title: Establishment of optimized in vitro assay methods for evaluating osteocyte functions.
abstract: Recent studies have revealed that osteocytes play multiple important physiological roles. To analyze osteocyte functions in detail, an in vitro experimental system for primary osteocytes would be useful. Unfortunately, osteocytes tend to dedifferentiate and acquire osteoblast-like features even when the cells are cultured in three-dimensional (3D) collagen gel. Therefore, it is desirable to establish osteocyte culture conditions that prevent dedifferentiation over longer periods. In this study, we obtained systematic information about the influence of culture conditions on osteocyte differentiation states. Fetal bovine serum (FBS) concentrations from 0.1 to 0.5 % in 3D culture matrix did not significantly influence the expression of osteocyte markers. On the other hand, addition of Matrigel to the culture matrix significantly enhanced the expression of Rankl and late osteocyte markers such as Sost and Fgf23. Matrigel addition also inhibited upregulation of Opg and early osteocyte markers such as Dmp1 and Gp38. These effects on osteocyte properties were maximal at a Matrigel culture matrix content of 50 %. Matrigel addition to the matrix also increased dendritic process extension by osteocytes. In addition, Matrigel addition significantly stimulated tartrate-resistant acid phosphatase activity in co-culture with bone marrow macrophages. Among the conditions tested, 50 % Matrigel and 0.2 % FBS in type I collagen matrix were optimal for culture of primary osteocytes.
authors.affiliation: Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan, mhonma-tky@umin.ac.jp.
authors.forename: Masashi
authors.initials: M
authors.lastname: Honma
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ikebuchi
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kariya
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000831:Animals, Newborn; D001854:Bone Marrow Cells / Q000166:cytology; D002417:Cattle; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D018920:Coculture Techniques; D003094:Collagen / Q000737:chemistry; D003470:Culture Media / Q000737:chemistry; D003713:Dendritic Cells / Q000166:cytology; D004338:Drug Combinations; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D007797:Laminin / Q000737:chemistry; D008264:Macrophages / Q000166:cytology; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D010011:Osteocytes / Q000166:cytology*; D011509:Proteoglycans / Q000737:chemistry; D053245:RANK Ligand / Q000378:metabolism; D044967:Serum / Q000737:chemistry; D000071681:Tartrate-Resistant Acid Phosphatase; D000098853:Podoplanin
chemical_list: D000135:Acid Phosphatase; D048868:Adaptor Proteins, Signal Transducing; D003094:Collagen; D003470:Culture Media; D004338:Drug Combinations; D016326:Extracellular Matrix Proteins; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D007527:Isoenzymes; D007797:Laminin; D008562:Membrane Glycoproteins; D011509:Proteoglycans; D053245:RANK Ligand; D000071681:Tartrate-Resistant Acid Phosphatase; C513308:Dmp1 protein, mouse; C000717428:Fgf23 protein, mouse; C490441:Pdpn protein, mouse; D000098853:Podoplanin; C486030:Sost protein, mouse; C505689:Tnfsf11 protein, mouse; C059485:matrigel