Plga Microshperes/Chondrocyte Extracellular Matrix Composite Scaffold With Controlled Release Of KGN For Cartilage Regeneration

Objective:Repair of articular cartilage defects has always been a difficult problem in clinical treatment.Studies have shown kartogenin(KGN)can effectively promote the differentiation of mesenchymal stem cells into chondrocytes for repair.This experiment aims to construct a scaffold based on chondrocyte extracellular matrix(CECM)that can slow release kartogenin(KGN),so as to extend the action time of KGN and achieve the advantage of good biocompatibility of CECM and the effect of promoting cartilage regeneration and repair of KGN menwhile.Methods:1.The PLGA microspheres and nanoparticles are prepared by emulsification method and ultrasonic method respectively.The particle size was observed through Nano zs90.Scanning electron microscopy or the projection electron microscope was used to observe the shape of them.Comparing drug loading capacity and envelope rate of KGN in the two particles by High Performance Liquid Chromatography,and choosing PLGA microspheres to go on.The CECM was extracted from pig kneew,which was made to 3% homogenate.The right amount of PLGA microspheres and CECM were fully mixed,freeze-drying and crosscrossings.The scaffold,which KGN was directly added to CECM,was made to be control to verify the solw release effect of KGN in PLGA(KGN)/CECM scaffold.2.Rabbit marrow stem stem cell(BMSCs)were extracted and cultured to the third generation(p3).Four kinds of scaffolds were made: CECM group,PLGA(KGN)/CECM group,TGF-β3/CECM group,KGN/TGF-β3/CECM group.BMSCs(P3)were seeded in these scaffolds.Cell adhesion,Live/dead staining,cck-8,chondrogenic induction was conducted,and histological staining,quantitative analysis were performed.3.Cartilage defects were prepared in the knee of New Zealand rabbits,which was4 mm in diameter,and 1.5mm in deepth.After 4 and 12 weeks of postoperative feeding,gross observation,histological staining and Western Blot detection of COL II and GAG were performed to compare the repair differences of cartilage defects between the groups.Results:1.The PLGA microsphere and nanoparticles have good shapes,with an average diameter of 2.186μm and 288 nm respectively.The packet ratios of KGN loaded in two particles were 78.97+1.95 and 5.55+1.69(the mean + standard deviation)respectively,the drug loading capacity were 9.47+2.3 and 0.42+0.28 respectively.Therefore,PLGA microspheres are selected for follow-up experiments.PLGA(KGN)/CECM scaffold has a good three dimensional structure,and KGN in it released slowly.2.The cell adhesion,Live/dead staining,cck-8 results indicated PLGA(KGN)/CECM scaffold had good biocompatibility to rabbit BMSCs.Histological staining and quantitative analysis indicated that the effect of PLGA(KGN)/CECM composite scaffold prepared in this study on promoting the differentiation of BMSCs into chondrocytes was similar to that of TGF-β3/CECM.3.The in vivo repair experiment results further verified that PLGA(KGN)/CECM had good biocompatibility and could effectively release KGN,which promoting cartilage defect repair.Besides,and the repair effect was close to that of TGF-β3/CECM scaffold.Conclusion:1.PLGA(KGN)/CECM composite scaffold can release KGN solwly and maintain the activity of cells in the scaffold,so as to promote the proliferation of mesenchymal stem cells and the differentiation into chondrocytes.2.PLGA(KGN)/CECM composite scaffold had good biocompatibility in vivo environment and promoted the regeneration of cartilage.3.There was no significant difference in the effect of promoting cartilage regeneration between TGF-β3 and KGN,and there was no synergistic effect between the two.KGN alone could achieve good effect in cartilage tissue engineering.