The Influence Of Collagen Hydrogel Substrate Cross-linked By Non-enzymatic Glycation On Corneal Fibroblasts Behavior In Vitro

Keratoconus is a clinical common eye disease.Corneal collagen cross-linking(CXL)is considered as one of the most efficient way to prevent keratoconus process.After CXL surgery,collagen fiber structure is rearranged,stress of corneal matrix is redistributed and cells survival mechanics microenvironment is changed.How cells adapt to this change and interact with those changes need to be solved.Constructing non-enzymatic glycation can cross-linked collagen hydrogels in advance.Thus it provides good carrier for studies CXL postoperative in vitro.In this paper,we use different ribose concentration to glycated collagen hydrogels as carriers of cell growth.We study cells changes of viability,morphology,proliferation,cytoskeleton and so on,in order to find the effects of matrix microenvironment changes on human corneal fibroblasts.The main research work and results are as follows:1.Using different ribose concentration(0,50,100,150,200 mM)to glycated type ?rat tail collagen hydrogels,setting up collagen samples mechanics test system and observing collagen microstructure through confocal laser scanning microscope and scanning electron microscope.Studies have shown that internal structure of the samples change with increase of concentration of ribose.Collagen fibers began to gather and crosslink.Meanwhile,diameters of collagen fibers become thicker,and mechanical properties of collagen hydrogels are improved from 180 Pa to 470 Pa.2.Primary extraction of keratoconus fibroblasts were seeded on collagen hydrogels surface,FDA-PI was used to study cellular viability.Cell morphology was observed by relief phase contrast microscope,and we also measured the area of cell through Image plus 5.1.Proliferation was measured through a way called WST-1.Cytoskeleton and cell nucleus was stained and observed and so on.Studies showed that cells and nucleus areas were decreased with increasing the concentration of ribose.Cells viability is almost not changed.However,cellular proliferation decreased.Cells on lower ribose concentration collagen hydrogels appeared much clearer cytoskeleton.Our experiment results suggest that,the mechanics microenvironment of non-enzymatic glycation collagen hydrogels have changed.These changes have effects on cells viability,morphology,proliferation,and cytoskeleton.The appropriate glycation can both improve the mechanical property of collagen hydrogels and be suitable to the growth of cells.This study may provide valuable reference for exploring the interactions between corneal stromal cells and substrate after CXL surgery.