Distinct effects of polyphenols and solvents on dentin collagen crosslinking interactions and biostability

ObjectiveTo evaluate the effects of different polyphenols and solvents on dentin collagen’s crosslinking interactions and biostabilization against MMPs and collagenase degradation.MethodsTwo polyphenols [proanthocyanidin (PA) and quercetin (QC)] with different water solubility were prepared as treatment solutions using ethanol (EtOH) or dimethyl sulfoxide (DMSO) as solvents. 6-um-thick dentin films were microtomed from dentin slabs of third molars. Following demineralization, films or slabs were subject to 60-s treatment (PA or QC) or no treatment (control) with subsequent extended-rinse with original solvent (EtOH or DMSO) or distilled water (DW). Collagen crosslinking interactions were assessed by FTIR. Biostability was assessed through endogenous MMPs activity via confocal laser scanning microscopy, and exogenous collagenase degradation via weight loss, hydroxyproline release and SEM. Finally, direct collagenase inactivation was also evaluated. Data were analyzed by three-way ANOVA and post-hoc tests (α = 0.05%).ResultsDistinct effects of two polyphenols and solvents on collagen crosslinking and biostabilization were observed. Higher crosslinking and biostability efficacy occurred with PA than QC (p < 0.001) that demonstrated negligible collagen interactions. With DMSO solvent, efficacy results were significantly reduced with both polyphenols (p < 0.05). DMSO-rinse further weakened interactions of PA with collagen, diminishing biostability (p < 0.05). Low biostability was detected with QC and DW-rinse, suggesting direct enzymatic inhibition due to physical presence in collagen.SignificanceCollagen crosslinking interactions and biostability depend on polyphenol chemical characteristics. Treatment-solution solvents may affect interactions between polyphenols and collagen, specifically, DMSO showed detrimental effects on collagen crosslinking and biostability and should be used with caution.     

Methacrylate-functionalized proanthocyanidins as novel polymerizable collagen cross-linkers – Part 1: Efficacy in dentin collagen bio-stabilization and cross-linking

ObjectiveThe aim of the study was to investigate the effects of methacrylate-functionalized proanthocyanidins (MAPAs) on dentin collagen’s bio-stabilization against enzymatic degradation and crosslinking capability.MethodsThree MAPAs were synthesized via varying methacrylate (MA) to proanthocyanidins (PA) feeding ratios of 1:2, 1:1, and 2:1 to obtain MAPA-1, MAPA-2, and MAPA-3, respectively. The three MAPAs were structurally characterized by proton nuclear magnetic resonance (¹H NMR) and Fourier transform infrared (FTIR) spectroscopic methods. 5-μm-thick dentin films were microtomed from dentin slabs of third molars. Following demineralization, films or slabs were treated with 1% MAPAs or PA in ethanol for 30 s. Collagen bio-stabilization against enzymatic degradation was analyzed by weight loss (WL) and hydroxyproline release (HYP) of films, as well as scanning electron microscopy (SEM) on dentin slabs. Crosslinking capacity and interactions of MAPAs with collagen were investigated by FTIR. Data were analyzed by ANOVA and Tukey’s test (α = 0.05%).ResultsMA:PA feeding ratios affected MAPAs’ chemical structures which in turn led to different collagen stabilization efficacy against degradation and varied collagen crosslinking capabilities. Higher collagen stabilization efficacy was detected using MAPA-1 (WL 10.52%; HYP 13.53 μg/mg) and MAPA-2 (WL 5.99%; HYP 11.02 μg/mg), which was comparable to that using PA (WL 8.79%; HYP 13.17 μg/mg) (p > 0.05), while a lower collagen stability occurred in MAPA-3 (WL 38.48%; HYP 29.49 μg/mg), indicating excessive MA-functionalization would compromise its stabilization efficacy. In comparison, complete digestion was detected for untreated collagen (WL 100%; HYP 102.76 μg/mg). The above results were consistent with collagen crosslinking efficacy of the three MAPAs revealed by SEM and FTIR.SignificanceA new class of novel polymerizable collagen cross-linkers MAPAs was synthesized and shown that, when appropriate MA:PA ratios were applied, the resulting MAPAs could render high collagen stability and the ability to copolymerize with resin monomers, overcoming the drawbacks of PA. These new polymerizable crosslinkers, when included in adhesives, could lead to long-lasting dentin bonding.     

