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

Chutirat Torsahakul,Watchareewan Rodprasert,Sirirat Nantavisai,Sirakarnt Dhitavat,Supaporn Khramchantuk,Chenphop Sawangmake,Juthamas Ratanavaraporn,Nipan Israsena,Panayiotis Maghsoudlou

doi:10.1371/journal.pone.0263141

Chutirat Torsahakul, Watchareewan Rodprasert + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0263141

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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 of tumor protein P63 (P63) and ATP-binding cassette super-family G member 2 (Abcg2) of cLESCs as well as Keratocan (Kera), Lumican (Lum), aldehyde dehydrogenase 3 family member A1 (Aldh3a1) and Aquaporin 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.

Highlights

The cornea is a transparent soft tissue located at the outermost layer of the eye
This study aimed to investigate silk modified with gelatin film and scaffold for culturing canine limbal epithelial stem cells and canine corneal stromal stem cells to fabricate 3-D synthetic corneal tissue
Results canine limbal epithelial stem cells (cLESCs) and canine corneal stromal stem cells (cCSSCs) isolation and characterization cLESCs and cCSSCs were successfully isolated from the canine cadaveric cornea with collagenase I. cLESCs were formed holoclones within 3 days after isolation

Summary

Introduction

Corneas consist of 5 recognized layers, including 3 cellular layers (epithelium, stroma, and endothelium) and 2 acellular layers (Bowman’s layer and Descemet’s membrane) [1]. Corneal blindness is the third most common cause of vision loss, reported in approximately 4.9 million cases for bilateral corneal blindness worldwide [3]. Among these cases in humans, trauma resulting in corneal ulcer, keratitis, and trachoma are the major causes [4]. Several critical disadvantages of corneal grafts remain, such as disease transmission, contamination, limited tissue shelf life, specific storage condition (−86 ̊C) and graft rejection associated with biological variability [7, 11]

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