A Novel Extracellular Matrix-Mimetic Hydrogel for Corneal Regeneration

Abstract

Purpose : Corneal stromal scarring is a serious cause of visual impairment and blindness worldwide. Human limbus-derived mesenchymal/stromal stem cells (hLMSCs) have been found to be effective in amelioration of superficial corneal scars. This study aimed to develop a novel extracellular matrix (ECM)-mimetic hydrogel formulation that can incorporate hLMSCs for use in deeper wounds to promote scar-less corneal healing.

Methods : Human cadaveric corneas were obtained from the eye-bank. The corneas were decellularized and the ECM was extracted as powder using freeze-miller and enzymatic digestion. After the ECM-powder was assessed for sterility and level of endotoxins, the ECM-mimetic hydrogel was developed by entrapping enzymatically-digested ECM-derived proteins and glycosaminoglycans within a semi-interpenetrating hydrogel network. The final product was analyzed by using scanning-electron microscopy (SEM) and dynamic light scattering for its physical and transmittance properties. The hLMSCs were then encapsulated and cultured inside this 3D hydrogel for 72-hours to test their viability and proliferation. Immunohistochemistry for hLMSCs markers (ABCG2, PAX-6, Vimentin, Collagen-III, CD73, CD90) and fibroblast marker α-SMA was performed to assess the phenotypic properties of encapsulated hLMSCs.

Results : The SEM analysis of ECM-powder showed intact collagen fibril structure. The ECM-derived proteins’ particle size averaged 2 (±0.5) µm on dynamic light scattering analysis. The encapsulated hLMSCs in the ECM-mimetic 3D hydrogel showed similar cell viability (92±3%) as compared to 2D hLMSCs culture (p=0.12). The hLMSCs were able to maintain phenotypic expression of ABCG2, PAX-6, Vimentin, Collagen-III, CD73 and CD90 biomarkers, in the presence of ECM-derived proteins within the hydrogel matrix. However, in 2D culture without ECM-derived components, the hLMSCs showed significantly higher expression of α-SMA (p<0.0001).

Conclusions : The findings of this study suggest that the novel ECM-mimetic hydrogel possesses the ability to maintain viability and phenotype of encapsulated hLMSCs. This opens the possibility of using hLMSCs encapsulated in the ECM-mimetic hydrogel for application in deeper corneal wounds, which needs to be tested in pre-clinical studies.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.