A novel culture system to generate osteoclasts and bone resorption using porcine bone marrow cells: role of M-CSF.
A novel osteoclast generation and bone resorption assay system is described in which enhanced osteoclastic generation and bone resorption is induced in porcine bone marrow cell cultures cultured in low-serum medium supplemented with fibroblastic cell (L929) conditioned medium (CM). Numerous osteoclasts, which could be identified by TRAP staining and specific labelling with 121F antibody, were generated in a time-dependent and L929-CM concentration-dependent fashion. A specific antibody against murine M-CSF/CSF-1 abolished osteoclast formation indicating that M-CSF is the essential component of the L929-CM driven osteoclast generation. Culturing on devitalized bone slices resulted in extensive osteoclast-mediated resorption as visualized microscopically. After 16 days in culture, practically the entire bone slice surface was excavated by the osteoclastic cells. Bone resorption could be monitored with time using a novel enzyme-linked immunoassay measuring type I collagen N-telopeptides in culture supernatants. Release of collagen fragments from the slices was paralleled by osteoclastic secretion of TRAP. Salmon calcitonin significantly inhibited collagen fragment and TRAP release. 1,25-Dihydroxyvitamin D3 greatly promoted osteoclast generation and subsequent bone resorption, but its presence was not essential for this process to occur.
Scheven
B A
BA
Rowett Research Institute, Division of Biochemical Sciences, Bucksburn, Aberdeen, Scotland, United Kingdom.
Milne
J S
JS
Robins
S P
SP
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001854:Bone Marrow Cells; D001862:Bone Resorption; D002116:Calcitonin / Q000494:pharmacology; D002117:Calcitriol / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D017077:Culture Media, Conditioned; D007527:Isoenzymes / Q000378:metabolism; D007739:L Cells; D016173:Macrophage Colony-Stimulating Factor / Q000502:physiology*; D051379:Mice; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D013552:Swine; D000071681:Tartrate-Resistant Acid Phosphatase
D017077:Culture Media, Conditioned; D007527:Isoenzymes; C028815:salmon calcitonin; D016173:Macrophage Colony-Stimulating Factor; D002116:Calcitonin; D003094:Collagen; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; D002117:Calcitriol