Quantitative analyses of matrices, osteoblasts, and osteoclasts during bone remodeling using an in vitro system
Quantitative analyses of matrices, osteoblasts, and osteoclasts during bone remodeling using an in vitro system
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DOI：
10.1007/s00774-022-01381-z
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发表时间：
2022-11
影响因子：
3.3
通讯作者：
Shuya Oguchi;Tomoaki Sakamoto;K. Hoshi;Atsuhiko Hikita
中科院分区：
文献类型：
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作者：
Shuya Oguchi;Tomoaki Sakamoto;K. Hoshi;Atsuhiko Hikita
前言骨重建在维持骨内环境平衡中起着核心作用。我们团队已经建立了一个体外系统，通过它可以纵向观察骨重建过程中的细胞事件。本研究使用该系统对成骨细胞、破骨细胞和基质进行定量分析，以阐明它们的时间变化和相互关系。材料和方法将EGFP小鼠的成骨细胞培养成钙化结节，然后与Tnfrsf11aCre/+x Ai14小鼠的骨髓巨噬细胞共同培养3周(吸收阶段)。然后用成骨细胞分化培养液培养3周(形成期)。每周使用双光子显微镜观察相同的部位。使用二次谐波产生来检测矩阵。对基质、成骨细胞和破骨细胞的相关参数进行量化和统计学分析。结果在双光子显微镜下观察到基质在同一部位的吸收和补充。大体定量显示基质和成骨细胞参数在吸收相减少，在形成期增加，而破骨细胞参数则呈现相反的模式。当将一个视野划分为16个感兴趣区(ROI)，并分析每个ROI中各参数之间的相关性时，矩阵体积减少和增加为中度相关。基质与成骨细胞、基质与破骨细胞的参数呈中度相关，而成骨细胞与破骨细胞的参数仅呈弱相关。结论细胞与基质在改建过程中存在一定的相关性。该系统有可能成为骨重建研究的有力工具。
IntroductionBone remodeling plays a central role in the maintenance of bone homeostasis. Our group has established an in vitro system by which the cellular events during bone remodeling can be observed longitudinally. This study used this system to quantitatively analyze osteoblasts, osteoclasts, and matrices to elucidate their temporal changes and correlations.Materials and methodsOsteoblasts from EGFP mice were cultured to form calcified nodules, followed by co-culture with bone marrow macrophages from Tnfrsf11aCre/+x Ai14 mice for 3 weeks (resorption phase). Then cells were cultured with osteoblast differentiation medium for 3 weeks (formation phase). The same sites were observed weekly using 2-photon microscopy. Matrices were detected using second harmonic generation. Parameters related to matrices, osteoblasts, and osteoclasts were quantified and statistically analyzed.ResultsResorption and replenishment of the matrix were observed at the same sites by 2 photon microscopy. Gross quantification revealed that matrix and osteoblast parameters decreased in the resorption phase and increased in the formation phase, while osteoclast parameters showed the opposite pattern. When one field of view was divided into 16 regions of interest (ROIs) and correlations between parameters were analyzed in each ROI, decreased and increased matrix volumes were moderately correlated. Parameters of matrices and osteoblasts, and those of matrices and osteoclasts exhibited moderate correlations, while those of osteoblasts and osteoclasts were only weakly correlated.ConclusionSeveral correlations between cells and matrix during remodeling were demonstrated quantitatively. This system may be a powerful tool for the research of bone remodeling.