Summary
Our laboratory has been involved in finding optimal conditions for producing dermal and skin equivalents. As an original approach, a Box-Behnken experimental design was used to study the effects of the initial collagen and fibroblast concentrations and the initial gel thickness on the contraction of dermal and skin equivalents. The final surface area of dermal equivalent varied significantly with the initial concentration of collagen and fibroblast, whereas the initial thickness of gel had no appreciable effect on the contraction of the dermal equivalent. When keratinocytes were grown on these dermal equivalents they produced a very severe contraction, to an extent that all skin equivalents had a similar final surface area. This severe contraction was independent of collagen and fibroblast concentrations. Models for the prediction of the final percentage contraction of dermal and skin equivalents as a function of the initial concentration of collagen, the logarithm of fibroblast concentration, and the initial gel thickness were obtained and analyzed. Keratinocytes grown at the lowest seeding density did not contract the equivalents. However, histologic analysis has shown an incomplete coverage by these cells of the equivalents. The extensive contraction of the skin equivalent presenting adequate morphology is a major drawback toward its clinical utilization for burn wound coverage.
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References
Allen, T. E.; Schor, S. L. The contraction of collagen matrices by dermal fibroblasts. J. Ultrastruct. Res. 83:205–219; 1983.
Bell, E.; Ivarsson, B.; Merrill, C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc. Natl. Acad. Sci. USA 76(3):1274–1278; 1979.
Bell, E.; Ehrlich, H. P.; Sher, S., et al. Development and use of a living skin equivalent. Plast. Reconstr. Surg. 67(3):386–392; 1981.
Bell, E.; Sher, S.; Hull, B. The living skin-equivalent as a structural and immunological model in skin grafting. Scam. Electr. Microsc. 4:1957–1962; 1984.
Box, G. E. P.; Behnken, D. W. Some new three level designs for the study of quantitative variables. Technometrics. 2(4):455–475; 1960.
Box, G. E. P.; Wilson, K. B. On the experimental attainment of optimum conditions. J. R. Stast. Soc. B. 13:1–45; 1951.
Chen, T. C. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 104:255–262; 1977.
Coulomb, B.; Saiag, P.; Bell, E., et al. A new method for studying epidermalization in vitro. Br. J. Dermatol. 114:91–101; 1986.
Coulomb, B.; Lebreton, C.; Dubertret, L. Influence of human dermal fibroblasts on epidermalization. J. Invest. Dermatol. 92(1):122–125; 1989.
Ehrlich, H. P. The role of connective tissue matrix in wound healing. In: Ehrlich, H. P., ed. Growth factors and other aspects of wound healing: biologial and clinical implications. New York: Alan R. Liss; 1988:243–258.
Emerman, J. T.; Enami, J.; Pitelka, D. R., et al. Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes. Proc. Natl. Acad. Sci. USA 74(10):4466–4470; 1977.
Emerman, J. T.; Pitelka, D. R. Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro 13:316–328; 1977.
Green, H.; Kehinde, O.; Thomas, J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc. Natl. Acad. Sci. USA 76(11):5665–5668; 1979.
Grinnell, F.; Lamke, C. R. Reorganization of hydrated collagen lattices by human skin fibroblasts. J. Cell Sci. 66:51–63; 1984.
Grinnell, F.; Takashima, A.; Lamke-Seymour, C. Morphological appearance of epidermal cells cultured on fibroblastreorganized collagen gels. Cell Tissue Res. 246:13–21; 1986.
Harris, A. K.; Stopak, D.; Wild, P. Fibroblast traction as a mechanism for collagen morphogenesis. Nature 290:249–251; 1981.
Hull, B. E.; Sher, S. E.; Rosen, S., et al. Structural integration of skin equivalents grafted to Lewis and Sprague-Dawley rats. J. Invest. Dermatol. 81:429–436; 1983.
Hill, W. J.; Hunter, W. G. A review of response surface methodology: a literature survey. Technometrics 8(4):571–590; 1966.
Lillie, J. H.; MacCallum, D. K.; Jepsen, A. Growth of stratified squamous epithelium on reconstituted extracellular matrices: long-term culture. J. Invest. Dermatol. 90(2):100–109; 1988.
Mackenzie, I. C.; Fusenig, N. E. Regeneration of organized epithelial structure. J. Invest. Dermatol. 81(1):189s-194s; 1983.
Michalopoulos, G.; Pitot, H. C. Primary culture of parenchymal liver cells on collagen membranes. Exp. Cell Res. 94:70–78; 1975.
Rheinwald, J. G.; Green, H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–344; 1975.
Rowling, P. J. E.; Raxworthy, M. J.; Wood, E. J., et al. The influence of keratinocyte seeding and keratinocyte growth medium on dermal equivalent contraction. J. Invest. Dermatol. 92(3):510a; 1989.
Saiag, P.; Coulomb, B.; Lebreton, C., et al. Psoriatic fibroblasts induce hyperproliferation of normal keratinocytes in a skin equivalent model in vitro. Science 230(4726):669–672; 1985.
Schor, S. L. Cell proliferation and migration on collagen substrata in vitro. J. Cell Sci. 41:159–175; 1980.
Sher, S. E.; Hull, B. E.; Rosen, S., et al. Acceptance of allogeneic fibroblasts in skin equivalent transplants. Transplantation 36(5):552–557; 1983.
SAS Institute Inc. SASR user's guide: Statistics, version 5 edition. Cary, NC: SAS Institute Inc., 1985.
Schafer, I. A.; Shapiro, A.; Kovach, M., et al. The interaction of human papillary and reticular fibroblasts and human keratinocytes in the contraction of three-dimensional floating collagen lattices. Exp. Cell Res. 183:112–125; 1989.
Souren, J. M.; Ponec, M.; van Wijk, R. Contraction of collagen by human fibroblasts and keratinocytes. In, Vitro Cell. Dev. Biol. 25:1039–1045; 1989.
Yoshizato, K.; Taira, T.; Yamamoto, N., et al. Remodeling of collagen: an in vitro model of connective tissue. Biomed. Res. 6(5):287–296; 1985.
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The financial supports for this project were received from Canadian NSERC postgraduate scholarship (P. Rompré), Québec FCAR postgraduate scholarship (C.A. López Valle), France-Québec research grant in Biotechnology (F.A. Auger), Canadian MRC grant (F.A. Auger), and NSERC grants (A. LeDuy and J. Thibault).
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Rompré, P., Auger, F.A., Germain, L. et al. Influence of initial collagen and cellular concentrations on the final surface area of dermal and skin equivalents: A box-behnken analysis. In Vitro Cell Dev Biol 26, 983–990 (1990). https://doi.org/10.1007/BF02624473
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DOI: https://doi.org/10.1007/BF02624473