Catechol-thiol-based dental adhesive inspired by underwater mussel adhesion

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Catechol–Thiol-based Dental Adhesive Inspired by Underwater Mussel Adhesion Dohoon Lee , Hyogeun Bae , Jinsoo Ahn , Taegon Kang , Deoggyu Seo , Dong Soo Hwang PII: DOI: Reference:

S1742-7061(19)30804-9 https://doi.org/10.1016/j.actbio.2019.12.002 ACTBIO 6485

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Acta Biomaterialia

Received date: Revised date: Accepted date:

4 September 2019 22 November 2019 2 December 2019

Please cite this article as: Dohoon Lee , Hyogeun Bae , Jinsoo Ahn , Taegon Kang , Deoggyu Seo , Dong Soo Hwang , Catechol–Thiol-based Dental Adhesive Inspired by Underwater Mussel Adhesion, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.12.002

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Catechol–Thiol-based Dental Adhesive Inspired by Underwater Mussel Adhesion Dohoon Lee a, Hyogeun Bae b, Jinsoo Ahn c, Taegon Kang d*, Deoggyu Seo e*, Dong Soo Hwang a,f* a

Division of Environmental Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, 37673, Korea b

Division of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang, 37673, Korea c

Dental Research Institute and Biomaterials Science, Dentistry, Seoul National University, Seoul, 110–749 South Korea d

Lotte Advanced Materials Corporation, Gocheon-Dong, Uiwang-Si, Gyeonggi-Do 16073, Korea e

Department of Conservative Dentistry and Dental Research Institute, School of Dentistry, Seoul National University, 101 Daehak-ro, Jongno-gu, Seoul, Korea f

Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, 37673, Korea *Corresponding authors E-mail address: [email protected] (T. Kang), [email protected] (D. Seo), [email protected] (D. S. Hwang).

Graphical abstract

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Abstract The critical problem associated with the underwater mussel adhesive catechol-based 3,4dihydroxy-L-phenylalanine (DOPA) is its sensitivity to oxidation. To overcome this problem, mussels underwent etching in the presence of acidic pH conditions (<3.0), and thiol chemistry was used to control the propensity of DOPA for oxidation. Similar strategies deployed by mussels are also actively utilized in dental adhesives which undergo etching in the presence of phosphoric acid derivatives to maximize the bonding strength and adapt thiol chemistries to minimize shrinkage stress. In view of the similarities between dental and underwater mussel adhesives, we employ in this study the strategy of mussel adhesion—the combination of DOPA and thiol chemistry with acid etching—to one of the most critical issues in dental adhesives, namely, the dentin bonding with zirconia. As a result, the adhesion bonding between zirconia and dentin, one of the most elusive problems in dentistry, has improved compared to the commercially available adhesive resin formulation. In addition, in view of the similar human oral and mussel adhesive environments, our findings will considerably contribute to the translation of the adhesive system inspired by mussels. Statement of Significance Mussels are effectively operated by creating an acidic environment when adhering with 3,4dihydroxy-L-phenylalanine (DOPA)–thiol redox chemistry for underwater bonding. Similarly, in dental adhesives, phosphoric acid-based etching is used for dentin-bonding materials. In view of the similarity between dental adhesives and underwater mussel adhesives, the combination of DOPA and thiol chemistry with acid etching can be used to overcome one of the most critical issues in dentin medical adhesives. The proposed adhesion method produces high adhesion strengths compared to those currently used in dentin and zirconia adhesives. Here, we extend and evaluate dentin and zirconia dental adhesives by mixing with mussel (DOPA)–thiol redox chemistry and acid etching.

Keywords: dental adhesive, self-healing, catechol, 3,4-dihydroxy-L-phenylalanine (DOPA), zirconia

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1. Introduction The adhesive system of mussels possesses many intriguing properties that can be studied to engineer superior multifunctional devices and materials that would be useful and beneficial to man [1]. Mussels can be attached tightly to wet and rough surfaces owing to their specialized holdfast called byssus in adverse and turbulent environments which include saline conditions, variations of fluid flow, dynamic changes in temperature and pH, and the exposure of the effects from other microbes and enzymatic activities [2, 3].The human oral environment possesses many similarities compared to the mussel living environment. The oral environment has natural salinity, and generates mechanical stresses from chewing food and the flow of saliva, stresses induced at varying temperatures and pH values, as well as biological stresses from the effect of plague by microbes [4]. While the tissues in the oral environment have slowly evolved from the biological functional point-of-view to better adapt to the aforementioned environments manifested at various levels of adversity compared to marine organisms, additional time is required for adaptation given the longer history of the marine organisms. Thus, the knowledge of numerous biological mechanisms in marine organisms provides useful information for emulating dental and other biomedical technologies. The byssus of the mussels are composed mainly of a variety of adhesive proteins that play important roles in their adhesiveness to endure all the aforementioned conditions [3]. As these proteins are mainly secreted from the foot of the mussel, they are also known as mussel foot proteins (mfps) [2, 3]. The key chemical functionality present in the mfps is the postranslationally modified amino acid 3,4-dihydroxy-L-phenylalanine (DOPA) [4]. The catecholic moiety of DOPA forms strong and reversible coordination complexes with metal ions and metal oxides, or undergoes covalent crosslinking with neighboring chemical moieties [5]. Given the aforementioned similarities of the underwater and the human oral

