Evaluation of in Vitro Bone Resorption: High-Performance Liquid Chromatography Measurement of the Pyridinolines Released in Osteoclast Cultures

✍ Scribed by F. Lorget; R. Mentaverri; B. Meddah; G. Cayrolle; A. Wattel; A. Morel; N. Schecroun; M. Maamer; M.C. de Vernejoul; S. Kamel; M. Brazier

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 120 KB
• Volume: 284
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4724

✦ Synopsis

None of the currently used methods to evaluate bone resorption by osteoclasts cultured on bone substrate measures directly the amounts of degraded bone collagen, which is a direct reflection of the osteoclast "work done." We therefore propose a reliable biochemical method to evaluate the in vitro collagenolysis process. Bone-resorbing activity was evaluated, after HPLC separation, by fluorimetric measurement of hydroxylysylpyridinoline (HP), a collagen cross-link molecule, released in culture supernatants. We first confirm 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 correlated with the lacunae area (r ‫؍‬ 0.68, P < 0.0001) and with the amounts of antigenic collagen fragments (Cross-laps for culture) released in culture medium (r ‫؍‬ 0.77, P < 0.0002). Using a cysteine protease inhibitor, we observed that lacunae areas are dramatically less inhibited (35% inhibition) than the release of bone-degraded products, including HP and antigenic collagen fragments (96 and 92% inhibition, respectively). Coupled to the resorbed area measurement, biochemical evaluations offer both quantitative and qualitative complementary measurements of the osteoclastic bone-resorbing process.