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. 2022 Nov 26;18:100506. doi: 10.1016/j.mtbio.2022.100506

Mussel-inspired polymer with catechol and cationic Lys functionalities for dentin wet bonding

Zunhan Hu a,1, Wenzhen Wu b,1, Meizhe Yu c,1, Zhi Wang a, Zhenyu Yang d, Xiaodong Xing c, Xiaofang Chen e, Lina Niu d, Fan Yu d,f,∗∗∗, Yuhong Xiao a,∗∗, Jihua Chen d,∗
PMCID: PMC9719097  PMID: 36471892

Abstract

Mussels can form tough and long-lasting adhesions to organic and inorganic surfaces in saline and impactive severe aquatic environments. Similar to mussel adhesion, dentin bonding occurs in a wet environment. However, unlike mussels, it is difficult to achieve long-lasting bonds with dentin. Moreover, water is considered a major hindrance in dentin bonding. Inspired by the synergistic effect of cationic lysine (Lys) and catechol on the elimination of the hydration layer during mussel adhesion, a catechol- and Lys-functionalized polymerizable polymer (catechol–Lys–methacrylate [CLM]) was synthesized to replicate the complex synergy between amino acids and catechol. The bond-promoting potential of 5 ​mg/mL CLM primer was confirmed using an in vitro wet dentin-bonding model, which was characterized by an improvement in bond strength and durability. CLM can adhere to wet demineralized dentin, with Lys acting as a molecular vanguard to expel water. Subsequently, a myriad of interfacial interactions can be obtained by introducing the catechol group into the interface. Additionally, tough and long-lasting adhesion, similar to that formed by mussels, can be achieved by grafting CLM onto type I collagen via covalent bonds, hydrogen bonds, Van der Waals interactions, and cation–π interactions, which can enhance the mechanical and chemical stability of collagen, increase the enzymatic resistance of collagen, and provide additional physical/chemical adhesion to dentin bonds. Catechol- and cationic Lys-functionalized polymers can improve the stability of the resin–dentin interface under wet conditions.

Keywords: Dentin, Adhesive, Mussel, Catechol, Lysine, Bionics

Graphical abstract

Image 1

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1. Introduction

Mussels can form tough and long-lasting adhesions to organic as well as inorganic surfaces in a saline and impactive severe aquatic environment [[1], [2], [3]]. Because of its fascinating and versatile bonding ability, several bionic designs based on mussel adhesion have been applied in various fields, including biomedical engineering, soft robotics, soft electronics, environmental science, and energy science [[3], [4], [5], [6], [7], [8]]. Although the intricate mechanism of mussel bonding is still being studied, many mussel-inspired polymers have been evaluated extensively in the field of underwater bonding, which remains a difficult task that was once considered impossible for a man-made adhesive [[9], [10], [11]].

At present, composite resin restoration based on dentin–resin bonding technology has become the primary method for repairing tooth defects because of its good immediate adhesion performance, excellent esthetic effect, and minimally invasive nature [12,13]. Similar to mussel adhesion, dentin bonding occurs in wet conditions in which water from saliva, dentinal tubules, rinse fluids, and dental equipment is in contact with the dentin substrate. However, unlike mussel bonding, long-lasting dentin bonding, to which water is considered the main barrier, is difficult to achieve [[14], [15], [16]]. In general, dentin bonding is a process in which exogenous resins penetrate the demineralized collagen network [12,13]. The presence of an adequate amount of water within the demineralized dentin substrate can help maintain the three-dimensional structure of the collagen matrix and facilitate adhesive infiltration; however, excess water may hinder close contact between the adhesive monomers, thereby impeding complete hybridization. Under clinical conditions, precisely controlling the water content in the dentin matrix is impossible. Moreover, after hybridization is complete, it is difficult to remove excess water from the bonding interface. This residual moisture in the cured bonding interface can induce a series of degradation processes and undermine the durability of the bonding interface [17]. Recently, chemical dehydration protocols and extrafibrillar demineralization techniques have been introduced to address water-associated issues during dentin bonding; however, limitations still exist [[16], [17], [18], [19], [20], [21]].

Considering the similarities between dentin bonding and mussel adhesion, researchers have attempted to use mussel-inspired polymers to achieve reliable dentin bonding. The dynamic underwater bonding process by mussels is initiated with the deposition of the vanguard mussel foot catecholic proteins; i.e., protein-3 (Mfp-3) and Mfp-5, on the wet surface, which form an interfacial adhesion layer [3,22]. These catecholic proteins play a vital role in mussel bonding as the catechol moiety can form various chemical bonds with the substrate, including hydrogen bonds, Michael additions, cation–π interactions, and π–π coupling [[23], [24], [25], [26], [27], [28]]. Accordingly, recombined mussel adhesive protein and catechol-functionalized polymers have been used as primers or etchant additives in dentin wet bonding [[29], [30], [31], [32], [33], [34]]. According to previous studies, these highly active monomers can improve the types of chemical bonding forces and reduce collagen degradation at the bonding interface, thereby improving the durability of dentin bonding [[29], [30], [31], [32], [33], [34]]. However, these previous studies did not involve in-depth research on the dynamic bonding process involved in water contamination. Mussel adhesion is a process involving multiple components, especially in the eviction of the interfacial hydration layer [23]. A previous study demonstrated that although Mfp-1 and Mfp-3 exhibited different adhesion performances, their catechol contents were similar [35]. Thus, it is reasonable to speculate that using only catechol moiety-functionalized polymers fails to achieve a bonding performance equal to that of natural mussels [23].

