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.

Assessment with the dermal torque meter of skin pliability after treatment of burns with cultured skin substitutes.

The assessment of visco-elastic (V-E) properties in cutaneous scars is critical to reduction of impairment and restoration of function after grafting of excised burns. Cultured skin substitutes (CSS) that consist of autologous keratinocytes and fibroblasts attached to biopolymer substrates are alternatives for permanent closure of excised, full-thickness burns, but assessment of scarring has been subjective. V-E properties of CSS were measured with a Dia-Stron Dermal Torque Meter (DTM 310, Dia-Stron, Ltd, Broomall, Pa), which applies a constant torque (10 mNm) for a fixed interval (10 seconds) and measures rotational deformation and recovery. Parameters of skin deformation were measured in patients (n = 10) after grafting of CSS or meshed skin autograft. Native human skin (NHS) of healthy volunteers (n = 13) served as the control. Skin healed after treatment with CSS or autograft was evaluated for 1 year or longer after grafting. Elastic stretch (Ue), viscous stretch (Uv), total extensibility (Uf), elastic recovery (Ur), total recovery (Ua), and residual plasticity (R) were measured as degrees of rotation, were tested for significance (P < .05) by Student t test comparisons between treatment groups and controls, and were subjected to regression analysis. Assessment of burn scar with the Dermal Torque Meter detected time-dependent increases of all individual parameters of V-E properties for both CSS and autograft during the first year after grafting. At 1 year or later, no statistical differences were found between CSS and autograft for individual parameters, but Ue and Ur for autograft were significantly lower than for NHS. At 1 year or longer, autograft was significantly different from CSS or NHS, with a greater ratio of Uv to Ue, and both graft types had a lower ratio of Ur to Uf than NHS had. These results suggest that instrumental measurement of scar pliability may increase objectivity in assessment of patient recovery and establish an absolute scale for quantitative analysis of V-E properties in skin after grafting of conventional or alternative skin substitutes.

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.

Topical sulfamylon reduces engraftment of cultured skin substitutes on athymic mice.

Sulfamylon (mafenide acetate) remains extremely valuable for the control of the bacterial contamination of burn wounds, but it is cytotoxic to cultured keratinocytes used for wound closure. Because composite skin substitutes develop a partial epidermal barrier in vitro, they may hypothetically tolerate the use of topical Sulfamylon. To test this hypothesis, cultured skin substitutes were prepared from cultured human fibroblasts; keratinocytes were attached to these collagen-based substrates, which were grafted to full-thickness wounds in athymic mice (n = 8 per group). Wounds were irrigated twice daily with 5% (wt/vol) Sulfamylon solution or with a formulation of noncytotoxic antimicrobials (0% Sulfamylon). On day 9 after grafting, the wounds were treated with dry dressings and assessed at 4 weeks for expression of human leukocyte antigens-A, B, C and at 2, 3, and 4 weeks for percentage of original wound area and surface electrical capacitance in picofarads (pF). Data were analyzed for statistical significance (P < .05) by Fisher's exact test, Student's t test, and repeated measures analysis of variance: [table: see text] The data demonstrate that irrigation of cultured skin substitutes with a solution of 5% Sulfamylon results in smaller wound area, fewer wounds that contain human cells, and greater surface hydration (higher surface electrical capacitance) than irrigation with noncytotoxic antimicrobial agents. These results support the conclusion that cultured skin substitutes of this type do not tolerate the chemical toxicity of Sulfamylon as well as skin autografts. Further improvements in the properties of the epidermal barrier of cultured skin substitutes may facilitate the use of Sulfamylon or other potent antimicrobial agents for the management of microbial contamination during engraftment of transplanted skin cells.

Incubation of cultured skin substitutes in reduced humidity promotes cornification in vitro and stable engraftment in athymic mice.

Cultured skin substitutes have been used successfully for adjunctive treatment of excised burns and chronic skin wounds. However, limitations inherent to all models of cultured skin include deficient barrier function in vitro, and delayed keratinization after grafting in comparison to native skin autografts. Experimental conditions for incubation of skin substitutes were tested to stimulate barrier development before grafting, and measure responses in function and stability after grafting. Cultured skin substitutes consisted of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan biopolymer substrates. Parallel cultured skin substitutes were incubated at the air-liquid interface in ambient (48-61%) or saturated (79-91%) relative humidity, and grafted to athymic mice on culture day 14. Additional cultured skin substitutes were incubated in the experimental conditions for a total of 28 days. Cadaveric human skin and acellular biopolymer substrates served as controls. Epidermal barrier was evaluated as the change in surface hydration by surface electrical capacitance with the NOVA Dermal Phase Meter. Cultured skin substitutes and cadaveric skin incubated in ambient humidity had lower baseline surface electrical capacitance and less change in surface electrical capacitance than parallel samples incubated in saturated humidity at all time points in vitro. Data from healing cultured skin substitutes at 2, 4, 8 and 12 weeks after grafting showed an earlier return to hydration levels comparable to native human skin, and more stable engraftment for skin substitutes from ambient humidity. The data indicate that cultured skin substitutes in ambient humidity have lower surface electrical capacitance and greater stability in vitro, and that they reform epidermal barrier more rapidly after grafting than cultured skin substitutes in saturated humidity. These results suggest that restoration of functional epidermis by cultured skin substitutes is stimulated by incubation in reduced humidity in vitro.

