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Objective: Investigate the bioactivity and stability of Rhodiola rosea (RR) fractions as a natural source of prodelphinidin gallate (PDg) on dentin collagen via analysis of the viscoelastic and resin-dentin adhesive properties of the dentin matrix., Methods: The biomimicry and stability of RR subfractions (F1, F2, F3 and F4) with collagen were determined by dynamic mechanical analysis (DMA). DMA used a strain sweep method to assess the dentin matrix viscoelastic properties [storage (E'), loss (E"), and complex (E*) moduli and tan δ] after treatment, 7-, 30- and 90-days of storage in simulated body fluids (SBF). Resin-dentin interface properties were assessed after 1 and 90-days in SBF by microtensile bond strength test and confocal laser scanning microscopy. Data were analyzed using two and one-way ANOVA and post-hoc tests (α = 0.05)., Results: RR fractions increased dentin matrix complex (96 - 69 MPa) and storage (95 - 68 MPa) moduli, compared to the control (∼9 MPa) in the ranking order: F2 ≥ F3 = F1 = F4 > control (p < 0.001). Treatment did not affect tan δ values. After 30- and 90-days, RR-treated dentin E*, E' and tan δ decreased (p < 0.001). F2 fraction yielded the highest microtensile bond strength (43.9 MPa), compared to F1, F4 (35.9 - 31.7 MPa), and control (29 MPa). RR-treated interfaces mediated stable surface modifications and enhanced collagen-methacrylate resin interactions at the bioadhesive interface., Significance: Prodelphinidin gallates from RR are potent and reasonably stable biomimetic agents to dentin. Higher potency of F2 fraction with the dentin matrix and the adhesive interface is associated with a degree of polymerization of 2-3 and gallo(yl) motifs., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In nature, proanthocyanidins (PACs) with A-type linkages are relatively rare, likely due to biosynthetic constraints in the formation of additional ether bonds to be introduced into the more common B-type precursors. However, A-type linkages confer greater structural rigidity on PACs than do B-type linkages. Prior investigations into the structure-activity relationships (SAR) describing how plant-derived PACs with B- and complex AB-type linkages affect their capacity for dentin biomodification indicate that a higher ratio of double linkages leads to a greater interaction with dentin type I collagen. Thus, A-type PACs emerge as particularly intriguing candidates for interventional functional biomaterials. This study employed a free-radical-mediated oxidation using DPPH to transform trimeric and tetrameric B-type PACs, 2 and 4 , respectively, into their exclusively A-type linked analogues, 3 and 5 , respectively. The structures and absolute configurations of the semisynthetic products, including the new all-A-type tetramer 5 , were determined by comprehensive spectroscopic analysis. Additionally, molecular modeling investigated the conformational characteristics of all trimers and tetramers, 1 - 5 . Our findings suggest that the specific interflavan linkages significantly impact the flexibility and low-energy conformations of the connected monomeric units, which conversely can affect the bioactive conformations relevant for dentin biomodification.

Flavan-3-ol monomers are the building blocks of proanthocyanidins (PACs), natural compounds from plants shown to mediate specific biologic activities on dentin. While the stereochemistry of the terminal flavan-3-ols, catechin (C) versus epicatechin (EC), impacts the biomechanical properties of the dentin matrix treated with oligomeric PACs, structure-activity relationships driving this bioactivity remain elusive. To gain insights into the modulatory role of the terminal monomers, two highly congruent trimeric PACs from Pinus massoniana only differing in the stereochemistry of the terminal unit (Trimer-C vs. Trimer-EC) were prepared to evaluate their chemical characteristics as well as their effects on the viscoelasticity and biostability of biomodified dentin matrices via infrared spectroscopy and multi-scale dynamic mechanical analyses. The subtle alteration of C versus EC as terminal monomers lead to distinct immediate PAC-trimer biomodulation of the dentin matrix. Nano- and micro-dynamic mechanical analyses revealed that Trimer-EC increased the complex moduli (0.51 GPa) of dentin matrix more strongly than Trimer-C (0.26 GPa) at the nanoscale length (p < 0.001), whereas the reverse was found at the microscale length (p < .001). The damping capacity (tan δ) of dentin matrix decreased by 70% after PAC treatment at the nano-length scale, while increased values were found at the micro-length scale (~0.24) compared to the control (0.18 ; p < .001). An increase in amide band intensities and a decrease of complex moduli was observed after storage in simulated body fluid for both Trimer-C and Trimer-EC modified dentin. The stereochemical configuration of the terminal monomeric units, C and EC, did not impact the chemo-mechanical stability of dentin matrix., (© 2023 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals LLC.)

Proanthocyanidins (PACs) are complex flavan-3-ol polymers with stunning chemical complexity due to oxygenation patterns, oxidative phenolic ring linkages, and intricate stereochemistry of their heterocycles and inter-flavan linkages. Being promising candidates for dental restorative biomaterials, trace analysis of dentin bioactive cinnamon PACs now yielded novel trimeric ( 1 and 2 ) and tetrameric ( 3 ) PACs with unprecedented o - and p- benzoquinone motifs (benzoquinonoid PACs). Challenges in structural characterization, especially their absolute configuration, prompted the development of a new synthetic-analytical approach involving comprehensive spectroscopy, including NMR with quantum mechanics-driven 1 H iterative functionalized spin analysis (HifSA) plus experimental and computational electronic circular dichroism (ECD). Vital stereochemical information was garnered from synthesizing 4-(2,5-benzoquinone)flavan-3-ols and a truncated analogue of trimer 2 as ECD models. Discovery of the first natural benzoquinonoid PACs provides new evidence to the experimentally elusive PAC biosynthesis as their formation requires two oxidative post-oligomerizational modifications (POMs) that are distinct and occur downstream from both quinone-methide-driven oligomerization and A-type linkage formation. While Nature is known to achieve structural diversity of many major compound classes by POMs, this is the first indication of PACs also following this common theme.

The aim of this study was to determine the immediate and sustained effect of a fluoride varnish and its combinations with toothpastes in preventing root caries development using a salivary microcosm in vitro model. Human root dentin specimens (n = 150) were randomly divided into 5 experimental protocols (n = 30): (1) Fluoride Varnish (V); (2) V followed by Paste One (V + PO); (3) V followed by Paste Plus (V + PP); (4) V followed by PO and PP (V + PO + PP); and (5) No treatment (control). One varnish layer was applied on the specimens (except for the control group) and kept for 18 h. Then, the varnish was removed and toothpaste treatments were initiated according to experimental groups. For the short-term incubation model (n = 15), the specimens were also immediately subjected to 7-day cariogenic challenge. For that, human saliva was used as bacterial inoculum and McBain artificial saliva containing 2% sucrose as growth medium. The other half of the specimens (n = 15) were used to study the varnish's sustained effect by long-term incubation (8 weeks) before cariogenic challenge. The protocols' anti-caries properties were evaluated by dentin porosity (rhodamine intensity; RI) and mineral density, while their anti-biofilm effects were evaluated using biofilm's biomass and viability assays. For short- and long-term incubation models, all experimental regimens resulted in statistically significant decreases (p &lt; 0.05) in the RI (up to 180 μm and 120 μm, respectively) as well as higher mineral density compared to No treatment (p &lt; 0.001). V + PO + PP and/or V + PO resulted in statistically lower RI compared to V for some depths (p &lt; 0.05) in both models. There were changes in RI and mineral density within groups over time. All experimental treatments exhibited anti-biofilm effects. All prevention protocols exhibited immediate and sustained anti-caries effect against root caries development. The combination of a fluoride varnish with PO resulted in superior additional anti-caries effects., (© 2023 S. Karger AG, Basel.)