Although catechol can provide abundant interaction force for the bonding interface, studies have indicated that interactions between other functional groups and a moist substance underlie the action of catechol in natural mussels. Recently, a study identified the synergistic effect of cationic lysine (Lys) and catechol on the eviction of the hydration layer. Maier et al. found that catechol- and Lys-functionalized siderophores, together with its analogs, achieved robust adhesion on wet mica surfaces, wherein Lys repelled the hydration layer from the mineral surface and enabled catechol binding to the underlying substance [36]. Subsequent studies have shed additional light on the optimal synergy obtained when coupling cationic amine residues and catechol within the same molecule [37,38]. However, to date, no catechol- or cationic Lys-functionalized polymers have been used in the field of dentin bonding. Therefore, to achieve reliable and durable dentin adhesion under wet conditions, a catechol- and Lys-functionalized polymerizable polymer (catechol–Lys–methacrylate [CLM]) was designed as inspired by the synergistic process of wet adhesion in mussels (Fig. 1). In this study, we synthesized a CLM monomer and evaluated its dentin-bonding potential. We also investigated the potential effects of CLM on the physicochemical properties of dentin collagen.

Fig. 1.

Fig. 1

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Schematic diagram of the synthesis of catechol–Lys–methacrylate (CLM). HOBt, 1-Hydroxybenzotriazole; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DIPEA, N, N-diisopropylethylamine; DEA, diethylamine; TEA, triethylamine.

2. Materials and methods

2.1. Materials

The materials used in this study are listed in Table 1.

Table 1.

Materials.

Reagents Manufacturer
Fmoc-L-Lys (Boc; 99.23%) and 2-hydroxyethyl methacrylate (HEMA, 97%) Bidepharm, Shanghai, China
2,3-dihydroxybenzoic acid Yuanye, Shanghai, China
Hydroxybenzotriazole (HOBt, >99%) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC–HCl, >99%) D&B, Shanghai, China
N, N-diisopropylethylamine (DIPEA, 99%), diethylamine (DEA, >99%), and triethylamine (TEA, 99%) Aladdin, Shanghai, China
Sodium bicarbonate (NaHCO3, >99.5%) and sodium chloride (NaCl, >99.5%) KESHI, Chengdu, China
Methanol (>99.5%) Hushi, Shanghai, China
Dichloromethane (DCM, >99.5%), anhydrous sodium sulfate (Na2SO4, >99%), tetrahydrofuran (THF, >99.5%), and 1,4-dioxane (>99.5) N&S, Nanjing, China
Bisphenol-A glycol dimethacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA), and diurethane dimethacrylate (UDMA) MilliporeSigma, St. Louis, MI, USA
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2.2. Synthesis and characterization of CLM

CLM was synthesized step-by-step (Fig. 1) as follows:

Step 1

Briefly, 5 ​g of Fmoc-L-Lys (Boc, 10.67 ​mmol) was completely dissolved in 25 ​mL of dichloromethane (DCM) in a three-necked flask, followed by the addition of hydroxybenzotriazole ([HOBt]; 1.59 ​g, 11.74 ​mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide ([EDC]; 2.25 ​g, 11.74 ​mmol) while stirring at room temperature. The resulting mixture was stirred at 0 ​°C for 30 ​min in a nitrogenous atmosphere. The mixture was washed with sodium bicarbonate and the collected DCM layer was washed with distilled water and saturated brine. The organic phases were dried over anhydrous sodium sulfate. After removing the DCM solvent, the raw product of 2-(methacryloyloxy)ethyl 2-[([(9H-fluoren-9-yl)methoxy]carbonyl)amino]-6-[(tert-butoxycarbonyl) amino] hexanoate (referred to as P1) was purified using silica gel column chromatography (ethyl acetate/petroleum ether, 1/2) to obtain the product (white solid, 4.2 ​g), with a yield of 67.79%.

Step 2

P1 was dissolved in 70 ​mL of DCM/diethylamine (v/v, 1/1) and stirred for 6 ​h at room temperature. DCM was added after the solvent evaporated. The crude product was purified using fast-gradient silica gel column chromatography (ethyl acetate/petroleum ether, 1/1; DCM/methanol, 15/1). After removing the solvent initially, all traces of the solvent were subsequently eliminated in a vacuum and 2.4 ​g of 2-(methacryloyloxy)ethyl 2-amino-6-[(tert-butoxycarbonyl)amino] hexanoate (referred to as P2, 93% yield) was obtained.

Step 3

P2 (6.71 ​mmol, 2.4 ​g), 2,3-dihydroxybenzoic acid (3.36 ​mmol, 0.518 ​g), and HOBt (3.36 ​mmol, 0.454 ​g) were shaken in 30 ​mL of tetrahydrofuran at room temperature for 5 ​min in a nitrogenous atmosphere. Subsequently, triethylamine (10.08 ​mmol, 1.40 ​mL) and EDC–HCl (10.08 ​mmol, 1.93 ​g) were added. The reaction was allowed to continue for 16 ​h at 35 ​°C. The crude material was extracted with DCM and the solution was washed with 0.1 ​N HCl, distilled water, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and the crude product of 2-(methacryloyloxy)ethyl 6-[(tert-butoxycarbonyl)amino]-2-(2,3-dihydroxybenzamido) hexanoate (P3) was purified using gradient column chromatography on a silica gel (DCM/methanol, 20/1). The solution was concentrated and 1.15 ​g of P3 (light yellow oil, 69% yield) was obtained.

Step 4

P3 was stirred with 50 ​mL of HCl-dioxan (4 ​N) for 1–2 ​h at room temperature. After the reaction was complete, the mixture was concentrated under a high vacuum and brown-colored products were obtained (0.81 ​g, oily liquid, 89% yield).

