Bioinspired Micro/nano Fabrication And Mechanical Coupling On Dental Implant-bone Interface

The past two decades is the rapid development period of oral implantologyundoubtedly. Dental implants has become the most ideal and important way to replace themissing teeth, which have been acceptable to more and more patients. However, failuresleading to dental implants’ removal still do occur. Several risk factors, such as loosetrabecular bone, excessive occlusal loading, tobacco use, diabetes, periodontitis,autoimmune disease, have been considered to contribute to implant failures. Among thesefactors, the peri-implantitis and peri-implant mucositis caused by bacteria microbialinfection is usually the most important reason of implant failures. Therefore, dentalimplant-bone interface is playing a key role in the osseointegration and survival of dentalimplants. Meanwhile, the surface modification and decoration of dental implants hasalways been the emphasis and difficulty of domestic and overseas scholars’ study. Thisaricle is precisely based on this background, the research was interested in design andbiomimetic fabrication of dental implant–bone interface with multiscaled surfacialarchitecture similar to hierarchical micro/nano structure in alveolar bone natively.Consequently, to acquire a Ti implant with a larger surface energy and roughness, a preferable hydrophilicity, a more adaptive mechanical property, a better bioactivity andbiocompatibity. This biomimetic fabrication on the surface of Ti implant can improve itsprimary osseointegration, and this bioinspired design idea can provide a novel thought andtheoretical basis to enhance the success rate of implanting.Study objectivesFirstly, we will analyze the size and structure characteristics of micro/nano unit inWistar rat’s alveolar bone through pathological section, SEM, AFM. Thus, we can acquirea standard and native reference model of dental implant-bone interface biomimeticfabrication.Secondly, a novel way that combined specific concentration binary acid treatmentwith constant potential anodizing will be used to fabricate a biomimetic micro/nanoarchitecture on Ti implant’s surface. Furthermore, we will utilize SEM, XRD and XPS toanalyze and compare three different Ti implant-bone interface （Smooth, Micro,Nano/micro）. Meanwhile, we observe and analyze three different Ti implant-boneinterface’s morphology and3D structure. In addition, three different Ti implant-boneinterface’s mechanical property and adhesion work is analyzed by surface contact forcecurve of AFM tip’s scanning. The dynamic change of hydrophilicity before and aftermicro/nano interface’s fabrication is measured by a statical goniometer.Finally, we observe three different Ti implant-bone interface’s bioactivity andbiocompatibility through SBF’s immersing experiment in vitro. Moreover, we utilize a cellculture and adhesion study to evaluate three different Ti implant-bone interface’sosteocompatibility. At each time point, a biological AFM and SEM were also used tocompare the osteoblastic cell morphology and response on three different Ti substratesurfaces. Then, at each time point, the fluorescence microphotograph of osteoblastic cellsadhesion on the three different Ti substrate specimens during2h,24h,48h of incubationis captured to analyze the discrepancy among them.Methods and Results:PartⅠ:Methods: The male SPF-level Wistar rats, age approximately9weeks were anaesthetized with an intraperitoneal injection of2%pentobarbital sodium. Then, we cut a roundalveolar bone block adjacent to the angle of mandible by a surgical micromotor andirrigated with0.9%sterile saline solution. The alveolar bone sample was then harvestedand fixed in10%buffered formalin for1week at4℃. Subsequently, the bone sampleswere embedded in polyester resin and mounted in a sawing microtome. Thus, we obtaineda30m-thick undecalcified section, which was stained with hematoxylin and eosin stain.Finally, we utilize SEM and AFM to analyze the size and feature of the micro and nanounit.Results:（1）Wistar rats’micro units （haversian canals） can be observed clearly in the pathologicalsection with hematoxylin and eosin stain （the diameter range from0.5m to2m）.Through SEM and AFM analysis, the microscale pit-like structure was aligned inorder.