Red Marine Algae Lithothamnion calcareum Supports Dental Enamel Mineralization.

The current management of oral conditions such as dental caries and erosion mostly relies on fluoride-based formulations. Herein, we proposed the use of the remaining skeleton of Lithothamnion calcareum (LC) as an alternative to fluorides. LC is a red macroalgae of the Corallinales order, occurring in the northeast coast of Brazil, whose unique feature is the abundant presence of calcium carbonates in its cell walls. Two experimental approaches tested the general hypothesis that LC could mediate enamel de-remineralization dynamics as efficiently as fluorides. Firstly, the effect of LC on enamel de-mineralization was determined in vitro by microhardness and gravimetric measurements to test the hypothesis that LC could either prevent calcium/phosphate release from intact enamel or facilitate calcium/phosphate reprecipitation on an artificially demineralized enamel surface. Subsequently, an in situ/ex vivo co-twin control study measured the effect of LC on the remineralization of chemical-demineralized enamel using microhardness and quantitative light-induced fluorescence. With this second experiment, we wanted to test whether outcomes obtained in experiment 1 would be confirmed by an in situ/ex vivo co-twin control model. Both experiments showed that LC exhibited equivalent or superior ability to modulate enamel de-remineralization when compared to fluoride solution. LC should be explored as an alternative to manage oral conditions involving the enamel demineralization.

In vitro assessment of antimicrobial potential of low molecular weight chitosan and its ability to mechanically reinforce and control endogenous proteolytic activity of dentine.

AIMS: Chitosan-based biomaterials exhibit several properties of biological interest for endodontic treatment. Herein, a low molecular weight chitosan (CH) solution was tested for its antimicrobial activity against Enterococcus faecalis (E. faecalis) and effects on dentine structure. METHODOLOGY: The root canal of 27 extracted uniradicular teeth were biomechanically prepared, inoculated with a suspension of E. faecalis and randomly assigned to be irrigated with either 5.25% sodium hypochlorite (NaClO), 0.2% CH or sterile ultrapure water (W). Bacteriologic samples were collected from root canals and quantified for of E. faecalis colony-forming units (CFUs). The effectiveness of CH over E. faecalis biofilms was further measured using the MBEC Assay®. Additionally, dentine beams and dentine powder were obtained, respectively, from crowns and roots of 20 extracted third molars. Dentine samples were treated or not with 17% EDTA and immersed in either CH or W for 1 min. The effects of CH on dentine structure were evaluated by assessment of the modulus of elasticity, endogenous proteolytic activity and biochemical modifications. RESULTS: The number of E. faecalis CFUs was significantly lower for samples irrigated with CH and NaClO. No significant differences were found between CH and NaClO treatments. Higher modulus of elasticity and lower proteolytic activity were reported for dentine CH-treated specimens. Chemical interaction between CH and dentine was observed for samples treated or not with EDTA. CONCLUSIONS: Present findings suggest that CH could be used as an irrigant during root canal treatment with the triple benefit of reducing bacterial activity, mechanically reinforcing dentine and inhibiting dentine proteolytic activity.

Effect of sealing infected dentin with glass ionomer cement on the abundance and localization of MMP-2, MMP-8, and MMP-9 in young permanent molars in vivo.

BACKGROUND: The study of MMPs' behavior in carious lesions contributes to the understanding of the mechanisms involved in dentin reorganization after restoration. AIM: To compare the abundance and localization of MMPs 2, 8, and 9 in infected dentin before and after restoration. DESIGN: The sample consisted of 23 young permanent molars with active deep carious lesions. Infected carious dentin samples were collected from the same tooth at baseline and 60 days after cavity lining with GIC and composite resin restoration and processed for immunohistochemistry assays. After digital images were obtained, two calibrated operators analyzed the samples according to the immunostaining intensity and the MMPs' localization. Chi-square test was used for statistical analysis. RESULTS: The intensity of immunostaining for MMP-8 was reduced after 60 days (P = 0.02), and no difference was observed for MMP-2 (P = 0.32) and MMP-9 (P = 0.14). The MMPs' distribution was generalized in the intertubular dentin and absent or located in the intratubular dentin, regardless of the period. CONCLUSION: The sealing of infected carious dentin in young permanent molars reduced the expression of MMP-8, which is consistent with the initial remodeling process of the dentin matrix.

