Repair Of Knee OA Cartilage Defect With MSCs Engrafted On Graphene Oxide Scaffold Based On The " Solid Skeleton And Pliable Tendons" Theory

Objective:1.To study whether GO particles are biocompatible with MSCs,and analyze whether they can be used as a cell culture scaffold for MSCs;2.Study whether the GO+HA lubricating solution is biocompatible with MSCs,and analyze whether it can be used as the cell culture fluid of MSCs;3.Under the guidance of"Solid Skeleton and Pliable Tendons"theory,study the effect of GO-carrying MSCs on cartilage repair and intra-articular inflammation control of New Zealand rabbit knee articular cartilage defect model.Methods:Experiment 1:1.Grind graphene oxide into fine particles.After sterilization,15?ggraphene oxide particles and MSCs with a cell concentration of 5.0×10~5 cells/ml were co-cultured in vitro,and compared with MSCs of the same concentration cultured separately.2.Take out some graphene oxide particles and mix with sodium hyaluronate at a certain ratio to make two kinds of lubricating solutions with different concentration gradients,respectively 30?g/ml GO+0.25%HA and 15?g/ml GO+0.25%HA.The prepared two concentrations of lubricating solution were co-cultured with MSCs with a cell concentration of 5.0×10~5 cells/ml in vitro.Experiment 2:The modified Hulth method and angle grinding drill were used to make a KOA New Zealand rabbit model featuring cartilage defects,and the model was tested 6 weeks after the model was made.Experiment 3:The KOA model rabbits were randomly divided into 4 groups,namelyMSCs+GO group,MSCs group,GO group,model group,and a blank control group without any intervention.2 in each group."MSCs+GO group"was injected with 0.5ml MSCs+GO suspension in joint cavity,GO concentration was 30?g/ml GO+0.25%HA,MSCsconcentration was 5×10~6 cells/ml;"MSCs group"joint cavity was injected with 0.5mlconcentration was 5×10~6 cells/ml Stem cell suspension of MSCs;"GO group"was injected into the articular cavity with 0.5 ml of 30?g/ml GO+0.25%HA solution.On the first day after the intervention for 8 weeks,the joint fluid and femoral condyle specimens were taken,and the joint fluid was tested for TNF-?and MMP-13 concentration.,Minkin score,Micro-CT imaging observation.Results:Experiment 1:1.Co-cultivation of 15?g GO and MSCs with a concentration of 5.0×10~5MSC/ml for 1 day and 7 days found that GO is not cytotoxic to umbilical MSCs,and GO also has a certain adsorption effect on MSCs.MSCs within the scope are wrapped andaggregated;2.Two concentration gradient GO solutions are not cytotoxic to umbilical MSCs,and for MSCs,GO in HA has a certain adsorption effect,which can wrap and aggregate MSCs within a certain range.The concentration of 30?g/ml GO+0.25%HA lubricating fluid can culture MSCs better than 15?g/ml GO+0.25%HA lubricating fluid.Experiment 2:After 6 weeks of modeling,the synovial tissue in the KOA model of New Zealand rabbit cartilage defects proliferated,and the synovial fluid increased compared with the blank group.The cartilage was dull and the damage area was obviously damaged,which met the requirements of the model.Experiment 3:After 8 weeks of intra-articular injection of a MSCs-loaded graphene oxide stent in a rabbit cartilage defect model,gross cartilage observation,CT imagingobservation,histological observation,and Minkin score found that the MSC+GO group had the best cartilage repair effect and MSCs The group was slightly better than the GO group for cartilage repair,the GO group was slightly better than the model group for cartilage repair,and the model group had the most severe cartilage damage.Based on the concentration ofMMP-13 and TNF-?in the joint cavity,the inflammation level in the joint cavity of the MSC+GO group was also lower.The GO group and the MSCs group also played a role incontrolling the inflammation level in the joint cavity,but the effect was worse than that of the MSC+GO group.The model group had the highest level of inflammation in the joint cavity.Conclusion:1.GO is biocompatible with MSCs,and GO can be used as a culture scaffold for MSCs.The GO+HA solution is also biocompatible with MSCs,and 30?g/ml GO+0.25%HA lubricating fluid has a slight advantage over 15?g/ml GO+0.25%HA lubricating fluid in culturing MSCs.2.Under the guidance of the"Solid Skeleton and Pliable Tendons"theory,the graphene oxide scaffolds loaded with MSCs have a stronger effect on repairing damaged articularcartilage and controlling the level of inflammation in the joint than simple MSCs and GO lubricants.