Evaluation of calcium (Ca2+) and hydroxide (OH-) ion diffusion rates of indirect pulp capping materials.

INTRODUCTION: The aim of this study was to evaluate and compare the calcium (Ca2+) and hydroxide (OH-) ion release of 4 artificially produced pulp capping materials (MTA, Biodentin, TheraCal LC, Calsimol) used for indirect pulp capping treatment. METHODS: In total, 70 freshly extracted human third molar teeth were used for the study. Cavities of extracted teeth were prepared by round burs. The remaining dentin thickness (1 ± 0.3 mm) tissue was measured by a micrometer and cone beam computerized tomography. Indirect pulp capping was performed in the cavities using Calcimol, MTA, TheraCal LC and Biodentin. The leached Ca2+ were measured using optical emission spectrometry and the release of OH- ions using a pH meter. The measurements were performed after 24 hours, 7 days and 28 days in saline solution. Statistical analysis was performed using 1-way and 2-way analysis of variance (ANOVA) tests (p<0.05). RESULTS: Ca2+ ions were detected in treated saline solution during the experimental period for all materials. All the measurements of Biodentin and Theracal LC levels for Ca2+ ions were higher than those of the other materials (p<0.05). For all materials, Ca2+-ion release increased during the first 7 days followed by a linear decrease during the subsequent study periods. The Biodentine group showed the highest OH- ion rates compared to the other materials in the 24-hour examination period, while the scores gradually decreased during the subsequent measurement periods (p<0.05). CONCLUSIONS: Tricalcium silicate materials such as Biodentine and TheraCal LC used in this study may be preferable for indirect pulp capping because of their stimulation of hard tissue formation and ion-releasing ability.

Unbound monomers do diffuse through the dentin barrier.

OBJECTIVES: Assessing the role of dentinal fluid proteins in trans-dentinal diffusion of free monomers in vitro. METHODS: An artificial pulp chamber (APC) topped human dentin disks was used. A simplified two-step etch-and-rinse adhesive was formulated with 2-hydroethyl-methacrylate (HEMA), Bisphenol-A-diglycidyl-methacrylate (BisGMA), using Camphorquinone/tertiary amine as initiators. Two extraction media were used: buffered saline (Control), buffered saline with 1% bovine serum albumin (BSA). Samples were acid-etched, rinsed, air dried. Simplified primer was used, adhesive applied then light cured with a LED curing. Monomer diffusion was assessed by reverse phase HPLC. RESULTS: Quantifiable amounts of HEMA were detected in both extraction media while BisGMA was present in quantifiable amounts in BSA medium only. Diffused monomers concentrations were significantly higher for both monomers in BSA extraction medium. SIGNIFICANCE: Albumin is sometimes referred to as taxi protein for its ability to bind and transport hydrophobic ligands. From our results, we hypothesized that albumin can also transport unbound monomers released from dental adhesive through the dentin barrier. However, dentinal fluid proteins like albumin could have significant effect on monomer diffusion through dentin to the dental pulp transporting highly hydrophobic molecules like BisGMA and enhancing diffusion of more hydrophilic ones like HEMA. These results demonstrate a new possible mechanism for cytotoxicity of resin monomers.

Evaluation of penetration effect of resin infiltrant using optical coherence tomography.

OBJECTIVE: The aims of this in vitro study were to investigate whether optical coherence tomography (OCT) could analyze infiltration of resin infiltrant (RI) into early dental caries (EC), and to confirm the correlation between the results of OCT and confocal laser scanning microscopy (CLSM) for evaluation of RI infiltration into EC. METHODS: Sound bovine permanent teeth were used to produce sixty specimens by making two windows on the teeth. Each 20 specimens were demineralized for 20, 30, and 40 days, and the RI was treated on one of the windows. As a result, the images of the fifty-two specimens were taken by OCT and CLSM. The demineralized lesion depth (LDOCT and LDCLSM) and the infiltrated depth of RI into lesion (IDOCT and IDCLSM) obtained from the OCT and the CLSM were analyzed. The correlations between the LDOCT and the LDCLSM, and between the IDOCT and the IDCLSM, were analyzed by Pearson correlation and intra-class correlation. Also, Bland-Altman plot was constructed to assess the agreement between the IDOCT and the IDCLSM, and the IDOCT divided by refractive index of RI and the IDCLSM. RESULTS: The Pearson correlation coefficient and intra-class correlation of 0.75 and 0.83 (95% CI: 0.71-0.91) respectively were confirmed between the LDOCT and the LDCLSM (p<0.001), and 0.59 and 0.71 (95% CI: 0.50-0.84) respectively were observed between the IDOCT and the IDCLSM (p<0.001). The lower bias was confirmed in Bland-Altman plot between adjusted IDOCT and the IDCLSM than between the IDOCT and the IDCLSM. CONCLUSION: The OCT was the promising quantitative evaluation method for RI penetrated into EC. CLINICAL SIGNIFICANCE: The OCT would be used as a nondestructive and real-time evaluation method for RI penetrated into EC on clinical procedure.

