Mechanical Stability and Proteolytic Activity of Resin-dentin Bonds Using the Cross-linked Dry Bonding Technique.

OBJECTIVE: To evaluate the mechanical stability and the proteolytic activity of bonds created by a two-step, etch-and-rinse adhesive applied to cross-linked and air-dried etched dentin. METHODS: Flat dentin surfaces were produced in 64 extracted sound human molars. The dentin was etched with 35% phosphoric acid for 15 seconds, and then the teeth were divided into groups according to the cross-linking solution applied on the etched dentin. Group 1: 5% grape seed extract (GSE), Group 2: 5% glutaraldehyde, Group 3: Gluma Desensitizer, or Group 4: deionized water (control). Solutions were applied for 60 seconds, followed by rinse and blot drying. Then, the teeth were separated into two subgroups where the etched dentin was kept moist or air-dried. The adhesive was applied followed by a composite resin buildup. After 24 hours, the teeth were cut into beams (0.81 mm²) that were tested for microtensile strength immediately or after 12 months of aging in a 37°C saliva-like buffer. Additional teeth (n=32) were bonded as described and cut into 0.5-mm-thick slabs. The slabs were prepared for nanoleakage (scanning electron microscopy) and in situ zymography (EnzChek Protease Assay Kit). Bond strength data were submitted to ANOVA and Tukey tests (α =0.05). RESULTS: Significant reduction in immediate bond strength (ca 65%) and increase in proteolytic activity was seen when the etched dentin was air dried without previous cross-linking biomodification. Conversely, bond strengths did not differ from those produced on wet dentin when collagen was cross-linked before air drying, irrespective of the solution applied. For both moist and air-dried etched dentin, collagen cross-linking resulted in mechanically stable bonds and reduced proteolytic activity after 12 months of storage. CONCLUSION: Bonds produced by the application of a two-step, etch-and-rinse adhesive to cross-linked, air-dried, etched dentin were mechanically stable and revealed reduced proteolytic activity after 1 year of aging.

Chitosan-Based Extrafibrillar Demineralization for Dentin Bonding.

Instability of resin-dentin bonds is the Achilles' heel of adhesive dentistry. To address this problem, a chelate-and-rinse extrafibrillar dentin demineralization strategy has been developed that keeps intrafibrillar minerals within collagen fibrils intact to prevent activation of endogenous proteases that are responsible for collagen degradation within hybrid layers. The objective of the present study was to evaluate the potential of using chitosan >40 kDa as an antimicrobial extrafibrillar dentin-chelating agent to enhance bond durability. Transmission electron microscopy provided evidence for retention of intrafibrillar minerals and smear plugs in dentin conditioned with 1 wt% chitosan. Analyzed by Kruskal-Wallis analysis of variance, Dunn's statistic, and separate Mann-Whitney tests, tensile bond strengths to wet- and dry-bonded dentin indicated that chelating dentin with chitosan for 60 s prior to bonding did not result in a significant decline in resin-dentin bond strength when compared with that of phosphoric acid etching ( P > 0.05). Gelatinolytic activity within the hybrid layers was examined via in situ zymography after 24-h storage or after thermomechanical cycling and analyzed with 3-factor analysis of variance. After 24 h, enzymatic activity was detected only within completely demineralized phosphoric acid-etched dentin, with values derived from dry bonding significantly higher than those derived from wet bonding ( P < 0.05). Negligible fluorescence was detected within hybrid layers when dentin was conditioned with chitosan, even after thermomechanical cycling, as compared with the controls. Reduction in water permeability in chitosan-conditioned dentin, attributed to smear plug retention, also fostered long-term bond stability. Antibacterial testing performed with live/dead staining indicated that the acetic acid-solubilized chitosan possessed antibacterial activities against 3 single-species biofilms: Streptococcus mutans, Actinomyces naeslundii, and Enterococcus faecalis. Taken together, the new chitosan-based extrafibrillar demineralization strategy retains intrafibrillar minerals, reduces endogenous protease-initiated collagen degradation, prevents water permeation within hybrid layers, and kills bacteria on dentin surfaces, which are crucial factors for enhancing resin-dentin bond durability.