Investigation of a pine bark extract for bioactive proanthocyanidin oligomers resulted in the isolation of structurally related dimeric seco B-type procyanidin derivatives, 1 - 5 . This includes scalemic mixtures of gambiriin A1 ( 1a ) and A2 ( 2a ) and their newly described optical antipodes, ent -gambiriin A1 ( 1b ) and ent -gambiriin A2 ( 2b ), respectively, as well as a racemic mixture of the newly described ( ent -)gambiriin A5 ( 3a / 3b ). Furthermore, the study now fully characterizes the previously reported optically pure dimers gambiriin B1 ( 4 ) and gambirflavan D1 ( 5 ), and characterized the novel seco B-type procyanidin trimer, 6 (gambirifuran C1). Thermal conversion of catechin in aqueous solution provided further evidence for the structures of 1 - 6 and led to the purification of semisynthetic 1a and 2a as well as additional dimers 7 - 10 . Elucidating the structures of the natural dimers, 1 - 5 , from comprehensive NMR and ECD data and synthetic evidence provided crucial reference points for establishing the structure of the seco B-type procyanidin trimer, 6 . Serving as assigned building blocks, data from the dimers supported the 3D structural assignment of 6 based on NMR substituent chemical shift differences (s.c.s., syn. Δδ C ) and component-based empirical ECD calculations. Within the newly characterized series of PAC-related molecules, 5 exhibited high dentin biomodification potential. In addition, considering the nomenclature issues and plausible biosynthetic pathways of this group of compounds led to a consolidated nomenclature of all currently known seco B-type procyanidins. These findings, thereby, expand the chemical space of bioactive catechin oligomers, which have promise as agents for the natural enhancement of dental biomaterials. Finally, the current knowledge of the chemical space of seco B-type procyanidin derivatives was compiled to the level of absolute configuration.

Objectives: To compare the biodegradability, mechanical behavior, and physicochemical features of the collagen-rich extracellular matrix (ECM) of artificial caries-affected dentin (ACAD), natural caries-affected dentin (NCAD) and sound dentin (SD)., Methods: Dentin specimens from human molars were prepared and assigned into groups according to the type of dentin: ACAD, NCAD, or SD. ACAD was produced by incubation of demineralized SD with Streptococcus mutans in a chemically defined medium (CDM) with 1% sucrose for 7 days at 37 °C under anaerobic conditions. Specimens were assessed to determine collagen birefringence, biodegradability, mechanical behavior, and chemical composition. Data were individually processed and analyzed by ANOVA and post-hoc tests (α = 0.05)., Results: CDM-based biofilm challenge reduced loss, storage, and complex moduli in ACAD (p < 0.001), while the damping capacity remained unaffected (p = 0.066). Higher red and lower green birefringence were found in ACAD and NCAD when compared with SD (p < 0.001). Differently to ACAD, SD and NCAD presented higher biodegradability to exogenous proteases (p = 0.02). Chemical analysis of the integrated areas of characteristic bands that assess mineral quality (carbonate/phosphate and crystallinity index), mineral to matrix (phosphate/amide I) and post-translational modifications (amide III/CH 2 , pentosidine/CH 2 , and pentosidine/amide III) (p<0.05) showed that NCAD was significantly different from SD while ACAD exhibited intermediate values., Conclusions: CDM-based biofilm challenge produced a dentin ECM with decreased mechanical properties and increased collagen maturity. The compositional and structural conformation of the ACAD suggested that CDM-based biofilm challenge showed potential to produce artificial lesions by revealing a transitional condition towards mimicking critical features of NCAD., Clinical Significance: This study highlights the importance of developing a tissue that mimics the features of natural caries-affected dentin ECM for in vitro studies. Our findings suggested the potential of a modified biofilm challenge protocol to produce and simulate a relevant substrate, such as caries-affected dentin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

To enable translational studies, a scalable preparative isolation scheme was developed for underivatized cocoa ( Theobroma cacao ) proanthocyanidins (PACs), affording six all-B-type oligomeric PACs, including a new tetramer 4 . Their structures, including absolute configuration, were unambiguously established by comprehensive spectroscopic and chemical methods. Evaluation of the PACs' dentin biomodification properties employed dynamic mechanical and infrared spectroscopic analyses in dentin bioassay models. PAC treatment enhanced the biomechanical strength of dentin by 5- to 15-fold compared to untreated dentin. Among the PAC agents, the pentamer, cinnamtannin A3 ( 6 ), led to the highest complex modulus value of 131 MPa, whereas the "branched" tetramer, 4 , showed the lowest, yet still significant bioactivity. This study of specifically singly linked medium-length oligomeric PACs indicates that the linkage site is paramount in determining the potency of these PACs as dentin biomodifiers.

Aim: Grape seed extract contains a complex mixture of proanthocyanidins (PACs), a plant biopolymer used as a biomaterial to improve reparative and preventive dental therapies. Co-polymerization of PACs with type I collagen mechanically reinforces the dentin extracellular matrix. This study assessed the biocompatibility of PACs from grape seed extract on dental pulp stem cells (DPSCs) in a model simulating leaching through dentin to the pulp cavity. The aim was to determine the type of PACs (galloylated vs. non-galloylated) within grape seed extract that are most compatible with dental pulp tissue., Methodology: Human demineralized dentin was treated with selectively-enriched dimeric PACs prepared from grape seed extract using liquid-liquid chromatography. DPSCs were cultured within a 2D matrix and exposed to PAC-treated dentin extracellular matrix. Cell proliferation was measured using the MTS assay and expression of odontoblastic genes was analyzed by qRT-PCR. Categorization of PACs leaching from dentin was performed using HPLC-MS., Results: Enriched dimeric fractions containing galloylated PACs increased the expression of certain odontoblastic genes in DPSCs, including Runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor (VEGF), bone morphogenetic protein 2 (BMP2), basic fibroblast growth factor (FGF2), dentin sialophosphoprotein (DSPP) and collagen, type I, alpha 1 (COLI). Galloylated dimeric PACs also exhibited minor effects on DPSC proliferation, resulting in a decrease compared to control after five days of treatment. The non-galloylated dimer fraction had no effect on these genes or on DPSC proliferation., Conclusions: Galloylated PACs are biocompatible with DPSCs and may exert a beneficial effect on cells within dental pulp tissue. The observed increase in odontoblastic genes induced by galloylated PACs together with a decrease in DPSC proliferation is suggestive of a shift toward cell differentiation. This data supports the use of dimeric PACs as a safe biomaterial, with galloylated dimeric PACs exhibiting potential benefits to odontoblasts supporting dentin regeneration., Competing Interests: Declaration of Conflicting Interests The Authors declare that there is no conflict of interest.

