Improved Bioresponses Of MC3T3-E1 To The Piezoelectric Signal Of Barium Titanate Polarized By Low-Intensity Pulsed Ultrasound

Objective:Developing bioactive materials for bone implants to enhance bone healing and bone growth,has for years been the focus of clinical and laboratory research.Titanium and titanium alloys are widely used as bone substitute materials because of their good biocompatibility,excellent mechanical properties and corrosion resistance.However,because they are considered bio-inert,they are not anticipated to augment bio-interaction with surrounding tissue.To enhance its surface bioactivity while retaining desirable inherent characteristics,titanium and its alloys are commonly surface-coated to shorten osseointergration periods.Various signals including biochemical,electrical and mechanical signals coordinate together in bone remodelling process.Natural bone tissues are known to exhibit piezoelectricity,which can convert mechanical forces to physiologically electrical signals.Piezoelectric materials mimic the physiologically loading process without external power supply.Barium titanate（BT）is a piezoelectric material with great biocompatibility and piezoelectric performance.In this study,a BT piezoelectric ceramic coating was synthesized in situ on the surface of Ti6Al4V（titanium-6aluminum-4vanadium,TC4）to form a BT/TC4 composite.The mechanical properties of TC4 and the piezoelectric properties of BT piezoelectric ceramics were combined.At the same time,low-intensity pulsed ultrasound（LIPUS）is a non-invasive mechanical wave transmitted to the human body in the form of high-frequency pressure waves.In this study,a BT piezoelectric ceramic coating was prepared by hydrothermal synthesis method,and a BT/TC4 composite was made.Firstly,the surface morphology,electrical properties,and biocompatibility of the material were evaluated.Then LIPUS was applied to the BT/TC4 composite material to evaluate biological response of mouse embryo osteoblast precursor cells（MC3T3-E1）on the surface of the materials,and its mechanism was discussed,which provide a clue for the clinic application of piezoelectric material combining with LIPUS loading.Methods:The first part:BT piezoelectric ceramic coating（BT/TC4 composite material）was fabricated on the surface of TC4 substrate by hydrothermal synthesis method;morphology and elements of BT/TC4 material surface were detected by scanning electron microscope（SEM）and energy dispersive X-ray spectrum（EDS）,used to evaluate the effect of BT piezoelectric ceramic coatings prepared by hydrothermal synthesis;X-ray crystallography was used to analysis BT/TC4 crystal phase structure;dielectric tester measures the dielectric constant of BT/TC4 material,electrochemical workstation measures the electronic value of TC4 and BT/TC4materials with or without ultrasound（frequency:1 MHz;intensity:30 m W/cm²）,in order to evaluate electrical properties of BT/TC4 materials.Cell proliferation kit-8（CCK-8）was used to measure the proliferation of MC3T3-E1 cells on the surfaces of two materials.The second part:MC3T3-E1 cells were cultured on the surface of the material and randomly divided into four groups:ultrasound BT/TC4 group,ultrasound TC4group,control BT/TC4 group and control TC4 group.Relative expression level of mRNA was used to detect alkaline phosphatese（ALP）,osteocalcin（OCN）,and Runt-related transcription factor 2（runt-related transcription factor 2,Runx-2）by Real-time quantitative PCR（qPCR）.The protein level expression of ALP,Runx-2 and type I collagen（Col1）were measured by western blot（WB）.These two methods were used to investigate the osteogenesis effect of LIPUS on MC3T3-E1 cells cultured on the surface of BT/TC4;chromatography-mass spectrometry is for screening differential metabolites to explore the difference of metabolites;KEGG Pathway functional enrichment analysis was done to measure differentiated expressing metabolites.The third part:MC3T3-E1 cells were compound cultured on the surface of the material and randomly divided into ultrasound BT/TC4 group,ultrasound TC4 group,control BT/TC4 group and control TC4 group.The calcium ion concentration of MC3T3-E1 cells in each group was detected by calcium ion imaging technology at day7,and the changes of calcium ion concentration among four groups were compared;each of the above groups was randomly divided into blank groups,L-type calcium channel block group（verapamil group）and calcium pool block group（SKF-96365 group）,calcium ion imaging technology was used to observe the calcium ionization at day 1,after adding blockers,whether the difference in concentration disappeared;the expression level of Ca _V1.2 protein of MC3T3-E1 cells in each group was detected by WB technology at day 7;and protein expression of L-type calcium channel protein Ca _V1.2 were compared among four groups.Results:1.SEM observed that the surfaces of BT/TC4have uniform particle distribution,continuous and no obvious gaps,consistent particle size,around 50nm in diameter,while TC4 surface cannot see the particle distribution;the presence of Ba element on the surface of BT/TC4 by observing the element waveform on EDS;XRD results showed that the main peak of Ba element was obvious and the coating was composed of tetragonal Ba Ti O₃;the dielectric constant of BT/TC4material was 800～1000;The BT/TC4 generates a microcurrent of about 10μA/cm²under LIPUS