Impacts Of Extracellular Matrix Adhesion And Stiffness On Microenvironment Of Osteosarcoma

OBJECTIVE:Osteosarcoma is one of the most malignant tumor with high lethality in Orthopaedics.However,the mechanism of how it happens and makes progress is still unknown.Extracellular matrix(ECM)is a complex network in tissue and supports the structure of the tissue and thus modulates the growth of tissue and controls the cell(normal cells and tumor cells)fates.METHODS:Osteosarcoma cells and osteoblasts were chosen for this study.Four natural materials(collagen,matrigel,alginate and agarose)were used for two-dimensional(2D)and three-dimensional(3D)culture for osteosarcoma cells and osteoblasts respectively.And then,cell viability was detected using a cell live/dead staining,specific markers of osteosarcoma cells and osteoblasts were measured using PCR,alkaline phosphatase(ALP)was performed through ELISA,specific protein of osteosarcoma cells were conducted using western blot,and immunofluorescent staining for osteosarcoma cells and osteoblasts was also performed.Xenografts of osteosarcoma cells with four natural materials(collagen,matrigel,alginate and agarose)in nude mice were conducted to evaluate the tumor volume,vascularization,and the histological statement.Furthermore,the physical and chemical properties(porosity,stiffness,adhesivity,swelling property and etc.)of the four natural materials(collagen,matrigel,alginate and agarose)were also measured.methacrylated gelatin(GelMA)and poly(ethylene glycol)diacrylate(PEGDA)were used to synthesize nine hydrogels with different adhesivity and stiffness.GelMA was synthesized using gelatin and MA,and ~1H-NMR was used to test the resonance peaks of GelMA,with gelatin as control.Characteristics(porosity,stiffness,adhesivity,swelling property and etc.)of the nine GelMA/PEGDA hydrogels was detected.Osteosarcoma cells and osteoblasts were cultured in 2D and 3D system using the nine GelMA/PEGDA hydrogels,and their proliferation,growth and development were evaluated as well.In addition,Inhibition of Focal Adhesion was used for 3D cultured osteosarcoma cells and Adherence Junction blocking was used for 3D cultured osteoblasts,further functional tests were also performed.RESULTS:When osteosarcoma cells were cultured on the four natural materials(collagen,matrigel,alginate and agarose),their viability followed the trends as:collagen>matrigel>alginate>agarose.And the mRNA expression of HIFA,VEGF,MMP2 and MMP9,protein expression of LN,FN,MMP2,MMP9and CD133 were consisted with the trends above.Similarly,when osteoblasts were cultured on the four natural materials(collagen,matrigel,alginate and agarose),their viability,mRNA expression of ALP,COL1,BMP2,RUNX2,ALP excretion and immunofluorescent staining of OCN were also followed the trends as:collagen>matrigel>alginate>agarose.However,the trends of osteosarcoma cells cultured in 3D hydrogels(collagen,matrigel,alginate and agarose)were different from those in 2D culture system,with the trends as follows:collagen>agarose>matrigel>alginate.And the cell viability,mRNA and protein expression trends(collagen>matrigel>alginate>agarose)of the 3D cultured osteoblasts were in accordance of those in 2D culture system.Moreover,xenografts of osteosarcoma cells in nude mice also indicated that collagen supported tumor development the best,and followed by agarose,matrigel and alginate.The physical and chemical properties of the four natural materials were different,especially in adhesivity and stiffness.And priority of adhesivity was followed trends as:collagen>matrigel>alginate>agarose.The priority of stiffness was followed trends as:collagen>agarose>matrigel>alginate.Thus,we gave a prospect that osteosarcoma cells were more sensitive with ECM stiffness and osteoblasts were more sensitive with ECM adhensivity.PEGDA and GelMA can be used to synthesize nine hydrogels with difference stiffness and adhesivity.PEGDA was used to control the stiffness of the hydrogels and so as GelMA to adhesivity.The behaviors of osteosarcoma cells and osteoblasts were similar in 2D culture system.For 2D cultured osteosarcoma cells,cell viability,mRNA expression of HIF,VEGF,MMP2 and MMP9,and the immunofluorescent staining of MMP2 and MMP9 were increased with the aggradation of ECM adhesivity.Similarly,for 2D cultured osteoblasts,cell viability,mRNA expression of ALP,COL1,BMP2 and RUNX2,and the immunofluorescent staining of ALP and OCN were also increased with the aggradation of ECM adhesivity.By contrast,the behaviors of osteosarcoma cells and osteoblasts were different in 3D culture system.For 3D cultured osteosarcoma cells,the proliferation,mRNA expression of HIF,VEGF,MMP2and MMP9,ALDH activity and CD133 expression were promoted with the increase of ECM stiffness.In contrast,3D cultured osteoblasts didn't influence by the ECM stiffness.When osteoblasts cultured in 3D hydrogels,the proliferation,mRNA expression of ALP,COL1,BMP2 and RUNX2 were aggrandized with the increase of ECM adhesivity.After inhibition of Focal adhesion pathway,the behavior of 3D cultured osteosarcoma cells didn't influence by ECM stiffness.After blocking the Adherens Junction pathway,the behavior of 3D cultured osteoblasts didn't influence by ECM adhesivity.CONCLUSION:When cultured in 3D system,which similar to their natural state,osteosarcoma cells are highly dependent on ECM rigidity via the integrin-mediated Focal Adhesion pathway,while osteoblasts are sensitive to ECM adhesion ligand density through regulation of the integrin-mediated Adherens Junction pathway.