Matrix metalloproteinases and other matrix proteinases in relation to cariology: the era of 'dentin degradomics'.

Dentin organic matrix, with type I collagen as the main component, is exposed after demineralization in dentinal caries, erosion or acidic conditioning during adhesive composite restorative treatment. This exposed matrix is prone to slow hydrolytic degradation by host collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins. Here we review the recent findings demonstrating that inhibition of salivary or dentin endogenous collagenolytic enzymes may provide preventive means against progression of caries or erosion, just as they have been shown to retain the integrity and improve the longevity of resin composite filling bonding to dentin. This paper also presents the case that the organic matrix in caries-affected dentin may not be preserved as intact as previously considered. In partially demineralized dentin, MMPs and cysteine cathepsins with the ability to cleave off the terminal non-helical ends of collagen molecules (telopeptides) may lead to the gradual loss of intramolecular gap areas. This would seriously compromise the matrix ability for intrafibrillar remineralization, which is considered essential in restoring the dentin's mechanical properties. More detailed data of the enzymes responsible and their detailed function in dentin-destructive conditions may not only help to find new and better preventive means, but better preservation of demineralized dentin collagenous matrix may also facilitate true biological remineralization for the better restoration of tooth structural and mechanical integrity and mechanical properties.

Chlorhexidine does not improve but preserves bond strength to eroded dentin.

PURPOSE: To evaluate the effect of aqueous solutions of chlorhexidine digluconate (CHX) in different concentrations on bond strength to eroded dentin up to 6 months, using normal dentin as a control. METHODS: Exposed flat dentin of extracted third molars was only ground with 600-grit SiC paper/1 minute (normal dentin - N), or subsequently eroded by a regular-cola soft-drink (eroded dentin - E). N and E were acid-etched, washed, dried and rehydrated with 1.5 µL, respectively, of distillated water (control - NC / EC); of 0.004% CHX (N0.004% / E0.004%); or of 2% CHX (N2% / E2%). Adper Single Bond 2 was applied in all specimens and resin composite buildups were constructed with Filtek Z350. Specimens were sectioned in beams, which were tested (µTBS) immediately or after 6 months of aging. RESULTS: Microtensile bond strength to eroded dentin was always significantly lower than that to normal dentin. Application of tested CHX solutions did not exert a significant effect immediately; however, after aging, the 2% CHX prevented abrupt bond strength loss both to eroded and normal dentin.

Use of marine occurrent extracts to enhance the stability of dentin extracellular matrix.

Chitosan (CS) and phloroglucinol (PhG), two extracts abundantly found in marine life, were investigated for their ability to biomodify demineralized dentin by enhancing collagen crosslinks and improving dentin extracellular matrix (ECM) mechanical and biochemical stability. Dentin obtained from non-carious extracted human molars were demineralized with phosphoric acid. Baseline Fourier-transform infrared (FTIR) spectra, apparent flexural elastic modulus (AE) and dry mass (DM) of each specimen were independently acquired. Specimens were randomly incubated for 5 min into either ultrapure water (no-treatment), 1% glutaraldehyde (GA), 1% CS or 1% PhG. Water and GA were used, respectively, as a negative and positive control for collagen crosslinks. Specimens' post-treatment FTIR spectra, AE, and DM were obtained and compared with correspondent baseline measurements. Additionally, the host-derived proteolytic activity of dentin ECM was assessed using hydroxyproline assay (HYP) and spectrofluorometric analysis of a fluorescent-quenched substrate specific for matrix metalloproteinases (MMPs). Finally, the bond strength of an etch-and-rinse adhesive was evaluated after application of marine compounds as non-rinsing dentin primers. Dentin specimens FTIR spectral profile changed remarkably, and their AE increased significantly after treatment with marine compounds. DM variation, HYP assay and fluorogenic substrate analysis concurrently indicated the biodegradation of CS- and PhG-treated specimens was significantly lesser in comparison with untreated specimens. CS and PhG treatments enhanced biomechanical/biochemical stability of demineralized dentin. These novel results show that PhG is a primer with the capacity to biomodify demineralized dentin, hence rendering it less susceptible to biodegradation by host-proteases.