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environments, it is logical to assume that DOPA can potentially serve as a good candidate that can be used for dental adhesives. However, there are several difficulties in introducing DOPA directly in more practical application fields. One of them is the poor oxidation stability of DOPA. Given that the two hydroxyl groups on its benzene ring can be easily oxidized, the molecule is prone to change to dopaquinone [6, 7]. Although dopaquinone has a crosslinking role in nature, it causes DOPA to lose its own adhesiveness. To circumvent this, two strategies have been adopted by mussels, i.e., applying a) adhesives in acidic pH and b) thiol-rich reducing environments [8, 9]. Mussels create a local acidic environment (pH~2) by using their foot proteins when they secrete adhesive proteins [10]. The acidic pH is beneficial in protecting DOPA in the adhesives from being oxidized and allows the extinction and removal of microbes from the adhesive surface. At the same time, the thiol-rich antioxidant proteins mfp-6s, are co-secreted with DOPA to minimize the DOPA oxidation. With these two strategies, the underwater adhesives of mussels can yield the best underwater-adhesion performance in the marine environment [11]. Interestingly, the aforementioned two strategies adopted to minimize DOPA from oxidation—acid etching and thiol chemistry—are applied in dental adhesive technologies [12, 13]. Firstly, dental adhesives in the clinical treatments for dental caries are generally applied after acid etching of the teeth with phosphoric acid derivatives, such as phenyl hydrogen phosphate (phenol-P), and 10-methacryloyloxy decyldihydrogen phosphate (10 MDP) [14]. The introduction of thiol-rich crosslinkers has been used as the strategy to minimize shrinkage stress in the dental adhesive during the photopolymerization of the dental resin and adhesives [15]. Indeed, pentaerythritol tetra(3-mercaptopropionate) (PETMP), which is a tetra-functional crosslinker with four thiol groups, dramatically reduced shrinkage stress, volume shrinkage, leachable species when it was used as a component in acyrl-based dental

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resin [16]. The acidic condition in the dental adhesive technology not only provides an ideal environment for DOPA-containing adhesives and their easy incorporation in the conventional dental adhesives, but also minimizes DOPA oxidation which causes DOPA lose its own adhesiveness.

In addition, the poly thiols used for the reduction of shrinkage stress in the

dental resin formulation ensure that the DOPA remains in its reduced state without losing its adhesiveness. Therefore, in view of the similarities between dental adhesion and mussel adhesion environments, we applied the strategy of mussel adhesion — the combination of DOPA and thiol chemistry with acid etching — to the design of dental adhesive formulation. We synthesized a crosslinker containing both DOPA and thiol moieties, and observed that the addition of the crosslinker to the currently used dental formulation yields an enhanced adhesion between dentin and Zircornia, compared to the currently used dental formation.

2. Materials and Methods 2.1 Synthesis of Silyl-protected Catechol (SPC): Eugenol (1.64 g, 1.0 equivalent) and triethylsilane (2.56 g, 2.2 equivalents) were placed into a 100 mL round bottomed flask and were stirred for 5 min to ensure a uniform environment with the condenser. The flask was immersed in tap water at room temperature (25 °C) to eliminate the influences of temperature changes. The catalyst, tris(pentafluorophenyl)borane (10.2 mg, 0.002 equivalents) was added to the mixture and stirred. After the vigorous formation of gas was observed, the reaction mixture was stirred for an additional period of 10–30 min. The reaction mixture was collected and diluted with dichloromethane, and tris(pentafluorophenyl)borane was then removed by filtering the mixture through a neutral alumina-filled syringe with dichloromethane as the eluent. The filtered mixture was maintained under decreased pressure to remove the

