In Vitro Study of Zirconia Surface Modification for Dental Implants by Atomic Layer Deposition.

Zirconia is a promising material for dental implants; however, an appropriate surface modification procedure has not yet been identified. Atomic layer deposition (ALD) is a nanotechnology that deposits thin films of metal oxides or metals on materials. The aim of this study was to deposit thin films of titanium dioxide (TiO2), aluminum oxide (Al2O3), silicon dioxide (SiO2), and zinc oxide (ZnO) on zirconia disks (ZR-Ti, ZR-Al, ZR-Si, and ZR-Zn, respectively) using ALD and evaluate the cell proliferation abilities of mouse fibroblasts (L929) and mouse osteoblastic cells (MC3T3-E1) on each sample. Zirconia disks (ZR; diameter 10 mm) were fabricated using a computer-aided design/computer-aided manufacturing system. Following the ALD of TiO2, Al2O3, SiO2, or ZnO thin film, the thin-film thickness, elemental distribution, contact angle, adhesion strength, and elemental elution were determined. The L929 and MC3T3-E1 cell proliferation and morphologies on each sample were observed on days 1, 3, and 5 (L929) and days 1, 4, and 7 (MC3T3-E1). The ZR-Ti, ZR-Al, ZR-Si, and ZR-Zn thin-film thicknesses were 41.97, 42.36, 62.50, and 61.11 nm, respectively, and their average adhesion strengths were 163.5, 140.9, 157.3, and 161.6 mN, respectively. The contact angle on ZR-Si was significantly lower than that on all the other specimens. The eluted Zr, Ti, and Al amounts were below the detection limits, whereas the total Si and Zn elution amounts over two weeks were 0.019 and 0.695 ppm, respectively. For both L929 and MC3T3-E1, the cell numbers increased over time on ZR, ZR-Ti, ZR-Al, and ZR-Si. Particularly, cell proliferation in ZR-Ti exceeded that in the other samples. These results suggest that ALD application to zirconia, particularly for TiO2 deposition, could be a new surface modification procedure for zirconia dental implants.

Visualization of osteocalcin and bone morphogenetic protein 2 (BMP2) secretion from osteoblastic cells by bioluminescence imaging.

The secretions of osteocalcin and bone morphogenetic protein 2 (BMP2) from living osteoblastic cells were visualized for the first time using a method of video-rate bioluminescence imaging. The fusion proteins with Gaussia luciferase (GLase) for mouse osteocalcin and BMP2 (OC-GLase and BMP2-GLase, respectively) expressed in osteoblastic MC3T3-E1 cells were correctly processed and secreted. In the video images of exocytotic secretion, the luminescence spots of OC-GLase and BMP2-GLase disappeared rapidly and gradually, respectively, indicating different manners of these proteins in diffusion. Notably, a deletion mutant of BMP2 (Δ3BMP2-GLase) lacking three basic amino acid residues in the N-terminal region for binding to heparan sulfate showed rapidly disappearing luminescence spots. In our imaging conditions, the half-life of luminescence for the spots of Δ3BMP2-GLase (1.61 ± 0.20 s) was similar to that of OC-GLase (1.22 ± 0.14 s) but not to that of BMP2-GLase (4.31 ± 0.41 s). These results suggest that, in contrast to osteocalcin, the diffusion of BMP2 from cells occurred slowly after exocytosis. Thus, our bioluminescence imaging method is useful to study the diffusion properties of secreted proteins in exocytosis.

Bone Tissue Engineering in Rat Calvarial Defects Using Induced Bone-like Tissue by rhBMPs from Immature Muscular Tissues In Vitro.