Intrafibrillar silicification of collagen scaffolds for sustained release of stem cell homing chemokine in hard tissue regeneration.

Traditional bone regeneration strategies relied on supplementation of biomaterials constructs with stem or progenitor cells or growth factors. By contrast, cell homing strategies employ chemokines to mobilize stem or progenitor cells from host bone marrow and tissue niches to injured sites. Although silica-based biomaterials exhibit osteogenic and angiogenic potentials, they lack cell homing capability. Stromal cell-derived factor-1 (SDF-1) plays a pivotal role in mobilization and homing of stem cells to injured tissues. In this work, we demonstrated that 3-dimensional collagen scaffolds infiltrated with intrafibrillar silica are biodegradable and highly biocompatible. They exhibit improved compressive stress-strain responses and toughness over nonsilicified collagen scaffolds. They are osteoconductive and up-regulate expressions of osteogenesis- and angiogenesis-related genes more significantly than nonsilicified collagen scaffolds. In addition, these scaffolds reversibly bind SDF-1α for sustained release of this chemokine, which exhibits in vitro cell homing characteristics. When implanted subcutaneously in an in vivo mouse model, SDF-1α-loaded silicified collagen scaffolds stimulate the formation of ectopic bone and blood capillaries within the scaffold and abrogate the need for cell seeding or supplementation of osteogenic and angiogenic growth factors. Intrafibrillar-silicified collagen scaffolds with sustained SDF-1α release represent a less costly and complex alternative to contemporary cell seeding approaches and provide new therapeutic options for in situ hard tissue regeneration.

Dentin Degradation: From Tissue Breakdown to Possibilities for Therapeutic Intervention.

Purpose of the Review: Presently, dental materials science is driven by the search for new and improved materials that can trigger specific reactions from the affected tissue to stimulate repair or regeneration while interacting with the oral environment to promote or maintain oral health. In parallel, evidence from the past decades has challenged the exclusive role of bacteria in dentin tissue degradation in caries, questioning our understanding of caries etiopathogenesis. The goal of this review is to recapitulate the current evidence on the host and bacterial contributions to degradation, inflammation, and repair of the dentin-pulp complex in caries. Recent Findings: Contrasting findings attribute dentin breakdown to the activity of endogenous enzymes, such as matrix metalloproteinases (MMPs) and cathepsins, while the role of bacteria and their by-products in the destruction of dentin organic matrix and pulp inflammation has been for decades supported as an incontestable paradigm. Aiming to better understand the mechanisms involved in collagen degradation by host enzymes in caries, studies have showed that these proteinases are expressed in the mature dentin (i.e., after dentin formation) and become activated by the low pH in the acidic environment resulted by bacterial metabolism in caries. However, different host sources other than dentin-bound proteinases seem to also contribute to caries progression, such as saliva and pulp. Interestingly, studies evaluating pulp responses to bacteria invasion and inflammation in caries report higher levels of MMPs and cathepsins in inflamed tissue, but also showed MMP potential to resolve inflammation and stimulate wound healing. Notably, as reported for other tissues, MMPs exert dual roles in the dentin-pulp complex in caries, participating or regulating both degradative and reparative mechanisms. Summary: The specific roles of host and bacteria and their by-products in caries progression have yet to be clarified. The complex interactions between inflammation and repair in caries pose challenges to a clear understanding of the dentin-pulp complex responses and changes to bacteria invasion. However, it opens new venues for the development of novel therapies and dental biomaterials based on the modulation of specific mechanisms to favor tissue repair and healing.

Differential analysis of the dentin soluble proteomic.