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dichloromethane solvent and unreacted triethylsilane to produce silyl-protected eugenol, which is a transparent or slightly yellowish liquid. The product was used for another synthesis without further purification steps. 2.2 Synthesis of SPC Thiol Compound: The SPC (3.74 g, 7.48 g, 11.22 g, 14.96 g, from 1 to 4 equivalents), pentaerythritol tetra (3-mercaptopropionate) (4.89 g, 1 equivalent) and photo-irradiation 2,2-dimethoxy-2-phenylacetophenone (DMPA) (25.6 mg, 51.2 mg, 76.8 mg, 102.4 mg, from 0.01 to 0.04 equivalents for SPC) were placed in a clean glass vial. The mixture was dissolved with 10 ml of acetone using vortexing. The solution was purged with argon gas for 5 min and was exposed to UV (350 nm, 15 W, Sylvania 350BL) for 30 min. Some impurities were filtered through a 0.2 µm syringe filter. Solvent was removed by decreasing the pressure. The products of this procedure were named PETMP–mono-SPC, PETMP–di-SPC, PETMP–tri-SPC, and PETMP–tetra-SPC, respectively. 2.3 Nuclear Magnetic Resonance (NMR) Measurements of Synthesized Compounds: The 1

H spectra of SPC and PETMP–SPCs were obtained via NMR using a Bruker Advance III

FT-NMR spectrometer at 7.05 T (resonant frequency of 300 MHz). Given that all the molecules were well dissolved in chloroform solvent, deuterium substituted chloroform was used as the NMR solvent. Each sample was scanned for 32 times and the results were averaged. Acquired free induction decay (FID) spectra were Fourier transformed to generate spectra as functions of chemical shifts for each specific chemical bond in unit of parts per million (ppm). 2.4 Test Surface Preparation Procedures: For the adhesion test, transparent acryl bars (1 cm  3 cm  0.5 cm) were prepared. Given that the surface of the original acryl bars was too smooth, it was sanded by sandpapers to make it rough and uneven. Sanded bars were rinsed with water to remove dust and were sonicated for 15 min. After additional water rinsing, they were dried under the ambient atmosphere.

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2.5 Lap Shear Bond Strength Testing Sample Preparation and Measurement: After loading 100 μl of the five types of samples on a sanded acryl bar, the bar was covered with another acryl bar that also sanded with an overlapping area of 1 cm × 1 cm. Fe3+ (with a concentration of 10 mM in acetone) was added at a molar ratio of 1:3 (Fe3+:catechol). The tensile strengths of the prepared acryl bars were tested after 24 h using the Instron Universal Testing Machine (UTM). The sample bars were loaded using a 10 N load cell and were stretched uniaxially at a rate of 0.5 mm/min until the cured samples failed. The experiments were repeated 10 times. 2.6 Preparation of Zirconia and Bovine Tooth Specimens and Surface Treatments: Zirconia specimens were prepared with 97% zirconium dioxide stabilized with 3% YttriaLava Frame (3M ESPE, St. Paul, MN). By cutting these blocks with a low concentration diamond blade (Allied High Tech Productions Inc., Compton, CA), zirconia disk specimens with a thickness of 4 mm were fabricated. The surfaces of each specimen were polished with 120 grit silicon carbide abrasive and ground with 600 grit silicon carbide abrasive (PACE Technology, Tucson, AZ, USA) in the presence of water used for cooling purposes. After polishing, the blocks were cleaned in an ultrasonic bath of distilled water for 3 min and were then sintered. Specimens were embedded in a polyethylene mold (19 mm inner diameter, 21 mm outer diameter, and 12 mm height) with one side of the disk exposed to the resin. Other types of zirconia block specimens were also fabricated for the bovine tooth-zirconia adhesive test. Zirconia blocks were cut into 3 mm squares after a sintering process with a lowconcentration diamond blade. Bovine tooth specimens were prepared using an anterior bovine tooth. The root of the anterior tooth was cut, the pulp was completely removed and washed with ethanol, and the pulp space was filled with a Guttapercha bar. To fix the bovine specimen with resin, these specimens were embedded in a polyethylene mold (19 mm inner diameter, 21 mm outer diameter, 12 mm height) with one side exposed to the labial side of the anterior tooth. The specimens were polished using 120 and 600 grit SiC abrasive papers

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(PACE Technology, Tucson, AZ, USA) to expose dentin in the presence of water used for cooling purposes. After polishing, bovine tooth specimens were cleaned in an ultrasonic bath of distilled water for 3 min. 2.7 Knife-edge Shear Bond test for Zirconia and resin cement Specimens in only PETMP catechol: Specimens were fabricated from Zirconia – resin cement to measure only PETMP catechol adhesion in two groups (n = 10 per group, zirconia–resin cement PETMP monocatechol and zirconia–resin cement PETMP mono-catechol–IO4). A total of 20 μl of PETMP – mono – catechol was applied to the zirconia specimen, and resin cement (RelyXTM U200 Automix, 3M ESPE, Seefeld, Germany) was mixed according to the manufacturer's instructions, poured into a #5 gel-cap (area 16.8 mm2), and placed on the surface and bonded with each specimen (n = 10). And polymerizing resin cement with a LED curing light (EliparTM S10, 3M ESPE, Seefeld, Germany) at 600 mW/cm2 on four sides for 20 s each. A radiometer (checkMARC®, BlueLight Analytics Inc., Halifax, NS, Canada) measured the LED curing light intensity as 600 mW/cm2. Then, tetrabutyl ammonium (meta)periodate (100 mM in ethanol) was added to the PETMP – mono – catechol IO4 specimen and treated in the same manner. The specimens were left to crosslink catechol at room temperature (25 °C) for 24 h. The knife-edge shear bond strength test was performed according to ISO/TS 11405: 2015 using an Instron Universal Testing Machine (UTM, 3344, Instron, Norwood, MA, USA) with a crosshead speed of 0.5 mm/min.

