Evaluation of adhesive properties and enzymatic activity at the hybrid layer of a simplified adhesive loaded with 0.2 % Cu and 5 % ZnO nanoparticles: A Randomized Clinical Trial and ex vivo analysis.

OBJECTIVE: The aim of this study was to evaluate the effect of an adhesive loaded with 0.2 % copper (Cu) and 5 % zinc oxide (ZnO) nanoparticles (Nps) on its adhesive properties and enzymatic activity at the hybrid layer ex vivo in a randomized clinical model. METHODS: Fifteen patients participated in this study, and a total of 30 third molars were used. Occlusal cavities (4 × 4 × 2 mm) were made in each tooth, and randomly divided into 2 groups: (i) Experimental group: commercial adhesive loaded with 0.2wt % CuNps and 5wt % ZnONps; and (ii) Control Group: non-loaded commercial adhesive. Teeth were restored with resin composite. Thirty days later, extractions were performed. Extracted teeth were longitudinally sectioned. Nps in powder were characterized by field emission scanning electron microscope (FE-SEM) and energy dispersive X-ray (EDX) analysis. Microtensile bond strength (µTBS), degree of conversion (DC), and nanoleakeage (NL) tests were executed. In situ zymography (Zym) was performed to evaluate the gelatinolytic activity at the hybrid layer. Student's t-test (α = 0.05) was applied for all tests. RESULTS: µTBS and DC did not show significant differences (p > 0.05) between both groups. However, NL and gelatinolytic activity at the hybrid layer showed significant values (p < 0.05) for experimental group in comparison with control group. CONCLUSION: The addition of 0.2 % CuNps and 5 % ZnONps to a universal adhesive decreases NL and gelatinolytic activity at the hybrid layer, without jeopardizing its adhesive properties. SIGNIFICANCE: This randomized clinical trial with ex vivo analysis demonstrate that a commercial adhesive modified with 0.2wt % Cu and 5wt % ZnO Nps that does not affect its adhesive properties, reducing gelatinolytic activity and nanoleakage at the hybrid layer, which should contribute to an improvement of long term bonding-dentine clinical performance.

Acid challenge exacerbates activation of matrix metalloproteinases in permanent teeth undergoing radiotherapy.

The aim of this study was to investigate the effect of acid challenge on the activation of matrix metalloproteinases (MMPs) in the Dentinoenamel junction of primary and permanent teeth submitted to radiotherapy. For this purpose, a total of 178 dental fragments obtained from molars were used, and randomly divided into 2 groups (primary and permanent teeth) / 4 experimental subgroups (irradiated and non-irradiated, demineralized and non-demineralized). The fragments were exposed to radiation, with a dose fraction of 2 Gy, for 5 consecutive days, until a total dose of 60 Gy was reached, with a total of 30 cycles, for 6 weeks. To determine the activity of MMPs on the dentinoenamel junction (DEJ), in situ zymography assays on 0.6mm dental fragments were performed. To assess whether MMP activity would be impacted by an acidic environment, the fragments were placed in a demineralizing solution (pH of 4.8). The finding was that irradiation activated MMPs in DEJ and these effects were more evident in permanent when compared with primary teeth. When the effect of an acid challenge on MMPs activity was investigated, demineralization was observed not to increase MMPs activity in non-irradiated teeth, but it did increase MMPs activity in irradiated teeth. In conclusion, an acid challenge was found to exacerbate activation of MMPs in DEJ of permanent teeth submitted to irradiation, but not in primary teeth.

The inhibitory effects of various ions released from S-PRG fillers on dentin protease activity.

This study investigates the effect of ions released from S-PRG fillers on host-derived enzymatic degradation of dentin collagen matrices. Dentin beams (n=80) were demineralized and distributed to eight groups following baseline dry mass and total MMP activity assessments. Each group treated with boron, fluoride, sodium, silicone, strontium, aluminium, or S-PRG eluate solutions for 5 min. Untreated beams served as control. After pre-treatment, MMP activity was reassessed, beams were incubated in complete medium for 1 week, dry mass was reassessed. Incubation media were analyzed for MMP and cathepsin-K-mediated degradation fragments. Data were analyzed with ANOVA and Tukey's test. All pretreatment groups showed significant reduction in total MMP activity (p<0.05) that was sustainable after incubation in all groups except for boron and silicone groups (p<0.05). Cathepsin-K activity did not differ between control or treatment groups. The results indicated that ions released from S-PRG fillers have the potential to partly inhibit MMP-mediated endogenous enzymatic activity.