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.     

Effect of Odanacatib on the release of NTX (Amino Terminal Telopeptide) from LPS contaminated type I dentin collagen

ObjectiveTo evaluated the Odanacatib inhibitor treatment on lipopolysaccharide (LPS) contamination effect on cathepsin-K mediated dentin degradation by analysis of type I collagen C- and N-termini telopeptides.MethodsPulverized and disks of human dentin were demineralized and LPS contaminated, or stored in deionized water (DW) for 12 h. Samples were challenged with lactic acid (LA). Aliquots of dentin powder were treated with 1 mL Odanacatib or stored in DW for 30 min. Dentin collagen degradation was determined by sub-product release of C-terminal (ICTP and CTX) and N-terminal (NTX) telopeptides, normalized to total protein (tp) concentration (n = 3). Dentin matrix was evaluated for gravimetric (n = 8) and ultrastructural changes. Data were analyzed by Student t-test, one-way ANOVA and Tukey’s test (α = 5 %).ResultsLA incubation significantly increased telopeptide release compared with DW (p < 0.05). In untreated groups, significantly higher CTXtp, NTXtp telopeptide rates were observed for LA+LPS samples compared with DW (p < 0.01). Odanacatib significantly reduced ICTPtp, CTXtp, and NTXtp telopeptide release for LPS, LA, and LA+LPS conditions. In untreated groups, LPS and LA+LPS challenge significantly increased dentin weight loss (p = 0.02). Within each storage condition, Odanacatib treatment did not affect weight change (p > 0.05) of dentin disks.SignificanceThis study showed that LPS contamination resulted in significantly higher rates of NTX than CTX from dentin matrix. Odanacatib significantly reduced telopeptide release rates of LPS contaminated dentin matrix.     

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.     

Grape seed proanthocyanidins increase collagen biodegradation resistance in the dentin/adhesive interface when included in an adhesive

Objectives

Contemporary methods of dentin bonding could create hybrid layers (HLs) containing voids and exposed, demineralised collagen fibres. Proanthocyanidins (PA) have been shown to cross-link and strengthen demineralised dentin collagen, but their effects on collagen degradation within the HL have not been widely studied. The purpose of this study was to compare the morphological differences of HLs created by BisGMA/HEMA model adhesives with and without the addition of grape seed extract PA under conditions of enzymatic collagen degradation.

Methods

Model adhesives formulated with and without 5% PA were bonded to the acid etched dentin. 5-μm-thick sections cut from the bonded specimens were stained with Goldner's trichrome. The specimens were then exposed to 0.1% collagenase solution for 0, 1, or 6 days. Following collagenase treatment, the specimens were analysed with SEM/TEM.

Results

Staining did not reveal a difference in the HLs created with the two adhesives. SEM showed the presence of intact collagen fibrils in all collagenase treatment conditions for specimens bonded with adhesive containing PA. These integral collagen fibrils were not observed in the specimens bonded with adhesive without PA after the same collagenase treatment. TEM confirmed that the specimens containing PA still showed normal collagen fibril organisation and dimensions after treatment with collagenase solution. In contrast, disorganised collagen fibrils in the interfacial zone lacked the typical cross-banding of normal collagen after collagenase treatment for specimens without PA.

Conclusions

The presence of grape seed extract PA in dental adhesives may inhibit the biodegradation of unprotected collagen fibrils within the HL.     

