Tatsuhide Hayashi

Methyl Methacrylate Activates the Gsta1 Promoter

Residual monomers in resin-based biomaterials cause cytotoxicity. We previously showed that methyl methacrylate (MMA) induced mRNA expression of the glutathione S-transferase alpha 1 gene (Gsta1) located downstream of the cis-acting anti-oxidant responsive element (ARE). Herein, we tested the hypothesis that MMA activated the Gsta1 promoter through the ARE. HepG2 cells were transfected with a luciferase reporter vector containing the ARE and the Gsta1 promoter (−990 to +46 bp) and cultured for 12 hrs with MMA (initial concentration, 10 mM). Analysis of the expressed luciferase activity indicated that MMA activated the promoter 2.6-fold. MMA (from 1 to 30 mM) dose-dependently increased the promoter activity, which reached a plateau between 6 and 12 hrs. In HepG2 cells transfected with a reporter vector containing 2 AREs and a TATA-like promoter, 10 mM MMA increased the reporter expression 2.8-fold. These results suggest that MMA increases Gsta1 transcription through ARE-mediated promoter activation.

The effect of zinc levels in a gold-based alloy on porcelain–metal bonding

OBJECTIVES The purpose of this study was to ascertain whether the amount of Zn in gold alloys contributes to porcelain-metal bonding. METHODS Experiments were carried out using a commercial Pd-free gold alloy with a nominal composition of 88.7 wt% Au, 9.49 wt% Pt, 1.5 wt% Zn, 0.1 wt% Mn, 0.2 wt% Rh, and 0.1 wt% Ir, which contains Zn and no other elements (In, Sn, Fe) known to affect porcelain-metal bond strength. To establish the effect of oxidation of the metal surface, porcelain was applied both to preoxidized and to non-preoxidized metal specimens. The bond strength was evaluated by means of the ISO 9693: 1999 crack initiation test. A conventional gold alloy was used as a control. The elemental distributions at the porcelain/alloy interfaces were analyzed in cross section by electron probe microanalysis. Additionally, after the bond strength test, cross-sections of the interfaces of the debonded specimens were microscopically analyzed to characterize the fracture mode. RESULTS The Pd-free gold alloy joints showed significantly higher bond strength values than joints made with conventional gold alloy. Preoxidation treatment significantly increases the bond strength, in the preoxidized joints Zn was highly localized at the interface and diffused into the porcelain up to about 10 μm from the interface, and the joint failed by cohesive fracture in the porcelain. In contrast, the non-preoxidized joint showed mainly adhesive fracture at the porcelain/alloy interface. SIGNIFICANCE The presence of Zn in gold alloys plays a part in establishing chemical bonding thus improving the bond strength between porcelain and alloy.

Antimicrobial efficacy of gutta-percha supplemented with cetylpyridinium chloride

To develop a root canal filling material with high antimicrobial activity, we prepared gutta-percha supplemented with the cationic surfactant cetylpyridinium chloride (CPC). Thermoplastic gutta-percha was supplemented with 0.05%, 0.2%, or 0.8% CPC. The gutta-percha containing CPC was tightly packed at the bottom of a 24-well plate. Its antimicrobial activity against eight representative endodontic pathogens-including gram-positive and gram-negative bacteria and fungi-was evaluated by adding 0.5 mL of liquid samples containing pathogens to the wells. After 24 h of cultivation under appropriate conditions, microbial growth was analyzed by counting colony-forming units (CFU). Gutta-percha alone (without CPC) partially inhibited microbial growth, probably through the antimicrobial effect of some of its components, such as zinc oxide. Addition of CPC dose-dependently increased the antimicrobial efficacy of gutta-percha. Addition of 0.05%, 0.2%, and 0.8% CPC reduced the viable microbial number to below the lower limit of detection (20 CFU/mL) for all tested pathogens except Pseudomonas aeruginosa, which was detected in 0.8% CPC-containing gutta-percha, although the viable number significantly decreased. Gutta-percha with CPC might be useful for preventing microbial infections during root canal therapy. (J Oral Sci 58, 277-282, 2016).

Proliferation of mouse fibroblast-like and osteoblast-like cells on pure titanium films manufactured by electron beam melting.

The physical characteristics and biological compatibility of surfaces produced by electron beam melting (EBM) are not well known. In particular, there are not many reports on biocompatibility qualities. In this study, pure Ti films were manufactured using EBM. While it is reported that moderately hydrophilic biomaterial surfaces display improved cell growth and biocompatibility, contact angle measurements on the EBM‐produced pure Ti films showed slight hydrophobicity. Nonetheless, we found the cell count of both fibroblast‐like cells (L929) and osteoblast‐like cells (MC3T3‐E1) increased on pure Ti films, especially the MC3T3‐E1, which increased more than that of the control. In addition, the morphology of L929 and MC3T3‐E1 was polygonal and spindle‐shaped and the cytoskeleton was well developed in the pure Ti surface groups. Upon staining with Alizarin red S, a slight calcium deposition was observed and this level gradually rose to a remarkable level. These results indicate that pure Ti films manufactured by EBM have good biocompatibility and could be widely applied as biomedical materials in the near future.

Improving biocompatibility of zirconia surface by incorporating Ca ions

Though zirconia has been used in dental implant fixtures, the biocompatibility of the zirconia surface is not optimal for the surrounding tissue, and many surface modifications have been attempted. We have developed a novel method for improving the biocompatibility of zirconia by incorporating Ca ions. Elemental analysis showed that calcium ions become thoroughly incorporated into the zirconia surface after firing with calcium acetate. Mechanical tests indicated that the Ca ions had little effect on the flexural strength and hardness. Moreover, incorporating Ca ions also dramatically improved the water wettability of specimens that had been soaked in a simulated body fluid. The surface of the Ca-modified zirconia demonstrated good initial cell attachment.

