Effects of Cathepsin K Inhibitors on Dentin Erosion: An in vitro Study.

Cathepsin K (catK) modulates the degradation of dentin collagen. This study aimed to evaluate the effects of catK inhibitors on dentin erosion. Dentin beams were eroded (4 times/d for 5 days) and immersed in deionized water (negative control), 0.1 M NaCl, 0.3 M NaCl, 0.5 M NaCl, or 1 µm odanacatib (each n = 16) for 30 min after each erosive challenge. Erosive dentin loss (EDL) and demineralized organic matrix (DOM) thickness were evaluated profilometrically. Additionally, dentin beams were demineralized, immersed in the respective solutions for 30 min each (n = 5), and then incubated in artificial saliva for 5 days. Dentin collage degradation was evaluated by quantifying the levels of the C-terminal peptide of type I collagen (CTX), C-terminal cross-linked telopeptide of type I collagen (ICTP), and hydroxyproline (HYP) in the incubation media. Significantly lower EDL and dentin collagen degradation (CTX, ICTP, and HYP) and thicker DOM layers were observed in the samples treated with 0.3 m NaCl and 1 µm odanacatib than in those treated with deionized water (all p < 0.05). The samples treated with 1 µm odanacatib showed significantly lower levels of CTX and HYP than those treated with 0.3 M NaCl (all p < 0.05). The present findings support the potential use of catK inhibitors in controlling dentin erosion.

Effects of two disinfection/sterilization methods for dentin specimens on dentin permeability.

OBJECTIVES: To investigate the effects of two disinfection/sterilization methods on the permeability of dentin specimens. MATERIALS AND METHODS: Forty intact human third molars were freshly extracted and cut, close to the pulp chamber, into dentin disks with a 500-µm thickness. The disks were randomized (n = 20 each) into a 70% ethanol group (acid-etched dentin disks soaked in 70% ethanol for 15 min) and a steam autoclaving group (acid-etched dentin disks autoclaved for 25 min). The permeability (Lp) of each dentin disk was measured before and after either treatment using a hydraulic device, and intra- and inter-group differences in values before and after treatment were analyzed using t tests. Field emission scanning electron microscopy (FE-SEM) micrographs of the dentin surface were acquired and examined. FE-SEM samples were prepared using the critical point drying (CPD) method. RESULTS: Immersion in 70% ethanol increased the Lp values of dentin specimens by 17%, which was not statistically significant. Steam autoclaving significantly reduced dentin permeability by 66% because the dentin collagen mesh became compact and collapsed, as detected by FE-SEM. CONCLUSIONS: The disinfection of acid-etched dentin disks using 70% ethanol for 15 min does not significantly affect dentin permeability, whereas sterilization of acid-etched dentin disks via autoclaving significantly reduces dentin permeability. CLINICAL RELEVANCE: Considering the influences of dentin permeability by disinfection/sterilization methods, the disinfection of the acid-etched dentin disks using 70% ethanol for 15 min could be used for the study related to dentin permeability, while the sterilization of autoclaving could not.

Theaflavin -3,3'-digallate/ethanol: a novel cross-linker for stabilizing dentin collagen.

