Role of proteoglycans on the biochemical and biomechanical properties of dentin organic matrix

ObjectiveProteoglycans (PGs) are multifunctional biomacromolecules of the extracellular matrix of collagen-based tissues. In teeth, besides a pivotal regulatory role on dentin biomineralization, PGs provide mechanical support to the mineralized tissue and compressive strength to the biosystem. This study assessed enzymatic protocols for selective PGs removal from demineralized dentin to determine the roles of these biomacromolecules in the bulk mechanical properties and biostability of type I collagen.MethodsSelective removal of glycosaminoglycans chains (GAGs) and PGs from demineralized dentin was carried out by enzymatic digestion protocols using chondroitinase ABC (c-ABC) and trypsin (Try). A comprehensive study design included assessment of dentin matrix mass loss, biodegradability of the PGs/GAGs-depleted dentin matrix, ultimate tensile strength (UTS) and energy to fracture tests. Quantitative data was statistically analyzed by two-way and one-way ANOVA followed by the appropriate post hoc tests (α = 0.05).ResultsTransmission electron microscopy images show effective GAGs removal by c-ABC and Try and both enzymatic methods released statistically similar amounts of GAGs from the demineralized dentin. Try digestion resulted in about 25% dentin matrix mass loss and increased susceptibility to collagenolytic digestion when compared to c-ABC (p = 0.0224) and control (p = 0.0901). Moreover, PGs digestion by Try decreased the tensile strengths of dentin. Statistically lower energy to fracture was observed in c-ABC-treated dentin matrix.ConclusionsGAGs plays a pivotal role on tissue mechanics and anisotropy, while the core protein of PGs have a protective role on matrix biostability.     

Effect of Proanthocyanidin-enriched extracts on the inhibition of wear and degradation of dentin demineralized organic matrix

ObjectivesThe aim of this study was to evaluate the effect of Cranberry and Grape seed-enriched extract gels in inhibiting wear and degradation of demineralized organic matrix (DOM).Design225 dentin specimens obtained from bovine incisors were randomly allocated into 5 groups (n = 45): 10% Grape seed extract gel (GSE), 10% Cranberry extract gel (CE), 0.012% Chlorhexidine gel (CX), 1.23% NaF gel (F), and no active compound gel (P, placebo). Before the treatments, samples were demineralized by immersion in 0.87 M citric acid, pH 2.3 (36 h). Then, the studied gels were applied once over dentin for 1 min. Next, the samples were immersed in artificial saliva containing collagenase obtained from Clostridium histolyticum for 5 days. The response variable for dentin wear was depth of dentin loss measured by profilometry and for collagen degradation was hydroxyproline determination. Data were analyzed by ANOVA followed by Tukey's test and Pearson Correlation Test (p < 0.05).ResultsGrape seed extract significantly reduced dentin wear compared to the other groups (p < 0.05). Cranberry extract and Chlorhexidine did not differ statistically and were able to reduce wear when compared to NaF and placebo treatments. The hydroxyproline analysis showed that there was no significant difference among groups for all treatments (p < 0.05). Correlation analysis showed a significant correlation between the amount of degraded DOM evaluated by profilometry and the determination of hydroxyproline.ConclusionCranberry extract was able to reduce the dentin wear and collagen degradation, likely due to the proanthocyanidin content and its action. Therefore, Cranberry could be suggested as an interesting natural-based agent to prevent dentin erosion.     

