Enhanced Bioactivity and Bacteriostasis of Surface Fluorinated

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Enhanced bioactivity and bacteriostasis of surface fluorinated polyetheretherketone Meiling Chen, Liping Ouyang, Tao Lu, Heying Wang, Fanhao Meng, Yan Yang, Congqin Ning, Jingzhi Ma, and Xuanyong Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 8, 2017

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ACS Applied Materials & Interfaces

Enhanced bioactivity and bacteriostasis of surface fluorinated polyetheretherketone Meiling Chena,1, Liping Ouyangb,c,1, Tao Lub, Heying Wangb, Fanhao Mengb, Yan Yanga, Congqin Ningb, Jingzhi Maa,*, Xuanyong Liub,* a

Department of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan, P. R. China b

State Key Laboratory of High Performance Ceramics and Superfine Microstructure,

Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China c

University of Chinese Academy of Science, Beijing 100049, P. R. China

1

ACS Paragon Plus Environment


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Abstract: Although polyetheretherketone (PEEK) has been considered as the potential orthopedic and dental application material due to its similar elastic modulus as bones, the inferior osseointegration and bacteriostasis of PEEK hamper its clinical application. In this work, fluorinated PEEK was constructed via plasma immersion ion implantation (PIII) followed by hydrofluoric acid treatment to ameliorate the osseointegration and antibacterial properties of PEEK. The surface microstructure, composition and hydrophilicity of all samples were investigated. Rat bone mesenchymal stem cells (rBMSCs) were cultured on their surfaces to estimate bioactivity. The fluorinated PEEK can enhance the cell adhesion, cell spreading, proliferation and ALP-activity compared to pristine PEEK. Furthermore, the fluorinated PEEK surface exhibits good bacteriostatic effect against Porphyromonas Gingivalis, which is one of the major periodontal pathogens. In summary, we provide an effective route to introduce fluorine and the results reveal that the fluorinated PEEK can enhance the osseointegration and bacteriostasis, which provides a potential candidate for dental implants. Keywords: PEEK; fluorination; plasma immersion ion implantation; osseointegration; bacteriostasis

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INTRODUCTION With the increasing demands for longevity and high quality of life, human implants have been considered as suitable substitutes for damaged human bones caused by disease, trauma and aging, especially for teeth missing caused by aging. Nevertheless, according to long-term clinical tracking and research, there are a few causes for the failure of human implants, including mismatch of traditional metal implants with bone tissues in mechanical properties, inferior osseointegration caused by poor bioactivity, and bacterial infection of peri-implant tissues1. Therefore, to obtain dental implants with superior clinical performance, it is essential to explore new materials with enhanced osseointegration and antibiotic properties. As a preferred substitute for traditional metal biomaterials, polyetheretherketone (PEEK) was highlighted in the 1980s2. PEEK provides a plenty of superior properties for biomedical implants, including relatively closer elastic modulus to cortical bone3, good chemical and sterilization resistance4, excellent mechanical properties5, non-toxicity6, and natural radiolucency. However, the chemical stability of PEEK leads to poor bioactivity and inferior osseointegration7. Various methods have been developed to modify PEEK surface for improved bioactivity without affecting its bulk properties, such as depositing apatite on NaOH-treated PEEK8, coating PEEK surface with hydroxyapatite (HA) and titanium9-10 and grafting specific functional groups on PEEK surface11. It has been proved that these methods are effective, whereas there still exist some inherent drawbacks such as poor bonding between substrates and modification layers, and complex and time-consuming chemical reactions. Besides, 3



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despite extensive studies on the osseointegration of PEEK, there is few report about the antibiotic property. Fluorine is the essential micro-element for human life and plays a crucial role on bone growth and physiological function maintenance. It has been demonstrated that trace amounts of fluorine can accelerate osteoblast proliferation, up-regulate alkaline phosphatase activity, and stimulate osteocalcin and collagen I synthesis12-14. On the other hand, fluoride possesses good antimicrobial properties. For instance, sodium fluoride is worldwide employed to inhibit streptococcus for caries prevention15. Besides, some researchers provided a useful approach to inhibit bacterial activity by fluorinating the biomaterial surface16-17. Therefore, it is a good choice to introduce fluorine to PEEK surface for enhanced bioactivity and antibacterial ability. PEEK cannot react with most of the chemicals due to its chemical inertness except for concentrated sulfuric acid3,

