Regulation of Keratocyte Phenotype and Cell Behavior by Substrate Stiffness.

Jialin Chen,Patrik Danielson,Ludvig J. Backman,Wei Zhang,Chen Ling

doi:10.1021/acsbiomaterials.0c00510

Jialin Chen, Patrik Danielson + Show 3 more

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https://doi.org/10.1021/acsbiomaterials.0c00510

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Abstract

Corneal tissue engineering is an alternative way to solve the problem of lack of corneal donor tissue in corneal transplantation. Keratocytes with a normal phenotype and function in tissue-engineered cornea would be critical for corneal regeneration. Although the role of extracellular/substrate material stiffness is well-known for the regulation of the cell phenotype and cell behavior in many different cell types, its effects in keratocyte culture have not yet been thoroughly studied. This project studied the effect of substrate stiffness on the keratocyte phenotype marker expression and typical cell behavior (cell adhesion, proliferation, and migration), and the possible mechanisms involved. Human primary keratocytes were cultured on tissue culture plastic (TCP, ∼106 kPa) or on plates with the stiffness equivalent of physiological human corneal stroma (25 kPa) or vitreous body (1 kPa). The expression of keratocyte phenotype markers, cell adhesion, proliferation, and migration were compared. The results showed that the stiffness of the substrate material regulates the phenotype marker expression and cell behavior of cultured keratocytes. Physiological corneal stiffness (25 kPa) superiorly preserved the cell phenotype when compared to the TCP and 1 kPa group. Keratocytes had a larger cell area when cultured on 25 kPa plates as compared to on TCP. Treatment of cells with NSC 23766 (Rac1 inhibitor) mimicked the response in the cell phenotype and behavior seen in the transition from soft materials to stiff materials, including the cytoskeletal structure, expression of keratocyte phenotype markers, and cell behavior. In conclusion, this study shows that substrate stiffness regulates the cell phenotype marker expression and cell behavior of keratocytes by Rac1-mediated cytoskeletal reorganization. This knowledge contributes to the development of corneal tissue engineering.

Highlights

Corneal blindness due to trachoma, onchocerciasis, and vitamin A deficiency is common worldwide.[1]
The expression of genes for keratocyte phenotype markers was significantly upregulated in the Collagen I Coated Plates (CCP) group at day 3 (Figure 1A aldehyde dehydrogenase 3A1 (ALDH3A1), 6.04-fold, p < 0.001; ALDH1A1, 3.05-fold, p < 0.001; CD34, 3.73-fold, p < 0.001) and day 7 (Figure 1B ALDH3A1, 3.15-fold, p < 0.001; CD34, 2.46-fold, p < 0.001), as compared to that in the TCP group
The protein expression of keratocyte phenotype markers ALDH3A1 and ALDH1A1 was much higher in the CCP group than in the TCP group (1.86-fold and 1.55-fold, respectively) at day 7 (Figure 1D,F)

Summary

Introduction

Corneal blindness due to trachoma, onchocerciasis, and vitamin A deficiency is common worldwide.[1]. The quiescent keratocytes are stimulated to become fibroblasts and myofibroblasts to facilitate wound healing.[4] This kind of self-repair is meaningful in terms of evolution, which protects the injured corneal tissue from further damage. The functional recovery of injured cornea is seldom achieved because of the phenotype drift of keratocytes.[5] During the transformation of keratocytes to fibroblasts and myofibroblasts, the expression of keratocyte phenotype markers is reduced or diminished, such as aldehyde dehydrogenase 3A1 (ALDH3A1), CD34, and keratocan (KERA).[4] Transplantation of tissue-engineered cornea can rapidly fill the injured area. The keratocyte phenotype in the transplant determines the quality of corneal repair. Preservation of the keratocyte phenotype in tissueengineered cornea is critical for the quality and function of the transplant

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