MMP Inhibitors and Dentin Bonding: Systematic Review and Meta-Analysis.

OBJECTIVES: Resin-dentin bond strength decreases over time. This reduction is related to the loss of hybrid layer integrity. Collagenolytic enzymes, especially matrix metalloproteinases (MMPs), are responsible for the degradation of the collagen matrix of the hybrid layer. Various MMP inhibitors with the ability to prevent enzymatic degradation have been identified. This study aimed to systematically review the literature for studies which evaluated the effect of MMP inhibitors on the immediate and aged dentin bond strengths. Study SelectionScreening and analysis were carried out by two reviewers. Two databases were searched, and from a total of 740 articles, 43 were accepted for full review. 21 articles with 0.2%-2% chlorhexidine (CHX) treatments were included for meta-analysis. A risk of bias assessment was performed on all studies chosen for meta-analysis. A variety of MMP inhibitors have been studied, CHX being the most widely used. CONCLUSIONS: A clear trend for a lower loss of dentin bond strength was observed with different MMP inhibitors. In meta-analysis, no significant difference was seen between the CHX and control in the immediate bond strengths. Bond strengths in the CHX group were significantly higher than the control group after aging (P < 0.001). The percentage of fractures occurring at the adhesive interface increased after aging. Five out of 21 studies included in the meta-analysis had high and the rest medium risk of bias. More long-term studies with lower risks of bias should be carried out to increase the reliability of results. Clinical RelevanceThe use of MMP inhibition with chlorhexidine can be recommended to increase the longevity of resin-dentin bond strength.

European Society of Endodontology position statement: endodontic management of traumatized permanent teeth.

This position statement represents a consensus of an expert committee convened by the European Society of Endodontology (ESE) on the endodontic management of traumatized permanent teeth. A recent comprehensive review with detailed background information provides the basis for this position statement (Krastl et al. 2021, International Endodontic Journal, https://doi.org/10.1111/iej.13508). The statement is based on current scientific evidence as well as the expertise of the committee. Complementing the recently revised guidelines of the International Association of Dental Traumatology, this position statement aims to provide clinical guidance for the choice of the appropriate endodontic approach for traumatized permanent teeth. Given the dynamic nature of research in this area, this position statement will be updated at appropriate intervals.

Endodontic management of traumatized permanent teeth: a comprehensive review.

The pulp plays a key role in the treatment of traumatic dental injuries (TDIs) and is strongly associated with the outcome, particularly in severe cases. A correct pulp diagnosis is essential as it forms the basis for developing the appropriate management strategy. However, many TDIs are complex, and their treatment requires a profound knowledge of the physiological and pathological responses of the affected tissues. This comprehensive review will look at the dentine-pulp complex and its interaction with the surrounding tissues following TDIs. The literature up to 2020 was reviewed based on several searches on PubMed and the Cochrane Library using relevant terms. In addition to the recently revised guidelines of the International Association of Dental Traumatology, this article aims to provide background information with a focus on endodontic aspects and to gather evidence on which a clinician can make decisions on the choice of the appropriate endodontic approach for traumatized permanent teeth.

Association between dental factors and mortality.

