Limraksasin Phoonsuk, Okawa Hiroko, Zhang Maolin, Kondo Takeru, Osathanon Thanaphum, Pavasant Prasit, Egusa Hiroshi
Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, Sendai, Miyagi 980-8575, Japan.
Weintraub Center for Reconstructive Biotechnology, UCLA School of Dentistry, Los Angeles, CA 90095-1668, USA.
Stem Cells Int. 2020 May 14;2020:7082679. doi: 10.1155/2020/7082679. eCollection 2020.
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 . 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 m (Elp400), 500/700 m (Elp500), and 900/700 m (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 , , , , , and 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.
微空间培养对于诱导多能干细胞（iPSC）的自我组织具有广阔前景。然而，用于成骨分化的微空间的最佳尺寸尚不清楚。我们推测特定的微空间尺寸可以促进iPSC自我组织分化形成类骨组织。本研究的目的是研究微空间尺寸的这种影响，并评估所得成骨构建体移植后的骨再生情况。将解离的小鼠牙龈成纤维细胞来源的iPSC接种到超低附着微空间培养孔中，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。这些微孔具有不同的孔径/深度（400/560μm（Elp400）、500/700μm（Elp500）和900/700μm（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。只有在Elp500条件下的细胞在成骨诱导期间紧密聚集并保持高活力。诱导10天后，与Elp400和Elp900中的构建体相比，Elp500细胞构建体显示出、、、、和的基因表达显著更高。在亚甲蓝复染的冯库萨染色和莫瓦特五色染色中，只有Elp500构建体在第35天显示出强大的类骨质形成，I型胶原蛋白（主要类骨质成分）和骨钙素蛋白表达高。将细胞构建体移植到大鼠颅骨骨缺损处，3周后的微CT分析显示，与Elp900组相比，Elp500组的骨修复更好，骨矿物质密度显著更高。这些结果表明微空间尺寸影响iPSC的自我组织成骨分化。Elp500微空间培养特异性地将小鼠iPSC诱导成具有高骨再生能力的富含类骨质的类骨组织。