To enable the further exploration of structure-activity relationships (SARs) of proanthocyanidins (PACs) with dentin biomodification abilities, Cinnamomum verum was selected for scaled-up purification of mixed A-/B-type, medium-size PAC oligomers. Sequential purification by centrifugal partition chromatography (CPC), Sephadex LH-20, and semiprep HPLC chromatography yielded four underivatized tetrameric ( 5 - 8 ) and two pentameric ( 9 - 10 ) PACs. Their unambiguous structural characterization involved extensive spectral and chemical degradation approaches to show that epicatechin units are connected by plant-specific combinations of doubly linked A- and singly linked B-type interflavanyl bonds. The biomechanical properties (via dynamic mechanical analysis) and physicochemical structure (via infrared spectroscopy) were assessed to evaluate the biomodification potency of PAC-treated collagen in a preclinical dentin model. This study revealed that (4→8) versus (4→6) bonds in PAC interflavan linkages have limited influence on biomechanical outcomes of dentin. By exhibiting a 25-fold increase in the complex modulus of treated dentin compared to control, aesculitannin E ( 5 ) was found to be the most potent PAC known to date for enhancing the mechanical properties of dentin in this preclinical model.

The dentin matrix is a collagenous scaffold structurally involved in anchoring resin-based materials to the tooth. Time-dependent degradation of this scaffold at the resin-dentin interface remains a core problem in adhesive dentistry, limiting the service life of dental fillings. This study explored the use of emergent materials termed metal-organic frameworks (MOFs)─formed by the self-assembly of metal ions and organic building blocks─to safeguard the collagen integrity in the functional dentin matrix. We demonstrate that collagen fibrils (from demineralized human dentin) can induce the biomimetic growth of MOF crystals as protective coatings to strengthen and stabilize the fibrils. Zeolitic imidazolate framework-8 (ZIF-8), a zinc-based microporous MOF, was used to fabricate the MOF composites via a "one-pot" reaction in water. The ZIF-modified dentin matrix presented superior mechanical strength and resistance to proteolysis, which can positively affect the longevity of collagen as an anchoring substrate. This work identifies a potential biomedical application of biomimetically synthesized MOFs in repairing dental tissues critical to restorative therapies.

Objectives: To determine the long-term effect on the stability of dentin-resin interfaces after the addition of polylactide (PLA) capsules containing proanthocyanidin (PAC) to adhesive resin., Methods: Sub-micron (SM) and micron (M) size capsules containing PACs were produced using a combination of emulsification and solvent evaporation techniques and characterized. Human dentin surfaces (n = 8) were etched (35% glycolic acid) and primed (15% enriched Vitis vinifera extract solution - VV e ), followed by the application of an experimental adhesive containing 0 (control), 1.5 wt% of SM or M PAC-filled PLA capsules light cured for 40 s. A crown was built using commercial composite. After 24 h-immersion (37 °C) in simulated body fluid, specimens were serially sectioned into resin-dentin beams. Microtensile bond strength (TBS), micro-permeability and fracture pattern were assessed immediately and after 1 and 2 years. Data were statistically analyzed using two-way ANOVA and post-hoc test (α = 0.05)., Results: Polydisperse capsules were manufactured with average diameter of 0.36 µm and 1.08 µm for SM and M, respectively. The addition of capsules did not affect TBS (p = 0.889). After 2 years, TBS significantly decreased in SM (p = 0.006), whereas M showed similar initial values (p = 0.291). Overall, less micro-permeability was found in M than the control and SM group (p < 0.001). After 2 years, fractured surfaces from capsule-containing groups failed within the adhesive layer while control fractured at the bottom of the hybrid layer., Significance: The addition of PAC-filled PLA microcapsules in a dental adhesive did not affect the bond strength while increased and sustained the protection against micro-permeability in the interface, likely due to release of PACs., (Copyright © 2021 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Aging is a physiological process with profound impact on the biology and function of biosystems, including the human dentition. While resilient, human teeth undergo wear and disease, affecting overall physical, psychological, and social human health. However, the underlying mechanisms of tooth aging remain largely unknown. Root dentin is integral to tooth function in that it anchors and dissipates mechanical load stresses of the tooth-bone system. Here, we assess the viscoelastic behavior, composition, and ultrastructure of young and old root dentin using nano-dynamic mechanical analysis, micro-Raman spectroscopy, small angle X-ray scattering, atomic force and transmission electron microscopies. We find that the root dentin overall stiffness increases with age. Unlike other mineralized tissues and even coronal dentin, however, the ability of root dentin to dissipate energy during deformation does not decay with age. Using a deconstruction method to dissect the contribution of mineral and organic matrix, we find that the damping factor of the organic matrix does deteriorate. Compositional and ultrastructural analyses revealed higher mineral-to-matrix ratio, altered enzymatic and non-enzymatic collagen cross-linking, increased collagen d-spacing and fibril diameter, and decreased abundance of proteoglycans and sulfation pattern of glycosaminoglycans . Therefore, even in the absence of remodeling, the extracellular matrix of root dentin shares traits of aging with other tissues. To explain this discrepancy, we propose that altered matrix-mineral interactions, possibly mediated by carbonate ions sequestered at the mineral interface and/or altered glycosaminoglycans counteract the deleterious effects of aging on the structural components of the extracellular matrix. STATEMENT OF SIGNIFICANCE: Globally, a quarter of the population will be over 65 years old by 2050. Because many will retain their dentition, it will become increasingly important to understand and manage how aging affects teeth. Dentin is integral to the protective, biomechanical, and regenerative features of teeth. Here, we demonstrate that older root dentin not only has altered mechanical properties, but shows characteristic shifts in mineralization, composition, and post-translational modifications of the matrix. This strongly suggests that there is a mechanistic link between mineral and matrix components to the biomechanical performance of aging dentin with implications for efforts to slow or even reverse the aging process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

Plant-derived proanthocyanidins (PACs) mediate physicochemical modifications to the dentin extracellular matrix (ECM). The structure-activity relationships of PACs remain largely unknown, mostly due to the varied complex composition of crude extracts, as well as the challenges of purification and mechanistic assessment. To assess the role of galloylated PACs as significant contributors to high yet unstable biomodification activity to the dentin ECM, we removed the galloyl moieties (de-galloylation) via enzymatic hydrolysis from three galloyl-rich PAC-containing extracts (Camellia sinensis, Vitis vinifera, and Hamamelis virginiana). The biomechanical and biological properties of dentin were assessed upon treatment with these extracts vs. their de-galloylated counterparts. An increase in the complex modulus of the dentin matrix was found with all extracts, however, the crude extract was significantly higher when compared to the de-galloylated version. Exhibiting the highest content of galloylated PACs among the investigated plants, Camellia sinensis crude extract also exhibited the biggest relapse in mechanical properties after one-month incubation. De-galloylation did not modify the damping capacity of dentin ECM. Moreover, PAC-mediated protection against proteolytic degradation was unaffected by de-galloylation. The de-galloylation experiments confirmed that gallic acid in galloylated rich-PAC extracts drive stronger yet significantly less sustained mechanical effects in dentin ECM., (© 2021 Wiley Periodicals LLC.)

