The interaction of the biocompatible scaffold containing nanochitosan-chitosan-polycaprolactone with telomerase activator and rock inhibitor in propagation and functional properties of corneal endothelial cells

Abstract

Background

Corneal endothelial disfunction, which is results from the injury or loss of corneal endothelial cells (CECs), is the major cause of corneal opacity and visual impairment. Corneal transplantation is the only therapeutic choice for endothelium deficiency, but global donor shortage has posed a serious challenge worldwide. Alternative treatments, especially regenerative medicine strategies including cell therapy and tissue engineering, can overcome this limitation. Hence, the aim of this study is the construction of transplantable grafts carrying functional CECs.

Methods

We fabricated and characterized scaffold containing chitosan nanoparticles, chitosan and polycaprolactone. HCECs were isolated from corneal endothelium and cultured on the fabricated scaffold. Cell proliferation was induced using the telomerase activator and ROCK inhibitor. Functional characteristics of the HCECs and cell-scaffold interactions were evaluated via evaluation of gene expression, flow cytometry, electron microscopy and MTT assay.

Results

The results showed that fabricated scaffold had a suitable transparency and grate biocompatibility. >98 % of the isolated cells had the CD166⁺/CD98⁺ phenotype. Telomerase activator and ROCK inhibitor increased the proliferation of HCECs by 1.5 and 2.3 times, respectively, and the simultaneous use synergistically increased the cell proliferation by 4.3 times. Culture of HCECs on scaffolds significantly increased their proliferation. Cell count, SEM images and H&E results showed proper cell-surface interaction with the formation of a cell monolayer on the scaffold. Flow cytometry results showed that >97 % of the cells cultured on scaffold maintained their CD166⁺/CD44⁻ phenotype.

Conclusions

According to the results, the designed scaffold and telomerase activator and ROCK inhibitor had the synergistic effects on cultured HCECs behaviors such as cell adhesion, proliferation and phenotypic maintenance, which is powerful evidence for application of this scaffold as a suitable carrier for HCECs transplantation.