Zhu Jingxian, Cai Qing, Zhang Xin, Hu Xiaoqing, Li La, Wang Weiping, Shao Zhenxing, Dai Linghui, Cheng Liyuan, Yang Xiaoping, Zhou Chunyan, Ao Yingfang
Institute of Sports Medicine, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, PR China.
J Biomed Nanotechnol. 2013 Oct;9(10):1757-67. doi: 10.1166/jbn.2013.1661.
The nanotopographical features of artificial scaffolds have complex effects on the biological characteristics of stem cells. They influence cell adhesion, spreading, proliferation, and differentiation; however we have limited knowledge on how these processes occur under nanotopographical cues. In this study, two kinds of electrospun nanofibrous meshes with different fiber arrangements (totally non-woven and lattice-like) were fabricated and used for in vitro culture of mesenchymal stem cells (MSCs). By comparing the characteristic marks related to osteogenic differentiation, we found that with prolonged culture time, osteopontin (OPN), osteocalcin (OCN) and alkaline phosphatase (ALP), as well as related genes (Runx2 and Colla genes), were all expressed at higher levels on lattice-like nanofibrous meshes than on non-woven ones. These results indicated that the lattice-like nanofibrous mesh activated the osteogenic differentiation of MSCs owing to changes in cell morphology directed by nanofiber orientations. Compared with pure non-woven nanofibrous meshes, lattice-like ones possessed a combined structure of parallel, magnetic-line-like, and non-woven regions. MSCs adhering onto them had upregulated expression levels of integrin subunits a5 and b1, and activated downstream signaling pathways of Ras homolog gene family member A (RhoA) and extracellular signal-regulated kinase (ERK). When the specific inhibitors PD98059 and Y27632 were used to inhibit phosphorylated ERK and p160 ROCKII activity, respectively, F-actin became disordered and the expression level of Runx2 was downregulated. Thus, we concluded that the scaffold nanotopography may modulate the microenvironment of MSCs and promote their osteogenic differentiation through the RhoA and ERK signaling pathways. These findings provided valuable information on the selection of artificial matrices suitable for MSCs application in bone tissue engineering.
人工支架的纳米拓扑特征对干细胞的生物学特性具有复杂影响。它们会影响细胞黏附、铺展、增殖和分化；然而，我们对于这些过程在纳米拓扑线索下如何发生的了解有限。在本研究中，制备了两种具有不同纤维排列方式（完全无纺和格子状）的电纺纳米纤维网，并用于间充质干细胞（MSCs）的体外培养。通过比较与成骨分化相关的特征标记，我们发现随着培养时间延长，骨桥蛋白（OPN）、骨钙素（OCN）和碱性磷酸酶（ALP）以及相关基因（Runx2和Colla基因）在格子状纳米纤维网上的表达水平均高于无纺纳米纤维网。这些结果表明，格子状纳米纤维网由于纳米纤维取向引导的细胞形态变化而激活了MSCs的成骨分化。与纯无纺纳米纤维网相比，格子状纳米纤维网具有平行、磁线状和无纺区域的组合结构。黏附在其上的MSCs整合素亚基a5和b1的表达水平上调，并且Ras同源基因家族成员A（RhoA）和细胞外信号调节激酶（ERK）的下游信号通路被激活。当分别使用特异性抑制剂PD98059和Y27632抑制磷酸化ERK和p160 ROCKII活性时，F-肌动蛋白变得紊乱，Runx2的表达水平下调。因此，我们得出结论，支架纳米拓扑可能通过RhoA和ERK信号通路调节MSCs的微环境并促进其成骨分化。这些发现为选择适用于骨组织工程中MSCs应用的人工基质提供了有价值的信息。
