| Background and ObjectionTheory of Osseointegrationwas put forward by professor Branemarkin 1960s;followed by the first titanium implant replacing a missing tooth in 1965.Since then,Theory of Osseointegrationlay theory foundation for the modern theory of stomatology and oral implantology.For the first time to carry on the artificial dental implant surgery and restorations and succeed.With the development of oral implantology,the surface treatment is gradually mature,titanium materials aremore and more used in the field of stomatology.And with the gradual development of demands for higher quality of life,oral implantology has become the preferred restorative option for dentition defect and edentulism.The properties of implant surface was influenced by hydrophily、surface morphology、roughness and the surface energy,In the past forty years,titanium materials are widely appliedin prosthodontics due to the evolution of oral implantology,and has a distinctive advantage inbiological safety、corrosion resistanceand long-term stability.The surface is biologically connected to the surrounding tissues determined by the the properties of implant surface and more and more applied in clinical practice.MAO(micro-arc oxidation)reported for the first time by Gunterschulze and Betz was one of anodizing technology and applied in surface treatment of oral implants successfully in recent years in the early 1930s.Micro and nano-scale structure are formed with biologic activityon the titanium surface by technology of MAO,Many study show thatwhich could stimulate the proliferation and spreading of osteoblasts."MAOed" ceramic surface possesses certain roughness、hydrophilyand surface energy etc.Furthermore,The study of Y.Li discovered that the layer of oxide coating modified by MAO significantly improves the bonding strength between the implant-bone interface and promotes the stability.Another experiments done by Ran et al indicated that oral implant modified by MAO promote osteoanagenesis around the implant and the stabilization of implant-bone interface in comparison with the untreated MAO group.The study by Huan Huan Jiang proved that the hydrophilic surface promoted early physical and chemical adhesion of cells and increased the effect of biological adhesion by promoting the realignment of the cytoskeleton,expression of adhesion related factor-gelling kinase-FAK.In addition,studies shows that the roughness and chemically modification of implant surface increases the probability of success in implant surgery.Surface roughening of implant is classified asmicron scale and nanoscalein terms of morphology.In comparison,micron scale morphology strengthens the mechanical cohesionof implant-boneinterface while nanoscale morphology stimulates bone-implant integration via biomolecular interaction.Studies have shown that the force of mechanical cohesion provided by morphology of micron scale is the basisto activating biologic activity of nanoscale morphology.Therefore,it has been accepted by scholars that the combination of micron and nanoscalemorphologyhasan advantageto accelate osseointegrationof implants.Titanium possesses favourable biocompatibility and forms a thinoxide coatingwhen exposed to the air.However,this thinoxide coatingdoes not better promote bone proliferation or shorten time of the osseointegration.In order to achieving higher osseointegration、more aesthetic、long term stabilizationand decline of bone absorption better surface with definite roughness and hydrophilic by implant surface modificationis necessary.A novel cortex-like TiO2 coating was prepared on Ti surface throughmicro-arc oxidation(MAO)by using sodium tetraborate as electrolyte,and the effects of the coating oncell attachment were testified by Liu et al.this coating possesses a structure that isdifferent from traditional crater-like MAO surface.A newly formed cortex-like surface,which consisted ofnanopores(80-200 nm diameter)and microslots(3-5 μm diameter),The coatingsshowed a distinct hierarchical structure as well as arough and porous surface morphology.Additionally,in vitro studies showed that it hadsuperhydrophilicity and good biocompatibility asevidenced by an excellent in-vitro performance alongwith MG-63 cells ingrowth into the slots of thecortex-like structure.The cortex-like MAO coating isexpected to have broad applications on implants.the coatinghas less micro crack,low internal stress,and higher interfacial bonding strength.In addition,anatase,rutile,and brookite constitute the polymorphs