Objective: To 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., Methods: Under 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)., Results: DP 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., Significance: Findings 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., (Copyright © 2021 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Purpose: To evaluate the in vitro protective effect of a mint formulation containing (-)-epigallocatechin-3-gallate (EGCg-mint) on root dentin exposed to a highly erosive environment in the presence and absence of proteolytic challenge., Methods: Root dentin specimens were subjected to an erosion-remineralization cycling model (6×/day; 5 days) that included 5-minute immersion in 1% citric acid and 60-minute immersion in remineralization solution (RS). At the remineralization half-time, the specimens were treated (n= 20) with EGCg-mint, RS (negative control) or sodium fluoride (1,000 ppm of NaF; positive control). Half of the specimens were kept overnight in RS (pH cycling) and the other half in RS with Clostridium histolyticum collagenase (pH-proteolytic cycling). Erosion depth was measured using optical profilometry and data analyzed by two-way ANOVA and Tukey tests (α= 0.05)., Results: Under pH-cycling, NaF resulted in statistically lower erosion depth compared to EGCg-mint (P= 0.020) and RS (P= 0.005). Under pH-proteolytic cycling, EGCg-mint and NaF significantly decreased the tissue loss (erosion depth, P< 0.001) compared to the RS. The EGCg-mint exhibited an anti-erosion property on root dentin under a proteolytic challenge. NaF presented an anti-erosion property regardless of the erosive cycling model., Clinical Significance: The anti-erosive action of an over-the-counter mint, containing active ingredients, including epigallocatechin-3-gallate, is likely by the protective mechanisms of the dentin extracellular matrix., Competing Interests: The authors declared no conflict of interest. This study was supported by a research contract with Dr. Heff's Products Ltd. Dr. Zamperini and Dr. Padron contributed equally to the study and publication., (Copyright©American Journal of Dentistry.)

Objectives: To test the demineralization potential, bonding performance, and dentin biostability when using hydroxy acids for etching enamel and dentin., Methods: Surface microhardness, roughness and depth of demineralization were investigated after etching enamel and dentin with 35 % glycolic acid (Gly), tartaric acid (Ta), gluconic acid (Glu), gluconolactone (Gln), or phosphoric acid (Pa) (n = 5/group). Dentin microtensile bond strength (μTBS) after 24 h or 1 year of bonding (n = 8 teeth/group) and enamel shear bond strength (SBS) after 24 h (n = 10 teeth/group) were obtained. In dentin, failure mode was classified as adhesive, cohesive in dentin/resin, or mixed. Dentin biostability was assessed by loss of dry weight and collagen degradation after 30-day incubation (n = 10 beams/group). Statistical analysis consisted of ANOVA with post-hoc Tukey's HSD, Tukey-Kramer test, Bonferroni correction, and Fisher's exact tests (α = 0.05)., Results: Gly showed better or similar results than Pa for enamel microhardness and dentin roughness, while no significant differences were observed among Ta, Glu, and Gln (p > .05). Hydroxy acids produced significantly shallower demineralization than Pa (p < .05). Gln resulted in the lowest SBS and μTBS, while Gly, Glu, Ta, and Pa showed no significant difference. There was no significant difference in μTBS between 24 h and 1 year of storage. The association between failure mode and etchant was statistically significant after 24 h only (p < .001). Hydroxy acids resulted in higher dentin biostability than Pa (p < .05)., Conclusions: Gly, Glu and Ta resulted in adequate bonding performance and reduced dentin degradation and are potential alternative etchants to improve long-term stability of adhesive restorations., Clinical Significance: This study supports the potential use of hydroxy acids as alternative etchants when bonding to enamel and dentin and demonstrates that specific acids are more suitable to be used in adhesion since they result in appropriate bond strength and less dentin degradation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The use of polymer additives that stabilize fluidic amorphous calcium phosphate is key to obtaining intrafibrillar mineralization of collagen in vitro. On the other hand, this biomimetic approach inhibits the nucleation of mineral crystals in unconfined extrafibrillar spaces, that is, extrafibrillar mineralization. The extrafibrillar mineral content is a significant feature to replicate from hard connective tissues such as bone and dentin as it contributes to the final microarchitecture and mechanical stiffness of the biomineral composite. Herein, we report a straightforward route to produce densely mineralized collagenous composites via a surface-directed process devoid of the aid of polymer additives. Simulated body fluid (1×) is employed as a biomimetic crystallizing medium, following a preloading procedure on the collagen surface to quickly generate the amorphous precursor species required to initiate matrix mineralization. This approach consistently leads to the formation of extrafibrillar bioactive minerals in bulk collagen scaffolds, which may offer an advantage in the production of osteoconductive collagen-apatite materials for tissue engineering and repair purposes.

Background: Zirconia is a relatively new dental material used for indirect dental restorations. Little is known about how dental practitioners are using this material in their practice., Methods: A survey on zirconia restorations was developed and administered electronically through e-mail communications to the American Dental Association Clinical Evaluators (ACE) Panel on August 31, 2020. Reminders were sent to nonrespondents, and the survey closed 2 weeks after the launch date., Results: When using zirconia for a restoration, respondents choose it to restore natural teeth (99%) more often than implants (76%). Almost all respondents (98%) use it for posterior crowns, whereas approximately two-thirds (61%) use it for anterior crowns. Restoration removal or replacement and shade matching and translucency were the top 2 cited disadvantages of zirconia, whereas most of the respondents (57%) cited flexural strength or fracture resistance as the biggest advantage. Fine diamonds and ceramic polishers are used most often to polish and adjust zirconia restorations, whereas coarse diamond rotary instruments and those made specifically for zirconia are most frequently used for removing these restorations. Compared with metal ceramic restorations, more than 50% of respondents experience debonding more often with zirconia restorations., Conclusions: Dentists recognize the favorable fracture resistance and flexural strength properties of zirconia, and most use similar techniques when adjusting and removing this material. Removing these restorations and shade matching are a struggle for many., Practical Implications: Dentists may benefit from tips on the best methods to remove, shade match, and adhesively bond zirconia restorations., (Copyright © 2021 American Dental Association. Published by Elsevier Inc. All rights reserved.)

The role of the host immune system in caries progression is mainly speculative, and it is believed that it entails the enzymatic degradation of the dentin organic matrix. The aim of this study was to evaluate the proteolytic effect of human neutrophil enzymes on root caries progression. For this, specimens of bovine root dentin were divided into 4 groups (n = 30): caries (C), caries + neutrophils (C + N), no caries (Control), and no caries + neutrophils (Control + N). Streptococcus mutans biofilm (105 CFU/mL) was grown on the root surface to artificially induce root carious lesions (C and C + N groups). Specimens were then exposed to neutrophils (5 × 106 cells/mL) for 48 h (C + N and Control + N groups). Caries development and neutrophil exposures were repeated a 2nd and 3rd time. Caries depth (CD) and dentin demineralization (DD) were assessed by infiltration of rhodamine B using fluorescence microscopy. Collagen fibril ultrastructure was characterized under a polarized microscope with Picrosirius red staining. There were no significant differences (p > 0.05) in CD and DD between the C and C + N groups for 1, 2, and 3 caries-neutrophil exposures. Immature collagen was significantly less present in the carious groups (C, p = 0.003; C + N, p = 0.01) than in the noncarious groups in the most superficial 200 µm. We thus concluded that human neutrophil enzymes did not influence short-term root caries progression, and immature collagen fibrils were more susceptible to degradation during S. mutans-induced root caries progression., (© 2021 S. Karger AG, Basel.)

