Design principles for composition and performance of cultured skin substitutes.
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Burns↗
Biomedical subjects
S T Boyce
Publications and source records attributed to S T Boyce.
Expression of insulin-like growth factor I by cultured skin substitutes does not replace the physiologic requirement for insulin in vitro.
Clinical efficacy of cultured skin substitutes may be increased if their carbohydrate metabolism is optimized by understanding whether endogenous insulin-like growth factor I can substitute for exogenous insulin. Cultured skin substitutes were prepared and incubated at the air-liquid interface for 4 wk in media containing 0.5 or 5 microg per ml insulin, 10 or 50 ng per ml insulin-like growth factor I, or 0 insulin and 0 insulin-like growth factor I (negative control). In situ hybridization showed that the epidermal and dermal cultured skin substitute components express insulin-like growth factor I mRNA throughout the 28 d interval. Immunohistochemistry confirmed the expression of insulin-like growth factor I protein by the human keratinocytes and fibroblasts in cultured skin substitutes. Insulin-like growth factor I at 10 or 30 ng per ml could partially replace insulin in a clonal assay of keratinocyte growth. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays showed significantly higher values in cultured skin substitutes incubated with insulin at incubation days 14 and 28 compared to negative control or the 10 ng per ml insulin-like growth factor I condition. Cultured skin substitutes incubated in 50 ng per ml insulin-like growth factor I had MTT values similar to the insulin-treated cultured skin substitutes at day 14, but were significantly lower by day 28. Light microscopy agreed with MTT data showing that cultured skin substitutes grown with insulin media had multiple layers of nucleated keratinocytes and stratum corneum at days 14 and 28. The negative control and 10 ng per ml insulin-like growth factor I exhibited poor cultured skin substitute epidermal morphology throughout the experiment. In contrast, the cultured skin substitutes in 50 ng per ml insulin-like growth factor I were similar to the insulin-treated cultured skin substitutes at day 14, but by day 28 had deteriorated to resemble the negative control. Bromodeoxyuridine incorporation at day 28 was significantly higher for 5 microg per ml insulin cultured skin substitutes versus all other treatment groups. These data suggest that medium containing 5 microg per ml insulin supports greater physiologic stability in cultured skin substitutes over time, and that expression of insulin- like growth factor I by keratinocytes and fibroblasts in cultured skin substitutes is not sufficient to fully replace the requirement for exogenous insulin in vitro.
Bromodeoxyuridine↗
Enhanced vascularization of cultured skin substitutes genetically modified to overexpress vascular endothelial growth factor.
Cultured skin substitutes have been used as adjunctive therapies in the treatment of burns and chronic wounds, but they are limited by lack of a vascular plexus. This deficiency leads to greater time for vascularization compared with native skin autografts and contributes to graft failure. Genetic modification of cultured skin substitutes to enhance vascularization could hypothetically lead to improved wound healing. To address this hypothesis, human keratinocytes were genetically modified by transduction with a replication incompetent retrovirus to overexpress vascular endothelial growth factor, a specific and potent mitogen for endothelial cells. Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates inoculated with human fibroblasts and either vascular endothelial growth factor-modified or control keratinocytes were prepared, and were cultured in vitro for 21 d. Northern blot analysis demonstrated enhanced expression of vascular endothelial growth factor mRNA in genetically modified keratinocytes and in cultured skin substitutes prepared with modified cells. Furthermore, the vascular endothelial growth factor-modified cultured skin substitutes secreted greatly elevated levels of vascular endothelial growth factor protein throughout the entire culture period. The bioactivity of vascular endothelial growth factor protein secreted by the genetically modified cultured skin substitutes was demonstrated using a microvascular endothelial cell growth assay. Vascular endothelial growth factor-modified and control cultured skin substitutes were grafted to full-thickness wounds on athymic mice, and elevated vascular endothelial growth factor mRNA expression was detected in the modified grafts for at least 2 wk after surgery. Vascular endothelial growth factor-modified grafts exhibited increased numbers of dermal blood vessels and decreased time to vascularization compared with controls. These results indicate that genetic modification of keratinocytes in cultured skin substitutes can lead to increased vascular endothelial growth factor expression, which could prospectively improve vascularization of cultured skin substitutes for wound healing applications.