of TiO2surface.XRD diagram showed that the MAOsample surface was mainly composed of rutile.Rutilepossesses higher chemical stability compared with anatase,Ti-OH groups inrutile were formed on the surface of the MAO implant.The amount of Ti-OH group in rutile was higher thanthat in anatase,which then further triggered the formation of carbonated hydroxyapatite.Thus,the rutilecoatings achieved notable osseointegration at thebone-implant interface.The present study aimed to examine the in-vivoosseointegration of the cortex-like MAO coating incomparison with the sand-blasting and acid-etching(SLA)coating,which has been widely used in clinics.The implants were inserted into the femoral condyles ofrabbits and evaluated by micro-CT,histomorphometricanalysis,and sequential fluorescent labeling.Objective1.Preparethe pure Ti plates(015 mm),compare the surface physicochemical property of the small platesprocessed by MAO(cortex-like)technology and SLA technology,and evaluate the feasibility of MAO process as implant surfacing.2.Design thread-shaped titanium implant,and establish the experimental animal models.Implants modified by MAO(cortex-like)and SLA are respectively inserted into rabbit femoral contlyles,carrying out Micro-CT test at different time periods.Thus the results could provide a basis of whether the MAO(cortex-like)technology could become a surface treatment technology.3.To compare osteoconductivity inside thread-shaped implants between MAO and SLA by using following indicators:includingBIC%(Bone-to-implant contact),BA%(Bone area),and Mineral appositionrate(MAR).In addition,the result provides anverificationon osseointegration treated by MAO(cortex-like).Part 1:The preparation of pure Ti samples with two types of surfacing and the study of its physicochemical properties.Materials and methodsImplants and Treatments:The biomedical pure Ti rods(TA2)are made into the Ti plates samples(diameter:15mm,thickness:10mm)through rough and fine grinding processes of CNC,and are polished with 200-800 grit SiC paper step by step.Afterwards,the treated implants are washed successivelyfor 20 minutes with ultrasoundin acetone,anhydrous ethanol and deionized water and dry at room temperatureafterprocessed byMAO(cortex-like)and SLA.Surface characterization:Surface electron microscopy(SEM)is used to observe the implant surface morphology.The elemental concentrations of the implant surfaces are quantified by energy dispersive X-ray spectrometer(EDS),the crystal structure of the implant surfaces is analyzed by X-ray Diffraction(XRD).The surface roughness Ra and Rq are measured by the step profiler(probe-type surface profiler).Lastly,the contact angles are measured by the contact angle meter(OCAH200),and the surface energies are calculated by using Owens-Wendt method.ResultsSEM examination clearly shows the effects of MAO and SLA treatment on the surface structure of the Ti implant.The micron-nanoscale structure of porous oxide coating,which consists of micro slots(3-5um diameter)and nanopores(80-200nm diameter)demonstrates a micron-nano bilayer and are distributed evenly.Additionally,the SLA surface of the Ti implant are presented in wave-like morphology(30-40 μm)with micron cavities(2-5μm).After etching procedure(acid),primary cavities(2-3μm)are formed;sandblasting procedure forms secondary cavities(10-40 μm),increasing specific surface area.The surface roughness Ra and Rq:EDS spectra indicates thatonly Ti appears in the SLA group,whereas a large amount of O exists in the MAO group with oxidation coating.In addition,XRD patterns detects that MAO coating mainly consists of rutile(the natural form of Ti02)with a small amount of anatase.Part 2:Animals and Surgical Procedures and Micro-CT analysis Materials and methodsPure Ti rods areprocessed into the thread-shaped implants(n=36;MAO(cortex-like)=18,SLA surface= 18.diameter 3.3mm、length 10mm)by CNC.After process of MAO and SLA,the implants are washed chronologicallywith ultrasound with acetone,anhydrous ethanol,and deionized water for 10 minutes and then stored in deionized water,dried,and packed.Afterwards,sterilizing them using 25.0 kGy y-ray(GM series y irradiation apparatus)for 12 hours prior to in-vivo use.All implants wereinserted into New Zealand rabbit femoral condyles and divided into two times intervals to evaluate osseointegration at4weeks and 8 weeks;For the double-fluorescent labeling,alizarin red(1 Omg· mL-1,30 mg·kg-1)was subcutaneously injected intothe rabbits at 13 and 14 days and calcein(10 