DEVELOPMENT AND CHARACTERIZATION OF A PORCINE MODEL TO STUDY OSTEOCLAST DIFFERENTIATION AND ACTIVITY

The study of osteoclast differentiation, function, and fate has been h ampered by the lack of nonavian, nonrodent models in which biochemical and molecular studies can be conducted, The present study was underta ken to determine if osteoclasts could be generated from porcine bone m arrow cells, Bone marrow from the long bones of neonatal female pigs w as enriched for mononuclear cells and cultured in the presence or abse nce of 1,25-(OH)(2)D-3, rhIL-11, or PGE(2), A confluent layer of strom al cells was observed after 4-8 days in culture and multinucleated gia nt cells formed after 6-10 days of culture, The multinucleated cells s tained positively for tartrate-resistant acid phosphatase and formed r esorption lacunae when exposed to bovine cortical bone slices, When ex amined by transmission electron microscopy, abundant mitochondria, per inuclear Golgi complexes, numerous variably sized vacuoles, prominent rough endoplasmic reticulum, and free polysomes were observed in the m utlinucleated cells, Stimulation of the in vitro generated osteoclasts with 10(-8) mol/L salmon calcitonin resulted in a three to fivefold i ncrease in cAMP production and in cell retraction, Although the osteoc lasts formed in the presence or absence of 1,25-(OH)(2)D-3, 10-50-fold more osteoclasts were observed in the cultures treated with 1,25-(OH) (2)D-3 in comparison to cultures without 1,25-(OH)(2)D-3. Osteoclast d ifferentiation was also stimulated by rhIL-11 and PGE(2); although, th e number of cells generated in 6-7 days was significantly less than th e number obtained with 1,25-(OH)(2)D-3 treatment, In addition, these m ultinucleated cells expressed high levels of Src kinase activity and r esponded to bafilomycin A(1), an inhibitor of the vacuolar type H+-ATP ase, treatment with a decrease in osteoclastic bone resorption, In sum mary, the porcine cells possess the major distinguishing characteristi cs of osteoclasts and provide an alternative mammalian model to study osteoclast differentiation and resorptive activity.