Migration of endothelial cells on the surface of anodized Ni-Ti stent strut.

Background: Stent is widely regarded as the main treatment for curing cardiovascular diseases such as stenosis. Previous research has revealed that the damage of endothelial cells (EC), i.e., the components of endothelium, during stent implantation, could lead to severe complications, such as restenosis. To prevent restenosis, enhancements have been made to surface biocompatibility to accelerate the stent endothelialization process. Anodization on the Ni-Ti is a simple and efficient surface modification method to improve the biocompatibility of the Ni-Ti stent surfaces by enhancing the surface hydrophilicity, leading to an increase in the EC activities. The EC activity is known to be affected by the blood flow. Flow change by stent structure may result in EC dysfunctions, thereby leading to restenosis. It is thus essential to investigate the EC activities resulting from the anodization on the Ni-Ti surface under flow conditions. Objective: To study the influence of the endothelialization process on the Ni-Ti stent surface through anodization. The EC attachment and morphology on the anodized stent strut were observed under both with and without the flow conditions. Method: A parallel plate flow chamber was designed to generate a constant wall shear stress (WSS) to study the flow effect on the EC behavior. The hydrophilicity of the Ni-Ti stent strut surface was enhanced by a TiO2 layer fabricated via anodization. The EC distribution on the surface of the anodized nitinol stent strut was observed after 24 h of static (without flow) and flow exposure (with flow) experiment. Results: Under the static condition, the EC density on the surface of the anodized Ni-Ti stent strut was higher compared with the control. Under the flow condition, the enhancement of the EC density on the surface of the stent strut with anodization was reduced. The EC demonstrates a long and thin spindle-shaped morphology under the flow condition. Conclusion: Unlike the static condition, the EC is demonstrating a long and thin morphology in response to the flow under the flow condition. By improving the surface hydrophilicity, the anodization could enhance the EC migration onto the strut surface, and subsequently, accelerate the Ni-Ti stent endothelialization process. The improvement of the surface hydrophilicity is lower under the flow conditions when compared with the static conditions.

A facile chemical process to form an ultrathin hydroxyapatite layer with a customizable silver-releasing function on a titanium implant.

Silver-containing hydroxyapatite (Ag/HAp) layer on a bioinert material provides both bioactive and antibacterial properties; however, the Ag release duration needs to be customized to a patient's age and metabolism for minimizing the toxic effects. Herein, we present a facile chemical process to produce an ultrathin Ag/HAp layer on a Ti implant with a customized Ag-releasing profile. The process involves the following steps: preparation of a slurry-type reagent by mixing calcium phosphate powder with an aqueous AgNO3 solution, burying a Ti substrate in the slurry, and heating the slurry in air. An HAp layer, approximately 50 nm thick, with Ag particle deposits was obtained using this process. The Ag-particle content can be varied by adjusting the concentration of AgNO3 solution used for slurry preparation, resulting in different Ag-release profiles in a physiological solution. For instance, Ag release was retained for up to 30 days when 100 mM AgNO3 was used, whereas the release lasted 15 days when 10 mM AgNO3 was used. The duration of the antibacterial activity varied accordingly, but Ag-release-derived cytotoxicity was not observed irrespective of the AgNO3 concentration. In addition, differentiation of osteoblast-like cells was facilitated owing to the formation of the HAp layer. Thus, the chemical process presented in this study allows the production-at a clinical site-of an Ag/HAp layer customized to the patient's needs.

Quantitative Analysis of Nitrogen by Atom Probe Tomography Using Stoichiometric γ'-Fe4N Consisting of 15N Isotope.

The nitrogen deficiency in steels measured by atom probe tomography (APT) is considered to arise from the obscurement of singly charged dimer nitrogen ions (N2+) by the iron-dominant peak (56Fe2+) at 28 Da. To verify this by quantifying the amount of N2+ ions, γ'-Fe4N consisting of the 15N isotope was prepared on iron substrates by plasma nitriding using a nitrogen isotopic gas (15N2). Although considerable amounts of 15N2+ were observed at 30 Da without overlap with any iron peak, the observed nitrogen concentrations of γ'-Fe4N were clearly lower than the stoichiometric composition (19­20 at%), using both pulsed voltage and pulsed laser atom probes. The origin of the missing nitrogen, excluding nitrogen obscured by other ion species, was predicted to be the occurrence of neutral nitrogen or nitrogen gas molecules in field evaporation. The generation rate of iron nitride ions (FeN2+) for 15N was significantly lower than that for 14N in γ'-Fe4N, which affected the amount of the missing nitrogen. The isotope effect suggests that the isotopic ratio cannot always be determined from only one ion species among the multiple species observed in the APT analysis. We discuss the mechanism of the isotope effect in FeN2+ formation by field evaporation.