A load was applied, and the maximum force

was measured until the specimen failed. 2.8 Knife-edge Shear Bond test for Zirconia – Bovine Tooth and Zirconia – resin cement Specimens in resin adhesive system: The adhesion of the resin adhesive system was measured on the zirconia – bovine tooth and zirconia–resin cement surfaces in all eight groups (n = 10 per group, bovine tooth–zirconia Bis-GMA resin, bovine tooth–zirconia PETMP resin, bovine tooth–zirconia PETMP mono-catechol resin, bovine tooth–zirconia PETMP mono-

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catechol -IO4 resin, zirconia–resin cement Bis-GMA resin, zirconia–resin cement PETMP resin, zirconia–resin cement PETMP mono-catechol resin and zirconia–resin cement PETMP mono-catechol -IO4 resin). Bovine tooth – zirconia adhesion was measured by applying 20 μl of each resin adhesive on the surface of bovine tooth specimen, placing the 3 mm zirconia block, and polymerizing (n = 10) separately to each specimen with a LED curing light (EliparTM S10, 3M ESPE, Seefeld, Germany) at 600 mW/cm2 on four sides for 20 s each. In zirconia–resin cement, 20 μl of each resin adhesive was applied on the surface of zirconia each specimen, and resin cement (RelyXTM U200 Automix, 3M ESPE, Seefeld, Germany) was mixed according to the manufacturer's instructions, poured into a #5 gel-cap (area 16.8 mm2), then gel-cap was placed on zirconia spacimen and polymerized using the same LED curing light. The specimens were left to polymerize resin adhesives at room temperature (25 °C) for 24 h. The knife-edge shear bond strength test was performed according to the above method. 2.9 Cytotoxicity Test: A cytotoxicity test was carried out using commercially available human fibroblast cells (CCL-1: ATCC, USA). Standard 24-well culture plates were coated with poly dimethylaminoethyl methacrylate. Polymer-coated flasks were sterilized with ethylene oxide. Specimens from each of the dental adhesive compounds (3M-SBU, Bis-GMA, PETMP, PETMP mono-catechol) were extracted, and were used to coat the cell culture plates separately. A total of 1 × 104 cells were cultured on coated and uncoated wells in 5% CO2 at 37 °C with essential medium-alpha (DMEM, Hyclone, USA) media supplemented with 10% (v/v) FBS (Hyclone) and 1% penicillin/streptomycin (Hyclone). Subconfluent cell cultures were detached using 0.25% trypsin-EDTA (Hyclone), and viable cells were identified with the trypan blue assay and were counted using a hemocytometer. Viable cells were determined using the cell counting kit (CCK-8; Dojindo Laboratories, Japan) assay. This solution produced a yellow formazan dye in the presence of viable cells. One to three days after cell

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seeding, 50 μL of CCK-8 was added to the wells for 3 h at 37 °C to allow formation of formazan crystals, and the absorbance was measured at 450 nm using a microplate reader (Bio-Rad). 2.10 Statistical Methods: All of the experimental data were presented as the mean ± standard deviation (SD) and lap shear bond strength and knife-edge shear bond tests were performed ten different times. Cell viability measurements were performed at four different times. The differences between the experimental data were examined using Student’s t-tests. A p-value < 0.05 was considered as statistically significant.

3. Results and Discussion 3.1 Syntheses of Catechol-Functionalized Adhesive Compounds Recently, cheap and efficient chemical mechanisms have been invented to protect the hydroxyl groups in DOPA with silane groups (SPC) [17]. Previous studies in which SPCs were used to combine acrylic groups at the ends showed that the adhesive strength increased even in the presence of saliva or water [18]. Additionally, acrylic and methacryl-conjugated catechols—used for silyl protection—have been shown to act as dental primers [19]. This has been suggested as a protection strategy to prevent the oxidation of DOPA-containing adhesives and the detachment of the silane group from SPC, which is usually achieved in dental clinics with acid treatments using 10-MDP [19, 20, 21]. Based on a light-induced radical medicated thiol-ene click reaction [22], the tetrafunctional molecule of PETMP provides numerous attachment options for varying amounts of DOPA. The benefit of attaching varying amounts of thiol group is attributed to the fact that the shear stress of the self-healing property can be controlled [23]. Figure 1 shows the chemical structure at each stage of the synthesis of our desired catechol-functionalized adhesive compound. By mimicking the mussel adhesive protein that exploits the benefit of

