Secondary cytotoxicity of cross-linked dermal sheep collagens during repeated exposure to human fibroblasts

• Published in: Biomaterials Vol. 13; no. 14; pp. 1017 - 1024
• Main Authors: van Luyn, M.J.A., van Wachem, P.B., Damink, L.H.H.Olde, Dijkstra, P.J., Feijen, J., Nieuwenhuis, P.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 1992; Elsevier Science
• Subjects: Animals; Binding Sites; Biocompatible Materials - chemistry; Biocompatible Materials - isolation & purification; Biocompatible Materials - toxicity; Biological and medical sciences; Biological effects of radiation; Cell Division - drug effects; Cell Line; Collagen; Collagen - chemistry; Collagen - isolation & purification; Collagen - toxicity; cross-linking; Cross-Linking Reagents - toxicity; cytotoxicity; fibroblasts; Fibroblasts - drug effects; Fibroblasts - ultrastructure; Fundamental and applied biological sciences. Psychology; Humans; Hydrolysis; Materials Testing; Microscopy, Electron; Molecular Structure; Radiocontamination; Sheep; Tissues, organs and organisms biophysics; fibroblasts; cross-linking; Collagen; cytotoxicity; Human; Cell culture; Secondary; Cytotoxicity; Experimental study; In vitro; Vertebrata; Gamma irradiation; Mammalia; Sheep; Artiodactyla; Fibroblast; Ungulata
• ISSN: 0142-9612; 1878-5905
• DOI: 10.1016/0142-9612(92)90153-F

We investigated commercially available dermal sheep collagen either cross-linked with hexamethylenediisocyanate, or cross-linked with glutaraldehyde. In previous in vitro studies we could discriminate primary, i.e. extractable, and secondary cytotoxicity, due to cell-biomaterial interactions, i.e. enzymatic actions. To develop dermal sheep collagen for clinical applications, we focused in this study on the release, e.g. elimination, of secondary cytotoxicity over time. We used the universal 7 d methylcellulose cell culture with human skin fibroblasts as a test system. Hexamethylenediisocyanate-cross-linked dermal sheep collagen and glutaraldehyde-cross-linked dermal sheep collagen were tested, with intervals of 6 d, over a culture period of 42 d. With hexamethylenediisocyanate-cross-linked dermal sheep collagen, cytotoxicity, i.e. cell growth inhibition and deviant cell morphology, was eliminated after 18 d of exposure. When testing glutaraldehyde-cross-linked dermal sheep collagen, the bulk of cytotoxic products was released after 6 d, but a continuous low secondary cytotoxicity was measured up to 42 d. As a control, non-cross-linked dermal-sheep collagen was tested over a period of 36 d, but no secondary cytotoxic effects were observed. The differences in release of secondary cytotoxicity between hexamethylenediisocyanate-cross-linked dermal sheep collagen, glutaraldehyde-cross-linked dermal sheep collagen and non-cross-linked dermal sheep collagen are explained from differences in cross-linking agents and cross-links obtained. We hypothesize that secondary cytotoxicity results from enzymatic release of pendant molecules from hexamethylene-diisocyanate-cross-linked dermal sheep collagen, e.g. formed after reaction of hydrolysis products of hexamethylenediisocyanate with dermal sheep collagen. Glutaraldehyde-cross-linked dermal sheep collagen contains residual cross-linking agents, which induce the bulk cytotoxicity. Apart from being sensitive to enzymatic degradation, glutaraldehyde-cross-linked dermal sheep collagen was also found to be sensitive to aqueous hydrolysis. Hydrolysis of cross-links may release cytotoxic products and introduce new pendant molecules within glutaraldehyde-cross-linked dermal sheep collagen, which in turn induce cytotoxicity after enzymatic attack.