Loss of mutual protection between human osteoclasts and chondrocytes in damaged joints initiates osteoclast-mediated cartilage degradation by MMPs.

Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during skeletal maintenance, development and in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcoma. This study explores the mechanisms behind the osteoclast-cartilage interaction. Human osteoclasts differentiated on acellular human cartilage expressed osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR), visibly damaged the cartilage surface and released glycosaminoglycan in a contact-dependent manner. Direct co-culture with chondrocytes during differentiation increased large osteoclast formation (p < 0.0001) except when co-cultured on dentine, when osteoclast formation was inhibited (p = 0.0002). Osteoclasts cultured on dentine inhibited basal cartilage degradation (p = 0.012). RNA-seq identified MMP8 overexpression in osteoclasts differentiated on cartilage versus dentine (8.89-fold, p = 0.0133), while MMP9 was the most highly expressed MMP. Both MMP8 and MMP9 were produced by osteoclasts in osteosarcoma tissue. This study suggests that bone-resident osteoclasts and chondrocytes exert mutually protective effects on their 'native' tissue. However, when osteoclasts contact non-native cartilage they cause degradation via MMPs. Understanding the role of osteoclasts in cartilage maintenance and degradation might identify new therapeutic approaches for pathologies characterized by cartilage degeneration.

Plasma Membrane Ca2+-ATPase in Rat and Human Odontoblasts Mediates Dentin Mineralization.

Intracellular Ca2+ signaling engendered by Ca2+ influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca2+ is exported by the Na+-Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA) to maintain Ca2+ homeostasis. We previously demonstrated a functional coupling between Ca2+ extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under physiological conditions. We also examined the role of PMCA in the Ca2+ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca2+ concentration ([Ca2+]i). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca2+]i was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor teeth and human odontoblasts. We detected mRNA encoding PMCA1-4 in human odontoblasts. The application of hypotonic or alkaline solutions transiently increased [Ca2+]i in odontoblasts in both rat and human odontoblasts. The Ca2+ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca2+]i increase was decreased by PMCA inhibitors in both cell types. Alizarin red and von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did not affect DMP-1 or DSPP mRNA levels at pH 7.4-8.8 and under isotonic and hypotonic conditions, respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These findings indicate that PMCA participates in maintaining [Ca2+]i homeostasis in odontoblasts by Ca2+ extrusion following [Ca2+]i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca2+ to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of high-pH dental materials.

Endogenous Enzymatic Activity in Dentin Treated with a Chitosan Primer.

The aim of this study was to evaluate the effect of different concentrations of chitosan polymer on dentinal enzymatic activity by means of gelatin and in situ zymography. Human dentin was frozen and ground in a miller. Dentin powder aliquots were demineralized with phosphoric acid and treated with three different concentrations of lyophilized chitosan polymer (1, 0.5 and 0.1 wt%) dissolved in distilled water. Dentin proteins were extracted from each experimental group and electrophoresed under non-reducing conditions in 10% SDS-PAGE containing fluorescein-labeled gelatin. After 48 h in the incubation buffer at 37 °C, proteolytic activity was registered under long-wave UV light scanner and quantified by using Image J software. Furthermore, additional teeth (n = 4) were prepared for the in situ zymographic analysis in unrestored as well as restored dentin pretreated with the same chitosan primers. The registered enzymatic activity was directly proportional to the chitosan concentration and higher in the restored dentin groups (p < 0.05), except for the 0.1% chitosan primer. Chitosan 0.1% only showed faint expression of enzymatic activity compared to 1% and 0.5% concentrations. Chitosan 0.1% dissolved in water can produce significant reduction in MMPs activity and could possibly contribute to bond strength preservation over time.

Novel Approach to Tooth Chemistry. Quantification of the Dental-Enamel Junction.

