Biomedical subjects

S Regauer

Publications and source records attributed to S Regauer.

45 records · Page 3Linked to original sources

PubMed1990

Epithelial origin of cutaneous anchoring fibrils.

Anchoring fibrils are essential structural elements of the dermoepidermal junction and are crucial to its functional integrity. They are composed largely of type VII collagen, but their cellular origin has not yet been confirmed. In this study, we demonstrate that the anchoring fibrils are primarily a product of epidermal keratinocytes. Human keratinocyte sheets were transplanted to a nondermal connective tissue graft bed in athymic mice. De novo anchoring fibril formation was studied ultrastructurally by immunogold techniques using an antiserum specific for human type VII procollagen. At 2 d after grafting, type VII procollagen/collagen was localized both intracellularly within basal keratinocytes and extracellularly beneath the discontinuous basal lamina. Within 6 d, a subconfluent basal lamina had developed, and newly formed anchoring fibrils and anchoring plaques subjacent to the xenografts were labeled. Throughout the observation period of the experiment, the maturity, population density, and architectural complexity of anchoring fibrils beneath the human epidermal graft continuously increased. Identical findings were obtained using xenografts cultivated from cloned human keratinocytes, eliminating the possibility of contributions to anchoring fibril regeneration from residual human fibroblasts. Immunolabeling was not observed at the mouse dermoepidermal junction at any time. These results demonstrate that the type VII collagen of human cutaneous anchoring fibrils and plaques is secreted by keratinocytes and can traverse the epidermal basal lamina and that the fibril formation can occur in the absence of cells of human dermal origin.

Animals↗

PubMed1990

Cultured keratinocyte sheets enhance spontaneous re-epithelialization in a dermal explant model of partial-thickness wound healing.

An in vitro model for partial-thickness cutaneous wound healing is described in which the influence of variables present in vivo, such as blood-borne factors and inflammatory cells, is eliminated. Dermal sheets of porcine skin are maintained in culture at the air-liquid interface in serum-free medium, and re-epithelialization from the keratinocytes of the hair infundibula can be studied. Dermal sheets of different thicknesses harvested from various depths were first evaluated for viability and regenerative potential in serum-supplemented medium. Mid-dermal explants, 20/1000 inch thick, showed the greatest epithelial outgrowth from the appendigeal keratinocytes and the longest viability in vitro. Explants of this type were used in all subsequent experiments. The effects of growth factors on re-epithelialization of the explants were studied in a serum-free environment. Epidermal growth factor, cholera toxin, bombesin, and insulin-like growth factor alone and in various combinations were applied to the explant surface in aqueous solutions by micropipette. Outgrowth was assessed by computerized morphometric analysis (RS/1 program by BBN) at days four and eight. Among all factors tested, cholera toxin alone and in combination with insulin-like growth factor produced the greatest epithelial outgrowth. Nevertheless, topical applications of growth factors failed to induce complete re-epithelialization within the experimental time frame. In contrast, explants to which cultured human keratinocyte sheets were topically applied regenerated a confluent and regularly stratified epidermis within 6 d.

Administration, Topical↗

PubMed1990

Cultured porcine epithelial grafts: an improved method.

An improved method of in vitro cultivation of porcine keratinocytes by which keratinocyte sheets suitable for grafting can be generated rapidly is described. Epidermis from split-thickness porcine skin is enzymatically separated from dermis with 0.25% Dispase solution (37 degrees C) within 3 h, and trypsinized to a single cell suspension. Keratinocytes are grown in Dulbecco-Vogt modified Eagle medium supplemented with 20 ng/ml hydrocortisone, 100 micrograms/ml penicillin, 100 micrograms/ml streptomycin, and 20% (cells from six-month-old pigs) or 10% fetal calf serum (cells from two-month-old pigs). Freshly isolated keratinocytes are plated at a density of 1.25 X 10(6) cells/ml since their plating efficiency is about 15 times lower than that of human keratinocytes grown under comparable conditions. Primary keratinocytes plated on plastic grow to confluence faster than those plated on lethally irradiated 3T3-J2 feeder layer cells. Porcine keratinocytes grown on plastic reach senescence in the third passage but, when subsequently cultivated on a lethally irradiated 3T3-J2 feeder layer, can be passaged up to seven times. Nevertheless, plating efficiency of second-passage porcine keratinocytes is only about 5%-7%, whereas that of human newborn foreskin keratinocytes is 20%-30%. Confluent stratified primary cultures grown on plastic, or secondary cultures grown on feeder layers, are used for grafting. The sheets are detached with Dispase solution and stapled to vaseline gauze to facilitate handling. Epidermal regeneration from porcine grafts produced by this method has been demonstrated after transplantation to full-thickness wounds excised to muscle fascia in donor animals.

Animals↗

PubMed1990

Human Merkel cell regeneration in skin derived from cultured keratinocyte grafts.

