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 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.
Lorget
F
F
Laboratoire de Pharmacie Clinique, 1 rue des Louvels, Amiens, France. florence.Lorget@sa.u-picardie.fr
Mentaverri
R
R
Meddah
B
B
Cayrolle
G
G
Wattel
A
A
Morel
A
A
Schecroun
N
N
Maamer
M
M
de Vernejoul
M C
MC
Kamel
S
S
Brazier
M
M
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
D011725:Pyridines