Elsevier

Dental Materials

Volume 37, Issue 12, December 2021, Pages 1794-1805
Dental Materials

Distinct effects of polyphenols and solvents on dentin collagen crosslinking interactions and biostability

https://doi.org/10.1016/j.dental.2021.09.009Get rights and content

Abstract

Objective

To evaluate the effects of different polyphenols and solvents on dentin collagen’s crosslinking interactions and biostabilization against MMPs and collagenase degradation.

Methods

Two polyphenols [proanthocyanidin (PA) and quercetin (QC)] with different water solubility were prepared as treatment solutions using ethanol (EtOH) or dimethyl sulfoxide (DMSO) as solvents. 6-um-thick dentin films were microtomed from dentin slabs of third molars. Following demineralization, films or slabs were subject to 60-s treatment (PA or QC) or no treatment (control) with subsequent extended-rinse with original solvent (EtOH or DMSO) or distilled water (DW). Collagen crosslinking interactions were assessed by FTIR. Biostability was assessed through endogenous MMPs activity via confocal laser scanning microscopy, and exogenous collagenase degradation via weight loss, hydroxyproline release and SEM. Finally, direct collagenase inactivation was also evaluated. Data were analyzed by three-way ANOVA and post-hoc tests (α = 0.05%).

Results

Distinct effects of two polyphenols and solvents on collagen crosslinking and biostabilization were observed. Higher crosslinking and biostability efficacy occurred with PA than QC (p < 0.001) that demonstrated negligible collagen interactions. With DMSO solvent, efficacy results were significantly reduced with both polyphenols (p < 0.05). DMSO-rinse further weakened interactions of PA with collagen, diminishing biostability (p < 0.05). Low biostability was detected with QC and DW-rinse, suggesting direct enzymatic inhibition due to physical presence in collagen.

Significance

Collagen crosslinking interactions and biostability depend on polyphenol chemical characteristics. Treatment-solution solvents may affect interactions between polyphenols and collagen, specifically, DMSO showed detrimental effects on collagen crosslinking and biostability and should be used with caution.

Introduction

Type I collagen is the most abundant protein in human body, and is essential constituent for support and stability of body tissues such as vessels, skin, tendons, bone and dentin in teeth [1]. In dentin, collagen accounts for 90% of organic matrix and presents the particular interest in dentistry because it is the base for interactions between organic matrix and restorative resin materials. Moreover, use of collagen-associated biomaterials is also a trend in regenerative medicine. Despite of excellent biological properties, collagen is susceptible to degradation by collagenolytic proteases and biomechanical stress, limiting its strengths and applications. Dentin collagen degradation is the key reason for breakage of the resin-dentin interface, leading to failed composite restorations and progression of secondary caries [2]. Recently, it was reported that replacement of restorations still accounts for more than 50% of the restorations placed by dentists [3]. Also, a recent systematic review demonstrates that secondary caries is the major reason for failure after 3 years of composite restorations placed [4], meaning high costs for oral health systems. There is a strong need to reinforce collagen, which not only benefits dentistry, but also regenerative medicine.

The basis for collagen biostability, strength and function is derived from crosslinks at molecular and microfibrillar levels, which are mediated by enzymatic reactions between lysyl oxidase on lysine and hydroxylysine amino acids in the specific telopeptides regions of collagen molecules [1,5]. However, the types and amount of crosslinks in collagen vary according to physiological and pathological conditions, leading tissues susceptible to degradation. In light of this, approaches to mimicking crosslinks have been widely investigated to increase collagen biostability for tissue repair and regeneration. Polyphenols have been highlighted in this task due to their biocompatibility and potential to interact with proteins [6,7]. Besides enhancing mechanical properties via mineralization of dental tissues [6], increasing collagen biostability against enzymatic degradation has been one of main approaches to attest crosslinking ability of polyphenols [[8], [9], [10]].

Although several natural polyphenols showed outstanding in vitro results, low bioavailability in vivo is still a limiting factor for clinical application due to their poor water solubility [11,12]. Thus, the use of a solvent is required for collagen treatment involving polyphenols. Most often used solvents include ethanol (EtOH), acetone and dimethyl sulfoxide (DMSO), either applied alone or in combination with water or phosphate-buffered saline (PBS). Besides the increase of mixture entropy and interaction between solute and solvent, some studies have recently demonstrated a synergistic effect promoted by the organic solvents in combination with polyphenols [13,14]. For examples, the combinations of baicalein [13,15], resveratrol [16], and epigallocatechin-3-gallate (EGCG) [17] with EtOH, most recently, EGCG with DMSO [14], were reported as a promising strategy to reduce degradation of dentin bond strength through increased collagen biostability against enzymatic activity.