Zymography of Hybrid Layers Created Using Extrafibrillar Demineralization.

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

Effect of dimethyl sulfoxide on dentin collagen.

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

Substantivity of Carbodiimide Inhibition on Dentinal Enzyme Activity over Time.

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

Role of indoleamine 2,3-dioxygenase in an inflammatory model of murine gingiva.

BACKGROUND AND OBJECTIVE: Indoleamine 2,3-dioxygenase (IDO) is one of the major pathways for metabolism of tryptophan in a variety of cells, including immune cells. Increasing evidence indicates that IDO is a critical player in establishing the balance between immunity and tolerance and ultimately in the maintenance of homeostasis. By inducing inflammation in gingival tissue, we tested the hypothesis that IDO is a pivotal player in regulating the immune and inflammatory responses of gingiva. MATERIAL AND METHODS: We utilized the IDO knockout mouse model in conjunction with lipopolysaccharide (LPS)-induced inflammation. Accordingly, wild-type and IDO knockout mice were injected with LPS or vehicle in the anterior mandibular gingiva, twice over a 2-wk period, which was followed by procurement of gingival tissue for histopathology and preparation of tissue for flow cytometry-based studies. RESULTS: Clinical and histological examinations revealed a marked adverse impact of IDO deficiency on gingival inflammation. These observations were consistent with a more marked increase in the number of cells positive for the proinflammatory cytokine interleukin (IL)-17, but no significant change in the number of cells positive for the anti-inflammatory cytokine IL-10, in LPS-treated IDO knockout mice. Consistent with the more marked proinflammatory impact of IDO deficiency, the percentage of regulatory T cells was much reduced in gingival tissue of LPS-treated IDO knockout mice than in gingival tissue of wild-type mice. These proinflammatory changes were accompanied with a prominent increase in apoptotic and necrotic cell death in gingival tissue of IDO knockout mice compared with wild-type mice. CONCLUSION: Collectively, our findings support a major role for IDO in the development of gingival inflammation, as an example of an inflammatory condition, and lay the foundation for subsequent studies to explore it as a novel immunotherapy target.

Phosphoric acid concentration affects dentinal MMPs activity.

OBJECTIVES: To evaluate whether the concentration of phosphoric acid (PA) has an effect on the proteolytic activity of sound human demineralized dentin. It is hypothesized that the activity of matrix-bound and extracted enzymes depends on the PA concentration used to demineralize dentin. METHODS: One-gram aliquots of mid-coronal human dentin powder were demineralized with 1wt%, 10wt% and 37wt% PA. Concentrations of released calcium were measured for each set of demineralization. Extracted MMP-2 was immunologically identified by western blot and its activity was determined by conventional gelatin zymography. Analysis of released hydroxyproline (HYP) and in situ zymography were performed to evaluate the activity of insoluble, bound-matrix enzymes. RESULTS: The amount of released calcium from dentin powder treated with 37wt% PA was significantly higher (p≤0.05) than that obtained by dentin demineralization with 10wt% and 1wt% PA. Expression and activity of endogenous enzymes, extracted from or bound to dentin matrix, were detected for all samples regardless of the PA concentration. However, the expression and activity of extracted MMP-2 were significantly higher when dentin was treated with 10wt% PA (p<0.05), followed by 1wt% and 37wt% PA. Similarly, the highest concentration of released HYP (i.e. meaning higher percentage of collagen degradation) and the highest activity in in situ zymography were observed when dentin samples were treated with 10wt% PA (p<0.05). CONCLUSIONS: It was confirmed that PA does not denature endogenous enzymes of dentin matrices, but it may somehow modulate the expression and activity of these enzymes in a concentration-dependent manner. CLINICAL SIGNIFICANCE: Endogenous proteases have been identified and suggested to be responsible for the digestion of dentin matrix when activated by the acidic components of dental adhesives. Proteolytic activity of dentinal MMPs showed to be dependent on phosphoric acid concentration. The clinically-used concentration (37%) does not inhibit MMPs activity, but slows it.

