nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties

Biomaterials xxx (2014) 1e18

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Polyetheretherketone/nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties Lixin Wang a, Shu He b, d, Xiaomian Wu c, d, Shanshan Liang e, Zhonglin Mu e, Jie Wei f, Feng Deng c, Yi Deng d, **, Shicheng Wei b, c, d, * a

Department of Stomatology, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, China Department of Oral and Maxillofacial Surgery, Laboratory of Interdisciplinary Studies, School and Hospital of Stomatology, Peking University, Beijing 100081, China c Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing Medical University, Chongqing 401147, China d Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China e The Affiliated Hospital, Hainan Medical College, Hainan 571199, China f Key Laboratory for Ultrafine Materials of Ministry of Education, Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 February 2014 Accepted 22 April 2014 Available online xxx

Lack of antibacterial activity and binding ability to natural bone tissue has significantly limited polyetheretherketone (PEEK) for many challenging dental implant applications. Here, we have developed a polyetheretherketone/nano-fluorohydroxyapatite (PEEK/nano-FHA) biocomposite with enhanced antibacterial activity and osseointegration through blending method. Smooth and rough surfaces of PEEK/ nano-FHA biocomposites were also prepared. Our results showed that in vitro initial cell adhesion and proliferation on the nano-FHA reinforced PEEK composite were improved. In addition, higher alkaline phosphatase activity and cell mineralization were also detected in cells cultured on PEEK/nano-FHA biocomposites, especially for rough PEEK/nano-FHA surfaces. More importantly, the as-prepared PEEK/ nano-FHA biocomposite could effectively prevent the proliferation and biofilm formation of bacterial. For in vivo test, the newly formed bone volume of PEEK/nano-FHA group was higher than that of bare PEEK group based on 3D microcomputed tomography and 2D histomorphometric analysis. These reports demonstrate that the developed PEEK/nano-FHA biocomposite has increased biocompatibility and antibacterial activity in vitro, and promoted osseointegration in vivo, which suggests that it holds potential to be applied as dental implant material in dental tissue engineering applications. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Polyetheretherketone Fluorohydroxyapatite Antimicrobial Bioactivity Osseointegration Implant

1. Introduction The most popular orthopedic/dental materials are metals such as titanium (Ti) and its alloys due to their excellent corrosion resistance, high mechanical strength, as well as cytocompatibility [1,2]. However, there are concerns regarding release of harmful metal ions and radiopacity of metal alloys in vivo. Moreover, the

* Corresponding author. School and Hospital of Stomatology, Peking University, No.22 Zhong-Guan-Cun South Road, Hai-Dian District, Beijing 100081, China. Tel./ fax: þ86 10 82195780. ** Corresponding author. Academy for Advanced Interdisciplinary Studies, Peking University, No. 5 Yi-He-Yuan Road Hai-Dian District, Beijing 100871, China. Tel./ fax: þ86 10 62753404. E-mail addresses: [email protected] (Y. Deng), [email protected], [email protected] (S. Wei).

elastic moduli of metal alloys mismatch mechanical properties between metals and human bones resulting in bone resorption [3,4]. In fact, serious post-operative complications such as osteolysis, allergenicity, and loosening as well as eventual implant failure may occur [5]. To overcome these limitations and minimize negative post-implantation biological reactions, substitutes for metals are extensively pursued. Polyetheretherketone (PEEK), a semi-crystalline and nonresorbable thermoplastic polymer, exhibits excellent mechanical properties, thermal stability and environmental resistance [6]. It also is non-toxicity and has low elastic modulus (3e4 GPa) compared to titanium and other metal alloys, which reduces the extent of stress shielding that is often observed in titanium-based metallic implants [7,8]. From the processing perspective, PEEK can be fabricated readily by conventional plastic processing equipments, repeatedly sterilized and heat contouring to fit the

http://dx.doi.org/10.1016/j.biomaterials.2014.04.085 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Wang L, et al., Polyetheretherketone/nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.085

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L. Wang et al. / Biomaterials xxx (2014) 1e18

