| Fracture nonunion remains a great challenge for orthopedic surgeons.Fracture repair comprises of three phases,stage of organization of hematoma,primary callus and remodeling.Extensive advancements have been made in the field of bone repair,including development of strategies to balance the M1/M2 macrophage populations,and to improve osteogenesis and angiogenesis.However,such developments focused more on the latter two phases,while ignoring the organization of hematoma phase during which cell recruitment occurs.In this study,we combined Stromal Cell-Derived Factor-1α(SDF-1α)and M2 macrophage derived exosomes(M2D-Exos)with a hyaluronic acid(HA)-based hydrogel precursor solution to synthesize an injectable,self-healing,adhesive HA@SDF-1α/M2 DExos hydrogel.The HA hydrogel demonstrated good biocompatibility and hemostatic ability,with the 4% HA hydrogels displaying great antibacterial activity against gramnegative E.coli and gram-positive S.aureus and Methicillin-resistant Staphylococcus aureus(MRSA).Synchronously and sustainably released SDF-1α and M2D-Exos from the HA@SDF-1α/M2D-Exos hydrogel enhanced proliferation and migration of human bone marrow mesenchymal stem cell(HMSCs)and Human Umbilical Vein Endothelial Cells(HUVECs),promoting osteogenesis and angiogenesis both in vivo and in vitro.Overall,the developed HA@ SDF-1α/M2D-Exos hydrogel was compatible with the natural healing process of fractures and provides a new modality for accelerating bone repair by coupling osteogenesis,angiogenesis,and resisting infection at all stages.Part I The fabrication and characterization of the hydrogelObjective: To design and prepare HA-based hydrogels generated from oxidized HA-quaternary ammonium(OHA-QA)and adipic acid dihydrazide-modified HA(HA-ADH)by Schiff base reactionMethods: HA-based hydrogels were prepared and tested for injectability,self-healing and tissue adhesion.The morphology of HA hydrogels was observed by scanning electron microscopy(SEM),and the effect of HA hydrogels on cell proliferation was examined by CCK-8 and Ed U proliferation assays.The swelling properties of HA hydrogels were observed and the rheological properties were examined by rotational rheometry.The antibacterial and hemostatic functions of HA hydrogels were evaluated by antibacterial assay and mouse liver bleeding model.In vivo and in vitro biocompatibility assays were achieved by HE staining and live-dead stainingResults: We successfully prepared three different concentrations of HA hydrogels at 2%,4% and 6% and evaluated them from various perspectives,including pore size,swelling properties,rheological properties,antibacterial properties and biocompatibility in vitro.The internal pore size of our HA hydrogels decreased with increasing polymer concentration,and the proliferation rate of both HMSCs and HUVECs decreased significantly when the hydrogel concentration reached 6%.The strain at break decreased from 259% to 63% when the hydrogel concentration was increased from 2% to 6%.Both 4% and 6% HA hydrogels had excellent antimicrobial properties.4% HA hydrogels had excellent hemostatic properties and in vivo biocompatibility.Conclusion: We have successfully prepared HA hydrogels with injectable,self-healing,and tissue adhesive properties.HA hydrogels exhibit excellent biocompatibility and hemostatic ability and can provide an antimicrobial microenvironment for fracture healing.Part II HA@SDF-1α hydrogel promotes migration of HMSCs and HUVECsObjective: To investigate the effects of hydrogels loaded with SDF-1α(HA@SDF-1α)and SDF-1α/CXCR4 axis on the migration of HMSCs and HUVECs cellsMethods: The optimal concentration of SDF-1α was confirmed by PCR and WB methods and the effect of HA hydrogels loaded with SDF-1α on the SDF-1α/CXCR4 axis was evaluated.The effect of HA@SDF-1α on the migration ability of HMSCS and HUVECs cells was evaluated by cell scratching assay and Transwell assay.Results: The optimum concentration of hydrogel-loaded SDF-1α was 500 ng/m L,and the hydrogel-loaded SDF-1α promoted migration of HMSCs and HUVECs.Conclusion: Sustained release of SDF-1α from HA@SDF-1α promoted migration of HMSCs and HUVECs.Part III HA@M2D-Exos hydrogel enhanced angiogenesis and osteogenesis in vitroObjective: To investigate the effect of hydrogels loaded with M2D-Exos(HA@M2D-Exos)on promoting osteogenic differentiation of HMSCs and angiogenesis of HUVECs in vitroMethods: Bone marrow-derived macrophages were extracted and induced to differentiate to M2 type.M2D-Exos were extracted and characterized by transmission electron microscopy(TEM),dynamic light scattering,nano-flowmetry and WB.Cellular up-take of exosomes and the distribution of hydrogel-loaded exosomes was visualized by confocal microscopy.The proliferation effect of HA@M2D-Exos on HMSCS and HUVECs was evaluated by CCK-8,cell cycle flow assay and Ed U proliferation assay.The effect of HA@M2D-Exos on osteogenic differentiation of HMSCs in vitro was also evaluated by WB,ALP staining and alizarin red staining,and the effect of HA@M2D-Exos on angiogenesis of HUVECs was evaluated by tube formation assay.Results: We have successfully extracted and characterized M2D-Exos,which has a particle size range of 50-150 nm and highly expressed tumor susceptibility gene 101(TSG101),CD9,CD63 and CD81 exosomal marker proteins.The hydrogel loaded with M2D-Exos had a pro-proliferative effect on HMSCs and HUVECs,and promoted osteogenic differentiation of HMSCS and angiogenesis of HUVECs,respectively.Conclusion: HA@M2D-Exos hydrogel enhanced angiogenesis and osteogenesis in vitroPart IV HA@ SDF-1α/M2D-Exos hydrogel enhanced bone repair in miceObjective: To investigate the modulation of fracture healing and angiogenesis in mice by an injectable antimicrobial hydrogel sustained release system(HA@SDF-1α/M2D-Exos)loaded with SDF-1 and M2D-Exos.Methods: Exosome loading of the hydrogel was clarified by in vivo fluorescence imaging of small animals.The effect of HA@SDF-1α/M2D-Exos on fracture healing in mice was examined and analyzed by means of X-ray and micro-CT imaging.The effect of HA@SDF-1α/M2D-Exos on bone and cartilage formation in fracture healing in mice was evaluated by means of HE,Alcian Blue and Masson pathological staining.The regulatory effects of HA@SDF-1α/M2D-Exos on angiogenesis in mice were evaluated by EMCN fluorescence staining and CD31 immunohistochemical assay and blood flow scatter imaging in small animals.Results: The HA@SDF-1α/M2D-Exos hydrogel group showed significantly reduced fracture gaps,greater bone volume(BV),tissue volume(TV),BV/TV and bone mineral density(BMD),reflecting the better fracture healing effect of HA@SDF-1α/M2D-Exos.Pathological tests showed that the HA@SDF-1α/M2D-Exos group had a higher proportion of bone tissue and a lower proportion of cartilage tissue in the fracture site,and the fracture healing was faster.EMCN fluorescence staining,CD31 immunohistochemical assay and blood flow scatter imaging in small animals suggested significantly enhanced neovascularization in mice of HA@SDF-1α/M2D-Exos group.Conclusion: Injectable antimicrobial hydrogels loaded with SDF-1α and M2D-Exos promote fracture healing and hemodynamic reconstruction in mice. |
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