转谷氨酰胺酶交联胶原凝胶构建三维角膜基质

Scaffold of transglutaminase cross-linked collagen gel for three-dimensional corneal stroma reconstruction

目的检测转谷氨酰胺酶交联胶原凝胶对三维培养的角膜基质细胞的影响,探讨可提高机械性能的组织工程角膜基质层新途径。方法胶原酶消化法获取原代兔角膜基质细胞,以加入转谷氨酰胺酶与胶原凝胶交联为实验组,不加酶交联为对照组。倒置显微镜下每日观察细胞生长情况、Alamar-Blue试剂检测细胞增生、免疫荧光法检测凝胶内细胞波形蛋白、检测透光度、酶消化法检测胶原凝胶抗消化能力。结果实验组细胞胶原凝胶内附着和生长优于对照组,细胞在凝胶内呈树枝状生长。2组细胞均随培养时间延长明显增生(P=0.000)。共焦显微镜下见2组细胞胞浆波形蛋白均阳性表达,实验组细胞伪足更丰富。实验组透光度稍差于对照组。实验组抵抗胶原酶消化的能力显著增强。结论酶交联的胶原凝胶对角膜基质细胞无毒性作用,重构的基质层结构更加稳定,有利于组织工程角膜基质层的构建。

Objective This study was to investigate the property of scaffold of transglutaminase cross-linked collagen gel and the change of seeded keratocytes in order to provide a new way of enhance of the mechanical strength of tissue engineering corneal stroma. Methods Primary rabbit corneal keratocytes were obtained by digesting corneal stoma using type II collagenase. The suspension of the third passage of keratocytes was mixed with collagen solution on an ice bath at a density of 5 × 10^4 eelis/mL,and three-dimensional corneal stroma was reconstructed. The collagen gel was prepared with transglutaminase and erosslinker in experimental group,and collagen gel without transglutaminase was used as control group. Phase contrast microscope examination was performed to observe the state of cell-seeded scaffolds daily. The proliferation of keratoeytes was assessed at 1,2, 3,5 days separately by Alamar-Blue reduction assay. Immunohistochemistry was carried out to examine the cellular response to vimentin antibody under the confocal microscope. Transmittance of the collagen samples was evaluated at 2 and 3 weeks. The cross-linked statue of cell-seeded collagen substrates was observed to assess the digestion procedure. Results Keratocytes elongated and showed triangle or dendritic shape with a stronger ability of forming network in cross-linked collagen compared with spindle-shape cells in the native collagen without transglutaminase. Proliferation ability of the cells was increased in both groups after culture. The keratocytes showed the positive response for vimentin after 14-day culture in both groups, and the cellular pseudopods were abundant in experimental group. Light transmission of transglutaminase crosslinked ceil-seeded scaffold was lower than that of native collagen cell-seeded scaffold,but the stability was superior to control group. The transglutaminase-treated collagen substrates showed a stronger resistance to collagenase in comparison with untransglutaminase-treated collagen substrates. Conclusion The cellular morphology and activity of keratocytes within transglutaminase crosslinked collagen are similar to natural cornea. The scaffold of transglutaminase cross-linked collagen gel is favorable for the three-dimensional corneal stroma reconstruction.