QUANTIFICATION OF THE COLLAGENOLYTIC ACTIVITY OF ISOLATED OSTEOCLASTSBY ENZYME-LINKED-IMMUNOSORBENT-ASSAY

Difficulties in the geometrical definition and measurement of resorpti on pits is a major problem for the quantitative analysis of bone resor ption by isolated osteoclasts cultured on bone or dentin substrates, I n this study we developed an enzyme-linked immunosorbent assay (ELISA) for quantification of bone resorption in vitro, which specifically qu antifies type I collagen fragments released into the culture medium by the resorptive action of bone cells cultured on slices of bone or den tin, A consistently high correlation between the formation of resorpti on pits and the release of antigenic collagen fragments was observed f or isolated rabbit osteoclasts seeded at various densities and culture d for various periods on bovine, elephant, and human substrates, In a further support of the osteoclastic nature of the collagenolytic effec ts, a high consistency between pit formation and collagenolysis was al so observed when the rabbit bone cells were cultured in the presence o f very differently acting but typical inhibitors of pit formation, i,e ., the carbonic anhydrase inhibitor acetazolamide, the cysteine protei nase inhibitor epoxysuccinyl-L-leucylamido-(4-guanodino)butane (E-64), the phosphatidyl-inositol 3-kinase inhibitor wortmannin, and the bisp hosphonate ibandronate (BM 21.0955), In conclusion, the ELISA represen ts a simple, precise, and objective way to dynamically monitor bone re sorption in vitro through quantification of the collagenolytic activit y of isolated osteoclasts.