Ultraviolet light induces binding of antibodies to selected nuclear antigens on cultured human keratinocytes.

Antibodies which bind to different nuclear antigens in tissue sections or in permeabilized cell cultures are useful markers of subsets of connective tissue disease, especially of lupus erythematosus (LE), but whether these antibodies are able to react with these intracellular sequestered antigens in vivo and cause immunologic tissue damage has been a matter of much debate. We report experiments which show that ultraviolet light-irradiated, cultured human keratinocytes bind IgG antibodies from the sera of LE patients with either monospecific anti-SSA/Ro, anti-RNP, or anti-Sm activity, which implies that these antigens have been made accessible on the cell surface by ultraviolet irradiation. Normal human sera or LE patient's sera with anti-double-stranded DNA, anti-single-stranded DNA, or antihistone activity do not bind to the surface of irradiated human keratinocytes. In control experiments, all antisera produced the expected patterns of nuclear and cytoplasmic staining of fixed permeabilized, unirradiated keratinocytes. Careful study of the viability and permeability of irradiated keratinocytes by several techniques showed that this apparent cell membrane expression of extractable nuclear antigens (SSA/Ro, RNP, and Sm) following irradiation was seen on injured keratinocytes whose cell membranes were intact, but not on dead cells. It is particularly significant that all six monospecific anti-SSA/Ro sera bound to irradiated keratinocytes, since this antibody antigen system is highly associated with photosensitive cutaneous LE.

Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.

An improved serum-free culture system has been developed for normal human epidermal keratinocytes (HK). Short-term clonal growth and differentiation studies are routinely performed in a defined medium consisting of optimized nutrient medium MCDB 153 supplemented with epidermal growth factor, insulin, hydrocortisone, ethanolamine, and phosphoethanolamine. A small amount of whole bovine pituitary extract (wBPE) is added for initiation of primary cultures, for frozen storage, and for serial culture. The need for feeder layers, conditioned medium, serum, and specialized culture surfaces has been eliminated entirely. With an optimal level of calcium ion (0.3 mM), colony-forming efficiency is about 30 percent and cellular multiplication rate is 0.96 doublings per day in the defined medium. A high-calcium concentration (1.0 mM) induces stratification and terminal differentiation, which can be quantified by counting cornified envelopes that are resistant to boiling in sodium dodecyl sulfate plus dithiothreitol. Under optimal conditions with wBPE present, cellular senescence occurs after about 40 population doublings. Scanning electron microscopy (SEM) has verified the occurrence of stratification during differentiation in the defined medium with high calcium. High-voltage electron microscopy (HVEM) after detergent extraction of human epidermal keratinocyte (HK) colonies grown in the defined medium with low and high calcium has revealed specific changes in the intermediate filament network and keratohyalin granules corresponding to changes in cellular differentiation. Indirect immunofluorescence studies have verified that the intermediate filament network observed with HVEM is composed of keratin proteins.

Lipid supplemented medium induces lamellar bodies and precursors of barrier lipids in cultured analogues of human skin.

Barrier function of cultured skin substitutes (CSS) is required for their effective use in clinical treatment of skin wounds, and for percutaneous absorption in vitro. Arachidonic, palmitic, oleic, and linoleic free fatty acids, in conjunction with the antioxidant alpha-tocopherol acetate (lipid supplements, "LS"), were added to nutrient media of CSS to provide precursors of epidermal barrier lipids. CSS were composed of human keratinocytes (HK), fibroblasts (HF), and collagen-glycosaminoglycan substrates, and were incubated for 14 d submerged or lifted to the air-liquid interface in media based on MCDB 153 +/- LS. Duplicate samples (30 cm2) were harvested and the epidermal analogue was analyzed for total protein, total DNA, total lipid, lipid fractions including acylglucosylceramide (AGC), and presence of lamellar bodies. Significant increases (p < 0.05) were detected between CSS incubated in +LS medium for total lipid, total DNA, ceramide, glucosylceramide, triglycerides, and diglycerides. AGC and lamellar bodies were detected only in epithelia of CSS incubated in +LS medium. These data show that free fatty acids, vitamin E, and lifting of CSS promote increased epithelial morphogenesis compared to CSS cultured submerged without lipid supplements. Presence of lamellar bodies and AGC suggests enhanced production in vitro of barrier-associated epidermal lipids.