Animals↗
Keratinocytes suppress transforming growth factor-beta1 expression by fibroblasts in cultured skin substitutes.
Transforming growth factor (TGF)-beta1 is a multipotent growth factor with an important role in tissue homeostasis. This growth factor regulates cell proliferation, adhesion, migration and differentiation, as well as extracellular matrix deposition. The temporal secretion and activation of latent TGF-beta1 is thus of major importance to physiological and pathological processes and in wound healing and tumour formation. Cultured skin substitutes, as used to treat extensive acute or chronic skin wounds, offer an attractive model to investigate cellular interactions in cytokine and growth factor expression and response in vitro. In the present investigation, expression of TGF-beta1 was analysed in keratinocyte, fibroblast and melanocyte monolayer cultures, as well as in the dermal vs. epidermal components of reconstituted human skin. Immunohistology, enzyme-linked immunosorbent assay (ELISA) and Northern blotting were used to demonstrate expression at the RNA and protein level. In the monolayer cultures, levels of TGF-beta1 synthesized by melanocytes were observed to be considerably elevated when compared with keratinocytes. Most TGF-beta1, however, was secreted by fibroblasts. The relative contribution of the epidermal and dermal components of the skin substitutes to overall TGF-beta1 levels was determined by comparing results obtained for either component in the presence and absence of fibroblasts and keratinocytes. From results obtained by ELISA it was apparent that TGF-beta1 levels generated predominantly by fibroblasts within the skin substitutes were greatly reduced over time in the presence of keratinocytes. Suppression of fibroblast TGF-beta1 expression in the presence of keratinocytes was also demonstrable at the RNA level by Northern blotting. Results obtained by immunohistochemistry suggest that most, if not all, of the growth factor was present in the latent form. It is therefore most likely that the observed effect results from a factor secreted by keratinocytes, which is capable of suppressing TGF-beta1 synthesis by fibroblasts. These results suggest that expression of TGF-beta1 by fibroblasts is downregulated by paracrine actions of keratinocytes in healing skin.
Blotting, Northern↗
Skin substitutes from cultured cells and collagen-GAG polymers.
Engineering skin substitutes provides a potential source of advanced therapies for the treatment of acute and chronic wounds. Cultured skin substitutes (CSS) consisting of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates have been designed and tested in preclinical and clinical studies. Cell culture techniques follow general principles of primary culture and cryopreservation in liquid nitrogen for long-term storage. Biopolymer substrates are fabricated from xenogeneic (bovine) collagen and glycosaminoglycan that are lyophilised for storage until use. At maturity in air-exposed culture, CSS develop an epidermal barrier that is not statistically different from native human skin, as measured by surface electrical capacitance. Preclinical studies in athymic mice show rapid healing, expression of cytokines and regulation of pigmentation. Clinical studies in burn patients demonstrate a qualitative outcome with autologous skin that is not different from 1:4 meshed, split-thickness autograft skin, and with a quantitative advantage over autograft skin in the ratio of healed skin to biopsy areas. Chronic wounds resulting from diabetes or venous stasis have been closed successfully with allogeneic CSS prepared from cryopreserved skin cells. These results define the therapeutic benefits of cultured skin substitutes prepared with skin cells from the patient or from cadaver donors. Future directions include genetic modification of transplanted cells to improve wound healing transiently or to deliver gene products systemically.
Biopolymers↗
Stratum corneum lipid composition and structure in cultured skin substitutes is restored to normal after grafting onto athymic mice.