During synthesis, the proton nuclear magnetic resonance (1H NMR) spectra of the intermediate products and CLM were acquired using the Bruker Avance Ⅲ HD 500-MHz system (Bruker BioSpin, Switzerland). In addition, the cytotoxicity of CLM was compared with that of commercially available bisphenol-A glycol dimethacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA), hydroxyethylmethacrylate (HEMA), and diurethane dimethacrylate (UDMA) using the Cell Counting Kit-8 reagent as previously described [39] Briefly, the monomers were dissolved in dimethyl sulfoxide and mixed with a serum-free α-modified essential medium (HyClone, Logan, UT, USA) to obtain increasing concentrations (20, 40, 60, 80, 100, 120, 140, 160, and 180 ​μg/mL) of the monomers. Human dental pulp stem cells were seeded in a 96-well plate at 1 ​× ​104 ​cells per well and cultured in an α-modified essential medium (HyClone) supplemented with 20% fetal bovine serum, 100 U/mL penicillin G, 100 ​mg/mL streptomycin, and 50 ​mg/mL ascorbic acid (Millipore Sigma, St. Louis, MI, USA) at 37 ​°C. After culturing for 24 ​h, the culture medium was replaced with a serum-free medium during 24-h starvation. The cells were then cultured with different monomer solutions (n ​= ​6) for an additional 24 ​h, followed by the addition of 10 ​μL of the Cell Counting Kit-8 reagent to each well. After a 4-h incubation period, the absorbance of each well under 450 ​nm was measured using a multiplate reader (BIO-TEK, Winooski, VT, USA).

2.3. Bonding performance of CLM

Noncarious intact human third molars were collected from the Department of Oral Surgery, School of Stomatology, Fourth Military Medical University, following the protocol approved by the university's Institutional Review Board. The teeth were stored in 5% chloramine-T solution at 4 ​°C and used within one month after extraction. To evaluate the bonding potential of CLM, we dissolved CLM in deionized water to obtain 1, 5, and 10 ​mg/mL CLM primers. Pure deionized water was used as the blank control. We used a two-step etch-and-rinse adhesive called Single Bond 2 ([SB2]; 3 ​M ESPE) in the ensuing bonding protocol.

The effects of CLM treatment on the degree of conversion (DC) of SB2 were evaluated using attenuated total reflectance Fourier-transform infrared spectroscopy (ATR–FTIR; Shimadzu, FTIR-8400S, Kyoto, Japan) as previously described [31,32]. Briefly, the top plate of the ATR–FTIR spectrometer was wiped with ethanol. Five microliters of the CLM solution were premixed with SB2 in a ratio of 1:9, and then 20 ​μL of the mixed adhesive were smeared onto the top plate of the ATR–FTIR spectrometer. After gently air drying for 5 ​s, a layer of celluloid was applied on the surface of the adhesive and then scanned 24 times in the range of 4000–500 ​cm−1 ​at a resolution of 4 ​cm−1 before curing. After light curing for 20 ​s using a light-curing unit (Elipar S10, 3 ​M ESPE), the specimen was scanned at 40 ​s, 60 ​s, 5 ​min, 10 ​min, and 30 ​min. The absorbance at 1638 ​cm−1 (aliphatic C Created by potrace 1.16, written by Peter Selinger 2001-2019 C) and 1608 ​cm−1 (aromatic C–C) before and after curing was recorded, and the DC was calculated using the following formula: DC (%) ​= ​[1 ​− ​(1638 ​cm−1/1608 ​cm−1)cured/(1638 ​cm−1/1608 ​cm−1)uncured] ​× ​100(%).

A total of 128 teeth were randomly divided into four groups of 32 each. The occlusal one-third of the crown was removed using a diamond-impregnated cutting machine (SYJ-150A, Kejing, Shenyang, China). To create a standard smear layer, the obtained dentin surface was polished with 600-grit silicon carbide paper for 60 ​s under running water. After etching with 37% phosphoric acid for 15 ​s and rinsing, the specimens in each group were treated with deionized water and 1, 5, and 10 ​mg/mL CLM primers for 60 ​s. The SB2 adhesive was then applied and cured per the manufacturer's instructions. A cured 6-mm-thick composite buildup was established with the Z250 resin (Filtek Z250, 3 ​M ESPE) for each specimen. The prepared bonding specimens in each group were divided into two subgroups: the immediate group (immersed in distilled water for 24 ​h) and the aging group (thermocycling, 5 ​°C for 1 ​min, followed by 55 ​°C for 1 ​min, over 10,000 cycles).

Four 10 ​× ​1 ​× ​1-mm sticks perpendicular to the long axis of each tooth were used in the bond strength test as previously described [16]. The microtensile bond strength (μTBS) value of each specimen was measured using a microtensile testing machine (EZ-TEST 500 ​N, Shimadzu Co.) at a crosshead speed of 1 ​mm/min. Four μTBS values for each tooth were averaged (n ​= ​10). The debonded interface was observed under a stereomicroscope (MLC-150, Motic, Decatur, GA, USA) to determine the failure modes as described in a previous study; the failure modes were adhesive failure, cohesive failure within the dentin, cohesive failure within a resin composite, and mixed failure involving both interfacial and cohesive fractures [40].

The bonding interface was observed using field-emission scanning electron microscopy (FE-SEM, S-4800, Hitachi, Tokyo, Japan) as previously described [16]. Briefly, 1-mm-thick specimens perpendicular to the bonding interface were polished with silicon carbide paper, etched with 37% phosphoric acid, immersed in 5% sodium hypochlorite, ultrasonic-irrigated, fixed, dehydrated, sputter-coated with gold, and examined using FE-SEM.

The nanoleakage level of bonding interface was evaluated using FE-SEM as previously described [16]. Four 4 ​× ​1 ​× ​1-mm slices along the long axis from each tooth were coated with nail varnish, leaving an uncoated region of approximately 1 ​mm around the bonding interface. All specimens were then immersed in 50 ​wt% ammoniacal silver nitrate solution for 24 ​h, ultrasonically cleaned, soaked in a photo-developing solution, and exposed to fluorescent light irradiation for 8 ​h. All specimens were subsequently cleaned ultrasonically, dried with filter paper, sputter-coated with gold, and examined via FE-SEM in the backscattered mode.

