Dentin Powder Research Articles

Bacterial killing by several root filling materials and methods in an ex vivo infected root canal model

To evaluate the ability of two root canal sealers (Epoxy resin-based AH Plus or polydimethylsiloxane-based GuttaFlow) and five root filling techniques (continuous wave of condensation, Thermafil, lateral condensation, matched taper single gutta-percha point, laterally condensed-matched taper gutta-percha point) to kill bacteria in experimentally infected dentinal tubules. An infected dentine block model was used. One hundred and twenty extracted, single-rooted human teeth were randomly divided into 10 test (n = 10) and 2 control (n = 10) groups. The roots, except negative controls, were infected with Enterococcus faecalis for 21 days. The root canals were then filled using the test materials and methods. Positive controls were not filled. Sterile roots were used as negative controls. Dentine powder was obtained from all root canals using gates glidden drills using a standard method. The dentine powder was diluted and inoculated into bacterial growth media. Total colony-forming units (CFU) were calculated for each sample. Statistical analysis was performed using the Kruskal-Wallis and Mann-Whitney U test. The epoxy resin-based sealer was effective in killing E. faecalis except when using Thermafil (P < 0.05), but the polydimethylsiloxane-based sealer was not effective in killing this microorganism except in the continuous wave group (P < 0.05). In the test model, AH Plus killed bacteria in infected dentine more effectively than GuttaFlow. The filling method was less important than the sealer material.

PH changes upon mixing of single-step self-etching adhesives with powdered dentin.

Single-step self-etching adhesives have been developed as a bonding material that adheres to the tooth surface. The adhesives contain acidic resin monomers that penetrate the dentin but can be neutralized to stop the reaction. This study aimed to improve understanding of the pH changes that occur when self-etching adhesives are mixed with dentin powder. Dentin disks obtained from extracted bovine incisors were milled and pulverized into a fine powder. The powder was mixed with diluted self-etching adhesives, and pH changes were measured by a solid-state pH sensor connected to a pH meter at various time points after the start of mixing. Data were analyzed by Tukey's Honestly Significant Difference (HSD) test and Dunnett's test at a significance level of 0.05. Precipitates from the adhesive/ dentin powder mixture were observed by scanning electron microscopy (SEM). The baseline pH values of the self-etching adhesives ranged from 0.97 to 2.83. After mixing with the dentin powder, the pH values significantly increased, ranging from 6.30 to 7.11 at 180 s after mixing. SEM observation revealed products of the reaction between dentin powder and self-etching adhesive which indicate a chemical reaction between the functional monomer and dentin. Dentin has a strong modulation effect against the acidity of self-etching adhesives.

MMP-2 assay within the hybrid layer created by a two-step etch-and-rinse adhesive: Biochemical and immunohistochemical analysis

ObjectiveDegradation of hybrid layers (HLs) within resin-infiltrated dentine results from multiple degradation factors, including collagenolytic activity of specific matrix metalloproteinases (MMPs). Inhibition of host-derived MMPs may, therefore, slow the degradation of HL. The null hypothesis tested is that the presence of MMP-2 is similar regardless of chlorhexidine (CHX) pre-treatment or the use of an adhesive. MethodsPowdered dentine prepared from extracted human teeth was divided into 4 groups: (G1) mineralised powder (control group); (G2) dentine powder treated with 1% phosphoric acid for 1min; (G3) 1% phosphoric acid-etched dentine treated with Adper Scotchbond 1 XT (SB1XT; 3M ESPE); (G4) 1% phosphoric acid-etched dentine treated with 0.2% CHX followed by SB1XT. The concentration of detectable pro-MMP-2 and MMP-2 was assayed using a colorimetric assay system (QuantiSir). In addition, the presence of MMP-2 in the HL was assessed in 1 year-aged adhesive–dentine interfaces using an immunohistochemical approach under FEI-SEM/TEM. ResultsIn dentine powder treated with 1% phosphoric acid (G2), MMP-2 level decreased compared to controls (G1); the application of SB1XT (G3) resulted in an increase of MMP-2, whilst 0.2% CHX before SB1XT application (G4), reduced MMP-2. The FEI-SEM/TEM analysis revealed MMP-2 distribution within the HL of aged interfaces showing increase MMP-2 patterns in the control group and minor labelling in the CHX-pretreated specimens. ConclusionThe results of this study support the use of non-toxic MMPs inhibitors, such as CHX, as an appropriate additional step in bonding procedures in order to increase the longevity of the adhesive restorations.

