Circulating Exosomes-Mediated MiR-301 Regulates Ossification Of Canine Mandibular Distraction Osteogenesis Through TGF-β Pathway

ObjectiveWe first analyze the different RNA expression of circulating exosomes in canine mandibular distraction osteogenesis(DO)and bone fracture(BF)models,to find the key genes which regulating bone formation in the process of distraction osteogenesis,and exploring its possible mechanism of regulating the rapid osteogenesis of distraction osteogenesis.Methods1.Six beagle dogs were randomly divided into 2 groups,and animal models of canine mandibular distraction osteogenesis and fracture defects were constructed respectively.Collecting circulating serum exosomes of these two groups at 5 t ime points(before operation,7 da y after operation,8 day after operation,14 day after operation,21 day after operation)for whole transcriptome sequencing and bioinformatics analysis.The animal specimens were taken 21 days after operation.We observed the general healing of the distraction/frature gap of the mandible,detected the imaging of new bone formation in the distraction zone by Micro-CT,and observed the neovasculogenesis and osteogenesis of the new tissue by HE staining and Masson trichrome staining.2.We cultured canine mandibular bone marrow mesenchymal stem cells(JBMMSCs),and identified it by flow cytometry,as well as osteogenic,adipogenic,chondrogenic differentiation and colony forming unit assay.After extraction of serum exosomes in the DO group and BF group 14 days after the operation,they were identified by transmission electron microscopy,nanoparticle tracking analysis,and Western blot.The exosomes uptake experiment proved that the two groups of exosomes can be endocytosed by JBMMSCs.The effects of serum exosomes in the DO and BF groups on the proliferation,migration and osteogenic differentiation of JBMMSCs were detected by CCK8,transwell,alkaline phosphatase staining,alizarin red staining(ARS),RT-PCR and Western blot.3.Endothelial colony-forming cells(ECFCs)were isolated and cultured from adult canie peripheral blood,identified by flow cytometry,Dil-Ac-LDL and FITC-UEA-1 dual staining experiments and tube-forming experiments.After extracting the exosomes secreted by ECFCs,transmission electron microscopy,nanoparticle tracking analysis and Western blot were used for identification.Then ECFC-exos stained with PKH26 was traced in vivo and in vitro.Observe whether ECFC-exos can be taken up by JBMMSC and observe its accumulation in the area of distraction/fracture gap.4.Overexpress and silence miR-301 a by transfecting ECFCs with lentivirus,extract the exosomes secreted by ECFCs after lentivirus transfection,and detect the expression of miR-301 a in the exosomes by RT-PCR;CCK-8,transwell,alkaline phosphatase staining,ARS,RT-PCR and Western blot were used to detect the effects of ECFC-exos on the proliferation,migration and osteogenic differentiation of JBMMSCs,and to detect changes in the downstream TGF-βsignaling pathway.In vivo experiments,27 beagle dogs were randomly used to construct an animal model of mandibular distraction osteogenesis.ECFCs exosomes that silenced miR-301a(miR-301a-i group),ECFCs exosomes transfected the negative virus(inhibit-NC group)and normal saline(DO group)were injected intravenously on the first day and the middle of the distraction.The cannies were sacrificed on 14 days,28 days,and 42 days after surgery.The tissues of the distraction gap were observed.Micro-CT was used to detect the osteogenesis in the distraction area,and the histological changes of the new bone were observed by HE staining,Masson trichrome staining and Immunohistochemistry staining(IHC).Results1.Through imaging and histological analysis of distraction and fracture gap21 days after surgery,the results show that the speed of bone regeneration in distraction osteogenesis is stronger than that in fractures;and the sequencing results indicate that differential expression miRNA is cfa-miR-301 and cfa-miR-615 in circulating serum exosomes between DO groups and BF groups.2.We successfully isolated and cultured JBMMSCs and identified these cells based on the positive express the surface markers of CD44,CD90 and CD146,and negative express the CD34,CD31 and CD45.Meanwhile,we use osteogenesis,adipogenesis,cartilage assay to verify their differentiation ability and colony forming unit assay to identify the proliferation ability.Next,we isolated the serum exosomes and authenticate by their morphology of cup holderlike,particle size of between 50 to150 nm,and expressed exosomal markers CD63,TSG101 and CD81.Exosomes uptake experiments showed that serum exosomes labeled with PKH26 red fluorescence can be endocytosed into JBMMSCs.There was no significant difference between the two groups of exosomes in the proliferation of JBMMSCs;the exosomes of the DO group had better ability to promote the migration and osteogenic differentiation of JBMMSCs than the BF group.3.ECFCs were successfully isolated and cultured,flow cytometric identification was positive for CD34 and CD105,negative for CD45 and CD133,positive for double staining experiment,and had the ability to form tubes.The exosomes from the cell supernatant were successfully extracted,and their morphology was cup holder-like,with a particle size of about 133.93±1.11 nm,and expressed exosomal markers CD63 and TSG101,but did not express the negative marker calninex.In vivo and in vitro tracing results show that ECFCExos labeled with PKH26 red fluorescence can be endocytosed into cells by JBMMSCs,and can be enriched in the distraction/fracture renewal area in vivo.4.Using bioinformatics,combining the sequencing data with the expression profile of ECFC and its exosomes in public databases,the differential transcript miR-301 a can be obtained.The RT-PCR results showed that the expression of miR-301 a decreased and increased correspondingly after transfection of miR-301 a overexpression and silencing lentivirus.After transfection with ECFC-exos,JBMMSCs were treated with ECFC-exos and found that there was no significant change in the proliferation of JBMMSCs;Transwell,ALP staining,Alizarin Red staining,RT-PCR and WB experiments showed that silencing miR-301 a exosomes can promote the migration and osteogenic differentiation of JBMMSCs;at the same time,WB results indicate that the effect of miR-301 a on osteogenic differentiation may be through the TGF-β signaling pathway.The results of in vivo experiments showed that the miR-301 a silent group formed more and stronger distraction space callus,and the bone density value was higher than that of the rest of the groups.Conclusion1.The transcripts carried by circulating serum exosomes during distraction osteogenesis and fracture healing are different,and the two have different effects on osteogenesis of jaw bone marrow mesenchymal stem cells,which may result in different healing speeds.2.Through analysis of circulating serum exosomes at different stages of distraction osteogenesis and fracture and comparison with public database ECFC exosomes expression profile,we speculate that exosomes miR-301 a released by peripheral blood ECFC may be promoting distraction osteogenesis and play a key role in the rapid formation of new bone.3.Inhibiting the expression of miR-301 a in ECFC-exos can promote new bone formation,and its effect is related to the up-regulation of the TGF-βsignaling pathway of jaw bone marrow mesenchymal stem cells.