猪脱细胞角膜基质组织结构特点与生物相容性研究

背景构建组织工程化角膜时，载体的选择十分重要。目前有多种载体可供选用，但脱细胞角膜基质是公认的较好载体。目的观察猪脱细胞角膜基质的组织结构特点，评价其与异种角膜基质和上皮细胞的生物相容性。方法取猪角膜组织进行组织块培养，经胰蛋白酶-EDTA酶消化角膜上皮、基质、内皮细胞，支架组织于-20℃冷冻干燥后灭菌保存。猪脱细胞角膜基质石蜡切片行常规苏木精一伊红染色，光学显微镜下观察组织的形态学特征；扫描电镜下观察其组织结构特点；并对其物理特性，如抗拉性、膨胀性、含水量和透明度进行评价。将猪脱细胞角膜基质移植到兔角膜基质层内，同时与体外培养的兔角膜上皮细胞共培养4周，分析其组织和细胞生物相容性。结果经酶消化处理后猪角膜组织中未见上皮、基质和内皮细胞，其胶原纤维直径大小、排列与正常角膜组织相同，抗拉性、膨胀性、含水量和透明度等物理特性与正常猪角膜相似。猪脱细胞角膜基质行异种兔角膜基质层间移植1周时可见轻度水肿，2周后水肿基本消退，4周时透明度较好。猪脱细胞角膜基质与兔角膜基质之间贴附良好，未见炎症反应及新生血管。兔角膜上皮细胞接种于猪脱细胞角膜基质上共培养4周后CK3表达阳性。猪脱细胞角膜基质脱水前与脱水2h、4h后及正常猪角膜基质间的抗拉性、膨胀性、含水量的差异均无统计学意义（P〉0．05），但脱水2h、4h后及正常猪角膜基质的透明度明显好于脱水前，差异均有统计学意义（P〈0．01）。结论猪脱细胞角膜基质组织结构与正常猪角膜相似，与兔角膜基质和上皮细胞具有良好的生物相容性。

Structure characteristics and biocompatibility of decellulal matrix of porcine

Background The select of supporter is critical for the construction of tissue engineering cornea.Many carrier carl be utilized in the construction of tissue engineering cornea,but de-cellular corneal matrix is known to be one of optimal supporters.Objective Present study was to investigate the characteristics of de-cellular corneal matrix of porcine of structure and biocompatibility for rabbit cornea stroma and limbal epithelial ceHs. Methods The porcine cornea was prepared as de-cellular corneal matrix of porcine by the application of detergent enzyme combined process.The corneal epithelial cells,keratocyte and endothelial cells of porcine were removed completely and stored in -20℃ refrigerator after sterilization.The morphology of de-cellular corneal matrix of porcine was examined by hematoxylin-eosin staining under the light microscope.The structure characteristics of de.cellular corneal matrix of porcine under the scan electron microscope,and its physics features were investigated by the evaluation of water content,strength,expansion and transparency.The de-cellular corneal matrix of porcine were implanted to cornea stroma of rabbit and co-cultured with rabbit corneal epithelial cells for 4 weeks in vitro to assess the keracyte compatibility. Results The epithelial cells,keratocyte and endothelial cells of porcine were removed completely by trypsogen digestion.The collagen fibril network and collagen plates paralleled to corneal surface under the light microscope.The water content,strength,expansion。Ratio of light transparency of de-cellular corneal matrix of porcine were similar to normal porcine cornea.After implantation of de.cellular comeal matrix of porcine into rabbits corneal stroma,the edema of tissue was found in one week,and edema disappeared on two weeks and became clear on four weeks after surgery.The de-cellular eorneal matrix attached to rabbit cornea stroma well.No inflammatory eell and new vessel were found after surgery.The co-cultured rabbit corneal epithelial cells differentiated and proliferated on the surfaee of de-cellular corneal matrix and showed positive response for CK3.No statistically significant differences were found in the water content,strength,expansion of de-cellular cornea matrix of porcine among the normal,before dehydration,2 and 4 hours after dehydration cornea matrix(P＞0.05).However,the transparency was much better in the corneal matrix with 2-hour,4-hour dehydration in comparison with non-dehydration one(P＜0.05). Conclusion The structure features of de-cellular cornea matrix of porcine are similar to normal porcine cornea.Good biocompatibility is proved for xenogenesis of rabbit cornea.