shape of bones [9]. In recent decade, much attention has been given to PEEK composite in the biomedical field, particularly in the area of load-bearing orthopedic/dental applications [6,10] due to its unique properties mentioned above. After being inserted, a PEEK implant can support the body weight without the side effects of stress protection and its mechanical properties, which include elasticity, stiffness, tensile strength and resistance to distortion, abrasion and fatigue, are within the proper range to coexist with human bone [11,12]. In addition, bone recovery around the implant can be easily observed from outside the body because of the radiolucency of PEEK [13]. As a result, interbody fusion cages made from PEEK are widely used to treat degenerative spine diseases, to maintain mechanical stability during segmental fusion, and often to act as a carrier for orthopedic graft materials [14,15]. Although PEEK implants have been used frequently in recent medical operations, PEEK itself does not directly bind to bone due to its chemical and biological inertness and displays much lower mechanical properties than those of human bone [6,16]. Subsequently, to improve the bioactivity of PEEK, many researchers have successfully made PEEK-hybrid materials using hydroxyapatite (HA), because HA is a constituent of living bone [17]. Simple HAfilled PEEK mixtures have shown improved cell and tissue responses than pure PEEK in previous studies [10,18e20], but critical implant failures also often occur due to lack of antibacterial activity on the implanteabutment interface. It is well-known that microbial infection is one of the main causes of implant failure [21,22]. These device-associated infections can progress rapidly as planktonic bacteria first adhere to an implant interface and ultimately evolve into biofilms. Lack of antibacterial activity on the implanteabutment interface often causes undesirable complications such as oral infections, inflammatory reactions, destruction of the adjacent tissue, implant loosening or even detachment [23]. To enhance the antibacterial property of PEEK, Ag-doped or Ag-decorated HA coating was deposited onto the surface of PEEK implant by other groups and some prospective results were achieved [24,25]. Excess release of silver nanoparticles, however, inhibits osteoblasts growth [26] and cause many severe side effects [27,28], such as cytotoxicity and internal organ injury, though silver ions are known to have a broad spectrum of antimicrobial activities. Therefore, there is a pressing need to reduce and even eliminate the infection on PEEK implants without impairing the cytotoxicity. Nano-fluorohydroxyapatite (nano-FHA) is a bioactive calcium phosphate with chemical and crystallographic similarity to that of natural apatite in bones and dentals, and has been currently used in hard tissue engineering for bone regeneration and as bioactive coatings on Ti-based alloys for orthopedic/dental applications to improve the integration between the implants [29,30]. Compared to pure HA, FHA has much higher physic-chemical stability and possesses higher osteogenic activity to bone cells [31]. Furthermore, the effects of FHA on oral bacteria and plaque due to the release of fluorine ions, which can act as an antimicrobial agent, are well documented by a considerable amount of literature [32,33]. Despite the attractive advantages and progress in preparation of novel PEEK composites, the employment of nano-FHA as nano reinforcement in bioactive PEEK-based composites conferring PEEK with both antimicrobial activity and osseointegration for loadbearing dental applications, to our knowledge, has not been reported. Moreover, based on the clinical outcome and histological evidence from retrieved implants, a rough surface can promote ingrowth of soft and hard tissue into the materials, thereby creating more biological anchorage to improve the stability of the implant [34]. Hence, in the present study, a pilot and comprehensive work was conducted on the fabrication of PEEK/nano-FHA implants with smooth and rough surfaces, and we also systematically evaluated

the biofunctionalities of the PEEK/nano-FHA in vitro and in vivo. The PEEK/nano-FHA biocomposites with enhanced antimicrobial activity and osseointegration could be a promising candidate for dental implant. 2. Materials and methods 2.1. Materials Calcium nitrate tetrahydrate (Ca(NO3)2$4H2O, 99%, Sinopharm Chemical Reagent Co. Ltd., Beijing, China), diammonium hydrogen phosphate ((NH4)2HPO4, 99%, Sinopharm Chemical Reagent Co. Ltd., Beijing, China) and ammonia fluoride solution (NH4F, 99%, Shanghai Sanaisi Reagent Co. Ltd., Shanghai, China) were selected as Caprecursor, P-precursor and F-precursor, respectively. Ammonia solution (NH3$H2O) was also supplied from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China), and medical graded polyetheretherketone (PEEK powders, 450G, diameter approximately 2e3 mm, molecular weight about 5000) with the density about 1.30 g/cm3 was purchased from Victrex Co. Ltd. (United Kingdom). All other chemicals were of analytical reagent grade and were used as received unless otherwise noted. All aqueous solutions were prepared with de-ionized water (D.I. water). 2.2. Synthesis of nano-FHA Nano-FHA crystals were prepared in our laboratory via chemical precipitation reaction between aqueous Ca(NO3)2$4H2O and (NH4)2HPO4, with NH4F as the precursor for F during which F would substitute OH. 5 wt% F content was proposed in this study. Briefly, Ca(NO3)2 and (NH4)2HPO4 were dissolved in D.I. water separately according to a Ca/P molar ratio of 1.67, and NH4F was added to (NH4)2HPO4 solution. The pH of each solution was adjusted to 10 by adding ammonia. Then, a solution of Ca(NO3)2 was slowly added drop-wise into (NH4)2HPO4-NH4F mixed solution with continuous stirring. Crystal growth occurred when kept at 60  C for 8 h, and the pH value of the supernatant was maintained in the range of 10e10.5 using ammonia in whole experiments. The reaction of FHA (Ca10(PO4)6Fx(OH)2x) can be expressed by the reaction:   10Ca2þ þ 6HPO2 4 þ ð8  xÞOH þ xF /Ca10 ðPO4 Þ6 ðOHÞ2x Fx þ 6H2 O

