Subtle Chemical Shifts Explain the NMR Fingerprints of Oligomeric Proanthocyanidins with High Dentin Biomodification Potency.

The ability of certain oligomeric proanthocyanidins (OPACs) to enhance the biomechanical properties of dentin involves collagen cross-linking of the 1.3-4.5 nm wide space via protein-polyphenol interactions. A systematic interdisciplinary search for the bioactive principles of pine bark has yielded the trimeric PAC, ent-epicatechin-(4ß→8)-epicatechin-(2ß→O→7,4ß→8)-catechin (3), representing the hitherto most potent single chemical entity capable of enhancing dentin stiffness. Building the case from two congeneric PAC dimers, a detailed structural analysis decoded the stereochemistry, spatial arrangement, and chemical properties of three dentin biomodifiers. Quantum-mechanics-driven (1)H iterative full spin analysis (QM-HiFSA) of NMR spectra distinguished previously unrecognized details such as higher order J coupling and provided valuable information about 3D structure. Detection and quantification of H/D-exchange effects by QM-HiFSA identified C-8 and C-6 as (re)active sites, explain preferences in biosynthetic linkage, and suggest their involvement in dentin cross-linking activity. Mapping of these molecular properties underscored the significance of high δ precision in both (1)H and (13)C NMR spectroscopy. Occurring at low- to subppb levels, these newly characterized chemical shift differences in ppb are small but diagnostic measures of dynamic processes inherent to the OPAC pharmacophores and can help augment our understanding of nanometer-scale intermolecular interactions in biomodified dentin macromolecules.

Mimicking the hierarchical functions of dentin collagen cross-links with plant derived phenols and phenolic acids.

Proanthocyanidins (PACs) are secondary plant metabolites that mediate nonenzymatic collagen cross-linking and enhance the properties of collagen based tissue, such as dentin. The extent and nature of cross-linking is influenced by the composition and specific chemical structure of the bioactive compounds present in certain PAC-rich extracts. This study investigated the effect of the molecular weight and stereochemistry of polyphenol compounds on two important properties of dentin, biomechanics, and biostability. For that, purified phenols, a phenolic acid, and some of its derivatives were selected: PAC dimers (A1, A2, B1, and B2) and a trimer (C1), gallic acid (Ga), its esters methyl-gallate (MGa) and propyl-gallate (PGa), and a pentagalloyl ester of glucose (PGG). Synergism was assessed by combining the most active PAC and gallic acid derivative. Mechanical properties of dentin organic matrix were determined by the modulus of elasticity obtained in a flexural test. Biostability was evaluated by the resistance to collagenase degradation. PACs significantly enhanced dentin mechanical properties and decreased collagen digestion. Among the gallic acid derivatives, only PGG had a significant enhancing effect. The lack of observed C1:PGG synergy indicates that both compounds have similar mechanisms of interaction with the dentin matrix. These findings reveal that the molecular weight of polyphenols have a determinant effect on their interaction with type I collagen and modulates the mechanism of cross-linking at the molecular, intermolecular, and inter-microfibrillar levels.

Effects of silane application on luting fiber posts using self-adhesive resin cement.

PURPOSE: To evaluate the effects of different glass-fiber post surface treatments on the bond strength to root dentin. MATERIALS AND METHODS: Fifty bovine incisors were used in this study. After removing the crowns, the teeth were endodontically treated. The roots were randomly divided into five groups according to post surface treatment. The groups were as follows: CO (Control) - no treatment; G1 - RelyX Ceramic Primer (silane) only; G2 - silane and Solobond M; G3 - silane and Scotchbond Adhesive; G4 - silane and Excite. For post cementation, RelyX Unicem was used according to the manufacturer's recommendation and the roots were stored in a light-proof container with 100% relative humidity for 24 h. The specimens were transversally sectioned. Subsequently, the cervical, middle, and apical regions of the root were positioned in a push-out device and tested at 0.5 mm/min using a universal testing machine (Instron). The data were statistically analyzed with two-way ANOVA and Tukey's post-hoc test. The fractured specimens were then observed under a stereoscopic loupe at 60X magnification. RESULTS: No significant difference in bond strength was found among the groups that received a silane or silane plus an adhesive system (p > 0.05). However, the CO (no silane) showed the lowest bond strength. Regarding G1, G2, G3, and G4, the cervical region of the root canal attained better bond strengths than did the middle or apical regions. The most frequent failure mode occurred at the cement/dentin interface. CONCLUSION: Silane application may be necessary to improve the adhesion of fiber posts luted with the self-adhesive resin cement evaluated here. The application of an adhesive layer between the fiber post and resin cement did not have any influence on the bond strength when the silane coupling was previously used.

