| Background and objectsDiabetes mellitus(DM)is a chronic inflammatory disease.It can impede osteocyte function and delays bone remodeling via sustained high blood glucose and release of inflammatory cytokines,which named as diabetes bone disease(DBD).Current way for bone augmentation shows poor prognosis for DBD patients and may cause secondary trauma.Therefore,advanced treatment is of urgent need to improve bone augmentation effect as well as relief patient’s pain.Among these new methods,tissue engineering is a promise alternative,it mainly works by the function of seed cells and effector cell.bone marrow mesenchymal stem cells(BMSCs)are common seed cells in bone tissue enginerring which are characterized by self-renew and multi-differentiation.Osteoblasts(OBs)are common effector cells in bone regeneration,they’re major cell components of human skeleton which are differentiated from BMSCs and possess the ability of osteogenesis.Yet,the regulations between BMSCs and rOBs are still in vague and requires further exploration and investigation,especially the impact of diabetic BMSCs-Exos to rOBs function and its underlying mechanism.Studies have shown that exosomes,a member of extracellular vesicles,could conduct intercellular communication and cell function regulation.Exosomes communicate via their membrane protein-cell membrane receptor binding or directly fusion with cell membrane,and regulate target cell function via their inner components like miRNAs,lncRNAs.They have many advantages like specific targeting,high stability,low toxicity,superior biocompatibility,accessibility,abundant resources and thus be used as nano-drugs to treat various diseases.In particular,MSC-derived exosomes are proved to improve tissue repair.microRNAs are a member of non-coding RNAs which exert function via inhibition of target gene.In particular,miR-17 has been proved to promote osteogenesis and reduce inflammation,and its expression has reduced in type 2 diabetes mellitus(T2DM)rat jaw.Therefore,miR-17-5p could be underlying genes to cure DBD.Smad7 is one of miR-17’s target gene and has been proved to inhibit osteogenesis via targeting wnt/β-catenin and TGF-βsignaling pathway.Therefore,miR-17 might promote osteogenesis via inhibiting Smad7.Currently,there’re few studies about diabetic exosomes’ influence on bone regeneration,and the underlying mechanisms are far from complete.Therefore,our study aims to explore the implication of diabetic BMSC-derived exosomes on osteogenesis of OBs and bone remodeling of diabetes rat,and whether miR-17/Smad7 plays important role in this mechanism.Material and methods1.Isolation,culture and identification of rBMSCs and rOBsprimary rBMSCs was isolated by whole bone marrow adherent method and identified by flowcytometry,multi-potency and colony-forming assay.rOBs were isolated by tissue mass-enzyme digestion method and identified by morphology,ALP staining and IHC.rBMSCs and rOBs were cultured and passaged.Cell cryopreservation and recovery were also conducted.2.Isolation,identification of NG-Exos and HG-Exos and cell-uptake assayHigh glucose medium was used to mimic DM in vitro.Diabetic/non-diavetic BMSCderived exosomes was isolated by canonical ultracentrifugation method and named as NGExos and HG-Exos,respectively.Exosomes were identified by marker protein,NTA and TEM.Lipophilic dye PKH67 was used to mark exosome membranes and detect rOBs’ uptake.3.Implication of NG-Exos and HG-Exos to osteogenesis and migration of rOBsrOBs were cocultured with NG or HG exosomes,while control group were given equal volume of PBS.Exosomes’ implication on rOBs osteogenic differentiation and bone remodeling of rat cranial defect were tested by ALP staining,ARS staining,PCR and WB.Their implication on cell migration were tested by wound sketch assay and transwell assay.4.Selection of differential expressed exosomal miRNAs and verification in rat sckull,exosome-cocultured rOBsDifferential expressed miRNAs between two group exosomes were screened by miRNA array.Volcano map and heat map were used to analyze differential expressed genes(DEGs).qPCR was used to verify expression level of DEGs’among normal rat&T2DM rat as well as rOBs cocultured with NG-Exos or HG-Exos.Finally,common DEG was explored in further experiment.5.Osteogenic effect of miR-17 and its rescue effect on exosome’s osteogenic impactOverexpression or knock down of rOBs’ miR-17 were conducted by transefection of miR-17 mimics or inhibitor,respectively.qPCR was used to verify transfection efficiency.By transfecting corresboding miRNA analogs to NG-Exos and HG-Exos,we verified the rescue effect of miR-17 on two group of exosomes.6.Osteogenic effect of Smad7 and its rescue effect on miR-17’s osteogenic impactThe interaction of miR-17 and Smad7 was by dual luciferase reporting assay Overexpression of rOBs’ Smad7 was by transfection of pcSmad7 plasmid into cells.Cotransfection of pcSmad7+miR-17 were used to measure miR-17’s rescue effect on osteogenic effect of Smad77.Osteogenic effect of NG-Exos and HG-Exos on bone remodeling of rat cranial defect,rescue effect of miR-17 on exosome’s osteogenic impactMethylacrylyl gelatin(GelMa)gel was used as scafford of exosomes.By topical injection of agomir/antagomir,we overexpress/knock down miR-17 levels in defect area.microCT was used to evalute bone regeneration effect of exosomes and rescue effect of miR17 to osteogenic effect of exosomes.HE staining and masson staining were used to evaluate their effect on new bone mass.Results1.successfully isolated and identified primary rBMSCs and rOBs&NG-Exos and HGExos.Both group of exosomes could be uptake by rOBs.3.NG-Exos promoted osteogenesis and migration of rOBs and bone remodeling of T2DM rats.On the contrary,HG-Exos inhibited cell function of rOBs and bone remodeling of non-diabetic rats.4.HG-Exos,HG-Exos cocultured rOBs and T2DM rats had down-regulated expression of miR-17.Overexpression of miR-17 could boost ostegenesis of rOBs.5.Smad7 was one of the downstream targets of miR-17 which impeded ostegenesis of rOBs and undermined miR-17’s osteogenic promotion effect.Conclusion1.non-diabetic BMSC-Exos promoted rOBs migration,and promoted osteogenesis of rOBs and T2DM rats via upregulating miR-17 and downregulating of Smad7.2.diabetic rBMSCs-derived exosomes inhibited migration of rOBs,and impede osteogenesis of rOBs and bone remodeling of rats by downregulating miR-17,which further promote Smad7 transciption.This miR-17/Smad7 axis provide new insights of treatment of DBD and optimizing application of BMSCs-rOBs interaction in bone regeneration... |
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