The effect of active components from citrus fruits on dentin MMPs

ObjectivesThis study was aimed to evaluate the anti-matrix metalloproteinases (MMPs) ability of active components from citrus fruits (hesperetin: Hst, hesperidin: Hsd and naringenin: Nge).MethodsInactivation effects of citrus flavonoids (Hst, Hsd, Nge) at different concentrations on soluble collagenase were measured using a fluorometric assay. Matrix-bound endogenous MMPs activity was evaluated via dry mass loss and hydroxyproline (HYP) release of demineralized human dentin. Demineralized dentin beams were pretreated with 500 μg/mL citrus flavonoids for 10 min. Chlorhexidine (CHX) was used as inhibitor control. Beams pretreated with distilled water served as blank control. Dentin slabs were used for in situ zymography and evaluated under confocal microscopy. Ultrastructure of demineralized collagen fibers was exhibited by Transmission Electron Microscopy (TEM).ResultsCitrus flavonoids exhibited inactivation function on soluble MMPs and the extent of inactivation increased in a dose-dependent manner. The inactivation percent of citrus flavonoids reached above 90% at the concentration of 500 μg/mL. Compared with control group, citrus flavonoids pretreated demineralized dentin beams exhibited less dry mass loss, lower hydroxyproline release and more intact collagen architecture after 15 days storage. Dentin samples pretreated with citrus flavonoids showed lower enzymes activities in in situ zymography.ConclusionsHst, Hsd or Nge have anti-MMPs ability and can preserve dentin collagen from degradation.Clinical Significance: Hst, Hsd and Nge may have the potential to be used in dentin bonding systems and improve the resin-dentin bonding durability.     

Effect of proanthocyanidin on ultrastructure and mineralization of dentine collagen

ObjectiveProanthocyanidin (PA) is a natural collagen cross-linker that has been used in dentine matrix biomodification for reparative and preventive therapies. This study evaluated the ultrastructure of collagen after its interaction with PA. Furthermore, the mineralization of PA-biomodified collagen matrix was observed.MethodsTen freshly extracted sound human molars were sectioned into 0.5 mm × 1.7 mm × 7 mm beams for ultrastructural evaluation of PA and dentine matrix under Field Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM). Specimens for TEM were completely demineralized and divided into three groups according to PA treatments: deionized water, 2% PA and 6.5% PA. The specimens were fixed, dehydrated, sectioned and examined using TEM. Specimens for FESEM were lightly conditioned with EDTA and similarly divided into the three groups for observation using FESEM. Type I collagen from calf skin was used to analyse the mineral interaction after treatment with 6.5% PA. Formvar- and carbon-coated 400-mesh Ni grids (EMS, Hatfiels, PA, USA) were placed over a 2 mg/mL collagen solution prepared from calf skin-derived Type I collagen to achieve self-assembly of collagen fibrils. Grids were treated with 6.5% PA and divided into two groups. One group was floated over a remineralization solution containing 20 mM HEPES, 2.25 mM CaCl₂-2H₂O, 1.35 mM KH₂PO4, 3.08 mM NaN3 and 130 mM KCl and the other group was over a CPP-ACP solution (Tooth mousse 1:100 dilution with deionized water). The floating samples were kept in a 37 °C and 100% humidity chamber. Grids were taken out at selected time durations (24 h, 48 h and 72 h for mineralization solution/24 h for CPP-ACP) and observed under TEM without staining. Selected area electron diffractions (SAEDs) were performed at 110 kV.ResultsFollowing treatment of demineralized dentine collagen matrix with PA, the size and number of interfibrillar spaces were reduced. The collagen fibrils aggregated together with a reduction in porosity. A characteristic banding pattern of collagen fibrils was observed under TEM. Treatment of PA-biomodified collagen fibrils with remineralization solution increased mineral aggregation along its long axis, when compared to the control group. Furthermore, treatment of PA-biomodified collagen fibrils with CPP-ACP solution enhanced mineral uptake and deposition as well as initiated apatite formation within 24 h.ConclusionProanthocyanidin alters the ultrastructure of demineralized dentine collagen matrix. The PA-biomodified collagen matrix promotes remineralization.     

