An experimental study on the resorption of living and devitalized bones by isolated osteoclasts in vitro. The effects of TIMP,E-64 and TGF-.ALPHA..

In this study, a new bone resorption model was developed by using living bone substrates prepared from mice calvaria. Devitalized bones made by freezing and thawing of living bones as well as dead cortical bone slices of monkey femur were also used as substrates for isolated osteoclasts to act upon. The extent of bone resorption was assessed by measuring both the area and the depth of resorption pits. Recombinant Human Tissue Inhibitor of Metalloproteinases (TIMP) and Cysteine-Proteinase Inhibitor (E-64) were used to find out the different mode and extent of resorption by isolated osteoclasts between living and devitalized bones. Transforming Growth Factor-α(TGF-α) was applied to this model to investigate the possible mechanisms involved in bone resorption induced by tumors.Both the area and the depth of pits made on living bones were more extended compared to those of pits made on devitalized bone substrates. TIMP (100μ/ml) reduced the resorption of living bone both in the area and in the depth to the same amount of resorption made on devitalized bone. But, TIMP did not inhibit the resorption of devitalized bone. E-64 (60μM) significantly inhibited the resorption of devitalized bones by 66%, while it had no effect on the resorption of living bone. TGF-α(100 ng/ml) did not show significant effect on the resorption of any substrate, but appeared to inhibit the osteoclast activity slightly (p<0.1). Indomethacin (100 ng/ml) reduced the resorption of living bone to the same level of that of devitalized bone, but did not, however, inhibit the resorption of devitalized bone. These results suggest that:1 The resorption of living bone appeared to be aided by osteocyte-synthesis of metalloproteinases, among them collagenase, to degrade bone collagen.2. The excess of resorption of living bone compared to that of devitalized bone also depended on prostaglandin synthesis by living cells, interior to the substrate.3. Thus, the mechanisms of resorption underlying living bone and devitalized bone are different.4 TGF-α(100 ng/ml) appeared to inhibit the activity of isolated osteoclast, but not significant (13, 1, 0.1). So, the stimulation of bone resorption by this factor observed in vivo may be caused first by living cells other than osteoclast.5. This new culture system of isolated osteoclasts using living bone substrate presents a useful model for the study of bone resorption.