The significance of melanotropic hormones as physiologic regulators of cutaneous pigmentation in humans is still controversial. Until recently, no direct effect for melanotropins could be demonstrated on human melanocytes.Here we present conclusive evidence that a!-melanotropin (a-melanocyte-stimulating hormone, ai-MSH) and the related hormone corticotropin (adrenocorticotropic hormone, ACTH) stimulate the proliferation and melanogenesis of human melanocytes maintained in culture in a growth medium lacking any AMP inducer. The minimal effective dose of either hormone is 0.1 nM. In time-course experiments, the increase in cell number and tyrosinase activity became evident after one treatment of the melanocytes with 100 nM a-MSH for 48 hr. The mitogenic effect gradually increased to 50-270S% above control, depending on the individual melanocyte strain, with continuous treatment with 100 nM a-MSH for 8 days, whereas the melanogenic effect became maximal (70-450% increase above control) after 4 days oftreatment. Western blot analysis of tyrosinase and the tyrosinase-related proteins TRP-1 and TRP-2 revealed that a-MSH increased the expression of those three melanogenic proteins. This was not accompanied by any change in their mRNA levels after brief (1.5-24 hr) or prolonged (6 days) treatment with 100 nM a-MSH, suggesting that the increased expression of these melanogenic proteins was due to posttranscriptional events. These results demonstrate both mitogenic and melanogenic effects of a-MSH and ACTH on human melanocytes. That both hormones are effective at subnanomolar concentrations, combined with the presence of melanotropin receptors on human melanocytes, strongly suggests that these melanotropins play a physiologic role in regulating human cutaneous pigmentation.a-Melanotropin (a melanocyte-stimulating hormone, a-MSH) is the physiologic hormone that regulates integumental pigmentation of many vertebrate species. For example, a-MSH induces rapid skin darkening in amphibians and reptiles and stimulates follicular eumelanogenesis in the mouse (1,2). In addition to the pigmentary effects, other functions for a-MSH and related melanotropins have been described, such as the antagonistic interaction with interleukin 1 (3, 4) and trophic effects on neurons (5, 6

Restoration of an epidermal barrier is a definitive requirement for wound closure. To determine formation of an epidermal barrier as a function of hydration of the stratum corneum, we measured surface electrical capacitance (SEC) of the epidermis in cultured skin substitutes (CSS) in vitro and after grafting to athymic mice. CSS were prepared from human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates. On culture days 3, 7, 14, 17, and 21, SEC was measured in situ. CSS (n = 18; mean +/- SEM) showed a time-dependent decrease of SEC (picoFarads, "pF") from 4721 +/- 28 pF on day 3 to 394 +/- 117 pF on day 14, and subsequent increase to 1677 +/- 325 pF on day 21. After 14-d incubation, parallel CSS samples (n = 5) or murine autografts (n = 5) were grafted orthotopically to athymic mice. After grafting, CSS showed decreases in SEC from 910 +/- 315 pF at 2 wk to 40 +/- 10 pF at 4 wk with no significant decreases thereafter. Control values for murine autograft were 870 +/- 245 pF at 2 wk, and 87 +/- 30 pF at 4 wk. SEC values for native murine skin (n = 10) were 91 +/- 18 pF, and for native human skin (n = 10) were 32 +/- 5 pF. The data demonstrate that SEC decreases with time in culture and that healed or intact skin has approximately 10- to 100-fold lower SEC than CSS in vitro. This noninvasive technique provides a quantitative index of epidermal barrier in CSS in vitro and demonstrates the development of functional epidermal barrier during healing of wounds treated with cultured skin substitutes.

Routine treatment of burns with cultured skin substitutes (CSS) has been limited by poor engraftment and by scarring. Hypothetically, topical application of essential nutrients and/or growth factors may support epithelial survival temporarily during graft vascularization. CSS, composed of human epidermal keratinocytes and dermal fibroblasts attached to collagen-glycosaminoglycan substrates, were incubated for 19 d in media optimized for keratinocytes. CSS, human xenografts, murine autografts, or no grafts were applied orthotopically to full-thickness skin wounds (2 x 2 cm) in athymic mice. Wounds were irrigated for 14 d with 1 ml/d modified cell culture medium or with saline containing epidermal growth factor, or were treated with dry dressings. After 6 weeks, treated sites were scored for percentage original wound area (mean +/- SEM) and percentage HLA-ABC-positive healed wounds [(number positive/n) x 100], and tested for significance (analysis of variance, p < 0.0001; Tukey test, p < 0.05). The data showed that CSS irrigated with nutrient medium were not statistically different in wound area (67.8 +/- 5.1%) from murine autografts (63.3 +/- 2.9%) but were statistically larger than human xenograft, no graft, or CSS treated with saline irrigation or dry dressings. HLA-ABC expression was 100% in CSS with nutrient irrigation, 86% in CSS with saline irrigation, 83% in CSS without irrigation, and 75% in xenografts with nutrient irrigation. These findings suggest that availability of essential nutrients supports keratinocyte viability during graft vascularization of CSS.

Collagen-based implants have been described as vehicles for transplantation of cultured skin cells for treatment of burn wounds. To optimize vascularization and repair of connective tissue, collagen solubility and glutaraldehyde crosslinking were evaluated. Cultured skin substitutes consisted of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates that were prepared from acid-insoluble, or partially soluble collagen. Substrates were crosslinked with 0% or 0.25% glutaraldehyde, populated with cells, and grafted to full-thickness wounds on athymic mice (n = 6/condition). After 6 weeks, the wound area was measured by planimetry, and healed wounds were scored by histochemistry for immunoreactivity to HLA-ABC and bovine collagen. Data analysis shows that crosslinking of collagen implants with glutaraldehyde is associated (p < 0.001) with detection of the implant. No association was found between solubility of bovine collagen and immunodetection. Epidermis of all wounds was positive for HLA-ABC, and no differences in wound areas were found. These results suggest that glutaraldehyde crosslinking of collagen implants decreases the rate of biodegradation. Delayed degradation of crosslinked collagen may result clinically in reduced engraftment of skin substitutes.