Masayuki Kaga

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.

Fluid Movement across the Resin-Dentin Interface during and after Bonding

This study evaluated the extent of water penetration through resin-dentin interfaces before and after being sealed with adhesives. Four adhesive resin systems (2 total-etch adhesives and 2 self-etching primer adhesives) were used in this study. Dentin disks were placed in a split-chamber device, and in situ fluid movement across dentin was measured, with and without physiological pressure, during bonding procedures or 24 hrs after bonding. The fluid movement across dentin occurs via dentin tubules after acid-etching. Large outward or inward fluid shifts across dentin were observed during air-drying and light-curing for resin application. The amount of fluid movement across resin-bonded dentin when total-etch adhesives were used was significantly greater than that with self-etching adhesives. The milder acid-etching effects of self-etching primers may retain hybridized smear plugs within the tubules that reduce outward fluid flow, resulting in superior dentin sealing.

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.

Components of Dentinal Adhesives Modulate Heat Shock Protein 72 Expression in Heat-stressed THP-1 Human Monocytes at Sublethal Concentrations

Few studies have investigated the ability of dental resins to induce cellular stress at sublethal concentrations. Cellular stress, especially in immune cells such as monocytes, may modulate the biological response to materials or the hosts ability to respond to bacterially mediated inflammation. The current study examined the ability of sublethal concentrations of 2-hydroxylethylmethacrylate (HEMA) and triethyleneglycol dimethacrylate (TEGDMA) to induce heat shock protein 72 (HSP72) in human monocytes. HEMA and TEGDMA significantly suppressed heat-induced HSP72 expression, even at sublethal levels, but did not induce HSP72 by themselves. The results of the current study suggest that components released from dental resin could modulate the HSP stress response without altering cellular metabolic activity.

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.

Sublethal, 2-week exposures of dental material components alter TNF-α secretion of THP-1 monocytes

OBJECTIVES The aim of this study was to investigate the hypothesis that dental material components alter cytokine secretion from monocytes if applied for several weeks at sublethal doses. The current study significantly extended exposure times of monocytes to the components over times published in previous studies. These exposure times approached the estimated average life span of monocytes in the bloodstream. METHODS Human THP-1 monocytes were exposed to 2-hydroxyethylmethacrylate (HEMA, 0-1.2mmol/l), triethyleneglycoldimethacrylate (TEGDMA, 0-0.75mmol/l), Hg(2+) (0-2 micromol/l), or Ni(2+) (0-20 micromol/l) for 2 weeks. The cells were then collected and additionally incubated for 24h, with or without bacterial lipopolysaccharide (LPS), a common component of dental plaque. TNF-alpha secretion from THP-1 was determined using by enzyme-linked immunosorbent assay. RESULTS None of the dental material components induced TNF-alpha from THP-1 by themselves, but LPS alone strongly induced TNF-alpha secretion as expected. HEMA and TEGDMA significantly suppressed (40-70%) TNF-alpha secretion from cells stimulated with LPS. Hg(2+) at 2.0 micromol/l doubled TNF-alpha secretion from THP-1s stimulated with LPS over LPS alone. Ni(2+) did not significantly affect TNF-alpha secretion, with or without LPS exposure. Significance. The results in this study suggest that sublethal, 2-week exposures of some dental material components may alter TNF-alpha secretion from THP-1 monocytes when the cells are challenged. These alterations may influence the biological response of tissues to materials in an inflammatory intraoral environment.

Comparison of antimicrobial and cytotoxic effects of glutaraldehyde and formocresol

The in vitro antimicrobial and cytotoxic concentrations of glutaraldehyde and formocresol were determined. Minimal antimicrobial concentrations of these two agents against selected microbial flora reported in carious primary teeth were 3.125% for glutaraldehyde and 0.75% for formocresol. At a doubling of these concentrations, most organisms, except Candida albicans, Staphylococcus epidermidis, and Streptococcus mutans, were killed by both substances in 30 seconds. Cytotoxicity was evaluated on tissue cultures of pulp fibroblasts and HeLa cells at minimal cidal concentrations and at 10- and 100-fold dilutions. Exposure of pulp fibroblasts and HeLa cells to formocresol and indirect exposure to vapors caused the cells to become atrophic and to form a less dense tissue pattern. Cells directly exposed to glutaraldehyde retained their normal cell shape and tissue pattern, whereas cells indirectly exposed to vapors continued to proliferate. These data showed effective antimicrobial activity at concentrations of 3.125% glutaraldehyde and 0.75% formocresol and suggested that glutaraldehyde may exert a less cytotoxic effect on the immediate and surrounding tissues when used as a pulpotomy agent.