USE OF DIPHENYLPHOSPHORYLAZIDE FOR CROSS-LINKING COLLAGEN-BASED BIOMATERIALS

Cross-linking of collagen-based biomaterials increases their strength and persistence invivo. Recently, we described an efficient cross-link ing process via the formation of acyl azide groups on methylated carbo xyl groups of collagen using hydrazine and nitrous acid (referred to h ere as the hydrazine method). In this report, we propose a simpler, fa ster way to prepare acyl azide groups and to cross-link collagen-based biomaterials, using diphenylphosphorylazide (DPPA) as a reagent. Afte r determining the optimal conditions of cross-linking with DPPA, we co mpared the efficiency of this protocol with that using hydrazine and w ith the classical glutaraldehyde treatment. In order to validate and q uantitate the extent of reaction, the degree of crosslinking was deter mined by the measure of the free primary amino group content of the sa mples. Treatment of native bovine pericardium with 0.5% DPPA for 24h l ed to efficient cross-linking, corresponding to a 50% decrease in the free primary amino group content of the sample and raising its thermal stability from 62.8 up to 81.3 degrees C. In comparison, the thermal stabilities of glutaraldehyde or hydrazine-treated pericardium were 85 +/- 0.4 degrees C and 83.4 +/- 0.1 degrees C. Similar decreases in fr ee primary amino group content and increases in thermal stability were obtained for collagen films treated with DPPA, glutaraldehyde, or hyd razine. These results were corroborated by resistance to bacterial col lagenase digestion: DPPA-treated pericardium had a resistance to colla genase digestion similar to that of glutaraldehyde- or hydrazine-treat ed pericardium. Residual DPPA content was measured by determining the phosphorus content: the concentration of phosphorus in tissue treated with 0.5% DPPA was not significantly different from that of untreated tissue. Treatment by DPPA thus appears to be an efficient, rapid metho d for cross-linking collagen-based biomaterials. (C) 1994 John Wiley a nd Sons, Inc.