This study evaluates the use of composite grafts of cultured human keratinocytes and de-epidermalized, acellular human dermis to close full-thickness wounds in athymic mice. Grafts were transplanted onto athymic mice and studied up to 8 wk. Graft take was excellent, with no instances of infection or graft loss. By 1 wk, the human keratinocytes had formed a stratified epidermis that was fused with mouse epithelium, and by 8 wk the grafts resembled human skin and could be freely moved over the mouse dorsum. Immunostaining for keratins 10 and 16 and for involucrin revealed an initial pattern of epithelial immaturity, which by 8 wk had normalized to that of mature unwounded epithelium. Mouse fibroblasts began to infiltrate the acellular dermis as early as 1 wk. By 8 wk fibroblasts had completely repopulated the dermis, and blood vessels were evident in the most superficial papillary projections. Dermal elements, such as rete ridges and elastin fibers, which were present in the starting dermis, persisted for the duration of the experiment. Grafts using keratinocytes from dark-skinned donors as opposed to light-skin donors had foci of pigmentation as early as 1 wk that progressed to homogenous pigmentation of the graft by 6 wk. These results indicate that melanocytes that persist in vitro are able to resume normal function in vivo. Our study demonstrates that composite grafts of cultured keratinocytes combined with acellular dermis are a useful approach for the closure of full-thickness wounds.

This study shows that single-stage treatment of ventral hernias in contaminated fields can be accomplished with a low recurrence rate and acceptable morbidity in these extremely challenging patients.

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 x 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.

This study evaluated the in vitro and in vivo function of composite skin equivalents based on two different dermal analogs. Keratinocytes derived from the same dark-skinned neonatal foreskins were seeded onto both acellular human dermis and fibroblast-contracted collagen gels. Each type of composite graft readily formed an epithelium in vitro. However, the undulating surface of the acellular dermis acted as a template and organized the seeded keratinocytes into a rete ridge-like pattern, whereas the smooth surface of the fibroblast-contracted collagen gels generated an epithelium with a linear basal layer. Moreover, when acellular dermis was used, the composite grafts demonstrated enhanced melanocyte proliferation. When transplanted to athymic mice, both composite grafts formed a fully differentiated human epidermis, but repigmentation of the grafts when acellular dermis was used was more extensive and only the epidermis on the fibroblast-contracted collagen gels showed signs of hyperproliferation at 6 weeks after grafting. These results demonstrate that the type of dermal analog incorporated into a composite skin graft can influence the subsequent functionality of the skin substitute.

No abstract

No abstract

To improve the performance of bioengineered skin, we used a recombinant retrovirus encoding FGF-7 to modify diploid human keratinocytes genetically. Control or FGF-7-expressing keratinocytes were seeded onto acellular human dermis to form bioengineered skin. Gene-modified skin secreted significant levels of FGF-7 and formed a thicker and hyperproliferative epidermis with about four times the number of cells per square centimeter. Secretion of an endogenous trophic factor, VEGF, was increased approximately 5-fold. Migration of FGF-7-expressing keratinocytes was stimulated as was the self-healing of bioengineered skin expressing FGF-7. When tested in a bacterial infection model, the antimicrobial properties of FGF-7-expressing skin were increased >500-fold against both gram-negative and gram-positive bacteria. After transplantation to full-thickness wounds on athymic mice, skin expressing FGF-7 was revascularized more rapidly. These results demonstrate that genetic modification can be used to enhance performance and that expression of FGF-7 augments several properties important to the wound-healing properties of bioengineered skin.

Amputation is still recommended to patients with a difficult wound of the lower extremity because limb salvage after free tissue transfer in these patients remains uncertain. During the past 3 years, the authors studied 15 patients (11 men, 4 women; age range, 17-71 years) with difficult wounds of the lower extremities who had free tissue transfers for limb salvage. Eleven patients had an extensive soft-tissue defect (nearly the entire length) of the legs or feet, and 4 had a composite-tissue defect of the legs or feet that required bony reconstruction. A total of 16 free tissue transfers (13 free muscle flaps, 2 osteomusculocutaneous flaps, and 1 adipofascial flap) were performed in 15 patients (1 patient had bilateral transfers). A saphenous vein loop or graft was used in 3 patients and a subsequent bone graft was done in 2 patients. Free tissue transfer was accomplished successfully in 14 patients (93%). Limb salvage was achieved ultimately in 12 patients (80%) who were able to ambulate during a 36-month follow-up. The authors believe that free tissue transfer for limb salvage in any patient with a difficult wound of the lower extremity is still a worthwhile procedure and should be attempted if possible. Meticulous preoperative preparation and intraoperative execution combined with the use of innovative microsurgical techniques are the keys for success.