The effects of ethanol on the size-exclusion characteristics of type I dentin collagen to adhesive resin monomers.

During dentin bonding with etch-and-rinse adhesive systems, phosphoric acid etching of mineralized dentin solubilizes the mineral crystallites and replaces them with bound and unbound water. During the infiltration phase of dentin bonding, solvated adhesive resin comonomers are supposed to replace all of the unbound collagen water and polymerize into copolymers. A recently published review suggested that dental monomers are too large to enter and displace water from tightly-packed collagen molecules. Conversely, recent work from the authors' laboratory demonstrated that HEMA and TEGDMA freely equilibrate with water-saturated dentin matrices. However, because adhesive blends are solvated in organic solvents, those solvents may remove enough free water to allow collagen molecules to come close enough to exclude adhesive monomer permeation. The present study analyzed the size-exclusion characteristics of dentin collagen, using a gel permeation-like column chromatography technique, filled with dentin powder instead of Sephadex beads as the stationary phase. The elution volumes of different sized test molecules, including adhesive resin monomers, studied in both water-saturated dentin, and again in ethanol-dehydrated dentin powder, showed that adhesive resin monomers can freely diffuse into both hydrated and dehydrated collagen molecules. Under these in vitro conditions, all free and some of the loosely-bound water seems to have been removed by ethanol. These results validate the concept that adhesive resin monomers can permeate tightly-bound water in ethanol-saturated collagen molecules during infiltration by etch-and-rinse adhesives. STATEMENT OF SIGNIFICANCE: It has been reported that collagen molecules in dentin matrices are packed too close together to allow permeation of adhesive monomers between them. Resin infiltration, in this view, would be limited to extrafibrillar spaces. Our work suggests that monomers equilibrate with collagen water in both water and ethanol-saturated dentin matrices.

Paucity of Nanolayering in Resin-Dentin Interfaces of MDP-based Adhesives.

Self-assembled nanolayering structures have been reported in resin-dentin interfaces created by adhesives that contain 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP). These structures have been hypothesized to contribute to bond durability. The objective of the present study was to determine the extent of nanolayering in resin-dentin interfaces after application of commercialized 10-MDP-containing self-etch and universal adhesives to human dentin. Seven commercialized adhesives were examined: Adhese Universal (Ivoclar-Vivadent), All-Bond Universal (Bisco, Inc.), Clearfil SE Bond 2, Clearfil S3 Bond Plus, Clearfil Universal Bond (all from Kuraray Noritake Dental Inc.), G-Premio Bond (GC Corp.), and Scotchbond Universal (3M ESPE). Each adhesive was applied in the self-etch mode on midcoronal dentin according to the respective manufacturer's instructions. Bonded specimens (n = 6) were covered with flowable resin composite, processed for transmission electron microscopy, and examined at 30 random sites without staining. Thin-film glancing angle X-ray diffraction (XRD) was used to detect the characteristic peaks exhibited by nanolayering (n = 4). The control consisted of 15%wt, 10%wt, and 5%wt 10-MDP (DM Healthcare Products, Inc.) dissolved in a mixed solvent (ethanol and water weight ratio 9:8, with photoinitiators). Experimental primers were applied to dentin for 20 s, covered with hydrophobic resin layer, and examined in the same manner. Profuse nanolayering with highly ordered periodicity (~3.7 nm wide) was observed adjacent to partially dissolved apatite crystallites in dentin treated with the 15% 10-MDP primer. Three peaks in the 2θ range of 2.40° (3.68 nm), 4.78° (1.85 nm), and 7.18° (1.23 nm) were identified from thin-film XRD. Reduction in the extent of nanolayering was observed in the 10% and 5% 10-MDP experimental primer-dentin interface along with lower intensity XRD peaks. Nanolayering and characteristic XRD peaks were rarely observed in specimens prepared from the commercialized adhesives. The sparsity of nanolayering in resin-dentin interfaces created by commercialized adhesives challenges its clinical effectiveness as a mechanism for improving bond longevity in dentin bonding.

Effect of pretreatment with collagen crosslinkers on dentin protease activity.