Centrifugal partition chromatography enables selective enrichment of trimeric and tetrameric proanthocyanidins for biomaterial development.

Proanthocyanidins (PACs) find wide applications for human use including food, cosmetics, dietary supplements, and pharmaceuticals. The chemical complexity associated with PACs has triggered the development of various chromatographic techniques, with countercurrent separation (CCS) gaining in popularity. This study applied the recently developed DESIGNER (Depletion and Enrichment of Select Ingredients Generating Normalized Extract Resources) approach for the selective enrichment of trimeric and tetrameric PACs using centrifugal partition chromatography (CPC). This CPC method aims at developing PAC based biomaterials, particularly for their application in restoring and repairing dental hard tissue. A general separation scheme beginning with the depletion of polymeric PACs, followed by the removal of monomeric flavan-3-ols and a final enrichment step produced PAC trimer and tetramer enriched fractions. A successful application of this separation scheme is demonstrated for four polyphenol rich plant sources: grape seeds, pine bark, cinnamon bark, and cocoa seeds. Minor modifications to the generic DESIGNER CCS method were sufficient to accommodate the varying chemical complexities of the individual source materials. The step-wise enrichment of PAC trimers and tetramers was monitored using normal phase TLC and Diol-HPLC-UV analyses. CPC proved to be a reliable tool for the selective enrichment of medium size oligomeric PACs (OPACs). This method plays a key role in the development of dental biomaterials considering its reliability and reproducibility, as well as its scale-up capabilities for possible larger-scale manufacturing.

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.

Potential role of surface wettability on the long-term stability of dentin bonds after surface biomodification.

Degradation of the adhesive interface contributes to the failure of resin composite restorations. The hydrophilicity of the dentin matrix during and after bonding procedures may result in an adhesive interface that is more prone to degradation over time. This study assessed the effect of chemical modification of the dentin matrix on the wettability and the long-term reduced modulus of elasticity (Er) of adhesive interfaces. Human molars were divided into groups according to the priming solutions: distilled water (control), 6.5% Proanthocyanidin-rich grape seed extract (PACs), 5.75% 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/1.4% n-hydroxysuccinimide (EDC/NHS) and 5% Glutaraldehyde (GA). The water-surface contact angle was assessed before and after chemical modification of the dentin matrix. The demineralized dentin surface was treated with the priming solutions and restored with One Step Plus (OS) and Single Bond Plus (SB) and resin composite. Er of the adhesive, hybrid layer and underlying dentin was evaluated after 24h and 30 months in artificial saliva. The dentin hydrophilicity significantly decreased after application of the priming solutions. Aging significantly decreased Er in the hybrid layer and underlying dentin of control groups. Er of GA groups remained stable over time at the hybrid layer and underlying dentin. Significant higher Er was observed for PACs and EDC/NHS groups at the hybrid layer after 24h. The decreased hydrophilicity of the modified dentin matrix likely influence the immediate mechanical properties of the hybrid layer. Dentin biomodification prevented substantial aging at the hybrid layer and underlying dentin after 30 months storage.

Nanomechanical properties, SEM, and EDS microanalysis of dentin treated with 2.5% titanium tetrafluoride, before and after an erosive challenge.

The aim was to assess the nanohardness (H) and the reduced modulus of elasticity (Er ) of 2.5% titanium tetrafluoride (TiF4 ) modified dentin, before and after an erosive challenge with 0.3% citric acid (CA). Exposed dentin surfaces were divided into two groups (n = 5): (1) Control-no dentin pretreatment with TiF4 prior to etching with CA, and (2) Experimental-dentin pretreatment with TiF4 + CA. The H and the Er of intertubular dentin were measured using a triboindenter at different time points: baseline for both groups, after using 2.5% TiF4 for the experimental group, and after using CA for both the experimental and the control groups. Scanning electron microscope and energy dispersive X-ray spectroscopy (EDS) analysis of the dentin surfaces were undertaken at the same time points for both groups. Two-way ANOVA for randomized block design was applied. There was significant interaction between the application of the TiF4 solution and different time points (p = 0.001 for H and p < 0.001 for Er ), identified by Tukey's test. Erosive challenge provided a significant decrease in H and Er mean values. The TiF4 solution caused a significant increase in H and Er values, but no significant differences were found between post-TiF4 and post-CA application. TiF4 application produced a precipitate surface layer on intertubular and intratubular dentin. EDS analysis indicated the presence of titanium. The H and Er of the dentin surface were greatly increased after application of 2.5% TiF4 . TiF4 may modify the micromorphology of the dentin surface and produces an erosive resistance surface.

