Polymerization of poly(PEGMA-r-Phosmer) and fluorescence labeled

The synthesis of 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-like 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 reaction. 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 2-1). After the reaction, a small sample of 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 weight (Mw) and polydispersity (Mw/Mn) of polymers were measured by size exclusion chromatography (SEC) (Showa Denko, Tokyo, Japan).

To label the polymer with fluorescence, the fluorescent monomer 4-acrylamido fluorescein (AFA) 0.1% was added to the monomers and polymerized in the same condition.

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Scheme 2-1. Synthesis of poly(PEGMA-r-Phosmer) (A) and polymer labeled with fluorescence (B).

2.2.3 Immobilization of ploymer on HAp powder and plates

HAp plates were cut into 0.5 × 0.5 cm squares for subsequent use. The plates were sonicated in acetone, ethanol and water each for 15 min to remove the organic contamination. Then cleaned plates were etched with 37% phosphoric acid for 10 s.

After sonicated with water for 30 s, both sides of plates were treated under ultraviolet (UV)-ozone (O3) (Filgen, Inc., Aichi, Japan) for 30 min and ready for use.

To find out the optimal concentration and time for immobilization, the plates were immersed in 250 L/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 250 L/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 ^oC 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.

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In order to evaluate the stability of immobilized polymers to brushing in reality, the pig real tooth was chose as experimental object, because of the weak wear-resistance of HAp model plate. Procedures of polymerization, measurement and immobilization on pig tooth surface were same with the steps described above. After immobilization, the tooth was observed by fluorescent microscope (EVOS fl AMF-4302, Advanced Microscopy Group, Bothell, WA, USA) directly. Brushing was operated manually with commercial tooth brush and moderate force. Tooth surface was swept 5, 15, 30, 50 times respectively. Images were took under same light intensity.

To examine the detachment of copolymers on the HAp surfaces during the protein adsorption experiments, AFA labeled 0.2, 0.5 and 0.8PEGMA copolymers were used to immobilize and the surfaces were observed directly by the fluorescent microscope.

Images were took under same light intensity and analyzed by image J software. AFA labeled copolymers immobilized HAp plates were immersed in the same protein solution in the former experiments (BSA, lysozyme 1 mg/mL dissolved in ten-fold diluted PBS buffer) at 37 ^oC for 1 h and one group was treated in buffer under the same condition as control.

Zeta potential of unmodified and 0.5PEGMA-immobilized HAp surfaces was measured in 10 mM NaCl solution by Zeta-potential Analyzer ELSZ-2000 (Otsuka Electronics Co., Ltd., Japan).

The Ca²⁺ ions dissolved in distilled water were detected using calcein to evaluate the demineralization of HAp may caused by polymer immobilization. Raw HAp as control and 0.5PEGMA-immobilized HAp plates (similar surface area) were immersed in distilled water at 37 ^oC. The Ca²⁺ ions were detected after 2, 4, 6, 18, 24 h immersion.

The polymers were also immobilized on HAp powder (< 200 nm particle size).

HAp powder was mixed with aqueous polymer solution (10 mg/mL) at a solid-liquid ratio of 300 mg to 1 mL at 37°C. After stirring for 1 h, the suspension was centrifuged at 3500 rpm for 10 min three times, with the supernatant replaced with water each time.

Sediment was collected by filtration and then completely dried at 70°C overnight. The

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polymers-immobilized HAp powder was used in protein adsorption experiments to evaluate the protein resistance property of polymers.

The existence and relative amount of polymers immobilized on the HAp plates and powder were evaluated using C(1s), O(1s), Ca(2p) and P(2p) XPS (AXIS-ultra;

Shimadzu/Kratos, Kyoto, Japan). Peaks of XPS were analyzed with PeakFit V4.12 (Systat Software, Inc, CA, USA).

2.2.4 Protein adsorption on polymer-immobilized HAp powder

3 mL of 1 mg/mL BSA and lysozyme dissolved in 1 mM PBS buffer was added to 100 mg of the polymer-immobilized HAp powder. Adsorption was carried out in a shaker at 280 rpm (Bioshaker, TAITEC BR23UM, Japan) at 37°C for 2.5 h. The suspensions were then centrifuged three times for 10 min at 3500 rpm (TOMY Digital Biology Co., Ltd, Tokyo, Japan). Finally, the supernatant was collected and the change of protein concentration was measured at 280 nm using ultraviolet-visible (UV-vis) spectroscopy (Agilent 8453, Agilent Technologies, CA, USA). To prevent the powder interfusing, all supernatants were filtered through a 0.45 m filter before measurement.

To examine the detachment of copolymers on the HAp surfaces during the protein adsorption experiments, fluorescent labeled 0.2, 0.5, and 0.8PEGMA copolymers were used to immobilize the surfaces that observed directly by fluorescence microscope (EVOS fl AMF-4302, Advanced Microscopy Group, Bothell, WA, USA) after adsorption. Images were took under same light intensity and analyzed by image J software.

2.2.5 Bacterial adhesion on polymer-immobilized HAp plate surfaces and pig teeth surfaces

S. epidermidis was cultured in a broth medium (polypeptone 5 g/L, beef extract 3 g/L, and NaCl 5 g/L) and incubated at 37°C and shaken constantly at 100 rpm (Shaker

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NTS-2100, Tokyo Rikakikai Co., Ltd, Tokyo, Japan) until the concentration reached around 10⁸ CFU/mL. Bacterial suspensions were centrifuged and washed with PBS buffer three times to remove the culture medium.

BSA, lysozyme, dextran ⁵ and cytochrome C at 1 mg/mL were added into bacterial suspensions as plaque model compounds. The HAp plates were immersed into the mixtures at 250 L/piece and incubated for 2.5 h at 37°C in 24 well plates to imitate initial stage of the bacterial adhesion. After incubation, plates were washed with PBS buffer three times and 2.5% glutaraldehyde solution was added to each well and incubated for 24 h at 37°C to fix the bacterial cell shape. To dehydrate the cells, after gently washing with water, the plates were immersed into 25, 30, 50, 70, 80, 90, and 100% ethanol for 5, 5, 10, 10, 10, 10 and 10 min (thrice), respectively. Finally, these plates were dried at 37°C overnight. Adhered bacteria on the surfaces were observed using a scanning electron microscope (SEM) (SU8000, Hitachi High-Technologies Co., Tokyo, Japan). In order to quantify the adhesion results, the number of adhered cells on surfaces was counted based on at least 25 images from separate 5 areas of plates. Similar approaches were used to analyze the cell adhesion on each surface ^6,7. All the bacteria adhered on HAp surfaces was counted statistically.

The morphology effect was excluded by the measurement of unmodified and modified HAp surfaces using scanning probe microscope (SPM) (Dimensionlcon, Bruker- AXS K.K. Co., Ltd., Japan).

The lysozyme activity of 1 g/L was also evaluated. Lysozyme was dissolved at 0.1, 0.5, 1, 2 g/L and freeze-dried Micrococcus lysodeikticus cells (Sigma-Aldrich) were resuspended at 250 μg/ml in PBS. The Micrococcus lysodeikticus cell suspension (300 μL) was added to lysozyme solution (300 μL), and cell lysis was followed at room temperature by measuring the decrease in OD at 450 nm (OD450) using ultraviolet-visible (UV-vis) spectroscopy. The decrease in OD450 for the first 1 minute was used as the measure of lysozyme activity ⁸.