Inhibition of endogenous human dentin MMPs by Gluma

ObjectiveThe objective of this study was to determine if Gluma dentin desensitizer (5.0% glutaraldehyde and 35% HEMA in water) can inhibit the endogenous MMPs of dentin matrices in 60 s and to evaluate its effect on dentin matrix stiffness and dry mass weight.MethodsDentin beams of 2 mm × 1 mm × 6 mm were obtained from extracted human third molars coronal dentin. To measure the influence of Gluma treatment time on total MMP activity of dentin, beams were dipped in 37% phosphoric acid (PA) for 15 s and rinsed in water. The acid-etched beams were then dipped in Gluma for 5, 15, 30 or 60 s, rinsed in water and incubated into SensoLyte generic MMP substrate (AnaSpec, Inc.) for 60 min. Controls were dipped in water for 60 s. Additional beams of 1 mm × 1 mm × 6 mm were completely demineralized in 37% PA for 18 h, rinsed and used to evaluate changes on the dry weight and modulus of elasticity (E) after 60 s of Gluma treatment followed by incubation in simulated body fluid buffer for 0, 1 or 4 weeks. E was measured by 3-pt flexure.ResultsGluma treatment inhibited total MMP activity of acid-etched dentin by 44, 50, 84, 86% after 5, 15, 30 or 60 s of exposure, respectively. All completely demineralized dentin beams lost stiffness after 1 and 4 weeks, with no significant differences between the control and Gluma-treated dentin. Gluma treatment for 60 s yielded significantly less dry mass loss than the control after 4 weeks.SignificanceThe use of Gluma may contribute to the preservation of adhesive interfaces by its cross-linking and inhibitory properties of endogenous dentin MMPs.     

Non-thermal atmospheric plasma brush induces HEMA grafting onto dentin collagen

ObjectiveNon-thermal atmospheric plasma (NTAP) brush has been regarded as a promising technique to enhance dental interfacial bonding. However, the principal enhancement mechanisms have not been well identified. In this study, the effect of non-thermal plasmas on grafting of HEMA, a typical dental monomer, onto dentin collagen thin films was investigated.MethodsHuman dentin was sectioned into 10-μm-thick films. After total demineralization in 0.5 M EDTA solution for 30 min, the dentin collagen films were water-rinsed, air-dried, treated with 35 wt% HEMA aqueous solution. The films were then subject to plasma-exposure under a NTAP brush with different time (1–8 min)/input power (5–15 W). For comparison, the dentin collagen films were also treated with the above HEMA solution containing photo-initiators, then subject to light-curing. After plasma-exposure or light-curing, the HEMA-collagen films were rinsed in deionized water, and then examined by FTIR spectroscopy and TEM.ResultsThe FITR results indicated that plasma-exposure could induce significant HEMA grafting onto dentin collagen thin films. In contrast, light-curing led to no detectable interaction of HEMA with dentin collagen. Quantitative IR spectral analysis (i.e., 1720/3075 or 749/3075, HEMA/collagen ratios) further suggested that the grafting efficacy of HEMA onto the plasma-exposed collagen thin films strongly depended on the treatment time and input power of plasmas. TEM results indicated that plasma treatment did not alter collagen's banding structure.SignificanceThe current study provides deeper insight into the mechanism of dental adhesion enhancement induced by non-thermal plasmas treatment. The NTAP brush could be a promising method to create chemical bond between resin monomers and dentin collagen.     

Stability of bonds made to superficial vs. deep dentin,before and after thermocycling