OBJECTIVES: This study evaluated the effect of dentin pretreatment with collagen crosslinkers on matrix metalloproteinase (MMP) and cathepsin K mediated collagen degradation. METHODS: Dentin beams (1mm×2mm×6mm) were demineralized in 10% H3PO4 for 24h. After baseline measurements of dry mass, beams were divided into 11 groups (n=10/group) and, were pretreated for 5min with 1% glutaraldehyde (GA); 5% GA; 1% grape-seed extract (GS); 5% GS; 10% sumac (S); 20µM curcumin (CR); 200µM CR; 0.l% riboflavin/UV (R); 0.5% R; 0.1% riboflavin-5-phosphate/UV (RP); and control (no pretreatment). After pretreatment, the beams were blot-dried and incubated in 1mL calcium and zinc-containing medium (CM, pH 7.2) at 37°C for 3, 7 or 14 days. After incubation, dry mass was reassessed and aliquots of the incubation media were analyzed for collagen C-telopeptides, ICTP and CTX using specific ELISA kits. Data were analyzed by repeated-measures ANOVA. RESULTS: The rate of dry mass loss was significantly different among test groups (p<0.05). The lowest 14 day mean dry mass loss was 6.98%±1.99 in the 200µM curcumin group compared to control loss of dry mass at 32.59%±5.62, p<0.05, at 14 days. The ICTP release over the incubation period (ng/mg dry dentin) ranged between 1.8±0.51 and 31.8±1.8. CTX release from demineralized beams pretreated with crosslinkers was significantly lower than CM (5.7±0.2ng/mg dry dentin). SIGNIFICANCE: The results of this study indicate that collagen crosslinkers tested in this study are good inhibitors of cathepsin K activity in dentin. However, their inhibitory effect on MMP activity was highly variable.

Effect of polyacrylic acid on dentin protease activities.

OBJECTIVE: This study tested whether treatment of demineralized dentin with polyacrylic acid (PAA) has any activatory or inhibitory activity on dentin matrix metalloproteinases (MMP)s or cathepsin K (CAT-K). METHODS: Dentin beams (1mm×2mm×6mm; n=10) were completely demineralized with EDTA. After initial dry mass assessment, the beams were dipped into 37% phosphoric acid (PA), PA+2% benzalkonium chloride (BAC), PA+2% chlorhexidine digluconate (CHX), 10% PAA, PAA+BAC or PAA+CHX for 20s. Demineralized beams without treatment served as control. All beams were incubated in simulated body fluid (SBF) for 1 week and the dry mass loss was evaluated. Aliquots of SBF were used to analyze solubilized telopeptide fragments using ICTP as indicator of MMP-mediated collagen degradation and CTX for CAT-K-mediated degradation. Additional demineralized beams (n=10) were used to measure the influence of different chemical treatments on total MMP activity of EDTA-demineralized dentin using generic MMP assay. Data were analyzed by ANOVA (α=0.05). RESULTS: Dry mass loss ranged from 6% (PA) to 2% for (PA-BAC) or (PAA-BAC) (p<0.05). ICTP release of PAA-treated group was significantly higher (p<0.05) than the control, and not significantly different from the PA group (p>0.05). PA+CHX or PAA+CHX and PAA+BAC showed significantly lower ICTP than PA or PAA groups (p<0.05). CAT-K activity increased significantly after 10% PAA treatment compared to control (p<0.05) or to PA postreatment. SIGNIFICANCE: Demineralized dentin treated with 10% polyacrylic acid activated CAT-K more than 37% phosphoric acid; 2% chlorhexidine digluconate seems to be a better inhibitor of MMPs and CAT-K than 2% benzalkonium chloride.

Increased Durability of Resin-Dentin Bonds Following Cross-Linking Treatment.

