Development and characterization of a human in vitro resorption assay: Demonstration of utility using novel antiresorptive agents

A human in vitro resorption assay has been developed using osteoclastoma-de rived osteoclasts and used to evaluate novel antiresorptive agents includin g antagonists of the alpha(v)beta(3) integrin, and inhibitors of cathepsin K and the osteoclast ATPase, The potency of novel compounds in the in vitro resorption assay correlates with functional assays for each class of inhib itor: the human alpha(v)beta(3)-mediated cell adhesion assay for the vitron ectin receptor antagonists (r(2) = 0.82), the chick osteoclast vacuolar ATP ase enzyme assay for the H+-ATPase inhibitors (r(2) = 0.77) ;and the recomb inant human cathepsin K enzyme assay for the cathepsin K inhibitors (r(2) = 0.80). Cell suspensions, rich in osteoclasts, are prepared by collagenase digestion of the tumor tissue. These cells can be stored long-term in liqui d nitrogen and upon thawing maintain their bone-resorbing phenotype, The cr yopreserved cells can be cultured on bovine cortical bone for 24-48 h and r esorption can be measured by either confocal microscopy or biochemical assa ys. The resorptive activity of osteoclasts derived from a number of tumors can be inhibited reproducibly using a number of mechanistically unique anti resorptive compounds. In addition, the measurement of resorption pits by la ser confocal microscopy correlates with the release of type I collagen C-te lopeptides or N-telopeptides, as measured by enzyme-linked immunosorbent as say. Resorption can be measured reproducibly using a 48-h incubation of ost eoclasts on bone slices, or a 24-h incubation,with bone particles. This in vitro human osteoclast resorption assay provides a robust system for the ev aluation of inhibitors of osteoclastic function that may be developed for t he treatment of metabolic bone diseases such as osteoporosis.