improved outcomes can be correlated to experience and repetition of the complex skills that microsurgery stresses. Although a required part of every plastic surgery training program, the volume and variety of microsurgical cases is highly variable. In addition, increasing constraints on training hours for residency programs limit the ability to practice technical skills on patients in the operative setting. Moreover, patient safety and operative efficiency preclude the learning of these skills in the apprenticeship-style model of learning during live operations. It is important for trainees to obtain exposure to the basic skills required of the microsurgeon and essential that progressive levels of microsurgery skills be acquired before performing in the operating room. Although numerous models have been described, none has defined a standard proficiency as an achievable goal or described a learning curve applicable to the acquisition of a specific task in an inanimate microsurgical training model. The purpose of this study is to help define the utility and improve the efficiency of currently employed microsurgical training models based on the performance of specific basic tasks deemed to be important in developing microsurgical skill. We plan to define a "standard proficiency" for two well-established inanimate laboratory training models and then determine whether novice microsurgeons can achieve this proficiency goal and, if so, to determine the number of repetitions needed to do so. Subsequently, we will determine a learning curve for each of the training models to show the number of repetitions needed to plateau in performance for novice microsurgeons. Development of standardized microsurgery training models will provide improved and more consistent training and serve as a foundation of a formal fundamentals of microsurgical skills curriculum.

Surgical education has traditionally been built on the apprenticeship model, where experts teach skills to novices using real patients. While this is a well-established process, there are limitations and areas for improvement to maximize the educational benefit of residents and the safety of patients. The overriding goal of this project is to assess the feasibility of a simulation-based microsurgery skills curriculum and to evaluate progress in cognitive and technical performance as applied to microsurgical models. Our first aim will be to validate the efficacy of an online training webpage as an educational adjunct for trainees, with the rationale that an individualized, self-directed website is ideal for our current cohort of technologically savvy residents to practice and learn on their own. The second aim of our study will assess the validity of various microsurgery training models (latex glove, PTFE graft, and chicken foot) to establish a readily available, cheap, and effective model that will quickly and efficiently strengthen the surgical skills of plastic surgery residents. Overall, the widespread implementation of our proposed curriculum will establish a strong foundation of training for all residents who require microsurgical expertise.

Vascularized bone marrow (VBM) transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and is considered to be superior to cellular bone marrow transplantation alone in the induction of tolerance and reduction of immunosuppression requirements in animal models. Despite promising results with isolated VBM allotransplantation and transplantation of VBM contained in composite tissue limb allografts, no study to date has investigated the cotransplantation of isolated VBM with a solid organ. This proposal aims to develop a rat model of simultaneous isolated femoral VBM/renal cotransplantation before exploring the ability of cotransplanted VBM to support renal allograft survival and function with full immunosuppression. The proposed research will then explore the ability of cotransplanted VBM to induce chimersim and tolerance once immunosuppression is withdrawn. If successful, this work would support research efforts in non-human primates exploring VBM/solid organ cotransplantation, as well as human clinical trials in patients undergoing renal transplantation.

In the setting of post-mastectomy radiation therapy delayed autologous reconstruction, favored by many attempts to avoid complications encountered with radiating the immediately reconstructed breast. The timing of delayed reconstruction is however not known. The goal of this proposal is to study the gross, structural and vascular changes in radiated mastectomy skin in addition to possible structural and flow changes of the underlying internal mammary vessels over time. We plan to use these objective findings as a basis for determining an ideal time frame for delayed autologous breast reconstruction. Sequential mastectomy skin specimens will be obtained from 20 patients undergoing post-mastectomy radiation therapy over a 12 month period and these specimens will be evaluated histologically. These same patients will also undergo serial examinations and photographic documentation of gross skin changes. Skin perfusion will be assessed by laser Doppler imaging and internal mammary vessel structure, and flow characteristics will be assessed by color Doppler sonography. Based on these studies, we will elucidate short and long term changes in radiated breast skin, showing inflammatory, structural and perfusion patterns that can be correlated with optimal conditions for reconstruction. There is also potential for improved outcomes of delayed breast reconstruction by decreasing the number of reconstructions performed too soon after completion of radiation.

Trigger finger is one of the most common hand ailments with an incidence of 2-3% in the general population and up to 10% in diabetics. This condition causes painful catching or popping of the involved tendon with normal use. It can progress to the affected digit being temporarily or permanently locked in flexion, inhibiting activities of daily living. Currently there are multiple accepted management options; however, none have been linked to a clinical classification of the disease state. Our study aims to create a staging system for trigger finger and show how the stage influences response to corticosteroid injection. This will be a prospective study of four groups that are distinguished by stage of disease (1-4). We will identify a total of 120 patients (40 in stage 1 and 2, 20 in stage 3 and 4). This group of patients will then undergo corticosteroid injection. Follow-up will be at one, three, and six months. Success will be defined as resolution of symptoms at six month follow-up. This pilot study will create a foundation for us to be able to expand our research project to include assessing the reliability and validity of the staging classification system and treatment algorithm recommendations, as well as assessing the clinical efficiency/cost impact of using our staging system.

Dupuytren's disease is an idiopathic, prevalent fibrocontractile disease that can be severely debilitating to hand function. Diseased fascia contains nodules and cords, and although it is unknown why these structures develop, it has been proposed that nodules are the primary epicenter of disease progression. All nodules contain fibroblasts rich in actin and myosin contractile proteins. Thus, it is proposed that myofibroblasts and fibroblasts within nodules cause Dupuytren's disease and palmar fascia contractures. Several upstream intracellular contractile signaling mediators lead to activation of actin and myosin. However, it is not known which signaling pathways predominate in Dupuytren's fibroblasts and myofibroblasts. The first aim of this project is to identify which contractile proteins are expressed in fibroblasts and myofibroblasts in Dupuytren's nodules. There are several different known proteins that cause cell contractility. It is unknown which proteins are expressed in Dupuytren's nodule fibroblasts and myofibroblasts. The plan is to characterize which of the known contractile proteins are expressed by fibroblasts and myofibroblasts wihin the nodules. The rationale is to identify the common motor proteins that cause contractility. The second aim is to evaluate the functional contribution of contractile proteins in the pathogenesis of Dupuytren's contracture. There are multiple points of regulation in the pathway of contractile proteins involving fibroblasts and myofibroblasts. Measurement of contractile force after inhibition of separate points of the pathway will characterize which are most important in the progression of contracture and their location within Dupuytren's tissue. Identification of the contractile pathways that are active in Dupuytren's disease and functional assessment of their contribution towards contraction will allow for future development of novel small molecule inhibitors to prevent disease progression.

Introduction: Diabetes negatively affects wound neovascularization. We hypothesize that diabetic (db) wound healing can be improved by correcting endothelial progenitor cell (EPC) mobilization and homing using combination therapy with AMD3100 (a stem cell mobilization agent) and PDGF (a growth factor and chemoattractant). Methods: Two full-thickness wounds were made on the dorsum of wild-type (wt) and type II db mice. Mice were randomized into 5 experimental arms: untreated wt (WT), untreated db (DB), AMD3100-treated db (A+), PDGF-treated db (P+), and AMD3100/PDGF-treated db (A+P+). Treated animals received daily AMD3100 (10mg/kg; i.p.) and/or PDGF (2μg; topical) beginning on post-wounding day 3 and continuing until wound closure. Wound closure was assessed photometrically. Circulating(c) EPC number was determined by FACS. Wound vascularity (vessels/hpf) was assessed by CD31 immunofluorescence. Wound fibroblast and EPC function were assessed in vitro in the presence of AMD3100 (50ng/mL) and/or PDGF (100ng/mL). Results: DB had significantly impaired wound healing compared to WT. A+P+ showed the greatest improvements in db wound healing at 7, 14, and 21 day time points (p=0.06, p<0.01, and p=0.02). FACS analysis demonstrated that AMD3100 increased cEPC levels (3.7±1.0-fold at 1 hour, 11.9±1.7-fold at day 7, and 19.6±1.9-fold at day 14). On day 21, wound vascularity increased most significantly with combined therapy (DB: 155.3±16.1, WT: 403.5±15.8, A+: 279.3±44.7, and A+P+: 431.8±19.3, p=1.3 x 10-11). AMD3100 did not affect EPC or fibroblast function, whereas PDGF significantly increased fibroblast proliferation (p<0.05). Conclusion: Combination AMD3100 and PDGF therapy improves bone marrow-derived EPC mobilization and trafficking, respectively, resulting in significantly improved db wound vascularity and healing.

Despite significant advances in the field of neuroprosthetics for restoring upper extremity function after amputation, currently available prostheses have yet to be ideally interfaced to nerves of the residual limb. Unlike forearm amputations, where myoelectric signals from remaining muscles can be isolated to power a prosthetic hand, high amputations of the upper arm remain a difficult challenge. Recent work strives to interface bioelectric and mechanical signals between the residual peripheral nerve and the prosthesis. Our group focuses on the potential of a living regenerative peripheral nerve interface (RPNI) which consists of a unit of free muscle that has been neurotized by a transected peripheral nerve. Our laboratory has previously demonstrated the in vivo stability, conductivity, fidelity, and recording capability of the RPNI. The next critical step is the application of techniques to transmit bioelectric signals from the RPNI in order to achieve successful control of a neuroprosthetic limb. We propose the novel use of bio-integrated thin-film polyimide electrode technology to facilitate signal transduction from the RPNI to the prosthesis. The goals of this research are: 1) to determine the sensing and recording capabilities of polyimide electrodes when applied to normal muscle or normal nerve; 2) to test the signal sensing and recording capabilities of polyimide electrodes in conjunction with an RPNI; and 3) to examine the durability, signal fidelity, and local tissue response of implanted polyimide electrodes after 4 weeks when applied to normal muscle, normal nerve, and RPNI. This work will elucidate the role of polyimide electrodes in peripheral nerve signal transduction and will contribute to the goal of restoring extremity function in patients with limb amputation.

Background: Partial facial allotransplantation is an emerging option in reconstruction of central facial defects, providing function and aesthetic appearance. Ethical debate partly stems from uncertainty surrounding identity aspects of the procedure. There is no objective evidence regarding the effect of donors' transplanted facial structures on appearance change of the recipients and its influence on facial recognition of donors and recipients. Methods: Full-face frontal view color photographs of 100 volunteers were taken at a distance of 150 cm with a digital camera (Nikon/DX80). Photographs were taken in front of a blue background, and with a neutral facial expression. Using image-editing software (Adobe- Photoshop-CS3), central facial transplantation was performed between participants. Twenty observers performed a familiar "facial recognition task", to identify 40 post-transplant composite faces presented individually on the screen at a viewing distance of 60 cm, with an exposure time of 5 seconds. Each composite face comprised of a familiar and an unfamiliar face to the observers. Trials were done with and without external facial features (head contour, hair and ears). Two variables were defined: "Appearance Transfer" refers to transfer of donor's appearance to the recipient. "Appearance Persistence" deals with the extent of recipient's appearance change post transplantation. A t-test was run to determine if the rates of Appearance Transfer differed from Appearance Persistence. Results: Average Appearance Transfer rate (2.6%) was significantly lower than Appearance Persistence rate (66%) (P<0.001), indicating that donor's appearance transfer to the recipient is negligible, whereas recipients will be identified the majority of the time. External facial features were important in facial recognition of recipients, evidenced by a significant rise in Appearance Persistence from 19% in the absence of external features to 66% when those features were present (P<0.01). Conclusion: This study may be helpful in the process of prospective recipients' informed consents. It is beneficial for education of donor families and is expected to positively affect their decision to consent for facial tissue donation.

Injured peripheral nerves have to capacity to regenerate but this does not always translate into functional recovery in patients. We have studied extensively in previous experimental studies the factors responsible for suboptimal return of function in patients with these types of injuries The overall objective of our experimental design is to develop treatment strategies to will improve return of arm or leg function in patients that have sustained nerve injuries The experiments designed in this project are aimed at analyzing the molecular mechanisms of the growth-promoting effect of a growth factor, called transforming growth factor with the ultimate goal being to identify key molecular and/or genetic targets that can be manipulated to maximize nerve regeneration after injuries, which translates into functional recovery. We use a well-established animal model of nerve injury and repair that mimic clinical nerve injuries. Following nerve injuries in the animals, nerve repairs are supplemented with treatment with the growth factor and its effect on nerve regeneration and expression of other key molecules important for nerve regeneration is analyzed using well established laboratory techniques such as immunohistochemistry, light and electron microscopy as well as novel techniques such as gene array. The application of the gene array may lead to discovery of novel genes, molecules and proteins that may turn out to be essential to the process of nerve regeneration and return of function after nerve injuries. Therefore, findings of this research project have strong potential in greatly improving our understanding of how to optimize functional recovery in patients with nerve injuries.

The replacement of osseous tissue lost through trauma, disease or congenital anomalies is a continuing clinical challenge. Craniofacial bone defects are often large and characterized by complex geometry. The ideal reconstructive implant should be a fully osteointegrative biomimetic replication of native bone, fashioned into defect-specific geometry. Current biomimetic implants have recapitulated only the bony matrix, hydroxyapatite. Such implant technology has limited bone in-growth, healing only small intercalary defects. The long term objectives of this study are to develop customized defect-specific implants, and to investigate strategies to ensure osteoinduction, osteoconduction, and osteointegration throughout the implant by optimization of biomimicry to include the cellular and lacunocanalicular fluid flow components of native bone. Firstly, we develop a CT-guided computer aided design microprinter to machine made-to-measure hydroxyapatite tricalcium phosphate matrices. We then establish a 'critical size implanted defect' in a rat calvaria model. Subsequently, our third aim is to improve healing of this defect through progenitor cell seeding of the matrix. Finally, we aim to show that suction mediated increases in lacunocanalicular flow through the cell-seeded matrix and surrounding bone upregulates the mechanotransductive-osteogenic pathway to create an implant that overcomes an otherwise critical size defect. We expect that this technology will be scalable to enable high throughput production of geometrically complex customized implants that successfully restore functionally debilitating and socially incapacitating craniofacial skeletal deficiencies. We aim to improve bone repair and regeneration in order to heal larger osseous defects than have ever been healed before. These implants are a full recreation of native bone and epitomize the next paradigm shift in osseous reconstruction.

Despite the success of distraction osteogenesis (DO) in treating craniofacial disease and extremity deformities, distraction protocols are limited by lengthy activation and consolidation phases. (McCarthy et al 1992). The long term objectives of this study are to create clinically useful strategies to improve vascularization at the distraction bony generate in order to accelerate osteogenesis and improve patient outcomes as well as to elucidate the biomolecular mechanisms involved in translation of mechanical stimulus to new bone formation. Firstly, we will develop 3D flow perfusion multicellular culture as an in vitro tissue model for DO. We aim to show that the BMP-SMAD osteogenesis signalling pathway in de novo osseous tissue is upregulated when osteogenic cells are co-cultured with vasculogenic cells compared with osteogenic cells cultured in isolation. Our second aim is to use fractured murine long bones in 3D flow perfusion explant culture to measure responses of the BMP-SMAD osteogenic pathway to changes in fluid flow. Thirdly, using a rat DO model we aim to show AMD3100, a partial CXCR4 agonist and mobilising agent of endothelial progenitor cells (EPCs) involved in new blood vessel formation, accelerates bony formation in both the activation and consolidation phases of DO by increasing numbers of circulating progenitor cells, improving vascularisation and maintaining upregulation of the BMP-SMAD osteogenesis signally pathway. Understanding molecular events leading to osteogenesis during successful DO has important clinical implications since it is a fundamental step towards evolution of targeted therapeutic interventions designed to accelerate osseous regeneration during DO and optimise patient outcomes. Furthermore, it may allow DO to be used in scenarios of impaired bony healing and holds therapeutic potentials for fracture healing treatment especially in those at risk of avascular necrosis. This study also tests AMD3100, a newly FDA-approved drug.

An estimated 185,000 Americans undergo limb amputation each year; one in 190 Americans has had one already. Modern robotics hold the potential for detailed functional replication of the human hand. The significant obstacle to using these advanced prosthetics, however, lies at the interface between the nerves of the patient the electronics of the prosthetic. This nerve-machine interface is notorious for scarring over the long-term, which ultimately halts electrical communication between the prosthetic and the patient. Importantly, a significant proportion of amputees suffer from neuromas, a tangled cluster of regenerating nerve endings that forms at the end of cut nerves. Neuromas are both painful and a source of action potential signal interference. Our group is interested in developing a peripheral nerve interface that allows the patient to move a prosthetic using his own motor nerves, and to experience sensation from the prosthetic using his own sensory nerves. Such an interface requires high fidelity, long-term biocompatibility, and minimal neuroma potential. We have already synthesized an organic polymer that is very conductive and has demonstrated promising biocompatible traits. This polymer is known as poly(3,4-ethylenedioxythiophene) (PEDOT). To address neuroma formation, we have shown that placing muscle cells at the interface can give regenerating peripheral nerves a target to grow towards, potentially reducing neuromas. Our model is the rat peroneal nerve, a nerve in the leg that carries both motor information from the brain to the foot and sensory information from the foot to the brain. The purpose of this study is to investigate the influence of PEDOT and myoblasts on neuroma formation in rat peroneal nerves.

Although peripheral nerves possess the capacity for axonal regeneration, both functional and behavioural recovery following nerve injury remain relatively poor and may be accompanied by debilitating neuropathic pain. This impairment is even more pronounced with proximal nerve injuries, which lead to massive injury-induced motorneuron cell death. P7C3, a novel aminopropyl carbazole, and its analogue (P7C3A20) have recently been shown to display proneurogenic, neuroprotective properties, and may promote neuronal survivial and enhance regeneration following nerve injury. However, this compound has not yet been meticulously tested in a model of nerve injury and repair. The OVERALL OBJECTIVE of the proposed project is to enhance motor neuron survival, regeneration, and behavioural recovery following administration of the neurotrophic compound P7C3/P7C3A20 in both a neonatal and adult rodent model of sciatic nerve injury. The overall objective will be addressed through the following three Aims: (1) we will examine the neuroprotective role of P7C3/P7C3A20 treatment on both motor neuron survival and axonal regeneration following sciatic nerve injury in both a neonatal and adult injury paradigm; (2) we will assess the utility of P7C3/P7C3A20 therapy to improve both functional regeneration and behavioural recovery in both a neonatal and adult model of sciatic nerve injury, and; (3) we will examine and determine potential candidate factors responsible for the mechanism behind P7C3/P7C3A20 mediated neuroprotection such as neuregulin (NRG) and calcitonin gene-related peptide (CGRP). Detailed anatomical, histological, electrophysiological, and skilled locomotor assessments will be employed in order to properly address these important questions. Our long term goal is to enhance nerve regeneration and subsequent behavioural recovery following nerve injury through the use of this novel compound. Both of these models are clinically viable and translatable, which may ultimately lead to wide scale medical administration of this compound following nerve injury in a patient population.

Following peripheral nerve injury and reconstruction, a major factor contributing to the limited return of motor function is the time required for the regenerating nerve to reach its target muscle. The calcineurin binding agent FK-506 (tacrolimus) has demonstrated the ability to accelerate nerve regeneration and accelerate restoration of function following peripheral nerve injury. However, its immunosuppressive properties make it undesirable for use in neurological situations not requiring immunosuppression. FK-506 acts directly on neurons to enhances regeneration by binding to heat shock protein 90 (Hsp-90) and disrupting the steroid receptor complex. In vitro studies have shown that agents that bind Hsp-90 enhance neurite extension in neuronal cell cultures. These Hsp-90 binging agents have yet to be extensively evaluated for there ability to enhance peripheral nerve regeneration in vivo, however other analogs of FK-506 that have shown promise in vitro have demonstrated short commings in vivo. Peripheral nerve regeneration in vivo is fundamentally different than simulating regeneration in in vitro because of Wallerian degeneration (WD). WD in vivo is necessary for peripheral nerve regeneration and is partially carried out through activation of the innate immune response. Recently, it was demonstrated that increased levels of calcineurin (the target of inhibition by FK-506 in vivo) reduces the activation of the innate immune response. Delays in WD in the distal stump have been shown to negatively affect nerve regeneration. It is likely that FK-506 not only stimulates nerve regeneration by acting on the neuron, but also prepares the distal stump for that regeneration by enhancing the innate immune response and thus WD through suppression of calcineurin. Here we propose to investigate the contributions of both these FK-506 derived mechanism of nerve regeneration by evaluating each mechanism independently in comparison to treatment with FK-506.

In the last few years, the focus in tissue engineering has become more complex. Instead of growing a single cell source on a uniform scaffold, one is now trying to mimic the natural tissue as close as possible by using the several cell types that make up a tissue and scaffolds with specific regions for specific organizations of the tissue. However, one of the major limitations of tissue engineering is still the inability to provide sufficient blood supply in the initial phase after implantation. Our studies of two-dimensional co-culture demonstrate, that out of ten different cell combinations, bone formation to be greatest when a more mature vasculogenic cell is grown with a less mature osteogenic cell, ie human umbilical veins endothelial cells (HUVECs) and osteoprogenitor cells (OPCs). Based on our previous experiments, the goal of the current study is to create an in vitro endothelial network in a bone tissue engineering construct by seeding HUVECs and OPCs on HA-TCP scaffolds, which has never been tried before (Aim1). We will compare the osteogenic markers (alkaline phosphatase, osteocalcin), angiogenic markers (CD 31, VEGF 165, Vegfr1), histology, uCT imaging and immunohistology of our novel model to HA-TCP scaffolds seeded with HUVECs or OPCs as a comparative control. In Aim 2, we will then investigate optimum cell seeding ratios and investigate the effect of time delay co-culture on vessel like structures formation and bone nodule formation (Aim3). In Aim 4, we will investigate the effect of addition of VEGF and PDGF BB to the culture's media.

Ventral hernia recurrence is associated with substantial patient morbidity and presents a pervasive surgical dilemma. Adipose-derived stem cells (ASCs) are shown to improve healing in many settings. We propose a pilot study to evaluate ASCs in ventral hernia repair with non-cross-linked porcine acellular dermal matrix (ncl-PADM) in vivo. Stem cell research requires definitive quantification of the extent to which ASCs augment neo-vascularization, cellular infiltration, and repair site strength, as preliminary studies support their utility in these outcomes. Our hypothesis is that seeding ASCs onto ncl-PADM will increase the neo-vascularization and cellularization within the material as well as the mesh incorporation strength at the musculofascial edge in hernia repairs. An established syngeneic rat model will be used. 4 Brown-Norway rats will provide ASCs for culture and transfection with green fluorescent protein (GFP). 4 x 2 cm elliptical mesh constructs (ncl-PADM) will be seeded 1 x 10^6 ASCs for 24 hours. An acute ventral hernia will be made and repaired via an inlay, bridged technique. Group 1 (n=30) will serve as the controls and receive repair with un-seeded ncl-PADM. Group 2 (n=30) will undergo repair with ASC-seeded ncl-PADM. Animals will be euthanized at week 2, 4, or 6. Analysis will include histologic and immunohistologic evaluation with H & E stain for cellularity and Factor 8 for neo-vascularity. Uniaxial mechanical testing to determine incorporation strength will be performed. Stress strain curves will determine ultimate tensile strength and elastic modulus. Confocal microscopy will show fluorescent immunostaining of the GFP-labeled cells within the repair site and biomaterial. If ASCs are found to quantitatively enhance in vivo repair with ncl-PADM, preliminary clinical trials in humans may be warranted, as this novel technique could ultimately have significant clinical utility in decreasing hernia recurrence and its associated morbidity.

Despite optimum immunosuppression in clinical composite tissue allotransplantation (CTA), long-term graft tolerance remains a hopeful goal. Immunomodulation rather than immunosuppression may prove to be the key to tolerance, with the goal of eliminating or reducing the need for long-term immunosuppression. Anti-lymphocyte serum (ALS) depletion along with cyclosporine (CsA) is widely used in transplantation, but it is not tolerogenic since T-regulatory cells (Tregs) require interleukin-2 (IL-2) for survival and CsA blocks IL-2 transcription and consequent IL-2 dependent regulation. Exogenous IL-2 can prolong graft survival in mouse islet cell transplantation1 and can be similarly used in CTA. Members of our lab previously developed an IL-2 fusion protein (IL-2Fc) with a longer half-life2, which we began testing in CTA. In preliminary studies, 40% of animals that received IL-2Fc+ALS+CsA began to reject the limb at day 40 but then spontaneously recovered and survived >150 days, while the other 60% rejected by day 60. We hypothesize that adding IL-2Fc to ALS+CsA enhances Treg survival and consequent survival of CTA, and the 40% rejection was due to an initial T-effector (Teff) cell response that varied between animals. Adding topical immunosuppressants at the first sign of rejection may thus lead to 100% survival. We will perform orthotopic hind limb transplants in rats to compare IL-2Fc+CsA+ALS versus that regimen plus topical FK506. We will analyze the ratio of T-cells and gene expression patterns to test for variable T-cell responses. These studies will elucidate the mechanism of IL-2Fc-induced graft survival and the efficacy of topical immunosuppression as a graft salvage strategy. The regimen we utilize is similar to that used in clinical transplantation and is thus directly relevant. These data will further our understanding of the complex immunologic reactions that occur with CTA and how the reconstructive surgeon can best manage the patient's graft.

Lymphedema is a chronic, debilitating condition that reconstructive surgeons frequently encounter in patients treated for breast cancer. There is no cure, and only limited treatment options. Lymphedema results from inadequate repair of lymphatic vessels following surgery. The literature as well as our own preliminary data have implicated the balance of pro and anti-lymphangiogenic growth factors in the modulation of lymphatic regeneration. Local stem cells have been implicated as sources of important growth factors in wound healing and angiogenesis. Transplanation of adipose derived stem cells (ADSCs) has been shown to improve wound healing and angiogenesis. Further, we have shown that radiation, a major risk factor for the development of post-operative lymphedema, causes local stem cell dysfunction. We therefore hypothesize that lymphatic regeneration following wounding is dependent on a balance of pro and anti-lymphangiogenic factors and that this balance is in part mediated by resident stem cells. We will test our hypothesis using two specific aims. Specific Aim 1: To determine the efficacy of ADSCs in preventing lymphedema and Specific Aim 2: To determine the efficacy of ADSCs in treating established lymphedema. Our experimental approach will be to use a mouse tail model of surgical lymphedema. ADSCs will be transplanted at the time of surgery to prevent lymphedema, or once lymphedema has been established, in order to treat it. We believe that ADSCs will improve lymphatic regeneration by altering the molecular balance of the wound in favor of pro-lymphangiogenic factors. Lymphatic regeneration and function will be evaluated at various time points. The use of ADSCs in the prevention and treatment of lymphedema is a pealing because of the safety and ease of the technique. This study could also potentially guide options for types and timing of breast reconstruction in breast cancer patients.

There are over 1.7 million people within the United States currently suffering from some type of limb loss, and this number continues to grow by 185,000 each year. Typical upper extremity replacement limbs are passive prosthetic devices and provide little functional recovery beyond basic grasping. Newer prostheses that add additional control through using muscle activity of the patient's remaining muscle groups increase the utility of these replacement limbs. The current state of the art treatment allows subjects to have the greatest restoration of function through targeted reinnervation of muscle groups using nerve from the amputated limb. However, these devices have limited control options for prostheses with multiple degrees of freedom and are generally difficult to master. We have developed a regenerative peripheral nerve interface (RPNI) that creates a biologically robust and functional connection to the nerve in an amputated limb through the use of a graft of free muscle tissue. The graft is then sutured to the severed residual nerve, and electrodes are affixed allowing signals to be recorded from the nerve (epineural electrode), or the muscle (epimysial electrode). These electrophysiological signals are then used for control of a replacement robotic arm. We propose to quantify the effect of neuromuscular amplification in regenerative peripheral nerve interfaces. Using a rodent model developed within our research group, we will quantify the information content that can be recorded by using a neuromuscular "amplifier". Nerve signals will be sampled after they have been "amplified" by the muscle, thereby providing a higher signal-to-noise ratio without signal loss from electrode encapsulation and tissue trauma from direct epineural electrode placement. Further, the use of epimysial electrodes should increase the overall long-term stability of the interface, compared to current methods utilizing penetrating electrodes either in the nerve or muscle.

The endothelial barrier is the frontline defense of the vascular network; it is the backbone of a tightly regulated gateway that controls nutrient/waste delivery, fluid balance, host defenses, and ultimately, tissue survival. Unfortunately, the failure of this gateway represents a common feature within many clinical challenges of plastic surgery, including chronic wounds, tissue edema, ischemia-reperfusion injury, flap failure, and even allograft rejection. Currently, there are few strategies to improve endothelial integrity; treatment focuses solely on combating the overarching pathology. Preliminary data by our lab suggest that mesenchymal stem cells (MSCs) can protect the vascular network. We propose that MSC-seeded vasculature can effectively mitigate endothelial injury in in-vitro and in-vivo models of ischemia-reperfusion and immune-mediated rejection. We further intend to demonstrate that this vasculoprotective effect occurs through synergistic crosstalk between endothelial cells and MSCs. Strategies to protect the vasculature at the time of (or prior to) cytotoxic injury represents a paradigm shift in the use of MSCs in regenerative medicine, and has direct clinical applicability in reconstructive microsurgery, composite tissue allotransplantation, and tissue engineering.

Approximately 35-50% of children with nonsyndromic, single-suture craniosynostosis (NSC) have neuropsychological (NP) disabilities. Currently, no reliable method with predictive value exists for identifying patients with NSC at risk for developmental delay. Studies using developmental quotients (DQ) and intelligence quotients (IQ) to assess the intellectual capacity of infants with NSC are not predictive of long-term NP outcomes. This critical finding may serve as an important source of error in the data gathered to date, as erroneous conclusions may be reached regarding these patients' intellectual function. Thus, there is a need for an objective tool used to predict NP outcomes, enabling us to assess how surgery impacts intellectual function. Recent studies demonstrated that event-related potentials (ERPs) in infancy have strong predictive value for NP outcomes. Measuring ERPs will permit the identification of infants with NSC at risk of future NP deficits. Moreover, ERPs can potentially serve as an objective, standardized measure to evaluate the impact of the age and type of surgery on NP outcomes. We hypothesize that surgery will improve sensory processing, correcting ERP abnormalities. Specifically, we hypothesize that the younger the infants with NSC undergo surgery, the fewer NP deficits will be observed. Also, those children undergoing more comprehensive whole-vault cranioplasty, will have a lower incidence of NP sequelae. This project aims to: 1) Establish baseline ERPs and identify differences in sensory processing in infants with NSC compared to controls; 2) Examine how surgery changes sensory processing in infants with NSC by comparing pre- and post-operative ERPs; and 3) Utilize ERPs to assess the impact of age and type of surgery on NP outcomes. If this study confirms the aforementioned hypotheses, ERP testing may become standard of care, identifying the optimal age and type of surgery to reduce potential long-term NP disabilities.

Autologous fat has the potential for being the ideal soft-tissue filler. It possesses a natural consistency, is easy and safe to harvest, readily obtainable and because it is autologous, antigenic and allergenic reactions are minimized. Nonetheless, fat grafting is imperfect, as it is limited by unpredictable survival and because of this unpredictable behavior clinicians are faced with uncertainty concerning the ultimate volume of the graft, so that overcorrection and re-transplantation may be necessary to achieve satisfactory results. At present the exact mechanisms that dictate fat graft survival and resorption remain relatively unclear. One potential mechanism that may be responsible for graft loss is the lack of adequate revascularization within the transplanted fat. This project aims to investigate the role of the graft itself in its vascularization and survival after transplantation. We postulate that autologous fat graft survival is critically dependent on survival of the intact vasculature surrounding the adipose microstructure. Furthermore, we believe that FDA-approved therapeutics targeting preservation of this vasculature during its hypoxic phase would result in more reliable and predicable outcomes following autologous fat grafting. Specifically, we plan to determine the patterns of vascularization of autologous fat following grafting (donor-site or recipient-site derived) and the specific contribution of an intact donor vascular network on survival using a novel transgenic murine model. Finally, we will investigate a rapidly translatable FDA approved therapeutic intervention that we expect will make autologous fat grafting a more reliable and predictable technique in clinical practice, hence leading to more widespread use.

Lymphedema is a chronic painful swelling and disfigurement of the limbs that commonly occurs after the removal of lymph nodes for the treatment of cancer. Lymphedema is characterized by persistent inflammation and progressive scarring that progressively leads to obstruction of lymphatic channels. We propose that lymphedema is a progressive disorder of abnormal scarring, or fibroproliferative disorder. Among fibroproliferative disorders, the immune system acts to regulate tissue fibrosis. In fact, activation of a subtype of T-lymphocytes in the immune system called T-helper 2 (Th2) cells leads to fibrosis and scarring. We hypothesize that, similar to mechanisms regulating other fibroproliferative disorders, the pathologic effects of chronic lymph stasis are modulated by immune responses. Our objective is to determine how these immune responses regulate the development and progression of severe lymphedema. Our specific aims are: Specific Aim 1: Determine the role of Th2 immune responses in the initiation and progression of fibrosis and lymphatic dysfunction secondary to sustained lymphatic stasis and Specific Aim 2: Determine the role of the pro-fibrotic cytokine, transforming growth factor-beta 1 (TGF-B1), and adaptive immune responses in the development of chronic lymphedema. Our experiments will evaluate the effect of inhibiting the Th2 response and the pro-fibrotic cytokine TGF-B1 on the initiation and maintenance of lymphedema. We anticipate that blockade of these factors will prevent establishment and reverse the effects of severe lymphedema. We will perform our experiments using a mouse model in which sustained lymphedema is surgically induced. Outcomes of lymphatic function, tissue fibrosis, and chronic inflammation will be measured. Knowledge derived from our study will potentially lead to novel and targeted therapeutic strategies for the prevention and treatment of chronic lymphedema and may broadly apply to the treatment of other fibrotic disorders.

