Evaluation of cytocompatibility and osteoconductivity of Zr-14Nb-5Ta-1Mo alloy with MC3T3-E1 cells.

The cytocompatibility and osteoconductivity of the Zr-14Nb-5Ta-1Mo alloy were investigated using a mouse osteoblastic cell line (MC3T3-E1) to promote the application of this newly developed alloy in dental/medical treatment. The initial cell-attached morphology was visualized by fluorescent staining, and cells cultured on the Zr alloy showed similar cell adhesion behavior to cells cultured on titanium (Ti). In our 5-day proliferation investigation, similar cell numbers were obtained with both Zr alloy and Ti. These results indicate that the cytocompatibility of Zr alloy is similar to that of Ti. In addition, the similar results in the evaluation of alkaline phosphatase (ALP) activity and staining of deposited calcium using alizarin red S with both Zr alloy and Ti indicated that the osteoconductivity of the Zr alloy is similar to that of Ti. Our results prove the good cytocompatibility and osteoconductivity of the Zr-14Nb-5Ta-1Mo alloy, enabling its promotion for use in dental/medical applications.

Time Transient of Calcium and Phosphate Ion Adsorption by Rutile Crystal Facets in Hanks' Solution Characterized by XPS.

For the elucidation of the mechanism of calcium phosphate formation on commercially pure titanium (CP Ti) in the human body, rutile TiO2 single crystal plates with (001), (110), and (111) facets, namely, TiO2(001), TiO2(110), and TiO2(111), and polycrystalline plates (TiO2(poly)) were immersed in a simulated body fluid, Hanks' solution (Hanks), for 100-105 s, and the adsorption of calcium and phosphate ions was precisely characterized employing X-ray photoelectron spectroscopy (XPS). Previously published CP Ti data were used for comparison. Prior to immersion in Hanks, oxygen content was more than twice as high as that of titanium due to the existence of hydroxyl groups and water on the oxides. After immersion in Hanks, the composition and chemical state of the TiO2 substrates remained unchanged. Among the electrolytes contained in Hanks, only calcium and phosphate ions were adsorbed by and incorporated onto TiO2 surfaces. Adsorption of calcium ions onto rutile did not exhibit any systematic increase of calcium with immersion time except TiO2(poly). Adsorption of phosphate ions was initially constant, followed by an increase with the logarithm of immersion time. The adsorption rate of phosphate ions decreased in the following order: TiO2(001), TiO2(poly), TiO2(111), CP Ti, and TiO2(110). The coordination number and band gap of each crystal facet of rutile is important for the adsorption and incorporation of phosphate ions. Regular calcium phosphate formation on CP Ti is possibly enabled by the surface oxide film, which consists chiefly of amorphous TiO2. However, calcium phosphate formation kinetics on CP Ti differed from those on the TiO2 crystalline phase. These findings may further the understanding of CP Ti hard tissue compatibility.

Enhancement of antibacterial property of titanium by two-step micro arc oxidation treatment.

A customized micro arc oxidation (MAO) treatment technique was developed to obtain desirable antibacterial properties on titanium surfaces. The two-step MAO treatment was applied to fabricate a specimen containing both Ag and Zn in its surface oxide layer. Surface analyses and metal-ion release tests were performed to evaluate the presence of Ag and Zn and the ion release behavior for simulating practical usage, respectively. Additionally, the antibacterial properties of the specimens were also evaluated using gram-negative facultative anaerobic bacteria. The MAO-treated specimens containing both Ag and Zn showed excellent antibacterial properties against Escherichia coli, and the properties were sustained even after 28 days of immersion in physiological saline to simulate the living environment.

Time-Transient Effects of Silver and Copper in the Porous Titanium Dioxide Layer on Antibacterial Properties.