Stabilization of dentin matrix after cross-linking treatments,in vitro

Objectives

To evaluate the effect of EDC on elastic modulus (E), MMPs activity, hydroxyproline (HYP) release and thermal denaturation temperature of demineralized dentin collagen.

Methods

Dentin beams were obtained from human molars and completely demineralized in 10 wt% H₃PO₄ for 18 h. The initial E and MMP activity were determined with three-point bending and microcolorimetric assay, respectively. Extra demineralized beams were dehydrated and the initial dry mass (DM) was determined. All the beams were distributed into groups (n = 10) and treated for 30 s or 60 s with: water, 0.5 M, 1 M or 2 M EDC or 10% glutaraldehyde (GA). After treatment, the new E and MMP activity were redetermined. The beams submitted to DM measurements were storage for 1 week in artificial saliva, after that the mass loss and HYP release were evaluated. The collagen thermal denaturation temperature (TDT) was determined by DSC analysis. Data for E, MMP activity and HYP release were submitted to Wilcoxon and Kruskal–Wallis or Mann–Whitney tests. Mass loss and TDT data were submitted to ANOVA and Tukey tests at the 5% of significance.

Results

EDC was able to significantly increase collagen stiffness in 60 s. 10% GA groups obtained the highest E values after both 30 and 60 s. All cross-linking agents decreased MMP activity and HYP release and increased TDT temperature. Significant differences were identified among EDC groups after 30 or 60 s of cross-linking, 1 M or 2 M EDC showed the lowest MMP activity.

Significance

Cross-linking agents are capable of preventing dentin collagen degradation. EDC treatment may be clinically useful to increase resin-dentin stability.     

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.     

Self-assembled peptide P11-4 interacts with the type I collagen C-terminal telopeptide domain and calcium ions

ObjectivesEvaluate molecularly the role of P₁₁-4 self-assembly peptide in dentin remineralization and its interaction with collagen I. Methods: The calcium-responsive P₁₁-4 peptide was analyzed by intrinsic fluorescence emission spectrum, circular dichroism spectrum (CD), and atomic force microscope (AFM). Differential light scattering was used to monitor the nucleation growth rate of calcium phosphate nanocrystals in the absence or in the presence of P₁₁-4. AFM was used to analyze the radial size (nm) of calcium phosphate nanocrystals formed in the absence or in the presence of P₁₁-4, as well as to verify the spatial structure of P₁₁-4 in the absence or in the presence of Ca²⁺. Results: The interaction of Ca²⁺ with the P₁₁-4 (K_D = 0.58 ± 0.06 mM) promotes the formation of β-sheet antiparallel structure, leads to its precipitation in saturated solutions of Ca/P = 1.67 and induces the formation of parallel large fibrils (0.6 – 1.5 µm). P₁₁-4 organized the HAP nucleation by reducing both the growth rate and size variability of nanocrystals, analyzed by the F test (p < 0.0001, N = 30). P₁₁-4 interacts (K_D = 0.75 ± 0.06 μM) with the KGHRGFSGL motif present at the C-terminal collagen telopeptide domain. P₁₁-4 also increased the amount of HAP and collagen in the MDPC-23 cells. Significance: The presented data propose a mechanism that will help future clinical and/or basic research to better understand a molecule able to inhibit structural collagen loss and help the impaired tissue to remineralize.     