OBJECTIVES: To perform a differential analysis of the dentin soluble proteomic and assess the effects of tissue health state and protocol for protein extraction. We hypothesized the dentin soluble proteomic varies according to the tissue physiopathological state (intact vs. caries-affected) and protocol used to extract its proteins. METHODS: Dentin from freshly extracted non-carious and carious teeth were randomly assigned for protein extraction using either guanidine-HCl/ethylenediaminetetraacetic acid (EDTA) or acetic acid. Protein extracts from intact and caries-affected dentin were processed and digested with trypsin for shotgun label-free proteomic analysis (nLC-ESI-MS/MS). Peptides identification was performed on a nanoACQUITY UPLC-Xevo Q-Tof MS system. Peptides identified with scores of confidence greater than 95% were included in the quantitative statistical analysis embedded in the PLGS software. Differences between experimental conditions were calculated using Student test-t with significance pre-set at α=0.05. RESULTS: A total of 158 human proteins were identified. Approximately one-sixth of proteins (24/158) were present in at least two different extracts. Conversely, the greatest number of proteins (134/158) was identified uniquely in only one of the extracts. Overall, a larger number of soluble proteins was retrieved from caries-affected than intact dentin (86/158). Likewise, a greater number of proteins was extracted by the guanidine-HCl/EDTA (106/158) in comparison to acetic acid protocol. Several proteins detected in dentin extracts, mainly those from caries-affected teeth, are biological and/or metabolically involved with tissue turnover/remodeling. CONCLUSION: The identity/abundance of soluble proteins retrieved from and remained in dentin noticeably depend on this tissue physiopathological state and protocol used to remove its minerals. CLINICAL SIGNIFICANCE: The present findings brought new insight into the proteomic phenotype of human dentin and may provide targets for the development of novel caries disease-prevention therapies.

Dentin primer based on a highly functionalized gelatin-methacryloyl hydrogel.

Gelatin-methacryloyl hydrogels (GelMA) have demonstrated their utility as scaffolds in a variety of tissue engineering applications. OBJECTIVES: In this study, a highly functionalized GelMA hydrogel was synthesized and assessed for degree of functionalization. As the proposed GelMA hydrogel was coupled to a visible-light photoinitiator, we hypothesized it might serve as base to formulate a model dentin primer for application in restorative dentistry. METHODS: GelMA was mixed with photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), photopolymerized for 0-40 s using a dental light-curing device and tested for extrudability, degree of photo-crosslinking (DPxlink), water sorption/solubility/swelling (WS/SL/SW) and apparent modulus of elasticity (AE). Model dentin primer was prepared by mixing GelMA+LAP with a primer of a commercial three-step etch-and-rinse adhesive. After application of GelMA-based primer to acid-etched dentin, samples were bonded with correspondent adhesive agent, photopolymerized and had their immediate bond strength compared to control samples primed and bonded with the same commercial material. RESULTS: Extrudability of hydrogel was confirmed using a microsyringe to write the acronym "CDMI". DPxlink of GelMA+LAP changed significantly as a function of photopolymerization time (20 s < 30 s ≤ 40 s). WS, SL and SW were significantly reduced in hydrogels polymerized for 30 and 40 s. AE of hydrogels varied significantly as a function of photopolymerization time (20 s < 30 s ≤ 40 s; 20 s ‡ 40 s). Bond strength of dentin primed with GelMA-based primer was lower (∼29.3 MPa) but not significantly of that of control (∼34.6 MPa). CONCLUSIONS: Optimization of a GelMA-based dentin primers can lead to the development of promising biomimetic adhesives for dentin rehabilitation.

Changes to dentin extracellular matrix following treatment with plant-based polyphenols.