After reaction, FHA slurry was aged at ambient temperature for 24 h, and the precipitates were obtained after washing with D.I. water at least three times to neutral pH. Finally, FHA precipitation was treated hydrothermally with stirring at 140  C (heating rate 10  C/min) under 0.3 MPa for 24 h in an autoclave. After hydrothermal treatment, nano-FHA particles were rinsed with D.I. water and dried in an oven at 60  C for 12 h before use and characterization. 2.3. Preparation of PEEK/nano-FHA biocomposite Polyetheretherketone/nano-fluorohydroxyapatite composite (PEEK/nano-FHA) was fabricated containing 40 wt% nano-FHA (approximately 29.6 vol% reinforcement level) by compression molding methods. In brief, 40 wt% nano-FHA powder and PEEK powder were dispersed in alcohol using an electronic blender to obtain a homogeneous powder mixture. After well dispersed, the mixture was dried in a forced convection oven at 90  C to remove the excess alcohol. The resulting powder mixture was placed in two specially designed molds, i.e. disks (15.0 mm diameter and 2.0 mm thick) for physical and chemical characterization and in vitro testing, and cylindrical implants (4.0 mm diameter and 7.0 mm length) for in vivo measurement (Fig. 1). The molds and powder mixtures were preheated to 150  C under a load of 35 MPa, and the temperature was increased to 375  C under a load of 15 MPa. After reaching the target temperature, the temperature and pressure were held for 10 min. Then the die and samples were air cooled to 150  C, and the samples were removed from the molds. Samples in disc shape and cylindrical implant were divided into two groups. Disk samples of PEEK/nano-FHA biocomposite were polished with a series of increasing SiC abrasive papers (400, 1000, 1500, 2000 grit), cleaned ultrasonically for 20 min in baths of acetone, anhydrous ethanol and D.I. water respectively, and dried at 50  C overnight and then tested by a mechanical profilometer (Dektek8 stylus profiler, Veeco, Plainview, USA). Disk samples with roughness average (Ra) below 0.2 mm were collected and designated as smooth groups. Some disk samples from the smooth groups were blasted by TiO2 particles (F ¼ 180e212 mm) and disks with Ra ¼ 2.0e3.0 mm were collected and designated as rough groups. In order to remove any potential free TiO2 nanoparticles, all samples were cleaned with D.I. water using an ultrasonic cleaner for 8 h. The D.I. water was changed every 20 min during the ultrasonic cleaning process. The bare PEEK samples were also prepared according to the same process and cut into the same shapes as control group. 2.4. Chemical and morphological characterization Fourier transform infrared spectrometry (FT-IR, Magna-IR 750, Nicolet, USA) was used to identify the functional groups of the as-prepared FHA powders and PEEK/nano-FHA composites. The spectra were recorded from 4000 cm1 to 400 cm1.

Please cite this article in press as: Wang L, et al., Polyetheretherketone/nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.085

L. Wang et al. / Biomaterials xxx (2014) 1e18

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PEEK PEEK/nano-FHA Bioactivity TiO2 blasting Disc

nano-FHA

Osteogenic activity

in vitro Melt blending

Compression Smooth molding

Rough

Antibaterial activity

PEEK

PEEK/nano-FHA

in vivo

biocomposite

Implant

O O

C

Osseointegration

PEEK PEEK/nano-FHA

n

Fig. 1. Schematic diagram of preparation and evaluation of PEEK/nano-FHA composite samples.

Raman measurement was performed at 633 nm by using a Raman Imaging Microscope System (Renishaw 1000) in a backscattering geometry. The 633 nm laser beam has a spot size of about 1 mm and the spectral resolution is 1.5 cm1. X-ray photoelectron spectroscopy (XPS, AXIS Ultra, Kratos Analytical Ltd., Japan) was employed to identify the chemical constituents and elemental states of the FHA powders and PEEK/nano-FHA composite. The binding energies were calibrated by the C 1s hydrocarbon peak at about 285 eV. The crystalline phases of the prepared nano-FHA powders and PEEK/nano-FHA composites were examined and compared by X-ray diffraction (XRD, Shimadzu, Japan) using a Cu target as radiation source (l ¼ 1.540598 Å) at 40 kV. The diffraction angles (2q) were set between 10 and 60 , increment with a step size of 4 min1. Identification of phases was achieved by comparing the obtained sample diffraction pattern with standard cards in the ICDD-JCPDS database. The transmission electron microscopy (TEM) images were taken by FEI Tecnai F20 instrument (Philips Electron Optics, Eindhoven, Netherlands) operated at an accelerating voltage of 200 kV. Samples for TEM imaging were prepared by placing a drop of the aged FHA composite suspensions (the suspensions were diluted in D.I. water and dispersed by ultrasonic waves before use) onto carbon-coated copper grids, dried in air and loaded into the electron microscope chamber. Moreover, in order to characterize the nano-FHA dispersion in PEEK/nano-FHA biocomposite, thin foil TEM specimens were prepared by microtome with a diamond knife and imaged using TEM. Selected area electron diffraction (SAED) and energy dispersive X-Ray spectroscopy (EDS) were also recorded using the same equipment. Static contact angles on bare PEEK and PEEK/nano-FHA composite surfaces were measured at room temperature by the sessile drop method using 2 mL D.I. water droplet in a contact angle measuring device (SL200B, Kono, USA). Six samples in each stage were used to provide an average and standard deviation. The surface morphology of bare PEEK and PEEK/nano-FHA composites was characterized by a field emission scanning electron microscope (FE-SEM, JSM-6701F, JEOL, Tokyo, Japan). All samples were coated by gold for 1 min before FE-SEM observation. Atomic force microscopy (AFM, PI3800/SPA400, Seiko Instruments, Japan) was used in tapping modes (15-mm scanner) in dry condition at ambient temperature to assess morphological characteristics of the PEEK-based substrates. For the tapping mode measurement, we used a Si3N4 cantilever with a spring constant of 0.12 N/m (Seiko Instruments) for resolution imaging. The scan range was 20 mm  20 mm, and scan rate was 1 Hz. Before AFM measurement, the different PEEK substrates were rinsed with ethanol and D.I. water, and allowed to air dry. 2.5. Mechanical properties tests The uniaxial tensile test was performed with an initial strain rate of 5  104 s1 on a mechanical tester (Instron5969, USA) at room temperature. The ultimate tensile strength (UTS) and elastic modulus (E) was obtained from the stressestrain curve. For each group, five duplicate specimens were tested. Hardness of the PEEK-based composite and pure Ti was determined by a digital Vickers microhardness tester (HMV-2T, Shimadzu, Japan) with a 1.961 N load and 15 s dwell time. Six points were chosen and measured in different positions of each sample to get an average value. 2.6. Formation of bone-like apatite The bare PEEK and PEEK/nano-FHA samples were immersed in a simulated body fluid (SBF, nearly equal to that of human blood plasma) at 37  C by water