This study aimed to induce bone-like tissue from immature muscular tissue (IMT) in vitro using commercially available recombinant human bone morphogenetic protein (rhBMP)-2, rhBMP-4, and rhBMP-7, and then implanting this tissue into a calvarial defect in rats to assess healing. IMTs were extracted from 20-day-old Sprague-Dawley (SD) fetal rats, placed on expanded polytetrafluoroethylene (ePTFE) with 10 ng/µL each of rhBMP-2, BMP-4, and BMP-7, and cultured for two weeks. The specimens were implanted into calvarial defects in 3-week-old SD rats for up to three weeks. Relatively strong radiopacity was observed on micro-CT two weeks after culture, and bone-like tissue, comprising osteoblastic cells and osteoids, was partially observed by H&E staining. Calcium, phosphorus, and oxygen were detected in the extracellular matrix using an electron probe micro analyzer, and X-ray diffraction patterns and Fourier transform infrared spectroscopy spectra of the specimen were found to have typical apatite crystal peaks and spectra, respectively. Furthermore, partial strong radiopacity and ossification were confirmed one week after implantation, and a dominant novel bone was observed after two weeks in the defect site. Thus, rhBMP-2, BMP-4, and BMP-7 differentiated IMT into bone-like tissue in vitro, and this induced bone-like tissue has ossification potential and promotes the healing of calvarial defects. Our results suggest that IMT is an effective tissue source for bone tissue engineering.

Accuracy of a titanium maxillary complete denture baseplate fabricated by using the electron beam melting technique: An in vitro study.

STATEMENT OF PROBLEM: Electron beam melting (EBM) is a promising additive manufacturing technique for fabricating denture baseplates; however, studies evaluating its accuracy are sparse. PURPOSE: The purpose of this in vitro study was to compare the fit accuracy of titanium maxillary complete denture baseplates fabricated by using the EBM technique with those fabricated by using the conventional casting technique and to evaluate the dimensional accuracy of the EBM baseplate by using a 3-dimensional inspection software program. MATERIAL AND METHODS: Definitive casts of an edentulous maxilla were prepared. After the casts were optically scanned, computer-aided designs for the EBM baseplate were created by using a software program for standard tessellation language file editing. The EBM baseplates were fabricated with an EBM machine by using a Grade II titanium powder as the raw material. The cast baseplates were fabricated with a lost-wax casting technique by using refractory casts duplicated from the definitive casts. After fitting the EBM and cast baseplates to their corresponding definitive cast, they were embedded in a Type IV stone. The embedded baseplates on the casts were sectioned at 3 regions: posterior palatal seal, molar, and premolar. The maximum gaps between the baseplate and definitive cast were measured in these 3 regions. The virtual casts obtained by scanning the EBM baseplate were superimposed on the computer-aided design to evaluate the dimensional accuracy. Distribution color maps were then generated, and the mean absolute deviations and root mean square deviations were calculated. One-way analysis of variance and t tests were used for statistical analysis (α=.05). RESULTS: No significant differences in the maximum gaps among the 3 regions were found in the cast or EBM baseplate groups (P>.05). The EBM baseplate group showed significantly lower values than the cast baseplate group in all regions (premolar: P=.008; molar: P=.003; posterior palatal seal: P=.004). The mean maximum gap for the 3 regions in the cast baseplates was 168.0 µm and that in the EBM baseplates was 60.7 µm. The distribution color map of the EBM baseplate showed a favorable dimensional accuracy. The mean absolute deviation value was 19.7 µm, and the root mean square deviation value was 25.1 µm. CONCLUSIONS: The EBM baseplates had a significantly higher fit accuracy than the cast baseplates. Thus, the fit accuracy of the EBM technique is suitable for fabricating metal baseplates.

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.

Enhanced bioactivity of polyvinylidene chloride films using argon ion bombardment for guided bone regeneration.

