Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate

Porous collagen matrices with defined physical, chemical and biological characteristics are interesting materials for tissue engineering. Attachment of glycosaminoglycans (GAGs) may add to these characteristics and valorize collagen. In this study, porous type I collagen matrices were crosslinked using dehydrothermal (DHT) treatment and/or 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), in the presence and absence of chondroitin sulphate (CS). EDC covalently attaches CS to collagen. DHT crosslinking preserved a porous matrix structure. However, attachment of CS to DHT-treated matrices using EDC, resulted in collapsed surfaces, CS located only at the matrix exterior. EDC crosslinking resulted in a partial matrix collapse. This could be prevented if crosslinking was carried out in the presence of ethanol. Matrix porosity was then preserved. The presence of CS during EDC crosslinking resulted in covalent immobilization of CS throughout the matrix. The amount of CS incorporated was increased if crosslinking was performed in the presence of ethanol. EDC-crosslinked matrices, with and without CS, had increased denaturation temperatures and decreased free amine group contents. The susceptibility of these matrices towards degradation by proteolytic enzymes was diminished. Immobilized CS increased the water-binding capacity and decreased the denaturation temperature and tensile strength. Immobilized CS bound anti-CS antibodies and was susceptible to chondroitinase ABC digestion, demonstrating its bioavailability. The modified matrices were not cytotoxic as was established using human myoblast and fibroblast culture systems. It is concluded that the use of ethanol during EDC crosslinking, offers an elegant means for the preparation of defined porous collagenous matrices containing bioavailable, covalently attached CS.