Despite a real need for solvents in treatment solutions, solvent effect on crosslinking interactions between polyphenols and collagen has not been explored. In a treatment solution, the solvent is the medium where interactions take place, which could influence the polyphenols’ interactions with collagen [7]. Furthermore, another potential issue, often found in many studies of crosslinking efficacy of polyphenols via enzymatic degradation resistance, is that treated collagen is not rinsed with the original treatment solution solvent or only rinsed with water. This approach becomes more problematic when polyphenols with poor water solubility are used. Without using a solvent rinse or using water rinse only, a water-insoluble polyphenol could improve collagen biostability against degradation due to its physical presence in collagen matrix, and inappropriately be categorized as “an efficient crosslinker”. However, a true polyphenol collagen crosslinker should withstand the solvent rinse and show the ability to form strong stable interactions between the polyphenol and collagen even after the rinse step.

Therefore, in this study two polyphenols with different molecular characteristics and water solubility [proanthocyanidin (PA) and quercetin (QC)] were dissolved in different solvents (EtOH and DMSO). To distinguish collagen biostabilization via crosslinking as compared to just the physical presence, treated collagen was thoroughly rinsed with either the original solvent of treatment solution or distilled water (DW). Collagen crosslinking interactions were assessed by Fourier-transform infrared (FTIR) spectroscopy. Collagen biostability against endogenous MMPs activity was evaluated by confocal laser scanning microscopy (CLSM), and biostability against exogenous collagenase degradation was evaluated by weight loss (WL) and hydroxyproline release (HYP) of dentin films as well as by scanning electronic microscopy (SEM) of dentin slabs. A measurement of direct inactivation of collagenase by PA and QC was also performed. The research hypotheses in this study were that the crosslinking interactions, biostability against endogenous MMPs and exogenous collagenase degradation of dentin collagen would be affected by 1) polyphenols; 2) solvents in treatment solutions; or 3) the rinse solutions used.

Section snippets

Reagents

QC (quercetin, ≥95%), DMSO (dimethyl sulfoxide, ≥99%), ethanol (absolute) and collagenase type I (from Clostridium histolyticum, 125 U/mg) were purchase from Sigma-Aldrich (St. Louis, MO, USA). PA (Proanthocyanidin, ≥90%, from grape seed) was donated by the manufacturer (Polyphenolics, Madera, CA, USA). 0.96% phosphate buffered saline (PBS, pH 7.4) was prepared using Sigma Life Science – Dulbeccos PBS packet, 0.002% sodium azide was added to prevent fungi or bacteria. TESCA buffer was prepared

FTIR characterization of PA and QC

Representative FTIR spectra of polyphenols PA (red line) and QC (green line) as well as their chemical characteristics including molecular weight, number of phenolic hydroxyl groups, water solubility are shown in Fig. 2. There are a few vibrational bands that are detected for both polyphenols since they are built on the similar flavan-3-ol unit containing three typical rings (A–C–B). These bands include phenolic OH groups (∼3280 cm−1), aromatic rings (Cdouble bondC stretching at ∼1608 cm−1 and ∼1520 cm−1, Csingle bondC

Discussion

The present study was designed to determine the effectiveness of polyphenols in collagen biostability imparted exclusively by crosslinking interactions as well as the potential solvent effect on polyphenols’ interactions with collagen. Two polyphenols (PA and QC) with different chemical characteristics and water solubility as well as two popular solvents (EtOH and DMSO) with different properties were used. The distinct effects of polyphenols and solvents on collagen crosslinking and

Conclusions

Within the limitations of this study, the following conclusions can be drawn: Collagen crosslinking interactions and biostability imparted by polyphenols depend on their chemical and structural characteristics, with PA showing much higher collagen crosslinking and biostabilization efficacy than QC.

Solvents used in treatment solutions may affect interactions between polyphenols and collagen, specifically, DMSO shows detrimental effects on the collagen interactions with PA.

Collagen biostability

Acknowledgments

This study was supported by Research Grant R01-DE027049 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

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