Enzyme-assisted Extraction of Bioactive Phytochemicals from Japanese Peppermint (Mentha arvensis L. cv. 'Hokuto').

In this study, we provide a method for obtaining essential oil from Mentha arvensis L. in large quantities. Three types of polysaccharide-degrading enzymes were investigated, namely cellulase A "Amano" 3, cellulase T "Amano" 4, and hemicellulase "Amano" 90. The optimum extraction conditions were the combined use of 2 wt% cellulase T and 2 wt% hemicellulase 90, and 3 h of incubation. Enzymeassisted extraction increased the amount of the essential oil from 2.2 mL to 3.0 mL, compared with the amount extracted without an enzyme.

Conversion and Hydrothermal Decomposition of Major Components of Mint Essential Oil by Small-Scale Subcritical Water Treatment.

Thermal stabilities of four major components (l-menthol, l-menthone, piperitone, and l-menthyl acetate) of Japanese mint essential oil were evaluated via subcritical water treatment. To improve experimental throughput for measuring compound stabilities, a small-scale subcritical water treatment method using ampoule bottles was developed and employed. A mixture of the four major components was treated in subcritical water at 180-240 °C for 5-60 min, and then analyzed by gas chromatography. The results indicated that the order of thermal resistance, from strongest to weakest, was: l-menthyl acetate, l-menthol, piperitone, and l-menthone. In individual treatments of mint flavor components, subsequent conversions of l-menthyl acetate to l-menthol, l-menthol to l-menthone, l-menthone to piperitone, and piperitone to thymol were observed in individual treatments at 240 °C for 60 min. As the mass balance between piperitone and thymol was low, the hydrothermal decomposition of the components was considered to have occurred intensely during, or after the conversion. These results explained the degradation of mint essential oil components under subcritical water conditions and provided the basis for optimizing the extraction conditions of mint essential oils using subcritical water.

Predominant surface property of an anodized titanium that enhances the cell response.

The aim of this study is to evaluate the predominant material property that enhances the biocompatibility of an anodized titanium (Ti) implant. A Ti surface was anodized in an H3PO4 electrolyte with various voltages. Then, the cell responses involving attachment, proliferation, and differentiation were evaluated. Anodization using various voltages formed TiO2 layers with various surface morphologies. All the anodized surfaces showed enhanced cell responses; however, the performance differences depending on the surface morphologies were minimal. In addition, enhanced cell responses were not observed on the thermally oxidized Ti surface, although a TiO2 layer was formed; therefore, the beneficial effect was derived from the TiO2 layer fabricated via anodization. Based on these findings, the topmost surface structure of the TiO2 layer predominantly influenced the cell behaviors because this property governed the important surface functions, such as hydrophilicity.

Utilization of the Japanese Peppermint Herbal Water Byproduct of Steam Distillation as an Antimicrobial Agent.

The present study provides valuable data that the herbal water byproduct of Japanese peppermint, produced during the steam distillation extraction of an essential oil, can be utilized as an antibacterial agent. The major ingredient in the herbal water from Japanese peppermint 'Hokuto' was menthol, with a concentration close to its water solubility. The herbal water produced showed excellent antibacterial efficacy against typical gram-negative and gram-positive bacterial strains of Escherichia coli and Staphylococcus aureus, respectively, and the antibacterial efficacy was maintained even when the herbal water was diluted up to an appropriate concentration of 50%. The efficacy of the herbal water against E. coli was higher than that against S. aureus, which is likely because of the difference in the efficacy of menthol against these two different bacterial strains. The excellent antibacterial efficacy of the herbal water is mainly attributed to the function of menthol, while other trace ingredients also contributed to the antibacterial efficacy. The Japanese peppermint herbal water byproduct, generally treated as industrial waste and disposed, can be easily commercialized as an antibacterial agent if efforts are made to maintain a constant menthol concentration throughout the steam distillation essential oil extraction process.

Promotion of bone regeneration on titanium implants through a chemical treatment process using calcium phosphate slurry: Microscopic analysis, cellular response, and animal experiment.

The present study provides scientific evidence that a new chemical treatment process using calcium phosphate slurry promotes bone regeneration on titanium (Ti) implants. The material's surface modified by the treatment was analyzed using microscopic observation and the bone regeneration efficacy was evaluated both in vitro and in vivo. Formation of a thin hydroxyapatite layer with a thickness of about 50 nm and an increase of surface roughness were confirmed by microscopic observations. Histological evaluation of rat femora implanted with the specimens showed that the areas of the specimens directly attached to bone tissue were significantly more extensive than those implanted with control Ti at 2 and 8 weeks. Likewise, on the treated Ti, ALP activity, osteopontin, osteocalcin, and calcium contents of rat bone marrow stromal cells were significantly higher than on the control Ti. Furthermore, reverse transcription polymerase chain reaction showed greater expression of messenger ribonucleic acid encoding Cbfa1 and collagen type1 on the treated Ti at 2 weeks. Based on these results, we concluded that the new process was effective to enhance the osteoconductivity of Ti. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2716-2724, 2018.

