Haruhisa Oguchi

In vivo Degradation of Resin-Dentin Bonds in Humans Over 1 to 3 Years

The longevity of resin restorations is currently an area of great interest in adhesive dentistry. However, no work has been conducted to investigate the durability of resin-dentin bond structures using human substrate in vivo. The purpose of this study was to investigate the degradation of the resin-dentin bond structures aged in an oral environment for 1, 2, or 3 years. Cavities were prepared in primary molars, and an adhesive resin system (Scotchbond Multi-Purpose) was applied to the cavity. After I to 3 years, following the eruption of the succedaneous permanent teeth, the resin-restored teeth were extracted. Immediately after extraction, those teeth were sectioned perpendicular to the adhesive interface and trimmed to produce an hourglass-shaped specimen. Then, a micro-tensile test was performed at a crosshead speed of 1.0 mm/min. The mean bond strengths were statistically compared with one-way ANOVA and Fishers PLSD test (p < 0.05). Further, all fractured surfaces were observed by SEM, and the area fraction of failure mode was calculated by means of a digital analyzer on SEM photomicrographs. There were significant differences in tensile-bond strength among all 3 groups (p < 0.05), with mean values ranging from 28.3 ± 11.3 MPa (control), to 15.2 ± 4.4 MPa (1 to 2 years), to 9.1 ± 5.1 MPa (2 to 3 years). Moreover, under fractographic analysis, the proportion of demineralized dentin at the fractured surface in specimens aged in an oral environment was greater than that in control specimens. Furthermore, degradation of resin composite and the depletion of collagen fibrils was observed among the specimens aged in an oral environment. Analysis of the results of this study indicated that the degradation of resin-dentin bond structures occurs after aging in the oral cavity.

In vitro degradation of resin–dentin bonds analyzed by microtensile bond test, scanning and transmission electron microscopy

Our knowledge of the mechanisms responsible for the degradation of resin-dentin bonds are poorly understood. This study investigated the degradation of resin-dentin bonds after 1 year immersion in water. Resin-dentin beams (adhesive area: 0.9mm(2)) were made by bonding using a resin adhesive, to extracted human teeth. The experimental beams were stored in water for 1 year. Beams that had been stored in water for 24h were used as controls. After water storage, the beams were subjected to microtensile bond testing. The dentin side of the fractured surface was observed using FE-SEM. Subsequently, these fractured beams were embedded in epoxy resin and examined by TEM. The bond strength of the control specimens (40.3+/-15.1MPa) decreased significantly (p<0.01) after 1 year of water exposure (13.3+/-5.6MPa). Loss of resin was observed within fractured hybrid layers in the 1 year specimens but not in the controls. Transmission electron microscopic examination revealed the presence of micromorphological alterations in the collagen fibrils after 1 year of water storage. These micromorphological changes (resin elution and alteration of the collagen fibrils) seem to be responsible for the bond degradation leading to bond strength reduction.

The effect of hybrid layer thickness on bond strength: demineralized dentin zone of the hybrid layer.

OBJECTIVES The purpose of this study was to evaluate the correlation between hybrid layer thickness and bond strength using specimens acid-conditioned for varying lengths of time. METHODS The dentin surfaces of human premolars, sectioned to remove the enamel from the labial surface, were conditioned with 35.0% phosphoric acid of an adhesive resin system (Scotchbond Multi-Purpose; 3M) for 15 (as directed by the manufacturer), 60, 120, or 180 s (experimental acid-conditioning times). The bonded specimens were then sectioned perpendicular to the adhesive interface to measure the hybrid layer thickness by SEM. The specimens for the micro-tensile test were sectioned perpendicular to the adhesive interface and trimmed to an hourglass-shape. Then, the micro-tensile test was performed at a crosshead speed of 1.0 mm/min. The bond strengths and hybrid layer thickness were statistically compared with Students t-test (p < 0.05). All fractured surfaces were also observed by SEM. RESULTS Significant differences between the groups exposed to acid for 15 and 60 s, and those exposed for 120 and 180 s were observed in hybrid layer thickness and bond strength (p < 0.05). SEM observation of the fractured surfaces revealed that a demineralized dentin zone without resin impregnation remained within the hybrid layer. SIGNIFICANCE A demineralized dentin zone was formed in the bond structures after prolonged acid-conditioning, resulting in low bond strength. The shrinkage of the hybrid layer due to desiccation during the SEM examination process provided evidence of the presence of the demineralized dentin zone within the hybrid layer.

Synchronization of circadian firing rhythms in cultured rat suprachiasmatic neurons.

