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

Abstract

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.

Keywords: Biodegradation; Collagen; Collagen cross-linkers; Dentinl; Polyphenols; Proanthocyanidins; Stiffness.

Figure 1.

Stacked Diol HPLC-UV profiles of methyl acetate/ water partitions of crude extracts. Pinus massoniana (PM), Cinnamomum verum (CV), Hamamelis virginiana (HV) and Vitis vinifera (VV). UP – upper phase; LP – lower phase. The chromatograms were stacked in OriginPro using Waterfall Z:color plot; * cinnamaldehyde; ** Hamamelitannin. Compounds (UV peaks) eluting with an increasing retention time indicate an increasing degree of polymerization (DP). The diol stationary phase is especially utilized to separate the extracts according to their DP. Each UV peak represents several PACs with the same DP grouped together.

Figure 2.

Results of the long-term evaluation of dentin matrix degradation via endogenous proteases (A) and exogenous bacterial collagenases (B) upon treatment with proanthocyanidin (PAC) sources with distinct phytochemistry (Pinus massoniana - PM, Cinnamomum verum - CV, Hamamelis virginiana - HV and Vitis vinifera - VV). In A, the * control group exhibited significantly higher degradation than all treatment groups (p < 0.001). Higher concentrations of extracts resulted in significantly increased resistance to degradation for all PAC sources (p < 0.001); statistical differences among sources of PACs is depicted by different letters (p < 0.05). In B, *indicates statistically significant higher resistance to degradation for the treatment groups, whereas **indicates higher degradation of control groups long-term when compared to short term storage (p < 0.001); ^¥ indicates that the controls exhibit statistically higher degradation than all other groups (p < 0.05); ^δ indicates that 6.5% CV treatment exhibits the lowest degradation of all experimental groups (p ≤ 0.004).