[Induced differentiation of human peripheral blood CD68+ mononuclear cells into osteoclasts in vitro].
OBJECTIVE
To establish a method for obtaining highly purified primary human osteoclast precursors for the biochemical and molecular biological research.


METHODS
CD68(+) mono/macrophages were separated from peripheral blood mononuclear cells (PBMCs) of healthy donors by means of immunomagnetic cell sorting for subsequent analysis with flow cytometry. The isolated cells were incubated on coverslips or bone slices in the presence of dexamethasone(10(-8) mol/L), macrophage colony-stimulating factor (25 microg/L ) and soluble receptor activator of nuclear factor (NF)-kappaB ligand (s-RANKL, 16 microg/L). Calcitonin receptor (CR) immunocytochemistry and tartrate-resistant acid phosphatase (TRAP) histochemistry were employed. The bone slices were also studied by scanning electron microscopy (SEM).


RESULTS
Fluorescence-activated cytometric analysis showed that 93.06%+/-0.61%  n=4  of the selected cells were CD68(+) cells. After 7 days of culture of the CD68(+) cells, VR+, TRAP+ multinucleated giant cells appeared, and resorption lacunae could be observed by SEM.


CONCLUSIONS
Highly purified CD68(+) cells can be obtained from human PBMCs as the osteoclast precursors, and mature osteoclasts can be induced from CD68(+) mono/macrophages by RANKL.
Zeng
Run-ming
RM
Department of Orthopedics and Spine Surgery, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China. hosward@21cn.com
Jin
Da-di
DD
Zhang
Zhong-min
ZM
D000328:Adult; D000818:Animals; D015703:Antigens, CD / Q000032:analysis*; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis*; D002454:Cell Differentiation; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D008297:Male; D051379:Mice; D008855:Microscopy, Electron, Scanning; D010010:Osteoclasts / Q000166:cytology*; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D018950:Lipopolysaccharide Receptors; C067980:CD68 antigen, human; D000097382:CD68 Molecule