Prodelphinidins enhance dentin matrix properties and promote adhesion to methacrylate resin.

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.

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.

Paradoxical effects of galloyl motifs in the interactions of proanthocyanidins with collagen-rich dentin.

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.

The stoic tooth root: how the mineral and extracellular matrix counterbalance to keep aged dentin stable.

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.

Unveiling structure-activity relationships of proanthocyanidins with dentin collagen.

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 (4ß â†’ 6 and 4ß â†’ 8); and (iii) the terminal monomeric flavan-3-ol unit plays a modulatory role in the viscoelasticity of dentin.

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.

Bonding crowns and bridges with resin cement: An American Dental Association Clinical Evaluators Panel survey.

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.

Removal of water binding proteins from dentin increases the adhesion strength of low-hydrophilicity dental resins.

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 (H6) and 18% HEMA (H18) 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 H6 (109% higher, p < 0.001) and H18 (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.

Long-term Evaluation of Dentin Matrix Stability and Gelatinolytic Activity after Dentin Pretreatment with Caffeic Acid Phenethyl Ester.

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.

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.

Long-term nanomechanical properties and gelatinolytic activity of titanium tetrafluoride-treated adhesive dentin interface.

OBJECTIVE: This study investigated the effects of dentin pretreatment with 2.5% titanium tetrafluoride (TiF4) 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 TiF4 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 (Er) 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 Er 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% TiF4 followed by self-etching adhesive systems did not influence nanomechanical properties or gelatinolytic activity of the adhesive-dentin interface layers, over time.

Evidence to the role of interflavan linkages and galloylation of proanthocyanidins at sustaining long-term dentin biomodification.

OBJECTIVES: The interactivity of proanthocyanidins (PACs) with collagen modulates dentin matrix biomechanics and biostability. Herein, PAC extracts selected based on structural diversity were investigated to determine key PAC features driving sustained effects on dentin matrices over a period of 18months. METHODS: The chemical profiles of PAC-rich plant sources, Pinus massoniana (PM), Cinnamomum verum (CV) and Hamamelis virginiana (HV) barks, as well as Vitis vinifera (VV) seeds, were obtained by diol HPLC analysis after partitioning of the extracts between methyl acetate and water. Dentin matrices (n=15) were prepared from human molars to determine the apparent modulus of elasticity over 18months of aging. Susceptibility of the dentin matrix to degradation by endogenous and exogenous proteases was determined by presence of solubilized collagen in supernatant, and resistance to degradation by bacterial collagenase, respectively. Data were analyzed using ANOVA and Games-Howell post hoc tests (α=0.05). RESULTS: After 18months, dentin matrices modified by PM and CV extracts, containing only non-galloylated PACs, were highly stable mechanically (p<0.05). Dentin matrices treated with CV exhibited the lowest degradation by bacterial collagenase after 1h and 18months of aging (p<0.05), while dentin matrices treated with PM showed the least mass loss and collagen solubilization by endogenous enzymes over time (p<0.05). SIGNIFICANCE: Resistance against long-term degradation was observed for all experimental groups; however, the most potent and long-lasting dentin biomodification resulted from non-galloylated PACs.

An Overview of Dental Adhesive Systems and the Dynamic Tooth-Adhesive Interface.

From the conception of resin-enamel adhesion to today's contemporary dental adhesive systems, clinicians are no longer afraid of exploring the many advantages brought by adhesive restorative concepts. To maximize the performance of adhesive-based restorative procedures, practitioners must be familiar with the mechanism of adhesion, clinical indications, proper handling, the inherent limitations of the materials and the biological challenges. This review provides an overview of the current status of restorative dental adhesives, their mechanism of adhesion, mechanisms of degradation of dental adhesive interfaces, how to maximize performance, and future trends in adhesive dentistry.

Role of proteoglycans on the biochemical and biomechanical properties of dentin organic matrix.

OBJECTIVE: Proteoglycans (PGs) are multifunctional biomacromolecules of the extracellular matrix of collagen-based tissues. In teeth, besides a pivotal regulatory role on dentin biomineralization, PGs provide mechanical support to the mineralized tissue and compressive strength to the biosystem. This study assessed enzymatic protocols for selective PGs removal from demineralized dentin to determine the roles of these biomacromolecules in the bulk mechanical properties and biostability of type I collagen. METHODS: Selective removal of glycosaminoglycans chains (GAGs) and PGs from demineralized dentin was carried out by enzymatic digestion protocols using chondroitinase ABC (c-ABC) and trypsin (Try). A comprehensive study design included assessment of dentin matrix mass loss, biodegradability of the PGs/GAGs-depleted dentin matrix, ultimate tensile strength (UTS) and energy to fracture tests. Quantitative data was statistically analyzed by two-way and one-way ANOVA followed by the appropriate post hoc tests (α=0.05). RESULTS: Transmission electron microscopy images show effective GAGs removal by c-ABC and Try and both enzymatic methods released statistically similar amounts of GAGs from the demineralized dentin. Try digestion resulted in about 25% dentin matrix mass loss and increased susceptibility to collagenolytic digestion when compared to c-ABC (p=0.0224) and control (p=0.0901). Moreover, PGs digestion by Try decreased the tensile strengths of dentin. Statistically lower energy to fracture was observed in c-ABC-treated dentin matrix. CONCLUSIONS: GAGs plays a pivotal role on tissue mechanics and anisotropy, while the core protein of PGs have a protective role on matrix biostability.

Oligomeric proanthocyanidins released from dentin induce regenerative dental pulp cell response.

Proanthocyanidins (PACs) are plant-derived, multifunctional compounds that possess high interactivity with extracellular matrix (ECM) components. The documented affinity of PACs for type-I collagen is directly correlated with their structural features and degree of polymerization. In this investigation, centrifugal partition chromatography (CPC) was used to sequentially deplete less active monomeric and polymeric PACs from a crude Pinus massoniana bark extract to create refined mixtures enriched in oligomeric PACs. The ability of these oligomeric PACs to modify the mechanical properties of the dentin collagen matrix and their biocompatibility with dental pulp cells (DPCs) was evaluated in an innovative biomimetic environment. The refined mixtures displayed high interactivity with dentin collagen as demonstrated by a significant increase (>5-fold) in the modulus of elasticity of the dentin matrix. In a simplified model of the dentin-DPC complex, DPCs embedded within their native ECM in the presence of PAC-treated dentin exhibited increased proliferation. Quantitative gene expression analyses indicated that exposure to PAC-treated dentin increased the expression of key biomineralization and odontogenic differentiation regulators, including RUNX2, BMP2, OCN, and DSPP. LC-MS/MS analysis revealed that PACs two to four units long (dimers, trimers, and tetramers) were being released from dentin into media, influencing cell behavior. Overall, the results suggested that PAC dimers, trimers, and tetramers are not only biocompatible, but enhance the differentiation of DPCs towards a phenotype that favors biomineralization. PAC-enriched refined mixtures can influence the field of biomaterials and regeneration by serving as renewable, non-cytotoxic agents that can increase the mechanical properties of biomaterials. STATEMENT OF SIGNIFICANCE: Pine bark extract is a renewable source of structurally diverse proanthocyanidins (PACs), multifunctional compounds whose interaction with collagen can be tailored to specific purposes by enrichment of selected PACs from the complex mixture. Oligomeric PACs were enriched from the extract and were shown here to sustain desired tissue modification and were thus assessed for cellular response in a model of the dentin-pulp interface. This model was developed to mimic leaching of potentially reactive compounds into pulp tissue. Dental pulp cells exposed to PAC-treated dentin showed increased proliferation and expression of genes necessary for extracellular matrix deposition and biomineralization, processes crucial for forming new dentin. Thus, collagen-interactive PACs may also enhance tissue regeneration and have broad impact in tissue engineering.

Encapsulation of grape seed extract in polylactide microcapsules for sustained bioactivity and time-dependent release in dental material applications.