Microstructures, mechanical properties and cytotoxicity of low cost beta Ti-Mn alloys for biomedical applications.

The microstructures, mechanical properties and biocompatibility of low cost ß-type Ti-(6-18)Mn alloys were investigated after solution treatment. Ti-9 Mn exhibits the best combination of tensile strength and elongation among the fabricated alloys, and its performance is comparable to or superior to those of Ti-6Al-4V ELI (Ti-64 ELI) in terms of every parameter evaluated. A hardness of 338 HV, a Young's modulus of 94 GPa, a 0.2% proof stress of 1023 MPa, an ultimate tensile strength of 1048 MPa and elongation of 19% were obtained for Ti-9 Mn. Furthermore, the cell viability and metallic ion release ratios are comparable to those of commercially pure titanium, making this alloy promising for biomedical applications. The Young's modulus is also lower than that of Ti-64 ELI (110 GPa), which can possibly reduce the stress shielding effect in implanted patients. STATEMENT OF SIGNIFICANCE: This study evaluates mechanical and biological performance of low cost solution treated ß-type Ti-(6, 9, 13 and 18 mass%)Mn alloys. It includes alloys containing a Mn content range higher than most previously published works (which is around or lower than 8 mass%). Furthermore, the effects of the ω phase and the ß phase stability of the alloys over some mechanical properties and microstructures are discussed. Ion release behavior under simulated body fluids and cell viability are also evaluated. For the case of the Ti-9 Mn, a mechanical and biological performance that is comparable to or superior than that of the widely used Ti-6Al-4V ELI and commercially pure Ti was observed.

In vitro and in vivo biocompatibility and corrosion behaviour of a bioabsorbable magnesium alloy coated with octacalcium phosphate and hydroxyapatite.

Octacalcium phosphate (OCP) and hydroxyapatite (HAp) coatings were formed on Mg-3 mass% Al-1 mass% Zn (AZ31) magnesium alloy by a single-step chemical solution deposition method. Chemically polished AZ31 (Cpol-AZ31) and HAp- and OCP-coated AZ31 (HAp- and OCP-AZ31) were immersed in a medium for 52 weeks or implanted in transgenic mice for 16 weeks to examine the long-term corrosion behaviour and in situ inflammation behaviour. In the medium, Mg-ion release was restricted for the initial several days and the corrosion rate thereafter was suppressed by approximately one-half with the HAp and OCP coatings. HAp-AZ31 showed a ∼20% lower corrosion rate than OCP-AZ31. Tissues of the transgenic mouse emit fluorescence in proportion to the degree of inflammation in situ. The luminescence intensity level was too low to be a problem regardless of the coatings. A thinner fibrous tissue layer was formed around OCP- and HAp-AZ31 than around Cpol-AZ31, indicating that the HAp and OCP coatings suppressed corrosion and foreign-body reaction in vivo. Visible pits were formed in filiform and round shapes in vitro and in vivo, respectively. Corrosion was observed underneath the coatings, and almost uniform corrosion took place in vitro, while local corrosion was predominant in vivo. These differences in corrosion morphology are attributed to the adhesion of tissues and the lower diffusivity on the surface in vivo than that in vitro. Dissolution behaviour of OCP crystals in vivo was different from that in vitro. It was demonstrated that the HAp and OCP coatings developed have great potential for a biocompatible and corrosion protection coating.

Response of osteoblast-like MC3T3-E1 cells on bioactive titanium fabricated by a chemical treatment process using a calcium-phosphate slurry.

We recently developed a chemical treatment process using a calcium-phosphate slurry for fabricating new layers consisting of hydroxyapatite and titanium dioxide (TiO2) on titanium (Ti) substrate. In this study, the response of osteoblast-like MC3T3-E1 cells on Ti substrate treated with a calcium-phosphate slurry was investigated to elucidate its behavior in a biological environment. The cellular adhesiveness and proliferation capacity did not differ significantly between the treated and untreated Ti substrates, suggesting that the slurry treatment did not cause cytotoxicity. The slurry treatment did not affect the increase in alkaline phosphatase activity after the induction of cell differentiation, whereas it was found to be significantly advantageous for the calcification behavior on the slurry-treated Ti substrate. In consequence, the hard-tissue compatibility of Ti is expected to be improved by the chemical treatment process using a calcium-phosphate slurry.

Hydrocarbon decomposition on a hydrophilic TiO2 surface by UV irradiation: spectral and quantitative analysis using in-situ XPS technique.