18-19

. Therefore, the combination of physical and

chemical methods would be an effective strategy to introduce fluorine onto PEEK surface. Among the various physical techniques, PIII precedes due to its non-light-of-sight feature, which is beneficial to modify the complex shaped biomaterials20-26. During PIII treatment, radiation disrupts the structure of polymers by displacement of the atoms or by excitation of the electrons27, resulting in a relatively active polymer surface, which is easier to be chemically modified. In this work, argon PIII followed by hydrofluoric acid treatment is employed to fluorinate PEEK surface. The in vitro responses of cells to the samples are evaluated by culturing rBMSCs on their surfaces. Porphyromonas Gingivalis (P. gingivalis), one 4


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of the periodontal pathogens, was used to determine the antibacterial ability of the samples. A craniofacial model, the same bone structure with mandible, is utilized to evaluate the in vivo osseointegration ability.

EXPERIMENTAL SECTION Fluorinated PEEK Preparation. The samples were one-side polished, and the square samples with the size of 10 mm were designed for surface characterization and in vitro studies , the 20 mm long and 10 mm wide rectangle samples were used for zeta potential measurements, and the size of 10 mm diameter wafer were used for in vivo evaluation. Before PIII treatment, the samples were ultrasonically cleaned to remove contaminations. Table 1 lists the parameters of the argon PIII. After argon PIII treatment, the samples were taken out from the vacuum chamber and immediately immersed in hydrofluoric acid aqueous solution (HF, AR, China National Medicines Co. Ltd.) for 24 hours, then followed by ultrasonically cleaned with deionized water (3 times, each time for 20 minutes). The obtained samples are designated as AF-PEEK. Meanwhile, the samples subjected to argon PIII treatment were conducted as the comparison set. The obtained samples are designated as A-PEEK. Morphology and chemistry characterization. The morphologies of the samples were observed by scanning electron microscopy (FE-SEM; S-4800, HITACHI, Japan). The surface chemical states were detected by X-ray photoelectron spectroscopy (XPS) (Perkin Elmer) with Mg Kα radiation. Zeta-potential measurements. The surface zeta-potential of the samples was 5



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measured by Surpass electrokinetic analyzer (Anton Parr, Austria). A KCl solution (0.001 M) was used as the medium. HCl and NaOH aqueous solution were used to adjust the pH value. The zeta-potential was measured and calculated according to the following formula,28

where ζ denotes the zeta-potential, and η, ε0 and ε denote the viscosity, vacuum permittivity and dielectric constant of the electrolyte solution. The measurement was conducted four times at each pH value.

Wettability measurement. Contact angle measurement (Automatic Contact Angle Meter Model SL200B, Solon information technology Co., Ltd, China) was applied to evaluate the surface wettability. After 1 µL of water droplet dropped on the specimen surface, the equipped camera system would capture a photo, according to which the contact angle was measured. In vitro cell response Cell adhesion and spreading assay. rBMSCs (Stem Cell Bank, Chinese Academy of Sciences) suspension with the density of 5.0×104 cells/mL was cultured on each surface at 37 oC in a 5% CO2 incubator and incubated for 60 minutes. At each time points, the cells were rinsed with the PBS solution and fixed with 2.5% glutaraldehyde aqueous solution. Before SEM observation, the cells were dehydrated using graded ethanol solutions for 10 minutes sequentially. Cell proliferation. The alamarBlueTM assay was applied to detect the proliferation of 6


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the cells cultured on the samples. At 1, 4 and 7 days, five specimens were tested. Accumulation of reduced alamarBlueTM was quantified on a microplate reader based on the following formula,29

where Aλ1 denotes the wavelength of the test wells at 570nm; Aλ2 denotes the wavelength of the test wells at 600 nm; A

