Use of glass slides coated with apatite-collagen complexes for measurementof osteoclastic resorption activity

This study was designed to evaluate the use of apatite-collagen complexes ( ACC) coated onto glass slides for measurement of osteoclastic resorption ac tivity. ACC-coated glass slides were prepared by immersion in P-glycerophos phate solution for 7-14 days after glass slides coated with type I collagen had been treated with alkaline phosphatase and phosvitin. Osteoclast-conta ining cell suspensions were prepared from the long bones of 1-day-old rabbi ts and were seeded in medium 199 (containing 10% FBS) onto ACC-coated glass slides. After allowing the cells to attach for 1.5 h, the glass slides wer e incubated for periods of up to 96 h. The cells were observed by scanning electron microscopy and cytochemically for tartarate resistant acid phospha tase (TRAP) activity. Some slides were treated with FITC-phalloidin and ant i-type I collagen antibody. TRAP-positive multinucleated cells were located in transparent spaces on the glass slides. These spaces did not stain immu nohistochemically with anti-type I collagen antibody. Podosome formation wa s observed in the multinucleated cells facing the edge of the transparent s paces. The scanning electron microscopy demonstrated well-spread large cell s located on the flattened surface on apatite particles covering the glass surface. Our results suggest that osteoclasts could resorb the apatite part icles and coated collagen on the glass slide. The resorption lacunae appear ed as transparent spaces, and the cytoskeleton of resorbing osteoclasts was observed in these spaces. ACC-coated glass slides could be useful for inve stigating the function and metabolic activities of osteoclasts. (C) 2000 Jo hn Wiley & Sons, Inc.