Abstract
Biodegradable scaffolds, along with cells, are important components of most tissue-engineered consructs. In the study, there is a comparison of the behaviour of human fibroblasts cultured for up to six weeks in four diffeeent collagen-based three-dimensional matrices, in the form of sponges composed of pure native type I collagen (control), of collagen-GAG-chitosan (CGC) and of collagen cross-linked by two concentrations of diphenylphosphorylazide (DPPA-2 and DPPA-3). Variations in size and weight of the sponges, as well as fibroblast growth and migration, and total protein and collagen synthesis, are determined with time in culture. Owing to their low thermal stability, the partial denaturation and dissolution of the control sponges after incubation at 37°C lead to considerable contraction and low cell proliferation. CGC sponges, stabilised by ionic interactions between the different components, show, after six weeks, limited contraction (20%) and weight increase (10% when seeded) and high growth (threefold increase). Similar results are obtained with weakly, cross-linked (DPPA-2) collagen sponges. Highly crosslinked (DPPA-3) sponges do not contract, whereas weight gain and cell proliferation are no different from those found with CGC and DPPA-2 sponges. Similar levels of total protein and collagen synthesis shown for fibroblasts seeded in different matrices, with a slight general decrease (twofold) after three weeks, a much lower value than that observed with fibroblasts in culture within a contracted collagen gel (sixfold). Furthermore, the fraction of neo-synthesised collagen deposited in the sponges after six weeks represents more than 60% of the total, compared with only 10% obtained with fibroblasts in monolayer culture or 30% within a collagen gel. These results indicate that the matrices, particularly the CGC and DPPA-2 sponges, provide excellent supports for fibroblast growth and the formation of dermal and skin equivalents.
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Augustin, C., andDamour, O. (1995): ‘Development of kit for predicting cutaneous toxicity,in vitro using three-dimensional dermal equivalent: phase 1 reproducibility of dermal equivalent’,Cell. Eng.,,1, pp. 58–62
Augustin, C., Collombel, C., andDamour, O. (1997a): “Use of dermal equivalent and skin equivalent models for identifying phototoxic compounds,in vitro’,Photodermatol. Photoimmunol. Photomel.,13, pp. 27–36
Augustin, C., Collombel, C., andDamur O. (1997b): ‘Use ofin vitro dermal equivalent and skin equivalent kits for evaluating cutaneous toxicity of cosmetic products’,In Vitro Toxicol.,10, pp. 21–29.
Augustin, C., Collombel, C., andDamour, O. (1997c): ‘Measurement of the protective effect of topically applied sunscreens usingin vitro dermal and skin equivalents’,Photochem. Photobiol.,66, pp. 853–859
Augustin, C., Collombel, C., andDamour, O. (1998): ‘Use of dermal equivalent and skin equivalent models forin vitro cutaneous irritation testing of cosmetic products: comparison within vitro humandata’,J. Toxicol. Cut. Ocular Toxicol.,17, pp. 5–17
Bell, E., Ivarsson, B., andMerrill, C. (1979) ‘Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potentialin vitro’,Cell Biol.,76, pp. 1274–1278
Berthod, F., Hayek, D., Damour O., andCollombel, C. (1993): ‘Collagen synthesis by fibroblasts cultured within a collagen sponge’,Biomaterials,14, pp. 749–754
Berthod, F., Sahuc, F., Hayek, D., Damour, O., andCollombel, C. (1996). ‘Deposition of collagen fibril bundles by long-term culture of fibroblasts in a collagen sponge’,J. Biomed. Mater. Res.,32, pp. 87–93
Boyce, J., Christianson, D. J., andHansborough, J. F. (1988): ‘Structure of a collagen GAG dermal skin substitute optimized for cultured epidermal keratinocytes’,J. Biomed. Mater. Res.,22, pp. 939–957