Plastic surgeons are often asked to help in the management of calciphylactic wounds. Calciphylaxis is a poorly understood and highly morbid syndrome of vascular calcification and skin necrosis. Factors implicated in the pathogenesis of calciphylaxis include chronic renal failure, obesity, diabetes, hyperphosphatenemia and secondary hyperparathyroidism. We wish to propose a novel hypothesis that leptin produced by the parathyroid gland is an important pathogenic factor in this complex disease. This is of particular importance since the reported mortality rate of calciphylaxis is between 60-80%. Calciphylaxis was originally thought to be secondary to elevated calcium and phosphate levels and hence parathyroidectomy was performed in an effort to treat this pathology. However decrease in calcium levels did not alter the course of the disease process thereby implicating the presence of another factor. Leptin is a well known adipocyte hormone which has pleiotropic functions including control of body weight, reproduction, angiogenesis and, as found recently, bone mass (1). This hormone has never been shown to be present in parathyroid tissue. We wish to propose a novel hypothesis that leptin is also an important calcium regulatory hormone secreted by the parathyroid gland which may influence the pathogenesis of calciphylaxis. To this end we will 1. Confirm the presence of leptin (ob) and its receptor (ob-R) (long and short forms) in normal and abnormal parathyroid tissue (adenomas and hyperplasia) 2. Measure the variations in serum leptin levels in humans following removal of pathologic parathyroid tissue, i.e. resection of solitary adenomas or near complete parathyroidectomy (3.5 gland resection) for hyperplasia or resection in the setting of surgical treatment of calciphylaxis 3. Identify the presence of leptin and leptin receptors in calciphylactic tissue 4. Identify pathways involved in the leptin mediated control of calcium regulation using whole tissue cult

Palatogenesis involves complex cellular movement and morphogenesis events that are difficult to study in a mammalian model. The zebrafish (Danio rerio) is a powerful model for studying palate development owing to its developmental, genetic, and practical characteristics. Several studies have established that the zebrafish embryonic ethmoid plate is analogous to mammalian palate structure. Zebrafish embryos develop ex vivo and are transparent, where migratory neural crest cells can be fate-mapped in real time. Lineage-specific labeling of migrating neural crest cells could be achieved by generating a transgenic fish where the sox10 promoter drives the expression of the green-to-red photoconvertable protein, kaede. Endogenous sox10 gene expression is restricted to neural crest cells, oligodendrocytes, and the otic epithelium. Therefore, the sox10:kaede transgenic will provide a powerful method of following neural crest cells migrating to form the face at single-cell resolution, in real time. Our first specific aim is to generate a sox10::kaede transgenic zebrafish. Our second specific aim is to use sox10::kaede to trace neural crest cell lineage in the context of gene knockdown and mutant zebrafish. The results of these lineage tracing experiments will be compared to previous work on zebrafish neural crest migration, to improve our understanding of this intricate process. In addition to being well suited for genetic manipulations, zebrafish have also been used to successfully identify chemical modifiers of vascular and hematopoietic phenotypes. Our third aim is to dissect the Wnt regulatory pathway in zebrafish oropharyngeal development, with functional and biochemical analysis of Wnt9a. Wnt signaling provides important cues regulating migration of neural crest cells that form the vertebrate craniofacial structures. We are exploiting the advantages of the zebrafish system to elucidate the role of Wnt signaling in palatogenesis, through morphogenetic studies of Wnt9a.

Since it's advent in the 1970's microsurgical repair has remained the gold standard approach to peripheral nerve injury. Microsurgical neurorrhaphy often results in a suboptimal functional recovery. Photochemical tissue bonding (PTB), a method of covalent bonding with a laser activated dye, combined with an amnion nerve wrap, has been shown in animal models of nerve repair to have improved functional outcome over standard repair. Sealing of the repair site isolates the endoneural environment, reduces axonal escape, adhesion/scar formation and disturbance from extrinsic inflammatory factors. Leukemia inhibitory factor (LIF), a potent neuromuscular growth factor has a powerful effect in enhancing the survival of both motor and sensory neurons, while reducing denervation-induced muscle atrophy following nerve transaction in many in vitro and in vivo studies. In addition, LIF attracts activate macrophages important in Wallerian degeneration and recycling of cholesterol to regenerating neurons, stimulates mitosis and trophic factor production by Schwann cells. Transcription and synthesis of LIF at the injured site takes 2-7 days and anterograde LIF transport ceases within 24 hours after nerve injury, suggesting that exogenous administration of LIF to a neurorrhaphy site would improve functional outcome. Hypothesis: Exogenous administration of LIF into photochemically sealed amnion-wrapped neurorrhaphy sites will enhance functional outcomes over PTB/amnion alone or microsurgical repair alone. Rationale: PTB with amnion wrapping and administration of LIF separately have been shown to improve functional outcome in nerve repair when evaluated individually. Administering LIF into a PTB-sealed amnion wrap at the repair site should optimize its delivery, retention, efficacy and the functional outcome. Aim: Compare the technique of delivering LIF into PTB/amnion wrapping with PTB/amnion wrapping alone and with standard microsurgical repair of peripheral nerve injuries.

Treatment options for ulnar neuropathy at the elbow (UNE) are extensive, but controversy exists over the effectiveness of these procedures, including simple decompression, decompression and medial epicondylectomy, anterior subcutaneous transposition, and anterior submuscular transposition. Reflected in the evolution and number of variations of surgical procedures for UNE is the intense debate among expert hand surgeons in the past decades regarding which of these procedures are optimal. To date, the literature remains surprisingly inconclusive despite the progressively debilitating nature of UNE. In the absence of evidence-based practices, a decision analysis and economic evaluation is a valuable tool for comparing treatment options. Decision analysis is a method of assigning a level of desirability, or utility, to various health states. In UNE, a progressively debilitating chronic condition, the patient's quality of life is affected. Thus understanding patients' preferences, in addition to the costs of the treatments, may be the most important factors in choosing among treatments. We thus propose to conduct a cost-utility analysis comparing conservative treatment and four common operative techniques (simple decompression, anterior subcutaneous transposition, anterior submuscular transposition, and medial epicondylectomy) for UNE. Utilities will be measured directly on a sample of community members that represent potential future patients who may develop UNE by means of a time trade-off survey supplemented with animations. A cost-utility analysis for UNE would be an informative decision aid for both patients and surgeons when choosing among all the different surgical options.

Functional motor recovery following limb allotransplantation is based on regeneration of the transected host and donor neurons. As a therapeutic agent,Schwann cells can successfully and safely be harvested,cultured,and reintroduced into peripheral nerves to aid in and favor neuroregeneration. This study aims to examine the mechanism and effects of neuroregeneration following limb allotransplantation by introducing isologous Schwann cells into the sciatic nerve of the donor leg prior to engraftment. A fully mismatched rat hind limb allotransplantation model where Brown Norway rats serve as limb donors for Lewis rat recipients will be utilized. All donor limbs will receive Schwann cells, suspension solution, or Schwann cells transfected with Green Fluorescent Protein (GFP). Neuroregeneration will be examined after 4 and 12 weeks. Recipient animals will undergo walking track analysis in order to assess the hind limb motor performance by video capture and analyzed to measure the stance factors and joint angles. Electrophysiology will assess the quality of neuroregeneration. This will provide detailed clinical information about evaluating peripheral nerve function after nerve injury and repair in this transplant model. In addition, the gastrocnemius muscle ratio for each rat will be measured. Those nerve samples treated with Schwann cells or suspension solution will then be embedded in epoxy and stained for Toluidine blue to characterize myelin of regenerated nerve fibers. The cellular counts and structural architecture will be analyzed with light microscopy. Those nerve samples treated with GFP-Schwann cells will be embedded in Paraffin and stained for the Schwann cell marker S-100 and further visualized for co-localization of GFP using fluorescent microscopy. The results of this study will significantly contribute to advance our knowledge of Schwann cells as a therapeutic option for improved nerve regeneration and functional outcome following limb allotransplantation.

The care of chronic wounds in the elderly in the U.S. costs approximately $25 billion annually. Wound healing is impaired in the elderly, and it is worsened by illnesses and conditions that reduce blood flow and decrease oxygen levels in wounds. These factors contribute to the generation of reactive oxygen species (ROS) in wounds, which further increases the chance that they will become chronic. The body's antioxidant defense system protects against ROS generation and damage, and the antioxidant superoxide dismutase is a key feature of this system. A significant gap in knowledge is the mechanism that makes the antioxidant system less efficient in the elderly. We will use a model of wound generation and healing that uses the commercially available skin matrix called Dermagraft®. Dermagraft® is used clinically to enhance wound healing, and it contains specialized cells from newborns that promote skin cell migration – a key feature of wound healing. In Aim 1 we will determine if ROS levels are higher in skin cells (fibroblasts and keratinocytes) from aged versus young donors, and if high ROS levels inhibit keratinocyte migration across Dermagraft®. In Aim 2 we will determine if levels of superoxide dismutase are lower in skin samples from aged versus young donors, and if low oxygen levels affect the ability of skin cells from the edge of a wound to migrate and initiate healing.

Currently, our ability to analyze the composition and physical maturity of stem cell graft-derived myelin is limited to the study of fixed, dead tissues. For translational study of graft-derived myelin in humans, this is obviously inadequate. Spectral imaging of environmentally sensitive myelin dyes, with their ability to detect physical and chemical state of their surroundings, may be the answer to this problem. SKP-SCs are a potentially autologous source of Schwann cell like cells that have improved nerve regeneration in both nerve gap and chronic denervation models. We propose that these cells can remyelinate demyelinated peripheral nerve axons, and that the maturity of this myelination can be characterized and analyzed by the fluorescent spectral characteristics of SKP-SC myelin, as stained with environmentally sensitive myelin dyes. Aim 1 - proposes that environmentally sensitive myelin dyes can detect changes in the structure and composition of myelin. We will therefore analyze myelin spectra during developmental time points of the rat sciatic nerve. Sciatic nerves from MBP and P0 knockout mice will also be examined. Aim 2 - proposes that SKP-SCs can indeed myelinate peripheral axons (demonstrated in preliminary data), and that maturity of this myelination can be followed in-vivo using the dyes elucidated above. We will use an adriamycin injury model (demyelination) to analyze labeled SKP-SC induced remyelination. Aim 3 - proposes that SKP-SC remyelination can be imaged (spectral 2-photon) in living rat sciatic nerve, and that the spectral signature of the SKP-SC myelin will correlate with its structure and composition.

Cleft lip and palate (CLP) is the most common craniofacial birth defect where improved understanding of the genetic and embryological basis that underlies the CLP malformation is necessary to identify potential treatments or prevention. IRF6 is a transcription factor that has been shown to play a role in epithelial morphogenesis. Human mutation of IRF6 results in syndromic cleft lip and palate including Van der Woude and Popliteal Pterygium syndromes. Recently, IRF6 has been confirmed as a key locus for non-syndromic cleft lip and palate from large population genome-wide association studies. The zebrafish is a powerful vertebrate model that can be used to study the genetic and embryological basis that underlie cleft lip and palate malformation. We aim to study the molecular and morphogenetic mechanisms of IRF6 function in vertebrate craniofacial development. We are generating IRF6 dominant negative mutants based on known human mutations to study the gain-of-function phenotype and generate a zebrafish model of orofacial clefting. Our second aim is to perform fate mapping analysis of cranial neural crest cells during palate formation in IRF6 dominant negative transgenic lines. We have generated IRF6 dominant negative transgenic lines in the background of sox10::kaede fish to be utilized for lineage tracing studies. Furthermore, we have shown the utility of the sox10::kaede model by photo-labeling cells that form the frontonasal process, maxillary process and mandibular process long before organogenesis has begun. To better understand the morphogenetic events that occur in cleft palate formation, we plan to utilize IRF6 dominant negative transgenic lines to photo-label precursor cells destined to form the maxiallary and frontonasal process and follow their course during palate formation by time-lapse photography. We believe the IRF6 dominant negative transgenic model will serve to better understand the complex migratory events that occur during palate formation. Impact Statement: The aim of this research project is to investigate the functional and molecular role of IRF6 in zebrafish via dominant negative transgenesis. Improved understanding of the genetic and embryological basis that underlies the cleft lip and palate malformation will give further mechanistic insight and help identify potential treatments or prevention.

Lymphedema is a common condition affecting nearly 5 million Americans. One third of patients who undergo lymph node removal for cancer treatment will develop lymphedema, which is characterized by scarring, inflammation and fat deposition. The pathogenesis of lymphedema is still largely unclear, which has led to a lack of effective and targeted treatment options. We have demonstrated that lymphedematous tissue is populated by increased adipose tissue, dilated and dysfunctional lymphatic vessels, dense collagen deposition and prominent inflammation. We have also shown that lymphatic fluid stasis potently upregulates the expression of fat differentiation markers (i.e. PPAR-y, CEBP-a). We hypothesize that lymphatic stasis induces chronic inflammation that drives adipose deposition by adipose cell hypertrophy, mesenchymal stem cell (MSC) migration and adipogenesis. Our objective is to determine how adipocytes are regulated in the setting of lymphedema. Our specific aims are: Aim 1: Determine how obesity and lymphatic stasis are related. Aim 2: Determine how CD4+ cells regulate adipogenesis. The objective of our first aim is to define the relationship between obesity and lymphedema. We anticipate that lymphatic fluid stasis will contribute to adipose deposition in lymphedema and obesity. Experiments will be performed using a microsurgical mouse tail model of sustained lymphedema and outcomes of lymphatic function, fat deposition and inflammation will be measured. The objective of our second aim is to determine if CD4+ cells can promote adipogenesis. We anticipate that CD4+ T cell differentiation resulting from lymphatic stasis will cause MSC migration, adipocyte hypertrophy and adipose differentiation. We will perform tail operations on nude mice (no T cells) and utilize adoptive T cell transfer to test our hypothesis. We hope to elucidate the role of adipose tissue in the development of lymphatic fluid stasis as a means to develop targeted therapies for lymphedema.

Since the 1980's, there has been an increased interest in autologous lipofilling for breast reconstruction after mastectomy. By providing excellent reconstruction results without severe donor site morbidity, autologous fat has been considered an ideal filler. However, concerns over graft survival and oncological safety have limited its application. Modified liposuction technique, various growth factors and cytokines have been used during fat grafting to increase graft survival. However, clinical results remain limited. Recently, an alternative approach relying on enhancement of the proportion of adipose-derived stem cells (ADSCs) within the fat has been found to promote long term graft survival. However, these cells have been suggested to play a role in carcinogenesis. There are studies showing that ADSCs may contribute to tumor growth or even differentiate into tumor related cells, but other studies also suggest an anti-tumor effect in these cells. Despite these controversies, there is a consensus that ADSCs can migrate to tumors in vivo, which gives them potential for use in gene therapy to treat cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) superfamily, which causes apoptosis of tumor cells through the extrinsic apoptosis pathway without damaging normal cells. It has been found effective in treating several malignancies including breast and gynecologic cancers. There are also studies showing that inducible TRAIL-expressing ADSCs can migrate to and kill both primary breast tumor and its metastases. Moreover, the "stemness" and the multi-differentiation potential of these cells are well reserved during transduction. In this study, lipofilling combined with genetically engineered TRAIL-expressing ADSCs will be applied to a breast cancer xenograft model. The survival of the fat graft and growth of the tumor will be evaluated after transplantation to test the hypothesis that TRAIL-expressing ADSCs can promote the survival of the fat graft and inhibit growth of the tumor. Although our studies will be performed in mice, this project has great translational potential. This is because ADSCs have already been used in humans and are proven to have promise for gene therapy in cancer treatment.

Autologous fat grafting is a simple and natural way of doing face reconstruction, but it is still not accepted as a standard procedure because of the unpredictable clinical results caused by low rate of fat graft survival. The main objective of this project is to improve fat graft survival by adding adipose-derived stem cells (ASCs) non-virally transfected with vascular endothelial growth factor (VEGF) and increasing graft vascularization. This project will be divided in three parts: 1) Transfecting ASCs using a non-viral delivery system based on nanobiohybrids, 2) In vitro characterization of signal efficiency and duration and 3) Evaluating the efficacy of this system on graft survival in animal model. The first phase of the study will include: fabrication of nanobiohybrids, ASCs transfection with GFP, and transfection efficiency and signal duration assessment. The second phase: ASCs transfection with VEGF and impact of this system on graft survival in animal model. Athymic nude mice will be divided in three groups (n=20): experimental group - lipoaspirate and VEGF-ASCs, positive control - lipoaspirate and GFP-ASCs and negative control - lipoaspirate. Animals will be followed-up for 12 weeks. Plasma VEGF levels, weight and volume displacement measurements, H&E and immunohistochemical staining for CD 31 will be performed. The exact number of newly formed blood vessels will be determined using software (Metamorph). The significance of this study is to implement a relatively simple, efficient and safe transfection technique to ASCs in order to increase the vascularization and survival of fat grafts. To the best of our knowledge, this is a novel approach to VEGF delivery. Up till now, VEGF has been efficiently delivered using a viral vector, which carries the risk of immunogenicity and insertional mutagenesis. This study is focused in establishing fat grafting as a reliable, standard clinical procedure for face reconstruction.

Composite Tissue Allotransplantation (CTA) is the umbrella term for transplantations composed of multiple tissues like the hand or face. However, a major drawback for CTA is the requirement for lifelong immunosuppression. The growing development of CTA highlights the need for tolerance induction protocols. CD4+ Foxp3+ regulatory T cells (Treg) have emerged as critical effectors in tolerance induction and maintenance. Evidence exists that Treg are selectively expanded by Rapamycin or T cell-depleting agents. Combination of Rapamycin and T-cell depletion treatment has been observed to promote long-term limb allograft survival in a mouse CTA model, but not sufficient to induce tolerance. The essential contribution of TGF-beta to the generation and function of Treg is reminiscent of its potential role for the control of transplant immunity. Thus, we hypothesize the synergistic action of TGF-beta, Rapamycin and T-cell depletion will further shift the balance of transplant immunity toward tolerance by strengthening the cadre of Treg. Moreover, we have developed a novel auto-active long-lived human mutant TGF-beta/ Fc fusion protein to overcome the problem associated with the short t1/2 and secreted latent form of TGF-beta. In this project we will explore a short course of TGF-beta/Fc fusion protein, Rapamycin and antilymphocyte serum combined therapy to induce donor-specific tolerance in rat hind climb allografts and further investigate their underlying mechanisms. Achieving tolerance will have a profound impact on the clinical application of CTA, likely greater than even solid organ transplantation. This study is likely to lead to a safe, effective and pro-tolerogenic approach for CTA.

Many factors contribute to the impairment of wound healing in diabetic patients. Diabetic wound healing may be impaired due to macro and micro angiopathy, decreased blood flow, decreased immune response, decreased availability of stem or progenitor cells involved in neovascularization and tissue regeneration, and decreased cellular activity due to glycosylation. The purpose of this study is to investigate the role of circulating endothelial progenitor cells in diabetic wound healing. With increased knowledge of the mechanisms resulting in impaired diabetic wound healing, we may be able to better treat these patients or even prevent the occurrence of chronic wounds all together. We hypothesize that impaired diabetic wound healing is in part a result of a decreased population of circulating endothelial progenitor cells (EPC) as well as an inability of these cells to migrate to ischemic tissues. We propose that improving these processes or circumventing them altogether will lead to improved diabetic wound healing.

Breast cancer affects one out of every eight American women in their lifetime. The primary goal of breast reconstruction after mastectomy is to re-establish a sense of physical normalcy in the breast cancer survivor. The autogenous transverse rectus abdominus musculocutaneous (TRAM) flap represents the gold standard in post-mastectomy breast reconstruction. Several studies, however, have shown high rates of abdominal wall morbidity including decreased strength, abdominal laxity, abnormal contour, and frank bulge or hernia formation. In recent years, development of new microsurgical techniques has introduced the deep inferior epigastric perforator (DIEP) flap and the superficial inferior epigastric artery (SIEA) flap. These operations spare the abdominal wall to varying degrees, and represent more technically demanding alternatives to the traditional TRAM. Our research group is involved in determining the functional significance of donor site morbidity related to these three methods of autogenous breast reconstruction. Although previous studies have revealed differences in the function of the rectus abdominus muscle following the free TRAM, DIEP and SIEA using EMG, MRI, ultrasound and dynamometer testing, none of these studies include information on the functional significance of these tests and their relation to patient reported outcomes. We believe that applying functional muscle testing and patient quality of life surveys in a prospective study of surgical modalities will allow us to distinguish the impact of these three operations on abdominal wall function. Preoperative assessment of abdominal wall integrity will be performed utilizing the "Manual Muscle Function Test" for trunk flexion and the Functional Independence Measure (FIM) for getting up out of bed. Patients will also complete a SF 36 quality of life survey, as well as a survey designed specifically for abdominal wall function in everyday life. At the 3, 6, and 12 month post-operative visits, a physical therapist will evaluate the patients' abdominal strength, function and range of motion using tests described above, and quality of life surveys will be repeated. ANOVA will be used to compare differences across subgroups and logistic regression analysis performed to determine which risk factors predict post-reconstruction complications.

To expand our knowledge of current options for post-mastectomy breast reconstruction, we propose to build a multicenter, international consortium of centers and a Web-based patient registry to track long-term outcomes of post-mastectomy reconstruction. Eleven centers in the United States, Canada, and Belgium with demonstrated expertise and high case volumes in these procedures have agreed to participate. The overall purpose of the proposed project is to establish the infrastructure and to pilot test the instruments and other data-gathering processes for a later Department of Defense Breast Cancer Research Program (DoD BCRP) Center of Excellence grant. Eventual DoD funding will enable the consortium to recruit and track breast reconstruction patients among the eleven sites for at least three years following reconstruction. Specific aims of the proposed PSEF/NEPS project are as follows: 1) To establish an international consortium of institutions and a central, on-line patient registry/database for tracking long-term outcomes in breast reconstruction. 2) To establish a standard panel of instruments for assessing the following outcomes of breast reconstruction: complication rates, patient satisfaction; health-related quality of life, including psychosocial and physical functioning; and resource costs. 3) To pilot test the survey instruments, on-line database, and other data-gathering infrastructure required for a long-term outcome study of common breast reconstruction procedures, including tissue expanders/implants; latissimus dorsi (LD) flaps; pedicle TRAM flaps with and without surgical delays; deep inferior epigastric perforator (OIEP) flaps; and gluteal artery perforator (GAP) flaps. The proposed PSEF/NEPS pilot project will run for one year and will include three phases: 1) Creation of the on-line survey process and database; 2) Obtaining institutional review board approvals from the eleven centers for both the PSEF/NEPS pilot study and for the upcoming DoD-funded IBROC outcome study; and 3) Pilot testing of the survey process with patients from the eleven IBROC members. The primary products of this research will be the establishment of a Web database, as well as mechanisms for recruiting and enrolling patients, obtaining their survey data via the Web, and analyzing the data on a regular basis to produce periodic reports comparing the long-term outcomes of breast reconstruction procedures. Ultimately, this information will support patients and plastic surgeons in evidence-based decision-making and may prove useful in negotiations with health payers to improve reimbursement for breast reconstruction.

Clinical observations suggested that the classification of the retaining system of the face was incomplete. Pilot work centered on the region of the platysma retaining system, where we observed boundary zones between adjacent regions of subcutaneous adipose tissue. The observation led to a hypothesis that subcutaneous tissue exists as separate regions. This was validated by dye injection studies in the anatomical laboratory. Continued work described multiple regions termed subcutaneous adipose compartments. Other findings became known as work progressed: septal barriers partition dye; blood vessels run along and are stabilized by these fascial membranes; and additional deep adipose tissue compartments exist as well. The clinical implications of the compartmentalization of adipose tissue are just beginning to emerge: not only does this work affect surgical techniques for facial rejuvenation, but also for the study of aging, health, and disease.

Recent trials have demonstrated superior locoregional control, disease-free survival, and overall survival in node-positive breast cancer patients who have undergone radiation in addition to mastectomy and chemotherapy (Gebski 2006). Based on these results, early-stage breast cancers are being treated with adjuvant radiotherapy in an attempt to increase survival. The effects of radiotherapy on outcomes after implant-based breast reconstruction can, however, be profound. To date, researchers have focused on the evaluation of post-operative complications such explantation and capsular contracture (Benediktsson 2005). While these are important issues, the overriding goal of postmastectomy reconstruction is to improve quality of life and to satisfy the patient with respect to her body image. Thus, it is critical that both the patient and surgeon have a clear understanding of the impact radiation treatment may have on body image and quality of life following expander/implant reconstruction. Unfortunately, there is little scientific literature that evaluates these outcome measures in this patient population. The objective of this project is to evaluate the impact of radiation treatment on both patient satisfaction and health-related quality of life following tissue expander/implant reconstruction. Patients who have completed two-stage, expander/implant reconstruction at least one year prior to study initiation will be eligible for participation. Three patient subgroups will be further identified as follows: i) patients with a history of pre-operative, chest wall irradiation follow who have completed tissue expander/ implant reconstruction (TE/I), ii) patients who completed TEll reconstruction and received post-operative, adjuvant radiotherapy, and iii) patients who completed TE/I reconstruction without radiation. A panel of self-administered questionnaires will be administered at one time point, including the 'MSKCC BREAST-Q' and the 'EORTC - Breast .Cancer Specific Quality of Life Questionnaire'. Survey subscale scores for each subgroup will be calculated and compared. Statistical regression techniques that adjust for potentially confounding variables such as age, timing of procedure and length of follow-up, will be employed.

This project is important for the following reasons. Firstly, GSK-3p is a key (negative) regulator of B-catenin, a well known transcriptional activating factor of the Wnp/B-catenin signaling pathway. Recent studies from our lab have shown that B-catenin is significantly elevated in Dupuytren's contracture (DC), leading us to hypothesize that a decreased GSK-3B activity may be responsible for the observed elevated levels of B-catenin. Secondly, because current GSK-3B assays are time consuming, require labile radioisotopes and significant amounts of patient material, there is a need for a more rapid, non-radioactive GSK-3p assay that requires minimal patient material to accurately assay GSK-3B activity. Lastly, GSK-3 (in a and p isoforms) is an extraordinary enzyme controlling several fundamental cellular functions that are linked to a number of prominent human diseases, including cancer and diabetes. Consequently, it is anticipated that a functional assay with many key advantages over current time-consuming radioisotope-based assays for GSK-3 would also be of broad fundamental importance to numerous other basic and clinical investigations. For our purposes, however, the development of this new GSK-3 assay will enable us to further study the molecular pathological events underlying Dupuytren's contracture. We expect that elucidation of the molecular pathology of DC may open up new rational avenues for the prevention and treatment of this disease for which currently no non-surgical treatment option exist.

The purpose of this study is to more fully characterize the resident microflora of chronic wounds of the lower extremity (CWLEs) and explore the role of quorum sensing (QS) systems in latent bacterial infections. Because QS has been shown to regulate proliferation and colonization within their hosts, it is hypothesized that bacterial QS systems may be altered when microflora is grown in conventional laboratory culture media, which may render many pathogenic bacteria nonculturable. Thus, disruption of the QS system through introduction of an agent such as Quorum Sensing Quenching (QSQTM; developed by Athena Biotechnologies, Inc.) to conventional culture media may facilitate growth, detection, and identification of pathogenic microflora of CWLEs when paired with 16S rRNA PCR. Additionally, identification of commensal bacteria that provide host protection through production of 'antibiotic' factors may also have future treatment implications. In the long-term, a more explicit characterization of nonculturable microorganisms, latent bacterial infections and biofilms, paired with a better understanding of QS could direct novel healing therapies for CWLEs and better management of tissue ulceration. Finally, this study may also offer a better understanding of the potentiality of antimicrobial use on clinically uninfected wounds.

Patients suffering from severe lower limb trauma (type III) are faced with the difficult decision of whether to have limb salvage by undergoing an extensive number of reconstructive surgical procedures and long period of immobility or early amputation to resume their rehabilitation programs. Plastic surgeons are faced with a similar dilemma of having to consider the cost of the reconstructive effort and the ultimate quality-of-life when confronted with these two treatment options. A cost-utility analysis by considering direct and indirect costs of treatment and quality-of-life after treatment using a decision analysis model is particularly suited to objectively evaluate these difficult treatment choices. We plan to assess the societal preferences from groups of informed subjects for the treatment of Type III tibial fractures by comparing amputation versus reconstruction. We will consider cost incurred by all members of society (patient, provider, insurer, etc.) to conduct a cost-utility analysis for the two treatments. In this era of cost containment and outcomes movement, a formal economic analysis is important in identifying a dominant treatment strategy to prescribe the most appropriate course of treatment for this complex problem. This economic analysis will address the immediate issues facing the clinical practice of plastic surgery by understanding the cost incurred and quality-of-life for those suffering from severe lower limb trauma. Additionally, we will determine the most optimal treatment algorithm based on modeling of various cost and outcomes variables. The data derived from this project will help patients and plastic surgeons in the critically difficult decision-making process when faced with devastating injuries of the lower limb.

This study's goal is to introduce a new murine model in order to facilitate research into the cellular and molecular pathways underlying capsular. The specific aims of this study are: Aim 1: To characterize the temporal differences in capsular formation and contracture between irradiated and non-irradiated wildtype mice using uCT, realtime RT-PCR, and histology. Rationale: The mouse is the only animal in which knockout and transgenic strains can be developed for molecular research. Currently, there is no published mouse model for the study of capsular contracture. We will develop a novel mouse model using livescan uCT, realtime RT-PCR, and histology to study and evaluate capsular contracture. Aim 2: To characterize the difference in severity of capsular contracture between irradiated and non-irradiated wildtype and Smad3-/- mice using realtime RT-PCR, uCT, and histology. Rationale: TGF-β has been linked with both scar formation and radiation injury, and Smad3 plays a critical role in TGF-β activation. We will elucidate the role of TGF-β and Smad3 signaling in capsular contracture. To test this, wildtype and Smad3-/- mice will be implanted with bilateral silicone gel implants (Mentor, Inc.) and receive slit beam radiation. The targeted radiation is designed to induce contracture and prevent systemic effects of radiation. Implant shape and orientation will be evaluated using μCT, gene expression will be quantified using realtime RT-PCR and capsule quality will be evaluated with histology. This study will develop a murine silicone implant model and test the hypothesis: Smad3 loss of function in the Smad3-/- mouse attenuates the TGF-β signaling pathway leading to reduced capsular contracture following silicone implant insertion and radiation therapy. This work will provide a model to further study molecular pathways involved in capsular contracture and will translate to molecular based therapies to reduce capsular contracture.

The primary objectives of this proposal are to determine the compatibility of FK506 with the blockade of costimulatory pathways in the composite tissue transplantation model with emphasis on allograft nerve regeneration as well as survival, and to define strategies that allow the utilization of the neuro-enhancing properties of FK506. Hand transplantation has been performed since 1998 but remains very controversial and no further procedures have been performed since 2003. The overall recovery of hand allograft function has been only fair and has not met expectations. Multiple and late episodes of rejection in almost all of the recipients are also concerning for a high risk of chronic rejection. Despite these issues and the risks of lifelong nonspecific immunosuppression, two partial face transplants have been performed since November of 2005 and are felt by most to be premature because the benefits do not clearly outweigh the long-term risks. Restoration of function is critical to the success of composite tissue transplantation and to justify non-vital transplantation. The use of FK506 remains essential to the composite tissue allograft because it enhances the rate of nerve regeneration and therefore the recovery of allograft muscle function. However, the immunosuppressive properties of calcineurin inhibitors have been shown to be both additive and abrogative depending on the context in which the therapies are used. We propose that selected strategies of costimulation blockade can be used in conjunction with FK506 to minimize the immunologic risks of transplantation and optimize functional outcome. The specific aims of this proposal are: 1) To determine the compatibility of FK506 and costimulation blockade when used as maintenance immunosuppression; and 2) To characterize the immunological mechanisms involved in the interactions between FK506 and selective strategies of costimulation blockade. We remain unique in the ability to perform limb and composite tissue transplantation in the murine model. As such, we are able to investigate the use of costimulation-blocking agents in limb and composite tissue transplantation, and to utilize transgenic mice that express fluorescent chromophores on nerve tissue to study nerve regeneration in the composite tissue allograft in novel ways.

Peripheral nerve injuries represent a significant source of morbidity and disability in the United States and abroad. When end-to-end repair is not possible, autologous nerve grafting remains the preferred method for reconstruction of nerve gaps. Such grafts are typically taken from superficial sensory nerves, such as the medial antebrachial cutaneous or sural nerves, in an effort to minimize donor-site morbidity. Though sensory nerves have long been assumed to be a suitable substrate for motoneuron regeneration, the limited functional recovery achieved with grafting compared to end-to-end repair suggests otherwise. A growing body of experimental evidence suggests that axonal regeneration improves when motor nerve gaps are reconstructed with motor grafts, and likewise for sensory. Termed modality-specific regeneration (MSR), this phenomenon seems to challenge the existing clinical dogma of repairing motor defects with sensory grafts. However, previous experiments demonstrating MSR utilized nerve grafts constructed of multiple cables or taken from spinal roots, which are not representative of nerve reconstructions performed in human patients. In our proposed study, we seek to demonstrate MSR in a more clinically relevant scenario. We hypothesize that matching of motor or sensory modality between donor and recipient nerves results in enhanced axonal regeneration. In order to more closely replicate nerve grafting as performed in human patients, our study will utilize only single-cable peripheral nerve grafts without the use of spinal roots. Our design also incorporates a more extensive analysis of grafted nerves than in prior studies, including retrograde labeling, stereology, and electron microscopy. If borne out by this study, modality matching of nerve grafts has the potential to significantly alter the practice of peripheral nerve reconstruction and enhance the clinical outcomes achieved in the nerve-injured patient.

