Investigation of a new implant surface modification using phosphorylated pullulan.

Various implant surface treatment methods have been developed to achieve good osseointegration in implant treatment. However, some cases remain impossible to treat with implants because osseointegration is not obtained after implantation, and the implants fail. Thus, this study focused on phosphorylated pullulan because of its adhesiveness to titanium (Ti) and bone, high biocompatibility, and early replacement with bone. In this study, the response of bone-related cells to phosphorylated pullulan was evaluated to develop a new surface treatment method. Saos-2 (human osteosarcoma-derived osteoblast-like cells), MC3T3-E1 (mouse calvaria-derived osteoblast-like cells), and RAW264.7 (mouse macrophage-like cells) were used. In evaluating cellular responses, phosphorylated pullulan was added to the culture medium, and cell proliferation and calcification induction tests were performed. The proliferation and calcification of cells on the surface of Ti disks coated with phosphorylated pullulan were also evaluated. In addition, bone morphogenetic protein-2 (BMP-2), an osteogenic factor, was used to evaluate the role of phosphorylated pullulan as a drug carrier in inducing calcification on Ti disks. Phosphorylated pullulan tended to promote the proliferation of osteoblast-like cells and the formation of calcification on Ti disks coated with phosphorylated pullulan. Ti disks coated with phosphorylated pullulan loaded with BMP-2 enhanced calcification. Phosphorylated pullulan inhibited osteoclast-like cell formation. These results are due to the properties of phosphorylated pullulan, such as adhesiveness to titanium and drug-loading function. Therefore, phosphorylated pullulan effectively promotes bone regeneration when coated on titanium implants and is useful for developing a new surface treatment method.

Aluminum-Doping Effects on the Electronic States of Graphene Nanoflake: Diffusion and Hydrogen Storage Mechanism.

Graphene nanoflakes are widely utilized as high-performance molecular devices due to their chemical stability and light weight. In the present study, the interaction of aluminum species with graphene nanoflake (denoted as GR-Al) has been investigated using the density functional theory (DFT) method to elucidate the doping effects of Al metal on the electronic states of GR. The mechanisms of the diffusion of Al on GR surface and the hydrogen storage of GR-Al were also investigated in detail. The neutral, mono-, di-, and trivalent Al ions (expressed as Al, Al+, Al2+, and Al3+, respectively) were examined as the Al species. The DFT calculations showed that the charge transfer interaction between Al and GR plays an important role in the binding of Al species to GR. The diffusion path of Al on GR surface was determined: the barrier heights of Al diffusion were calculated to be 2.1-2.8 kcal mol-1, which are lower than Li+ on GR (7.2 kcal/mol). The possibility of using GR-Al for hydrogen storage was also discussed on the basis of the theoretical results.

Bone Formation Ability and Cell Viability Enhancement of MC3T3-E1 Cells by Ferrostatin-1 a Ferroptosis Inhibitor of Cancer Cells.

Recently, ferroptosis has gained scientists' attention as an iron-related regulated necrosis. However, not many reports have investigated the effect of ferroptosis on bone. Therefore, with the present study, we assessed the effect of ferroptosis inhibition using ferrostatin-1 on the MC3T3-E1 pre-osteoblast cell. Cell images, cell viability, alkaline phosphatase activity test, alizarin red staining, and RUNX2 gene expression using real-time PCR were applied to investigate the effects of ferrostatin and erastin on MC3T3-E1 osteoblast cells. Erastin was used as a well-known ferroptosis inducer reagent. Erastin with different concentrations ranging from 0 to 50 µmol/L was used for inducing cell death. The 25 µmol/L erastin led to controllable partial cell death on osteoblast cells. Ferrostatin-1 with 0 to 40 µmol/L was used for cell doping and cell death inhibition effect. Ferrostatin-1 also displayed a recovery effect on the samples, which had already received the partially artificial cell death by erastin. Cell differentiation, alizarin red staining, and RUNX2 gene expression confirmed the promotion of the bone formation ability effect of ferrostatin-1 on osteoblast cells. The objective of this study was to assess ferrostatin-1's effect on the MC3T3-E1 osteoblast cell line based on its ferroptosis inhibitory property.

Ion Capture and Release Ability of Glass Ionomer Cement Containing Nanoporous Silica Particles with Different Pore and Particle Size.