Rate of stratum corneum formation in the perinatal rat.

During late gestation, the fetal rat exhibits marked hyperplasia of the interfollicular epidermis and accelerated cornification in preparation for birth. In this study, we utilized simple morphometric techniques to provide quantitative estimates of the rate of stratum corneum (SC) formation during the perinatal period. Cryostat sections of dorsal epidermis from rat pups between -48 and +72 h of age were expanded under alkaline conditions and the number of corneocyte interfaces counted from photomicrographs. This method yielded the following regression lines: prenatal, y = 0.19x + 13.07, r = 0.93; postnatal, y = 0.13x + 13.00, r = 0.93, where y = number of SC layers and x = age in hours. Lack of desquamation was assured by the postnatal persistence of the granular periderm. Adhesive stripping of the epidermis followed by phase-contrast microscopy revealed intact monolayer sheets of SC. Quantitation using a computerized image analysis system gave an average corneocyte surface area in the newborn rat of 1908 +/- 36 mu 2 (mean +/- SEM). Treatment of neonatal rats with epidermal growth factor (500 ng/g BW) increased the number of SC layers over the first 24 h of life (p < 0.05) and resulted in marked hyperkeratosis by one week of age. These results allow the following conclusions: 1) in the prenatal rat, SC forms at a rate of one layer every 5 h; 2) postnatally, SC formation slows to one layer every 8 h; 3) to support normal corneum synthesis at birth, dorsal keratinocytes must enter transition at a rate of approximately 3 cells/second/cm2 of body surface; and 4) treatment with exogenous EGF augments the rate of terminal differentiation of perinatal rat epidermis.

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.

Reconstitution of the histologic characteristics of a giant congenital nevomelanocytic nevus employing the athymic mouse and a cultured skin substitute.

This study addresses the development of an animal model for human giant congenital nevomelanocytic nevi (GCNN). Skin grafts were made from 1) non-involved split-thickness skin from a 12-month-old GCNN patient, 2) nevus split-thickness skin from the same GCNN patient, 3) nevus full-thickness skin, and 4) cadaveric human split-thickness skin. For groups 1) and 2), human epidermal and dermal cells were enzymatically isolated and expanded in tissue culture. Composite grafts were made by placing the cultured dermal cells into a collagen-glycosaminoglycan (GAG) matrix, followed by placement of the epidermal cells onto the opposite, laminated side of the matrix. All grafts were placed onto full-thickness wounds of athymic mice and biopsies were obtained from 6 to 38 weeks later for light microscopy including S-100 immunoperoxidase staining, and electron microscopy. The GCNN cultured skin mice (group 2) developed black, raised skin in the healed wounds. None of the group 1 mice developed lesions, grossly or histologically. All of the nevus full-thickness mice retained the nevus grossly. Histopathologic examination at 38 weeks of the black, raised plaques of group 2 demonstrated a reconstituted dermis similar to group 3. Nevus cells were larger and more epithelioid in the upper dermis, as seen with true GCNN. These nevomelanocytes were not seen in the dermis at 24 weeks, suggesting that the nevus cells migrated from the epidermal component of the cultured graft to the dermis during this time frame (24-38 weeks). The melanocyte identity of these cells was confirmed with S-100 immunoperoxidase staining and electron microscopy. These findings are unique to this composite cultured graft system. The ability to culture specific types of melanocytes and place them int skin substitutes on athymic mice provides a basis for the study of GCNN and melanocyte biology in vivo.

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.

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.

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.

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.

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.

Study of HLA-DR synthesis in cultured human keratinocytes.