Restoration of an epidermal barrier is a definitive requirement for wound closure. Cultured skin substitutes grafted onto athymic nude mice were used as a model for a long-term study of stratum corneum barrier lipid metabolism and organization. Samples of stratum corneum collected after 12 and 21 d in vitro and 6, 11, and 24 mo postgrafting were examined for their lipid and fatty acid composition, and their lipid organization and structure using electron microscopy and small angle X-ray diffraction, respectively. All of these methods confirm the impaired barrier function of cultured skin substitutes in vitro, as judged from the deviations in lipid composition and from poor organization of the stratum corneum lipids that show no lamellar structure. At 6 mo postgrafting, the total stratum corneum lipid profiles of the epidermal grafts is close to that of the human stratum corneum with the exception of the presence of mouse specific lipids. The increase of ceramides 4-7 in cultured skin substitutes after grafting indicates restored activity of processes involved in the hydroxylation of fatty acids and sphingoid bases. Conversely, the ceramide profile still reveals some abnormalities (elevated content of ceramide 2 and slightly lower content of ceramide 3) and the content of long-chain fatty acids remains below its physiologic level at 6 mo postgrafting, but normalizes by 2 y postgrafting. The ultramicroscopic observations revealed the formation of lamellar extracellular lipid domains by 4 mo postgrafting. Despite these findings, the X-ray diffraction showed differences in the diffraction pattern at 2 y after grafting, suggesting that the organization of stratum corneum lipids in all epidermal grafts differs from that of the native skin.
Animals↗
Endotoxemia in mice stimulates production of complement C3 and serum amyloid A in mucosa of small intestine.
We examined the effect of endotoxemia in mice on protein and mRNA levels for the acute phase proteins complement C3 and serum amyloid A (SAA) in jejunal mucosa. Endotoxemia was induced in mice by the subcutaneous injection of 250 microg lipopolysaccharide per mouse. Control mice were injected with saline. C3 and SAA were measured by ELISA. Messenger RNA levels were determined by Northern blot analysis or competitive PCR. Immunohistochemistry was performed to determine in which cell type(s) C3 and SAA were present. Mucosal C3 and SAA protein and mRNA levels were increased in endotoxemic mice. Immunohistochemistry showed that C3 was present in both enterocytes and cells of the lamina propria, whereas SAA was seen mainly in lamina propria cells. Results suggest that endotoxemia stimulates production of C3 and SAA in small intestinal mucosa. The response may be regulated at the transcriptional level and probably reflects increased synthesis of the acute phase proteins in both enterocytes and cells of the lamina propria.
Animals↗
Glutaraldehyde crosslinking of collagen substrates inhibits degradation in skin substitutes grafted to athymic mice.
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.
Animals↗
Reduced engraftment and wound closure of cryopreserved cultured skin substitutes grafted to athymic mice.
Cryopreservation of cultured skin substitutes is a requirement for establishment of banks of alternative materials for treatment of acute and chronic skin wounds. To determine whether cryopreservation of skin substitutes that contain cultured cells reduces their efficacy for wound closure, cell-biopolymer grafts were frozen, recovered into culture, and grafted to wounds on athymic mice. Grafts consisted of cultured human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates that were frozen in cell culture medium with 20% serum and 10% DMSO at a controlled rate and stored overnight in liquid nitrogen. After recovery into culture for 24 h, frozen or unfrozen (control) skin substitutes were grafted to full-thickness wounds on athymic mice. Wound area and surface electrical capacitance were measured at 2, 3, and 4 weeks after grafting at which time animals were sacrificed. Wounds were scored for presence of human cells by direct immunofluorescence staining with a monoclonal antibody to HLA-ABC. The data demonstrate that cell-biopolymer grafts are less efficacious after controlled-rate cryopreservation using 10% DMSO as a cryoprotectant. Frozen grafts at 4 weeks after surgery have significantly smaller wound areas, higher capacitance (wetter surface), and fewer healed wounds that contain human cells. The results suggest that these conditions for cryopreservation of cultured grafts reduce graft viability. Improved conditions for cryopreservation are required to maintain viability and efficacy of cultured skin substitutes after frozen storage.