Breast reconstruction after mastectomy is an important treatment for women with breast cancer. However, there is virtually no information about whether there is unmet need for reconstructive surgery after the initial treatment period. The goal of this study is to examine the decision-making process of breast cancer reconstruction after the initial diagnosis and treatment period. The specific aims of the proposed study are: 1 ) to perform a survey of a population-based cohort of women diagnosed with breast cancer from August 2005 to June 2006 in the metropolitan areas of Los Angeles and Detroit who received a mastectomy, and to measure the proportion of women undergoing immediate and delayed breast reconstruction; 2) to describe correlates of receipt of immediate and delayed breast reconstruction compared to those women who have not been reconstructed; 3) to evaluate decisional satisfaction and health-related quality of life in women who did and did not receive breast reconstruction. The methodology will be a cross-sectional survey of 650 breast cancer patients from the Los Angeles and Detroit SEER registries who were treated with a mastectomy. The results of this exploratory study will improve informed decision-making regarding breast cancer reconstruction by evaluating barriers to and unmet need for treatment. The results will also be used to design a larger study across SEER registries.

Dupuytren's disease (DD) is a common problem encountered by the hand and plastic surgeon. It is characterized by nodular fibroblastic proliferation of the palmar fascia leading to contracture of the hand. The pathogenesis of DD remains unclear, but studies have shown that DD is more common in males of northern European ancestry, suggesting a genetic link. Recent work in our laboratory using DNA microarray analysis has identified upregulation of known genes including the TGF-Beta family. In addition, microarray analysis has identified upregulation of a novel gene, musculoaponeurotic fibrosarcoma B [MafB], in diseased cords taken from DD patients. MafB is a transcription factor represented in a class of basic leucine zipper transcription factors. The Maloncogene was initially identified in an oncogenic avian retrovirus that induces musculoaponeurotic fibrosarcomas in vivo and also cellular transformation of fibroblasts in chicken embryos in vitro. While the function of MafB is not fully known, its purported role in initiating cellular transformation of fibroblasts makes MafB upregulation in Dupuytren's cords a novel mechanism. The purpose of the present study is to confirm and investigate the role of MafB in Dupuytren's disease.

Plastic surgeons replace tissues which have been lost secondary to trauma, malignancy, or other disease processes. Therefore, surgeons would benefit from having available engineered tissues which can be used to replace missing tissue such as bone, cartilage, and fat. Currently, autologous adipose tissue is used as a graft to correct deformities but this technique has limited success because graft survival and quality is unpredictable. There are many factors likely responsible for graft loss including lack of vasculogenesis, mechanical disruption, lipid induced membrane damage, apoptosis, and lack of a mechanical scaffold. One useful strategy to obtain adipose tissue for reconstruction is to employ tissue engineering principles. Adipose-derived adult stem cells (ADAS) or pre-adipocytes can be isolated from the stromal vascular fraction (SVF) of adipose tissue, and then combined with biological scaffolds and growth factors to form constructs suitable for implantation. We propose to use polyethylene-glycol (PEG) as the biological scaffold because of its favorable mechanical properties. Our long term goal is to better understand the interactions between pre-adipocytes and biological scaffolds which can lead to engineered adipose tissue. The specific aims of the current proposal are to manipulate the interactions between the PEG scaffold and human pre-adipocytes to induce adipose tissue formation first in vitro. Once experimental conditions have been optimized in vitro, we will place the human pre-adipocytes / PEG scaffold in vivo in a nude mouse model. According to the ASPS, 5.4 million reconstructive procedures were performed in the United States in 2005. Plastic surgeons would benefit from having reliable methods of engineering adipose tissue for use in reconstructive and cosmetic procedures. Our project combines specialists in tissue engineering, angiogenesis, and plastic surgery. We propose to use easily available and hearty adipose derived stem cells combined with a scaffold to accomplish this goal.

Soft tissue augmentation is frequently used in plastic surgery, and while autogenous fat grafts offer many ideal properties, its volume maintenance has proven unpredictable and temporary. Multiple clinical techniques and strategies have been proposed to minimize fat graft resorption, employing variations in harvesting, processing, donor site, and fat storage. Current clinical strategies are largely based on anecdotal evidence. The goal of this project is to systematically analyze the critical steps in clinical fat grafting at the histologic and molecular biological levels, in order to produce rigorous, objective findings that can drive clinical fat grafting protocols and strategy. Using specialized histological/immunochemical staining techniques, a refined animal model of autogenous fat grafting, and novel imaging modalities, we will analyze and evaluate strategies that have been suggested to improve the efficacy and safety of autogenous fat grafting. The specific aims of this project are: (1) to compare the various fat harvesting and processing techniques that are used in clinical practice, to attempt to identify those technique(s) that yield maximal efficacy and safety to the patient; (2) to evaluate fat obtained from different anatomic sites, to determine whether regional donor site differences affect later fat graft resorption; and (3) to analyze various storage methods for fat grafts, including conventional refrigeration, storage in cell media, as well as experimental fluorohydrocarbon-based strategies that can augment stored graft tissue oxygenation. Ultimately, this project will attempt to identify those techniques in current clinical practice that can offer the patient the most efficacious and most safe outcomes. The demand for soft tissue augmentation is rising sharply, and autogenous fat grafting will always be a prime tool in the armament of the plastic surgeon. Through the systematic evaluation of the clinical techniques currently in use, this project will identify optimal clinical strategies through investigation at the biochemical and histological levels. Most important, the findings of this study will have immediate clinical impact, and can guide optimal and safe clinical practices in autogenous fat grafting.

Biological spatial patterning of growth factors plays a critical role directing cell fate during embryo development and wound healing. The capability to engineer specified and persistent spatial patterns of endogenous growth factors within biologically-relevant substrates would be useful to direct differentiation of cells towards a desired tissue type with a precise shape. In pursuing this goal, we plan to mimic the pericellular microenvironment present during endogenous bone healing with the ultimate goal of spatially controlling bone and blood vessel formation. A custom-built inkjet printer will persistently immobilize BMP-2, Noggin, and VEGF on and within a biologically-relevant matrix. Analogous to how growth factors are normally anchored to extracellular matrices during bone healing, immobilization permits local delivery of endogenous growth factors in physiological doses to control cell behavior. Using computer-based designs, growth factors will be patterned onto a matrix, which will then be implanted subcutaneously in adult mice. Specific shapes will be bio-printed to test the hypothesis that physical immobilization of growth factors will induce tissue formation in spatial register to the bio-printed pattern (e.g., Can we induce formation of a Y-shaped blood vessel?). We hope to stimulate, in vivo, spatially controlled blood vessel formation in regenerated bone in mice using the bio-printing methodology. We also plan to test the hypothesis that bio-printed VEGF will enhance spatial control of bio-printed BMP-induced bone healing in mice in vivo. We envision this work fostering novel techniques for 3-dimensional spatial control of tissue regeneration. Our rationale relates to the current suboptimal treatment of complex structure abnormalities such as those that may occur following trauma, surgical intervention, or congenital malformation. Spatial control of bone and blood vessel formation would enable customized patient treatment for these insults.

Replacement organs are critically needed for patients suffering from a multitude of diseases. At present, the only option for these patients is organ transplantation with the attendant problems of donor scarcity and life-long immunosuppression. Tissue engineering holds the promise of creating replacement organs outside the human body. However, two major obstacles have hindered the development of techniques to fabricate replacement organs: 1.) the inability to adequately vascularize tissue constructs in vitro and 2.) the inability to re-integrate these tissues into the systemic circulation. Tissue engineering approaches based on the implantation of cells onto resorbable matrices have had success in replicating simple, relatively avascular structures such as cartilage and bone, but have been unable to create more complex parenchymal organs, such as liver. Despite the obvious promise that stem cell technology holds to "patch" or "regenerate" partially damaged organs in vivo, it is difficult to envision the creation of new organs in vitro using existing methodologies. To solve this problem we have taken explanted microcirculatory beds (EMBs) as the foundation for the fabrication of autologous, vascularized neo-organs in vitro. This microsurgical approach starts with the vascular system as the scaffold and builds tissue from the "inside-out" when compared to existing paradigms of tissue engineering. In this grant, we will use this approach in combination with embryonic stem cells to create a functional autologous liver in vivo which can reliably replace hepatic function in an animal model of liver failure.

Although effective techniques exist for cleft palate closure, post-repair complications of an oronasal fistula may be problematic. AlloDerm or acellular dermis has been used successfully in a cleft palate animal model and in a few clinical cases (1). Improvement of this procedure with cellular seeding of AlloDerm may maximize the chance for healing. In our two-part study, we will attempt to develop a superior tissue engineering scaffold that can ultimately promote osteogenesis and heal cleft palate defects. 1. In our in vitro study, we will determine if AlloDerm may act as suitable matrix for adipose derived stem cells (ASCs) in promoting cell growth, aggregation, and differentiation using DNA proliferation assays and RT-PCR for osteogenic factors. 2. In our in vivo study, we will determine if an AlloDerm/ASC construct may be used to heal oronasal cleft defects in athymic rat model with histologic analysis. Our goal is to determine the efficacy of lipoaspirate stem cells (PLA cells) seeded in AlloDerm, an acellular collagen-based dermal matrix, in healing alveolar cleft defects. We hypothesize that AlloDerm will ensure homogenous ASC proliferation and distribution and promote bone growth in order to treat cleft-palatal defects. This tissue engineering strategy may then be used clinically in the operating room without the need for creation of a laboratory bioreactor.

It is estimated that 1 in 7 women in the United States will develop breast cancer, and 270,000 women will be diagnosed in 2005. As a result of earlier detection and improved treatments - surgery, hormonal therapies, chemotherapy, and radiotherapy - the mortality rate for breast cancer patients is at its lowest since the 1950s. This means that more breast cancer patients will survive and live with the sequelae of their treatment. Post-mastectomy breast reconstruction restores body image and psychological well-being and is recognized as an integral part of breast cancer survivorship. However, complications with resultant poor outcomes occur with tissue expander and implant breast reconstruction in patients requiring radiotherapy - and the indications for adjuvant radiotherapy are increasing. The main complication is fibroproliferation of the capsular tissue around the expander with resultant capsular contracture. This leads to poor expansion, breast distortion with poor aesthetic outcome and pain, and often requires additional surgery. Understanding the pathogenesis of radiotherapy induced capsular fibroproliferation and contracture will allow us to design pharmacological intervention against the development of capsular contracture in patients requiring radiotherapy who may be limited in their options for breast reconstruction. Recently, experimental evidence is accumulating to indicate that the canonical Wnt (Wingless) signaling pathway plays a pivotal role in the pathogenesis of fibromatosis and hyperplastic skin wounds. It is most likely that intervention at the mediator or effector level in this signal transduction pathway will attenuate fibroproliferation. Therefore, the purpose of this research project is to test our hyporthesis that the Wnt signaling pathway also plays a central role in the regulation of radiotherapy-induced fibroproliferation in capsular tissue around tissue expanders in postmastecotmy breast reconstruction. To this end, experiments are designed to study the phosphorylation states and protein and/or gene expression of biochemical components known to be involved in the Wnt signaling pathway, and the collagen I and III protein expression in capsular tissue around tissue expanders in patients with or without radiotherapy prior to mastectomy.

Photochemical Tissue Bonding (PTB) is a new technique in which a photosensitizing dye is irradiated with visible laser light to create covalent bonds between tissue surfaces. Unlike conventional laser welding that generates heat in the tissue; PTB is a non thermal system that initiates immediate chemical bonds without collateral damage to surrounding tissues. This technique has been used experimentally in a number of tissue repair models including corneal lesion repair, nerve repair and enhancement of skin graft adhesion. It has also been used with success in the repair of achilles tendon rupture in a rat model. PTB repair of peripheral nerve also demonstrated improved functional outcome following augmentation of the technique with an amnion wrap.

The past 30 years have provided us with a great deal of experimental data on neuroscientific elements of nerve repair. Unfortunately this has not translated into improved clinical outcomes. We are still in search of the ideal method of nerve repair. Photochemical tissue bonding is an exciting new method of tissue adhesion that uses visible laser light to create bonds between tissue surfaces that have been pretreated with a photosensitizing dye. This innovative use of laser technology has the potential to revolutionize many aspects of microsurgery. In this project we will combine photochemical technology with the previously described technique of nerve wrapping in an attempt to enhance peripheral nerve regeneration by reducing scar formation at the repair site. The objects of this study are: (1) To determine if photochemical sealing of the peripheral nerve repair site can improve functional recovery following nerve injury and repair (2) To determine if natural or bioengineered nerve wrapping materials are more effective in enhancing peripheral nerve regeneration.

Transplantation of composite tissue allografts (CTA) such as human hand, larynx, face, muscles, tendons nerves would have unlimited reconstructive applicability in plastic surgery if life-long immunosuppression would not be required for allograft survival. Different tolerance inducing strategies are tested in experimental models to bring CTA closer to clinical reality. Stem cell-based therapies included hematopoietic stem cell transplantation (HSCT) and adoptive immunotherapy constitutes promising strategies for tolerance induction in CTA and solid organ transplants. We have developed a model of tolerance induction in limb allograft transplants across major histocompatibility (MHC) barrier using 7-day protocol of combined anti-ab T-cell receptor (TCR) monoclonal antibody and CsA therapy (ab-TCRlCsA). Remarkably stable hematopoietic chimerism and a long-term tolerance (over 750 days) were achieved, suggesting that the presence of certain tissue compartments (stromal cells) may facilitate efficient engraftment of the HSC. We hypothesize that augmentation of the HSCT with stromal cells of donor origin is essential for improved engraftment and function of the HSCT. The intact bone marrow (BM) compartment of the vascularized graft (VG) model will be compared with non-vascularized bone marrow grafts (NVG) augmented with donor bone marrow stromal cell (BMSC) co-transplantation for chimerism induction. Once stable chimerism is induced, chimeric cells can be isolated, purified and propagated for adoptive transfer that will serve as a new therapeutic modality for allograft transplant recipients without the need for chronic immunosuppression. This innovative therapy may be directly applied to reconstructive procedures in Plastic Surgery, where shortage of autologous tissues will justify composite allografts transplantation.

Recently, we have focused on immunomodulation protocol of selective targeting of alpha/beta T-cell receptor (alpha/beta TCR). Selective inhibition of alloreactive T-cells via application of 7- day protocol of alpha/beta TCR/CsA induced stable chimerism and tolerance (over 750 days) in rat limb allografts. Further face transplant under alpha/beta TCR/CsA supported with donor bone marrow transplantation (BMT) resulted in long-term facial allograft survival (495 days) and presence of regulatory T-cells (Treg) CD41 CD25. Selective, short acting alpha/beta TCR/CsA protocol temporarily eliminate alpha/beta TCR positive cells, and preserves gamma/delta positive T-cells, which are known to carry tolerogenic properties. We hypothesize that presence of Treg cells and/or specific population of gamma/delta dendritic epidermal T cells (OETC) is prerequisite for tolerance induction in face transplants. We will assess tolerogenic properties and immunomodulatory function of CD4+/CD25+ or gamma/delta DETC under: Specific Aim 1. To establish tolerogenic properties and efficacy of engraftment of Treg (CD4/CD25) cells of donor origin used as supportive therapy in face allograft transplantation. Specific Aim 2. To test immunomodulatory effect and migratory potential of donor Dendritic Epidermal gamma/delta T Cells (DETC) in facial allografts across MHC barrier. To test our hypothesis under S.A. 1 and S.A. 2 face transplant recipients under 7 day alpha/beta TCR/CsA and BMT augmented with tolerogenic CD4+/CD25+ or DETC will be tested. Methods of assessment include: (i) Flow cytometry for donor chimerism, lymphoid markers: CD4+/CD25+, gamma/delta T-cells, and myeloid markers; (ii) Annexin V-FITC and TUNEL technique for apoptosis; (iii) immunohistochemistry for donor-cell engraftment; (iv) Real-Time PCR for intragraft cytokine gene expression of Th1 (IL-2, IFNgamma) and Th2 (IL-4, IL-10, TGF-beta) and chemokines RANTES, MIP-1, SDF-1, CXCR4 in rejected versus tolerant allografts.

Chronic ischemic wounds continue to frustrate surgeons because few therapies exist that induce the repair of compromised endothelium or damaged vasculature. One approach to this challenge is to stimulate vasculogenesis, where endothelial progenitor cells (EPCs) home to sites of ischemia to restore adequate tissue perfusion. While hypoxia and inflammation are known to affect EPC homing, other stimuli are important to investigate as potential therapeutic targets. One such stimulus may be radiation therapy. Although radiation has traditionally been thought to be harmful on a cellular level (e.g. oncologic therapy), recent data demonstrates that low dose radiation may stimulate neovascularization, suggesting a potential role in revascularization therapy. Therefore, we propose that low dose radiation can exert a vasculogenic effect by stimulating EPCs to home to a site of vascular ischemia. We hypothesize that radiation stimulates EPC mobilization through the stabilization of HIF-I, a protein that transcriptionally activates chemokines responsible for initiating vasculogenesis.

Tissue injury resulting from radiation therapy is a significant problem. We have previously shown that radiation causes dysfunction of tissue stem cells by preventing their self-renewal and differentiation into adult cell types. This response is dependent on p21, a cell cycle regulator. In the proposed studies we aim to evaluate the potential of inhibiting p21 on preventing radiation induced stem cell injury. Furthermore, we aim to optimize timing and methods of stem cell delivery as a means of treating established radiation injury of local tissue stem cells. Optimization of stem cell incorporation has significant clinical implications as these techniques may increase the effectiveness of adult stem cell delivery, such as those derived from fat, for tissue regeneration. In addition, the identification of the mechanisms responsible for stem cell homing has broad implications on other clinically relevant topics for plastic surgeons including tissue engineering and wound healing. The expected outcome of this study is that we will identify and prevent the effects of radiation therapy on resident tissue stem cells. In addition, we expect to optimize engraftment of stem cells in order to treat radiation damaged tissues.

Partial or complete flap loss can have devastating consequences during reconstructive and cosmetic surgery. The incidence of these complications may be related to comorbid factors such as obesity, smoking, and prior history of radiation therapy. Although a number of techniques have been developed to address this issue, to date, no non-invasive measures have been proven effective. Recently, a number of studies have demonstrated that administration of culture expanded endothelial progenitor cells (EPCs) is capable of improving ischemic flap survival. The circulating concentrations of these cells can be manipulated using commonly available oral medications such as statins. Statins (eg. simvastatin) have been used effectively in millions of patients for cholesterol reduction. However, recent studies have shown that these medications also increase circulating EPC levels 2 to 3 fold. In addition, treatment with statins has been effective in diminishing neointimal hyperplasia in a carotid artery injury model. Therefore, the purpose of these studies is to evaluate the effect of presurgical treatment with statins on ischemic flap survival. This will be performed using the following specific aims: 1. Evaluate effect of oral statin therapy on concentrations of circulating EPCs in mice. In this specific aim we will identify the optimal dose and timing of oral simvastatin on mobilization of bone marrow derived PCs. 2. Evaluate the effect of oral statin therapy on ischemic flap healing in mice. This specific aim will assess the effects of oral Simvastatin on ischemic flap survival using the optimal oral dose identified in specific aim 1. 3. Demonstrate that alterations in ischemic flap survival are due to bone marrow stimulation. In this specific aim we will demonstrate that statins augment EPC production by changes in bone marrow production of these cells. In addition, we will track the newly formed EPCs to the wound bed using transgenic animals.

Persons with extreme obesity are often motivated to undergo bariatric surgery with the goal of improving their physical health. Many patients also anticipate that the significant weight loss associated with bariatric surgery will lead to improvements in their quality of life and, more specifically, their physical appearance and body image. A large body of research suggests that bariatric surgery frequently confers these physical and psychosocial benefits. An estimated 140,000 Americans underwent bariatric surgery in 2004. According to the American Society of Plastic Surgeons, 55,927 massive weight loss patients underwent plastic surgical procedures in the same year. Taken together, these numbers suggest that a sizable percentage of bariatric surgery patients subsequently elect to undergo body contouring procedures. To our knowledge, no study has comprehensively investigated the psychosocial and physical outcomes of body contouring surgery among massive weight loss patients. Furthermore, little is known about the factors that influence the decision to undergo plastic surgery. Some patients likely undertake these procedures to address body image dissatisfaction not resolved with the massive weight loss associated with bariatric surgery. Others may be motivated to lessen the physical problems associated with loose, sagging skin that often develops following significant weight loss. The proposed study will investigate changes in psychosocial and physical functioning in 100 individuals who present for body contouring surgery at one of four practices across the country. These individuals will be compared to changes in bariatric surgery patients who do not elect to undergo body contouring surgery. The study also will investigate which pre- and post-bariatric surgery variables are most strongly associated with undergoing body contouring surgery. The study is innovative, as it will provide new information on the psychosocial and physical changes experienced by the substantial number of patients who elect to undergo plastic surgery following bariatric surgery.

Mechanical forces can affect cellular behavior. Distraction osteogenesis (DO) applies mechanical force to cells in the distraction gap, stimulating new bone formation. In vivo experiments demonstrate that in the bone generate, a collagen framework is constructed, new blood supply is recruited and then the framework is ossified. Building and then ossifying the collagen framework is a delicate balance of complex interactions (e.g. mechanobiology, cell recruitment, cytokine production). Perturbations of this complex system can lead to fibrous union. Alternatively, external manipulation of this system could improve bone formation and shorten therapy. While there are many factors to investigate, elucidating the mechanism of matrix formation (e.g. collagen type I secretion) and ossification (e.g. alkaline phosphatase) in response to tensile strain is essential to improving our understanding of this valuable technique. Evidence suggests that the intracellular signaling molecules: mitogen activated protein kinase (MAPK), phosphoinostitol-3 kinase (PI3K), and the 90 kDa ribosomal S6 kinases (p90RSK), can translate a mechanical stimulus into a cellular response in osteoblasts. However, there is a paucity of data linking strain-induced activation of intracellular pathways to bone formation. The central hypothesis of this proposal is that strain-dependent collagen I and alkaline phosphatase gene expression are regulated by the MAPK and PI3K pathways in osteoblasts.

Pilot experiments support the notion that blockade of non muscle myosin II prevents wound contraction and that myosin II is robustly expressed in scar versus normal skin. Unfavorable scarring and fibrosis affects millions of people worldwide. Current treatments are frequently unsuccessful and preventative agents do not exist. Therefore, there is a large unmet need for an effective therapeutic agent to prevent fibrosis. This proposal describes a novel strategy fundamentally different from past efforts. The rationale is to suppress the remodeling phase of repair, targeting the mechanisms fibroblasts and myofibroblasts use to incrementally contract a scar, to prevent fibrocontractile disease progression. The hypothesis is that non muscle myosin II is the motor protein that primarily mediates fibrocontractile disease. The primary objective of this proposal is to validate whether non muscle myosin /I is the motor protein that primarily regulates scar contracture.

Dupuytren's disease is a fibrocontractile disease that can be severely debilitating. Diseased fascia contains nodules and cords, and although it is unknown why these structures develop, it has been proposed that nodules are the primary epicenter of disease progression. While some investigators believe that myofibroblasts are putatively responsible for causing Dupuytren's disease progression, it is interesting to note that not all nodules contain myofibroblasts. Then what causes Dupuytren's disease? All nodules contain fibroblasts that, like myofibroblasts, are rich in actin and myosin contractile proteins. Thus, it is proposed that fibroblasts and myofibroblasts within nodules cause Dupuytren's disease and it is the putative interaction between actin and myosin II within these cells that causes palmar fascia contractures. There are several well-characterized myosin II isoforms and upstream intracellular contractile signaling mediators that lead to activation of actin-myosin to cause cellular contractility. However, it is not known which signaling pathway predominates in Dupuytren's fibroblasts and which predominates in myofibroblasts. The long term goal of this work is to identify the contractile proteins that are active in Dupuytren's disease to allow for development of novel small molecule inhibitors to prevent disease progression. It is hypothesized that nodule fibroblasts are rich in MLCK and SMMHC, and nodule myofibroblasts are rich in ROCK1, MYPT1, and NMMHC. Both fibroblasts and myofibroblasts contain MRLC. The first specific aim studies protein expression in Dupuytren tissue by immunostaining human tissue and the second specific aim investigates the functional role of contractile proteins in myofibroblast rich and myofibroblast poor Dupuytren tissue by applying contractile agonists and protein antagonists to Dupuytren tissue strips attached to a tensiometer in a tissue culture bath.

Pressure ulcers CPU) are a devastating and common comorbidity for all immobilized patients. PU are complicated by pain, infection, and increased hospital stay. The wounds represent a significant health care burden. In 2003, the average hospital acquired PU prevalence was 9.2% in the in-patient health care setting with 2.5 million PU treated each year. The cost for treating one full thickness PU is estimated to be $70,000 with total US expenditures equaling approximately $11 billion annually. Thus, there is great need for prophylaxis against PU formation. The primary aim of this study is to investigate the use of hypoxically driven heme oxygenase-1 (HO-l) gene therapy to prevent PU formation.

During wound healing under ischemic conditions, tissues have certain ingredients in their environmental milieu that vary both in content and amount from the surrounding normal tissue. These changes are the tissues' way of compensating for or trying to overcome the deleterious conditions that otherwise leads to poor or incomplete wound healing. For instance, hypoxiainducible factor 1 (HIP -1) is produced in large amounts by ischemic cells and is known to promote neovascularization via induction of vascular endothelial growth factor (VEGF) production. We would like to take advantage of these known differences in environmental make up, and specifically the presence of increased levels of HIP-l to increase the expression of genes that are known to promote more rapid skin wound closure, including VEGF and keratinocyte growth factor-2 (KGF-2). Our method is to link one of these genes to a sequence of DNA, known as a promoter region, that, when activated, turns up gene production of genes that are downstream from the promoter. By linking one of these genes to a promoter that is activated by HIP-l and introducing it to cells in an ischemic environment, we can increase cellular production of this gene by allowing HIP -1 to bind to the promoter region and activate cellular production of the gene. Furthermore, we will be able to control exactly where the upregulation occurs, since only a hypoxic environment will possess elevated HIP-l levels, thus affording targeted gene expression. We have generated a viral vector with the promoter region hypoxia-response element (HRE) linked to genes known to be active in wound healing. In addition, we have created a novel wound healing model in the rat that provides ischemic and non-ischemic wounds on the same animal, allowing for direct comparisons between the two wound healing conditions. We now are ready to introduce the viral vector into wounds to see if we can drive increased gene expression and wound healing in the presence of HIP -1.

The overall goal of this research project is to perform composite tissue allotransplantation without the use of long-term systemic immunosuppression through induction of tolerance. Special attention will be given to examining the immunogenicity of skin. The focus of our laboratory has been to explore the interactions between the immune system and allogeneic musculoskeletal tissues (skin, muscle, bone, nerve, etc.). Induction of donor-specific tolerance would permit significant advances in reconstructive surgery and bum treatment, where autologous sources of tissue are inadequate or absent. Dendritic cells (DCs) are highly specialized, bone marrow-derived antigen presenting cells (APCs) that induce and regulate immune responses. DCs can be manipulated, either in vivo or in vitro to promote antigen specific tolerance. We hypothesize that pulsing "tolerogeneic" DCs with donor skin antigen in addition to donor major histocompatibility complex (MHC) alloantigens will promote long-term survival of skin as well as the other components of a CTA.

A composite tissue allograft (CTA) is unique compared to solid organs in that it contains multiple tissue types and specialized immune cells. It is well established in animal models that a CTA, in particular the skin component, is more immunogenic than solid organs. This fact has manifested clinically with multiple acute rejection episodes reported in the majority of the human CTA cases. Our body of knowledge currently lacks appropriate identification of the immune cells involved in CTA rejection and their trafficking. Some distinct characteristics of a CTA include the potential to include a vascularized bone marrow compartment and the presence of Langerhans cells (LC) and other potent antigen presenting cells (APC). The association of blood and lymphoid tissue chimerism (the coexistence of donor and recipient cells) with graft prolongation has generated many papers on the trafficking of bone marrow cells in CTA. Their focus has been to identify where donor derived hematopoietic cells engraft, what is level of chimerism in various tissues, and how these levels change over time. What they do not address is what phenotype of immune cells are involved in acute rejection immediately after transplantation, where they interact with host cells (local versus systemic lymphoid organs), and how they get there (intravascular versus lymphatics). This basic knowledge will provide a more solid foundation in developing new strategies for immunomodulation in CTA. Clinical composite tissue allotransplantation (CTA) is a young and evolving field in plastic surgery. Current protocols employ systemic immunosuppression that have resulted in significant morbidity in many of the human CTA recipients worldwide, including metabolic disturbances, opportunistic infection, and femoral head necrosis requiring hip replacement . Our laboratory is investigating several novel cell-based and drug-based therapies in an attempt to decrease or eliminate the need for systemic immunosuppression. In order to accurately target specific pathways of rejection at appropriate time points, fundamental questions of immune cell trafficking after CTA must be answered.

Physicians and scientists have been looking for a single molecule or pathway that can be targeted to diminish scars. The mammalian target of rapamycin (mTOR) has surfaced as a key molecule to potentially affect this change. Over the last few years, mTOR has proven to be an important regulator of cell growth. The drug, rapamycin, specifically and safely inhibits mTOR's function. It has been used as a therapeutic agent against cancer and restenosis of cardiac stents, where uncontrolled regrowth of tissue is the hallmark of the disease process. Uncontrolled and excessive collagen deposition and growth are the defining characteristics of keloids and hypertrophic scars, which appear and act like benign tumors. mTOR has been shown to control collagen production in human skin cells. However, the role of mTOR in wound healing and abnormal scarring has not been explored. Our proposal hypothesizes that the key regulatory protein, mTOR, exists in higher quantities in hypertrophic and keloid scars and is responsible for their development. We plan to obtain keloid, hypertrophic scar and normal skin from patient donors. Levels of mTOR and collagen will be evaluated from these samples. We will then evaluate the effect of rapamycin on collagen production and regulation of growth factors that cause abnormal scars to form. Identifying the roles that mTOR and collagen levels play in abnormal scarring is critical for establishing methods to prevent keloids and hypertrophic scars. We can then develop pilot studies using topical rapamycin to treat these scars, as well as to treat surgical wounds prophylactically. Everyone is affected by varying degrees of scarring. The general public invests enormous amounts of resources and money on treatments to minimize scars. These include ointments, gels, creams, oils, specialized dressings, silicone sheeting, steroids, and laser therapy. Given the lack of overall success of surgical revision, and topical and intralesional therapies, the field of wound healing is a huge market for any pharmaceutical product that diminishes scarring. The goal of all surgeons, especially plastic surgeons, is to heal wounds with minimal scarring. Scarless healing is the "Holy Grail" that all plastic surgeons seek. Keloids and hypertrophic scars keep us from attaining this goal. It is imperative that we develop new therapies whose effectiveness is based on actual scientific data.

Complex musculoskeletal defects resulting from cancer, congenital absence and trauma represent a unique reconstructive challenge. Autologous tissue is often not available to reconstruct these deformities. Composite tissue allograft (CTA) transplantation offers a solution to this problem. Examples of these procedures include hand, limb, and potentially, face transplantations. Technical aspects of these procedures are known. However, the chronic immunosuppression that patients would require to tolerate these transplants would create side effects such as drug toxicity, opportunistic infections and malignancies. Novel techniques to limit immunosuppression for recipients of CTA transplantation are needed. Dendritic cells (DCs) are unique antigen presenting cells that have been shown to be inducers of tolerance in both in-vitro and in-vivo settings. In our plastic surgery lab we have a developed a protocol for the harvesting and phenotypic characterization of rat dendritic cells. In addition, an orthotopic rat hind-limb transplantation model has been optimized to study rejection phenomena. The hypothesis of this study is that the intravenous (IV) administration of donor derived immature myeloid DCs into a recipient prior to orthotopic hind-limb CT A transplantation along with a short course of low dose immunosuppression will prolong the hind-limb allograft survival and induce tolerance across MHC mismatched rat models.

The central hypothesis of our project is that fetal and adult keratinocytes (FKs, AKs) interact with fetal and adult fibroblasts (FFs, AFs) through TGF-B in promoting scarless repair. Scarless fetal wound healing is a paradigm for ideal tissue repair. However, the biology responsible for this phenomenon is poorly understood Understanding scarless healing has profound implications in plastic surgery, in diverse areas such as bum, cleft lip and breast implant surgery. Surgical treatment is limited by scarring and need for additional surgery. Developing methods to promote scarless healing will play a critical role in improving treatment modalities. Until recently, the mechanisms and interactions regulating scarless wound healing have remained largely unknown. Our previous work suggests that the fetal fibroblast, which produces collagen and the extracellular matrix, is the cell type primarily responsible for scarless skin repair. In addition, we have shown that keratinocytes, in co-culture with fibroblasts, influence fibroblast cell biology. However, the function of the fetal keratinocyte during the fetal repair process has not been investigated and remains unknown. Based on our preliminary data, we have focused on TGF-B as a prototype growth factor which has function both during scarless and scarring repair that is determined by differential expression of ligand, receptors, and modulators. Our research will examine TGF-B biology in a co-culture model containing FKs, AKs, FFs and AFs in mix and match combinations. We hypothesize that FK-derived and AK derived TGF-B is responsible for the changes in FF and AF cell biology found when co-cultured Furthermore, we anticipate that fetal keratinocytes can influence adult fibroblasts to behave more fetal-like through TGF- B expression resulting in increased ratio of Type III to Type I Collagen and an altered extracellular matrix profile. There are three specific aims for this project. They are: 1) To determine TGF-B expression in fetal and adult keratinocytes (FKs, AKs) cultured in isolation; 2) To determine TGF-B expression in FKs and AKs co-cu1tured with fetal and adult fibroblasts (FFs, AFs); and 3) To determine the extent of TGF-B activation in these culture sets and its influence on fetal and adult fibroblasts with regards to collagen expression and the extracellular matrix profile. By examining the interaction of fetal fibroblasts and fetal keratinocytes compared to their adult counterparts, we troy begin to understand ways to promote Scarless healing. The implications of understanding fetal repair have tremendous appeal for physicians and scientists, because this knowledge may one day allow us to manipulate adult wound healing to decrease scarring and fibrosis in plastic surgery as well as in surgery of other organs of the body.

