Platelet Rich Fibrin Composite Granule Cartilage Up Regulation Repair Of Articular Cartilage Defects In Rabbits By AMPK/Sox-9 Pathway

Background and purposeThe ideal goal of articular cartilage defect repair is to reshape the dual structure of cartilage and subchondral bone,restore the normal shape and stress distribution of articular surface.At present,there is no gold standard treatment method.The commonly used surgical methods have the disadvantages of limited supply,immune rejection,secondary surgery and high cost;Therefore,it is more urgent and necessary to explore an economic,applicable,efficient and stable repair scheme.Cartilage formation depends on the synergistic effect of various growth factors and their physical and chemical factors mediated by related signal pathways.Studies have shown that growth factors promote cartilage formation by up regulating specific cartilage genes and target proteins;Sox-9 is a transcription factor necessary for cartilage formation.It activates cartilage specific cytokines through transcription,promotes cell differentiation and proliferation,and significantly enhances the repair ability after cartilage granulation,but the specific mechanism is not completely clear;A variety of growth factors participate in the regulation of related signal pathways and receptor proteins in the form of complexes,which may become potential therapeutic targets in the process of cartilage formation.In this study,granular cartilage was used as seed cell,growth factor as signal molecule and platelet rich fibrin as carrier to explore the feasibility of platelet rich fibrin composite granular cartilage in repairing rabbit cartilage defect,so as to provide experimental basis for clinical application.Method1.Platelet rich fibrin was prepared by uniform centrifugation.The specimens were observed,the microstructure was observed by scanning electron microscope;The concentrations of TGF-β3,PDGF-AA and IGF-1 were detected in platelet rich fibrin by ELISA,and the release law of growth factor was analyzed.2.CCK-8 was used to detect the effect of different concentrations of platelet rich fibrin on the activity of chondrocytes;Adult chondrocytes were transfected with AMPK siRNA to detect the effect of PRF mediated AMPK /Sox-9 pathway on chondrocyte proliferation.The experiment was divided into four groups: adult chondrocyte group,juvenile chondrocyte group,100% PRF +adult chondrocyte group and siRNA + 100% PRF + adult chondrocyte group.The regulatory effect of PRF on inhibited and non-inhibited AMPK/Sox-9signaling pathway was analyzed by PCR,Western blot and immunofluorescence,and downstream ACAN and Col2a1 gene transcription and protein expression.3.The rabbit articular cartilage defect model was constructed and divided into four groups: A.blank control group,B.allogeneic juvenile granular cartilage group,C.allogeneic adult granular cartilage group,D.platelet rich fibrin + allogeneic adult granular cartilage group.Different materials were implanted into the cartilage defect area of each group.Specimens were obtained at 6,12 and 24 weeks after operation for gross observation,micro CT scanning,degree of subchondral bone repair;HE,toluidine blue and safranine solid green were used to evaluate the repair effect of cartilage defects,the elastic capacity of new cartilage was measured by compressive modulus,and the gene expression of Sox-9 and Col2a1 were observed by immunohistochemistry.Result1.Platelet rich fibrin is generally light-yellow jelly,with smooth surface,uniform texture and elasticity;Scanning electron microscope showed that collagen fiber bundles were regular,intertwined and intertwined,showing a loose and porous network structure;TGF-β3,PDGF-AA and IGF-1 were released continuously and slowly within 3 weeks in platelet rich fibrin.The release curves of the three growth factors were consistent,and there were different release peak time points.2.The results of CCK-8 showed that 25%,50% and 100% platelet rich fibrin could promote the proliferation of chondrocytes,and 100% concentration had the highest ability to promote the proliferation of chondrocytes,and its proliferation promoting effect was concentration dependent;PCR,Western blot and Immunofluorescence detection showed that the expression of AMPK,Sox-9,ACAN and Col2a1 genes in 100% PRF + adult chondrocytes group was increased,which was significantly different from that in adult chondrocytes group and siRNA + 100% PRF + adult chondrocytes group(P < 0.05).PRF up-regulated the expression of AMPK and Sox-9 genes by regulating AMPK/Sox-9 signal pathway,enhanced the expression of ACAN and Col2a1 genes,and silenced the upstream expression of AMPK genes,ACAN and Col2a1 genes was inhibited and the up-regulated expression was reversed.3.Specimens were obtained at 6,12 and 24 weeks after operation.The cartilage defects in each group were repaired to varying degrees.The effect of PRF combined with adult granular cartilage was the best;At 24 weeks after operation,the gross morphological scores in groups A,B,C and D were 6.50 ±1.52,14.17 ± 0.75,9.67 ± 1.21 and 14.33 ± 0.82 respectively.Compared with group A,the difference was significant(P < 0.05);Micro CT scan showed that PRF combined with adult granular cartilage group had more bone trabeculae and higher bone volume fraction;Histopathology showed that typical chondrocytes and lacunae could be seen in PRF combined with adult granular cartilage group,and the morphology,composition,structure and thickness of new cartilage were closer to normal cartilage;O’Driscoll histological scores in groups A,B,C and D were 10.67 ± 1.03,21.00 ± 1.10,15.17 ± 0.75 and 21.83 ± 0.75 respectively,which were significantly different from those in group A(P < 0.05);Immunohistochemistry showed strong positive expression of Sox-9 and Col2a1 in PRF combined with adult granular cartilage;The compressive elastic modulus of cartilage in groups A,B,C and D were 0.219 ± 0.057,0.584 ± 0.048,0.375 ±0.059 and 0.683 ± 0.082 Mpa respectively,and that of normal cartilage was0.716 ± 0.075 Mpa.Compared with group A,the difference was significant(P <0.05).The elastic property of new cartilage in PRF combined with adult granular cartilage group was the closest to that of normal cartilage.Conclusion1.Platelet rich fibrin is rich in TGF-β3,PDGF-AA and IGF-1;The reticular loose structure composed of interwoven collagen fiber bundles is conducive to the storage and release of growth factors.2.Platelet rich fibrin promotes chondrocyte proliferation by regulating AMPK/Sox-9 pathway.3.Granular cartilage can effectively repair articular cartilage defects in rabbits.Platelet rich fibrin enhances the repair promoting effect of granular cartilage by up regulating AMPK/Sox-9 pathway.