The aim of the study was to investigate the effects of methacrylate-functionalized proanthocyanidins (MAPAs) on dentin collagen’s bio-stabilization against enzymatic degradation and crosslinking capability.
Three MAPAs were synthesized via varying methacrylate (MA) to proanthocyanidins (PA) feeding ratios of 1:2, 1:1, and 2:1 to obtain MAPA-1, MAPA-2, and MAPA-3, respectively. The three MAPAs were structurally characterized by proton nuclear magnetic resonance ( 1 H NMR) and Fourier transform infrared (FTIR) spectroscopic methods. 5-μm-thick dentin films were microtomed from dentin slabs of third molars. Following demineralization, films or slabs were treated with 1% MAPAs or PA in ethanol for 30 s. Collagen bio-stabilization against enzymatic degradation was analyzed by weight loss (WL) and hydroxyproline release (HYP) of films, as well as scanning electron microscopy (SEM) on dentin slabs. Crosslinking capacity and interactions of MAPAs with collagen were investigated by FTIR. Data were analyzed by ANOVA and Tukey’s test ( α = 0.05%).
MA:PA feeding ratios affected MAPAs’ chemical structures which in turn led to different collagen stabilization efficacy against degradation and varied collagen crosslinking capabilities. Higher collagen stabilization efficacy was detected using MAPA-1 (WL 10.52%; HYP 13.53 μg/mg) and MAPA-2 (WL 5.99%; HYP 11.02 μg/mg), which was comparable to that using PA (WL 8.79%; HYP 13.17 μg/mg) (p > 0.05), while a lower collagen stability occurred in MAPA-3 (WL 38.48%; HYP 29.49 μg/mg), indicating excessive MA-functionalization would compromise its stabilization efficacy. In comparison, complete digestion was detected for untreated collagen (WL 100%; HYP 102.76 μg/mg). The above results were consistent with collagen crosslinking efficacy of the three MAPAs revealed by SEM and FTIR.
A new class of novel polymerizable collagen cross-linkers MAPAs was synthesized and shown that, when appropriate MA:PA ratios were applied, the resulting MAPAs could render high collagen stability and the ability to copolymerize with resin monomers, overcoming the drawbacks of PA. These new polymerizable crosslinkers, when included in adhesives, could lead to long-lasting dentin bonding.
Resin-dentin bonding relies on the formation of hybrid layer in which there is micromechanical interlocking among demineralized collagen fibrils and infiltrated methacrylate adhesive resin. In the hybrid layer, minerals surrounding collagen fibrils are removed by acid etching and replaced/infiltrated by resin monomers. The bio-stability of both the underlying demineralized collagen and infiltrated resin is considered one of the most important factors for success of dental restorations [ , ]. Despite recent advances, limited durability of resin-dentin interfaces is still an unsolved problem. Hydrolysis of sub-optimally polymerized adhesive resin and enzymatic degradation of unprotected demineralized collagen by host-derived enzymes (MMPs and cysteine cathepsins) lead to interface breakdown, lowering service life of restorations [ , ]. Developing strategies that can reinforce both dentin collagen and resin from degradation is therefore a viable approach to enhancing the stability of the resin-dentin interface [ , ].
Collagen bio-modification mediated by bioactive agents has emerged as a powerful tool for improving dentin’s mechanical, anti-enzymatic and bonding properties [ ]. Among various bio-modification approaches, the exogenous collagen cross-linkers, especially natural polyphenolics such as proanthocyanidins (PA), have shown some promising applications due to their high bioactivity, absence of toxicity and wide availability [ , , ]. However, in spite of encouraging collagen stabilization results with a treatment time even as short as tens of seconds, the use of PA in a clinically acceptable setting remains a challenge [ ]. For example, the use of PA as a primer has been widely investigated [ , , ]. However, due to its antioxidant nature, PA needs to be rinsed off after priming, which is time-consuming and adds an extra step in bonding protocols. The inclusion of PA in etchants has also been tried [ , ]; however, PA has poor solubility in etchant acids, requiring the addition of volatile solvents such as ethanol or acetone which results in short shelf life of etchants due to fast solvent evaporation. In addition, PA, like most polyphenolics in general, is susceptible to auto-oxidation reactions, constantly altering its structures and properties [ , ]. Consequently, preparation of fresh solutions is required before every application. Furthermore, all those strategies, whether using PA as a primer or in an etchant, limit PA’s usage to the etch-and-rinse mode only, and cannot be applied to the self-etch mode.
