Indiana University-Purdue University Indianapolis (IUPUI) / In the US, more than 1 million burn injuries are reported annually. About 45,000 injuries due to fires and burns result in hospitalization and ten percent of these result in death every year. Advances in burn treatment have led to a reduction in mortality rate over the last decades. Since more patients are surviving the initial resuscitation phase even with very large areas of skin being burned away, wound care has become increasingly important to ensure continued patient survival and improvement. While currently a common treatment for third degree burn wounds, skin grafts have several drawbacks. The availability of donor sites for autografts may be limited, especially in incidences of extensive skin loss. The rejection associated with the use of allografts and xenografts may render them inadequate or undesirable. Even if a suitable graft is found, poor retention due to infection, hematoma, and low vascularity at the recipient site are other drawbacks associated with the use of skin grafts as a primary treatment for severe burn wounds. As such, research has been done into alternative treatments, which include but are not limited to artificial skin, cell therapy, and growth factor application. We propose the delivery of adipose derived stem cells (ASC) in combination with endothelial progenitor cells (EC) via Integra Dermal Regenerative Template (DRT) to promote faster graft vascularization and thus faster healing of wounds. Integra DRT is an acellular skin substitute that consists of a dermal layer composed of bovine collagen and chondroitin-6-sulfate glycosaminoglycan, and an "epidermal" layer, which consists of silicone polymer. This silicone layer is removed after the collagen matrix is adequately vascularized (usually takes 2-3 weeks), and then a thin layer autograft is applied to the top of the neo-dermis. ASC are derived from the stromal-vascular fraction (SVF) of adipose tissue and are a readily available, pluripotent, mesenchymal cell known to promote angiogenesis. They are being explored as a treatment for a myriad of diseases and conditions, including wound healing. In combination with ECs, they form stable microvessel networks in vitro and in vivo. In our work, we found that ASC+EC form stable microvessel networks when cultured on Integra DRT. Also, ASC and ASC+EC conditioned media promoted both survival and migration of human epidermal keratinocytes compared to control medium. In a full thickness wound healing model, using healthy NSG mice, the ASC+EC case showed a significantly higher rate of wound closure compared to control. Based on best linear unbiased estimates (BLUE), the difference between the healing rates of ASC alone treatment and the Control treatment group is -0.45 +/- 0.22 mm²/day (p=0.041), which is not less than 0.025 and thus not statistically significant (Bonferroni Adjusted). However, the BLUE for the difference between the ASC+EC group and the Control group healing rates is -0.55 +/- 0.28 mm²/day (p = 0.017<0.025, Bonferroni Adjusted), which is statistically significant. Histology revealed a significantly higher number of vessels compared to control in both ASC alone and ASC+EC case. CD31 staining revealed the presence of human vessels in ASC+EC treatment scaffolds. We conclude that the combination of ASC and EC can be used to accelerate healing of full-thickness wounds when delivered to site of the wound via Integra. This result is especially compelling due to the fact that the mice used were all healthy. Thus our treatment shows an improvement in healing rate even compared to normal wound healing.

Indiana University-Purdue University Indianapolis (IUPUI) / Solid organ transplantation is limited by available donor allografts. Pig to human transplantation, xenotransplantation, could potentially solve this problem if physiologic and immunologic incompatibilities are overcome. Genetic modifications of pigs have proven valuable in the study of xenotransplantation by improving pig to human compatibility. More genetic targets must be identified for clinical success. First, this study examines platelet homeostasis incompatibilities leading to acute thrombocytopenia in liver xenotransplantation. Mechanisms for xenogeneic thrombocytopenia were evaluated using liver macrophages, Kupffer cells, leading to identification of CD18, beta-2 integrin, as a potential target for modification. When disruption of CD18 was accomplished, human platelet binding and clearance by pig Kupffer cells was inhibited. Further, human and pig platelet surface carbohydrates were examined demonstrating significant differences in carbohydrates known to be involved with platelet homeostasis. Carbohydrate recognition domains of receptors responsible for platelet clearance Macrophage antigen complex-1 (CD11b/CD18) and Asialoglycoprotein receptor 1 in pigs were found to be different from those in humans, further supporting the involvement of platelet surface carbohydrate differences in xenogeneic thrombocytopenia. Second, immunologic incompatibilities due to antibody recognition of antigens resulting in antibody-mediated rejection were studied. Identification of relevant targets was systematically approached through evaluation of a known xenoantigenic protein fibronectin from genetically modified pigs. N-Glycolylneuraminic acid, a sialic acid not found in humans, was expressed on pig fibronectin and was identified as an antigenic epitope recognized by human IgG. These studies have provided further insight into xenogeneic thrombocytopenia and antibody-mediated rejection, and have identified potential targets to improve pig to human transplant compatibility.