This study prepared glass ionomer cement (GIC) containing nanoporous silica (NPS) (GIC-NPS) at 5 wt% concentrations using 3 types of NPS with different pore and particle sizes and evaluated the differences in their cationic ion capture/release abilities and mechanical properties. The cationic water-soluble dye was used as cationic ion. The test GIC-NPS complexes captured dyes by immersion in 1 wt% dye solutions. All the GIC-NPS complexes released dyes for 28 d, and the amount of dye released from the complexes increased with decreasing pore size; however, the particle size of NPS did not affect the amount of dye released. Additionally, GIC-NPS was able to recharge the dye, and the amount of released the dye by the complexes after recharge was almost identical to the amount released on the first charge. Although not significantly different, the compressive strength of GIC-NPS was slightly greater than that of GIC without NPS regardless of the type of NPS. These results suggest that the degree of capture and release of cationic molecules, such as drugs, can be controlled by optimizing the pore size of NPS without sacrificing its mechanical strength when its content is 5 wt%.

A novel multi-structural reinforced treatment on Ti implant utilizing a combination of alkali solution and bioactive glass sol.

OBJECTIVE: Alkali treatment and bioactive glass (BG) sol dip-coating are well-known individual methods for titanium (Ti) surface modification. In this study, a unique combination of alkali treatment and bioactive glass sol dip coating was applied to the Ti substrate, then the mechanical properties and cell responses were investigated. METHODS: Based on the methods introduced above, the Ti substrate was treated by 6 mL of an NaOH 5 M aqueous solution for 24 h at 60 ̊C; this was followed by adding 1.2 mL of a BG 58S sol to form a novel combined nanostructure network covered by a thin BG layer. For the assessment of the formed coating layer, the morphology, elemental analysis, phase structure, adhesion property and the cell response of the untreated and treated surfaces were investigated. RESULTS: The BG coating layer was reinforced by the nanostructure, fabricated through the alkali treatment. The results obtained by applying the combined modification method confirmed that the mechanical and biological properties of the fabricated surface demonstrated the highest performance compared to that of the unmodified and individually modified surfaces. SIGNIFICANCE: The achieved upgrades for this method could be gained from the demanded porous nanostructure and the apatite transformation ability of the alkali treatment. Therefore, the hybridized application of the alkali-BG treatment could be introduced as a promising surface modification strategy for hard-tissue replacement applications.

A novel coating layer on zirconia using modified zinc phosphatizing method.

Yttria doped ZrO2 was deposited using an acidic zinc phosphatizing solution and the hydrothermal treatment. The coating was analyzed using a field emission-scanning electron microscope (FE-SEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS). A piston on three balls (ISO 6872) was used for the measurement of biaxial flexural strength. MC3T3-E1 cells attachment was evaluated by SEM, and cell proliferation were assessed using MTS assay™. SEM images confirmed that the zinc phosphate coating layer was successfully prepared and fully covered the surface. The measured adhesive strength of the coating was 79.11 MPa. In vitro cell study indicated that the coated sample had better cell morphology and proliferation. XRD and EDS analysis revealed that the crystalline coating structure indexed as zinc phosphate (hopeite) and the substrate was assigned as zirconia. The flexural strength test showed that the strength of zirconia before and after hydrothermal treatment was not affected.

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.

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.

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.

Size- and Morphology-Controlled Preparation of Surface-Modified Water-Dispersible Fullerene Nanoparticles for Bioapplications.

In this study, we investigated water-dispersible surface modification for size- and shape-controlled fullerene nanoparticles (C60P) based on a condensation reaction with di-amino alkane. This modification provided for water dispersibility of C60P and the capability for secondary modification as well. The resultant C60P particles have several useful physical properties: water-dispersibility for ease of injection; fluorescence for detection and quantification; and a characteristic morphology to assist identification. These properties will widely extend the applications of these particles, especially into the biological fields of bioimaging and drug delivery.

Surface-Engineered Design of Efficient Luminescent Europium(III) Complex-Based Hydroxyapatite Nanocrystals for Rapid HeLa Cancer Cell Imaging.

