Residual stresses in glass crowns generated by polymerization and water sorption of resin cements.

This study investigated residual stresses in glass crowns cemented with resin cements. Glass caps were cemented to cylindrical cores using a conventional resin composite cement, a self-adhesive resin cement, or a methyl methacrylate (MMA)-based cement in dual-cure or self-cure mode. The cemented caps were stored in 37°C water for 28 days, and stresses on the cap surface were repeatedly measured. The water sorption, water solubility, and elastic modulus of the cements were also measured. Polymerization of the cements initially generated compressive stresses on the surfaces. Dual-curing or a greater modulus yielded greater initial stress. The stresses gradually decreased over time and lingered on the surfaces at 28 days with all the cements. Greater sorption tended to lead to greater stress reduction; however, the MMA-based cement exhibited less stress reduction despite exhibiting the greatest sorption. The use of a resin composite cement or dual-curing is recommended to reinforce crown restorations.

Mutational analysis of the transmembrane α4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin.

Cry4Aa, produced by Bacillus thuringiensis subsp. israelensis, exhibits specific toxicity to larvae of medically important mosquito genera. Cry4Aa functions as a pore-forming toxin, and a helical hairpin (α4-loop-α5) of domain I is believed to be the transmembrane domain that forms toxin pores. Pore formation is considered to be a central mode of Cry4Aa action, but the relationship between pore formation and toxicity is poorly understood. In the present study, we constructed Cry4Aa mutants in which each polar amino acid residues within the transmembrane α4 helix was replaced with glutamic acid. Bioassays using Culex pipiens mosquito larvae and subsequent ion permeability measurements using symmetric KCl solution revealed an apparent correlation between toxicity and toxin pore conductance for most of the Cry4Aa mutants. In contrast, the Cry4Aa mutant H178E was a clear exception, almost losing its toxicity but still exhibiting a moderately high conductivity of about 60% of the wild-type. Furthermore, the conductance of the pore formed by the N190E mutant (about 50% of the wild-type) was close to that of H178E, but the toxicity was significantly higher than that of H178E. Ion selectivity measurements using asymmetric KCl solution revealed a significant decrease in cation selectivity of toxin pores formed by H178E compared to N190E. Our data suggest that the toxicity of Cry4Aa is primarily pore related. The formation of toxin pores that are highly ion-permeable and also highly cation-selective may enhance the influx of cations and water into the target cell, thereby facilitating the eventual death of mosquito larvae.

Random Mutational Analysis Targeting Residue K155 within the Transmembrane ß-Hairpin of the Mosquitocidal Mpp46Ab Toxin.

Mpp46Ab is a mosquito-larvicidal pore-forming toxin derived from Bacillus thuringiensis TK-E6. Pore formation is believed to be a central mode of Mpp46Ab action, and the cation selectivity of the channel pores, in particular, is closely related to its mosquito-larvicidal activity. In the present study, we constructed a mutant library in which residue K155 within the transmembrane ß-hairpin was randomly replaced with other amino acid residues. Upon mutagenesis and following primary screening using Culex pipiens mosquito larvae, we obtained 15 mutants in addition to the wild-type toxin. Bioassays using purified proteins revealed that two mutants, K155E and K155I, exhibited toxicity significantly higher than that of the wild-type toxin. Although increased cation selectivity was previously reported for K155E channel pores, we demonstrated in the present study that the cation selectivity of K155I channel pores was also significantly increased. Considering the characteristics of the amino acids, the charge of residue 155 may not directly affect the cation selectivity of Mpp46Ab channel pores. Replacement of K155 with glutamic acid or isoleucine may induce a similar conformational change in the region associated with the ion selectivity of the Mpp46Ab channel pores. Mutagenesis targeting the transmembrane ß-hairpin may be an effective strategy for enhancing the ion permeability of the channel pores and the resulting mosquito-larvicidal activity of Mpp46Ab.

Crystal structure of the in-cell Cry1Aa purified from Bacillus thuringiensis.

In-cell protein crystals which spontaneously crystallize in living cells, have recently been analyzed in investigations of their structures and biological functions. The crystals have been challenging to analyze structurally because of their small size. Therefore, the number of in-cell protein crystals in which the native structure has been determined is limited because most of the structures of in-cell crystals have been determined by recrystallization after dissolution. Some proteins have been reported to form intermolecular disulfide bonds in natural protein crystals that stabilize the crystals. Here, we focus on Cry1Aa, a cysteine-rich protein that crystallizes in Bacillus thuringiensis (Bt) and forms disulfide bonds. Previously, the full-length structure of 135 kDa Cry1Ac, which is the same size as Cry1Aa, was determined by recrystallization of dissolved protein from crystals purified from Bt cells. However, the formation of disulfide bonds has not been investigated because it was necessary to replace cysteine residues to prevent aggregation of the soluble protein. In this work, we succeeded in direct X-ray crystallographic analysis using crystals purified from Bt cells and characterized the cross-linked network of disulfide bonds within Cry1Aa crystals.