The effect of universal adhesives on dentine collagen

ObjectivesThe purpose of the study was to evaluate the integrity of dentine type I collagen after self-etching (SE) treatments with strong and mild universal adhesives.MethodsCoronal dentine specimens (n = 10/product) were imaged by optical microscopy and analyzed by ATR-FTIR spectroscopy before and after treatment with 32% phosphoric acid gel (PA-negative control), 17% neutral EDTA (ED-positive control) conditioners and Adhese Universal (AD), Clearfil Universal Bond Quick (CQ), G-Premio Bond (GP), Prelude One (PR) and Scotchbond Universal (SB) adhesives. From the spectroscopic analysis the following parameters were determined: a) Extent of dentine demineralization (DM%) and b) percentage area of the Amide I curve-fitted components of β-turns, 3₁₀-helix/β-turns, α-helix, random coils, β-sheets and collagen maturation (R) index. Statistical analysis was performed by one-way ANOVA (DM%), paired t-test/Wilcoxon test (Amide I components) and Spearman correlation coefficient (DM% vs Amide I components) at an a = 0.05 level.ResultsPA, ED and GP removed the smear-layer and opened tubule orifices, whereas all other treatments removed only the intratubular smear-layer fraction. The ranking of the statistically significant differences in DM% was PA > GP > ED > AD, SB, CQ, PR, with AD being significantly different from PR. Regarding the Amide I components, PA demonstrated a significant reduction in β-turns, α-helices and an increase in β-sheets, GP a reduction in β-turns, AD an increase in β-turns and random coils, and CQ an increase in β-turns. PR, SB and ED showed insignificant differences in all the Amide I components. Significant correlations were found between DM%-random coils and DM%-R.SignificanceThe universal adhesives used in the SE mode induced none to minimal changes in dentine collagen structure, without evidence of the destabilization pattern observed after conventional phosphoric acid treatments.     

In vitro evaluation of plant-derived agents to preserve dentin collagen

Objective

Biomodification of dentin by a natural crosslinker has been recommended to improve a mechanical property of demineralized dentin. This study investigated the effect of various plant-derived agents (hesperidin, proanthocyanidin, epigallocatechin gallate and genipin) on the stability of dentin collagen matrix to resist collagenase degradation.

Methods

The dentin specimens were treated with glutaraldehyde (0.5% and 5.0%) and each plant-derived test solution (0.5%). They were subjected to ultimate tensile strength (UTS) and swelling ratio measurements. Demineralized human dentin powder was incubated with 0.02%, 0.1% and 0.5% of each test agent and followed by bacterial collagenase digestion. The extent of collagen degradation was investigated using hydroxyproline assay.

Results

The UTS and swelling ratio measurements revealed that the mechanical property of dentin was improved by the use of these natural agents. The greatest reduction in collagen degradation was shown following the use of hesperidin, proanthocyanidin, and epigallocatechin gallate at 0.5%.

Significance

The use of hesperidin, proanthocyanidin, and epigallocatechin gallate could improve the mechanical properties of collagen and resist enzymatic degradation, leading to functional repair of pathological dentin lesion.     

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.     

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.     

Proanthocyanidins’ efficacy in stabilizing dentin collagen against enzymatic degradation: MALDI-TOF and FTIR analyses

Objectives

To investigate grape seed extract proanthocyanidins’ (PA) capability in improving dentin collagen's sustainability in an enzymatic environment, given that the size and shape of the collagen samples, and the manner to apply PA are both clinically relevant.

Methods

Human dentin was sectioned into 6-μm-thick films. After demineralisation in 35 wt% phosphoric acid for 15 s, the films were subject to 30 s of treatment at PA concentrations of 0% (control), 0.5%, 1%, 2%, 3.75%, 7.5% and 15% (w/w), respectively. The films were then digested in 0.1 wt% collagenase for 1 h and 24 h. The amount of degraded collagen in the liquid digests was determined by MALDI-TOF mass spectroscopy. The trend of PA's incorporation into dentin collagen was analysed by ATR-FTIR.

Results

The control exhibited complete digestion in 1 h. In contrast, collagen treated with 0.5% and 1% PA afforded 13.84 ± 4.69% and an undetectable level of degradation, respectively in the first 1 h of digestion, and additional 17.48 ± 4.38% and 4.50 ± 1.68%, respectively in the following 23 h. Collagen treated with ≥2 wt% PA was not significantly digested regardless of digestion time. FTIR spectroscopy revealed that PA incorporation was saturated at ≥2 wt% PA.

Conclusion

Thirty seconds of PA treatment at 2 wt% and above could provide optimal protection for dentin collagen against collagenase digestion.

