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.

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.

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 stern 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 remodeling 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 stern 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 stern cells. We hypothesize that differentiation of stern cells into tenocytes. We will study notch signaling in bioreactor that induces differentiation of stern cells to tenocytes by mechanical stimulation. Our research might contribute to develop new strategies to overcome the major clinical problems of tendon surgery today.

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.

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 Thyl-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 Thyl-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.

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 auto grafts 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 rum 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.

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-Iox cassette. This trans gene 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.

In this study we will concomitantly compare the inhibition/reduction of TGFfr and fibroblast contraction by 5-Fluorouracil, Methotrexate, Paclitaxel, Tamoxifen, Imiquimod, Mitomycin-C, and Bleomycin using fibroblast-populated collagen lattices (FPCLs) and TGFfr immunoassays. The presumptive mechanism of reduced fibroblast contraction is inhibition of transforming growth factor beta (specifically TGFfrl and/or TGFfr2) produced by scar fibroblasts from scar-forming and skin fibrosing disease processes. Decreasing or inhibiting the effects of these TGFfr 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 TGFfr 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 TGFfrl and TGFfr2 present following exposure. It will be determined which agents lead to the greatest decrease in TGFfrl and TGFfr2 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 TGFfr and decreasing fibroblast contraction will direct future research.

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.

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.