Assessment of replication rates of human keratinocytes in engineered skin substitutes grafted to athymic mice.

Stable closure of skin wounds with engineered skin substitutes (ESS) requires indefinite mitotic capacity to generate the epidermis. To evaluate whether keratinocytes in ESS exhibit the stem cell phenotype of label retention, ESS (n = 6-9/group) were pulsed with 5-bromo-2'-deoxyuridine (BrdU) in vitro, and after grafting to athymic mice (n = 3-6/group). Pulse and immediate chase in vitro labeled virtually all basal keratinocytes at day 8, with label uptake decreasing until day 22. Label retention in serial chase decreased more rapidly from day 8 to day 22, with a reorganization of BrdU-positive cells into clusters. Similarly, serial chase of labeled basal keratinocytes in vivo decreased sharply from day 20 to day 48 after grafting. Label uptake was assessed by immediate chases of basal keratinocytes, and decreased gradually to day 126, while total labeled cells remained relatively unchanged. These results demonstrate differential rates of label uptake and retention in basal keratinocytes of ESS in vitro and in vivo, and a proliferative phenotype with potential for long-term replication in the absence of hair follicles. Regulation of a proliferative phenotype in keratinocytes of ESS may improve the biological homology of tissue-engineered skin to natural skin, and contribute to more rapid and stable wound healing.

Improvement of epidermal barrier properties in cultured skin substitutes after grafting onto athymic mice.

Barrier function in cultured skin substitutes (CSS) prepared from human cell sources was measured by noninvasive (surface hydration, transepidermal water loss) and invasive methods (water permeation, niacinamide flux) before and after grafting onto athymic mice. In vitro measurements were made on days 7 and 14. Although three of the four measures of barrier function improved markedly from day 7 to 14, the values obtained were still far from those obtained with native human skin controls. Additional CSS were grafted onto athymic mice on day 14, and skin was harvested 2 and 6 weeks after grafting. Grafting brought about a substantial decrease in all measurements by 2 weeks and almost complete normalization of barrier function after 6 weeks. The most sensitive measure of this recovery was niacinamide permeability, which decreased from (280 +/- 40) x 10(-4) cm/h in vitro to (17 +/- 30) x 10(-4) cm/h 2 weeks after grafting and (5 +/- 2) x 10(-4) cm/h 6 weeks after grafting, versus control values of (2 +/- 2) x 10(-4) cm/h in human cadaver skin and (0.6 +/- 0.4) x 10(-4) cm/h in human epidermal membrane prepared from freshly excised breast skin. These results demonstrate the reformation of epidermal barrier function after transplantation and provide insights for the development of a functional epidermal barrier in CSS in vitro.

Increased expression of integrins and decreased apoptosis correlate with increased melanocyte retention in cultured skin substitutes.

Losses of human melanocytes (HM) in transplantation of cultured skin substitutes (CSS) may result from poor cellular attachments. To test this hypothesis, HM integrin expression was measured in four culture media: (a) melanocyte growth medium (MGM), an HM proliferation medium; (b) UCMC 160, a CSS maturation medium; (c) mMGM, modified MGM with 1.8 mM calcium; and (d) modified UCMC 160 with HM supplements (mUCMC 160). HM grew well in all media except UCMC 160. Increased expression of beta1, beta4, alpha3beta1 and alpha5 integrins on HM cultured in MGM and mMGM versus UCMC 160 was found by flow cytometry. Annexin V-allophycocyanin (APC) labeled HM in apoptosis and increased significantly in UCMC 160 (31.1%) compared with MGM (11.9%) or mMGM (13.9%). CSS were incubated in UCMC 160, mMGM or mUCMC 160 media, and grafted to athymic mice. In the mMGM group, grafts were darker as measured with a chromameter through 6 weeks and the average number of basal HM per field was greater at 12 weeks post-grafting. Increased graft loss was observed in the mMGM group which corresponded with the poor epidermal morphology in vitro. Although HM retention improved in vivo using mMGM to culture the CSS, the stability of the epidermis decreased. These results indicate that expression of integrins on HM in vitro correlates with HM retention in CSS and short-term survival after transplantation, but that long-term survival depends also on stable epithelium.

Evaluation of cytotoxicity and antimicrobial activity of Acticoat Burn Dressing for management of microbial contamination in cultured skin substitutes grafted to athymic mice.

Cultured skin substitutes (CSS) have become a useful adjunctive treatment for closure of burn wounds, but CSS are avascular and remain susceptible to microbial destruction longer than split-thickness skin grafts. Irrigation of CSS grafted to burn wounds with a topical antimicrobial solution (TAS) has been shown to promote engraftment of CSS, but TAS usage has potential limitations. Acticoat Burn Dressing (Acticoat; Westaim Biomedical, Exeter, NH) is a silver-coated barrier dressing reported to exhibit antimicrobial activity and to reduce infection in partial-thickness and full-thickness wounds. This study evaluated the cytotoxicity of Acticoat with CSS and the efficacy of Acticoat for the management of microbial contamination in CSS grafted to full-thickness wounds in athymic mice. The cytotoxicity of Acticoat was assessed in preliminary studies after 1 week of exposure to CSS during in vitro maturation or healing on wounds in athymic mice. Histologies were analyzed and cellular viability in the CSS was determined by MTT conversion on days 0, 1, and 7 of Acticoat exposure. At 1, 2, 3, and 4 weeks after grafting, wounds were traced, and areas of healing CSS were calculated by image analysis. At 4 weeks, wound biopsies were evaluated and scored for engraftment of human cells. In a subsequent study, wounds were inoculated with strain SBI-N of Pseudomonas aeruginosa at 1 x 10(5) cfu/wound before the application of CSS or inoculated onto the surface of Acticoat. At 4 weeks, swab cultures were collected from the surface of CSS and scored for the presence of SBI-N. Statistical significance was accepted at the 95% confidence level (P <.05). The data show that exposure in vitro of CSS to Acticoat was cytotoxic within 1 day, but 1 week of exposure in vivo did not injure CSS or inhibit wound healing. Contaminated wounds treated with Acticoat healed similarly to control treatments, with comparable rates of engraftment, and detection of SBI-N on the surface of only one graft. No SBI-N was detected on CSS after inoculation onto the surface of Acticoat. These results suggest that Acticoat may be suitable as a protective dressing to reduce environmental contamination of CSS, if used in conjunction with additional antimicrobials to control organisms present in the wound.