MMP-Inhibitory Effect of Chlorhexidine Applied in a Self-etching Adhesive

To evaluate the MMP-inhibitory effect of chlorhexidine when incorporated in the primer of a two-step selfetching adhesive (Clearfil SE Bond). Powdered dentin made from human teeth was treated with Clearfil SE Bond primers containing chlorhexidine of different concentrations for 20 s or for 2 min. The collagenolytic activity of the dentin powder was assayed using fluorescein-labelled collagen. Untreated dentin powder contains a low but measurable level of intrinsic activity, which was significantly inhibited by 0.05% chlorhexidine. Treating dentin powder for 20 s with SE Bond primers containing chlorhexidine (0.5%, 1.0%, and 2.0%) reduced the collagenolytic activities by 15.6%, 44.9%, and 56.7% respectively. When treated for 2 min, only SE Bond primer containing 2.0% chlorhexidine could inhibit the collagenolytic activity by 41.1%. When incorporated in a two-step self-etching adhesive primer (Clearfil SE Bond primer), chlorhexidine can partially maintain its inhibitory effect on MMPs.

Immunohistochemical and biochemical assay of MMP-3 in human dentine

ObjectiveThe function of endogenous MMP-3 and its distribution within the human dentine is unclear. Thus, the aim of the present study was to assay the presence and distribution of MMP-3 within human sound dentine by means of biochemical and immunohistochemical assays. MethodsPowdered dentine from extracted human teeth was prepared and (1) partially demineralised with 1% H3PO4 for 10min or (2) untreated (control). The presence of MMP-3 was measured using a colorimetric assay system (QuantiSir™, Epigentek, USA). Additional cryo-fractured dentine fragments were processed for immunohistochemical identification of MMP-3 under FEI-SEM. Casein-zymography was used to investigate MMP-3 activity. ResultsMMP-3 detected level was 2.732ng/μL in partially demineralised dentine powder, whilst it increased to 3.280ng/μL in mineralised dentine. The FEI-SEM analysis revealed positive immunolabelling patterns for MMP-3, predominantly localized on the intertubular collagen fibrillar network showing MMP-3 directly or indirectly bound to the collagen fibrils. Casein-zymograms showed positive proteolytic activity for MMP-3 in demineralised dentine powder. ConclusionThe results of the study clearly revealed the presence and distribution of MMP3 in human sound dentine. Whilst the presence was verified, its role is still unclear. Future studies are needed to investigate the possible involvement of MMP-3 in physiological and pathological condition of the dentine–pulp complex.

Biochemical and immunohistochemical analysis of tissue inhibitor of metalloproteinases-1 (TIMP-1) in human sound dentin