Animals↗
Regulation of pigmentation in cultured skin substitutes by cytometric sorting of melanocytes and keratinocytes.
Unpredictable pigmentation in cultured skin substitutes (CSS) is an anatomic deficiency after wound treatment and can require years to normalize. Variable numbers of human melanocytes (HM) survive in cultures of human keratinocytes (HK) as demonstrated by focal areas of pigmentation in CSS after healing. The purposes of this study were to deplete HM from HK cultures and to regulate the numbers of HM contained in CSS. A highly pigmented HM cell strain was chosen for these studies to emphasize the differences in light scattering between HK and HM by flow cytometry. Cytometric gates were set with selective cultures of HM and HK and were used to sort a mixed population of HK + 4% HM. After sorting, CSS were prepared from human fibroblasts attached to collagen-glycosaminoglycan sponges combined with cells from the HK + 4% HM (pre-treatment control), the sorted HK (experimental), or sorted HK + 3% HM (post-treatment positive control) subpopulations and grafted to athymic mice. Grafted wounds were assessed for 6 wk by planimetry for area of pigment and by a Minolta Chromameter for color density and hue in situ. Histology and staining of HLA-ABC were performed at 6 wk. Data from percent pigmented area and chromameter measurements identified quantitative and statistically significant decreases in color of healed skin after flow cytometric separation of HK and HM. Therefore, a purified HK subpopulation depleted of HM was isolated by flow cytometry that generated healed skin with reduced pigmentation. These results suggest that HM can be selectively depleted from HK cultures and then added to cultured skin substitutes at specific densities to generate predictable pigmentation for improved function and cosmesis in healed wounds.
Animals↗
Formulation of 'idealized' topical antimicrobial mixtures for use with cultured skin grafts.
In order to develop antimicrobial mixtures which provide broad-spectrum antimicrobial activity for use with cultured cell autografts, several individual antimicrobial agents, in concentrations non-toxic for cells in culture, were tested against a variety of bacteria and Candida spp. isolated from burn patients. An agar well diffusion topical assay was used. Antimicrobials active against Gram-positive and Gram-negative bacteria and antifungal agents, individually, were uniformly effective against their respective spectra of organisms. Broad-spectrum antibacterials were uniformly effective against Gram-negative bacteria but their activity varied against Gram-positive bacteria. Adding an agent active against Gram-positive bacteria to all broad spectrum antibacterial agents conferred uniform Gram-positive activity to the mixture. One mixture consisting of specific Gram-negative, Gram-positive and broad spectrum antibacterial agents, was uniformly active against all bacteria tested and the addition of antifungal agents extended the activity to cover Candida spp. without interfering with the mixture's overall antibacterial activity. Another mixture showed either additive or antagonistic activities against the battery of microorganisms tested. Thus, these methods can be used to identify mixtures of antimicrobials, in concentrations non-toxic for cells in culture, that have very broad spectra of antimicrobial activity. Such mixtures should be evaluated in patients when cultured skin grafts are used.
Administration, Topical↗
Expression of interleukin-1alpha, interleukin-6, and basic fibroblast growth factor by cultured skin substitutes before and after grafting to full-thickness wounds in athymic mice.