Assessment of a silver-coated barrier dressing for potential use with skin grafts on excised burns.

Acticoat burn dressing is a silver-coated dressing with antimicrobial activity purported to reduce infection from environmental organisms in partial and full-thickness wounds. Acticoat was tested for activity as an antimicrobial treatment and as an antimicrobial barrier dressing in three in vitro assays. It was found that a modified disc assay method gave false negative results but in an assay in which bacteria were inoculated on top of samples of Acticoat, bacterial numbers were reduced, over time, with all microorganisms tested. Acticoat served as a barrier for bacteria, inoculated onto it, from contaminating the surface of an agar plate under the Acticoat. The data show that Acticoat has: antimicrobial capabilities, but to be effective hours of contact between Acticoat and the microorganisms are required; and the capacity to serve as an antimicrobial barrier dressing. These findings support the conclusion that Acticoat has activity to reduce microbial contamination of wounds from environmental sources.

Regulation of cutaneous pigmentation by titration of human melanocytes in cultured skin substitutes grafted to athymic mice.

Pigmentation of healed cultured skin substitutes in burn patients is frequently irregular and unpredictable which compromises solar protection and the patient's self-image. To address these morbidities, human fibroblasts were inoculated on a collagen-glycosaminoglycan substrate followed 1 day later by the addition of keratinocytes at 1.1 x 10(6)/cm2 combined with either 0, 1.1 x 10(2), 1.1 x 10(3), or 1.1 x 10(4) melanocytes/cm2. The skin substitutes were incubated in vitro for 3 weeks and grafted to athymic mice. In vitro, the number of L-Dopa-positive melanocytes in the skin substitutes increased proportionately to the number of melanocytes inoculated. The melanocytes localized to the basal epidermis when labeled for MEL-5. The skin substitutes with 1.1 x 10(4) melanocytes/cm2 were significantly darker than other groups in vitro by chromameter evaluation. By 12 weeks after grafting, the cultured skin ranged from no pigment in the control group, to 75% pigmented area in the 1.1 x 10(3) melanocytes/cm2 group, to complete pigmentation in the 1.1 x 10(4) melanocytes/cm2 group. In vivo, the mean chromameter values were significantly darker for the grafts with 1.1 x 10(3) and 1.1 x 10(4) melanocytes/cm2. These results suggest that complete restoration of cutaneous pigmentation can be accomplished by addition of between 0.1 and 1.0 x 10(4) melanocytes/cm2 to skin substitutes.

Vitamin C regulates keratinocyte viability, epidermal barrier, and basement membrane in vitro, and reduces wound contraction after grafting of cultured skin substitutes.

Cultured skin substitutes have become useful as adjunctive treatments for excised, full-thickness burns, but no skin substitutes have the anatomy and physiology of native skin. Hypothetically, deficiencies of structure and function may result, in part, from nutritional deficiencies in culture media. To address this hypothesis, vitamin C was titrated at 0.0, 0.01, 0.1, and 1.0 mM in a cultured skin substitute model on filter inserts. Cultured skin substitute inserts were evaluated at 2 and 5 wk for viability by incorporation of 5-bromo-2'-deoxyuridine (BrdU) and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion. Subsequently, cultured skin substitute grafts consisting of cultured human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates were incubated for 5 wk in media containing 0.0 mM or 0.1 mM vitamin C, and then grafted to athymic mice. Cultured skin substitutes (n = 3 per group) were evaluated in vitro at 2 wk of incubation for collagen IV, collagen VII, and laminin 5, and through 5 wk for epidermal barrier by surface electrical capacitance. Cultured skin substitutes were grafted to full-thickness wounds in athymic mice (n = 8 per group), evaluated for surface electrical capacitance through 6 wk, and scored for percentage original wound area through 8 wk and for HLA-ABC-positive wounds at 8 wk after grafting. The data show that incubation of cultured skin substitutes in medium containing vitamin C results in greater viability (higher BrdU and MTT), more complete basement membrane development at 2 wk, and better epidermal barrier (lower surface electrical capacitance) at 5 wk in vitro. After grafting, cultured skin substitutes with vitamin C developed functional epidermal barrier earlier, had less wound contraction, and had more HLA-positive wounds at 8 wk than without vitamin C. These results suggest that incubation of cultured skin substitutes in medium containing vitamin C extends cellular viability, promotes formation of epidermal barrier in vitro, and promotes engraftment. Improved anatomy and physiology of cultured skin substitutes that result from nutritional factors in culture media may be expected to improve efficacy in treatment of full-thickness skin wounds.