基于新型压缩胶原支架的组织工程板层角膜构建及移植研究

Corneal dysfunction account for 40% of all ophthalmic diseases. Corneal transplantation is a major part of the strategies currently used to correct corneal blindness, a common endpoint of disorders associated with the cornea. The construction of a tissue engineered cornea for use in the replacement of dysfunctional corneal tissue would resolve the problem of the shortage of corneal graft material. Collagen is a very suitable biomaterial for the assembly of an artificial cornea, as collagen type I is the most abundant stromal protein in cornea. Collagen also possesses excellent biocompatibility, an appropriate biodegradation rate and low immunogenicity. The highly hydrated nature of conventional collagen gels,however, indicates that collagen fibres are highly dispersed, resulting in a gel that is inherently weak and difficult to manipulate. By plastic compression of collagen hydrogels, dense, mechanically strong collagen scaffolds with controllable micro-scale features were produced. In order to further mimic the normal corneal stroma, keratocytes were embedded into the collagen scaffold. Polyvinyl alcohol (PVA) was incorporated into collagen hydrogels to further increase the mechanical stability of the compressed scaffold. We aim to use this engineered structure as a substrate to support the expansion and stratification of limbal epithelial stem cells, to produce a novel biomaterial for application in ocular tissue engineering. At the same time, investigate the effect of different levels of compression affect the corneal stromal progenitor cells growth, proliferation and differentiation which were embedded in collagen scaffold, in order to provide references for the individual collagen scaffold design and production. On the other hand, the transplanted cells were labeled by fluorescence to monitoring the proliferation, migration and repositioning process of the exogenous cells on the corneas of receptor rabbits, in order to enrich the research theory of stem cell reconstruction of the ocular surface of limbal stem cell deficiency.

角膜病占眼科所有疾病的40%，角膜移植是"角膜盲人"复明的唯一手段。然而，角膜移植材料极度匮乏，角膜支架联合干细胞构建组织工程角膜是从根本上解决这一矛盾的有效途径。由于角膜的结构蛋白主要为胶原蛋白, 胶原蛋白具有优良的生物相容性、适宜的降解率及弱抗原性，因此非常适合制作成角膜生物支架。然而，该材料力学强度差的缺点又限制了其应用。本研究拟以I型胶原溶液为主要材料，并添加聚乙烯醇和角膜基质祖细胞，通过机械压缩联合毛细原理，构建力学强度高的微米级压缩胶原支架，承载角膜上皮干细胞后，进行角膜板层移植，并评价移植效果，以期为临床角膜移植提供材料；同时，研究不同程度压缩对包埋在胶原支架中角膜基质祖细胞生长、增殖及分化的影响，为进一步个性化胶原支架的设计和构建提供参考；另外，对外源移植上去的角膜细胞荧光标记，动态监测外源角膜干细胞在被移植兔眼表增殖、迁移及重新定位过程，丰富干细胞重建角膜上皮理论。

角膜移植是“角膜盲人”复明的唯一手段，角膜移植材料极度匮乏，利用干细胞构建组织工程角膜是从根本上解决这一矛盾的有效途径。角膜基质主要由I型胶原构成，I型胶原本身又有优良的生物特性，因此I型胶原非常适合用于构建组织工程角膜支架。然而，I型胶原水凝胶力学强度低的缺点又限制了其应用。本项目通过机械压缩联合毛细原理，将传统I型胶原水凝胶中的水分快速排出，构建出力学强度高的微米级压缩胶原支架。为了模拟正常角膜的细胞组织结构，在胶原溶液中添加角膜基质细胞，培养箱中塑形后压缩，可以得到透明度很高的新型压缩胶原支架。进一步研究不同程度压缩（未压缩、部分压缩、完全压缩），对包埋在胶原支架中细胞活力的影响，live-dead染色结果显示：压缩过程本身并没有影响压缩胶原支架中角膜基质细胞的活力。为了进一步提升压缩胶原支架的力学性能，在胶原溶液中混入聚乙烯醇（3%），支架力学性能可提高48%（n=5）。采用角膜基质囊袋法将压缩胶原支架和去上皮羊膜移植于家兔角膜基质中，结果表明，两种支架均具有良好的生物相容性。将角膜上皮细胞分别接种到未压缩胶原支架、压缩胶原支架和去上皮羊膜支架，结果显示：细胞可在三种支架上粘附增殖并形成复层，并保持角膜免疫表型（CK3阳性）。将上述后两种力学强度好的角膜植片进行动物移植实验，结果显示：两种植片均可重建兔角膜受损病理眼表，和去上皮羊膜相比，压缩胶原支架降解时间更长，更适合用于组织工程角膜植片的构建。