Durable Urushiol-Based Nanofilm with Water ... - ACS Publications

Article pubs.acs.org/journal/abseba

Durable Urushiol-Based Nanofilm with Water Repellency for Clear Overlay Appliances in Dentistry Hyejoong Jeong,† Young-Ah Cho,‡ Younghyun Cho,§ Eunah Kang,† Hyo-won Ahn,*,∥ and Jinkee Hong*,† †

School of Chemical Engineering and Material Science, Chung-Ang University, 84 Heukseok-ro, Dongjak-Gu, Seoul 06974, Republic of Korea ‡ Department of Oral and Maxillofacial Pathology and ∥Department of Orthodontics, School of Dentistry, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea § Department of Materials Science and Engineering, University of Pennsylvania, 3231 Walnut Street, Philadelphia, Pennsylvania 19104, United States S Supporting Information *

ABSTRACT: With increased esthetic needs, orthodontics is an indispensable medical treatment in dentistry, and transparent clear overlay appliances (COAs) are in general use to fix teeth. However, COAs are easily worn out because of the lack of durability. Here, we applied a nanofilm onto COAs using urushiol (U), a durable coating material from plant via a layer-by-layer assembly technique. In particular, polymerized urushiol (PU) provided COAs with higher mechanical strength in the large-scale assessment, lower cytotoxicity, and intrinsic hydrophobicity for antimicrobial use. In this report, we inceptively attempted to functionalize COAs with nanofilm for advanced biomedical use. KEYWORDS: clear overlay appliances, urushiol, nanofilm, intrinsic hydrophobicity, durability manufacturing processes.8 Because of intraoral aging, polymers eventually undergo thermal, mechanical, and chemical degradation, and these changes are visible in their molecular structure and orientation.8 From a clinical point of view, major limitations of COAs are their poor wear resistance and subsequent cracking and breakage. Intraoral use leads to an increase in the surface roughness of COAs, which show different characteristics depending on the exposure time and evaluation site.8,9 COAs show poor durability along the incisal and occlusal surfaces and a decrease in vertical height after only a few months of use.3,10 Consequently, frequent replacement increases economic burden on patients. To reduce the wear rate and increase the replacement interval of COAs, functionalization of thermoplastic polymers is essential. However, there has been a lack of improved materials in terms of surface alteration, structural conformation, and changes in mechanical properties. Although multilayered devices formed using different polymer sheets11,12 and polymer blending techniques6 have been reported, they present various problems such as increases in the thickness of COAs or changes

1. INTRODUCTION Esthetics is critical in the field of dentistry, and invisible appliances using thermoplastic polymer sheets have become popular, replacing conventional devices that use acrylic resin. For example, patients prefer clear overlay retainers compared to Hawley retainers for retention of orthodontic treatments.1 Moreover, clear overlay appliances (COAs) have many advantages, such as improvements in oral hygiene, costeffectiveness, good formability, and good optical characteristics.2,3 Currently, various types of thermoplastic polymers have been used for COAs. They are classified as “amorphous polymers”, including polycarbonate and polyethylene terephthalate glycol (PETG)-modified, or “partly crystalline polymers”, including polypropylene, polyethylene, and ethylene vinyl acetate (EVA).4 The characteristics of these polymers such as glass transition temperature, wear resistance, durability, and water absorption rate vary with molecular structure.4,5 Among the polymers, PETG plays an important role due to its good mechanical strength, formability, optical qualities, fatigue resistance, and dimensional stability.6,7 Thermoplastic polymers exhibit different mechanical properties compared with those indicated by the manufacturers because they are highly viscoelastic and sensitive to temperature, humidity, time elapsed after elastic deformation, and © XXXX American Chemical Society

Received: October 15, 2015 Accepted: February 2, 2016

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DOI: 10.1021/acsbiomaterials.5b00440 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Scheme 1. (Left) Chemical Structure and Full Name of Main Materials for the Films and (Right) Illustration of the LbL Process To Produce Multilayer Nanofilms and Post-thermal Treatmenta

a

In the box, the urushiol is converted to polyurushiol (red line) described by chemical structures during the thermal process.