Introduction. Matrix metalloproteases (MMPs) are a family of enzymes mainly produced by cells of connective tissue as proenzymes and secreted in the extracellular matrix. MMPs are calcium/zinc-dependent proteinases and operate a specific proteolytic activity on most constituents of the extracellular matrix. The effects of MMPs are regulated by tissue inhibitor of metalloproteinases (TIMPs), small regulatory proteins that can bind ubiquitously different enzyme forms. Different studies showed that MMPs are also involved in dentinogenesis. Additionally it has been shown that some enzymes such as MMP-2 and MMP-9 are involved in autodegenerative processes, such as the degradation of dentin matrix in caries lesions. Purpose. The purpose of this study was to identify different enzyme isoforms in association with TIMP-1, through co-immunoprecipitation/immunoblotting and the evaluation of enzyme localization by immunohistochemical techniques and light and electron microscopy analysis. Methods. Proteins were extracted from human dentin powder previously demineralized with 1%H3PO4 for 10min. Extraction buffer is composed of 5mM CaCl2, 100mM NaCl, 0.1% Triton X-100, 0.1% NONIDET (non ionic detergent), 0.1mM ZnCl2, 0.02% NaN3 and protease inhibitors in 50mM Tris-HCl pH6. The extracted proteins were subjected to co-immunoprecipitation and subsequent immunoblotting against MMPs and TIMP-1. Additional samples were prepared to analyze the presence of TIMP-1 in light microscopy using diaminobenzidine colorimetric assay. The evaluation of TIMP-1 localization within the fine dentinal structure was performed also by immunogold labeling in FEI-SEM microscopy (Field Emission In lens Scanning Electron Microscopy). Results. Co-immunoprecipitation/immunoblotting analysis show the association TIMP1/MMP-2 and TIMP1/MMP-9 in human sound dentin. Light and electron microscopy revealed a strong presence of TIMP-1 in intratubular dentin rather than in the intertubular. Their association with MMPs is finely adjusted and an imbalance could lead to caries pathology and failure of adhesive systems.

The anti-MMP activity of benzalkonium chloride

Objective This study evaluated the ability of benzalkonium chloride (BAC) to bind to dentine and to inhibit soluble recombinant MMPs and bound dentine matrix metalloproteinases (MMPs). Methods Dentine powder was prepared from extracted human molars. Half was left mineralized; the other half was completely demineralized. The binding of BAC to dentine powder was followed by measuring changes in the supernatant concentration using UV spectrometry. The inhibitory effects of BAC on rhMMP-2, -8 and -9 were followed using a commercially available in vitro proteolytic assay. Matrix-bound endogenous MMP-activity was evaluated in completely demineralized beams. Each beam was either dipped into BAC and then dropped into 1 mL of a complete medium (CM) or they were placed in 1 mL of CM containing BAC for 30 days. After 30 days, changes in the dry mass of the beams or in the hydroxyproline (HYP) content of hydrolysates of the media were quantitated as indirect measures of matrix collagen hydrolysis by MMPs. Results Demineralized dentine powder took up 10-times more BAC than did mineralized powder. Water rinsing removed about 50% of the bound BAC, whilst rinsing with 0.5 M NaCl removed more than 90% of the bound BAC. BAC concentrations 0.5 wt% produced 100% inhibition of soluble recombinant MMP-2, -8 or -9, and inhibited matrix-bound MMPs between 55 and 66% when measured as mass loss or 76–81% when measured as solubilization of collagen peptide fragments. Conclusions BAC is effective at inhibiting both soluble recombinant MMPs and matrix-bound dentine MMPs in the absence of resins.

In vitro study of the inactivation by dentine of some endodontic medicaments and their bases

The aim of this study was to investigate the antimicrobial effect of endodontic medicaments and their bases in the presence of dentine powder. The medicaments tested were Ledermix paste, Pulpdent paste, a 50:50 combination of the Pulpdent:Ledermix and their bases. The test organism was Enterococcus faecalis ATCC 29212. The presence or absence of dentine was examined as well as the effect of autoclaving dentine. Serial dilutions of samples at 1 hour, 1 day and 3 days were used for colony counting. The effects of dentine powder on pH for saturated Ca(OH)2 solution and Pulpdent paste at 1 hour and 24 hours were also measured. Pulpdent and the 50:50 combination of Pulpdent:Ledermix completely inhibited the growth of E. faecalis from 1 hour onwards, and these results were not affected by the presence/absence of dentine powder, pre-incubation period, timing of autoclaving, or exposure time. Saturated solutions of Ca(OH)2 are prone to inactivation by dentine powder unlike Pulpdent paste. Ledermix paste took 3 days to exert a significant effect on the growth of E. faecalis. In this laboratory study, both Pulpdent and the 50:50 mixture of Pulpdent with Ledermix were effective medicaments against E. faecalis in the presence of dentine powder.

