Stability of trypsin immobilized on inorganic orthopedic biomaterials

Biochemical surface modification of biomaterials utilizes immobilized biomolecules to induce preferred tissue responses. Although several techniques are available for immobilizing biomolecules on organic substrates, comparatively few are available for use with inorganic materials, such as those used in many orthopedic applications. The present study investigated the stability/elutability of a model enzyme immobilized on Co-Cr-Mo and Ti-6Al-4V alloys using p-nitrophenyl chloroformate (p-NPC). Trypsin-conjugated biomaterials were incubated in cell culture medium at 37 degrees C for up to 96 hr, and the residual immobilized activity was measured. Although all samples initially bound enzymatically active trypsin, significant decreases were observed within the first 2 hr of incubation. Immobilization of trypsin on Co-Cr-Mo treated with 0.65 mg p-NPC/cm2 of nominal surface area gave significantly higher residual activity than on untreated samples at 24-96 hr of incubation and prevented the nearly complete loss of enzymatic activity that was observed with free (not immobilized) enzyme. Derivatization of Ti-6Al-4V with p-NPC was not beneficial to the level of immobilized enzymatic activity after incubation in medium for longer than 6 hr.