INTRODUCTION: Radiation-induced skin fibrosis is one of the main adverse effects of radiation therapy for cancer treatment. Radiation fibrosis syndrome is caused by the overactivation of TGF-B that promotes fibroblast that induces collagenases and breaks down type III collagen and replaces it with type I collagen. The inhibition of P53 and the accumulation of ROS are the main mechanisms of radiation-induced damage. To fully understand these mechanisms, we tested the use of our novel human skin perfusion model to recreate the damage caused by radiation and as a platform to test possible therapeutic and/or prophylactic treatments to prevent fibrosis. METHODS: We use our perfusion model, which consists of a human tissue sample recovered from abdominoplasty. We dissect the superficial inferior epigastric artery and cannulated it and perfuse it with special culture media. On the first day after cannulation, we exposed the skin to a single targeted irradiation dose of 20gy and 40gy and took punch biopsies on days 3, 6, 12, and 16 for histological and gene expression analyses. The histological samples were stained with H&E, and Masson´s Trichrome stain to determine the morphology changes and extracellular matrix deposition respectively. TUNEL and DAPI immunofluorescent staining was performed to analyze for apoptotic changes in the epidermis/dermis. Expression of inflammatory, fibrotic and apoptotic genes was analyzed by real-time quantitative PCR. RESULTS: The morphological changes in the skin were significant. The radiation-exposed skin started peeling compared to the control group. The H&E staining showed an increase of inflammation in the dermis along with epidermis/dermis separation as well as papillary dermis containing fibrin deposition, accumulation of inflammatory cells, reactive changes in the endothelial cells, and abundant necrotic keratinocytes. Masson’s Trichrome staining revealed a increased deposition of extracellular matrix between the papillary and reticular dermis in the irradiated skin. The TUNEL and DAPI stain shows an increase in apoptotic cells in the radiation group that correlates with the damage induced by radiation. Gene expression analyses revealed upregulation of inflammatory and anti-apoptotic genes expression. CONCLUSIONS: Our perfusion model was stable for 19 days and was able to recreate the radiation-induced deposition of collagen shown in the H&E and Masson´s trichrome as well as the radiation-induced damage in the TUNEL and DAPI staining. Our system is reliable and can be used as a platform for developing therapeutics to mitigate the effects of radiation on the skin.

Background: To address the lack of non-cytotoxic, non-surgical options to treat undesirable focal adiposity of the face, we propose use of the anti-glaucoma medication and prostaglandin F2α analogue latanoprost, which has a well-described side effect of periorbital adipose shrinkage. Objective: To evaluate the safety and efficacy of soluble and liposomal latanoprost for focal fat reduction. Approach: To compare efficacy, single administrations of either the FDA-approved cytolytic drug deoxycholic acid (DOCA), latanoprost, or liposomal latanoprost were injected into ob/ob mouse inguinal fat pads. Study outcomes included mouse weight, inguinal fat pad volume, architecture, and cytotoxicity. Results: Both DOCA and soluble latanoprost significantly reduced inguinal fat pad volume whereas liposome encapsulation reduced inguinal fat pad volume insignificantly over the 14-day study period. Hematoxylin and eosin demonstrated effective reduction in adipocyte volume without histologic evidence of cytolysis or inflammation whereas DOCA caused dermal ulcerations, adipocyte lysis, and increased tissue inflammation. Conclusion: Latanoprost reduced fat volume without inducing cell lysis or inflammation.