Objectives: To develop a protocol for assessment of the bulk viscoelastic behavior of dentin extracellular matrix (ECM), and to assess relationships between induced collagen cross-linking and viscoelasticity of the dentin ECM., Methods: Dentin ECM was treated with agents to induce exogenous collagen cross-linking: proanthocyanidins (PACs) from Vitis vinifera - VVe, PACs from Pinus massoniana - PMe, glutaraldehyde - (GA), or kept untreated (control). A dynamic mechanical strain sweep method was carried out in a 3-point bending submersion clamp at treatment; after protein destabilization with 4 M urea and after 7-day, 6-month, and 12-month incubation in simulated body fluid. Tan δ, storage (E'), loss (E"), and complex moduli (E*) were calculated and data were statistically analyzed using two-way ANOVA and post-hoc tests (α = 0.05). Chemical analysis of dentin ECM before and after protein destabilization was assessed with ATR-FTIR spectroscopy., Results: Significant interactions between study factors (treatment vs. time points, p < 0.001) were found for all viscoelastic parameters. Despite a significant decrease in all moduli after destabilization, PAC-treated dentin remained statistically higher than control (p < 0.001), indicating permanent mechanical enhancement after biomodification. Covalently crosslinked, GA-treated dentin was unaffected by destabilization (p = 0.873) and showed the lowest damping capacity (tan δ) at all time points (p < 0.001). After 12 months, the damping capacity of PMe and VVe groups decreased significantly. Changes in all amide IR resonances revealed a partial chemical reversal of PAC-mediated biomodification., Significance: Viscoelastic measurements and IR spectroscopy aid in elucidating the role of inter-molecular collagen cross-linking in the mechanical behavior of dentin ECM., (Copyright © 2020 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

The present study elucidated the structures of three A-type tri- and tetrameric proanthocyanidins (PACs) isolated from Cinnamomum verum bark to the level of absolute configuration and determined their dental bioactivity using two therapeutically relevant bioassays. After selecting a PAC oligomer fraction via a biologically diverse bioassay-guided process, in tandem with centrifugal partition chromatography, phytochemical studies led to the isolation of PAC oligomers that represent the main bioactive principles of C. verum : two A-type tetrameric PACs, epicatechin-(2β→ O →7,4β→8)-epicatechin-(4β→6)-epicatechin-(2β→ O →7,4β→8)-catechin ( 1 ) and parameritannin A1 ( 2 ), together with a trimer, cinnamtannin B1 ( 3 ). Structure determination of the underivatized proanthocyanidins utilized a combination of HRESIMS, ECD, 1D/2D NMR, and 1 H iterative full spin analysis data and led to NMR-based evidence for the deduction of absolute configuration in constituent catechin and epicatechin monomeric units.

Background: Bonding crowns and bridges with resin cement can improve retention and reinforcement of the restoration. However, there is variation in the steps taken by different practitioners to achieve this goal., Methods: The authors developed a survey on bonding dental crowns and bridges with resin cement and distributed it electronically to the American Dental Association Clinical Evaluators (ACE) Panel on May 22, 2020. The survey remained open for 2 weeks. Descriptive data analysis was conducted using SAS Version 9.4., Results: A total of 326 panelists responded to the survey, and 86% of respondents who place crowns or bridges use resin cements for bonding. When placing a lithium disilicate restoration, an almost equal proportion of respondents etch it with hydrofluoric acid in their office or asked the laboratory to do it for them, and more than two-thirds use a silane primer before bonding. For zirconia restorations, 70% reported their restorations are sandblasted in the laboratory, and 39% use a primer containing 10-methacryloyloxydecyl dihydrogen phosphate. One-half of respondents clean their lithium disilicate or zirconia restorations with a cleaning solution. Resin cements used with a primer in the etch-and-rinse mode are the most widely used. The technique used to cure and clean excess resin cement varies among respondents., Conclusions: The types of resin cements used, tooth preparation, crown or bridge preparation, and bonding technique vary among this sample., Practical Implications: Although many dentists bond crowns and bridges on the basis of best practices, improvement in the process may be achieved by dentists communicating with their laboratory to confirm the steps performed there, ensuring an effective cleaning technique is used after try-in and verifying that the correct primer is used with their chosen restorative material., (Copyright © 2020 American Dental Association. Published by Elsevier Inc. All rights reserved.)

Objectives: To investigate the role of proteoglycans (PGs) on the physical properties of the dentin matrix and the bond strength of methacrylate resins with varying hydrophilicities., Methods: Dentin were obtained from crowns of human molars. Enzymatic removal of PGs followed a standard protocol using 1 mg/mL trypsin (Try) for 24 h. Controls were incubated in ammonium bicarbonate buffer. Removal of PGs was assessed by visualization of glycosaminoglycan chains (GAGs) in dentin under transmission electron microscopy (TEM). The dentin matrix swelling ratio was estimated using fully demineralized dentin. Dentin wettability was assessed on wet, dry and re-wetted dentin surfaces through water contact angle measurements. Microtensile bond strength test (TBS) was performed with experimental adhesives containing 6% HEMA (H 6 ) and 18% HEMA (H 18 ) and a commercial dental adhesive. Data were statistically analyzed using ANOVA and post-hoc tests (α = 0.05)., Results: The enzymatic removal of PGs was confirmed by the absence and fragmentation of GAGs. There was statistically significant difference between the swelling ratio of Try-treated and control dentin (p < 0.001). Significantly lower contact angle was found for Try-treated on wet and dry dentin (p < 0.002). The contact angle on re-wet dentin was not recovered in Try-treated group (p = 0.9). Removal of PGs significantly improved the TBS of H 6 (109% higher, p < 0.001) and H 18 (29% higher, p = 0.002) when compared to control. The TBS of commercial adhesive was not affected by trypsin treatment (p = 0.9)., Significance: Changing the surface energy of dentin by PGs removal improved resin adhesion, likely due to more efficient water displacement, aiding to improved resin infiltration and polymerization., (Copyright © 2020 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Plant-derived compounds incite applications virtually on every biomedical field due to the expedient antioxidant, anti-inflammatory and antimicrobial properties in conjunction with a natural character. Here, quercetin (QCT), a flavonoid with therapeutic potentials relevant to the oral environment, was encapsulated within metal-organic frameworks (MOFs) to address the concept of on-demand release of phytochemicals at the biointerface. We verified the applicability of a microporous MOF (ZIF-8) as a controlled-release system for QCT, as well as investigated the incorporation of QCT@ZIF-8 microparticles into a dental adhesive resin for desirable therapeutic capabilities at the tooth-restoration interface. QCT was encapsulated within the frameworks through a water-based, one-step synthetic process. The resulting QCT@ZIF-8 microparticles were characterized with respect to chemical composition, crystal structure, thermal behavior, micromorphology, and release profile under acidic and physiological conditions. A model dental adhesive formulation was enriched with the bioactive microparticles; both the degree of conversion (DC) of methacrylic double bonds and the polymer thermal behavior were accounted for. The results confirm that crystalline QCT@ZIF-8 microparticles with attractive loading capacities, submicron sizes, high thermal stability and responsiveness to environmental pH change were successfully manufactured. The concentration of QCT@ZIF-8 in the resin system was a key factor to maintain an optimal DC plateau and rate of polymerization. Essentially, one-step encapsulation of QCT in biocompatible ZIF-8 matrices can be easily achieved, and QCT@ZIF-8 microparticles proved as smart platforms to carry bioactive compounds with potential use to prevent microbial and enzymatic degradation of hard tissues and extracellular matrix components.