2. RESULTS AND DISCUSSION 2.1. Characterization of Thin Films. The films produced on a silicon wafer were used for characterization because a completely uniform surface on the wafer is appropriate for analysis at the nanoscale. In the mechanical and cellular tests, the films on the PETG substrate were used to assess the practical effect of thin films on COA. In the urushiol-based multilayers, small urushiol molecules were assembled with BPEI uniformly onto a silicon wafer. Figure S1A indicates a linear growth curve increasing at approximately 20 nm per bilayer due to properties of intermolecular binding forces without repulsion. An amine group in BPEI and a hydroxyl group in urushiol interacted by hydrogen bonding, and urushiol molecules were bound together with hydrophobic forces of the long alkyl chain as a form of agglomeration. To determine the assembly of the multilayered film more precisely, alterations in surface wettability in an LbL manner were measured by a static contact angle (SCA) (shown in Figure S1B). The BPEI surface marked with an odd number of points was determined as a hydrophilic surface, with an average contact angle of 33.15 ± 1.63°. On the contrary, the urushiol surface which was marked with an even number of points indicated a hydrophobic property with a contact angle of 96.91 ± 4.43° on average. This is because the amine groups of BPEI induced the hydrophilic property, while the long alkyl chain of urushiol generated intrinsic hydrophobicity on the films. From this result, we were able to assemble multilayered films successfully using urushiol as a building block and to control the wettability of the film simply by changing the outermost layer. Furthermore, the multilayered (BPEI/U)n film was capable of exhibiting antibiofouling with controlled wettability proportional to the number of layers and the effect of unsaturated alkyl groups.14,20 The thermal stability of the (BPEI/U)20 film is indispensable to produce a proper form of customized COA. The test was performed at 150 °C, which is a temperature similar to that of practical molding processes, and (BPEI/U)20 was converted to a polymerized form of (BPEI/PU)20 . We determined polymerized urushiol within the films by measuring Fourier transform infrared (FT-IR) spectroscopy, as shown in Figure

in the optical characteristics. As an alternative, we have developed nanofilm-coated thermoplastic polymer sheets. The nanofilm does not increase the thickness, an important factor for patient compliance, but effectively increases the mechanical strength of COAs. Nanofilms were fabricated on COAs using the layer-by-layer (LbL) assembly technique, a simple method involving sequential adsorption of interactive molecules in aqueous solutions.13 The LbL technique regulates nanoscale thickness by controlling the number of layers with various materials on any kind of substrate. Using this approach, nanofilms applied on COAs can enhance mechanical strength by durable materials and control optical properties while providing an extremely thin coating for patient’s convenience. In previous research, we reported the LbL-assembled urushiol multilayer nanofilms including antibacterial effect and durability.14 In this report, for practical use, we applied our urushiol-based durable nanofilms on COAs. Urushiol is the major component of ancient Korean lacquer, which is a durable coating material extracted from plants (Toxicodendron vernicifluum). In Scheme 1, urushiol has a small molecular weight (MW) less than 500 and is composed of a catechol group substituted with long alkyl chains. Alkyl chains are irregularly distributed as saturated or unsaturated forms with 1−3 double bonds, as shown in Scheme 1.15 Though urushiol is very small, it can produce nanofilms with BPEI (branched poly(ethylene imine), MW ∼ 25 000) by hydrogen bonding. With an illustration of the LbL process shown in Scheme 1, urushiol existing as a form of nanoparticle can produce durable intrinsic hydrophobic LbL multilayer films with BPEI. Through postthermal treatment, urushiol can be converted into polyurushiol (PU) to hydrogenate unsaturated alkyl chains.16−18 PU indicates higher mechanical properties and loses mild toxicity, causing skin allergy that is induced by unsaturated alkyl chains and catechol groups.19 In the result of this process, urushiolbased nanofilms are more biocompatible and durable, retaining intrinsic hydrophobicity throughout the indispensable heating process for molding appliances. Moreover, we investigated the cytotoxic effect of the film using periodontal ligament (PDL) cells in the applied area. B


