Mimicking the hierarchical functions of dentin collagen cross-links with plant derived phenols and phenolic acids

Abstract

Proanthocyanidins (PACs) are secondary plant metabolites that mediate nonenzymatic collagen cross-linking and enhance the properties of collagen based tissue, such as dentin. The extent and nature of cross-linking is influenced by the composition and specific chemical structure of the bioactive compounds present in certain PAC-rich extracts. This study investigated the effect of the molecular weight and stereochemistry of polyphenol compounds on two important properties of dentin, biomechanics, and biostability. For that, purified phenols, a phenolic acid, and some of its derivatives were selected: PAC dimers (A1, A2, B1, and B2) and a trimer (C1), gallic acid (Ga), its esters methyl-gallate (MGa) and propyl-gallate (PGa), and a pentagalloyl ester of glucose (PGG). Synergism was assessed by combining the most active PAC and gallic acid derivative. Mechanical properties of dentin organic matrix were determined by the modulus of elasticity obtained in a flexural test. Biostability was evaluated by the resistance to collagenase degradation. PACs significantly enhanced dentin mechanical properties and decreased collagen digestion. Among the gallic acid derivatives, only PGG had a significant enhancing effect. The lack of observed C1:PGG synergy indicates that both compounds have similar mechanisms of interaction with the dentin matrix. These findings reveal that the molecular weight of polyphenols have a determinant effect on their interaction with type I collagen and modulates the mechanism of cross-linking at the molecular, intermolecular, and inter-microfibrillar levels.

Figure 1

Collagen fibril hierarchical structure and possible dentin biomodification mechanisms. PACs, gallic acid and its derivatives were scaled to the dimensions of tissue. The collagen fibril shows the 67 nm periodicity, due to the staggering of the collagen molecules in the axial direction. One single collagen fibril is formed by smaller entities grouped in bundles, known as microfibrilar bundles, with about 10 – 25 nm diameter. The microfibrils are 4 – 5 nm diameter structures, spaced 3 – 4.5 nm from each other. Thus, inter-microfibrillar cross-linking is likely to occur mediated by C1 and PGG. The inter-microfibrillar cross-links may significantly affect the mechanical properties. Each microfibril contains five 1D staggered, rope-like right hand twisted collagen molecule. The distance between the collagen molecules (1.26 – 1.33 nm) allows inter-molecular cross-link by A1, A2, B1 and B2 compounds. On the molecular scale, the collagen molecule is formed by one N and one C-terminus along with a triple helix with 1014 amino acid residues. Available cross-linking sites are found in both N and C-terminus that allows for intra-molecular cross-link by smaller compounds. A hydroxyproline-depleted area constitutes the thermally labile domain in the C-terminus. Undertwisted regions of the triple helix and a collagenase binding site on the C-terminus are primary sites for bacterial collagenase binding. A1: procyanidin A1; A2: procyanidin A2; B1: procyanidin B1; B2: procyanidin B2; C1: procyanidin C1. Ga: gallic acid; MGa: methyl-gallate; PGa: propyl-gallate; PGG: penta-O-galloyl-β-D-glucose.

Figure 2

Chemical structure, formulae, and molecular weights of naturally occurring phenolic compounds used in this study. Two and three dimensional models for PACs (A and B) and gallic acid and its derivatives (C and D) were performed using Chem3D Pro of ChemBioDraw® package (ver. 13.0.2, PerkinElmer, Waltham, MA, USA). The distance between two hydrogen atoms was determined with MM2 function during the molecular dynamics calculation for each molecule in angstroms (Å). The dynamic ranges for the compounds were: between 13.5 and 14.1 Å for A1, 13.9 and 15.1 Å for A2, 9.5 and 10.9 Å for B1, 10.0 and 11.2 Å for B2, 12.8 and 18.2 Å for C1, 7.1 and 7.7 Å for Ga, 8.1 and 8.9 Å for MGa, 10.8 and 11.4 Å for PGa, and 14.9 and 18.0 Å for PGG (β-D-glucose conformation confirmed by NMR). The mean distances for each compound are shown in B and D. The minimize Energy for each molecule were −17.32 Kcal/mol for A1, −15.40 Kcal/mol for A2, −28.66 kcal/mol for B1, −33.69 Kcal/mol for B2, −43.66 Kcal/mol for C1, −10.76 Kcal/mol for Ga, −1.88 Kcal/mol for MGa, −0.68 Kcal/mol for PGa, and −25.01 Kcal/mol for PGG. Calculations were done using the following parameters: intervals of step and frame were set as 2.0 fs and 10 fs, respectively; iteration was stopped after 10000 steps, rate of heating/cooling was set as 0.1 Kcal/atom/ps; and target temperature of 300 Kelvin. Blue and white dashed lines indicate intra-molecular hydrogen bonds. The high intensity in blue color represents less than ideal geometry.

Figure 3

Mean (standard deviation) of modulus of elasticity and biodegradation rates of dentin beams treated with different PACs, gallic acid and its derivatives and association of C1 and PGG. Different symbols represent statistical significant difference (p < 0.05). No statistically significant difference was observed between groups at baseline (p > 0.05). A1: procyanidin A1; A2: procyanidin A2; B1: procyanidin B1; B2: procyanidin B2; C1: procyanidin C1. Ga: gallic acid; MGa: methyl-gallate; PGa: propyl-gallate; PGG: penta-O-galloyl-β-D-glucose.

Figure 4

Pearson correlation between modulus of elasticity (E) (MPa) after treatment and biodegradation rates (%) with number of hydroxyl groups (A and C) and molecular weight (Da) (B and D) of PACs and gallic acid and its derivatives.