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Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient.

Three consecutive studies were performed in 58 patients evaluating the effect of occlusion on the healing of partial-thickness wounds. Mirror-image donor sites were covered with the occlusive hydrocolloid dressing (HCD) (DuoDerm) and compared to fine mesh gauze, and the HCD was subsequently compared to a semi-occlusive dressing of polyurethane film, (Op-site). In addition, partial-thickness burn wounds were covered with the HCD and the remaining burn wound was treated with silver sulfadiazine. The donor sites and burn wounds treated with HCD healed significantly faster than those covered with fine mesh gauze or silver sulfadiazine (p less than 0.001) and with less pain. The HCD and polyurethane film were equivalent. There were no clinical infections with the wounds that were occluded. The exudate collected beneath the DuoDerm and Op-site on donor sites was added to the tissue culture system and resulted in a modest increase in keratinocyte proliferation. However, the exudate from burn wounds under HCD resulted in a marked increase in cell proliferation (p less than 0.001).

We treated five adult individuals with six full-thickness chronic ulcerations in the skin caused by venous insufficiency, sickle cell anemia, or surgical wounds. Each patient received applications to the ulcerations of sheets of autologous epidermal cells grown in culture. All patients experienced relief of pain after grafting. Four of the six ulcers healed completely in 21 to 35 days, and three of the four remained healed for up to 2 years. One ulceration recurred within 2 months. Our experience suggests that cultured autologous epidermal grafts can provide continuous covering, relief from pain, and rapid healing of chronic debilitating ulcerations of the skin.

Results of previous in vivo experiments indicated that the presence of arterial endothelium modifies cholesteryl ester (CE) metabolism and the retention of low density lipoproteins (LDL) in injured arteries. We describe herein the effects of bovine arterial endothelial cells (ENDO) on the CE cycle, fluid phase endocytosis, and cell proliferation in co-cultured bovine arterial smooth muscle cells (SMC). Following several days of cultivation on confluent SMC, ENDO were removed from SMC by treatment of the co-cultures with 1.0% collagenase (type II). Removal of only ENDO from the co-culture dishes was confirmed by immunofluorescent staining for Factor VIII antigen, hemotoxylin-eosin staining, and biochemical analyses. We observed that ENDO grown to 75% confluency on confluent SMC induced: 1) a reduction of CE hydrolysis as a result of decreased lysosomal CE hydrolytic activity in SMC as compared to SMC cultured alone; and 2) an increase in the rate of incorporation of labeled oleate into CE as a result of increased acyl CoA:cholesterol O-acyltransferase activity in SMC as compared to SMC cultured alone. Neither endothelial cell-derived culture media (ECDM) nor fibroblasts modulated CE metabolism in co-cultured SMC. Additional experiments showed that the presence of endothelial cells or ECDM decreased the proliferation of co-cultured SMC by 50%, but enhanced the endocytotic rate of labeled sucrose into SMC threefold. Results of experiments described herein demonstrate that, in addition to providing a thrombo-resistant surface and regulating permeability, endothelial cells may also serve to modulate cholesteryl ester metabolism in smooth muscle cells derived from the arterial wall.

These studies present evidence that when human epidermal cells are grown in culture they lose both the ability to stimulate the proliferation of allogeneic T lymphocytes in vitro and their expression of HLA-DR antigens. Our results also show that epidermal cells incubated with anti-HLA-DR serum lose their ability to stimulate the proliferation to allogeneic T lymphocytes in a mixed skin cell-lymphocyte reaction.

Flow cytometry revealed that, in the presence of tritiated thymidine, a greater percentage of phytohemagglutinin-stimulated lymphocytes from old human donors were arrested in the G2 or M phase than were cells from young donors. Furthermore, lymphocytes from old donors showed significantly more chromosomal damage than did lymphocytes from young donors. Lymphocyte cultures from old or young donors not exposed to tritiated thymidine had the same percentage of cycling lymphocytes in G2 or M, although the number of lymphocytes stimulated by phytohemagglutinin to enter the cell cycle was significantly lower in cultures from old donors. Thus, the impaired incorporation of tritiated thymidine by phytohemagglutinin-exposed lymphocytes from old humans reflects both an impaired proliferative response to phytohemagglutinin and an increased sensitivity to the radiobiological effects of tritiated thymidine.