INTRODUCTION: The main challenge of autologous fat transfer procedures in patients is the requirement of repeat grafting to compensate for the resorbed fat over time. On average about 40-50% of the grafted fat resorb in 3-6 months post grafting requiring repeat graft procedure. Repeat harvest is a painful and expensive procedure exerting a traumatic and financial burden on the patient. In addition, it also led to reduced productivity of the surgeons. The development of strategies that eliminate the requirement of repeat harvest is the need of time. In this direction, we have designed a cryopreservation device and optimized the protocol that enables on-site storage of the excess harvested fat for repeat graft procedures thus eliminating the need for repeat harvest. METHODS: We designed a device that can connect seamlessly to current devices used in fat harvest and grafting. We tested different approaches and methods combinations of cryoprotectant and freezing temperatures, and measured cell viability up to 3 months using viability stains Tryptan blue and Calcin-Am. For in-vivo validation, we used Nu/Nu athymic mice injected with human fat cryopreserved for 7 days, 21 days, 3 months, and 11 months. Each group was compared to a fresh fat graft. We analyzed the graft for weight, volume retention, histology, vacuole formation, and inflammation markers after 9 weeks. Using our method we determined the optimal time range for cryopreserving the fat post-harvest. RESULTS: In vitro viability analyses showed a combination of 10% DMSO, 2% human serum albumin, and storage temperature of -80˚C demonstrated optimal viability of cryopreserved fat comparable to fresh fat. In vivo, Nude mice studies showed no significant changes in the graft weight and volume retention between the comparison groups up to 11 months. The histological scoring index for inflammation and vacuole formation also showed no significant changes. Our time range analyses showed the best outcome when the fat is cryopreserved within 5 hours post-harvest. CONCLUSIONS: This study shows that the clinical adaptation of our device and protocol can reduce multiple harvest sessions along with the complications of this procedure e.g. ecchymosis, swelling, hematoma, and infections. Fat can be preserved without any morphological, weight, or volume changes for up to 1 year.

BACKGROUND: Free tissue transfer stands at the apex of the reconstructive ladder, however, despite decades of surgical innovation, free flaps remain limited by physiologic need for rapid and sustained reperfusion to maintain viability. This need can cause a particular limitation in cases of prolonged ischemia periods and restricted availability of recipient vessels. To overcome this limitation, paradigm shifts towards device-assisted and/or ex vivo perfusion supported flap technology are necessary to expand capabilities of free flap surgery. However, in developing these technologies, there is a need for a replicable, docile, and anatomically relevant large-animal model from which we could easily assess and modify free tissue transfer with external analytic and/or treatment devices. Here we aim to describe the novel ovine model of fasciocutaneous free flap transfer, which we have developed for this purpose. METHODS: Female Suffolk sheep cadavers weighing 50-65 kg were studied to identify a saphenous system-based fasciocutaneous free flap model. Anatomical measurements and photography were performed to standardize flap harvest and feasibility of cadaveric flap perfusion was assessed via ex vivo fluorescein angiography. Following flap identification, the autologous microsurgical transfer of the flap to the neck was performed in a female Hampshire sheep weighing 65 kg. The sheep was followed for 2 weeks with daily photography. Extracellular tissue lactate and glucose levels were characterized during the transfer and weekly with a custom microdialysis probe placed in the flap tissue. Doppler ultrasonography was performed weekly to assess pedicle viability. Fluorescein angiography was used to assess flap viability at the initial surgery as well as the endpoint. Tissues were collected for histology at the endpoint. RESULTS: Saphenous vessels branching off the femoral system were identified to have up to 2.5 mm vein and 2.0 mm artery diameter, with the total pedicle length reaching up to 6 cm. The cadaveric flap tissue demonstrated feasible inflow and outflow with fluorescein angiography. Upon autologous transfer, the transferred flap of 9x6 cm provided complete coverage of the neck defect and demonstrated viability for the entire duration of the experiment. Viability of the free flap was confirmed with doppler ultrasonography and fluorescein angiography. The donor site was covered with a skin graft from the neck with bolster placement. CONCLUSION: We identified a novel model of free flap transfer to serve as a testbed for clinically translatable approaches to improve outcomes in reconstructive microsurgery. Ovine docility, relative to other quadrupeds, improves daily flap accessibility and supports survival. These initial studies serve to set the foundation for the use of complex extracorporeal membrane oxygenation (ECMO)-like devices for enhancing uses of the free flaps.