A galloylated dimeric proanthocyanidin from grape seed exhibits dentin biomodification potential.

Grape seeds are a rich source of polyphenols, especially proanthocyanidins (PACs), and are also known for the presence of galloylated oligomeric PACs (OPACs). The present study focuses on the phytochemical methodology for grape seed (O)PACs and their potential role as dentin biomodifiers to be used in restorative and reparative dentistry. A new method using centrifugal partition chromatography (CPC) was developed for the preparative separation of the grape seed (O)PACs. Orthogonal phytochemical profiling of the resulting CPC fractions was performed using C18 and diol HPLC, normal phase HPTLC, and IT-TOF MS analysis. A galloylated procyanidin dimer (1) was isolated from a CPC fraction in order to evaluate its potential to enhance dentin bio-mechanical properties. Moreover, it helped to evaluate the impact of the galloyl moiety on the observed bioactivity. Structure elucidation was performed using ESI-MS, 1D and 2D NMR analyses. For the first time, (1)H iterative full spin analysis (HiFSA) was performed on this type of molecule, enabling a detailed proton chemical shift and coupling constant assignment. The CPC fractions as well as 1 showed promising results in the dentin stiffness bioassay and indicate that they may be used as dental intervention biomaterial.

Dentin biomodification: strategies, renewable resources and clinical applications.

OBJECTIVES: The biomodification of dentin is a biomimetic approach, mediated by bioactive agents, to enhance and reinforce the dentin by locally altering the biochemistry and biomechanical properties. This review provides an overview of key dentin matrix components, targeting effects of biomodification strategies, the chemistry of renewable natural sources, and current research on their potential clinical applications. METHODS: The PubMed database and collected literature were used as a resource for peer-reviewed articles to highlight the topics of dentin hierarchical structure, biomodification agents, and laboratorial investigations of their clinical applications. In addition, new data is presented on laboratorial methods for the standardization of proanthocyanidin-rich preparations as a renewable source of plant-derived biomodification agents. RESULTS: Biomodification agents can be categorized as physical methods and chemical agents. Synthetic and naturally occurring chemical strategies present distinctive mechanism of interaction with the tissue. Initially thought to be driven only by inter- or intra-molecular collagen induced non-enzymatic cross-linking, multiple interactions with other dentin components are fundamental for the long-term biomechanics and biostability of the tissue. Oligomeric proanthocyanidins show promising bioactivity, and their chemical complexity requires systematic evaluation of the active compounds to produce a fully standardized intervention material from renewable resource, prior to their detailed clinical evaluation. SIGNIFICANCE: Understanding the hierarchical structure of dentin and the targeting effect of the bioactive compounds will establish their use in both dentin-biomaterials interface and caries management.

Distance and protective barrier effects on the composite resin degree of conversion.

CONTEXT: The food wrap films are used to cover the tip of curing light units in order to avoid contamination and prevent damage to the light guide. However, their effects on resin polymerization are not fully known. AIMS: We investigated the effects on restoration efficiency of a food wrap protective barrier used on the tip of curing light units. MATERIALS AND METHODS: For each treatment, five replications were performed, a total of 60 bovine incisor. The degree of conversion (%DC) of restorations with the composite resin Opallis EA2 was evaluated using 3 curing light devices (Optilux 501, Optilight and Ultra LED) and 2 curing distances (0 and 5 mm). The composite resin was tested for restoration of cavities in bovine crowns. %DC values were measured by the Fourier transform infrared spectroscopy-attenuated total reflectance technique. STATISTICAL ANALYSIS USED: The data were analyzed using 3-way ANOVA and Tukey's test. RESULTS: Use of the protective film lowered %DC (F = 4.13; P = 0.05), and the effects of curing distance were associated to the curing light device (F = 3.61; P = 0.03). CONCLUSIONS: The distance from the light curing tip and use of a translucent protective barrier on the light-cure device can both impair composite resin %DC.

Hydrolytic degradation of the resin-dentine interface induced by the simulated pulpal pressure, direct and indirect water ageing.