OBJECTIVES: Cultured skin substitutes (CSSs), consisting of human keratinocytes and human fibroblasts attached to collagen-glycosaminoglycan substrates, have been demonstrated to cover wounds, and may release detectable quantities of growth factors that promote wound healing. MATERIALS AND METHODS: Basic fibroblast growth factor (bFGF), interleukin-1alpha (IL-1alpha), and interleukin-6 (IL-6) were assayed by enzyme linked immunosorbent assay and immunohistochemistry in CSSs in vitro and at days 1, 3, 7, 14, and 21 after grafting to full-thickness wounds in athymic mice. MEASUREMENTS AND MAIN RESULTS: When isolated cells were tested, IL-1alpha was found to come primarily from the keratinocytes, whereas bFGF was from the fibroblasts. Combinations of both cell types in the CSSs resulted in a synergistic enhancement of IL-6 expression. Quantities of all three cytokines from CSSs were greater in vitro compared with in vivo levels at all time points after grafting. bFGF increased from day 1 to day 7, and then remained relatively constant until day 21. At day 3 maximal levels of IL-1alpha were observed. By day 7, IL-1alpha decreased to approximately 40% of maximal levels, and subsequently increased until day 21. IL-6 levels were highest at day 7 after grafting. All cytokines had reached elevated levels during the time of wound revascularization (days 3-7). CONCLUSIONS: The sequence of cytokine synthesis in the wounds (i.e., rapid IL-1alpha increase followed by IL-6 expression) parallels serum levels reported after a septic challenge. These findings support the hypothesis that the wound is a source of systemic cytokines.
Animals↗
Effects of the angiotensin converting enzyme inhibitor enalapril on bacterial translocation after thermal injury and bacterial challenge.
Burn injury and sepsis produce acute gastrointestinal derangements that may predispose patients to bacterial translocation. We studied the effects of enalapril, an angiotensin converting enzyme inhibitor (ACEI), on gastrointestinal anatomic alterations, bacterial translocation, and related mortality during gut-derived sepsis in burned mice that had received a prior bacterial challenge. BALB/c mice (n = 111) were treated with enalapril 10 or 1 mg/kg body weight or sterile saline as control twice daily for 3 days. They were then gavaged with 10(a)111 in radiolabeled or unlabeled Escherichia coli and given a 20% total body surface area (TBSA) burn injury. Animals gavaged with unlabeled bacteria were observed for survival (n = 60). Survival was significantly higher in the group receiving enalapril 10 mg/Kg compared with control (75% vs. 10%). Mice treated with enalapril maintained small intestine weight, measured 4 h postburn, and ileal mucosal height was preserved, whereas burned untreated animals lost intestinal weight and mucosal height. Bacterial translocation was decreased in mice treated with enalapril, but killing was unaffected. This study suggests that treatment with enalapril positively affects the outcome in gut-derived sepsis by ameliorating gastrointestinal structural and functional damage and decreasing bacterial translocation.
Angiotensin-Converting Enzyme Inhibitors↗
Surface electrical capacitance as a noninvasive index of epidermal barrier in cultured skin substitutes in athymic mice.
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.
Animals↗
Pigmentation and microanatomy of skin regenerated from composite grafts of cultured cells and biopolymers applied to full-thickness burn wounds.
Rapid coverage and epithelial closure of extensive burns remains a major requirement for patient recovery. Although many skin substitutes have been described, permanent regeneration of both epithelial and connective tissues after a single surgical application of a skin substitute has not become routine. To replace both dermal and epidermal skin, cultured skin substitutes (CSS) were prepared from autologous keratinocytes and fibroblasts seeded onto collagen-glycosaminoglycan (C-GAG) substrates. CSS were applied to excised, full-thickness burns on 5 patients. Histologic analysis showed a fully stratified, hyperkeratotic epidermis within 12 days of grafting with little to no evidence of an inflammatory reaction. Epidermal and connective tissues are interdigitated in analogy to rete pegs and dermal papillae, and the neovascular plexus approximates the dermal-epidermal junction. Transmission electron microscopy identified a continuous basement membrane with hemidesmosomes and anchoring fibrils that connected the epidermis with the underlying connective tissue. Within 14-28 days, the C-GAG had been degraded and replaced by newly synthesized collagen in regenerated connective tissue. Spontaneous repigmentation of healing CSS from passenger melanocytes in keratinocytes culture was observed within 2 months after grafting. Electron microscopy revealed the presence of numerous melanosomes within the keratinocytes, illustrating pigment transfer between melanocytes and keratinocytes after wound closure. These results demonstrate that the CSS develop into functional permanent skin tissue capable of spontaneous repigmentation after grafting onto burn wounds.
Adolescent↗
Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides.