This study investigated whether treatment with plant-based polyphenols (PB-P) affected the biochemical and/or biomechanical properties of dentin extracellular matrix (ECM). Three PB-Ps were evaluated: luteolin (LT), galangin (GL), and proanthocyanidin (PAC). Because dentin ECM requires demineralization before treatment, this study also assessed the effect of these PB-Ps on dentin demineralized by two different chemicals. Dentin samples from extracted third molars were obtained, sectioned, and randomly assigned for demineralization with either phosphoric acid (PA) or ethylenediaminetetraacetic acid (EDTA). Following demineralization, baseline infrared (IR) spectra and apparent elastic modulus (AE) of each specimen were independently acquired. Based upon these initial tests, samples were randomly assigned to one of the PB-P treatments to ensure that distribution of baseline AE was similar across treatment groups. IR and AE specimens were individually immersed in either 0.2% LT, 0.4% GL or 1% PAC for 2 min. IR spectra of treated samples were compared to baseline IR spectra, looking for any interaction of PB-Ps with the demineralized dentin. The IR spectrum and AE of each PB-P-treated specimen were compared with their own correspondent baseline measurement. The ability of PB-Ps to inhibit proteolytic activity of dentin ECM was assessed by the hydroxyproline assay. Finally, the effect of PB-Ps on immediate bond strength of a dental adhesive to PA- or EDTA-etched dentin was also evaluated. PB-Ps exhibited distinctively binding affinity to dentin ECM and promoted significant increase in AE. PB-P treatment reduced the degradation rate of dentin ECM without causing detrimental effect on immediate bond strength to dentin. Our work represents the first-time that LT and GL have been assessed as dentin ECM biomodifiers.

Laser Photobiomodulation 808 nm: Effects on Gene Expression in Inflammatory and Osteogenic Biomarkers in Human Dental Pulp Stem Cells.

The tissue engineering of dental oral tissue is tackling significant advances and the use of stem cells promises to boost the therapeutical approaches of regenerative dentistry. Despite advances in this field, the literature is still scarce regarding the modulatory effect of laser photobiomodulation (PBM) on genes related to inflammation and osteogenesis in Postnatal Human Dental Pulp Stem cells (DPSCs). This study pointedly investigated the effect of PBM treatment in proliferation, growth and differentiation factors, mineralization, and extracellular matrix remodeling genes in DPSCs. Freshly extracted human third molars were used as a source for DPSCs isolation. The isolated DPSCs were stimulated to an inflammatory state, using a lipopolysaccharide (LPS) model, and then subjected or not to laser PBM. Each experiment was statistically evaluated according to the sample distribution. A total of 85 genes related to inflammation and osteogenesis were evaluated regarding their expression by RT-PCR. Laser PBM therapy has shown to modulate several genes expression in DPSCs. PBM suppressed the expression of inflammatory gene TNF and RANKL and downregulated the gene expression for VDR and proteolytic enzymes cathepsin K, MMP-8 and MMP-9. Modulation of gene expression for proteinase-activated receptors (PARs) following PBM varied among different PARs. As expected, PBM blocked the odontoblastic differentiation of DPSCs when subjected to LPS model. Conversely, PBM has preserved the odontogenic potential of DPSCs by increasing the expression of TWIST-1/RUNEX-2/ALP signaling axis. PBM therapy notably played a role in the DPSCs genes expression that mediate inflammation process and tissue mineralization. The present data opens a new perspective for PBM therapy in mineralized dental tissue physiology.

Performance of MDP-based system in eroded and carious dentin associated with proteolytic inhibitors: 18-Month exploratory study.

The aim of this study was to explore the impact of the interaction between an MDP-based universal adhesive system in etch-and-rinse mode and two proteolytic inhibitors on the longevity of restorations bonded to artificially-affected-dentin substrates. 90 sound human third molars were randomly distributed into three groups according to the substrate: N-no challenges-control (stored in artificial saliva), ACD-artificial caries dentin (6 h DE + 18 h-RE/5 days + 48 h RE) and ERO-artificial erosion dentin (3 × 5 min/5 days with orange juice). They were further redistributed according to dentin pretreatment: W- water (control), CHX-2% digluconate chlorhexidine and E64- 5 µM E64-Trans-Epoxysuccinyl-L-Leucylamido-(4-guanidino) butane, which resulted in the following 9 groups (n = 10): N-W, N-CHX, N-E64, ACD-W, ACD-CHX, ACD-E64, ERO-W, ERO-CHX and ERO-E64. All specimens were restored with Adper Single Bond Universal (Etch-and-rinse mode)/Filtek Z250. Sticks (0.64 mm2) were obtained and subjected to microtensile test (µTBS) in a universal testing machine at 0.5 mm/min for 7-days, 6 and 18-month analyses. Failure modes were classified using optical microscopy (40X). Data were statistically analyzed by three-way ANOVA and Tukey tests (p < 0.05). All individual factors (p < 0.0001) and interaction between factors were statistically significant (substrate X pretreatment (p = 0.00093); substrate X time (p = 0.01035) and pretreatment X time (p = 0.0035). Caries-affected substrate was the most compromised one, disregarding the pretreatment. CHX was mostly affected compared with E64 up to 18 months, possibly due to its calcium-dependent mechanism.

