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Xylitol is a five-carbon sugar alcohol, i.e. polyol that has beneficial health properties such as noncariogenicity and a preventive agent against osteoporosis by increasing bone density. However, its potential to induce odontogenic differentiation on dental pulp stem cells (DPSCs) remains unclear. Thus, this study aimed to investigate the capacity of xylitol to induce odontogenic differentiation and mineralization of DPSCs. Dental pulp stem cells (DPSCs) were isolated from human third molars and cultured in complete medium (CM) Minimum Essential Medium Eagle—Alpha Modification (α-MEM) with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin through several successive subcultures. The cells from passage 3 were used for the entire experiment. To evaluate the effect of xylitol on odontogenic differentiation, DPSCs were treated with 0.5 mM, 1 mM or 10 mM Xylitol in odontogenic medium containing CM with 50 μg/mL ascorbic acid, 5 mM β-glycerol phosphate, and 10−7 M dexamethasone. Control group was treated with odontogenic medium only (OSs) Cell viability assay was used to detect the cytotoxicity of xylitol on DPSCs and was determined by 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Odontogenic activity and mineralization of DPSCs was assessed by alkaline phosphatase (ALP) assay and alizarin red staining (ARS), respectively. Odontogenic differentiation of DPSCs was evaluated by real-time quantitative polymerase chain reaction (RT–qPCR) for odontogenic markers: dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), alkaline phosphatase (ALP) and collagen type I (COL1), on day 7 and 14 which were normalized to the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Untreated DPSCs were used as a control throughout the study. Data were analyzed with one-way ANOVA at α=0.05 Cells treated with xylitol at all concentrations tested maintained viability and there was no statistically significant difference in cell viability between the control and xylitol-treated groups, indicating that used concentration of xylitol were biocompatible (p>0.05). DPSCs treated with 1 mM xylitol showed the highest ALP activity (p< 0.05). The amount of culture mineralization and formation of mineralized nodules on DPSCs was higher for the group treated with 1 mM xylitol compared to control. These findings suggest that Xylitol induces odontogenic differentiation and mineralization of DPSCs. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the degradation of completely demineralized dentin specimens in contact with a filler-free or 2 ion-releasing resins containing micrometer-sized particles of Bioglass 45S5 (BAG) or fluoride-containing phosphate-rich bioactive glass (BAG-F). Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were also used to evaluate the remineralization induced by the experimental ion-releasing resin-based materials. Dentin beams were totally demineralized in H3PO4 (10%) and placed in direct contact with a filler-free (RESIN) or 2 experimental ion-releasing resins (BAG or BAG-F) and immersed in artificial saliva (AS) up to 30 d. Further specimens were also processed and submitted to FTIR and SEM analysis to evaluate the remineralization induced by such ion-releasing resins before and after AS immersion. BAG and BAG-F alkalinized the incubation media. A significant decrease of the dry mass was observed between the specimens of all groups stored for 3 and 30 d in AS. However, the fluoride-containing phosphate-rich bioactive glass incorporated into a resin-based material (BAG-F) showed greater ability in reducing the solubilization of C-terminal cross-linked telopeptide (ICTP) and C-terminal telopeptide (CTX) after prolonged AS storage. Moreover, after 30 d of AS storage, BAG-F showed the greatest remineralizing effect on the stiffness of the completely demineralized dentin matrices. In conclusion, fluoride-containing phosphate-rich bioactive glass incorporated as micrometer-sized filler in dental composites may offer greater beneficial effects than Bioglass 45S5 in reducing the enzyme-mediated degradation and remineralization of demineralized dentin. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

This study determined if dentin proteases are denatured by phosphoric acid (PA) used in etch-and-rinse dentin adhesives. Dentin beams were completely demineralized with EDTA for 30 days. We “acid-etched” experimental groups by exposing the demineralized dentin beams to 1, 10, or 37 mass% PA for 15 sec or 15 min. Control beams were not exposed to PA but were incubated in simulated body fluid for 3 days to assay their total endogenous telopeptidase activity, by their ability to solubilize C-terminal crosslinked telopeptides ICTP and CTX from insoluble dentin collagen. Control beams released 6.1 ± 0.8 ng ICTP and 0.6 ± 0.1 ng CTX/mg dry-wt/3 days. Positive control beams pre-incubated in p-aminophenylmercuric acetate, a compound known to activate proMMPs, released about the same amount of ICTP peptides, but released significantly less CTX. Beams immersed in 1, 10, or 37 mass% PA for 15 sec or 15 min released amounts of ICTP and CTX similar to that released by the controls (p > 0.05). Beams incubated in galardin, an MMP inhibitor, or E-64, a cathepsin inhibitor, blocked most of the release of ICTP and CTX, respectively. It is concluded that PA does not denature endogenous MMP and cathepsin activities of dentin matrices. [ABSTRACT FROM PUBLISHER]

