Study Of Osteogenesis And Angiogenesis Effects Of BaTiO₃ Coated Porous Ti6Al4V Scaffold

BackgroundPorous titanium alloys have a lower elastic modulus and porous characteristics which can not only effectively maintain the mechanical stability of the bearing parts of the skeleton,but also provide sufficient space for the growth of bone tissue and blood vessels,thus becoming an advantage material in the field of bone repair replacement product.With the emergence of metal 3D printing technology,which can not only realize the precise spatial configuration design and manufacturing of porous titanium alloy materials,but also prepare personalized metal bones for simulation,so as to achieve accurate repair and reconstruction of bones and joints.Therefore,it has a well prospect.However,porous titanium alloys are still a kind of biological inert material,and their pore interior lack of effective bone growth characteristics due to stress shielding,leads to the difficulty of bone growth inside the material,which affects the service life of porous titanium alloy implant material.However,at present,most modification methods focus on the bone-like components and activity simulation of the coating,while the“biological functions”related to the force and electricity of bone tissue under physiological conditions are not paid enough attention,resulting in the unsatisfactory bone formation at pore interior.As a kind of biological piezoelectric body,human skeleton has the characteristic of“force-electricity conversion”,which can generate endogenous electric field through force stimulation.This endogenous electric field can effectively promote the complex biological process of bone integration.Therefore,more and more scholars believe that in the design of bone repair materials in the future,the importance of force electrical signal conversion to bone growth should be fully paid attention to.Piezoelectric ceramic materials have attracted much attention because of their similar properties to bone piezoelectric effect,which can realize force-electrical signal conversion.BaTiO₃is the lead-free piezoelectric ceramic which first discovered and applied in the medical field.It has excellent biocompatibility and safety,and has been widely studied in the field of orthopedic grafts.However,the high brittleness and the obvious lack of mechanical strength of BaTiO₃ limit its application in the replacement of bone repair at the bearing site.ObjectiveIn this study,we fabricated the uniformly BaTiO₃ coating on porous Ti6Al4V scaffold surface,expected to get a graft that not only have sufficient mechanical strength but also could generate endogenous electric field.And we hypothesized the composites can effectively promote the combination of material and bone through the piezoelectric effect of BaTiO₃ on angiogenesis and osteogenesis.Methods1.Electron beam melting（EBM）technology was used to prepare porous titanium alloy scaffolds.The scaffolds（?12 mm×H2 mm）were used in vitro cell experiments,and the scaffolds（?12 mm×H6 mm）were used in vivo animal experiments.The BaTiO₃piezoelectric ceramic coating was prepared on the surface of the porous titanium alloy scaffolds by hydrothermal synthesis,then subjected to polarization treatment.Scanning electron microscopy（SEM）,energy spectroscopy（EDS）,and X-ray diffraction（XRD）were used to detect the surface morphology,surface elements,and elemental composition of the materials.Micro-CT was used to detect the pore structure of the materials.The water contact angle of the materials was tested to determine its hydrophilicity.Piezoelectricity microscopy（PFM）,quasi-static d₃₃ meter,and Zeta potential analyzer were used to test the piezoelectric properties of the materials.2.The effects of different scaffolds on MSCs and HUVECs cells were observed using dynamic pressure culture in vitro.According to different materials,they were divided into:（1）simple porous Ti6Al4V scaffold（pTi）;（2）porous Ti6Al4V scaffold modified with BaTiO₃ piezoelectric ceramic coating,unpolarized（pTi/BaTiO₃（unpoled））;（3）Porous Ti6Al4V scaffold modified by BaTiO₃ piezoelectric ceramic coating,polarized（pTi/BaTiO₃（poled））.CCK-8 was used to detect the proliferation ability of cells on different scaffolds.Flow cytometry was used to detect the apoptosis rate of cells on different scaffolds.SEM was used to observe the morphology of cells on different scaffolds.Transwell assay was used to detect cell migration ability.Alkaline phosphatase（ALP）activity,extracellular matrix（ECM）mineralization ability and RT-PCR was used to detect the osteogenic differentiation ability of MSCs.The VEGF and PDGF-bb protein secretion of HUVECs on different scaffolds were