The collagenase activity within the bonding interface after aging was also evaluated using confocal laser scanning microscopy (Fluoview FV1000; Olympus, Japan). SB2 was premixed with tetramethylrhodamine B isothiocyanate (Millipore Sigma, St. Louis, MI, USA) and three teeth from each group were bonded and aged as described above. Two-millimeter thick slabs that contained the bonding interface were obtained and subjected to an in situ zymography test using the EnzChek collagenase kit (E−12055; Molecular Probes, Eugene, OR, USA) as previously described [41].

2.4. Interaction between CLM and collagen

To evaluate the binding affinity of CLM to dentin collagen, molecular docking was performed using Molecular Operating Environment (Chemical Computing Group, Montreal, QC, Canada) [34,42]. Three crystal structures of type I collagen were selected from the Protein Data Bank database (IDs: 4OY5, 1CGD, and 1QSU). The two-dimensional structure of CLM was converted into a three-dimensional structure in Molecular Operating Environment according to the energy minimization process. The protonation state of the target and the orientation of hydrogen were optimized using LigX at a pH of 7 and temperature of 300 ​K. Docking was performed in the force field of AMBER10:EHT and the implicit solvation model of R-field. The binding site of collagen was identified using the Site Finder module in Molecular Operating Environment. Before the docking workflow, an “induced-fit” protocol was used to make the side chains of the collagen pocket mobilizable to ligand conformations, with a constraint on their positions. A weight of 10 was used for tethering sidechain atoms to their original positions. All docked conformations were ranked by London dG scoring, followed by force field refinement on the top 30 poses and rescoring based on the GBVI/WSA dG bonding free-energy scoring method [34].

To further characterize the interaction between CLM and collagen, we used time-resolved FTIR detection. A 1-mm-thick dentin slab was prepared perpendicular to the long axis of the tooth using a low-speed diamond saw and polished using 600-grit silicon carbide paper under running water. The slabs were etched with 10% phosphoric acid for complete demineralization, in which the endpoint of demineralization was determined by the dropwise addition of a 30% potassium oxalate solution [39]. The slabs were scanned using ATR–FTIR in the range of 4000–500 ​cm−1 with a resolution of 4.0 ​cm−1. The specimens were then immersed in CLM solution for 5 ​min. After ultrasonic cleaning, the specimens were scanned using ATR–FTIR.

2.5. Effects of CLM on the physicochemical properties and enzymatic stability of collagen

Tooth enamel was removed using a high-speed handpiece, and 1 ​× ​1 ​× ​6-mm dentin slabs parallel to the long axis of the tooth were prepared using a low-speed diamond saw. In total, 200 dentin specimens were etched with 10% phosphoric acid for 24 ​h and thoroughly rinsed with distilled water. All specimens were further treated with distilled water (blank control), 1 ​mg/mL CLM, 5 ​mg/mL CLM, 10 ​mg/mL CLM, or 2.5% glutaraldehyde ([GD]; positive control) for 5 ​min. Subsequently, 20 specimens in each group were evaluated for ultimate tensile strength (UTS), while the other 20 specimens were treated with 0.1 ​mg/mL collagenase (type IV Clostridium histolyticum, 160 units/mg, Aladdin) for 24 ​h. The UTS test was performed using a universal testing machine (EZ-TEST 500 ​N; Shimadzu) at a crosshead speed of 1 ​mm/min.

Thermogravimetric analysis was performed to evaluate the effects of CLM on the thermostability of demineralized dentin collagen. Briefly, dentin specimens without enamel, cementum, and pulp tissue were processed in liquid nitrogen for 30 ​min, pulverized, etched with 10% phosphoric acid for 48 ​h, and lyophilized. A total of 150 ​mg demineralized dentin powder was divided into five groups and treated with distilled water (blank control), 1 ​mg/mL CLM, 5 ​mg/mL CLM, 10 ​mg/mL CLM, or 2.5% GD for 5 ​min (n ​= ​5). The lyophilized powder for each group was processed using a thermogravimetric analyzer (TA Instruments, New Castle, DE, USA) from 20 ​°C to 200 ​°C at a heating rate of 10 ​°C/min.

One gram of the dried dentin powder was divided into five groups and treated with the same abovementioned solutions for 5 ​min. After being rinsed with distilled water, the powder for each group was placed in Eppendorf tubes containing 2 ​mL of 0.1 ​mg/mL type IV collagenase solution for 24 ​h at 37 ​°C. Subsequently, 500 ​μL of the supernatant was collected from each tube for hydroxyproline (HYP) estimation using an HYP detection kit (Solarbio, Beijing, China) per the manufacturer's instructions. The remaining powder was dried in a vacuum, and the dry mass loss of each group was calculated (n ​= ​5 for each group).

Zymography of demineralized dentin treated with CLM was evaluated using the EnzChek collagenase kit. The reaction solution was prepared by mixing 1 ​mg/mL DQ gelatin, 1 ​× ​buffer, and an antifluorescence quenching agent (DAPI H-1200) in a ratio of 1:8:1. Further, 2-mm-thick dentin slabs were etched with 37% phosphoric acid for 15 ​s and treated with distilled water, 1 ​mg/mL CLM, 5 ​mg/mL CLM, 10 ​mg/mL CLM, or 2.5% GD for 5 ​min (n ​= ​5). Mineralized dentin slabs served as blank controls. All slabs were treated with the reaction solution for 24 ​h in the dark (37 ​°C, 100% humidity), rinsed thoroughly, and examined using confocal laser scanning microscopy (Fluoview FV1000; Olympus, Japan) as previously described [16].

