Establishment of optimized in vitro assay methods for evaluating osteocyte functions
Establishment of optimized in vitro assay methods for evaluating osteocyte functions
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DOI：
10.1007/s00774-013-0555-5
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发表时间：
2015-01-01
影响因子：
3.3
通讯作者：
Suzuki, Hiroshi
中科院分区：
文献类型：
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作者：
Honma, Masashi;Ikebuchi, Yuki;Suzuki, Hiroshi
近年来的研究表明，骨细胞具有多种重要的生理功能。为了详细分析骨细胞的功能，原代骨细胞的体外实验系统将是有用的。不幸的是，骨细胞倾向于去分化并获得成骨细胞样特征，即使当细胞在三维（3D）胶原凝胶中培养时。因此，需要建立在较长时间内防止去分化的骨细胞培养条件。在这项研究中，我们获得了系统的信息，培养条件对骨细胞分化状态的影响。3D培养基质中胎牛血清（FBS）浓度为0.1%至0.5%，对骨细胞标志物的表达无显著影响。另一方面，向培养基质中添加Matrigel显著增强了Rankl和晚期骨细胞标志物如Sost和Fgf 23的表达。基质胶的加入也抑制了Opg和早期骨细胞标志物如Dmp 1和Gp38的上调。这些对骨细胞特性的影响在50%的Matrigel培养基质含量时最大。基质胶除了基质也增加了树突状突起延伸的骨细胞。此外，Matrigel除了显着刺激抗酒石酸酸性磷酸酶活性与骨髓巨噬细胞共培养。在所测试的条件中，50%Matrigel和0.2%FBS在I型胶原基质中是原代骨细胞培养的最佳条件。
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.