Objectives: To study the ability of theaflavin-3,3'-digallate (TF3)/ethanol solution to crosslink demineralized dentin collagen, resist collagenase digestion, and explore the potential mechanism. Methods: Fully demineralized dentin blocks were prepared using human third molars that were caries-free. Then, these blocks were randomly allocated into 14 separate groups (n = 6), namely, control, ethanol, 5% glutaraldehyde (GA), 12.5, 25, 50, and 100 mg/ml TF3/ethanol solution groups. Each group was further divided into two subgroups based on crosslinking time: 30 and 60 s. The efficacy and mechanism of TF3's interaction with dentin type I collagen were predicted through molecular docking. The cross-linking, anti-enzymatic degradation, and biomechanical properties were studied by weight loss, hydroxyproline release, scanning/transmission electron microscopy (SEM/TEM), in situ zymography, surface hardness, thermogravimetric analysis, and swelling ratio. Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy were utilized to explore its mechanisms. Statistical analysis was performed using one and two-way analysis of variance and Tukey's test. Results: TF3/ethanol solution could effectively crosslink demineralized dentin collagen and improve its resistance to collagenase digestion and biomechanical properties (p < 0.05), showing concentration and time dependence. The effect of 25 and 50 mg/ml TF3/ethanol solution was similar to that of 5% GA, whereas the 100 mg/mL TF3/ethanol solution exhibited better performance (p < 0.05). TF3 and dentin type I collagen are mainly cross-linked by hydrogen bonds, and there may be covalent and hydrophobic interactions. Conclusion: TF3 has the capability to efficiently cross-link demineralized dentin collagen, enhancing its resistance to collagenase enzymatic hydrolysis and biomechanical properties within clinically acceptable timeframes (30 s/60 s). Additionally, it exhibits promise in enhancing the longevity of dentin adhesion.

Dentin Collagen versus Er:YAG Laser as Surface Biomodifiers for Intact Root Slices Simulating Delayed Replanted Roots.

Objective: To evaluate effects of dentin collagen versus Er:YAG laser application through enhancing human periodontal ligament fibroblast (PDLF) cells to attach to intact root surfaces imitating delayed replanted roots. Background Data: Accidental traumatic injuries with teeth avulsion are managed by replantation. Root resorption, poor conditioning, and non-viable fibroblasts are factors responsible for failure. Methods: Thirty six human healthy single-rooted premolars were collected. Six teeth were used for PDLF, six teeth used for dentin collagen, whereas the remaining 24 teeth (48 root slices) were used for PDLF cell density and morphology. Each root was soaked in 5.25% NaOCl. Three groups (n = 16 slices/each) were planned as follows: I: Control (untreated); II: dentin collagen application; III: Er:YAG laser irradiation (4 mm distance, 40 mJ/pulse, under coolant). Following incubation, cell density and morphology of PDLF were investigated under SEM. Statistical analysis was performed using analysis of variance with Scheffé's test, and p < 0.05 was considered significant. Results: All groups showed increased cultured PDLF following incubation. Regarding cell density, attached PDLFs were significantly lower in untreated controls (36.5 ± 6.36) (p < 0.00001 i.e., <0.05) in negative empty and/or light cellular areas, compared with dentin collagen (65 ± 6) and laser-irradiated (66.75 ± 5.77) groups that did not show significant differences (p = 0.940 i.e., >0.05) and showed intermediate and/or heavy cellular areas. Regarding cell morphology, controls showed round and/or oval appearance with less lamellipodia, whereas dentin collagen and laser groups showed flat morphology with cytoplasmic processes. Conclusions: Both dentin collagen and Er:YAG laser showed comparable effectiveness as biomodification tools with good biocompatibility for human PDLF cell attachment on intact root slices imitating delayed replantation. Dentin collagen as a natural bioactive material is considered an alternative to Er:YAG laser to enhance the regenerative effects.

Effect of chitosan-oleuropein nanoparticles on dentin collagen cross-linking.

BACKGROUND: The integrity and stability of collagen are crucial for the dentin structure and bonding strength at dentin-resin interface. Natural plant-derived polypehenols have been used as collagen crosslinkers. OBJECTIVE: The aims of the study were to develop novel chitosan oleuropein nanoparticles (CS-OL-NPs), and to investigate the CS-OL-NPs treated dentin's the resistance to enzymatic degradation and mechanic property. METHODS: CS-OL-NPs were developed using the ionotropic gelation method. Release and biocompatibility of the CS-OL-NPs were tested. Twenty demineralized dentin collage specimens were randomized into four interventions groups: A, Deionized Water (DW); B, 5% glutaraldehyde solution (GA); C, 1 mg/ml chitosan (CS); and D, 100 mg/L CS-OL-NPs. After 1-min interventions, dentin matrix were evaluated by the micro-Raman spectroscopy for the modulus of elasticity test. Collagen degradation was assessed using hydroxyproline (HYP) assay. RESULTS: CS-OL-NPs were spherical core-shape with a size of 161.29 ± 8.19 nm and Zeta potential of 19.53 ± 0.26 mV. After a burst release of oleuropein in the initial 6 h, there was a long-lasting steady slow release. CS-OL-NPs showed a good biocompatibility for the hPDLSCs. The modulus of elasticity in the crosslinked groups were significantly higher than that in the control group (P< 0.05 for all). The specimens treated with CS-OL-NP showed a greater modulus of elasticity than those treated with GA and CS (P< 0.05 for both). The release of HYP in the crosslinked group was significantly lower than that in the non-crosslinked groups (P< 0.05 for all). CONCLUSION: CS-OL-NPs enhanced the dentin mechanical property and resistance to biodegradation, with biocompatibility and potential for clinical application.

