Immobilization effect of bone morphogenetic protein-2 on collagen membrane via photoreactive gelatin derivatives: Biocompatibility and preservability of osteoinductive activity

Abstract

To photo-immobilize bone morphogenetic protein-2 (BMP-2), UV light-reactive gelatin (azidophenyl gelatin, Az-gel), which is introduced with an N-(4-azidobenzoyloxy) succinimide-containing an azide group, was prepared as a carrier of BMP-2. Characterization of the photo-reactivity of Az-gel was evaluated by the curing ratio and it was micropatterned using a photomask. The cross-linking ratio and an enzyme-linked immunosorbent assay, enzymelinked immunospecific assay (ELISA)-based BMP-2-release test were used to determine the optimal density of Azgel (1, 3, and 5%) and UV treatment time (10, 30, 60 s) of the BMP-2 carrier in collagen matrix. The results showed that 5% Az-gel with 60 s of UV treatment was the most efficient condition for cross-linking. Cytotoxicity assays using the C2C12 mouse myoblast cell line showed that there were no cytotoxic effects of Az-gel. Alkaline phosphatase activity and calcium ion detection assays to evaluate the osteoinductive activities were conducted in assumed optimal condition of Az-gel (5% and 60 s UV treatment) 14 days post-treatment. In our study, the osteoinductive activities were the highest in cells treated with BMP-2 and the photo-reactive Az-gel carrier compared to BMP-2 alone or untreated control cells in collagen matrix at all time points (3, 7, and 14 days).