We tested the hypothesis that prostacyclin (PGI2), 6-keto-prostaglandinF1 alpha(6-keto-PGF1 alpha), and several E series prostaglandins (PG) may affect the activity of cholesteryl ester (CE) hydrolase since our previous experiments indicated that smooth muscle cells (SMC) in neointima of injured rabbit aorta (a) acquire the capacity to produce PGI2 and (b) have increased lysosomal CE hydrolytic (acid cholesteryl ester hydrolase [ACEH])activity. Using cultured SMC from rabbit thoracic aorta, we demonstrated that PGI2, 6-keto-PGF1 alpha, and 6-keto-PGE1 enhanced ACEH activity fourfold. No significant effects on ACEH activity were observed with PGE1 or PGE2. Preincubation of SMC with an inhibitor of adenylate cyclase activity (dideoxyadenosine) abolished the effect of these PG on CE hydrolytic activity. Addition of dibutyryl cAMP to these SMC significantly increased ACEH activity. Although concentrations of PGI2 used significantly increased cAMP levels, proliferation of these SMC was not observed. In related experiments, we determined if the addition of PGI2, 6-keto-PGF1 alpha, or 6-keto-PGE1 to cultured aortic SMC would enhance the egress of unesterified cholesterol and CE from these SMC. A significant loss of total cholesterol from PG-treated SMC was observed at the end of 14 d. Results suggest that increased synthesis of PGI2 by neointimal SMC in the injured aortic wall may, at least in part, explain the changes in CE catabolism and accumulation following injury. These PG may also be important in CE metabolism and accumulation in human arteries.

Arterial endothelial cells (EC) or their conditioned medium (ECCM) can alter the proliferation of cocultured arterial smooth muscle cells (SMC). Previously, we have shown, as have others, that EC regulate the growth of cocultured SMC depending on the density of both cell types. To ascertain the rate of cell-cycle traverse in preconfluent arterial SMC cocultured with arterial EC or ECCM (derived from preconfluent EC), we have conducted a series of stathmokinetic experiments using flow cytometry to determine where specific changes may occur in the cell cycle. Results of our experiments indicate for the first time that ECCM stimulates the proliferation of preconfluent SMC by significantly shortening the residence times in the G1 and S phases of the cell cycle. The predominant relative effect occurs within the early G1 (G1A) compartment where pretreatment with ECCM shortens the residence time by approximately 55%. Furthermore, we have observed that preincubation of serum-free ECCM with antiplatelet-derived growth factor (PDGF) antibody abolishes any mitogenic effect on SMC. This suggests that EC secrete PDGF-like molecules which enhance the proliferation rate of preconfluent, cocultured SMC. These findings support the hypothesis that arterial EC may secrete mitogens which stimulate arterial SMC proliferation in the vascular wall.

Stratification of human epidermal cells into multilayered sheets composed of basal and suprabasal layers (resembling the stratum germinativum and stratum spinosum of the epidermis) was studied in a dermal component-free culture system. Although no stratum corneum developed in vitro, this culture system provided a method to study early events in human keratinocyte differentiation. Multiparameter flow cytometric analysis of acridine orange-stained epidermal cells from these cultures revealed three distinct subpopulations differing in cell size, RNA content, and cell cycle kinetics. The first subpopulation was composed of small basal keratinocytes with low RNA content and a long generation time. The second subpopulation consisted of larger keratinocytes, having higher RNA content and a significantly shorter generation time. Finally, the third subpopulation contained the largest cells, which did not divide, and represent the more terminally differentiated keratinocytes. This in vitro approach provides discriminating cytochemical parameters by which the maturity of the epidermal cell sheets can be assessed prior to grafting onto human burn patients.

Twenty-six individuals with second- and third-degree burn wounds have been grafted with cultured allogeneic epidermal cells. These epidermal cell grafts were grown in culture from cadaver skin according to a technique which we have developed. After being grafted with cultured allogeneic epidermal cells, superficial wounds, e.g., donor sites, healed within 7 days, compared to 14 days for mirror image control sites. Deep second-degree burn wounds which were excised before grafting with cultured cells healed in a mean time of 10 days. Deep second-degree burn wounds which were not excised before grafting healed in a mean time of 14 days. The cultured cells produced rapid healing in 11 of the 12 patients with deep second-degree burn wounds. The deep second-degree wounds grafted with cultured allogeneic epidermal cells healed with results which were comparable to the deep second-degree wounds which were autografted. Grafts of cultured allogeneic epidermal cells placed on full-thickness, or third-degree burn, wounds did not grow well.