Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Key Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong Province, China.
ACS Biomater Sci Eng. 2022 Mar 14;8(3):1301-1311. doi: 10.1021/acsbiomaterials.2c00039. Epub 2022 Mar 1.
Tissue-engineered cornea endothelial sheets (TECES), created using a biocompatible thin and transparent carrier with corneal endothelial cells, could alleviate the shortage of donor corneas and provide abundant functional endothelial cells. In our previous clinical trials, the effectiveness and safety of the acellular porcine corneal stroma (APCS) applied in lamellar keratoplasty have been confirmed. In this study, we optimized the method to cut APCS into multiple 20 μm ultrathin lamellae by a cryostat microtome and investigated the feasibility of TECES by seeding rabbit corneal endothelial cells (RCECs) on ultrathin APCS. Cell adhesion, proliferation, and functional gene expression of RCECs on tissue-culture plastic and APCS of different thicknesses were compared. The results indicated that ultrathin lamellae were superior in increasing cell viability and maintaining cell functions. Analyzing with histology, electron microscopy, and immunofluorescence, we found that RCECs cultured on 20 μm ultrathin APCS for 5 days grew into a confluent monolayer with a density of 3726 ± 223 cells/mm and expressed functional biomarkers Na/K-ATPase and zonula occludens. After 14 days, RCECs formed an early stage of Descemet's membrane-like structure by synthesizing collagen IV and laminin. Human corneal endothelial cells were also used to further validate the supportive effect of ultrathin APCS on cells. The resulting constructs were flexible and tough enough to implant into rabbits' anterior chambers through small incisions. TECES adhered to the posterior corneal stroma, and the thickness of cornea gradually reduced to normal after grafting. These results indicate that the ultrathin APCS can serve as a tissue engineering carrier and might be a suitable alternative for endothelial cells expansion in endothelial keratoplasty.
组织工程化角膜内皮片（TECES）是使用具有角膜内皮细胞的生物相容性薄而透明的载体构建的，它可以缓解供体角膜的短缺，并提供丰富的功能性内皮细胞。在我们之前的临床试验中，已证实脱细胞猪角膜基质（APCS）在板层角膜移植中的有效性和安全性。在这项研究中，我们通过冷冻切片机将 APCS 切成多个 20μm 的超薄薄片，优化了方法，并研究了在超薄 APCS 上接种兔角膜内皮细胞（RCEC）构建 TECES 的可行性。比较了 RCEC 在组织培养塑料和不同厚度的 APCS 上的细胞黏附、增殖和功能基因表达。结果表明，超薄薄片更有利于提高细胞活力并维持细胞功能。通过组织学、电子显微镜和免疫荧光分析，我们发现 RCEC 在培养 5 天后，在 20μm 的超薄 APCS 上生长成具有 3726±223 个细胞/mm2 密度的致密单层，并表达功能性生物标志物 Na/K-ATPase 和紧密连接蛋白。培养 14 天后，RCEC 通过合成胶原 IV 和层粘连蛋白形成早期类似 Descemet 膜的结构。进一步用人角膜内皮细胞验证了超薄 APCS 对细胞的支持作用。构建的组织具有足够的柔韧性和强度，可通过小切口植入兔的前房。TECES 黏附于后角膜基质，在移植后角膜厚度逐渐恢复正常。这些结果表明，超薄 APCS 可用作组织工程载体，可能是内皮角膜移植中内皮细胞扩增的合适替代品。