Chevallay, B., Abdul-Malak, N., andHerbage, D. (2000): ‘Mouse fibroblasts in long-term culture within collagen three-dimensional scaffolds: matrix reorganization, growth, biosynthetic and proteolytic activities’,J. Biomed. Mater. Res.,49, pp. 448–459
Collombel, C., Damour, O., Gagnieu, C., Poinsignon, F., Echinard, C., andMarichy, J. Peau artificielle et son proćdé de fabrication’. Brevet d'invention français n0 87-08752, 15 juin 1987, Brevet d'invention européean no 88-4201948, 14 juin 1988
Damour, O., Gueugniaud, P. Y., Bertin-Maghit, M., Rousselle, P., P. Bethod, F., Sahuc, F., andCollombel, C. (1994): ‘A dermal substrate made of collagen-GAG-chitosane for burns coverage: first clinical uses’,Clin. Mater.,15, pp. 273–276
Ehrlich, H. P., Buttle, D. J., andBernanke, D. H. (1989): ‘Physiological variables affecting collagen lattice contraction by human dermal fibroblast’,Exp. Mol. Path.,50, pp. 220–229
Ehrmann, R., andGey, G. (1956): ‘The growth of cells on a transparent gel of reconstitued rat-tail collagen’,J. Natl. Cancer Inst.,16, pp. 1375–1403
Grinnel, F. (1994): ‘Fibroblasts, myofibroblasts, and wound contraction’,J. Cell. Biol.,124, pp. 401–404
Gudry, C., andGrinnel, F. (1986): ‘Contraction of hydrated collagen gels by fibroblasts: evidence for two mechanisms by which collagen fibrils are stabilized”,Collgen Rel. Res.,6, pp. 515–529
Lange, R., andVacanti, J.P. (1993): ‘Tissue engineering’,Science,260, pp. 920–926
Mauch, C., Hatamochi, A., Scharffetter, K., andKrieg, T. (1988): ‘Regulation of collagen synthesis in fibroblasts within a three-dimensional collagen gel’,Exp. Cell Res.,178, pp. 493–503
Mossman, T. (1993): ‘Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay’,J. Immunol. Methods,65, pp. 55–63
Nakagawa, S., Pawelek, P., andGrinnel, F. (1989): ‘Long term culure of fibroblasts in contracted collagen gel: effect on cell growth and biosynthetic activity’,J. Invest. Dermatol.,93, pp. 792–798
Nusgens, B., Merrill, C., Lapiere, C., andBell, E. (1984): ‘Collagen biosynthesis by cells in a tissue equivalent matrix in vitro’,Collagen Rel. Res.,4, pp. 351–364
Peterkofsky, B., andDiegelmann, R. F. (1971): ‘Collagen biosynthesis during connective tissue development in chick embryo’,Dev. Biol,28, pp. 443–453
Petite, H., Raut, I., Huc, A., andHerbage, D. (1994): ‘Use of DPPA for crosslinkng collagen-based biomaterials’,J. Biomed. Mater. Res.,28, pp. 159–165
Rault I., Frei V., andHerbage D. (1996): ‘Evaluation of different chemical methods for crosslinking collagen gel, film and sponges’,J. Mater: Sci. Mater: Med.,7, pp. 215–221
Sahuc, F., Nakazawa, K., Berthod, F., Collombel, C., andDamour, O. (1996): ‘Mesenchymal-epithelial interaction of type IV collagen and kalinin in keratinocytes and dermal-epidermal junction formation in a skin equivalent model’,Wound Rep. Reg.,4, pp. 93–102
Saintigny, G., Bonnard, M., Damour, O., andCollombel, C. (1993): ‘Reconstruction of an epidermis on a chitosan-cross-linked collagen-GAG-lattice: effect of fibroblasts’,Acta. Derm. Venereol. (Stockh.),73, pp. 175–180
Thie, M., Schlumberger, W., Rauterberg, J., andRobenek, H. (1989): ‘Mechanical confinement inhibits collagen synthesis in gelcultured fibroblasts’,Eur. J. Cell Biol.,48, pp. 294–302
Yannas, I. V., Burke, J. F., Gordon, P. L., Huang, C., andRubenstein, J. K. (1980a): ‘Design of an artificial skin 1: basic design principles’,J. Biomat. Mater: Res.,14, pp. 65–81.
Yannas, I. V., Burke, J. F., Gordon, P. L., Huang, C., andRubenstein, J. K. (1980b): ‘Design of an artificial skin II: Control of chemical composition’,J. Biomat. Mater Res.,14, pp. 107–131
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Vaissiere, G., Chevallay, B., Herbage, D. et al. Comparative analysis of different collagen-based biomaterials as scaffolds for long-term culture of human fibroblasts. Med. Biol. Eng. Comput. 38, 205–210 (2000). https://doi.org/10.1007/BF02344778
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DOI: https://doi.org/10.1007/BF02344778