mg·mL-1,5mg·kg-1)at 3 and 4 days before the sacrifice,respectively.Each group has two rabbits died after the surgery,the rest of the experiment rabbits are fine in terms of mental state,noopenor slit wounds,inflammation,or infectionwasappeared.The number of effectiveimplants for each group is respectively 14(MAO = 14,SLA = 14),thus with the total of 28.The experimental animals are executed afterfour and eightweeks of surgery.Micro-CT Evaluation:The fixed specimens(n=7 for each of MAO and SLA group)were scannedusing a micro-CT system(MicroXCT-400,Xradia,USA).The scanning system was set at 80 kV and 10W with anisotropic resolution of 10μm.The 3D imagesreconstructed from microtomographic images wereutilized for quantitative evaluation.These data were analyzed by CTAn software torebuild the tissues surrounding the implants in the regionof interest(ROI),which was defined as a rectangleextending 1.5 mm in the opposite direction from theimplant surface.The ROI used for analysis was withinthe 2-mm thread part of the implants.Thefollowing parameters were used to monitormorphometric changes within ROI:bone volume pertotal volume(BV/TV),mean trabecular thickness(Tb.Th),mean trabecular number(Tb.N),meantrabecular separation(Tb.Sp),and structural model index(SMI),bone surface area/volume(BS/BV).Statistical AnalysisData were expressed as x±s and analyzed usingone-way ANOVA and Student’s t-test.Statistical analysis was conducted using the statistics package SPSS 20.0(Chicago,USA).Differences were considered to be statistically significant when the P value was less than 0.05.ResultsThe 2D micro-CT images of the transverse and coronal planes at four and eight weeks after implantation are shown,which clearly showed the effects of MAO-andSLA-modified implants on osseointegration andperi-implant trabecular microstructure.Similar resultswere observed for the ROI.The induced biological tissue(trabecular bone)showed point-like contact around the Tiimplant surface.Quantitative evaluation provided furtherdetailed information onfollowing parameters.The experimental group MAO(cortex-like)has more dense trabecular boneat fourweeks,while SLA group has relatively smaller,sparse,less osseointegration.As period of healing prolongs,the ratio of bone deposition increases;both groups also have augmented at BV/TV、Tb.Th、Tb.N,but decreased at BS/BV、Tb.Sp、SMI.Both groups indicate thick,dense,and noticeable trabecular bone formationat eight weeks.The parameters of BV/TV、SMIand BS/BV have statistical significance between two groupsat four weeks(P<0.05).However,BV/TV、Tb.N、Tb.Sp and BS/BV values become statistically significant at eight weeks(P<0.05),Other values have no statistical differenceat at four and eight weeks.This result showedthat the bone-implant contact of MAO was consistentwith that of SLA.Part 3:The comparison of MAO and SLA withmicron and nanocoating on histomorphometric analysis after implantion Materials and methodsMeasurement of Mineral appositionrate(MAR):the specimens were fixed in 10%neutralformalin-buffered solution,dehydrated in ethanol atgraded concentrations,and subsequently embedded inmethyl methacrylate resin withoutdecalcification.Perpendicular to the femoral shaft,eachspecimen was sectioned into 150-200 μm thick slicesusing a rotary diamond saw and ground to approximately 20 μm byEXAKT E400CS.The double-fluorescent labeledsections were directly observed under a fluorescencemicroscope(OLYMPUS BX51,Japan).DP2-BSW software was used to analyze the fluorescence diagrams;andthe distance between two fluorescent strips was measured via IPP6.0.Within each area,values were taken five times to obtain the average value.Mineral appositionrate(MAR)was the distance between the two fluorescentbands(μm)/dosing interval(days),which was calculatedas the evidence of bone regeneration.Average mineralization rate(Mineral Apposition Rates MAR um/d)= space of dual color fluorescence band/10.Quantitativehistological analysis:Histological sections werestained in 1%toluidine blue for bone histology analysis.The histological examination was performed under amicroscope(BX43,OLYMPUS,Japan)with normallight sourcewith magnificationof 10x,40x/50x,100x,and 200x.Images were captured using a digital cameraattached to the microscope.Bone-to-implant contact(BIC%)was defined as the length of mineralized bone in direct contact with all thethreads.Bone area(BA%)was defined as thepercentage of mineralized bone found in the area insideall the threads.Image-Pro Plus6.0(IPP6.0)microscopy image