The dentin-enamel junction (DEJ) is known for its special role in teeth. Several techniques were applied for the investigation of the DEJ in human sound molar teeth. The electron (EPMA) and proton (PIXE) microprobes gave consistent indications about the variability of elemental concentrations on this boundary. The locally increased and oscillating concentrations of Mg and Na were observed in the junction, in the layer adhering to the enamel and covering roughly half of the DEJ width. The chemical results were compared with the optical profiles of the junction. Our chemical and optical results were next compared with the micromechanical results (hardness, elastic modulus, friction coefficient) available in the world literature. A strong correlation of both result sets was proven, which testifies to the self-affinity of the junction structures for different locations and even for different kinds of teeth and techniques applied for studies. Energetic changes in tooth strictly connected with crystallographic transformations were calculated, and the minimum energetic status was discovered for DEJ zone. Modeling of both walls of the DEJ from optical data was demonstrated. Comparing the DEJ in human teeth with the same structure found in dinosaur, shark, and alligator teeth evidences the universality of dentin enamel junction in animal world. The paper makes a contribution to better understanding the joining of the different hard tissues.

[The Effect of PAMAM Dendrimer on the Dentin Remineralization and Matrix Metalloproteinases Activity].

OBJECTIVE: The purpose of this study was to investigate the effect of G4.5 carboxyl-terminated poly dendrimer (PAMAM-COOH) on the dentin remineralization and the matrix metalloproteinases (MMPs) activity. METHODS: The dentine samples were averagely divided into four groups: 100 mg/mL PAMAM-COOH group (A group), 10 mg/mL PAMAM-COOH group (B group), 2% (wt) chlorhexidine (CHX) group (C group) and deionized water group (Control group). MMP Activity Assay Kit was used to detect the activity of dentin endogenous MMPs in the four groups. The loss of dry mass of dentin after 30 d were measured. In situ zymography analysis was performed to detect the effects of PAMAM dendrimer in each group (except A group) on gelatinase activity in dentin. After incubation in artificial saliva for 7 and 14 d incubated, the remineralization of each group (except A group) in dentin surfaces were examined using a field emission-scanning electron microscope (FESEM). RESULTS: Compared with the control group, the dentin endogenous MMPs activity in A, B and C groups were all decreased ( P<0.05). The activity of endogenous MMPs in C group was lower than that of A and B groups ( P<0.001), but the difference between A and B groups was not statistically significant. The loss of dry mass in A, B and C groups were lower than that in control group ( P<0.05), but there were no significant difference in A, B and C groups. The in situzymography analysis showed that 48 h later, the dentin gelatinase activity in B group was inhibited compared with the control group, but the inhibitory effect was weaker than that of CHX. After 7 d and 14 d, there were no obvious mineralization in the control group, while distinct mineralization were observed in B group. The mineralization effect in group B was better than group C. CONCLUSION: G4.5 PAMAM-COOH could introduce remineralizationin and demineralizeddentin by effectively inhibiting endogenous MMPs and gelatinase, thus contributes as novel material to enhancing durability of adhesion.

A new method for dentine matrix metalloproteinase extraction.

In addition to their involvement in tissue remodelling, matrix metalloproteinases (MMPs), in the oral environment, are linked to leakage in the resin-dentine interface through their involvement in the proteolytic degradation of the resin-dentine hybrid layer. Numerous studies have evaluated dentine MMP activity and the vast majority of those studies have used an MMP extraction protocol to semi-purify the MMPs from dentine, first described around 20 years ago. This is a protocol that requires 32 days for completion. The technique is based on the three-step sequential use of NaCl solution for pulverised dentine washing, then guanidine HCl and EDTA to demineralise the pulverised dentine to extract the MMPs. In this study, a new one-step dentine MMP extraction protocol was adapted to extract dentine MMPs in only four days. This was achieved by eliminating the NaCl washing step and combining the guanidine HCl and EDTA into one extraction solution that also contained proteinase inhibitors. Fifty-two dentine MMP extracts were obtained utilising the two different extraction methods. The amount of total and endogenously active MMP-2 in the specimens was assayed utilising a human MMP-2 activity enzyme-linked immunosorbent assay (ELISA). The study results showed that the new extraction method is as effective as the traditional three-step extraction method in semi-purifying dentine MMP-2.

Carbodiimide inactivation of matrix metalloproteinases in radicular dentine.

