Chemical Transformation of B- to A-type Proanthocyanidins and 3D Structural Implications.

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.

Endothelial cell invasion is controlled by dactylopodia.

Sprouting angiogenesis is fundamental for development and contributes to cancer, diabetic retinopathy, and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on how tip cells invade into tissues. Here, we show that endothelial tip cells use dactylopodia as the main cellular protrusion for invasion into nonvascular extracellular matrix. We show that dactylopodia and filopodia protrusions are balanced by myosin IIA (NMIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of NMIIA promotes excessive dactylopodia formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents dactylopodia development and leads to excessive filopodia formation. We further show that NMIIA inhibits Rac1-dependent activation of Arp2/3 by regulating the maturation state of focal adhesions. Our discoveries establish a comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way toward strategies to block invasive tip cells during sprouting angiogenesis.

Unprecedented Benzoquinone Motifs Reveal Post-Oligomerizational Modification of Proanthocyanidins.

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 1H 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.

Immediate and Sustained Root Caries Prevention of Fluoride Varnish Combined with Toothpastes.

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 < 0.05) in the RI (up to 180 µm and 120 µm, respectively) as well as higher mineral density compared to No treatment (p < 0.001). V + PO + PP and/or V + PO resulted in statistically lower RI compared to V for some depths (p < 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.

[Prevalence and geographical distribution of bovine sexually transmitted diseases in the province of Formosa, Argentina]. / Prevalencia y distribución geográfica de las enfermedades de transmisión sexual de los bovinos en la provincia de Formosa, Argentina.

Bovine genital campylobacteriosis (BGC) and bovine trichomonosis (BT) are sexually transmitted diseases (STDs) that affect bovine breeding herds, decreasing their reproductive efficiency. The objective of this work was to estimate the prevalence of these diseases and their temporal-spatial distribution in the province of Formosa, Argentina. The cross-sectional study conducted between 2018 and 2021 included a total of 15,571 bulls, inter-herd prevalence being 29.62% and 17.23% for BGC and BT, respectively. The prevalence of positive animals was 2.05% for BGC and 0.43% for BT. The temporal-spatial analysis of BGC showed two distinct spatial groupings, one group had a low risk of contracting the disease (RR = 0.13; p < 0.001; 2018-2021) while the other group had a high risk (RR = 2.84; p < 0.001; 2020-2021). BT had a high-risk group for the disease (RR = 35.24; p < 0.001; 2019). This study shows that STDs are endemic in the region, providing updated and valuable information as a tool for the health management of these diseases.

Biomimetic Growth of Metal-Organic Frameworks for the Stabilization of the Dentin Matrix and Control of Collagenolysis.

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.

Proanthocyanidin Tetramers and Pentamers from Cinnamomum verum Bark and Their Dentin Biomodification Bioactivities.

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.

Seco B-Type Oligomers from Pinus massoniana Expand the Procyanidin Chemical Space and Exhibit Dental Bioactivity.

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. ΔδC) 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.

Heat-related mortality amplified during the COVID-19 pandemic.

Excess mortality not directly related to the virus has been shown to have increased during the COVID-19 pandemic. However, changes in heat-related mortality during the pandemic have not been addressed in detail. Here, we performed an observational study crossing daily mortality data collected in Portugal (SICO/DGS) with high-resolution temperature series (ERA5/ECMWF), characterizing their relation in the pre-pandemic, and how it aggravated during 2020. The combined result of COVID-19 and extreme temperatures caused the largest annual mortality burden in recent decades (~ 12 000 excess deaths [~ 11% above baseline]). COVID-19 caused the largest fraction of excess mortality during March to May (62%) and from October onwards (85%). During summer, its direct impact was residual, and deaths not reported as COVID-19 dominated excess mortality (553 versus 3 968). A prolonged hot spell led mortality to the upper tertile, reaching its peak in mid-July (+ 45% deaths/day). The lethality ratio (+ 14 deaths per cumulated ºC) was higher than that observed in recent heatwaves. We used a statistical model to estimate expected deaths due to cold/heat, indicating an amplification of at least 50% in heat-related deaths during 2020 compared to pre-pandemic years. Our findings suggest mortality during 2020 has been indirectly amplified by the COVID-19 pandemic, due to the disruption of healthcare systems and fear of population in attending healthcare facilities (expressed in emergency room admissions decreases). While lockdown measures and healthcare systems reorganization prevented deaths directly related to the virus, a significant burden due to other causes represents a strong secondary impact. This was particularly relevant during summer hot spells, when the lethality ratio reached magnitudes not experienced since the 2003 heatwaves. This severe amplification of heat-related mortality during 2020 stresses the need to resume normal healthcare services and public health awareness.

The Dynamics of Patient Visits to a Public Hospital Pediatric Emergency Department: A Time-Series Model.

BACKGROUND: The overcrowding of emergency departments (EDs) is an increasingly relevant public health problem. The main aims of this study were to identify and analyze temporal periodicities of a self-referred pediatric ED (PED), correlate them with meteorological and calendar variables and build a robust forecasting model. METHODS: An 8-year administrative data set (2010-2017) of the daily number of admissions to the PED of a public hospital in Lisbon, Portugal, was used (n = 670,379). A time-series model of the daily number of visits was built, including temporal periodicities, the Portuguese school calendar, and a meteorological comfort index (humidex). RESULTS: Several temporal cycles were identified: 1 year (peak in January/February related to respiratory infections in younger children and infants), 6 months (peaks in May and October with an increase in the admissions of older children and adolescents with trauma, gastrointestinal infections and atopic symptoms), 4 months (related to annual school vacations), 1 week (lower admission values on Saturday), and half a week (low from Friday to Monday morning). School calendar and humidex were significantly correlated with daily admissions. The model yielded a mean absolute percentage error of 10.7% ± 1.10% when cross-validation was performed for the full data set. CONCLUSION: Although PED visits are multifactorial, they may be predicted and explained by a relatively small number of variables. Such a model may be easily reproduced in different settings and represents a relevant tool to improve quality in EDs through correctly adapting human resources to ED demand.