λ1

denotes the wavelength of control wells

at 570 nm; A λ2 denotes the wavelength of control wells at 600 nm. In terms of cell morphology observation, rBMSCs was rinsed by PBS, fixed by 2.5% glutaraldehyde solution at 4 oC, and then subjected to step dehydration in graded ethanol solutions prior to SEM observation. ALP activity assay. The ALP activity of rBMSCs incubated for 14 days was estimated using the ALP reagent. The amount of p-nitrophenol, which was produced by incubating the supernatant with p-nitrophenyl phosphate for 30 minutes at 37 oC, was spectrophotometrically quantified by microplate reader at 405 nm. The BCA protein assay was applied to estimate total protein content and normalized with graded BSA (bovine serum albumin, Sigma) standard at 570 nm. Besides, the ALP activity was qualitatively observed by a staining method. The samples were rinsed with the PBS solution, followed by fixation with citrate buffered acetone. Then an alkaline-dye mixture composed of naphthol AS-MX phosphate and fast blue RR salt solution was added to each sample. After incubation for 30 minutes, the samples were stained with Mayer’s Hematoxylin Solution for 10 minutes and 7



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finally examined by fluorescence microscopy.

Assessment of bacteriostatic activity. P. gingivalis is one of the major periodontal pathogens, therefore the bacteriostatic activity of the samples against Porphyromonas Gingivalis (ATCC 3377) was examined by the bacterial counting method. Also, Staphylococcus aureus (ATCC 25923), the usual bacteria in water, was used to evaluated the bacteriostatic activity. The specimens were sterilized with 75% ethanol for 2 hours and then 60 µL solution with the bacteria at the density of 107 CFU/mL was dropped onto the samples’ surfaces. After 24h, the solution with detached bacteria was inoculated into a standard agar culture medium and incubated for another 24h. For statistical accountability, four petri dishes of bacteria were incubated and counted for each sample set. Live/Dead Kits was applied to observe the fluorescent staining of the samples attached with bacteria. After rinsing twice with the PBS solution, 500 mL of the Live/Dead staining reagent was added. After incubation for 15 minutes in the dark, the stained samples were observed by fluorescence microscopy.30

In vivo osseointegration Surgical procedures. All the animal procedures were approved by the Experimental Animal Center of Huazhong University of Science and Technology. 24 male Sprague Dawley rats, weighed 200 ~ 250g, were separated into three groups. To let the skull be exposed, an arc incision was made between two ears and the mucoperiosteum was 8


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separated carefully. Afterwards, a cycle area with a diameter of 10 mm on the skull was grinded away, then the samples were put into the aforementioned cycle area and the wound was closed carefully. Sequential fluorescent labeling. The newly formed bone and mineralization was characterized by polychrome sequential fluorescent labeling method. Different fluorochromes were administered intraperitoneally at a sequence of 30 mg/kg alizarin red S and 20 mg/kg calcein at 2 and 6 weeks after operation. Sample preparation. All the rats in the three groups were sacrificed after surgery for 8 weeks. The micro-CT observation and histomorphometric measurement were carried out after harvesting the skull of the rats with the implants. Micro-CT assay. The new bone formation was determined by imaging the fixed samples with micro-CT (Scanco-medical micro-CT 100 system; Scanco Medical, Bassersdorf, Switzerland).

The two-dimensional (2D) models were reconstructed

using the MeVisLab 2.8.2 software. Histological analysis. After micro-CT, the samples were dehydrated with graded ethanol and embedded in polymethylmethacrylate (PMMA). The specimens were cut into 60 µm thick slices perpendicular to the bone using Leica SP1600 saw microtome. After polishing the specimens to the thickness of 50 µm, the observation of fluorescence labeling was conducted under the confocal laser scanning microscope. The excitation/emission wavelengths of 543/620 nm and 488/520 nm were used to observe chelating fluorochromes for alizarin red S (red) and calcein (green) respectively. Subsequently, the sections were stained with methylene-fuchsin. The 9



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mineralized bone tissue (red) were recorded by a fluorescence microscope.