Objectives: Proanthocyanidins (PACs) are biocompounds mimicking native collagen cross-links. The effective and practical delivery of any biocompound is pivotal for clinical usage. The aim was to investigate the dentin biomodification and effective formation of dentin-resin biointerfaces of two highly bioactive PAC-rich extracts, Vitis vinifera (Vv) and Camellia sinensis (Cs), delivered using neutral (NP) or acidic (AP) rinse-out primer approaches., Methods: The depth of dentin demineralization (optical profilometry), dentin biomodification (apparent modulus of elasticity, collagen auto-fluorescence) and properties of dentin-resin interfaces (microtensile bond strength - μTBS, and micro-permeability) were investigated. NP consisted of either 15% Vv or Cs applied for 60 s after surface etching; while AP contained 15% Vv or Cs in either 35% glycolic acid or tartaric acid applied for 30 s or 60 s. Data were analyzed using ANOVA and post-hoc tests (α = 0.05)., Results: The depth of demineralization was statistically higher when applied for 60 s, regardless of rinse-out primer approach (p < 0.001). Compared to the AP strategy, NP exhibited statistically higher apparent modulus of elasticity, regardless of PAC extract (p < 0.001). Highest μTBS were obtained for NP Vv , which were statistically similar to AP GAVv , when applied for 60 s (p < 0.001); both resulted in a dramatic decrease of the interfacial permeability. NP Cs group showed the lowest μTBS (p < 0.001)., Conclusions: A combination of high bond strength and low micro-permeability can be accomplished using glycolic acid with the mid- and high-PAC oligomer enriched extract (Vv). Cs extract containing mostly catechins and dimeric PACs, was found unsuitable for resin-dentin adhesion despite exhibiting high initial dentin biomodification., Clinical Significance: This study provides a new conceptual delivery of PAC-mediated dentin biomodification and conservative dentin surface etching using rinse-out primers. The strategy requires a specific combination of PAC source, α-hydroxy acid, and application time., Competing Interests: Declaration of Competing Interest The authors report no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

An investigation of the dental bioactive proanthocyanidin (PAC) oligomer fractions led to three structurally distinct new PACs ( 1 - 3 ) from pine bark. Pinutwindoublin ( 1 ) is the first reported trimer with double A-type interflavanyl linkages (2α→O→5,4α→6 and 2α→O→7,4α→8). Pinuspirotetrin ( 2 ) represents the first reported PAC tetramer with a heterodimeric framework consisting of one spiro-type and one A-type dimer. Pinumassohexin ( 3 ) was elucidated as a mixed A + B-type hexamer that consists of a peanut-derived tetramer, peanut procyanidin E, and an A-type dimer ( 5 ). Compound 3 increased the modulus of elasticity of dentin by an impressive 4.3 times at a concentration of 0.65%.

Guided by dentin biomechanical bioactivity, this phytochemical study led to the elucidation of an extended set of structurally demanding proanthocyanidins (PACs). Unambiguous structure determination involved detailed spectroscopic and chemical characterization of four A-type dimers ( 2 and 4-6 ), seven trimers ( 10-16 ), and six tetramers ( 17-22 ). New outcomes confirm the feasibility of determining the absolute configuration of the catechol monomers in oligomeric PACs by one-dimensional (1D) and two-dimensional (2D) NMR. Electronic circular dichroism as well as phloroglucinolysis followed by mass spectrometry and chiral phase high-performance liquid chromatography (HPLC) analysis generated the necessary chiral reference data. In the context of previously reported dentin-bioactive PACs, accurately and precisely assigned 13 C NMR resonances enabled absolute stereochemical assignments of PAC monomers via (i) inclusion of the 13 C NMR γ-gauche effect and (ii) determination of differential 13 C chemical shift values (Δδ C ) in comparison with those of the terminal monomer (unit II) in the dimers 2 and 4-6 . Among the 13 fully elucidated PACs, eight were identified as new, and one structure ( 11 ) was revised based on new knowledge gained regarding the subtle, stereospecific spectroscopic properties of PACs.

Background: The ability to polymerize light-activated dental materials with dental light-curing units (DLCUs) has revolutionized dentistry. However, proper DLCU use is essential for ensuring the effectiveness and performance of these materials., Methods: The authors developed an electronic cross-sectional survey in the American Dental Association Qualtrics Research Core platform. The survey included questions about DLCU use, unit type and selection, training, maintenance, technique, and safety measures. The authors deployed the survey to 809 American Dental Association Clinical Evaluators (ACE) panelists on October 9, 2019, and sent reminder links to nonrespondents 1 week later. They conducted exploratory and descriptive analyses using SAS software Version 9.4., Results: Of the 353 ACE panelists who completed the survey, most used a DLCU in their practices (99%), and light-emitting diode multiwave units were the most common type of DLCU units (55%). Dentists use DLCUs for over one-half of their appointments each day (mean [standard deviation], 59% [22%]). Regarding technique, respondents reported that they modify their curing technique on the basis of material thickness (79%) and material type or light tip-to-target distances (59%). Maintenance practices varied, with two-thirds of respondents reporting that they periodically check their DLCUs' light output., Conclusions: DLCUs are an integral part of a general dentist's daily practice, but maintenance, ocular safety, and technique varied widely among this sample., Practical Implications: Because clinical effectiveness requires delivery of an adequate amount of light energy at the appropriate wavelength, variation in DLCU maintenance, safety, and techniques suggest that dentists could benefit from additional guidance and training on DLCU operation., (Copyright © 2020 American Dental Association. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to evaluate the effects of glycolic acid (GA) (with pH 1.2 and 5) and ethylenediaminetetraacetic acid (EDTA) on the chemical and mechanical properties of dentin to investigate the potential use of GA as final irrigant in the root canal therapy. Specifically, changes in microhardness, smear layer removal, erosion, mineral content distribution, apatite/collagen ratio and flexural strength of mineralized dentin treated with GA were assessed. Saline solution was used as a negative control. Knoop microhardness (KHN) was measured on the root canal lumen of root segments. Dentin beams were used for 3-point flexural strength (σ) test. Scanning electron microscopy (SEM) images of root sections were obtained for evaluation of smear layer removal and dentin erosion on root segments and energy dispersive X-ray spectroscopy (EDS) was used for mineral content distribution. The apatite/collagen ratio (A/C) in dentin powder were examined by Fourier transform infrared (FTIR) spectroscopy. KHN, σ and A/C results were statistically analyzed with ANOVA and Tukey tests (α = 0.05). Smear layer and dentin erosion scores were analyzed with Kruskal-Wallis and Dunn tests (α = 0.05). Root dentin treated with EDTA and GA presented similar KHN regardless of the pH (p > 0.05). However, KHN was significantly reduced in EDTA and GA groups when compared to control group (p<0.001). GA showed the same ability to remove the smear layer and to cause dentin erosion as EDTA. EDS results showed that the GA and EDTA solutions did not alter the dentin mineral content distribution. The apatite/collagen ratio reduced with all irrigant solution and was the lowest with GA pH 5 (p<0.001), while σ was not significantly affected by the experimental solutions (p = 0.559). It can be concluded that GA has similar ability to remove the smear layer than EDTA. GA does not affect negatively the chemical/mechanical properties and it does not increase dentin erosion. The use of GA with low pH seems to promote less change in collagen/apatite ratio, but further studies are needed to establish an ideal clinical protocol. Therefore, this study supports the potential use of GA as an alternative final irrigation solution for root canal preparation.

