Enhanced bioactivity and bacteriostasis of surface fluorinated

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 ...
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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*,‡

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Department of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China ‡ State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China § University of Chinese Academy of Science, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: Although polyetheretherketone (PEEK) has been considered as a potential orthopedic and dental application material due to its similar elastic modulus as bones, inferior osseointegration and bacteriostasis of PEEK hampers 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 alkaline phosphatase (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

As a preferred substitute for traditional metal biomaterials, polyetheretherketone (PEEK) was highlighted in the 1980s.2 PEEK provides a plenty of superior properties for biomedical implants, including relatively closer elastic modulus to that of cortical bone,3 good chemical and sterilization resistance,4 excellent mechanical properties,5 nontoxicity,6 and natural radiolucency. However, the chemical stability of PEEK leads to poor bioactivity and inferior osseointegration.7 Various methods have been developed to modify PEEK surface for improved bioactivity without affecting its bulk properties, such as depositing apatite on NaOH-treated PEEK,8 coating PEEK

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 periimplant tissues.1 Therefore, to obtain dental implants with superior clinical performance, it is essential to explore new materials with enhanced osseointegration and antibiotic properties. © 2017 American Chemical Society

Received: February 20, 2017 Accepted: May 5, 2017 Published: May 5, 2017 16824

DOI: 10.1021/acsami.7b02521 ACS Appl. Mater. Interfaces 2017, 9, 16824−16833

Research Article

ACS Applied Materials & Interfaces surface with hydroxyapatite (HA) and titanium9,10 and grafting specific functional groups on PEEK surface.11 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 timeconsuming chemical reactions. Besides, despite extensive studies on the osseointegration of PEEK, there are few reports about the antibiotic properties. Fluorine is the essential microelement 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, upregulate alkaline phosphatase activity, and stimulate osteocalcin and collagen I synthesis.12−14 On the other hand, fluoride possesses good antimicrobial properties. For instance, sodium fluoride is employed worldwide to inhibit streptococcus for prevention of caries.15 Besides, some researchers provided a useful approach to inhibit bacterial activity by fluorinating the biomaterial surface.16,17 Therefore, it is a good choice to introduce fluorine to the 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 acid.3,18,19 Therefore, the combination of physical and chemical methods would be an effective strategy to introduce fluorine onto the PEEK surface. Among the various physical techniques, PIII stands out due to its non-light-of-sight feature, which is beneficial to modify the complex shaped biomaterials.20−26 During PIII treatment, radiation disrupts the structure of polymers by displacement of the atoms or by excitation of the electrons,27 resulting in a relatively active polymer surface, which is easier to modify chemically. 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, one of the periodontal pathogens, was used to determine the antibacterial ability of the samples. A craniofacial model, the same bone structure with a mandible, is utilized to evaluate the in vivo osseointegration ability.

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h, followed by ultrasonically cleaning with deionized water (three times, each time for 20 min). 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) (PerkinElmer) with Mg Kα radiation. Zeta-Potential Measurements. The surface zeta-potential of the samples was measured by using a 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 formula28

ζ=

where ζ denotes the zeta-potential, and η, ε0, and ε denote the viscosity, vacuum permittivity, and dielectric constant of the electrolyte solution, respectively. 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 was 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 °C in a 5% CO2 incubator and incubated for 60 min. At each time point, the cells were rinsed with PBS solution and fixed with 2.5% glutaraldehyde aqueous solution. Before SEM observation, the cells were dehydrated using graded ethanol solutions for 10 min sequentially. Cell Proliferation. The alamarBlue assay was applied to detect the proliferation of the cells cultured on the samples. At 1, 4, and 7 days, five specimens were tested. Accumulation of reduced alamarBlue was quantified on a microplate reader based on the following formula29

117216Aλ1 − 80586Aλ2 × 100% 155677A′λ2 − 14652A′λ1 where Aλ1 denotes the wavelength of the test wells at 570 nm, Aλ2 denotes the wavelength of the test wells at 600 nm, A′λ1 denotes the wavelength of control wells at 570 nm, and A′λ2 denotes the wavelength of control wells at 600 nm. In terms of cell morphology observation, rBMSCs were rinsed with PBS, fixed with 2.5% glutaraldehyde solution at 4 °C, and then subjected to step dehydration in graded ethanol solutions prior to SEM observation. ALP Activity Assay. The alkaline phosphatase (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 min at 37 °C, was spectrophotometrically quantified by using a 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 via 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 min, the samples were stained with Mayer’s hematoxylin solution for 10 min and finally examined by fluorescence microscopy. Assessment of Bacteriostatic Activity. P. gingivalis is one of the major periodontal pathogens; therefore, the bacteriostatic activity of

EXPERIMENTAL SECTION

Fluorinated PEEK Preparation. The samples were one-side polished, and the square samples with size of 10 mm were designed for surface characterization and in vitro studies; 20 mm long and 10 mm wide rectangle samples were used for zeta potential measurements, and size 10 mm diameter wafers 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

