Study Of A Porous Ti6Al4V Scaffold Modified With Barium Titanate Combined With Low-intensity Pulsed Ultrasound To Repair Large Segmental Bone Defects

BackgroundIn clinical practice,many bone defects are caused by bone trauma,infection,or tumors.When the length of the bone defect exceeds a critical size（1.5 times the diameter of the tubular bone）,it is defined as a large bone defect.Clinical healing of large segmental bone defects is a challenging problem for the international orthopedic community because the bone’s innate healing process is unable to complete bone regeneration at the deteriorated defect site.Supportive therapeutic intervention must also be provided.Therefore,methods using biomaterials and tissue engineering have been developed and utilized to support bone regeneration.However,due to a lack of essential vascularization and mechano-electric effects and poor biomechanical properties at the site of a large bonedefect,the application of these methods is limited in clinical treatment.In this context,a comprehensive treatment strategy should be adopted.Furthermore,many requirements must be met,including a good material（biocompatible with adequate pore size and interconnectivity and stiffness similar to that of the surrounding tissue）,a good coating（biocompatible with a mechano-electric effect）,and additional auxiliary measures（vascularization）.For large segmental bone defects,porous titanium（Ti）alloy scaffolds can provide appropriate mechanical strength to maintain initial mechanical stability in load-bearing areas.In addition,three-dimensional（3D）porous Ti scaffolds fabricated by electron beam melting（EBM）show the advantages of appropriate pore size and adjustable interconnectivity.Thus,nutrients are more likely to diffuse into the center of scaffold,which is also good for new bone ingrowth.Due to their lack of biological activity or mechano-electric effect,however,porous Ti scaffolds may fail to repair large bone defects.Barium titanate（BaTiO₃）piezoelectric ceramic is commonly employed to promote bone regeneration,which can mimic the stress-generated potentials（SGPs）of natural bone,which is itself a piezoelectric object,to produce micro-electric currents and promote calcium salt deposition at the bone defect site.When deformation of native bone occurs,it can generate piezoelectric polarization stimuli and adjust the growth of bone,shaping and rebuilding the tissue.For piezoelectric ceramics,the piezoelectric effect can make them convert between mechanical energy and electrical energy,which is similar to the behavior of natural bone.Therefore,BaTiO₃ piezoelectric ceramic can be combined with porous Ti alloy scaffolds in the form of a coating by a wet chemical method to repair small bone defects.However,for large segmental defects,due to the lack of conditions promoting vascularization,the combination of BaTiO₃ with the Ti scaffold may not be sufficient.Low-intensity pulsed ultrasound stimulation（LIPUS）is a non-invasive physical therapy used to accelerate bone repair and promote bone healing.In 1994 and 2000,LIPUS was clinically approved for the treatment of fresh fracture and bone nonunion,respectively,by the US Food and Drug Administration（FDA）.Firstly,LIPUS can directly influence a series of cellular signal changes involved in the bone healing process,andtherefore cause calcium ions to enter the cell and promote bone mineralization.Secondly,LIPUS can increase the mRNA level of vascular endothelial growth factor A（VEGF-A）,an important regulatory factor of angiogenesis.Lastly,LIPUS can stimulate cartilage cell proliferation,increase cartilage matrix production（type II collagen,glycosaminoglycan sugars,etc.）,improve the maturity of newly formed bone,and ultimately promote the occurrence of endochondral ossification.Additionally,LIPUS as an external mechanical force can act on the BaTiO₃ piezoelectric ceramic coating,make it produce a piezoelectric effect,and further promote bone regeneration.ObgectiveIn this study,we deposited the BaTiO₃ composite as a piezoelectric coating onto the surface of porous Ti6Al4 V scaffolds to form a composite Ti implant（Ba TiO₃/pTi）.The surface performance of the BaTiO₃/pTi implant was assessed by scanning electron microscopy（SEM）,X-ray photoelectron spectroscopy（XPS）,water contact angle measurement,and surface roughness analysis.Then,four groups were compared with each other,the pure titanium implant（pTi）,the barium titanate/pure titanium implant（BaTiO₃/pTi）,the pure titanium implant + low-intensity pulsed ultrasound（pTi+LIPUS）,and the barium titanate/pure titanium implant + low-intensity pulsed ultrasound（BaTiO₃/pTi+LIPUS）.The in vitro proliferation,apoptosis,and osteogenic differentiation of rabbit bone marrow mesenchymal stem cells（BMSCs）on the scaffolds were evaluated.The in vivo osteointegration and osteogenesis properties also were assessed using a large segmental defect model of the radius in New Zealand White rabbits.Methods1)The electron beam melting technique was used to fabricate two different specifications