Visualizing preparation using asymmetrical choline-like ionic liquids for scanning electron microscope observation of non-conductive biological samples.

In this study, we investigated conductivity preparation for scanning electron microscope (SEM) observation that used novel asymmetrical choline-type room temperature ionic liquids (RTIL). By immersion in only an RTIL solution, clear SEM images of several types of biological samples were successfully observed. In addition, we could visualize protozoans using RTILs without any dilution. These results suggested that the asymmetrical choline-type RTILs used in this study are suitable for visualizing of biological samples by SEM. Treatment without the need for dilution can obviate the need for adjusting the RTIL concentration and provide for a rapid and easy conductivity treatment for insulating samples.

Photophysical properties and biocompatibility of Photoluminescent Y2O3:Eu nanoparticles in polymethylmetacrylate matrix.

In this study, we produced europium-doped yttoria (Y2O3:Eu) nanoparticles and investigated their photoluminescent properties and biocompatibility. The Y2O3:Eu nanoparticles showed excellent photoluminescent properties and cytocompatibility. We also analyzed the photophysical properties of the nanoparticles in PMMA films. When the Y2O3:Eu nanoparticles were incorporated in the polymer film, they showed a strong red emission spectrum, similar to that seen with the particles alone. Energy dispersive X-ray spectroscopy (EDS) measurements indicated that the particles were distributed homogeneously in the PMMA film. Such materials could be applied not only to optoelectronic devices but also to biomedical applications such as bioimaging tools or luminescent medical/dental adhesive materials.

Physical and mechanical characteristics of short fiber-reinforced resin composite in comparison with bulk-fill composites.

PURPOSE: The objective of this study was to evaluate the physical and mechanical properties of a short fiber-reinforced resin composite: everX-Posterior and compare it with two bulk-fill composites, namely, Filtek Bulk-fill and Beautifil-Bulk, which are intended for large posterior restorations. METHODS: Investigated properties were flexural strength, flexural modulus, surface roughness, volumetric shrinkage and depth of cure. Scanning electron microscopy images of each specimen after the flexural test were used for cross-sectional comparison. Results were analyzed using ANOVA following Tukey post-hoc test. RESULTS: Flexural strength of everX-Posterior was comparable with two other resin composites, showing higher flexural modulus. EverX-Posterior showed the highest surface roughness after polishing and the lowest volumetric shrinkage (2.29%) among all composites used in this study. Data also showed that the everX-Posterior depth of cure was 4.24 mm, which was the highest among the three groups. CONCLUSION: Based on the results of this study, it was concluded that everX-Posterior as a short fiber-reinforced composite showed improvements and satisfactory performance in mechanical and physical properties, which make it a reliable base material candidate for large posterior restorations.

Fabrication of bioactive glass coating on pure titanium by sol-dip method: Dental applications.

This study aimed to assess the mechanical and biological properties of bioactive glass (BG) coating on titanium (Ti). Bioinert Ti substrates were coated by BG to induce bioactivity to the surface. The sol-gel derived BG 58S sol was successfully prepared and coated on the abraded and blasted Ti surface using the sol-dip method. The characterization and cell study for all substrates' surface was carried out. Adhesion test confirmed that a firmly adhered BG coating layer was formed on the abraded and blasted Ti. The measured bonding strength between the coating and the blasted Ti substrate was the highest among all samples, which was 41.03±2.31 MPa. In-vitro cell viability and alkaline phosphatase activity (ALP) tests results also showed that BG coating on the Ti substrate improved the biological properties of the surface. The BG sol-dip coating method could be used to fabricate Ti substrate with a bioactive surface.

Bonding Performance of a Hydrophilic Amide Monomer Containing Adhesive to Occlusal and Cervical Dentin.