Statistical analysis. Statistically significant differences (P) between the various groups were calculated using one-way analysis of variance and Tukey’s multiple comparison tests. All the data are expressed as means ± SD.

RESULTS AND DISCUSSION Surface characterizations. The surface morphologies of the PEEK samples before and after treatment are exhibited in Fig. 1. The surface of pristine PEEK is flat (Fig. 1a), whereas the nanostructure emerges on the A-PEEK surface after argon PIII (Fig. 1b), which is believed to result from the polymer chain scission during PIII treatment 31

. In contrast, the surface morphology of hydrofluoric acid treated PEEK without PIII

treatment is similar to that of the pristine PEEK (Fig. S1). The morphology of AF-PEEK is well inherited from A-PEEK as shown in Fig. 1c, indicating that hydrofluoric acid treatment has negligible effect on the morphology of A-PEEK. The results demonstrate that argon PIII induces the formation of nanostructure on PEEK surface, whereas hydrofluoric acid treatment brings no changes on the morphology of A-PEEK. PIII bodes well for the modification of biomedical implants with complex shape due to its non-light-of-sight feature. In our previous work, water PIII was employed to modify the surface of PEEK for enhanced osteoblast responses and argon PIII was found to be the main cause of scission of polymer chain and the formation of 10


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nanostructure32. Upon argon PIII, the occurrence of polymer chain scission and the generation of free radicals on the surface of samples provide an active surface for the subsequent modification. Moreover, the argon PIII can avoid the introduction of undesirable impurity components. Therefore, it is an effective approach to obtain an active and uncontaminated surface of polymer through argon PIII for the subsequent modification. XPS is applied to characterize the chemical states of all samples surfaces and the results are exhibited in Fig. 2. Only the peak of C1s and O1s can be observed on PEEK and A-PEEK (Fig. 2a and b), while a significant peak representing fluorine (F1s) emerges in the spectrum of AF-PEEK at a binding energy of 687 eV 33 (Fig. 2c), demonstrating that the surface of AF-PEEK is fluorinated. However there is a small peak of fluorine in the spectrum of F-PEEK (Fig. S2). According to the semiquantitative results, the fluorine content on the surface of F-PEEK and AF-PEEK are 0.79% and 9.01% respectively, indicating that the fluorine on F-PEEK is much less than that on AF-PEEK. Fig. 2d is the high-resolution spectrum of carbon (C1s) on AF-PEEK, from which a fitted peak representing C*-F bond (288.5 eV 34) is clearly observed. Therefore, the combined argon PIII and hydrofluoric acid treatment is a technical knowhow to introduce a plenty of fluorine on PEEK surface. The nanostructure on the surface of samples can be fabricated through argon PIII due to the scission of polymer chain. Meanwhile, the argon PIII provides an active surface to interact with F-, whereas the pristine PEEK is inertness and cannot interact with F-. Consequently, the incorporation of argon PIII and hydrofluoric acid treatment 11



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is pivotal to the introduction of fluorine on the surface of PEEK. The electrical state near the surface is one of the factors that influence the biological performance. The surface zeta-potential is used to detect the electrical state near the surface and the results are displayed in Fig. 2e. Each sample has a descending zeta-potential with the ascending pH values of the electrolyte solution. The zeta-potential of A-PEEK and AF-PEEK at the pH value of 7.4, the pH closed to that in physiological environment, is more positive than PEEK. Compared with A-PEEK, the zeta-potential of AF-PEEK is relatively more negative owing to the existence of fluorine. The surface hydrophilicity of the samples surfaces is characterized by static contact angle measurement (Fig. 2f). The contact angle of pristine PEEK is 81o, whereas the contact angle of PEEK treated by argon PIII increases significantly to 125o on account of the hydrophobic recovery of polymers treated by high energy particles

35

. In

contrast, the contact angle of AF-PEEK decreases dramatically to 32o, indicating highly improved hydrophilicity of the surface of fluorinated PEEK.