bath to examine the bioactivity. After 3 and 14 days, the specimens were removed from the given solution, gently rinsed with distilled water, and quickly dried and kept in a drying oven at room temperature. The chemical composition of surface deposits was characterized by XPS and XRD. After sputter-coated with gold, the microstructures of specimens were examined by SEM equipped with EDS. 2.7. Cell culture Human osteoblast-like MG-63 cells (American Type Culture Collection, VA, USA) were cultured in Dulbecco’s modified eagle’s medium (DMEM, Gibco, Carlsbad, CA) containing 10% fetal calf serum (Gibco), 100 mg/mL streptomycin (Amresco, Cleveland, USA) and 100 mg/mL penicillin (Amresco) at 37  C in a humidified atmosphere of 5% CO2. The culture media was changed at 3-day intervals. 2.8. In vitro cell viability assay Human osteoblast-like cells (MG-63) were adopted to evaluate the cytotoxicity of the bare PEEK and PEEK/nano-FHA composite, and the cell viability was tested by a direct assay and indirect method according to the instruction of ISO 1099312:2007 in this study. In the direct assay, prior to in vitro testing, the samples were sterilized using gamma radiation at a total dose of 25 KGg. Cell attachment and viability of MG-63 cells were assessed using the cell proliferation reagent WST-1 (Roche Diagnostics, Germany). After cell counting, MG-63 cells were seeded in 24well plates (Costar, USA) at a density of 1  105 cells/well. After seeding 24 h, cells were exposed to the PEEK-based biocomposites, and bare PEEK was used as a material control. After incubating for 4 h, 3, 7 and 14 days respectively, the culture media were removed and the specimen were rinsed with PBS buffered three times in order to remove the unattached cells. Cell viability of the adherent cells was measured by adding 1 mL/well culture medium containing 100 mL WST-1 for 4 h incubation. Then 100 mL of supernatant from each well was transferred to new 96well cell culture plates. Optical density (OD value) of the supernatant was measured with a microplate reader (Model 680, Bio-Rad, CA) at 450 nm with the reference wavelength at 630 nm. In the indirect method, extraction media of the studied materials were prepared using serum free cell culture medium (DMEM), with the extraction ratio (the ratio of specimen surface area to extraction medium) of 3 cm2/mL, and then incubated in a humidified atmosphere with 5% CO2 at 37  C for 72 h. Cell culture medium (DMEM) was used as a negative control. Cells were incubated in 96-well cell culture plates at 5  103 cells per 100 mL in each well and incubated for 24 h to allow attachment. Then culture media were substituted by the extracts obtained from the studied materials and incubated for 3, 7 and 14 days, respectively. After the culturing period, the cell viability was measured by the same approach using WST-1 as the aforementioned discussion. 2.9. FE-SEM observation of cells The morphologies of MG-63 cells co-cultured on the PEEK-based biocomposites were observed using the FE-SEM after 3, 7 and 14 days culture. All samples were washed with PBS buffer, fixed in 2.5% glutaraldehyde solution for 30 min and then dehydrated with graded ethanol solutions (25%, 50%, 75%, 95%, and 100%, 15 min each concentration). Dehydrated samples were dried by a vacuum dryer before sputter-coating with gold using a sputter coater.

Please cite this article in press as: Wang L, et al., Polyetheretherketone/nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.085

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L. Wang et al. / Biomaterials xxx (2014) 1e18

Fig. 2. Chemical characterization and morphology of the prepared nano-FHA crystals: FT-IR spectrum (a), XPS wide spectrum (b), XRD pattern (c), and TEM image with EDS pattern (d).

2.10. Immunofluorescence After 7 days incubation, cells were washed with PBS and fixed with 4% (w/v) paraformaldehyde in PBS for 15 min at ambient temperature. The samples were then washed with PBS and permeabilized with 0.1% (v/v) Triton X-100 (Sigma) for 5 min, before being incubated with 1% bovine serum albumin/PBS at 37  C for 30 min to block nonspecific binding. This was followed by adding 5 mg/mL rhodaminephalloidin (Molecular Probes, Eugene, OR) to stain MG-63 cells for 30 min. The stained signals in the cells were observed by a fluorescent microscopy (Carl Zeiss, Oberkochen, Germany). The number of adherent cells was determined by counting the number of nuclei at projected area (magnification 100), and stained images were obtained from five different areas per sample (n ¼ 3). 2.11. Flow cytometric analysis Cell cycle and cell apoptosis of MG-63 osteoblasts were analyzed by flow cytometry using propidium iodide (Invitrogen, Carlsbad, CA) and using an Annexin V-FITC apoptosis detection kit (Beyotime, Shanghai, China), respectively. Briefly, the cells were initially cultured on different samples at 1  105 cells/well for 3, 7, and 14 days. Then cells were digested with 0.05% pancreatic enzyme for 3 min. The digested cells were washed with the collected culture medium, and then suspended in PBS buffer. As for cell cycle test, the cells were fixed in 75% ethanol and kept in ethanol at 20  C for 16 h. Finally, they were collected and washed again, and re-suspended in 1 mL RNaseA (1 mg/mL) at room temperature for 30 min. Each sample was then centrifuged at 1200g for 4 min, re-suspended in 190 mL of binding buffer and 10 mL of propidium iodide working solution was added. As for cell apoptosis test, at 7 days cells were stained with the apoptosis detection kit according to the manufacturer’s instructions. Briefly, the cells from each sample were suspended in 195 mL of Annexin V-FITC binding buffer, and 5 mL of Annexin V-FITC. The cells were incubated at room temperature for 10 min. Each sample was then centrifuged at 1200g for 4 min, re-suspended in 190 mL of binding buffer and 10 mL of propidium iodide working solution was added. For both tests, the samples were analyzed by a

