This study aimed to investigate the underlying mechanisms of corneal endothelial cells (CECs) differentiation and identify the extracellular matrix (ECM) compositions using chitosan/polycaprolactone (PCL) blended membrane, hence exploring the potential use of chitosan/PCL blends in tissue engineering of CECs. We utilized the chitosan/PCL blends named as PCL25 consisting of PCL at 25% by weight. The surface characteristics of PCL25 were confirmed by using Fourier Transform Infrared Spectroscopy (FTIR) and Atomic Force Microscope (AFM). Bovine CECs were cultured on the blends, compared with TCPS and pure chitosan membrane. Cell behaviors in terms of cell attachment, proliferation, differentiation phenotype and expression of differentiation proteins were examined. Furthermore, ECM protein productions were also analyzed. From the experiments, we found the topography (roughness) of PCL25 membrane examined by AFM was greater than pure chitosan membrane. FTIR results confirmed the functional groups of C=O bond of PCL. The CECs displayed hexagonal morphology and similar proliferation rate on both PCL25 membrane and TCPS. In addition, the immunofluorescence evidence showed well-localized ZO-1 and Na+/K+ ATPase expression of membrane proteins. ECM protein productions of CECs on PCL were no inferior to TCPS. Moreover, western blot results verified the higher amount of collagen type IV, and reduced TGF-β2 expression on PCL25 membrane compared to TCPS substrate. In conclusions, chitosan/PCL blends membrane provided a favorable environment for CECs in terms of ECM compositions, therefore enhancing the growth and differentiation. Accordingly, for CEC tissue engineering applications, PCL 25 might be a suitable alternative for cadaveric cornea transplantation in the near future.
Keywords: Chitosan; Collagen type IV; Corneal endothelial cells; ECM compositions; PCL blends.
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