analysis software was usedto collect the data.Statistical AnalysisData were expressed asx ±s and analyzed using one-way ANOVA and Student’s t-test.Statistical analysis was conducted using the statistics package SPSS 20.0(Chicago,USA).Differences were considered to be statistically significant when the P value was less than 0.05.ResultsResults of Mineral apposition rate(MAR):The cortex-like coated implants displayed acontinuous bone apposition,in accordance with the SLAimplants.Wave-like fluorescent strips of alizarin red andcalcein were distributed on the threads in both groups,and new bone labeled in different fluorochromes was observed,which indicated a directbone-implant contact in the two groups.Result shows that the MAR is respectively 4.22±1.27μm/dand 5.11 ± 1.14μm/din MAO group at four and eight weeks,MAR is respectively 3.99 ± 0.85μm/dand 4.17 ± 1.17μm/d in SLA group at four and eight weeks,MAR in MAO group was higher than that in SLA group.Nevertheless,nosignificantdifference was observed between the MAO and SLAgroups(P>0.05).This result was consistent with the histomorphometric analysis,thereby suggesting that MAO coating possesses an excellent bone apposition capability asthe SLA coating.BIC%and BA:The intact interface between the trabecular and implant was observed.Structures of the new bonestained with toluidine blue appeared around the implantthread of MAO and SLA.The site with new bone formation was stained dark blue,whereas the old bones were inlight blue.Notable osteons,osteoblasts,and osteocyteswere observed in the new bone around the implants.MAO(cortex-like)group has more new bone trabeculaat four weeks compared to SLA group,while all implants showed excellent direct contactwith the surrounding bone tissue without the signs ofinflammatory response at eight weeks.Data were expressed asx±s and according to one-way ANOVA and Student’s t-test,the comparison of BIC%and BA%between the two groups is following at four weeks.BIC%:MAO(51.16%±6.65%)>SLA(48.61%±5.04%);BA%:MAO(38.04%±5.43%)>SLA(36.93%±7.76%);at eight weeks,BIC%:MAO(63.24%±7.51%)>SLA(55.99%±12.14%);BA%:MAO(49.43%±8.79%)>SLA(44.42%±11.77%).In cortical bone area,at four weeks,BIC%:MAO(56.57%±11.93%)>SLA(52.63%±15.1%);BA%:MAO(24.78%±6.44%)>SLA(23.12%±6.24%);at eight weeks,BIC%:MAO(61.00%±5.61%)>SLA(58.70%± 6.71%);BA%:MAO(35.86%± 9.40%》>SLA(34.24%± 4.49%).The difference between the two groups in each parameter was nostatistically significant in the two periods,which is consistent with MAR.Therefore,at an early stage,MAO could achieve similar results of bone induction as SLA.thereby suggesting that the osseointegration ability of MAO was consistent with thatof SLA.Conclusion1.From the perspective of bionics,the design of superior hydrophilic micron-nano structure is proposed,a novel cortex-like TiO2 coating was prepared on Ti surface throughmicro-arc oxidation(MAO)by using sodium tetraborate as electrolyte.Results showed that the cortex-like coating had microslots and nanopores and itwas superhydrophilic,the cortex-like MAO coating causes excellentosseointegration,holding a promise of an application to implant modification.2.Simulating the surface structure of moss leaves,A newly formed cortex-like surface,which consisted ofnanopores(80-200 nm diameter)and microslots(3-5μm diameter)was observed.The coatingsshowed a distinct hierarchical structure as well as arough and porous surface morphology.A tilted view ofthe cortex-like coating showed that the interconnectedtubes were formed on the coating,which indicated that MAO treatment in sodium tetraborate electrolyte can promote the growth of the porous structure onthe coating surface,thereby playing a critical role in theimplant-bone interactions.3.The formation mechanism of MAO micron-nano scale structure of TiO2coatingwas provided and as a theoretical guidance for the technology of MAO coating.The animal study aimedto investigate the effects of this cortex-like MAOcoating on osseointegration for first time.4.The roughness of MAO was similarto that of SLA.The MAO and SLA implants were implanted into the femoral condyles of New Zealandrabbits to evaluate their in-vivo performance through micro-CT,histological analysis,and fluorescentlabeling at the bone-implant interface for the first time.The results show thatthe cortex-like MAO coating withmicro/nanostructure and superhydrophilicity causes excellentosseointegration,holding a promise of an application to implant modification. |
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