AIM: To study whether oral parameters such as endodontic infections, root canal fillings, number of teeth or wearing removable dentures at baseline are associated with cardiovascular- and all-cause mortality in a follow-up of approximately 8 years. METHODOLOGY: The Finnish Parogene cohort consists of 508 Finnish adults (mean age 63.3 years, SD 9.1) with cardiac symptoms, all of whom had undergone coronary angiography for accurate baseline coronary status. Extensive clinical and radiographic oral examinations were performed, and additional data were acquired from medical records and questionnaires. Root canal fillings and endodontic lesions, as well as their co-occurrence, were determined from panoramic radiographs. The mortality data were assessed via record linkage with the Finnish Causes of Death register (mean follow-up time 7.81 years, SD 1.45 years). A total of n = 471 dentate patients were included in the statistical analyses. RESULTS: A total of n = 69 deaths were recorded, of which n = 41 were due to cardiovascular diseases (CVDs, ICD-10 I00-I99). The deceased had fewer root canal fillings (mean 1.57; SD 1.64 vs. mean 2.30; SD 2.34, P = 0.03) than the survivors. The number of missing teeth was associated with smoking, occluded coronary arteries and diabetes. Cox regression with Firth's penalized maximum-likelihood method using age as timescale revealed an inverse association (HR; 95%CI) between mortality and number of teeth (all-cause 0.91; 0.86-0.96, CVD mortality 0.89; 0.83-0.96), use of removable dentures (all-cause 0.24; 0.09-0.62, CVD mortality 0.20; 0.06-0.72), root canal fillings (all-cause 0.82; 0.70-0.94, CVD mortality 0.79; 0.63-0.96) and having root canal fillings in all teeth with apical rarefactions (all-cause 0.27; 0.06-0.79, CVD mortality 0.09; 0.01-0.63), when gender, smoking, occluded coronary arteries, periodontal inflammatory burden index and the number of teeth were adjusted for. CONCLUSIONS: The number of missing teeth appeared to be the strongest predictor of mortality in this study, whereas endodontic infections per se had no independent association. Nevertheless, signs of professional intervention in these problems, such as root canal fillings and removable dentures, appeared to be associated with improved survival, which might partly be explained by the utilization of healthcare services.

The effect of chlorhexidine and dimethyl sulfoxide on long-term sealing ability of two calcium silicate cements in root canal.

OBJECTIVES: To evaluate the long-term effect of chlorhexidine (CHX) and dimethyl sulfoxide (DMSO) on the sealing ability and biomineralization of two different calcium silicate cements (CSC) in root canal. METHODS: Sixty human third molar root canals were obturated with ProRoot MTA or Biodentine. Before obturation the canals were irrigated with saline (control), 2% CHX or 5% DMSO. Microleakage was tested after three days and after six months. After additional six months (12 months after root filling) the roots were cut into 2 mm thick dentine discs. The discs were stored in artificial saliva for one year. The bond strength was measured with the push-out method, and the failure mode was evaluated with a stereomicroscope. The most apical disc of each tooth was used for Vickers hardness test. RESULTS: No significant differences between the groups was found in initial microleakage. The leakage increased significantly during the 6-month storage in all groups except in Biodentine-CHX group and Biodentine-DMSO group. CHX and DMSO irrigation significantly increased the leakage with ProRoot MTA with time, but there was no statistically significant difference compared to the ProRoot MTA-control group at six months' time point. CHX significantly reduced the push-out bond strength of ProRoot MTA. With Biodentine irrigation with CHX or DMSO resulted with significantly higher push-out strength compared to the Biodentine control group. Fracture analysis showed statistically significant difference in the distribution of the fractures between the groups, but neither CHX nor DMSO change the fracture pattern statistically significantly. With Vickers hardness test ProRoot MTA with and without DMSO as the final irrigant showed significantly higher dentin hardness than any Biodentine-group. SIGNIFICANCE: Considering that aging increased the leakage in all groups except with Biodentine-DMSO and the differences in the push-out strength and surface microhardness data, it appears that the time-related biomineralizing effect of MTA and Biodentine does not improve sealing to dentin. CHX significantly reduced ProRoot MTA bond strength and increased pure adhesive failures with both cements.

The Effect of Chlorhexidine and Dimethyl Sulfoxide on Long-Term Microleakage of Two Different Sealers in Root Canals.

