Fabrication And Evaluation Of Bifunctional Antibacterial/Bioactive Implant Coatings Based On Titania Nanotubes

[Background]Dental implant has become an important strategy to restore missing tooth, but associated infection still poses serious threat leading to possible complications. There are two main reasons for the high incidence of infection: biofilm colonization and compromised host defence on the implant surface. Hence, implant coating with dual functions of antibacterial ability and high bioactivity is supposed to prevent infection and elongate the service life of the dental implants.Considering that the natural tissues are composed of nanoscale blocks, nanostructure may give rise to better bioactivity from the biomimetic viewpoint. Titania nanotubes can be easily fabricated and readily applied on the dental implants with complex shapes thanks to the electrochemical fabrication process. In addition, the titania nanotubes with tube size ranging from tens to hundreds of nanometers can be fabricated, faciliating screening the optimal size for tissue integration and the titania nanotubes can mimic the size and arrangement of collagen fibrils in bone and the elastic modulus of bone. Therefore, it's expected that the titania nanotubes with optimized structure can show better bioactivity.The most effective way to prevent biofilm formation is to prohibit the initial bacterial adhesion, because the biofilm is hard to remove once occurs. To this end, antibacterial coatings especially antibacterial agent loading coatings are good choices. One good merit of the titania nanotubes is that they can serve as carriers for drugs such as antibacterial agents thereby acquiring antibacterial ability. Various kinds of antibiotics have been studied, but development of new antibiotic resistant strains after prolonged use reduces the effectiveness. We believe that silver is better for antibacterial coating application because it owns many advantages such as broad antibacterial spectrum, good bioactivity at suitable dose, good stability and low risk of resistance development. Because the effective dose of silver is every low, long-term antibacterial ability can be obtained by loading enough amount of silver and controlling its release.[Aims]To determine factors influencing the bioactivity of titania nanotubes and fabricate structures with satisfying bioactivity based on them; To uncover the mechanism through which the nanostructures modulate cell functions, rationaling implant surface design in the following study; To find the feasible way to load silver into the titania nanotubes for long-term antibacterial ability; To study the interaction of titania nanotubes with mesenchymal stem cells (MSCs) considering their important roles in osseointegration to provide more evidence for implant surface optimization; To observe their in vivo performance.[Methods]1) The titania nanotubes are fabricated by anodization in 0.5 wt% HF and field-emission scanning electron microscopy (FE-SEM) is used to observe their morphology. The effects of anodization potential and duration on the titania nanotube formation are studied; 2) The bioactivity of 5 and 20 V titania nanotubes is evaluated by in vitro primary rat osteoblast culture. The surface free energy of the titania nanotubes is monitored. DAPI staining of the nucleus followed by cell counting is used to evaluate cell adhesion. MTT method is used to determine cell activity. Fluorescence staining and SEM are used to show the cell morphology. Intracellular alkaline phosphatase (ALP) activity is measured by commercial kit and qRT-PCR is used to monitor the gene expressions; 3) The surface free energy of the titania nanotubes sterilized by different methods is monitored. The protein adsorption ability of the samples experiencing different sterilization processes is determined using a MicroBCA protein assay kit and their toxicity is evaluated by measuring the lactate dehydrogenase (LDH) activity in the culture medium. Cell adhesion, cell activity, intracellular ALP activity and total protein synthesis, and gene expressions are also measured as above-mentioned. Sirius red staining is used to determine collagen secretion and alizarin red staining is used to display extracellular matrix (ECM) mineralization; 4) Acid-etching treatment combined with anodization is used to fabricate biomimetic hierarchical micropits/nanotubes textures, whose bioactivty is evaluated using in vitro primary osteoblast culture as above; 5) Cell behaviors on the qusi-aligned nanowire arrays (QANWAs) of different surface chemistries (TiC/ core/shell structure or TiO₂) are observed to elucidate the mechanism by which nanotopographies modulate cell functions; 6) AgNO3 solution immersion followed by UV irridation is used to load silver into titania nanotubes. In vitro antibacterial assay and biocompatibility assays are performed; 7) The effects of titania nanotubes, acid-etched texture and hierarchical micropits/nanotubes textures on MSCs differentiation are observed. CCK-8 assay is used to measure the cell proliferation. Flow cytometry is used to determine the cell cycle propagation. Intracellular ALP activity and total protein synthesis, collagen secretion, ECM mineralization and gene expressions are also monitored as above-mentioned. Collagen secretion and ECM mineralization after blocking the signal pathways of ERK1/2, JNK and PI3K/Akt are also observed; 8) The differential roles of OS on differentiation of MSCs between on titanium and tissue culture plate (TCP) are compared; 9) In vivo performance of selected coatings is observed in rats.