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.

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.

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.

Effective management of microbial contamination in cultured skin substitutes after grafting to athymic mice.

Cultured skin substitutes have become therapeutic alternatives for treatment of acute and chronic skin wounds, but all models of these substitutes are avascular and susceptible to microbial destruction during vascularization. To develop a practical management protocol for increased survival of skin substitutes, experimental wounds were contaminated with Pseudomonas aeruginosa and treated with a formulation of noncytotoxic antimicrobial agents (polymyxin B, neomycin, ciprofloxacin, mupirocin, amphotericin B) in a nutrient medium (vehicle). Cultured skin substitutes consisting of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan sponges were grafted to 2 x 2 cm full-thickness wounds on athymic mice that were contaminated with the strain SBI-N of P. aeruginosa at 1 x 10(4), 1 x 10(5), and 1 x 10(6) organisms/wound. Experimental wounds were irrigated with 1 ml/day topical antimicrobial solution for 10 days, and controls received vehicle only. Two, three, and four weeks after grafting, wounds were traced and swabbed for microbial culture and areas were measured with planimetry. At 4 weeks, biopsy samples were scored histochemically for immunoreactivity to HLA antigens. Data analysis by chi-square, analysis of variance, and Tukey's test shows that treatment of contaminated wounds with noncytotoxic topical antimicrobials is associated with an increased area of healed wounds, positive detection of HLA antigens, and negative cultures for P. aeruginosa. These results show that microbial contamination of cultured skin substitutes on full-thickness wounds may be managed effectively during graft vascularization. However, this formulation of antimicrobial agents is not currently approved for human use and is investigational only. Effective management of microbial contamination suggests that clinical efficacy of avascular tissue analogs may be increased by local application of noncytotoxic antimicrobial agents.

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.

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.

Surface electrical capacitance as an index of epidermal barrier properties of composite skin substitutes and skin autografts.

Restoration of the epidermal barrier is a requirement for burn wound closure. A rapid, reliable, and noninvasive measure of the rate of restoration of the epidermal barrier is not readily available. To monitor the reformation of the epidermal barrier, we measured surface electrical capacitance on cultured skin substitutes (human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates) and split-thickness skin autografts grafted to patients. Data were collected from four patients with burns and one pediatric patient with a congenital hairy nevus comprising > 60% total body surface area. Capacitance measurements were performed at days 7, 10, 12, 14, and 28 by direct contact of the capacitance probe for 10 seconds to the cultured skin substitutes or split-thickness autograft. On postoperative days 7, 10, 12, 14, 21, and 28, the surface electrical capacitance of cultured skin substitutes after 10 seconds of sampling was 2468 +/- 268, 1443 +/- 439, 129 +/- 43, 200 +/- 44, 88 +/- 20, and 74 +/- 19 picofarads (mean +/- standard error of the mean), respectively. Surface electrical capacitance for split-thickness autograft on the same days was 1699 +/- 371, 1914 +/- 433, 125 +/- 16, 175 +/- 63, 110 +/- 26, 271 +/- 77 picofarads, respectively. Surface electrical capacitance in all of the grafts decreased with time. Cultured skin substitutes had approximately the same 10-second capacitance values as split-thickness autograft during 3 weeks of healing and approached values for uninjured skin (32 +/- 5 picofarads) by 12 days. Measurement of surface electrical capacitance is a direct, inexpensive, and convenient index for noninvasive monitoring of epidermal barrier formation.

Topical nutrients promote engraftment and inhibit wound contraction of cultured skin substitutes in athymic mice.

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.

Delivery and activity of antimicrobial drugs released from human fibrin sealant.