In light of this, many attempts have been made to directly include PA into adhesives. Unfortunately, a negative effect of PA on the photo-polymerization of adhesive monomers [ , , ] has been frequently observed. PA is a well-known free radical scavenger, hindering molecular chain initiation and propagation during polymerization [ , ]. Poor polymerization of adhesive monomers results in reduced resin quality, breakdown of bonding interfaces as demonstrated in laboratorial studies [ , ]. A recent two-year clinical evaluation showed that PA included in adhesives adversely affected polymerization of adhesive resin, causing higher loss rate of restorations and jeopardizing the longevity of composite restorations [ ], further confirming the limitations of using PA directly in a clinical setting.
Grafting of additional functional unit onto methacrylate-based monomers has led to the development of multifunctional dental monomers. In addition to the polymerization function, these monomers may possess other functions such as self-etching ability, chemical interactions to dental substrates [ , ], antibacterial and anti-MMPs activity [ , ], etc. Specific functional unit is often incorporated into methacrylate (MA) through the ester linkage, thus maintaining polymerization ability of the vinyl moiety in MA groups [ , ]. This strategy inspired us to develop a novel class of polymerizable collagen crosslinker methacrylate-functionalized proanthocyanidins (MAPAs). Following a one-step synthesis reaction, MAPAs have been synthesized aiming to combine the collagen cross-linking ability of PA and MA’s capability to co-polymerize with resin monomers into the same MAPA molecule. This strategy would reinforce both dentin collagen (via crosslinking) and resin (via co-polymerization) at the same time, therefore is a novel approach to enhancing stability of the resin-dentin interfaces.
During functionalization, the phenol hydroxyl (OH) groups in PA may be consumed and replaced by MA. Since the cross-linking capability of PA is attributed to its phenolic structures such as catechol with ortho-phenol OH groups, it is unclear if the MA grafting/replacement would affect/sacrifice MAPA’s collagen crosslinking ability. Effects of three MA:PA feeding ratios on the chemical structures of MAPAs and the stabilization and cross-linking of dentin collagen after MAPA treatment have therefore been evaluated in this study. The bio-stability of dentin collagen against collagenase degradation was assessed quantitatively by weight loss (WL) and hydroxyproline release (HYP), and qualitatively by scanning electron microscopy (SEM). The cross-linking efficacy and chemical interactions with collagen were studied by Fourier-transform infrared (FTIR) spectroscopy. The null hypotheses tested were that regardless of MA:PA feeding ratios, the synthesized MAPAs would not be able to (1) induce collagen bio-stabilization against degradation or (2) promote cross-linking and chemical interactions with collagen.
Material and methods
All chemicals used were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. The PA from grape seed extract (GSE) was generously donated by Mega Natural (Madera, CA, USA). Three polymerizable collagen crosslinkers derived from PA (MAPA-1, MAPA-2 and MAPA-3) were synthesized as further described below (Scheme 1). Catechin is the major monomeric unit for PA and the mole of PA samples was thus calculated as the mole of catechin units. All the treatment solutions (PA, MAPA-1, MAPA-2 and MAPA-3) were prepared at 1% concentration in pure ethanol (w/w). Collagenase (type I, from Clostridium histolyticum, ≥125 CDU/mg) solution was made at 0.1% (w/v) in TESCA buffer (50 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 0.36 mM CaCl 2 , pH = 7.4).
Synthesis of polymerizable collagen crosslinkers MAPAs
The MAPAs were synthesized from grape seed extract through a one-step reaction. Sample of PA (0.90 g, 3.1 mmol of catechin) was suspended in N,N-dimethylformamide (10 mL)/triethylamine (0.31 g, 3.1 mmol for MAPA-1; 0.63 g, 6.2 mmol for MAPA-2; 1.25 g, 12.4 mmol for MAPA-3) at 0 °C. To the above solution, methacryloyl chloride (0.16 g, 1.5 mmol for MAPA-1; 0.32 g, 3.1 mmol for MAPA-2; 0.65 g, 6.2 mmol for MAPA-3) was added dropwise with the MA:PA (based on catechin unit) molar ratio being 1:2, 1:1 and 2:1 (0.5 eq., 1 eq, and 2 eq) for MAPAs-1, -2, and -3, respectively. The resulting reaction mixture was stirred for another 1 h at 0 °C, and then stirred at room temperature overnight. Water was then added to the reaction mixture. After stirring under N 2 for at least 2 h, the mixture was centrifuged, rinsed with distilled water twice, and the final product was collected and dried under vacuum for at least 48 h. Yield: 0.25 g, 24.3% for MAPA-1; 0.50 g, 44.8% for MAPA-2; 0.79 g, 59.7% for MAPA-3. The lower yields for MAPA-1 and MAPA-2 are due to their higher solubility in water, which led to some product loss during the water rinsing step. The final products were characterized by 1 H NMR ( Fig. 1 ) and FTIR ( Fig. 2 ).