Within the normal human epidermis only Langerhans and indeterminate cells express HLA-DR. Human keratinocytes (HK), however, may also express HLA-DR in certain disease states characterized by mononuclear cell infiltrates. Previous studies have shown that HK synthesize HLA-DR in response to stimulation by interferon gamma (INF-gamma). The purposes of this study were to define conditions under which cultured HK might express HLA-DR and to compare the HLA-DR synthesis of HK with that of monocytes. HLA-DR expression by HK as determined by indirect immunofluorescence of HK cultures was absent under standard low calcium conditions and remained absent with the addition of calcium, serum, mitogens, and supernatants from Pam-212 cells containing epidermal thymocyte-activating factor. HLA-DR expression in HK was induced by cocultivation with concanavalin A-stimulated peripheral blood mononuclear cells (PBMC), but not unstimulated PBMC. This effect was time-dependent and directly related to the number of PBMC. HLA-DR expression was also induced in a time- and dose-dependent manner by addition of supernatant from stimulated PBMC (SS) or by addition of recombinant INF-gamma but not by addition of interleukin (IL)-1 or IL-2. Induction by either SS or INF-gamma was blocked by an antiserum to INF-gamma. As determined by a semiquantitative immunoprecipitation technique, HLA-DR synthesis by HK was directly related to INF-gamma concentration. The pattern of HLA-DR peptides produced by HK was similar to that of monocytes, but the relative quantity synthesized was far less than that of monocytes.

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.

Genetically modified skin to treat disease: potential and limitations.

Molecular definition of disease at the level of the gene and advances in recombinant DNA technology suggest that many diseases are amenable to correction by genes not bearing the defective elements that result in disease. Many questions must be answered before this therapy can be used to correct chronic diseases. These questions fall into safety and efficacy categories. Experience with transplanting cellular elements of skin or skin substitutes (defined as skin that possess the cell types and a dermal structure to develop into a functioning skin) to athymic rodents is considerable and is seen as a system where these questions can be answered. This paper reviews these questions and presents our early analysis of genetically modified cells in skin substitutes in vivo and in vitro. Experimental data demonstrate that both a matrix of woven nylon, housing a fibroblast generated collage, and dead dermis can be utilized to shuttle genetically modified human fibroblasts from the laboratory to an in vivo setting. Genetically modified fibroblasts do not migrate from the shuttle to the surrounding tissue. The survival of significant numbers, approximately 70%, of genetically modified fibroblasts for at least 6 weeks in these shuttles, supports this general approach as having clinical utility. It is also concluded that skin substitute systems can be used to generate a genetically modified skin in vitro that has the capacity to develop into functional skin in vivo. Further, as genetically modified keratinocytes differentiate there is increased production by the transgene, supporting the concept that keratinocytes have true potential as shuttles for therapeutic genes. This work demonstrates that transplantation of systems containing genetically modified cells of the skin can be used to experimentally define many aspects of gene therapy using skin before this technology is taken to the clinic. Examples include determining the effect of gene transduction and expression on structure and function of the genetically modified skin as well as on distant skin and an assessment of the translational capacity of the transgene as function of time and cell number.

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.

Absence of tumorigenicity in athymic mice by normal human epidermal keratinocytes after culture in serum-free medium.

The very rapid growth rate (1 population doubling/day) of normal human epidermal keratinocytes (HK) cultured in serum-free medium can be utilized for wound closure in burn treatment. However, rapid growth in vitro may present the possibility of neoplastic transformation. To investigate this possibility, HK were cultured from primary isolation to large populations in MCDB 153 medium supplemented with epidermal growth factor (EGF, 10 ng/ml), insulin (5 micrograms/ml), hydrocortisone (0.5 micrograms/ml), and Bovine Pituitary Extract (BPE, 70 micrograms/ml). HK were studied for their ability to form tumors in athymic mice after subcutaneous inoculation. Sixteen separate HK strains were inoculated from primary cultures, or from secondary cultures either before or after storage in liquid nitrogen. Transformed cell lines, SCC 13 and FL, derived from human epithelial carcinomata were used as controls for tumor formation. HK formed no tumors (0/79) after 26 weeks incubation, SCC 13 formed nodular tumors (3/5) after 20 weeks incubation, and FL formed tumors (5/5) after 4 weeks incubation. HK cells were not found by histological examination of inoculation sites of keratinocyte cultures derived from primary culture from skin. In contrast, palpable tumors from both SCC 13 and FL were returned to tissue culture and continued to proliferate. These results support the conclusion that the rapid growth rate of human epidermal keratinocytes in vitro can be attributed to permissive culture conditions, and not to neoplastic transformation.