OBJECTIVES: This study evaluated the long-term effect of carbodiimide treatments of acid-etched dentin on resin-dentin bond strength of a simplified etch-and-rinse adhesive system. METHODS: Forty-eight sound third molars were divided into three groups (n=16) according to the dentin treatment: G1: deionized water; G2: 0.5 mol/L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) applied for 30 seconds; and G3: 0.5 mol/L EDC applied for 60 seconds. Flat dentin surfaces were produced, etched with 37% phosphoric acid for 15 seconds, and then treated with deionized water for 60 seconds or with 0.5 mol/L EDC for 30 or 60 seconds prior to the application of Single Bond 2. Crowns were restored with resin composite, and beam specimens were prepared for microtensile testing. The beams from each group were tested 24 hours or 6 or 12 months after the adhesive procedures. One slab from each tooth was prepared and analyzed for nanoleakage. Bond strength (MPa) data were submitted to analysis of variance and Tukey test (α=0.05). RESULTS: The treatment of dentin with 0.5 mol/L EDC for 30 seconds (24.1±6.2 MPa) and 60 seconds (25.5±5.1 MPa) did not negatively affect the immediate bond strength of Single Bond 2 when compared to the control group (24.6±7.3 MPa). Additionally, EDC prevented resin-dentin bond degradation after 12 months in artificial saliva for both periods of treatment. An increased accumulation of silver ions was seen for the control group over time, while a much lower amount of silver grains was observed for the EDC-treated groups. CONCLUSIONS: 0.5 mol/L EDC was able to prevent resin-dentin bond degradation after 12 months, especially when applied for 60 seconds.

Bonding of universal adhesives to dentine--Old wine in new bottles?

OBJECTIVE: Multi-mode universal adhesives offer clinicians the choice of using the etch-and-rinse technique, selective enamel etch technique or self-etch technique to bond to tooth substrates. The present study examined the short-term in vitro performance of five universal adhesives bonded to human coronal dentine. METHODS: Two hundred non-carious human third molars were assigned to five groups based on the type of the universal adhesives (Prime&Bond Elect, Scotchbond Universal, All-Bond Universal, Clearfil Universal Bond and Futurabond U). Two bonding modes (etch-and-rinse and self-etch) were employed for each adhesive group. Bonded specimens were stored in deionized water for 24h or underwent a 10,000-cycle thermocycling ageing process prior to testing (N=10). Microtensile bond testing (µTBS), transmission electron microscopy (TEM) of resin-dentine interfaces in non-thermocycled specimens and scanning electron microscopy (SEM) of tracer-infused water-rich zones within hybrid layers of thermocycled specimens were performed. RESULTS: Both adhesive type and testing condition (with/without thermocycling) have significant influences on µTBS. The use of each adhesive in either the etch-and-rinse or self-etch application mode did not result in significantly different µTBS to dentine. Hybrid layers created by these adhesives in the etch-and-rinse bonding mode and self-etch bonding mode were ∼5µm and ≤0.5µm thick respectively. Tracer-infused regions could be identified within the resin-dentine interface from all the specimens prepared. CONCLUSION: The increase in versatility of universal adhesives is not accompanied by technological advances for overcoming the challenges associated with previous generations of adhesives. Therapeutic adhesives with bio-protective and bio-promoting effects are still lacking in commercialized adhesives. CLINICAL SIGNIFICANCE: Universal adhesives represent manufacturers' attempt to introduce versatility in product design via adaptation of a single-bottle self-etch adhesive for other application modes without compromising its bonding effectiveness.

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

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

Transdentinal cytotoxicity of carbodiimide (EDC) and glutaraldehyde on odontoblast-like cells.