Dentin films preparation and MAPAs treatment
Sixteen non-carious human third molars were collected under the approved protocol by the University of Missouri-Kansas City Adult Heath Sciences Institutional Review Board (IRB). After storage in phosphate-buffered saline containing 0.002% sodium azide at 4 °C, the occlusal enamel and roots were removed from the teeth using a water-cooled diamond saw (Buehler, Lake Bluff, IL, USA). Six teeth were sectioned into dentin blocks (6 × 6 × 5 mm), which were further processed into 350 ultrathin dentin films (5 μm thick) with a tungsten carbide knife mounted on SM2500S microtome (Leica, Deerfield, IL, USA). These films were randomly assigned into 5 groups (n = 70/group) according to the cross-linkers investigated: PA, MAPA-1, MAPA-2, MAPA-3 and an untreated group as a negative control (solvent only). All the cross-linkers were prepared as treatment solutions at 1% in ethanol (w/w). Each of the dentin films was first demineralized with 10% phosphoric acid for 30 min, rinsed with deionized water for 30 min and spread on a plastic cover slip (Fisher Scientific, Pittsburgh, PA, USA) using a paintbrush. After blotting away excessive water, 30 μL of selected treatment solution was dropped onto each of the demineralized dentin films. After 30 s, the films were rinsed with absolute ethanol for 30 min (3 times ×10 min) in order to remove any residual treatment solution. The films were then dried for 48 h under vacuum. The treated and dried films were subsequently evaluated for collagen biostability – weight loss (WL) and hydroxyproline release assay (HYP) (n = 60 per treatment group) and the remaining 10 films per group was further analyzed by FTIR.
Collagen biostability against collagenase digestion by weight loss and HYP release measurements of dentin films treated with MAPAs
The 60 treated dentin films from each treatment group were randomly assigned into 6 specimen sub-groups containing 10 films in each group. The films were immersed in 300 μL of 0.1% bacterial collagenase solution in TESCA at 37 °C for 1 h. The WL percentage was determined by the dry weight change before (W 0 ) and after (W 1 ) collagenase digestion for each specimen using an analytical balance (d = 0.01 mg, Mettler Toledo AG285, Zurich, Switzerland), and was calculated by the following equation: WL%=(W 0 -W 1 )/W 0 ×100%.
For the HYP assay, the digestion solution was collected and hydrolyzed in 6M HCl at 110 °C for 24 h. The dry residue (free hydroxyproline and other amino acids) of each specimen was pre-treated by neutralization, oxidation and subjected to 5% Ehrlich’s reagent to develop the color. The absorbance was measured at 555 nm with a microplate reader (Biotek Instruments, Winooski, VT, USA). The trans-4-hydroxy- l -proline (analytical standard, Sigma-Aldrich) was used as the standard to obtain the working curve for quantifying the HYP release (n = 6) from each micro gram of demineralized dentin films during the digestion.
SEM morphology of demineralized dentin layer treated with MAPAs
Ten non-carious human third molars were processed into dentin slabs as follows. After crown removal, a uniform smear layer was created on the dentin surface utilizing wet 600-grit SiC sandpaper (Buehler) for 60 s. Further sections were made in the occlusal-apical direction at increments of 1 mm, followed by one cut parallel to and ∼1.5 mm below the abraded surface to free the slabs. Slabs for SEM were notched at the middle position from the side opposite to the abraded surface for the purpose of subsequent fracturing and visualization. The abraded surfaces of notched slabs were etched with 32% phosphoric acid gel (Scotchbond Universal Etchant, 3M ESPE, St. Paul, MN, USA) for 15 s and rinsed with deionized water for 30 s. After being blot-dried, the demineralized dentin surfaces were treated for 30 s with each of the treatment solutions and rinsed three times (10 min each time) with ethanol. Eight dentin slabs were used for each treatment group, 4 of which were not subject to collagenase digestion, whereas the other 4 experienced 1 h of digestion at 37 °C in 0.1% collagenase solution. After rinsing, all the slabs were fixed in 2.5% glutaraldehyde buffered with 0.1M sodium cacodylate for 1 h, dehydrated in graded solutions of ethanol (33%, 67%, 85%, 95% and 100%) for 2 h each and dried overnight in vacuum desiccator. The slabs were fractured, mounted on aluminum stubs, and coated with carbon. The fractured cross-sections were examined in a FEI/Philips XL30 Field-Emission Environmental SEM (Philips, Eindhoven, Netherlands) at 2500, 5000 and 10,000 x magnification.