Normal wound healing is characterized by three phases: inflammation, proliferation, and maturation (remodeling). Angiogenesis, the process of new blood vessel formation, is an important component of the proliferative phase of wound healing and is crucial to support the healing wound environment. Chronic wounds, such as diabetic wounds, fail to complete all stages of wound healing; these wounds are likely ischemic in nature as hypoxia and inflammation play key roles in their failure to progress to healing. Macrophages secrete numerous growth factors and mediators vital to all phases of wound healing, particularly the inflammatory and proliferative phases. In chronic wounds, however, macrophages appear to be senescent (Moore, 1997), staying at the wound edge. This altered chemotaxis may be a result of the accumulation of advanced glycation end-products (AGEs) within diabetic skin (Goova, 2001). Elevated levels of AGEs and the concomitant increased expression of receptor for advanced glycation end-products (RAGE), lead to increased free radical formation and inflammatory cytokine production (Simm, 2004). Wound healing studies have suggested that the nonhealing diabetic wound is in a pro-inflammatory state. Subsequently, we hypothesize that increased expression of RAGE results in wound macrophage dysfunction by decreasing macrophage-mediated angiogenesis and prolonging macrophage-mediated inflammation.

Composite tissue allografts (CTA) have emerged as an option for hand and even face reconstruction following major traumas, tumor ablation or congenital malformations. As of now, CTA survival requires continued immunosuppression, which brings along increased susceptibility to infections, cytotoxicity, and carcinogenesis. Furthermore, clinical and experimental evidences show that cutaneous components, a significant portion of most CTAs, are more prone to be rejected. By manipulating the immune system, less or no immunosuppressant drugs would be required to prolong allograft survival and allogeneic skin would also be more tolerated by recipients, leading to a considerable improvement in quality of life of these patients. The work in our laboratory is concentrated on the induction of immunological tolerance to CTAs using rodent model of hind-limb or vascularized skin. We have shown significant prolongation in hind-limb allograft survival using donor-pulsed bone marrow (BM)-derived dendritic cells (DCs) and a short course of anti-lymphocyte serum (ALS)/cyclosporine (CsA). Our preliminary data demonstrate that stromal stem cells (SSC) regulate allogeneic T cell responses and inhibit stimulatory function of heist DCs in vitro. Administration of host SSC delays onset of graft versus host disease (GVHD), enhances engraftment of allogeneic rat BM cells, and Induces survival of vascularized skin allograft. Based on these studies, we have hypothesized that prolonged survival of allogeneic skin flaps in rats can be achieved by utilizing a conjoint approach of different tolerogeneic techniques during the perioperative period.

Repair of large abdominal wall defects pose a difficult surgical problem with limited operative options. Ideal repair of hernias would involve using native tissue, maintaining the dynamic nature of the abdominal wall and preventing recurrence. A commonly applied method (components separation), requires cutting the external oblique muscles to achieve mechanical release and thus defect closure. This method is invasive and has a significant recurrence rate. Loss of function of the external oblique muscles may be a factor for recurrence since the abdominal wall can no longer properly counteract periods of increased intra-abdominal pressure, as occurs during sneezing or coughing. Botulinum toxin has been used clinically to paralyze selected muscles. Using a swine model, we will examine the effect of Botulinum toxin on the external and internal oblique muscles. Under general anesthesia and using a minimally invasive approach, the space between the external and internal oblique will be accessed on each side. A preparation of Botulinum toxin will be administered on one side and saline solution on the other side. The operating surgeon will be blinded to the treatment (Botulinum toxin) side. After 2 weeks, we will examine the abdominal wall for elasticity and muscle activity. We will also evaluate the safety of administering Botulinum toxin to a large muscle group by monitoring and recording all adverse effects. The proposed use of Botulinum toxin may help a large group of patients. It may obviate the more invasive approach used in components separation and its associated complications. An advantage of replacing permanent surgical transection of the abdominal wall muscles with Botulinum toxin is that its effects are temporary but of long enough duration to allow for tissues involved in the repair to achieve near maximum strength. Recuperation of normal muscle function will provide patients with the dynamic function often lost in current major abdominal wall reconstruction.

Breast cancer affects one in eight women. Patients may undergo a variety of surgical oncologic treatment options including local excision, partial mastectomy, and complete mastectomy. Reconstruction options also vary depending on surgical defect. There are a rapidly growing number of plastic surgeons restoring post-mastectomy defects using fat injection techniques. In any given fat harvest sample site (abdomen, flank, buttock, etc), there is an ample supply of adipocytes. Furthermore, in these same anatomical areas, there is also a subpopulation of adipocyte derived stem cells (ADSC). ADSCs are reported to have potent angiogenic and regenerative properties capable of treating numerous medical and surgical problems. However from a safety standpoint, it is not known whether these beneficial properties of ADSCs for reconstruction could have detrimental effects on any remaining breast cancer cells post-resection, or if ADSCs could promote transformation of normal breast epithelium. The purpose of this simple "proof of principle" study is to investigate the safety of ADSCs when exposed to breast cancer cells. We hypothesize that ADSCs will alter breast cancer cell proliferation, apoptosis, and migration all of which may contribute cancer progression and metastasis. The reciprocal effects of cancer cells on ADSCs (i.e. cancer cells may alter ADSC differentiation and therefore affect reconstructive efficacy) are equally important but beyond the scope of this proposal.

Skeletal muscle is one of the most sensitive tissues to ischemic injury and the success of free flap and replantation surgery is dependent on the survival of this tissue following brief periods of ischemia. Tissue injury subsequent to ischemia-reperfusion (IR) insults is thought to be due to a cascade of cellular events that ultimately destroy skeletal muscle cells. While many of the cellular events of this cascade have been described, the molecular events that initiate this destructive cascade have not been elucidated in skeletal muscle tissue. The transcription factor early growth response protein-1 (Egr1) is a crucial mediator of inflammatory gene expression and has been shown to regulate IR injury in various organ models of IR injury. We propose that Egr1 contributes to skeletal muscle IR injury and will test this hypothesis using mice lacking a single copy or both copies of the Egr1 gene (Egr1 heterozygous, +/- and homozygous, -/- mice, respectively; Taconic). Further understanding of the mechanisms involved in mediating the effects of IR injury in skeletal muscle will aid in our ability to create a positive environment for muscle survival during procedures requiring free flap and replantation surgeries.

Breast surgery aims to modify breast size, shape, and contour, while achieving bilateral symmetry. The present day approach to augmentation or reduction mammaplasty remains largely subjective, as surgeons base operative strategies on visual size estimates and predicted change. Thus, the overall success of aesthetic breast surgery is limited by the inability of plastic surgeons to objectively assess breast volume and shape. A potential solution to this subjective obstacle may be three-dimensional (3D) technology, which can provide unique objective clinical data that was previously unattainable to plastic surgeons. In the following study, we hypothesize that 3D-surface imaging technology can provide novel objective data with clinical relevance for surgical planning and outcomes analysis in aesthetic breast surgery.

Chronic non-healing wounds and ulcers account for up to 20% of all diabetic patient hospitalizations. Wound complications make a significant contribution to the growing cost of diabetes, which has reached an annual economic burden of $132 billion (NDIC. 2002). Currently a wide range of approaches are used to treat non-healing diabetic wounds, but most are strictly palliative. The following study offers a unique approach to wound healing by investigating the in vivo self-assembly of locally delivered mesenchymal stem cells (MSCs). Recent research has demonstrated that MSCs from the bone marrow traffic to healing wounds and are able to differentiate into mature endothelial cells to form new blood vessels. Previous work by our group and others suggests that bone marrow-MSC trafficking to diabetic wounds is impaired. The central hypothesis to be tested in this study is whether the delivery of non-diabetic (wildtype) lineage negative bone marrow cells to diabetic wounds will improve healing.

Our laboratory was the first to establish the rabbit orbito-zygomatic complex as a reliable and replicable model for studying craniofacial bone growth inhibition following therapeutic radiation (O'Donovan, et al. 200 I) and to demonstrate the efficiency of cytoprotection in its prevention (Forrest, et al. 2002). The purpose of the current study is to investigate the molecular and genetic mechanisms of radiation damage to osteoblasts in vitro and the efficiency of cytoprotection with WR-1065, the active metabolite of Amifostine (Capizzi, 1999), in MC3T3-E, mouse osteoblastic cell line. We hypothesize that (I) inhibition of osteoblast function following radiation occurs in a dose dependent way; (2) cytoprotection with WR-I 065 prevents radiation-induced inhibition of osteoblast function; (3) the mechanism of cytoprotection lies in cell cycle arrest, inhibition of apoptosis and activation of p53.

A method by which the internal neural state could be assessed, in terms of architectural preservation, would allow for an early grading of the level of injury, a more rapid and accurate determination of the prognosis, and an earlier, more informed decision about whether to continue observation or to proceed with nerve reconstruction. The objective of this study is to assess the ability of a novel high resolution imaging technique (Coherent Anti-stokes Raman Scattering Microscopy) to categorize peripheral nerve damage, and to monitor the recovery process of a damaged nerve. Features in these images will be compared with known anatomy and histology in real time in order to assess the ability of this technology to evaluate peripheral nerve damage. Changes in these features will be used to observe regrowth or atrophy of the nerves.

Onlay cranioplasty, the placement of material on top of cranial bone, is used for craniomaxillofacial reconstruction. The skeleton is augmented in order to correct asymmetry or to improve function. Common indications for cranial augmentation include congenital anomalies, trauma, and deformities following operative procedures. Approximately 8,000 cranial bone grafts are used per year in the United States, with and additional number of grafts from alloplastic materials. Cranioplasty in children is particularly difficult because I) the supply of bone graft is limited and 2) alloplastic materials are not advocated because they may inhibit cranial growth. In children, autologous bone is the cranioplasty material of choice because alloplastic substances may become unstable or inhibit the growth of the skull. Despite the advantages of autologous bone for pediatric cranioplasty, it is difficult to obtain in children. The iliac crest and ribs are small and the preferred donor site, the cranium, is unable to be split before 4 years of age when a diploic space has formed. In situ harvest of cranial bone is not recommended until 9 years of age, when the parietal region is at least 6 mm thick. Not only is autologous bone difficult to harvest in children, but when used as an onlay material, it does not maintain its volume as well as alloplastic material. In order to overcome the problem of limited bone graft supply in the pediatric population, we have developed a technique for harvesting particulate cranial bone graft in children. Clinically, we have shown that this graft effectively heals full-thickness cranial defects (inlay cranioplasty).

Excessive and hypertrophic scarring are problems that continue to confront surgeons. Clinical and experimental attempts to reduce scarring have met with limited success. There remains a need for the development of an effective anti-scarring therapy. The purpose of this project is to use a molecular therapy approach to reduce scar formation in a rabbit ear model of hypertrophic scarring. We hypothesize that specific inhibition of various gene products integral to the scarring process, namely, TGFbeta-l, TGFbeta-2, TGFbeta receptor I, and CTGP, will cause a reduction in cutaneous scarring. We aim to achieve this goal using antisense oligonucleotides that block the mRNA of these genes.

The purpose of the proposed study is to attempt to induce immunological tolerance and prolonged graft survival by modification of the local immuno-modulatory environment of a composite tissue allograft. We will attempt to modify the generation of a cellular immune response to the transplanted hindlimb by the expression of tolerance inducing cytokines in the transplanted flap. By modifying the initial immune response from reactive to suppressive we hope to block or reduce the process of rejection and enhance flap survival. We will attempt to induce tolerance by over-expressing transforming growth factor beta (TGF beta) and Interleukin 10 (IL- 10); alone and in combination; in a heterotopic hindlimb transplantation model across a major histocompatibility barrier in rats.

Trauma and disease related upper extremity amputation causes substantial disability due to functional and tactile deficits while performing basic activities of daily living, Restoration of mechanical and sensory function following amputation is critically important. To date, the development of a biointegrated, durable, high fidelity, permanent prosthesis with bidirectional neural motor and sensory function, combined with refined robotics to effect or trigger these responses remains elusive, We have developed a novel construct using the biocompatible electroconductive polymer PEDOT (poly(3,4-ethylenedioxythiophene)) on acellular muscle (ACM) scaffolds, Preliminary data has shown that this construct is capable of propagating action potentials across at least 20mm nerve gaps, We hypothesize that an efferent action potential in the peripheral nerve can be electrically detected and delivered to downstream electronic circuitry using PEDOT-ACM scaffolds with longevity and fidelity. Furthermore, we hypothesize that afferent sensory signals can be delivered centrally using the same device architecture. Our specific aims are to investigate the bioelectrical construct in regards to the following: 1) in vivo PEDOT-ACM characteristics including physical and electrical interactions with native axons, in vivo biocompatibility, and durability. 2) ability to detect an efferent motor action potential 3) ability to initiate an afferent sensory action potential through electrical stimulation.

Our laboratory is currently involved in engineering a bone substitute, which can be utilized to repair segmental osseous defects. Utilizing a biologic scaffold seeded with autologous adipose-derived adult stem (ADAS) cells we hypothesize that these ADAS cells will colonize the bone matrix and differentiate into osteoblasts accelerating incorporation of the allograft into the host skeleton. This may reduce the complications associated with conventional bone allografts. The assembly and surgical implantation of such a construct is expected to be simpler than conventional vascularized bone transfer methods and will permit its use at medical facilities outside major medical centers. This technique may reduce amputation and complication rates in patients who otherwise have no access to technically demanding reconstructive surgery. To achieve the long-term goal of creating an engineered bone substitute capable of immediate mechanical support, it will be necessary to deliver viable osteoprogenitor cells to the defect for osseo integration. In order to assure the viability of these oseoprogenitor cells it is necessary to understand the metabolic requirements of the implanted cells. Therefore the aim of this proposal is to characterize the metabolic requirements and their effects on the differentiation capacity of human adipose-derived adult stem (ADAS) cells. The results of this study will allow for the development of techniques designed to maximize the viability and utility of implanted ADAS cells for bone and other tissue engineering applications.

Pediatric craniofacial surgery is complicated by a shortage of autologous bone for grafting in the repair of osseous defects after trauma or congenital anomaly reconstruction. In the pediatric population, because of an under-developed diploic space, local calvarial bone graft sources are non-existent, and distant sources are limited by practical concerns ranging from donor site morbidity to low tissue yield. Infants, however, benefit from highly osteoinductive dura mater, and can spontaneously heal large calvarial defects. It is in this context of a shortage of bone graft material for craniofacial reconstruction that the objective of this project is to create a new source of osteoprogenitor cells by utilizing the osteoinductive potential of infant dura mater. We hypothesize that the co-culture of adipose-derived stem cells (ASCs) with infant dura mater-derived cells will yield enhanced and accelerated osteoblastic differentiation of the ASCs, as infant dura is likely "programmed" to produce the signaling molecules required for the maintenance of an ideal osteoinductive microenvironment. Confirming this hypothesis would allow craniofacial surgeons to use readily available adipose derived stem cells as an alternative to autologous bone grafts for the reconstruction of bone defects in the pediatric craniofacial skeleton.

Despite huge interest in the facial nerve by many disciplines, its extratemporal microanatomy remains controversial, especially in the periorbital and midbrow regions. The purpose of this study is to combine electrophysiology and microanatomy to better define the extratemporal microanatomy of the facial nerve. Special interest is going to be paid to the periorbital area and glabella, and mapping of the innervation of the orbicularis oculi, procerus and corrugator supercilii muscles. The intention is for this study to serve as a guide of the facial nerve microanatomy and its variations for facial rejuvenation surgery, especially useful both for the aggressive and minimally invasive approaches to the face.

Adult wounds are repaired by proliferation and migration of multiple cell types that form a scar. Scar lacks skin appendages such as hair follicles, sweat glands and sebaceous glands; and dermal structure is contracted with variable collagen deposition. Undifferentiated cells can be found in the healing wound and can be recruited for skin structure regeneration rather than repair with scar. During embryogenesis, fate of undifferentiated cells are guided by multiple morphogens, including the Wnt gene family. The undifferentiated cells found in adult wounds can similarly be guided by Wnt genes transcription. The purpose of this application is to evaluate the effect of Wnt gene transcription on skin appendage regeneration of healing wounds. We hypothesize that Wnt gene expression will guide undifferentiated skin wound cells toward skin appendage regeneration during wound repair morphogenesis.

Adipose tissue derived stem cells (ADSCs) have enormous potential for use in tissue engineering and cell-based regenerative medicine. Unfortunately, like all adult cells, ADSCs can only undergo a finite number of cell divisions prior to entering senescence. We hypothesize that by manipulating molecular regulators of senescence, such as Bmi-l or telomerase (hTERT), we will enable improved ex-vivo expansion of ADSCs and thereby enhance their potential clinical utility. Furthermore, by characterizing a large panel of independently derived ADSC lines for the expression of senescence markers such as Bmi-l, p 16Ink4a , and p 19Arf , we may be able to pre-select ADSC populations that are optimal for use in future tissue engineering applications or clinical protocols.

Plastic surgeons rely on free tissue transfer for complex reconstructions. The greatest risk in free tissue transfer is occlusion of the blood vessels supplying the tissue. Vessel occlusion occurs in up to five percent of free tissue transfers, potentially leading to complete tissue loss. Fortunately, if blood flow is restored within four hours of occlusion the tissue transfer can be salvaged. Methods exist to detect occlusion, but they are limited by low sensitivity, poor specificity, delay in detection, and cost. Monitoring of metabolites inside the transferred tissue offers a new frontier in the detection of vessel occlusion. These metabolites provide continuous updates of tissue perfusion. We demonstrated in a pilot study that tissue glucose is 97% sensitive for vessel occlusion, detecting tissue ischemia within one hour of the occlusion. Cost and technical barriers prevented previous implementation of glucose monitoring in clinical practice. Due to advances in biosensor design, inexpensive and reliable continuous glucose monitors are now commercially available. We hypothesize that continuous glucose monitors can provide rapid and accurate detection of vessel occlusion following free tissue transfer. To test our hypothesis, we will use a continous glucose monitor to measure tissue glucose levels following vessel occlusion in a rodent model of free tissue transfer. Our study will also identify the time delay between vessel occlusion and detection of tissue ischemia. Further, we will determine the sensitivity and specificity of specific glucose thresholds for vessel occlusion. This study investigates the use of glucose monitoring to detect vessel occlusion rapidly and accurately, allowing surgeons to repair the occlusion and salvage the tissue transfer. This research may dramatically improve patient outcomes after free tissue transfer, changing both how surgeons monitor for vessel occlusion and eliminating the devastating consequences of undetected occlusion.

Targeted reinnervation (TR) is a novel, revolutionary strategy to achieve a functioning neural-machine interface that allows upper extremity amputees to voluntarily control motorized prosthetic arms just as they would control their native arms. Developed and pioneered by Todd Kuiken and Gregory Dumanian at Northwestern University, TR employs a process called neurotization, in which the stumps of amputated nerves of the brachial plexus (that had originally innervated the arm) are connected to denervated, otherwise functionless, remnant muscles in the shoulder, chest, and/or proximal arm. After several months, the newly reinnervated muscle segments act as biological amplifiers of the nerve action potentials that had previously controlled the arm. For example, if the radial nerve is connected to one portion of the pectoralis major and the musculocutaneous nerve is connected to pectoralis minor, then when the patient "thinks" to extend his/her elbow, the pectoralis major muscle will contract. Likewise, when the patient "thinks" to flex his/her elbow, the pectoralis minor will contract. The EMG Signals of these reinnervated muscle segments are then detected by electrodes placed on the skin and can be used to control myoelectric prostheses in an intuitive way that requires less training compared to conventional prostheses to achieve greater degrees of freedom. 1 So far TR has been successfully implemented in nine human subjects. Since the beginning of the war in Iraq, with many US soldiers returning stateside after suffering from blast amputations, the US Government has placed more of a focus on the treatment of amputations through the Defense Advanced Research Projects Agency (DARPA) of the Department of Defense (DoD). There is now a more visible, everyday need to improve upon the management of human amputees. One problem often experienced by people who have suffered from amputations or other forms of trauma is the formation of painful neuromas that are responsible for various uncomfortable sensory disturbances.6 We hypothesize that TR can also be an effective technique for the prevention and treatment of painful neuromas, which is supported empirically by the fact that none of the ten patients in our series who have undergone TR has developed a painful neuroma, and TR has even eliminated pre-existing painful neuromas in several of these patients. Animal studies will help provide a better understanding of neuroma formation in the setting of TR. My overall goal for my upcoming research year is to help refine strategies for Targeted Reinnervation and to investigate the utility of TR as a way to prevent or treat neuroma formation.

Rabbit adipose-derived stem cells (ASCs) can be used to generate clinically useful autologous bone and cartilage constructs when seeded on appropriate biocompatible scaffolds. Specific Goals: 1. To generate osteocytes and chondrocytes from rabbit ASCs in vitro on biocompatible tissue scaffolds, both biodegradable and soluble (poly (lactide-co-glycolide acid) (PLGA)), and biodegradable but insoluble (MEDPOR®). 2. To apply the above techniques to an in vivo model through the implantion of cell-impregnated scaffolds in rabbits, and then study the effects of the in vivo microenvironment on the biologic behavior of the implants. This research will provide valuable data on engineering connective tissue constructs from autologous tissue. Our ultimate goal is to extend these techniques to future projects in humans, in order to improve outcomes in patients requiring reconstructive surgery following cancer resection, traumatic injuries, and degenerative diseases.

Over the last several decades, numerous augmentation agents have been employed in attempts to offset the soft tissue loss that occurs in the aging face. Methods used include fat autografting procedures and the injection of collagen and hyaluronic acid derivatives. However, all share the disadvantages of variable incorporation and graft resorption over time. Human adipose derived stem cells (ADSC) are an ideal source of autogenous tissue that may overcome many of the limitations of both auto grafted whole adipose tissue and extracellular matrix derivatives. ADSCs comprise the most viable population of cells residing within adipose tissue, and may be injected as a cellular suspension devoid of intervening tissue, allowing for rapid access to diffusing nutrients and oxygen, thus enhancing viability. Increasing evidence is revealing the great dependence of grafted tissue upon neovascularization to overcome diffusion limitations. Vascular endothelial growth factor (VEGF) has been shown to significantly enhance the process of angiogenesis, though its application to tissue engineering strategies remains limited. The purpose of this study is to determine to role of ADSCs as agents for soft tissue augmentation. A comprehensive tissue engineering based approach combining adipose derived stem cells with commercially available hyaluronic acid (Restylane) and VEGF will be utilized to determine the most effective pairing of these agents. Our specific objectives are: 1) To assess the cellular viability of grafted fat versus ADSC constructs, and 2) to compare the volumetric persistence of injected agents based on histological area analysis. The development of a more durable agent for soft tissue augmentation would contribute greatly to the field of aesthetic plastic surgery.

Vascularized skin transplantation holds great potential for reconstruction of defects for which there are no other adequate surgical solutions. This has been demonstrated by the recent face transplant and by human abdominal wall and scalp transplants that have taken place. Currently, the planned solution in the event of failure of the skin transplant is to replace it with another one. However, it is not known whether this would even be possible: the rejecting skin paddle may affect the underlying tissues and vessels to such an extent that it may be too difficult to repeat the procedure. Even if it is possible to retransplant, there may be an increased risk of rejection and underlying tissue changes, restricting the function of the transplant and making any further procedures more complex to perform. This study intends to address these important problems: whether it is possible to retransplant following a transplant rejection, and if so, whether the function of the underlying tissues be significantly compromised. The findings of this study will have immediate direct clinical application: of central importance for planning the best possible surgical management in such cases, as well as counseling the patient before a procedure.

The concept of scarless wound healing became popular nearly twenty years ago when the discovery was made that fetal skin, when wounded, will regenerate with normal dermal and epidermal components. This scarless wound healing phenomena has been witnessed in many different mammals, including humans. Much of the recent work on this subject has been directed toward discovering the mechanism behind this wonderful process. It has been clearly shown that factors intrinsic to fetal tissue are responsible for healing without scar. What has not been made clear is what these factors are. We have identified a unique stem cell population from the fetal mouse dermis. These particular cells, called Dot cells (due to their morphology), are blood derived and can be found in bone marrow and blood of fetal and adult mice as well as in adult human blood. These cells have been isolated and studied in our lab. They are approximately 7 um in diameter, are made almost completely of nuclear material, travel in large numbers, and have multilineage differentiation potential. They are almost 20 times more abundant in fetal circulation. Injection of fetal mouse Dot cells into injured postnatal mice results in Dot cell differentiation into normal dermal components that participate in the restoration of normal skin structure. Tagged Dot cells, after being injected into wounded postnatal mice, migrate into the site of wound repair and are present at the wound site until skin regeneration is concluded. We believe that Dot cells are a unique group of blood-derived stem cells, have a high proliferation rate, demonstrate a multi-lineage differentiation potential during tissue repair, and are the key elements for scarless fetal skin repair. Although some preliminary work on Dot cell activity has been done, the mechanism for scarless skin regeneration in postnatal wounds is unknown. Observation suggests that the tissue regeneration function of Dot cells is dependent on the Dot cells themselves and not on local wound differentiated cells. Our plan is to assess the wound repair mechanisms of Dot cells by studying their migration, their cellular differentiation, and their regulatory factors during wound repair.

As tissue engineered cartilage moves closer to clinical practice for soft tissue craniofacial remodeling and joint surfacing, finding sources for adequate number of chondrocytes remains a primary obstacle. Ear, nose and joint cartilage can be used as sources of chondrocytes but can cause unwarranted donor site morbidity if large quantities of cells are needed. Bone Marrow is a potential source of chondrogenic cells (Mesenchymal Stem Cells, MSC) and has been shown to produce cartilage in vitro under the appropriate culture conditions (dexamethasone, TGFp, etc.) (Pittenger, 1999). In vivo, however, it appears that a three dimensional scaffold is required for chondrogenic differentiation of cells (Martin I, 1998). Additionally, evidence of in vivo chondrodifferentiation of human MSC has not been demonstrated. Our previous studies have shown that swine MSC chondrodifferentiate in vivo when encapsulated in certain hydrogel scaffolds (PEG, Alginate). Additionally, in vitro pretreatment of swine MSC enhances the chondrodifferentiation of these cells in vivo (Mesa, 2004, 2005). Based on these data we hypothesize that human MSC encapsulated in hydrogel scaffolds chondrodifferentiate in vivo. Additionally, we hypothesize that chondrodifferentiation of human MSC in vivo can be enhanced by in vitro pretreatment with chondrogenic factors. To test hypothesis we will use commercially available human MSC, and chondrogenic hydrogel scaffolds previously tested with chondrocytes and MSC from swine. We believe that these studies will yield valuable preclinical data for future clinical applications of using tissue engineered cartilage with human MSC especially for craniofacial and joint surgery.

The fibula osteofasciocutaneous free flap, a vascularized composite graft composed of a segment of fibula and peroneal artery, plus a skin island derived from the lateral calf, is among the most widely used and versatile flaps for composite reconstruction of the mandible and soft tissue of the jaw and neck. The skin island is perfused by septocutaneous perforators that emanate from the peroneal artery then course transversely in the posterolateral intermuscular septum. Challenges in preparing the fibula free flap include determining the number and location of the perforator vessels within posterolateral intermuscular septum, and incorporating them into the flap without injury. A separate body of information is required about the tibioperoneal vessels, to prevent an attempt at flap harvest from a patient who has severe peripheral vascular disease, or an anatomic anomaly that could jeopardize the extremity or the flap. We will study the perforators in healthy volunteers and patients undergoing fibula free flaps. We hypothesize that magnetic resonance angiography (MRA) can provide anatomic information about the septocutaneous perforators while simultaneously providing an image of the tibioperoneal trunk sufficient to obviate the conventional arteriogram obtained by many surgeons prior to performing flap surgery.

Peripheral nerve injuries are a common and debilitating problem for which we still have inadequate solutions and suboptimal functional outcomes. For this reason, the field of peripheral nerve research is indispensible. The specific aim of this project is to demonstrate that the novel transgenic rat model, the Thy1-GFP rat, which expresses green fluorescent protein (GFP) in peripheral nerves, can be used to evaluate nerve injury and introduce new outcome measures that will complement the already validated measures established in the non-fluorescing rat model. This study will examine three common peripheral nerve surgical paradigms, i.e. nerve crush, transection and repair, and nerve graft placement, in the Thy1-GFP rats. We will assess nerve regeneration with in vivo serial imaging, confocal microscopy, histomorphometry and walking track analysis. The underlying rationale is that no previous rat model exists that has GFP expression isolated to the central and peripheral nervous system. GFP expression allows for direct visualization of nerve regeneration and muscle reinnervation. Although mouse models with this expression exist, there are many limitations to their application in peripheral nerve research. Thus, in this feasibility and model development study we hope to show that this novel transgenic rat line can be used as a validated model in the advancement of peripheral nerve research. Nerve injuries are devastating. As hand and microsurgeons, plastic surgeons have a large impact in the treatment of these injuries. With the advances in neurobiology and the creation of transgenic animals to assess nerve regeneration, enhanced understanding of nerve regeneration will translate to improved clinical outcomes.

Previous work in our laboratory has demonstrated reduced FGFR2 signaling in the palate in a mouse model for Crouzon syndrome. Mice with the mutation have high arched or cleft palates. One of the findings associated with this work has been the demonstration of reduced levels of B-catenin (Catnb) in the palate. B-catenin is a key mediator for WNT signaling. Proteins of the Wnt family are secreted factors regulating cell proliferation, fate, and behavior during development, and are required for early craniofacial and tooth development (Kim-Weroha et aI., 2006). Microarray screening of palatal tissue from mice heterozygous for the Crouzon mutation revealed that transcripts for several genes of the Wnt family were decreased. A DermolCre; B-catenin c/c mouse construct available in our laboratory was also analyzed for a cleft palate phenotype. Analysis of these mice at E18.5 revealed cleft palate in 5/5 specimens. The palate of DermolCre; B-catenin c/c mice is much narrower than that of WT mice. It is also steeply angulated and further resembles the Byzantine arch-type palate frequently seen in humans with different types of syndromic craniosynostosis. We hypothesize that loss of Fgfr 2 signaling affects the Wnt signaling pathway and that disruption of Wnt signaling is in part responsible for the associated phenotype. Our specific objective is to characterize Wnt signaling and p-catenin levels and subsequent effects on downstream signaling pathways during palatogenesis and to link these findings to specific developmental anomalies seen in individuals with syndromic craniosynostosis.

Muenke syndrome is the most common form of syndromic craniosynostosis, which is caused by a mutation in the fibroblast growth factor receptor 3 (FGFR3). Unexpected from a monogenetic syndromic craniosynostosis, the suture phenotype of Muenke syndrome shows incomplete penetrance and variability; some patients show normal patent sutures, whereas others present with unicoronal or bicoronal craniosynostosis. Notably, the incidence of unicoronal synostosis, which is a characteristic feature of non-syndromic cases and not usually present in a syndromic craniosynostosis, is high in Muenke syndrome. This finding implicates the contribution of a localized non-genetic factor to the etiology of Muenke craniosynostosis. Earlier studies including ours have suggested that mechanical stress in the form of intrauterine constraint may be one of the critical factors involved in non-syndromic (unilateral) craniosynostosis. The purpose of this study is to delineate the potential contribution of mechanical stress to Muenke craniosynostosis and the underlying cellular mechanisms.

A vascularized, neurotized, tissue engineered contractile skeletal muscle would be of benefit to patients with disorders such as longstanding facial paralysis or upper extremity muscle loss or dysfunction. Both in vitro and in vivo techniques have been used to develop skeletal muscle constructs, which have been shown to resemble the behavior and structure of native skeletal muscle. Furthermore, vascularization of these tissues has been attempted, in order to achieve clinically significantly-sized constructs with their own vascular pedicles, which can subsequently be transferred micro surgically. We hypothesize that neurotization of vascularized in vivo muscle constructs will allow for directed mechanical action, and will direct their development and phenotypic maturity.

Notch signaling regulates adult adipocyte derived stem cell fate decisions and restricts osteoblast differentiation of these cells. Specific Aim 1: To determine the effect of notch signaling on adipocyte derived adult stem cell proliferation and osteoblast differentiation potential. Reconstructive surgery using engineered tissue constructs is a rapidly growing therapeutic field. Adipose-derived adult stem cells (ADSCs) have been touted as an easily accessible, readily expandable, multipotential cell population that could serve as one of the key components to a successful tissue engineering strategy. ADSCs can be induced in culture to differentiate, under specific culture conditions, into adipogenic, osteogenic, chondrogenic, myogenic, endothelial and neural lineages. However, the mechanism(s) regulating the maintenance of this cell population in vivo is not known. Many investigators, including us, are interested in using this population in bone reconstruction procedures and the mechanisms regulating the osteogenic potential of ADSCs is similarly poorly defined. The Notch signaling pathway is an attractive target for regulating both the stem cell maintenance in the adult and cell fate decisions of the uncommitted progenitors. Therefore, the goal of this study is to begin to elucidate the role of Notch signaling on ADSCs in vivo and in vitro. Understanding how this cell population is regulated may allow the development of strategies that allow for increasing the expansion, osteogenic, and in vivo recruitment potential of ADSCs to aid in tissue engineering and endogenous repair mechanisms.

Some of the most challenging problems in plastic surgery include the management of chronically infected wounds in vasculopathic patients and the reconstruction of large, contaminated, complex defects with exposed bone and/or hardware following lower limb trauma. Microvascular free tissue transfer is often required to fill resulting soft tissue and osseous defects. Free flaps provide a reconstructive role by transferring well vascularized tissue to deficient areas. As they have an intact microvasculature supplied by an afferent artery and efferent vein, they have the potential to provide an additional therapeutic role. We intend to exploit the vascular properties of free flaps to deliver targeted genes capable of producing therapeutic proteins. This dual therapeutic as well as reconstructive role holds enormous potential in the field of reconstructive surgery and provides a framework for expanding the efficacy of free flaps.

