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

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

doi:10.1371/journal.pone.0263141

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

PLoS One. 2022 Feb 4;17(2):e0263141. doi: 10.1371/journal.pone.0263141. eCollection 2022.

Authors

Chutirat Torsahakul^{1

2

3}, Nipan Israsena^{4

5}, Supaporn Khramchantuk⁶, Juthamas Ratanavaraporn^{7

8

9}, Sirakarnt Dhitavat¹⁰, Watchareewan Rodprasert^{2

3}, Sirirat Nantavisai^{2

3

11}, Chenphop Sawangmake^{2

3

12

13}

Affiliations

¹ Graduate program in Veterinary Bioscience, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
² Veterinary Stem Cell and Bioengineering Research Unit, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
³ Veterinary Stem Cell and Bioengineering Innovation Center (VSCBIC), Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
⁴ Stem Cell and Cell Therapy Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
⁵ Department of Pharmacology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
⁶ Excellence Center for Stem Cell and Cell Therapy, King Chulalongkorn Memorial Hospital, The Thai Red Cross Society, Bangkok, Thailand.
⁷ Biomedical Engineering Program, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand.
⁸ Biomedical Engineering Research Center, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand.
⁹ Biomedical Engineering for Medical and Health Research Unit, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand.
¹⁰ Biochemistry Unit, Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
¹¹ Academic Affairs, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
¹² Department of Pharmacology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
¹³ Center of Excellence for Regenerative Dentistry (CERD), Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

3T3 Cells
ATP Binding Cassette Transporter, Subfamily G, Member 2 / metabolism
Animals
Aquaporin 1 / metabolism
Biocompatible Materials / chemistry*
Cell Proliferation
Collagen Type I / metabolism
Corneal Transplantation
Dogs
Epithelium, Corneal / pathology*
Eye Proteins / metabolism
Fibroblasts / cytology
Fibroins / chemistry
Gelatin / chemistry
Genes, Tumor Suppressor
Immunohistochemistry
In Vitro Techniques
Lumican / metabolism
Mice
Regeneration*
Stem Cells / cytology*
Stromal Cells / cytology*
Tensile Strength
Tissue Engineering
Tissue Scaffolds

Substances

ATP Binding Cassette Transporter, Subfamily G, Member 2
Biocompatible Materials
Collagen Type I
Eye Proteins
Lumican
Aquaporin 1
Gelatin
Fibroins

Grants and funding

CT was supported by the 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship; the 90th Anniversary Chulalongkorn University Fund; and Veterinary Stem Cell and Bioengineering Research Unit, Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University. JR was supported by Chulalongkorn University: CU_GR_62_98_21_15. CS was supported by research supporting grant of the Faculty of Veterinary Science; Chulalongkorn Academic Advancement into Its 2nd Century Project; Center of Excellence for Regenerative Dentistry(CERD), Faculty of Dentistry, Chulalongkorn University; Veterinary Stem Cell and Bioengineering Research Unit, Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University; and Government Research Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript.