A.A. Leme-Kraus

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-(2β→O→7,4β→8)-epicatechin-(2β→O→7,4β→8)-catechin (5) and epicatechin-(2β→O→7,4β→8)-epicatechin-(2β→O→7,4β→6)-epicatechin-(2β→O→7,4β→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.

Biostability of the Proanthocyanidins-Dentin Complex and Adhesion Studies

Oligomeric proanthocyanidins (OPACs) are potent and renewable natural bioactives possible to be refined into chemically standardized mixtures for biological applications. Herein, we found that multiscale interactions of OPACs with the dentin matrix create tight biointerfaces with hydrophobic methacrylate adhesives on wet surfaces. An enriched mixture of OPACs, with a known phytochemical profile, was produced from grape seed crude extract (Vitis vinifera; enriched grape seed extract [e-GSE]) and applied to dentin matrices to determine changes to the mechanical properties and biodegradability of the dentin matrix and favorable resin adhesion mechanisms. Methods included a 3-point flexural test, quantification of hydroxyproline (collagen solubilization), static and dynamic nanomechanical analyses, resin-dentin microtensile bond strength, and micropermeability at the adhesive interface. The e-GSE-modified dentin matrix exhibited remarkably low collagen solubilization and sustained the bulk elastic properties over 12 mo. Tan δ findings reveal a more elastic-like behavior of the e-GSE-modified dentin matrix, which was not affected by H-bond destabilization by urea. Dentin-methacrylate biointerfaces with robust and stable adhesion were created on e-GSE-primed dentin surfaces, leading to a dramatic decrease of the interfacial permeability. Standardized OPAC mixtures provide a new mechanism of adhesion to type I collagen–rich tissues that does not rely on hydrophilic monomers. The bioadhesion mechanism involves physicochemical modifications to the dentin matrix, reduced tissue biodegradation, and bridging to methacrylate resins.

Effect of Bioactive Primers on Bacterial-Induced Secondary Caries at the Tooth-Resin Interface

Secondary caries at the tooth-resin interface is the primary reason for replacement of resin composite restorations. The tooth-resin interface is formed by the interlocking of resin material with hydroxyapatite crystals in enamel and collagen mesh structure in dentin. Efforts to strengthen the tooth-resin interface have identified chemical agents with dentin collagen cross-linking potential and antimicrobial activities. The purpose of the present study was to assess protective effects of bioactive primer against secondary caries development around enamel and dentin margins of class V restorations, using an in vitro bacterial caries model. Class V composite restorations were prepared on 60 bovine teeth (n=15) with pretreatment of the cavity walls with control buffer solution, an enriched fraction of grape seed extract (e-GSE), 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide/N-hydroxysuccinimide, or chlorhexidine digluconate. After incubating specimens in a bacterial model with Streptococcus mutans for four days, dentin and enamel were assessed by fluorescence microscopy. Results revealed that only the naturally occurring product, e-GSE, significantly inhibited the development of secondary caries immediately adjacent to the dentin-resin interface, as indicated by the caries inhibition zone. No inhibitory effects were observed in enamel margins. The results suggest that the incorporation of e-GSE into components of the adhesive system may inhibit secondary caries and potentially contribute to the protection of highly vulnerable dentin-resin margins.

An Overview of Dental Adhesive Systems and the Dynamic Tooth–Adhesive Interface

From the conception of resin-enamel adhesion to todays 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.

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.

Long-term evaluation of the stability of dentin matrix following treatments with aqueous solutions of titanium tetrafluoride at different concentrations

OBJECTIVE The purpose of this study was to investigate the effects of aqueous solutions of different concentrations of titanium tetrafluoride (TiF4) on dentin matrix stability up to six months. DESIGN Dentin specimens prepared from fifteen nonerupted molars were demineralized and randomly distributed into groups: 2.5% TiF4, 4% TiF4, 1000 ppm NaF, and control (distilled water). The modulus of elasticity (ME) and dry masses of the dentin matrix were determined at baseline, and up to 6 months following treatment. Collagen solubilization was estimated by hydroxyproline (HYP) quantification in the simulated body fluid used to store the dentin specimens. RESULTS The 2.5% TiF4 group presented higher ME immediately after treatment, and at 3 and 6 months storage, whereas 4% TiF4 showed higher means at 3 and 6 months (p < 0.001). No significant differences were observed among the groups over time (p = 0.9325). However, the 2.5% TiF4 group showed significantly higher ME than the control group, immediately after treatment. All the groups presented significantly higher mass change immediately, compared with 3 and 6 months (p < 0.0001). Except for the 4% TiF4 group, HYP release was higher in the first quarter (p = 0.0152), when no significant differences were found among the groups. In the second quarter, the means were significantly higher in the 2.5% TiF4 and 4% TiF4 groups. The group treated with 2.5% TiF4 had a statistically higher HYP release than the control group. CONCLUSION An aqueous solution of 2.5% TiF4 increases the immediate stiffness values, but does not stabilize the collagenous dentin matrix.

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.

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.