OBJECTIVE: To sustain the bioactivity of proanthocyanidins-rich plant-derived extracts via encapsulation within biodegradable polymer microcapsules. METHODS: Polylactide microcapsules containing grape seed extract (GSE) were manufactured using a combination of double emulsion and solvent evaporation techniques. Microcapsule morphology, size distribution, and cross-section were examined via scanning electron microscopy. UV-vis measurements were carried out to evaluate the core loading and encapsulation efficiency of microcapsules. The bioactivity of extracts was evaluated after extraction from capsules via solvent partitioning one week or one year post-encapsulation process. Fifteen human molars were cut into 7mm×1.7mm×0.5mm thick mid-coronal dentin beams, demineralized, and treated with either encapsulated GSE, pristine GSE, or left untreated. The elastic modulus of dentin specimens was measured based on three-point bending experiments as an indirect assessment of the bioactivity of grape seed extracts. The effects of the encapsulation process and storage time on the bioactivity of extracts were analyzed. RESULTS: Polynuclear microcapsules with average diameter of 1.38µm and core loading of up to 38wt% were successfully manufactured. There were no statistically significant differences in the mean fold increase of elastic modulus values among the samples treated with encapsulated or pristine GSE (p=0.333), or the storage time (one week versus one year storage at room temperature, p=0.967). SIGNIFICANCE: Polynuclear microcapsules containing proanthocyanidins-rich plant-derived extracts were prepared. The bioactivity of extracts was preserved after microencapsulation.

Absolute Configuration of Native Oligomeric Proanthocyanidins with Dentin Biomodification Potency.

The structurally complex oligomeric proanthocyanidins (OPACs) are promising biomimetic agents, capable of strengthening the macromolecular backbone of teeth via intermolecular and intermicrofibrillar cross-linking. This study establishes analytical methods capable of determining the absolute configuration of the catechin-type monomeric units of underivatized OPACs. This preserves the capacity of their biological evaluation, aimed at understanding the inevitably stereospecific interactions between the OPACs and dentin collagen. Guided by dental bioassays (modulus of elasticity, long-term stability), two new trimeric and tetrameric A-type OPACs were discovered as dentin biomodifiers from pine (Pinus massoniana) bark: epicatechin-(2ß→O→7,4ß→8)-epicatechin-(2ß→O→7,4ß→8)-catechin (5) and epicatechin-(2ß→O→7,4ß→8)-epicatechin-(2ß→O→7,4ß→6)-epicatechin-(2ß→O→7,4ß→8)-catechin (6), respectively. Combining 1D/2D NMR, HRESIMS, ECD, 1H iterative full spin analysis (HiFSA), and gauge-invariant atomic orbital (GIAO) δ calculations, we demonstrate how 13C NMR chemical shifts (diastereomeric building blocks (A-type dimers)) empower the determination of the absolute configuration of monomeric units in the higher oligomers 5 and 6. Collectively, NMR with ECD reference data elevates the level of structural information achievable for these structurally demanding molecules when degradation analysis is to be avoided. Considering their numerous and deceptively subtle, but 3D impactful, structural variations, this advances the probing of OPAC chemical spaces for species that bind selectively to collagenous and potentially other biologically important biomacromolecules.

Experimental primers containing synthetic and natural compounds reduce enzymatic activity at the dentin-adhesive interface under cyclic loading.

OBJECTIVE: To evaluate the effect of experimental primers (chlorhexidine, enriched mixture of proanthocyanidins, and doxycycline) on the adhesive properties and gelatinolytic activity at dentin-resin interfaces of occlusal Class I restorations. METHODS: The inactivation of enzymes by the experimental primers was assessed by fluorescence assay and gelatin zymography. To assess the adhesive properties, occlusal Class I cavities were prepared in sound human molars, etched with phosphoric acid and restored with one of the primers and an etch-and-rinse adhesive system (Adper Single Bond Plus-3M ESPE). After the restorative procedures, specimens were divided into two subgroups (n=6) consisting of storage in incubation buffer or axial cyclic loading at 50N and 1,000,000 cycles. Then, the specimens were sectioned and slices were assigned to in situ zymography assay and microtensile bond strength (TBS) test. RESULTS: Fluorescence assay and gelatin zymography revealed that the experimental primers inactivated rMMPs. In situ zymography (2-way ANOVA, Tukey, p<0.05) showed that cyclic loading increased the gelatinolytic activity at the resin-dentin interface and the experimental primers decreased the gelatinolytic activity at the adhesive interface. The experimental primers had no significant effects on dentin-adhesive bond strengths with or without cyclic loading (2-way ANOVA, p>0.05). SIGNIFICANCE: The use of experimental primers impaired the enzymatic activity at the dentin-adhesive interface after cyclic loading and the activity of rMMPs. Cyclic loading did not have a significant effect on the bond strength.