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Figure 1. Analysis of the (BPEI/U)20 film after thermal treatment. (A) Bar graphs correspond to the left y-axis, indicating the thickness of each film; dots correspond to the right y-axis, indicating the contact angle of each film. Inset: SCA of each film. (B) Mechanical properties of (BPEI/U)20 films before and after thermal treatment analyzed by atomic force microscopy (AFM) nanoindentation. Surface morphology of the films obtained by AFM (C) before and (D) after thermal treatment.

determined that the thickness of both (BPEI/U)20 and (BPEI/ PU)20 films could be maintained without disintegration for more than 27 days (Figure 2). Although the thickness of

S2. In Figure 1A, we compared the thickness and wettability of the (BPEI/U)20 film with respect to thermal treatment. The thickness and SCA decreased slightly by 7.14 and 8.15%, respectively. The reason for these changes is that urushiol was thermally polymerized between the hydroxyl group of catechol and the unsaturated point of the alkyl chain.15 Films were converted to a more condensed form and a slightly less hydrophobic surface due to a cross-linking network of urushiol molecules.18 The polymerization of urushiol had a substantial effect on the mechanical properties of the nanofilms, including hardness and Young’s modulus, as shown in Figure 1B, of which (BPEI/ PU)20 films exhibited values that were 3.55 and 2.12 times, respectively, greater than those of (BPEI/U)20 films. From this result, better rigidity and elasticity were obtained in the polymerized form of the film. With the full advantage of PU, (BPEI/PU)20 films were much more biocompatible for removing the toxicity of urushiol and providing higher mechanical stability. To demonstrate topographical change, we compared the root mean square (rms) values of each film (shown in Figure 1C,D, respectively). There was no major difference between the rms values before and after thermal treatment, which were 61.98 ± 1.29 and 65.589 ± 1.22 nm, respectively. From this result, the decrease in SCA was not due to the change in microstructure but was due to the presence of functional groups on the surface of the films. The SCA decreased relatively, but the film still maintained hydrophobic. The unique properties of urushiol have been substantially studied in Asia, and one of these properties is resistance to aqueous solutions and salts. Kim et al. reported in 2009 the anticorrosive property of urushiol micron films in a 5 wt % aqueous sodium chloride solution.21 To determine the physiological stability of urushiol nanofilms in the oral cavity, the test was performed with artificial saliva (pH 7.0) containing a large number of salts (the chemical composition is provided in the Supporting Information). With heavy salt conditions, we

Figure 2. Physiological stability testing of films in the model oral environment. Inset: Each axis is equal to the outer one. The graph magnifies the range of 0 to 50 h.

(BPEI/U)20 films was maintained, we could observe a relative thickness variation and certain damage with the naked eye in comparison to the thermally treated film. 2.2. Mechanical Testing. Figure S3 represents the superimposed load−elongation curves for a series of specimens with varying notch lengths. Each area under the load− elongation curves, that is, the normalized total work of fracture (Wf), was plotted on a graph of Wf versus notch length (Figure 3). Essential work of fracture (EWF) is the energy that is essential for generating new fracture surfaces, and it depends only on the fracture surface area. It is an intrinsic material property for the evaluation of resistance to crack initiation.22,23 The plastic work of fracture (PWF) is nonessential work, which is plastically dissipated energy throughout the zone surrounding C


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cells onto the (BPEI/PU)20 film indicated cell morphologies similar to that of the control. We determined noncytotoxic effects of the raw materials including BPEI and urushiol through MTT assay. Furthermore, the attachment assay demonstrated the initial attachment of PDL fibroblasts when exposed to the (BPEI/PU) films. Initial cell attachment to the materials is possible when the materials have no deleterious effects on cells and possess the capacity to encourage cell spreading and proliferation.25

3. CONCLUSION Our study is significant as it is the first attempt at applying nanofilms to orthodontic devices to enhance mechanical properties. First of all, we demonstrated that the urushiolbased nanofilms had better durability and physiological stability in a model oral cavity through thermal treatment at the nanoscale. Furthermore, urushiol nanofilms enhanced mechanical strength of COAs in the large-scale assessment. As future follow up studies, urushiol nanofilm-coated COAs will be assessed for its mechanical strength and antimicrobial effects by in vivo study. From this report, the nanotechnology delineated herein paves the way to assign functionality not only onto orthodontic appliances but also onto entire medical devices with superior performance for actual applications.

