Development of a new method for obtaining osteoclasts from endosteal surfaces.

Abstract

Techniques for the isolation of a highly pure population of viable osteoclasts are limited. For this reason, we developed an isolation procedure that results in a high yield of osteoclast-like cells, up to 92% pure, from 3-wk-old chicken tibias. The unique feature of the method is the migration of cells from marrow-free endosteal surfaces to vitronectin-coated plates. The cells retain the osteoclast phenotype and remain viable in culture for a minimum of 1 wk. The cells were characterized and compared to two populations of authentic avian osteoclasts, which were isolated on the basis of association with fibronectin-coated plates. The cells contained substantial amounts of tartrate-resistant acid phosphatase. Alkaline phosphatase levels were negligible, suggesting little contamination by osteoblasts. Response to parathyroid hormone, dibutyryl cyclic adenosine monophosphate, calcitonin, acetazolamide, 17 beta-estradiol, and prostaglandin E2 was evident, as detected by measuring acid production. The vitronectin-associating cells contained numerous mitochondria, had the ability to resorb bone in an in vitro bone slice assay, and specifically bound biotinylated vitronectin. At 5 d of culture, the cells demonstrated marginal multinuclearity, having two to three nuclei. A large number (approximately 1 x 10(6) cells/tibia) of viable cells that exhibit characteristics of authentic osteoclasts can be obtained by the method described. Potentially, this method could be applied to other species.