OBJECTIVE: To evaluate the transdentinal cytotoxicity of three different concentrations of carbodiimide (EDC) or 5% glutaraldehyde (GA) on MDPC-23 cells. METHODS: Seventy 0.4-mm-thick dentin disks obtained from human molars were adapted to artificial pulp chambers. MDPC-23 cells were seeded on the pulpal surface of the disks. After 48 hours, the occlusal dentin was acid-etched and treated for 60 seconds with one of the following solutions (n=10): no treatment (negative control); 0.1 M, 0.3 M, or 0.5 M EDC; 5% GA; Sorensen buffer; or 29% hydrogen peroxide (positive control). Cell viability and morphology were assessed by methyltetrazolium assay and scanning electron microscopy (SEM), respectively. The eluates were collected after the treatments and applied on MDPC-23 seeded in a 24-well plate to analyze cell death, total protein (TP), and collagen production. The last two tests were performed 24 hours and seven days after the challenge. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (p<0.05). RESULTS: EDC at all test concentrations did not reduce cell viability, while 5% GA did increase cell metabolism. Cell death by necrosis was not elicited by EDC or 5% GA. At the 24-hour period, 0.3 M and 0.5 M EDC reduced TP production by 18% and 36.8%, respectively. At seven days, increased TP production was observed in all groups. Collagen production at the 24-hour period was reduced when 0.5 M EDC was used. After seven days, no difference was observed among the groups. SEM showed no alteration in cell morphology or number, except in the hydrogen peroxide group. CONCLUSIONS: Treatment of acid-etched dentin with EDC or GA did not cause transdentinal cytotoxic effects on odontoblast-like cells.

Role of dentin MMPs in caries progression and bond stability.

Dentin can be described as a biological composite with collagen matrix embedded with nanosized hydroxyapatite mineral crystallites. Matrix metalloproteinases (MMPs) and cysteine cathepsins are families of endopeptidases. Enzymes of both families are present in dentin and collectively capable of degrading virtually all extracellular matrix components. This review describes these enzymes and their presence in dentin, mainly focusing on their role in dentin caries pathogenesis and loss of collagen in the adhesive hybrid layer under composite restorations. MMPs and cysteine cathepsins present in saliva, mineralized dentin, and/or dentinal fluid may affect the dentin caries process at the early phases of demineralization. Changes in collagen and noncollagenous protein structure may participate in observed decreases in mechanical properties of caries-affected dentin and reduce the ability of caries-affected dentin to remineralize. These endogenous enzymes also remain entrapped within the hybrid layer during the resin infiltration process, and the acidic bonding agents themselves (irrespective of whether they are etch-and-rinse or self-etch) can activate these endogenous protease proforms. Since resin impregnation is frequently incomplete, denuded collagen matrices associated with free water (which serves as a collagen cleavage reagent for these endogenous hydrolase enzymes) can be enzymatically disrupted, finally contributing to the degradation of the hybrid layer. There are multiple in vitro and in vivo reports showing that the longevity of the adhesive interface is increased when nonspecific enzyme-inhibiting strategies are used. Different chemicals (i.e., chlorhexidine, galardin, and benzalkonium chloride) or collagen cross-linker agents have been successfully employed as therapeutic primers in the bonding procedure. In addition, the incorporation of enzyme inhibitors (i.e., quaternary ammonium methacrylates) into the resin blends has been recently promoted. This review will describe MMP functions in caries and hybrid layer degradation and explore the potential therapeutic role of MMP inhibitors for the development of improved intervention strategies for MMP-related oral diseases.

Zoledronate and ion-releasing resins impair dentin collagen degradation.

This study analyzed the amounts of solubilized telopeptides cross-linked carboxyterminal telopeptide of type I collagen (ICTP) and C-terminal crosslinked telopeptide of type I collagen (CTX) derived from matrix-metalloproteinases (MMPs) and cysteine cathepsins (CTPs) subsequent to application of a filler-free (Res.A) or an ion-releasing resin (Res.B) to ethylenediaminetetraacetic acid (EDTA)-demineralized dentin with or without zoledronate-containing primer (Zol-primer) pre-treatment. The chemical modification induced following treatments and artificial saliva (AS) storage was also analyzed through attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Totally EDTA-demineralized specimens were infiltrated with Res.A or Res.B with or without Zol-primer pre-treatment, light-cured, and immersed in AS for up to 4 wk. ICTP release was reduced following infiltration with Res.B and further reduced when Res.B was used with Zol-primer; remarkable phosphate mineral uptake was attained after AS storage. CTX release was increased in Res.A- and Res.B-treated dentin. However, when Zol-primer was used with Res.A, the CTX release fell significantly compared to the other tested resin-infiltration methods. In conclusion, zoledronate offers an additional inhibitory effect to the ion-releasing resins in MMP-mediated collagen degradation. However, Zol-primer induces a modest reduction in CTX release only when used with resin-based systems containing no ion-releasing fillers.