The effects of collagenase inhibition of CLM were evaluated using the EnzChek collagenase kit. Briefly, 0.4 U/mL collagenase solution, 1 ​× ​buffer, and 100 ​μg/mL DQ gelatin solution were prepared and kept in the dark before use. Further, 20 ​μL of the DQ gelatin solution and 100 ​μL of the collagenase solution (type IV collagenase, 0.4 U/mL) were added to each well of a 96-well plate. Finally, 80 ​μL of deionized water, 1 ​mg/mL CLM, 5 ​mg/mL CLM, 10 ​mg/mL CLM, or 1,10 phenanthroline was added into each well (n ​= ​5). The fluorescence intensities of each well before and after 24 ​h of incubation in the dark were measured using a GloMax® Discover Microplate Reader (Promega, Madison, WI, USA) at absorption and emission wavelengths of 495 and 515 ​nm, respectively.

2.6. Statistical analyses

All quantitative data are expressed as the mean ​± ​standard deviation. All data analyses were performed using SPSS 20.0 and the threshold for statistical significance was set at α ​= ​0.05. The data of failure mode distribution was analyzed using the chi-square test. A two-way analysis of variance was performed to characterize the effects of conditioning and aging on the dentin bond strength of the bonding interface and on the UTS of collagen. Least significant difference tests were used for pairwise comparisons. We also used one-way analysis of variance and post-hoc Fisher's least significant difference test after ascertaining normality and homoscedasticity.

3. Results

3.1. Synthesis and characterization of CLM

Fig. 2 presents the 1H NMR image of P1, P2, and CLM. The signals at 5.5–6.25 ​ppm were attributed to the protons of C Created by potrace 1.16, written by Peter Selinger 2001-2019 C of HEMA (Fig. 2a), indicating the interaction between the –OH of HEMA and the –OH of Fmoc-L-Lys (Boc). The removal of Fomc was confirmed by the disappearance of the signals at 7.25–8.0 ​ppm in the 1H NMR spectra of P2 (Fig. 2b) [43,44]. The signals at 6.42–7.04 ​ppm and 2.73 ​ppm confirmed the presence of catechol and Lys groups, respectively, in CLM (Fig. 2c) [43,44].

Fig. 2.

Fig. 2

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1H NMR spectra of intermediate products and catechol–Lys–methacrylate (CLM). (a)1H NMR spectra of P1. 1H NMR (500 ​MHz, chloroform-d) ​= ​δ 7.77 (d, 2H); 7.61 (dd, 2H); 7.40 (t, 2H); 7.32 (t, 2H); 6.12 (s, 1H); 5.59 (t, 1H); 4.63–4.31 (m, 8H); 3.10 (t, 2H); 1.93 (s, 3H); 1.89–1.68 (m, 2H); 1.49 (q, 2H); 1.43 (s, 9H); 1.40–1.32 (m, 2H). (b)1H NMR spectra of P2. 1H NMR (500 ​MHz, chloroform-d) ​= ​δ 6.10 (s, 1H); 5.58 (t, 1H); 4.44–4.22 (m, 4H); 3.43 (dd, 1H); 3.08 (q, 2H); 1.92 (s, 3H); 1.72–1.68 (m, 2H); 1.46(tt, 2H); 1.41 (s, 9H); 1.38–1.07 (m, 2H). (c)1H NMR spectra of CLM. 1H NMR (500 ​MHz, deuterium oxide) ​= ​δ 7.04–6.96 (t, 1H); 6.80–6.71 (d, 1H); 6.56–6.42 (m, 1H); 5.83–5.56 (t, 1H); 5.39–5.15 (t, 1H); 4.44–3.93 (m, 5H); 2.73 (q, 2H); 1.84–1.49 (m, 5H); 1.44 (m, 2H); 1.30–1.12 (m, 2H).

Fig. 3 shows the viability of human dental pulp stem cells exposed to different monomers after 24 ​h. Inhibited cell viabilities were observed for Bis-GMA, UDMA, and CLM at test concentrations. However, CLM showed a median lethal concentration (between 140 and 160 ​μg/mL) similar to that of UDMA, both of which were higher than that of Bis-GMA, thereby indicating the safety of CLM when used as a dental monomer.

Fig. 3.

Fig. 3

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Cytotoxicity analysis. Cell viability of human dental pulp stem cells exposed to (a) CLM, (b) Bis-GMA, (c) UDMA, (d) TEGDMA, and (e) HEMA at various resin monomer concentrations. The red dotted line represents 50% cell viability. Bis-GMA, bisphenol-A glycol dimethacrylate; TEGDMA, triethylene glycol dimethacrylate; UDMA, diurethane dimethacrylate; HEMA, 2-hydroxyethyl methacrylate.

3.2. Bonding performance of CLM

Fig. 4a shows the effects of CLM primers on the DC of SB2. At each time point, the DC values of the 10-mg/mL group were lower than those of the control group (all P ​< ​0.05). Compared with the control group, the DC decreased in the 1- and 5-mg/mL CLM groups; however, the differences were not statistically significant (P ​> ​0.05).

Fig. 4.

Fig. 4

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Bonding performance of CLM-based adhesion schemes. (a) Degree of conversion. Data are expressed as the mean ​± ​standard deviation. ∗P ​< ​0.05 vs. control. (b) μTBS values of the different bonding schemes. Data are expressed as the mean ​± ​standard deviation. For μTBS evaluated before aging, groups labeled with the same uppercase letter were not significantly different (P ​> ​0.05). After aging, groups labeled with the same lowercase letter were not significantly different (P ​> ​0.05). (c) Distribution of failure modes.

