Article pubs.acs.org/journal/abseba
Inhibition of Bacterial Adhesion on Hydroxyapatite Model Teeth by Surface Modification with PEGMA-Phosmer Copolymers Xinnan Cui,† Yuki Koujima,† Hirokazu Seto,† Tatsuya Murakami,‡ Yu Hoshino,† and Yoshiko Miura*,† †
Department of Chemical Engineering, Graduate School of Engineering, Kyushu University 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan ‡ Center for Nano Materials and Technology, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan S Supporting Information *
ABSTRACT: Modification of the interface properties on hydroxyapatite and tooth enamel surfaces was investigated to fabricate bacterial resistance in situ. A series of copolymers containing pendants of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and ethylene glycol methacrylate phosphate (Phosmer) were polymerized by conventional free radical polymerization and changing the feed ratio of monomers. The copolymers were immobilized on hydroxyapatite and tooth enamel via the affinity of phosphate groups to hydroxyapatite to form the stable and durable polymer brushes on the surfaces. The amounts of polymer immobilized depended on the phosphate group ratio in the copolymers. Surface modification altered the interfacial properties of hydroxyapatite and inhibited bacterial adhesion. Copolymers containing 40−60% PEGMA segments showed a significant inhibitory effect on bacterial adhesion of S. epidermidis both in the presence and absence of plaque model biomacromolecules. KEYWORDS: hydroxyapatite, copolymers, surface modification, antibacterial reduce bacterial adhesion (by S. epidermidis and E. coli).6 Silicon surfaces modified by PEGMA films showed effective depression of both plasma protein adsorption and cell attachment to the modified surfaces.7 PEGylated silicon surfaces retained their protein and cell repulsive properties after at least 4 weeks of immersion in phosphate buffer, indicating the durability of PEG coatings in a marine environment.8 The conventional approach to attach the PEG onto the surface is the introduction of an anchoring group to the terminal of PEG.9,10 However, this method is limited by the functional groups of PEG. Therefore, a polymerizable PEG monomer such as PEGMA can be utilized to introduce the other functional groups acting as the bonding sites for substrates via copolymerization and surface initiated polymerization.11 The experimental substrate in this study is hydroxyapatite (HAp) (Ca10(PO4)6(OH)2), which is a main component of tooth enamel. The modification of hydroxyapatite (HAp) with organic compounds was attained in the presence of phosphate groups. It has been reported that phosphate and bisphosphates, such as 10-methacryloyloxydecyl dihydrogen phosphate (MDP), bind strongly and readily to bone and teeth minerals.12,13 The high affinity of phosphate groups toward hydroxyapatite bonding is attributed to Ca2+ and PO43− sites in
1. INTRODUCTION Tooth decay is one of the most prevalent human diseases. Conventional treatments recommended for tooth decay are essentially supportive care, such as removal of soft decay or repair of damage with a filling or crown. Tooth decay is considered to begin with bacterial adhesion. These microorganisms universally attach to tooth surfaces and then generate organic acids, such as lactic, propionic, and acetic acids, especially in the presence of sugars and other fermentable carbohydrates. These resulting acids lead to localized demineralization of tooth enamel.1 Accordingly, the inhibition of bacterial adhesion in the oral environment is a key element in preventing dental problems. The initial process of bacterial adhesion occurs by various interactions such as electrostatic interaction, hydrophobic interaction, and specific interactions of ligands−receptors.2−4 For this reason, surface modification to control the biointerface through an antifouling layer formed directly on the teeth becomes an effective strategy to protect teeth from decay. Such antibiofouling materials are expected to prevent the approach from bacteria. Numerous poly(ethylene glycol) (PEG)-based systems have been described to date, among many popular antibiofouling materials, because these offer steric exclusion, hydrophilicity with low values of polymer−water interfacial energy,5 and excellent biocompatibility. For example, poly(ethylene glycol) methyl ether methacrylate (PEGMA)modified polyurethane surfaces were shown to significantly © XXXX American Chemical Society
Received: August 13, 2015 Accepted: December 28, 2015
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DOI: 10.1021/acsbiomaterials.5b00349 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering Scheme 1. Synthesis of Poly(PEGMA-r-Phosmer)
National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. The water used in this study was purified using a Direct-Q Ultrapure Water System (Merck, Ltd., Darmstadt, Germany). Calcein (Kanto Chemical Co., Inc.) was used to detect the Ca2+ dissolved from HAp in solution. Pig tooth samples were pulled from adult pig mandibles purchased from Tokyo Shibaura Internal Organs Co., Ltd. 2.2. Synthesis of Poly(PEGMA-r-Phosmer). The synthesis of the poly(PEGMA-r-Phosmer) proceeds via free radical polymerization, involving the vinyl groups of PEGMA and Phosmer, where the poly(ethylene oxide) chains extend in the brush conformation in an aqueous medium. A series of copolymers were prepared by changing the feed ratio of PEGMA and Phosmer monomers. PEGMA was first purified by an alumina column to remove inhibitors before reactions. Monomers (1 mmol) were then dissolved in 10 mL of water and bubbled under a nitrogen atmosphere at 70 °C for 30 min. Polymerization began with the decomposition of initiator V-501 (0.0128 mmol) dissolved in a modicum of methanol and then reacted for 11 h at 70 °C (Scheme 1). After the reaction, a small sample of the reaction solution was taken to measure the conversion, and the remainder was dialyzed (MWCO: 3500 Da) with water for 3 days. Unrefined and refined polymers were analyzed by 1H NMR (400 Hz, D2O) (JEOL Ltd., Tokyo, Japan) to confirm the conversion and calculate the ratio of components in the resultant polymers. The weight-average molecular weights (Mw) and polydispersities (Mw/Mn) of polymers were measured by size exclusion chromatography (SEC) (Showa Denko, Tokyo, Japan). 2.3. Immobilization of Poly(PEGMA-r-Phosmer) on HAp Powder and Plates. HAp plates were cut into 0.5 × 0.5 cm squares for subsequent use. Both sides were treated under ultraviolet (UV)ozone (O3) (Filgen, Inc., Aichi, Japan) for 30 min, then rinsed by ethanol and air-dried thoroughly. To find out the optimal concentration and time for immobilization, the plates were immersed in a 250 μL/piece of 0.05, 0.1, 0.5, 1, 5, and 10 mg/mL polymer solution for 1 h at 37 °C. Moreover, plates were then immersed in a 250 μL/piece of 10 mg/mL polymer solution for 5, 15, 30, 45, or 60 min at 37 °C. The HAp plates used in bacterial adhesion experiments were prepared by immersing the plates into the 10 mg/mL aqueous polymers solution at 37 °C for 1 h. To evaluate the durability of polymers on HAp plates, unmodified and polymer-immobilized HAp plates were immersed in phosphate buffered saline (PBS) (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 1.8 mM) for 2.5 h at 37 °C and then washed gently with water. In order to evaluate the stability of immobilized polymers to brushing in reality, the pig real tooth was chosen as the experimental object. The fluorescent labeled polymer immobilized pig tooth surface was brushed with moderate force in water. The zeta potential of unmodified and 0.5PEGMA-immobilized HAp surfaces was measured in10 mM NaCl solution by Zeta-potential Analyzer ELSZ-2000 (Otsuka Electronics Co.,Ltd.,Japan). The Ca2+ ion dissolution of unmodified and 0.5PEGMA-immobilized HAp plates was measured at 2, 4, 6, 18, and 24 h using calcein. The polymers were also immobilized on HAp powder (