Influence of the Surface Chemical Composition Differences between Zirconia and Titanium with the Similar Surface Structure and Roughness on Bone Formation.

The osseointegration of zirconia (ZrO2) implants is still controversial. In this study, we aimed to make clear the influence of surface chemical composition, Ti or ZrO2, to osseointegration. First, a roughened Ti surface was prepared with a combination of large-grit sandblasting and acid treatment. Then, we applied molecular precursor solution containing Zr complex onto roughened Ti surface and can deposit thin ZrO2 film onto roughened Ti surface. We can change surface chemical composition from Ti to ZrO2 without changing the surface structure and roughness of roughened Ti. The tetragonal Zr was uniformly present on the ZrO2-coated Ti surface, and the surface of the ZrO2-coated Ti showed a higher apparent zeta potential than Ti. Ti and ZrO2-coated Ti rectangular plate implant was placed into the femur bone defect. After 2 and 4 weeks of implantation, histomorphometric observation revealed that the bone-to-implant contact ratio and the bone mass values for ZrO2-coated Ti implants inserted into the femur bone defects of the rats at 2 weeks were significantly higher than those for Ti implants (p < 0.05). It revealed that ZrO2 with a similar surface structure and roughness as that of roughened Ti promoted osteogenesis equivalent to or better than that of Ti in the early bone formation stage.

Color changes of dental zirconia immersed in food and beverage containing water-soluble/lipid-soluble pigments.

The present study examined color changes in the tooth-colored restorative materials, zirconia (3Y-TZP), resin composite, and porcelain. The colors (CIELab) of these materials were measured using a spectrophotometer. Specimens were immersed in black tea or curry for 1 and 7 days, after which colors were re-assessed. Color differences (∆E*ab) before and after immersion were calculated. Specimens after the 7-day immersion were ultrasonically cleaned, and colors were measured again to assess the color recovery rate. The surface free energy, roughness, and water sorption/solubility of each material were also evaluated. Specimens were observed under a scanning electron microscope. The ∆E*ab of 3Y-TZP was the smallest with both immersions. Resin composite had the smallest recovery rate. The surface free energy and roughness of 3Y-TZP were smaller than those of porcelain. 3Y-TZP and porcelain showed almost no sorption during the 7-day period. The present results revealed that 3Y-TZP exhibited the strongest resistance to discoloration.

Application of multi-directionally forged high-strength titanium to dental implants in beagle dogs.

Pure titanium is widely used as a material in dental implants. However, it possesses inferior mechanical strength. This study aimed to elucidate the efficacy of acid treated multi-directionally forged (MDF) pure titanium in vivo. We verified the temporal changes until osseointegration in beagle dogs. Using two types of experimental materials (conventional pure titanium or MDF pure titanium), new bone formation was assessed using morphological examinations, and the bone-to-implant contact (BIC) value was evaluated at each time point (14, 30, and 90 days after the operation). As such, new bone formation was observed around the acid-etched MDF group, in which the BIC value was highest, followed by that in the acid-etched pure titanium group. MDF pure titanium implants showed early promotion of new bone formation compared to conventional titanium implants. The new acid-treated MDF made of pure titanium could be applied to humans in the future to prove its practicality.

Fitness accuracy and retentive forces of milled titanium clasp.

Since cast titanium prostheses have many drawbacks, multi-directionally forged titanium grade 2 (MDF) was developed, and the application of the milling process was proposed for improving the titanium clasp. This in vitro study evaluated milled titanium clasps, including MDF titanium. Milling clasps were manufactured with commercially pure (CP) titanium grade 2 (CP 2), grade 4 (CP 4), Ti-6-Al-4V, and MDF. As a control, a CP 2 cast titanium clasp was fabricated in the conventional manner. No porosities and catastrophic failures were observed in the four milled titanium clasps. Fitness accuracy and retentive forces of milled CP 2 and CP 4 tended to be worse, and the milled MDF showed the higher retentive forces (12.45 N) than did cast and milled CP 2 clasps (9.32 N and 4.42 N). Milled titanium clasps can be recommended for longer-term clinical use as compared to cast clasps.