Polyvinylidene chloride (PVDC) is a long chain carbon synthetic polymer. The objective of this study was to improve the bioactivity of PVDC films through surface modification using argon (Ar) ion bombardment to create Ar-modified PVDC films (Ar-PVDC) to address the clinical problems of guided bone regeneration (GBR), which is technique-sensitive, and low bone regenerative ability. First, the effects of Ar ion bombardment, a low temperature plasma etching technique widely used in industry, on PVDC film wettability, surface chemistry, and morphology were confirmed. Next, fibroblast-like and osteoblast-like cell attachment and proliferation on Ar-PVDC were assessed. As a preclinical in vivo study, Ar-PVDC was used to cover a critical-sized bone defect on rat calvaria and osteoconductivity was evaluated by micro-computed tomography analysis and histological examinations. We found that the contact angle of PVDC film decreased by 50° because of the production of -OH groups on the PVDC film surface, though surface morphological was unchanged at 30 min after Ar ion bombardment. We demonstrated that cell attachment increased by about 40% and proliferation by more than 140% because of increased wettability, and 2.4 times greater bone regeneration was observed at week 3 with Ar-PVDC compared with untreated PVDC films. These results suggest that Ar ion bombardment modification of PVDC surfaces improves osteoconductivity, indicating its potential to increase bone deposition during GBR.

Automatic point detection on cephalograms using convolutional neural networks: A two-step method.

This project aimed to develop an artificial intelligence program tailored for cephalometric images. The program employs a convolutional neural network with 6 convolutional layers and 2 affine layers. It identifies 18 key points on the skull to compute various angles essential for diagnosis. Utilizing a custom-built desktop computer with a moderately priced graphics processing unit, cephalogram images were resized to 800×800 pixels. Training data comprised 833 images, augmented 100 times; an additional 179 images were used for testing. Due to the complexity of training with full-size images, training was divided into two steps. The first step reduced images to 128×128 pixels, recognizing all 18 points. In the second step, 100×100 pixels blocks were extracted from original images for individual point training. The program then measured six angles, achieving an average error of 3.1 pixels for the 18 points, with SNA and SNB angles showing an average difference of less than 1°.

Clinically applicable antianginal agents suppress osteoblastic transformation of myogenic cells and heterotopic ossifications in mice.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by progressive heterotopic ossification. FOP is caused by a gain-of-function mutation in ACVR1 encoding the bone morphogenetic protein type II receptor, ACVR1/ALK2. The mutant receptor causes upregulation of a transcriptional factor, Id1. No therapy is available to prevent the progressive heterotopic ossification in FOP. In an effort to search for clinically applicable drugs for FOP, we screened 1,040 FDA-approved drugs for suppression of the Id1 promoter activated by the mutant ACVR1/ALK2 in C2C12 cells. We found that that two antianginal agents, fendiline hydrochloride and perhexiline maleate, suppressed the Id1 promoter in a dose-dependent manner. The drugs also suppressed the expression of native Id1 mRNA and alkaline phosphatase in a dose-dependent manner. Perhexiline but not fendiline downregulated phosphorylation of Smad 1/5/8 driven by bone morphogenetic protein (BMP)-2. We implanted crude BMPs in muscles of ddY mice and fed them fendiline or perhexiline for 30 days. Mice taking perhexiline showed a 38.0 % reduction in the volume of heterotopic ossification compared to controls, whereas mice taking fendiline showed a slight reduction of heterotopic ossification. Fendiline, perhexiline, and their possible derivatives are potentially applicable to clinical practice to prevent devastating heterotopic ossification in FOP.

Effect of crystal orientation on flexural strength of pressable lithium disilicate glass-ceramics.

This study examined the crystal orientation of four kinds of pressable lithium disilicate glass-ceramics and evaluated the effect of crystal orientation on flexural strength. Bar-shaped (24 mm in length, 1.2 mm in thickness, 4.0 mm in width), disk-shaped (12 mm in diameter, 0.5 mm in thickness), and crown-shaped (maxillary first molar) specimens were prepared according to the manufacturer's instructions. Three-point and biaxial flexural strengths were measured for bar- and disk-shaped specimens. Microstructure analysis was performed using X-ray diffractometry and scanning electron microscopy. Three-point flexural strength was improved by parallel crystal orientation along the longitudinal direction of the bar-shaped specimen. There was no relationship between two-dimensional crystal orientation and biaxial flexural strength. The results of this study assumed that biaxial flexural strength was improved by the crystal orientation in the cross-sectional direction. Pressed restorations are expected to possess higher strength than milled restorations due to their crystal orientation.