Tridimensional surface roughness analysis after resin infiltration of (deproteinized) natural subsurface carious lesions.

OBJECTIVES: The objectives of this study were to evaluate ex vivo the effects of resin infiltration on the areal surface roughness of natural non-cavitated proximal subsurface lesions with or without previous deproteinization and to determine differences between E2 and D1 lesions or between premolars and molars. MATERIALS AND METHODS: Forty premolars and 40 molars with proximal carious lesions and macroscopically intact surfaces (International Caries Detection and Assessment System (ICDAS) II; code 2) were radiologically assessed and randomly allocated to four groups (with 20 E2 and 20 D1 lesions, respectively). In each group, 10 lesions were deproteinized (NaOCl; 1%) before etching (HCl; 15%) and resin infiltration (Icon). Areal surface roughness (Sa) at the most demineralized lesion part (DIAGNOdent) was evaluated topometrically before and after deproteinization, after etching, and after infiltration using focus variation 3D scanning microscopy. RESULTS: Pretreatment with NaOCl (n = 40) had no significant effects on Sa (p = 0.208), but resulted in significantly differing Sa values between premolars and molars after etching (p = 0.011). Regarding the effects between etching and baseline, significantly differing Sa values (p = 0.0498) were found for premolars and molars (n = 40/40); Sa after resin infiltration (compared to etching) differed significantly between premolars and molars (p = 0.009). No treatment regimen lead to differences among the radiological grades (E2 vs. D1; p > 0.106). CONCLUSIONS: Resin infiltration showed only minor effects on Sa values of etched subsurface lesions (p < 0.170) and did neither equal nor improve baseline surface roughness (p > 0.401) of the different tooth types. CLINICAL RELEVANCE: Deproteinization should be recommended before etching and infiltration, even if surface roughness of infiltrated advanced (pre-)molar lesions will not be improved.

Kinetics of polymerization and contraction stress development in self-adhesive resin cements.

OBJECTIVES: The aim of the study was to evaluate the contraction stress, microhardness and polymerization kinetics of three self-adhesive cements vs. conventional dual-cure resin cement. METHODS: Cements tested were: RelyX Unicem (3M ESPE, St. Paul, MN, USA), MaxCem Elite (Kerr, Orange, CA, USA), Clearfil SA Cement (Kuraray, Tokyo, Japan) and Duolink (Bisco Inc., Schaumburg, IL, USA). Cements were irradiated with a LED-curing unit (bluephase, IvoclarVivadent) for 20 or 40 s and the contraction forces (N) generated during polymerization were continuously recorded for 6 h with a universal testing machine. Polymerization kinetics were monitored using micro-Raman spectroscopy and degree of conversion was calculated. Vickers microhardness was also recorded. All measurements were performed at 10 min and 6h. Data were statistically analyzed by three-way ANOVA with repeated measures and Tukey's post hoc test (α=0.05). RESULTS: Irrespective of exposure time, stress analysis ranked in the following order: Clearfil SA Cement

Sorption and solubility characteristics of self-adhesive resin cements.

OBJECTIVE: The aim of this in vitro study was to evaluate some sorption characteristics [sorption (S), solubility (SL) and the percentages of mass change (M(g)%), solubility (SL%) as well as sorbed liquid (S%)] of self-adhesive resin cements when immersed in distilled water and lactic acid. METHODS: A disc-shaped specimen of each self-adhesive resin cements [G-Cem (GC), SmartCem™ 2 (SC2), RelyX™ U100 (R1), RelyX™ Unicem 2 (RU2)] were prepared in a split-Teflon mold and irradiated by an Optilux 501 light cure at 580mW/cm(2) for 40s in eight overlapping sections each side. The volume of each specimen was calculated and placed inside a desiccator containing anhydrous calcium chloride, then weighed on an analytical electronic balance. Two independent groups were established according to the immersion media or liquids (distilled water and 0.01M lactic acid) maintained at 37°C for the time intervals: 1, 6, 12, 24, 48 and 168h, where the sorption (S) property (µg/mm(3)) was calculated. However, the SL, M(g)%, SL% and S% were measured after 168h of immersion. The data were statistically analyzed by repeated measures ANOVA, one-way ANOVA and post hoc Tukey's test (p<0.05). RESULTS: Analysis of variance revealed highly significant differences between the materials for the sorption and solubility values examined with some exceptions (p<0.05). However, independent samples T-test expressed significant differences of all the sorption values between both water and lactic acid media for the resin cements with some border significances (p>0.05). The highest liquid's sorption was exhibited by GC material after immersion in lactic acid for 168h period followed by SC2 (37.83 and 34.15µg/mm(3), respectively), while the lowest sorption was presented by RU2 cement after 1h immersion period in water (3.89µg/mm(3)). Stereomicroscope showed homogenous surface topography in RU2 and R1 samples, while some striated cracks and microvoids were observed in GC and SC2 materials, respectively. The SL values followed this order: RU2

