The Reseach Of Mechanism And Application Of 3D-printed Magnesium-doped β-tricalcium Phosphate Bioceramics Scaffolds For Bone Repair

Objective:Bone defect is one of the common clinical diseases in orthopaedics.β-tricalcium phosphate(β-TCP)is a widely used bone defect repairing material due to its outstanding osteoconductivity,biocompatibility,and similar chemical composition to the mineral component of natural bones.However,β-TCP displays disadvantages such as poor osteoinductive properties and low mechanical strength.Magnesium(Mg)is an essential inorganic component in bone tissue and plays an important role in bone metabolism and bone development,and magnesium ion was reported to promote osteogenesis and angiogenesis.This study intends to combine the advantages of magnesium and β-TCP,by incorporating different mass fractions of magnesium oxide(Mg O)into β-TCP powder,and fabricates β-TCP scaffolds with gyroid biomimetic structures doped with different contents of Mg O by 3D printing method.The osteogenic,angiogenic and immunomodulatory effects of the Mg-doped β-TCP bioceramics(Mg-TCP)were studied in vitro,and the bone defect repair capability of the Mg-doped β-TCP bioceramics scaffolds were verified in vivo.Methods:1.Different mass fractions(0 wt%,1 wt%,3 wt%,5 wt%)of Mg O were mixed into the β-TCP powders,and β-TCP bioceramics scaffolds with gyroid structures doped with different amounts of Mg O were printed by digital light processing(DLP)technology,named TCP,1Mg-TCP,3Mg-TCP and 5Mg-TCP,respectively.The bioceramics scaffolds were characterized by scanning electron microscope(SEM),X-ray diffraction analysis(XRD),and electronic dynamic and static fatigue testing machine.2.Methods including sirius red staining,alizarin red staining,osteogenesis-related genes expression detection and ALP activity detection were used to study the effects of Mg-TCP on the osteogenic differentiation of bone marrow stromal cells(BMSCs).Methods including wound healing assay,transwell assay,tube formation assay,and angiogenesis-related genes expression detection were used to study the effects of Mg-TCP on the angiogenic differentiation of endothelial progenitor cells(EPCs).Methods including immunofluorescence staining and macrophage polarization-related genes expression detection were used to study the effects of Mg-TCP on macrophage polarization.An indirect cell co-culture model was constructed,and Mg-TCP derived macrophage conditioned medium was used to culture BMSCs or EPCs to study the immunomodulatory effects of Mg-TCP on osteogenesis and angiogenesis.3.The bioceramics scaffolds were implanted into the femoral condyle bone defect areas of rabbits.After 6 and 12 weeks of scaffolds implantation,the bone defect repair properties of Mg-TCP were evaluated by Micro-computed tomography and hard tissue section staining.Results:1.In this study,various β-TCP bioceramics doped with different amounts of Mg O with gyroid structures were printed by DLP technology.SEM results showed that as the increase of doping amounts of Mg,the surface of the bioceramics became denser and the grains became smaller.XRD results confirmed that Mg O was dissolved into β-TCP bioceramics.Mg-doping enhanced the mechanical strength of β-TCP bioceramics,and3Mg-TCP had the highest strength.The degradation rate of β-TCP bioceramics decreased with the increase of the doping amounts of Mg.2.When the bioceramics scaffolds or their extracts were used to directly intervene BMSCs or EPCs,compared with TCP,Mg-TCP promoted the osteogenic differentiation of BMSCs and the angiogenic differentiation of EPCs,and 5Mg-TCP exhibited the best results.Compared with TCP,both Mg-TCP can promote the transformation of RAW264.7cells to M2 phenotype,especially 3Mg-TCP.When Mg-TCP derived macrophage conditioned medium was used to culture BMSCs or EPCs,it was found that 3Mg-TCP had the best osteogenic and angiogenic properties.3.Animal experiments showed that compared with pure TCP,Mg-TCP showed better bone defect repair performance,and 3Mg-TCP exhibited the optimal bone defect repair capability both after 6 and 12 weeks of implantation.Conclusion:Mg-doping could enhance the mechanical properties of β-TCP bioceramics scaffolds and improve their osteogenesis,angiogenesis,and immunomodulatory properties.3Mg-TCP displays the best immunomodulatory properties,promotes the transformation of macrophages to M2 phenotype to regulate the process of osteogenesis and angiogenesis,and thus exhibits the optimal osteogenesis and angiogenesis properties.In addition,3Mg-TCP possesses the optimal bone defect repair capability in vivo.Therefore,gyroidβ-TCP bioceramics doped with 3 wt% Mg O have good application prospects as orthopaedic implants.