Microspace culture is promising for self-organization of induced pluripotent stem cells (iPSCs). However, the optimal size of microspaces for osteogenic differentiation is unclear. We hypothesized that a specific microspace size could facilitate self-organizing iPSC differentiation to form bone-like tissue in vitro. The objectives of this study were to investigate such effects of microspace size and to evaluate bone regeneration upon transplantation of the resulting osteogenic constructs. Dissociated mouse gingival fibroblast-derived iPSCs were plated in ultra-low-attachment microspace culture wells containing hundreds of U-bottom-shaped microwell spots per well to form cell aggregates in growth medium. The microwells had different aperture diameters/depths (400/560 μm (Elp400), 500/700 μm (Elp500), and 900/700 μm (Elp900)) (Kuraray; Elplasia). After 5 days of aggregation, cells were maintained in osteogenic induction medium for 35 days. Only cells in the Elp500 condition tightly aggregated and maintained high viability during osteogenic induction. After 10 days of induction, Elp500 cell constructs showed significantly higher gene expression of Runx2, Osterix, Collagen 1a1, Osteocalcin, Bone sialoprotein, and Osteopontin compared to constructs in Elp400 and Elp900. In methylene blue-counterstained von Kossa staining and Movat’s pentachrome staining, only Elp500 constructs showed robust osteoid formation on day 35, with high expression of type I collagen (a major osteoid component) and osteocalcin proteins. Cell constructs were transplanted into rat calvarial bone defects, and micro-CT analysis after 3 weeks showed better bone repair with significantly higher bone mineral density in the Elp500 group compared to the Elp900 group. These results suggest that microspace size affects self-organized osteogenic differentiation of iPSCs. Elp500 microspace culture specifically induces mouse iPSCs into osteoid-rich bone-like tissue possessing high bone regeneration capacity.

中文翻译：

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尺寸优化的微空间培养有助于将小鼠诱导的多能干细胞分化为富含类固醇的骨构建体。

微空间培养有望用于诱导多能干细胞（iPSC）的自组织。但是，用于成骨分化的微空间的最佳大小尚不清楚。我们假设特定的微空间大小可以促进自组织iPSC分化在体外形成骨样组织。这项研究的目的是研究微空间大小的这种影响，并评估所得成骨构建物移植后的骨再生。将解离的小鼠牙龈成纤维细胞衍生的iPSC铺在超低附着的微空间培养孔中，每个孔包含数百个U形微孔斑点，以在生长培养基中形成细胞聚集体。微孔具有不同的孔直径/深度（560分之400  μ米（Elp400），500/700  μ M（Elp500），和七百分之九百 μ M（Elp900））（可乐丽; Elplasia）。聚集5天后，将细胞在成骨诱导培养基中维持35天。在成骨诱导过程中，只有Elp500条件下的细胞紧密聚集并保持高活力。诱导10天后，Elp500细胞构建体显示Runx2，Osterix，胶原1a1，骨钙蛋白，骨唾液蛋白和骨桥蛋白的基因表达明显升高。与Elp400和Elp900中的构建体相比。在亚甲基蓝复刻的von Kossa染色和Movat的五色染色中，只有Elp500构造物在第35天显示出强劲的类骨质形成，并具有高表达的I型胶原蛋白（主要的类骨质成分）和骨钙蛋白。将细胞构建体移植到大鼠颅骨缺损中，与Elp900组相比，三周后的micro-CT分析显示Elp500组的骨修复效果更好，骨矿物质密度明显更高。这些结果表明，微空间大小会影响iPSC的自组织成骨分化。Elp500微空间培养可以特异性地诱导小鼠iPSC进入具有高骨再生能力的富含类骨质的骨样组织。