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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.

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., (© 2021. The Author(s).)

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.

Intracellular Ca 2+ signaling engendered by Ca 2+ influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca 2+ is exported by the Na + -Ca 2+ exchanger (NCX) and plasma membrane Ca 2+ -ATPase (PMCA) to maintain Ca 2+ homeostasis. We previously demonstrated a functional coupling between Ca 2+ 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 Ca 2+ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca 2+ concentration ([Ca 2+ ] i ). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca 2+ ] 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 [Ca 2+ ] i in odontoblasts in both rat and human odontoblasts. The Ca 2+ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca 2+ ] 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 [Ca 2+ ] i homeostasis in odontoblasts by Ca 2+ extrusion following [Ca 2+ ] i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca 2+ 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.

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.

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 -C 18 H 37 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., (Published by Elsevier Ltd.)

Introduction: One of the possible mechanisms for the gradual destruction of bond strength in dentin-resin interface, could be due to the demineralized unstable collagen matrix. Use of protease inhibitors, such as tannic acid (TA) could prevent destruction of collagen fibers. The aim of this study was to compare the TA effect on bond strength of etch and rinse and self-etch adhesive systems in the dentin of primary teeth., Materials and Methods: This in vitro study was done on 40 extracted primary molar teeth. The teeth were sectioned in the mesiodistal direction, and enamel of buccal and lingual surfaces was removed. Samples were randomly divided into four groups: Single bond (SB) + TA, SB, Clearfil SE Bond (CSB) + TA, and CSB. Then, Z250 and Clearfil AP-X composites were cured on the surfaces of SB and CSB groups respectively. After that, all samples were divided into aging and non-aging groups. For 3 months, samples were placed under 1,000 thermal cycles in aging group. Subsequently, the shear bond strengths of all groups were measured by the International testing machine, and failure mode was evaluated by an optical stereomicroscope. Data were analyzed with paired t-test and independent t-test., Results: Tannic acid induced a significant reduction in the immediate bond strength of adhesive SB. Meanwhile, TA had no significant effect on shear bond strength of the CSB system., Conclusion: Based on our findings, use of TA is not recommended with SB and CSB adhesives on primary teeth., Clinical Significance: Tannic acid may not be considered in resin restorations of primary teeth.

Aim: The aim of this study was to comparatively assess the gelatinolytic activity of matrix metalloproteinases(MMPs) and Cysteine Cathepsins (CCs) in the adhesive interface using etch and rinse adhesive at different time intervals using zymographic technique., Methodology: Twenty freshly extracted non-carious human third molars were used in this study. Occlusal surfaces were ground flat and 1mm thick horizontal dentin slabs were obtained from each tooth using a diamond disc. The dentin surface was polished with 600-grit silicon-carbide paper. Five out of 20 samples were directly pulverized. In the remaining fifteen samples, the dentin was etched and adhesive was applied and light cured according to the manufacturer's instructions. A 1mm thick flowable composite was build up and light cured. Bonded specimens were cut vertically into 3 to 4 dentin slabs by means of diamond disc to expose the adhesive/dentin interfaces. These were then ground down to 500 µm thick resin-dentin interface using a hard tissue microtome. These sections were then pulverised into powder. Following this, every five samples were subjected to zymographic analysis after 1 day, 7 days and 21 days., Results: Zymograms showed clear, thicker bands on all three isoforms in the etched samples compared to control samples at 1st and 7th day intervals and became inactive at 21st day for all three isoforms. MMP 9 activity was relatively higher when compared to CCs and MMP 2., Conclusion: Etch and rinse adhesive activated MMPs and CCs within the hybrid layer that remained active till 7th day and no gelatinolytic activity was found on 21st day and MMPs are more active compared to CCs and MMP-2.

