Transplantation of tissue-engineered skin grafts has delivered new medical benefits to patients with acute skin wounds arising from extensive, full-thickness burns, but applications of these devices to elective surgery (i.e., burn scars) is compromised by their limited lifespan in vitro, and by anatomic deficiencies, including absence of hair, glands and nails. The investigators have demonstrated stable wound closure in burns, chronic wounds and giant nevi with autologous cultured skin substitutes (CSS) consisting of a collagen-based sponge populated with cultured human fibroblasts and keratinocytes. This model of engineered human skin develops a keratinized epithelium, basement membrane, and has a dermal substitute, but requires high inoculation densities, has a limited lifespan in vitro, and has no epidermal adnexal structures. The investigators hypothesize that: a) the clinical efficacy of CSS will improve, and elective surgery and adnexal structures will be enabled by selection for the somatic stem cells (SSC) of the epidermis, and preservation of the SSC phenotype during keratinocyte culture; and, b) keratinocytes in CSS can be induced to form hair under regulation of the Wnt/p-catenin signaling pathway. To address these hypotheses, three specific aims are proposed: 1) Selection of epidermal SSCs by high expression of 06 integrin, CD90, low expression of receptors for EGF and transferrin (CD71), and preservation of the SSC phenotype by low amounts of TGF-01 in medium. The SSC phenotype will be tracked by label-retaining cells (LRCs) in CSS and healed human skin. 2) Induction of hair morphogenesis in CSS by expression in keratinocytes of stabilized p-eatenin, either constitutively, or under an estrogen-responsive element; or, by expression in fibroblasts of Wnt 3a proteins. 3) Grafting of CSS with a SSC phenotype to excised, full-thickness burns, and evaluations of rates of engraftment, area ratios of closed wounds to donor skin, and qualitative outcome by the Vancouver Scale. These studies will be performed in well-characterized, preclinical models of cell cultures, skin substitutes, and grafting of CSS to full-thickness skin wounds in athymic mice. Improvements in regulation of the cellular proliferation and differentiation for preparation of CSS will be studied in skin structure (e.g., histology, expression of protein & mRNA), physiology (LRCs in situ), and function (e.g., epidermal barrier, pliability) in vitro and in vivo. Results from specific Aim 1 are expected to promote the success of Aim 2, and will be tested clinically in Specific Aim 3. Preclinical studies in Specific Aim 2 are expected to open possibilities for replacement of hair and glands in burned skin with the potential to address conditions such as burn alopecia and thermal regulation. These advances require a paradigm shift from past studies of wound healing to future studies of developmental biology. Only by making this change in approach will the ultimate goal of skin regeneration replace the dogma of wound repair which leads to scar and disability. The investigators expect fully that the proposed studies of CSS can further reduce the donation of tissue required for grafting, make initial demonstrations of adnexal development in engineered skin, improve efficacy of CSS for treatment of burn scars, and increase the probability of saving the lives of patients with life-threatening burns. ? ? ?