Galloylated proanthocyanidins in dentin matrix exhibit biocompatibility and induce differentiation in dental stem cells.

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.

In vitro Study of the Role of Human Neutrophil Enzymes on Root Caries Progression.

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.

In vitro anti-erosive property of a mint containing bioactive ingredients.

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.

Tri- and Tetrameric Proanthocyanidins with Dentin Bioactivities from Pinus massoniana.

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 13C NMR resonances enabled absolute stereochemical assignments of PAC monomers via (i) inclusion of the 13C NMR γ-gauche effect and (ii) determination of differential 13C chemical shift values (ΔδC) 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.

Targeting Trimeric and Tetrameric Proanthocyanidins of Cinnamomum verum Bark as Bioactives for Dental Therapies.

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-(2ß→O→7,4ß→8)-epicatechin-(4ß→6)-epicatechin-(2ß→O→7,4ß→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 1H iterative full spin analysis data and led to NMR-based evidence for the deduction of absolute configuration in constituent catechin and epicatechin monomeric units.

Heat-related mortality at the beginning of the twenty-first century in Rio de Janeiro, Brazil.

Temperature record-breaking events, such as the observed more intense, longer-lasting, and more frequent heat waves, pose a new global challenge to health sectors worldwide. These threats are of particular interest in low-income regions with limited investments in public health and a growing urban population, such as Brazil. Here, we apply a comprehensive interdisciplinary climate-health approach, including meteorological data and a daily mortality record from the Brazilian Health System from 2000 to 2015, covering 21 cities over the Metropolitan Region of Rio de Janeiro. The percentage of absolute mortality increase due to summer extreme temperatures is estimated using a negative binomial regression modeling approach and maximum/minimum temperature-derived indexes as covariates. Moreover, this study assesses the vulnerability to thermal stress for different age groups and both genders and thoroughly analyzes four extremely intense heat waves during 2010 and 2012 regarding their impacts on the population. Results showed that the highest absolute mortality values during heat-related events were linked to circulatory illnesses. However, the highest excess of mortality was related to diabetes, particularly for women within the elderly age groups. Moreover, results indicate that accumulated heat stress conditions during consecutive days preferentially preceded by persistent periods of moderate-temperature, lead to higher excess mortality rather than sporadic single hot days. This work may provide directions in human health policies related to extreme climate events in large tropical metropolitan areas from developing countries, contributing to altering the historically based purely reactive response.

Biomimetic strategy to stabilize the mechanical properties of caries-affected dentin matrix: A 12-month in vitro study.

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.

Proanthocyanidin Dimers and Trimers from Vitis vinifera Provide Diverse Structural Motifs for the Evaluation of Dentin Biomodification.

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 13C 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.

Structural and biomechanical changes to dentin extracellular matrix following chemical removal of proteoglycans.

Proteoglycans are biomacromolecules with significant biomineralization and structural roles in the dentin extracellular matrix. This study comprehensively assessed the mechanical properties and morphology of the dentin extracellular matrix following chemical removal of proteoglycans to elucidate the structural roles of proteoglycans in dentin. Dentin extracellular matrix was prepared from extracted teeth after complete tissue demineralization. Chemical removal of proteoglycans was carried-out using guanidine hydrochloride for up to 10 days. The removal of proteoglycans was determined by dimethylmethylene blue colorimetric assay and histological staining analyses using transmission electron microscopy and optical microscopy. The modulus of elasticity of dentin matrix was determined by a 3-point bending test method. Partial removal of proteoglycans induced significant modifications to the dentin matrix, particularly to type I collagen. Removal of proteoglycans significantly decreased the modulus of elasticity of dentin extracellular matrix (p < 0.0001). In conclusion, the subtle disruption of proteoglycans induces pronounced changes to the collagen network packing and the bulk modulus of elasticity of dentin matrix.

Remineralization Potential of Mints Containing Bioactive Agents in Artificially Induced Root Caries.

This study investigated the remineralization effect of experimental mint formulations containing bioactive agents (xylitol; green tea extract, GT; and amorphous calcium phosphate, ACP) in the progression of artificially induced root caries. Root caries lesions were induced by demineralization solution (pH 4.6; 96 h; 37°C). The lesions were treated with mint A, mint B, mint C, xylitol, GT, ACP, or remineralization solution (RS; negative control). Specimens were pH-cycled through treatments (5×/day; 3 min) and 6 cycles of acidic (pH 5.0; 30 min) and neutral (pH 7.0; 10 min) buffers for 8 days. Bacterial collagenase (Clostridium histolyticum) was used overnight to simulate proteolytic challenge. Caries depth and porosity as well as mineral density were estimated using fluorescence microscopy (n = 15) and microcomputed tomography (n = 6). Analysis of variance (ANOVA, α = 0.05) showed no statistically significant difference in caries depth among all groups (p = 0.172). The highest fluorescence intensity decrease was observed for GT followed by mint C, with no significant difference between them (p = 0.868). There were significant differences among GT and mints A, B, and C when compared to RS (p < 0.001). No statistically significant differences in fluorescence intensity were observed among ACP, xylitol, and RS (p > 0.05). The mineral density of the lesions in GT, mints A, B, and C, and ACP was statistically similar (p > 0.05) and significantly higher than that in RS (p < 0.05). No significant difference was observed between xylitol and RS (p = 0.728). The experimental mints showed remineralization action on artificial root caries, and GT was found to be the main active ingredient in the investigated formulations.