Abstract Introduction Complex burns are highly morbid injuries that can cause severe disfigurement and can be devastating to one’s quality of life and psychosocial well-being. Currently, there are no simple, single-stage procedures available for extensive or multifocal burns to address full-thickness trilaminar defects. Our team has previously demonstrated the viability of an adipose-first reconstruction to address hypodermal defects. In this study, we demonstrate the efficacy of a combined adipose plus finely minced skin to achieve a single-stage trilaminar skin reconstruction. Methods Full-thickness burns were created on female Yorkshire swine. After 48-hours, escharectomies were performed to the level of fascia. The wounds were layered with adipose harvested from female Yorkshire swine. In one group, autologous split-thickness skin grafts were cut into pixel size (0.3x0.3 mm) grafts and layered on top of the adipose. Pigs were maintained for 4-weeks with weekly photography, ultrasound, followed by endpoint histology and tension measurements. Results At the end of the 4-week period, adipose combined with pixel graft demonstrated improved epithelialization and less contracture (p< 0.01). Thickness and mobility measurements were consistent in both groups. This findings were similar to our previous approaches using adipose following surgical debridement. Tissue pliability in the pixel grafting group was maintained to a high degree. Cross sections were performed which showed the persistence of fat graft at the base of the wounds. Conclusions Immediate, single-stage trilaminar reconstruction of full-thickness complex burns reduces contracture, mitigates adhesion, and restores normal soft-tissue thickness, therefore, presenting a paradigm changing approach in the current practice of burn injuries to the mobile surfaces. Applicability of Research to Practice Our results support the therapeutic potential of adipose-based soft-tissue reconstruction of burns to both enhance hypodermal augmentation and mitigate fibrotic complications including contracture and adhesions. Our data suggest that this approach is compatible with the standard of care skin grafting, and in fact provides improved epidermal thickness and pliability.

Abstract Introduction Complex burns are a challenge, often requiring prolonged reconstruction. Management requires consideration not only to the skin but to underlying structures and often requires staged-reconstruction and revision for increasingly unsatisfactory results. This standard is expensive, limited by donor-site availability, and often impacts quality of life. Here we demonstrate the efficacy of single-staged minced skin grafting in an incubator-like microenvironment with a negative-pressure moist wound chamber device to achieve rapid epithelialization after burns. Methods Full-thickness burns were induced to female Yorkshire swine. Escharectomies were performed to the level of fascia after seventy-two hours. One group received standard of care skin grafting with a bolster dressing. In another group, split-thickness skin was cut into pixel size (0.3x0.3 mm) grafts, followed by application of either bolster or negative-pressure moist wound chamber dressing. Wounds were followed for 4-weeks with serial photography, ultrasound, and biopsies for histology. Results As early as one week, epithelialization started in the group with pixel grafts with negative-pressure moist wound chamber dressings with visible epithelioid islands on the wound bed granulation tissue. This progressed with a similar trend throughout the 4-week period eventually leading to near-complete epithelization and keratinization. There was reconstruction of trilaminar cutaneous architecture demonstrated by the presence of distinct, viable epidermal, dermal and hypodermal elements as well as viability of adipose on histology. Distinct differences in contour were noted between the bolster and negative-pressure moist wound chamber groups. Conclusions Minced grafting minimized donor burden and alleviated the need for graft orientation. In both the bolstered and negative-pressure moist wound chambers, pixel-grafts viably survived to form a viable basal layer. Use of positive-pressure (bolster) vs. negative-pressure moist wound chamber dressings demonstrated distinct differences in the convexity/concavity and topography of the singe-stage skin graft with critical implications for aesthetic reconstruction. Applicability of Research to Practice This study introduces the efficacy of a single-staged trilaminar reconstruction with the use of a negative-pressure moist wound chamber for complex burns. These findings support the potential for a new paradigm in the treatment of complex burns which allows for single-stage reconstruction with minimal donor site morbidity.

