Department of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwan.
Department of Surgery, Fu Jen Catholic University Hospital, and Fu Jen Catholic University College of Medicine, New Taipei City, Taiwan.
J Tissue Eng Regen Med. 2018 Apr;12(4):e1852-e1864. doi: 10.1002/term.2616. Epub 2017 Dec 12.
The effects of the stiffness of substrates on the cell behaviours of human bone marrow-derived mesenchymal stem cells (hBMSC) have been investigated, but the effects of the secondary structures of proteins in the substrates on the morphological transformation and differentiation of hBMSC have yet been elucidated. To investigate these issues, silk fibroin-poly(ε-caprolactone) SP cardiac patches of poly(ε-caprolactone; P), on which is grafted by silk fibroin (SF) with various β-sheet contents (or crystallinity) to provide various degrees of stiffness, were produced to examine the in vitro behaviours of hBMSC during proliferation, and cardiomyogenesis on the SP patches. β-sheet contents of SF from 20% to 44% (SP20 to SP44, respectively) were induced on patches, which were examined by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, and analysed using the Fourier self-deconvolution method. The stiffness of the SP patches, quantified by their Young's moduli and elasticities, increased with the crystallinity of the SF. During 3 days of proliferation, hBMSC migrated and morphologically transformed into 3D microtissues with diameters of approximately 150-200 μm on low-stiffness SP20 and SP30 patches, whereas 2D monolayers were observed on the SP37 and SP44 patches. The 3D microtissues/patch yielded more extensive in vitro cardiomyogenesis of hBMSC than the 2D cell monolayer with significantly higher expressions of all examined cardiac-specific proteins after induction by 5-aza. Notably, in vivo subcutaneously growing 3D microtissues on SP20 patches and a 2D monolayer on SP44 patches were preliminarily demonstrated in a rat model. Morphological transformations of hBMSC from a 2D monolayer to a 3D microtissue by low-stiffness SP cardiac patches, promoting cardiomyogenesis, provide a new opportunity for cardiac tissue engineering.
研究了基底硬度对人骨髓间充质干细胞（hBMSC）细胞行为的影响，但基底中蛋白质二级结构对 hBMSC 形态转化和分化的影响尚不清楚。为了研究这些问题，制备了丝素蛋白-聚（ε-己内酯） SP 心脏补片，其中聚（ε-己内酯）（P）接枝有不同β-折叠含量（或结晶度）的丝素蛋白（SF），以提供不同程度的硬度，用于研究 hBMSC 在增殖过程中的体外行为，以及在 SP 补片上的心肌发生。通过衰减全反射傅里叶变换红外（ATR-FTIR）光谱对 SF 的β-折叠含量从 20%到 44%（分别为 SP20 到 SP44）进行了检测，并使用傅里叶自解卷积法进行了分析。通过杨氏模量和弹性来量化 SP 补片的硬度，其随 SF 结晶度的增加而增加。在增殖的 3 天内，hBMSC 在低硬度 SP20 和 SP30 补片上迁移并形态转化为直径约 150-200μm 的 3D 微组织，而在 SP37 和 SP44 补片上观察到 2D 单层。与 2D 细胞单层相比，3D 微组织/补片上的 hBMSC 进行了更广泛的体外心肌发生，经 5-氮杂胞苷诱导后，所有检查的心脏特异性蛋白的表达均显著升高。值得注意的是，在大鼠模型中初步证明了 SP20 补片上的 3D 微组织和 SP44 补片上的 2D 单层在体内皮下生长。低硬度 SP 心脏补片将 hBMSC 从 2D 单层形态转化为 3D 微组织，促进心肌发生，为心脏组织工程提供了新的机会。