OBJECTIVE: The aim of root canal obturation is to prevent leakage and inhibit microbial invasion. This study aimed to determine the effect of chlorhexidine (CHX) and dimethyl sulfoxide (DMSO) as final irrigants on microleakage of root filling immediately and after 18 months. The hypothesis was that either CHX or DMSO would not affect the immediate or long-term microleakage. METHODS: A total of 120 human third molar root canals were obturated with RealSeal SE or Topseal and gutta percha. Before obturation, the canals were irrigated with saline (control), 2% CHX or 5% DMSO. Microleakage of half of each groups (n=10) was measured after 3 days, and of the other half was measured after 18 months with fluid filtration method. RESULTS: In immediate measurements, RealSeal SE performed significantly better in CHX-irrigated group (p=0.035; Mann-Whitney test). For both sealers, DMSO had the lowest mean microleakage values, which were also statistically significantly lower than with CHX irrigation within sealers (p<0.009 for Topseal and p=0.04 for RealSeal SE; Mann-Whitney test). With RealSeal SE, the microleakage with CHX was significantly higher than that in controls (p=0.022; Mann-Whitney test). CONCLUSION: Neither final irrigant showed statistically significant differences in the immediate microleakage within the two sealers. Irrigation with DMSO caused significantly less microleakage than CHX for both sealers after 18 months.

European Society of Endodontology position statement: the use of antibiotics in endodontics.

This position statement represents a consensus of an expert committee convened by the European Society of Endodontology (ESE) on Antibiotics in Endodontics. The statement is based on current scientific evidence as well as the expertise of the committee. The goal is to provide dentists and other healthcare workers with evidence-based criteria for when to use antibiotics in the treatment of endodontic infections, traumatic injuries of the teeth, revascularization procedures in immature teeth with pulp necrosis, and in prophylaxis for medically compromised patients. It also highlights the role that dentists and others can play in preventing the overuse of antibiotics. A recent review article provides the basis for this position statement and more detailed background information (International Endodontic Journal, 2017, https://doi.org/10.1111/iej.12741). Given the dynamic nature of research in this area, this position statement will be updated at appropriate intervals.

Role of Proteolytic Enzyme Inhibitors on Carious and Eroded Dentin Associated With a Universal Bonding System.

OBJECTIVES: The aim of this study was to evaluate the effect of proteolytic inhibitors on the bond strength of a universal adhesive system (etch-and-rinse mode) applied to artificial carious and eroded dentin. METHODS: Ninety molars were prepared and randomly divided into three groups according to the substrate: N, no challenges; ACD, artificial carious dentin simulation and ERO, artificial erosion simulation with orange juice. All groups were redivided into three subgroups according to the dentin pretreatment: W, water; CHX, 2% digluconate chlorhexidine; and E-64 (trans-epoxysuccinyl-L-leucylamido-[4-guanidino] butane), 5 µM E-64 inhibitor. They constituted a total of nine groups (n=10): N-W, N-CHX, N-E64, ACD-W, ACD-CHX, ACD-E64, ERO-W, ERO-CHX, and ERO-E64. All specimens were restored with Adper Single Bond Universal/Filtek Z250. Beams (0.64 mm2) were obtained and subjected to the microtensile test (µTBS) in a universal testing machine at 0.5 mm/min. The failure mode of the interfaces was determined by optical microscopy (40× magnification). Data were statistically analyzed by three-way analysis of variance and Tukey tests (p<0.05). RESULTS: All individual factors (p<0.0001) and the interaction between substrate and treatment (p=0.0011) and between substrate and time (p=0.0003) were statistically significant. The caries substrate contributed negatively to bond strength. Chlorhexidine reduced bond strength for normal and eroded conditions. Only the normal substrate was negatively affected by time despite the pretreatment. CONCLUSIONS: The universal bonding system appears to be a promising bonding strategy for the maintenance of bond strength to affected dentin. E-64 did not affect bonding to the dentin in contrast to the use of chlorhexidine, which, when associated with the universal system, did affect the microtensile bond strength for artificial carious dentin.

PAR-1 and PAR-2 Expression Is Enhanced in Inflamed Odontoblast Cells.

