Use of p-nitrophenyl chloroformate chemistry to immobilize protein on orthopedic biomaterials

Biochemical surface modification involves covalently immobilizing biomolecules onto biomaterial surfaces to induce specific biological responses. This approach may be useful for enhancing the fixation of orthopedic implants. p-Nitrophenyl chloroformate (p-NPC) was used to immobilize protein on bulk samples of Co-Cr-Mo and Ti-6Al-4V. Activation of both materials was dependent on the concentration of p-NPC, with a maximum of approximately 1.5 active groups/nm2 of nominal surface area. Trypsin was used as a model protein because much is known about its structure and mode of action. Derivatization with 0.65 mg p-NPC/cm2 resulted in significantly greater enzymatic activity (7.4 BAEE [N-(alpha)-benzoyl-L-arginine ethyl ester hydrochloride] units) on the Co-Cr-Mo samples compared with higher concentrations of p-NPC (5 BAEE units) and with simple adsorption of trypsin (1.5 BAEE units). An activity of 10.5 BAEE units was measured on both adsorbed and p-NPC-activated Ti-6Al-4V, with the exception of samples derivatized with 1.95 mg p-NPC/cm2, on which activity was significantly lower (4 BAEE units). In probing the linkages between trypsin and biomaterial by treatment with chaotropic agents, guanidine hydrochloride (GuHCl) was observed to eliminate more enzymatic activity than was urea. On Co-Cr-Mo samples, GuHCl removed nearly all the trypsin activity, while urea significantly decreased the activity only at a concentration of 0.65 mg p-NPC/cm2. Treatment of Ti-6Al-4V samples with GuHCl caused a trend of decreasing activity with increasing concentration of p-NPC, whereas urea had no effect on immobilized trypsin activity.