Recombinant human collagen type I hydrogels as superior cell carriers for corneal epithelial stem cells

Abstract

Purpose : Cultivated limbal epithelial transplantation (CLET) can successfully regenerate the anterior cornea in Limbal Stem Cell Deficiency. The cell carrier most commonly used in CLET is Amnion. The aim of this study is to investigate recombinant human collagen type I (RHCI) hydrogels as a substitute carrier.

Methods : RHCI and Amnion were tested for ultrastructure (SEM, TEM, AFM), optical properties (Refractometry - Transparency), bulk mechanical properties (Rheology), microbial susceptibility (P. aeruginosa challenge) and genotoxicity (VITOTOX). Hydrogels were nano-surface modified by Fibronectin Micro-ContactPatterning (F-µCP). Composite grafts were generated using primary limbal epithelial stem cells (LESC) and characterized using immunohistochemistry, EM and qPCR analysis. To test bio-integration and degradation, acellular hydrogels were implanted subcutaneously in rats and as a corneal implant in mini-pigs.

Results : RHCI generates transparent (Fig.1) and ultrathin (<100µm) hydrogels that withstand manipulation. The collagen bundles are randomly aligned, contributing to the inherent strength of the hydrogel. Hydrogels show no genotoxic effect, and are a 100-fold more resistant to microbial growth. When cultivated on RHCI, primary LESC keep expressing their undifferentiated adherent stem cell genotype and phenotype(Fig.2). In vivo, RHCI hydrogels show good biocompatibility as hydrogels remain intact, elicit minimal inflammation and integrate within the surrounding tissue.

Conclusions : Primary LESCs can successfully be cultivated on RHCI hydrogels using a standardized xeno-free GMP-grade cultivation protocol. Its favorable optical characteristics, relative microbial resistance, successful composite graft generation and biocompatibility prove that RHCI is a highly promising scaffold for ocular tissue engineering. Further animal studies are ongoing to confirm the in vivo benefit of Fibronectin nano-lithography.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Fig.1: Image of an RHCI cornea (A). Relative transparency testing indicates that RHCI hydrogels are more transparent than amnion (B).

 

Fig.2: Immunohistochemistry of primary cells cultivated on RHCI. Cultivated cells form a confluent monolayer with high expression of adherence and stem cell markers ΔNp63α, Cytokeratin14, Integrin-β4, Laminin and CollagenIV; and low expression of differentiation markers Cytokeratin3/12 and Desmoglein3. These findings are confirmed by qPCR* analysis.