Morphologic characterization of organized extracellular matrix deposition by ascorbic acid-stimulated human corneal fibroblasts
Morphologic characterization of organized extracellular matrix deposition by ascorbic acid-stimulated human corneal fibroblasts
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DOI：
10.1167/iovs.06-1216
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发表时间：
2007-09-01
影响因子：
4.4
通讯作者：
Ruberti, Jeffrey W.
中科院分区：
文献类型：
--
作者：
Guo, Xiaoqing;Hutcheon, Audrey E. K.;Ruberti, Jeffrey W.
目的。观察未转化培养的人角膜成纤维细胞在长期培养中合成的细胞外基质（ECM）的结构和形态。人角膜基质角质细胞在胎牛血清和稳定的维生素c衍生物的存在下进行transwell培养，细胞被允许合成纤维状ECM长达5周。通过光学显微镜（相衬显微镜和差示干涉对比显微镜）和透射显微镜（标准显微镜和快速冷冻/深蚀刻显微镜）对构建体进行评估。电子显微照片显示了分层结构，包括多个平行的细胞层和细胞外基质，包括小的、多分散的纤维（27-51 nm）的平行阵列，这些纤维经常在方向上交替。差示干涉对比图像显示定向的ECM纤维阵列平行于结构的平面，而速冻、深染显微图像通过膜表面结构阵列显示基质与成纤维细胞相互作用的细节。在稳定的维生素C衍生物中培养的人角化细胞能够组装细胞外基质，其中包括平行排列的ECM原纤维。由此产生的结构是高度细胞化的，在形态上类似于发育中的哺乳动物基质，其中有组织的基质是衍生的。细胞膜上结构阵列的出现表明在合成ECM的局部组织中起作用。该模型可以为组织结缔组织（如角膜）形成的基本过程提供关键的见解，并可能为组织工程提供一个适合的仿生基质支架。
PURPOSE. To characterize the structure and morphology of extracellular matrix (ECM) synthesized by untransformed, cultured human corneal fibroblasts in long-term cultures.METHODS. Human corneal stromal keratocytes were expanded in transwell culture in the presence of fetal bovine serum and a stable derivative of vitamin C. The cells were allowed to synthesize a fibrillar ECM for up to 5 weeks. Constructs were assessed by light (phase-contrast and differential interference-contrast) and transmission (standard and quick freeze/deep etch) microscopy.RESULTS. Electron micrographs revealed stratified constructs with multiple parallel layers of cells and an extracellular matrix comprising parallel arrays of small, polydisperse fibrils (27-51 nm) that often alternate in direction. Differential interference contrast images demonstrated oriented ECM fibril arrays parallel to the plane of the construct, whereas quick-freeze, deepetch micrographs showed the details of the matrix interaction with fibroblasts through arrays of membrane surface structures.CONCLUSIONS. Human keratocytes, cultured in a stable vitamin C derivative, are capable of assembling extracellular matrix, which comprises parallel arrays of ECM fibrils. The resultant constructs, which are highly cellular, are morphologically similar to the developing mammalian stroma, where organized matrix is derived. The appearance of arrays of structures on the cell membranes suggests a role in the local organization of synthesized ECM. This model could provide critical insight into the fundamental processes that govern the genesis of organized connective tissues such as the cornea and may provide a scaffolding suitable for tissue engineering a biomimetic stroma.