Adsorption behaviors of salivary pellicle proteins onto denture base metals using 27-MHz quartz crystal microbalance.

BACKGROUND: The adsorption of salivary pellicle proteins onto the material surface is key for denture plaque formation. OBJECTIVE: We aimed to investigate the adsorption of bovine serum albumin (BSA) and mucin (MCN) onto denture base metal materials using a 27-MHz quartz crystal microbalance (QCM) method. METHODS: A gold (Au), titanium (Ti), and cobalt chromium alloy (Co-Cr) sensors were employed for QCM measurements. Adsorbed amounts of BSA or MCN were calculated by observing the frequency decrease, and the apparent reaction rate, kobs, was obtained by the curve fitting of the frequency shift against the adsorption time. RESULTS: The adsorbed amounts of BSA on Ti were significantly lower than those on Au and Co-Cr. For MCN adsorption, Au showed significantly greater amounts of adsorption than Co-Cr. The kobs of Ti for BSA adsorption was significantly smaller than for the Co-Cr. The kobs of Ti, and Co-Cr for MCN adsorption were significantly smaller than for the Au. A clear correlation was not determined between adsorbed amounts of BSA or MCN onto each sensor and the surface topography or contact angles. CONCLUSIONS: The difference of denture base metals and the difference of salivary proteins influences the adsorption behavior of salivary proteins.

Cellular responses of histatin-derived peptides immobilized titanium surface using a tresyl chloride-activated method.

Effects of histatin-derived peptides immobilization by tresyl chloride-activation technique for MC3T3-E1 cellular responses on titanium (Ti) were evaluated. MC3T3-E1 were cultured on sandblasted and acid-etched Ti disks immobilized with histatin-derived peptides, including histatin-1, JH8194, and mixed histatin-1 with JH8194. Surface topography and cellular morphology were examined using a scanning electron microscope. Elemental composition and conformational peptides on Ti surface were examined using energy dispersive X-ray and fourier transform infrared spectroscopy, respectively. Cellular adhesion, proliferation, osteogenesis-related genes, and alkaline phosphatase activity were evaluated. The results showed that peptides were successfully immobilized on Ti surface. Cell attachments on histatin-1 and mixed peptides coated groups are higher than control. Histatin-1 achieved the significantly highest cellular proliferation. Histatin-derived peptides improved the osteogenesis related-gene expression and alkaline phosphatase activity (p<0.05). This study suggested that histatin-1 immobilization by tresyl chloride-activation technique enhanced cellular responses and might be able to promote cellular activities around the dental implants.

Application of multi-directional forged titanium for prosthetic crown fabrication by CAD/CAM.

Titanium are often used as dental materials, pure titanium present low strength and titanium alloy is reported poor biocompatibility, respectively. To overcome the problem, we fabricated high-strength multi-directional forged (MDF) titanium with improved mechanical properties without changing the chemical composition and evaluated its applicability in prosthetic crowns. Cutting tests: the average absolute value of the difference before and after cutting was calculated as the uncut amount. Surface evaluations: MDF titanium, pure titanium, and the Ti-6Al-4V alloy were the surface properties (the surface roughness, the contact angles, glossiness) of the samples were evaluated. The fitness test used digital data. These demonstrated that the good workability of high-strength MDF titanium. The surface-roughness and contact-angle properties of MDF titanium and pure titanium were similar. The fitness test showed no significant differences between MDF titanium and pure titanium crowns. These results suggest that MDF titanium is promising for fabricating prosthetic crowns in dental applications.

A Lipid Bilayer Formed on a Hydrogel Bead for Single Ion Channel Recordings.

Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, α-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.

Channel-pore cation selectivity is a major determinant of Bacillus thuringiensis Cry46Ab mosquitocidal activity.

Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type architecture, and it is expected to be used as a novel bioinsecticide. Cry46Ab acts as a functional pore-forming toxin, and characteristics of the resulting channel pores, including ion selectivity, have been analyzed. However, the relationship between channel-pore ion selectivity and insecticidal activity remains to be elucidated. To clarify the effects of charged amino acid residues on the ion permeability of channel-pores and the resulting insecticidal activity, in the present study, we constructed Cry46Ab mutants in which a charged amino acid residue within a putative transmembrane ß-hairpin region was replaced with an oppositely charged residue. Bioassays using Culex pipiens mosquito larvae revealed that the mosquitocidal activity was altered by the mutation. A K155E Cry46Ab mutant exhibited toxicity apparently higher than that of wild-type Cry46Ab, but the E159K and E163K mutants exhibited decreased toxicity. Ions selectivity measurements demonstrated that the channel pores formed by both wild-type and mutant Cry46Abs were cation selective, and their cation preference was also similar. However, the degree of cation selectivity was apparently higher in channel pores formed by the K155E mutant, and reduced selectivity was observed with the E159K and E163K mutants. Our data suggest that channel-pore cation selectivity is a major determinant of Cry46Ab mosquitocidal activity and that cation selectivity can be controlled via mutagenesis targeting the transmembrane ß-hairpin region. KEY POINTS: • Cry46Ab mutants were constructed by targeting the putative transmembrane ß-hairpin region. • Charged residues within the ß-hairpin control the flux of ions through channel pores. • Channel-pore cation selectivity is correlated with insecticidal activity.

Influence of mucin pre-adsorption for lipoteichoic acid adsorption on titanium surfaces.

We investigated the adsorption of lipoteichoic acid (LTA) on titanium surfaces by using the quartz crystal microbalance (QCM) method, and confirmed the influence of mucin (MUC) pre-adsorption on LTA adsorption. Two injection methods were evaluated. Namely, Method A: single-step injection of LTA solution to the Ti sensor, and Method B: MUC solution was injected to the Ti sensor followed by LTA injection on the MUC pre-adsorbed surface. QCM measurement revealed that the adsorbed amount of LTA by Method A was low and constant regardless of the increase in LTA concentration. In contrast, the adsorption amount of LTA in Method B increased according with increasing concentration of LTA and was significantly higher than that of Method A. The hydrophobic surface after MUC pre-adsorption was presumed to contribute to the enhancement of LTA adsorption. Our results revealed that MUC pre-adsorption to Ti is necessary for LTA adsorption in living tissue.

Bond strength and computational analysis for silane coupling treatments on the adhesion of resin block for CAD/CAM crowns.

The purposes of this study were to evaluate the shear bond strength and perform the computational analysis for silane coupling treatments on the adhesion between CAD/CAM composite resin and resin cement. As silane coupling agents, γ-methacryloyloxy propyltrimethoxysilane, 8-methacryloyloxyoctyl trimethoxysilane, and 3-methacryloyloxypropyl trichlorosilane were used. The shear bond strengths were influenced by the silane coupling agent used, its application method, and acid addition. There was no correlation between the contact angle and shear bond strengths. The steric energy difference between a silane coupling agent and its corresponding hydrolyzed trisilanol compound, ∆E, was calculated by the molecular mechanics method. There was a moderate or strong linear correlation between ∆E and shear bond strengths in treatment without acid addition and a weak correlation between them in treatment with acid addition. Computational analysis could suggest the different path of silane coupling treatments of CAD/CAM composite resin in the presence or absence of acid.

A nitrogen-containing bisphosphonate inhibits osteoblast attachment and impairs bone healing in bone-compatible scaffold.

Compromised osteoblast attachment on hydroxyapatite could be involved in the development of bone healing failure. We developed a bone-compatible scaffold that mimics bone structure with sub-micron hydroxyapatite (HA) surfaces, so that we could evaluate the effects of nitrogen-containing bisphosphonate (N-BP) on osteoblast behavior and bone healing. Human osteoblasts were seeded onto the bone-compatible scaffold with or without N-BP, and cell attachment and spreading behavior were evaluated 4 and 24 h after seeding. Then, mineralization was evaluated at 7 and 14 days. The osteoconductive activity of the scaffold was evaluated by implantation for 3 and 6 weeks into a rat cranial bone defect. The numbers of osteoblasts and their diameters were significantly less in N-BP-binding scaffolds than in untreated scaffolds at 4 and 24 h. Mineralization were also significantly less in the N-BP-binding scaffolds than in controls at 7 and 14 days. In vivo study revealed bone formation in N-BP-binding scaffolds was significantly less than in untreated scaffolds at 3 and 6 weeks. These results suggest that N-BP-binding to HA inhibited osteoblast attachment and spreading, thereby compromising bone healing process in the injured bone defect site.

Molecular precursor method for thin carbonate-containing apatite coating on dental implants.

