Toda Hiroyuki, Yamamoto Masaya, Uyama Hiroshi, Tabata Yasuhiko
Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Division of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka, Japan.
Acta Biomater. 2017 Aug;58:312-322. doi: 10.1016/j.actbio.2017.03.016. Epub 2017 Mar 12.
The objective of this study is to design the manner of ephrinB2 immobilized onto polyacrylamide (PAAm) hydrogels with varied elasticity and evaluate the effect of hydrogels elasticity and the immobilized manner of ephrinB2 on the Runx2 expression of human mesenchymal stem cells (hMSC). The PAAm hydrogels were prepared by the radical polymerization of acrylamide (AAm), and N,N'-methylenebisacrylamide (BIS). By changing the BIS concentration, the elasticity of PAAm hydrogels changed from 1 to 70kPa. For the bio-specific immobilization of ephrinB2, a chimeric protein of ephrinB2 and Fc domain was immobilized onto protein A-conjugated PAAm hydrogels by making use of the bio-specific interaction between the Fc domain and protein A. When hMSC were cultured on the ephrinB2-immobilized PAAm hydrogels with varied elasticity, the morphology of hMSC was of cuboidal shape on the PAAm hydrogels immobilized with ephrinB2 compared with non-conjugated ones, irrespective of the hydrogels elasticity. The bio-specific immobilization of ephrinB2 enhanced the level of Runx2 expression. The expression level was significantly high for the hydrogels of 3.6 and 5.9kPa elasticity with bio-specific immobilization of ephrinB2 compared with other hydrogels with the same elasticity. The hydrogels showed a significantly down-regulated RhoA activity. It is concluded that the Runx2 expression of hMSC is synergistically influenced by the hydrogels elasticity and their immobilized manner of ephrinB2 immobilized.
Differentiation fate of mesenchymal stem cells (MSC) is modified by biochemical and biophysical factors, such as elasticity and signal proteins. However, there are few experiments about combinations of them. In this study, to evaluate the synergistic effect of them on cell properties of MSC, we established to design the manner of Eph signal ligand, ephrinB2, immobilized onto polyacrylamide hydrogels with varied elasticity. The gene expression level of an osteogenic maker, Runx2, was enhanced by the immobilized manner, and significantly enhanced for the hydrogels of around 4kPa elasticity with bio-specific immobilization of ephrinB2. This is the novel report describing to demonstrate that the Runx2 expression of MSC is synergistically influenced by the hydrogels elasticity and their manner of ephrinB2 immobilized.
本研究的目的是设计将ephrinB2固定在具有不同弹性的聚丙烯酰胺（PAAm）水凝胶上的方式，并评估水凝胶弹性和ephrinB2的固定方式对人间充质干细胞（hMSC）Runx2表达的影响。PAAm水凝胶通过丙烯酰胺（AAm）和N，N'-亚甲基双丙烯酰胺（BIS）的自由基聚合制备。通过改变BIS浓度，PAAm水凝胶的弹性从1kPa变化到70kPa。对于ephrinB2的生物特异性固定，利用Fc结构域与蛋白A之间的生物特异性相互作用，将ephrinB2与Fc结构域的嵌合蛋白固定在蛋白A偶联的PAAm水凝胶上。当hMSC在具有不同弹性的ephrinB2固定的PAAm水凝胶上培养时，与未偶联的PAAm水凝胶相比，无论水凝胶弹性如何，hMSC在固定有ephrinB2的PAAm水凝胶上的形态均为立方形。ephrinB2的生物特异性固定增强了Runx2的表达水平。与具有相同弹性的其他水凝胶相比，对于弹性为3.6kPa和5.9kPa且具有ephrinB2生物特异性固定的水凝胶，其表达水平显著更高。水凝胶显示出RhoA活性显著下调。结论是hMSC的Runx2表达受到水凝胶弹性及其ephrinB2固定方式的协同影响。
间充质干细胞（MSC）的分化命运受到生化和生物物理因素的影响，如弹性和信号蛋白。然而，关于它们组合的实验很少。在本研究中，为了评估它们对MSC细胞特性的协同作用，我们设计了将Eph信号配体ephrinB2固定在具有不同弹性的聚丙烯酰胺水凝胶上的方式。成骨标志物Runx2的基因表达水平通过固定方式得到增强，对于弹性约为4kPa且具有ephrinB2生物特异性固定的水凝胶，其表达水平显著增强。这是一篇新颖的报道，证明了MSC的Runx2表达受到水凝胶弹性及其ephrinB2固定方式的协同影响。
