Genetically Modified Human Keratinocytes Overexpressing PDGF-A Enhance the Performance of a Composite Skin Graft

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Abstract
Skin loss due to burns and ulcers is a major medical problem. Bioengineered skin substitutes that use cultured keratinocytes as an epidermal layer with or without analogues of the dermis are one strategy for skin repair. However, none can achieve definitive wound closure, function, or cosmesis comparable to split-thickness autografts. Moreover, autograft donor sites, which require time to heal, may be limited or have attendant problems such as infection or functional/cosmetic deficiencies. To determine if the performance of composite skin grafts of keratinocytes on a dermal analogue could be enhanced, human keratinocytes were genetically modified to overexpress platelet-derived growth factor A chain (PDGF-A). Composite grafts of modified keratinocytes seeded onto acellular dermis, prepared from cryopreserved cadaver skin, secreted PDGF-AA protein in vitro [90 ng/graft (1.5 × 1.5 cm)/24 hr]. To test their performance in a wound healing model, composite grafts were transplanted to full-thickness excisional wounds on the back of athymic mice. PDGF-A grafts formed a stratified differentiated epidermis similar to control grafts. The acellular dermis was repopulated with host fibrovascular cells and by day 7, the PDGF-A grafts had significantly more cells in the dermis and increased staining for murine collagen types I and IV. At this early time point, wound contraction was also significantly inhibited in PDGF-A grafts versus control grafts. Thus, PDGF-A overexpression improves graft performance during the first critical week after transplantation. Cultured human keratinocytes have been used as part of various skin substitutes for the repair of skin defects, such as burns and ulcers. We show here that the performance of a skin substitute can be enhanced if the keratinocytes are genetically modified to overexpress platelet-derived growth factor A chain (PDGF-A). Composite skin substitutes of PDGF-A-expressing keratinocytes seeded on acellular human dermis were prepared and tested in an animal model. Graft revascularization was stimulated and wound contraction was inhibited by those grafts expressing PDGF-A. This study illustrates how a gene therapy approach can be used to help achieve an important goal in tissue engineering, namely, the repair of defective tissues.

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