Dual-functional etchants that simultaneously demineralize and stabilize dentin render collagen resistant to degradation for resin bonding.

OBJECTIVES: To develop dual-functional etchants that could demineralize and stabilize dentin collagen simultaneously, and to assess the effects of these etchants on collagen crosslinking, biostability and resin bonding properties under clinically relevant conditions. METHODS: Dual-functional etchants were prepared by mixing 56% glycolic acid and 17% phosphoric acid and adding 1% of theaflavins (TF) or proanthocyanidins from grape seed extract (GSE). The etchant without crosslinker was used as control. The prepared human dentin specimens were treated with the 3 etchants for 30 s and analyzed for chemical interaction using Fourier transform infrared spectroscopy and resistance of the demineralized layer to collagenase degradation using electron microscopy (EM). Resin-dentin interfacial bonding properties were evaluated after 24 h and after 10,000 thermocycling through microtensile bond strength (µTBS), nanoleakage and matrix metalloproteinases (MMPs) activity via in situ zymography. Statistical analysis was done using ANOVA and post- hoc Tuckey's test. RESULTS: Compared to control, TF and GSE dual-functional etchants were able to demineralize dentin, induce collagen crosslinking and protect the demineralized layer from collagenase degradation within 30 s. High resolution EM images showed better protection with TF etchant compared to GSE. There was a significant reduction in µTBS and an increase in nanoleakage and MMPs activity in control after thermocycling (p < 0.05) while these changes weren't seen in dual-functional etchants. SIGNIFICANCE: Dual-functional etchants, especially TF containing, provide collagen protection against degradation and result in stable µTBS and less nanoleakage and MMPs activity under clinically relevant conditions.

Characterization of cold atmospheric plasma-modified dentin collagen.

The aim of this study was to evaluate the crosslinking effect of the radio-frequency atmospheric-pressure glow discharge (RF-APGD) plasma jet treatment on dentin collagen. The dentin collagen was treated by an RF-APGD plasma jet with the gas temperature of 4°C under different treatment times, while the control was a non-treatment group. The dentin collagen was characterized in terms of atomic force microscopy-based nanoindentation, differential scanning calorimeter, Raman analysis and X-ray photoelectron spectroscopy (XPS) measurement. The crosslinking effect of the plasma-treated dentin collagen was found compared to that of the control group. The elastic modulus and denaturation temperature of the dentin collagen after plasma treatment for 30 s were significantly higher than those in the control group (p<0.05). The RF-APGD plasma jet treatment can promote the crosslinking of the dentin collagen, which is of great significance to improve its mechanical and thermal stabilities.

Changes to dentin extracellular matrix following treatment with plant-based polyphenols.