detected by ELISA.3.Sheep interbody fusion cage implantation model was used to judge the osteogenesis and angiogenesis ability of different scaffolds.The experimental grouping was the same as the vitro experiment.Specimens were taken at 4 and 8 months after surgery.Micro-CT scan,VG staining of histological section,and EDS analysis of histological section were used to judge the internal bone invasion of the materials.Angiography of sheep cervical spine using Microfil contrast medium infusion,Micro-CT scan was used to judge the angiogenesis around the material,and histological section fluorescence microscope was used to observe the angiogenesis inside the material.Results1.SEM revealed that the surface of the pTi scaffolds were smooth,and spherical particles with a diameter of about 200μm were uniformly distributed on the surface of the pTi/BaTiO₃ scaffolds.EDS and XRD proved that the BaTiO₃ piezoelectric ceramic coating was successfully prepared on the surface of porous Ti6Al4V scaffolds.Water contact angle results prove that the pTi/BaTiO₃ scaffolds were more hydrophilic.Micro-CT scans and general images of the scaffolds proved that the construction of the BaTiO₃ piezoelectric ceramic coating on the surface of the porous Ti6Al4V scaffolds had no effect on the overall structure and pore structure,and both groups of materials have open pore structures.The piezoelectric constant(d₃₃)of the pTi/BaTiO₃scaffolds were 0.7 p C/N,and the surface potential were about-30m V.PFM found that with the change in the output voltage of the probe（-10V～10V）,the amplitude of the BaTiO₃ crystal on the surface of the material gradually increased from 0pm,and the phase also changed with the output voltage.2.MSCs and HUVECs in the pTi/BaTiO₃（poled）group show the most activity of cell proliferation（P<0.05）,meanwhile the cell proliferation activity of pTi/BaTiO₃（unpoled）group was better than pTi group（P<0.05）.All the normal living cells in the 3 groups were at a higher rate,which proved that the materials had little toxicity to MSCs and HUVECs.MSCs and HUVECs were successfully attaching to the surface of the scaffolds.The MSCs morphology in the BaTiO₃/pTi（poled）group presents a varied line osteogenesis shape,and the cell surface area was bigger than that in the BaTiO₃/pTi（unpoled）and pTi group.The migration ability of MSCs and HUVECs were more obvious on the BaTiO₃/pTi（poled）scaffolds.The ALP activity extracellular matrix（ECM）mineralization ability and RT-PCR showed that the pTi/BaTiO₃（poled）group had the most significant effect on promoting osteogenic differentiation of MSCs,followed by pTi/BaTiO₃（unpoled）.HUVECs cells in the pTi/BaTiO₃（poled）group had the highest ability to secrete VEGF and PDGF-bb,which was significantly higher than the other two groups（P<0.05）.At the same time,pTi/BaTiO₃（unpoled）had a higher ability to secrete PDGF-bb than the pTi group（P<0.05）,but the ability to secrete VEGF was similar to the pTi group（P>0.05）.3.In vivo experiments,the results of Micro-CT,histological section VG staining,and EDS showed that the bone growth inside the cages of the pTi/BaTiO₃（poled）group were significantly better than the other two groups（P<0.05）,and the bone growth in the pTi/BaTiO₃（unpoled）group was better than that in the pTi group（P<0.05）.Micro-CT was used to observe the angiogenesis around the materials.It was found that the pTi/BaTiO₃（poled）group significantly promoted angiogenesis around the material compared with the other two groups,and the pTi/BaTiO₃（unpoled）group was better than the pTi group.The angiogenesis inside the materials were also consistent with the outside of the material.Conclusion1.BaTiO₃piezoelectric ceramic coating was successfully prepared on the surface of porous Ti6Al4V without changing the original structure of the scaffolds.This material has both the mechanical characteristics and an open pore structure of a porous titanium alloy,as well as a force-electric conversion characteristic.2.In the condition of dynamic pressure culture in vitro,the pTi/BaTiO₃（poled）material with piezoelectric effect can effectively promote cell proliferation and migration,and also promote osteogenic differentiation of MSCs and secretion of angiogenic related proteins of HUVECs.Meanwhile,pTi/BaTiO₃（unpoled）material without piezoelectric effect also promotes cell proliferation,osteogenesis,and angiogenesis due to its increased surface roughness and hydrophilicity.3.In vivo study,the pTi/BaTiO₃（poled）material can effectively promote the bone regeneration and angiogenesis around and inside the material through the piezoelectric effect,pTi/BaTiO₃（unpoled）material was also better than pTi for bone regeneration and angiogenesis.