Preparation Of Graphene/Zinc Silicate /Calcium Silicate Composite Microspheres And Its Osteogentic Performance

Owing to its excellent bioactivity and osteoinductivity,calcium silicate(CS)has attracted considerable research interest in recent years.However,the degradation rate of the pure CS is too fast,it can not only result in overhigh microenvironmental pH value,but also induce the mechanical strength of CS to rapidly decrease,which will greatly affect its following clinical applications.Therefore,the graphene/zinc silicate/calcium silicate(RGO/ZS/CS)composite microspheres with a core-shell stucture would be prepared in this study,and we tried to use the graphene surface layer and zinc silicate interlayer to slow down the degradation rate of the CS.Furthermore,we tried to further improve the bone repair effect of the RGO/ZS/CS composite microspheres from the following three ways,including minimizing the inflammatory reactions;promoting the osteogenesis and the angiogenesis as well as reducing the osteoclastogenesis;improving the cell affinity of the surface of the RGO/ZS/CS composite microspheres.In this study,the antibiotics loading efficiency of the RGO/ZS/CS composite microspheres and its corresponding releasing characteristics were investigated in detail.Besides,the effect of the exogenous electrical stimulus and the zinc ions(Zn2+)on osteogenic differentiation as well as the effect of the released functional inorganic ions on vascularization and osteoclastogenic differentiation were also evaluated.In additon,the cell affinity of the bovine serum albumin(BSA)covalent bonded graphene was carefully studied.The detailed results about this work were described as below.Through the crosslinking reactions between sodium alginate and calcium ions,the RGO/ZS/CS composite microspheres with a core-shell structure were successfully prepared via the liquid droplet method,and the vancomycin hydrochloride(VH)was also in situ loaded into the RGO/ZS/CS microspheres.The in vitro mineralization experimental results indicated that no hydroxyapatite(HA)crystals formed on the RGO/ZS/CS composite microspheres surface after they were soaked in the SBF for 28 days,but gel-like amorphous calcium phosphate could be found in this case,implying RGO/ZS/CS composite microspheres still possessed bioactive.For the RGO/ZS/CS composite microspheres,the VH loading efficiency was approximately 30.66%.In addition,the RGO surface layer and the ZS interlayer could effectively slow down the release rate of VH.In summary,the core-shell structured RGO/ZS/CS composite microspheres exhibit good VH sustained release ability.In this study,the four-point probe method was used to determine the surface electrical conductivity of the RGO/ZS/CS disks,and the influences of the concentration of the RGO suspensions,the spin coating times as well as the annealing temperature on surface electrical conductivity were investigated in detail.When the RGO/ZS/CS were prepared by spin coating twice with 3 mg/mL of the RGO suspensions,and then heated at 600°C for 2 h,its surface electrical conductivity was 5656 S/m.The results of the in vitro biomineralization experiments indicated that the RGO surface layer and the ZS interlayer would not retard HA forming on its surface after RGO/ZS/CS were calcined.As demonstrated by the cell culture experiments,the exogenous electrical stimulus(3 ?A for 1 hour per day)and the released Zn2+ could significantly promote the alkaline phosphatase(ALP)activity of the mouse bone mesenchymal stem cells(mBMSCs),and the expression levels of the osteogenic-related genes including the Collagen-I(Col-I),Osteocalcin(OC)and Runt-related transcription factor 2(Runx-2)were remarkablely up-regulated,suggesting the exogenous electrical stimulus really could promote the osteogenic differentiation of mBMSCs.As the human umbilical vein endothelial cells(HUVECs)were co-cultured with varying concentrations of RGO/ZS/CS extracts,the results indicated that the proliferation of the HUVECs was significantly promoted when the RGO/ZS/CS extracts were separately diluted to 1/16,1/32 and 1/64.Furthermore,the expressions of the angiogenic-related genes including Vascular endothelial growth factor(VEGF),Fibroblast growth factor(FGF)and Endothelial nitric oxide syntase(eNOS)were remarkablely up-regulated.The relusts of the in vitro vascularization experiments also proved the RGO/ZS/CS extracts with the dilution rate ranging from 1/16 to 1/64 benefited vascularization.As the mouse leukaemic monocyte macrophages(RAW264.7)were co-clutured with the RGO/ZS/CS extracts,it was found that the released Si and Zn could significantly decrease the tartaric-resistant acid phosphatase(TRAP)activity of RAW264.7.The expressions of the osteoclastogenic-related genes including TRAP,Carbonic anhydrase 2(Car2),Matrix metalloproteinases 9(MMP-9)and nuclear factor 1 of active T cells(NFATc1)were remarkablely down-regulated,implying the released Si and Zn really could suppress the osteoclastogenic differentiation of RAW264.7.The carboxyl on the graphene oxide(GO)surface was successfully activated into NHS active ester(GO-EDC-NHS)with N-(3-Dimethylaminopropyl)-N?-ethylcarbodiimide hydrochloride(EDC)and N-hydroxysuccinimide(NHS).Approximately 12.75 mg of bovine serum albumin(BSA)could be covalent bonded onto the GO-EDC-NHS surface(BSA-CB-GO),whereas only 6.83 mg of BSA physical absorbed onto the GO surface(BSA-NB-GO).With a 168 h of phosphate buffer saline(PBS)soaking,the BSA accumulative desorption ratio,which was accordingly assigned to the BSA-NB-GO and the BSA-CB-GO,was separately 29.91 wt.% and 2.95 wt.%.Consequently,it proved GO-EDC-NHS exhibited more stable and stronger BSA absorption capacity.As compared to mBMSCs cultivated on the BSA-NB-GO surface,the immunofluorescence staining images showed that more vinculins and integrin ?5 were visible in the mBMSCs cultivated on the BSA-CB-GO surface,implying the BSA-CB-GO possessed better cell adhesion and cell spreading behaviors,which can be used to improve the cell affinity of bone repair materials.