Kyosuke Ueda

Effects of niobium ions released from calcium phosphate invert glasses containing Nb2O5 on osteoblast-like cell functions.

The effects of niobium ions released from 60CaO-30P(2)O(5)-(10-x)Na(2)O-xNb(2)O(5) (mol %, x = 0-10) glasses on MC3T3-E1 cell functions were evaluated by culture tests with two systems; cell culture on glass plates, or in culture media containing glass extracts. Alkaline phosphatase (ALP) activity in the cells cultured on the glass plates containing 3 and 5 mol % of Nb(2)O(5) was significantly higher than that on the Nb(2)O(5)-free glass, although proliferation was not enhanced on all glasses containing Nb(2)O(5). Cells cultured in the medium containing 3 × 10(-7) M niobium ions showed the highest ALP activity in comparison with other Nb-containing media or normal medium, regardless of the presence of osteogenic factors (ascorbic acid, β-glycerophosphate and dexamethasone) in the media. Calcium deposition by the cells cultured in the medium containing 3 × 10(-7) M niobium ions was twice as high as those cultured in medium containing no niobium ions. The effects of niobium ions were thought to depend on ion concentration, and to enhance differentiation and mineralization of osteogenic cells rather than their initial adhesion or proliferation.

Synthesis and characterization of Ag-containing calcium phosphates with various Ca/P ratios

Ag-containing calcium phosphate (CaP) powders were synthesized by a precipitation method using aqueous solutions of calcium nitrate, silver nitrate, and ammonium phosphate. The powders were sintered at temperatures ranging from 1173 to 1473 K. The charged atomic ratios of (Ca+Ag)/P and Ag/(Ca+Ag) in solution were varied from 1.33 to 1.67 and from 0 to 0.30, respectively. The Ag content in the as-precipitated CaP powders increased with the charged Ag/(Ca+Ag) atomic ratio in solution and was lower than the charged Ag/(Ca+Ag) value. The as-precipitated CaP powders consisted of hydroxyapatite (HA) as the main phase. Ag nanoparticles were observed on the as-precipitated HA particles under all conditions of Ag addition. After the sintering, HA, β-TCP (tricalcium phosphate), α-TCP, and β-CPP (calcium pyrophosphate) were mainly detected as CaPs on the basis of the Ca/P atomic ratio of the as-precipitated powders. The addition of Ag stabilized the β-TCP phase, and the distribution of Ag in β-TCP was homogeneous. A metallic Ag phase coexisted with HA. The solubility of Ag in HA was estimated to be 0.0019-0.0061 (Ag/(Ca+Ag)) atomic ratio, which was lower than that in β-TCP (higher than 0.0536) and higher than that of β-CPP (below the detection limit of analyses).

NKG2D⁺ IFN-γ⁺ CD8⁺ T cells are responsible for palladium allergy.

Nickel, cobalt, and chromium are well known to be causal agents of allergic contact dermatitis. Palladium (Pd) can also cause allergic disease and exposure results from wide use of this metal in dental restorations and jewelry. Metal allergy is categorized as a delayed-type hypersensitivity, and metal-responsive T cell clones have been isolated from allergic patients. However, compared to nickel, little is known about the pathology of allergic disease mediated by Pd, and pathogenic T cells are poorly understood. To identify the pathogenic T cells that are responsible for onset of Pd allergy, we enriched metal-responsive lymphocytes by sequential adoptive transfer of involved lymph node cells. Here we show that sequential adoptive transfer gradually increased the incidence and the intensity of Pd allergy, and CD8+ T cells are responsible for the disease as CD8+ T cell-depleted mice and β2-microglobulin-deficient mice did not develop Pd allergy. In addition, we found that draining lymph node cells skewed toward CD8+ T cells in response to Pd challenge in 8th adoptive transferred recipient mice. The CD8+ T cells expressed NKG2D, a costimulatory molecule involved in the production of IFN-γ. NKG2D ligand was also induced in Pd-injected tissues. Furthermore, both NKG2D ligand-transgenic mice, where NKG2D is downmodulated, and IFN-γ-deficient mice showed impaired Pd allergy. Taken together, these results indicate that IFN-γ-producing NKG2D+ CD8+ T cells are responsible for Pd allergy and suggest that NKG2D is a potential therapeutic target for treatment of metal allergy.

Evaluation of thin amorphous calcium phosphate coatings on titanium dental implants deposited using magnetron sputtering.