This paper presents valuable evidence in support of the removal of a contaminant hydrocarbon layer adsorbed on photocatalytic TiO2 film by the latter's photo-oxidization. UV light was radiated on an anodic TiO2 film in an atmosphere-controlled chamber, and the film was then transferred to an ultrahigh-vacuum chamber for X-ray photoelectron spectroscopic (XPS) analysis; during the transfer, care was taken to ensure that the film was not exposed to air. This "in situ" setting eliminates the influence of carbon and water adsorptions during the transfer, thus enabling the accurate analysis of the UV-induced surface reaction. The spectral and quantitative results clearly revealed that the adsorbed hydrocarbon was removed from the photocatalytic TiO2 film when the film was irradiated in an oxygen atmosphere. Such removal occurs only in the case of TiO2 films that exhibit superhydrophilicity. This indicates that the removal of hydrocarbon is dependent on the UV-induced hydrophilicity on the film. In situ XPS measurement also presented evidence for UV-induced hydroxide group adsorption, where the adsorption was observed on both the surfaces showing superhydrophilicity and devoid of it.

Investigation for analytical procedure for determination of trace metallic ions in simulated body fluids by inductively coupled plasma atomic emission spectrometry (ICP-AES).

Influences of matrix elements and high viscosity in three kind of simulated body fluids (SBFs) on determination of trace metallic elements (Co, Cr, Ni, Al and V) by inductively coupled plasma atomic emission spectrometry (ICP-AES) were investigated. In addition, decreases of these effects were attempted by H(2)SO(4) fume treatment. Calibration lines of the elements were constructed by the standard solutions made of elemental solutions and HCl or the SBFs. Gradients of calibration lines constructed by the each standard solution were different. Therefore, for accurate determination, calibration curve must be constructed by the elemental standard solution and the analytical solution. Limit of detection (LOD) of each element in the solutions was measured by a blank test. Although LODs of microg [Symbol: see text] L(-1) (ppb) order were nominal instrumental data, because of influences of the matrix elements and the high viscosity, the measured LODs of the elements in the SBFs were higher than those. However, the LODs were lowered by employing the H(2)SO(4)-fume treatment and approached to the nominal instrumental data. Therefore, H(2)SO(4)-fume treatment is extremely effective treatment in order to reduce the influences.

CaTiO(3) coating on titanium for biomaterial application--optimum thickness and tissue response.

The objectives of this study were to determine the optimum thickness of a CaTiO(3) film for biomaterial applications and to investigate the biocompatibility and bone formation of titanium with a CaTiO(3) film. First, CaTiO(3) films of 10, 20, 30, and 50 nm in thickness were deposited on titanium substrates using radiofrequency magnetron sputtering followed by annealing at 873 K in air for 7.2 ks. The optimum thickness of the CaTiO(3) film for bone formation was determined by comparison with its performance regarding calcium phosphate formation in Hanks' balanced saline solution (HBSS). Regarding calcium phosphate formation, the performance of the specimen with a 50-nm-thick CaTiO(3) film was superior to those of specimens with other thicknesses. A titanium prism with a CaTiO(3) film of 50-nm in thickness was surgically inserted in both soft and hard rat tissues. The biocompatibility of CaTiO(3)-deposited titanium and bone formation on it was investigated by histological observations. A slight inflammatory reaction was observed around the titanium with the 50-nm-thick CaTiO(3) film, while no severe response, such as degeneration and necrosis, was observed in either soft or hard rat tissue. New bone formation on the titanium plate with the CaTiO(3) film was more active than that without the film. The 50-nm-thick CaTiO(3) film has biocompatibility and can facilitate new bone formation in vivo, and, consequently, it is an excellent surface modification method for biomaterial applications.

Calcium phosphates formation on CaTiO3 coated titanium.

In this study, performance of calcium phosphate formation of CaTiO3 coating film on Ti in Hanks' balanced saline solution (HBSS) was investigated. CaTiO3 thin films with a thickness of 50 nm were deposited on Ti using radiofrequency (RF) magnetron sputtering. The temperature of Ti substrate was adjusted to room temperature (RT) and 873 K. Thereafter, the specimens deposited at RT were annealed at 873 K in air for 7.2 ks. The films were characterized by grazing incident angle X-ray diffractometry (GI-XRD) and X-ray photoelectron spectroscopy (XPS). After immersion in HBSS for 60 d, on CaTiO3 coated Ti, the formation of hydroxyapatite (HAP) was observed. Furthermore, HAP layer formed was thicker on the specimen on which CaTiO3 film was deposited at RT and annealed than that prepared at 873 K. The major difference between both specimens was the chemical properties of the outermost surface. In summary, CaTiO3 thin film deposited at RT and followed by annealing at 873 K for 7.2 ks in air enhances calcium phosphate formation ability on Ti.