Collagen-collagen interactions mediated by plant-derived proanthocyanidins: A spectroscopic and atomic force microscopy study

Abstract

Collagen cross-linkings are determinant of biological tissue stability and function. Plant-derived proanthocyanidins (PACs) mimic different hierarchical levels of collagen cross-links by non-enzymatic interactions resulting in the enhancement to the biomechanics and biostability of collagen-rich tissues such as dentin. This study investigated the interaction of PACs from Vitis vinifera grape seed extract with type I collagen in solubilized form and in the demineralized dentin matrix (DDM) by fluorescence spectral analysis; collagen-collagen binding forces in presence of cross-linking solutions by atomic force microscopy (AFM); and spectroscopic analysis of the DDM using attenuated total reflectance Fourier transform-infrared spectroscopy (ATR-FTIR). Glutaraldehyde (GA) and carbodiimide hydrochloride (EDC) with known cross-linking mechanisms were selected for comparative analyses. Changes in fluorescence upon interaction of solubilized type I collagen with PACs, EDC and GA reflected pronounced modifications in collagen conformation. PACs also promoted stronger collagen-collagen fibrils interaction than EDC and GA. A new feature was observed using ATR-FTIR spectroscopic analysis in PACs-treated collagen and DDM. The findings suggest covalent interactions between collagen and PACs. The mechanisms of interaction between PACs-collagen hold attractive and promising tissue-tailored biomedical applications and the binding forces that potentially drive such interaction were characterized.

Statement of significance: Connective tissues such as skin, bone and dentin are mainly composed of type I collagen, which is cross-linked to promote tissue stability, strength and function. Novel therapies using substances that mimic cross-links have been proposed to promote repair of collagen-based-tissues. In dentistry, naturally occurring proanthocyanidins (PACs) have the potential to enhance dentin mechanical properties and reduce its enzymatic degradation, but their mechanisms of cross-linking are unclear. The present study investigated the specific interactions between PACs-type I collagen in purified and dentin collagen and compared to the well described cross-linking mechanisms promoted by synthetic chemical substances. Findings reveal that covalent-like bonds are induced by plant PACs in type I collagen as well as in complex dental native tissue, promoting strong collagen-collagen interactions.

Keywords: ATR-FTIR spectroscopy; Atomic force microscopy; Collagen cross-linking; Dentin; Proanthocyanidins.

Figure 1

Collagen fluorescence spectra (nm) and changes in peak fluorescence intensity when treated with different concentrations of PACs, EDC/NHS and GA. (A) Collagen fluorescence peak positions and (B) fluorescence intensities when incubated with 0.0065 – 0.065% PACs. (C) Collagen fluorescence intensities after incubation with 0.0575 – 0.575% EDC containing 0.014 – 0.14% NHS. (D) Fluorescence intensities when collagen was incubated with 0.005 – 0.5% GA.

Figure 2

Coating of Au substrate with different concentrations of type I collagen characterized by atomic force microscopy and used to determine collagen-collagen molecular interactions.

Figure 3

Retract traces between collagen-tip and collagen-substrate treated with (A) PBS, (B) 0.65% PACs in PBS pH 7.4, (C) 0.1%/0.03% EDC/NHS in PBS pH 7.4 and (D) 1% GA in PBS pH 7.4. AFM tip velocity 1000 nm/s. Dwell time 2 s.

Figure 4

Representative force histograms obtained for PBS (control), PACs, EDC/NHS and GA with dwell 2s. Increased number of interactions in the force range 500–1000 pN indicates the cross-linking between collagen fibrils due to the presence of cross-linker.

Figure 5

Representative ATR-FTIR spectra of (A) type I collagen treated with 0.065% PACs. Black solid line: PACs; red short dash line: collagen; green dash dot line: collagen + PACs; blue short dot line: difference spectrum; (B) type I collagen treated with 5.75% EDC/1.4% NHS. Black solid line: EDC/NHS; red short dash line: collagen; green dash dot line: collagen + EDC/NHS; and (C) type I collagen treated with 5% GA. Black solid line: 5% GA; red short dash line: collagen; green dash dot line: collagen + GA; blue short dot line: difference spectrum.

Figure 6

Representative ATR-FTIR spectra of (A) demineralized dentin matrix (DDM) treated with PACs. Black solid line: 6.5% PACs; red short dash line: DDM; green dash dot line: DDM + PACs; blue short dot line: difference spectrum; (B) DDM treated with 5.75% EDC/1.4% NHS. Red short dash line: DDM; green dash dot line: DDM + EDC/NHS; blue short dot line: difference spectrum; and (C) DDM treated with 5% GA. Red short dash line: DDM; green dash dot line: DDM + GA; blue short dot line: difference spectrum (multiplied by three).