Immature muscular tissue differentiation into bone-like tissue by bone morphogenetic proteins in vitro, with ossification potential in vivo

The objective of this study was to induce bone formation from immature muscular tissue (IMT) in vitro, using bone morphogenetic proteins (BMPs) as a cytokine source and an expanded polytetrafluoroethylene (ePTFE) scaffold. In addition, cultured IMTs were implanted subcutaneously into Sprague-Dawley (SD) rats to determine their in vivo ossification potential. BMPs, extracted from bovine cortical bones, were applied to embryonic SD rat IMT cultures, before 2 weeks culture on ePTFE scaffolds. Osteoblast-like cells and osteoid tissues were partially identified by hematoxylin-eosin staining 2 weeks after culture. Collagen type I (Col-I), osteopontin (OP), and osteocalcin (OC) were detected in the osteoid tissues by immunohistochemical staining. OC gene expression remained low, but OP and Col-I were upregulated during the culture period. In vivo implanted IMTs showed slight radiopacity 1 week after implantation and strong radiopacity 2 and 3 weeks after implantation. One week after implantation, migration of numerous capillaries was observed and ossification was detected after 2 weeks by histological observation. These results suggest that IMTs are able to differentiate into bone-like tissue in vitro, with an ossification potential after implantation in vivo.

In vitro biocompatibility of dextrin: the addition of a low concentration of dextrin in the medium promotes the cell activity of L929 mouse fibroblasts

To develop a bone substitute with shape‐generating properties, we focused our attention on dextrin, which has a low viscosity. After considering methods of evaluation for research and development, we started by using cells that are widely used for safe biological evaluations in the field of dentistry and conducted in vitro evaluations. In this experiment, we variously added concentrations of 0.1, 1.0 and 10 mmol/l of dextrin to a culture medium in order to examine the effects on L929 mouse fibroblasts in vitro. As a result, the proliferative activity of the L929 cells was promoted during the culture period as the concentration of added dextrin became lower, and in particular, the 0.1 and 1 mmol/l addition group showed higher values than those of the control group. From the above results, it was revealed that the addition of a low concentration of dextrin in a medium promotes the cell proliferative activity.

Influence of Newly Developed Resin and MMA Resin on Mouse Fibroblasts Cellular Viability

The aim of this study is evaluate to the cellular viability of elution from the newly developed resin and Osteobond® in vitro. The basis of the newly developed resin are methacryloyloxyethyl methyl succinate and 1,6-Hexanediol dimethacrylate. The basis of Osteobond is methyl methacrylate. The concentrations of basis in each elution were determined by high-performance liquid chromatography (HPLC). Cellular viabilities of L-929 mouse fibroblasts were evaluated by direct cells counting, and then, each IC50 value was calculated. Moreover, patterns of cell death were analyzed using annexin V/propidium iodide staining with the phase-contrast microscope and flow cytometry. The concentration of Osteobond elution was 2.16 mM of MMA, and the newly developed resin elution was 1.02 mM of TA and 1.87 x 10-2 mM of HX. Until 72 hours of incubation, treatment with each elution impaired the viability of L-929 cells in a dose-dependent manner. IC50 value of Osteobond was 6.48 x 10-4 mM of MMA. However, IC50 of the newly developed resin was not calculated. Treatment with Osteobond elution showed more necrotic cells than with the newly developed resin elution. In conclusion, the results demonstrated much more excellent cellular viability of the newly developed resin than that of MMA resin. Thus, it is suggested that the newly developed resin will be more useful as an implantation material for dentistry and orthopaedics.

Rheological approach for determining yield stresses in flowable resin composites prior to setting

The purpose of this study is to develop a method for quantifying the fluidity of flowable resin composites using determinations of yield stress. Five commercially available composites (AliteFlo LV, Flow-it ALC, Venus flow, Tetric N-flow, Revolution Formula2) were investigated. Yield stress values were obtained by plotting shear stresses for a range of shear rates, followed by fitting of Casson fluid models to flow curve data and extrapolation to the stress axis. To confirm that yield stress reflected fluidity, apparent viscosity at the lower shear rate (0.2 s-1) was calculated from flow curves. Yield stresses ranged from 5.4 to 43.1 Pa, and were found to capture differences in the fluidity of composites that were not captured by viscosity measurement at the low shear rate. Yield stress is directly proportional to fluidity, and could serve as a simple and precise indicator for selecting flowable resin composites for use in various clinical applications.

rhBMP-2 Induces Immature Muscular Tissue to Differentiate into Bone-Like Tissue In Vitro

The aim of this study is to induce bone from immature muscular tissue in vitro using recombinant human BMP (rhBMP)-2 and expanded polytetrafluoroethylene (ePTFE) as a scaffold. Commercially available rhBMP-2 was used in this experiment. IMTs were harvested from the forelimbs of 20th Sprague-Dawley embryonic rats and placed into a homogenizer with 10ng/μl of rhBMP-2 and then homogenized. The homogenized IMT was placed on ePTFE and cultured for 2 weeks. The analyses of histological observation, electron probe micro analyzer (EPMA), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) were carried out following culture. The bone-like tissue, which was made up of osteoblast-like cells and osteoids, was partially observed by H-E staining. Moreover, strong mineral deposition was observed in the extracellular matrix by von Kossa staining. Ca, P and O were detected in the extracellular matrix by EPMA and were confirmed to be at almost the same position based on the findings of synchronized images. XRD patterns and FTIR spectra of specimen were found to have typical hydroxyapatite crystal peaks and spectra, respectively. These results suggest that rhBMP-2 induced IMT differentiation into bone-like tissue in vitro.