Substrate stiffness- and topography-dependent differentiation of annulus fibrosus-derived stem cells is regulated by Yes-associated protein
Substrate stiffness- and topography-dependent differentiation of annulus fibrosus-derived stem cells is regulated by Yes-associated protein
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Yes相关蛋白调节纤维环来源干细胞的基质硬度和地形依赖性分化
DOI：
10.1016/j.actbio.2019.05.013
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发表时间：
2019-07-01
影响因子：
9.7
通讯作者：
Li, Bin
中科院分区：
文献类型：
--
作者：
Chu, Genglei;Yuan, Zhangqin;Li, Bin
纤维环（AF）组织工程作为一种有前途的治疗椎间盘退行性疾病（DDD）的方法越来越受到人们的关注。然而，AF的再生仍然面临着许多挑战，由于该组织的巨大复杂性，并且缺乏对AF内细胞水平的结构-功能关系的深入理解是非常必要的。鉴于AF是由多种类型的细胞组成的，并且沿着径向具有梯度的力学、地形学和生化特征。在这项研究中，我们的目的是实现定向分化的AF衍生干细胞（AFSCs）通过模仿天然AF组织的机械和地形特征。AFSCs培养在四种类型的静电纺丝聚（醚碳酸酯氨基甲酸酯）脲（PECUU）支架具有不同的刚度和纤维尺寸（软，小尺寸;硬，小尺寸;软，大尺寸和硬，大尺寸）。结果表明，在纤维尺寸不变的情况下，随着支架刚度的增加，外AF（oAF）表型标记基因的表达水平增加，而内AF（iAF）表型标记基因的表达水平则相反。当支架刚度固定时，AFSC中oAF表型标记基因的表达随着纤维尺寸的增加而增加。而iAF表型标记基因的表达量下降。这种基质硬度和地形依赖性的AFSCs的变化是符合AF组织的遗传和生化分布从内到外的区域。此外，我们发现，在AFSCs培养增加刚度和纤维尺寸的支架，Yes相关蛋白（雅普）易位到细胞核中，但它仍然主要是磷酸化和胞质中的细胞在软支架与小纤维尺寸。抑制雅普下调肌腱/韧带相关基因的表达，而软骨相关基因的表达上调。结果表明，矩阵刚度是AFSC分化的一个有力的调节器。此外，我们发现，纤维尺寸的支架诱导细胞粘附的变化，并确定细胞的形状，铺展面积和细胞外基质的表达。总之，支架的力学性能和形貌特征调节AFSC的分化，可能通过YAP依赖的mechanotransduction mechanism.Statement的显著性物理线索，如机械性能，地形和几何特征被证明深刻影响培养的干细胞的生长和分化。以前，我们已经发现纤维环来源的干细胞（AFSCs）的分化可以通过支架的刚度来调节。在这项研究中，我们制作了四种类型的聚醚碳酸酯氨基甲酸酯脲（PECUU）支架与控制刚度和纤维的大小，探索AFSCs诱导分化的潜力。我们发现，AFSCs能够呈现不同的基因表达模式，这仅仅是支架材料的刚度和纤维尺寸的结果。这项工作首次证明了较大尺寸和较高刚度的底物增加了黏着斑蛋白组装的量，并激活了预分化AFSC中的雅普信号传导。本研究有助于深入理解材料生物学，并有助于解释AF中雅普机械传感响应材料生物物理效应的机制。(C)2019 Acta Materialia Inc.由爱思唯尔有限公司出版。保留所有权利。
Annulus fibrosus (AF) tissue engineering has attracted increasing attention as a promising therapy for degenerative disc disease (DDD). However, regeneration of AF still faces many challenges due to the tremendous complexity of this tissue and lack of in-depth understanding of the structure-function relationship at cellular level within AF is highly required. In light of the fact that AF is composed of various types of cells and has gradient mechanical, topographical and biochemical features along the radial direction. In this study, we aimed to achieve directed differentiation of AF-derived stem cells (AFSCs) by mimicking the mechanical and topographical features of native AF tissue. AFSCs were cultured on four types of electrospun poly(ether carbonate urethane)urea (PECUU) scaffolds with various stiffness and fiber size (soft, small size; stiff, small size; soft, large size and stiff, large size). The results show that with constant fiber size, the expression level of the outer AF (oAF) phenotypic marker genes in AFSCs increased with the scaffold stiffness, while that of inner AF (iAF) phenotypic marker genes showed an opposite trend. When scaffold stiffness was fixed, the expression of oAF phenotypic marker genes in AFSCs increased with fiber size. While the expression of iAF phenotypic marker genes decreased. Such substrate stiffness- and topography-dependent changes of AFSCs was in accordance with the genetic and biochemical distribution of AF tissue from the inner to outer regions. Further, we found that the Yes-associated protein (YAP) was translocated to the nucleus in AFSCs cultured with increasing stiffness and fiber size of scaffolds, yet it remained mostly phosphorylated and cytosolic in cells on soft scaffolds with small fiber size. Inhibition of YAP down-regulated the expression of tendon/ligament-related genes, whereas expression of the cartilage-related genes was upregulated. The results illustrate that matrix stiffness is a potent regulator of AFSC differentiation. Moreover, we reveal that fiber size of scaffolds induced changes in cell adhesions and determined cell shape, spreading area, and extracellular matrix expression. In all, both mechanical property and topography features of scaffolds regulate AFSC differentiation, possibly through a YAP-dependent mechanotransduction mechanism.Statement of SignificancePhysical cues such as mechanical properties, topographical and geometrical features were shown to profoundly impact the growth and differentiation of cultured stem cells. Previously, we have found that the differentiation of annulus fibrosus-derived stem cells (AFSCs) could be regulated by the stiffness of scaffold. In this study, we fabricated four types of poly(ether carbonate urethane)urea (PECUU) scaffolds with controlled stiffness and fiber size to explore the potential of induced differentiation of AFSCs. We found that AFSCs are able to present different gene expression patterns simply as a result of the stiffness and fiber size of scaffold material. This work has, for the first time, demonstrated that larger-sized and higher-stiffness substrates increase the amount of vinculin assembly and activate YAP signaling in pre-differentiated AFSCs. The present study affords an in-depth comprehension of materiobiology, and be helpful for explain the mechanism of YAP mechanosensing in AF in response to biophysical effects of materials. (C) 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.