The circadian clock in mammals is located in the suprachiasmatic nucleus (SCN) which consists of multiple oscillating neurons. Integration of the cellular oscillations is essential for the generation of a single circadian period in the SCN. By using a multielectrode dish (MED), we measured circadian firing rhythms in individual SCN neurons for more than 2 weeks continuously, and examined the involvement of synaptic communication in the synchronization of circadian rhythms. Cross‐correlation analysis of spontaneous action potentials revealed that a neuron pair was functionally connected by synapses when their circadian rhythms were synchronized. No correlation was found between the paired neurons whose circadian rhythms were not synchronized. Calcium (Ca2+)‐dependent synaptic transmission in the cellular communication was indicated by dose‐dependent lengthening of an intercellular spike interval and loss of spike correlation with a Ca2+ channel blocker. Approximately 60% of the SCN neurons in culture were immunoreactive to antibodies against γ‐aminobutyric acid (GABA) or glutamic acid decarboxylase (GAD). Spontaneous firing of all the neurons tested was either increased or decreased by bicuculline, the GABAA receptor antagonist. These findings indicate that synaptic communication plays a critical role in the synchronization of circadian rhythms in individual SCN neurons and the GABAergic transmission is involved in the synchronization mechanism.

The Extent to which Resin can Infiltrate Dentin by Acetone-based Adhesives

The combined methodologies of fractography and laser-Raman spectroscopic analysis were used for evaluation of the resin-dentin bonds made with wet and dry bonding. Resin-dentin-bonded beams were produced by means of 2 acetone-based adhesives (One-Step and Prime & Bond NT). The micro-tensile bond test was conducted, and the fractured surfaces of all specimens were examined by SEM and an image analyzer. The amount of resin infiltration within the hybrid layer was quantified by means of a laser-Raman spectroscope. In Raman analysis, the amount of resin impregnation within the hybrid layer of the dry bonding was found to be significantly lower (approximately 50%) than that in the wet one. Under fractographic analysis, a correlation was found between the bond strength and the failure mode. Based on those findings, it was suggested that the integrity between the bonding resin and the top of the hybrid layer played a major role in bond strength.

Over-etching effects on micro-tensile bond strength and failure patterns for two dentin bonding systems

OBJECTIVES The purpose of this study was to determine (1) the weakest zone of resin-dentin bonds and (2) the relation between bond strength and failure mode to clarify the effect of demineralized dentin. METHODS Human premolars were sectioned to expose the dentin surfaces, and the dentin surfaces were conditioned with phosphoric acid for 15, 60, 120, or 180s. Resin-dentin bonded specimens were produced using two adhesives: One-Step (Bisco) and OptiBond Solo (Kerr). Each sample was sectioned to produce a beam (adhesive area: 0.9mm(2)). Microtensile bond tests were then conducted, and the mean bond strengths (n=12 for each group) were statistically compared using two-way ANOVA and Duncans multiple-range test (p<0.05). The fractured surfaces of all specimens were examined using SEM, and the areas of failure were measured using an image analyzer. RESULTS For One-Step, the bond strength decreased with increase in acid-conditioning time (15s: 50.7+/-9.7, 60s: 40.8+/-11.0, 120s: 23.6+/-4.9 and 180s: 12.1+/-4.6MPa) (p<0.05). For OptiBond Solo, the bond strength in the case of 15s acid-conditioning time (42.6+/-7.9MPa) was significantly greater than that for the other times (60s: 31.9+/-10.3, 120s: 31.8+/-14.4 and 180s: 31.8+/-7.4MPa) (p<0.05). Fractography showed that the area percentage of the hybrid layer increased with increase in etching time for both systems. CONCLUSIONS The integrity of the hybrid layer, especially the top part, has an effect on bond strength.

The in vitro cytotoxicity of eluates from dentin bonding resins and their effect on tyrosine phosphorylation of L929 cells.