FACSCalibur (Becton Dickinson, NY, USA) flow cytometry with at least 10,000 events recorded for each condition. 2.12. Hemocompatibility assessment 2.12.1. Hemolysis tests Healthy human blood from volunteers containing sodium citrate (3.8 wt%) in the ratio of 9:1 was taken and diluted with normal saline (4:5 ratio by volume). The bare PEEK and PEEK/nano-FHA surfaces were dipped in a standard tube containing 10 mL of normal saline that were previously incubated at 37  C for 30 min. Then 0.2 mL of diluted blood was added to this standard tube, and the mixtures were incubated at 37  C for 60 min. Similarly, normal saline solution was used as a negative control and D.I. water as a positive control. After the incubation, all the tubes were centrifuged for 5 min at 3000 rpm, and the supernatant was carefully removed and transferred to the a new 96-well plate for spectroscopic analysis by a microplate reader (Model 680, Bio-Rad, CA) at 545 nm. In addition, the hemolysis was calculated based on the average of three replicates. hemolysis ¼

ODðtestÞ  ODðnegative controlÞ  100% ODðpositive controlÞ  ODðnegative controlÞ

2.12.2. Platelet adhesion Platelet-rich plasma (PRP) was prepared by centrifuging the whole blood for 10 min at a rate of 1000 rpm/min. The PRP was overlaid atop the PEEK-based plates and incubated at 37  C for 1 h. After incubating, all samples were rinsed with PBS buffer three times to remove the non-adherent platelets. The adhered platelets were also fixed in 2.5% glutaraldehyde solutions for 1 h at room temperature followed by dehydration in a gradient ethanol/distilled water mixture (from 25% till 100%) for 15 min each and dried. The surfaces of platelet attached the PEEK-based sheets were observed by FE-SEM. Different fields were randomly counted and values were expressed as the average number of adhered platelets per mm2 of surface.

Please cite this article in press as: Wang L, et al., Polyetheretherketone/nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.085

L. Wang et al. / Biomaterials xxx (2014) 1e18 2.13. In vitro bacterial attachment assay In the present work, a semi-quantitative measurement of bacterial adhesion on bare PEEK and PEEK/nano-FHA surfaces was investigated via Microbial Viability Assay Kit-WST. Streptococcus mutans (S. Mutans UA159, obtained from American type Culture Collection, USA) were cultivated in Brian Heart Infusion (BHI) medium (containing 10 g/L proteose peptone, 2 g/L dextrose, 5 g/L sodium chloride and 2.5 g/ L disodium phosphate). After an overnight culture at 37  C, Colony-forming Unit (CFU) counts were determined from the absorbance at 600 nm wavelength using previously established standard curves. The concentration of S. Mutans in broth were adjusted to give an initial optical density (OD), reading of 0.175 at the wavelength of 600 nm, which corresponded to the concentration of 1  106 CFU/mL. Cells were then centrifuged at 10,000 rpm at 4  C (5804R Centrifuge; Eppendorf, Hamburg, Germany) and the pellets re-suspended in sterile saline solution. For the bacterial attachment assay, bare PEEK and PEEK/nano-FHA were sterilized under UV irradiation for 30 min each side, then were placed in a 24-well tissue plate (Corning, USA) and covered with 40 mL bacterial solution with about 106 CFU/mL of the desired strain and 760 mL prepared medium for 1 h, 4 h, 12 h, and 24 h respectively. The pure medium was used as the control and all samples were kept in an incubator containing 5% CO2 for desired time at 37  C. At scheduled time, substrates were taken out, gently rinsed with PBS for three times to remove non-attached bacteria, and placed into new 24-well tissue plate and incubated with 20 mL WST, which produced a water-soluble formazan dye upon reduction by dehydrogenase in cells, and 380 mL medium for another 2 h. OD value (l ¼ 450 nm) of the suspension in each well was measured on a microplate reader (Elx808, Bio-Tek, USA). Each test was carried out in triplicate.

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and neutralized with 10% ammonium hydroxide (Sigma Aldrich, Germany). 100 mL of each sample was added to 96-well plates and the OD405 was read using a microplate reader (Elx808, Bio-Tek, USA). 2.17. In vivo study Surgical implantation was performed on six male beagle dogs aged 1.5 years and weighing 11.4  2.1 kg for observation period of 4 and 8 weeks after surgery. The in vivo study was conducted according to the ethical principles of the Peking University Institutional Animal Care and Use Committee. The animals were maintained on a normal, solid lab diet and regular tap water. The design of the implants after blasting used in the present study was the same as that in our previous study [35], which consisted of a 7-mm-long and 4-mmdiameter cylindrical bar with screw thread (Fig. 1). The sample implants were sterilized prior to surgery for in vitro testing. The animals were anesthetized using an intravenous injection of 1% pentobarbital 80 mg/kg. The third and fourth premolars of dogs were bilaterally extracted in the mandibles and implants were inserted immediately. The antagonistic teeth of the upper jaw were not removed and all implants were covered with the mucoperiosteal flaps to allow for submerged healing. The order of implantation was predetermined by block-randomization. For every group, two cylindrical implants were placed on each animal in the premolar socket region. After surgery, three fluorochromes (Sigma, St Louis, MO), i.e. calcein (100 mg/kg), calcein blue (100 mg/kg), and tetracycline (100 mg/kg) were administered to assess the osteogenic activity at 1, 2, and 4 weeks, respectively. Meanwhile, after surgery the animals were maintained in their normal cages without any limitation and three dogs sacrificed at 4 and 8 weeks, respectively, with intravenous injection of 10% kalium chloratum (0.5 mL/kg).