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thiol chemistry to achieve a strong, DOPA-based underwater adhesive (Figure 1a–1), PETMP—a tetra-functional molecule—and a poly thiol were used to synthesize the SPC intermediate (Figure 1a–3). This led to the corresponding SPC-functionalized pentaerythritol tetra(3-mercaptopropionate) (Figure 1a–5). The DOPA mimicking compound, SPC, was synthesized from eugenol. Eugenol is the main component of clove’s essential oil, it is inexpensive, and it is an easily available phenylpropene. Eugenol was synthesized on both phenolic and methyl ether units with triethylsilane at room temperature conditions (25 °C) with a single-step catalysis of the compound tris(pentafluorophenyl)borane (TPFPB), as depicted in Figure 1a and 2. The reaction mixture was then collected to remove TPFPB by filtering using a neutral aluminafilled syringe with dichloromethane as the eluent. The crude silyl-protected derivative (Figure 1a–3) was the pure product which remained after filtering, as verified by the peak of the 1H NMR spectra (Supplementary Figure S1). The spectra show the existence of a sharp, single proton peak from the methoxy of eugenol that appears at 3.9 ppm that indicates a complete reaction. Given that there are no other peaks near 3.9 ppm, it is reasonable to conclude that all of the eugenol was converted into SPC. The terminal of the alkene group that retains the SPC can be used as a reactive handle for the attachment to the commercially available PETMP through the thiol-ene coupling reaction. PETMP—as the thiol-functionalized precursor—and adhesive catechol-functionalized SPC were mixed in acetone, and were exposed to ultraviolet (UV) radiation in the presence of 2,2-dimethoxy-2-phenylacetophenone (DMPA) at room temperature for 30 min, and led to the final SPC-functionalized PETMP form (Figure 1a–5). The final PETMP–SPC products were easy to acquire because the thiol-ene reaction normally results in a high yield, and is not severely affected by environmental conditions. Supplementary Figure S2 shows the 1H NMR spectra for a series of thiol functional groups in the case of the PETMP conversion with SPC based on the incorporation of four different

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types of compounds. Depending on the varying levels of SPC incorporation, the combined compound was named as PETMP–mono-SPC, PETMP–di-SPC, PETMP–tri-SPC, or PETMP–tetra-SPC. Among these four compounds, the synthesis of PETMP–mono-SPC was the most effective. In the spectral plot, all the peaks of the protons of the SPC molecule are clearly depicted except the proton of the “ene.” Additionally, the propionate peaks of the PETMP molecule are also distinctly observed in the spectral plot. The ratio of the integrals of these peaks matched closely the expected ratio. In the case of PETMP–mono-SPC, the group of peaks that represents the thiols in the compound is clearly depicted because it involves large thiol molecules compared to those of the other PETMP SPCs. However, as the catechol quantity grows, the ability to distinguish the thiol peaks diminishes. Notably, the silyl protecting groups were stable throughout the synthetic process. Therefore, the handling of the final silyl-protected derivative, PETMP–SPC (as indicated by the resonances at 0.98 and 0.74 ppm in the 1H NMR spectra), ensured that the catechol groups would not participate in any undesirable, premature adhesion, and that this compound would be stable from unwanted side reactions that involve oxidative pathways. Furthermore, they were found to be stable in both solutions and in a solid state in the presence of normal atmospheric conditions. More importantly, the silyl groups could be removed to form the surface-active catechol unit based on the simple treatment with Amberlyst (a solid-type acid catalyst) for the evaluations of the shear bond strengths and cytotoxicity. 3.2 Bulk Adhesion of PETMP–SPCs Based on the Bulk Lap Shear Bonding Test PETMP–SPCs were deprotected and converted into PETMP catechols based on their simple treatment with Amberlyst or acidic buffers (Figure 2a, left panel). To test the bulk adhesion abilities of the PETMP catechols, adhesion was tested with low-surface-energy poly(methyl methacrylate) (PMMA, 41 mJ/m2) as substrate panels because acryl-based resin is one of the main components in commercially available dental resins. As the