OBJECTIVES: The present in vitro study evaluated the effect of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), a cross-linking agent used as an additional therapeutic primer for luting fiber posts to radicular dentine to prevent hybrid layer degradation. METHODS: Root canal treatment was performed on 80 extracted single-rooted human teeth. A 10-mm post space was prepared and pecimens were randomly assigned to four groups (n=20) according to the bonding system: 1) All Bond 3 (Bisco); 2) All Bond 3 + 0.3M EDC; 3) Prime&Bond XP (Dentsply Sirona); 4) Prime&Bond XP + 0.3M EDC. In groups 2 and 4, EDC was applied on phosphoric acid-etched dentine for 1 min. Fiber posts (RelyX Fiber Post, 3M ESPE) were luted with a dual-cured resin cement (Core-X flow, Dentsply Sirona). Slices were prepared for micro push-out test and interfacial nanoleakage evaluation of the coronal and apical region of the canal space after 24 h and 1 year storage in artificial saliva. In-situ zymography was performed to investigate endogenous matrix metalloproteinase activities within the hybrid layer. Results were statistically analysed with three-way ANOVA test or Chi Square test. Statistical significance was set at α=0.05. RESULTS: No significant influence was identified between the two adhesives. The use of EDC significantly improved fiber post bond strength at 1 year but not at 24 h. Application of 0.3 M EDC prior to bonding significantly reduced gelatinolytic activities within the radicular hybrid layers. CONCLUSIONS: Carbodiimide was effective in preserving fibre post bond strength over time, through reducing the activities of intra-radicular endogenous proteases. CLINICAL SIGNIFICANCE: Inhibition of matrix metalloproteinases using EDC over radicular dentin could play an important role in bond strength preservation. However, the clinical relevance of these findings needs to be proven.

Radiotherapy Activates and Protease Inhibitors Inactivate Matrix Metalloproteinases in the Dentinoenamel Junction of Permanent Teeth.

The objectives of this study were to investigate changes in the activity and expression of matrix metalloproteinase (MMP)-2 and MMP-9 in permanent teeth with or without exposure to radiotherapy, and the role of proteinase inhibitors in their inactivation. In situ zymography and immunofluorescence assays were performed to evaluate the activity and expression of two key gelatinases (MMP-2 and MMP-9) in sections of permanent molars, assigned to irradiated and nonirradiated subgroups. Dental fragments were exposed to radiation at a dose of 2 Gy fractions for 5 consecutive days until a cumulative dose of 60 Gy was reached. To evaluate the effect of protease inhibitors on MMPs, teeth were immersed in 0.5 mL of 0.12% chlorhexidine digluconate (CHX), 0.05% sodium fluoride (NaF), 400 µM polyphenol epigallocatechin-3-gallate (EGCG), or distilled water (control) for 1 h. Fluorescence in the dentinoenamel junction (DEJ) was evaluated in 3 areas of the tooth: cervical, cuspal, and pit. These regions were photographed using a fluorescence microscope at 1.25× and 5× magnifications. Results were analyzed using the D'Agostino-Person normality test, and the Kruskal-Wallis, Dunn, and Wilcoxon tests for intergroup and paired comparisons (α = 0.05). The fluorescence intensity/mm2 in the DEJ at the three regions studied was higher in the irradiated teeth (p < 0.05) than in the nonirradiated teeth, revealing regions of expression of MMP-2 and MMP-9 by immunofluorescence. Postradiotherapy treatment with different solutions (CHX, NaF, and EGCG) led to lower fluorescence intensity/mm2 in irradiated teeth than in the control group (distilled water; p < 0.05), as a result of MMP inactivation. In conclusion, irradiation increased gelatinase activity in all regions of the DEJ. Treatment with 0.12% CHX, 0.05% NaF, and 400 µM polyphenol EGCG postradiotherapy inactivated enzyme activity.

Quaternary ammonium silane-based antibacterial and anti-proteolytic cavity cleanser.

OBJECTIVE: Secondary caries and degradation of hybrid layers are two major challenges in achieving durable resin-dentin bonds. The objectives of the present study were to investigate the effects of a 2% quaternary ammonium silane (QAS) cavity cleanser on bacteria impregnated into dentin blocks and the gelatinolytic activity of the hybrid layers. METHODS: Microtensile bond strength was first performed to evaluate if the 2% QAS cavity cleanser adversely affected bond strength. For antibacterial testing, Streptococcus mutans and Actinomyces naeslundii were impregnated into dentin blocks, respectively, prior to the application of the cavity cleanser. Live/dead bacterial staining and colony-forming unit (CFU) counts were performed to evaluate their antibacterial effects. Gelatinolytic activity within the hybrid layers was directly examined using in-situ zymography. A double-fluorescence technique was used to examine interfacial permeability immediately after bonding. RESULTS: The cavity cleanser did not adversely affect the bond strength of the adhesives tested (p>0.05). Antibacterial testing indicated that 2% QAS significantly killed impregnated bacteria within the dentin blocks compared with control group (p<0.05), which was comparable with the antibacterial activity of 2% chlorhexidine (p>0.05). Hybrid layers pretreated with 2% QAS showed significant decrease in enzyme activity compared with control group. With the use of 2% QAS, relatively lower interfacial permeability was observed, compared with control group and 2% chlorhexidine (p<0.05). SIGNIFICANCE: The present study developed a 2% QAS cavity cleanser that possesses combined antimicrobial and anti-proteolytic activities to extend the longevity of resin-dentin bonds.