A chemical phosphorylation-inspired design for Type I collagen biomimetic remineralization

Objectives Type I collagen alone cannot initiate tissue mineralization. Sodium trimetaphosphate (STMP) is frequently employed as a chemical phosphorylating reagent in the food industry. This study examined the feasibility of using STMP as a functional analog of matrix phosphoproteins for biomimetic remineralization of resin-bonded dentin. Methods Equilibrium adsorption and desorption studies of STMP were performed using demineralized dentin powder (DDP). Interaction between STMP and DDP was examined using Fourier transform-infrared spectroscopy. Based on those results, a bio-inspired mineralization scheme was developed for chemical phosphorylation of acid-etched dentin with STMP, followed by infiltration of the STMP-treated collagen matrix with two etch-and-rinse adhesives. Resin–dentin interfaces were remineralized in a Portland cement-simulated body fluid system, with or without the use of polyacrylic acid (PAA) as a dual biomimetic analog. Remineralized resin–dentin interfaces were examined unstained using transmission electron microscopy. Results Analysis of saturation binding curves revealed the presence of irreversible phosphate group binding sites on the surface of the DDP. FT-IR provided additional evidence of chemical interaction between STMP and DDP, with increased in the peak intensities of the P O and P–O–C stretching modes. Those peaks returned to their original intensities after alkaline phosphatase treatment. Evidence of intrafibrillar apatite formation could be seen in incompletely resin-infiltrated, STMP-phosphorylated collagen matrices only when PAA was present in the SBF. Significance These results reinforce the importance of PAA for sequestration of amorphous calcium phosphate nanoprecursors in the biomimetic remineralization scheme. They also highlight the role of STMP as a templating analog of dentin matrix phosphoproteins for inducing intrafibrillar remineralization of apatite nanocrystals within the collagen matrix of incompletely resin-infiltrated dentin.

Enzymatic degradation of adhesive–dentin interfaces produced by mild self‐etch adhesives

Endogenous matrix metalloproteinases (MMPs) released by adhesive procedures may degrade collagen in the hybrid layer and so compromise the bonding effectiveness of etch-and-rinse adhesives. In this study, endogenous enzymatic degradation was evaluated for several simplified self-etch adhesives. In addition, primers were modified by adding two MMP inhibitors: chlorhexidine, a commonly used disinfectant, but also a non-specific MMP inhibitor; and SB-3CT, a specific inhibitor of MMP-2 and MMP-9. Gelatin zymography of fresh human dentin powder was used to identify the enzymes released by the adhesives. Micro-tensile bond strength (μTBS) testing was used to assess the mechanical properties of resin-dentin interfaces over time. In none of the experimental groups treated with the mild self-etch adhesives was MMP-2 and/or MMP-9 identified. Also, no difference in the μTBS was measured for the inhibitor-modified and the control inhibitor-free adhesives after 6 months of water storage. It is concluded that in contrast to etch-and-rinse adhesives, the involvement of endogenous MMP-2 and MMP-9 in the bond-degradation process is minimal for mild self-etch adhesives.

Chlorhexidine binding to mineralized versus demineralized dentin powder

Objectives The purposes of this work were to quantitate the affinity and binding capacity of chlorhexidine (CHX) digluconate to mineralized versus demineralized dentin powder and to determine how much debinding would result from rinsing with water, ethanol, hydroxyethylmethacrylate (HEMA) or 0.5 M NaCl in water. Methods Dentin powder was made from coronal dentin of extracted human third molars. Standard amounts of dentin powder were tumbled with increasing concentrations of CHX (0–30 mM) for 30 min at 37 °C. After centrifuging the tubes, the supernatant was removed and the decrease in CHX concentration quantitated by UV-spectroscopy. CHX-treated dentin powder was resuspended in one of the four debinding solutions for 3 min. The amount of debound CHX in the solvents was also quantitated by UV-spectroscopy. Results As the CHX concentration in the medium increased, the CHX binding to mineralized dentin powder also increased up to 6.8 μmol/g of dry dentin powder. Demineralized dentin powder took up significantly ( p < 0.01) more CHX, reaching 30.1 μmol CHX/g of dry dentin powder. Debinding of CHX was in the order: HEMA < ethanol < 0.05 M NaCl < water. The highest CHX binding to demineralized dentin occurred at 30 mM (1.5 wt.%). Significance As CHX is not debound by HEMA, it may remain bound to demineralized dentin during resin–dentin bonding. This may be responsible for the long-term efficacy of CHX as an MMP inhibitor in resin–dentin bonds.