Recently, silver (Ag) and copper (Cu) have been incorporated into a titanium (Ti) surface to realize their antibacterial property. This study investigated both the durability of the antibacterial effect and the surface change of the Ag- and Cu-incorporated porous titanium dioxide (TiO2) layer. Ag- and Cu-incorporated TiO2 layers were formed by micro-arc oxidation (MAO) treatment using the electrolyte with Ag and Cu ions. Ag- and Cu-incorporated specimens were incubated in saline during a period of 0-28 days. The changes in both the concentrations and chemical states of the Ag and Cu were characterized using X-ray photoelectron spectroscopy (XPS). The durability of the antibacterial effects against Escherichia coli (E. coli) were evaluated by the international organization for standardization (ISO) method. As a result, the Ag- and Cu-incorporated porous TiO2 layers were formed on a Ti surface by MAO. The chemical state of Ag changed from Ag2O to metallic Ag, whilst that of Cu did not change by incubation in saline for up to 28 days. Cu existed as a stable Cu2O compound in the TiO2 layer during the 28 days of incubation in saline. The concentrations of Ag and Cu were dramatically decreased by incubation for up to 7 days, and remained a slight amount until 28 days. The antibacterial effect of Ag-incorporated specimens diminished, and that of Cu was maintained even after incubation in saline. Our study suggests the importance of the time-transient effects of Ag and Cu on develop their antibacterial effects.

Investigation of antibacterial effect of copper introduced titanium surface by electrochemical treatment against facultative anaerobic bacteria.

This study investigated the efficacy of copper (Cu) as an antibacterial element incorporated on titanium (Ti) surface by electrochemical treatment. Cu was incorporated onto Ti surface by micro-arc oxidation (MAO). A small amount of Cu was incorporated into the oxide layer and was found to be in oxidized states. Cu-incorporated samples exhibited no-harmful effect on the proliferation of osteoblastlike cells. Moreover, the difference in antibacterial property between fresh and incubated samples was evaluated using gram-positive and gram-negative facultative anaerobic bacteria. The specific antibacterial property of Cu incorporated into the Ti surface were confirmed. The antibacterial property prolonged upon immersion in physiological saline for 28 days. In other words, MAO-treated Ti containing Cu in this study is expected to achieve long-term antibacterial property in practical usage.

Three-dimensional quantification of magnetic resonance imaging artifacts associated with shape factors.

Differences in the volumes of artifacts caused by variously shaped titanium objects on magnetic resonance imaging (MRI) were evaluated. Spherical-, square cubic-, and regular tetrahedron-shaped isotropic, and elongated spherical-, elongated cubic-, and elongated tetrahedron-shaped anisotropic objects, with identical volumes, were prepared. Samples were placed on a nickel-doped agarose gel phantom and covered with nickel-nitrate hexahydrate solution. Three-Tesla MR images were obtained using turbo spin echo and gradient echo sequences. Areas with ±30% of the signal intensity of the standard background value were considered artifacts. Sample volumes were deducted from these volumes to calculate the total artifact volumes. Isotropic samples had similar artifact volumes. For anisotropic samples, the artifact volume increased in proportion with the normalized projection area. MRI artifact size can be reduced by high anisotropic designs, and by positioning the long axis of the metal device as parallel as possible to the magnetic field axis.

Surface characterization of commercially available yttria-stabilized tetragonal zirconia polycrystalline in water and Hanks' solution using X-ray photoelectron spectroscopy.

To elucidate the mechanism of adhesion of soft and hard tissues to yttria-stabilized tetragonal zirconia polycrystalline (Y-TZP), Y-TZP and titanium disks were immersed in ultrapure water and in Hanks' solution, and the changes in the surface compositions and chemical states were characterized using X-ray photoelectron spectroscopy. After immersion in ultrapure water for 60 days, the concentration of hydroxyl groups on the Y-TZP surface increased. In addition, only phosphate ions were incorporated into the surface during immersion in Hanks' solution, while other ions did not react. On the other hand, the surface of Ti was also hydrated in ultrapure water; however, calcium phosphate formed on it during immersion in Hanks' solution. Therefore, the reactivity of Y-TZP with electrolytes was lower than that of Ti. We conclude that the formation of the phosphate on the Y-TZP surface in physiological conditions possibly enhances the adhesion of soft and hard tissues to Y-TZP.

Investigation of Realizing Both Antibacterial Property and Osteogenic Cell Compatibility on Titanium Surface by Simple Electrochemical Treatment.

