Study On The Construction And Transplantation Effect Of Tissue Engineering Meniscus With Punched Matrix Scaffold And BMSCs

Meniscus tear is one of the most common knee injuries.Although surgical methods can be used to suture and repair torn meniscus,they are only effective in the peripheral vascular area and usually have a 30% chance of relacing.Partial or total meniscectomy can reduce joint pain and improve function in a short time.But it can increase the pressure on the articular cartilage,leading to cartilage degeneration,osteoarthritis and instability of the knee joint.Meniscus tissue engineering aims to regenerate damaged tissue and provides a feasible solution for meniscus repair.Scaffold is a key element of tissue engineering.The acellular matrix of the meniscus retains the biochemical composition,biological activity and three-dimensional structure,and has the same biomechanical properties as the natural meniscus.In addition,it has low immunogenicity and regeneration potential,making it an ideal scaffold for the construction of tissue engineering meniscus.However,the tight arrangement of collagen fiber network structure in the meniscus makes the matrix density too high and shows low porosity,which limits the proliferation,migration and matrix remodeling of cells in the damaged.Neither exogenously inoculated seed cells nor adjacent synovial cells in the articular cavity can infiltrate into the scaffold,and then redistribute and synthesize the necessary extracellular matrix,which has no long-term ability to remodeling and maintain tissue homeostasis.Therefore,the natural meniscus was first punched uniformly in the direction perpendicular to the surface through whole layer in this experiment.Then matrix scaffolds were prepared by repeated freeze-thaw SDS washing and ultrasonic cleaning.The effect of decellularization was confirmed by DNA quantitative assay and histological staining,And the effects of punch method on the treatment time and the changes of porosity and mechanical properties of scaffolds after punching were examined;the bone marrow mesenchymal stem cells were co-cultured on the punched scaffold,and the test of scanning electron microscopy,cell proliferation assay,glycosaminoglycan quantification assay and collagen type II western blot assay were performed to detect the adhesion proliferation and chondrogenic differentiation of stem cells on the punched scaffold;finally BMSCs-loaded punched acellular matrix scaffolds were induced and cultured in vitro for 4 weeks and implanted into the knee joint of the meniscus defect model dogs.Clinical observation,Lysholm score,anatomical observation,the width retraction rate detection of meniscus implant,histological and immunohistochemical observation of meniscus implant and histological observation of tibial plateau and femoral condyle cartilage platform were performed to evaluate the effect of tissue engineered meniscus on knee joint function recovery and protection of articular cartilage at 12 weeks postoperatively.The results are as follows:1.The DNA content of the punched scaffold was significantly reduced after decellarization.And good collagen fiber structure was observed histologically,but the nucleus was not.The decellularization time of meniscus in the punched group was significantly shorter than that in the unpunched control group.After punching,the porosity of the scaffolds increased significantly and the compressive modulus did not change significantly.The number of BMSCs proliferation and adhesion on the scaffold in the punching group was significantly higher than that in the control group.With the increase of in vitro culture time,the GAG content and type II collagen protein expression of BMSCs-loaded punched scaffolds are on the rise and higher than the control group.2.The tissue engineering meniscus graft was implanted into the knee joint of the model dogs with meniscus defect.The experimental dogs recovered well in the 12 weeks after the operation,and the Lysholm score was not significantly different from that of the sham operation group.Anatomical observation of the knee joint showed slight wear on the surface of the femoral condyle and tibial plateau cartilage,but it was significantly better than that of the remove group.Fresh fibrous cartilage was found on the surface of the meniscus graft.Histological and immunohistochemical staining of meniscus grafts revealed more cellular infiltration and matrix secretion through the introduced channels in the punched group than in the control group.Histological observation of the femoral condyle and tibial plateau cartilage showed that the cartilage surface of the punched group and the control group still maintained a certain integrity,showing the protective effect of cartilage.In summary,this study demonstrated that punch method can increase the porosity of the meniscus acellular matrix scaffold and maintain good biomechanical properties,and at the same time can improve the cell adhesion and matrix secretion on the scaffold when combined with BMSCs in vitro.In vitro remodeling of tissue-engineered meniscus grafts using BMSCs-loaded punched acellular matrix scaffolds has a positive effect on repairing meniscus injury,protecting articular cartilage,and restoring knee function.