OBJECTIVES: The aim of this study was to compare the hydrolytic effects induced by simulated pulpal pressure, direct or indirect water exposure within the resin-dentine interfaces created with three "simplified" resin bonding systems (RBSs). METHODS: A two-step/self-etching (CSE: Clearfil SE Bond), one-step/self-etching (S3: Clearfil S3) and etch-and-rinse/self-priming (SB: Single-bond 2) adhesives were applied onto dentine and submitted to three different prolonged (6 or 12 months) ageing strategies: (i) Simulated Pulpal Pressure (SPP); (ii) Indirect Water Exposure (IWE: intact bonded-teeth); (iii) Direct Water Exposure (DWE: resin-dentine sticks). Control and aged specimens were submitted to microtensile bond strength (µTBS) and nanoleakage evaluation. Water sorption (WS) survey was also performed on resin disks. Results were analysed with two-way ANOVA and Tukey's test (p < 0.05). RESULTS: The µTBS of CS3 and SB dropped significantly (p < 0.05) after 6 months of SPP and DWE. CSE showed a significant µTBS reduction only after 12 months of DWE (p = 0.038). IWE promoted no statistical change in µTBS (p > 0.05) and no evident change in nanoleakage. Conversely, SPP induced a clear formation of "water-trees" in CS3 and SB. WS outcomes were CS3 > SB = CSE. CONCLUSION: The hydrolytic degradation of resin-dentine interfaces depend upon the type of the in vitro ageing strategy employed in the experimental design. Direct water exposure remains the quickest method to age the resin-dentine bonds. However, the use of SPP may better simulate the in vivo scenario. However, the application of a separate hydrophobic solvent-free adhesive layer may reduce the hydrolytic degradation and increase the longevity of resin-dentine interfaces created with simplified adhesives.

Adaptação à dentina de materiais forradores sob restauração de resina composta / Liner adaptation to dentin under composite resin restoration

Introdução: Um dos principais problemas na utilização de resina composta é a sua contração de polimerização e consequente tensão transmitida às paredes aderidas e ao material forrador. Foi analisada a adaptação de agentes protetores à dentina associadosà restauração de resina composta fotopolimerizável. Material e Métodos - CavidadesClasse I oclusal foram confeccionadas com profundidade 1,5 mm, em molares humanos hígidos (n=40). Os dentes foram divididos em 4 grupos, segundo o material de forramento utilizado: FF - resina de baixa viscosidade (Fill Magic Flow), HP - cimento de hidróxidode cálcio fotopolimerizável (Hi-Cal Photo), VLC: cimento de ionômero de vidro fotopolimerizável (Vitro Fill LC), VB: cimento de ionômero de vidro fotopolimerizável(Vitrebond). As cavidades foram restauradas com resina composta (Adper Single Bond2/Filtek Z250), inserida em incrementos oblíquos. Após armazenamento em ambiente úmido a 37°C por 7 dias, os molares foram seccionados no longo eixo do dente e obtidas réplicas em negativo de polivinilsiloxana Adsil e em positivo com resina epóxica. Foi utilizada microscopia óptica para mensuração da porcentagem de adaptação linear domaterial forrador e a parede pulpar. Resultados - Os testes de Kruskal-Wallis e Dunnapontaram diferença estatisticamente significante (p<0,05) entre VB (97,7±1,30) e FF(59,3±33,7), e entre HP (5,3±4,9) e VLC (44,3±24,2). Conclusões - A tensão de contração da resina composta causada à interface adesiva durante sua fotoativação provoca fendas entre o material forrador e a parede pulpar, em magnitudes distintas, mesmo quando ainserção da resina se dá por incrementos. O cimento de ionômero de vidro fotoativado Vitrebond obteve a melhor adaptação.

Introduction: One of the main problems when using composite resin is its polymerization shrinkage and resultant stress transmitted to the adhered walls and to the cavity lining material. This work analyzed the adaptation of protective agents to the dentin associated to the composite resin restoration. Material and Methods - Class I occlusalcavities − 1.5 mm deep − were performed in healthy human molars (n=40). The teethwere divided into four groups according to the lining material used: FF – flowable resin(Fill-Magic Flow); HP – light-cured calcium hydroxide cement (Hi-Cal Photo); VLC –resin-modified glass-ionomer liner (Vitro Fill LC); and VB – resin-modified glass-ionomer liner (Vitrebond). The cavities were restored with composite resin (Adper Single Bond2/Filtek Z250) inserted into oblique increments. After storage in a humid environmentat 37°C for seven days, the molars were sectioned to the long axis of the tooth. Negative replicas were obtained through polyvinyl siloxane (Adsil), and positive ones by epoxyresin. Optical microscopy was used for measuring the percentage of linear adaptationof the cavity liner to the pulp wall. Results - Kruskal-Wallis and Dunn tests showed a statistically significant difference (p<0,05) between VB (97,7 ± 1,30) and FF (59,3 ±33,7), and between HP (5,3 ± 4,9) and VLC (44,3 ± 24,2).Conclusions - The shrinkage stress of the composite resin polymerization causes gaps between the cavity liner and the pulp wall even when restored in increments, in distinct magnitude. The resin-modified glass-ionomer liner Vitrebond proved to have the best adaptability.