Penetration of experimental infiltrants with different penetration coefficients and ethanol addition into natural caries lesions in primary molars.

This study evaluated the influence of the penetration coefficient (PC) and ethanol addition on the penetration depth (PD) of experimental infiltrants into proximal caries lesions in primary molars. Caries lesions (n = 45) were randomly treated with 1 of 4 experimental infiltrants (PC63; PC185; PC204; PC391) for 5 min. Lesion depths and PDs were analysed using dual fluorescence confocal microscopy. Lesions were almost completely infiltrated in all groups. Median PDs and percentage penetrations were not significantly different between groups (p > 0.05). When applied for 5 min, all tested infiltrants were able to infiltrate proximal caries in primary molars nearly completely.

MMP activity in the hybrid layer detected with in situ zymography.

Dentinal proteases are believed to play an important role in the degradation of hybrid layers (HL). This study investigated the HL gelatinolytic activity by in situ zymography and functional enzyme activity assay. The hypotheses were that HLs created by an etch-and-rinse adhesive exhibit active gelatinolytic activity, and MMP-2 and -9 activities in dentin increase during adhesive procedures. Etched-dentin specimens were bonded with Adper Scotchbond 1XT and restored with composite. Adhesive/dentin interface slices were placed on microscope slides, covered with fluorescein-conjugated gelatin, and observed with a multi-photon confocal microscope after 24 hrs. Human dentin powder aliquots were prepared and assigned to the following treatments: A, untreated; B, etched with 10% phosphoric acid; or C, etched with 10% phosphoric acid and mixed with Scotchbond 1XT. The MMP-2 and -9 activities of extracts of dentin powder were measured with functional enzyme assays. Intense and continuous enzyme activity was detected at the bottom of the HL, while that activity was more irregular in the upper HL. Both acid-etching and subsequent adhesive application significantly increased MMP-2 and -9 activities (p < 0.05). The results demonstrate, for the first time, intrinsic MMP activity in the HL, and intense activation of matrix-bound MMP activity with both etching and adhesive application.

Biodegradation of resin-dentin interfaces increases bacterial microleakage.

Bis-GMA-containing resin composites and adhesives undergo biodegradation by human-saliva-derived esterases, yielding Bis-hydroxy-propoxy-phenyl-propane (Bis-HPPP). The hypothesis of this study is that the exposure of dental restorations to saliva-like esterase activities accelerates marginal bacterial microleakage. Resin composites (Scotchbond, Z250, 3M) bonded to human dentin were incubated in either buffer or dual-esterase media (pseudocholinesterase/cholesterol-esterase; PCE+CE), with activity levels simulating those of human saliva, for up to 90 days. Incubation solutions were analyzed for Bis-HPPP by high-performance liquid chromatography. Post-incubation, specimens were suspended in a chemostat-based biofilm fermentor cultivating Streptococcus mutans NG8, a primary species associated with dental caries, for 7 days. Bacterial microleakage was assessed by confocal laser scanning microscopy. Bis-HPPP production and depth and spatial volume of bacterial cell penetration within the interface increased with incubation time and were higher for 30- and 90-day PCE+CE vs. buffer-incubated groups, suggesting that biodegradation can contribute to the formation of recurrent decay.

Effect of bacterial collagenase on resin-dentin bonds degradation.

