The Effect Of Substrate Stiffness On The Biological Behavior And Mechanical Property Of Hepatocytes

Hepatic fibrosis is a pathological change which associated with the development of chronic liver injury and cirrhosis.The pathological feature of liver fibrosis is the excessive deposition of extracellular matrix(ECM),which influences the function of liver.Previous studies showed that hepatocytes play an important role in the process of liver fibrosis,and their apoptosis,abnormal migration and mechanical signal transduction can initiate and participate in the process of hepatic fibrosis.However,the relationship between substrate stiffness change and hepatocyte function is not clear at present,and the mechanism is also not clear.Therefore,in this research,hepatocytes are used as research object,and substrate stiffness as the research condition,we investigated the effect and mechanism of substrate stiffness on the biological behavior and mechanical property of hepatocytes at two dimensions/ three dimensions(2D/3D)scales.The main research contents and results are as follows:(1)Gene expression profiling of human hepatocytes grown on differing substrate stiffnessStiffness controllable polyvinyl alcohol(PVA)hydrogels were fabricated by chemical cross-linking method to be used as in vitro cell culture model.Three levels of stiffness(Soft,Moderate and Stiff)were used in our experiment that corresponded to the stiffness levels found in normal liver tissue,the early and late stages of fibrotic liver tissues.Results showed that hepatocytes seeding on the three stiffness of hydrogel were in good condition and low ratio of apoptosis.High qualities of RNA could be extracted from these cells for subsequent RNA-Seq and bioinformation analysis.GO and KEGG pathway were applied to analyze the DEGs obtained from the RNA-Seq,result showed that different substrate stiffness affected bio-functions and signaling pathways such as hepatocytes growth,apoptosis,actin cytoskeleton remodeling,epithelial-mesenchymal phenotype transition,cell-cell connecting and cell-substrate interaction.RNA sequencing results were further verified by a quantitative real-time reverse transcriptase polymerase chain reaction(qRT-PCR),the results suggested that RNA-seq results have high accuracy and reference significance.Based on the bioinformatics analysis,next,we investigated the effect and mechanism of 2D substrate stiffness on hepatocytes function from the experimental level.(2)The effect and mechanism of different 2D substrate stiffness on the biological behavior and mechanical property of hepatocytesWith the aid of the 2D in vitro cell culture model in section(1),we explore the effects and mechanism of 2D substrate stiffness on the morphology,apoptosis,migration ability and mechanical properties.The results showed that Stiff group promoted hepatocyte cytoskeleton rearrangement,pseudopod generation and cell polarization.The expression of F-actin and ?-tubulin were significantly up-regulated,and the expression of protein Albumin was significantly down-regulated by the Stiff group.The Stiff group can induce the nucleation of ?-catenin,and then significantly increase the proportion of dead cells in hepatocytes,suggesting that the pathological stiffness of later stage hepatic fibrosis can damage the function of hepatocytes and induce the apoptosis of hepatocytes.Substrate stiffness and the concentration of ECM coating protein had a biphasic regulation effect on hepatocellular migration.In the experimental range,hepatocytes seeded on the Soft group with coated with 0.01mg/m L fibronectin(FN)showed the maximum migration trajectory,displacement and velocity.For confluent growth hepatocytes,cell migration were regulated by substrate stiffness and culture time.In a short time period(6 h),Soft group significantly increased the migration ability,while for long time periods(48 h and 72 h),Stiff group increased the migration significantly.Substrate stiffness regulated the cellular Young's modulus and the mechanical stress distribution of single and confluent growth hepatocytes.AFM results showed that confluent growth cells have higher Young's modulus than single growth hepatocyte,while hepatocytes on the Moderate group showed the maximum Young's modulus.Finite element three dimensional cell model analysis revealed that with the same uniform distribution of force,cellular stress distribution in Soft and Moderate group were similar,there was a higher stress distribution in cellular border and the Mises stress of Soft group was significant higher than that of the Moderate group.In vivo and in vitro experimental results showed that cellular Young's modulus could be modulated by the balancing process of Integrin-?1 mediated cell-cell interaction and the ?-catenin mediated cell-substrate adhesion.We believe that there is a critical point of substrate stiffness,at which,the cell-cell and cell-substrate adhesion maintained balance and the cellular mechanical property exhibited a maximum value.In our experiment,the cellular Young's modulus of hepatocyte showed the highest value on moderate substrate,whose stiffness is more close to the critical point than the soft and stiff substrates.(3)The effect of different 3D substrate stiffness on the biological behavior of hepatocytesOn the basis of the above study,we further investigated the effect of 3D substrate stiffness on the biological behavior of hepatocytes.Results showed that 3D-Stiff group significantly increased the proportion of dead cells,while there was no significant effect on the hepatocyte migration.Similar to the results in 2D situation,the 3D-Stiff group could induce beta-catenin to enter the nucleus and regulate downstream gene expression.The results suggested that the pathological stiffness of later stage hepatic fibrosis can increase the progression of hepatic fibrosis by inducing the apoptosis of hepatocytes.Moreover,3D-Stiff group can induce the activation of the nucleation signal pathway of ?-catenin,which indicates the direction of the further study of 3D substrate stiffness regulating the changes of hepatocytes apoptosis,migration and mechanical properties.In conclusion,we successfully constructed an in vitro cell culture model which simulating the stiffness levels found in normal liver tissue,the early and late stages of fibrotic liver tissues.It is found that the 2D/3D Stiff group may be involved in the process of hepatic fibrosis by regulating hepatocytes cytoskeleton rearrangement,polarization,down-regulation of hepatocyte functional protein,up-regulation of apoptosis and migration.Preliminary confirmed that the balancing process of Integrin-?1 mediated cell-substrate interaction and ?-catenin mediated cell-cell interaction maybe responsible for the mechanical property changes modulated by substrate stiffness.This study help to understand the important role of substrate stiffness in the process of hepatic fibrosis.Meanwhile,it provides a new perspective and thought for the treatment of clinical liver fibrosis from the perspective of mechanobiology.