Responses of rBMSCs. To estimate the primal cell adhesion of the samples, the cell morphologies are observed by SEM (Fig. 3a, b and c). Most of the cells on PEEK exhibit a round morphology with pseudopodia (Fig. 3a). The cell morphology on A-PEEK tends to become fusiform (Fig. 3b) and the lamellipodia extensions can be observed on AF-PEEK, (Fig. 3c). Hence, the fluorinated PEEK surface is favorable for cell adhesion, offering superior conditions for the subsequent cell proliferation. 12


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Assessment of cell proliferation activity is conducted to further investigate the cytocompatibility of the various surfaces. Cell subsistence on AF-PEEK surface is obviously improved as revealed in Fig. 3d, demonstrating the favorable role of fluorinated PEEK in cell proliferation. Argon PIII treated PEEK can also accelerate cell viability to some extent, which is attributed to the nanostructure on the surface of Argon PIII treated PEEK. There is no significant difference in cell proliferation activity between F-PEEK and PEEK (Fig.S3), indicating that the low fluorine content on the PEEK surface immersed in HF solution has a negligible effect on acceleration of cell proliferation. The cell morphology examination (Fig. S4) agrees well with the results of the cell proliferation activity assay. The ALP activity of rBMSCs cultured on the surfaces of samples for 14 days is displayed in Fig. 4. Fig. 4a, b and c show the fluorescent images of cells cultured on the three sets of samples with ALP dyed dark red. The surface of AF-PEEK is almost covered with dyed areas (Fig. 4c), while A-PEEK has larger dyed areas than unmodified samples (Fig. 4b). The quantitative ALP activity of rBMSCs cultured on the samples surfaces for 14 days is displayed in Fig. 4d. The ALP activity is up-regulated both on A-PEEK and AF-PEEK surface, especially on AF-PEEK. In comparison, the ALP activity of rBMSCs on F-PEEK is almost at the same level as PEEK (Fig. S5). It is known that ALP is the marker for the early stage of osteogenic differentiation

38

. Our results indicate that fluorinated PEEK could be a promising

alternative medical implant as it can accelerate the osteogenic differentiation of rBMSCs. 13



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The observation of the enhanced adhesion, proliferation and osteogenic differentiation of rBMSCs can be found on the fluorinated PEEK. Yoon et al. found that human osteoblastic cells grown on the fluorapatite-collagen composites exhibited significantly enhanced proliferation and differentiation activity than those on the hydroxyapatite-collagen composite37. The combined advantages of fluorine and nanostructure account for the cell responses38. Though the nanostructure on A-PEEK is favorable for cell proliferation and differentiation, the effect is reduced due to the hydrophobic recovery of argon PIII treated PEEK. The introduction of fluorine in AF-PEEK provide a more hydrophilic surface than A-PEEK, which is favorable for cell adhesion. Therefore, it can be deduced that the incorporation of argon PIII and hydrofluoric acid treatment plays an important role to enhance cell response of PEEK.

In vitro bacteriostatic effect. Except for superior osseointegration, the antibacterial ability against periodontal pathogen is of equal importance for the success of dental implants. Considering that P. gingivalis is one of the major periodontal pathogens, P. gingivalis were used to determine in vitro bacteriostatic effect by the Live/Dead staining and bacterial counting method (Figs. 5 and 6). The bacteriostatic effect evaluated by bacterial counting method is displayed in Fig. 5. The number of bacteria on A-PEEK and AF-PEEK is less than PEEK, especially on AF-PEEK. The fluorescent images of P. gingivalis on various surfaces after applying the Live/Dead staining kits are exhibited in Fig. 6. The total amounts of red stains on AF-PEEK is remarkably more than that on PEEK and A-PEEK, indicating that more 14


