Purpose

To identify the feasibility of reconstructing corneal endothelial sheets by seeding non-infected monoclonal human corneal endothelial cells (HCECs) onto porcine Descemet's membrane (DM) and verifying the function in vitro and in vivo.

Methods

Denuded porcine DM was decellularized for haematoxylin and eosin staining, and DNA was removed via incubation with ethylene glycol diglycidyl ether (EGDE). The physical properties of the incubated DMs were evaluated and compared to those of unincubated DMs. The non-infected monoclonal HCECs were examined by chromosome analysis and the cell proliferation was evaluated by BrdU-labelling. Then HCECs at passage 30 were then seeded on the DM and cultured for approximately 5 days. The cell growth, density and expression of the sodium-potassium adenosine triphosphatase (Na⁺/K⁺-ATPase), the tight-junction-associated protein zonula occludens (ZO-1) and acetylated alpha tubulin were examined by electron microscopy and immunocytochemistry to compare HCECs cultivated on porcine DM and those cultured in vitro. Cells on the reconstructed HCEC sheets were labeled with DiI, and the sheets were subsequently transplanted into cat eyes via DM endothelial keratoplasty (DMEK). The corneal transparency, thickness, anterior segment, and HCEC density were monitored in vivo, and the corneal endothelial cell morphology and histological structure were examined ex vivo 98 days after surgery.

Results

No significant differences were observed in the elongation at break of the DMs and the thickness of the DMs incubated with EGDE compared to those of the unincubated DMs (P > 0.05). Results of chromosome analysis shown the number of the HCEC cell line was still 46 and no abnormal chromosome structure was found. BrdU-labelling shown the HCECs stopped proliferating after 5 days and the cells formed a single layer. The cells transferred to porcine DM formed tight connections with the substrate and generated layers of hexagonal cells on day 5. Adjacent cells cultivated on DM were closely attached to each other, tightly adhered to the porcine DM and expressed the Na⁺/K⁺-ATPase, ZO-1 protein and acetylated alpha tubulin, as did HCECs cultured in vitro. In addition, the HCEC density on DMs was 3020.14 ± 52.30 cells/mm². After surgery, the corneas gradually became transparent, and the thickness decreased to 525.33 ± 56.23 μm at day 98 after the transplantation, while the control corneas showed consistent oedema during the monitoring period. The HCEC density was 2521.60 ± 78.24 cells/mm² in vivo 98 days after transplantation. The histological results showed that the DiI-labeled cells were dense in the transplanted area and had a hexagonal or polygonal morphology and a normal ultrastructure; adjacent cells were closely attached to each other and tightly adhered to the porcine DM.

Conclusions

Seeding non-infected monoclonal HCECs on porcine DM could reconstruct functional corneal endothelial sheets. These results may help uncover new applications for tissue-engineered endothelium in endothelial keratoplasty.

中文翻译：

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用猪Descemet膜制成的体外和体内评估重建的人角膜内皮片。

目的

通过将未感染的人类人角膜内皮细胞（HCEC）播种到猪Descemet膜（DM）上并在体外和体内验证其功能，确定重建角膜内皮层的可行性。

方法

将裸露的猪DM脱细胞以进行苏木精和曙红染色，并通过与乙二醇二缩水甘油醚（EGDE）孵育除去DNA。评价了孵育的DM的物理性质，并将其与未孵育的DM的物理性质进行比较。通过染色体分析检查未感染的单克隆HCEC，并通过BrdU标记评估细胞增殖。然后将第30代的HCECs接种在DM上，并培养约5天。钠钾腺苷三磷酸酶（Na ⁺ / K ⁺-ATPase），紧密连接相关的小带闭合蛋白（ZO-1）和乙酰化的α微管蛋白通过电子显微镜和免疫细胞化学检查，以比较在猪DM和体外培养的HCEC。重建的HCEC薄片上的细胞用DiI标记，然后通过DM内皮角膜移植术（DMEK）将薄片移植到猫眼中。体内监测角膜透明度，厚度，前节和HCEC密度，并在术后98天离体检查角膜内皮细胞的形态和组织学结构。

结果

与未孵育的DM相比，在DMDE的断裂伸长率和DM的厚度上没有观察到显着差异（P> 0.05）。染色体分析的结果表明，HCEC细胞系的数目仍然是46，并且没有发现异常的染色体结构。BrdU标记显示HCEC在5天后停止增殖，并且细胞形成单层。转移到猪DM的细胞与底物形成紧密连接，并在第5天产生六边形细胞层。在DM上培养的相邻细胞彼此紧密附着，紧密粘附在猪DM上并表达Na ⁺ / K ⁺-ATPase，ZO-1蛋白和乙酰化的微管蛋白，与体外培养的HCEC一样。另外，DMs上的HCEC密度为3020.14±52.30个细胞/ mm ²。手术后，角膜逐渐变得透明，在移植后第98天厚度降至525.33±56.23μm，而对照角膜在监测期间显示出一致的水肿。移植后98天的体内HCEC密度为2521.60±78.24个细胞/ mm ²。组织学结果表明，DiI标记的细胞在移植区致密，具有六边形或多边形的形态和正常的超微结构。相邻的细胞彼此紧密附着并紧密粘附于猪DM。

结论

在猪DM上播种未感染的单克隆HCEC可以重建功能性角膜内皮层。这些结果可能有助于发现组织工程内皮在角膜移植术中的新应用。