Injury to the facial nerve can occur after different etiologies and range from simple transection of the branches to varying degrees of segmental loss. Management depends on the extent of injury and options include primary repair for simple transections and using autografts, allografts, or conduits for larger gaps. Tissue engineering plays an important role to create artificial materials that are able to mimic the nerve itself without extra morbidity in the patients. The use of neurotrophic factors or stem cells inside the conduits or around the repair site is being increasingly studied to enhance neural recovery to a greater extent. Preclinical studies remain the hallmark for development of these novel approaches and translation into clinical practice. This review will focus on preclinical models of repair after facial nerve injury to help researchers establish an appropriate model to quantify recovery and analyze functional outcomes. Different bioengineered materials, including conduits and nerve grafts, will be discussed based on the experimental animals that were used and the defects introduced. Future directions to extend the applications of processed nerve allografts, bioengineered conduits, and cues inside the conduits to induce neural recovery after facial nerve injury will be highlighted. Impact statement Recovery after facial nerve injury is a complex process, which involves different management options such as primary repair or the use of nerve grafts or conduits. Various tissue-engineered approaches are increasingly studied on preclinical models with limited, but promising, translation to the clinical setting. Herein, preclinical models focusing on different recovery methods after facial nerve injury are comprehensively reviewed based on the experimental animals used. The review provides key insights into current developments and future directions on this highly relevant topic to help researchers further expand the field of tissue engineering and facial nerve recovery.

Purpose: Burn and blast injuries to the face and extremities are highly morbid injuries affecting quality of life, ability to work, and psychosocial well-being. Without exception, extensive burn injuries require surgical debridement, with standard of care reconstruction involving autologous skin grafting to restore cutaneous integrity. This treatment modality is limited in extensive burns or in highly visible areas by lack of donor site and/or soft tissue deficits resulting in significant disfigurement. Hypodermal restoration via autologous adipose transplantation provides padding for the overlying skin, helps restore native features, and enhances contour and texture. However, this technique is limited by graft retention and often requires multiple rounds of grafting and consequently, multiple rounds of surgery, each with separate anesthesia, to achieve adequate results. The goal of this study was to demonstrate the therapeutic validity and efficacy of utilizing cryopreserved adipose to avoid multiple liposuction events when serial skin and fat grafting procedures are performed to restore epidermal, dermal, and hypodermal integrity after full-thickness burn. Methods: Adipose was collected from female Yorkshire swine and processed day-of-collection for immediate cryopreservation. This adipose was preserved for 3 months prior to initiation of the next stage of the experiment. After three-month elapse, female Yorkshire swine received 16, 4x4 cm full-thickness burns using an electric brand. After 48 hours, eschar was removed down to fascia. Skin grafts were collected as split-thickness skin grafts. The pigs were maintained for 8 weeks from time of engraftment and interval serum, photography, ultrasound, and biopsies were collected. At 8 weeks post-engraftment animals were sacrificed and all wounds were collected for histology and proteomic evaluation. Results: Split thickness skin graft take was greater than 95% in all injuries. Adipose grafts from Group B, were noted to remain present and incorporated into the granulation tissue in absence of skin graft with viability confirmed on biopsy. Initial increase in granulation layer thickness was noted in presence of fat graft with Group B vs. Group A. On serial ultrasound assessment, penetrating adipose grafts from Group D were noted to be present without gross resorption at all time points. Cryopreserved adipose remained viable throughout the duration of the experiment with histologic evidence of incorporation at 8 weeks post-operative. Conclusion: Burn and blast injuries predominantly affect military personnel and first responders for whom improvement in protective equipment has decreased mortality without resolving the risk of morbidity to the face or extremities. Hypodermal augmentation with lipografting as part of a strategy of autologous skin grafting addresses contour deficits and skin quality, however is limited by the need for multiple liposuction and grafting procedures, requiring multiple trips to the operating room with increased surgical and anesthetic risk and high economic burden. Here we demonstrate cryopreservation of adipose as an avenue to alleviate that burn.