Cartilage possesses limited capacity for repair and self-regeneration. Consequently, injury to cartilage in the articulating joints results in scar formation and possible arthritic changes that can lead to loss of structure and function. For these reasons, more than one million surgical procedures are performed in the United States each year involving cartilage replacement. Numerous attempts to repair damaged cartilage by applying tissue engineering concepts have failed and to date there are no methods that can be employed to create cartilage constructs that fully integrate with adjacent cartilage. Meanwhile, my collaborators have shown that collagen can be crosslinked using photoreactive dyes. Crosslinking a gel in situ could induce molecular interactions with the native cartilage surrounding the lesion to stabilize the gel during cartilage formation. Pilot studies have demonstrated the cartilage-forming potential of the type I collagen solution with chondrocytes. Based on these results, the hypotheses of this work are: 1) Photochemical crosslinking of collagen can be used to generate a hydrogel that permits cartilage formation by encapsulated chondrocytes; 2) Chondrocytes in hydrogels have the capacity to form neocartilage with improved biochemical and biomechanical properties for cartilage repair; 3) The neocartilage will integrate with existing native tissues.

The role of chondrocyte biology in the development and treatment of craniofacial disorders remains largely unstudied. Current advances in both genetic and bioengineering have significant potential for the field of craniofacial and pediatric plastic surgery. Recent studies indicate that the genetic sex of a cell has fundamental consequences for the success of certain cell therapy strategies (l). Dr. Boyan's group has shown that chondrocytes from male and female rats respond to estrogen and testosterone in distinctly different ways, although both sexes possess receptors for both hormones. Her group has now shown that human chrondrocytes also exhibit sexual dimorphism in their responses to 17-beta estradiol (2). A study from another group showed that male predominance in metopic and sagittal craniosynostosis and in nonsynostotic plagiocephaly, suggesting a role for circulating androgens in early craniofacial development skeleton (3). Based on these observations we would like to investigate the role of sexual dimorphism in chondrocyte differentiation in vitro and in vivo. My overall goal is to develop a method for reproducible and effective delivery of cells for tissue regeneration and repair. I will use an alginate microencapsulation method developed in Dr. Boyan's lab to produce microcapsules 150-180 microns in diameter containing approximately 80 chondrocytes each. These microcapsules are small enough to permit percutaneous delivery using a standard gauge needle; they permit rapid diffusion of nutrients; and cell viability and phenotype expression are preserved. I will assess whether there is a difference in the behavior of male and female chondrocytes with respect to their response to 17- beta-estradiol and 5-hydroxytestosterone when encapsulated, and to circulating hormones when implanted in same or opposite sex mice. We will use nude mouse model muscle as a site for implantation and will analyze the quality and quantity of the cartilage produced by histomorphometric techniques. The last part of this study will be to determine if sexual dimorphism extends to responses of cells to growth factors typically used in cartilage tissue engineering: TGF-beta-l and 3, IGF-l, and PDGF-BB. In vitro studies (dose response and time course) will indicate if male and female cells exhibit differences in phenotypic expression or matrix synthesis. Based on these results, we will pretreat microencapsulated chondrocytes and then assess chondrogenesis in vivo using similar techniques. Since cartilage and soft tissue defects extend not only to craniofacial disorders but also to the trauma and aging population, the ability to expand a patient's own cartilage or mesenchymal stem cells using a micro-delivery system would have a plethora of applications in the field of plastic surgery. Such a delivery system could be applied to fat or muscle injection as well.

Brachial plexus palsies, whether obstetrical or traumatic, are devastating injuries with significant impairment of the affected limb, resulting in functional paralysis, sensory deficits, as well as deep psychological scars. Because of the prolonged delay in nerve regeneration, chronic muscle atrophy and fibrosis continues to be a severe, irreversible impediment to recovery. Current microsurgical techniques have reached their limit in terms of improving the results of brachial plexus reconstruction, we hypothesize that stem cell transplantation may be able to further enhance outcomes by better supporting the biological integrity of the injured muscle and nerve. The requirements for optimal functional recovery after a brachial plexus injury are that (1) the regenerating axons make functional connections with their original muscle cells and (2) the number and size of the motor units in those muscles are restored. To prevent prolonged denervation atrophy and eventual fibrosis, we have previously transplanted embryonic stem (ES) cell derived motor neurons (MNs) into denervated muscles (Craff, et. at., PRS in press). These MNs can provide trophic support to the muscle by forming neo-neuromuscular junctions and upregulating specific growth factors, preserving motor unit integrity for a period of one week. In the current study, we propose to (1) examine the effect of this transplant on the functional outcome after nerve repair and (2) characterize the interaction on a molecular level. To better understand the interaction of the stem cell derived MNs and muscle cells, an in vitro co-culture model of stem cell derived MNs and myotubes will be used to perform a microarray analysis. If this interaction can be better understood, perhaps a method of preserving motor unit health in the denervated state could be achieved, ultimately improving outcomes in brachial plexus reconstruction.

Obstetric brachial plexus palsies are devastating birth injuries with an incidence of 0.5-2.5 per 1000 live births. Twenty to twenty-five percent suffer permanent impairment of the affected limb, resulting in functional paralysis, sensory deficits, as well as deep psychological scars. Treatments continue to be complex, requiring a multidisciplinary approach and often several operative interventions. Nevertheless, prognoses remain very poor in the more severe cases, emphasizing the importance of research in this field. The requirements for functional recovery are (1) that the regenerating axons make functional connections with their original muscle cells and (2) the restoration of the number and size of the motor units in those muscles. A motor unit is a single motor neuron and the group of muscle fibers innervated by it. Chronic muscle atrophy and fibrosis is a problem in situations where there is a prolonged delay of nerve regeneration, since nerves only regenerate at a rate of 1 mm/day. Brachial plexus injuries therefore are at significant risk of progressive, irreversible atrophy of the denervated target muscles. Therefore, to prevent denervation atrophy, we propose that transplanting embryonic stem (ES) cell derived motor neurons (MNs) into denervated muscles can provide trophic support to the muscle by forming neo-neuromuscular junctions and up-regulating specific growth factors until the original nerve fibers regenerate to restore and maximize functional recovery. Preliminary studies in rodent models have shown that at day 7, transplanted motor neurons remain viable in vivo in denervated muscle and are able to maintain muscle mass, consistent with the concept of preventing denervation atrophy (Plast Reconstr Surg, in press). Using a GFPIHB9+ mouse embryonic stem cell line (Cell, 2002), this study will further investigate the potential of ES cell derived MN transplantation. These cells express Green Fluorescent Protein, which auto-fluoresces, when fully differentiated into post-mitotic MNs, thereby allowing us to monitor them both in vitro and in vivo.

With the advent of distraction osteogene3is a new era of craniofacial reconstruction began. New bone is created to correct deficiencies, in effect representing in vivo tissue engineering in the craniofacial skeleton. Since its inception the biomolecular and cellular mechanisms occurring in the distraction zone have undergone rigorous investigation. Recently it has been demonstrated that injected, expanded adult stem cells, endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC), are incorporated into the vascular and bony elements of healing bone. Utilization of these progenitor cells could lead to therapeutics which could overcome the ongoing limitations of distraction such as lengthy periods of activation and consolidation as well as the possibility of complete failure and production of a fibrous non-union. While these purified stem cell populations have been shown to be active participants within the healing callus, the contribution of native, marrow derived stem cells to tissue generation in fracture healing and distraction osteogenesis has yet to be defined. The central hypothesis to be tested in this proposal is that marrow derived stem cells are up-regulated in response to craniofacial fractures and distraction osteogenesis, homing to the nascent callus via the peripheral circulation, and becoming incorporated into critical tissue systems involved in new bone formation.

The ability to affect gene expression via topical therapy has profound therapeutic implications for dysfunctional wound healing. Specifically targeting local gene expression topically avoids systemic toxicity and simplifies treatment. We have recently developed a novel method of topical gel-based siRNA application to precisely and effectively silence local gene expression in non delimited areas. In this project we will employ this technology to treat non-healing and slow healing diabetic wounds. Recent work has begun to elucidate the multiple genetic aberrations in this disease state that lead to dysfunctional wound healing. Two particularly interesting derangements in the diabetic state that may contribute to alter wound healing are dysregulation of the apoptotic pathways and berated matrix metalloproteinase (MMP) activity in the wound bed. We propose that by silencing various genetic targets, we will be able to restore normal gene patterning and in-turn, normal wound healing. To accomplish this, we will utilize a novel, topical gel based matrix to deliver small interfering RNA directly to the wound bed, allowing for local gene modulation without systemic effects.

Cell based therapies using autologous adipose tissue may represent the future of regenerative medicine. In the most basic and time tested form, mature adipocytes and small parcels of surrounding tissue are harvested by standard suction lipectomy and transplanted to another anatomic location. In recent years, the regenerative potential of adipose precursor cells that reside in adipose tissue has been demonstrated and holds promise for clinical reconstructive strategies. These adipose derived stem cells (ASC's) can be isolated from whole fat tissue or lipoaspirate. Also in recent years, the popularity of ultrasonic liposuction techniques has increased. Newer generation devices are able to apply energy in a more efficient and focused manner. Since ultrasonic liposuction devices rely on cavitation to extract adipose tissue, the viability and function of harvested cells has been questioned. Preliminary data from our laboratory suggests that high numbers of viable mature adipocytes and ASC's can be harvested by ultrasonic techniques. We hypothesize that ultrasonic fat extraction will yield cells equivalent in number and function to standard liposuction.

This study is designed to determine whether adipose derived stem cells are primarily derived in adipose tissue during embryogenesis and are a distinct cell population, or whether the multipotent cells in adipose tissue represent bone marrow stem cells that have migrated into adipose depots. We hypothesize that adipose derived stem cells are indeed a distinct population and not simply migratory bone marrow cells. This knowledge has broad applications to the development of cell based therapies, especially therapies for the treatment of hematopoietic malignancies. Autologous adipose stem cells (ACS's) could be used in place of allogeneic bone marrow transplantation in the setting of bone marrow dyscrasias. This would greatly decrease the morbidity and mortality of using bone marrow transplants.

The identification of molecules that promote the formation of apoptosis-resistant myofibroblasts is of clinical importance for the development of treatments for scarring. We have identified the protein periostin as an up-regulated component of hypertrophic scarring. We have shown that periostin induces myofibroblast differentiation and avoidance of apoptosis in vitro, that periostin is transiently expressed during normal cutaneous wound repair in mice and that it is not evident in unwounded skin. In contrast, periostin is abundant in human hypertrophic scar tissue and is localized to the dermal side of the basement membrane. Keratin-14 positive basal keratinocytes adjacent to the basement membrane exhibit the highest expression of POSTN mRNA, encoding periostin. We hypothesize that sustained POSTN mRNA expression by keratin-14 positive basal keratinocytes results in secretion of periostin into the dermis, where it induces myofibroblast development, avoidance of apoptosis and scarring. The aim of this study is to generate a transgenic mouse featuring inducible, basal keratinocyte-specific POSTN mRNA expression. The transgene consists of a POSTN cDNA downstream of a lox-stop-lox cassette. This transgene will be inserted in the Gt(ROSA)26Sor locus, a widely used, reliable system for expressing transgenes. This mouse will be mated with a Keratin-14 Cre mouse and the offspring are predicted to express POSTN mRNA specifically in K-14 expressing basal keratinocytes. These animals can then be used for wound repair studies to determine if sustained expression of POSTN mRNA by basal keratinocytes is sufficient to induce abnormal scar formation. This model will also be amenable to treatments designed to inhibit periostin secretion, such as POSTN siRNA injection, to further confirm the wound repair phenotype(s). We believe this model will allow us to determine the role of periostin in cutaneous repair and its potential as a therapeutic target.

Non-healing diabetic ulcers are characterized by low levels of endogenous growth factors, including Insulin-like Growth Factors (IGFs) and Transforming Growth Factor-B (TGF -B), as well as altered proliferative responses to exogenous growth factors, suggesting that decreased bioavailability of growth factors may be a primary contributing cause non-healing wounds. The IGF-III Mannose 6-Phosphate Receptor (IGF -II/M6PR) IS a cell surface receptor that has been shown to mediate IGF-II and TGF-B levels. It is our hypothesis that the IGF-II1M6PR receptor complex contributes to the observed limited growth factor bioavailability in the healing wound and that a transient repression of IGF-II/M6PR expression by siRNA in primary dermal fibroblasts derived from diabetic ulcers will restore growth factor bioavailability leading to enhancement of their proliferation, migration, collagen invasion and contraction in vitro. To test this hypothesis, our goal is to develop an siRNA approach in cultured cell lines to specifically down-regulate IGF-II/M6PR expression. Primary human fibroblasts derived either from skin biopsies collected from unrelated surgical procedures (controls) and tissue biopsies from nonhealing ulcers will be isolated and treated with siRNA to IGF-II1M6PR. In vitro assays designed to closely monitor the effects of altering IGF-II1M6PR expression in vivo will determine the potential for this molecule as a target for the treatment of non-healing diabetic ulcers. Ultimately, this project is aimed at further elucidating the molecular mechanisms that govern growth factor availability in non-healing wounds and may lead to the identification of novel targets for clinical therapy.

There has recently been significant progress in the field of fetal gene therapy, as evidenced by recent success with in utero gene delivery in monogenetic animal models of congenital blindness, hemophilia B and various other metabolic diseases. These studies have made real the potential of fetal gene therapy as a means of preempting disease onset and exacting cure in utero. To date, however, there has not been a study of fetal gene therapy for the treatment of craniofacial disease. Congenital anomalies such as craniosynostosis and orofacial clefting carry a lifelong burden for those affected, often necessitating multiple surgeries, corrective procedures, and years of speech and developmental therapy. The great promise, then, of applying fetal gene therapy to craniofacial anomalies lies in its potential to prevent such anomalies, thereby eliminating the associated burden of care. TGF-b3 is the major temporal and spatial regulator of palatogenesis, orchestrating fusion of the palatal shelves. In turn, the TGF-b3 (-/-) mouse model displays 100% phenotypic expression of cleft palate. In this study, we propose the rescue of cleft palate in TGF-b3 (-/-) mice by in utero gene delivery of TGF-b3 as a first means of assessing fetal gene therapy treatment for craniofacial disease. Our first aim involves characterization of the transduction kinetics and levels of viral vector in the developing palatal shelves. Using an organ culture system, we will compare gene delivery of GFP tagged adeno- and lentiviruses to the midline epithelial edge of the palatal shelves, in order to determine the better vector for the rescue experiment. The vector with the highest and most rapid level of induction will be chosen for the second aim, to test the hypothesis that in utero delivery of TGF-b3 can rescue cleft palate in the TGF-b3 (-/-) mice. The TGF-b3 viral vector will be injected into the amniotic sac to restore TGF-b3 expression in E12-15 palatal epithelium of TGF-b3 (-/-) embryos, harvested at E19/E20, and analyzed by stereomicroscopic and immunohistochemical analysis for gene transduction and rescue of cleft palate.

Over 2 million limb amputees need prosthesis or reconstruction in the United States each year (Langer R, Science 1993). Initial concerns regarding the risk-benefit ratio of non-life-sustaining transplants such as composite tissue allograft (CTA) as a reconstructive option have been partially circumvented by the initial results of human hand transplantations. Chronic rejection (CR) is the most significant cause for solid organ loss following transplantation burdening the healthcare with $300,000 per re-transplant per organ (Kasiske B, JAMA 2000). Besides the myriad of side effects, current immunosuppressants cannot prevent CR. A similar occurrence in CTA cannot be ignored. However, there is no data or description of CR in a CT A. Due to this, the current clinical hand transplant patients are being monitored with great caution. With current lack of knowledge of CR in CT A, it would not be possible to predict the long-term outcome of such transplants precluding their routine use for reconstruction. In this study, we propose to create the first model of CR in CTA enabling the precise recognition of mechanism and pathology of CR. Following this; we would assess safe, drug-based strategies to prevent CR that would fuel preclinical studies before being implemented in clinical CTA.

Tendons are made up of densely packed connective tissues that are organized to transmit the high forces between muscle and bone. They are a complex tissue composed of fibroblasts and parallel fibrils of collagen that interact with an extracellular matrix (ECM). The ECM is composed of many proteins, glycosaminoglycans, and proteoglycans that control the assembly of the collagen fibrils and contribute to the formation of the tissue hierarchy inherent to its functional properties. Tissues such as tendon, cornea and cartilage are ideal candidates for three dimensional tissue engineering due to the relative avascular environments in which they exist. Due to the lack of requirement of a vascular supply, several researchers have begun to investigate tendon engineering in vitro. By inducing tendon fibroblasts to secrete and generate their own ECM, members of our lab have had success with the in vitro development of scaffold free tendon constructs. These constructs, although consisting of the prime ingredients present in native tendon, demonstrate significantly decreased tensile strength and collagen content when compared to their natural counterparts. We hypothesize that by 'conditioning' these constructs manufactured in vitro within an in vivo environment, that they will improve both their structural and mechanical properties, thus making them better candidates for use as three dimensional tissue replacement analogues.

Patients with muscle loss and/or localized paralysis could benefit from tissue-engineered skeletal muscle. Using an in vivo model of directed angiogenesis in the rat, we have previously engineered vascularized, neurotized skeletal muscle flaps that exhibit structural and functional characteristics of native muscle, and have the potential for microsurgical transfer on a dedicated vascular pedicle. Being able to influence the phenotypic development (slow- or fast-twitch) of a developing muscle construct would be highly desirable, in order to engineer a specific muscle type for a specific deficit. It is well-known that specific neural innervation patterns are responsible for directing skeletal muscle fiber type development. Therefore, we hypothesize that differential neural input can direct the fiber type of tissue engineered skeletal muscle constructs.

The project described below aims to characterize the functional adaptation of osteoblastic cells in response to fluid shear stress. Specifically, we intend to demonstrate that osteoblasts form meaningful networks in 3D dynamic culture - i.e., they communicate with other cells via podocytic extensions and gap-junction formation. We hypothesize that gap-junction intercellular communication (GJIC) is necessary for cellular growth and proliferation, and that abrogation of gap junctions will prevent osteoblasts from responding normally to stimulatory fluid shear forces. In order to test the central hypothesis, we propose the following specific aims: 1. To characterize the structure and function of osteoblastic gap junctions in 3D dynamic cell culture 2. To study the effect of increasing fluid shear force on cellular proliferation, function, and formation of gap junctions 3. To determine the role of gap junctions in effecting the osteoblastic functional adaptation to fluid shear force

Congenital abnormalities, trauma and cancer can cause deformities that are functionally debilitating and unsightly. The technical and financial constraints of autogenous tissue transplantation used in craniofacial reconstruction call for alternative methods of reconstruction. An ideal technique would be to completely avoid the need to sacrifice healthy tissue by instead bioengineering replacement tissue. To date, in vitro tissue engineering has been widely successful at culturing a variety of tissues, including skin, fat, cartilage, and bone. However, in vivo applicability of these technologies has not progressed as anticipated, suffering mainly from limitations in tissue viability. For example, bioengineered tissues, once implanted, can only survive in minute volumes due to limitations of chemotransportation (oxygen delivery and waste removal) by diffusion. Thus, the only way to ensure viability of a bioengineered tissue construct is to create a reliable, dedicated blood supply. Since EPCs and OPCs create vascularized bone in vivo, we speculate that these cell populations can work in vitro to recapitulate ontogeny. The central hypothesis to be tested in this proposal is that EPCs and OPCs seeded together on a scaffold can be guided by fluid-shear forces to form vascularized bone.

Native bone is a living tissue composed of multiple cell types that are organized in such a way as to optimize cellular communication and ensure adequate chemotransportation necessary for cell survival. This project will study the relationship between osteo-progenitor cells (OPCs) and endothelial progenitor cells (EPCs) during simultaneous bone and blood vessel formation. To this end, OPCs, EPCs and mesenchymal stem cells (MSCs) will be seeded on a 3D scaffold and cultured under the influence of fluid-shear forces in a flow-perfusion culture system. We hypothesize that, by simulating the in vivo microenvironment, a 3D dynamic culture system can induce EPCs, OPCs and MSCs to form mature bony constructs with a dedicated microvascular network.

The proposed study will utilize the only existing congenital cleft palate model that can be repaired in utero, which has been developed and characterized over the past ten years. The long-term objectives of this study are: (1) to elucidate the mechanism of cleft palate development in humans; (2) to improve the clinical management of cleft palate and treatment of resultant secondary midface hypoplasia; and (3) develop tissue-engineered gene and cell based therapies to treat patients with cleft palate; and 4) to prevent cleft palate development in humans. The specific aims of this study are: (1) to assess the bone-producing potential of palatal shelf mucoperiosteum (lining) following in utero cleft repair in a caprine model; and (2) to evaluate and compare midfacial growth and palatal development following in utero surgical repair and after in utero BMP gene therapy. To accomplish these goals, gene therapy with BMP will be compared to surgical repair of in utero cleft palate in a caprine model. Our hypothesis is that the composite DNA plasmid and biodegradable delivery system with rhBMP-4 will promote bone regeneration equivalent if not superior to that after cleft palate repair in a fetal caprine model.

The purpose of our project is to develop a clinically feasible protocol that harnesses the unique immune privilege (IP) of skin appendages in limb composite tissue allografts (CIA's) and establishes tolerance while obviating need for prolonged immunosuppression. Currently, major tissue reconstruction is associated with poor functional aesthetic outcomes and significant morbidity/high expense of revision surgery - issues that can potentially be avoided by utilizing CIA's. CIA are inherently unique from solid organ transplants due to presence of multiple tissues. The skin component of CIA's is highly immunogenic due to potent antigen presenting cells called Langerhan's cells (LC). In clinical CIA's like hand transplants, this has mandated higher doses of immunosuppression to avoid skin rejection and graft loss. Recent evidence suggests that skin adnexae like the growing (anagen) hair follicles (HF) exhibit IP whereby they are protected from immune attack Keratinocytes (KC's) of anagen HF's are Class I neg and are not attacked by LC's of skin. KC's also secrete immunosuppressive cytokines that maintain the anagen state. Further, the CD200 molecule on KC's upon binding to its receptor (CD200R) on LC's, suppresses LC maturation and antigen presenting ability. Our overall hypothesis is that generating and maintaining IP in the skin of limb CIA and promoting critical molecular interactions between LC's and KC's will down-regulate LC maturation, suppress antigen presentation and promote a tolerogeneic milieu without requirement for prolonged immunosuppression.

Although composite tissue allotransplantation (eTA) is of major current interest for reconstruction a variety of defects, there is an urgent need for minimizing toxicity of the required immunosuppressive treatment. The purpose of this project is to develop a novel immunosuppressive regime specifically designed for eTA. The immunosuppressive protocol described here shall prevent acute as well as chronic rejection while avoiding the severe side effects associated with high dose long-term systemic immunosuppression currently used for clinical hand transplantation. The effect of local immunosuppression in the skin as the component representing the primary target for rejection in composite tissue allotransplantation is addressed. Our goal is the inhibition of lymphocyte infiltration and not for inactivation of previously infiltrated lymphocytes while avoiding an immunosuppressive "overkill" and interference with the known tolerogenic effect resulting from the continuous antigen presentation to the recipient of a composite tissue allograft. For this purpose, two novel inhibitors of lymphocyte migration (Efomycine M and B~15-42) have been selected and will be studied for their efficacy in prevention of rejection in experimental models for vascularized skin and limb transplantation. Both compounds act on lymphocyte adhesion, rolling or transmigration. Inhibition of lymphocyte migration into the skin shall ultimately allow for profound reduction or avoidance of systemically applied long term immunosuppression in composite tissue transplantation involving the skin. Along these lines, inhibition of lymphocyte trafficking will be combined with a steroid free immunosuppressive protocol using a depletional polyclonal antibody (ALG) followed by very low dose monotherapy with Tacrolimus for 50 days. This approach provides a clinically oriented protocol for immunosuppression with minimal toxicity in hand transplantation and any other type of eTA that includes the skin.

Non-healing wounds and ulcers are a major cause of morbidity and mortality in diabetic patients. It is well know that diabetics exhibit impaired neovascularization and thus are at risk for the formation of diabetic ulcers and non healing wounds. Despite our understanding of this critical relationship, there currently is no effective pro-angiogenic therapy for the treatment of diabetic wounds. Recently, there have been advances in the field of vascular biology that offer new insights into mechanisms of neovascularization, and stem cell therapy has been proposed as a possible technique for augmenting diabetic revascularization. Unfortunately, stem cell therapy may confer less benefit to diabetic patients, as recent literature indicates that diabetic progenitor cells are dysfunctional. The objective of this study is to locate the dysfunction in the diabetic Endothelial Progenitor Cells (EPCs) and to test whether ex vivo expansion of diabetic EPCs restores vasculogenic potential in vitro and in vivo.

Periosteal grafts can aid in fracture repair and promote healing of bone defects by providing bone progenitor cells and acting as a barrier to scar tissue. Unfortunately, these grafts have many of the same disadvantages as bone grafts (i.e. donor site morbidity and limited donor sites). We hypothesize that a tissue engineered construct composed of acellular human dermis (Alloderm) and bone progenitor cells would provide a periosteum-like material with the potential to deliver cells and growth factors thus promoting osseous regeneration.

Nerve grafting is the proposed current solution to overcome peripheral nerve deficits. An alternative to this "classical" strategy is the use of synthetic conduits. The purpose of this experimental project is to identify if the combination of a sub immunosuppressive dose of FK 506 and a synthetic tube can enhance nerve regeneration especially in cases of long nerve gaps (above 4 cm). If this is bearded out, then future clinical applications of this combination for patients with severe nerve injuries could be extensive. We will use two different 4 cm synthetic tubes, a polycaprolactone tube (Neurolac ®) and a collagen tube (Integra TM) in rats which receive FK 506. These will be compared with regeneration achieved through the same tubes in rats which do not receive FK 506. In two more groups a saphenous nerve graft will be used in bridging a 4 cm nerve gap: one group will receive FK 506 and the other will not receive it. As it is critical to test regeneration across a distance of 4 cm, a cross-chest nerve regeneration model is established in the rodent using the contralateral C7 nerve root as the motor donor for musculocutaneous nerve neurotization.

Regeneration of human tissue from muitipotential stem cells is a promising option for the treatment of a wide array of diseases. The mesenchymal stem cells derived from human adipose tissue have the potential to differentiate into a variety of mature tissues including muscle, bone, nerve and fat. While there has been rapid expansion in the understanding of stem cell induction there remains little knowledge of the factors which govern adipose derived stem cells (ADSC's) interaction with the surrounding extracellular matrix (ECM). Furthermore, the plasticity of these interactions as stem cells differentiate is not known. The purpose of this focused study is to investigate the role that transmembrane proteins, known as integrins, play in ADSC attachment and migration. We hypothesize that integrin expression in these cells is dynamic, and will vary dependent on degree and direction of differentiation. Moreover, the timing and specificity of integrin expression will govern critical interactions with the extracelular environement, specifically, ECM dependent cell migration and attachment. This knowledge would contribute to the efforts to design targeted delivery of ADSC's to wounded tissue. To date, there has been little understanding of the expression and role of integrins in human ADSC' s.

Cosmetic body contouring surgery is one of the most rapidly growing fields within plastic surgery. In addition, large numbers of bariatric surgery patients are electing to have body contouring following massive weight loss. In both cosmetic and post-bariatric surgery patients, body shape deformities can have a significant negative impact on a patient's physical and mental well-being. Body contouring procedures have the potential to improve or restore body image and health-related quality of life (QOL). Demonstration of the potential benefits of body contouring procedures requires careful evaluation of all surgery-related outcomes from the patients' perspective. Thus, well-developed, psychometrically sound, patient-reported outcome measures (PROMs) are needed. We therefore seek to develop a new PROM that can be used to measure patient satisfaction and QOL in patients who undergo cosmetic or post-bariatric body contouring surgery. For this study, we have assembled a research team of QOL experts with expertise in questionnaire development and plastic surgeons with clinical expertise in the treatment of body-contouring patients. We propose to complete the qualitative component of the questionnaire development process - formation of a conceptual model and item generation. The conceptual model and preliminary versions of the questionnaire will be developed from three sources: 1) In-depth qualitative interviews with approximately 50 patients who have had or are waiting to have body-contouring procedures of the abdomen, back, arms, and legs; 2) our systematic review of the body-contouring literature(already performed); and 3) expert panel including plastic surgeons and QOL researchers. Once completed, a PROM for body contouring will provide surgeons and patients with high-quality data on surgical outcomes. This will lead to improvements in surgical techniques, patient selection, patient education and advocacy.

African-American women are significantly less likely to undergo breast reconstruction following mastectomy than their Caucasian counterparts. Whereas lower utilization rates had previously been attributed to disparities in insurance coverage for breast reconstruction, utilization patterns remain unchanged since the adoption of the Women's Health and Cancer Rights Act (Alderman 2006). Few studies have attempted to identify potential causes for existing disparities in utilization, or examined in depth the reasons African-American women have for electing to have or not have reconstruction. Researchers have suggested that the cultural value of the breast may differ across ethnic groups (Alderman 2003), a plausible hypothesis given findings that African-American women may construe body and beauty ideals differently than Caucasian women (Rubin 2004). On the other hand, Morrow et al. (2005) found that African-American patients receiving mastectomy were more likely to report that the procedure was not recommended or was discouraged by their physician, or that they did not know enough about reconstruction. Further research is needed to understand African-American women's decision-making regarding breast reconstruction. To this end, we are proposing a qualitative study that will provide information about the reasons African American women have for choosing to receive or not to receive breast reconstruction. Through semistructured patient interviews, this study will explore factors influencing women's decision-making, including the role of cultural factors that shape women's surgical preferences, as well as their access to information and referrals. African-American women who have recently undergone mastectomy will be recruited in equal numbers from Memorial Sloan-Kettering Cancer Center (MSKCC) and the Queens Cancer Center (QCC). We plan to interview 50 women (25 MSKCC - 10 reconstruction, 15 non-reconstruction; 25 QCC - 10 reconstruction, 15 non-reconstruction). We will over-sample women who have had mastectomy without reconstruction, as our primary goal is to understand the under-utilization of these procedures. Interviews will be taped and transcribed, and data analysis will be conducted using grounded theory methodology. Results will be used to inform larger-scale quantitative studies of women's decision-making regarding breast reconstruction.

The overriding goal of breast reconstruction is to satisfy the patient's expectations with respect to her body image and quality of life (Sarwer, 1998). Studies have revealed a strong correlation between expectations and satisfaction, suggesting that unrecognized or unfulfilled expectations are associated with patient ratings of poor outcome and dissatisfaction with the result of surgery (Mancuso, 2003). Understanding individual patient expectations would allow surgeons to identify patients who have unrealistic expectations and to address their issues preoperatively. Despite this, no instruments have been developed to accurately measure patient expectations about breast reconstruction. Thus, the objective of this project is to develop a new patient-reported measure of expectations that will serve as a preoperative assessment tool. It will provide patients with a way to specifically indicate what they anticipate from surgery, and provide surgeons with a template to guide discussion about realistic and unrealistic goals for individual patients. Measure development will be conducted in accordance with an internationally recognized sequence of steps recommended by quality of life experts and psychometricians (Medical Outcomes Trust, 2002). Breast reconstruction patients will be recruited from MSKCC for questionnaire development, field-testing and psychometric evaluation. Preliminary versions of the scale will be developed from three sources: literature review, patient interviews and expert opinion. Semi-structured interviews (n=30) with open-ended questions will be performed by a trained interviewer to generate categories of expectations. The item pool will be pretested on a small sample of patients (n=20) to clarify ambiguities, confirm appropriateness, determine acceptability and determine completion time. The instrument will be field-tested in a sample of 150 preoperative patients to determine items that demonstrate the best psychometric properties. This questionnaire will be clinically useful and will aid surgeons as they endeavor to better understand and to fulfill their patients' expectations. Within a broader program of research, this new instrument will facilitate future studies to examine the interface between expectations and satisfaction and to help identify patients at risk for dissatisfaction. This knowledge will also guide the establishment of new educational materials specifically designed to address and modify expectations.

The objective of this study is to develop and validate a patient-reported measure of satisfaction with facial appearance for head and neck cancer patients. The intent is to develop a short questionnaire that can be easily incorporated in routine practice to measure patient outcomes. The instrument will offer a means to evaluate postoperative satisfaction with appearance from a patient perspective and will aid research directed towards the following specific aims: 1. Assessing differences in patient satisfaction comparing various reconstructive techniques 2. Identification of patient groups at risk for poor outcome and dissatisfaction 3. Investigating the relationship between facial appearance and quality of life outcomes 4. Examining the extent to which education and psychosocial interventions may influence satisfaction with appearance and specific quality of life outcomes.

Ischemia-reperfusion injury (IRI) is an inevitable complication of many surgical and microsurgical procedures. Previous work in our laboratory has shown that when delivered prior to ischemic insult, hydrogen sulfide (H2S) has a protective effect against IRI both in vitro and in vivo. We propose subsequent studies that will continue our in vivo evaluation, with the iterative advancements being an improved method of drug delivery (intravenous systemic administration) and the implementation of an additional in vivo model involving intestinal IRI. Furthermore, we will explore whether a similar protective effect is observed when H2S is delivered after the ischemic insult has occurred. This question is especially relevant because if the protective effect of H2S is as great when delivered post-ischemia as it is when delivered pre-ischemia, then the potential applications of H2S broaden to include the treatment of all ischemic tissue, not just "prophylaxis" against the unavoidable IRI that occurs during free tissue transfer. Finally, we intend to elucidate the intracellular biochemical pathways that underlie the protective effects of H2S.

