DEGRADATION OF INSOLUBLE BOVINE COLLAGEN AND HUMAN DENTIN COLLAGEN PRETREATED IN-VITRO WITH LACTIC-ACID, PH-4.0 AND PH-5.5

The purpose of the study was to test the hypothesis that both insolubl e pure type I collagen from bovine Achilles tendon and dentine collage n in root dentine powder from human teeth required acid pretreatment f or subsequent degradation by trypsin, a non-specific protease. Pure ty pe I collagen or dentine powder was treated with lactic acid, at pH 4 or 5.5, or distilled, deionized water (pH 7) as a negative control. Af ter incubation at 37 degrees C for 24 h, extracts of pure type I colla gen solutions were analysed for soluble collagen with the hydroxyproli ne assay. Extracts of dentine powder solution were analysed for Ca2+, total protein, final pH, and hydroxyproline. Residual, undegraded pell ets were washed and then treated with trypsin or collagenase. After 24 h of incubation, the soluble fractions from the enzyme-treated pure t ype I collagen and dentine powder solutions were analysed for hydroxyp roline. Results showed that almost no pure type I collagen was degrade d during acid pretreatment. Trypsin degraded significantly more pure t ype I collagen in the pH 4-treated group than in the other groups. Col lagenase degraded about 70% of the pure type I collagen irrespective o f acid preatreatment. While acid pretreatment at pH 4 did not degrade dentine collagen, data from Ca2+ analyses and collagen breakdown by tr ypsin suggested that pretreatment at pH 4 demineralized and denatured dentine collagen so that the collagen could be subsequently degraded b y enzymes. After pretreatment at pH 4, about 27 and 57% of the dentine collagen was degraded by trypsin and collagenase, respectively, in co ntrast to minimal degradation of non-acid-treated dentine collagen by the same enzymes. These results indicate that acid pretreatment is imp ortant for subsequent degradation of pure type I collagen and dentine collagen by non-specific proteases such as trypsin.