Effect of calcium fluoride on the activity of dentin matrix-bound enzymes.

OBJECTIVE: Matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs) are two distinct enzymatic pathways responsible for the degradation of collagen fibrils in demineralized dentin. NaF and KF have been shown to inhibit salivary MMP-2, -9 and CCs. This study investigated the inhibitory effect of calcium fluoride (CaF2) on the dentin matrix-bound MMPs and CCs. DESIGN: Phosphoric acid (10%)-demineralized dentin beams (1 × 2×6 mm) were incubated at 37 °C in an 1 ml of artificial saliva (AS, control), or AS with 6, 12, 24, 48, 120. 179 and 238 mM F containing CaF2 (n = 10/group) for 1, 7 and 21 days. All groups were further incubated in AS only for 6 months. Total MMP activity, dry mass loss, CTX and hydroxyproline (HYP) analyses were performed after each incubation. The beams were examined under scanning electron microscopy (SEM). MMP-2 and MMP-9 activities were screened with gelatin zymography. Data were analyzed by using ANOVA and Tukey HSD tests (p = .05). RESULTS: The total MMP activity was similar for all groups after 21 days and 6 months. After 21 days, the cumulative mass loss and CTX levels were lower compared to control for the CaF2 ≥48 and CaF2≥120 mM, respectively (p < .05). After 6 months, no significant difference was detected in the dry mass loss and CTX compared to the control (p > .05), whereas HYP level was higher with F 24 and 238 mM groups. CaF2-like minerals were observed on the beams under SEM. There was no gelatinase inhibition in zymography. CONCLUSION: CaF2 does not prevent the degradation of demineralized dentin matrices due to the catalytic activity of MMPs and CCs.

Optimizing resin-dentin bond stability using a bioactive adhesive with concomitant antibacterial properties and anti-proteolytic activities.

Secondary caries and hybrid layer degradation are two major challenges encountered in long-term resin-dentin bond stability. As a link between resin and dentin, adhesives that possess both antimicrobial and anti-proteolytic activities are in demand for eliminating bacteria-induced secondary caries and preventing hybrid layers from degradation. In the present study, a new quaternary ammonium methacryloxy silane (QAMS) prepared from sol-gel chemistry was incorporated into experimental adhesives to examine their antimicrobial effect and anti-proteolytic potential. This functional methacrylate resin monomer contains polymerizable methacryloxy functionalities as well as a positively-charged quaternary ammonium functionality with a long, lipophilic -C18H37 alkyl chain for puncturing the cell wall/membrane of surface-colonizing organisms. Antibacterial testing performed using agar diffusion test, live/dead bacterial staining and colony-forming unit counts all indicated that the QAMS-containing adhesives killed Streptococcus mutans and Actinomyces naeslundii in a dose-dependent manner via a predominant contact-killing mechanism. Gelatinolytic activity within the hybrid layers created by these adhesives was examined using in-situ zymography. Hybrid layers created with 0% QAMS-containing adhesive exhibited intense green fluorescence emitted by the hydrolyzed fluorescein-conjugated gelatin, with 4-fold increase in enzymatic activity compared with an experimental adhesive containing 5% QAMS. Taken together, incorporation of 5% QAMS in the experimental adhesive provides simultaneous antimicrobial and anti-proteolytic activities that are crucial for the maintenance of long-term resin-dentin bond integrity. STATEMENT OF SIGNIFICANCE: Durability of resin-dentin interfacial bond remains a clinically-significant challenge. Secondary caries caused by bacteria and the degradation of hybrid layers via endogenous dentin proteases are two important contributors to the poor resin-dentin bond durability. The present study developed a new 5% QAMS-containing adhesive that provides simultaneous antimicrobial and dentin protease inhibition functions to extend the longevity of resin-dentin bonds.

Inhibitory effect of curcuminoid pretreatments on endogenous dentin proteases.