Protease-activated receptors (PARs) are G protein-coupled receptors, which are activated by proteolytical cleavage of the amino-terminus and act as sensors for extracellular proteases. We hypothesized that PAR-1 and PAR-2 can be modulated by inflammatory stimulus in human dental pulp cells. PAR-1 and PAR-2 gene expression in human pulp tissue and MDPC-23 cells were analyzed by quantitative polymerase chain reaction. Monoclonal PAR-1 and PAR-2 antibodies were used to investigate the cellular expression of these receptors using Western blot, flow cytometry, and confocal microscopy in MDPC-23 cells. Immunofluorescence assays of human intact and carious teeth were performed to assess the presence of PAR-1 and PAR-2 in the dentin-pulp complex. The results show for the first time that human odontoblasts and MDPC-23 cells constitutively express PAR-1 and PAR-2. PAR-2 activation increased significantly the messenger RNA expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MMP-14 in MDPC-23 cells ( P < 0.05), while the expression of these enzymes decreased significantly in the PAR-1 agonist group ( P < 0.05). The high-performance liquid chromatography and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis showed the presence of MMP-13 activity cleaving PAR-1 at specific, noncanonical site TLDPRS42↓F43LL in human dental pulp tissues. Also, we detected a presence of a trypsin-like activity cleaving PAR-2 at canonical site SKGR20↓S21LIGRL in pulp tissues. Confocal microscopy analysis of human dentin-pulp complex showed intense positive staining of PAR-1 and PAR-2 in the odontoblast processes in dentinal tubules of carious teeth compared to intact ones. The present results support the hypothesis of activation of the upregulated PAR-1 and PAR-2 by endogenous proteases abundant during the inflammatory response in dentin-pulp complex.

Effect of dimethyl sulfoxide on dentin collagen.

OBJECTIVES: Infiltration of adhesive on dentin matrix depends on interaction of surface and adhesive. Interaction depends on dentin wettability, which can be enhanced either by increasing dentin surface energy or lowering the surface energy of adhesive. The objective was to examine the effect of dimethyl sulfoxide (DMSO) on demineralized dentin wettability and dentin organic matrix expansion. METHODS: Acid-etched human dentin was used for sessile drop contact angle measurement to test surface wetting on 1-5% DMSO-treated demineralized dentin surface, and linear variable differential transformer (LVDT) to measure expansion/shrinkage of dentinal matrix. DMSO-water binary liquids were examined for surface tension changes through concentrations from 0 to 100% DMSO. Kruskal-Wallis and Mann-Whitney tests were used to test the differences in dentin wettability, expansion and shrinkage, and Spearman test to test the correlation between DMSO concentration and water surface tension. The level of significance was p<0.05. RESULTS: Pretreatment with 1-5% DMSO caused statistically significant concentration-dependent increase in wetting: the immediate contact angles decreased by 11.8% and 46.6% and 60s contact angles by 9.5% and 47.4% with 1% and 5% DMSO, respectively. DMSO-water mixtures concentration-dependently expanded demineralized dentin samples less than pure water, except with high (≥80%) DMSO concentrations which expanded demineralized dentin more than water. Drying times of LVDT samples increased significantly with the use of DMSO. SIGNIFICANCE: Increased dentin wettability may explain the previously demonstrated increase in adhesive penetration with DMSO-treated dentin, and together with the expansion of collagen matrix after drying may also explain previously observed increase in dentin adhesive bonding.

Substantivity of Carbodiimide Inhibition on Dentinal Enzyme Activity over Time.