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surfaces of the original acryl bars were generally smooth, sandpapers were used to roughen them. Initially, the adhesion strengths of the PETMP catechols were tested in the absence of the crosslinker. PETMP mono-catechol exhibited a tensile strength of ~0.2 MPa, whereas the other PETMP catechols exhibited a bonding strength of ~0.4 MPa (Figure 2b). Interestingly, the PETMP catechols which were not cured remained as viscous gels even after 24 h of exposure to air. This indicates that the PETMP catechols require assistance from the crosslinking agents to be cured as solids. Presumably, the thiol groups in PETMP prevent catechols from oxidation. Therefore, the adhesion of each PETMP catechol was tested in the presence of the crosslinkers. As a crosslinking strategy, periodate-mediated oxidative covalent crosslinking (Figure 2a) [24, 25, 26, 27] and iron-mediated coordinative crosslinking schemes (Figure 3a) were tested [28, 29, 30]. Addition of periodate ions (IO4-) induced significant increases in the bonding strength and cured the PETMP catechols. PETMP mono-catechol exhibited a tensile strength of ~1.2 MPa, whereas the other PETMP catechols exhibited a bonding strength of ~2.0 MPa (Figure 2c). These data indicate that the optimal number of catechol residues per PETMP for maximizing the adhesion is two. As an alternative crosslinking mechanism, PETMP catechols were mixed with Fe3+ ions to crosslink the adhesive via coordinative complexes. Fe3+ ions form reversible and strong bis- or tris-DOPA–Fe complexes with DOPA in neutral pH environments with a cumulative stability constant (Ks) of ~40, and crosslink with the mussel adhesive protein in water. To emulate the DOPA mimicking PETMP–SPC adhesive, each of the PETMP–SPC solutions were mixed with ferric ions (Fe3+) when they were applied on the acryl surface. Fe3+ was added at a molar ratio of 1:3 (Fe3+:catechol).

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The shear strength of the 24 h curing time of the PETMP mono-catechol was 184 kPa and it increased significantly to 915 kPa with PETMP–tetra-catechol (Figure 3b). These results suggest that as the number of catechols in the PETMP increases, and that stronger shear strengths are achieved when it is crosslinked via Fe–DOPA complexes. The observed increase of the shear strength bears similar characteristics with those of the mussel byssal thread, whereby increased amounts of DOPA are utilized by mussels for them to achieve strong adhesion and cohesion to their byssal threads. The shear strength of the PETMP– catechol compounds was measured again after 24 h to test their self-healing abilities. For the PETMP mono-catechol, the measured strength was 63 kPa, while the measured strength for the PETMP tetra-catechol was 558 kPa. Accordingly, similar, yet smaller increases were observed compared to those observed 24 h before these measurements were conducted. This indicates the existence of the self-healing property derived from the DOPA–Fe3+ complex. Incorporating Fe3+ ions in the byssal threads has been shown to impart strength and selfhealing properties based on the formation of strong, reversible, tris-catechol–Fe3+ crosslinks [29, 30, 31, 32]. As such, in our study, as the amount of catechol increased, the number of coordinating catechol monomer units per metal atom increased, as the stoichiometry of the complex bond changed from mono- to bis-, tris-, and then to tetra-complexes when Fe3+ was used. Accordingly, the mechanical strength increased in a respective manner. Regarding the self-healing properties, Harrington et al. proposed a model where the catechol–Fe3+ complexes in the cuticle functioned as sacrificial load-bearing crosslinkers to facilitate the extensibility of the material [32]. In comparison to covalent bonds, metal–catechol bonds can spontaneously reform after breaking. Furthermore, the model predicts that the damage accumulated in the cuticle could self-heal via the reformation of the broken catechol–Fe3+ complexes.

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The self-healing property of the PETMP catechol with metal ions would be important in dental resin systems because many metal oxides such as silica, titania, and zirconia, have used as dental implant materials. In addition, the 2,2-bis[4-(2-hydroxy-3-methacryloxyprop1-oxy) phenyl] propane (Bis-GMA) [33] and the 1,6-bis(methacryloxy-2ethoxycarbonylamino)-2,4,4-trimethylhexane (UDMA) [34] are the two most commonly employed monomers in dental resins. The fundamental drawbacks associated with this class of chain-growth polymerized dimethacrylate resin are incomplete conversion, high-shrinkage stress, and weak bonding to metal oxides in the dental formulation or materials [35 ,36, 37]. In addition, because Bis-GMA and UDMA-based polymers have numerous ester groups that are susceptible to chemical and enzymatic hydrolysis in the oral environment, the hydrolysis of the ester group may limit the service time of the resin [38]. These drawbacks are the main factors that restrict the duration and application of the polymeric dental composites [39, 40]. This incomplete polymerization in methacrylate-based resins leads to the situation whereby the residual monomers will leak out as time progresses, and saliva would then flow in the empty space, and will cause cracks and secondary corrosion. Therefore, the linkage of the monomers cured via polymerization can be prevented by mixing the PETMP–catechols with the self-healing bonding and the metal oxides in the dental resin compositions. PETMP, a tetra-armed crosslinker, has been used in the acryl-based resins to reduce the shrinkage stress of the acryl-based resin without compromising mechanical properties. Thus, the PETMP– catechols that contain the self-healing property can be used for filling cavities and for reconstructing the dental structures. 3.3 Synergetic Effect of PETMP Mono-catechol with Dental Adhesive Resin Formulation on the Bonding of Dentin–Zirconia and Zirconia–Resin Cements One of the most critical issues in medical dental adhesives is the dentin bonding with zirconia. Patients choose three-dimensionally (3D) printed zirconia blocks over gold crowns owing to

