Physicochemical Properties Of TGF-?3-induced ADSCs Constructing ECM-PCL-HA Scaffolds

Objective: Adipose-derived mesenchymal stem cells(ADSCs)have become the focus of seed cells in tissue processes due to their simple extraction,high yield and high cell dryness.In this experiment,TGF-?3 was used as a target gene to transfect adipose-derived mesenchymal stem cells.After the extracellular matrix of the cartilage,the decellularized cells only retain the extracellular matrix scaffold material,and the physical and chemical properties of the ECM-PCL-HA scaffold are detected,which provides a better choice for tissue engineering scaffold materials.Methods:(1)Isolation and culture of rabbit adiposederived mesenchymal stem cells in vitro,and multi-directional differentiation and identification;(2)lentiviral-mediated transfection of TGF-?3 to rabbit adipose-derived mesenchymal stem cells;(3)flow cytometry and laser Confocal detection of transfection efficiency;(4)detection of cytotoxicity of different titers of lentivirus-infected ADSCs in combination with CCK-8;(5)detection of cartilage-associated genes by real-time quantitative PCR detection,western blot and protein expression;(6)3D printing scaffold material,preparation of chondrocyte-rich extracellular matrix scaffold material;(7)physical and chemical properties of cartilage-rich extracellular matrix scaffold material.Results: The adipose tissue is rich in cells and convenient to obtain.The characteristics of ADSCs stem cells are remarkable,and the adipogenic,osteogenic and chondrogenic differentiation time is short and the ability is strong.The results of real-time quantitative PCR showed that the expression levels of Aggrecan,SOX-9 and COL2 genes induced by lentivirustransfected TGF-?3 adipose-derived mesenchymal stem cells at 7 days,14 days and 21 days were significantly higher than those in the blank group(P<0.01).The levels of Aggrecan,SOX-9 and COL2 genes induced by 21 days were significantly higher than those of 7 days and 14 days(P<0.05),and the higher the virus transfection titer,the more Aggrecan,SOX-9 and COL2 were synthesized.To form more extracellular matrix,construct a good microenvironment for chondrogenic differentiation.The ADSCs were inoculated into the PCL-HA scaffold after transfection,and the cells were eluted after 2 weeks,and only the ECM was retained to construct a composite material ECM-PCL-HA.The swelling ratio and porosity were higher than those of PCL-HA(P<0.05).Conclusion: The physicochemical properties of the composite scaffold ECM-PCL-HA is higher than that of the simple polymer scaffold material,which meets the requirements of cartilage tissue engineering,and provides an experimental basis for the clinical application of cartilage tissue engineering to repair articular cartilage defects.