USE OF P-NITROPHENYL CHLOROFORMATE CHEMISTRY TO IMMOBILIZE PROTEIN ONORTHOPEDIC BIOMATERIALS

Biochemical surface modification involves covalently immobilizing biom olecules onto biomaterial surfaces to induce specific biological respo nses. This approach may be useful for enhancing the fixation of orthop edic implants. p-Nitrophenyl chloroformate (p-NPC) was used to immobil ize protein on bulk samples of Co-Cr-Mo and Ti-6Al-4V. Activation of b oth materials was dependent on the concentration of p-NPC, with a maxi mum of approximately 1.5 active groups/nm(2) of nominal surface area. Trypsin was used as a model protein because much is known about its st ructure and mode of action. Derivatization with 0.65 mg p-NPC/cm(2) re sulted 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) a nd 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-6 Al-4V, with the exception of samples derivatized with 1.95 mg p-NPC/cm (2), on which activity was significantly lower (4 BAEE units). In prob ing the linkages between trypsin and biomaterial by treatment with cha otropic agents, guanidine hydrochloride (GuHCl) was observed to elimin ate more enzymatic activity than was urea. On Co-Cr-Mo samples, GuHCl removed nearly all the trypsin activity, while urea significantly decr eased the activity only at a concentration of 0.65 mg r-NPC/cm(2). Tre atment of Ti-6Al-4V samples with GuHCl caused a trend of decreasing ac tivity with increasing concentration of p-NPC, whereas urea had no eff ect on immobilized trypsin activity. (C) 1996 John Wiley & Sons, Inc.