Influence of dentin on pH of 2% chlorhexidine gel and calcium hydroxide alone or in combination

The aims of endodontic treatment in cases of apical periodontitis are to reduce as much as possible the number of microorganisms inside the root canal system and to inactivate toxins produced by them. Most of the times, these objectives are not achieved solely by chemomechanical preparation, and intracanal dressing may be necessary. In these cases, calcium hydroxide is used as a root canal dressing due to its well-known and recognized antimicrobial activity. Chlorhexidine has a wide spectrum of antimicrobial activity and its association with calcium hydroxide has been recommended in an attempt to amplify antimicrobial effects of calcium hydroxide. It is also known that dentin exerts a buffering effect under wide pH variations, and may be responsible for decreasing the antimicrobial activity of drugs inside the root canal. The objectives of this study were to assess the pH of 2% chlorhexidine gel and calcium hydroxide alone or in combination, as well as the influence of dentin on the pH of these compounds. Dentin powder was obtained from bovine teeth and added as 1.8% to the volume of the medications. All substances were individually stored in plastic flasks, in triplicate. A pH meter was used at five different moments to assess pH in viscous medium: immediately after preparation and after 24 h, and 7, 14, and 21 days. Results were analyzed by paired Student's t-test. Statistically significant differences were observed in the 2% chlorhexidine gel group alone or associated with calcium hydroxide and added of dentin powder (P < 0.05). Mean pH values indicated the influence of dentin powder because of a significant increase in pH. Calcium hydroxide with propylene glycol as the vehicle always showed high pH, demonstrating that this compound was not affected by the presence of dentin.

An in vitro investigation of the mechanical–chemical and biological properties of calcium phosphate/calcium silicate/bismutite cement for dental pulp capping

The properties of new calcium phosphate/calcium silicate/bismutite (CPCSBi) cement were compared with those of calcium hydroxide (CH) and Dycal cements in dental pulp-capping applications. CPCSBi is composed of hydroxyapatite, tetracalcium phosphate, bismutite, and calcium silicate, which was analyzed by SEM, FTIR, and XRD. The results of ion release from CPCSBi showed that the concentrations of Bi(3+), Ca(2+), PO4(2-), and Si(4+) increased with time in deionized water solutions. The setting time of CPCSBi and Dycal was 13 min 50 s and 2 min 25 s, respectively. There were no statistical differences in compressive strength and solubility between CPCSBi and Dycal (p > 0.05). The pH of CPCSBi (10.9) was lower than that of CH (11.6) and Dycal (12.5) after immersion for 24 h. Only slight cytotoxicity appeared for CPCSBi, whereas both CH and Dycal produced moderate discoloration and lysis. In antimicrobial tests against Sm, Av, La, and Sa, the antimicrobial potency of the CPCSBi was approximately 5-10 times greater than that of Dycal and CH groups. The dissoluble dentin matrix components (DDMCs) extracted from CPCSBi exposed to dentin powder demonstrated increased expression of dentin sialophosphoprotein (DSPP) and soteocalcin (OCN) dramatically in human pulp cells by RT-PCR. These results suggest that CPCSBi will be a good candidate for use as a dental pulp-capping agent in future.