Figure 3. Linearity of work of fracture × notch length. Slope = relative plastic work of fracture; intercept = relative essential work of fracture. Relative Wf of (BPEI/PU)50 nanofilm-coated sheet compared to the control is indicated in purple.

the fracture. It is not an intrinsic material property because it depends on specimen volume, geometry, and loading configuration. It is regarded as a measure of resistance to crack propagation and an indicator of ductility.22,24 According to the curve-fitted equations, the EWF value in the (BPEI/ PU)50 nanofilm-coated group was 0.539 (arbitrary unit), which was greater than that in the control group (0.426). The PWF value was 0.266 in the control group and 0.206 in the (BPEI/ PU)50 nanofilm-coated group. The (BPEI/PU)50 nanofilm contributed to the increase in resistance to crack initiation, which is a critical factor for the long-term use of COAs. 2.3. Cytotoxicity Testing. 2.3.1. MTT Assay. Changes in the viability of PDL fibroblast were not detected after exposure for up to 100 nM BPEI and up to 200 μM urushiol compared with the control (Figure S4). In the film-fabricating process, 40 μM BPEI and 2 mM urushiol were used. The reason why we investigated the use of lower concentrations was that very small quantities of materials were incorporated in the nanofilms. 2.3.2. Attachment Assay. Cell attachment data are presented as mean ± standard deviation in Figure 4A,C. The cell number on the (BPEI/PU)20 film cultured 48 h after seeding (20.0 ± 3.2) was similar to that on the control (22.6 ± 1.7). Figure 4B shows the microscopic view of spindle-shaped human PDL cells that attached to the materials, showing the typical morphology of fibroblasts. In comparison, the attached

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.5b00440.

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experimental section, thickness growth curve, contact angle hysteresis, FT-IR transmittance, load−elongation curve, and cell viability (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

Figure 4. (A) Histogram, (B) photomicrographs, and (C) number of human PDL fibroblasts that have attached to the materials 48 h after seeding. Scale bar = 20 μm. D