Human Merkel cell regeneration in epidermis derived from cultured keratinocyte autografts was studied from 6 days to 6 years after transplantation. Cultured keratinocyte sheets derived from skin of the sole, axilla, groin, or scalp were transplanted to full-thickness wounds in 20 pediatric patients treated for massive burns or giant congenital nevi. Normal age- and site-matched skin as well as meshed split-thickness autografts from the same patients served as controls. Merkel cells were identified by immunohistochemistry using antibodies to cytokeratins #8 and #18. Cultured keratinocytes in vitro expressed no neuroendocrine markers, but nonspecific, simple-epithelial cytokeratin expression was observed in about 20% of cells. After transplantation, Merkel cells were identified only in cultured grafts derived from sole skin and appeared in the epidermis as early as 21 days postgrafting. Dermal Merkel cells were rarely observed, but their appearance invariably succeeded that of intraepidermal Merkel cells. Regenerated Merkel cells were never innervated, and their emergence was unrelated either spatially or temporally to epidermal reinnervation. In skin bridges of meshed split-thickness grafts, Merkel cells survived after degeneration of associated neurites, but no Merkel cells appeared within re-epithelialized interstices. Among the neuroendocrine markers tested, Merkel cells in cultured grafts, meshed skin grafts or normal pediatric skin expressed only neuron-specific enolase. They failed to stain for calcitonin, chromogranin A, Leu-7, synaptophysin, bombesin, or vasoactive intestinal polypeptide by immunohistochemistry. These findings suggest that: (a) Merkel cells derive from keratinocyte precursors which undergo neuroendocrine differentiation in the epidermis; (b) that keratinocyte stem cells are capable of undergoing Merkel cell differentiation postnatally; (c) that postnatal Merkel cell differentiation may be body-site dependent; and (d) that Merkel cell development and maintenance is independent of neural induction.

Antibodies, Monoclonal↗

PubMed1989

Cytokeratins and cytokeratin filaments in subpopulations of cultured human and rodent cells of nonepithelial origin: modes and patterns of formation.

Using immunofluorescence microscopy, we observed that in several established cell culture lines derived from different nonepithelial tissues and species, cells spontaneously emerge, usually at low frequencies, which contain cytoplasmic structures decorated by antibodies specific for cytokeratins 8 and 18. This phenomenon was further examined at both the protein (gel electrophoreses of cytoskeletal proteins, followed by immunoblotting) and the RNA (Northern blots, "nuclear run-on" analysis, in situ hybridization) level. Positive cell lines included simian virus (SV40)-transformed human fibroblasts (HF-SV80, WI-38 VA13), human astrocytic glioma cells (U333 CG/343MG), rat (RVF-SMC) and hamster (BHK-21/13) cells derived from vascular smooth muscle and murine sarcoma MS-180 cells. In two cell lines (HF-SV80 and BHK-21/13), the frequency of the cytokeratin-containing cells and of the cytokeratin fibril arrays per cell was drastically increased upon treatment with 5-azacytidine. The structural appearance of the cytokeratins was variable in the different cell lines but could also differ among cells of the same culture: While small granular or comma-shaped structures or bizarrely shaped filament arrays prevailed in WI-38, RVF and normally grown BHK-21 cells, most of the other lines revealed extended normal-looking, fibrillar arrays. In one line (MS-180), the appearance of cytokeratins was associated with a morphological change, as it was only found in a subpopulation of cells that had lost their typical elongated and spindle-shaped phenotype and assumed a rounded ("coccoid") shape. Our results show that the expression of the genes encoding cytokeratins 8 and 18 is not necessarily restricted to programs of epithelial differentiation and that factors stochastically effective appear in cultured cell lines that allow the synthesis of these cytoskeletal components. Mechanisms possibly involved in this spontaneous and selective advent of cytokeratins 8 and 18 and implications for tumor diagnosis are discussed.

Animals↗

PubMed1989

Skin regenerated from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting. A light, electron microscopic and immunohistochemical study.

Regeneration of skin from cultured keratinocyte autografts used in the treatment of full-thickness burn wounds was studied in 21 pediatric patients from 6 days to 5 years after grafting. Findings were compared both to controls of age- and site-matched normal skin and to controls for epithelial wound-healing, re-epithelialized interstices of meshed split-thickness skin grafts of comparable postgrafting age. Six days after transplantation, a mildly hypertrophic, flat epidermis with all normal strata had regenerated, and the process of de novo dermal-epidermal junction formation had begun. Hemidesmosomes, basal lamina, and anchoring fibrils reformed conjointly in punctate fashion along the attachment face of the grafts. Within 3 to 4 weeks, the dermal-epidermal junction was complete, but full maturation of anchoring fibrils required more than a year. The process was comparable to that observed in meshed graft interstices. Rete ridges regenerated from 6 weeks to 1 year after grafting. The subjacent connective tissue initially healed to form normal scar, but it remodeled dramatically, regenerated elastin, and resembled a true dermis within 4 to 5 years. Meshed-graft interstice controls showed no rete ridge regeneration, subepithelial connective tissue remodeling, or elastin production up to 5 years after grafting. Langerhans cells repopulated grafts within 1 week, and normal population densities were reached within 2 to 6 months. After 1 year, Langerhans cell densities were increased compared with normal skin but were lower than those in age-matched meshed graft controls. Melanocytes were present in cultures at the time of transplantation, but functional epidermal melanin units were not seen in groin- or axilla-derived grafts for 6 to 8 weeks or in sole-derived epidermis until a year or more after transplantation. Normal histologic features were maintained for years after grafting. Transitory pathologic changes including parakeratosis, dyskeratosis, and intraepithelial friction blister formation were infrequently observed. No dysplastic or premalignant changes were seen.