ObjectivesBonding stability of resinous adhesives to dentin is still problematic and may involve regional variations in dentin composition. This study is to evaluate the effect of dentin depth on the stability of resin-dentin bonds under thermocycling challenge.MethodsDentin slabs with two flat surfaces parallel to the tooth axis were obtained from extracted human third molars. The slabs were randomized into eight groups according to the location of dentin [deep dentin (DD) or superficial dentin (SD)], the adhesive treatment (Single Bond 2 or Clearfil S³ Bond), and the storage treatment (thermocycling for 5000 times vs. no). After the adhesive treatment and composite buildup on the dentin slabs, the micro-shear bond strength (μSBS) of each group was detected. The concentrations of cross-linked carboxyterminal telopeptide of type I collagen (ICTP) were also evaluated using an immunoassay to detect the degree of collagen degradation in each group.ResultsDentin depth, adhesive treatment and storage treatment all showed significant effects on both the μSBSs and the ICTP values (P < 0.05). Regardless of the adhesive type, thermocycling decreased the μSBSs and increased the ICTP values (P < 0.05). The DD groups showed significantly lower μSBSs and higher ICTP values than SD groups after thermocycling aging (P < 0.05). The treatment with Single Bond 2 significantly increased the ICTP values (P < 0.05), whereas Clearfil S³ Bond showed no effect on the ICTP values (P > 0.05).SignificanceDeep dentin showed significantly more bond degradation after thermocycling than did superficial dentin.     

Alternative model for cathepsin K activation in human dentin

ObjectiveTo evaluate the protease activity in dentin matrices subjected to lactic acid (LA) in comparison to polyacrylic acid (PAA) challenge model at cathepsin K (CT-K) optimum pH 5.5 to assess effectiveness of inhibitors in dentin collagen degradation.MethodsDentin disks measuring 0.5 mm prepared from human molars were completely demineralized in 10% H₃PO₄. Demineralized dentin disks were challenged with 0.1 M LA, 1.1 mM PAA, artificial saliva (AS), or deionized water (C) for 24 h or 7-days. Dentin collagen properties were tested by measurement of %dry mass change, and ultimate tensile strength (UTS). Degradation of dentin type I collagen was measured by telopeptide assays measuring the sub-product release of C-terminal cross-linked telopeptides (ICTP) and C-terminal peptide (CTX) in the incubation media in relation to total protein concentration, which correlates with matrix metalloproteinases (MMPs) and CT-K activities.ResultsGravimetric analysis showed statistically significant difference between C and other groups (p < 0.04) at 24 h. LA specimens showed significantly higher weight loss from 24 h to 7-days (p = 0.02). UTS revealed statistically significant difference between AS and LA at 24 h and 7-days. UTS at 24 h and 7-days for C and AS had significantly higher mean values compared to LA and PAA. Telopeptide assays reported that CTX_tp results showed that LA at 24 h had significantly higher mean values compared to C and AS.SignificanceLA has the ability to activate endogenous CT-K in dentin as measured by the release of CTX (CT-K specific telopeptide). This LA based model has the potential application for further investigations on the activity and possible inhibitors of CT-K in human dentin.     

The effect of different cooling procedures on mechanical properties of denture base materials measured by instrumented indentation testing

PurposeTo evaluate the different cooling procedures on the mechanical properties of five heat-cured polymethyl methacrylate (PMMA) denture materials.Methods250 specimens were made equally from Meliodent (ME), Paladon 65 (PA), Probase Hot (PB), Stellon QC–20 (QC) and Vertex Rapid Simplified (VE) implementing five different cooling procedures (n = 10/procedure): A) removal from water bath, bench-cooling (10 min) and cooling under water (15 min), B) remain in water bath till room temperature, C) removal from water bath and cooling in water for 15 min, D) removal from water bath and bench cooling till room temperature and E) removal from water bath, bench cooling for 30 min and cooling under water for 15 min. The specimens were immersed in distilled water (15 days/37 ºC) and then subjected to Instrumented Indentation Testing for Martens Hardness (HM), indentation modulus (E_IT) and elastic index (η_ΙΤ). Results were statistically analyzed by two- and one-way Analysis of variance (ANOVA) plus Tukey post hoc tests (α = 0.05).ResultsThe highest values for HM were recorded for QC, PA, VE with B cooling procedure, PB with A and ME with E, for E_ΙΤ for QC, PB with A, for PA, VE with B and ME with E, and for η_IT for QC, PB with B, PB with E, ME with C and VE with D.ConclusionsThe cooling procedures recommended for PB resulted in the lowest mechanical properties. A and B may be considered as universal short- and long-cooling procedures respectively providing the highest mechanical properties for the materials tested.     

