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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]

Objectives This study evaluated the endogenous matrix metalloproteinase (MMP) activity of demineralized dentin matrix following 1 or 5 min pretreatment by various collagen crosslinkers. Generic MMP activity assay, total protein analysis, in situ zymography, gelatin zymography and multiplex bead technology were used to evaluate matrix-bound MMP activity. Methods Six different crosslinkers; glutaraldehyde, riboflavin/UVA, riboflavin-5-monophospate/UVA, sumac berry extract, grape seed extract, and curcumin were used. Demineralized dentin beams were pretreated with respective crosslinkers for 1 or 5 min. Demineralized dentin beams with no crosslinker pretreatment served as control. The reduction in the total activity of dentin matrices were measured using generic MMP activity assay. Dentin slabs were used for in situ zymography and evaluated by using hydrolysis of self-quenched fluorescein-conjugated gelatin under confocal microscopy. Dentin beam extracts were used for total protein assay and multiplex analysis and powder extracts were used for gelatin zymography. Results MMP activity in crosslinker pretreated samples decreased significantly between 21% and 70%, whereas untreated control samples’ activity increased up to 84%. Zymograms confirmed a decrease in the gelatinolytic activity and in the amount of extractable total protein content. Multiplex analysis of extracts of crosslinker-treated dentin showed a reduction in the MMP-8, MMP-2 and MMP-9 release. Significance The result of this work suggests that the effect of the crosslinkers is source-dependent. The use of crosslinkers for as little as 1 min on demineralized dentin can inactivate the endogenous protease activity of dentin matrices. [ABSTRACT FROM AUTHOR]

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 (1 mm × 2 mm × 6 mm) were demineralized in 10% H 3 PO 4 for 24 h. After baseline measurements of dry mass, beams were divided into 11 groups ( n = 10/group) and, were pretreated for 5 min with 1% glutaraldehyde (GA); 5% GA; 1% grape-seed extract (GS); 5% GS; 10% sumac (S); 20 μM curcumin (CR); 200 μM 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 1 mL 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 200 μM 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 ± 0.2 ng/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. [ABSTRACT FROM AUTHOR]

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 (1 mm × 2 mm × 6 mm; 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 20 s. 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. [ABSTRACT FROM AUTHOR]

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]