Experimental Study Of Progressive Gradient Aperture Stent Combined With BMP2-ADSCS And Autogenous PRP Gel To Repair Osteochondral Defects

Objective: Based on the successful preparation of gradient aperture SF-CS-n HA osteochondral scaffold.BMP2-ADSCs and autologous PRP gel were used to construct osteochondral grafts to repair Beagle dog knee osteochondral defects and complete the performance test of the graft.It is hoped to provide a more suitable graft for repairing osteochondral defects.Methods: ADSCs from Beagle dogs were extracted by type I collagenase digestion.ADSCs were cultured to the third generation,and the proliferation activity of ADSCs was detected by the CCK-8 method.The phenotype of ADSCs surface antigen was identified by immunofluorescence and flow cytometry to explore the optimal MOI value of LV-BMP2-GDF transfected ADSCs.After subculture of BMP2-ADSCs,the level of BMP-2 gene in seed cells was detected by RT-PCR.PRP was prepared by comparing centrifugal venous blood by centrifugation.Bovine thrombin was used to activate PRP into a gel state.The release levels of TGF-β1 and PDGF-BB in PRP gel were detected by ELISA.Osteochondral grafts were constructed using gradient aperture SF-CS-n HA osteochondral scaffolds,BMP2-ADSCs,and autologous PRP gel.The graft was implanted into the central osteochondral defect of the distal femur of the Beagle dog’s knee joint.Twenty-four beagle dogs were randomly divided into group A(cell-PRP-scaffold group),group B(PRP-scaffold group),group C(scaffold group)and group D(blank control group),group A: BMP2-ADSCs + PRP gel + scaffold;Group B: PRP gel + scaffold;Group C: scaffold;Group D: blank control group.The effect of SF-CS-n HA scaffold on inflammation in joint cavity was analyzed.X-ray diagnosis was performed in the cell-PRP-stent group before,after,1st month after surgery,and 2nd month after surgery.To analyze the effect of bone regeneration in osteochondral defects.The tissue from the repair site of osteochondral defect in the cell-PRP-stent group was smeared 2 months after the operation.Observe the specimen smear for immunofluorescence under an inverted fluorescence microscope.Samples of osteochondral defect repair in the 4th month of each group were used for immunohistochemical detection of ColⅠ and ColⅡ.H & E staining,toluidine blue staining,alixin blue staining and alkaline phosphatase staining were used to observe the osteochondral regeneration.Results: Beagle dog ADSCs extracted by type I collagenase digestion.ADSCs of Beagle dogs grow in a spiral shape,with uniform cell morphology,obvious spindle and spindle shapes,and good proliferation activity.The phenotypic results of ADSCs identified by immunofluorescence and flow cytometry were consistent with CD90(+),CD44(+),CD29(+),CD34(-),HLA-DR(-),Blank(-),Isotype control(-).The optimal MOI value of LV-BMP2-GFP transfected ADSCs was 60.RT-PCR detection of BMP-2 gene level showed that the level of BMP-2 gene in LV-BMP2-60 group was significantly higher than that of LV-BMP2-NC group and Mock group.The ELISA results of extracts after PRP activation showed TGF-β1 release: 214.39 ± 35.41 ng / m L;PDGF-BB release: 33.159 ± 2.16 ng / m L.Samples of the osteochondral defects repaired in the 2nd and 4th month after operation were taken and observed.In the 2nd and 4th months after surgery,the defect repair of the cell-PRP-stent group was significantly better than that of the PRP-stent group,the simple stent group and the blank control group.The X-rays of the cell-PRP-stent group were taken before,after,1st month after surgery,and 2nd month after surgery.The results showed that the bone regeneration of osteochondral defects showed a good progressive repair trend.Tissue smears were taken at the repair site of osteochondral defect in the cell-PRP-stent group 2 months after the operation.The results showed that a large amount of green fluorescent tissue was present.Immunohistochemical detection of ColⅠ and ColⅡ in osteochondral defect repair samples at 4th month after operation in each group.The results showed that the expression levels of ColⅠ and ColⅡ in the cell-PRP-scaffold group were significantly higher than those in the PRP-scaffold group,the simple scaffold group and the blank control group.There was almost no expression of ColⅡ in the blank control group.H & E staining,toluidine blue staining,alixin blue staining,and alkaline phosphatase staining were performed on the osteochondral defect repair samples in each group at 4th month after surgery.The results showed that the cartilage layer repaired by the cell-PRP-scaffold group was similar in shape and thickness to the surrounding normal cartilage.The shape and thickness of cartilage repaired in the PRP-stent group and the simple scaffold group were worse than those in the cell-PRP-stent group.The blank control group had no cartilage layer.Conclusion: In this study,osteochondral grafts were successfully constructed by extracting highly pure ADSCs,LV-BMP2-GFP transfected ADSCs,preparing PRP gels,and gradient pore SF-CS-n HA osteochondral scaffolds.Various osteochondral related detection methods have confirmed that the graft has a good regeneration and repair effect on osteochondral defects.The osteochondral graft constructed in this research is expected to become a bionic composite material for repairing osteochondral defects.