Aim: This review aims to summarise our understanding of the destructive role of acid environment and metalloproteinases in dentin caries progression using a review process., Method: The acids resulting from consumption of sugars by acidogenic and aciduric bacteria can cause demineralisation of the tooth surface, but are not able to cause caries-like lesions. The appearance of such lesions requires the activation of enzymatic proteolysis in an acidic environment for degradation of the dentin organic matrix, leading to cavity formation. Bacterial collagenases have long been considered responsible for organic matrix destruction; host cell-derived matrix metalloproteinases (MMPs) have recently been considered to be involved in the dentinal matrix destruction of carious lesions., Discussion and Conclusion: MMPs are initially synthesised as inactive zymogens to be activated in acid environment of dentinal fluid during the carious process, resulting in destruction of the collagenous matrix. The role of acid environment on enamel and dentin demineralisation and the role of salivary and dentinal MMPs in dentin progression of caries has encouraged general dentists to include the monitoring of oral environment not only by control of bacterial oral flora in caries treatment protocol, but mainly by inhibition of dentinal and salivary MMPs through the use of toothpaste and/or mouthwash containing specific active agents.

The immunoexpression profile of matrix metalloproteinase-13 was investigated for the first time in dentin of human caries and healthy teeth. Twelve permanent premolars (10 caries and 2 sound) were decalcified in ethylenediaminetetraacetic acid and processed for embedding in paraffin wax. Sections 3-4 µm in thickness were cut and processed for immunohistochemistry. A mouse monoclonal anti-metalloproteinase-13 antibody was used for localisation using an immunoperoxidase technique. Dentinal immunoreactivity was detected in all teeth; it was weak in sound teeth and strong close to the caries area. These in vivo findings suggest a role for metalloproteinase-13 in the development and progression of adult human dental tissue disorders.

Background: There is evidence that dentine matrix gelatinases are involved in the destruction of carious dentine after demineralization by bacterial acids. It has also been observed that chlorhexidine, in very low concentrations, inhibits the activity of these enzymes in mammalian cells. The goal of this study was to determine if the gelatinase activity of carious dentine may be inhibited by chlorhexidine in clinical use concentrations., Methods: Gelatinolytic activity was evaluated through zymography and identified by Western blot. The inhibitory effects of chlorhexidine at concentrations of 0.01%, 0.04%, 0.08% and 1% on the enzymatic activity of softened carious dentine samples were determined., Results: In carious dentine, five bands of gelatinolytic activity were detected, with molecular sizes of 86, 75, 38, 33 and 32 kDa. The two bands of the greatest molecular size corresponded to latent and active metalloproteinase-9, respectively. Concentrations of chlorhexidine that were greater than or equal to 0.04% were sufficient to inhibit gelatinolytic activity in the observed bands of carious dentine., Conclusions: These results support the use of chlorhexidine in clinical use concentrations for the treatment and control of dentine caries. Our study demonstrates for the first time the inhibitory effect of chlorhexidine on gelatinases from carious human dentine., (© 2014 Australian Dental Association.)

The presence of matrix metalloproteinase-2 (MMP-2) in dentin has been reported, but its distribution and activity level in mature human coronal dentin are not well understood. The purpose of this study was to determine the MMP-2 distribution and relative activity in demineralized dentin. Crowns of twenty eight human molars were sectioned into inner (ID), middle (MD), and outer dentin (OD) regions and demineralized. MMP-2 was extracted with 0.33 mol x L(-1) EDTA/2 mol xL(-1) guanidine-HCl, pH 7.4, and MMP-2 concentration was estimated with enzyme-linked immunoabsorbant assay (ELISA). Further characterization was accomplished by Western blotting analysis and gelatin zymography. The mean concentrations of MMP-2 per mg dentin protein in the dentin regions were significantly different (P = 0.043): 0.9 ng (ID), 0.4 ng (MD), and 2.2 ng (OD), respectively. The pattern of MMP-2 concentration was OD > ID > MD. Western blotting analysis detected -.66 and -72 kDa immunopositive proteins corresponding to pro- and mature MMP-2, respectively, in the ID and MD, and a -66 kDa protein in the OD. Gelatinolytic activity consistent with MMP-2 was detected in all regions. Interestingly, the pattern of levels of Western blot immunodetection and gelatinolytic activity was MD > ID > OD. The concentration of MMP-2 in human coronal dentin was highest in the region of dentin that contains the dentinoenamel junction and least in the middle region of dentin. However, levels of Western blot immunodetection and gelatinolytic activity did not correlate with the estimated regional concentrations of MMP-2, potentially indicating region specific protein interactions.