This study aimed to evaluate the bonding performance of a new one-step self-etching adhesive system containing a novel hydrophilic amide monomer. Clearfil Universal Bond Quick (CUB) and Clearfil Megabond 2 (CMB) were used as the one-step and two-step adhesive systems, respectively. Flat dentin surfaces of human premolars were exposed using #600 SiC (silicon carbide) and bonded with the respective adhesives of each system. The teeth were sectioned to obtain beams (1 mm × 1 mm) after 24 h of water storage. The mean bond strength and standard deviations (MPa) on an occlusal surface were as follows: CUB: 45.9 ± 19.7 and CMB: 67.9 ± 25.3. The values for cervical ones were CUB: 56.0 ± 20.3 and CMB: 67.6 ± 16.0, respectively. In both conditions, the microtensile bond strength (µTBS) value was lower than that of CMB. As seen during the microscopic observation, no adhesive failure was observed after µTBS testing because CUB formed a firm and tight adhesive interface.

Multiwalled carbon nanotube coating on titanium.

Carbon nanotubes (CNTs) have excellent chemical durability, mechanical strength and electrical properties. Therefore, there is interest in CNTs for not only electrical and mechanical applications, but also biological and medical applications. We coated titanium, a common material for dental implants, with multiwalled carbon nanotubes (MWCNTs). First, titanium was aminated and covered with collagen. Then, the carboxylated MWCNTs were coated onto the collagen attached to the titanium plate. The collagen-coated titanium plate had a homogeneous MWCNT coating, which showed strong attachment to the titanium surface as a thin layer. The surface roughness was significantly increased with the MWCNT coating. MC3T3-E1 cells were cultured on the MWCNT-coated Ti plate, and showed good cell proliferation and strong cell adhesion. Therefore, the MWCNT coating for titanium could be useful for improvement of cell adhesion on titanium implants.

Adhesion of human osteoblast-like cells (Saos-2) to carbon nanotube sheets.

Carbon nanotubes (CNTs) exhibit excellent cell proliferation properties, which can serve as a scaffold for cell culturing. However, there are only a few reports on adhesion of osteoblast-like cells to a CNT sheet. In this study, we investigated adhesion of osteoblast-like cells to single-walled carbon nanotube (SWNT) and multi-walled carbon nanotube (MWNT) sheets and compared these adhesions with that on a cell culture polystyrene dish by using a cell adhesion test and a scanning electron microscope. The MWNT sheets exhibited faster adhesion of cells at an initial stage than SWNT sheets and cell culture polystyrene dish. The number of attached cells on the MWNT sheets seemed to be greater than on SWNT sheets and cell culture polystyrene. Moreover, the MWNT sheets exhibited both high speed and good capacity for cell adhesion. However, the surface of the MWNT sheets was such that it facilitated cell adherence but hindered the spreading of the attached cells. Interestingly, cell adhesion to CNT sheets was significantly influenced by pre-coating with serum. These results indicate that CNT sheets would play an important role in adsorption of serum proteins, which would consequently facilitate cell adhesion, and that the MWNT sheets have a high cell adhesiveness.

In vivo internal diffusion of several inorganic microparticles through oral administration.

We observed the internal diffusion behavior of inorganic micro/nano particles through oral administration. By oral exposure, the fed particles were absorbed through the digestive system then reached some organs after internal diffusion in the body. For example, TiO2 particles fed to mice were detected in the lung, liver, and spleen after 10 days of feeding. Whereas, the absorption efficiency was extremely low compared with intravenous injection. In a comparison of the simple amount of administration, oral exposure required 102 times or more amount by intravenous injection for detection by an X-ray scanning analytical microscope. During dental treatment, micro/nano particles from tooth or dental materials would generate in the oral cavity, and some of the particles had a possibility to be swallowed, absorbed through the digestive system, and then diffuse into the body. However, our results suggest that biocompatible microparticles that are naturally taken orally affect animals only rarely because of the low absorption efficiency.

Development of a multiwalled carbon nanotube coated collagen dish.