of porous Ti6Al4 V scaffolds.The scaffolds（?12 mm × H2 mm）were used in in-vitro cell experiments,and the scaffolds（?5 mm × H13 mm）were used in in-vivo animal experiments.For all the scaffolds,aperture size was 700 μm,porosity is 70%and the beam diameter is 380 μm..By hydrothermal synthesis method barium titanate piezoelectric ceramic coating was used to modify the surface of scaffolds.SEM、EDS and XPS were used to detect whether BaTiO₃ coating was successfully fabricated on thesurface of porous Ti6Al4 V scaffolds.Through the water contact angle and surface roughness analysis,changes of surface properties of porous Ti6Al4 V scaffolds were detected.Micro-CT scanning analyzed the changes of the aperture,connected diameter,porosity and beam size of the modified scaffolds.The changes of elastic modulus and strength of the modified scaffolds was evaluated by mechanics test.2)Rabbit bone marrow mesenchymal stem cells were seeded onto the surface of scaffolds,and LIPUS was used to stimulate them.According to the conditions,with or without LIPUS,the experiment was divided into four groups: BaTiO₃/pTi+LIPUS group、BaTiO₃/pTi group、LIPUS+pTi group 和 pTi group.When cells had been cultured on the samples under different conditions for 4 and 7 days,their proliferation was investigated using the cell counting kit-8.At 4 days,SEM was used and at 4 and 7 days,the fluorescent staining was used to observe the basic morphology and adhesion activity of cells.At 4days,flow cytometry instrument was used to analyzed apoptosis of cell.At 4 and 7 days,fluorescent dye was used to detect cell death.At 7 days and 14 days,alkaline phosphatase activity and osteogenesis related gene respectively were detected to evaluate the osteogenetic differentiation of cells.3)To establish large bone defects model of New Zealand white rabbit radius,the scaffolds with or without BaTiO₃ piezoelectric ceramic coating were implanted into bone defect site.After surgery,low intensity pulsed ultrasound was used to stimulated implantation area.Like the above cell experiment,the experiment was divided into BaTiO₃/pTi+LIPUS group、BaTiO₃/pTi group、LIPUS+pTi group 和 pTi group.At 6 and 12 weeks after surgery,X-ray and Micro-CT scanning to analyzed the internal bone tissue regeneration of scaffolds;by fluorescent tags,bone mineralization apposition rate were calculated;through histological section VG stain further analyzed the growth of new bone tissue and bone integration of bone-scaffold interface;biomechanical tests were used to detect the peak pull-out load.Results1)SEM 、 EDS and XPS showed that BaTiO₃ piezoelectric ceramic coating wassuccessfully immobilized on the surface of pTi.The average water contact angle of the Ti6Al4 V scaffolds with a BaTiO₃ coating was 40.22° ± 1.38°,while that of the bare Ti6Al4 V scaffolds without a BaTiO₃ coating was 77.28° ± 3.47°.These results had statistical significance,indicating that BaTiO₃/pTi was significantly more hydrophilic than pTi.That is,the BaTiO₃ coating successfully enhanced the wettability of pTi.The Ba TiO₃ coating significantly improved the surface roughness（Fig.2 A2,B2,and D）,as the data showed that the average surface roughness of BaTiO₃/pTi was 5.78 ± 1.36 μm while that of pTi was 8.69 ± 1.22 μm.Micro-CT scanning results showed that the aperture,connected diameter,porosity and beam size of the scaffolds with BaTiO₃ had no significant change compared with pTi.Mechanical test results indicated that elastic modulus and mechanical strength of BaTiO₃/pTi had no obvious change.2)At 4 days,the cell viability was significantly higher in the BaTiO₃/pTi,pTi+LIPUS,and Ba TiO₃/pTi+LIPUS groups than in the pTi group.The cell viability in the BaTiO₃/pTi+LIPUS group was also much greater than that in the Ba TiO₃/pTi and pTi+LIPUS groups.Although the cell viability was higher in the pTi+LIPUS group than in the BaTiO₃/pTi group,no statistical difference was found in the cell proliferation between the two groups.After incubation for 7 days,the result was similar to that seen at 4 days.At4 days,statistical analysis showed that cells in the BaTiO₃/pTi+LIPUS group had significantly higher CN ratios and vinculin fluorescence intensity than cells in the other three groups,that cells in the pTi+LIPUS group had higher CN ratios than those in the BaTiO₃/pTi and pTi groups,that there was no statistical significance in the CN ratios between the BaTiO₃/pTi and pTi groups,and that there was no significant difference in the vinculin fluorescence intensity among the three groups（pTi+LIPUS,BaTiO₃/pTi,and pTi）.At 7 days,statistical analysis of the CN ratio and vinculin fluorescence intensity further indicated that these parameters were significantly higher in the BaTiO₃/pTi+LIPUS group than in the other three groups,that they were higher in the pTi+LIPUS and BaTiO₃/pTi groups than in the pTi group,and that there was no statistical difference between the pTi+LIPUS and BaTiO₃/pTi groups.The DC/TC percentage in the BaTiO₃/pTi+LIPUS group was statistically lower than that in the other three