2.14. Biofilm formation assay Biofilm formation on the surface of the PEEK-based composites was observed using confocal laser scanning microscope (CLSM). A LIVE/DEAD BacLight bacterial viability kit (L-7007, Invitrogen, USA) was used to obverse the long-term bacterial cell viability on the surfaces. In this assay, the red-fluorescent nucleic acid staining agent propidium iodide, which only penetrates damaged cell membrane, was used to label dead bacterial cells. In contrast, the SYTOÔ9 green-fluorescent nucleic acid staining agent, which can penetrate cells both with intact and damaged membranes, was used to label all the bacterial cells. 20 mL of the S. Mutans bacteria solution and 2 mL prepared medium were seeded onto bare PEEK and PEEK/nano-FHA followed by incubation at 37  C for 14 days. The supernatant was removed, and the substrate was washed with PBS buffer at least three times. They were then incubated in a 24well plate with 400 mL of a dye-containing solution, which was prepared by adding 6 mL of SYTO (3.34 mM) and 6 mL of propidium iodide (20 mM) to 4 mL of PBS buffer, at room temperature in the dark for 15 min, according to the manufacturer’s protocol. The stained bacterial cells were examined under a Zeiss LSM510 laser scanning confocal microscope (Germany). Images were obtained using an oil immersed 40 object lens under the same conditions.

2.18. Micro-CT analysis High-resolution images of all specimens were obtained from a micro-CT scanner (Skyscan 1076, Aartselaar, Belgium) running at 40 kV (X-ray source voltage), 200 mA (beam current), 900 msec (exposure time), 9 mm (resolution), 0.8 (rotation step), and 180 (rotation angle). A polygonal region of interest (ROI) in 100 slices with approximate 1 mm wide ring around implant surface was chosen, which represented the regenerated bone only. After scanning, the two-dimensional (2D) and three-dimensional (3D) models were reconstructed from the volume of interest, where an optimized threshold was used to isolate the bone and materials from the background using the NRecon (Skyscan Company) and CTVol (Skyscan Company, Belgium). The percent bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) around the implant were calibrated and determined by the CTAn program (Skyscan Company, Belgium). Radiographs were also synthesized using the CTvol software package (Skyscan) to reconstruct a three-dimensional (3D) regenerated bone of 0.5 mm width in the marrow bonding to the implants. 2.19. Histology and histomorphometric analysis

2.15. Alkaline phosphatase activity (ALP) assay Alkaline phosphatase activity (ALP) of MG-63 cells was evaluated by an assay reagent kit (Nanjing Jiancheng Bioengineering Institute, China). At the end of the incubation, cells were exposed to the different PEEK-based biocomposite samples for 3, 7 and 14 days. Briefly, the supernatant was removed and 100 mL of lysis solution (1% Triton X-100) was added into each well and incubated for 1 h. Afterwards, 30 mL of MG-63 cell lysates at each well was transferred to new 96-well cell culture plates, and cultivated with 50 mL of carbonated buffer solution (pH 10) and 50 mL of substrate solution (4-amino-antipyrine) at 37  C for 15 min. Then 150 mL of potassium ferricyanide was added into the above solution, and the production of pnitrophenol was determined by the absorbance at 405 nm. For normalization, the total protein concentration was measured by a Bicinchoninic Acid (BCA) protein assay kit (Beijing Biosea Biotechnology, China). Thus, alkaline phosphatase activity was normalized and expressed as the total protein content (U/mg protein).

The bone samples with the implants were harvested and fixed in 10% buffered formalin for 3 days, dehydrated in a series of solutions with different ethanol concentrations over a time period of 6 weeks. The samples were embedded in methyl methacrylate resin without decalcification and micron sections were made after polymerization with a microtome (SP1600; Leica, Wetzlar, Germany) along the longitudinal axis of the bone-implant interface. Afterwards, the embedded tissue samples were cut into sections with a thickness of about 30 mm. The sectioned samples were stained by toluidine blue-fuchsine at 8 weeks. Optical microscopy (Olympus, WILD MP5, Japan) and confocal laser scanning microscopy (LSM710 NLO, Zeiss, Oberkochen, Germany) were conducted to observe bone ingrowth and integration with the host tissue and the percentage of bone-implant contact (BIC) was calculated by histometric analysis (GSA Image Analyser, Rostock, Germany) for each implant type. 2.20. Statistical analysis

