Collagen-like-peptide nano-implants as stem cell loaded substitutes to human cornea transplantation

Abstract

Purpose : The consistent global shortage in donor corneas, indicate the need of lab-grown alternatives. Collagen-like-peptides (CLP) have been proposed as alternatives to full-length collagen for tissue regeneration. The aim of this study is to investigate nano-refined CLP as a stem cell-loaded corneal substitute.

Methods : CLP hydrogels were tested for physical properties (DMA), optical properties (Refractometry – Transparency), microbial susceptibility (P. aeruginosa challenge) and genotoxicity (VITOTOX). Hydrogels were nano-surface modified with 3D topography or fibronectin micro-contactprinted patterns (F-µCP). Cell viability and cell proliferation of immortalized limbal epithelial stem cells (LESC) cultivated on the hydrogel were investigated using PrestoBlue, BrdU, and Live cell imaging assays. In vitro biocompatibility was tested by analyzing composite grafts using primary LESC (EM, immunohistochemistry and qPCR). Acellular hydrogels were implanted in the cornea of mini-pigs to test in vivo biocompatibility.

Results : CLP forms transparent hydrogels that withstand manipulation (fig.1). CLP shows no genotoxic effect, and hydrogels are 100-fold more resistant to microbial growth when compared to amnion. No significant difference in cell viability and active proliferation were noted when compared to tissue culture plastic. Cultivated primary LESC (fig.2) keep expressing their adherent stem cell phenotype (low CK3/12 & GJA1 – high ΔNp63α, ITGA6 & ITGB1). In vivo, CLP hydrogels show stable and functional integration promoting regeneration of corneal epithelium and stromal nerves (fig.1).

Conclusions : The favorable optical characteristics, relative microbial resistance, successful composite graft generation and in vivo biocompatibility prove that CLP is a highly promising alternative for human donor corneas. Furthermore, the benefits of nano-surface modification will enable production of custom-built cell loaded cornea grafts.

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

 

1. Example of a CLP hydrogel before (A) and after (B) animal implantation. In vivo confocal microscopy shows that CLP implanted corneas have regenerated their epithelium (C), stroma (D) and sub-epithelial nerve plexus (E).

 

2. Cells cultivated on Amnion, unmodified CLP, F-µCP CLP and 3D grooved CLP form a confluent monolayer. Primary cells preferentially first attach to nano-modified patterns (i.e. fibronectin stripes or 3D grooves) before spreading over the rest of the hydrogel.