Effects Of Substrate Stiffness On The Behavior Of Corneal Fibroblasts

The cornea belongs to the load-bearing tissue,and has typical viscoelastic characteristic.Diseases(such as keratoconus)or surgery(such as corneal cross-linking,corneal refractive surgery,etc.)change the structure of the cornea,which in turn influence the mechanical properties of the tissue.How stromal cells adapt to this change and interact with extracellular matrices are closely related to disease development and postoperative corneal stroma remodeling.PDMS is a commonly used polymeric polymer material.In this paper,the biological behavior of corneal fibroblasts in different mechanical environments were studied by simulating the mechanical properties of corneal stroma matrix with different stiffness PDMS elastomer.The main research works are summarized as follows:1.The mechanical properties and surface properties of PDMS were studied.The mechanical properties of corneal stroma were controlled by changing the ratio of PDMS main agent to curing agent.The elastic modulus PDMS wasmeasured by micro-indentation method,and the viscosity were analyzed.To promote the adhesion of corneal fibroblasts on the surface of the substrates,the surface modification of PDMS substrates were carried out.The contact angle and surface roughness of the modified substrates were also detected.The results showed that the elastic modulus of PDMS substrates increased and the viscosity decreased with the proportion of curing agent.After type I collagen modifying,the hydrophilicity of PDMS substrates increased,and the surface roughness basically unchanged.2.The effects of different PDMS substrates stiffness on the behavior of corneal fibroblasts were investigated.The normal and keratoconus corneal fibroblasts were seeded on the surface of different stiffness PDMS substrates after collagen modifying,and the cell viability was detected by FDA-PI staining.The spreading area of cells on different stiffness substrates was measured.The proliferation of cells on different stiffness substrates was detected by WST-1 cell proliferation kit.The gene expressions were detected by real-time PCR.The results showed that the PDMS substrates with different stiffness after collagen modifying had no significant effect on the normal and keratoconus corneal fibroblasts viability within 5 days.The spread area of normal and keratoconus corneal fibroblasts on stiff substrate was significantly larger than those on soft substrate.The normal corneal fibroblasts had the highest proliferation rate on stiff substrate,while the keratoconus fibroblasts had the lowest proliferation rate on stiff substrate.The gene expressions of collagen and a-SMA in cornealfibroblasts on stiff substrates were significantly higher than those on other substrates.The gene expression of LUM and ITGB1 decreased with the decrease of substrate stiffness.The results showed that substrate stiffness had significant impact on the spreading,proliferation and gene expression of normal and keratoconus corneal fibroblasts.This research could provide us references to understand the mechanism of keratoconus development and the corneal matrix remodeling after corneal collagen cross-linking and corneal refractive surgery.