Bioengineering Organized, Multilamellar Human Corneal Stromal Tissue by Growth Factor Supplementation on Highly Aligned Synthetic Substrates
Bioengineering Organized, Multilamellar Human Corneal Stromal Tissue by Growth Factor Supplementation on Highly Aligned Synthetic Substrates
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DOI：
10.1089/ten.tea.2012.0545
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发表时间：
2013-09-01
影响因子：
4.1
通讯作者：
Wagner, William R.
中科院分区：
文献类型：
--
作者：
Wu, Jian;Du, Yiqin;Wagner, William R.
概述天然人角膜基质组织的微结构被认为是成功设计角膜组织的关键特征。层状的多层胶原原纤维具有正交取向，这决定了该组织具有良好的生物力学性能，而均匀的胶原原纤维尺寸和纤维间距是其光学透明性的关键。本研究的目的是开发一种由人角膜基质干细胞(HCSSCs)分泌的高度组织化的胶原纤维结构，以模拟人角膜基质组织。在由聚氨基甲酸酯尿素制成的高度排列的纤维基质上培养时，成纤维细胞生长因子-2(10 ng/m L)和转化生长因子-β3(0.1 ng/m L)影响胶原纤维基质的组织和分泌的丰度。HCSSCs分化为角质形成细胞，典型的基因标志物KERA、B3GnT7和CHST6显著上调。成纤维细胞生长因子-2刺激hCSSCs分泌单向排列的胶原纤维，而转化生长因子-β3诱导的胶原层呈垂直纤维取向。联合应用成纤维细胞生长因子-2和转化生长因子-β3诱导的多层板层具有垂直取向的胶原纤维，呈类似于人角膜基质组织的模式。构建物厚度为60-70微米，并增加了角膜特异性细胞外基质成分的含量，包括硫酸角蛋白、鲁米肯和角质形成蛋白。结合基质提示和生长因子增强的方法提供了一种新的方法来设计组织良好的、基于胶原的结构，并具有合适的纳米结构用于角膜修复和再生。
Recapitulating the microstructure of the native human corneal stromal tissue is believed to be a key feature in successfully engineering the corneal tissue. The stratified multilayered collagen fibril lamellae with orthogonal orientation determine the robust biomechanical properties of this tissue, and the uniform collagen fibril size and interfibrillar spacing are critical to its optical transparency. The objective of this investigation was to develop a highly organized collagen-fibril construct secreted by human corneal stromal stem cells (hCSSCs) to mimic the human corneal stromal tissue. In culture on a highly aligned fibrous substrate made from poly(ester urethane) urea, the fibroblast growth factor-2 (FGF-2, 10 ng/mL) and transforming growth factor-beta 3 (TGF-beta 3, 0.1 ng/mL) impacted the organization and abundance of the secreted collagen fibril matrix. hCSSCs differentiated into keratocytes with significant upregulation of the typical gene markers, including KERA, B3GnT7, and CHST6. FGF-2 treatment stimulated hCSSCs to secrete collagen fibrils strongly aligned in a single direction, whereas TGF-beta 3 induced collagenous layers with orthogonal fibril orientation. The combination of FGF-2 and TGF-beta 3 induced multilayered lamellae with orthogonally oriented collagen fibrils, in a pattern mimicking the human corneal stromal tissue. The constructs were 60-70 mu m thick and had an increased content of cornea-specific extracellular matrix components, including keratan sulfate, lumican, and keratocan. The approach of combining substrate cues with growth factor augmentation offers a new means to engineer well-organized, collagen-based constructs with an appropriate nanoscale structure for corneal repair and regeneration.