Cyclic mechanical stretch contributes to network development of osteocyte-like cells with morphological change and autophagy promotion but without preferential cell alignment in rat.
Cyclic mechanical stretch contributes to network development of osteocyte-like cells with morphological change and autophagy promotion but without preferential cell alignment in rat.
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DOI：
10.1016/j.bbrep.2017.04.018
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发表时间：
2017-09
影响因子：
2.7
通讯作者：
Sawase T
中科院分区：
文献类型：
--
作者：
Inaba N;Kuroshima S;Uto Y;Sasaki M;Sawase T
骨细胞在控制骨质量和生物磷灰石c轴/胶原纤维优先排列中发挥重要作用。然而，由于骨细胞体外三维（3D）培养的困难，骨细胞与机械应力之间的关系尚不清楚。本研究的目的是探讨循环机械拉伸对3d培养的骨细胞样细胞的影响。骨细胞样细胞是用大鼠颅骨成骨细胞在三维培养系统中培养的。连续24、48和96小时进行循环机械拉伸（幅度为8%，速率为2 cycles min - 1）。形态学、细胞数目和优先细胞排列进行了评价。采用定量PCR检测细胞凋亡和自噬相关基因的表达水平。3d培养的成骨细胞成为骨细胞样细胞，表达Dmp1、Cx43、Sost、Fgf23、RANKL等骨细胞特异性基因，形态变化与骨细胞相似。在无负荷条件下，细胞数量以时间依赖性的方式显著减少，而循环机械拉伸通过增加抗凋亡相关基因的表达显著防止细胞数量减少。此外，循环机械拉伸显著降低细胞大小和椭圆度，增加自噬相关基因LC3b和atg7的表达。有趣的是，无论机械拉伸如何，都没有发生优先细胞排列。这些发现表明抗凋亡作用有助于负载条件下骨细胞样细胞的网络发育。机械拉伸诱导的骨细胞样细胞球形变化可能与自噬上调有关。在成骨细胞分化成骨细胞并嵌入骨基质之前，体内机械载荷诱导的骨细胞优先排列可能是部分预先确定的。建立了新型骨细胞样细胞三维培养体系。机械拉伸影响细胞形态，没有优先的细胞排列。机械拉伸上调自噬相关基因的表达。机械拉伸防止了电池在无载荷状态下的收缩。
Osteocytes play important roles in controlling bone quality as well as preferential alignment of biological apatite c-axis/collagen fibers. However, the relationship between osteocytes and mechanical stress remains unclear due to the difficulty of three-dimensional (3D) culture of osteocytes in vitro. The aim of this study was to investigate the effect of cyclic mechanical stretch on 3D-cultured osteocyte-like cells. Osteocyte-like cells were established using rat calvarial osteoblasts cultured in a 3D culture system. Cyclic mechanical stretch (8% amplitude at a rate of 2 cycles min−1) was applied for 24, 48 and 96 consecutive hours. Morphology, cell number and preferential cell alignment were evaluated. Apoptosis- and autophagy-related gene expression levels were measured using quantitative PCR. 3D-cultured osteoblasts became osteocyte-like cells that expressed osteocyte-specific genes such as Dmp1, Cx43, Sost, Fgf23 and RANKL, with morphological changes similar to osteocytes. Cell number was significantly decreased in a time-dependent manner under non-loaded conditions, whereas cyclic mechanical stretch significantly prevented decreased cell numbers with increased expression of anti-apoptosis-related genes. Moreover, cyclic mechanical stretch significantly decreased cell size and ellipticity with increased expression of autophagy-related genes, LC3b and atg7. Interestingly, preferential cell alignment did not occur, irrespective of mechanical stretch. These findings suggest that an anti-apoptotic effect contributes to network development of osteocyte-like cells under loaded condition. Spherical change of osteocyte-like cells induced by mechanical stretch may be associated with autophagy upregulation. Preferential alignment of osteocytes induced by mechanical load in vivo may be partially predetermined before osteoblasts differentiate into osteocytes and embed into bone matrix. Novel three-dimensional culture system for osteocyte-like cells were developed. Mechanical stretch affected cell morphologies without preferential cell alignment. Mechanical stretch upregulated expression of autophagy-related genes. Mechanical stretch prevented cell decreases under non-loaded condition.