This study investigated whether treatment with plant-based polyphenols (PB-P) affected the biochemical and/or biomechanical properties of dentin extracellular matrix (ECM). Three PB-Ps were evaluated: luteolin (LT), galangin (GL), and proanthocyanidin (PAC). Because dentin ECM requires demineralization before treatment, this study also assessed the effect of these PB-Ps on dentin demineralized by two different chemicals. Dentin samples from extracted third molars were obtained, sectioned, and randomly assigned for demineralization with either phosphoric acid (PA) or ethylenediaminetetraacetic acid (EDTA). Following demineralization, baseline infrared (IR) spectra and apparent elastic modulus (AE) of each specimen were independently acquired. Based upon these initial tests, samples were randomly assigned to one of the PB-P treatments to ensure that distribution of baseline AE was similar across treatment groups. IR and AE specimens were individually immersed in either 0.2% LT, 0.4% GL or 1% PAC for 2 min. IR spectra of treated samples were compared to baseline IR spectra, looking for any interaction of PB-Ps with the demineralized dentin. The IR spectrum and AE of each PB-P-treated specimen were compared with their own correspondent baseline measurement. The ability of PB-Ps to inhibit proteolytic activity of dentin ECM was assessed by the hydroxyproline assay. Finally, the effect of PB-Ps on immediate bond strength of a dental adhesive to PA- or EDTA-etched dentin was also evaluated. PB-Ps exhibited distinctively binding affinity to dentin ECM and promoted significant increase in AE. PB-P treatment reduced the degradation rate of dentin ECM without causing detrimental effect on immediate bond strength to dentin. Our work represents the first-time that LT and GL have been assessed as dentin ECM biomodifiers.

Effects of the Combined Application of Trimethylated Chitosan and Carbodiimide on the Biostability and Antibacterial Activity of Dentin Collagen Matrix.

The structural integrity of a dentin matrix that has been demineralized by the clinical use of etchants or calcium-depleting endodontic irrigants, such as endodontic ethylenediaminetetraacetic acid (EDTA), is often deteriorated due to the collagenolytic activities of reactivated endogenous enzymes as well as the infiltration of extrinsic bacteria. Therefore, the biomodification of dentin collagen with improved stability and antibacterial activity holds great promise in conservative dentistry. The purpose of this study was to evaluate the effects of the combined application of trimethylated chitosan (TMC) and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) on the biostability and antibacterial activity of the demineralized dentin collagen matrix. The morphological changes in the collagen matrix were observed by scanning electron microscopy (SEM), the amount of TMC adsorbed on the collagen surface was detected by X-ray photoelectron spectroscopy, and the elastic modulus was measured by a three-point bending device. Dry weight loss and amino acid release were detected to evaluate its anti-collagenase degradation performance. The antibacterial performance was detected by confocal microscopy. The TMC-treated group had less collagen space and a more compact collagen arrangement, while the untreated group had a looser collagen arrangement. The combined application of TMC and EDC can increase the elastic modulus, reduce the loss of elastic modulus, and result in good antibacterial performance. The current study proved that a dentin collagen matrix biomodified by TMC and EDC showed improved biodegradation resistance and antibacterial activities.

Collagen stabilization by natural cross-linkers: A qualitative and quantitative FTIR study on ultra-thin dentin collagen model.

Due to its low tolerance to external factors such as enzymes, dentin collagen often requires stabilization, which can be achieved through cross-linking. In this study, qualitative and quantitative Fourier transform infrared (FTIR) analyses were used to assess dentin collagen stabilization effects of three structurally-different flavonoids -A-type linkage proanthocyanidins (A-PA), B-type linkage proanthocyanidins (B-PA), and epigallocatechin gallate (EGCG), all from natural extracts. Particularly, transmission FTIR spectroscopy was used for the first time to quantitatively assess the biodegradation of fresh ultra-thin (10 µm) dentin collagen films caused by collagenase digestion. Two traditional analytical methods, namely the hydroxyproline assay and weight loss analysis, were also used for comparison purposes. The results from all three methods showed consistently that A-PA and B-PA provide better collagen stabilization than EGCG at concentrations of 0.65% and 1.3% (p<0.01). FTIR is demonstrated to be a valuable and reliable analytical tool for qualitative and quantitative evaluation of ultra-thin collagen films.

Dentin collagen denaturation status assessed by collagen hybridizing peptide and its effect on bio-stabilization of proanthocyanidins.