Salivary esterases dramatically reduce biostability of dentin collagen treated with galloylated polyphenols

ObjectiveTo investigate the effects of salivary esterases on biostability of collagen treated by galloylated polyphenols.MethodsHuman dentin was microtomed into 6-μm-thick films, which were demineralized and treated for 60 s using solutions containing 0.6% and 2% of one of the crosslinkers: tannic acid (TAC), epigallocatechin gallate (EGCG), epigallocatechin (EGC), and N-[3-dimethylaminopropyl]-N′-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS), and for 1 h using EDC/NHS. Half of the treated and untreated (control) films were subjected to human saliva incubation. Collagen biostability was assessed via exogenous protease biodegradation by weight loss and hydroxyproline release, and endogenous MMPs by in situ zymography. The degradation products of galloylated polyphenols (TAC and EGCG) by saliva were monitored using proton nuclear magnetic resonance (¹H NMR) and gel permeation chromatography (GPC). The esterase activity of saliva induced by the crosslinkers was also assessed.ResultsCollagen films treated with TAC and EGCG exhibited significantly improved biostability (p < 0.05); however, the enhanced biostability was severely reduced after saliva incubation (p < 0.001). For EDC/NHS treated collagen, saliva incubation showed negligible effect on the biostability. ¹H NMR studies confirmed the esterase-catalyzed hydrolysis of the galloyl. GPC measurements showed decreased molecular weight of TAC in saliva indicating its chemical degradation. Both TAC and EGCG showed much higher esterase activity than other treatment groups.SignificanceThe galloyl group plays important role in collagen crosslinking, inducing higher biostability. However, galloylated polyphenols crosslinked on collagen are highly susceptible to metabolism of human saliva by salivary esterase, dramatically compromising the enhanced biostability.     

Evidence to the role of interflavan linkages and galloylation of proanthocyanidins at sustaining long-term dentin biomodification

Objectives

The interactivity of proanthocyanidins (PACs) with collagen modulates dentin matrix biomechanics and biostability. Herein, PAC extracts selected based on structural diversity were investigated to determine key PAC features driving sustained effects on dentin matrices over a period of 18 months.

Effect of laser activated bleaching on the chemical stability and morphology of intracoronal dentin

ObjectivesTo evaluate the effect of the bleaching with 35% hydrogen peroxide either activated or not by a 970 nm diode laser on the chemical stability and dentin surface morphology of intracoronary dentin.MethodsTwenty-seven slabs of intracoronary dentin specimens (3 × 3 mm) were distributed into three groups (n = 9), according to surface treatment: HP – 35% hydrogen peroxide (1 × 4’), DL – 970 nm diode laser (1 × 30”/0,8W/10 Hz), HP + DL – 35% HP activated with 970 nm diode laser (1 × 30”/0,8W/10 Hz leaving the gel in contact to the surface for 4′ after activation). Three Raman spectra from each fragment were obtained to calculate the mean intensity of peaks of inorganic component (a.u.), organic collagen content (a.u.), and the ratio of inorganic/organic content, before and after treatment. Analyses of the samples by confocal laser microscopy were performed to evaluate the surface roughness, percentage of tubules, perimeter and area percentage of tubules, before and after treatment. Data were analyzed by Kruskal-Wallis, Dunn’s, and Wilcoxon test (P < 0.05).ResultsData analysis showed that HP + DL did not change the inorganic content peaks 8.31 [29.78] or the inorganic/organic ratio 3.37 [14.67] (P > 0.05). Similarly, DL did not affect the chemical stability of the dentin surface (P > 0.05). However, HP significantly increased inorganic content peaks 10.87 [22.62], as well as the inorganic/organic ratio 6.25 [27.78] (P < 0.05). Regarding the morphological alterations, all surface treatments increase tubules exposure; HP treatment significantly increases perimeter and area percentage; and HP + DL increases surface roughness.ConclusionsBleaching HP combined with DL offers an improvement in terms of intracoronal dentin surface protection, yielding better maintenance of dentin chemical stability and morphology.     

