Preparation Of Three Dimension Decellularizd Scaffolds With Different Stiffness And Their Effects On The Biological Behavior Of Breast Tumor Cells

Objective: In this study,three dimension(3D)decellularized extracellular matrix(DECM)scaffolds with different stiffness were prepared to mimick the microenvironment of human breast tumor tissue in vitro.Subsequently,the effect of stiffness of the scaffolds on cell biological behavior such as cell viability and cell infiltration of breast tumor cell(MDA-MB-231)were explored.Methods: The lentivirus vectors of interference or over-expression of lysyl oxidase(LOX)gene were constructed,and then were transfected into MDA-MB-231 respectively.By means of the methods quantitative real time polymerase chain reaction(qPCR)and Western blot to detect the expression levels of LOX mRNA and protein.The cells with different LOX expression levels were injected into the subcutaneous of the nude mouse armpit.Once the tumor tissue formed,decellularized approach was applied to harvest the 3D DECM scaffolds and the elastic modulus was measured by material testing machine.The microstructure of DECM was examined by scanning electron microscopy(SEM).Furtherly paraffin section and histological staining were utilized to detect the major components of extracellular matrix(ECM).After the repopulation and culture of DECM scaffolds with MDA-MB-231,Live/dead staining and MTS assay were applied to detect cell viability at 1,4,7,and 10 day.Based on the results of MTS assay,hematoxylin-eosin(HE)staining was applied to verify the cell infiltration of MDA-MB-231 after cultured for 7 days.Finally,the mRNA expression levels of epithelial-mesenchymal transition(EMT)relative markers(vimentin and Snail 1),type I collagen,fibronectin and LOX in MDA-MB-231 were verified after cultured for 7 days through qPCR.Results: 3D DECM scaffolds with different stiffness were harvested by means of decellularized approach,preserving the ECM components and structure.Repopulation experiments indicated that 3D DECM scaffolds with different stiffness had a significant influence on biological behavior of MDA-MB-231.(1)The results of qPCR and Western blot showed that the mRNA and protein expression levels of LOX in MDA-MB-231 were inhibited after transfected by lentiviral vectors with interference of LOX,but increased when transfected by lentiviral vectors with over-expression of LOX effectively in a long time.(2)The histological staining results showed that the total cells were removed completely after decellularization and the intact structure and major components of the ECM were kept,such as collagen and glycosaminoglycan.(3)The results of SEM and mechanical propeties test showed that the porosity of control group was 48.47 ±5.27% and stiffness was 3.24 ± 0.39 kPa,and the porosity of low stiffness group was increased to 68.12 ± 5.74% but stiffness was decreased to 2.57 ± 0.26 kPa.Conversely,the porosity of high stiffness group was decreased to 32.68 ± 4.30% but stiffness was increased to 4.91 ± 0.31 kPa.(4)The repopulation results showed that MDA-MB-231 cells could infiltrate into the 3D DECM scaffolds with different stiffness and cell survival of cells was confirmed to be well.(5)The results of qPCR showed that the high stiffness group could promote the mRNA expression levels of LOX in MDA-MB-231.Besides,the mRNA expression level of vimentin in MDA-MB-231 was improved but fibronectin was inhibited in the low stiffness group.Conclusion: The results confirmed that the expression levels of LOX in MDA-MB-231 cells could be effectively regulated after transfected with the lentivirus vectors of over-expression or interference of LOX.Furthermore,the stiffness of DECM scaffolds were changed through the regulatory effect of LOX on the crosslinking degrees of collagen.After repopulation of DECM scaffolds,the scaffolds showed no cytotoxicity on the MDA-MB-231 cells.All above results suggested that the DECM scaffolds could be used as 3D models for studying the influence of stiffness on properties of tumor cells in vitro in this study.