The effect of ageing on the elastic modulus and degree of conversion of two multistep adhesive systems.

During the curing reaction, the monomers of dentine bonding systems should cross-link sufficiently to strengthen an adhesive so that it is clinically reliable. This study evaluated how different storage conditions (air vs. water storage) affect the elastic modulus (E-modulus) and degree of conversion (DC) of a three-step etch-and-rinse adhesive and a two-step self-etch adhesive. The biaxial flexural test and Raman microscopy were performed on resin disks made from the bonding agents Adper Scotchbond Multi-Purpose (SBMP; 3M ESPE) and Clearfil Protect Bond (CPB; Kuraray). The measurements were repeated after storage in either air or water for 15 and 30 min and for 1, 24, and 72 h. At time 0, the E-modulus was not affected by the adhesive system, whilst the degree of cure of CPB was higher than that of SBMP. Air storage increased the E-modulus at each ageing interval. Storage in water increased the E-modulus until it reached a maximum at 24 h, after which it decreased significantly at 72 h. No linear correlation between the percentage DC and E-modulus of the two adhesives was found when stored in water. The results of this study indicate that the mechanical properties and polymerization kinetics of SBMP and CPB are affected by storage time and medium.

Effect of oxalate desensitizer on the durability of resin-bonded interfaces.

Potassium oxalate desensitizers were previously shown to effectively reduce the immediate permeability of resin-bonded dentin. The current study evaluated whether the effect of the combined application of oxalate with etch-and-rinse adhesives interferes with the durability of resin-dentin bonds when using etch-and-rinse adhesives. The bond strength of resin-bonded dentin specimens composed of two-step or three-step etch-and-rinse adhesives (Single Bond, One-Step and Scotchbond Multi-Purpose, respectively) was tested immediately (24 hours) and after 12 months of water storage. The adhesives were used either according to the manufacturers' instructions (control groups) or after treating acid-etched dentin with a potassium oxalate gel (BisBlock, BISCO, Inc). The treatment of dentin with potassium oxalate was shown to negatively affect the baseline bond strength of resin-bonded dentin specimens, regardless of the adhesive used (p < 0.05). After storage, the bond strength of the resin-bonded interfaces was significantly reduced for all the tested groups (p < 0.001). Nevertheless, the rate of decreasing bond strength was significantly lower for oxalate-treated specimens than for the controls (p < 0.05).

Effect of protease inhibitor specificity on dentin matrix properties.