Purpose: To evaluate the effect of dentin biomodification on the long-term strength of sound and caries-affected (CA) dentin as a strategy to stabilize the dentin matrix. The biomodification strategy utilized a naturally occurring proanthocyanidin-rich Vitis vinifera grape seeds (Vv), and compared with glutaraldehyde (GD)., Methods: Dentin from sound and carious human molars were sectioned from mid-coronal dentin. The temperature denaturation (Td) was assessed using differential scanning calorimetry in sealed pans. The inhibitory effect of the agents on the activity of recombinant MMP-2 and -9 were assessed using colorimetric assay. The ultimate tensile strength (UTS) of demineralized dentin were determined 24 hours after treatment and after 12 months storage in simulated body fluid. Data were statistically analyzed using ANOVA and post-hoc tests ( α= 0.05)., Results: There was no statistically significant difference in the Td between sound and CA dentin (P= 0.140); however, Vv and GD significantly increased the Td of both substrates (P< 0.001), indicating formation of collagen cross-linking. The activity of MMP-2 and MMP-9 were reduced by Vv and GD in a concentration dependent manner. The UTS of dentin matrix was significantly affected by treatments and storage times (P< 0.001). After a 12-month period, a significant decrease in UTS was observed for sound and CA, with complete solubilization of the CA dentin matrix. Vv and GD stabilized the UTS of both dentin substrates (P< 0.05). Sound and CA dentin matrix were susceptible to degradation after the 12-month period. Degradation of dentin matrix due to endogenous proteases activity was more pronounced in CA dentin. Dentin biomodification strategies increased the thermal stability and enhanced the long-term mechanical properties of both sound and CA dentin matrix., Clinical Significance: Carious dentin matrix is more susceptible to breakdown over time than sound dentin; however, the degradation process can be impaired by dentin biomodification. This biomimetic strategy increases the long-term tensile strength of the dentin matrix. Reinforcement of caries-affected dentin may increase longevity of adhesive interfaces., Competing Interests: The authors declared no conflict of interest. This work was supported by the National Institutes of Health (DE021040)., (Copyright©American Journal of Dentistry.)

Background: Human papillomavirus (HPV) is a sexually transmitted oncovirus associated with several malignancies, including oropharyngeal squamous cell carcinoma. The 9-valent HPV vaccine can help protect against the high-risk HPV strains most commonly associated with HPV-related cancers., Methods: The authors used an electronic survey to assess the roles of dentists and their team members in discussing the HPV vaccine, as well as administering the vaccine in a dental setting. On December 6, 2019, the authors e-mailed a survey link to the American Dental Association Clinical Evaluators (ACE) Panel (n = 813), a sample of American Dental Association member dentists. After 1 e-mail reminder, the survey closed on December 19, 2019, and the authors conducted exploratory and descriptive data analyses using SAS Version 9.4 (SAS)., Results: A total of 329 dentists responded to the survey, and 83 (25%) of them reported that they or their team members discuss the implications of the HPV vaccine with age-eligible patients or their parents or guardians. Dentists lead two-thirds (n = 218) of the discussions, and the clinical examination is the most frequent moment during the patient visit in which HPV-related topics are discussed. Some of the top reasons respondents mentioned for not discussing the vaccine in their practice were the perception that these discussions are best left to other health care professionals and not knowing how to address the topic with patients. If the scope of dental practice is expanded to include administering the vaccine, 125 (38%) of respondents would feel uncomfortable administering the vaccine. The most common potential barriers to administering the vaccine in a dental setting include obtaining reimbursement and vaccine management and preservation., Conclusions: The survey results suggest that dentists' comfort levels and perceived roles in discussing and administering the HPV vaccine vary., Practical Implications: There is a need to further define the role of dentists and their team members in the promotion and administration of the HPV vaccine. Resources for dentists and dental team members may be helpful to support professional education and communication about the HPV vaccine., (Copyright © 2020 American Dental Association. Published by Elsevier Inc. All rights reserved.)

This study investigated the contribution of small leucine rich proteoglycans (SLRPs) to the fracture toughness of the dentin extracellular matrix (ECM) by enzymatically-assisted selective removal of glycosaminoglycan chains (GAGs) and proteoglycans (PGs) core protein. We adapted the Mode III trouser tear test to evaluate the energy required to tear the dentin ECM. Trouser-shaped dentin specimens from crown and root were demineralized. Depletion of GAGs and PGs followed enzymatic digestion using chondroitinase ABC (c-ABC) and matrix metalloproteinase 3 (MMP-3), respectively. The legs from specimen were stretched under tensile force and the load at tear propagation was determined to calculate the tear energy (T, kJ/m 2 ). SLRPs decorin and biglycan were visualized by immunohistochemistry and ECM tear pattern was analyzed in SEM. Results showed T of crown ECM was not affected by PGs/GAGs depletion (p = 0.799), whereas the removal of PGs significantly reduced T in root dentin ECM (p = 0.001). Root dentin ECM exhibited higher T than crown (p < 0.03), however no regional difference are present after PG depletion (p = 0.480). Immunohistochemistry confirmed removal of GAGs and PGs. SEM images showed structural modifications after PGs/GAGs removal such as enlargement of dentinal tubules, increased interfibrillar spaces and presence of untwisted fibrils with increased diameter. Findings indicate that the capacity of the PGs to unfold and untwist contribute to the dentin ECM resistance to tear, possibly influencing crack growth propagation. The regional differences are likely an evolutionary design to increase tooth survival, that undergoes repetitive mechanical loading and load stress dissipation over a lifetime of an individual., Competing Interests: Declaration of Competing Interest The authors declared that there is no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Collagen glycation takes place under physiological conditions during chronological aging, leading to the formation of advanced glycation end-products (AGEs). AGEs accumulation induces non-enzymatic collagen cross-links increasing tissue stiffness and impairing function. Here, we focused on determining the cumulative effect of induced glycation on the mechanical behavior of highly collagen cross-linked dentin matrices and assess the topical inhibition potential of aminoguanidine. Bulk mechanical characterization suggests that early glycation cross-links significantly increase the tensile strength and stiffness of the dentin matrix and promote a brittle failure response. Histologically, glycation yielded a more mature type I collagen in a densely packed collagen matrix. The time-dependent effect of glycation indicates cumulative damage of dentin matrices that is partially inhibited by aminoguanidine. The regional dentin sites were differently affected by induced-glycation, revealing the crown dentin to be mechanically more affected by the glycation protocol. These findings in human dentin set the foundation for the proposed in vitro ribose-induced glycation model, which produces an early matrix stiffening mechanism by reducing tissue viscoelasticity and can be partially inhibited by topical aminoguanidine., Competing Interests: Declaration of competing interest None., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A dentin biomodification strategy with selective proanthocyanidin (PAC)-enriched extracts reinforces dentin and dentin-resin interfaces. Enrichment of the extracts according to the degree of polymerization allows exploration of bioactive principles of PACs and structure-activity relationships. This study investigated the sustained dentin matrix biomodification and dentin-resin bioadhesion of 2 fractions consisting exclusively of B-type PAC dimers with or without a single galloyl motif (specifically, DIMER G and DIMER NG ) and their precursor material, enriched grape seed extract (e-GSE; Vitis vinifera ). The biomodification potential was determined by long-term evaluation of the apparent modulus of elasticity and collagen solubility (hydroxyproline release). Chemical characterization of the dentin matrix was performed by attenuated total reflectance-Fourier-transform infrared spectroscopy. The bioadhesive properties were assessed by a microtensile bond strength test at different time points, and macro-hybrid layers were produced to verify the degree of conversion of the adhesive resin. Fractions consisting of DIMER G , DIMER NG , and their precursor, e-GSE, increased the modulus of elasticity at all time points and reduced collagen degradation. Specimens treated with DIMER NG remained stable throughout 12 mo of storage, whereas a significant drop in the modulus of elasticity was observed for the DIMER G and e-GSE groups at 6 mo. The fractions and precursor did not affect the degree of resin conversion at the hybrid layer. Changes in infrared resonances corresponding to collagen cross-links in the dentin matrix occurred for all treatments. Higher bond strength was observed for dentin treated with e-GSE as compared with DIMER G and DIMER NG ; all biointerfaces remained stable after 12 mo. Nongalloylated PACs mediate stable dentin biomodification, which includes protective activity against collagen degradation and reinforcement of the anchoring dentin matrix. Collectively, PACs with a higher degree of oligomerization offer a robust bioadhesion between the hydrophilic dentin matrix and the hydrophobic adhesive.