Recently, the problem of infection on implanted devices caused by the formation of biofilms has been recognized. Surface treatment to prevent the initial stages of bacterial adhesion and subsequent bacterial growth is the only possible solution against such infection. In this study, simple electrochemical treatment was used for introducing silver, an antibiotic agent, on the titanium surface. A porous oxide layer containing small amounts of silver was formed on the metal of the substrate. This was done by microarc oxidation using the electrolyte with silver nitrate. The porous oxide layer was almost amorphous with a small fraction of anatase phase. The samples prepared using the electrolyte containing 0.04 mM or a higher concentration of silver nitrate showed an excellent antibacterial effect against both E. coli and S. aureus. However, the proliferation of osteoblast-like cells in the samples was not affected when a concentration of 0.5 mM or lower was used. Moreover, samples containing silver showed no harmful effects on the process of bone differentiation. Furthermore, the calcification process of the cells on the samples treated with and without silver were more promoted than that on untreated Ti. Thus, we found that it is possible to use this optimum concentration of silver to realize the conflicting biofunctions: its antibacterial property and osteogenic cell compatibility.

Adhesion and differentiation behaviors of mesenchymal stem cells on titanium with micrometer and nanometer-scale grid patterns produced by femtosecond laser irradiation.

To clarify the effects of grid topographies with different scales on cell morphology and functionalization, we investigated the adhesion and differentiation of human mesenchymal stem cells (hMSCs) to titanium surfaces with micron, nano, and micron/nano (hybrid) grid topographies created by femtosecond laser irradiation. The results showed that cellular adhesion and differentiation strongly depended on the scales of the grid topography. hMSCs cultured on micron and hybrid grid topographies showed regulation of cellular adhesion plaques following the surface topography and were vinculin-positive, whereas filamentous vinculin was evident at the filopodia of hMSCs cultured on nanogrids. The findings indicate that the micron grid topography was beneficial for cell colonization by anchoring the cells to the substrate surface, whereas the nanogrid topography was beneficial for cell locomotion. With the superposition effect of the micron and nanogrids, micro/nanohybrid grid topography strongly promoted cell adhesion. This differential adhesion induced differences cell differentiation. Nanogrids promoted differentiation of hMSCs, particularly osteogenic differentiation. These findings provide a basis for the design of novel biomaterial surfaces that can regulate specific cellular functions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2735-2743, 2018.

Micron/Submicron Hybrid Topography of Titanium Surfaces Influences Adhesion and Differentiation Behaviors of the Mesenchymal Stem Cells.

To clarify the effects of micron/submicron hybrid topography on cell morphology and functionalization, we investigated the adhesion and differentiation of human mesenchymal stem cells (hMSCs) to titanium (Ti) surfaces with three different topographies: micron, submicron, and hybrid grooves created using a femtosecond laser. hMSCs cultured on Ti specimens showed high alignment on micron and hybrid surfaces after 6 h of incubation, whereas cells attached to submicron and hybrid surfaces were elongated. An examination of vinculin-positive adhesion plaques indicated that micron grooves affected cellular alignment by modifying the initial cell polarization, whereas submicron grooves affected cellular extension. A superposition effect of topography was evidenced by the highly aligned and elongated morphology of hMSCs grown on the hybrid surface, which promoted osteogenic and chondrogenic differentiation. These findings provide a basis for the design of novel biomaterial surfaces that can control specific cellular functions.

Fatigue strength of Co-Cr-Mo alloy clasps prepared by selective laser melting.