Use of a specific MMP-inhibitor (galardin) for preservation of hybrid layer

Objective Dentinal MMPs have been claimed to contribute to the auto-degradation of collagen fibrils within incompletely resin-infiltrated hybrid layers and their inhibition may, therefore, slow the degradation of hybrid layer. This study aimed to determine the contribution of a synthetic MMPs inhibitor (galardin) to the proteolytic activity of dentinal MMPs and to the morphological and mechanical features of hybrid layers after aging. Methods Dentin powder obtained from human molars was treated with galardin or chlorhexidine digluconate and zymographically analyzed. Microtensile bond strength was also evaluated in extracted human teeth. Exposed dentin was etched with 35% phosphoric acid and specimens were assigned to (1) pre-treatment with galardin as additional primer for 30 s and (2) no pre-treatment. A two-step etch-and-rinse adhesive (Adper Scotchbond 1XT, 3M ESPE) was then applied in accordance with manufacturer's instructions and resin composite build-ups were created. Specimens were immediately tested for their microtensile bond strength or stored in artificial saliva for 12 months prior to being tested. Data were evaluated by two-way ANOVA and Tukey's tests ( α = 0.05). Additional specimens were prepared for interfacial nanoleakage analysis under light microscopy and TEM, quantified by two independent observers and statistically analyzed ( χ 2 test, α = 0.05). Results The inhibitory effect of galardin on dentinal MMPs was confirmed by zymographic analysis, as complete inhibition of both MMP-2 and -9 was observed. The use of galardin had no effect on immediate bond strength, while it significantly decreased bond degradation after 1 year ( p < 0.05). Interfacial nanoleakage expression after aging revealed reduced silver deposits in galardin-treated specimens compared to controls ( p < 0.05). Conclusions This study confirmed that the proteolytic activity of dentinal MMPs was inhibited by the use of galardin in a therapeutic primer. Galardin also partially preserved the mechanical integrity of the hybrid layer created by a two-step etch-and-rinse adhesive after artificial aging.

The effect of initial irrigation with two different sodium hypochlorite concentrations on the erosion of instrumented radicular dentin

Objective This study evaluated the effects of different NaOCl concentrations and contact times on removal of the organic phase from mineralized dentin with and without the adjunctive use of EDTA, and the effect of NaOCl concentrations on canal wall erosion after the use of EDTA as the final active irrigant. Methods Dentin powders were immersed in 5.25% or 1.3% NaOCl for different contact periods and then rinsed with 17% EDTA for 2 min. Before and after the use of 17% EDTA as the final rinse, the NaOCl-treated dentin powders were examined using ATR–FT-IR spectroscopy to analyze the relative loss of organic and inorganic components. Scanning (SEM) and transmission electron microscopy (TEM) were used to examine the erosion of instrumented canal walls irrigated with 5.25% NaOCl/EDTA or 1.3% NaOCl/EDTA. Results Compared with 1.3% NaOCl, less intact collagen remained within the subsurface of the mineralized dentin powder after the use of 5.25% NaOCl, irrespective of subsequent rinsing with 17% EDTA. Canal wall erosion was apparent only under SEM when root canals were irrigated 5.25% NaOCl followed by 17% EDTA. Under TEM examination, subsurface erosion extended 10–15 μm beneath the sealer-bonded dentin surface after the use of 5.25% NaOCl for 20 min. Conclusion The superficial destructive effect of NaOCl on mineralized dentin is irreversible and is present irrespective of whether EDTA is subsequently employed as the final active irrigant. The EDTA removes the collagen-depleted apatite phase to expose the underlying cause of destruction that is morphologically perceived as canal wall erosion.