Purpose: To investigate whether interfacial enzymatic activity and adhesion receptiveness of artificial caries-affected dentin (ACAD) simulate those of natural caries-affected dentin (NCAD)., Materials and Methods: Thirty dentin specimens were prepared from human molars to determine interfacial gelatinolytic activity using in situ zymography and adhesion experiments (micropermeability and bond strength [µTBS]). Groups were formed according to the type of dentin: artificial caries-affected dentin (ACAD), natural caries-affected dentin (NCAD), or sound dentin. ACAD was produced by incubating dentin with Streptococcus mutans in a chemically defined medium (CDM) with 1% sucrose for 7 days at 37°C under anaerobic conditions. CDM was replaced daily, and the sterility as well as the pH of the culture was monitored. Adhesion experiments employed Single Bond Universal (3M Oral Care) in self-etch mode. Data were individually processed and analyzed using ANOVA and post-hoc tests (α = 0.05)., Results: The enzymatic activity of ACAD was similar to that of sound dentin, but was lower than that of NCAD, which elicited the highest activity (p < 0.05). Interfacial micropermeability intensity at the hybrid layer or in underlying dentin (5 µm below the interface) was similar in all types of dentin (p > 0.05). On the other hand, substrate permeability was higher for NCAD than for ACAD. The highest sealing ability was detected in sound dentin. Bond strengths to ACAD were higher than to NCAD. However, the highest µTBS was observed in sound dentin (p < 0.05)., Conclusion: Artificial caries-affected dentin simulated neither the gelatinolytic activity nor bonding receptiveness of natural caries-affected dentin.

Purpose: To investigate the long-term effect of 0.05% or 0.1% caffeic acid phenethyl ester (CAPE) on dentin matrix stability and hybrid layer stability, using an etch-and-rinse (Adper Scotchbond Multipurpose/ASB) or a self-etch adhesive (Clearfil SE Bond/CSE)., Materials and Methods: Dentin matrix specimens were assigned to five groups: 0.05% or 0.1% CAPE, green tea (GT), and the controls distilled water (DW) and dimethyl sulfoxide (DMSO). Following immersion of specimens for 1 h, modulus of elasticity (ME) and dentin mass change (MG) were determined at 3 post-treatment time points: immediately afterwards and at 3 and 6 months. Collagen solubilization (CS) was estimated by hydroxyproline (HYP) quantification. Resin-dentin interfaces with both adhesives were assessed with in situ zymography tests to evaluate gelatinolytic activity (GA). The dentin pretreatments were actively applied for 60 s. The sealing ability of aged resin-bonded slices was assessed by nanoleakage tests., Results: GT increased immediate ME, which decreased significantly after 3 months (p < 0.0001). The CAPE groups did not differ from the control groups. GT provided a significant increase in dentin matrix mass after treatment (p < 0.0001). No significant differences regarding MG were observed for CAPE 0.1%, CAPE 0.05%, DW, and DMSO groups after 3 and 6 months. Cumulative HYP release revealed that CAPE groups and GT were statistically similar to DW and DMSO; the GT group exhibited statistically significantly less HYP release than did CAPE groups (p = 0.0073). Treatment with 0.05% or 0.1% CAPE presented lower GA when applied to ASB before acid conditioning (p < 0.05), but no differences were detected when the CAPE groups were applied to CSE. CAPE at 0.1% significantly reduced nanoleakage for CSE, and 0.05% CAPE with CSE presented levels of nanoleakage similar to those of the CSE control group., Conclusion: CAPE at 0.05% or 0.01% did not influence ME, MG, or CS, but reduced GA when applied to ASB before acid conditioning. CAPE at 0.1% with CSE promoted adhesive layer integrity.

Objective: This study investigated the effects of dentin pretreatment with 2.5% titanium tetrafluoride (TiF 4 ) on nanomechanical properties, and the in situ gelatinolytic activity of the dentin-resin interface, for up to 6 months., Methods: Twenty-four human teeth were prepared by exposing occlusal flat dentin surfaces, and were randomly assigned to experimental groups, according to application or non-application of a TiF 4 pretreatment, and to the adhesive systems (Clearfil SE Bond or Scotchbond Universal). Resin composite (Filtek Supreme Ultra) was built up incrementally on the teeth in all the groups. Then, the specimens were sectioned and randomly selected for evaluation at 24h, 3 months and 6 months of storage time. The reduced modulus of elasticity (E r ) and the nanohardness of the underlying dentin, as well as the hybrid layer and the adhesive layer were measured using a nanoindenter. Gelatinolytic activity at the dentin-resin interfaces was assessed by in situ zymography using quenched fluorescein-conjugated gelatin at 24h and 6 months. Statistical analyses were performed with ANOVA and Tukey's tests., Results: There were no differences in E r and nanohardness values between adhesives systems and pretreatment (p=0.1250). In situ zymography showed significantly higher gelatinolytic activity after 6 months for all the experimental groups (p=0.0004), but no differences between the adhesive systems (p=0.7708) and the surface pretreatment (p=0.4877)., Significance: Dentin pretreatment with 2.5% TiF 4 followed by self-etching adhesive systems did not influence nanomechanical properties or gelatinolytic activity of the adhesive-dentin interface layers, over time., (Copyright © 2019 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Aimed at exploring the dentin biomodification potential of proanthocyanidins (PACs) for the development of dental biomaterials, this study reports the phytochemical and dental evaluation of nine B-type PACs from grape seed extract (GSE). Out of seven isolated dimers ( 1 - 7 ), four new compounds ( 2 , 3 , 5 , and 6 ) involved relatively rare ent -catechin or ent -epicatechin monomeric flavan-3-ol units. Low-temperature NMR analyses conducted along with phloroglucinolysis and electronic circular dichroism enabled unequivocal structural characterization and stereochemical assignment. Additionally, one known ( 8 ) and one new ( 9 ) B-type trimer were characterized. Differential 13 C NMR chemical shifts (Δδ) were used to determine the absolute configuration of 9 , relative to the dimers 1 and 2 as the possible constituent subunits. Compared to the dimers, the trimers showed superior dentin biomodification properties. The dimers, 1 - 7 , exhibited pronounced differences in their collagenase inhibitory activity, while enhancing dentin stiffness comparably. This suggests that PAC structural features such as the degree of polymerization, relative and absolute configuration have a differential influence on enhancement of dentin biomechanical and biostability. As mechanical enhancement to dentin and resistance to proteolytic biodegradation are both essential properties functional and stable dentin substrate, the structurally closely related PACs suggest a new metric, the dentin biomodification potential (DBMP) that may rationalize both properties.