The molecular precursor method is an easy and simple method for coating thin carbonate-containing apatite (CA) films onto titanium surfaces. A molecular precursor solution containing ethanol, calcium-EDTA complex, and phosphate salt was dropped onto a titanium surface and then heated at 600°C for 2 h. An adherent thin CA coating was achieved. Animal implantation experiments showed that CA-coated implants had significantly higher bone-to-implant values than non-coated implants (p<0.05). The molecular precursor method was also used to coat three-dimensional titanium webs (TWs). Thin CA films could be coated inside the center area, as well as the surface of the TW, with excellent bone formation inside the CA-coated TW. Furthermore, the molecular precursor method was used to coat partially stabilized zirconia with CA. Better bone response was observed for CA-coated zirconia. From this, it is concluded that the molecular precursor method is useful for producing thin CA coatings on implant materials.

In vitro evaluation of a removable partial denture framework using multi-directionally forged titanium.

PURPOSE: This study evaluated the availability of multi-directionally forged (MDF) titanium (Ti) as a component of removable partial dentures (RPDs). MDF-Ti remarkably improved the mechanical properties of RPDs due to its ultrafine-grained structure. MATERIALS AND METHODS: The wear resistance, plaque adhesion, and machinability of MDF-Ti were tested. As controls, commercially pure (CP) titanium was used for wear, plaque adhesion, and machinability tests. For wear resistance, the volume losses of the titanium teeth before and after wear tests were evaluated. Plaque adhesion was evaluated by the assay of Streptococcus mutans. In the machinability test, samples were cut and ground by a steel fissure bur and carborundum (SiC) point. An unpaired t-test was employed for the analysis of the significant differences between MDF-Ti and the control in the results for each test. RESULTS: Wear resistance and plaque adherence of MDF-Ti similar to those of CP-Ti (P>.05) were indicated. MDF-Ti exhibited significantly larger volume loss than CP-Ti in all conditions except 100/30,000 g/rpm in machinability tests (P<.05). CONCLUSION: Although the wear resistance and plaque adherence of MDF-Ti were comparable to those of controls, MDF-Ti showed better machinability than did CP-Ti. MDF-Ti could be used as a framework material for RPDs.

Surrounding Tissue Response to Surface-Treated Zirconia Implants.

Yttria-stabilized tetragonal zirconia polycrystals (Y-TZP), which are partially stabilized zirconia, have been used for fabricating dental implants. This study investigated the soft tissue attachment, the collagen fiber orientation to zirconia at different surface conditions, and the bone response using implantation experiments in animals. The zirconia implant surfaces were treated with ultraviolet irradiation (UV), a combination of large-grit sandblasting and hydrofluoric acid etching (blastedHF), and a combination of blastedHF and UV (blastedHF+UV). The surface treated with blastedHF and blastedHF+UV appeared rough and hydrophilic. The surface treated with blastedHF+UV appeared to be superhydrophilic. Subsequently, tapered cylindrical zirconia implants were placed in the alveolar sockets of the maxillary molars of rats. The bone-to-implant contact ratio of blastedHF and blastedHF+UV implants was significantly higher than that of the non-treated controls and UV-treated implants. The four different surface-treated zirconia implants demonstrated tight soft tissue attachments. Perpendicularly oriented collagen fibers towards zirconia implants were more prominent in blastedHF and blastedHF+UV implants compared to the controls and UV-treated implants. The area of the soft tissue attachment was the greatest with the perpendicularly oriented collagen fibers of blastedHF+UV-treated implants. In conclusion, blastedHF+UV treatment could be beneficial for ensuring greater soft-tissue attachment for zirconia implants.

Antimicrobial Activity of Protamine-Loaded Calcium Phosphates against Oral Bacteria.

Protamine is an antimicrobial peptide extracted from fish. In this study, we loaded protamine onto dicalcium phosphate anhydride (DCPA), a dental material. Protamine was loaded by stirring DCPA into a protamine solution. To explore the antimicrobial activity of the materials, we cultivated Streptococcus mutans on fabricated discs for 24 h. When S. mutans was cultivated on the discs under no sucrose conditions, the loaded protamine was not released, and the ratio of dead bacteria increased on the surface of P (125) DCPA (half of the saturated level of protamine (125 ppm protamine) was loaded). Aside from P (500) DCPA (saturated level of protamine was loaded), some protamine was released, and the number of planktonic bacteria in the supernatant decreased. Using medium containing 1% sucrose, the release of protamine was promoted from P (125) DCPA due to lowered pH. However, lowering of the pH decreased the antimicrobial activity of protamine. On the other hand, P (500) DCPA released protamine before the pH was lowered, and biofilm formation was inhibited. The loaded protamine expressed antimicrobial activity, both on the surface of the materials and in the surrounding environment. The interaction of loaded protamine with calcium phosphates could promote the application of protamine in the dental field.