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(9) Ahn, H. W.; Ha, H. R.; Lim, H. N.; Choi, S. J. Effects of Aging Procedures on the Molecular, Biochemical, Morphological, and Mechanical Properties of Vacuum-Formed Retainers. J. Mech. Behav. Biomed. Mater. 2015, 51, 356−366. (10) Jäderberg, S.; Feldmann, I.; Engström, C. Removable Thermoplastic Appliances as Orthodontic Retainers-A Prospective Study of Different Wear Regimens. Eur. J. Orthod. 2012, 34, 475−479. (11) Takeda, T.; Ishigami, K.; Mishima, O.; Karasawa, K.; Kurokawa, K.; Kajima, T.; Nakajima, K. Easy Fabrication of a New Type of Mouthguard Incorporating a Hard Insert and Space and Offering Improved Shock Absorption Ability. Dent. Traumatol. 2011, 27, 489− 495. (12) Miyahara, T.; Dahlin, C.; Galli, S.; Parsafar, S.; Koizumi, H.; Kasugai, S. A Novel Dual Material Mouthguard for Patients with Dental Implants. Dent. Traumatol. 2013, 29, 303−306. (13) Decher, G. Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites. Science 1997, 277, 1232−1237. (14) Jeong, H.; Heo, J.; Son, B.; Choi, D.; Park, T. H.; Chang, M.; Hong, J. Intrinsic Hydrophobic Cairnlike Multilayer Films for Antibacterial Effect with Enhanced Durability. ACS Appl. Mater. Interfaces 2015, 7, 26117−26123. (15) Watanabe, H.; Fujimoto, A.; Takahara, A. Characterization of Catechol-Containing Natural Thermosetting Polymer “Urushiol” Thin Film. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3688−3692. (16) Johnson, R. A.; Baer, H.; Kirkpatrick, C. H.; Dawson, C. R.; Khurana, R. G. Comparison of the Contact Allergenicity of the 4 Pentadecyl Catechols Derived from Poison-Ivy-D Urushiol in Human Subjects. J. Allergy Clin. Immunol. 1972, 49, 27−35. (17) Auerbach, R.; Baer, H. Comparison of the Potency of Poison Ivy Extracts with Synthetic Pentadecyl Catechol in Sensitive Humans. J. Allergy 1964, 35, 201−205. (18) Zheng, X.-L.; Weng, J.-B.; Huang, Q.-M.; Hu, B.-H.; Qiao, T.; Deng, P. Fabrication of a Stable Poly(vinylpyrrolidone)/poly(urushiol) Multilayer Ultrathin Film through Layer-by-layer Assembly and Photo-induced Polymerization. Colloids Surf., A 2009, 337, 15−20. (19) McGovern, T. W.; Barkley, T. M. Botanical Dermatology. Int. J. Dermatol. 1998, 37, 321−334. (20) Suk, K. T.; Baik, S. K.; Kim, H. S.; Park, S. M.; Paeng, K. J.; Uh, Y.; Jang, I. H.; Cho, M. Y.; Choi, E. H.; Kim, M. J.; Ham, Y. L. Antibacterial Effects of the Urushiol Component in the Sap of the Lacquer Tree (Rhus Verniciflua Stokes) on Helicobacter Pylori. Helicobacter 2011, 16, 434−443. (21) Kim, H. S.; Yeum, J. H.; Choi, S. W.; Lee, J. Y.; Cheong, I. W. Urushiol/Polyurethane−Urea Dispersions and Their Film Properties. Prog. Org. Coat. 2009, 65, 341−347. (22) Karger-Kocsis, J. Toward Understanding the Morphologyrelated Crack Initiation and Propagation Behavior in Polypropylene Systems as Assessed by the Essential Work of Fracture Approach. J. Macromol. Sci., Part B: Phys. 1999, 38, 635−646. (23) Pardoen, T.; Marchal, Y.; Delannay, F. Essential Work of Fracture MechanicsTowards a Thickness Independent Plane Stress Toughness. Eng. Fract. Mech. 2002, 69, 617−631. (24) Peres, F. M.; Schon, C. G. Application of the Essential Work of Fracture Method in Ranking the Performance in Service of HighDensity Polyethylene Resins Employed in Pressure Pipes. J. Mater. Sci. 2008, 43, 1844−1850. (25) Grinnell, F. Cellular Adhesiveness and Extracellular Substrata. Int. Rev. Cytol. 1978, 53, 65−144.

ACKNOWLEDGMENTS This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation of Korea (NRF) funded by the Korean Government (Grant 2012M3A9C6050104) and supported by the National Research Foundation of Korea (NRF), funded by the Korean Government Ministry of Science, ICT & Future Planning (Grant 2013R1A1A1076126) and supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2014R1A1A1038263). Additionally, this research was also supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grants HI14C-3266 and HI15C-1653). This work was also carried out with the support of Cooperative Research Program for Agriculture Science & Technology Development (Grant PJ00998601) Rural Development Administration, Republic of Korea. This research was supported by the Chung-Ang University Excellent Student Scholarship in 2015. J.H. thanks Dr. K. Kwon for thoughtful help in this research.

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ABBREVIATIONS COA, clear overlay appliance PETG, polyethylene terephthalate glycol LbL, layer-by-layer assembly U, urushiol PU, polymerized urushiol BPEI, branched polyethylene imine SCA, static contact angle AFM, atomic force microscopy PDL, periodontal ligament Wf, work of fracture EWF, essential work of fracture PWF, plastic work of fracture

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Durable Urushiol-Based Nanofilm with Water ... - ACS Publications

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