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their good mechanical properties and for aesthetic reasons [41, 42]. However, commercially available dental resins result in weak adhesions with zirconia substrates. To test the bonding strength of the PETMP mono-catechol to zirconia, the zirconia adhesion test was prepared as depicted in Figure 4 (ISO/TS 11405:2015) [43]. Periodate-mediated oxidative crosslinking of PETMP mono-catechol showed a bonding strength of ~1 MPa in between zirconia and dental resin. The microtensile bond strength was tested by mixing each of the compounds with the commercially available adhesive resins with 10-MDP, which is one of the most commonly used functional monomers to zirconia substrate [44]. This 10-MDP is the hydrophilic phosphate monomer that increases resin diffusion and adhesion by causing acidic decalcification and binding to calcium ions or amino groups of the tooth structure [45]. The adhesive compounds that have been used for the test are Bis-GMA, PETMP, PETMP monocatechol, and PETMP mono-catechol-IO4- (Table 1). Each of the mixed compounds is then attached either to the bovine dentin surface with the zirconia block or the zirconia with the resin cement. A schematic of the dentin specimens is depicted in Figure 5a. After the application of the adhesive resin to each of the bonding surfaces, they were left to set using blue-light curing. Note that the addition of 10-MDP, one of the most commonly used functional monomers in self-etching approaches, decapped the silyl-protected group of the PETMP procedures. The tooth substrate was first etched with 10-MDP. This self-etching processes are also encountered in marine mussels wherein they impose an acidic pH (pH~2) under the foot during the formation of adhesive plaque prior to its encounter with seawater [46, 47, 48]. Depositing adhesive proteins at acidic pH values has important implications for mussel biology and mussel-inspired dental adhesives. For the mussels and dental adhesives, low pH values stabilize the catecholic moiety of DOPA, and enable the formation of bidentate H-

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bonds and coordination complexes with surface oxides, which also remove the applied silylprotected group [47, 48]. Furthermore, at increased pH, such as the case of seawater (pH = 8.2) or saliva (pH = 7.4) in mussel and oral environments, respectively, serve as a switch for initiating quinone-based crosslinking and catechol-mediated metal chelation. We first tested the bond strength at the bovine dentin–zirconia interface. The bonding stress was evaluated with the use of the mechanical shear test with the UTM, and a shear force was applied until the tested specimens fractured. The load was applied at the dentin/adhesive interface as close as possible to the surface of the tooth. For comparison purposes, the bond strength of the commercially used dental adhesive Bis-GMA was measured to be ~2.5 MPa, the dental resin with PETMP was measured to be ~3.0 MPa, the dental resin with PETMP mono-catechol was ~4.0 MPa, and the dental resin with PETMP mono-catechol in the presence of periodate (IO4-) was measured at 4.5 MPa (Figure 5a). In the second test, the bond strength between the zirconia and the resin cement were measured. Similar to the first test, the dental adhesive Bis-GMA and the compound dental adhesive BisGMA plus PETMP were compared with several of our proposed PETMP adhesive combinations. The bond strength of the adhesive applied to zirconia using resin cement mixed with Bis-GMA was measured to be ~6.4 MPa, while the use of PETMP as the adhesive resulted in a bond strength of ~7.7 MPa, i.e., an increase of ~1.3 MPa. The adhesive strength further increased by ~2.3 MPa relative to that of the commercial dental adhesive BisGMA when the additional functional groups of thiols and catechols were added. Regarding the treatment of IO4- for oxidation, the measured bond strength showed that the adhesiveness of PETMP mono-catechol was similar with that of the nontreated PEMPT mono-catechol. This indicates that DOPA oxidation compromises the bonding performance of the adhesive resin (Figure 5b). The bond strength between the zirconia and the resin cement was higher than that between the zirconia and bovine dentin, suggesting catechol and thiol moieties in

