Evaluation of in vitro bone resorption: High-performance liquid chromatography measurement of the pyridinolines released in osteoclast cultures

None of the currently used methods to evaluate bone resorption by osteoclas ts cultured on bone substrate measures directly the amounts of degraded bon e collagen, which is a direct reflection of the osteoclast "work done." We therefore propose a reliable biochemical method to evaluate the in vitro co llagenolysis process. Bone-resorbing activity was evaluated, after HPLC sep aration, by fluorimetric measurement of hydroxylysylpyridinoline (HP), a co llagen cross-link molecule, released in culture supernatants. We first conf irm previous data reporting that HP is released in the culture medium in a peptide-conjugated form. After acid hydrolysis, we show that HP is highly c orrelated with the lacunae area (r = 0.68, P < 0.0001) and with the amounts of antigenic collagen fragments (Cross-laps for culture) released in cultu re medium (r = 0.77, P < 0.0002). Using a cysteine protease inhibitor, we o bserved that lacunae areas are dramatically less inhibited (35% inhibition) than the release of bone-degraded products, including HP and antigenic col lagen fragments (96 and 92% inhibition, respectively). Coupled to the resor bed area measurement, biochemical evaluations offer both quantitative and q ualitative complementary measurements of the osteoclastic bone-resorbing pr ocess. (C) 2000 Academic Press.