Intracellular stress caused by composite resins: An in vitro study using a bioluminescent antioxidant-responsive element reporter assay.

Context: Elucidating the effects of leachates from composite resins (CRs) on cells by examining the transcription level of detoxification genes and the antioxidant-responsive element (ARE), would be helpful in clinical practice. Aims: The aim of the study is to investigate the cytotoxicity of commercially available CRs, we used a reporter assay system to evaluate intracellular stress based on ARE-mediated transcription. Setting and Design: The study design was an in vitro study. Materials and Methods: Seven kinds of CRs were each placed in four-well plates to which culture medium was added and then light-cured. The prepared samples were used either immediately (sample A) or after incubation at 37°C for 24 h (sample B) in the subsequent ARE-luciferase reporter assay, in which HepG2 cells stably expressing an ARE-regulated luciferase reporter gene (HepG2-AD13 cells) were cultured for 6 h in culture media with the CR eluate (samples A or B) or without (control) (n = 4). In the cell viability assay, cell viability in various solutions with the same incubation time was confirmed by MTT assay (n = 4). Statistical analysis was performed using the paired t-test and one-way analysis of variance. Results: All CR solutions showed an increase in ARE activation rate; a CR with spherical nanofillers showed the highest ARE activation rate of 108.5-fold in sample A. Cell viability was not significantly reduced for any of the CRs in sample A. However, the CR-containing bisphenol A-glycidyl methacrylate (Bis-GMA) caused a significant decrease in cell viability in sample B. Conclusions: The intracellular stress in the viable cells differed among the CRs, depending on the type of monomer used. In particular, Bis-GMA-containing hydroxyl groups showed high cytotoxicity.

Bonding Characteristics of Silane Coupling Agent and MMA-Containing Primer to Various Composite CAD/CAM Blocks.

This study evaluated the bonding characteristics of a silane coupling agent (SCA) and a methyl methacrylate (MMA)-containing primer (MCP) for 11 types of commercial composite blocks (CBs) for sandblasted and non-sandblasted surfaces. The shear bond strength (SBS) was measured according to ISO 29022: Notched-edge shear bond strength test. The SBS results demonstrated statistically significant differences between the CBs under all identical conditions. For the non-sandblasted groups, the SBSs of MCP-treated specimens were significantly higher than those of SCA-treated specimens for all but two CBs. Comparing the two treatments in sandblasted groups, the SBS was significantly higher for seven out of 11 MCP-treated RCB specimens, in contrast with three cases for the SCA-treated group. Two-way ANOVA for SBS showed the interaction effect between sandblasting and primer type for specific CBs, indicating that the sandblasting treatment improved SBS more effectively for SCA-treated specimens. Moreover, the effect of the SCA treatment was more material-dependent compared to that of the MCP treatment, which did not achieve a strong bond in all CBs but proved more effective than the SCA treatment, especially for non-sandblasted surfaces.

Identification method for dental alloy type using a cosine similarity program: A preliminary investigation.

Although dental evidence is frequently used for the identification of unidentified persons, information about the many types of alloys used in prosthetics is not utilized. If the type of alloy can be identified from a small amount of material, the scope of the search could be narrowed. In this experiment, a method was investigated for identifying the alloy type using 3 kinds of cutting points (a white point and 2 types of silicone points). Wavelength-dispersive X-ray spectroscopy (WDS) was used for elemental analysis. The elements were translated into multidimensional vectors, and the cosine similarity was calculated to compare vectors of the WDS results and vectors of the official data of alloys. According to the results, cosine similarity showed a concordance of more than 0.8. The developed program is expected to be useful as a method for identifying alloy types using only a small amount of grinding dust.

