Cross-linking of collagen-based biomaterials increases their strength and persistence in vivo. Recently, we described an efficient cross-linking process via the formation of acyl azide groups on methylated carboxyl groups of collagen using hydrazine and nitrous acid (referred to here as the hydrazine method). In this report, we propose a simpler, faster way to prepare acyl azide groups and to cross-link collagen-based biomaterials, using diphenylphosphorylazide (DPPA) as a reagent. After determining the optimal conditions of cross-linking with DPPA, we compared the efficiency of this protocol with that using hydrazine and with the classical glutaraldehyde treatment. In order to validate and quantitate the extent of reaction, the degree of crosslinking was determined by the measure of the free primary amino group content of the samples.
Treatment of native bovine pericardium with 0.5% DPPA for 24 h led 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"C. In comparison, the thermal stabilities of glutaraldehydeor hydrazine-treated pericardium were 85 2 0.4"C and 83.4 ? 0.1"C. Similar decreases in free primary amino group content and increases in thermal stability were obtained for collagen films treated with DPPA, glutaraldehyde, or hydrazine. These results were corroborated by resistance to bacterial collagenase digestion: DPPA-treated pericardium had a resistance to collagenase digestion similar to that of glutaraldehyde-or hydrazine-treated 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 method for cross-linking collagenbased biomaterials.