through an electrochemical workstation,while the TC4 material has no microcurrent under the same loading;CCK-8 assay showed that MC3T3-E1 cell numbers increased with time.At day 4 and day 7,the growth rate of osteoblasts on the surface of BT/TC4material was significantly higher than that of TC4 material（p<0.05）.2.qPCR results:the relative expression levels of ALP,Runx-2,and OCN in BT/TC4 group were higher than those in TC4 group（p<0.05）,and the relative mRNAs of ALP,Runx-2,and OCN in the ultrasound TC4 group were higher than those in the control TC4 group（p<0.05）,and the relative expression levels of ALP,Runx-2,and OCN in the ultrasound BT/TC4 group were higher than those in the control BT/TC4 group（p<0.05）,ultrasound TC4 group（p<0.05）and control TC4group（p<0.05）;WB results:The protein expression levels of ALP,ColI,Runx-2 in the control BT/TC4 group were higher than those in the control TC4 group（p<0.05）,and the ultrasound TC4 group had ALP,ColI,Runx-2 protein levels higher than those of the control TC4 group（p<0.05）,and the protein expression levels of ALP,ColI,and Runx-2 in the ultrasound BT/TC4 group were higher than those in the control BT/TC4 group（p<0.05）,ultrasound TC4 group（p<0.05）and control TC4 group（p<0.05）;LC-Mass non-targeted metabolite test results:In negative ion mode,compared with the ultrasonic TC4 group,45 metabolites were up-regulated and 46metabolites were down regulated compared in the ultrasonic BT/TC4 group.In the positive ion mode,compared with the ultrasonic TC4 group,the ultrasound BT/TC4group had 49 metabolites up-regulated and 40 metabolites down-regulated;Masshunter software was used to identify differential metabolites selected from the Metlin database,and 29 differences were finally confirmed.6 differentially expressed metabolites were analyzed by KEGG enrichment to obtain 7 metabolic pathways that may be related to osteogenesis,including calcium ion metabolism pathway and energy supply metabolism pathway.3.Calcium imaging:At day 7,the intracellular concentration of the ultrasound TC4 group was significantly higher than the control TC4 group（p<0.05）,and the control BT/TC4 group was significantly higher than the control TC4 group（p<0.05）.The ultrasound BT/TC4 group was significantly higher than the control BT/TC4group（p<0.05）,the ultrasound TC4 group（p<0.05）and the control TC4 group（p<0.05）.When the blocker was applied for 1 d,the calcium concentration in MC3T3-E1cells of the four groups in the blank group were different:the intracellular concentration in the ultrasound TC4 group was significantly higher than that in the control TC4 group（p<0.05）,and the control BT/TC4 group was significantly different compared with control TC4 group（p<0.05）,ultrasound BT/TC4 group was significantly higher than control BT/TC4 group（p<0.05）,ultrasound TC4 group（p<0.05）and control TC4 group（p<0.05）;After using Verapamil to block L-type calcium ion channel,compared with the blank group,the differences of calcium ion concentration in the MC3TC-E1 cells among the four groups in the verapamil group disappeared;after SKF-96365 inhibited the calcium pool,differences still existed in the SKF-96365 group:the intracellular concentration of the ultrasound TC4 group was significantly higher than the control TC4 group（p<0.05）;the control BT/TC4group was significantly higher than the control TC4 group（p<0.05）;the ultrasound BT/TC4 group was significantly higher than the control BT/TC4 group（p<0.05）,the ultrasound TC4 group（p<0.05）,and the control TC4 group（p<0.05）.qPCR and WB results:The relative mRNA and protein expression levels of Ca _V1.2 in MC3T3-E1 cells were significantly higher in the ultrasound TC4 group than in the control TC4 group,the ultrasound TC4 group was significantly higher than the control TC4 group.the control BT/TC4 group and the control TC4 had no difference.Conclusion:1.Barium titanate ceramic coatin on TC4 was successfully prepared by hydrothermal synthesis method.The surface coating of BT/TC4 material is dense and the structure is consistent.It has good electrical property:microcurrent of about10μA/cm²were generated under LIPUS（1MHz,30m W/cm²）;BT/TC4 material has good biocompatibility.2.Both LIPUS and BT/TC4 can promote osteogenic differentiation of MC3T3-E1 cells.LIPUS loaded on BT/TC4 materials can generate piezoelectric effect and further promotes osteogenic differentiation of MC3T3-E1 cells;this piezoelectric effect caused differential expression of 29 metabolites,of which 6metabolites may be related to 7 osteogenic metabolic pathways,including calcium ion metabolism pathway and energy supply metabolism pathway.3.Both LIPUS and BT/TC4 can promote the influx of calcium ions in MC3T3-E1 cells.The piezoelectric effect of LIPUS on BT/TC4 further promotes this effect.The potential reason may be that LIPUS loading on BT/TC4 causing electrical effect which promotes the opening of L-type calcium ion channels;LIPUS can promote the expression of L-type calcium ion channel proteins in MC3T3-E1 cells,and the piezoelectric effect of BT/TC4 under LIPUS further promotes the expression of L-calcium channel proteins.LIPUS-stimulated BT/TC4 has the ability to generate a piezoelectric signal which then enhances the biological responses of MC3T3-E1 cells.The combination of BT/TC4 material and LIPUS could be a potential method for forming early bone–implant contact in bone regeneration.