The use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) has recently been investigated for its effectiveness in the prevention of collagen degradation over time and the improvement of resin-dentin bond durability. The objective of the present study was to evaluate the effects of a 0.3 M EDC-containing conditioner on endogenous enzymatic activities within the hybrid layer (HL) created by a self-etch or an etch-and-rinse adhesive after 1 y. The activity within the HL was examined using in situ zymography and confocal laser scanning microscopy after 24 h or 1-y storage in artificial saliva. Dentin specimens were bonded with Clearfil SE Bond (CSE) or XP Bond (XPB). For CSE, the self-etching primer was applied and treated with 0.3 M EDC for 1 min, and then the bonding agent was applied. For XPB, dentin was etched and treated with 0.3 M EDC for 1 min and then bonded with the primer-bonding agent. Control specimens were prepared without EDC treatment. Slices containing the adhesive-dentin interface were covered with fluorescein-conjugated gelatin and observed with a multiphoton confocal microscope. Fluorescence intensity emitted by hydrolyzed fluorescein-conjugated gelatin was quantified, and the amount of gelatinolytic activity was represented by the percentage of green fluorescence emitted within the HL. After 24 h of storage, enzymatic activity was detected by in situ zymography within the HLs of both tested adhesives, with XPB higher than CSE ( P < 0.05). Almost no fluorescence signal was detected when specimens were pretreated with EDC compared to controls ( P < 0.05). After 1 y of storage, enzymatic activities significantly increased for all groups (excluding XPB control) compared to 24-h storage ( P < 0.05), with EDC pretreated specimens exhibiting significantly lower activity than controls ( P < 0.05). The present study showed, for the first time, that the use of EDC for both the self-etch and the etch-and-rinse approaches results in the reduction but not complete inhibition of matrix-bound collagenolytic enzyme activities over time in the HL.

2-Hydroxyisocaproic acid is bactericidal in human dental root canals ex vivo.

AIM: To compare the activity of 2-hydroxyisocaproic acid (HICA), calcium hydroxide (Ca(OH)2 ) and chlorhexidine digluconate (CHG) against Enterococcus faecalis T-75359 (root canal isolate) in the root canals of extracted human teeth. METHODOLOGY: Bacterial suspensions (108  cfu mL-1 ) were incubated in root canals with 0.9 mm diameter root blocks (n = 73) for 21 days. Bacterial penetration into dentine was analysed by the Brown and Brenn method (n = 5). Canals (n = 17/group) were medicated with 40% of HICA paste, 40% of Ca(OH)2 paste, 2% of CHG solution or 0.9% of saline solution for 7 days. Samples taken from the inner (first 0.1 mm) and deeper (second 0.1 mm) dentine, and residual roots were cultured in broth for 24 h. Bacterial growth was detected by spectrophotometry (optical density, OD) and confirmed by culture on agar. The OD data were analysed with Kruskal-Wallis and Friedman with Wilcoxon signed-rank test between and within groups, respectively, and agar culture data with Pearson chi-square with Mann-Whitney and Cochran with McNemar tests, respectively (P < 0.05). RESULTS: Bacterial invasion into dentine tubules was confirmed. In deeper dentine, HICA inhibited >90% of bacterial growth in comparison with saline. No bacterial growth was observed in 82-100% of inner and deeper dentine samples. CHG prevented the growth in 88%, Ca(OH)2 in 59-76% and saline in 65-71%, respectively. HICA was significantly more active than Ca(OH)2 (P = 0.008) in the residual roots. The viability testing on agar showed essentially the same result. CONCLUSION: HICA paste exerted superior activity against E. faecalis and could have potential for root canal medication.

Antibiotics in Endodontics: a review.

