Engineering a Corneal Stromal Equivalent Using a Novel Multilayered Fabrication Assembly Technique.
Citation:
Fernández-Pérez J, Madden PW, Ahearne M., Engineering a Corneal Stromal Equivalent Using a Novel Multilayered Fabrication Assembly Technique., Tissue engineering. Part A, 2020Download Item:
Abstract:
To overcome the serious shortage of donor corneas for transplantation, alternatives based on tissue engineering need to be developed. Decellularized corneas are one potential alternative, but their densely packed collagen architecture inhibits recellularizationin vitro. Therefore, a new rapid method of recellularizing these constructs to ensure high cellularity throughout the collagen scaffold is needed. In this study, we developed a novel method for fabricating corneal constructs by using decellularized porcine corneal sheets assembled using a bottom-up approach by layering multiple sheets between cell-laden collagen I hydrogel.Corneal lenticules were cut from porcine corneas by cryosectioning, then decellularized with detergents and air-dried for storage as sheets. Human corneal stromal cells were encapsulated in collagen I hydrogel and cast between the dried sheets. Constructs were cultured in serum-free medium supplemented with ascorbic acid and insulin for 2 weeks. Epithelial cells were then seeded on the surface and cultured for an additional week.Transparency, cell viability, and phenotype were analyzed by qPCR, histology, and immunofluorescence.Constructs without epithelial cells were sutured onto anex vivoporcine cornea and cultured for 1 week. Lenticules were successfully decellularized, achieving dsDNA values of 13–1.2 ng/mg dry tissue, and were more resistant to degradation than the collagen I hydrogels. Constructs maintained high cell viability with akeratocyte-like phenotype with upregulation of keratocan, decorin, lumican, collagen I, ALDH3A1, and CD34and the corneal epithelial cells stratified with a cobblestone morphology. The construct was amenable to surgical handling and no tearing occurred during suturing. After 7 daysex vivo, constructs were covered by aneoepithelium from the host porcine cells and integration into the host stroma was observed.This study describes a novel approach toward fabricating anterior corneal substitutes in a simple and rapid manner, obtaining mature and suturable constructs using only tissue-derived materials.
Sponsor
Grant Number
Science Foundation Ireland (SFI)
15/ ERC/3269
Author's Homepage:
http://people.tcd.ie/ahearnmDescription:
PUBLISHED
Author: Ahearne, Mark
Type of material:
Journal ArticleSeries/Report no:
Tissue engineering. Part AAvailability:
Full text availableKeywords:
Cornea, Tissue engineering, Decellularization, Keratocytes, LenticulesDOI:
http://dx.doi.org/10.1089/ten.tea.2020.0019ISSN:
1937-3341Metadata
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