Despite the ubiquity of microvascular free tissue transfer, a major limitation and source of morbidity in these procedures is related to reperfusion injury of the transferred tissue. Currently, the only accepted way to minimize ischemic injury is by rapid re-establishment of blood flow within the flap. Although some authors have reported improved tolerance to ischemia by rapidly cooling the flap during the ischemic interval, this practice is not universally accepted. Even the swiftest of microsurgical transfers exposes the flap to nearly one hour of ischemia time. Although the flap tissues generally "tolerate" this ischemic interval, even this limited period contributes to significant post-operative edema, especially in the most ischemia-sensitive tissues such as muscle and intestine. In more difficulty microsurgical transfers, longer periods of ischemia time may result in significant reperfusion injury ultimately leading to partial flap loss or flap failure. We propose a unique approach to significantly improving flap tolerance to ischemia by inducing a "suspended animation" like state within the flap tissue by exposure to H2S, which can bind to cytochrome c oxidase and reversibly interrupt the process of oxidative phosphorylation. If we are successful in this endeavor then we will be able to significantly reduce the morbidity associated with ischemia reperfusion injury. Furthermore, this technique of cytoprotection may be applied beyond the field of microsurgery into other medical disciplines where ischemia reperfusion injury remains a significant obstacle to progress (e.g. transplant surgery, aneurysm repair, etc.).

Numerous studies in other surgical disciplines have demonstrated that the most important predictor of patient satisfaction is patients' preoperative expectations. Thus, in order to maximize patient satisfaction with postmastectomy breast reconstruction. Surgeons must be able to improve patients' understanding of what may realistically be expected from surgery. Existing educational tools for breast reconstruction patients focus on reconstructive techniques and potential complications, but do not typically address patients' expectations. By focusing on concerns that are important to the surgeon, current teaching fails to address those issues that are most significant to the patient. For example, unilateral implant reconstruction patients may be disconcerted to discover that breast symmetry may only be achieved when wearing a bra. Similarly, a patient may look forward to nipple reconstruction without realizing that sensation will not be restored. On the other hand, educational materials and teaching modules that respond to patient-derived expectations can uncover problems that have been overlooked or considered irrelevant by the surgeon. Thus, educational programs must instead address patient-derived expectations and include issues that are important to both breast reconstruction patients and their surgeons. The objective of this project is thus to: 1. Develop an interactive, web-based, multimedia educational program to address patient expectations about breast reconstruction; and, 2. Pilot the interactive program on a representative sample of preoperative breast reconstruction patients to clarify ambiguities and ensure acceptability. Future NCI grant funding will be sought to formally evaluate the program's efficacy in addressing patient expectations and, ultimately, improving patient satisfaction with postmastectomy reconstruction.

The routine use of AlloDerm (Life Cell, N.J.) in tissue expander/implant (TE/I) reconstruction is advocated by some. Proponents suggest that the use of the implantable dermal matrix affords multiple advantages when compared to traditional techniques. Firstly, by using AlloDerm in the creation of the infero-Iateral expander pocket, elevation of the serratus fascia/muscle is avoided. It is hypothesized that the pain and sensory morbidity experienced due to the surgical disruption and subsequent expansion of the lateral intercostal nerves is therefore minimized (Spear 2005; 2006). Secondly, because of the pliability of the dermal matrix, it is suggested that the rate of postoperative expansion can be accelerated. Thirdly, by facilitating expansion in the lower pole of the breast, it is theorized that a breast(s) with greater ptosis and more natural contours can be created. While the proponents of AlloDerm encourage its use, however, there is a paucity of scientific data which supports its clinical effectiveness in the setting of TE/l reconstruction. Before a practice is established as routine care, it is imperative that it is tested under controlled conditions to generate evidence for its efficacy. The high cost of AlloDerm and the widespread use of tissue expander/implant reconstruction in postmastectomy patients underscore the need to objectively evaluate its effectiveness. A double-blind, multicenter, randomized, controlled-trial is designed. Patients who elect to undergo immediate TE/I reconstruction will be considered eligible for participation. Women will be randomized to undergo expander placement with or without the use of AlloDerm. In patients who are randomized to receive AlloDerm, the dermal matrix will be used to create the infero-Iateral expander pocket thereby eliminating the elevation of the serratus musculature/fascia. Patients and evaluators will remain blinded to the treatment received. The primary endpoint of this study will be patient-reported pain and sensory morbidity. The secondary endpoint will be the rate of post-operative tissue expansion. Tertiary outcomes will include long-term patient satisfaction and quality of life, capsular contracture grade and overall aesthetic results.

Our overall goal is to understand the molecular bases of bone healing. Bone diseases and injuries, such as tumors and fractures, pose formidable threats to the public health and clinical care for these diseases often results in enormous cost on our health care system. Thus, understanding the molecular mechanisms of bone formation would likely lead to the development of novel therapies for these diseases. With that in mind, we are interested in the molecular biology of bone formation and regeneration. Clinical efforts over the years have been directed towards replacing bone made absent by tumor resection, trauma, infection, or congenital malformation. Trauma or cancer resection, for example, can lead to large (critical-sized) defects of bone from the cranium (skull), mandible (lower jaw) or maxilla (upper jaw), necessitating major reconstructive efforts. Bone replacement has involved materials such as autogenous or cadaveric bone graft, demineralized bone matrix, and other bone substitutes. Large defects from trauma or cancer resection, however, require almost a limitless source of bone, as autogenous graft donor sites (skull, hip, or rib) can be readily exhausted and result in serious morbidity. Large quantities of alloplastic (synthetic) material, in turn, are relatively contraindicated in cancer reconstruction, because a well vascularized recipient bed is required to accept such materials. In post-oncologic cases, which often involve radiation, a poorly vascularized recipient site is not suitable for synthetic replacement. An attractive modality for bone regeneration and healing bony defects involves the use of pluripotent mesenchymal stem cells. Such cells can be engineered to differentiate into bone forming cells; however, long-term viability and replication of engineered cells are essential to stable and successful regeneration of missing tissue. In other words, these cells must be optimized for longevity to replicate, differentiate, and deliver critical growth factors for defect healing. Engineering of these cells with particular gene products that extend the lifespan of this cell population, such as human telomerase (hTER1), may potentiate their capacity to heal critical-sized bony defects. Such research may benefit our knowledge of bone regeneration and approach to trauma and cancer reconstruction in humans.

Autologous fat represents perhaps the ideal filler for use in aesthetic surgery. It is natural, abundant, easily harvested, and potentially permanent. In 1980, liposuction presented plastic surgeons with a fatty solution that could be easily harvested and refined for re-injection. Since that time, interest in fat grafting has grown exponentially. However, results to date have been conflicting and equivocal. Despite these results, fat grafting continues to be used for breast augmentation, facial and hand rejuvenation, scar revision, lip augmentation, buttock augmentation, as well as for a host of other aesthetic cases. Further studies of harvesting, processing, and lipoinfiltration techniques are needed to better understand the mechanisms underlying long term fat graft survival. Increased knowledge of this subject will help to improve aesthetic outcomes and will potentially decrease patient morbidity by limiting the number of procedures needed to reach a desired appearance.

Ischemic damage to tissue is a common problem for plastic surgeons. Ionizing radiation damage is one of the many etiologies of ischemic injury. Recent clinical studies and case reports have observed dramatic improvement in clinical appearance of ischemic wounds that have been treated with human lipoaspirate. The use of adipose derived substrates for tissue repair and regeneration has been a rapidly evolving field over the past two decades. The concept that adipose tissue contains a host of metabolically active cells within the stromal compartment was first described in 1968, with subsequent studies noting the regenerative and differentiation capacity of these cells. The revolution in stem cell technology has reshaped the scientific landscape identifying the possibility that stem cells can be harvested from within the stromal component of the adipose compartment and used for tissue repair, gene therapy and tissue engineering. Clinical experience continues to grow while the molecular mechanisms involved in this process remain unknown. Our lab has an extensive background developing and using animal wound models to study ischemic and radiation damage. We propose utilizing our well established animal models to examine the potential of lipoaspirate to treat ischemic and irradiated wounds and tissue. While much clinical evidence suggests a beneficial effect of lipoaspirate in the treatment of these problems, the data remains sparse and anecdotal. Once the model has been well established we will examine and elucidate the specific mechanisms and cellular components accounting for the clinical improvement. Our specific aims include altering our established murine model to adapt it to study the effects of human lipoaspirate injections on ischemic and irradiated tissue beds. We will accomplish this through a nude mouse model and will use the well described Coleman technique to harvest lipoaspirate. We will analyze the histological changes in the tissue as well as angiogenic and inflammatory mediators. All experiments will be compared to controls. The potential clinical impact of this project is far reaching. Radiation and ischemic damage are common problems facing the plastic surgeon. The animal model will enable us to study and elucidate the beneficial role of lipoaspirate, which then could translate to an easily accessible treatment that plastic surgeons in the community could employ in their every day practice.

Activating mutations in FGFR2 have been identified as the cause of several craniofacial anomalies, including Crouzon, Pfeiffer, and Apert syndromes (Cohen 2000, Wilkie and Morriss-Kay 2(01). Most mutations underlying Crouzon and Pfeiffer syndromes involve gain or loss of a cysteine residue in the alternatively spliced domain of the FGFR2c protein; the most common of these mutations is FGFR2C342Y (Wilkie and Morriss-Kay 2001, Ornitz and Itoh 2(01). Recently a mouse model with an Fgfr2c342Y mutation has been constructed (Eswarakumar et al., 2004), and my project involves an in depth analysis of craniofacial development in this mouse. Like the human cases, Fgfr2c342Y mice are viable and have premature fusion of the cranial sutures, proptotic eyes, shortened midface, and high arched or cleft palate. Fgfr2c342YIC342Y mice, which die shortly after birth, show a more severe Crouzon-like phenotype with rounder cranium, shorter nasomaxillary region, and cleft palate. Anomalies of the trachea, lungs, axial skeleton, and joints are found. The aim of my research is to understand the role of Fgfr2c in craniofacial development through investigating 3 developmental processes in the Fgfr2c342Y mouse model: (1) Palatogenesis and the etiology of cleft palate; (2) Tracheal ring development and etiology of the cartilaginous sleeve anomaly; (3) Skull growth and cranial suture development.

The overall purpose of this study is to investigate the effect of carbamylated erythropoietin (CEPO) for its effect on nerve regeneration and function after peripheral nerve injury and delayed repair. Erythropoietin has been shown to profoundly improve nerve regeneration after injury in the central and peripheral nervous system, however, its hematopoietic potential poses the risk of unwanted thrombotic events. We hypothesize, that CEPO - a derivative of erythropoietin devoid of hematopoietic potential - will provide a beneficial profile of peripheral nerve regeneration after injury and delayed repair. More specifically, we investigate the effect of CEPO on the quality and velocity of nerve regeneration when the nerve endings are repaired with a delay of 3, 7 and 14 days after nerve injury.

Clinical studies demonstrate that 10-11 % of all abdominal wall closures fail to heal, leading to incisional hernia formation. This failure to heal results in approximately 200,000 incisional hernia repairs performed each year in the United States.2 Despite technically adequate repairs, recurrent incisional hernia formation is common. Prospective randomized studies that compared the outcomes of suture and mesh herniorraphy found that the incidence of recurrent incisional hernia formation was 43% after standard suture repair and decreased to 23% with implant of a mesh prosthesis. Despite increased prosthesis utilization with hernia repair in the modem clinical era, mesh has an increased risk of serious complications and significant recurrence rates. Synthetic materials to date still have inadequate incorporation, remodeling, and long-term stability often leading to late mechanical failure. Biocompatible acellularized materials such as Alloderm have recently become popular but hold many disadvantages such as immunogenic reactions, infectious transmissions, slow remodeling, and variable success. Due to the limited intrinsic capabilities of "conventional" prostheses and synthetic material, we propose using acellularized myofascial (AM) constructs tissue engineered in our own lab for abdominal wall reconstruction and incisional hernia repair. Tissue prostheses should mimic "native" abdominal wall physiology that is have high compliance, low stiffness, submaximal breaking strength, and high biocompatibility7. The purpose of this study is to develop and characterize a new scaffold based on AM construct material seeded with autologous dermal fibroblasts in cell culture thus tissue engineering of a "new" abdominal wall construct. In theory, fibroblasts in the short term might improve extracellular matrix deposition and local cell recruitment and in the long term they would influence scaffold resorption and remodeling. This study will determine the physical and biological properties of AM constructs in comparison to mesh, will formally evaluate their utility for incisional hernia repair, and will establish a rational basis directed at developing AM constructs for future large-animal and clinical studies. We propose that AM constructs with fibroblasts have BOTH 1) better mechanical compliance matching to native abdominal wall AND 2) allow cellular ingrowth. The primary outcome variable for this experiment is the hernia recurrence rate. In this proposal, patented acellularization methods are employed and allografts are tissue engineered from rat abdominal walls to decrease immune reactions. The technique allows preservation of the collagenous matrix to maintain long term mechanical strength allowing repopulation and remodeling of the grafts by ingrowing cells. We use an established rat "giant" incisional hernia model8 in which the laparotomies reliably progress to form incisional hernias. When used as a tissue prosthesis for incisional hernias, acellularized myofascial (AM) constructs impregnated with fibroblasts result in reduced recurrence rates compared to alloplastic mesh or AM constructs alone.

The use of Mandibular Distraction Osteogenesis (MDO) as a reconstructive option for deformations secondary to irradiated bone could have immense therapeutic ramifications. Currently free tissue transfer is the preferred reconstructive option. These complex operations entail attendant complications that can often lead to delays in initiation of therapy jeopardizing prognosis as well as quality of life.(Hidalgo) MDO generates replacement of deficient tissue from local substrate and therefore would seemingly be an attractive alternative to the use of free tissue transfer. Radiation drastically impairs bone healing, however potentially precluding the utilization of MDO as a durable reconstructive method for head and neck cancer. Although a small number of anecdotal clinical cases have been reported utilizing MDO following XRT (Raghoebar, Taub) the results have been mixed, lacking objective measures of analysis to critically determine outcomes. The main goal of this proposal is to analyze the injury in the adult mandible following radiation, using micro-CT and histology, in a rat model. Several studies have described that radiation injury may cause minimal increases in bone mineral density (BMD) (Chen, Marguiles). This puzzling increase is at odds with the myriad of bony morbidities reported after radiation, such as pathologic fractures, non-union, and osteoradionecrosis (Brown, Marx). Our hypothesis is that although radiation injury induces increased BMD, the new bone formed is of less quality. Quantitative measure of this decrease in quality will allow us to both formulate therapeutic interventions and to measure outcomes in order to enhance DO, potentially allowing clinical use in the future.

Nerve injuries are a complex and challenging surgical problem. Though a variety of causative factors lead to such peripheral nerve injuries including trauma and obstetric complications, the ongoing military operations in the Middle East and Afghanistan have and will continue to lead to numerous nerve injuries. The treatment of nerve injuries can be approached via several therapeutic measures. The success of nerve autografts is known, but also the limitations. For instance, the nerve harvest locations may be inaccessible by concomitant injuries, such as thermal disruption due to blast injuries. Nerve allografts are of unlimited supply but have the requirement and risk of systemic immunosuppression. Recently, decellularized nerve allografts have become an alternative to the two prior techniques, as they are easily available and the decellularization process reduces the immunogenicity of the grafts. Our group performed a comparative study of a decellularized allograft in a rat sciatic nerve model and found that the endoneurial conduits remained intact, and laminin was present in the decellularized grafts. While axonal regeneration was not equivalent to an isograft, the rat decellularized allograft performed better than the most commonly used commercially available nerve conduit at both 14 mm and 28 mm gap lengths (Neuragen, Integra, Inc.). We hypothesize that the addition of a motor specific neurotrophic factor, GDNF, to the decellularized allograft will enhance axonal regeneration and improve end organ function to the level of an isograft. It has been shown that GDNF has a significant long-term survival effect on motoneurons in the setting of nerve injury and models of motoneuron disease. As the need for an "off the shelf'" alternative to autografting becomes more apparent, especially in the light of our war victims, the findings of this study could certainly translate to improved clinical treatment of peripheral nerve injuries.

Nonspecific immunosuppression is the only established method currently available for sustaining a hand transplant that des not differ fundamentally from immunosuppression for organ transplantations. However, the requirement of lifetime immunosuppression to prevent composite tissue allotransplantation (CTA) rejection and the toxicity of immunosuppressive drugs has impeded the widespread clinical application. These complications can be particularly troublesome in CTA recipients who do not suffer from a life-threatening disease. This makes the decision for a risky chronic immunosuppression ethically debatable, and the ideal solution would be to get reliable protocols for inducing CTA tolerance. Recently emerged importance of regulatory T cells (Treg) in allograft tolerance has lead to the concept that the balance between allospecific T effectors and Treg is crucial to tolerance induction. Enhanced allospecific Treg function has been reported to be necessary to induce allograft tolerance in experimental tolerogenic regimens. Thus, vigorous evaluation of the impact of conventional immunosuppressive drugs on Treg activation, proliferation and function is critical to develop new strategies for CTA tolerance. Cyclosporine (CsA) is a major contributor to improved short-term allograft outcome. However, CsA blocks interleukin-2 (IL-2) transcription and consequent IL-2 dependent regulation, we hypothesize that the addition of IL-2/Fc fusion protein, a long-lasting form of IL-2, to widely used clinic strategy of depletion and CsA will induce immunomodulation and promote hind-limb allograft tolerance. In deed our prelimanary data indicate IL-2/Fc plus anti-lymphcyte serum and CsA permit long-term hind-limb allograft survival. In this project we will i) further define the mechanisms by which this novel strategy promotes CTA tolerance; ii) develop a clinic relevant immune monitoring system for rejection and tolerance; iii) optimize the treatment protocol to induce CTA tolerance.

In this study we will concomitantly compare the inhibition/reduction of TGFâ and fibroblast contraction by 5-Fluorouracil, Methotrexate, Paclitaxel, Tamoxifen, Imiquimod, Mitomycin-C, and Bleomycin using fibroblast-populated collagen lattices (FPCLs) and TGFâ immunoassays. The presumptive mechanism of reduced fibroblast contraction is inhibition of transforming growth factor beta (specifically TGFâ1 and/or TGFâ2) produced by scar fibroblasts from scar-forming and skin fibrosing disease processes. Decreasing or inhibiting the effects of these TGFâ at the scar site will decrease contraction of fibroblasts and thus collagen, leading to a decreased overall scar formation. The extent to which these agents decrease/inhibit TGFâ and fibroblast/collagen contraction is not well defined. This experiment will utilize FPCLs to investigate fibroblast contraction inhibition by each of the listed antiproliferative/antimetabolite agents, and will employ the supernatants from these lattices to determine levels of TGFâ1 and TGFâ2 present following exposure. It will be determined which agents lead to the greatest decrease in TGFâ1 and TGFâ2 and fibroblast contraction of abnormal scars (keloid, hypertrophic scar) and skin fibrosing conditions (Dupuytren's contracture, rhinophyma). Data may be utilized to determine the likelihood of successful treatment of scarring/cutaneous fibrosing diseases with the test substances, or as a prelude to clinical trial. Improved function, range of motion, and cosmesis are some of the positive clinical effects of less scarring. Data from these investigations also has the potential to help reduce the number of surgical wound failures with subsequent re-operation, in the end, reducing healthcare cost. Determining which agent is superior at down-regulating TGFâ and decreasing fibroblast contraction will direct future research.

The goal of this proposal is to study how localized mechanical forces stimulate wound healing with special focus on the cell mediators of local wound angiogenesis. The project will have two focuses each taking approximately six months to accomplish. Since transient hypoxia stimulates angiogenesis, which is necessary for tissue regeneration, the currently used continuous tissue stretching by the Vacuum Assisted Closure (VAC) system may not be the optimal mechanical force to induce wound healing. The first six months will focus on using a servo-controlled stepping motor to alter the waveform, amplitude and frequency of mechanical force applied to a mouse model wound and monitor through immunohistochemistry and biochemical methods which variation of mechanic force leads to more robust angiogenesis. After determining the ideal mechanical force to apply to the mouse wound, the second six months will apply this mechanical force on eNOS and VEGF isoform-specific knockout mice and evaluate whether the absence of eNOS or the angiogenic isoform of VEGF will affect angiogenesis induced by stretch when compared to wild-type mice. This will determine whether VEGF and hypoxia induced eNOS are critical in stretch-induced angiogenesis. Through these simple experiments, we hope to clarify both the impact of continued versus waveform mechanical force on a wound and how mechanical forces interact with the angiogenesis pathway critical to sustain wound healing in order to further improve the VAC system for wound healing.

The long-term goals of our research are to understand and control the process of postoperative adhesion formation and to develop a transplantable tendon using host-derived adult stem cells in order to overcome the two major clinical problems in tendon surgery today: postoperative adhesions and lack of suitable graft material. Aim 1: Our first goal is to investigate the role of the highly conserved Notch signaling pathway in the context of tendon healing. Knowledge about the molecular mechanisms of adhesion formation is growing constantly, but so far research has focused on growth factors and signaling pathways that are related to wound healing. Notch signaling has been associated with cell cycle control, differentiation and migration of cells in various cell lines. In contrast to the previous approaches studied in tendon healing and engineering, the pathway is depending on cell-to-cell interaction rather than signal transduction by soluble ligands. The involvement of the Notch signaling pathway in collagen production and remodelling will be first investigated in vitro in rabbit derived tendon and tendon sheath cells. Then the role of the pathway will be characterized and ultimately modulated after tendon surgery in a rabbit model in vivo. Aim 2: We will characterize the role of Notch signaling in the process of tendon differentiation of human adipose derived stem cells. Although there are reports on successful engineering of tendon and tendon-like tissue, the knowledge about molecular mechanisms in tendon differentiation is scarce. As there is increasing evidence that Notch signaling contributes to the maintenance and proliferation of adult stem cells. we hypothesize that differentiation of stem cells into tenocytes. We will study notch signaling in bioreactor that induces differentiation of stem cells to tenocytes by mechanical stimulation. Our research might contribute to develop new strategies to overcome the major clinical problems of tendon surgery today.

Our objective is to investigate the effectiveness and clinical use of rhBMP-2 to augment the repair of primary alveolar cleft defects. With the use of presurgical nasoalveolar molding (NAM), gingivoperiosteoplasty (OPP) may be done effectively with limited dissection so there is not detrimental effect on maxillary growth and 60% of patients subsequently do not require an alveolar bone graft. Bone morphogenic protein-2 is clinically available and may offer improved healing in these cases. Our hypothesis is that rhBMP-2 used in combination with NAM and OPP will improve bony healing across the alveolar cleft and further reduce the need for secondary bone grafting. Our first aim is to compare alveolar cleft healing with and without BMP-2 in a rat alveolar cleft model using a OPP with histologic and radiographic bone density assessment. Our second objective is to evaluate midface growth after primary alveolar cleft defect repair with BMP-2 in a rat model using serial cephalograms. Our final objective is to assess clinical bone healing in newborns after NAM and OPP with BMP-2 using serial radiographic studies. Our strategy of employing the osteoinductive properties of BMP-2 may improve clinical outcomes.

Obese patients struggle with impaired wound healing. Thousands of obese patients' postoperative courses are complicated by wound infection, seroma, dehiscence, delayed closure and necrosis. Despite the large numbers of patients affected, no substantive research has been performed to examine the etiologies of impaired wound healing in obesity. We and others have shown that wound healing defects in diabetics are due in part to impaired endothelial progenitor cell (EPC) function. EPCs migrate to wounded tissue, where they create 35% of new blood vessels formed in a healing wound, therefore their impairment contributes to wound healing dysfunction. We hypothesize that obese patients share similar EPC dysfunction to diabetics, and that this contributes to impaired wound healing. Assays of human endothelial progenitor cell function will be performed in vitro, comparing EPC number and function in obese versus non-obese subjects and controls. Additionally, using a wound healing model established in our laboratory, we will examine the EPC response during wound healing in obese mice versus wild type controls. We hypothesize that EPC function is impaired by suppression of the HIF-1/SDF-1 hypoxic signaling pathway. This signaling cascade serves to recruit and mobilize EPCs from the bone marrow to the bloodstream in response to tissue hypoxia. When dampened it can significantly hinder EPC function. By investigating the role of endothelial progenitor cells during wound healing in obese humans and mice, this study will provide valuable insight into the etiology of impaired wound healing in the obese, as well as open the door for discovery of novel therapeutic options in this population.

The purpose of these experiments is to evaluate the effect of scarring secondary to surgery or radiation (XRT) on lymphatic regeneration. Specifically, we will evaluate the role of transforming growth factor beta 1 (TGF-Bl) expression during this process. Our central hypothesis is that inhibition of local TGF-B I action following surgery and XRT will reduce scarring and improve lymphatic regeneration. TGF-B 1 an important growth factor with numerous functions during wound repair. In addition, a number of studies have demonstrated that TGF-B 1 is an important regulator of scarring following wounding both in tissues that have been irradiated, and those that have not. In a surgical mouse tail model of lymphatic regeneration we have previously shown that increased TGF -B 1 expression correlates temporally and spatially with increased scarring and impaired lymphatic repair. Previous studies have also demonstrated that radiation therapy is associated with tissue fibrosis and a significant increase in the risk of lymphedema. The tissue fibrosis secondary to radiation therapy, similar to scarring, is associated with increased TGF -B 1 expression. Based on these findings, we propose that abrogation of local TGF-BI activity may lead to improved lymphatic repair after surgical excision in both non-irradiated and irradiated tissues. These experiments are highly relevant since they may provide a means to decrease the incidence of lymphedema, a devastating condition with no viable cure.

INTRO: Tissue engineered bone (TEB) holds great clinical promise. Understanding interspecies variation between animal models and humans is essential to translate experimental results from bench to bedside. Using two-and three-dimensional culture models, we studied the species variation of osteogenic and angiogenic marker expression in human and murine osteoblasts and mesenchymal stem cells (MSCs). METHOD: Three human and three murine cell lines were used. Human cells included human mesenchymal stem cells, multi-lineage progenitor cells and normal human osteoblasts. Murine cells included MC3T3, C3H10T1/2 and M2-10B4 cells. Cells were seeded directly on to Poly-Lactide-Glycolic-Acid (PLGA) coated tissue culture plates or three-dimensional PLGA scaffolds, incubated in osteogenic media and harvested at 1, 4 and 7 days. mRNA expression was analyzed with quantitative real-time RT-PCR for osteogenic and angiogenic markers including Alkaline Phosphatase (ALP), Osteocalcin (OCN), Bone Sialoprotein (BSP), Core Binding Factor Alpha-1 (CBFA-1), VEGF and Interleukin-8 (IL-8). Data were analyzed using ANOVA. RESULTS: All human cells had significantly increased expression of osteogenic markers in 3-D compared to 2-D (ALP by 220%, OCN by 323%, BSP by 534%, and CBFA-1 by 357%). However, all murine cells exhibited significant decreases in the expression of osteogenic markers in 3-D compared to 2-D (ALP by 89%, OCN by 64%, BSP by 76%, and CBFA-1 by 73%). In contrast to this pattern of osteogenic markers, all human and murine cells showed markedly elevated expression of angiogenic factors IL-8 and VEGF in 3-D compared to 2-D cultures. IL-8 expression increased significantly by 845% in human cells and 168% murine cells. VEGF expression was increased by 906% and 332% respectively. Measurement of ALP activity confirmed this pattern of osteogenic differentiation. CONCLUSION: While both human and mouse cells showed increased activity in angiogenesis when cultured in 3-D, they exhibited distinct pattern in osteogenesis. Osteogenic differentiation is increased significantly in 3-D human MSCs while decreased in 3-D murine MSCs. Since 3-D cultures represent in vivo conditions more closely, this species variation has important translational implications to TEB research.

Healthcare will be a decisive issue in the 2008 US presidential election. Over the upcoming months, the candidates' visions for future healthcare policies will be discussed, debated and dissected. In a December 2007 poll by the Kaiser Family Foundation, healthcare ranked second on a list of voters' most important issues. It is so important that 21% of Americans named healthcare as the single most important issue in their choice for president in this election.1 In contrast, in 2004, healthcare ranked only fourth among decisive issues, with only 14% of those polled considering it the most important. The issue of healthcare has remained a major concern because restrictions and lack of access to affordable care have eroded the standard of living expected by many Americans. Any discussion of healthcare is likely to touch upon a trio of topics: cost, access and quality. These topics weigh on the minds of healthcare consumers as well. A September 2007 CBS News poll found that 66% of registered voters reported that they were unsatisfied with the quality of healthcare in the US. Another recent poll by the Kaiser Family Foundation found that 80% of respondents were worried about the worsening of the quality of the healthcare services they receive. Furthermore, 81% of Americans reported that they were dissatisfied with the cost of healthcare in the US, up from 62% in 2004. There is much to be dissatisfied about. US healthcare spending is among the highest in the world, averaging $7,026 per person, or $2.1 trillion in 2006 and is growing at a rate of over 6.7% per year. Despite continually increasing expenditures, the US has not enjoyed the quality that should be accompanied by this enormous investment. The stakes are high for various special interests groups to protect their "turf" in this battle for healthcare allocations. These interest groups, which include the government, insurance companies, health maintenance organizations, consumer groups (such as the American Association of Retired Persons), employees of corporations, and ordinary consumers have competing interests that conflict with hospitals and physician organizations in their efforts to extract as much as possible from a fixed pie of healthcare expenditure. The current system of US medical care is based on the free-market economic model in which supply and demand will create a mutually-beneficial market for both buyers and sellers. Noted Princeton Health Economist Dr. Uwe Reinhardt supports this free-market model. He believes that competition in medicine is healthy and has the potential to give consumers the ability to choose among various providers for the highest quality of care. However he also realizes that medicine is a unique field that is influenced by government regulation, consumer norms and market prices. In addition to not holding price in check, the existing US provider reimbursement system does not pay much attention to quality, but bases payment instead on volume and intensity of services provided. As the US moves towards a single payor system like those that have been adopted by many industrialized nations, quality metrics will be instituted to improve efficiency of service delivery by focusing on preventive care measures and minimizing costly complications.

Composite tissue allotransplantation (CTA) is an ideal means of reconstructing devastating traumatic and congenital soft tissue defects. The main obstacle to widespread application of CTA is the potential harm to patients secondary to the use of immunosuppressive agents needed to prevent graft rejection. This problem can be combated by focusing on and incorporating immunomodulatory cell-based therapeutic modalities instead of sole immunosuppressive regimens. Based on prior murine studies, isolation and administration of double negative T regulatory cells has enabled prolonged graft survival in skin and pancreatic islet allotransplants. In this project, double negative T regulatory cells will be used to tip the balance towards immunomodulation and hopefully achieve graft tolerance. The first aim is to stimulate rat CD4 cells thereby enabling them to convert into double negative T regulatory cells. The next aim will be to harvest these cells and administer them to allograft recipient rats of an orthotopic rat hindlimb transplant. This project can have a profound impact on the practice of plastic surgery. By favoring such cell-based methods of immunomodulation, patients will be less threatened by the risks of infection, diabetes, malignancy, etc. associated with current immunosuppression regimens. Furthermore, achieving tolerance in this rat model will elucidate further methods on how to accomplish such a feat in humans.

The 1rf6 gene has been cloned in zebrafish and preliminary expression analysis has been described. We plan to carry out a detailed time-course expression analysis of 1rf6, in the context of other genes involved in vertebrate pharyngeal arch development (such as sonic hedgehog, wnt9b, dlx2, dHand, runx2, Msxl, endothelin-l) using whole mount RNA in situ hybridization. We will also analyze 1116 expression in existing zebrafish pharyngeal arch mutations (such as sucker/endothelin-l) in order to place 1rf6 in a genetic pathway. We hypothesize that 1rf6 has expression pattern overlaps with subsets of cranial neural crest progenitors that express genes involved in neural crest induction and specification (such as dlx2 and MsxI) but is activated later during pharyngeal morphogenesis. We anticipate that 1rf6 expression will be perturbed by zebrafish mutations in genes regulating jaw formation. We will exploit the ability to carry out rapid gene knockdown in zebrafish in order to assess the functional requirement for 1rf6 during zebrafish embryogenesis. We will examine the morphological effects of 1rf6 deficiency by Alcian blue staining of the head cartilages and investigate molecular defects by surveying known markers of the developing jaw (such as dlx-2, and msx genes). From this analysis we should be able to compare the zebrafish craniofacial phenotype caused by 1rf6 deficiency to the known mouse gene-targeting and human mutation phenotypes. Complementary gene disruption approaches are also planned, such as expression of dominant negative 1rf6 mutant proteins by mRNA microinjection and stable transgenics. We hypothesize that 1rf6 is required for proper differentiation of first pharyngeal derivatives. Inactivation of 1rf6 may create a defect in that palatquadrate, a teleost jaw structure analogous to mammalian maxilla. There may also be epidermal differentiation defect, as 1rf6 has been implicated to regulate epithelial-mesenchymal transition in the mouse model.

We intend to evaluate Staphylococcus aureus adhesion and biofilm formation on both synthetic and biologically-derived surgical mesh materials. We hypothesize that S. aureus will adhere more readily to acellular dermal matrix than to synthetic materials. There might also be differences in S. aureus' propensity to adhere to various acellular dermal matrix preparations. Furthermore, we will determine if different substrate material differentially activate S. aureus virulence and biofilm factors. We hypothesize that different subsets of S. aureus genes might be activated upon contact with biotic versus abiotic surfaces and will employ transcriptional profiling to examine this possibility.

Composite Tissue Allotransplantation (CTA) presents a viable option for reconstructing difficult defects resulting from traumatic injury, congenital deformities, and surgical extirpation. The main obstacles to the wide spread application of CTA are overcoming the risks associated with systemic immunosuppression and obtaining consistent and optimal functional outcome. In 2003, W.P. Andrew Lee investigated the functional outcome of nearly all of the hand transplant patients in the world. Motor function was good enough to perform daily tasks in most patients and sensory outcome was inconsistent. One patient described no sensation nearly a year after transplant, while another claimed he had sensation almost immediately, with actual cortical activity on functional MRI. (Lee in press) Similarly, the first face transplant has shown promising results, with reported sensation on her entire allograft, and some cortical activity on functional MRI. Her motor function however has been slower to recover. (Devauchalle 2006) Although these results are promising, the experience thus far has shown variable and inconsistent functional outcome in CT A. Thus, there is still a need for well-designed animal models to explore functional outcome in CT A. My overall goal for my next research year is to study functional outcome in Composite Tissue Allotransplantation using the rat hemi-face transplant model.