OBJECTIVE: To assess dentin collagen denaturation from phosphoric acid and enzyme treatments using collagen hybridizing peptide (CHP) and to investigate the effect of collagen denaturation on bio-stabilization promoted by proanthocyanidins (PA). METHODS: Human molars were sectioned into 7-µm-thick dentin films, demineralized, and assigned to six groups: control with/without PA modification, H3PO4-treated collagen with/without PA modification, enzyme-treated collagen with/without PA modification. PA modification involved immersing collagen films in 0.65% PA for 30 s. H3PO4 and enzyme treatments were used to experimentally induce collagen denaturation, which was quantitated by fluorescence intensity (FI) from the fluorescently-conjugated-CHP (F-CHP) staining (n = 4). FTIR was used to characterize collagen structures. All groups were subject to collagenase digestion to test the bio-stabilization effect of PA on denatured collagen using weight loss analysis and hydroxyproline assay (n = 6). Data were analyzed using two-factor ANOVA and Games-Howell post hoc tests (α = 0.05). RESULTS: FTIR showed collagen secondary structural changes after denaturation treatments and confirmed the incorporation and cross-linking of PA in control and treated collagen. F-CHP staining indicated high-degree, medium-degree, and low-degree collagen denaturation from H3PO4-treatment (FI = 83.22), enzyme-treatment (FI = 36.54), and control (FI = 6.01) respectively. PA modification significantly reduced the weight loss and hydroxyproline release of all groups after digestion (p < 0.0001), with the results correlated with FI values at r = 0.96-0.98. SIGNIFICANCE: A molecular method CHP is introduced as a sensitive technique to quantitate dentin collagen denaturation for the first time. PA modification is shown to effectively stabilize denatured collagen against collagenase digestion, with the stabilization effect negatively associated with the collagen denaturation degree.

The effect of universal adhesives on dentine collagen.

OBJECTIVES: The 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. METHODS: Coronal 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 ß-turns, 310-helix/ß-turns, α-helix, random coils, ß-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. RESULTS: PA, 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 ß-turns, α-helices and an increase in ß-sheets, GP a reduction in ß-turns, AD an increase in ß-turns and random coils, and CQ an increase in ß-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. SIGNIFICANCE: The 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.

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

OBJECTIVE: The 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. METHODS: Three 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%). RESULTS: MA: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. SIGNIFICANCE: A 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.

Dataset on the effect of flavonoids on the stabilization of the resin-dentin interface.

Data in this article are associated with our research article "Effect of Myricetin on Odontoblast-like Cells and its Potential to Preserve Resin-Dentin Bonds." Both a poor infiltration of resin monomers into the demineralized dentin matrix and hydrolytic degradation of the adhesive could lead to the instability of the resin-dentin interface. The degradation of collagen is caused by matrix metalloproteinases (MMP) and cysteine cathepsins. These collagenolytic enzymes are contained in their latent form as pro-MMPs in the dentinal structure, and undergo activation during the adhesive process. Given that the integrity of the collagen matrix is essential for the preservation of the dentin bond strength in both the medium and long term, the inhibition of these proteases is necessary to improve the durability of adhesive restorations. Among the different strategies suggested to improve both the behavior of the substrate against enzymatic degradation and the biomechanical behavior of the adhesive interface, the use of protease inhibitors and collagen crosslinking agents has been recommended, such as polyphenols. Research has focused on flavonoids such as proanthocyanidins (PAC), a class of phenolic compounds found in a variety of plants such as blueberry and grape whose chemical structure favors their action as cross-linking agents. However, the focus has recently shifted towards myricetin (MYR) due to its chemical structure: a greater amount of hydroxyl groups at the substitution positions, which form bonds with the carbonyl groups of the side chains of collagen amino acids and generate interfiber bonds. Our previous study has shown the efficacy of MYR both as a cross-linking agent and as a MMP inhibitor without any immediate effects on microtensile bond strength (µTBS) and preserving it for six months after storage, and maintaining the odontoblastic phenotype without affecting cell viability. The objective of this article is to present a dataset on the effect of flavonoids PAC and MYR on the resin-dentin interface. Given that durability of the resin-dentin bond holds great importance for the clinical longevity of adhesive restorations, our data aims to show the effects of these flavonoids on resin-dentin µTBS after 18-month storage. Test groups for the µTBS assay were set as follows: G1 (negative control), conventional adhesion technique; G2 (vehicle control), 100% ethanol (EtOH) for 120 s; G3, 0.2% chlorhexidine (CHX) for 60 s; G4, 1% glutaraldehyde (GA) for 60 s; and G5, 600 µM myricetin (MYR) for 120 s. Datasets were exported to SPSS software, version 21.0 (SPSS, Chicago, IL, USA) for analysis using the Shapiro-Wilk, a two-way analysis of variance including factor interactions (treatment and storage time). Data are presented as mean ± standard deviation (SD). Differences with p-values < 0.05 were considered significant. Our data can be used as a basis for comparison among other natural and synthetic substances that could work as MMP inhibitors and crosslinking agents. These findings could be useful for designing an effective strategy towards the stabilization of the hybrid layer in a relevant clinical protocol.