Dentin sialophosphoprotein (Dspp) is critical for proper dentin biomineralization because genetic defects in DSPP cause dentin dysplasia type II and dentinogenesis imperfecta types II and III. Dspp is processed by proteases into smaller subunits; the initial cleavage releases dentin phosphoprotein (Dpp). We incubated fluorescence resonance energy transfer (FRET) peptides containing the amino acid context of the Dpp cleavage site (YEFDGKSMQGDDPN, designated Dspp-FRET) or a mutant version of that context (YEFDGKSIEGDDPN, designated mutDspp-FRET) with BMP-1, MEP1A, MEP1B, MMP-2, MMP-8, MMP-9, MT1-MMP, MT3-MMP, Klk4, MMP-20, plasmin, or porcine Dpp and characterized the peptide cleavage products. Only BMP-1, MEP1A, and MEP1B cleaved Dspp-FRET at the G-D peptide bond that releases Dpp from Dspp in vivo. We isolated Dspp proteoglycan from dentin power and incubated it with the three enzymes that cleaved Dspp-FRET at the G-D bond. In each case, the released Dpp domain was isolated, and its N-terminus was characterized by Edman degradation. BMP-1 and MEP1A both cleaved native Dspp at the correct site to generate Dpp, making both these enzymes prime candidates for the protease that cleaves Dspp in vivo. MEP1B was able to degrade Dpp when the Dpp was at sufficiently high concentration to deplete free calcium ion concentration. Immunohistochemistry of developing porcine molars demonstrated that astacins are expressed by odontoblasts, a result that is consistent with RT-PCR analyses. We conclude that during odontogenesis, astacins in the predentin matrix cleave Dspp before the DDPN sequence at the N-terminus of Dpp to release Dpp from the parent Dspp protein., (© 2011 American Society for Bone and Mineral Research.)

This study has examined the use of polyvinylphosphonic acid (PVPA) as a potential matrix metalloproteinase (MMP) inhibitor and how brief cross-linking of demineralized dentin matrix that did not affect its mechanical properties enhanced the anti-MMP activity of PVPA. The anti-MMP potential of five PVPA concentrations (100-3000 microgml(-1)) was initially screened using a rhMMP-9 colorimetic assay. Demineralized dentin beams were treated with the same five concentrations of PVPA to collagen and then aged for 30 days in a calcium- and zinc-containing medium. The changes in modulus of elasticity, loss of dry mass and dissolution of collagen peptides were measured via three-point bending, precision weighing and hydroxyproline assay, respectively. All tested PVPA concentrations were highly effective (P<0.05) in inhibiting MMP-9. Ageing in the incubation medium did not significantly alter the modulus of elasticity of the five PVPA treatment groups. Conversely, aged dentin beams from the control group exhibited a significant decline in their modulus of elasticity (P<0.05) over time. Mass loss from the dentin beams and the corresponding increase in hydroxyproline in the medium in the five PVPA treatment groups were significantly lower than for the control (P<0.05). PVPA is a potent inhibitor of endogenous MMP activities in demineralized dentin. It may be used as an alternative to chlorhexidine to prevent collagen degradation within hybrid layers to extend the longevity of resin-dentin bonds., (2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Dentine matrix metalloproteinases (MMPs) may participate in the destruction of dentine following demineralization by bacterial acids. This study investigated the localization of MMPs in carious dentine., Methods: Frozen sections of dentine caries were prepared without demineralization and immersed in monoclonal antibody against MMP-2, -8, -9 and -20. The sections were labelled by IgG conjugated with gold colloidal particles, and observed under FE-SEM. Labelling indexes (number of gold particles/mum(2)) of outer and inner carious dentine, respectively, with and without bacterial infection, were compared with that of normal dentine., Results: MMP-2 was distributed in both carious and normal dentine; the level of MMP-2 showed no significant difference among the outer caries, inner caries, and normal dentine. The labelling indexes of MMP-8 and MMP-9 both significantly decreased at the inner carious dentine compared with the level of normal dentine, but intensified again at the outer caries region. The labelling index of MMP-20 was the highest at normal dentine., Conclusions: The localization of MMPs was visibly detected using immunogold labelling. The localization of MMP-2 showed no significant difference among the three regions, while MMP-8 and MMP-9 showed significant reduction at the inner caries layer, and MMP-20 reduced toward the outer caries.