Dehydration and the dynamic dimensional changes within dentin and enamel

ObjectivesThe objectives of this study were to quantify the dimensional changes in dentin and enamel during dehydration, and to determine if there are differences between the responses of these tissues from young and old patients.MethodsMicroscopic digital image correlation (DIC) was used to evaluate deformation of dentin and enamel as a function of water loss resulting from free convection in air. Dimensional changes within both tissues were quantified for two patient age groups (i.e. young 18 ≤ age ≤ 30 and old 50 ≤ age) and in two orthogonal directions (i.e. parallel and perpendicular to the prevailing structural feature (dentin tubules or enamel prisms)). The deformation histories were used to estimate effective dehydration coefficients that can be used in quantifying the strains induced by dehydration.ResultsBoth dentin and enamel underwent contraction with water loss, regardless of the patient age. There was no significant difference between responses of the two age groups or the two orthogonal directions. Over 1 h of free convection, the average water loss in dentin was 6% and resulted in approximately 0.5% shrinkage. In the same time period the average water loss in the enamel was approximately 1% and resulted in 0.03% shrinkage. The estimated effective dehydration coefficients were ?810 μm/m/(% weight loss) and ?50 μm/m/(% weight loss) for dentin and enamel, respectively.SignificanceThe degree of deformation shrinkage resulting from dehydration is over a factor of magnitude larger in dentin than enamel.     

Effect of biomimetic mineralization on enamel and dentin: A Raman and EDX analysis

ObjectiveTo investigate the effect of an experimental biomimetic mineralization kit (BIMIN) on the chemical composition and crystallinity of caries-free enamel and dentin samples in vitro.MethodsEnamel and dentin samples from 20 human teeth (10 for enamel; 10 for dentin) were divided into a control group without treatment and test samples with BIMIN treatment. Quantitative analysis of tissue penetration of fluoride, phosphate, and calcium was performed using energy-dispersive X-ray spectroscopy (EDX). Mineralization depth was measured by Raman spectroscopy probing the symmetric valence vibration near 960 cm⁻¹ as a marker for crystallinity. EDX data was statistically analyzed using a paired t-test and Raman data was analyzed using the Student’s t-test.ResultsEDX analysis demonstrated a penetration depth of fluoride of 4.10 ± 3.32 μm in enamel and 4.31 ± 2.67 μm in dentin. Calcium infiltrated into enamel 2.65 ± 0.64 μm and into dentin 5.58 ± 1.63 μm, while the penetration depths for phosphate were 4.83 ± 2.81 μm for enamel and 6.75 ± 3.25 μm for dentin. Further, up to 25 μm of a newly mineralized enamel-like layer was observed on the surface of the samples. Raman concentration curves demonstrated an increased degree of mineralization up to 5–10 μm into the dentin and enamel samples.SignificanceBiomimetic mineralization of enamel and dentin samples resulted in an increase of mineralization and a penetration of fluoride into enamel and dentin.     

Effect of residual solvent on performance of acrylamide-containing dental materials