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.

Cultured skin substitutes: a review.

Skin substitutes composed of cultured cells and biopolymers provide alternative materials for study of skin biology and pathology, treatment of skin wounds, safety testing of consumer products, and therapeutic delivery of gene products. Most frequently, substitutes for epidermis consist of cultured keratinocytes and dermal substitutes consist of resorbable biopolymers populated with cultured fibroblasts. Preclinical models characterize cellular morphogenesis, antigen expression, and barrier properties in vitro, and recovery of tissue function after grafting. Clinical considerations include time required to prepare cultured autografts, time required for graft vascularization, management of microbial contamination in wounds, mechanical fragility of cultured grafts, and high cost. Safety in graft preparation generally requires the use of materials and procedures that comply with standards for quality assurance. Efficacy of engineered skin substitutes has been evaluated predominantly by subjective criteria, but evaluation may become more objective and quantitative by application of noninvasive biophysical instrumentation. Future directions with engineered skin substitutes are expected to include gene therapy by addition or deletion of selected gene products and establishment of international standards for fabrication and assessment of engineered skin.

Biologic attachment, growth, and differentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-sulfate substrate.

Repair of full-thickness burns requires replacement of both the dermal and the epidermal components of the skin. Use of tissue culture methods allows very large expansions of surface area to be covered by cultured normal human epidermal keratinocytes (HK). Porous and resorbable materials, such as collagen and chondroitin-6-sulfate membranes, may be expected to adhere to wounds and promote fibrovascular ingrowth better than grafts of cultured epidermal keratinocytes alone. This article demonstrates the in vitro formation of biologic attachments between HK and a collagen and chondroitin-6-sulfate dermal skin replacement. Dermal membranes are prepared as generic acellular sheets and stored in the dry state for extended periods. Subconfluent HK cultures in logarithmic phase growth can attach quickly to dermal membranes in vitro, form a confluent epithelial sheet on the surface of each membrane, and exhibit mitotic cells for at least 1 week in vitro. Transmission electron microscopy demonstrates the formation of hemidesmosomes, extracellular matrix, and banded collagen at the interface of the epidermal cells and the dermal membrane. By comparison, HK cultures as confluent sheets released enzymatically with Dispase do not attach to the dermal membranes in vitro, under the conditions tested, although complete coverage of the membrane by the cell sheets is obtained. Growth assays show that subconfluent HK cells retain sufficient growth potential to maintain logarithmic phase growth, but that HK cells disaggregated from confluent sheets become growth arrested in comparison. The composite material has discrete dermal and epidermal compartments, has total thickness comparable to split-thickness skin graft, and can be applied to full-thickness skin defects in a single procedure.

Skin wound closure in athymic mice with cultured human cells, biopolymers, and growth factors.

Skin wound closure remains a major problem in acute and reconstructive skin grafting after large burns because of limited availability of donor skin. This report evaluates six protocols for preparation in vitro of skin substitutes composed of cultured human cells, biopolymers, and growth factors for wound closure. Full-thickness wounds in athymic mice treated in a single procedure with cultured skin substitutes were compared directly to treatments with murine skin autograft, human skin xenograft, or no graft. Rectilinear planimetry of healed wounds 6 weeks after surgery showed that skin substitutes cultured in serum-free medium, and for 24 hours before surgery in defined medium with basic fibroblast growth factor (100 ng/ml), were not statistically different (p less than 0.05) in size from treatment with human skin xenograft. Acceptance and persistence of skin substitutes cultured in serum-free media were 70% at 6 weeks after surgery, as determined by staining of healed skin with a fluorescein-labeled monoclonal antibody against human HLA-ABC antigens. Ultrastructural examination of wounds with cultured human skin 6 weeks after treatment showed complete basement membrane, including anchoring fibrils, presence of melanocytes and pigment transfer to keratinocytes, and innervation of healed skin adjacent to basement membrane. These findings demonstrate effectiveness of cultured skin substitutes for closure of skin wounds and illustrate important capabilities to modulate the natural processes of wound repair, to increase supply of materials used for wound repair, and to enhance quality of wound healing.