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Osteoclasts isolated from the endosteum of 2.5 to 3-week chick tibia were cultured on glass coverslips or natural CaC03 (Tridacna) wafers for 2 and 4 days. The cells were exposed to the pH-dependent dye, acridine orange, and fluorescence was measured by a light microscope photometer. Fluorescence intensity values were higher in cells adherent to Tridacna wafers than in those incubated on glass after 2 and 4 days of culture (three and two-fold, respectively). Moreover, osteoclasts on Tridacna wafers were more flattened and were found to produce resorption pits. Acid production by osteoclasts cultured on Tridacna wafers was stimulated with 10-8 M parathyroid hormone and inhibited with 10-7 M acetazolamide or 10-7 M hydroxybenezoyl thiophene sulfonamide, as shown by changes in intensity of acridine orange fluorescence after 30, 60 and 120 minutes of treatment. These results indicate that osteoclasts cultured on natural CaC03 wafers mimic the behavior of osteoclasts cultured on other substrates. Further, the capacity to acidify was enhanced in cells cultured on CaC03 wafers. These results indicate that natural CaC03 Tridacna wafers provide a suitable substrate for osteoclasts in culture and demonstrate that carbonic anhydrase plays a role in carbonated substrate resorption.

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