The objective of this study is to evaluate the effect of a bacterial collagenase on the degradation of resin-dentin bonds. Human dentin surfaces were bonded with: an etch-&-rinse self-priming adhesive (SB), a two-step self-etching primer/adhesive (SEB), and a 1-step self-etching adhesive (OUB). Composite build-ups were constructed. The bonded teeth were stored (24 h, 3 months, 1 year) in distilled water or in a buffered bacterial collagenase solution. Half of the specimens were stored as intact bonded teeth (Indirect Exposure/IE). The other half were sectioned into beams prior to storage (Direct Exposure/DE). After storage the intact teeth were sectioned into beams and all specimens were tested for microtensile bond strengths (MTBS). ANOVA and multiple comparisons tests were performed. Fractographic analysis was performed by scanning electron microscopy. The inclusion of bacterial collagenase in the storing solution did not lower the MTBS values over those seen in specimens stored in water. SB and SEB bonds strength were equal, and were superior to OUB. After 3 months of DE, SB and OUB bonded specimens showed decreases in MTBS; similar reductions required 1 year for SEB/DE. MTBS did not decrease in IE specimens except for OUB. Resin and collagen dissolution were evident in DE groups after storing.

Migration and particle clearance from hard-setting Ca(OH)2 and self-etching adhesive resin following direct pulp capping.

PURPOSE: To evaluate the migration and particle clearance from a hard-setting calcium hydroxide (HSCH) and self-etching adhesive resin (SEAD) following direct pulp capping using the light and electron microscope. METHODS: Exposed monkey pulps were capped with a hard-setting calcium hydroxide (Dycal) or adhesive resin (Clearfil SE Bond), and histopathologically evaluated at 14 and 21 days using light and transmission electron microscopy (n = 14). RESULTS: At 14 days, both HSCH and SEAD materials showed no severe inflammatory reactions of the pulp (necrosis and abscess formation). The main reaction was slight inflammatory cell infiltration consisting of leukocytes. A number of HSCH particles were entrapped by macrophages and observed in the small capillaries similar to blood or lymphatic vessels. For SEAD, slight hemorrhage was observed at the exposed surface. At 21 days, for both HSCH and SEAD, a few cases showed minimal inflammatory response which was limited to the area beneath the exposure. Some macrophages entrapping the HSCH particles in vacuoles within the cytoplasm were arranged at the surface of the capping layer. HSCH particles were also observed in the vessels similar to blood or lymphatic vessels. A few macrophages entrapped filler-like particles of SEAD adjacent to the capping material, but there was no evidence of any SEAD in the vessels.

[The penetration of various adhesives into early enamel lesions in vitro]. / Penetration verschiedener Adhäsive in initiale Schmelzläsionen in vitro.

The aim of the present study was to evaluate the penetration depth (PD) and the thickness of the oxygen inhibition layer (OIL) of a fissure sealant (Helioseal, Vivadent) and various adhesives (Heliobond, Excite, Vivadent; Resulcin, Merz; Solobond M, Voco; Prompt L-Pop, 3M-Espe) applied to enamel lesions in vitro. From 27 bovine teeth 54 enamel specimens were prepared and covered with nail varnish (control) thus obtaining three windows for treatment. After demineralisation (pH 5.0, 14 d) two of the windows were etched with phosphoric acid (20%, 5 s), whereas the third area served as control. The specimens were divided randomly into six groups (n = 9) and the respective adhesive was applied (90 s), either once or twice. Light-curing followed each application. Enamel slabs (perpendicular to the surface) were cut and studied after infiltration with a fluorescent low-viscous resin using confocal microscopy (CLSM). The image of the lesion was divided into two areas with different grey values. Lesion depths were calculated (ImageJ) from the surface to that point in the lesion where the grey value clearly changed to a darker grey value. The zone with the darker grey values marked the front of demineralisation. Mean lesion depths (+/- SD) after demineralisation were measured at 105 (+/- 21) microm. After single application, Resulcin [89 (+/- 22)%] and Helioseal [98 (+/- 6)%] had almost completely penetrated the lesion. Heliobond [126 (+/- 33)%] and Excite [184 (+/- 40)%] penetrated even deeper than the defined lesion. For Excite double application decreased the OIL significantly (p = 0.03; adjusted paired t-test). Adhesives are capable to penetrate artificial initial enamel lesions completely. Follow-up studies are needed to confirm this effect for natural lesions.

Detection of acid diffusion through bovine dentine after adhesive application.