Peripheral nerve injuries are frequently seen by plastic surgeons. While it is known that the best result is obtained when those nerve gaps are repaired with auto grafts, auto grafts sometimes may not provide adequate amounts of nerve for reconstruction of long or complex defects (Rodriguez et al. 2000). Tissue-engineered grafts, such as acellularized peripheral nerve grafts, provide a possible solution for reconstruction of gaps less than 2 cm in length (Haase et al. 2003). Unfortunately, no tissue-engineered constructs are efficacious for long (>6 cm) nerve gaps. Peripheral nerve allografts are a potential alternative for reconstruction of long gaps but are limited in their clinical utility due to the need for nonspecific, global immunosuppression. Specifically, the allograft Schwann cells (SCs) behave as antigen-presenting cells leading to cell death and rejection without immunosuppression (Trumble and Shon 2000). Significant attention has been directed towards targeting co-stimulatory pathways as inductive therapy for reducing alloimmune responses and preventing allograft rejection. The CD40-CD40L pathway has received the most academic scrutiny and seems to play a prominent role in T-cell-mediated rejection (Harlan 1999). Blockade of this pathway has been found to produce a permissive state in murine peripheral nerve allografts (Brenner et al. 2004). Strategies to induce tolerance or long-term immunologic hyporesponsiveness following nerve allografting require a better understanding of which SCs populate a nerve allograft in the setting of costimulatory pathway blockade with MRl after nerve-gap reconstruction: donor or recipient SCs. Our hypothesis is that donor nerve SCs do not survive allotransplantation and are rejected if they are not given immunosuppression and that the transplanted nerve graft is repopulated with autogenous or recipient SCs. Under conditions of CD40lCD40L co-stimulatory pathway blockade, there will likely exist a combination of autogenous and donor SCs within the transplanted nerve. If this hypothesis is correct, successful nerve regeneration in long nerve grafts will then require that tolerance or a permissive state must be established in the recipient through co-stimulatory pathway blockade to prevent rejection of the remaining SCs. If the entire allograft ultimately is replaced with autogenous SCs, then only temporary immunosuppression is required until all allograft SCs have been eliminated. Once the source of the SCs is clear, variables can then be manipulated to effect nerve regeneration.

Cartilage possesses limited capacity for repair and self-regeneration. Consequently, injury to cartilage in the articulating joints results in scar formation and possible arthritic changes that can lead to loss of structure and function. Many approaches have been explored over the years to heal damaged cartilage. Traditional therapies include autogenic and allogenic implants in which the former is limited mainly by donor site morbidity, whereas the latter poses a risk for disease transmission. Autologous chondrocyte implantation is a promising therapy but its success is limited by the defect size and location. A new approach is tissue engineering where cells on or in a degradable scaffold can permit new matrix formation specific to the defect site. Previous studies have evidenced the production of extracellular matrix by chondrocytes photoencapsulated in fibrin hydrogels. More recently, we have studied the use of various degradable, photoreactive and crosslinkable polymers as scaffolds for cartilage tissue engineering such as poly (ethylene glycol) (PEG). Based on these results, the hypotheses of this work are: 1) Photochemical crosslinking of PEG can be used to generate a hydrogel that permits cartilage formation by encapsulated chondrocytes and 2) Chondrocytes in PEG hydrogels have the capacity to form neocartilage with improved biochemical and biomechanical properties for cartilage repair. To test these hypotheses, we have designed experiments to address the following aims: Aim 1: To evaluate novel biomimetic hydrogels formed from PEG that permit encapsulation of chondrocytes. This first step will test various concentration ratios of degradable and nondegradable forms of the polymer tailoring the degradation of resulting hydrogels on cell survival in the gels. Aim 2: To employ these photocrosslinked hydrogels to stimulate chondrogenesis by chondrocytes in vivo. These photocrosslinked hydrogels will be used to encapsulate chondrocytes to evaluate their ability to form new cartilage matrix in mice and to validate biocompatibility and their potential for chondrogenesis in vivo.

Regeneration of facial nerve axons following injury critically depends on support provided by the Schwann cells (SCs). Following nerve injury, SCs dedifferentiate, proliferate, and provide support for eventual axonal regrowth. The molecular mechanisms regulating the response of SCs to loss of axonal contact are unclear. Merlin is the product of the nf2 tumor suppressor gene which mediates SC-axon contact information to regulate cell proliferation and survival. Phosphorylation of serine 518 (S518) inactivates the growth suppressive function of merlin, facilitating proliferation. Protein kinase A (PKA) phosphorylates merlin S518 during SC proliferation. We hypothesize that denervation results in merlin inactivation by phosphorylation of serine 518 (S518) by protein kinase A (PKA) and that this is necessary for SC proliferation. Aim 1: Determine whether PKA activity is required in vivo for merlin S518 phosphorylation and FN SC proliferation following axotomy. In preliminary studies, we show that merlin becomes phosphorylated on S518 following axotomy and that this correlates with SC proliferation. We will test the requirement of PKA activity in mediating these responses. Following facial nerve axotomy, rats will be treated with KT5720, a specific PKA, inhibitor delivered via a minipump. Merlin phosphorylation will be assessed by Western blot and immunofluorescence to quantify the SC proliferation in treated vs. control nerves in vivo. Aim 2: Determine if merlin S518 phosphorylation by PKA is necessary for SC proliferation. In preliminary studies, we show that activation of PKA with forskolin (FSK) results in merlin S518 phosphorylation and increases SC proliferation. To establish a causative relationship between PKA-induced merlin S518 phosphorylation and SC proliferation, we will use adenovirus-mediated gene transfer to introduce wild-type or S518-mutated merlin isoforms into primary schwannomas cells which lack merlin. S518A mutations render merlin unable to be phosphorylated while S518D mutations are phosphomimetic. We will compare the percent of proliferating cells expressing the merlin isoforms in the different conditions. Facial nerve regeneration following injury depends on support by SCs. Understanding the molecular events that regulate the response of SCs to injury may lead to novel methods to enhance regeneration. Using in vivo and in vitro models, we endeavor to define the requirement of merlin phosphorylation by PKA in promoting facial nerve SC proliferation following loss of axonal contact.

The creation of a bony defect results in a hypoxic gradient wherein the oxygen tension at the center of the defect is very low. Previously, our lab has demonstrated that osteoblasts exposed to a hypoxic environment alter their expression of cytokines, bone-specific extracellular matrix molecules, and their regulators. Bone morphogenetic protein (BMP) expression has been found to decrease significantly in hypoxia. We hypothesize that the expression of noggin, a BMP inhibitor, is unaltered or increased in hypoxia, thereby altering the noggin to BMP ratio and prohibiting bone formation. Consequently, application of small interfering RNA (siRNA) to suppress noggin may restore BMP activity to normal (normoxic) levels over the long-term, restoring the balance between BMPs and BMP inhibitors and ultimately increasing the rate of new bone formation.

The specific aim of this research is to test the hypothesis that administration of the androgen-receptor blocker, flutamide, to New Zealand White rabbits with delayed onset coronal suture synostosis will inhibit suture fusion and improve craniofacial growth. The rabbits, because they possess susceptible phenotypes, provide a unique opportunity for investigating the relationship between systemic variations in circulating androgen and congenital suture pathology. Research has shown that androgens increase both osteoblast proliferation and TGF-j32 expression, and that androgen receptors are present in both cranial sutures and underlying dura mater. Concerning treatment, individuals with subclinical or clinical levels of excess androgen levels may be at greater risk for post-operative resynostosis following suturectomy, a common clinical complication. With evidence of a relationship between androgen and suture synostosis, research may determine that a molecular intervention is effective. If so, it is conceivable that a suture might be "rescued" using an androgen receptor blocking agent delivered in a slow-release collagen gel in rabbits with post-gestational or delayed-onset coronal suture synostosis.

The peripheral nervous system regulates wound healing during all its phases, i.e., inflammation, proliferation, and remodeling. It has been reported that there is a significant reduction in the inflammatory response to tissue injury as well as slower wound healing in the absence of innervation. Although some studies suggest that neuropeptides released by functional nerves within and around the wound site play an important role during the wound healing process, there is little understanding of the underlying mechanisms. A better understanding would help devise strategies that enhance wound healing when working with free flaps, or when treating extremity injuries with or without neuropathies. We hypothesize that the lack of innervation and associated neuropeptide stimuli down regulates the metabolic processes, such as energy production, protein and DNA synthesis, that are critical for wound healing. We propose to develop an animal model consisting of a tubed superficial epigastric flap where the epigastric branch of the femoral nerve to the flap can be severed at will. Furthermore, these flaps can be perfused ex vivo to obtain a detailed map of the metabolic rates throughout the tissue. Wounds will be created to the flaps and the metabolic response with or without a functional nerve will be compared.

The results of ongoing work by our research group demonstrate over 92% improvement or elimination of migraine headaches triggered from the frontal, temporal, or occipital region. The hypothesis is that the terminal branches of the trigeminal and greater occipital nerves (GON) are irritated by contraction of adjacent muscles, namely the corrugator group, temporalis, and semispinalis capitis, respectively, leading to central sensitization and migraine headaches. The long-term objective of this study is to establish the enduring effectiveness of surgical treatment of migraine headaches, a disabling condition affecting millions of Americans. The specific aim is to demonstrate the efficacy of surgical release of peripheral branches of the trigeminal nerve and GON on migraine headaches through a double-blind, placebo-controlled clinical study for each of the three common trigger sites. Patients who experience migraine headaches, predominantly in one of the trigger sites (frontal, temporal, or occipital), will be examined by neurologists of the research team to confirm the diagnosis of migraine headaches according to the criteria set forth by the International Headache Society. Patients will complete headache questionnaires: Migraine Specific Quality of Life (MSQ) and SF36. A positive response to injection of botulinum toxin type A into the compressing muscles will determine patient eligibility for this study and predict the likely success of surgery. Thirty patients will be included in each group (90 patients in all). Selected on a random basis, 20 patients in the group with frontal trigger sites will undergo removal of the corrugator supercilii, depressor supercilii, and procerus muscles. Twenty patients with temporal trigger sites will undergo detachment of the zygomaticotemporal branch of the trigeminal nerve. In the group with occipital trigger sites, 20 patients will undergo removal of a small portion of the semispinalis capitis muscle surrounding the GON, and the nerve will be shielded with a small subcutaneous flap. The 10 patients in each group who will undergo the placebo surgery will be treated in a similar manner as those of the actual surgery group except that neither the muscles will be removed nor will the nerves be detached. Neither the patients nor the research team analyzing the results will be aware of the assignment. All patients will maintain headache diaries and will complete standardized migraine questionnaires after surgery every third month. After one year of follow-up, the questionnaires will be repeated including the SF36 and MSQ, and the results will be analyzed using standard statistical methods. The patients in the placebo surgery group will have the option to undergo the definitive surgery after serving as a control for one year.

There are an estimated 10.8 million wounds in the United States every year, many of which are chronic and difficult to heal. Wound healing is critical to all plastic surgical interventions and novel strategies, such as micro-mechanical forces to stimulate healing, should lead to better cost-effective therapies. Mechanical forces are commonly used in plastic surgery, yet understanding the optimal application is lacking. We hypothesize that refined mechanical stretch of skin tissue in vivo, induces cell proliferation and angiogenesis as a consequence of structural deformations and hypoxic stress. The optimized mechanical input of stretch on cells, indeed, can be transformed into a chemical mitogenic output that induces protein synthesis and ultimately cell proliferation. Moreover, stretch alters blood flow and determines hypoxic areas that can act as potent inducers of VEGF/NO pathway, leading to a powerful angiogenic effects. Both cell proliferation and angiogenesis are crucial in wound healing and we believe that the study and optimization of two simple triggers will bring the-needed innovation into wound healing management. In the first set of experiments we will try to optimize the mechanical stretch Signal in order to obtain maximal proliferation effects. Using a servo-controlled tension device that allows us to apply precise loads in a predetermined wave-form, we will stretch rat ears following different protocols and study the effects on vasculature (lHC: CD31, CD34, Mecca32) and vascular-related growth factor expression (VEGF). The stretch protocols will include variations in magnitude and intervals of applied forces, as well as, waveform. In the second set of experiments we will try to demonstrate that optimized cyclical stretch determines angiogenesis also by inducing hypoxic stress. We will use the tension device on the back of different type of mice (VEGF -/-, eNOS-/-, db/db, wyld type) in the attempt to show the correlation between cyclical stretch and hypoxia, and the correlations with angiogenesis. We will study the expression of VEGFNEGF-R2 and eNOS with immunohistochemistry and elisa assays. This project aims ultimately to acquire the needed knowledge to design the next generation wound healing device to help surgeons to successfully face the increasing number of complicated wound of everyday practice.

Using recent advances in tissue engineering and the principles behind the prefabrication of flaps, this research will compare various methods of bone formation in a capsular flap. The ultimate goal is to provide a technique to reconstruct bony defects by using a composite tissue flap engineered at a distant location and in a specific shape and then transferred to the site of the defect, thereby minimizing donor site morbidity and avoiding the limitations presented by a finite supply of autogenous bone available for grafting. From previous experiments on prefabrication and prelamination of different cell types, including urothelial cells, tracheal epithelial cells, and chondrocytes, we have found that transposing a vascular pedicle to a subcutaneously placed silicone block will result in a vascular capsule, that can be mobilized and transferred based solely on the pedicle. The capsule provides the necessary blood supply for cell growth. No study to date has investigated osteoinduction in a prefabricated vascularized capsular flap. In a clinical setting, this technique may provide another option in reconstruction of bony defects of the upper and lower extremities and the mandible due to trauma, tumor resection, or congenital abnormalities. The use of the patient's own bone marrow or adipose tissue as sources for cultured mesenchymal stem cells may represent the best option for creating new bone in the prefabrication of flaps without the morbidity associated with the harvesting of autogenous bone graft and may also prove to be more reliable in the osteoinductive process than bone graft substitutes or genetically engineered products.

Currently described regimens based on costimulation blockade for the induction of tolerance have been developed utilizing experimental models of organ transplantation. A nerve allograft differs from an organ allograft because it acts as a temporary conduit to guide nerve regeneration whereas an organ allograft is permanent. Once the host peripheral nerve has regenerated through the allograft and the donor cells have been replaced, immunosuppression is no longer necessary. Anti-CD40 ligand costimulation blocking antibody is able to induce an extended period of immune unresponsiveness without additional dosing following antibody administration. We hypothesize that a single dose of costimulation-blocking antibody(s) is sufficient for successful regeneration in the murine peripheral nerve allograft model. The dosage may be further reduced by combination with additional treatments such as T-cell depletion and donor bone marrow transplantation. Anti-CD40L antibody has been used in clinical trials for bone marrow transplantation and is potentially an essential part of strategies that will be used clinically for organ transplantation. The findings of this study will determine the ability for successful clinical nerve transplantation using lower doses and shorter courses of antibody therapy than are required for organ transplantation.

Cranial sutures are sites of bone deposition in the growing craniofacial skeleton. Through the addition of bone at osteogenic fronts, the cranial sutures eventually fuse (Opperman 2000). However, sutures also assist in the active maintenance of suture patency during development and act as shock absorbers during birth (Moss 1959, Warren 2003). Thus, it is likely that, in response to mechanical strain, the normal cell biology of sutures and the underlying dura represents a balance of both osteoinductive and counter-regulatory signals. The purpose of these experiments is to investigate changes in dural cell biology and intact calvarial sutures caused by tensile strain. More specifically, we propose to investigate: 1) the effect of intermittent tensile strain on the biology of dural cell cultures derived from tissue underlying both fusing and patent calvarial sutures, and 2) changes in the cell biology of normally patent calvarial sutures in response to tensile strain exerted across their structure. In all of these experiments special attention will be paid to both the osteoinductive (increased rates of osteoblast differentiation and increased expression of osteoinductive genes) and counter-regulatory effects (the expression of factors known to restrict osteogenesis) that accompany the exertion of exogenous tensile strain. We hypothesize that the exertion of both intermittent tensile strain on dural cell cultures, and static tensile strain on calvarial sutures in organ culture, results in increased rates of osteoblast differentiation, increased expression of osteoinductive genes, and decreased expression of counter-regulatory signals required to maintain suture patency. A growing body of evidence has implicated various forms of biomechanical force in altered dural cell biology, sutural morphogenesis, and the pathogenesis of craniosynostosis. Clinical reports and experimental evidence suggest that intrauterine constraint (likely resulting in increased intermittent compressive force) may play a causal role in the pathogenesis of craniosynostosis (Kirschner 2002, Hunenk02001). Recent laboratory reports also suggest that static tensile strain produces increased rates of dural cell proliferation, growth factor production (TGFbeta-l, FGF-2), and increased rates of osteoblast differentiation (Fong 2003). Furthermore, a growing body of research suggests that tensile strain exerted upon calvarial sutures in organ culture results in increased rates of osteoblast differentiation, and increased expression of osteoinductive proteins (Ikegame 2001, Yu 2001). Despite these advances, the effect of intermittent tensile strain on dural cell cultures has not been investigated. Furthermore, the role of counter-regulatory factors (Noggin, TGFbeta-3) in maintaining suture patency in response to mechanical perturbation has not been examined.

Options for reconstruction of peripheral nerve gaps are currently limited. In clinical practice, long nerve gaps following injury or tumor resection that cannot be repaired primarily are repaired with autologous nerve grafts. This procedure requires sacrifice of healthy nerve with permanent functional impairment. In some clinical situations, there may not be sufficient autologous nerve available for reconstruction of larger, more complex nerve defects. As a result some investigators and clinicians have turned to peripheral nerve allografting as an alternative. The main obstacle to widespread clinical use of peripheral nerve allografts however has been T cell mediated immune rejection. Although potent immunosuppressive drugs are currently available to prevent graft rejection, they are associated with many toxicities. In addition, chronic global suppression of the immune system with these medications may result in opportunistic infections or secondary malignancies. Therefore more selective immunomodulatory strategies to prevent graft rejection of peripheral nerve allografts such as induction of specific immune tolerance will need to be developed before this technique can be routinely used clinically. The CD40-CD40L costimulatory pathway has been shown to play a crucial role in allograft rejection. CD40 is a 50-kDa membrane glycoprotein found on a variety of antigen-presenting cells of grafted tissue. The ligand for this membrane receptor is CD40L, a 39-kDa glycoprotein that is preferentially expressed on activated CD4+ T helper cells of the recipient. The CD40-CD40L interaction mediates T cell immune responses by enhancing the costimulatory pathway which leads to rejection. Manipulation of the CD40-CD40L co-stimulatory pathway may be beneficial in preventing allograft rejection. Indeed, consistent with its central role in cell mediated immunity, blockade of CD40- CD40L by anti-CD40L monoclonal antibody (mAb) has been shown to prevent rejection of solid organ transplants such as cardiac and renal allografts. However, the effect of blocking the CD40-CD40L pathway on the fate of peripheral nerve allografts has not been determined. The purpose of this project is to determine the effectiveness of anti-CD40L mAb treatment as a method to induce tolerance to peripheral nerve allografts.

Regardless of etiology, a common feature of chronic wounds is tissue ischemia and its sequelae. This is compounded in elderly individuals who have poor tissue perfusion due to peripheral vascular disease or heart disease. Induction of angiogenesis in the ischemic wound bed is critical for tissue repair. Both hypoxia and hyperoxia have been shown to induce angiogenesis, yet the mechanism whereby these two opposing stimuli can produce the same result remains obscure. We will utilize the aortic ring assay to test the hypothesis that both hypoxia and hyperoxia induce oxidative stress, resulting in a series of molecular signals that promote angiogenesis. This ex vivo assay is well established in our laboratory and is well suited for studying the molecular mechanisms of angiogenesis. Upon completion of this proposal we will have identified the molecular pathway(s) used in redox sensitive signaling. Further resolution of the mechanisms of oxygen sensitive signal transduction will lead to strategies, such as gene therapy, to induce angiogenesis in the wound bed.

The successful reconstruction of motor peripheral nerves depends upon the selective reinnervation of motor targets and the receptiveness of the neuromuscular junction to reinnervation. Noting that denervated muscle is receptive to inputs from regenerating axons for up to one year following nerve injury in humans and shorter time periods in rodents, a means of preserving denervated muscle for delayed reinnervation needs to be devised for an acceptable functional outcome to be rendered from primary nerve repair techniques. Otherwise, proximal or latently diagnosed peripheral nerve injuries must be reconstructed with tendon and nerve transfers that necessarily downgrade donor neuromuscular function to recover lost function. Embryonic stem (ES) cells are pluripotential cells that can serve a duel role in our approach to preserving, or "baby-sitting" denervated muscle. Firstly, ES cells provide a source of inducible cells that can be transplanted to replace lost tissue. Secondly, ES cells are an extremely versatile research tool that can be genetically manipulated to upregulate or down regulate numerous proteins and transcription factors and allow us to study the mechanisms involved with muscle innervation and denervation. We hypothesize that ES cells, transplanted near the neuromuscular junction, wiJ1 keep the neuromuscular junction receptive to regenerating motor axons for significantly longer periods compared with experimental subjects not receiving ES cell transplants. From a mechanistic perspective, we hypothesize that denervated muscles and Schwann cel1s wi11 maintain patterns of transcription factor expression that more closely resemble innervated than denervated muscle in the presence of motor neuron-induced ES cell transplants. To test these hypotheses, our goals are to demonstrate that muscle cannot be reinnervated in a mouse model after 3 months of denervation. We will demonstrate this with functional techniques including walking track analysis, immunohistochemical techniques including a-Bungarotoxin and acetyl choline receptor (AChR) staining, and molecular techniques by quantifying the expression of the muscle regulatory factors myogenin, and Id-l and the neurotrophins NT -3 and NT -4. Transplants of ES cells, induced down a motor neuron lineage are then introduced into this denervated mouse model. Transplants of motor neuron-induced ES cells will enable denervated muscle to maintain an innervated phenotype as characterized by our immunohistochemical and molecular markers. Moreover, nerve repair conducted at least 3 months after injury, with latent reinnervation of motor targets will result in significant functional recovery only in subjects pretreated with motor neuron-induced ES cells. Findings from these studies will have applicability not only to proximal or latently diagnosed peripheral nerve injuries, but also injuries to the spinal cord where reconstructive strategies will also require a receptive neuromuscular junction.

A tissue engineered, biocompatible source of small diameter vessels would be of significant clinical benefit to patients requiring vein grafting for free tissue transfer, replantation, lower extremity vascular bypass, or coronary artery bypass. The current practice of autologous vessel harvest is limited by the availability of suitable vessels, operative time, and donor site morbidity. We hypothesize that a chemically acellularized arterial graft seeded with endothelial cells will have mechanical properties suitable for clinical use and remain patent in vivo.

Photochemical tissue bonding (PTB) is a novel tissue repair technique that combines photosensitizing dyes with visible laser light to produce covalent cross-linking of collagen at a molecular level (nanosutures). Unlike previous laser tissue welding techniques that relied on thermal energy to melt and re-anneal collagen, PTB is non-thermal and avoids the tissue damage inherent with a photo-coagulation approach. PTB has been explored in a variety of tissues including cornea, skin and peripheral nerve, and is ideally suited to surgical techniques requiring a watertight seal and precise coaptation without the introduction of foreign material. Although blood vessel repair stands to benefit from these effects, it has yet to be evaluated with PTB as the repair technique. The current gold standard for vascular anastomosis is primary suture repair, which is both time and labor intensive and technically demanding. Furthermore, repeated handling of the vessel wall can damage the endothelial layer promoting thrombus formation or aneurysm. In contrast, PTB repair requires minimal manipulation and can be accomplished with a light dosage lasting approximately 3-6 minutes. For these reasons, we feel that PTB assisted blood vessel repair could provide favorable results in terms of repair quality and procedure time when compared to primary repair using sutures.

Muscle undergoes rapid atrophy after injury to its supplying peripheral nerve, and frequently fibroses before being reached by regenerating motor neurons. After 12 to 18 months, the severe atrophy and fibrotic scarring prevent any chance of recovery of function even after reinnervation. This is particularly problematic for proximal peripheral nerve injuries, where neurons regenerating at a rate of about 1mm/day may take years to cover the distance between the proximal nerve stump and the target muscle. As a result, upper brachial plexus injuries generally lead to permanent weakness or paralysis of the muscles of the shoulder, arm and wrist. We hypothesize that transplantation of neural stem cells into denervated muscle will provide trophic support to the muscle and preserve the capacity for motor recovery. Furthermore, we hypothesize that stem cell derived motor neurons will form motor end-plates with denervated adult muscle fibers. The ability to form appropriate chemical connections with target cells would support the potential future use of neural stem cells to engineer peripheral nerve. We are encouraged by the recent demonstration of muscle innervation and motor end plate formation by transplanted stem cell derived motor neurons in an avian embryo (Wichterle et al. 2002 Cell 110: 385-97). We propose to investigate whether stem cell derived motor neuron progenitors will innervate paralyzed adult muscle in a mammalian model of peripheral nerve injury.

Many research studies have assessed the short term health risks and quality of life associated with breast implants. However, little data exist about long term quality of life after breast augmentation. The Women's Health Initiative (WHI) study provides a unique opportunity to evaluate quality of life of women many years after breast augmentation. This federally funded investigation was established to examine the mortality, morbidity and quality of life in postmenopausal women. Initiated in 1993, 161,000 women aged 50-79 are enrolled in this 15 year study. Comprehensive health and quality of life data have been collected in a highly controlled manner in over 40 clinical centers nationwide and updated annually. Importantly, specific data were collected about breast augmentation history and type of implant. For most WHI subjects, decades have elapsed between breast augmentation and data collection. A preliminary search of the WHI database reveals 2673 participants who underwent breast augmentation. We plan a detailed analysis of the database to accomplish the following specific aims: 1. Assess general health characteristics of subjects with a history of breast augmentation and compare to the rest of the study population with specific attention to cancer, autoimmune diseases, chronic illness, and psychiatric disorders. 2. Evaluate quality of life measures in women with a history of breast augmentation compared with women who did not undergo breast augmentation matched for age and general health characteristics.

Acquired bone deficiencies are a significant clinical problem. Failure to adequately reconstruct these defects may lead to loss of function and impairment of quality of life. Traditionally, small bone defects have been treated with non-vascularized bone grafts. These grafts, however, undergo resorption or may become infected. Larger defects and bone deficiencies in contaminated wounds require alternative reconstructive methods. The use of microsurgical bone transfer has enabled reliable transfer of large segments of bone without resorption or infection in these settings. Unfortunately, vascularized bone flaps are not always available, require significant manipulation to match the defect, and can be a source of donor site morbidity. Tissue engineering may provide an alternative method by which osseous tissues can be synthesized. Recent studies in our lab and by other investigators have demonstrated that osteoblasts can be seeded on synthetic on naturally occurring matrices and that these cells retain their ability to differentiate and mineralize. Although these findings are exciting, numerous limitations of in vitro tissue synthesis must be overcome for successful free tissue transfer. For example, reliable vascular ingrowth must occur in a timely fashion to allow oncologic reconstruction. In addition, in order to, avoid rejection and immunologic complications, the cells used for seeding of the construct should ideally be autogenous or syngeneic cells. Thus, a reliable method by which a rapidly vascularized, autogenous cell seeded osseous constructs can be synthesized would represent a significant advance in bone tissue engineering. The purpose of these experiments is to combine tissue engineering principles with recombinant DNA technology to augment cell adhesion, vascular growth, and bone synthesis.

According to the American Society of Plastic Surgeons, 236,888 women underwent cosmetic breast augmentation surgery in 2002. This represents an increase of approximately 600% since 1992, the year that the Food and Drug Administration, in response to concerns about the safety and efficacy of silicone-gel breast implants, issued a moratorium on their use. Then-FDA Commissioner David Kessler called for further study of the physical safety and psychological benefits of breast implants. Several studies and literature reviews since have suggested that silicone implants are not associated with significant, long-term health problems. Nevertheless, several issues remain unresolved. A limited number of studies have investigated the motivational factors related to the pursuit of cosmetic breast augmentation or the psychological benefits that may occur postoperatively. Similarly, few studies have investigated the relationship of postoperative complications, experienced by approximately 10-25% of women, and psychological outcomes. The proposed study will assess the psychological status of 3 groups of women: 40 women who undergo cosmetic breast augmentation surgery, 40 women who are considering breast augmentation surgery, and 40 physically similar women who undergo other cosmetic procedures. We predict that women interested in cosmetic breast augmentation, as compared to those who undergo other cosmetic procedures, will report greater motivation for and knowledge about breast augmentation. They also will report fewer concerns about the safety of breast implants and report greater awareness and internalization of sociocultural ideals of beauty. Women who undergo cosmetic breast augmentation, as compared to women who do not receive breast implants, will report greater improvements in quality of life, body image, self-esteem as well as relationship and sexual satisfaction postoperatively. The experience of an implant-related complication, however, will be related to decreased levels of satisfaction with the procedure and smaller improvements in psychological status. If confirmed, these results will provide important information on the psychological benefits of breast implants.

Bone marrow stem cells have been found to play a critical role in orchestrating the healing process. The transdifferentiation of multipotent cells has been studied by systemically delivering these cells in various injury models. Recently, an alternative mesenchymal source for progenitor cells has been isolated from adipose tissue. Adipose-derived multipotent mesenchymal cells (AdMMCs) have the capacity to differentiate into various cell lineages; however, their potentially important role in cutaneous wound healing has not been well characterized. In this proposal, we hypothesize that systemically administered AdMMCs will preferentially traffic to skin wound sites and facilitate repair. In Specific Aim #1, the homing pattern of these cells to cutaneous wound sites using a novel in vivo bioluminescence imaging technique will be performed. In Specific Aim #2, utilizing this luminescent modality, we will study the migration of AdMMCs in a mouse injury model by adding specific growth factors to the skin wound site. Engraftment of AdMMCs will be confirmed by immunolocalization, and wound area will be used to quantitate repair rate. We believe AdMMCs are a novel progenitor cell source for clinical transplantation in cutaneous wounds that may enhance wound repair and also be useful for tissue engineering.

Scar formation is the result of uncontrolled wound healing and leads to poor functional recovery after trauma and surgery. In the hand, tendon adhesions formed after flexor tendon injury and repair result in decreased post-operative range of motion and hand function. The costs in terms of disability and inability to return to work: are enormous. The purpose of this proposal is to investigate whether biochemical modulation of transforming growth factor-Beta [TGF-B], a growth factor implicated in scar formation, will decrease adhesions in flexor tendon wound healing. Hypotheses: TGF-B isoforms and TGF-B receptors are differentially expressed in a specific temporal and regional pattern after flexor tendon injury and repair. These expression patterns may be manipulated to modulate flexor tendon wound healing. Natural inhibitors of TGF-B, Decorin and Mannose-6-phosphate, may decrease collagen production in vitro and post-operative adhesions in vivo in models of flexor tendon injury & repair. Quantifiable models of flexor tendon wound healing [in vitro tendon & tendon sheath cultures and in vivo tendon range of motion & tensile strength] can be used to test the anti-scarring candidates Decorin and Mannose-6-phosphate.

The concept of tissue engineering cartilage to generate new tissue is one of the most promising avenues for creating suitable tissue for plastic and reconstructive purposes (Passaretti, Tissue Eng 2001). Using small cartilage biopsy specimens, cartilage cells can be isolated and expanded in culture to generate large numbers for engineering new tissue. Also, chondrogenic cells obtained from bone marrow aspiration (Mesenchymal Stem Cells) can be differentiated into chondrocytes and used as alternative cartilage cell source (Pittenger, Science 1999). By seeding dissociated swine chondrocytes into biodegradable scaffold polymers in vivo, our laboratory and others have shown that it is possible to produce autologous neocartilage that resembles the native cartilage for restoring both form and function (Pannossian, PRS 2001). Although the tissue engineering of cartilage has a full potential of restoring shape and function of cartilage defects, it remains an imperfect approach that has to be improved prior to clinical trials. The overall objective of this study is to evaluate specific aspects of the neocartilage formation process that could ultimately aid in the development of safe, effectual, and predictable cartilage tissue engineering therapies for plastic and reconstructive purposes.

Normal cartilage is immunologically privileged by virtue of the absence of blood and lymphatic vessels. Chondrocytes that are embedded in lacunae within the extracellular matrix obtain nutrients through diffusion from the synovial fluid in the joint cavity but are excluded from interacting with cells of the immune system by their protective extracellular matrix. Under pathologic conditions, cartilage cells are exposed and can interact with lymphocytes and other cells present in the synovial tissue and fluid. For example, human articular cartilage chondrocytes from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) express class II major histocompatibility (MHC) antigens, a potent activator of immune cells. These antigens are not known to be expressed in the absence of pathology, and their presence in RA/OA may underlie an immune response to exposed chondrocytes in these conditions. As the potential for tissue engineering approaches expands for cartilage repair, chondrocytes can be transplanted across syngeneic, allogeneic and xenogeneic barriers. During the process of engineering cartilage, chondrocytes are removed from their native immunoprotective matrix and the cell surface antigens are exposed to immunologic or antigen-presenting cells. This may lead to immune reaction against the naked chondrocytes, which may lead to pathologic conditions like those associated with arthritis. However, little is known about the expression of MHC molecules (class I or II) in engineered cartilage and their propensity to stimulate an immune response by the host. Our laboratory has extensive experience in the field of tissue engineering cartilage using swine chondrocytes obtained from various anatomical sites (e.g. articular joints, ribs, ears, etc.) and encapsulated in biological or synthetic hydrogels. The Transplantation Biology Research Center (TBRC) of the Massachusetts General Hospital has developed a unique population of swine in which the immunogenetics have been mapped in order to perform well-controlled organ and tissue transplantation studies in a large animals. We propose to use these swine and the numerous cell-marker antibodies available to study the immunogenicity of chondrocytes in large animals that will serve as a valuable preclinical cartilage repair model.

