Effects Of Exosome-graphene-porous Titanium Alloy Scaffold To Promote ADSCs Repair Jaw Defects

Objective:Maxillofacial bone defects caused by trauma and tumor are a great challenge for plastic surgeons,but satisfactory repair materials have not been found yet.In recent years,the rapid development of tissue engineering has brought new hope for the clinical treatment of bone defects.Widely used bone repair materials Titanium and Titanium alloy(Ti6Al4V,Ti)have good biocompatibility,mechanical strength and corrosion resistance.However,Titanium and Titanium alloy are biologically inert,and are mainly bonded in the body through physical integration,which is also different from the elastic modulus of bone.The combination of material and tissue is unstable.These problems need to be solved,so different titanium surface treatment method were used to meet clinical application.The common surface modification methods of implants mainly include two aspects:One is to loosen the structure of the implant;Second,implant surface coating,loading bioactive molecules or drugs to modify the implant surface.These methods can effectively improve the bioactivity and biocompatibility of implants and promote osseointegration at the implant-bone interface.Graphene(Gr)has good biocompatibility performance,which can be used as a culture substrate for specific cells.Graphene’s unique mechanical properties,such as high elongation,flexibility and adaptability to regular or irregular surfaces,lend themselves to cell adhesion and migration.Graphene and graphene oxide have been found to be used in bone tissue field.Materials coated with graphene or graphene oxide or even 3D graphene can promote stem cell viability and induce osteogenic differentiation of stem cells compared to traditional substrates or scaffolds.ADSCs have multidirectional differentiation potential.Recent studies have shown that the function of tissue repair and regeneration of stem cells is not only due to the proliferation of stem cells at injured sites,but also largely due to their paracrine function.Extracellular vesicles(EVs)are tiny vesicles secreted into the Extracellular environment by cell membrane fusion after cells in the body form polyvesicles through endocytosis.Exosomes(Exos),as EVs with a diameter of 30~150nm,can participate in the regulation of normal physiological functions and abnormal pathological processes of the body,and are considered as a new mechanism of communication between cells.Adipose Stem cells-derived exosomes(ADSCs-Exos)play a role in information exchange between cells and have been widely proven to important in tissue regeneration.Some studies have shown that it can act as an "inducer" to promote bone tissue regeneration and repair.Scaffold materials can provide a carrier for inducing factors to promote stem cell differentiation.Titanium alloy can be endowed with good mechanical properties and biological functions through surface modification technology.Graphene can promote the adhesion and differentiation of stem cells due to its unique structure and properties.The co-culture method of seed cells and three-dimensional scaffold material composite culture after implantation has been recognized by academic circles.These results suggest that it is possible to fabricate graphene-coated porous titanium alloy scaffolds that can load adipose stem cells and their exosomes in vivo to construct tissue-engineered bone with better properties.In order to make up for the deficiency of traditional titanium alloy materials in repairing bone tissue defects,in this study,the composite graphene-coated porous titanium alloy scaffold(Gr-Ti)was prepared by micro-arc oxidation(MAO)technology with porous titanium alloy as the substrate from both structure and surface.Through compound adipose stem cells and exosomes,the effects of composite scaffold materials on bone repair and bone integration were evaluated,providing theoretical basis for the combined application of bone tissue engineering and stem cell therapy,and experimental basis for the development and application of new clinical bone defect repair materials.The related studies have not been reported.Methods: 1.A 3D printed porous titanium alloy(Ti)scaffold was designed and prepared by Selective Laser Melting(SLM)technology,and graphene coating was coated on its surface by MAO technology.Scanning electron microscopy(SEM)was used to observe the changes of surface morphology.Its porosity was measured;The existence of characteristic peak of graphene was verified by energy spectrum analysis.3D measurement laser confocal microscope was used to detect the surface roughness of the scaffold.Mechanical properties of titanium alloy were tested by mechanical compression test.2.ADSCs from human adipose tissue obtained through liposuction were extracted to verify the cytotoxicity and proliferation activity of scaffold materials in vitro,and to explore the effect of Exos combined with Gr-Ti scaffold on the osteogenic differentiation of ADSCs.3.Explore the role of Wnt in Exos in promoting osteogenic differentiation of Gr-Ti scaffold in vitro.4.Rabbit ADSCs and their exosomes were extracted,and the effect of Exos and Gr-Ti scaffold on the osteogenic differentiation of ADSCs was verified in vivo by constructing the critical bone defect model of rabbit mandible.Results: 1.The porosity of porous titanium alloy was 69±3% and the pore size was 546±21μm.SEM and surface composition analysis showed that the graphene coating on 3D porous titanium alloy scaffold was successfully prepared.The surface roughness results show that the surface roughness of Gr-Ti group increases obviously.The mechanical test results showed that the scaffold had elastic modulus suitable for the bone tissue,and the graphene coating had no obvious effect on the overall mechanical properties of titanium alloy.2.In vitro experiments confirmed that Ti and Gr-Ti scaffolds had no cytotoxicity,and the Gr-Ti scaffolds with composite graphene-coated scaffolds could better promote cell adhesion and proliferation than the Ti scaffolds without coating.3.In vitro qPCR and WB method indicate that the level of osteogenic related genes and proteins Runx2,ALP and Osterix were up-regulated in the Gr-Ti /Exos group,and the expression of key proteins Wnt-1,Axin2 and β-catenin were also significantly increased in the Wnt signaling pathway.Adding Wnt pathway inhibitor DKK reduced the expression of osteogenic related proteins and key proteins of Wnt pathway.4.In vivo experiments,the osteogenic ability and bone defect repair of the Gr-Ti /Exos group were superior to that of the Gr-Ti group.It was confirmed by tissue staining that the regeneration of bone tissue in Gr-Ti/Exos group was significantly better than that in Gr-Ti group.Conclusion: 1.SLM technology can prepare 3D porous titanium alloy scaffolds,whose elastic modulus is close to bone tissue and reduces stress shielding.The MAO method can prepare graphene coating on the surface of the scaffold,which is stably bonded to the titanium alloy substrate.The graphene coating enhanced surface roughness of the scaffold,but did not change its mechanical properties.2.Gr-Ti has good biocompatibility.Gr-Ti scaffolds combined with exosomes can promote cell adhesion and proliferation.3.Gr-Ti scaffolds with combined exosomes can promote the osteogenic differentiation of ADSCs through Wnt signaling pathway.4.Gr-Ti scaffold combined with exosome can better promote the repair of critical bone defect of rabbit mandible.