We synthesized hydroxyapatite nanocrystals under the existence of tris(2,2,6,6-tetramethyl-3,5-heptanedionato)europium(III) (EuTH) complex to form inorganic/organic hybrid nanocrystal (EHA). Then, the folic acid derivative (folate N-hydroxysuccinimidyl ester (FA-NHS)) as the targeting ligand for the HeLa cancer cells was immobilized on the EHA by the mediation of both 3-aminopropyltriethoxysilane and methyltriethoxysilane molecules. Here, we investigated the photofunctions based on the interfacial interactions between the FA-NHS and EHA nanohybrids for preparing the novel bioimaging nanomaterials. As a result, the photofunctions could be changed by the FA-NHS molecular occupancy on the EHA. When the molecular occupancy ratio to the EHA surfaces is at around 3-5%, the intense luminescence from the f-f transition of the Eu3+ ions as well as the charge transfer between the EuTH-FA-NHS was observed to exhibit higher quantum efficiency. Moreover, effective dispersibility in phosphate-buffered saline was confirmed with immobilizing the positively charged FA-NHS. The cytotoxicity against the HeLa cells was also evaluated to verify whether the nanohybrids can be the candidate for cell imaging. The affinity and noncytotoxicity between the FA-NHS-immobilized EHA nanohybrids and cells were monitored for 3 days. Red luminescence from the cells could be observed, and the labels with following the cellular shapes were achieved by an additional culture time of 1 h after injecting the FA-NHS-immobilized EHA nanohybrids to the spheres, indicating the rapid bioimaging process. Therefore, this is the first successful report to describe the synthesis of inorganic-organic nanohybrid systems for controlling the EuTH-FA-NHS interactions. The photofunction of the interfacial interactions was successfully designed to provide "efficient luminescent ability" as well as "rapid targeting to the cancer cells" in one particle.

Calcium Charge and Release of Conventional Glass-Ionomer Cement Containing Nanoporous Silica.

The aim of this study was to evaluate calcium charge and release of conventional glass-ionomer cement (GIC) containing nanoporous silica (NPS). Experimental specimens were divided into two groups: the control (GIC containing no NPS) and GIC-NPS (GIC containing 10 wt % NPS). The specimens were immersed in calcium chloride solutions of 5 wt % calcium concentration for 24 h at 37 °C, whereupon the calcium ion release of the specimens was measured. The calcium ion release behavior of GIC-NPS after immersion in the calcium solution was significantly greater than that of the control. Scanning electron microscopy and electron-dispersive X-ray spectroscopy results indicated that calcium penetrated inside the GIC-NPS specimen, while the calcium was primarily localized on the surface of the control specimen. It was demonstrated that NPS markedly improved the calcium charge and release property of GIC.

Electric Charge Dependence of Controlled Dye-Release Behavior in Glass Ionomer Cement Containing Nano-Porous Silica Particles.

We investigated in controlled dye-release behavior of nanosized silica particles containing nanocavities (Nanoporous silica, NPS). To determine this, NPS were mixed with glass ionomer cement (GIC), which is a medical material used as a matrix. The dye-release behavior was observed using a UV-visible spectrometer. After cationic dye was charged into GIC pellet containing NPS, the pellet could gradually release cationic dye for up to two weeks. To understand the dependence of electric charge on the dye-release behavior, three types of dyes with different charge were also investigated. Dyes having a neutral or negative electric charge were quickly released from the pellet within a couple of days. These results suggest that the nanocavities present in NPS can selectively bind cationic dyes and allow for their gradual release. This result reveals the excellent sustained dye-release property of NPS.

Cytocompatibility of HeLa Cells to Nano-Sized Ceramics Particles.

In this study, we investigated the behaviors and cytocompatibility response of human cervical carcinoma (HeLa) cells expose to nano-sized particles. Cultivated cells exposed to titanium oxide and indium oxide nanoparticles remained highly viable. In the presence of copper oxide (CuO); however, the cells became seriously inflamed. To understand the mechanism by which CuO causes cell death, we evaluated cell death and apoptosis cytometry. CuO induced cells apoptosis more strongly than exposure to titania nanoparticles. Confocal fluorescence microscopy revealed that the nano-sized particles penetrate the cells.

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.

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.

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.

Scanning Electron Microscope Observation of Carbon Nanotubes with Room Temperature Ionic Liquids: Effect of Their Hydrophilicities.

In this study, we succeeded in acquiring scanning electron microscope (SEM) images of carbon nanotube (CNT) derivatives with different surface properties based on an electro-conductive pretreatment using a room temperature ionic liquid (IL). The quality of the obtained SEM images depended on their surface properties and the hydrophilicities of IL used. When the hydrophilicities of both the sample surface and the IL were close, the obtained SEM images had a high resolution. In contrast, SEM imges of samples pretreated with an IL, which had different hydrophilicities from the sample, was observed with low resolution and low quality. This result suggests that the relationship between both hydrophilicities is the dominant factor for this visualization method.

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.

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.