Effects of heated tobacco products and conventional cigarettes on dental implant wound healing: experimental research.

Smoking affects wound healing and is associated with dental implant failure. Heated tobacco products (HTPs) appear to be less harmful than conventional cigarettes (CCs); however, there is limited analytical data to support this claim. This study aimed to compare HTPs and CCs for their impact on wound healing using L929 mouse fibroblast cells and evaluate whether HTPs also lead to failure in implant therapy. Materials and methods: Cigarette smoke extract (CSE) was obtained from CCs (Marlboro, Philip Morris) and HTPs (Marlboro Heat Sticks Regular for IQOS, Philip Morris) and initiated a wound-healing assay with a cell-free area created in the centre of a titanium plate by sticking a 2-mm-width line tape. The L929 mouse fibroblast cells were exposed with 2.5 and 5% CSE from HTPs and CCs and then seeded in the titanium plate. A scratch wound-healing assay was initiated when all samples were at 80% confluence. The number of cells migrating to the wound site was counted after 12, 24, and 48 h. Results: Cell migration decreased after CSE exposure from both CCs and HTPs. At each time-point with 2.5% CSE, cell migration in the HTP group was less than that of the CC group. There were significant differences between the 2.5% CC and 2.5% HTP groups and the 5% CC and 5% HTP groups after 24 h. HTPs and CCs had similar effects in the wound-healing assay. Conclusion: Therefore, HTP use may be a risk factor for poor dental implant healing.

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.

Effect of degradation of filler elements on flexural strength for dental CAD/CAM resin composite materials in water.

Purpose This study aimed to evaluate the effects of degradation on the strength of computer-aided design/computer-aided manufacturing (CAD/CAM) resin composite blocks (RCBs) by subjecting them to accelerated degradation in water and conducting biaxial flexural strength tests.Methods Six commercial RCBs were tested. The RCBs were cut into disks, after which the disks were immersed in purified water. For the aging experiment, the samples were subjected to heat treatment at 37, 60, 70, and 80 °C, in a constant temperature oven and stored statically for 30 d. After the aging experiment, the elements released from the RCB fillers were measured by inductively coupled plasma atomic emission spectroscopy. In addition, the biaxial flexural strength of the RCB fillers was measured after accelerated degradation at 70 °C.Results Si (the main component of the filler) was detected in all the RCB solutions after the aging experiment; however, the type and amount of other elements differed considerably among the RCBs. The flexural strength of some of the RCBs decreased by approximately 20-40% after the accelerated degradation. For most materials, the Weibull coefficient decreased or remained unchanged after the test, whereas it increased in some materials.Conclusions The strength of all the RCBs decreased after the accelerated degradation tests; however, this behavior differed among the materials. In addition, the release of elements from the filler of some of the materials into the water correlated with the decrease in the strength of these materials. These findings indicate that the evaluation of the degradation behavior of RCBs in water is essential for their long-term usage.

Effect of self-cured acrylic resin added with an inorganic antibacterial agent on Streptococcus mutans.

In order to develop an self-cured acrylic resin having an antibacterial property, three types of commercially available inorganic antibacterial agents were added, at 1% each, to UNIFAST III to evaluate the antibacterial property. The antibacterial test evaluated the amount of Streptococcus mutans attached to UNIFAST III, the residual viable count of Streptococcus mutans cultured on UNIFAST III. And the color tone changes evaluated immediate and temporal color tone changes of UNIFAST III caused by the addition of antibacterial agents. As a result, compared to UNIFAST III without any added inorganic antibacterial agent, a significant decrease was observed in the attachment amount and the residual viable count of Streptococcus mutans. In addition, when adding NOVARON from among the added inorganic antibacterial agents, UNIFAST III exhibited little change in the temporal color tone. Thus, these results suggest that the addition of antibacterial agents to resins is effective first step toward developing self-cured acrylic resins having an antibacterial property.