Bioinspired catechol chemistry for dentin remineralization: A new approach for the treatment of dentin hypersensitivity.

OBJECTIVE: Dentin remineralization is of considerable clinical interest for dentin hypersensitivity and developing biomimetic analogs that can regulate hydroxyapatite (HAp) nucleation and growth remains a challenge. This study aimed to evaluate in vitro the potential for dentin remineralization using the following biomimetic in situ prepared poly(catechols): poly(dopamine), poly(DOPA), poly(caffeic acid) and a synthesized DOPA-peptide possessing collagen and calcium-binding domains (DOPA-Ahx-(Gly)3-(Glu)5). METHODS: Dentin samples were immersed in a freshly prepared phosphate-buffered saline (PBS) containing the respective catechol and laccase. After the reaction, they were immersed in calcium and phosphate remineralization solution, which was changed every day for 10 days. Samples of intact and demineralized dentin were used as control groups and kept in deionized water under the same experimental conditions. The remineralized dentin was characterized by scanning electron microscopy (SEM), Micro-energy dispersion X-ray fluorescence spectroscopy (µEDX) and X-ray diffraction (XRD). RESULTS: The application of different poly(catechols) and DOPA-peptide promoted crystal nucleation and the formation of HAp, which partially covered both the dentin surface and dentinal tubules walls. SIGNIFICANCE: By mimicking the role of charged non-collagenous proteins in vivo, polymers consisting of catechol groups showed the ability to modify demineralized dentin surface properties, promoting mineral formation. The use of poly(catechols) may be encouraged for the development of a therapeutic technique for dentin hypersensitivity.

Effect of tea root-derived proanthocyanidin fractions on protection of dentin collagen.

OBJECTIVES: Proanthocyanidins (PAs) have been widely used as effective agents for dentin collagen cross-linking to enhance the biomechanics and biostability of dentin in vitro. However, the effects and protective mechanisms of various tea root-derived PA components on dentin remain undefined. This study evaluated the effects of these tea root-derived PA components on dentin biomechanics and biostability. METHODS: In this study, ethyl acetate and n-butyl alcohol were used to extract PAs with different degrees of polymerization from tea roots; the effects of these PA extracts on dentin were evaluated. RESULTS: Dentin was treated with glutaraldehyde, ethyl acetate, n-butyl alcohol, or water. PAs with a high degree of polymerization, extracted using n-butyl alcohol, were able to more effectively improve dentin collagen cross-linking, increase resistance to bacterial collagenase digestion, and enhance dentin elasticity, relative to treatment with glutaraldehyde or PAs with a low degree of polymerization (extracted using ethyl acetate). Additionally, treatment with aqueous extract of tea roots was detrimental to dentin stability and function. CONCLUSIONS: PAs with a high degree of polymerization were effective for dentin protection and restoration in vitro, suggesting clinical treatment potential for tea root-derived PAs.

Dentine collagen cross-linking using tiopronin-protected Au/EDC nanoparticles formulations.