Fig. 4b presents the μTBS values of SB2 under different primer conditions. The bond strength was affected by the concentration of CLM and the aging mode (P ​< ​0.05). Before aging, treatment with 5 ​mg/mL CLM yielded the highest μTBS value among the groups (P ​< ​0.05). After aging, the μTBS values of all groups decreased significantly (P ​< ​0.01) and dentin conditioned with 5 ​mg/mL CLM showed the highest μTBS value (P ​< ​0.01). The μTBS value of the control group showed a drastic decrease of 45.03%, whereas that of the 5-mg/mL CLM group decreased by only 17.54%. Fig. 4c shows the distribution of the failure mode. The most common failure mode among the groups was mixed failure, and neither the aging mode nor the conditioning agent had any significant effect on the distribution of faliure modes (P ​> ​0.05).

As shown in Fig. 5a, an intact hybrid layer with abundant underlying resin tags was found in all groups before aging. There was no discernible visual difference in terms of bonding interfaces created by the different conditioning modes. After the aging process, a rough-looking hybrid layer and fractured resin tags were observed in the control group, whereas a more integrated hybrid layer with intact resin tags was preserved in the 5-mg/mL group. Fig. 5b presents representative images of the nanoleakage level. Before aging, only a few white silver depositions could be identified in the hybrid layer in the three CLM-conditioned groups, whereas distinct linear silver depositions were noted in the control group. After aging, all groups exhibited silver deposition at the bottom of the hybrid layer, and the 5-mg/mL CLM group had the lowest silver deposition of all groups.

Fig. 5.

Fig. 5

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Bonding interface observations. (a) SEM observation of the bonding interface before and after aging. Images on the right are magnified images of those on the left. Bar ​= ​10 ​μm. (b) Nanoleakage levels of the bonding interface before and after aging. Bar ​= ​10 ​μm. (c)In situ zymography of the bonding interface. Bar ​= ​50 ​μm.

We performed an in situ zymography test to reveal the collagenase activity within the bonding interface (Fig. 5c). The adhesive mixed with tetramethylrhodamine B isothiocyanate appeared as a red fluorescent region, while the enzymatic degradation product of exogenous gelatin substrates formed by the action of endogenous collagenase appeared as green fluorescence. In the control group, the bottom of the bonding interface showed marked green fluorescence; however, this phenomenon was rare in other CLM-treated groups.

3.3. Interactions between CLM and collagen

Fig. 6a illustrates the docking patterns for targeted type I collagen variants. As shown by molecular docking, CLM can form a suitable steric complementarity with the binding site of 1QSU, 1CGD, and 4OY5, separately. The binding free-energy between CLM and type I collagen 1QSU was −3.83 ​kcal/mol. Both hydrogen bonds and covalent interactions were formed between CLM and 1QSU. The nitrogen atom in CLM, which is regarded as a hydrogen acceptor, formed a hydrogen bond with the backbone nitrogen atom of Gly12. The sidechain nitrogen atom of Lys14 formed a covalent bond with CLM. Van der Waals (VDW) interactions were formed between CLM and the surrounding residues. These interactions mainly contributed to the binding energy between CLM and the protein 1QSU. The binding affinity between 1CGD and CLM was −4.71 ​kcal/mol. A hydrogen bond was formed between CLM and 1CGD. The oxygen atom in CLM, which is considered the hydrogen acceptor, formed a hydrogen bond with the backbone nitrogen atom of Gly24. The other oxygen atom in CLM formed a hydrogen bond with the sidechain oxygen atom of Hyp26. VDW interactions were formed between CLM and the surrounding residues. The binding affinity between 4OY5 and cpd2 was −4.60 ​kcal/mol. Both hydrogen bonds and cation–π interactions were formed between CLM and 4OY5. The oxygen atom in CLM, considered a hydrogen acceptor, formed a hydrogen bond with the backbone nitrogen atom of Gly4. The benzene ring in CLM formed a cation–π interaction with the nitrogen atom of Gly1. VDW interactions were formed between CLM and the surrounding residues.

Fig. 6.

Fig. 6

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Interaction between collagen and CLM. (a) Representative illustrations of the docked collagen–CLM complex. Left: two-dimensional bonding pattern. Right: three-dimensional visualization. (b) FTIR spectra of CLM-treated collagen.

As shown in Fig. 6b, the inherent Amide A (3297 ​cm−1), Amide I (1632 ​cm−1), Amide II (1551 ​cm−1), and Amide III (1239 ​cm−1) of collagen was found in untreated as well as CLM-treated collagen. After CLM treatment, the Amide I peak shifted to a lower wave number. After the reaction, the appearance of peaks at 1638 ​cm−1, 1722 ​cm−1, and 669 ​cm−1 originating from the –C Created by potrace 1.16, written by Peter Selinger 2001-2019 C, –C Created by potrace 1.16, written by Peter Selinger 2001-2019 O, and –OH groups in the phenol ring of CLM indicated the grafting of CLM onto collagen.

3.4. Effects of CLM on the physicochemical properties and enzymatic stability of collagen

Fig. 7a presents the results of the UTS test of collagen. Regardless of the aging process, CLM treatment increased the UTS of collagen in a concentration-dependent manner. The UTS increased in the CLM and GD groups before aging (P ​< ​0.05). There was no significant difference between the 2.5% GD and 1-mg/mL CLM groups (P ​> ​0.05). After collagenase-mediated aging, the UTS decreased significantly in all groups (P ​< ​0.01). The T5% value of demineralized dentin collagen increased significantly after CLM and GD treatment (P ​< ​0.01), indicating improved thermal stability of collagen (Fig. 7b).

Fig. 7.