The aim of this study was to evaluate the effect of curcuminoids on the dentin endogenous protease activity. Demineralized dentin were pretreated with 50 or 100 µM of three different curcuminoids for 60 s and incubated up to 3 months. Untreated beams served as controls. Dry dentin mass was measured after incubation. Aliquots were analyzed for the quantity of ICTP and CTX releases for MMP and cathepsin-K mediated degradation, respectively. The effect of curcuminoids on matrix-bound MMP and soluble rhMMP-9 were measured using an activity assay. Data were subjected to repeated-measures-ANOVA (α=0.05). Gelatinolytic activity was analyzed using zymography. ICTP and CTX release and dry mass loss of curcuminoid-treated groups were significantly lower than the control. Inhibition of rhMMP-9 varied from 29-49% among curcumonoid-treated groups, whereas no inhibition was observed at untreated control (p>0.05). Results were confirmed by zymography. The study showed that the pretreatment of dentin matrices by curcuminoids decreases endogenous protease activity-mediated degradation in dentin.

The Effect of Chemically Modified Tetracycline-3 on the Progression of Dental Caries in Rats.

OBJECTIVE: Matrix metalloproteinases (MMPs) exist in human saliva and dentin and play an important role in the degradation of organic matrix in teeth. Chemically modified tetracycline-3 (CMT-3) is an inhibitor of MMPs. CMT-3 has been used experimentally to treat caries since 1999, but no distinction between dental caries prevalence and dentin caries prevalence has been described. METHODS: A total of 65 Sprague-Dawley rats were randomly divided into three groups. The positive control group (25 rats) was inoculated with Streptococcus mutans (ATCC700610) and fed the cariogenic feed of improved Keyes Diet 2000. The CMT-3 group (25 rats) was also inoculated with S. mutans and fed the cariogenic feed of improved Keyes Diet 2000; the surfaces of rats' molars were daily treated with 0.02% CMT-3. The negative control group (15 rats) was only fed the standard rodent chow. At the end of the 10th week, the dental caries prevalence and dentin caries prevalence of each group were calculated, and the regions of caries were assessed. RESULTS: No caries was found in the negative control group. The dental caries prevalence of the CMT-3 and the positive control group was 75.0 and 83.3%, respectively (p > 0.05, Table 2). The dentin caries prevalence of the CMT-3 and the positive control group was 33.3 and 70.8%, respectively (p < 0.05, Table 2). The Keyes scoring of dentin caries in the CMT-3 group was significantly lower than that in the positive control group (p < 0.05, Table 3). CONCLUSIONS: CMT-3 had no effect on the prevalence of dental caries, but could lower the prevalence and slow down the progression of dentin caries.

Dynamic Influence of pH on Metalloproteinase Activity in Human Coronal and Radicular Dentin.

The aim of this study was to evaluate the effect of pH on the activation of matrix metalloproteinases (MMPs) of human coronal (CD) and radicular dentin (RD). CD and RD were pulverized to powder, and proteins were extracted with 1% phosphoric acid. The extracted proteins and the demineralized powder were separately incubated in the following solutions: 4-aminophenylmercuric acetate (control) or a buffer solution at different pHs (2.5, 4.5, 5.0, 6.0, and 7.0). After incubation, proteins were separated by electrophoresis to measure MMP activities by zymography. To assess the solubilized dentin collagen, the demineralized dentin powder was sustained in incubation buffer, and the amount of hydroxyproline (HYP) released was measured. Zymography revealed MMP-2 gelatinolytic activities for CD and RD in all experimental groups. For both substrates, the lowest pH solutions (2.5, 4.5, and 5.0) yielded higher gelatinolytic activity than those obtained by the highest pH solutions (6.0 and 7.0). For HYP analysis, no detectable absorbance values were observed for pHs of 2.5 and 4.5. The amount of HYP was higher for pH 7.0 than those of all other groups (p < 0.05), except for pH 6.0. No statistical differences were found between pHs 6.0 and 5.0 and control (p > 0.05). The MMP-2 enzyme from human CD and RD is dynamically influenced by pH: at low pH, the extracted enzyme activates this latent form, whereas collagen degradation by the matrix-bound enzyme is only observed when pHs are close to neutral.

In situ analysis of gelatinolytic activity in human dentin.