Differences in the microstructure and fatigue properties of dentine between residents of North and South America.

UNLABELLED: Spatial variations in the microstructure of dentine contribute to its mechanical behaviour. OBJECTIVE: The objective of this investigation was to compare the microstructure and fatigue behaviour of dentine from donors of two different countries. METHODS: Caries-free third molars were obtained from dental practices in Colombia, South America and the US to assemble two age-matched samples. The microstructure of the coronal dentine was evaluated at three characteristic depths (i.e. deep, middle and superficial dentine) using scanning electron microscopy and image processing techniques. The mechanical behaviour of dentine in these three regions was evaluated by the fatigue crack growth resistance. Cyclic crack growth was achieved in-plane with the dentine tubules and the fatigue crack growth behaviour was characterized in terms of the stress intensity threshold and the Paris Law parameters. RESULTS: There was no difference in the tubule density between the dentine of patients from the two countries. However, there were significant differences (p≤0.05) in the tubule lumen diameters between the two groups in the deep and peripheral regions. In regards to the fatigue resistance, there was a significant increase (p≤0.05) in threshold stress intensity range, and a significant decrease in fatigue crack growth coefficient with increasing distance from the pulp in teeth from the US donors. In contrast, these properties were independent of location for the dentine of teeth from the Colombian donors. CONCLUSIONS: The microstructure of dentine and its mechanical behaviour appear to be a function of patient background, which may include environmental factors and/or ethnicity.

Carbodiimide inactivation of MMPs and effect on dentin bonding.

The use of protein cross-linking agents during bonding procedures has been recently proposed to improve bond durability. This study aimed to use zymography and in situ zymography techniques to evaluate the ability of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linker to inhibit matrix metalloproteinase (MMP) activity. The hypotheses tested were that: (1) bonding procedures increase dentin gelatinolytic activity and (2) EDC pre-treatment prevents this enzymatic activity. The zymographic assay was performed on protein extracts obtained from dentin powder treated with Optibond FL or Scotchbond 1XT with or without 0.3M EDC pre-treatment. For in situ zymography, adhesive/dentin interfaces were created with the same adhesives applied to acid-etched dentin slabs pre-treated or not with EDC conditioner. Zymograms revealed increased expression of dentin endogenous MMP-2 and -9 after adhesive application, while the use of EDC as a primer inactivated dentin gelatinases. Results of in situ zymograpy showed that hybrid layers of tested adhesives exhibited intense collagenolytic activity, while almost no fluorescence signal was detected when specimens were pre-treated with EDC. The correlative analysis used in this study demonstrated that EDC could contribute to inactivate endogenous dentin MMPs within the hybrid layer created by etch-and-rinse adhesives.

The importance of size-exclusion characteristics of type I collagen in bonding to dentin matrices.

The mineral phase of dentin is located primarily within collagen fibrils. During development, bone or dentin collagen fibrils are formed first and then water within the fibril is replaced with apatite crystallites. Mineralized collagen contains very little water. During dentin bonding, acid-etching of mineralized dentin solubilizes the mineral crystallites and replaces them with water. During the infiltration phase of dentin bonding, adhesive comonomers are supposed to replace all of the collagen water with adhesive monomers that are then polymerized into copolymers. The authors of a recently published review suggested that dental monomers were too large to enter and displace water from collagen fibrils. If that were true, the endogenous proteases bound to dentin collagen could be responsible for unimpeded collagen degradation that is responsible for the poor durability of resin-dentin bonds. The current work studied the size-exclusion characteristics of dentin collagen, using a gel-filtration-like column chromatography technique, using dentin powder instead of Sephadex. The elution volumes of test molecules, including adhesive monomers, revealed that adhesive monomers smaller than ∼1000 Da can freely diffuse into collagen water, while molecules of 10,000 Da begin to be excluded, and bovine serum albumin (66,000 Da) was fully excluded. These results validate the concept that dental monomers can permeate between collagen molecules during infiltration by etch-and-rinse adhesives in water-saturated matrices.