Fig. 7

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Effects of CLM on the physicochemical properties and enzymatic stability of collagen. (a) Ultimate tensile strength. (b) 5% mass loss temperature. (c) Dry mass loss. (d) HYP release. (e) Zymography of the dentin matrix under different treatments. Bar ​= ​50 ​μm. (f) Quantification of collagenase activity. Data are expressed as the mean ​± ​standard deviation. Groups designated with different letters were significantly different (P ​> ​0.05). (g) Inhibitory effect of CLM on type IV collagenase. All data are expressed as the mean ​± ​standard deviation. Groups labeled with the same letter were not significantly different (P ​> ​0.05).

After collagenase-mediated aging, collagen in the control group exhibited the highest loss of dry mass among all the groups (P ​< ​0.01; Fig. 7c). As evidenced by the lower mass loss values than those of controls, both CLM and GD treatment can prevent degradation by enzymolysis (all P ​< ​0.01). To detect the degree of collagen degradation, we performed an HYP release test (Fig. 7d), and its results were consistent with those of the dry mass loss test in which a lower amount of HYP could be found in the CLM and GD groups than in the control group (P ​> ​0.05).

After etching, the host-derived enzymes embedded in the dentin mineral were released and activated in an acidic environment, initiating a cascade of the proteolytic degradation of collagen [45]. As shown in Fig. 7e and f, green fluorescence represents the activity of the endogenous enzymes of dentin. A lower intensity of green fluorescence can be observed in the CLM- and GD-treated groups, indicating inhibited collagenase activity. Our results also indicated that CLM can inhibit the activity of type IV collagenase in a concentration-dependent manner (Fig. 7g; P ​< ​0.01).

4. Discussion

Water has long been considered a double-edged sword in dentin bonding. A certain amount of water during dentin demineralization is a prerequisite for the penetration of adhesive resin monomer to keep the collagen network from collapsing. However, the water that cannot be removed completely from the bonding interface negatively affects the interaction between collagen and resin monomers, adhesive polymerization, and other hazards of bonding durability. Inspired by the remarkable underwater bonding efficiency of mussels, we synthesized a water-expelling dentin adhesive monomer and confirmed its bonding-promoting potential in an in vitro wet dentin-bonding model characterized by improved bond strength and bonding durability. In addition, the potential benefits of CLM on the physicochemical properties of collagen were verified.

To address the disadvantages of the presence of water on dentin adhesion, researchers have proposed a few mussel-inspired catechol-containing polymers for use in dentin bonding that showed promising results [[30], [31], [32],34]. However, these simplified polymers failed to capture the complex synergy between amino acids and catechol in natural Mfps. Inspired by the synergistic effects of catechols and cationic amino residues within vanguard Mfp-3 and Mfp-5 proteins on mussel underwater bonding, we synthesized a multifunctional CLM monomer containing a functional catechol group and cationic Lys to achieve dynamic water-expelling properties and promote robust adhesion during dentin bonding. To ensure the capacity of copolymerization with other resin monomers, we also grafted a methacrylate group onto CLM. The structure of CLM was confirmed via NMR spectroscopy (Fig. 2c). In addition, CLM exhibited comparable cytotoxicity to common commercially used dental monomers, including Bis-GMA, TEGDMA, and HEMA, indicating that it can be used in a dental adhesive formula.

To evaluate the boding potential of CLM, we formulated an experimental primer containing CLM and established an in vitro dentin-bonding model. The μTBS test revealed that pretreatment with 5 ​mg/mL CLM achieved the highest μTBS among all groups, regardless of aging (Fig. 4b). Compared with the control group, the value of immediate bond strength increased by 12.64% in the 5-mg/mL CLM group. After aging, the control group showed a drastic decrease of 45.03%, which was consistent with the results of previous studies [29,[31], [32], [33], [34]]. However, the bond strength in the 5-mg/mL CLM group decreased by only 17.54%, indicating improved bonding durability. In addition, a more integrated bonding interface was observed in the 5-mg/mL CLM group via FE-SEM and the nanoleakage assay, indicating adequate adhesive penetration into the demineralized collagen matrix. Our results were in accordance with those of previous mussel-inspired dentin-bonding studies in which catechol-functionalized adhesives exhibited improved bond strength and durability [[31], [32], [33], [34]]. However, it is difficult to make exact comparisons among these works owing to different solvent types, adhesive types, treatment times, and test conditions.

There may be several reasons for the improved bonding performance of the experiment primer. The water-expelling effect of Lys and the high chemical affinity of catechol provide a “one–two punch,” thereby remarkably increasing the strength of the bonding interface [[36], [37], [38]]. The cationic Lys of CLM acted as a vanguard moiety to break through the hydration layer on the surface of the substance. Additionally, the hydrophobic groups of CLM, such as ester and alkyl groups, may move spontaneously toward the hydrophobic amino residues in demineralized collagen via hydrophobic interactions, further facilitating the eviction of water [46]. Accordingly, CLM can achieve close contact with wet demineralized dentin and a myriad of interfacial interactions can be obtained by introducing the catechol group into the interface, including hydrogen bonding, metal-ion coordination bonds, Michael additions, π–π interactions, cation–π interactions, and electrostatic interactions [[23], [24], [25], [26], [27], [28], [47]]. In addition, to remove the hydration layer, Lys can amplify the cation–π interactions and enhance cohesion [48,49]. These abovementioned interfacial interactions of CLM may play a vital role in the efficiency of dentin bonding, as additional chemical adhesion within the bonding interface was reported to contribute to the high quality of the hybrid layer against harmful biological and chemical activities [39,50,51].