Carbon nanotubes (CNTs) are one of the most interesting nanomaterials because of their excellent characteristics. In this study, a transparent CNTs coating for cell culture dishes was developed and its properties for cell culture were estimated. Carboxylated multiwalled carbon nanotubes (MWCNTs) were dispersed in aqueous sodium cholate solution and applied on a collagen type I-coated cell culture dish (cover glass). The dish surface was homogeneously covered by MWCNTs without aggregation. The MWCNT-coated dish was slightly gray and had good transparency, so conventional optical microscopic observation of the cells on the MWCNT-coated dish was possible. Rat osteoblast-like cells cultured on the MWCNT-coated dish showed slightly lower viability and proliferation compared to the collagen-coated dish. The cell adhesion on the MWCNT-coated dish was much higher than that on the collagen-coated dish. Therefore, MWCNT-coating for dishes will be a useful new material for cell culture.

Assessment of the inhibitory effects of fissure sealants on the demineralization of primary teeth using an automatic pH-cycling system.

In this study, we examined the effects of fissure sealants on inhibition of demineralization of primary teeth using an automatic pHcycling system. Three fissure sealants were used: Teethmate F-1 2.0 (TM), BeautiSealant (BS), and Fuji III LC (IIILC). Using an automatic pH-cycling system, the specimens (n=12) were repeatedly demineralized and remineralized. Specimens were subjected to transverse microradiography (TMR), and changes in integrated mineral loss (IML) and lesion depth (Ld), indicated as ΔIML and ΔLd, respectively, were calculated. In addition, fluoride levels in the enamel were assessed using microparticle-induced gamma-ray emission/particle-induced X-ray emission (n=3). IIILC showed the lowest values for ΔIML and ΔLd, followed by BS and then TM. The highest amount of fluorine in the enamel was observed for IIILC, followed by TM and BS. All fissure sealants inhibited demineralization in primary teeth.

Estimation of Biocompatibility of Nano-Sized Ceramic Particles with Osteoblasts, Osteosarcomas and Hepatocytes by Static and Time-Lapse Observation.

We assessed the biocompatibility of nano-sized ceramic particles with several cells types. Though these particles have less than 100 nm in diameter, they act as submicron-sized particles in saline by aggregation that was estimated using laser diffraction particle size analysis (LDS). they act as submicro-sized particles in saline by aggregation based on laser diffraction particle size analysis (LDS). Several types of cells (osteoblasts, osteosarcoma and hepatocyte cells) were exposed to these particles and their cytocompatibility was estimated. Not only the cytotoxic assay but also their static and dynamic morphology under nanoparticles exposure were investigated. The intercellular uptake of particles was determined using a confocal fluorescence microscope. The particles used in this study did not inhibit cellular activity or growth even when their concentrations were high. Only copper oxide particles caused acute cytotoxicity depending on the particle size. The cytotoxicity assay, dynamic behavior of the nanoparticle-exposed cells and their examination under a confocal fluorescence microscope suggests that the irritative reaction was induced by contact between the cells and particles, whereas eluted copper ions are not dominant factor. These results indicate that nano-sized particles used in this study have excellent biocompatibility except copper oxide ones.

Effect of remaining dentin thickness on microtensile bond strength of current adhesive systems.

The purpose of this study was to evaluate the effect of remaining dentin thickness (RDT) on the bond strength of current adhesive systems. Third molars were randomly allocated among four groups depending on the adhesive system used: Clearfil SE Bond ONE (SE1), G-Bond PLUS (GB), BeautiBond (BB), and Clearfil Mega Bond (MB). Bonded specimens were stored in water at 37°C for 24 h. Teeth were then sectioned perpendicular to the adhesive interface to produce beams. After measuring RDT of each beam, microtensile bond strength test was carried out using a universal testing machine at a crosshead speed of 1 mm/min. All data were analyzed by linear regression analysis. Bond strengths of one-step self-etch materials used in this study increased with an increase in RDT. In contrast, that of two-step self-etch adhesive system was not affected by RDT.

Polyglycerol-coated nanodiamond as a macrophage-evading platform for selective drug delivery in cancer cells.