The overuse of antibiotics and the emergence of antibiotic-resistant bacterial strains is a global concern. This concern is also of importance in terms of the oral microbiota and the use of antibiotics to deal with oral and dental infections. The aim of this paper was to review the current literature on the indications and use of antibiotics and to make recommendations for their prescription in endodontic patients. Odontogenic infections, including endodontic infections, are polymicrobial, and in most cases, the prescription of antibiotics is empirical. This has led to the increasing use of broad-spectrum antibiotics even in cases where antibiotics are not indicated, such as symptomatic irreversible pulpitis, necrotic pulps and localized acute apical abscesses. In case of discrete and localized swelling, the primary aim is to achieve drainage without additional antibiotics. Adjunctive antibiotic treatment may be necessary in the prevention of the spread of infection, in acute apical abscesses with systemic involvement and in progressive and persistent infections. Medically compromised patients are more susceptible to complication arising from odontogenic infections and antimicrobials have a more specific role in their treatment. Therefore, antibiotics should be considered in patients having systemic diseases with compromised immunity or in patients with a localized congenital or acquired altered defence capacity, such as patients with infective endocarditis, prosthetic cardiac valves or recent prosthetic joint replacement. Penicillin VK, possibly combined with metronidazole to cover anaerobic strains, is still effective in most cases. However, amoxicillin (alone or together with clavulanic acid) is recommended because of better absorption and lower risk of side effects. In case of confirmed penicillin allergy, lincosamides such as clindamycin are the drug of choice.

Association of Endodontic Lesions with Coronary Artery Disease.

An endodontic lesion (EL) is a common manifestation of endodontic infection where Porphyromonas endodontalis is frequently encountered. EL may associate with increased risk for coronary artery disease (CAD) via similar pathways as marginal periodontitis. The aim of this cross-sectional study was to delineate the associations between EL and CAD. Subgingival P. endodontalis, its immune response, and serum lipopolysaccharide were examined as potential mediators between these 2 diseases. The Finnish Parogene study consists of 508 patients (mean age, 62 y) who underwent coronary angiography and extensive clinical and radiographic oral examination. The cardiovascular outcomes included no significant CAD ( n = 123), stable CAD ( n = 184), and acute coronary syndrome (ACS; n = 169). EL was determined from a panoramic tomography. We combined data of widened periapical spaces (WPSs) and apical rarefactions to a score of EL: 1, no EL ( n = 210); 2, ≥1 WPS per 1 apical rarefaction ( n = 222); 3, ≥2 apical rarefactions ( n = 76). Subgingival P. endodontalis was defined by checkerboard DNA-DNA hybridization analysis, and corresponding serum antibodies were determined by ELISA. In our population, 50.4% had WPSs, and 22.8% apical rarefactions. A total of 51.2% of all teeth with apical rarefactions had received endodontic procedures. Subgingival P. endodontalis levels and serum immunoglobulin G were associated with a higher EL score. In the multiadjusted model (age, sex, smoking, diabetes, body mass index, alveolar bone loss, and number of teeth), having WPSs associated with stable CAD (odds ratio [OR] = 1.94, 95% confidence interval [95% CI] = 1.13 to 3.32, P = 0.016) and highest EL score were associated with ACS (OR = 2.46, 95% CI = 1.09 to 5.54, P = 0.030). This association was especially notable in subjects with untreated teeth with apical rarefactions ( n = 59, OR = 2.72, 95% CI = 1.16 to 6.40, P = 0.022). Our findings support the hypothesis that ELs are independently associated with CAD and in particular with ACS. This is of high interest from a public health perspective, considering the high prevalence of ELs and CAD.

Use of crosslinkers to inactivate dentin MMPs.

OBJECTIVES: This study evaluated the endogenous matrix metalloproteinase (MMP) activity of demineralized dentin matrix following 1 or 5 min pretreatment by various collagen crosslinkers. Generic MMP activity assay, total protein analysis, in situ zymography, gelatin zymography and multiplex bead technology were used to evaluate matrix-bound MMP activity. METHODS: Six different crosslinkers; glutaraldehyde, riboflavin/UVA, riboflavin-5-monophospate/UVA, sumac berry extract, grape seed extract, and curcumin were used. Demineralized dentin beams were pretreated with respective crosslinkers for 1 or 5 min. Demineralized dentin beams with no crosslinker pretreatment served as control. The reduction in the total activity of dentin matrices were measured using generic MMP activity assay. Dentin slabs were used for in situ zymography and evaluated by using hydrolysis of self-quenched fluorescein-conjugated gelatin under confocal microscopy. Dentin beam extracts were used for total protein assay and multiplex analysis and powder extracts were used for gelatin zymography. RESULTS: MMP activity in crosslinker pretreated samples decreased significantly between 21% and 70%, whereas untreated control samples' activity increased up to 84%. Zymograms confirmed a decrease in the gelatinolytic activity and in the amount of extractable total protein content. Multiplex analysis of extracts of crosslinker-treated dentin showed a reduction in the MMP-8, MMP-2 and MMP-9 release. SIGNIFICANCE: The result of this work suggests that the effect of the crosslinkers is source-dependent. The use of crosslinkers for as little as 1min on demineralized dentin can inactivate the endogenous protease activity of dentin matrices.