Matrix metalloproteinases (MMPs) such as gelatinases are differentially expressed in human tissues. These enzymes cleave specific substrates involved in cell signaling, tissue development and remodeling and tissue breakdown. Recent evidences show that gelatinases are crucial for normal dentin development and their activity is maintained throughout the entire tooth function in the oral cavity. Due to the lack of information about the exact location and activity of gelatinases in mature human dentin, the present study was designed to examine gelatinolytic levels in sound dentin. In situ zymography using confocal microscopy was performed on both mineralized and demineralized dentin samples. Sites presenting gelatinase activity were identified throughout the entire biological tissue pursuing different gelatinolytic levels for distinct areas: predentin and dentinal tubule regions presented higher gelatinolytic activity compared to intertubular dentin. Dentin regions with higher gelatinolytic activity immunohistochemically were partially correlated with MMP-2 expression. The maintenance of gelatinolytic activity in mature dentin may have biological implications related to biomineralization of predentin and tubular/peritubular dentinal regions, as well as regulation of defensive mechanisms of the dentin-pulp complex.

Zymography of Hybrid Layers Created Using Extrafibrillar Demineralization.

A chelate-and-rinse extrafibrillar calcium chelation dentin bonding concept has recently been developed and investigated for its effectiveness in improving resin-dentin bonding by bridging the gap between wet and dry dentin bonding. The objective of the present study was to evaluate the gelatinolytic activity of hybrid layers (HLs) created using the chelate-and-rinse bonding technique. Gelatinolytic activity within the HL was examined using in situ zymography and confocal laser-scanning microscopy after 24-h storage or after thermomechanical cycling. Dentin specimens were bonded with Prime&Bond NT (Dentsply Sirona) after conditioning with 15 wt% phosphoric acid for 15 s (control) or 15 wt% polymeric chelators (sodium salt of polyacrylic acid; PAAN) of 2 different molecular weights for 60 s. For each reagent, bonding was performed using dry-bonding and wet-bonding techniques ( n = 10). Slices containing the adhesive-dentin interface were covered with fluorescein-conjugated gelatin and examined with a confocal laser-scanning microscope. Fluorescence intensity emitted by the hydrolyzed fluorescein-conjugated gelatin was quantified. Gelatinolytic activity was expressed as the percentage of green fluorescence emitted within the HL. After storage for 24 h, enzymatic activity was only detected within the completely demineralized phosphoric acid-etched dentin, with values derived from dry bonding higher than those from wet bonding ( P < 0.05). Almost no fluorescence signals were detected within the HL when dentin was conditioned with PAANs compared with the controls ( P < 0.05). After thermomechanical cycling, enzymatic activities significantly increased for the phosphoric acid-conditioned, drying-bonding group compared with 24-h storage ( P < 0.05). The present study showed that the use of the chelate-and-rinse bonding concept for both dry-bonding and wet-bonding approaches results in the near absence of matrix-bound collagenolytic activities in the HL even after aging. This may be attributed to fossilization of endogenous proteases via preservation of intrafibrillar minerals within the dentin collagen matrix.

Effect of non-thermal atmospheric plasma on the dentin-surface topography and composition and on the bond strength of a universal adhesive.

This study investigated the effect of application of non-thermal atmospheric plasma (NTAP) on the topography and composition of the dentin surface, as well as the microtensile bond strength (µTBS) of a universal adhesive to NTAP-treated dentin. Exposed flat dentin surfaces from human third molars were either treated with NTAP for 10 and 30 s or untreated (control). The dentin-surface topography and chemical composition were characterized by atomic force microscopy (n = 3) and Raman confocal spectroscopy (n = 5), respectively. The µTBS (n = 8) of Scotchbond Universal to dentin was determined after storage for 24 h and 1 yr, either by direct water exposure or under simulated pulpal pressure. In-situ zymography was used to evaluate the influence of NTAP on the dentin-enzymatic activity. Non-thermal atmospheric plasma produced no remarkable topographical or chemical alterations at the dentin surface; only the amount of phosphate decreased following 10 s of treatment with NTAP. After 1 yr of direct water exposure, the µTBS of NTAP-treated specimens did not differ statistically significantly from that of untreated controls, whereas simulated pulpal pressure-aging resulted in a significantly higher µTBS for NTAP-treated dentin. The dentin-enzymatic activity appeared to be treatment-dependent, but the untreated controls showed more intense fluorescence within the hybrid layer. Scotchbond Universal maintained its µTBS strength after 1 yr of direct water exposure and simulated pulpal pressure, although remarkable statistical differences between treatments were observed depending on the aging condition.