Antibacterial effect of bioactive glass in combination with powdered enamel and dentin

In endodontics, various intracanal medications have been advocated to eliminate bacteria after root canal instrumentation. A recent study has revealed that addition of powdered dentin to bioactive glass (BAG) led to increased glass dissolution, and an increased antibacterial efficacy. Therefore, the present study was undertaken to compare the effect of enamel and dentin powder on the antibacterial efficacy of a commercially available BAG. Dentin blocks (dbs) were prepared from single rooted human teeth. These dbs were infected with Enterococcus faecalis for two weeks in Tryptic Soy Broth (TSB), while negative controls were kept in sterile TSB. In group I, the infected dbs were filled with BAG, in group II with BAG + Enamel powder and group III with BAG + Dentin powder. Dentin samples were harvested from the dbs and cultured. Kruskal-Wallis ANOVA was used for multiple group comparison followed by Scheffe's post hoc test for pair-wise comparisons. All the combinations of BAG evaluated significantly reduced the bacterial counts compared to the control group. However, at the end of 24 hours, three days, and five days BAG + Dentin powder showed significant reduction ( P < 0.01) in bacterial counts compared to the other experimental groups. Among the various materials evaluated, it appeared that though BAG exhibits antimicrobial efficacy, the addition of powdered enamel and dentin in aqueous suspension definitely enhanced this property. However, the addition of enamel powder BAG did not significantly alter its antimicrobial efficacy compared to BAG + dentin powder.

Different enzyme extraction methods for human dentin

Introduction Matrix metalloproteases (MMPs) are a family of enzymes mainly produced by cells of connective tissue as proenzymes and secreted in the extracellular matrix. MMPs are calcium/zinc-dependent proteinases and operate a specific proteolytic activity on most constituents of the extracellular matrix. Different studies showed that MMPs are also involved in dentinogenesis. Additionally it has been shown that some enzymes such as MMP-2 and MMP-9 are involved in auto-degenerative processes, such as the degradation of dentin matrix exposed during the dentin bonding procedures. Objective The purpose of this study was to analyze different enzymes extraction methods in relation to the assay to be performed, i.e. zymography and western blotting (WB) for MMP-2 and -9. Methods Proteins were extracted from human dentin powder and demineralized with 1% phosphoric acid for 10min. Two different extraction buffers with different extraction ability were tested. Buffer A: 7M urea, 2M thiourea, 4%CHAPS in 20mM TrisHCl pH7.4. Buffer B: 5mM CaCl2, 100mM NaCl, 0.1% Triton X-100, 0.1% NONIDET, 0.1mM ZnCl2, 0.02% NaN3 and protease inhibitors in 50mM Tris-HCl pH 6. Bradford assay was performed to quantify concentration and MMP-2 and -9 were analyzed with two different biochemical techniques: WB and zymography. Results The protein quantity extracted with buffer B was 10-fold less than buffer A. WB analysis of dentin protein extracted with buffer A showed presence of both MMP-2 and -9, while no gelatinolytic activity was found in zymography. Conversely, after the use of buffer B, despite a low rate of protein detection in WB, extracted enzymes showed intense gelatinolytic activity. Conclusion To preserve the enzymatic activity, enzymes must retain their native form. Buffer A has strong reducing properties with high extraction ability, while buffer B is a non-reducing agent with lower extraction ability. The results of this study showed that despite lower extraction capability, buffer B allows to retain enzymatic activity of MMP-2 and -9 in human dentin.