Outcomes for pediatric reconstructive surgeries are measured over decades and subject to a higher level of activity and addition than in adults (Eppley 2001). Current tissue engineered products such as Integra have acknowledged problems: material failure, increased rate of infection, and immune system destruction of foreign material. Furthermore, available bioengineered tissues do not grow or withstand a rapidly changing environment in pediatric patients. Thus, the future of tissue engineering in pediatric reconstructive surgery is to create readily available off the shelf bioengineered products that will satisfy the need for expansive growth. Human tissues possess distinctive biomechanical properties based on unique cell composition and extracellular matrix in three dimensional arrangements. Knowledge of 3-D Dimensional matrix of normal and scar tissue is limited. Furthermore, the mechanical stimulation on scar remodeling and biomechanical response of tissues to external forces are not well understood. Therefore, an optimal design of engineered tissue implants requires detailed understanding of the spatially varying requirements of tissue function in order to produce implants with appropriate response. Currently, the bioengineered products are a result of a trial-and-error process due to the lack of predictive mechanistic models. Therefore, this project aims develop and critically validate rigorous micromechanic models for structure/property relationships in engineered tissues based on investigations of mechanical driving forces on fibroblast function and activity within engineered tissues. We will also examine the mechanical functioning and structure of normal human tissue and hypertrophic scar tissue. The results of these investigations will provide a tissue engineer with the knowledge to design the optimal combination of structural and biochemical performance that will be applicable to pediatric patients. Within the given time frame of one year, the main goal for this project is to investigate the roles of biomechanical stimulation and structure characterization of normal and hypertrophic human skin and bioengineered tissues called fibroblast populated tissue analog (FPTA).

This study has clinical relevance to the management of all forms of traumatic peripheral nerve injury. Optimizing care of patients with peripheral nerve injury is an ongoing challenge for reconstructive surgeons and scientists alike. FK-506 (tacrolimus) has emerged as a prototype for future candidate therapies. Treatment with FK-506 results in enhanced nerve regeneration and accelerated functional recovery. Unfortunately, due to FK-506's potent immunosuppressive properties and significant toxicity profile, it is not acceptable to use this drug solely to promote nerve regeneration. It would therefore be highly desirable to isolate the neuroregenerative effects of FK-506. To do so requires a better understanding of the mechanism of action of this drug in regenerating nerve. The immediate goal of this study is to use cDNA microarrays to identify genes that are upregulated in injured nerve after treatment with FK-506. The experiment has been carefully designed to control for effects on genes that are influenced by immunosuppression, thereby enriching for those genes specifically involved in nerve regeneration. This study is a key step towards the long-term goal of developing therapeutic drugs that exploit FK-506's beneficial effects on injured nerve.

The purpose of this study is to investigate the role of cell cycle arrest and inhibition of apoptosis as the mechanisms of cytoprotection in radiation-induced craniofacial bone growth inhibition through in vitro methodology. Specifically, cultured osteoblasts obtained from newborn rabbit calvaria will undergo a single dose orthovoltage radiation and will be used to study the expression of important bone markers, cell cycle arrest and apoptosis with and without cytoprotection. We hypothesize that: (1) inhibition of craniofacial bone growth following radiation occurs at the level of osteoblasts in a dose-dependent way; (2) the level of apoptosis in osteoblasts is significantly elevated following radiation; and (3) cytoprotection is exerted through cell cycle arrest and inhibition of apoptosis. Our objectives are to: (1) test the dose-dependent effect of radiation on osteoblast proliferation and function using cultured rabbit calvarial cells; (2) investigate the mechanisms of radiation injury to osteoblasts based upon (a) basic osteoblast parameters (proliferation, alkaline phosphotase activity, collagen type I and mineralization); (b) apoptosis and (c) cell cycle analysis; and (3) investigate the effectiveness of cytoprotection in this model using Amifostine (WR -2721) and its active metabolite (WR-I065) as probes. This study is clinically relevant since craniofacial bone growth inhibition is a consequence of radiotherapy in over 90 % of survivors of pediatric head and neck cancers, leading to severe functional and cosmetic deformities. Reconstruction of these conditions is challenging at best or impossible to achieve with conventional surgical techniques in the worst circumstances. Cytoprotection seems to be a viable strategy in the prevention of radiation-induced craniofacial bone growth inhibition in children.

Ischemia and reperfusion (I/R) in skeletal muscle is unavoidable in many vascular and musculoskeletal traumas, in disease, and during vascular and musculoskeletal reconstructive surgeries. It can result in significant complications including muscle dysfunction and necrosis. Protecting tissue injury from I/R has been one of the goals for many years, but except for the early restoration of blood flow to the area at risk, there is little else that can be used as a protective intervention. Our recent study has shown that local intra-arterial infusion of vascular endothelial growth factor (VEGF) protein into natural blood flow of cremaster muscle during reperfusion after 4-hr warm ischemia provided a significant microvascular protection in the cremaster muscle and promoted mRNA expression of eNOS, but not inducible NOS (iNOS) and neuron NOS (nNOS) in the cremaster muscle. However, this protection was blocked by a non-selective nitric oxide synthase (NOS) inhibitor suggesting VEGF-induced microvascular protection is endothelial NOS (eNOS) dependent. It is unknown how VEGF promoting microvascular protection and eNOS expression. However, what we known is that the activity of VEGF is mainly mediated by two receptors; VEGFR-l and VEGFR-2. Both receptors are expressed mainly in endothelial cells and are essential for angiogenesis. We hypothesize that one of these receptors or both are required for VEGF to promote microvascular protection and eNOS expression. The purpose for present study is to determine the concentration ofVEGFR-1 and VEGFR-2 in the VEGF-treated cremaster muscle and to see whether the blockage of VEGFR-I or VEGFR-2 could eliminate VEGF-induced microvascular protection and eNOS upregulation. By combining physiologic, pharmacological and molecular means, the present proposal explores the microcirculatory mechanism of VEGF intervention in vivo. The potential of clinical application for using VEGF as an intervention approach may hold promise for attenuating I/R injury in the cases like replantation, free flap transfer, organ transplantation and cardiovascular surgeries, etc. More importantly, the elucidation of microvascular mechanisms of VEGF intervention will be of great value in developing future therapeutic strategies and pharmacological manipulations aimed at exploiting this protective mechanism for the treatment of patients with ischemia in the skeletal muscle.

Reconstruction of osseous craniofacial defects often requires bone harvest, incurring significant donor site morbidity, cost and time. Examples include autografting for cleft palate and osteocutaneous free tissue transfer for mandibular defects. Tissue engineering employing gene therapy for bone regeneration would be a highly desirable alternative. One method utilizes bioresorbable polymer scaffolding at the bony defect site containing plasmid DNA of bone growth factors. Previous studies have demonstrated bone growth in the rat calvarial critical-sized defect model with bone morphogenetic protein-4 (BMP-4) plasmid DNA. Other research suggests a synergistic effect of BMP-4 and vascular endothelial growth factor (VEGF) on bone regeneration. Objectives: 1) To confirm bone regeneration with a polymer scaffold containing BMP-4 plasmid DNA in the rat calvarial critical-sized defect model. 2) To quantify bone regeneration with VEGF plasmid DNA in the above model. 3) To investigate a possible synergistic effect of BMP-4 and VEGF plasmid DNA in the above model. Methods: Calvarial critical sized-defects will be created in 110 male Sprague-Dawley rats. Animals will be divided into 5 groups of 22 by defect treatment: none (negative control); VEGF plasmid DNA; BMP-4 plasmid DNA; VEGF and BMP-4 plasmid DNA; and autograft (positive control). Animals will be sacrificed at 4 and 8 weeks (11 animals per group at each time point); calvaria will be radiographed and histologically sectioned. Outcomes: Histomorphometry and radiomorphometry will measure bone growth within calvarial defects as a function of total defect area to quantify bone regeneration effects of BMP-4 and VEGF plasmid DNA alone and in combination.

As the potential for tissue engineering approaches expands for cartilage repair, chondrocytes could be transplanted across syngeneic, allogeneic, and xenogeneic barriers. During the process of engineering cartilage, chondrocytes are removed from their native immunoprotective matrix and the cell surface antigens are exposed to immunologic or antigen-presenting cells. This may lead to immune reaction against the naked chondrocytes, which may lead to pathologic conditions like those associated with arthritis. However, little is known about the expression of MHC molecules (class I or II) in engineered cartilage and their propensity to stimulate an immune response by the host. I propose to use tissue from MGH inbred swine and the numerous cell-marker antibodies available to study the immunogenicity of chondrocytes in large animals that will serve as a valuable preclinical cartilage repair model. I will also isolate chondrocytes from human cartilage in parallel studies.

Congenital microtia not only leaves deep psychological scars on affected children, it is also one of the most challenging pediatric reconstructive problems. It requires precise technique and artistic creativity, and the surgery is unforgiving. Total reconstruction of the external ear currently requires a subcutaneously placed ear-shaped framework of either alloplastic material or autologous costal cartilage to recreate the complex three-dimensional shape of the auricle. Tissue engineering of cartilaginous ear-shaped frameworks has also been attempted; however, the resultant cartilage is small, brittle, and inflexible. Our previously published work has shown that it is possible to engineer flexible neo-cartilage through the use of support materials simulating perichondrium (Xu, 2001). Recently, we combined fresh swine ear chondrocytes with xenogeneic lyophilized perichondrium (XLP) to engineer flexible cartilage in the shape of an adult human ear (unpublished data, see background). The large amount of harvested tissue needed for adequate chondrocyte numbers, however, makes this technique unsuitable as a clinical model. Ideally, a small number of chondrocytes obtained from an auricular or costal cartilage biopsy could be expanded in culture to produce the large numbers of cells needed for engineering an entire ear. In an effort to bring these techniques closer to a clinical scenario, we hypothesize that both auricular and costal chondrocytes, when expanded in culture and combined with either XLP or deep fascia as a pseudoperichondrium can be utilized to produce a flexible, ear-shaped, cartilaginous framework. Alternatively allogeneic or xenogeneic cells could be used.

Chronic muscle atrophy and fibrosis following denervation is a significant limiting factor in the ultimate restoration of limb function following peripheral nerve reconstruction. Muscle that is denervated for a prolonged period of time (> 12-18 months) results in atrophy and irreversible fibrosis. The clinical outcome after peripheral nerve repair in middle to proximal lesions such as brachial plexus injuries is limited. Since the 1970's microsurgical repair has been the standard of care, since that time no new advances have developed to better the quality of motor function following nerve repair. We hypothesize that transplantation of neural stem cells into denervated muscle will provide trophic support to the muscle and improve the capacity for motor recovery. Furthermore, we hypothesize that stem cell derived motor neurons will form motor end-plates with denervated adult muscle fibers. The ability to form appropriate chemical connections with target cells would support the potential future use of neural stem cells to engineer peripheral nerve. We are encouraged by the recent demonstration of muscle innervation and motor end plate formation by transplanted stem cell derived motor neurons in an adult mammalian model of motor nerve injury (Craff et al., submitted for publication). Initial results show that the transplanted cells remained viable and prevented muscle atrophy up to 7 days. We believe that these cells provided the trophic support and stimulus necessary to prevent atrophy of the denervated muscle. In this study we propose a syngeneic transplant model. By changing the recipient animal to mouse we will gain the advantage of using transgenic knockout mice to manipulate specific variables in our future studies.

Macrophages play a key role in wound repair and fibroproliferative diseases by regulating fibroblast proliferation, connective tissue synthesis and angiogenesis. Macrophages produce angiogenic factors, of which Vascular Endothelial Growth Factor (VEGF) is a major component, in an exquisitely magnified manner. It has been shown recently in Dr. Leibovich's lab here at UMDNJ, that VEGF expression is strongly and synergistically regulated by an interaction between Toll-like receptor (TLR) agonists and agonists of adenosine A2A receptors (A2ARs). TLR agonists are products of micro-organisms, such as endotoxin (LPS). Adenosine is produced as a result of hypoxia, ischemia and inflammation. Both receptor classes are strongly expressed on wound macrophages as well as other cell types. To determine whether the synergistic up-regulation of VEGF expression by TLR and adenosine agonists plays a role in wound healing in vivo, we plan to study wound healing in the skin of mice that lack a critical mediator of the TLR signaling pathway, namely MyD88. MyD88 is the immediate intracellular down-stream mediator of TLR signaling, and has been found in Dr. Leibovich's lab to be absolutely required for the synergistic up-regulation of VEGF expression by TLR agonists with adenosine agonists. The proposed studies in MyD88 knockout mice will illuminate the role of this pathway in vivo in skin wound repair. To this end, we will study various parameters of wound healing in full-thickness excisional skin wounds in MyD88- /- versus MyD88+/+ mice. Establishing the relevance of this synergistic pathway to the healing of wounds will pave the way for approaches to modulate and regulate this pathway using pharmacological agents, which will potentially be of major significance for the clinical modulation of wound healing and fibrosis.

Composite tissue allografts (CTA) represent a unique model in the context of transplantation. Increasing interest in the clinical application of CTA transplantation is highlighted by the recent hand transplantation. Similar to organ transplantation, the optimal approach to achieve permanent allografts survival would be the induction of donor-specific tolerance. Successful tolerance induction in the animal model will provide a means for the future development of a clinical protocol. We are interested to identify subsets of dendritic cells (DC) derived from rat bone marrow cells (BM) with distinct phenotype and function that have abilities to regulate T cell responses following hind limb composite tissue transplantation. In addition, these studies will enhance our understanding of rat DC biology in the context of transplantation. Most approaches using DC-induced tolerance have targeted the role of donor DC in the direct pathway of allorecognition and only few studies have addressed the potential for the manipulation of recipient rat DC for tolerance induction. The latter approach is of considerable importance because of the critical role of recipient DC in the indirect pathway of allorecognition that underlies the pathogenesis of chronic vascular rejection. DC are heterogeneous population of bone marrow-derived antigen presenting cells (APC) resident within virtually all tissues. The development of protocols involving recipient DC pulsed with allopeptide in conjunction with transient immunosuppression, make this an attractive and clinically important model. The overall goal of this project is to develop rat DC-based protocols that could be utilized to induce tolerance to the allogeneic hind limb composite tissue transplantation. The hypothesis for this study is that the allopeptide-pulsed recipient DC subsets with distinct phenotype and function are capable of inducing T cell tolerance via induction of regulatory mechanisms. These functions are mediated through the differential ability of DC subsets to secret cytokines as well as the level of costimulatory molecules expression. We will identify conditions that would allow for the generation of rat DC subsets with defined characteristics that provide desired therapeutic effects in the area of CTA transplantation.

Melanoma accounts for 4% of all diagnosed cancers. Whilst early surgical removal may be curative, currently no effective medical therapy exists for disseminated cancer. Using mainly ex vivo approaches, substantial progress has been made in preventing melanoma formation, as well as reducing tumor burden, in various mouse models that exist for the disease. The common principle employed is that of ex vivo transfection of melanoma cells by vectors expressing immunomodulator molecules (e.g. IL-2, IL-12, GM-CSF), followed by vaccination of mice with the irradiated transfected cells either prophylactically or therapeutically. In terms of clinical application, this method suffers from the significant drawback of having to establish a cell line from each individual tumor, losing valuable time in the treatment of the patient concerned. Our goal is to develop a vector based on Herpes Simplex Virus Type-1 (HSV-1) that will express the gene for murine Granulocyte Macrophage Colony Stimulating Factor (mGM-CSF) in transfected cells. The vector will be based on a novel HSV-1 mutant that, due to two gene deletions and addition of dominant-negative mutant polypeptide of HSV-1 UL9 gene, is replication defective, as well as capable of inhibiting Wild-type virus from replicating. This represents a significant safety enhancement over any HSV-1 based vectors for gene delivery to date. The vector will be used both for in vivo and ex vivo approaches of prophylactic and therapeutic vaccination in a mouse melanoma model. If found to be both safe and efficacious, this would present a novel vector for in vivo vaccination as adjuvant therapy in the treatment of cutaneous melanoma.

Adipose tissue engineering is the three-dimensional growth of adipose tissue by a process involving cells, growth factors, and bioresorbable scaffolds. In one method, cells are seeded into a scaffold material and exposed to growth factors. After an incubation period, the scaffold is then implanted to fill a soft tissue defect. The field of tissue engineering has been exploring the development of adipose derived stem cells (ADSCs) to meet the need for adipose tissue augmentation in soft tissue reconstruction. ADSCs are a promising new source of stem cells that may be easily harvested with suction-assisted lipectomy. Gene therapy of ADSCs may become an important tool in sustained expression of desired growth factors in adipose tissue-engineered constructs. Insulin growth factor-1 (lGF-1) is a potent stimulator of adipocyte growth and differentiation that has been used to augment adipose tissue in animal models. IGF-1 gene therapy of ADSCs may provide increased durability to adipose tissue engineered implants. To date, only viral methods of gene therapy have been investigated in ADSCs and are the preferred method. However, the limitations to viral gene therapy include immunogenicity, possible genetic mutation, and social stigma. There are several methods of safe and effective ex vivo non-viral gene therapy. The best known of which is the use of liposomes. The long term objective of this proposal is to evaluate non-viral gene uptake and expression in ADSCs for use in adipose tissue engineering therapies.

The goal of this proposal is to use stem cells with skin substitute transplantation to improve chronic wound healing in diabetes. The hypothesis to be tested is that the performance of marrow-derived progenitor cells in accelerating diabetic wound healing may be enhanced by genetic modification to overproduce a vasculogenic cytokine. The following are the two specific aims of the proposed study: 1. To develop adeno-associated viral (AA V -2) vectors with exogenous vascular endothelial growth factor (VEGF) gene for use in transducing bone marrow progenitor cells. These genetically modified bone marrow derived progenitors will then be seeded into an FDA approved artificial skin substitute. 2. To evaluate effects of skin substitutes loaded with AA V2-VEGF gene modified marrow progenitor cells on promotion of wound healing in chronic wounds in db+/db+ diabetic mice. This work is the extension of our ongoing efforts to promote soft tissue healing with gene transfer of growth factors by extending the target cells to bone marrow progenitors.

The past 30 years have provided us with a great deal of experimental data on neuroscientific elements of nerve repair. Unfortunately this has not translated into improved clinical outcomes. We are still in search of the ideal method of nerve repair. Photochemical tissue bonding is an exciting new method of tissue adhesion that uses visible laser light to create bonds between tissue surfaces that have been pretreated with a photosensitizing dye. This innovative use of laser technology has the potential to revolutionize many aspects of microsurgery. We believe it will prove particularly useful in the area of nerve repair. It offers a sutureless method of repairing this fragile tissue thus minimizing scarring and foreign body reaction. It can provide an immediate seal with subsequent protection of the regenerating axons within their specific environment. Among the most challenging aspects of peripheral nerve surgery is the bridging of the nerve gap. This project seeks to demonstrate that combining photochemical tissue bonding with autologous tissue conduits can enhance neural regeneration across a nerve gap.

Despite the many recent advances in peripheral nerve repair, synthetic nerve guides are still unable to repair long gap peripheral nerve injuries. For spinal cord injuries, the challenges are even greater, due to the progressive destruction of neural tissue and to the inhibitory nature of the spinal cord, which limits regeneration. The proposed study will investigate the use of human muscle-derived stem cells (MDSCs) as a potential cell source for neural tissue repair. The potential of MDSCs to differentiate into neurons will be assessed in a rat transection model of spinal cord injury, whereas the MDSCs' capacity to differentiate into glia will be assessed in both peripheral nerve and spinal cord.

In reconstructive and cosmetic breast surgery, patient satisfaction is the predominant determinant of surgical success. Irrespective of this, few instruments have been developed to accurately measure breast surgery outcomes from a patient perspective. The objective of this project is to develop and validate a new questionnaire that can be used to measure patient satisfaction following breast surgery. The intent is to develop a short questionnaire that can be easily incorporated into routine practice and administered before and after surgery. This questionnaire has the potential to impact strongly on national and international research endeavors. It would provide a reference point for comparison of different studies and various surgical populations. Insight into regional variations in patient satisfaction would be gained that might guide future decisions concerning standards of care. To develop this measure, we will adhere to the following specific sequence of stages recommended by quality of life experts (Fayers, 2000, Streiner, 2001): (1) to define the intended purpose of the survey and develop an exhaustive list of all issues relevant to the topic under investigation; (2) convert these issues into questions that are easily understood and pretest the questionnaire on a small patient sample; and (3) field test the questionnaire on a large heterogeneous sample of patients to select the best indicators of patient satisfaction to form the final instrument (item reduction) and determine acceptability, reliability, validity, and responsiveness. The goal is to include a large group of surgical patients to increase the applicability of the new measure in future outcome research and allow for comparability studies. The study will performed in three sites in the United States and Canada to ensure that differences in language, culture or education do not diminish the reliability or validity of the instrument. The sensitivity of the questionnaire to regional variations will also be investigated.

Bone grafts are the "Gold Standard" for reconstructing bone defects in the craniofacial (CF) skeleton, causing increased morbidity and cost (Tessier, 1982 & Wolfe, 1982). It would be beneficial to design a construct capable of supporting and stimulating bone growth and vascularization, which would be replaced by host bone allowing growth and remodeling. To design such a construct it is crucial to account for local bone strain patterns and soft tissue forces. Knowledge of strain patterns in the CF skeleton would also help elucidate the development of the CF skeleton, and to confirm or re-direct plating techniques, which are mostly based on intuitive, and sometimes mistaken, approaches to the strain patterns. The objectives of the proposed studies are to examine a new biodegradable foam construct as an option for repairing CF bone defects, and to define strain patterns in the human CF skeleton.

Extensive peripheral nerve injury requires reconstruction with interpositional grafts. Nerve autografting has the drawbacks of donor site morbidity and limited nerve material and nerve allografting requires systemic immunosuppression with potential significant side effects (Mackinnon 2001). Alternatives to immunosuppression include manipulation of the patient's immune system to induce tolerance or graft pretreatment to render it less antigenic (Evans 1994, Trumble 2000). Cold preservation of nerve allografts in University of "Wisconsin solution has emerged as a promising strategy for minimizing antigenicity, thereby creating an acellular scaffold that is conducive to nerve regeneration. Schwann cells are the primary antigenic target in nerve grafts (pollard 1981). Increasing duration of cold preservation diminishes the expression of Schwann cell antigens (Atchabahian 1999) and reduces Schwann cell viability (Levi 1994 ) while preserving a scaffold of Schwann cell tubes. Part I of the study investigates the effects of cold preservation on nerve antigenicity. Part II examines how these changes in antigenicity affect nerve regeneration and functional recovery. In Part I of the study, a mouse model is employed to characterize the immune response to cold preservation. Mice will be randomly assigned to 4 groups of 8 animals each and will undergo corresponding inlay sciatic nerve grafting. Peripheral blood, spleen, and nerve material will be harvested on day 10. Cellular immune responses will be quantified using the enzyme-linked immunosorbent spot assay (Elispot). This assay allows for detection of T cell sub-populations in the recipient mice spleens that have been stimulated to produce cytokines (Hutchings 1989). Both interferon gamma secretors, which represent the T helper 1 subset involved in the inflammatory response, and interleukin 4 secretors, which represent the T helper 2 subset involved in regulation of the cellular immune response, will be measured (Benichou, 1999). The humoral immune response will be evaluated by measuring for donor specific antibodies in the recipient animal's serum (Whitley 1989) using flow cytometry (Rodey2000). In Part II of the study, a rat model is used to optimize for functional and histomorphometric analysis (Dellon 1989, Mackinnon 1985). As in Part I, animals will be randomly assigned to 4 groups of 8 animals each. Rats will undergo 2.5 cm posterior tibial nerve grafting according to group. Walking track analysis, a functional assay previously described (Bain 1989), will be performed preoperatively, at 6 weeks and pre-sacrifice. At the 9-week end-point, chosen to maximize differences between groups (Calvert 2001), animals will be sacrificed and the posterior tibial nerve harvested. After tissue processing, cross sections of nerve will be analyzed for cellular infiltration, Wallerian degeneration and quality of nerve regeneration. The immunologic, functional and histomorphometric data generated in this study will provide a more complete understanding of the effects of cold preservation on nerve allografts. These findings will serve as a basis for future studies combining nerve allograft cold preservation with autologous Schwann cell injection in a large animal model. The long-term goal of this research is to provide a safe alternative to immunosuppression-dependent allografting that can be readily applied to human nerve reconstruction .

Reconstructive surgeons are frequently confronted with significant bony defects that result from trauma, ablative surgery, or congenital abnormalities. Current treatment options include the use of autogenous bone grafts or distraction osteogenesis; however, both of these techniques are limited in their application by anatomic considerations and operative and perioperative morbidity. In order to avert these problems, tissue engineering research has sought a biosynthetic bone substitute that could be used to treat critical-sized bony defects. The current experimental paradigm involves the seeding of a biodegradable scaffold with osteoblast precursor cells that will proceed to produce new bony tissue as the scaffold is gradually absorbed after implantation. The majority of scaffolds studied have been constructed from aliphatic polyester polymers such as poly (lactide-co-glycolide) (PLGA) and polycaprolactone (PCL), both of which are in current medical use as suture material and in drug delivery systems. Several studies have successfully grown osteoblasts on these polymer scaffolds with varying degrees of subsequent mineralization; osteoblasts cultured on PLGA display a greater amount of mineralization than osteoblasts cultured on PCL. Since PCL is superior to PLGA as a scaffold material, (it is cheaper and easier to mold into three-dimensional forms), it is our hypothesis that bone morphogenetic protein-2 (BMP-2) that has been covalently bound to PCL will retain its stimulatory effect on bone synthesis and increase both the amount and efficiency of new bone tissue formation. BMP-2 is currently one of the strongest known growth factors that induces the generation of new bone. The specific aim of this project is to determine if BMP-2 can be successfully bound in a covalent manner to PCL, and if so, whether or not it retains its biological activity in influencing osteogenesis. If indeed scaffolds made of PCL with bound BMP-2 display augmented bone formation, then BMP-2 treated PCL may prove to be the material of choice for biosynthetic replacement implants for bony reconstruction.

The hypothesis of this project is that hyperbaric oxygen (HBO) inhibits ischemia-reperfusion (IR) induced neutrophil-I CAM adherence by preventing polarization of expressed surface CD 18 molecules. This HBO induced reduced adherence and polarization in neutrophils is due to a systemic increase in plasma nitric oxide (NO). The effect of HBO on systemic NO production is through up regulation of eNOS. Specific Objectives: To use a new quantitative method to evaluate the percent of CD 18-polarized and adherent neutrophils formed when reacted with Sham and IR plasma. To manipulate the percent ofCDl8-polarized and adherent neutrophils formed by treatment with hyperbaric oxygen a nitric oxide scavenger, and a nitric oxide synthase (NOS) inhibitor. This research should provide insight into the mechanisms of HBO in IR injury, which could significantly impact limb salvage and could have even broader implications in the treatment of IR injury in other organ systems.

Hyperbaric oxygen (HBO) inhibits ischemia-reperfusion (IR) induced neutrophil-ICAM adherence by preventing polarization of expressed surface CD18 molecules through a nitric oxide (NO) mediated pathway. The hypothesis of this project is: 1) to determine if the effect of HBO on systemic NO production is through upregulation of vascular endothelial growth factor (VEGF) mRNA and 2) to determine if the increase in VEGF results in increased activity of NOS. Specific Objectives: 1. To use rt-PCR to evaluate the differences in upregulation of VEGF mRNA between Sham, IR, IR-HBO and Sham-HBO tissue. 2. To use a radioactive assay to evaluate the differences in NOS activity between Sham, IR, IR-HBO and Sham-HBO tissue. 3. To use a monoclonal antibody to block VEGF and repeat the radioactive NOS activity assay to determine if the differences in NOS activity between Sham, IR, IR-HBO and Sham-HBO tissue are affected. This research should provide insight into the mechanisms of HBO in IR injury, which could significantly impact limb salvage and could have even broader implications in the treatment of IR injury in other organ systems.

An ideal soft tissue substitute for reconstructive and aesthetic surgery has still not been identified. Alloplastic implants are prone to complications such as extrusion, infection, and capsular contracture. Autologous fat transplantation will undergo resorption. Tissue flap reconstruction is effective in many cases, but still has inherent donor site morbidity. A durable tissue engineered implant using a patient's own cells may represent a better clinical option. While preadipocytes can be induced to differentiate in-vitro with the use of an adipogenic medium, these cells would not be exposed to the same concentration of growth factors when implanted in-vivo. Therefore, many cells may survive as undifferentiated precursors instead of forming new fat tissue. Our group has developed a novel polymer scaffold that incorporates the adipogenic growth factors from our differentiation medium and releases these factors to the adherent cells during degradation. This would allow implantation of preadipocytes on a scaffold that not only provides structural support, but also stimulates the surrounding cells to an adipocyte phenotype. The polymer is designed so that the release of growth factors during degradation correlates with the time period required for adipocyte differentiation. The purpose of this study is to engineer a soft tissue implant using human preadipocytes seeded on a biodegradable polymer scaffold that integrates growth factors in its chemical structure.

Wound healing complications after oncologic surgery can have devastating consequences. These difficulties may delay adjuvant therapy and impair aesthetics and functional reconstruction. Numerous studies have shown the efficacy of hyperbaric oxygen therapy (HBOT) in the treatment of difficult wounds. For example, HBOT is effective in the treatment of osteoradionecrosis, ischemic flaps, gas gangrene, chronic wounds in diabetics, crush injuries, refractory chronic osteomyelitis, burns, and soft tissue radiation necrosis. However, fear of cancer cell activation or biologic alteration has led to a reluctance to use this therapeutic modality in cancer patients. For these patients, oncologic resections resulting from breast and head and neck malignancies represent the most common defects requiring reconstruction. Therefore, the purpose of these studies is to rigorously assess the effects of HBOT on breast and head and neck cancer cells as well as to determine the safety of this modality in the treatment of difficult wounds in this setting.

Composite tissue allografts (CTAs) are our best option in reconstructing complex tissue defects, including situations of extremity loss. Previous studies have investigated the relationship between dendritic cells (DCs) and the induction of tolerance to cardiac and skin allografts. We propose to further this model, utilizing composite tissues allografts to examine the extent and flexibility of this tolerance regimen. Previous experiments by other authors have shown that the transfer of immunodominant allopeptidepulsed host dendritic cells have tolerized recipient rats to allogeneic solid organ transplants. Garrovillo and colleagues have shown that the intravenous administration of host dendritic cells (DCs) expressing donor MHC molecules have induced tolerance to donor cardiac allografts, in a WF-to-ACI rat combination. We propose to adopt this functioning model and to extend its use to a more stringent histocompatibility barrier, utilizing a WF-to-Lewis rat combination. In addition, we propose the use of a hind-limb transplant model, to examine the protocol's potential for tolerance to the diverse elements of a composite tissue allograft (CTA). If successful, this experiment would represent a significant step towards developing an elegant preclinical model for the eventual use of composite tissue allografts in the reconstruction of complex tissue defects, including situations of traumatic limb loss and amputation.

Currently all popular methods of cleft palate closure rely on the extensive mobilization and transposition of palatal soft tissue flaps in order to close a combined bony and soft tissue defect. Problems associated with this approach include: 1. Extensive scarring along planes of dissection which subsequently restrict facial growth leading to midfacial deformity (Bardach 1990), and 2. A lack of an intact bony palatal arch which leads to unstable dentoalveolar arch form. To minimize these complications related to extensive soft-tissue surgery, closure of cleft palates by sutural distraction osteogenesis has been tested in a canine model (Liu 2000). Successful approximation of the hard palate edges was achieved, however, by a combination of new bone deposition at the palatomaxillary suture (desired) and dentoalveolar arch collapse (undesired). The latter is a complication that limits the clinical feasibility of this approach to cleft palate disease, as it hinders later orthodontic care and significantly deteriorates the final occlusal result. We, however, support the belief that sutural osteodistraction could be a viable therapeutic option pending elimination of associated arch collapse. Our goal is to demonstrate that arch collapse during distraction can be prevented by utilizing a passive maxillary acrylic splint to anchor the alveolus in place while tension forces are applied across the cleft palate. Furthermore, we will investigate the ultrastructural details of distracted palatal suture using state-of-the-art imaging technology (PQ CT and DXA, see below), in addition to standard histological and radiological techniques. Expression of bone morphogenetic proteins (BMPs) and their receptors, known mediators of osteogenesis in non-sutural distraction, will also be characterized temporally and spatially by our basic science collaborator. These new technologies have not previously been used to analyze sutural distraction osteogenesis, and will provide valuable information regarding the structural integrity of generated bone, in addition to a better understanding of the peptide mediators and potential pharmacologic enhancements of this process. In summary, achieving closure of a palatal cleft by suture distraction without arch collapse may have exciting clinical implications for human infant care by minimizing extensive soft tissue surgery. The benefits would include significantly decreased infant surgical morbidity and reduced growth abnormalities contributed to by scarring along surgical planes.

Since the late 1800s, many methods for reconstructing non-ossifying adult calvarial defects have been developed. Yet, the search for the source of donor materials continues. Recent findings have suggested that adipose tissue, capable of in vitro adipogenic, osteogenic, chondrogenic, and neurogenic differentiation, could contain multi potent stromal progenitor cells. These findings opened new possibilities for reconstruction of calvarial defects using tissue engineering and cell-based therapies. While much interest has been generated over the multilineage potential of these adipose-derived mesenchymal stromal cells (ADMSCs), very little is known about the effect of tissue donor age on the biology or function of ADMSCs. We hypothesize that juvenile and adult ADMSCs have different biologic profiles and thus different abilities to re-ossify calvarial defects. In the first Specific Aim, we will determine the differences in the molecular biology of juvenile and adult ADMSCs undergoing osteogenesis differentiation. Alterations in osteogenic gene expression will be assessed using micro array technology and quantitative, real-time RT-PCR. In the Second Specific Aim, we will utilize an established mouse critical size calvarial defect model to determine the ability of juvenile and adult ADMSCs to re-ossify critical size calvarial defects. The research proposed in this application will help to direct future strategies in cell-based therapies for calvarial healing. This will facilitate the development of novel, clinically applicable techniques with which to reconstruct non-healing calvarial defects.