17

the adhesive effectively formed chemical crosslinking with acryl moieties in the surface of the resin cement. The thiol group in the PETMP catechols has a tendency toward facile auto-oxidation, which often renders DOPA unreliable for adhesion. To limit the DOPA oxidation, mussels impose an acidic, reducing regime, based on the thiol-rich mfp-6 protein during the adhesive plaque formation [49, 50]. In addition, this imposition restores DOPA by coupling the oxidation of thiols to the reduction dopaquinone. Thus, by emulating the thiol characteristic of mfp-6 [51], our synthesized PETMP catechols also induce this thiol-mediated redox modulation. 3.4 Cytotoxicity The standard toxicity test of CCL1 was used as a first assessment test for the cytotoxicity of the polymer applied to the oral tissues to assess the adhesion and viability of the human fibroblast cell. Herein, we evaluated the viability of the cells with or without catechol on each of the dental adhesive compounds (3M-SBU, Bis-GMA, PETMP, PETMP mono-catechol) that were coated on the surface together with the control experiment after 3 days, as shown in Figure 6. In the case at which the cells were analyzed when they were cultured on the polymer-coated substrates, we observed slightly higher cell proliferation on the PETMP mono-catechol surface than on the PETMP surface, and the commercial adhesive resin 3MSBU (Figure 6). Given that the above results indicate a lower toxicity for cells adhered to the PETMP mono-catechol than the commercially available resin formulation, they could facilitate further development and clinical implementation of the proposed polymer adhesives.

18

4. Conclusions In the view of the similar dental adhesion and mussel adhesion strategies, we applied the wetadhesion strategy found in mussel — the combination catechol and thiol moieties — to dental adhesive formulation. We synthesized the PETMP catechols, crosslinker molecules with catechol and thiol moieties, and evaluated the potential of the PETMP catechols as a component in the dental adhesive formulation. As a result, the adhesion bonding between zirconia and dentin, one of the most elusive problems in dentistry, has improved when the PETMP-catechol was added on the commercially available dental adhesive formulation. In addition, from our cytotoxicity analysis, the synthesized polymers of PETMP catechol with 10-MDP did not inhibit the proliferation of human fibroblast cells. In summary, based on our synthesized polymers, we are able to increase significantly the bonding strength between the dentin with zirconia, our findings will considerably contribute to the translation of the adhesive system inspired by mussels.

Acknowledgements This work was supported by the Marine Biotechnology Program (Marine BioMaterials Research

Center)

funded

by

the

Ministry

of

Oceans

and

Fisheries,

Korea

(D11013214H480000110) and the National Research Foundation of Korea Grant funded by the Korean Government (NRF–2016M1A5A1027592 & NRF–2019M3C1B7025093). Declaration of interests 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.

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Figure 1. Synthetic design and nuclear magnetic resonance (NMR) spectra. a) Synthesis of pentaerythritol tetra(3-mercaptopropionate)–silyl-protected catechol (PETMP–SPC) undergoing the thiol-ene reaction between the ene of the SPC and the thiol of the PETMP. b) 1

H NMR spectrum of PETMP–SPC in deuterated chloroform (CDCl3).

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Figure 2. Adhesive force exhibited by PETMP catechol as a function of different catechol moieties. a) Periodate-mediated oxidative covalent crosslinking strategy and PETMP catechol chemical structure. b) The adhesive strength is measured by a lap shear test according to the number of catechols in the acrylic bars. c) Periodate (100 mM) is used in the PETMP catechols, and a lap shear test is performed (n = 10; mean ± standard error of the mean, * P < 0.05).

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Figure 3. Crosslinking catechols with Fe3+ to measure re-adhesion of PETMP catechols. a) Iron-mediated coordinative crosslinking strategy. b) Crosslinking was performed using iron ions. The catechol molar ratio is 1:3, and the adhesion is measured with PETMP catechols in acrylic bars (n = 10; mean ± standard error of the mean, * P < 0.05).

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Figure 4. Measurement of the adhesive strength of PETMP catechol in zirconia–resin cement. The adhesion between zirconia and resin was measured. PETMP mono-catechol was treated with 100 mM periodate to measure the adhesion of crosslinked PETMP monocatechol (n = 10; mean ± standard error of the mean, * P < 0.05).

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Figure 5. Adhesion strength measurement between bovine tooth–zirconia and zirconia– resin cement. a) Adhesive strength of the PETMP mono-catechol resin system of bovine tooth–zirconia surface. b) Adhesive strength of the PETMP mono-catechol resin system in the zirconia–resin cement. Both a) and b) were treated with periodate (100 mM) (n = 10; mean ± standard error of the mean, * P < 0.05).

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Figure 6. Cell viability measurement in a resin adhesive system. Cell viability was determined based on the measurement of absorbance at 450 nm in L929 cell lines and is compared with the group which contains a catechol group (n = 4; mean ± standard error of the mean, * P < 0.05).

30

Table 1. Resin adhesive composition

wt.% Bis-GMA PETMP

Bis-GMA Resin 50

PETMP Resin 40 10

PETMP mono-catechol

10-MDP Ethanol Tertiary amine2) Camphorquinone

10 36.5 2.5 1

10 36.5 2.5 1

1) tetrabutylammonium periodate (100 mM) 2) ethyl 4-(N,N-dimethylamino)benzoate

31

PETMP–catechol Resin 40

PETMP–catechol IO41) Resin 40

10 10 36.5 2.5 1

10 10 36.5 2.5 1