（2）Wistar rats’ nano units （collagen fibril molecules） can be seen imbedding on themicropits by AFM （the diameter range from60nm to80nm）. The tightly packedcollagen fibril molecules were stretched in the one direction similar toelectrospinning by SEM.PartⅡ:Methods: We utilize a binary acid （specific concentration HNO3&HF） treatment to builda microscale structure on Ti implant’s surface. Then, the vertical aligned TiO₂nanotubeswere fabricated on micro-treated Ti substrate surface by a three-electrodes’ anodization.Thus, a hierarchical aligned micro/nano structure can be fabricated on Ti implant’s surface.A morphology difference on nano/micro Ti implant’s surface can be observed by SEM andAFM as compared to smooth and micro counterpart. The diameter and characteristics ofTiO₂nanotubesµpits were analyzed by SEM. The roughness and averagepeak-to-valley height of three different Ti implant-bone interfaces were analyzed by AFMdomain scanning. We utilize a dynamic AFM tip contact scanning to analyze and couplethree different Ti implant-bone interfaces, which can acquire relative data （adhesion workand mechanical property）. Chemical characteristics and function groups on the three different Ti implant’s surfaces were also surveyed by XRD and XPS analysis. A staticalgoniometer was used to compare the discrepancy among three different Ti implant-boneinterfaces’s hydrophilicity.Results:（1）Our study has successfully fabricated a200nm-height TiO2nanotubes on micropits’sstructure. Their diameter （range from30nm to50nm） was similar to nano units （rangefrom60nm to80nm） in Wistar rats’ alveolar bone.（2）Three different Ti implant-bone interfaces’ roughness and average peak-to-valleyheight has a significant difference （P<0.05）（Smooth<Micro<Nano/micro）. Thehydrophilicity among three different Ti implant-bone interfaces also has a significantdifference （P<0.05）（Nano/micro>Micro> Smooth）.（3） XRD curves showed that mineralized crystal structure on three different Tiimplant-bone interfaces has a obvious discrepancy, which the peaks of TiO2and TiCin particular. XPS curves also demonstrated a significant difference in the peaks of Tiand O （Ti: Smooth>Micro>Nano/micro, O: Smooth<Micro<Nano/micro）.（4） The contact force curves of AFM tip scanning on three different Ti implant-boneinterfaces showed a obvious difference. The adhesion work among them has asignificant difference （P<0.05）（Nano/micro<Micro<Smooth）.PartⅢ:Methods: The bioactivity of three different Ti substrate surfaces was assessed through theimmersion of each sample into a polypropylene tube containing30ml of SBF at37C for7and14days. The difference of mineral deposits on three different Ti implant’s surfacewas analyzed by SEM, and we utilize XPS and Raman spectroscopy to affirm that thesemineral deposits were HA kind material. Then, we utilize a cell culture and adhesion studyto evaluate three different Ti implant’s osteocompatibility. At each time point （after2h,24h, and48h）, a Bio-AFM and SEM were used to observe osteoblasts’ adhesion on eachspecimen. Meanwhile, at each prescribed time point, the nonadherent cells were removedby rinsing with PBS. Cells were fixed and stained with DAPI. The cell numbers in five random fields were counted under a fluorescence microscope, and analyzed differenceamong three different Ti implant’s surfaces.Results:（1）After immersing in SBF for14days, a obvious difference in HA’s formation can beobserved between three different Ti substrate surfaces. On the smooth Ti substratesample, we can hardly observe mineral deposits. However, some small, granular,nonuniform mineral particles’ deposits were observed obviously on the micro-treatedTi substrate sample. Furthermore, we can observe a uniform tightly aligned minerallayer consisting of large, global particles on the nano/micro Ti substrate sample.（2）Three different Ti implant’s surface displayed a remarkable difference in SEM andAFM images after culturing and adhering osteoblasts for2h,24h,48h. On smooth Tisubstrate surface, the osteoblastic cells were highly elongated, presenting a rod-likeshape. However, the cells on micro-treated Ti samples were flattened and had morefilopodia, showing polygonal shape. Meanwhile, on nano/micro samples’ surfacerevealed that cell nucleus and several cell pseudopodia adhered together more closelyon it.（3） At each time point （after culturing for2h,24h,48h）, the cell number adhered onthree different interfaces have a significant difference （P<0.05）（Smooth<Micro<Nano/micro）.Conclusions:（1） Our study acquired the data about the size and structure characteristics of thenanoscaleµscale units in rat’s native alveolar bone. Thus, our results haveconfirmed that the multiscale hierarchical structures were existed and aligned orderlyin oral cavity.（2） Our study successfully obtained a novel way to biomimetic fabricate a nano/microarchitecture on Ti implant’s surface. Furthermore, this bioinspired structure is veryresemblant to hierarchical structure in native alveolar bone. This innovativetechnique has many superiority （such as simplicity, safety, excellent effectiveness）,which can apply to clinical research in near future. （3） This multiscale biomimetic fabrication on Ti implant’s surface can provide Tiimplant with a larger surface energy and roughness, a preferable hydrophilicity, amore adaptive mechanical property and adhesion work, a better bioactivity andbiocompatibity, a superior attachment and growth capacity of osteoblasts.（4） This biomimetic multiscale fabrication creates a new consideration to dentalimplant’s surface modification from bionics. In addition, this study will also providedental clinicians with a ingenious idea and theory to enhance the success rate ofdental implantation.