Relations between in vitro cytotoxicity and crosslinked dermal sheep collagens

Collagen-based biomaterials have found various applications in the biomedical field. However, collagen-based biomaterials may induce cytotoxic effects. This study evaluated possible cytotoxic effects of (crosslinked) dermal sheep collagen (DSC) using a 7-d-methylcellulose cell culture with human skin fibroblasts. Non-crosslinked DSC (NDSC), hexamethylene-diisocyanate-crosslinked DSC (HDSC), and glutaraldehyde-crosslinked DSC (GDSC), their extracts (1 x 10 d to 4 x 10 d extracts), or the corresponding extracted DSC samples were tested. Cell growth was evaluated by cell counting, while cell morphology was assessed by light microscopy and transmission-electron microscopy. Both GDSC and, to a lesser extent, HDSC, induced cytotoxicity, observed as inhibited cell growth and deviant cell morphology. The deviant morphology consisted of extensive accumulations of lipid, reduction in the amount and dilatation of rough endoplasmatic reticulum, increased inclusions of cell remnants, and relatively rounded cell membranes. With HDSC, both primary cytotoxicity, due to extractable products from the material, and secondary cytotoxicity, possibly due to a release of cytotoxic products resulting from enzymatic cell-biomaterial interactions, could be discriminated. With GDSC, however, no clear distinction between primary and secondary cytotoxicity could be made. With NDSC, only primary cytotoxicity, measured as low inhibition of cell proliferation, but without deviant morphology, was observed. These remarkable differences in cytotoxicity are discussed in relation to residual agents and specific crosslinks present in DSCs as a consequence of processing and the crosslinking agents used. The residual agents and the specific crosslinks give rise to differences in direct release of products and in sensitivity to hydrolysis and enzymatic breakdown.