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The Chinese Journal of Dental Research

Year 2001
Volume 4 , Issue 3

Pages: 26 - 31

Effects of 1,25-Dihydroxyvitamin D3 on Stimulation of Osteoclast-like Cell Formation and Bone Resorption in Murine Marrow Cell Culture

Tie Jun Li, DDS, MS, PhD/Shi Feng Yu, DDS, MS

Objective: To clarify the events involved in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced osteoclast-like cell formation and bone resorption in vitro, to compare this culture system with direct isolation of osteoclasts from long bones of newborn rabbits, and to evaluate their properties for in vitro detection of osteoclastic bone resorption. Methods: Marrow cells from NH mice were harvested and cultured in a-MEM with 10% fetal bovine serum. The appearance of tartrate-resistant acid phosphatase (TRAP)-staining multinuclear cells or osteoclast-like cells and formation of bone resorption pits were measured after 3, 6, 9, and 12 days of exposure to various concentrations of 1,25(OH)2D3 in culture. Osteoclasts isolated from endosteal surfaces of long bones of newborn rabbits were also co-cultured with glass coverslips and bovine cortical bone slices, and quantification of TRAP-positive multinuclear cells and bone resorbing pits was carried out in the same manner as that for murine marrow culture. Results: 1,25(OH)2D3 (concentration higher than 10-9 mol/L) could induce recruitment of murine osteoclast-like cells and their bone resorption activity in a dose-dependent manner. Formation of TRAP-positive osteoclast-like cells was first observed on day 6, reached its peak on day 9, and reduced in number thereafter. The number of bone resorption pits on the bone slices in culture showed an increase on day 9 and on day 12. Osteoclasts isolated from the unfractionated bone cells of newborn rabbits showed identical morphology, tartrate-resistant acid phosphatase (TRAP) reactivity, and the ability to excavate pits on cortical bone slices. The number of osteoclasts was stable for the first 6 days of culture, and resorption pits on the bone slice surface began to appear on day 3 and increased with time through day 12. Conclusion: While the classical method of isolating osteoclasts from endosteal surfaces of long bones maintains its practical usefulness in observing mature or functional osteoclasts in vitro, the 1,25(OH)2D3-modulated mouse bone marrow culture system appears to provide a better adjunct in investigation of the differentiation process of osteoclast-like cells from their hematopietic precursors.



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