Matrix metalloproteinases (MMPs) cause collagen degradation in hybrid layers created by dentin adhesives. This in vitro study evaluated the feasibility of using a cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), to inactivate soluble rhMMP-9, as an example of dentin MMPs, and matrix-bound dentin proteases. The inhibitory effects of 5 EDC concentrations (0.01-0.3 M) and 5 incubation times (1-30 min) on soluble rhMMP-9 were screened with an MMP assay kit. The same EDC concentrations were used to evaluate their inhibitory effects on endogenous proteinases from completely demineralized dentin beams that were incubated in simulated body fluid for 30 days. Decreases in modulus of elasticity (E) and dry mass of the beams, and increases in hydroxyproline content of hydrolysates derived from the incubation medium were used as indirect measures of matrix collagen hydrolysis. All EDC concentrations and pre-treatment times inactivated MMP-9 by 98% to 100% (p < 0.05) compared with non-cross-linked controls. Dentin beams incubated in 0.3 M EDC showed only a 9% decrease in E (45% decrease in control), a 3.6% to 5% loss of dry mass (18% loss in control), and significantly less solubilized hydroxyproline when compared with the control without EDC cross-linking (p < 0.05). It is concluded that EDC application for 1 min may be a clinically relevant and effective means for inactivating soluble rhMMP-9 and matrix-bound dentin proteinases if further studies demonstrate that EDC is not toxic to pulpal tissues. [ABSTRACT FROM PUBLISHER]

To evaluate whether the cytotoxicity of a resin-modified calcium silicate material (RMCSM) on odontoblast-like cells would be affected by dentin pretreatments containing dimethyl sulfoxide (DMSO) and/or ethanol. Dentin discs (n=8 discs/group) were prepared from deep dentin, (500 μm in thickness/10 mm diameter), using a precision saw (Isomet 1000; Buehler) under water-cooling. Discs were polished with 320-grit SiC abrasive paper to produce a final thickness of 300 μm, etched with 50% citric acid for 30 s, rinsed for 15 s. The permeability of each disc was measured, followed by sterilization by autoclave (121 ℃ for 25 min). Discs were then distributed into 6 balanced groups. Groups consisted of dentin pretreatments with different concentration of DMSO and/or EtOH or no pretreatment. The pretreatment solutions were composed of pure ethanol (EtOH), pure DMSO, 50% DMSO dissolved in water (DMSO/H 2 O) or ethanol (DMSO/EtOH), and an aqueous 50% ethanolic solution (EtOH/H 2 O). 3-D cultures of odontoblast-like cells (SV40 transfected pulp derived cells) were grown in polyamide meshes and transferred to the pulpal aspect of dentin slices (n=8) inside individual perfusion split-chambers designed for the dentin barrier cytotoxicity test. After applying 1.5 µL of dentin pretreatments for 10 s, a resin-modified calcium silicate material (TheraCal LC, Bisco) was applied in a 1 mm-thick increment and light cured for 20 s. As negative control (100% cell viability), a polyvinylsiloxane impression material was used. Cell viability (%) was analyzed spectrometrically and assessed by MTT essay. Data were analyzed by Kruskal-Wallis test (α=0.05). Cell viability produced by the untreated RMCSM (92.5%) was not affected by DMSO/H 2 O (89.07%) or EtOH/H 2 O (82.16%) pretreatments (p>0.05). The remaining dentin pretreatments, DMSO/EtOH (56.3%), pure DMSO (39.76%) and pure EtOH (30.9%), produced significantly lower cell viabilities (p0.05). While the use of non-aqueous polar solvents as dentin pretreatments may increase the cytotoxicity of resin-modified calcium silicate materials, 50% dilution in water seems to be a safe threshold for the tested solvents. Therefore, the use of ethanol or DMSO to improve the performance of resin-modified calcium silicate material must be performed with caution, strictly avoiding their undiluted use in deep clinical cavities. [ABSTRACT FROM AUTHOR]