A successful targeted drug delivery device for cancer chemotherapy should ideally be able to avoid non-specific uptake by nonmalignant cells, particularly the scavenging monocyte-macrophage system as well as targeting efficacy to bring the drug preferentially into tumor cells. To this purpose, we developed a platform based on detonation nanodiamond (dND) with hyperbranched polyglycerol (PG) coating (dND-PG). dND-PG was first demonstrated to evade non-specific cell uptake, particularly by macrophages (U937). RGD targeting peptide was then conjugated to dND-PG through multistep organic transformations to yield dND-PG-RGD that still evaded macrophage uptake but was preferentially taken up by targeted A549 cancer cells (expressing RGD peptide receptors). dND-PG and dND-PG-RGD showed good aqueous solubility and cytocompatibitlity. Subsequently, the anticancer agent doxorubicin (DOX) was loaded through acid-labile hydrazone linkage to yield dND-PG-DOX and dND-PG-RGD-DOX. Their cellular uptake and cytotoxicity were compared against DOX in A549 cells and U937 macrophages. It was found that dND-PG-DOX uptake was substantially reduced, displaying little toxicity in either type of cells by virtue of PG coating, whereas dND-PG-RGD-DOX exerted selective toxicity to A549 cells over U937 macrophages that are otherwise highly sensitive to DOX. Finally, dND-PG was demonstrated to have little influence on U937 macrophage cell functions, except for a slight increase of TNF-α production in resting U937 macrophages. dND-PG is a promising drug carrier for realization of highly selective drug delivery in tumor cells through specific uptake mechanisms, with minimum uptake in and influence on macrophages.

Bone enhancing effect of titanium-binding proteins isolated from bovine bone and implanted into rat calvaria with titanium scaffold.

Based on our previous finding that a chromatography with titanium beads selectively binds phosphoproteins, including caseins, phosvitin and dentin phosphoproteins, we investigated whether bone phosphoproteins also bind to titanium. Bovine bone matrix proteins were extracted with 2 M urea/PBS after demineralization. The 2 M urea extract was directly applied to the titanium chromatography column as reported. The chromatogram showed an initial large peak at breakthrough position (non-binding fraction) and a smaller second peak eluted later (titanium-binding fraction). Both peaks were analyzed by SDS polyacrylamide gel electrophoresis. Stains-all staining which preferentially identifies phospho-proteins revealed that the first peak contained no positively stained band, while the second peak showed 4 or 5 distinctive bands indicative of bone phosphoproteins. To investigate the biological functions of the titanium-binding bone proteins (TiBP), we implanted them into calvaria of rats, combined with titanium web (TW), a highly porous titanium scaffold of thin titanium-fibers. Bone TiBP induced significantly enhanced bone formation, and new bone appeared connected directly to titanium fibers, accompanied by active blood vessel formations. Control TW alone did not induce bone formation within the titanium framework. These results demonstrate that the bone titanium-binding proteins include phosphoproteins which enhance bone formation when implanted into bone with titanium.

Interaction between titanium and phosphoproteins revealed by chromatography column packed with titanium beads.

The biochemical mechanism behind the strong binding between titanium and living bone has not been fully elucidated, in spite of worldwide clinical application of this phenomenon. We hypothesized that one of the core mechanisms may reside in the interaction between certain proteins in the host tissues and the implanted titanium. To verify the interaction between titanium and proteins, we chose the technique of chromatography in that titanium spherical beads (45 µm) were packed into a column to obtain a bed volume of 16×50 mm, which was eluted with phosphate buffered saline (PBS) and a straight gradient system made by using PBS and 25 mM NaOH. Fetal calf serum, albumin, lysozyme, casein, phosvitin and dentin phosphoprotein (phosphophoryn) were applied to the column. Most part of albumin and lysozyme eluted with the breakthrough peak, indicating practically no affinity to titanium. Fetal bovine serum also eluted mostly as the breakthrough peak, but distinct retained peak was observed. On the other hand, α-casein, phosvitin and phosphophoryn exhibited a distinct retained peak separated from the breakthrough peak. We proposed that phosphate groups (phosphoserines) in the major phosphoproteins, α-casein, phosvitin and phosphophoryn may be involved in the binding of these proteins with titanium.