Identification of MMP-8 in human sound dentin

Objective Matrix metalloproteinases (MMPs), a class of Ca/Zn-dependent endopeptidases, are secreted by odontoblasts as proenzymes that remain trapped within the mineralized dentin matrix. Activation of these proenzymes is a critical step that leads to dentin extracellular matrix breakdown. The study aimed to assess presence, localization and distribution of MMP-8 in human sound dentin by biochemical and immunohistochemical assays. Methods The presence of MMP-8 in human sound dentin was assayed using a colorimetric assay system that allows direct measuring of MMP-8 levels (QuantisirTM, Epigentek, USA): powdered dentin prepared from extracted human teeth was prepared and 1) partially demineralized with 1% H3PO4 for 10 min or 2) untreated (control). To further detect the localization and distribution of MMP-8 within the dentin organic matrix, dentin cryo-fractured fragments were obtained from human sound teeth, partially decalcified in 0.5 M EDTA pH 7.4 for 30min and submitted to a pre-embedding immunolabeling technique, using primary monoclonal antibodies anti-MMP-8 and exposed to a secondary antibody conjugated with gold nano-particles. Observation was performed under FEI-SEM. Results In mineralized dentin the amount of MMP-8 was 3.32 ng/μL, while the MMP-8 level in the partially demineralized dentin powder significantly decreased (2.45 ng/μL) compared to levels obtained in mineralized dentin. FEI-SEM analyses confirmed that MMP-8 is an endogenous component of the human dentin organic matrix. The labelling pattern of the gold particles bound to the antibody anti-MMP-8 was clearly detectible in the dentin fragments as spherical, highly electron reflective spots localised in the peritubular dentin, revealing the three-dimensional relationship between these proteinase and the collagen fibrils. Conclusion The role and function of dentin MMPs is not well known, but they have shown to contribute to auto-degenerative processes in dentin, such as inflammation of dental pulp, progression of caries lesions and, more recently, degradation of dentin matrix exposed during dental bonding procedures. This study demonstrated that MMP-8 is an intrinsic constituent of the fibrillar network of the human dentin organic matrix, thus supporting that this enzyme may play important roles in the degradation of the dentin organic matrix.

Chlorhexidine stabilizes the adhesive interface: A 2-year in vitro study

Objectives This study evaluated the role of endogenous dentin MMPs in auto-degradation of collagen fibrils within adhesive-bonded interfaces. The null hypotheses tested were that adhesive blends or chlorhexidine digluconate (CHX) application does not modify dentin MMPs activity and that CHX used as therapeutic primer does not improve the stability of adhesive interfaces over time. Methods Zymograms of protein extracts from human dentin powder incubated with Adper Scotchbond 1XT (SB1XT) on untreated or 0.2–2% CHX-treated dentin were obtained to assay dentin MMPs activity. Microtensile bond strength and interfacial nanoleakage expression of SB1XT bonded interfaces (with or without CHX pre-treatment for 30 s on the etched surface) were analyzed immediately and after 2 years of storage in artificial saliva at 37 °C. Results Zymograms showed that application of SB1XT to human dentin powder increases MMP-2 activity, while CHX pre-treatment inhibited all dentin gelatinolytic activity, irrespective from the tested concentration. CHX significantly lowered the loss of bond strength and nanoleakage seen in acid-etched resin-bonded dentin artificially aged for 2 years. Significance The study demonstrates the active role of SB1XT in dentin MMP-2 activation and the efficacy of CHX inhibition of MMPs even if used at low concentration (0.2%).

Effects of Different Exposure Times and Concentrations of Sodium Hypochlorite/Ethylenediaminetetraacetic Acid on the Structural Integrity of Mineralized Dentin

Introduction This study tested the null hypothesis that there is no difference between the use of 1.3% NaOCl/17% ethylenediaminetetraacetic acid (EDTA) and 5.25% NaOCl/17% EDTA irrigation regimens on the collagen degradation and flexural strength reduction in mineralized dentin. Methods Dentin powder and mineralized dentin sections were immersed in 1.3% or 5.25% NaOCl for 10–240 minutes and then rinsed with 17% EDTA as the final irrigant for 2 minutes. Untreated mineralized dentin powder/sections served as controls in the respective experiments. Dentin powders were examined by using Fourier transform infrared (FT-IR) spectroscopy to analyze their relative subsurface intact collagen content with the apatite/collagen ratio. Hydrated dentin sections were subjected to 3-point flexure under water for determining their flexural strengths. Results Collagen degradation was significantly increased and the flexural strength of mineralized dentin was significantly reduced after the use of 5.25% NaOCl as the initial irrigant for more than 1 hour ( P < .05). Conversely, changes were insignificant when 1.3% NaOCl was used as the initial irrigant for up to 4 hours (Kruskal-Wallis analysis of variance, n = 10, P < .05). Conclusions The null hypothesis was rejected. The deleterious effects attributed to the use of NaOCl on dentin are concentration-dependent and time-dependent and are not associated with the demineralization caused by the use of EDTA as the final active irrigant.