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.

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.

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.

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.

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.

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.

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.

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 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.