Adolescent↗

PubMed1985

Intermediate filament cytoskeleton of amnion epithelium and cultured amnion epithelial cells: expression of epidermal cytokeratins in cells of a simple epithelium.

Using immunofluorescence microscopy and two-dimensional gel electrophoresis, we compared the cytoskeletal proteins expressed by human amnion epithelium in situ, obtained from pregnancies of from 10-wk to birth, with the corresponding proteins from cultured amnion epithelial cells and cultures of cells from the amniotic fluid of 16 week pregnancies. Epithelia of week 16 fetuses already display tissue-specific patterns of cytokeratin polypeptides which are similar, although not identical, to those of the corresponding adult tissues. In the case of the simple amnion epithelium, a complex and characteristic complement of cytokeratin polypeptides of Mr 58,000 (No. 5), 56,000 (No. 6), 54,000 (No. 7), 52,500 (No. 8), 50,000 (No. 14), 46,000 (No. 17), 45,000 (No. 18), and 40,000 (No. 19) is present by week 10 of pregnancy and is essentially maintained until birth, with the addition of cytokeratin No. 4 (Mr 59,000) and the disappearance of No. 7 (Mr 54,000) at week 16 of pregnancy. In full-term placentae, the amnion epithelium displays two morphologically distinct regions, i.e., a simple and a stratified epithelium, both of which express the typical amnion cytokeratin polypeptides. However, in addition the stratified epithelium also synthesizes large amounts of special epidermal cytokeratins such as No. 1 (Mr 68,000), 10 (Mr 56,500), and 11 (Mr 56,000). In culture amnion epithelial cells obtained from either 16-wk pregnancies or full-term placentae will continue to synthesize the amnion-typical cytokeratin pattern, except for a loss of detection of component No. 4. This pattern is considerably different from the cytokeratins synthesized by cultures of cells from amniotic fluids (cytokeratins No. 7, 8, 18, and 19, sometimes with trace amounts of No. 17) and from several so-called "amnion epithelial cell lines." In addition, amnion epithelial cells in situ as well as amnion epithelial cell cultures appear to be heterogeneous in that they possess some cells that co-express cytokeratins and vimentin. These observations lead to several important conclusions: In contrast to the general concept of recent literature, positively charged cytokeratins of the group No. 4-6 can be synthesized in a simple, i.e., one-layered epithelium. The change from simple to stratified amnion epithelium does not require a cessation of synthesis of cytokeratins of the simple epithelium type, but in this case keratins characteristic of the terminally differentiated epidermis (No. 1, 10, and 11) are also synthesized.(ABSTRACT TRUNCATED AT 400 WORDS)

Amnion↗

PubMedDate not supplied

Hyaluronan and wound healing: a new perspective.

Hyaluronan has long been associated with the remodelling extracellular matrix. Such remodelling occurs in development, growth and wound healing. This role has been thought to be related to the physical structure and chemical composition of the pure glycosaminoglycan chain. We question this proposition and present evidence which suggests that proteins associated with hyaluronan may be more critical determinants of tissue remodelling.

Amino Acids↗

PubMedDate not supplied

The effect of bromocryptine-induced hypoprolactinemia on xenogeneic and allogeneic skin graft survival in a mouse model.

Bromocryptine-induced hypoprolactinemia produces immunosuppression capable of extending allograft survival in solid organ transplantation models, but its potential for delaying either xenogeneic or allogeneic skin graft rejection is as yet undefined. In this study the ability of bromocryptine to prolong the survival of either cutaneous human xenografts or murine allografts was compared with cyclosporin A in a mouse model. In xenograft experiments cryopreserved human cadaver skin was grafted to B6D2F1 mice. In allograft experiments skin graft donors (A/J mice) differed genetically from recipients (Balb-C mice) at the major histocompatibility locus. In the untreated control group xenograft survival averaged 7 days and allograft survival 8 days. Xenograft rejection was delayed significantly and nearly equally by both bromocryptine and cyclosporin A (mean 15 and 16 days, respectively). However, allograft rejection was delayed longer by cyclosporin A (mean 24 days; p = 0.0002) than by bromocryptine (mean 15 days; p = 0.066). Administration of porcine prolactin (1 mg) partially reversed the bromocryptine effect (mean xenograft survival, 11 days; mean allograft survival, 10 days). The results of this study suggest that the immunosuppression of prolactin release by bromocryptine may be an effective, benign means of prolonging the survival of biologic dressings (e.g., banked cadaver allograft or porcine xenograft) used for temporary wound coverage in massively burned patients.

Animals↗