OBJECTIVE: To evaluate protease activity of dentin matrices subjected to treatment with non-specific (chlorhexidine - CHX), cysteine cathepsin specific (E-64), and cysteine cathepsin-K (CT-K) specific (Odanacatib - ODN) inhibitors. METHODS: Pulverized dentin powder obtained from human dentin disks (0.5 mm thickness) completely demineralized with 10% H3PO4 were challenged in 1 mL lactic acid (LA) (0.1M, pH 5.5) or stored in deionized water for 30 min. Aliquots of dentin powder were then immersed in 1 mL of CHX (2%), E-64 (10 µM and 20 µM) or Odanacatib (0.2 nM and 1 µM) for 30min. Degradation of dentin collagen was determined by telopeptide assays measuring the sub-product release of C-terminal cross-linked telopeptides (ICTP) and C-terminal peptide (CTX) in incubation media, which correlates with matrix metalloproteinases (MMP) and CT-K activities respectively (n = 3). The ICTP and CTX data were normalized to concentration of total protein (ICTPtp and CTXtp) in the media, measured by bicinchoninic acid assay. Dentin matrix properties were also measured by gravimetric change (n = 8) and ultimate tensile strength (UTS) (n = 10). Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test and independent t-test (α = 5%). RESULTS: Telopeptide assays showed significantly lower CTXtp values after treatment with E-64 and Odanacatib. E-64 and Odanacatib at all tested concentrations significantly reduced the release of ICTPtp. Gravimetric analysis showed no significant difference between the tested inhibitors and control except for CHX after lactic acid challenge. UTS results showed significantly higher values for E-64 (20 µM) and Odanacatib (0.2 nM and 1 µM) groups in deionized water. SIGNIFICANCE: Dentin therapies targeting enzymes such as CT-K by specific inhibitors may provide superior pharmacokinetics and optimum efficacy due to precise protein binding, consequently limiting collagen degradation directly or indirectly by enzyme related pathways.

Insights into cathepsin-B activity in mature dentin matrix.

OBJECTIVE: Cysteine proteases are lysosomal enzymes that, under specific circumstances, may be secreted into the extracellular space and participate in protein turnover. This study investigated the involvement of cathepsin B in the gelatinolytic activity of mature dentin matrices at neutral pH. DESIGN: Human dentin fragments were made into powder and enzymes were extracted using guanidine-HCl/EDTA. Host-derived dentin proteases (cathepsin B, MMP-2 and MMP-9) were identified by immunoblotting, and their activities were evaluated spectrofluorimetrically using fluorogenic substrates. Proteases activities were monitored by measuring the rate of hydrolysis of substrates in the presence/absence of MMP- or cysteine cathepsin inhibitors, at neutral pH (7.4). Mass spectroscopy was used to determine the substrates' cleavage points. Reverse zymography was performed to examine the gelatinolytic activity of cathepsin B. RESULTS: Western-blots of dentin extracts yielded strong bands at 95, 72 and 30 kDa, corresponding respectively to MMP-9, MMP-2 and Cathepsin B. Greater fluorogenic substrates hydrolysis occurred in the absence of MMP and cysteine cathepsin inhibitors than in their presence. Cathepsin B exhibited significant gelatinolytic activity. CONCLUSIONS: Together with MMP-2 and MMP-9, cathepsin B also account for the host-derived gelatinolytic activity and matrix turnover of mature dentin at physiological, neutral pH.

Profile of a 10-MDP-based universal adhesive system associated with chlorhexidine: Dentin bond strength and in situ zymography performance.

The incorporation of functional monomers and proteolytic inhibitors into adhesive systems have shown to be promising strategies to improve the longevity of adhesive restorations. The aim of this study was to evaluate the long-term bonding performance and anti-gelatinolytic effect of a 10-MDP-based universal adhesive system applied in combination with 2% chlorhexidine digluconate (CHX). For that, this study assessed the resin-dentin bond strength and the in situ gelatinolytic activity profile at the adhesive interface at initial and after 6 month of storage. One hundred and two sound human third molars were prepared and randomly divided into 3 groups according to the adhesive strategy: SB (two-step etch-and-rinse adhesive, Adper Single Bond 2, 10-MDP-free control group); SU-ER (Adper Single Bond Universal, 10-MDP containing universal adhesive applied on etch-and-rinse mode); and SU-SE (SU applied on self-etching mode). The groups were subdivided into two according to the dentin pretreatment: W - water or CHX- 2% chlorhexidine digluconate aqueous solution (SB-W; SB-CHX; SU-ER-W; SU-ER-CHX; SU-SE-W; SU-SE-CHX) and subsequently restored according to the manufacturer's instructions. Bond strength (n = 12) was assessed by a microtensile test (µTBS) (500N/0.5 mm/min) after 24h or after 6 months of storage. In situ zymography was performed to evaluate anti-gelatinolytic activity (n = 5). Resin-dentin samples were incubated with fluorescein-conjugated gelatin for 24 h at 37 °C and analyzed by confocal laser scanning microscopy. Fluorescence indicating gelatinolytic activity at hybrid layer zone and adjacencies was quantified using Image J. Data was analyzed by three-way ANOVA and Tukey post-hoc tests (p < 0.05). Results: SU-SE showed the highest bond strength values, while similar results were observed for SU-ER and SB. No statistical significant differences were observed between pretreatment (CHX vs. W) or storage time (initial vs. 6 months of aging). For in situ zymography, fluorescence was detected in all groups and CHX pre-treatment was able to inhibit the gelatinolytic activity in all conditions. The 10-MDP-based universal adhesive system in self-etching mode was the strategy that showed the best bonding performance irrespective of its combination with chlorhexidine. Pre-treatment with CHX did not impair the bond strength when used in combination with 10-MDP and it may promote collagen stability overtime.