groups at 4 days.The DC/TCpercentages in the pTi+LIPUS and BaTiO₃/pTi groups were lower than that in the pTi group.Meanwhile,the DC/TC percentage in the pTi+LIPUS group was statistically similar to that in the BaTiO₃/pTi group.At 7 days,the results were similar to those found at 4 day.The proportion of apoptotic cells in the BaTiO₃/pTi+LIPUS group was statistically lower than that in the other three groups at 4 days.The proportion of apoptotic cells in the pTi+LIPUS and BaTiO₃/pTi groups was lower than that in the pTi group.Meanwhile,the proportion of apoptotic cells in the pTi+LIPUS group was statistically similar to that in the BaTiO₃/pTi group.ALP activity was significantly higher in the BaTiO₃/pTi+LIPUS,pTi+LIPUS,and Ba TiO₃/pTi groups than in the pTi group at 7 and 14 days.Additionally,ALP activity was significantly higher in the BaTiO₃/pTi+LIPUS group than in the pTi+LIPUS and BaTiO₃/pTi groups.However,there was no statistical difference in ALP activity between the pTi+LIPUS and BaTiO₃/pTi groups.The gene expression of COL-A1,ALP,OPN,and Runx2 at the mRNA level in the BaTiO₃/pTi+LIPUS group was significantly higher than that in the other three groups after7 and 14 days.The ALP,COL-A1,and Runx2 gene expression was significantly higher in the pTi+LIPUS and BaTiO₃/pTi groups than in the pTi group at 7 and 14 days.There was no significant difference in the ALP,COL-A1,and Runx2 gene expression at 7 and 14 days between the pTi+LIPUS and BaTiO₃/pTi groups,while the OPN gene expression was significantly higher in the pTi+LIPUS and Ba TiO₃/pTi groups than in the pTi group at 14 days.3)The BV/TV values were significantly higher in the BaTiO₃ / pTi + LIPUS,pTi +LIPUS,and Ba TiO₃/pTi groups than in the pTi group at 6 and 12 weeks.Meanwhile,the BV/TV values of the BaTiO₃ / pTi + LIPUS group were statistically higher than those of the pTi + LIPUS and BaTiO₃/pTi groups at 6 and 12 weeks.However,there were no significant differences in the BV/TV values between the pTi + LIPUS and BaTiO₃/pTi groups at 6 and 12 weeks.The MAR values in the pTi + LIPUS and BaTiO₃/pTi groups were significantly higher than that in the pTi group.However,there was no statistical difference in the MAR values between the pTi + LIPUS and BaTiO₃/pTi groups.At 12 weeks,the change in the MAR values was similar to that seen at 6 weeks in all groups.The percentages of newly formed bone in the BaTiO₃ / pTi + LIPUS,pTi + LIPUS,and BaTiO₃/pTi groups were significantly higher than in the pTi group at 6 and 12 weeks.The percentages of the BaTiO₃ / pTi + LIPUS group were statistically higher than those of the pTi + LIPUS and Ba TiO₃/pTi groups at 6 and 12 weeks.However,there were no significant differences in the percentage of newly formed bone between the pTi + LIPUS and BaTiO₃/pTi groups at 6 and 12 weeks.At 6 weeks,the BaTiO₃ / pTi + LIPUS,pTi +LIPUS,and Ba TiO₃/pTi groups had significantly higher peak pull-out load in the loading modes of the tension test than the pTi group.It is worth noting that the BaTiO₃ / pTi +LIPUS group achieved a more significant increase in peak pull-out load than the pTi +LIPUS and BaTiO₃/pTi groups.However,no significant difference was found between the pTi + LIPUS and BaTiO₃/pTi groups.By 12 weeks,peak pull-out loads of the three conditions（BaTiO₃ / pTi + LIPUS,pTi + LIPUS,and Ba TiO₃/pTi）had further increased compared with that of the pTi condition.Meanwhile,the trend in the differences in peak pull-out load among the four groups at 12 weeks was the same as that seen at 6 weeks.ConclusionIn the present study,a BaTiO₃ coating was successfully fabricated by a wet chemical method on the surface of porous Ti6Al4 V scaffolds,which showed better surface hydrophilicity and roughness than pure porous Ti6Al4 V scaffolds.In vitro studies,including the CCK-8 assay,live/dead cell staining,an apoptosis assay,immunofluorescent labeling,an ALP activity assay,and RT-PCR,suggested that the BaTiO₃ coating and LIPUS promoted adhesion,proliferation,and differentiation of BMSCs and that LIPUS activated the piezoelectric effect of the BaTiO₃ coating,further enhancing cell viability.In addition,the results of micro-CT,MAR,histological analysis,and biomechanical testing revealed that the BaTiO₃/pTi implants had greater osteointegration and osteogenesis properties with or without LIPUS in vivo than pTi implants.Especially with the combined conditions of BaTiO₃/pTi+LIPUS,large segmental bone defects were better repaired in this model than with the pTi,BaTiO₃/pTi,and pTi+LIPUS conditions.We concluded that the Ba TiO₃ coating and LIPUS improved cell viability in vitro and increased new bone formation in vivo,that LIPUS activated the piezoelectric effect of the BaTiO₃ coating tofurther strengthen its role both in vitro and in vivo,and that pTi,the BaTiO₃ coating,and LIPUS were all necessary factors in the complete bone regeneration of a large segmental bone defect.Therefore,the Ba TiO₃/pTi+LIPUS conditions showed promising potential as a method to repair long bone defects for clinical application.