ObjectiveMethacrylamide-based monomers are being pursued as novel, hydrolytically stable materials for use in dental adhesives. The impact of residual solvents, due to the chemical synthesis procedures or the need for solvated adhesives systems, on the kinetics of polymerization and mechanical properties was the aim of the present investigation.MethodsTwo base monomers (70 wt% BisGMA or HEMAM-BDI — newly synthesized secondary methacrylamide) were combined with 30 wt% N,N-dimethylacrylamide. Eethyl acetate (EtOAc), or 75 vol% ethanol/25 vol% water (EtOH/H₂O) were added as solvents in concentrations of 2, 5, 15 and 20 wt%. The resins were made polymerizable by the addition of 0.2 wt% 2,2-dimethoxy-2-phenyl acetophenone (DMPA) and 0.4 wt% diphenyliodonium hexafluorophosphate (DPI-PF6). Specimens (n = 3) were photoactivated with a mercury arc lamp (Acticure 4000, 320–500 nm, 250 mW/cm²) for 5 min. Degree of conversion (DC, %) was tracked in near-IR spectroscopy in real time and yield strength and modulus of elasticity were measured in three-point bending after dry and wet storage (n = 6). The data was subject to one-way ANOVA/Tukey’s Test (p ≤ 0.05), or Student’s t-test (p ≤ 0.001).ResultsIn all groups for both BisGMA and HEMAM-BDI-based materials, DC and DC at Rp_max increased and maximum rate of polymerization decreased as solvent concentration increased. Despite the increased DC, BisGMA mixtures showed a decrease in FS starting at 5 wt% EtOAc or 15 wt% EtOH/H₂O. Yield strength for the HEMAM-BDI groups was overall lower than that of the BisGMA groups, but the modulus of elasticity was significantly higher.SignificanceThe presence of residual solvent, from manufacturing or from practitioner’s handling, affects polymerization kinetics and mechanical properties of resins. Methacrylates appear to be more strongly influenced than methacrylamides.     

Long-term nanomechanical properties and gelatinolytic activity of titanium tetrafluoride-treated adhesive dentin interface

ObjectiveThis study investigated the effects of dentin pretreatment with 2.5% titanium tetrafluoride (TiF₄) on nanomechanical properties, and the in situ gelatinolytic activity of the dentin–resin interface, for up to 6 months.MethodsTwenty-four human teeth were prepared by exposing occlusal flat dentin surfaces, and were randomly assigned to experimental groups, according to application or non-application of a TiF₄ pretreatment, and to the adhesive systems (Clearfil SE Bond or Scotchbond Universal). Resin composite (Filtek Supreme Ultra) was built up incrementally on the teeth in all the groups. Then, the specimens were sectioned and randomly selected for evaluation at 24 h, 3 months and 6 months of storage time. The reduced modulus of elasticity (E_r) and the nanohardness of the underlying dentin, as well as the hybrid layer and the adhesive layer were measured using a nanoindenter. Gelatinolytic activity at the dentin–resin interfaces was assessed by in situ zymography using quenched fluorescein-conjugated gelatin at 24 h and 6 months. Statistical analyses were performed with ANOVA and Tukey’s tests.ResultsThere were no differences in E_r and nanohardness values between adhesives systems and pretreatment (p = 0.1250). In situ zymography showed significantly higher gelatinolytic activity after 6 months for all the experimental groups (p = 0.0004), but no differences between the adhesive systems (p = 0.7708) and the surface pretreatment (p = 0.4877). Significance: Dentin pretreatment with 2.5% TiF₄ followed by self-etching adhesive systems did not influence nanomechanical properties or gelatinolytic activity of the adhesive–dentin interface layers, over time.     

Nanoleakage,ultramorphological characteristics,and microtensile bond strengths of a new low-shrinkage composite to dentin after artificial aging