Collagen cross-linking effect and chemical interaction of MAPAs by FTIR
The remaining 10 treated and dried dentin films from each group were submitted to FTIR analysis (Spectrum One, Perkin-Elmer, Waltham, MA, USA) on a BaF 2 disc, in order to detect collagen cross-linking effect and chemical interaction of MAPAs. Each FTIR spectrum was collected in the 650–4000 cm −1 wavenumber range at a resolution of 4 cm −1 and 64 scans. For spectral analysis, the spectrum of untreated dentin film was subtracted from the spectra of treated films to elucidate the interactions between the cross-linkers and collagen. The integration areas of the band at 1604 cm −1 (associated with PA components) of the resultant difference spectra were calculated to show the extent of chemical interactions with collagen, while the integration band area at 1235 cm −1 (amide III) from untreated collagen was used as an internal standard/reference. Thus, a band ratio (1604 cm −1 /1235 cm −1 ) was calculated for each of the treatment groups.
After normality of distribution and homogeneity of variances (Komolgorov Smirnov and Levene tests, respectively), the averages were subjected to one-way analysis of variance and Tukey post hoc test (5%).
The synthesis of MAPAs was shown in Fig. 1 A. Functionalization of PA with MA was achieved in one step by reacting PA with varied amounts of methacryloyl chloride. Depending on the mole ratios of MA applied, various OH groups in the PA structure could react to form methacrylate ester groups (OMA) as shown as X, Y, Z in Fig. 1 A.
The 1 H-NMR spectra of PA and MAPAs in DMSO-d6 were shown in Fig. 1 B. Characteristically broad multiple-peak signals associated with the phenol OH protons (labeled as “a”), aryl protons (labeled as “b”), and the protons on the C2 and C3 of the C-ring (labeled as “d”) were identified for PA. While for MAPAs, the clear appearance of a new broad peak at δ = 6.0–6.4 ppm attributed to the MA vinyl protons (labeled as “c”) confirms the successful incorporation of MA into MAPAs. The integration ratios of signals a:b:c:d for MAPA-1, MAPA-2, and MAPA-3 were estimated to be 3.75:4.19:0.99:2.00, 2.02:3.23:1.10:2.00, and 0.68:2.14:1.25:2.00, respectively.
Representative FTIR spectra of PA and MAPAs are shown in Fig. 2 . Compared to the spectrum of PA, the appearance of new bands at ∼1720 cm −1 (CO, ester), ∼1650 cm −1 (CC , aliphatic), and 1284 cm −1 (ester CO stretching) of all three MAPAs is attributed to the MA functional group, confirming the grafting of MA onto PA. Meanwhile, the band intensities at 3200 cm −1 (OH stretching) for the three MAPAs were all reduced, reflecting the partial consumption of hydroxyl groups after reacting with MA. Among the three MAPAs, the relative intensities of the bands associated with MA functional group (i.e., 1720 cm −1 ) increased from MAPA-1 to MAPA-2 and to MAPA-3, indicating increased amount of MA grating, which was consistent with the 1 H NMR studies.
The stability results of dentin films, after treated with PA and MAPAs, against collagenase digestion were presented in Fig. 3 . The weight loss (WL) results ( Fig. 3 A) showed that all the cross-linkers led to significantly improved collagen biostability when compared to the untreated control (p < 0.001). While the collagen in the untreated group was completely digested (WL 100%), those treated with MAPAs showed significantly lower weight loss. Collagen treated with MAPA-1 (WL 10.52%) and MAPA-2 (WL 5.99%) showed the highest bio-stability, statistically comparable to that of PA (WL 8.79%) (p > 0.05). Collagen treated with MAPA-3, however, exhibited much lower biostability (WL 38.48%) (p < 0.001). Similar results were observed using HYP release measurements ( Fig. 3 B). HYP release is the highest in the untreated control (102.76 μg/mg), followed by MAPA-3 (29.49 μg/mg), while MAPA-2 (11.02 μg/mg) and MAPA-1 (13.53 μg/mg) showed the lowest (p < 0.001) and comparable HYP release that was statistically similar to that of PA (13.17 μg/mg) (p > 0.05).