OBJECTIVE: The aim of this study was to investigate EDC-assisted collagen crosslinking effect with different concentrations of tiopronin-protected gold (TPAu) nanoparticles on demineralized dentine. METHODS: TPAu nanoparticles were fabricated from 0.31-g tetrachloroauric acid and 0.38-g of N-(2-mercaptopropionyl) glycine (2.4-mmol). Then co-dissolved using 35-mL of 6:1 methanol/acetic acid and mixed using NaBH4. EDC (0.3-M) was conjugated to TPAu nanoparticles at TPAU/EDC-0.25:1, and TPAU/EDC-0.5:1 treatment formulations ratios. Dentin specimens treated with 0.3-M EDC solution alone or left untreated were used as control. Nanoparticles formulations were characterized in term of particles morphology and size, Zeta potential, thermogravimetric analysis and small-angle X-ray scattering. Dentin substrates were characterized in term of TEM investigation, dentin proteases characterization, hydroxyproline liberation, elastic modulus measurement, Raman analysis and confocal microscopy viewing. RESULTS: TEM evaluation of tiopronin protected gold nanoparticles dispersion revealed nano-clusters formations in both groups. However, based on our TEM measurements, the particle-size was ranging from ˜20 to 50 nm with spherical core-shape which were almost similar for both TPAu/EDC ratios (0.5:1 and 0.25:1). Zeta potential measurements indicate negative nanoparticles surface charge. SAXS profiles for both formulations, suggest a typical profile for uni-lamellar nanoparticles. Superior dentin collagen cross-linking effect was found with the TPAu/EDC nanoparticles formulations compared to the control and EDC treated groups. SIGNIFICANCE: Cross-linking of dentin collagen using TPAu coupled with EDC through TPAu/EDC nanoparticles formulations is of potential significance in improving the biodegradation resistance, proteases inhibition, mechanical and structural stability of demineralized dentin substrates. In addition, the cross-linking effect is dependent on TPAu/EDC ratio, whereas higher cross-linking effect was found at TPAu/EDC ratio of 0.5:1.

Potential of high-intensity focused ultrasound in resin-dentine bonding.

OBJECTIVE: This study introduced the potential and proof-of-concept of high intensity focused ultrasound (HIFU) technology for dentin-surface treatment for resin-dentin bonding without acid-aided demineralization. This new strategy could provide a way to enhance interface-integrity and bond-durability by changing the nature of dentin-substrate; bonded-interface structure and properties; and minimizing denuded-collagen exposure. METHODS: The interaction between HIFU waves and dentin-surface in terms of structural, mechanical and chemical variations were investigated by SEM, TEM, AFM, nano-indentation and Raman-analysis. The bonding between HIFU-treated dentin and two-step, etch-and-rinse, adhesive was preliminary explored by characterizing dentin-bound proteases activities, resin-dentin interfacial morphology and bond-durability with HIFU exposure at different time-points of 60, 90 and 120 s compared to conventional acid-etching technique. RESULTS: With the increase in HIFU exposure-time from 60-to-120 s, HIFU waves were able to remove the smear-layer, expose dentinal-tubules and creating textured/rough dentin surface. In addition, dentin surfaces showed a pattern of interlocking ribbon-like minerals-coated collagen-fibrils protruding from the underlaying amorphous dentin-background with HIFU exposure for 90 s and 120 s. This characteristic pattern of dentin-surface showing inorganic-minerals associated/aligned with collagen-fibrils, with 90-to-120 s HIFU-treatment, was confirmed by the Raman-analysis. HIFU-treated specimens showed higher nano-indentation properties and lower concentrations of active MMP-2 and Cathepsin-K compared to the acid-etched specimens. The resin-dentin bonded interface revealed the partial/complete absence of the characteristic hybrid-layer formed with conventional etch-and-rinse bonding strategy. Additionally, resin-infiltration and resin-tags formation were enhanced with the increase in HIFU exposure-time to 120 s. Although, all groups showed significant decrease in bond-strength after 12 months compared to 24 h storage in artificial saliva, groups exposed to HIFU for 90 s and 120 s showed significantly higher µTBS compared to the control acid-etched group. SIGNIFICANCE: The implementation of HIFU-technology for dental hard-tissues treatment could be of potential significance in adhesive/restorative dentistry owing to its ability of controlled, selective and localised combined tissue alteration/ablation effects.

