Mi Shengli, Connon Che J
Graduate School at Shenzhen, Tsinghua University, Shenzhen, People's Republic of China.
Methods Mol Biol. 2013;1014:143-55. doi: 10.1007/978-1-62703-432-6_9.
Collagen has excellent biocompatibility, is biodegradable, and possesses low immunogenicity. Therefore, this protein is a very suitable substrate for the formation of a corneal scaffold for therapeutic use. The highly hydrated nature of conventional collagen gels, however, results in a gel that is structurally weak and difficult to manipulate. In this chapter, we describe a novel method to cultivate limbal epithelial cells (LEC) on a compressed collagen scaffold. The compressed collagen scaffold can be rapidly constructed using a cell-independent process, which produces dense and mechanically strong collagen constructs with controllable microscale features.We embedded corneal keratocytes in a collagen gel, which we subsequently compressed and coated with laminin. The resulting construct supported the physiological morphology and stratification of LEC. The expression of a specific marker for differentiated LEC, cytokeratin 3 (CK3), and a marker for undifferentiated LEC, cytokeratin 14 (CK14), were similar in LEC expanded on both the compressed collagen construct and the leading conventional scaffold, denuded amniotic membrane (AM). We therefore demonstrate that a laminin-coated, compressed collagen gel containing keratocytes can support LEC expansion, stratification, and differentiation to a degree that is comparable to denuded AM. Our novel compressed collagen/keratocyte construct has potential for use as a tissue-engineered artificial cornea.
胶原蛋白具有优异的生物相容性、可生物降解性，且免疫原性低。因此，这种蛋白质是用于治疗用途的角膜支架形成的非常合适的基质。然而，传统胶原蛋白凝胶的高度水合性质导致凝胶结构薄弱且难以操作。在本章中，我们描述了一种在压缩胶原蛋白支架上培养角膜缘上皮细胞（LEC）的新方法。压缩胶原蛋白支架可以通过与细胞无关的过程快速构建，该过程产生具有可控微观特征的致密且机械强度高的胶原蛋白构建体。我们将角膜基质细胞嵌入胶原蛋白凝胶中，随后对其进行压缩并用层粘连蛋白包被。所得构建体支持LEC的生理形态和分层。在压缩胶原蛋白构建体和领先的传统支架——脱细胞羊膜（AM）上扩增的LEC中，分化的LEC的特异性标志物细胞角蛋白3（CK3）和未分化的LEC的标志物细胞角蛋白14（CK14）的表达相似。因此，我们证明含有角膜基质细胞的层粘连蛋白包被的压缩胶原蛋白凝胶能够在一定程度上支持LEC的扩增、分层和分化，其程度与脱细胞羊膜相当。我们新的压缩胶原蛋白/角膜基质细胞构建体具有用作组织工程人工角膜的潜力。