ObjectivesTo study the microtensile bond strengths and nanoleakage of low-shrinkage composite to dentin. The null hypotheses tested were (1) aging does not affect the bonding of low-shrinkage composite; (2) there is no difference in microtensile bond strengths and nanoleakage using different bonding strategies.Methods32 extracted molars were assigned to one of four groups: LS System Adhesive (LS, 3M ESPE); dentin etched for 15 s with phosphoric acid + LS System Adhesive (LSpa); Adper Single Bond Plus (SB, 3M ESPE); SB + LS Bond (SBLS). Occlusal dentin was exposed and restored with Filtek LS (3M ESPE). The samples were tested after 24 h or after 20,000 thermocycles and 6 months of aging. Teeth were sectioned with a cross-section of 0.8 ± 0.2 mm² and fractured at a crosshead speed of 1 mm/min. The data were submitted to ANOVA/Duncan's post hoc test, at p < 0.05. Five slabs from each group were selected and immersed in 50 wt% ammoniacal silver nitrate. Then, specimens were processed for SEM, the silver penetration was measured and data analyzed with Kruskal–Wallis at p < 0.05.ResultsNo statistically significant difference was found among the experimental groups for the factor dentin treatment (p = 0.165) and aging (p = 0.091). All experimental groups exhibit some degree of nanoleakage. There was no adhesion of Filtek LS applied directly over dentin surfaces treated with SB.SignificanceThe new low-shrinkage resin composite showed compatibility only with its dedicated adhesive. Pre-etching did not improve the bond strengths to low-shrinkage resin composite. Some degree of nanoleakage was evident in all groups.     

Simplified conditions for storing and cryopreservation of dental pulp stem cells

ObjectivesThis study aimed to simplify the collection, isolation and cryopreservation procedure of human dental pulp stem cells (DPSCs) to ease the establishment of dental stem cell banking.DesignExtracted third molars were collected and stored either in growth medium or in gentamicin-saline (480 μg/ml) for 6, 9 or 12 h. DPSCs were isolated and subjected to cryopreservation by a controlled-rate or rapid freezing method in 5 or 10% DMSO. Flow cytometry and growth pattern of DPSCs before and after cryopreservation were conducted.ResultsRate of contamination by which the extracted teeth were stored in control and gentamicin-saline were 9.1% (N = 33) and 2.3% (N = 43), respectively. Successful cell isolation rate of teeth preserved in gentamicin-saline at 6 h (92.9%) was comparable to those of growth media group (90.3%). At 9 and 12 h, the rates dropped significantly to 75% and 54%, respectively. Cryopreservation by controlled-rate freezing either in 5 or 10% DMSO resulted in a significantly higher percentage of viable cells than by rapid freezing. Cells conserved by controlled-rate freezing in 5% DMSO showed a pattern of growth similar to control unfrozen cells; 10% DMSO significantly deteriorated the growth pattern of the cells. After thawing, DPSCs conserved by controlled-rate freezing still expressed stemness characteristics, although hematopoietic stem cell markers were slightly increased.ConclusionGentamicin-saline was effective in preserving human teeth for DPSC isolation. Controlled-rate freezing in 5% DMSO gave the highest rate of cell viability. This study simplifies the storage conditions and proposes a simple method for cryopreservation of DPSCs.     

Wettability and surface morphology of eroded dentin treated with chitosan

ObjectiveTo assess the effect of chitosan, at concentrations of 2.5% and 5.0%, on the wettability of the eroded dentin, followed by analysis of surface morphology by SEM.Methods104 bovine dentin slabs were ground, polished and then immersed in 20 mL of citric acid (pH = 3.2) under continuous stirring for 2 h. Specimens were randomly divided according to the dentin substrate: sound and eroded, and then, subdivided into 4 groups (n = 10): without rewetting (control), 1% acetic acid, 2.5% chitosan and 5.0% chitosan. Then, a drop of the adhesive system Single Bond 2 (3M) was deposited onto surface of each specimen. The contact angle between dentin surface and the adhesive system was measured by using a goniometer. The other 24 specimens were subjected to analysis under SEM. Statistical analysis was performed using the normality test (Kolmogorov-Smirnov) and Analysis of Variance (ANOVA) (p > 0.05).ResultsNo differences were found between the angles produced on the eroded dentin rewetting with chitosan at the concentrations of 2.5% and 5%.ConclusionThe chitosan, regardless of the concentration used, did not influence the eroded dentin wettability. Through SEM analysis, it was found particles of chitosan deposited on the surface and within the dentinal tubules.