In addition, the high bond strength and integrated hybrid layer in the 5-mg/mL group indicated an acceptable polymerization of the adhesive resin. This was evidenced by the results of DC. Our results showed that low concentrations of CLM (1 and 5 ​mg/mL) did not affect resin polymerization, whereas a high concentration of CLM (10 ​mg/mL) showed an inhibitory effect. This might be attributed to catechol's reducibility. Catechol acts as a free radical scavenger and eliminates oxygen free radicals that are generated during polymerization. Previous studies have shown that catechol-functionalized dopamine methacrylamide decreases the DC of Adper SB2 at high concentrations [31,33]. In addition, high concentrations of catechol-rich proanthocyanidin were also reported to interfere with polymerization [52,53]. These findings are in agreement with the results of our study in which 10 ​mg/mL showed decreased DC and minimal satisfactory bonding performance regarding the bond strength values and silver nanoleakage levels.

The biodegradation of collagen components within the bonding interface mediated by matrix metalloproteinases (MMPs) has been well acknowledged as one of the main causes of bonding failure. Strategies including the use of collagen cross-linkers and MMP inhibitors have been recommended to improve the stability of collagen within the bonding interface [54,55]. It has been well documented that catechol-containing agents, including proanthocyanidin and DMA, have the ability to crosslink collagen and inhibit MMP activity [[31], [32], [33], [34],56,57]. In this study, in situ zymography revealed that the activity of endogenous MMPs was inhibited significantly in the CLM group, indicating that CLM might have similar effects on the enzyme-mediated collagen degradation process to those of proanthocyanidin and DMA. Therefore, we further explored the interaction between CLM and collagen and its effects on collagen stability.

The reactivity between CLM and collagen was confirmed by molecular docking and FTIR spectroscopy. The results of molecular docking indicated that CLM could bind to type I collagen through covalent bonds, hydrogen bonds, VDW interactions, and cation–π interactions. The appearance of C Created by potrace 1.16, written by Peter Selinger 2001-2019 O (1724 ​cm−1) and C Created by potrace 1.16, written by Peter Selinger 2001-2019 C (1638 ​cm−1) bonds in CLM-treated collagen indicated the grafting of CLM (Fig. 5b). In addition, the shifts of Amide I, Amide II, and Amide III could be attributed to the formation of hydrogen bonds, Michael additions, electrostatic interactions, and cation–π interactions between catechol and collagen. The abovementioned results are consistent with the findings of previous studies [58,59]. Catechol on Mfps can form not only hydrogen bonds with the O, F, and N atoms on the surface of organic and inorganic materials but also cation–π interactions with cationic amino acid residues in collagen, such as Lys and arginine [34,60]. Furthermore, the catechol moiety of DMA has been confirmed to form hydrogen bonds, covalent bonds, and VDW interactions with collagen, which is in agreement with the results of our study [34]. Accordingly, the collagen–CLM complexes formed can chemically bond to other adhesive resin monomers via the polymerization of methacrylate bonds. Although mechanical interlocking has always been regarded as the main bonding mechanism for dentin bonding, additional chemical bonding forces between collagen and adhesive resin may strengthen the bonding interface [39,40,50,51]. In this study, the high chemical affinity of binding of the catechol structure on CLM to collagen and other methacrylic resins may facilitate the fomation of a more perfect bonding interface.

Our results also showed improvement in the mechanical strength, thermal stability, and enzymatic stability of CLM-treated collagen. These results are consistent with the findings of previous studies in which catechol-functionalized DMA improved the physicochemical stability of collagen due to its collagen-crosslinking effects [34,40]. Similar to proanthocyanidin and DMA, CLM also showed a collagenase inhibitory effect, as evidenced by the collagenase inhibition test. This was further confirmed by zymography of the demineralized dentin, in which endogenous collagenase activities were inhibited in the CLM-treated groups. This might be attributable to the metal-chelating ability of the catechol structure on CLM, which competitively “preys” on Zn+ and Ca2+ (both of which are necessary for the activation of collagenase) in the surrounding environment.

5. Conclusion

We propose that CLM can act, similarly to the natural mussel protein Mfp-3, as a “molecular vanguard” in the bonding process, with catechol and Lys providing a synergistic “one–two punch” to destroy the hydrated layer on its surface and create a water-free microlayer. This creates a local water-free microenvironment, facilitating the introduction of the bonding agent to form a more perfect bonding interface. The polymerizable C Created by potrace 1.16, written by Peter Selinger 2001-2019 C bond on CLM enables copolymerization between the CLM–collagen complex and other resin monomers, leading to the formation of a monoblock. Furthermore, the interactions between CLM and collagen can enhance the mechanical and chemical stability of collagen, increasing its enzymatic resistance and providing additional physical/chemical adhesion to the dentin bonds, which results in the formation of a tough and long-lasting bond, similar to that formed by natural mussels.

Author contributions

Z.H. Hu: Conceptualization, Methodology, Investigation Validation, Formal analysis, Writing - Original Draft. W.Z Wu: Methodology, Investigation Validation, Formal analysis, Writing - Original Draft. M.Z. Yu: Methodology, Investigation Validation, Formal analysis, Writing - Original Draft. Z. Wang: Validation, Formal analysis, Resources, Visualization. Z.Y. Yang: Validation, Resources. X.D. Xing: Conceptualization, Validation, Resources. X.F. Chen: Formal analysis. L.N. Niu: Validation, Resources. F. Yu: Conceptualization, Methodology, Investigation Validation, Formal analysis, Writing - Original Draft, Writing - Review & Editing, Supervision. Y.H. Xiao: Conceptualization, Writing - Review & Editing, Supervision, Funding acquisition. J.H. Chen: Conceptualization, Writing - Review & Editing, Supervision, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (81720108011, 81970972, 82201112).

Contributor Information

Fan Yu, Email: yufanfmmu@163.com.

Yuhong Xiao, Email: xiaoyuhong56@126.com.

Jihua Chen, Email: jhchen@fmmu.edu.cn.

Data availability

Data will be made available on request.

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