RGD-Modified Hydrogel Maintains Cell Growth in Mechanically-Induced Limbal Stem Cell Deficient Mouse Model

Abstract

Purpose : To replicate limbal stem cell deficiency (LSCD) and assess arginylglycylaspartic acid-modified polymer nanoparticle (NP) hydrogels' (RGD-PNP) biocompatibility and impact on cell growth and maintenance in a mechanically-induced LSCD mouse model.

Methods : Firstly, PNP and RGD-PNP hydrogels were created with three polymer-to-NP ratios (1:5, 1:10, and 2:10). Limbal epithelial cells (LECs) were isolated from human corneal rims, and a viability assay was conducted in vitro for each hydrogel composition and followed by a confocal microscope for seven days. In vivo, tarsorrhaphy (three interrupted sutures) of the right eyelids of 12 B6 mice (six months old) was performed and injected with PNP or PNP-RGD under the sutured eyelids using a McIntyre cannula. Hydrogel fluorescence intensity was measured with the In Vivo Imaging System (IVIS) for four days.
Secondly, five eyes of 10 NSG mice (seven months old) underwent injury by Algerbrush II followed by tarsorrhaphy using a single continuous suture. Fluorescently labeled LECs were mixed with 1:5 RGD-PNP and injected under the sutured eyelids immediately after injury. The mice were followed using IVIS for four days.

Results : In vitro, LECs remained viable in hydrogels with a 1:5 ratio longer than other hydrogels (and more viable cell count in 1:5 RGD-PNP than in 1:5 PNP, p< 0.05) (Fig1). In vivo, 1:5 RGD- PNP remained the longest on the cornea (Fig1), so it was selected for the second experiment. The fluorescence intensity from fluorescent LECs within 1:5 RGD-PNP decreased from Day 0 to Day 1 and then plateaued (Fig2). Hydrogel stayed longer using the continuous versus interrupted suture method.

Conclusions : The Algerbrush injury adequately created LSCD in our mouse model. Continued suture offered a safer method for maintaining hydrogel on the cornea. 1:5 RGD-PNP provided an ample environment for stem cells to grow and remain longer on the cornea. These results demonstrate the use of a novel scaffold for stem cell delivery and grafting in injured cornea.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Fig1. Bar charts showing viable cell count in different hydrogel compositions in vitro (left) and fluorescent intensity illuminated from the same hydrogels injected under the sutured mice eyelids in vivo (right).

 

Fig2. IVIS image (left) and a line graph (right) showing fluorescent intensity illuminated from a mixture of fluorescent LECs and 1:5 RGD-PNP hydrogel injected under the sutured mice eyelids.