Enzymatic isolation of viable human odontoblasts.

AIM: To improve an enzymatic method previously used for isolation of rat odontoblasts to isolate viable mature human odontoblasts. METHODOLOGY: Collagenase I, collagenase I/hyaluronidase mixture and hyaluronidase were used to extract mature human odontoblasts from the pulp chamber. Detachment of odontoblasts from dentine was determined with field emission scanning electron microscopy (FESEM) and to analyse the significance of differences in tubular diameter, and the t-test was used. MTT-reaction was used to analyse cell viability, and nonparametric Kruskal-Wallis and Mann-Whitney post hoc tests were used to analyse the data. Immunofluorescent staining of dentine sialoprotein (DSP), aquaporin-4 (AQP4) and matrix metalloproteinase-20 (MMP-20) and quantitative PCR (qPCR) of dentine sialophosphoprotein (DSPP) were used to confirm the odontoblastic nature of the cells. RESULTS: MTT-reaction and FESEM demonstrated collagenase I/hyaluronidase resulted in more effective detachment and higher viability than collagenase I alone. Hyaluronidase alone was not able to detach odontoblasts. Immunofluorescence revealed the typical odontoblastic-morphology with one process, and DSP, AQP4 and MMP-20 were detected. Quantitative PCR of DSPP confirmed that the isolated cells expressed this odontoblast-specific gene. CONCLUSION: The isolation of viable human odontoblasts was successful. The cells demonstrated morphology typical for odontoblasts and expressed characteristic odontoblast-type genes and proteins. This method will enable new approaches, such as apoptosis analysis, for studies using fully differentiated odontoblasts.

Antimicrobial 2-hydroxyisocaproic acid and chlorhexidine resist inactivation by dentine.

AIM: To compare the antibacterial activity of 2-hydroxyisocaproic acid (HICA) with currently used root canal medicaments and to examine their interactions with potential inhibitors in nutrient-deficient and nutrient-rich conditions. METHODOLOGY: First, the antibacterial activity of single concentrations of HICA, calcium hydroxide solution or slurry, chlorhexidine digluconate or acetate was tested against Enterococcus faecalis with and without potential inhibitors: dentine powder (DP), hydroxyapatite or bovine serum albumin, in a low concentration of peptone water. Relative viable counts were determined by culture at 1, 24 and 48 h. In the second set of experiments, the activity of three concentrations of HICA was evaluated against two isolates of E. faecalis with and without potential inhibitors in nutrient-rich thioglycollate broth using a modification of a standard microdilution method. The minimum bactericidal concentration was determined by culture at 1, 24 and 48 h. RESULTS: Concentrations of ≥33 mg mL(-1) of HICA were found to be bactericidal against E. faecalis in both nutrient-deficient and nutrient-rich environments at 24- to 48-h incubation, whereas the initial activity of Ca(OH)2 slurry was lost at 48-h incubation. HICA tolerated well all tested potential inhibitors up to 19 mg mL(-1) . DP concentrations higher than this inhibited its activity in a dose-dependent manner in both environments. DP demonstrated moderate antibacterial activity, and it enhanced the otherwise limited activity of Ca(OH)2 slurry and solution. DP did not impact on the activity of chlorhexidine. CONCLUSIONS: These results support the long-term antibacterial activity of HICA and indicate its tolerance to clinically relevant concentrations of dentine and other inhibitors commonly present in the root canal system. Therefore, HICA may have potential as an interappointment medication in the treatment of root canal infections.

