A dynamic mechanical method to assess bulk viscoelastic behavior of the dentin extracellular matrix.

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.

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.

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

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

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

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