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 alpha-melanotropin (alpha-melanocyte-stimulating hormone, alpha-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 alpha-MSH for 48 hr. The mitogenic effect gradually increased to 50-270% above control, depending on the individual melanocyte strain, with continuous treatment with 100 nM alpha-MSH for 8 days, whereas the melanogenic effect became maximal (70-450% increase above control) after 4 days of treatment. Western blot analysis of tyrosinase and the tyrosinase-related proteins TRP-1 and TRP-2 revealed that alpha-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 alpha-MSH, suggesting that the increased expression of these melanogenic proteins was due to posttranscriptional events. These results demonstrate both mitogenic and melanogenic effects of alpha-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.
Adrenocorticotropic Hormone↗
Comparative assessment of cultured skin substitutes and native skin autograft for treatment of full-thickness burns.
OBJECTIVE: Comparison of cultured skin substitutes (CSSs) and split-thickness autograft (STAG) was performed to assess whether the requirement for autologous skin grafts may be reduced in the treatment of massive burns. SUMMARY BACKGROUND DATA: Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates populated with autologous fibroblasts and keratinocytes have been demonstrated to close full-thickness skin wounds in athymic mice and to express normal skin antigens after closure of excised wounds in burn patients. METHODS: Data were collected from 17 patients between days 2 and 14 to determine incidence of exudate, incidence of regrafting, coloration, keratinization, and percentage of site covered by graft (n = 17). Outcome was evaluated on an ordinal scale (0 = worst; 10 = best) beginning at day 14, with primary analyses at 28 days (n = 10) and 1 year (n = 4) for erythema, pigmentation, epithelial blistering, surface roughness, skin suppleness, and raised scar. RESULTS: Sites treated with CSSs had increased incidence of exudate (p = 0.06) and decreased percentage of engraftment (p < 0.05) compared with STAG. Outcome parameters during the first year showed no differences in erythema, blistering, or suppleness. Pigmentation was greater, scar was less raised, but regrafting was more frequent in CSS sites than STAG. No differences in qualitative outcomes were found after 1 year, and antibodies to bovine collagen were not detected in patient sera. CONCLUSIONS: These results suggest that outcome of engrafted CSSs is not different from STAG and that increased incidence of regrafting is related to decreased percentage of initial engraftment. Increased rates of engraftment of CSSs may lead to improved outcome for closure of burn wounds, allow greater availability of materials for grafting, and reduce requirements for donor skin autograft.
Animals↗
Isolation of a unique melanogenic inhibitor from human skin xenografts: initial in vitro and in vivo characterization.
Previously, split-thickness human skin grafted onto athymic mice has been shown to become markedly hyperpigmented, but the factor(s) responsible for this hyperpigmentation had not been isolated. The present study describes the isolation and characterization of a potent melanogenic inhibitor from grafted human skin. Extracts from grafted skin inhibited, in a concentration-dependent manner, tyrosinase activity of normal human melanocytes and of Cloudman S91 murine melanoma in culture. Sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis of extracts from pre- and post-grafted skin demonstrated the presence of a protein doublet of approximately 14 kD exclusively in the post-grafted skin. This protein inhibited both tyrosinase activity and cellular proliferation in a concentration-dependent manner. The inhibition of tyrosinase activity in normal human melanocytes was 53% at 0.5 microgram/ml concentration, whereas this inhibition was almost complete in murine melanoma cultures at 1.0 microgram/ml. The protein did not inhibit either cellular proliferation or protein synthesis in normal human fibroblast cultures, and therefore may act specifically on melanocytes. Injections of the inhibitor corresponded with a delay and reduction in the quantity of pigment in human skin 2 weeks after grafting. Multiple injections of the inhibitor into the hyperpigmented xenografts (20 weeks after grafting) reversed the hyperpigmentation with no observable inflammatory or toxic responses. The results indicate that hyperpigmented human skin xenografts contain a potent inhibitor of melanogenesis and melanocyte proliferation.
Animals↗