Development of image analysis using Python: Relationship between matrix ratio of composite resin and curing temperature.

The aim of this study was to establish a measurement method for filler and matrix in cured resin composite (RC) using Python programming and to investigate the correlation between matrix ratio and curing temperature rise. Eight kinds of RCs were used. Backscattered electron images were taken for each cured specimen. Matrix and filler contents were calculated using Python programming with the K-means or area segmentation method. Volume measurement methods were assessed for comparison. Heat released during the polymerization reaction was measured. The matrix ratio was calculated without human intervention. Three specimens contained only inorganic filler, and other specimens contained multiple types of fillers. Almost the same values of the matrix ratio were obtained by programming and the volume measurement methods for specimens containing a single type of inorganic filler. Moreover, a strong correlation was found between the matrix ratio obtained by the programming method and curing temperature rise (R=0.9826).

A novel identification method using perceptual degree of concordance of occlusal surfaces calculated by a Python program.

One of the important issues during the response to a mass disaster is the identification of victims. In this study, we verified the use of the occlusal morphology of molars for individual identification. The aim of this study was to establish a simple new method for identifying individuals from molar data. Using Python, we developed programming that included the perceptual Hash (pHash) function and the Hamming distance (HD) between antemortem data (AMD) and postmortem data (PMD). The AMD comprised 2,215 dental models. The PMD were selected from the AMD set and comprised 17 models from the same individual with changes over time. As a result, 16 PMD models (over 90%) were ranked in the top 5%. Although identification using only a single molar is difficult, there is the possibility of narrowing down victims' identity with high accuracy through verification using multiple teeth. This system is expected to be useful as a very simple method of identification.

Study on fabrication of orthodontic brackets with the photocatalytic function of titanium dioxide.

We sought to investigate whether the photocatalytic function of rutile-type titanium dioxide is applicable for orthodontic brackets. To this end, TiO(2 )specimens were compressed and sintered. Then, each specimen was analyzed by X-ray diffraction to confirm that no compositional changes had taken place. Next, the surface of each specimen was observed with a colorimeter, scanning electron microscope (SEM), and atomic force microscope (AFM). Fundamental material properties after calcination were measured. The photocatalytic function was evaluated using the decolorization method with toluidine blue. The following were observed with the increase of calcinations temperature: growth of crystal particles, decrease in surface roughness and surface brightness, but increase in shrinkage percentage and compressive strength. The aesthetic and mechanical properties of TiO(2 )material were adequate when compared with other bracket materials. In addition, it had a satisfactory photocatalytic function after high-temperature calcination. Based on the favorable results obtained, rutile-type titanium dioxide seemed to be applicable for the fabrication of self-cleaning orthodontic brackets.

Application of alpha-tricalcium phosphate coatings on titanium subperiosteal orthodontic implants reduces the time for absolute anchorage: a study using rabbit femora.

We are currently developing a small perforated titanium subperiosteal implant specifically for orthodontic therapy, which can be placed anywhere on the bone surface. In the present study, we coated this implant with hydroxyapatite (HA) or alpha-tricalcium phosphate (alpha-TCP) in an attempt to shorten the initial stabilization period relative to the few months that is usually required. The coated implants were placed beneath the periosteum in rabbit femora. The implants were observed by radiographically and histologically, and measured the tensile strength of the bone-implant interface. Two weeks after placement, the volume of new bone formed in the perforations of the implant was significantly greater for the alpha-TCP-coated implants than for the HA-coated implants. Our findings indicate that new bone is formed faster in the surrounding area with alpha-TCP- and HA-coated subperiosteal implants than with uncoated implants, and that alpha-TCP is a particularly effective stimulator of new bone formation.