Maturation of osteoblast-like SaoS2 induced by carbon nanotubes.

Osteogenic maturation of the osteoblast is crucial for bone formation. In this study, multi-walled carbon nanotubes (MWCNTs) and graphite (GP) were pressed as compacts. The greater ability of carbon nanotubes to adsorb proteins, compared with graphite, was shown. Human osteoblast-like SaoS2 cells were cultured and the cell response to the two kinds of compacts was compared in vitro. Meanwhile, we used cell culture on the culture plate as a control. Assays for osteonectin, osteopontin and osteocalcin gene expression, total protein (TP) amount, alkaline phosphatase activity (ALP) and DNA of cells cultured on the samples were done. During the conventional culture, significantly higher osteonectin, osteopontin and osteocalcin gene expression level, ALP/DNA and TP/DNA on carbon nanotubes were found. To confirm the hypothesis that the larger amount of specific proteins adsorbed on the carbon nanotubes was crucial for this, the compacts were pre-soaked in culture medium having additional recombinant human bone morphogenetic protein-2 (rhBMP-2) before cell culture. Compared with GP, osteonectin, osteopontin and osteocalcin gene expression level, ALP/DNA and TP/DNA of the cells tested increased more on the MWCNTs after the compacts were pre-soaked in the culture medium with rhBMP-2. The results indicated that the carbon nanotubes might induce osteogenic maturation of the osteoblast by adsorbing more specific proteins.

Material nanosizing effect on living organisms: non-specific, biointeractive, physical size effects.

Nanosizing effects of materials on biological organisms was investigated by biochemical cell functional tests, cell proliferation and animal implantation testing. The increase in specific surface area causes the enhancement of ionic dissolution and serious toxicity for soluble, stimulative materials. This effect originates solely from materials and enhances the same functions as those in a macroscopic size as a catalyst. There are other effects that become prominent, especially for non-soluble, biocompatible materials such as Ti. Particle size dependence showed the critical size for the transition of behaviour is at approximately 100 microm, 10 microm and 200 nm. This effect has its origin in the biological interaction process between both particles and cells/tissue. Expression of superoxide anions, cytokines tumour necrosis factor-alpha and interleukin-1 beta from neutrophils was increased with the decrease in particle size and especially pronounced below 10 microm, inducing phagocytosis to cells and inflammation of tissue, although inductively coupled plasma chemical analysis showed no dissolution from Ti particles. Below 200 nm, stimulus decreases, then particles invade into the internal body through the respiratory or digestive systems and diffuse inside the body. Although macroscopic hydroxyapatite, which exhibits excellent osteoconductivity, is not replaced with natural bone, nanoapatite composites induce both phagocytosis of composites by osteoclasts and new bone formation by osteoblasts when implanted in bone defects. The progress of this bioreaction results in the conversion of functions to bone substitution. Although macroscopic graphite is non-cell adhesive, carbon nanotubes (CNTs) are cell adhesive. The adsorption of proteins and nano-meshwork structure contribute to the excellent cell adhesion and growth on CNTs. Non-actuation of the immune system except for a few innate immunity processes gives the non-specific nature to the particle bioreaction and restricts reaction to the size-sensitive phagocytosis. Materials larger than cell size, approximately 10 microm, behave inertly, but those smaller become biointeractive and induce the intrinsic functions of living organisms. This bioreaction process causes the conversion of functions such as from biocompatibility to stimulus in Ti-abraded particles, from non-bone substitutional to bone substitutional in nanoapatite and from non-cell adhesive to cell adhesive CNTs. The insensitive nature permits nanoparticles that are less than 200 nm to slip through body defence systems and invade directly into the internal body.