Dentin matrix metalloproteinases (MMPs) are a family of host-derived proteolytic enzymes trapped within mineralized dentin matrix, which have the ability to hydrolyze the organic matrix of demineralized dentin. After bonding with resins to dentin there are usually some exposed collagen fibrils at the bottom of the hybrid layer owing to imperfect resin impregnation of the demineralized dentin matrix. Exposed collagen fibrils might be affected by MMPs inducing hydrolytic degradation, which might result in reduced bond strength. Most MMPs are synthesized and released from odontoblasts in the form of proenzymes, requiring activation to degrade extracellular matrix components. Unfortunately, they can be activated by modem self-etch and etch-and-rinse adhesives. The aim of this review is to summarize the current knowledge of the role of dentinal host-derived MMPs in dentin matrix degradation. We also discuss various available MMP inhibitors, especially chlorhexidine, and suggest that they could provide a potential pathway for inhibiting collagen degradation in bonding interfaces thereby increasing dentin bonding durability.

In situ zymography is a very important technique that shows the proteolytic activity in sections and allows researchers to observe the specific sites of proteolysis in tissues or cells. It is normally performed in non-fixed frozen sections and is not routinely performed in calcified tissues. In this study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in conventional microtome and cryostat sections. We used adult rat hemimandibles, which presented bone, enamel, and dentine matrices; the substrate used was dye-quenched-gelatin. Gelatinolytic activity was colocalized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, matrix metalloproteinase-2 was colocalized to the sites of green fluorescence in dentine. In conclusion, the technique presented here will allow in situ zymography reactions in fixed, decalcified, and paraffin-embedded tissues, and we showed that paraformaldehyde-lysine-periodate-fixed cryostat sections are suitable for colocalization of gelatinolytic activity and protein labeling with antibodies.

Matrix metalloproteinases (MMPs) have been expressed during root development and periodontal tissue formation, whereas it is not known if these MMP molecules are enzymatically active to degrade the extracellular matrices (ECMs). The present study was designed to investigate the gelatinolytic and collagenolytic activity in rat molar root and incisor development. Three-week old rat mandibles were frozen and cut without fixation or decalcification and processed for in situ zymography using substrates gelatin and collagen. The enzymatic activity was assessed according to the intensity of fluorescence due to the lysis of the substrates. Odontoblasts, predentin, cementum, bone and the enamel matrix showed the high activity. The present study demonstrated MMP activity in calcified tissues using in situ zymography for the first time and the possible involvement of the MMP activity in molar root and incisor development and periodontal tissue formation.

A collagenase cleaving native type I [14C]collagen but inactive against the synthetic substrate Pz-Pro-Leu-Gly-Pro-D-Arg was extracted from mineralized human dental tissue. The enzyme specifically degrades native collagen into characteristic products (3/4) and (1/4). Its apparent molecular mass of 68 kDa is relatively high in comparison with collagenases from other oral tissues. The enzyme is a metalloproteinase inhibited by low concentrations of the chelating agents EDTA, 1, 10-phenanthroline, alpha alpha'-dipyridyl, and not affected by diisopropylfluorophosphate, soybean trypsin inhibitor, and p-chloromercuribenzoate. It is stable to lyophilization and can be stored at-20 degrees C for at least 6 months.