Objective:Calcium phosphate is used for dental material because of its biocompatibility and osteoconductivity. Amorphous calcium phosphate (ACP) coatings deposited by magnetron sputtering can control their thickness and absorbability. This study aimed to evaluate and characterize ACP coatings deposited via magnetron sputtering. It was hypothesized that ACP coatings would enhance bone formation and be absorbed rapidly in vivo. Methods:ACP coatings that are 0.5 &mgr;m in thickness were deposited via magnetron sputtering on dental implants. Uncoated implants served as controls. The effect of the ACP coatings in vivo was investigated in New Zealand white rabbit. To evaluate the effect of the ACP coatings on the bone response of the implants, the removal torque, implant stability quotient, and histomorphometric analysis were performed on the implants at 1, 2, and 4 weeks after implantation. Results:Results of the x-ray diffraction analyses confirmed the deposition of ACP coatings. Images from the scanning electron microscopy revealed that the coatings were dense, uniform, and 0.5 &mgr;m in thickness and that they were absorbed completely. Mechanical stability and bone formation in the case of the ACP-coated implants were higher than those of control. Conclusion:These results suggest that implants coated with thin ACP layers improve implant fixation and accelerate bone response.

Precipitates in Biomedical Co-Cr-Mo-C-Si-Mn Alloys

The phase and dissolution behavior of precipitates in biomedical ASTM F75 Co-Cr-Mo-C-Si-Mn alloys were investigated. Alloys of five different compositions, Co-28Cr-6Mo-0.25C-1Si, Co-28Cr-6Mo-0.25C-1Mn, Co-28Cr-6Mo-0.25C-1Si-1Mn, Co-28Cr-6Mo-0.15C-1Si, and Co-28Cr-6Mo-0.35C-1Si, were heat-treated from 1448 to 1548 K. The precipitates observed in the as-cast and heat-treated alloys were carbides (M23C6 type, h-phase, and p-phase) and an intermetallic compound (c-phase). The main precipitates observed after heat treatment at high temperatures such as 1548 K were p-phase and M23C6 type carbide. At these high temperatures, two types of starlike precipitates—dense and stripe-patterned—were observed. The starlike-dense precipitate was the p-phase, and the starlike precipitate with a stripe pattern was identified as the M23C6 type carbide and metallic fcc g-phase. In the alloys heat-treated at 1448 to 1498 K, blocky-dense M23C6 type carbide was primarily observed. c-phase was detected in the Co-28Cr-6Mo-0.15C-1Si alloy under as-cast condition and after heat treatment at 1448–1523 K for a short holding time. The addition of Si seemed to increase the holding time for complete precipitate dissolution because of the effects of Si on the promotion of p-phase formation at high temperatures and the increased carbon activity in the metallic matrix.

Enhancement of nickel elution by lipopolysaccharide-induced inflammation

BACKGROUND Implantations of metallic biomedical devices into bodies are increasing. The elution of Ni ions from these devices can lead to metal allergies. However, the molecular mechanisms of the elution have not been fully examined. Furthermore, it is not clear whether infection and inflammation affect the corrosion of metals. OBJECTIVE We examined whether the elution of Ni from metal wires and plates was enhanced by inflammation in vivo and in vitro. METHODS A Ni or SUS316L wire was implanted subcutaneously in the dorsum of mice. Lipopolysaccharide (LPS) was injected at the site immediately following the implantation. After 8, 24, and 72 h, the tissue around the wire was excised. RAW 264 cells were seeded on a Ni plate and incubated for 24h in medium containing LPS. The amount of Ni in the tissue or conditioned medium was determined fluorometrically. RESULTS The release of Ni ions from the wire was significantly increased from 8 to 72 h, and further increased by LPS. LPS also enhanced the release of Ni ions by the cells, but only when they were attached to the Ni plate. Chloroquine, bafilomycin A(1) and amiloride markedly inhibited the effects of LPS. CONCLUSION The activation of inflammatory cells on metals enhanced the elution of Ni probably via the release of protons at the interface of the cells and material.

The antihistamine olopatadine regulates T cell activation in palladium allergy.

