Collagen-based films and sponges are widely used as biomaterials. The rate of their biodegradation can be reduced by treating them with different cross-linking agents. The efficiency of different reticulation procedures on thermal stability (measured by differential scanning calorimetry) and susceptibility to bacterial collagenase digestion of the final material (films or sponges) is compared. The chemical agents used on collagen gels or directly on collagen sponges and films were glutaraldehyde (GTA), hexamethylene diisocyanate (HMDC), cyanamide, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and the two acyl azide methods (with hydrazine or diphenylphosphorylazide (DPPA)) developed in the authors' laboratory. Under these experimental conditions, collagen stabilization by the different agents increased in the following order: cyanamide < EDC < HMDC < hydrazine < DPPA < GTA. Sponges with the highest thermal stability were obtained by treating collagen gel with 0.5% GTA for 1 h at pH 5.5 (initial T~=75.8°C; after 3 months, Ta=75.6°C). Direct treatment of collagen films with DPPA gave similar thermal stability (Ta=72.6 °C) and collagenase resistance when compared with treatment with 0.6% GTA for 96 h (Td =74.6 °C). It is demonstrated that collagen sponges and films can be prepared with a wide range of thermal stability (49-75.8 °C) and collagenase digestion resistance (10-100%). Recent biocompatibility studies show, however, that DPPA and EDC are the best choices for preparing cross-linked collagen sponges and films.
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 cross-linking 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 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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
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