Table 1. Part of the Parameters for Plasma Immersion Ion Implantation of PEEK pressure flow rate bias voltage radiofrequency power pulsing frequency duty ratio treatment time

dU η K dP ε ε0

5 × 10−3 Pa 30 sccm −800 V 300 W 30 kHz 30% 60 min 16825

DOI: 10.1021/acsami.7b02521 ACS Appl. Mater. Interfaces 2017, 9, 16824−16833

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Figure 1. Surface morphologies of the samples: (a) PEEK, (b) A-PEEK, and (c) AF-PEEK. (d) Schematic of fluorination. 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 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.

the samples against P. gingivalis (ATCC 3377) was examined by using 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 h and then 60 μL solution with the bacteria at the density of 107 CFU/mL was dropped onto the sample surfaces. After 24 h, the solution with detached bacteria was inoculated into a standard agar culture medium and incubated for another 24 h. For statistical accountability, four Petri dishes of bacteria were incubated and counted for each sample set. A Live/Dead kit was applied to observe the fluorescent staining of the samples with bacteria attached. After rinsing twice with the PBS solution, 500 mL of the Live/Dead staining reagent was added. After incubation for 15 min 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. Twenty-four 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 separated carefully. Afterward, 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 8 weeks after surgery. The micro-CT observation and histomorphometric measurement were carried out after harvesting the skulls of the rats with the implants. Micro-CT Assay. The new bone formation was determined by imaging the fixed samples with a micro-CT (Scanco-medical micro-CT 100 system; Scanco Medical, Bassersdorf, Switzerland). The twodimensional (2D) models were reconstructed using MeVisLab 2.8.2 software. Histological Analysis. After micro-CT, the samples were dehydrated with graded ethanol and embedded in poly(methyl methacrylate) (PMMA). The specimens were cut into 60 μm thick slices perpendicular to the bone using a Leica SP1600 saw microtome.

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RESULTS AND DISCUSSION Surface Characterizations. The surface morphologies of the PEEK samples before and after treatment are exhibited in Figure 1. The surface of pristine PEEK is flat (Figure 1a), whereas the nanostructure emerges on the A-PEEK surface after argon PIII (Figure 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 (Figure S1). The morphology of AF-PEEK is well inherited from A-PEEK as shown in Figure 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 nonlight-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 nanostructure.32 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. There16826

DOI: 10.1021/acsami.7b02521 ACS Appl. Mater. Interfaces 2017, 9, 16824−16833

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Figure 2. XPS full spectra of the samples: (a) PEEK, (b) A-PEEK, and (c) AF-PEEK. (d) High-resolution spectrum of C 1s on AF-PEEK. (e) Zetapotential 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.

cannot interact with F−. Consequently, the incorporation of argon PIII and hydrofluoric acid treatment 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 Figure 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 pH 7.4, the pH close to that in physiological environment, is more positive than that of PEEK. Compared with A-PEEK, the zetapotential 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 (Figure 2f). The contact angle of pristine PEEK is 81°, whereas the contact angle of PEEK treated by argon PIII increases significantly to 125° 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 32°, 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

fore, it is an effective approach to obtain an active and uncontaminated surface of polymer through argon PIII for subsequent modification. XPS is applied to characterize the chemical states of all samples surfaces and the results are exhibited in Figure 2. Only the peak of C 1s and O 1s can be observed on PEEK and APEEK (Figure 2a and b), while a significant peak representing fluorine (F 1s) emerges in the spectrum of AF-PEEK at a binding energy of 687 eV33 (Figure 2c), demonstrating that the surface of AF-PEEK is fluorinated. However, there is a small peak of fluorine in the spectrum of F-PEEK (Figure 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. Figure 2d is the high-resolution spectrum of carbon (C 1s) on AF-PEEK, from which a fitted peak representing C*−F bond (288.5 eV34) 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 16827

DOI: 10.1021/acsami.7b02521 ACS Appl. Mater. Interfaces 2017, 9, 16824−16833

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Figure 3. SEM images at different magnifications showing the initial adhesion and spreading of rBMSCs incubated on the samples: (a) PEEK, (b) APEEK, and (c) AF-PEEK. (d) Reduction percentages of alamarBlue 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).

Figure 4. ALP activities of rBMSCs cultured on the samples for 14 days. Fluorescence microscopy images of rBMSCs: (a) PEEK, (b) A-PEEK, and (c) AF-PEEK. (d) 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).