[Results]1) Titania nanotubes with tube size ranging from 20 to 100 nm are successfully fabricated; 2) Initial cell adhesion is not obviously affected by the titania nanotubes. With the exception of slightly higher intracellular ALP activity and more ECM deposition on the titania nanotubes, cell growth and cell differentiation represented by the expressions of osteogenesis-related genes are impaired on both 5 and 20 V anodized surfaces. The cells closely attach to the polished surface and are connected with each other nearly fully covering the substrate. The cells spread out with robust lamellopodia on the 5 V surfaces whereas the cells on the 20 V surfaces show a large amount of thin fillopodia; 3) UV and ethanol sterilization results in higher surface free energy and induces higher initial cell adhesion and proliferation than autoclaving, whereas UV irradiation leads to the best cell functions including adhesion, proliferation, as well as differentiation represented by ECM deposition and mineralization and osteogenesis-related gene expressions. Higher initial cell adhesion is observed on titania nanotubes after autoclaving. However, this difference disappears after sterilization by UV irradiation or ethanol immersion. The 20 V anodized surface shows higher cell proliferation than the other two surfaces after sterilization by ethanol, but no obvious cell proliferation difference can be found among the three topographies after sterilization by UV irradiation or autoclaving; 4) Biomimetic hierarchical micropits/nanotubes textures are successfully fabricated. The microtopography induces inconsistent osteoblast functions with initial cell adhesion and osteogenesis-related gene expressions dramatically enhanced while other cell behaviors depressed. In comparison, addition of nanotubes to the microtopography leads to enhancement of multiple osteoblast functions with nearly all the cell functions investigated in this study retained or promoted; 5) TiC/C and TiO₂ QANWAs are fabricated successfully. The TiC/C QANWAs show superhydrophobicity whereas the TiO₂ ones are more hydrophilic than the control. Both QANWAs impair cell adhesion leading to cell apoptosis. Other cell functions such as proliferation and differentiation are also obviously inhibited. The cell-repelling property is mainly ascribed to the structure of the QANWAs irrespective of the surface chemistry and wettability. The cell-repelling capability does not origin from reduced protein adsorption; 6) Silver nanoparticles are incorporated into titania nanotubes through AgNO3 immersion and UV irradiation. The silver nanoparticles adhere strongly to the inner walls of the nanotubes, and the size and amount of silver nanoparticles can be regulated by adjusting the AgNO3 concentration and immersion time. The silver loading nanotubes shows the ability to kill all the planktonic bacteria in the culture medium during the first week and the ability to prevent bacterial adhesion is maintained in the second week; 7) The effects of titania nanotubes, acid-etched texture and hierarchical micropits/nanotubes textures on MSC differentiation without OS are systemically compared and we find that all the surface topographies concerned are capable to induce MSC osteogenic commitment and their abilities are differential. The 20VNT and Micro/20VNT surfaces show the best ability to induce MSC osteogenic differentiation; 8) On TCP, OS slightly inhibits MSC proliferation and significantly promotes MSC differentiation represented by enhanced collagen secretion and ECM mineralization, while on titanium surfaces OS still inhibits MSCs proliferation which is a little more significant than on TCP but cell differentiation isn't enhanced like on TCP but decreased dramatically indicated by the lower intracellular ALP activity and total protein synthesis, collagen secretion and ECM mineralization. This inhibitory effect of OS on stem cell differentiation on Ti surfaces shows little relation to the surface microtopography; 9) The 20 V nanotubes enhance osseointegration in vivo.[Conclusions]1) Titania nanotubes of different tube sizes (20-100 nm) can be easily fabricated through anodization.2) Some cell functions such as osteogenesis-related gene expressions are impaired on the anodized surfaces, especially the 20 V anodized surface with larger tube diameter, which should be attributed to the compromised focal contact formation on the anodized surfaces. The difference in the phenotypes of the primary osteoblasts and osteoblastic cell lines may account for previous conflicting reports in osteoblast cytocompatibility of titania nanotubes. 3) The sterilization process significantly influences the surface characteristics of the nanostructures and subsequent osteoblast behavior, and UV irradiation leads to the best cell functions. The different sterilization methods influence the cytocompatibility evaluation of the surface nanotopographies relative to control, confirming that the different sterilization methods partly account for the conflicting biological behavior reported on titania nanotubes. From the viewpoint of removing surface contamination, UV sterilization may be the optimal sterilization method.4) The microtopography formed by acid etching induces inconsistent osteoblast functions, while addition of nanotubes to the microtopography leads to enhancement of multiple osteoblast functions. Our study reveals a synergistic role played by the micro and nanotopography on osteoblast functions and helps to design better implant surfaces.5) QANWAs impair cell adhesion leading to cell apoptosis possibly by impeding the focal adhesion assembly, indicating that one important mechanism through which nanostructures, including the titania nanotubes, regulate cell function is the modulation of cell adhesion, involving integrin clustering and focal adhesion assembly.6) Silver loading titania nanotubes are successfully fabricated by AgNO3 immersion and UV irradiation, which can kill all the planktonic bacteria in the suspension during the first week but the efficacy diminishes rapidly thereafter. However, the ability of the NT-Ag to prevent bacterial adhesion is maintained without obvious decline during the two weeks observation, indicating its potential long-term antibacterial effect. It's a pity that the silver loading nanotubes show cytotoxicity, and in the further study we will diminish it by controlling the sivler releasing rate. 7) The 20VNT and Micro/20VNT surfaces show the best ability to induce MSC osteogenic differentiation. The osteoinductivity of these topographies may be related to their modulation on focal adhesion at the micro or nanoscale and the following mechanotransduction. ERK1/2, JNK and PI3K/Akt are involved in the signal transdunction but their roles are differential.8) The effect of OS are various related to the biomaterials involved, possibly because that the soluble chemical cues and the biomaterial surface cues affect cell behaviors through some shared mechanisms. Our result also suggests that we should give a re-consideration of the concept of OS and its application in biomaterial research.9) The 20 V nanotubes enhance osseointegration in vivo, suggesting good in vivo performance of the titania nanotubes.