To evaluate whether the cytotoxicity of a mineral trioxide aggregate (MTA) on odontoblast-like cells would be affected by dentin pretreatments containing dimethyl sulfoxide (DMSO) and/or ethanol. Dentin discs (n=8 /group) were prepared from deep dentin, (500 μm in thickness), using a precision saw under water-cooling. Discs were reduced to a final thickness of 300 μm with 320-grit SiC abrasive paper, etched with 50% citric acid for 30 s, rinsed for 15 s. Discs were sterilized by autoclave (121℃ for 25 min), and then distributed into 6 balanced groups according to their permeability. Groups consisted of dentin pretreatments with different concentration of DMSO and/or EtOH or no pretreatment. The pretreatment solutions were composed of pure ethanol (EtOH), pure DMSO, 50% DMSO dissolved in water (DMSO/H2O) or ethanol (DMSO/EtOH), and an aqueous 50% ethanolic solution (EtOH/H2O). Polyamide meshes were used to grow 3-D cultures of odontoblast-like cells (SV40 transfected pulp derived cells) and transferred to the pupal aspect of dentin slices inside individual perfusion split-chambers designed for the dentin barrier cytotoxicity test. After applying 1.5 µL of dentin pretreatments for 10 s, a mineral trioxide aggregate (MTA, Orbis) was applied in a 1mm-thick increment. As negative control (100% cell viability), a polyvinylsiloxane impression material was used. Cell viability (%) was analyzed spectrometrically and assessed by MTT essay. Data were analyzed by Kruskal-Wallis test (α=0.05). Cell viability produced by MTA (98.53%) was not affected by the pretreatments DMSO/H 2 O (98.47%), EtOH/H 2 O (95.88%), DMSO/EtOH (94.58%) and pure DMSO (84.17%) (p>0.05). However, pure EtOH produced significantly lower cell viability (52.98%) when used along with MTA (p<0.05). The tested polar solvents used as dentin pretreatments had no substantial effects on the cytotoxicity of MTA up to 50% aqueous dilution, which may serve as a safe threshold regardless of solvent type. The use of DMSO constitutes a safer option, compared to ethanol, to potentially improve MTA performance in deep clinical cavities, especially at higher concentrations. [ABSTRACT FROM AUTHOR]

Highlights • The novel mini-interfacial fracture-toughness approach (mini-iFT) was applied to evaluate if collagen cross-linkers can improve bond durability of adhesives to dentin. • Any benefit from the use of cross-linking agents in the dentin-bonding protocol appeared dependent on the adhesive/adhesive mode and the cross-linker used. • Applied following clinically relevant conditions, proanthocyanidin enhanced the collagen fibrillar resistance against enzymatic degradation. Abstract Objectives Dentin biomodification using collagen cross-linkers has been proposed as one of the strategies to improve bond durability of adhesives to dentin. However, literature is not very consistent regarding their benefit, in particular when cross-linkers are applied in clinically realistic application times. This study investigated the effect of three cross-linkers on the mini-interfacial fracture toughness (mini-iFT) of four adhesives bonded to dentin following either etch&rinse (E&R) or self-etch (SE) modes. Methods 60 molars were randomly divided in accordance with the three variables: cross-linker, adhesive and bonding mode (n = 5). The cross-linkers glutaraldehyde (5 wt%; GA), proanthocyanidin (6.5 wt%; PA), or UVA-activated riboflavin (0.5 wt%; RB), and distilled water (control) were applied on dentin for 60 s after acid-etching (E&R) or before self-etching (SE). The 3-step E&R adhesive (3E&Ra) OptiBond FL (Kerr), the 2-step SE adhesive (2SEa) Clearfil SE Bond 2 (Kuraray Noritake) and the universal adhesives G-Premio Bond (GC) and Prime&Bond Active (Dentsply), the latter two employed in both E&R and SE modes, were applied following the respective manufacturer's instructions. Composite buildups (8 × 8 × 8 mm) were made using Filtek Supreme XTE (3M) prior to 1-week storage in artificial saliva. After the teeth were sectioned into mini-specimens (1.5 × 2.0 × 18 mm), a single notch was prepared at the adhesive–dentin interface. Half of the specimens were immediately loaded until failure by 4-point bending to determine the mini-iFT, while the remaining specimen set was tested upon 6-month aging. Data were statistically analyzed with a linear model (p < 0.05). Results No significant decrease in mini-iFT was noted only for PA (p < 0.05), while the mini-iFT decreased for both other cross-linkers and in quite a similar way as when solely water (Wa) was applied. Significance The cross-linker proanthocyanidin (PA) applied in clinically relevant conditions was able to maintain a stable mini-iFT after 6-month aging. The incorporation of UVA-activated riboflavin (RB) and glutaraldehyde (GA) in the dentin-bonding protocol appeared not effective to improve the stability of adhesive–dentin interfaces. [ABSTRACT FROM AUTHOR]