Collagen cross-linkers on dentin bonding: Stability of the adhesive interfaces, degree of conversion of the adhesive, cytotoxicity and in situ MMP inhibition.

OBJECTIVE: To investigate the effect of collagen cross-links on the stability of adhesive properties, the degree of conversion within the hybrid layer, cytotoxicity and the inhibition potential of the MMPs' activity. METHODS: The dentin surfaces of human molars were acid-etched and treated with primers containing: 6.5wt% proanthocyanidin, UVA-activated 0.1wt% riboflavin, 5wt% glutaraldehyde and distilled water for 60s. Following, dentin was bonded with Adper Single Bond Plus and Tetric N-Bond; and restored with resin composite. The samples were sectioned into resin-dentin "sticks" and tested for microtensile bond strength (µTBS) after immediate (IM) and 18-month (18M) periods. Bonded sticks at each period were used to evaluate nanoleakage and the degree of conversion (DC) under micro-Raman spectroscopy. The enzimatic activity (P1L10 cross-linkers, P1L22 MMPs' activities) in the hybrid layer was evaluated under confocal microscopy. The culture cell (NIH 3T3 fibroblast cell line) and MTT assay were performed to transdentinal cytotoxicity evaluation. Data from all tests were submitted to appropriate statistical analysis (α=0.05). RESULTS: All cross-linking primers reduced the degradation of µTBS compared with the control group after 18M (p>0.05). The DC was not affected (p>0.213). The NL increased after 18M for all experimental groups, except for proanthocyanidin with Single Bond Plus (p>0.05). All of the cross-link agents reduced the MMPs' activity, although this inhibition was more pronounced by PA. The cytotoxicity assay revealed reduced cell viability only for glutaraldehyde (p<0.001). SIGNIFICANCE: Cross-linking primers used in clinically relevant minimized the time degradation of the µTBS without jeopardizing the adhesive polymerization, as well as reduced the collagenolytic activity of MMPs. Glutaraldeyde reduced cell viability significantly and should be avoided for clinical use.

Dental adhesives and strategies for displacement of water/solvents from collagen fibrils.

OBJECTIVES: To evaluate the influence of temperature of evaporation in adhesive systems with different solvents on the apparent modulus of elasticity and mass change of macro-hybrid layers modified by proanthocyanidins (PACs). METHODS: Adhesive resin beams (A) from Single Bond Plus (SB), Excite (EX) and One Step Plus (OS) were prepared after solvent evaporation at 23°C or 40°C (n=12). Macro-hybrid layers (M) (n=12) were prepared using demineralized dentin beams sectioned from extracted human third molars. The demineralized dentin specimens were infiltrated with each one of the three adhesive systems at 23°C or 40°C; with or without prior dentin treatment with PACs for 10min. The apparent modulus of elasticity (E) and mass change (Wmc, %) of adhesives beams and resin-infiltrated specimens were assessed in dry and wet conditions after immersion in water (24h, 1, 3 and 6 months). The E was statistically analyzed by Tukey-Kramer test and the Wmc, % by Kruskal Wallis, and Dunn (α=0.05). RESULTS: Solvent evaporation at 40°C resulted in higher E values for adhesive resin beams at all storage conditions, regardless of the adhesive system (p<0.05). Increased mass loss (3 months: -0.01%; 6 months: -0.05%) was observed in One Step resin beams (p≤0.05). In the macro-hybrid layer models the pretreatment with PACs along with solvent evaporation at 40°C increased E and decreased the Wmc, % (3 months: -2.5; 6 months: 2.75%) for adhesives evaluated over time (p<0.05). No significant differences in ratio (resin/dentin) were found for the macro-hybrid layers (p>0.05). SIGNIFICANCE: Improved solvent evaporation at higher temperature, and increased collagen cross-linking induced by PACs, enhanced the mechanical properties resulting in highly stable macro-hybrid layers over 6 months storage.