Enzyme-Pretreatment Removes In Vitro Cytoxic Effects of Dermal Sheep Collagen
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ENZYME-PRETREATMENT REMOVES IN VITRO CYTOXIC EFFECTS OF DERMAL SHEEP COLLAGEN M.J.A. van Luyn, P.B. van Wachem, L.H.H. Olde Damink*, P.J. Dijkstra*, J. Feijen* and P. Nieuwenhuis. Department of Histology and Cell Biology, Section Biomaterials Research, University of Groningen, Oostersingel 69/2, 9713 EZ Groningen, The Netherlands. *Department of Chemical Technology, Twente University, p.o. Box 217, 7500 AE Enschede, The Netherlands.
ABSTRACT We investigated the in vitro cytotoxicity of commercially available hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC). HDSC was found to induce medium cytotoxic effects, as measured with methylcellulose cell culture. Apart from primary cytotoxicity, due to direct release of (extractable) cytotoxic products, HDSC was found to contain secondary cytotoxicity, possibly released by enzymatic interactions. In this study we found proof for this hypothesis, by exposing extracted HDSC to enzyme-containing medium. Furthermore we observed, that enzymatic pre-treatment can remove all secondary cytotoxic products, possibly due to detachment of pendants, which are still coupled to fragments of collagen molecules. The possibility of enzymatic pretreatment of HDSC, to obtain a non-cytotoxic/biocompatible material, may be important for in vivo applications.
INTRODUCTION Collagen-based materials intended for use in in vivo applications may be crosslinked to increase their strength and persistence. However, crosslinked collagen, or their degradation products may induce cytotoxicity [1-4]. At present, several types of crosslinked collagens are commercially available. Previously, we studied the in vitro cytotoxicity of commercially available hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC) [5-7]. In these studies we discriminated primary cytotoxicity, due to direct leakage of cytotoxic products from HDSC, and secondary cytotoxicity, probably due to release of cytotoxic products from cell-biomaterial interactions, i.e. enzymatic actions. We used a new and advanced in vitro cytotoxicity test system for biomaterials, i.e. methylcellulose cell culture [5]. With this test system interactions between biomaterials or their extracts and human fibroblasts can be examined over a period of 7 days without refreshing culture medium. Possible cytotoxic effects are measured by counting cell numbers, and cell morphology can be evaluated by phase-contrast light-microscopy in situ, and by electron- microscopy [5,6]. The aim of the present study was to focus on the secondary cytotoxicity of HDSC. On one hand to find proof for our hypothesis of secondary cytotoxicity being due to cellular (i.e. enzymatic) actions. On the other hand to investigate, whether enzymatic pretreatment can remove secondary cytotoxicity, which might be important for in vivo applications of HDSC. Mat. Res. Soc. Symp. Proc. Vol. 252. '1992 Materials Research Society
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MATERIALS and METHODS Materials Human fibroblasts (established cell-line PK 84) were routinely cultured in RPMI 1640 medium (Gibco Biocult
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