AIM: Acidic diffusion through bovine dentine was investigated by measuring pH changes on dentine surfaces after applying three adhesive systems. METHODOLOGY: Coronal incisor bovine dentine discs, 0.5 mm thick, were prepared from dentine close to the pulp chamber. A single-bottle adhesive system-Single Bond, a self-etching primer system-Clearfil SE Bond and an 'all-in-one' adhesive system-AQ Bond were used. The labial dentine surfaces were conditioned as follows: Single Bond groups: (SB-1) 35% phosphoric acid etchant was applied and left in place; (SB-2) the etchant was applied for 15 s and rinsed off for 10 s; (SB-3) application of adhesive agent and light curing following step SB-2; Clearfil SE Bond groups: (SE-1) SE primer was applied for 20 s and dried; (SE-2) application of adhesive agent and light curing following step SE-1; AQ Bond groups: (AQ-1) AQ Bond adhesive was applied for 20 s and dried, applied for additional 5 s and dried again; (AQ-2) light curing following step AQ-1. The pH change on the pulpal dentine surface was measured using a pH-imaging microscope. RESULTS: All the Single Bond groups revealed a lower pH on the pulpal surface (pH 6.25, 6.59 and 6.64 for SB-1, SB-2 and SB-3, respectively) compared with intact dentine. Clearfil SE Bond and AQ Bond groups showed no significant deference in pH value from intact dentine. CONCLUSIONS: Acid diffusion from phosphoric acid etching was observed when placed on 0.5 mm-thick dentine discs; however, there was only limited evidence of acid diffusion from SE primer and AQ Bond.

Relationship between dentine hydraulic conductance and the cytotoxicity of four dentine bonding resins in vitro.

OBJECTIVES: Dentine modifies pulpward diffusion of monomers leaching from restorative materials. Thus, remaining dentine thickness must be taken into account during in vitro cytotoxicity tests. This in vitro study was designed to determine the influence of dentine permeability on the outcome of a cytotoxicity test. METHODS: Dentine slices were made from 36 human third permanent molar teeth. The 36 dentine slices were divided into two groups according to their hydraulic conductance: high or low hydraulic conductance. The cytotoxicity of four dentine bonding agents of similar cytotoxicity was tested on dentine slices from each group. Four dilutions of the experimental culture medium were tested: undiluted, 1:2, 1:10 and 1:100. An analysis of variance was used to compare the cytotoxicity of the dentine bonding agents tested on high versus low hydraulic conductance. RESULTS: The cytotoxicity of the high hydraulic conductance (Lp) group was higher than that of low Lp group when tested with the undiluted test culture medium (p = 0.001). No difference was obtained with the 1:2, 1:10, 1:100 dilutions. CONCLUSIONS: Under the conditions of the study, the dentine bonding resins were more cytotoxic when applied onto dentine slices of high hydraulic conductance.

The influence of dentine permeability on cytotoxicity of four dentine bonding systems, in vitro.

Dentine adhesives are often placed directly on dentine from which the smear layer has been removed, the thickness of the dentine is minimal and the potential for diffusion of adhesive components into the pulp is greatest. The permeability of the dentine is one factor that should be critical to whether sufficient diffusion of adhesive components occurs to cause damage to pulpal cells. Dentine discs were prepared and divided into those with low-, medium-, and high-permeability. They were then treated with four different dentine adhesives, after which the pulpal side of the dentine was placed in contact with 1 mL of cell-culture medium. The medium was collected at 24 h intervals for 168 h, and was then placed on monolayers of human pulpal fibroblasts for 24 h. The response of the cells was assessed by succinic dehydrogenase activity (MTT method). The results showed that four dentine adhesive systems released sufficient components to cause suppression of cellular metabolism through dentine. High-permeability dentine generally allowed more diffusion of these components, but the effect of dentine permeability depended on the material. On the other hand, the time interval between the application of the bonding agent and collection of the eluant was consistently important for all materials. Materials were most cytotoxic at early intervals, and were generally less cytotoxic at later intervals, although there were exceptions and there was persistent (> 15%) suppression of cellular metabolism even at late (168 h) intervals. The results suggest that application of these materials to dentine, and particularly dentine with high permeability, poses a potential risk to the health of pulpal tissues.

The comparative evaluation of release profiles of aluminium, fluoride, sodium and strontium by resin modified and conventional glass polyalkenoate cements in neutral and acidic medium--an in vitro study.

The present study was carried out to evaluate the Al, F, Na and Sr release profiles of conventional and resin modified glass polyalkenoate cements in neutral (deionized water) and acidic medium (lactic acid). Twelve pellets of each material were prepared under standardised conditions and were immersed in their respective solutions for a study period of 90 days. Fluoride analysis was carried out by Orion Electrode and the other elemental analysis were done by Atomic Absorption Spectrophotometer. Statistical analysis revealed no significant difference in the Al, F and Na release profiles between the two materials in both neutral and acidic media.