Development and characterization of a porcine model to study osteoclast differentiation and activity.

ABSTRACT

The study of osteoclast differentiation, function, and fate has been hampered by the lack of nonavian, nonrodent models in which biochemical and molecular studies can be conducted. The present study was undertaken to determine if osteoclasts could be generated from porcine bone marrow cells. Bone marrow from the long bones of neonatal female pigs was enriched for mononuclear cells and cultured in the presence or absence of 1,25-(OH)2D3, rhIL-11, or PGE2. A confluent layer of stromal cells was observed after 4-8 days in culture and multinucleated giant cells formed after 6-10 days of culture. The multinucleated cells stained positively for tartrate-resident acid phosphatase and formed resorption lacunae when exposed to bovine cortical bone slices. When examined by transmission electron microscopy, abundant mitochondria, perinuclear Golgi complexes, numerous variably sized vacuoles, prominent rough endoplasmic reticulum, and free polysomes were observed in the multinucleated cells. Stimulation of the in vitro generated osteoclasts with 10(-8) mol/L salmon calcitonin resulted in a three to fivefold increase in cAMP production and in cell retraction. Although the osteoclasts formed in the presence or absence of 1,25-(OH)2D3, 10-50-fold more osteoclasts were observed in the cultures treated with 1,25-(OH)2D3 in comparison to cultures without 1,25-(OH)2D3. Osteoclast differentiation was also stimulated by rhIL-11 and PGE2; although, the number of cells generated in 6-7 days was significantly less than the number obtained with 1,25-(OH)2D3, treatment. In addition, these multinucleated cells expressed high levels of Src kinase activity and responded to bafilomycin A1, an inhibitor of the vacuolar type H(+)-ATPase, treatment with a decrease in osteoclastic bone resorption. In summary, the porcine cells possess the major distinguishing characteristics of osteoclasts and provide an alternative mammalian model to study osteoclast differentiation and resorptive activity.