Clinical significance

This study demonstrated PA's extraordinary efficiency in stabilizing demineralised dentin collagen when it is applied in a clinical relevant manner, and identified the optimal conditions for its utilization.     

Effect of silver-containing agents on the ultra-structural morphology of dentinal collagen

ObjectiveThis study investigated the effect of silver diammine fluoride (SDF), nano-silver (AgNPs) and nano-silver/potassium fluoride (AgNPs/KF) on the morphology of dentin collagen and color staining on the dentin. Also, we investigated the anti-caries properties in terms of anti-bacterial effects and potentiality of dentin remineralization at collagen level.MethodsDentin specimens obtained from bovine tooth roots were finished with #2000 SiC and sonicated for 5 min. Specimens were divided into two main groups: sound and demineralized dentin. Each group was subdivided into five subgroups according to treatment solution: control (no treatment), SDF, nano-silver (AgNPs), AgNPs/KF and KF. Specimens were analyzed using transmission electron microscope (TEM), spectrophotometer, scanning electron microscope (SEM) and antibacterial test. Repeated Measures ANOVA was used for statistical analysis of color change while one-way ANOVA was used for CFU analysis.ResultsRegarding color change, SDF showed the highest values while the other groups exhibited insignificant changes. SDF showed alteration in the collagen fibrils while the other groups showed normal intact fibrils. Regarding antibacterial activity, SDF and AgNPs/KF showed the greatest antibacterial effect.SignificanceSDF altered the dentin collagen morphology and inferior ethetics, but still providing intrafibrillar remineralization. Nano-silver fluoride preparations (AgNPs/KF) can be an alternative material for SDF as it can preserve the dentin collagen, showing no color change overtime, and provide a high anti-caries property including intrafibrillar remineralization with a significant anti-bacterial effect.     

Dry bonding to dentin: Broadening the moisture spectrum and increasing wettability of etch-and-rinse adhesives

ObjectiveTo determine whether the effect of dentin moisture on the etch-and-rinse bonding may be minimized by dry-bonding protocols utilizing aqueous or ethanolic dimethyl sulfoxide (DMSO) pretreatments.MethodsH₃PO₄-etched mid-coronal dentin surfaces from human molars were randomly blot- or air-dried for 30 s and pretreated with DMSO/H₂O or DMSO/EtOH solutions. Untreated samples served as control. Moisture control was performed by either blot- or air-drying. Samples were bonded with a multistep etch-and-rinse adhesive. Restored crown segments (n = 8/group) were stored in distilled water for 24 h and sectioned for microtensile bond strength testing. Resin-dentin beams (0.8 mm²) were tested under tension until fracture (0.5 mm/min) after 24 h and two years of storage in artificial saliva at 37 °C.SEM nanoleakage evaluation was performed on aged samples. Collagen wettability was also measured by sessile drops of the hydrophilic and hydrophobic bonding resins (n = 8/group). Data were examined by factorial ANOVA followed by the Tukey test (α = 0.05).ResultsDry bonding to untreated collagen produced inferior immediate and long-term bond strengths than wet bonding (p < 0.05). Regardless of initial hydration and moisture control, DMSO-dry bonding produced initially higher and stable bond strengths after aging (p < 0.05). DMSO-pretreated groups presented improved collagen wettability with lower silver uptake (p < 0.05).SignificanceDespite the common belief that etch-and-rinse adhesives must be applied onto moist collagen, DMSO-dry bonding protocols not only improved bonding performance and hybrid layer integrity, but also brought more versatility to collagen hybridization by reducing overdrying-related issues.     

