Bio-fabrication of stem-cell-incorporated corneal epithelial and stromal equivalents from silk fibroin and gelatin-based biomaterial for canine corneal regeneration

Abstract

Corneal grafts are the imperative clinical treatment for canine corneal blindness. To serve the growing demand, this study aimed to generate tissue-engineered canine cornea in part of the corneal epithelium and underlying stroma based on canine limbal epithelial stem cells (cLESCs) seeded silk fibroin/gelatin (SF/G) film and canine corneal stromal stem cells (cCSSCs) seeded SF/G scaffold, respectively. Both cell types were successfully isolated by collagenase I. SF/G corneal films and stromal scaffolds served as the prospective substrates for cLESCs and cCSSCs by promoting cell adhesion, cell viability, and cell proliferation. The results revealed the upregulation oftumor protein P63(P63) andATP-binding cassette super-family G member 2(Abcg2) of cLESCs as well asKeratocan(Kera),Lumican(Lum),aldehyde dehydrogenase 3 family member A1(Aldh3a1) andAquaporin 1(Aqp1) of differentiated keratocytes. Moreover, immunohistochemistry illustrated the positive staining of tumor protein P63 (P63), aldehyde dehydrogenase 3 family member A1 (Aldh3a1), lumican (Lum) and collagen I (Col-I), which are considerable for native cornea. This study manifested a feasible platform to construct tissue-engineered canine cornea for functional grafts and positively contributed to the body of knowledge related to canine corneal stem cells.