Chemical and Topographical Effects on Cell Differentiation and Matrix Elasticity in a Corneal Stromal Layer Model
Chemical and Topographical Effects on Cell Differentiation and Matrix Elasticity in a Corneal Stromal Layer Model
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DOI：
10.1002/adfm.201200655
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发表时间：
2012-09
影响因子：
19
通讯作者：
S. Wilson;I. Wimpenny;M. Ahearne;S. Rauz;A. E. El Haj;Ying Yang
中科院分区：
文献类型：
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作者：
S. Wilson;I. Wimpenny;M. Ahearne;S. Rauz;A. E. El Haj;Ying Yang
角膜细胞表型的控制和维持对于开发体外组织工程角膜修复策略至关重要。在这项研究中，地形和化学线索对成人角膜基质（AHDCS）细胞在三维（3D）多层组织结构中的机械、表型和基因型行为的影响进行了研究。地形线索通过多个对齐的静电纺纳米纤维网提供，这些网在整个结构中垂直排列，能够对齐单个细胞并允许细胞在层之间迁移。在培养基中使用不同的补充物来检查化学线索的影响。采用非破坏性压痕技术和光学相干层析成像技术分别测定了基体弹性（弹性模量）和尺寸变化。这些测量表明在实验期间细胞表型从收缩成纤维细胞到静止角化细胞的变化，并通过qPCR证实。与没有纳米纤维的结构相比，含有纳米纤维的结构具有更高的初始模量，减少收缩和有组织的细胞方向。在含血清培养基中培养的细胞种子构建体在整个培养期间的模量增加，并且与在无血清和含胰岛素培养基中培养的构建体相比，经历了明显更多的收缩。这表明血清中存在的生长因子促进成纤维细胞样表型；qPCR数据进一步验证了这些观察结果。这些结果表明，纳米纤维和无血清培养基加上胰岛素补充的协同效应为在3D结构中将角膜成纤维细胞恢复为角质细胞表型提供了最合适的地形和化学环境。
Control and maintenance of the keratocyte phenotype is vital to developing in vitro tissue engineered strategies for corneal repair. In this study the influence of topographical and chemical cues on the mechanical, phenotypical and genotypical behaviour of adult human derived corneal stromal (AHDCS) cells in three dimensional (3D) multi‐layered organised constructs is examined. Topographical cues are provided via multiple aligned electrospun nanofiber meshes, which are arranged orthogonally throughout the constructs and are capable of aligning individual cells and permitting cell migration between the layers. The influence of chemical cues is examined using different supplements in culture media. A non‐destructive indentation technique and optical coherence tomography are used to determine the matrix elasiticity (elastic modulus) and dimensional changes, respectively. These measurements were indicative of changes in cell phenotype from contractile fibroblasts to quiescent keratocytes over the duration of the experiment and corroborated by qPCR. Constructs containing nanofibers have a higher initial modulus, reduced contraction and organised cell orientation compared to those without nanofibers. Cell‐seeded constructs cultured in serum‐containing media increased in modulus throughout the culture period and underwent significantly more contraction than constructs cultured in serum‐free and insulin‐containing media. This implies that the growth factors present in serum promote a fibroblast‐like phenotype; qPCR data further validates these observations. These results indicate that the synergistic effect of nanofibers and serum‐free media plus insulin supplementation provide the most suitable topographical and chemical environment for reverting corneal fibroblasts to a keratocyte phenotype in a 3D construct.