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 Figure 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

by SEM (Figure 3a−c). Most of the cells on PEEK exhibit a round morphology with pseudopodia (Figure 3a). The cell morphology on A-PEEK tends to become fusiform (Figure 3b), and the lamellipodia extensions can be observed on AF-PEEK (Figure 3c). Hence, the fluorinated PEEK surface is favorable for cell adhesion, offering superior conditions for subsequent cell proliferation. 16828

DOI: 10.1021/acsami.7b02521 ACS Appl. Mater. Interfaces 2017, 9, 16824−16833

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Figure 5. Photographs of recultivated P. gingivalis colonies on agar which were previously dissociated from the samples: (a) PEEK, (b) A-PEEK, and (c) AF-PEEK. (d) Results of the bacteria counting method. Data in (d) are expressed as means ± SD and n = 4.

cells grown on the fluorapatite-collagen composites exhibited significantly enhanced proliferation and differentiation activity than those on the hydroxyapatite-collagen composite.37 The combined advantages of fluorine and nanostructure account for the cell responses.38 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 (Figures 5 and 6). The bacteriostatic effect evaluated by bacterial counting method is displayed in Figure 5. The number of bacteria on APEEK 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 Figure 6. The total amounts of red stains on AF-PEEK is remarkably more than that on PEEK and A-PEEK, indicating that more

treated PEEK. There is no significant difference in cell proliferation activity between F-PEEK and PEEK (Figure 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 (Figure 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 Figure 4. Figure 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 (Figure 4c), while A-PEEK has larger dyed areas than unmodified samples (Figure 4b). The quantitative ALP activity of rBMSCs cultured on the samples surfaces for 14 days is displayed in Figure 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 (Figure S5). It is known that ALP is the marker for the early stage of osteogenic differentiation.36 Our results indicate that fluorinated PEEK could be a promising alternative medical implant as it can accelerate the osteogenic differentiation of rBMSCs. 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 16829

DOI: 10.1021/acsami.7b02521 ACS Appl. Mater. Interfaces 2017, 9, 16824−16833

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to the limited fluorine content (Figures 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 types of bacteria. Other than superior osseointegration, the antibacterial ability against periodontal pathogens is of equal importance for the success of dental implants.24,39,40 The interface between the implants surfaces and tissues is easy to be infected by bacteria and serious infection leads to postsurgical implant failure.41 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 ADP at the reaction centers of enzymes.42 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 Figure 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 to the surfaces 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 6 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 procedure. After 8-week implantation, the histological sections are processed by methylene-fuchsin staining and the results are shown in Figure 8. The basic fuchsin stains bone red and methylene blue stains fibrous tissue blue.43−46 From the low magnification views, fibrous tissue can be observed around all

Figure 6. Confocal micrographs of P. gingivalis cultured on both samples for 24 h on the samples: the green fluorescence indicates live bacteria and the red fluorescence indicates dead bacteria. Scale bars for insets = 75 μm.

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

Figure 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) Schematic of implants. (e) Histogram of percentage for the area of fluorochromes stained bone. ***p < 0.01 when compared with PEEK. 16830

DOI: 10.1021/acsami.7b02521 ACS Appl. Mater. Interfaces 2017, 9, 16824−16833

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Figure 8. Methylene-fuchsin staining of samples: (a) PEEK, (b) A-PEEK, and (c) AF-PEEK.

samples, whereas the distance between the newly formed bone and PEEK is more than A-PEEK and 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 that of 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 sample 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.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86 2152412409. Fax: +86 21 52412409. *E-mail: [email protected]. ORCID

Xuanyong Liu: 0000-0001-9440-8143 Author Contributions ∥

M.C. and L.O. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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) is acknowledged.

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

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including cell proliferation, cell adhesion, and ALP activity evaluation; in vivo antibacterial properties against S. aureus; supporting figures and text (PDF)

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REFERENCES

(1) Lee, J. H.; Jang, H. L.; Lee, K. M.; Baek, H.-R.; Jin, K.; Hong, K. S.; Noh, J. H.; Lee, H.-K. In Vitro and in Vivo Evaluation of the Bioactivity of Hydroxyapatite-Coated Polyetheretherketone Biocomposites Created by Cold Spray Technology. Acta Biomater. 2013, 9 (4), 6177−6187. (2) Williams, D. F.; McNamara, A.; Turner, R. M. Potential of Polyetheretherketone (Peek) and Carbon-Fiber-Reinforced Peek in Medical Applications. J. Mater. Sci. Lett. 1987, 6 (2), 188−190. (3) Kurtz, S. M.; Devine, J. N. Peek Biomaterials in Trauma, Orthopedic, and Spinal Implants. Biomaterials 2007, 28 (32), 4845− 4869. (4) Godara, A.; Raabe, D.; Green, S. The Influence of Sterilization Processes on the Micromechanical Properties of Carbon FiberReinforced Peek Composites for Bone Implant Applications. Acta Biomater. 2007, 3 (2), 209−220. (5) Harsha, A. P.; Tewari, U. S. The Effect of Fibre Reinforcement and Solid Lubricants on Abrasive Wear Behavior of Polyetheretherketone Composites. J. Reinf. Plast. Compos. 2003, 22 (8), 751−767.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b02521. Surface characterizations including SEM and XPS of the samples only through HF treatment; in vivo cell response 16831

DOI: 10.1021/acsami.7b02521 ACS Appl. Mater. Interfaces 2017, 9, 16824−16833

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DOI: 10.1021/acsami.7b02521 ACS Appl. Mater. Interfaces 2017, 9, 16824−16833