Methacrylic monomer derived from cardanol incorporated in dental adhesive as a polymerizable collagen crosslinker

ObjectivesThe aim of this study was to evaluate the influence on MMP inhibition, dentin adhesion and physicochemical properties of an adhesive system incorporated with polymerizable collagen crosslinker monomer derived from cardanol.MethodsThe intermediary cardanol epoxy (CNE) was synthesized through cardanol epoxidation, followed by synthesis of cardanol methacrylate through methacrylic acid solvent-free esterification. Zymographic analysis was performed to evaluate the substances’ ability to inhibit gelatinolytic enzymes. Collagen crosslinkers were added into adhesives systems according to the following groups: Ybond Universal® (Control), Ybond® + 2 % proanthocyanidin (PAC), Ybond® + 2 % unsaturated cardanol (Cardanol) and Ybond® + 2 % cardanol methacrylate (CNMA). Degree of conversion (DC) of the adhesives was assessed by FT-IR. Disk-shaped specimens were prepared for water sorption (WS) and solubility (SL) tests. Human third molars were sectioned to expose medium dentin and restored according to the different adhesives used (n = 5). Then, the specimens were cut into 1 mm² sticks to evaluate, after 24 h and 6-month aging, microtensile bond strength (µTBS) and nanoleakage by scanning electron microscopy. Data were analysed with ANOVA and Tukey's post-test (α = 0.05).ResultsCNMA and PAC completely inhibited all forms of gelatinolytic enzymes. Cardanol achieved a significantly lowest DC, while the other groups did not differ from each other (p > 0.05). PAC achieved significantly higher water sorption, while CNMA solubility was significantly lower when compared to the other adhesives (p < 0.05). PAC provided a statistically higher 24 h and 6-month aging bond strength. Intermediary similar µTBS were presented by control and CNMA (p = 0.108). All adhesives applied attained significantly reduced bond strength after aging (p < 0.05). Interfaces created using CNMA were almost devoid of silver deposits initially, however all groups showed large amounts of silver deposits on resin-dentin interface subjected to water aging.SignificanceAlthough CNMA was effective in inhibiting gelatinolytic enzymes, when incorporated into a universal adhesive it could not promote less degradation of the adhesive interface after water aging. Since it is a hydrophobic monomer, CNMA did not interact well with dentin collagen, however it reduced the solubility of the adhesive system besides not interfering in its polymerization.     

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.     

Unveiling structure–activity relationships of proanthocyanidins with dentin collagen

ObjectiveTo elucidate the structure–activity relationships (SARs) of proanthocyanidins (PACs) with type I collagen using sixteen chemically defined PACs with degree of polymerization (DP) 2–6.MethodsUnder a dentin model, the biomimicry of PACs with type I collagen was investigated by dynamic mechanical analysis (DMA) and infrared spectroscopy. The dentin matrix was modified with PACs from Pinus massoniana [monomers (Mon-1 and Mon-2), dimers (Dim-1-Dim-4), trimers (Tri-1-Tri-4), tetramers (Tet-1-Tet-5), and hexamer (Hex-1)]. A strain sweep method in a 3-point bending submersion clamp was used to assess the viscoelastic properties [storage (E’), loss (E”), and complex moduli (E*) and tan δ] of the dentin matrix before and after biomodification. Biochemical analysis of the dentin matrix was assessed with FTIR spectroscopy. Data were statistically analyzed using one-way ANOVA and post-hoc tests (α = 0.05).ResultsDP had a significant effect on modified dentin moduli (tetramers ≈ trimers > hexamers ≈ dimers > monomers ≈ control, p < 0.001). Trimers and tetramers yielded 6- to 8-fold increase in the mechanical properties of modified dentin and induced conformational changes to the secondary structure of collagen. Modifications to the tertiary structure of collagen was shown in all PAC modified-dentin matrices.SignificanceFindings establish three key SARs: (i) increasing DP generally enhances biomimicry potential of PACs in modulating the mechanical and chemical properties of dentin (ii) the secondary structure of dentin collagen is affected by the position of B-type inter-flavanyl linkages (4β → 6 and 4β → 8); and (iii) the terminal monomeric flavan-3-ol unit plays a modulatory role in the viscoelasticity of dentin.