Department of Ophthalmology, Xiang'an Hospital of Xiamen University; Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China.
Xiamen University Affiliated Xiamen Eye Center, Xiamen, Fujian, China.
Invest Ophthalmol Vis Sci. 2019 Feb 1;60(2):517-527. doi: 10.1167/iovs.18-24869.
To investigate a novel strategy in constructing tissue-engineered corneal stromal equivalent based on amniotic membrane and keratocytes.
The ultrathin amniotic membrane (UAM) was laminated, with corneal stromal cells (CSCs) distributed between the space of the layered UAMs. Calcein AM staining was used to evaluate cellular viability, morphology, and arrangement. Immunostaining, qRT-PCR, and Western blot were performed to detect gene and protein expression in keratocytes. Optical coherence tomography visualized the cross sections and thickness of the UAM construction. The microstructure of the CSC-secreted extracellular matrix (ECM) was investigated by scanning electron microscopy and transmission electron microscopy (TEM). To evaluate the feasibility of the multilayer UAM-CSC lamination for surgery, the corneal substitute was used to perform lamellar keratoplasty. Slit lamp microscopy and corneal fluorescein staining were performed in postsurgery observation.
The CSCs maintained their keratocyte phenotype and secreted well-organized ECM on the aligned UAM surface. The four-layer UAM-CSC lamination attained half thickness of the human cornea (250 ± 18 μm) after 8 weeks' culture, which also showed promising optimal transparency. In TEM images, the CSC-generated ECM displayed stratified, multilayered lamellae with orthogonal fibril arrangement, which was similar to the human cornea microstructure. Furthermore, the stromal equivalent was successfully preformed in lamellar keratoplasty. Four weeks post surgery, the substitute was well integrated into the recipient cornea and completely epithelialized without myofibroblast differentiation.
Our study established a novel 3D biomimetic corneal model to replicate the corneal stromal organization with multilayer UAM, which was capable of promoting the development of corneal stroma-like tissues in vitro, establishing a new avenue for basic research and therapeutic potential.
研究基于羊膜和角膜基质细胞构建组织工程化角膜基质等效物的新策略。
将超薄羊膜（UAM）分层，角膜基质细胞（CSC）分布在分层 UAM 的间隙之间。使用钙黄绿素 AM 染色评估细胞活力、形态和排列。免疫染色、qRT-PCR 和 Western blot 用于检测角膜基质细胞中的基因和蛋白表达。光相干断层扫描可视化 UAM 构建的横截面和厚度。扫描电子显微镜和透射电子显微镜（TEM）用于研究 CSC 分泌的细胞外基质（ECM）的微观结构。为评估多层 UAM-CSC 层压用于手术的可行性，使用角膜替代物进行板层角膜移植。术后观察行裂隙灯显微镜和角膜荧光素染色。
CSC 保持其角膜基质细胞表型，并在对齐的 UAM 表面分泌排列整齐的 ECM。经过 8 周培养，四层 UAM-CSC 层压达到人眼角膜厚度的一半（250±18μm），同时也显示出有希望的最佳透明度。在 TEM 图像中，CSC 产生的 ECM 显示出分层、多层的薄片，具有正交纤维排列，类似于人眼角膜的微观结构。此外，在板层角膜移植中成功构建了基质等效物。术后 4 周，替代物与受体角膜完全整合，无成纤维细胞分化，完全上皮化。
本研究建立了一种新的 3D 仿生角膜模型，通过多层 UAM 复制角膜基质组织的结构，能够促进体外角膜基质样组织的发育，为基础研究和治疗潜力开辟了新途径。
