Inhibition of calcification in vivo by acyl azide cross-linking of a collagen-glycosaminoglycan sponge.

A collagen-glycosaminoglycan sponge composed of collagen (80%), chondroitin-4-sulfate (13.3%) and heparan sulfate (6.6%) was cross-linked using the acyl azide method or glutaraldehyde (0.0075%). Under optimal conditions, the denaturation temperature (Td) was raised to 69 degrees C (+23 degrees C) for the sponge treated by the acyl azide method and to 68 degrees C (+22 degrees C) for that treated with glutaraldehyde. The biocompatibility of the treated and control sponges was studied up to 3 months after subcutaneous implantation in rats by analysing cellular responses and calcification by histological and ultrastructural methods. A control collagen-glycosaminoglycan sponge was rapidly invaded by mononuclear cells (8 days), with the formation of granulation tissue. Calcification was observed at the periphery of the implant after 8 days, and the implant was entirely calcified after 15 days; it was degraded progressively after 30 days. Acyl azide treatment increased the persistence of the sponge in vivo up to 90 days and inhibited its calcification. A glutaraldehyde-treated sponge was completely calcified after 15 days, and calcified nodules persisted after 90 days. Thus, acyl azide method efficiently cross-linked a collagen-glycosaminoglycan sponge and inhibited calcification after subcutaneous implantation in rats (at least up to 90 days after implantation).