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dead bacteria exist on AF-PEEK when compared to PEEK and A-PEEK. Besides, the red stains on PEEK are negligible compared with those on A-PEEK and AF-PEEK, indicating that the inability of PEEK killing bacteria and the enhanced antibacterial ability of AF-PEEK samples against P. gingivalis. The results are consistent with the results of bacterial counting method. Consequently, we can conclude that the introduction of fluorine is favorable for bacteriostatic effect against P. gingivalis of PEEK. S. aureus, the usual bacteria in water, was used to determine the antibacterial ability of all samples too. The hydrofluoric acid treatment can introduce a small amount of fluorine to the surface of PEEK and the samples have a bacteriostatic effect against S. aureus, whereas the bacteriostatic effect against S. aureus is negligible compared to AF-PEEK due to the limited fluorine content (Fig. S6 and 7). Consequently, the argon PIII plays an imperative role in the introduction of enough fluorine content to improve the bacteriostatic effect of PEEK against both two types of bacteria. Other than superior osseointegration, the antibacterial ability against periodontal pathogen is of equal importance for the success of dental implants24,

39-40

. The

interface between the implants surfaces and tissues is easy to be infected by bacteria and serious infection leads to post-surgical implant failure41. Therefore, an antibacterial surface of implant is imperative. Fluorine is an effective antibacterial reagent due to its effect on the bacterial metabolism as an enzyme inhibitor. The formation of metal fluoride complexes results in fluoride-induced inhibition of proton-translocating F-ATPases by mimicking phosphate to form complexes with 15



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ADP at the reaction centers of enzymes42. In this work, the antibacterial effect of samples against P. gingivalis, one of the major periodontal pathogens, is enhanced by introducing fluorine.

In vivo osseointegration. The results of bone formation evaluated by micro-CT and sequential fluorescent labeling are displayed in Fig.7. The micro-CT evaluation shows that the newly formed bone of PEEK cannot seal the wound manufactured by craniofacial model and that of A-PEEK can almost seal the wound. However, the wound of AF-PEEK manufactured by craniofacial model can seal absolutely. The newly formed bone is more tightly anchored to the surface of AF-PEEK than those of PEEK and A-PEEK. The right column represents the results of sequential fluorescent labeling. Two weeks later, alizarin red is layered on the surfaces of all samples, whereas calcein can scarcely be observed on the PEEK surface at six weeks. However, obvious green fluorescence can be found from the A-PEEK and AF-PEEK samples and the fluorescent area ratio of AF-PEEK is more than A-PEEK. In summary, the results testify that fluorinated PEEK stimulates more newly formed bone through the whole procedures. After 8-week

implantation,

the

histological

sections are processed

by

methylene-fuchsin staining and the results are shown in Fig. 8. The basic fuchsin stains bone red and methylene blue stains fibrous tissue blue43-46. From the low magnification views, fibrous tissue can be observed around all samples, whereas the distance between the newly formed bone and PEEK is more than A-PEEK and 16


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AF-PEEK. From the high magnification views, it can be found that the newly formed bone is tightly anchored onto the surface of AF-PEEK. Besides, the fibrous tissue encompassing the PEEK and A-PEEK is significantly thicker than AF-PEEK, which indicates an enhanced osseointegration of fluorinated PEEK because the thick fibrous tissue implies the weak bone-bonding between the samples and tissue. Consequently, the samples through argon PIII followed by hydrofluoric acid treatment is favorable for bone integration. In

vivo

evaluation

by

micto-CT,

sequential

fluorescent

labeling

and

methylene-fuchsin staining shows that the samples through argon PIII followed by hydrofluoric acid treatment possess an enhanced osseointegration. And the results in vivo correspond to the results in vitro, indicating that the introduction of fluorine and nanostructure are essential for the osseointegration of PEEK.

CONCLUSION The argon PIII followed by hydrofluoric acid treatment is utilized to introduce fluorine to PEEK surface. Argon PIII is crucial for the introduction of fluorine. Meanwhile, the nanostructured surface fabricated by argon PIII and the fluorine introduced by hydrofluoric acid treatment have a synergistic effect on cell proliferation and differentiation. And the in vivo results present a good osseointegration of fluorinated PEEK. Additionally, the antibacterial determination against P. gingivalis in vitro, one of the major periodontal pathogens, reveals that fluorinated PEEK exhibits a better bacteriostatic effect than PEEK. The argon PIII 17



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followed by hydrofluoric acid treatment provides an effective approach to introduce fluorine to PEEK surface, and the superior properties of osseointegration and bacteriostasis give an easier access to future dental applications.