Increased glycated calmodulin in the submandibular salivary glands of streptozotocin-induced diabetic rats.

Non-enzymatic glycosylation, a post translational protein modification may be implicated in the diabetes complications. Calmodulin is an important calcium binding protein that complexed with Ca(2+) may be implicated in salivary gland secretory process. Glycated calmodulin has shown to be less effective in binding calcium. The aim of this study was to determine whether the concentration of glycated-calmodulin may be elevated in the submandibular salivary glands of streptozotocin-induced diabetic rats. Diabetes was induced by an intraperitoneal injection of spreptozotocin, and hyperglycemia was confirmed 72 h after injection using a glucosimeter. Thirty days after the induction of diabetes, submandibular salivary glands were used for the analysis of glycated and non-glycated calmodulin, using a glycogel B columns for separation. Glycated and non-glycated calmodulin were assayed by an enzymatic method and by ELISA. The overall concentration of CaM (non-glycated + glycated) in induced diabetic rats was significantly lower than in controls (p < 0.05). The concentration of non-glycated CaM in controls was significantly higher than in experimental group (p < 0.05), while the concentration of glycated calmodulin between these groups was statistically similar (p > 0.05).

Influence of chlorhexidine concentration on the durability of etch-and-rinse dentin bonds: a 12-month in vitro study.

PURPOSE: To investigate the effect of 0.2% and 2% chlorhexidine (CHX) used as a therapeutic primer on the long-term bond strengths of two etch-and-rinse adhesive systems. MATERIALS AND METHODS: Adper Scotchbond 1XT (SB1) and XP-Bond (XPB) were evaluated. Etched dentin substrates were assigned to 6 treatment groups: (1) 0.2% CHX + SB1; (2) 2% CHX + SB1; (3) SB1 (control); (4) 0.2% CHX + XPB; (5) 2% CHX + XPB; (6) XPB (control). Composite buildups were made and beams prepared for microtensile bond strength testing. Beams were divided into 3 subgroups and either immediately pulled to failure or stored in artificial saliva for 6 or 12 months prior to testing. Data were evaluated with three-way ANOVA. Additional adhesive interfaces were prepared to investigate nanoleakage expression by TEM. RESULTS: SB1 and XPB showed similar immediate bond strength values with or without CHX pretreatment (p > 0.05). After 12 months, bonds fell from 43.9 +/- 9.5 MPa to 20.1 +/- 5.4 MPa and from 39.6 +/- 9.4 MPa to 14.2 +/- 5.0 MPa in control specimens for SB1 and XPB respectively, while bond fell only from 41.9 +/- 9.6MPa to 33.2 +/- 8.3 MPa and 38.3 +/- 8.9 MPa to 26.5 +/- 10.9 (for SB1 and XPB, respectively) when 0.2% CHX was previously used. CHX concentration did not affect bond strength values (0.2% vs 2%, p > 0.05). Nanoleakage increased during aging in controls, but reduced silver deposits were found in CHX-treated specimens. CONCLUSION: Chlorhexidine significantly reduced the loss of bond strength seen in control bonds. Since no bacterial growth was present in the aging conditions, the results of this study suggest that endogenous factors thought to degrade the adhesive interface can be inhibited by CHX. Further in vivo trials should confirm the role of CHX in bond durability.