OBJECTIVES The aim of this study was to examine the relationship between the monomers eluted from dentin-bonding systems and their cytotoxicities, and to investigate the biochemical effect of the monomers on tyrosine phosphorylation, especially relating to the cell growth activity, of L929 cells in vitro. METHODS The primers, uncured or cured adhesives (3M and Kuraray) were tested to determine the cytotoxicity of confluent L929 cells cultured by Eagles MEM medium supplemented with 10% FCS. The area of cells affected by the eluted monomers were evaluated with an image analyzer and the concentrations of monomers eluted into the medium were measured with high performance liquid chromatography (HPLC) after 24h incubation. The protein composition of the stimulated cells was compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tyrosine phosphorylation was detected by Western blot. RESULTS The primer and uncured adhesives revealed variable cytotoxicities. 2-hydroxyethyl-methacrylate (HEMA) was the major component eluted from uncured primers and adhesives. Small amounts of triethylene glycol dimethacrylate (TEGDMA) were also detected from the uncured adhesives. The cytotoxicities of the adhesives decreased as photo activation time increased. The amount of monomers eluted from the cured adhesives was almost undetectable and did not reach a sufficient concentration to suppress cell viability or cell growth. The cytotoxicities of the primers and adhesives correlated well with the amounts of either HEMA or TEGDMA eluted. Moreover, a high concentration of HEMA (4 mg/ml medium) affected intracellular tyrosine phosphorylation, which is related to cellular activities. SIGNIFICANCE Although the monomers present in dentin bonding resins are cytotoxic to L929 cells, the amount from cured bonding resin is very small and does not provide a cytotoxic dose. This data does however suggest that clinical exposure to the uncured primers and adhesives of dentin bonding resins should be minimized.

Autosomal-dominant Hypoplastic Form of Amelogenesis Imperfecta Caused by an Enamelin Gene Mutation at the Exon-Intron Boundary

Amelogenesis imperfecta (AI) is currently classified into 14 distinct subtypes based on various phenotypic criteria; however, the gene responsible for each phenotype has not been defined. We performed molecular genetic studies on a Japanese family with a possible autosomal-dominant form of AI. Previous studies have mapped an autosomal-dominant human AI locus to chromosome 4q11-q21, where two candidate genes, ameloblastin and enamelin, are located. We studied AI patients in this family, focusing on these genes, and found a mutation in the enamelin gene. The mutation detected was a heterozygous, single-G deletion within a series of 7 G residues at the exon 9-intron 9 boundary of the enamelin gene. The mutation was detected only in AI patients in the family and was not detected in other unaffected family members or control individuals. The male proband and his brother showed hypoplastic enamel in both their deciduous and permanent teeth, and their father showed local hypoplastic defects in the enamel of his permanent teeth. The clinical phenotype of these patients is similar to that of the first report of AI caused by an enamelin gene mutation. Thus, heterogeneous mutations in the enamelin gene are responsible for an autosomal-dominant hypoplastic form of AI.

Fractured surface characterization: wet versus dry bonding

OBJECTIVE Fractographic analysis was conducted to evaluate the resin-dentin bond structures made under wet and dry conditions. METHODS Resin-dentin bonded specimens were prepared using two adhesive resin systems (Single Bond/SB; 3M and All Bond 2/AB2; Bisco Inc) under wet and dry conditions. The specimens were sectioned perpendicular to the adhesive interface to produce a square bar-shaped specimen (adhesive area: 0.9 mm(2)) by means of a diamond saw. The mean bond tensile test was then conducted at a crosshead speed of 1.0 mm/min. The mean bond strengths were statistically compared with two-way ANOVA and Fishers PLSD test (p<0.05). Subsequently, the fractured surfaces of all specimens were examined using SEM and the area fractions of failure modes (%) were measured using an image analyzer on SEM microphotographs. RESULTS No significant differences in tensile-bond strength were observed between SB (60.1+/-16.4MPa) and AB2 (69.8+/-17.4MPa) (p>0.05) under wet conditions. However, the bond strength either of SB or AB2 made under wet conditions was significantly greater than those made under dry conditions (SB: 26.2+/-12.5MPa and AB2: 6.8+/-3.3MPa) (p<0.05). Under fractographic analysis, the major portion at the fractured surface was occupied by the cohesive failure of bonding resin and the resin composite for the wet conditions, and the top of the hybrid layer for the dry conditions in both systems. SIGNIFICANCE The interaction between the top of the hybrid layer and the bonding resin influenced the bond integrity.

Circadian rhythm of nitric oxide production in the dorsal region of the suprachiasmatic nucleus in rats

Extracellular concentration of nitrite (NO2-), an oxidized product of nitric oxide (NO), was measured consecutively in the dorsal region of the rat suprachiasmatic nucleus (SCN) by means of in vivo microdialysis. The NO2- concentrations in the dialysates showed robust circadian rhythm under a 12:12 h light/dark cycle and were higher during the dark phase than during the light phase. When the rats were transferred to constant darkness, the 24 h rhythm of NO2- persisted without damping the amplitude. The NO2- level was significantly lowered by an injection of NO synthase inhibitor (NG-monomethyl-L-arginine, 10 mg/kg i.p.). These findings indicate that the daily fluctuation of NO2- in the dorsal region of the SCN, which represents endogenous rhythm of NO, is regulated independently of photic inputs into the SCN and may be related to the circadian clock functions.