Isolation of highly enriched rabbit osteoclasts from collagen gels: A new assay system for bone-resorbing activity of mature osteoclasts

Abstract

When unfractionated rabbit bone cells were seeded and incubated on a collagen gel, tartrate-resistant acid phosphatase (TRAP)-positive multinucleate cells (MNCs) adhered to the surface of the collagen gel more readily than other types of cells. Based on the differences in adherence of the MNCs onto the collagen gel, we developed a new method for the isolation of functionally mature osteoclasts with high purity. Sequential treatments with pronase E/EDTA and collagenase at a low concentration (0.01%) released most of the nonosteoclasts from the gel surface, whereas only MNCs still remained on the surface. After washing off the released cells, we could harvest a cell population highly enriched for osteoclasts from the collagen gel by a second digestion with collagenase. The yield of TRAP-positive MNCs was 40000–50000 cells per long bones from one rabbit. The purity of the TRAP-positive MNCs was reproducibly more than 95%. These cells, cultured on dentine slices, showed typical ultrastructural features of osteoclasts, i.e., a highly developed ruffled border and clear zone, and they excavated the dentine to form pits. In addition, the isolated MNCs showed specific binding to calcitonin. The dentine-resorbing activity appeared as early as 2h after plating and reached a plateau at 24h. The pit area increased in direct proportion to the number of osteoclasts plated. Calcitonin inhibited the dentine-resorbing activity, but parathyroid hormone or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] did not have any effect on it. Though these cells exhibited dentine-resorbing activity even in the absence of any support from stromal cells, this activity was enhanced by rabbit stromal cells in response to 1,25(OH)2D3. In addition, these stromal cells elongated the life span of the isolated osteoclasts without 1,25(OH)2D3. To assess the functions of authentic osteoclasts, we can now culture highly purified and functional osteoclasts under the desired conditions and in controlled cell numbers. This isolation method is also applicable to numerous molecular studies.