2.16. Alizarin Red S staining Mineralized nodule formation was determined on 14 and 21 days of culture following the manufacturer’s instruction by staining with Alizarin Red S (ARS) solution (2%, pH 4.2; Sigma, A5533). hMSC cells were seeded in 6-well plates (2  106 cells/well) and cultured in osteogenic induction medium (containing 107 M dexamethasone, 10 mM b-glycerophosphate and 50 mg/mL ascorbic acid) for osteoblast differentiation. At different time points (14 and 21 days), cells were fixed with 10% paraformaldehyde in PBS for 15 min and then washed three times with PBS. The fixed cells were further washed with distillated water in order to remove any salt residues and then a solution of 2% (wt/v) Alizarin Red S (ARS, Sigma Aldrich, Germany) with a pH adjusted to 4.5 was added so that it covered the entire surface of the wells containing cells. After an incubation of 30 min at room temperature, the excess ARS was washed with distillated water. The ARS staining was imaged using a Zeiss Discovery V8 Stereo Microscope (DISV8). To quantify the orange-red coloration of ARS, 10% acetic acid (Sigma Aldrich, Germany) was added to the cells. After an overnight incubation, the cells with the acetic acid were transferred to tubes and centrifuged for 15 min at 20,000 g. The supernatant was removed to other tubes

All quantitative data are expressed as mean  standard deviations with n ¼ 3. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc tests using SPSS 19.0 and p-values less than 0.05 are considered statistically significant.

3. Results and discussion 3.1. Characterization of nano-FHA crystals Fourier transform infrared spectroscopy confirmed the presence of an apatite phase in nano-FHA crystals as shown in Fig. 2a. The strong characteristic peak of PO3 4 (triply degenerate n4) vibrations appeared at 568 cm1 along with other non-degenerate symmetric stretching mode n1, doubly degenerate bending mode n2 and triply degenerate antisymmetric stretching band n3 phosphate peaks at

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Fig. 3. XRD patterns (a), FT-IR spectra (b), XPS wide spectra (c), and water contact angles (d) of bare PEEK and PEEK/nano-FHA composite. * represents p < 0.05.

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Fig. 4. Typical SEM images of the surface morphologies for smooth PEEK (a), smooth PEEK/nano-FHA (b) (* shows nano-FHA particles exposed to the surface of smooth PEEK/nanoFHA composite in (b)), rough PEEK (c) and rough PEEK/nano-FHA (d). Typical AFM images for smooth PEEK (e), smooth PEEK/nano-FHA (f). Surface roughness data for all PEEKbased groups is shown in (g). * represents p < 0.05.

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Table 1 Mechanical properties for bare PEEK and PEEK/nano-FHA composites showing the mean (standard deviation) hardness (H), elastic modulus (E) and ultimate tensile strength (UTS) for each group. Samples

UTS (MPa)

Elastic modulus (GPa)

Hardness (MPa)

PEEK/nano-FHA PEEK Ti Cortical bone [44]

137.6  9.1 82.5  10.5 965.8  24.5 50w150

12.1  0.4 3.9  0.5 116.3  1.2 6w23

141.0  6.4 89.1  3.2 3110.2  300.6 e

961, 467, 1094 and 1039 cm1, respectively [17], which was consistent with the results from Raman analysis (Fig. S1a). The typical broad peak at 3435 cm1 and 1640 cm1 were associated with the adsorbed water. The weak band at 3570 cm1 could be attributed to the presence of OH group of apatite implying OH was partially superseded by F ion in apatite [36]. Existence of CO2 3 originating from atmosphere was observed at around 1468,

1413 cm1 (n3) and 871 cm1 (n2), showing the formation of B-type carbon-substituted apatite, which was similar to the apatite found in the bone [37]. XPS analysis demonstrated the presence of F element (F KLL and F 1s) in the apatite (Fig. 2b), indicating the forming of FHA particles. The XRD patterns of the resulting samples were shown in Fig. 2c, and the Bragg diffraction peaks of the asprepared nano-FHA, matched quite well with those of FHA (PDF # 15-0876) at 2q values of 25.8 , 31.7, 32.9 , 46.7 and 49.5 , which were indexed to be (002), (211), (300), (222) and (213) planes, respectively [38]. The morphologies of the obtained FHA nanocrystals were observed by TEM. Fig. 2d showed the typical rod-like particles, which was the characteristic structure of FHA after hydrothermal treatment. Due to the high surface area and surface energy, the pure FHA nanoparticles with a length of 85  10 nm and a width of 22  4 nm had a strong tendency to form agglomerates. The lattice orientation of the FHA nanoparticles in the HRTEM image was

Fig. 5. SEM photographs of bare PEEK acquired after soaking in SBF for 3 days (a) and 14 days (c), and PEEK/nano-FHA for 3 days (b) and 14 days (d). The inset in (d) stands for EDS spectra of the particle sediments on the surface of PEEK/nano-FHA sample. XPS Ca2p and P2p spectra of the particle sediments on the surface of PEEK/nano-FHA sample at 14 days are shown in Fig. 5eef.

