The Effect Of NGF On SF/CS-BMSCs In Repairing Osteochondral Defect Of Rabbit Knee Joint

Objective: To explore the repair effect of NGF on SF/CS scaffold combined with BMSCs on osteochondral defect of rabbit knee joint.Methods:1.Prepare SF/CS scaffold by vacuum drying and chemical cross-linking.observe the general appearance of the scaffold and observe the internal structure of the scaffold by scanning electron microscope and HE staining.2.BMSCs were isolated and cultured by density gradient centrifugation and adherent screening.Loaded onto SF/CS scaffolds and cultured in osteogenic differentiation medium and chondrogenic differentiation medium.The osteogenic effect was identified by the pigment red S staining and COL1a1 immunohistochemical staining.The cartilage effect was identified by Alcian blue staining and COL2a1 immunohistochemical staining.3.The BMSCs on the SF/CS scaffolds were continuously cultured with ordinary medium containing NGF at concentrations of 0 ng/m L,10 ng/m L,100 ng/m L and 200ng/m L for 8 days.The effects of different concentrations of NGF on the proliferation of BMSCs on SF/CS scaffolds were observed by CCK8 method.4.BMSCs on SF/CS scaffolds were cultured with chondrogenic differentiation medium containing NGF at concentrations of 0 ng/m L,10 ng/m L,100 ng/m L and 200ng/m L.Western blot was used to detect the expression of COL2a1 and AGG.RT-q PCR was used to detect the m RNA expression of AGG,SOX9 and COL2a1.5.NGF was prepared into sustained-release microspheres by double-emulsion solvent evaporation method.The appearance of NGF sustained-release microspheres was observed by light microscope and electron microscope.The particle size of NGF sustained-release microspheres was measured by Malvern particle size analyzer.Elisa detects the concentration of NGF in the sustained-release microsphere solution,and calculates the entrapment rate,drug loading rate and sustained-release effect.And the biological activity of NGF embedded in the sustained-release microspheres was detected by PC12 cells.6.NGF sustained-release microspheres,SF/CS scaffolds,and BMSCs were prepared into NGF-SF/CS-BMSCs complexes.which were implanted into a rabbit knee osteochondral defect model.The control group did not add NGF sustained-release microspheres,and the blank group was a simple SF/CS scaffolds.At 4 weeks,8 weeks and 12 weeks.The repair effect of articular osteochondral was observed from gross specimens,imaging studies,and pathological staining.The ICRS cartilage gross repair score and the modified Wakitani method histological score were used to evaluate the cartilage repair status,and analyze possible mechanisms of repair.Results:1.The SF/CS scaffold prepared by vacuum drying and chemical crosslinking has a loose and porous structure.2.The isolated and cultured BMSCs can be massively expanded in vitro,and can be directed to differentiate into osteoblasts expressing COL1a1 and calcium nodules under the condition of osteogenic medium;and can be directed to differentiate into chondrocytes expressing COL2a1 and cartilage matrix under the condition of chondrogenic medium.3.There was no statistical difference in the OD value of BMSCs proliferation assay on SF/CS scaffolds with different concentrations of NGF ordinary medium.4.The chondrogenic differentiation medium containing different concentrations of NGF induced the directional chondrocyte differentiation of BMSCs on SF/CS scaffolds,and proteins expression of COL2a1 and AGG had no statistical difference,and the m RNA expression of AGG,SOX9 and COL2a1 genes had no statistical difference.5.The encapsulation rate of the prepared NGF sustained-release microspheres was31.15%.The drug loading rate was 31.36%.The total amount of NGF sustained-release in 90 days reached 90.49%.The NGF released by the sustained-release microspheres could differentiate PC12 cells into neural cells.6.Compared with the SF/CS-BMSCs group without sustained-release microspheres and the SF/CS group,the NGF-SF/CS-BMSCs group added with sustained-release microspheres showed higher ICRS scores and lower modified Wakitani scores for repair of knee osteochondral defect in rabbits.Conclusion:1.SF/CS scaffolds and BMSCs can be used as good tissue engineering materials,and the prepared NGF-loaded sustained-release microspheres have good sustainedrelease effect and biological activity;2.NGF did not promote or inhibit the proliferation of BMSCs on SF/CS scaffolds and the expression of COL2a1,AGG proteins in chondrocytes after BMSCs differentiation.3.Compared with the SF/CS-BMSCs group and the SF/CS group,the NGFSF/CS-BMSCs group loaded with NGF sustained-release microspheres has a better repair effect on the osteochondral defect of the rabbit knee joint.The effect may be related to the early promotion of subchondral bone repair.