The role of matrix metalloproteinases and cysteine-cathepsins on the progression of dentine erosion.

OBJECTIVE: To evaluate in vitro the effect of the inhibition of endogenous dentinal enzymes (matrix metalloproteinases-MMPs and cysteine cathepsins-CCs) on dentine erosion. DESIGN: Dentine blocks (4mm×4mm×2mm) from sound human teeth were randomly divided into 7 groups (n=17) according to the treatment: MMP- and CC-inhibitor chlorhexidine digluconate (CHX, 10mM); MMP-inhibitor galardin (G, 0.2mM); specific cathepsin B inhibitor (CCB, 0.2mM); non-specific CC inhibitor (CCE-64, 0.5µM); fluoride (F, 1.23% NaF); placebo (P) and untreated (UT). Inhibitors were applied as gels once for 1min. Specimens were submitted to 5 days of pH cycling including the erosive challenge (Coke, pH 2.64, 90s/day) and remineralisation (artificial saliva). Demineralised organic surface loss was determined profilometrically. Demineralised organic matrix (DOM) was removed with collagenase and the profile was re-evaluated in the absence of collagen fibrils. The differences in profilometric results and DOM thickness among the groups were analysed with ANOVA and Tukey's test (p<0.05). RESULTS: Loss of demineralised tissue (µm, mean±SD) was: CHX 8.4±1.7 b, G 8.6±1.9 b, CCB 9.6±1.4 a, CCE-64 9.9±1.3 a, F 9.9±1.7 a, P 10.9±2.2 a, UT 11.0±1.5 a. Loss of mineralised tissue was: CHX 15.4±2.2 b, G 16.0±1.8 b, CCB 17.6±2.4 a, CCE-64 17.6±2.0 a, F 17.3±2.8 a, P 19.1±2.1 a, UT 18.9±2.4 a. MMP-inhibitors significantly reduced organic matrix and mineral loss in comparison to all the other groups (p<0.05). No statistically significant differences were found in the thickness of the remaining DOM (p=0.845). CONCLUSION: Dentine endogenous MMPs seem to be the main enzymes responsible for DOM loss and erosion.

Cytotoxicity of dimethyl sulfoxide (DMSO) in direct contact with odontoblast-like cells.

OBJECTIVES: To evaluate the cytotoxicity of dimethyl sulfoxide (DMSO) on the repair-related activity of cultured odontoblast-like MDPC-23 cells. METHODS: Solutions with different concentrations of DMSO (0.05, 0.1, 0.3, 0.5 and 1.0 mM), diluted in culture medium (DMEM), were placed in contact with MDPC-23 cells (5 × 104 cells/cm(2)) for 24 h. Eight replicates (n = 8) were prepared for each solutions for the following methods of analysis: violet crystal dye for cell adhesion (CA), quantification of total protein (TP), alizarin red for mineralization nodules formation (MN) and cell death by necrosis (flow cytometry); while twelve replicates (n = 12) were prepared for viable cell number (Trypan Blue) and cell viability (MTT assay). Data were analyzed by ANOVA and Tukey or Kruskal-Wallis and Mann-Whitney's tests (p < 0.05). RESULTS: Cell viability, adhesion and percentage of cell death by necrosis were not affected by DMSO at any concentration, with no statistical significant difference among the groups. A significant reduction in total protein production was observed for 0.5 and 1.0 mM of DMSO compared to the control while increased mineralized nodules formation was seen only for 1.0 mM DMSO. SIGNIFICANCE: DMSO caused no or minor cytotoxic effects on the pulp tissue repair-related activity of odontoblast-like cells.