Engraftment and healing of native or cultured skin grafts depend on adherence, vascularization, and control of microbial contamination in the wound bed. Fibrin sealant is a biocompatible polymer that may be used to promote skin engraftment by serving as a delivery vehicle for antimicrobial drugs. Human fibrin sealant (25 mg/ml) was polymerized with antibacterial agents (mupirocin [32 micrograms/ml], nitrofurazone [0.02% wt/vol], polymyxin B [400 U/ml], or norfloxacin [20 micrograms/ml]) on nitrocellulose (nc) backing and was prepared as 6 mm diameter discs with skin punches. Discs (n = 6) were applied in the Wet Disc Assay to clinical isolates of Staphylococcus aureus (mupirocin, nitrofurazone) or Pseudomonas aeruginosa (polymyxin B, norfloxacin). Controls included drug applied to 6 mm paper discs (25 microliter) and nitrocellulose discs submerged in each drug, blotted, and applied to bacterial cultures on agar in petri dishes. Data were expressed as zone of clearing (mm diameter +/- SEM) after overnight incubation at 35 degrees C. Significant differences (ANOVA and Turkey's test, p < 0.05) were found for each drug released from the disc of fibrin sealant compared with other vehicles. Release from filter paper discs compared with nitrocellulose was significant for nitrofurazone and norfloxacin. Serial transfer of fibrin discs to fresh bacterial cultures after 24 hours showed no zones of clearing. The data show that fibrin sealant releases topical drugs with no inhibition of antimicrobial activity on burn organisms. Greater zones of clearing from fibrin sealant may result from passive fluid retention or from active binding to fibrin followed by protease digestion by burn organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Attachment of an aminoglycoside, amikacin, to implantable collagen for local delivery in wounds.

Cultured skin substitutes consisting of implantable collagen (COL) and cultured human skin cells often fail clinically from destruction by microbial contamination. Hypothetically, addition of selected antimicrobial drugs to the implant may control microbial contamination and increase healing of skin wounds with these materials. As a model for drug delivery, bovine skin COL (1 mg/ml) and amikacin (AM; 46 micrograms/ml) were modified by covalent addition of biotin (B-COL and B-AM, respectively) from B-N-hydroxysuccinimide and bound together noncovalently with avidin (A). B-COL was incubated with A and then with B-peroxidase (B-P) or by serial incubation with B-AM and B-P, before P-dependent chromogen formation. Colorimetric data (n = 12 per condition) from spot tests on nitrocellulose paper were collected by transmission spectrophotometry. Specificity of drug binding in spot tests was determined by (i) serial dilution of B-COL; (ii) reactions with COL, AM, or P that had no B; (iii) removal of A; or (iv) preincubation of B-COL-A with B before incubation with B-P. Binding of B-AM was (i) dependent on the concentration of B-COL; (ii) specific to B-COL, A, and B-P (P < 0.05); and (iii) not eluted by incubation in 0.15 or 1.0 M NaCl. B-AM was found to block binding of B-P to the B-COL-A complex and to retain bacteriocidal activity against 10 clinical isolates of wound bacteria in the wet disc assay. Antimicrobial activity of B-AM was removed from solution by treatment with magnetic A and a permanent magnet. These results suggest that selected antimicrobial drugs can be biotinylated for attachments to COL-cultured cell implants without loss of pharmacologic activity. Because this chemistry utilizes a common ligand, any molar ratio of agents may be administered simultaneously and localized to the site of implantation.

Pigmentation and inhibition of wound contraction by cultured skin substitutes with adult melanocytes after transplantation to athymic mice.

Wound closure with cultured skin substitutes results in epithelium that is consistently hypopigmented. Hypothetically, addition of human melanocytes to cultured skin grafts may result in normal pigmentation of healed skin. Skin substitutes were composed of human epidermal keratinocytes and melanocytes, dermal fibroblasts, and collagen-glycosaminoglycan substrates, and were incubated for 12 d in media for keratinocyte growth (KG, n = 4), for keratinocyte differentiation containing four fatty acids and vitamin E with basic fibroblast growth factor (KDF, n = 6) or epidermal growth factor (KDE, n = 6), or for melanocyte growth (MG, n = 6) with phorbol ester and 5% fetal bovine serum. Skin substitutes were grafted orthotopically to full-thickness skin wounds (2 x 2 cm) on athymic mice, and scored for percent original wound size (+/- SEM), visible pigmentation (number pigmented/n), and positive staining for human leukocyte antigens (HLA)-ABC after 6 weeks on the mice. The data show that cultured skin grafts containing human melanocytes that are incubated in KDE or MG media have statistically significant reduction in wound contraction, 1:1 correlation of expression of pigment and HLA-ABC, and increased frequency of pigmentation after healing compared to incubation in KG or KDF media. Transmission electron microscopy confirmed the presence of melanocytes, melanosomes, and pigment transfer to keratinocytes in pigmented skin. These results suggest that survival and differentiated function of cultured epithelium can support melanization of skin, and that skin analogues exposed to phorbol ester in vitro can support skin pigmentation after wound healing.