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ASSOCIATED CONTENT Supporting information The surface characterizations including SEM and XPS of the samples only through HF treatment; the in vivo cell response including cell proliferation, cell adhesion and ALP activity evaluation; the in vivo antibacterial properties against S. aureus; supporting figures and text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Prof. Xuanyong Liu. E-mail: [email protected]. Tel: +86 2152412409. Fax: +86 21 52412409. Prof. Jingzhi Ma. Email: [email protected] Author Contributions 1

These authors contributed equally to this work

Notes The authors declare no competing financial interest. Acknowledgements Financial support from the National Science Foundation for Distinguished Young Scholars of China (51525207), Shanghai Committee of Science and Technology, China (16441904600, 14JC1493100, 14XD1403900) and National Natural Science Foundation of China (81641035) are acknowledged.

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22


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Table 1 Part of the parameters for plasma immersion ion implantation of PEEK. pressure

5×10-3 Pa

flow rate

30 sccm

bias voltage

-800 V

radiofrequency power

300 W

pulsing frequency

30 kHz

duty ratio

30%

treatment time

60 minutes

23



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Fig. 1 Surface morphologies of the samples: (a) PEEK, (b) A-PEEK and (c) AF-PEEK; (d) schematic of fluorination. 42x30mm (300 x 300 DPI)


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Fig. 2 XPS full spectra of the samples: (a) PEEK, (b) A-PEEK, (c) AF-PEEK, (d) high-resolution spectrum of C1s on AF-PEEK; (e) Zeta-potential variation versus pH of the potassium chloride solution acquired from the samples; (f) Water contact angles of the samples: all the data are expressed as means ± SD and n=5, The insets in the figure are the optical images of water droplet on the corresponding sample surfaces. 58x66mm (300 x 300 DPI)



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Fig. 3 SEM images at different magnifications showing the initial adhesion and spreading of rBMSCs incubated on the samples: (a) PEEK, (b) A-PEEK, (c) AF-PEEK; and (d) Reduction percentages of alamarBlueTM showing the proliferation and viabilities of rBMSCs cultured on the sample surfaces. (* p < 0.05, *** p < 0.001; All the data are expressed as means ± SD and n=5) 44x31mm (300 x 300 DPI)


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Fig. 4 ALP activities of rBMSCs cultured on the samples for 14 days. Fluorescence microscopy images of rBMSCs: (a) PEEK, (b) A-PEEK, (c) AF-PEEK; and (d) the quantitative activity of rBMSCs cultured on the surface of samples. (* p < 0.05, ** p < 0.01. All the data are expressed as means ± SD and n=4) 48x37mm (300 x 300 DPI)



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Fig. 5 Photographs of re-cultivated P. gingivalis colonies on agar which were previously dissociated from the samples: (a) PEEK, (b) A-PEEK and (c) AF-PEEK; (d) the results of the bacteria counting method. The data in (d) are expressed as means ± SD and n=4. 52x53mm (300 x 300 DPI)


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Fig. 6 Confocal micrographs of P. gingivalis cultured on both samples for 24 h on the samples: the green fluorescence referred to live bacteria and the red fluorescence referred to dead bacteria. Scar bars for insets = 75µm. 101x101mm (300 x 300 DPI)



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Fig.7 Characterization of implants and the surrounding bones by Micro-CT and sequential fluorescent labeling: (a) PEEK, (b) A-PEEK and (c) AF-PEEK; (d) The schematic of implants, (e) Histogram of percentage for the area of fluorochromes stained bone. *** (p < 0.01) when compared with PEEK. 127x73mm (300 x 300 DPI)


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Fig.8 Methylene-fuchsin staining of samples: (a) PEEK, (b) A-PEEK and (c) AF-PEEK 127x85mm (300 x 300 DPI)



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Table of Content 83x37mm (300 x 300 DPI)


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Enhanced Bioactivity and Bacteriostasis of Surface Fluorinated

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