We aimed to investigate the fatigue strength of Co-Cr-Mo clasps for removable partial dentures prepared by selective laser melting (SLM). The Co-Cr-Mo alloy specimens for tensile tests (dumbbell specimens) and fatigue tests (clasp specimens) were prepared by SLM with varying angles between the building and longitudinal directions (i.e., 0° (TL0, FL0), 45° (TL45, FL45), and 90° (TL90, FL90)). The clasp specimens were subjected to cyclic deformations of 0.25mm and 0.50mm for 10(6) cycles. The SLM specimens showed no obvious mechanical anisotropy in tensile tests and exhibited significantly higher yield strength and ultimate tensile strength than the cast specimens under all conditions. In contrast, a high degree of anisotropy in fatigue performance associated with the build orientation was found. For specimens under the 0.50mm deflection, FL90 exhibited significantly longer fatigue life (205,418 cycles) than the cast specimens (112,770 cycles). In contrast, the fatigue lives of FL0 (28,484 cycles) and FL45 (43,465 cycles) were significantly shorter. The surface roughnesses of FL0 and FL45 were considerably higher than those of the cast specimens, whereas there were no significant differences between FL90 and the cast specimens. Electron backscatter diffraction (EBSD) analysis indicated the grains of FL0 showed preferential close to <001> orientation of the γ phase along the normal direction to the fracture surface. In contrast, the FL45 and FL90 grains showed no significant preferential orientation. Fatigue strength may therefore be affected by a number of factors, including surface roughness and crystal orientation. The SLM process is a promising candidate for preparing tough removable partial denture frameworks, as long as the appropriate build direction is adopted.

Differences in the calcification of preosteoblast cultured on sputter-deposited titanium, zirconium, and gold.

In this study, osteogenic differentiation and calcification of preosteoblast (MC3T3-E1) cultured on sputter-deposited titanium (Ti), zirconium (Zr), and gold (Au) on cover glasses were evaluated to understand the differences in bone formation ability among these three metals; these metals show the same high corrosion resistance, but Ti and Zr are covered by surface passive oxide film while Au is not covered by the oxide film. Ti and Zr promoted cellular proliferation without osteogenic differentiation. Cells cultured on Ti and Zr expressed higher levels of Runx2, Col1α1, and Akp2 at an earlier stage, which indicated faster promotion of osteogenic differentiation, as compared to those cultured on Au. Moreover, after 21 days of culture, the Bglap1 and Ifitm5 expression peaks in cells cultured on Ti and Zr were higher than those in cells cultured on Au, which indicated faster promotion of calcification. Cells cultured on Ti showed an advantage in osteogenic differentiation at an early stage, while cells on Zr showed better calcification promotion with a long-term culture. The amount of extracellular calcified deposits was in good agreement with the gene expression results. On the other hand, the intracellular calcium content of cells on Au specimens was higher than that of cells on Ti and Zr specimens. The results indicate that preosteoblasts on Ti and Zr showed faster osteogenic differentiation and calcification than those on Au, whereas Au improved the intracellular calcium content. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 639-651, 2016.

Calcification of MC3T3-E1 cells on titanium and zirconium.

To confirm similarity of hard tissue compatibility between titanium and zirconium, calcification of MC3T3-E1 cells on titanium and zirconium was evaluated in this study. Mirror-polished titanium (Ti) and zirconium (Zr) disks and zirconium-sputter deposited titanium (Zr/Ti) were employed in this study. The surface of specimens were characterized using scanning electron microscopy and X-ray diffraction. Then, the cellular proliferation, differentiation and calcification of MC3T3-E1 cells on specimens were investigated. The surface of Zr/Ti was much smoother and cleaner than those of Ti and Zr. The proliferation of the cell was the same among three specimens, while the differentiation and calcification on Zr/Ti were faster than those on Ti and Zr. Therefore, Ti and Zr showed the identical hard tissue compatibility according to the evaluation with MC3T3-E1 cells. Sputter deposition may improve cytocompatibility.

Formation of white oxide layer on Zr-14Nb alloy using thermal treatment.

This study aimed to develop a novel abutment material with good esthetic and mechanical properties by producing a white oxide layer on a zirconium-14 mass% niobium (Zr-14Nb) alloy substrate using a thermal oxidation process. Oxidation temperatures ranged 700-1000°C and oxidation time ranged 30-180 min. The color of the oxide layer varied depending on temperature and time. A white oxide layer was obtained under appropriate conditions. The oxide layer thickness increased with increased temperature and time, as revealed by scanning electron microscopy with energy-dispersive X-ray spectroscopy. Moreover, X-ray diffraction and X-ray photoelectron spectroscopy revealed that the oxide layer was predominantly monoclinic ZrO2, tetragonal ZrO2, and Nb2O5. The oxide layer revealed good abrasion resistance and high adhesion to the substrate. This novel process for producing white materials with good mechanical properties will be useful for abutments and prostheses in dental implant treatment.