Because of its corrosion resistance palladium (Pd) has been widely used in many consumer products ranging from fashion accessories to dental materials. Recently, however, an increase in Pd allergy cases has been reported. Metal allergy is categorized as a Type IV allergy, which is characterized as a delayed-type hypersensitivity reaction in which T cells are known to play an important role; however, the precise mechanism of their action remains unclear. Here we defined the relationship between histamine and the Pd allergic reaction specifically with respect to T cell responses. To verify the effects of histamine on T cells, we examined whether there is a change in IFN-γ production following stimulation of histamine or the antihistamine, olopatadine hydrochloride (OLP), in vitro. In addition, we assessed whether OLP administration affected the degree of footpad swelling or IFN-γ production during the Pd allergy response in mice. We found that histamine stimulation increased IFN-γ production in T cells, specifically enhancing IFN-γ production in CD8(+) T cells compared with CD4(+) T cells. Interestingly, OLP suppressed the production of IFN-γ in CD8(+) T cells, and this compound inhibited footpad swelling and IFN-γ production in mice with Pd allergy. These results suggest that histamine promotes the Type IV allergic reaction and thus, the histamine 1 receptor (H1R) might be useful therapeutic target for treatment of metal allergy.

In vitro evaluation of Ag-containing calcium phosphates: Effectiveness of Ag-incorporated β-tricalcium phosphate

Development of bioceramics with antibacterial activity and without cytotoxicity would be beneficial for preventing infection associated with implants. This study aimed to capitalize on the antibacterial properties of silver (Ag) incorporated in or coexisting in metallic form with calcium phosphates (CaPs). The in vitro dissolution behavior, antibacterial activity, and cytotoxicity of Ag-containing CaPs with different phase fractions of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) were evaluated. The antibacterial activity of Ag-containing CaPs depended on the main phase of CaP, the chemical state of Ag, and the amount of incorporated Ag. Superior antibacterial activity was obtained from sustained release of Ag ions through continuous dissolution of Ag-incorporated β-TCP compared to that obtained for HA coexisting with metallic Ag particles. Ag-containing CaPs did not exhibit any toxic effect on V79 fibroblasts. Thus, these results demonstrated the effectiveness of Ag-incorporated β-TCP in preventing infection, with respect to long-term applications.

In vitro performance of Ag-incorporated hydroxyapatite and its adhesive porous coatings deposited by electrostatic spraying

Bacterial infection of implanted materials is a significant complication that might require additional surgical operations for implant retrieval. As an antibacterial biomaterial, Ag-containing hydroxyapatite (HA) may be a solution to reduce the incidences of implant associated infections. In this study, pure, 0.2mol% and 0.3mol% Ag incorporated HA powders were synthesized via a precipitation method. Colloidal precursor dispersions prepared from these powders were used to deposit porous coatings onto titanium and stainless steel substrates via electrostatic spraying. The porous coating layers obtained with various deposition times and heat treatment conditions were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Scratch tests were conducted to assess the adhesion strength of the coating. Antibacterial activity of Ag-incorporated HA was tested towards Escherichia coli (E. coli) at various incubation times. Osteoblast adhesion on Ag-incorporated HA was evaluated to assess biocompatibility. Improvement in adhesion strength of the coating layer was observed after the heat treatment process due to mutual ionic diffusion at the interface. The Ag-incorporated HA killed all viable E. coli after 24h of incubation, whereas no antibacterial activity was detected with pure HA. In addition, in vitro cell culture tests demonstrated osteoblast adhesion similar to pure HA, which indicated good cytocompatibility. In summary, results of this study provided significant promise for the future study of Ag-incorporated HA for numerous medical applications.

Hydroxyapatite formation on calcium phosphate coated titanium

Calcium phosphate films were prepared by MOCVD using Ca(dpm)2 and (C6H5O)3PO precursors. The phases, composition and morphology of films changed depending on the molar ratio of Ca to P precursors (RCa/P), total pressure (Ptot) and substrate temperature (Tsub). α-tricalcimu phosphate (α-TCP, α-Ca3(PO4)2) films in a single phase were obtained at Tsub = 1073 K, 0.1 < RCa/P < 0.4 and Tsub = 973 K, RCa/P < 0.4. Hydroxyapatite (HAp, Ca10(PO4)6(OH)2) films in a single phase were obtained at Tsub = 1073 K, 0.8 < RCa/P < 1.0 and Tsub = 973 K, 0.5 < RCa/P < 0.6. Ca-P-O films had a dense and smooth surface. HAp formed within 1 day on the α-TCP film and wholly covered the specimens within 2 weeks in a Hanks’ solution. The surface of CVD-HAp film was covered by precipitated HAp within 6 hours.