Ultrastructural studies on corneal regeneration one year after implantation of biosynthetic cell-free hydrogel implants in mini pig

Abstract

Purpose : To understand the underlying endogenous cellular mechanisms involved in the integration and regeneration of cell-free hydrogel biosynthetic artificial corneas in a mini pig model.

Methods : Mini pigs received non-penetrating implants of cell-free collagen-like peptide (CLP-PEG) or recombinant human collagen type III (RHCIII) hydrogels. After one year, the animals were euthanized and the corneas were examined using 3D serial block face scanning electron microscopy (SBF SEM). They were fixed and processed through a series of staining solutions prior to embedding in epoxy resin. Polymerized resin blocks were trimmed and transferred to a Zeiss Sigma VP FEG scanning electron microscope equipped with a Gatan 3View system, where data sets of up to 2000 images were acquired of the block surface every 50nm through automated sectioning. Selected serial image sequences were extracted and 3D reconstructions were generated using Amira software. Transmission electron microscopy (TEM) was also carried out.

Results : Both CLP-PEG and RHCIII implants were extensively remodeled with keratocyte-like cells and banded collagen. SBF SEM further revealed that the cells were arranged in stratified layers within the regenerated corneas. The anterior surfaces of both the RHCIII and CLP-PEG regenerated corneas were re-epithelialized. Whilst the RHCIII epithelium appeared similar to controls, CLP-PEG regenerated corneal epithelial cells exhibited a basal surface with numerous invaginations. Below this, exosomes were present in a layer within the remodeled anterior stroma. No large pieces of the CLP-PEG or RHCIII implant were recognizable within the central portion of the regenerated cornea by either TEM or SBF SEM. However, abundant electron-dense deposits, likely to be degraded remnants of the original implants, were observed interspersed between the collagen fibers within the remodeled tissue.

Conclusions : Both CLP-PEG and RHCIII implants were found to be extensively remodeled with keratocyte-like cells and collagen fibrils. Whilst original degraded implant material was still present within the regenerated corneas, both implant types appeared fully integrated, forming a regenerated cornea almost indistinguishable from normal host cornea. The presence of exosomes within the regenerated CLP-PEG cornea suggests active endogenous intercellular communication within the regenerated cornea.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.