Regulatory Effect And Mechanism Of Adipose Stem Cells On Immune Inflammatory Response Of Tissue Engineered Cartilage

Background:Congenital microtia is a common disease and difficulty in plastic surgery.Tissue engineering technology provides a new strategy for auricle reconstruction.One of the key problems is how to maintain the stability of three-dimensional shape and function of tissue engineered cartilage in large animals with complete immunity.Among them,the immune inflammatory response,mainly involving macrophages,is one of the key factors affecting the stability of tissue engineered cartilage,but the specific mechanism is not clear.CD47SIRPa pathway plays an important role in macrophage-mediated immune inflammatory response,but it is not clear whether this pathway mediates oxidative damage between chondrocytes and macrophages.Adipose stem cells have good immune regulation effect.However,it is not clear whether adipose stem cells can regulate macrophage-mediated immune inflammation by regulating CD47-SIRPa pathway,which may be beneficial to the stability of tissue engineering cartilage implantation.Objective:1.To determine the effects of subculture in vitro and subcutaneous heterotopic transplantation on CD47 of chondrocytes;2.To construct a model of oxidative damaged chondrocytes and to clarify the mechanism of CD47-SIRPa pathway between oxidative damaged chondrocytes and macrophages;3.To determine the regulatory effect of adipose stem cells on CD47-SIRPa pathway in vitro;4.To determine the regulatory effect of adipose stem cells on macrophage-based immune inflammatory response and cartilage extracellular matrix metabolic balance after tissue engineered cartilage implantation in large animals;Methods and Results:1.Effects of subculture in vitro and subcutaneous heterotopic transplantation on CD47 molecules of chondrocytes.Methods:We selected different generations of human chondrocytes cultured in vitro and chondrocytes digested by subcutaneous implantation for one year,and identified the expression and conformational changes of CD47 on the surface of chondrocytes by flow cytometry.Results:Subculture in vitro and heterotopic subcutaneous transplantation,which are the key steps in the construction of tissue engineering cartilage repair,lead to the conformational change of CD47 in chondrocytes,but do not lead to the loss of CD47 expression.2.Study on CD47-SIRPa pathway of oxidative damage of chondrocytes.Methods:The model of oxidative injury of chondrocytes was established.Flow cytometry,immunofluorescence and other immunological methods were used to determine the specific mechanism and role of CD47-SIRPa pathway in oxidative damage of chondrocytes.Results:The model of oxidative injury of chondrocytes was successfully established.Oxidative damage could lead to conformational changes of CD47 on the surface of chondrocytes.The conformational changed CD47 bound to SIRPα under the promotion of TSP-1 binding.CD47-SIRPa pathway mediated phagocytosis of macrophages.3.Regulation of SIRPa expression by adipose stem cells in vitro.Methods:Human acute monocytic leukemia cells(THP-1)were used as a model to induce M1 macrophages.We used isolated co-culture method to explore the regulatory effect of adipose stem cells on the expression of SIRPa in macrophages.Results:THP-1 cells were successfully induced into M1 macrophages in vitro.Adipose stem cells could inhibit the expression of SIRPa and promote M2 polarization of M1 macrophages.4.The effect of local injection of adipose stem cells on the regulation of immune inflammatory response and the promotion of cartilage maturation after autologous implantation of tissue engineered cartilage.Methods:The chondrocyte cell sheets were constructed in vitro and implanted into the subcutaneous tissue of pigs.The experimental group was locally injected with autologous adipose stem cell suspension around the implant,while the control group was injected with the same amount of saline.Two weeks later,samples were collected from the chondrocyte cell sheets,tissues around the implants and peripheral blood.Immune and cartilage-related detection methods were used to compare the difference of immune inflammatory reaction and cartilage extracellular matrix synthesis between the two groups.Results:Local injection of adipose stem cells could reduce the local immune inflammatory response dominated by macrophages after chondrocyte cell sheets implantation in large animals,and promoted the metabolic balance of cartilage extracellular matrix.Conclusion:1.All chondrocytes expressed CD47.The key steps in the repair of tissue engineered cartilage-heterotopic subcutaneous transplantation and subculture in vitro would lead to conformational changes of CD47 in chondrocytes;2.Oxidative damage could lead to the conformational change of CD47 on the surface of chondrocytes,and the conformational changed CD47 binds to SIRPa under the promotion of TSP-1 binding,which led to phagocytosis of macrophages;3.Adipose stem cells regulate CD47-SIRPα pathway and promote M2 polarization by inhibiting the expression of SIRPa in M1 macrophages;4.Local injection of adipose stem cells could reduce the local immune inflammatory response dominated by macrophages after tissue engineered cartilage implantation,and promoted the metabolic balance of cartilage extracellular matrix.