title: Evaluation of in vitro bone resorption: high-performance liquid chromatography measurement of the pyridinolines released in osteoclast cultures.
abstract: 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.
authors.affiliation: Laboratoire de Pharmacie Clinique, 1 rue des Louvels, Amiens, France. florence.Lorget@sa.u-picardie.fr
authors.forename: F
authors.initials: F
authors.lastname: Lorget
authors.forename: R
authors.initials: R
authors.lastname: Mentaverri
authors.forename: B
authors.initials: B
authors.lastname: Meddah
authors.forename: G
authors.initials: G
authors.lastname: Cayrolle
authors.forename: A
authors.initials: A
authors.lastname: Wattel
authors.forename: A
authors.initials: A
authors.lastname: Morel
authors.forename: N
authors.initials: N
authors.lastname: Schecroun
authors.forename: M
authors.initials: M
authors.lastname: Maamer
authors.forename: M C
authors.initials: MC
authors.lastname: de Vernejoul
authors.forename: S
authors.initials: S
authors.lastname: Kamel
authors.forename: M
authors.initials: M
authors.lastname: Brazier
mesh_terms: D000818:Animals; D001862:Bone Resorption; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D010010:Osteoclasts / Q000737:chemistry* / Q000166:cytology; D011725:Pyridines / Q000032:analysis*; D011817:Rabbits
chemical_list: D011725:Pyridines