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.

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.

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.

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 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.

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.

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.

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.

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.

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.