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shown in Fig. S1b, with an interplanar distance of 0.281 nm, corresponding to the (211) planes of the FHA hexagonal structure [37]. To further verify the chemical composition of nano-FHA, selected area electron diffraction (SAED) patterns and EDS analysis were carried out. The observed strong concentric ring patterns (the inset in Fig. S1B) could be assigned to the (002), (211) and (222) planes of FHA, respectively. The Ca/P ratio of FHA determined by EDS analysis (Fig. 2d), was about 1.61, slightly lower than the stoichiometric ratio of Ca/P in FHA (z1.67). Additionally, the presence of F element was detected in EDS study, further proving the formation of FHA powders, in accordance with the results from XPS analysis. 3.2. Composition, morphology and mechanical properties of PEEK/ nano-FHA composite The XRD patterns of the resulting samples were shown in Fig. 3a, confirming that FHA crystals incorporated into PEEK composite successfully. The peaks of 2q at 18.7, 20.8 , and 22.8 were characteristic peaks of PEEK, while the characteristic peaks of FHA showed up at 25.9 , 31.8 , 40.0 , 46.7 and 49.5 in PEEK/nano-FHA biocomposite. Moreover, for PEEK/nano-FHA composite the intensity of PEEK major peaks decreased due to FHA beads partially covering the PEEK surface. No new peak appeared in the PEEK/ nano-FHA pattern except the inherent peaks of PEEK and FHA themselves. This suggested that there were no new crystalline phase formation and no change in crystal structure of both PEEK and FHA during composite preparation process. The strong peak at 1652 cm1 originated from the C]O carbonyl stretching vibration and the bands at 1597 cm1 and 1501 cm1 were in-plane vibration band of benzene (Fig. 3b). Two bands of CeH vibration at 840 cm1 and 764 cm1 belonged to the divided bands of the out-of-plane bending vibration absorption of benzene. Among them 840 cm1 was oppositesubstituted aromatic ring [39,40]. These were all characteristic peaks of PEEK. Furthermore, in PEEK/nano-FHA composite the bands of PeO stretch and vibration at 1042 cm1 resulted from the PO4 group. The absorption peaks at about 3570 cm1 could be attributed to the stretching vibration of hydroxyl resulted from FHA. XPS analysis further affirmed the results obtained from FTIR and XRD. As shown in Fig. 3c, bare PEEK exhibited carbon and oxygen peaks as the main atomic elements, whereas fluorine peaks newly appeared in the XPS spectra of PEEK/nano-FHA samples, indicating the presence of nano-FHA in PEEK/nanoFHA composite. Water contact angle is a convenient way to assess the hydrophilic-hydrophobic properties of composite surfaces, and the measurement results were shown in Fig. 3d. The presence of FHA particles on PEEK surfaces might help to improve the bioactive properties of the composite, especially the hydrophilicity. According to Fig. 3d, the bare PEEK possessed the highest contact angle of approximately 83.5 corresponding to the lowest surface hydrophilicity, owing to its hydrophobic aromatic ring and polyester functional groups. After mixed with FHA, the highlight was that the surface of PEEK/nano-FHA showed a significantly smaller contact angle decreasing to about 71.5 , which indicated nano-FHA has significantly induced hydrophily of PEEK because of its hydroxyl groups. This result agreed with the XPS result (Fig. 3c) from which the elements of nano-FHA crystals were detected on the surface of PEEK/nano-FHA composite. To gain further information and understand the microstructures of PEEK/nano-FHA composite, SEM, TEM and AFM observations were carried out on bare PEEK and PEEK/nano-FHA composite, and the results were shown in Fig. 4 and Fig. S2. Particle size and size distribution as well as dispersion play critical roles in the mechanical performance of nanocomposites as well as their bioactivity

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[35]. TEM image of our PEEK/nano-FHA composite showed relatively uniform dispersion of the nano-FHA in PEEK matrix (Fig. S2), even though occasionally some big agglomerates could be observed. As shown in Fig. 4, it could be seen that compared with bare PEEK group, there were many white FHA particles visualized on the smooth PEEK/nano-FHA surface, and the general dispersion of FHA nanopowder was homogeneous in PEEK/nano-FHA composite (Fig. 4b). In addition, a dramatic difference in surface morphology/tomography was observed between the smooth and rough samples after blasting by TiO2 particles (Fig. 4ced). Many studies in the literature have suggested that the roughness of implants can improve the rapid osseointegration, such as osteoblastlike cell adhesion, gene expression and alkaline phosphatase activity and osteocalcin production [41e43]. AFM image analysis was carried out for further investigation of the surface morphology and roughness. It was observed that bare PEEK surface was smooth as shown in Fig. 4e, however, after addition with nano-FHA, the surface morphology for PEEK/nano-FHA (Fig. 4f) became rougher in accordance with the SEM images. Surface of smooth PEEK/nanoFHA composite showed higher roughness (both Ra and RMS) than that of smooth PEEK possibly due to FHA nanopowder exposed to the surface, but both of them were below 0.2 mm. Nevertheless, after the same blasting process, rough PEEK/nano-FHA composites displayed the similar value of Ra and RMS to rough PEEK counterparts.

Fig. 6. Cell attachment (a) and proliferation (b) of smooth PEEK, rough PEEK, smooth PEEK/nano-FHA, and rough PEEK/nano-FHA samples after 3, 7 and 14 days measured by the direct method via WST-1 assay. * represents p < 0.05.

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Fig. 7. SEM images of MG-63 (a) on the surfaces of smooth PEEK (a-1), smooth PEEK/nano-FHA (a-2), rough PEEK (a-3) and rough PEEK/nano-FHA samples (a-4) after 3, 7 and 14 culturing. Adhesion morphology and actin cytoskeletal organization (red, labeled with rhodamine-phalloidin) of MG-63 (b) after incubation with smooth PEEK (b-1), smooth PEEK/ nano-FHA (b-2), rough PEEK (b-3) and rough PEEK/nano-FHA samples (b-4) at 7 days (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Table 1 presented the mechanical properties of bare PEEK and PEEK/nano-FHA composites, with pure Ti as control. It could be seen that bare PEEK showed extremely lower elastic modulus (E) and UTS, while UTS of pure Ti were approximately 6e20 times greater than that of cortical bones, which resulted in stress

shielding. The use of materials stiffer than bone issue can lead to mechanical mismatch problems between the implant and the adjacent bone tissue, where the integrity of the bone/implant interface may be compromised due to the resorption of bone tissue [4,44]. However, the addition of nano-FHA into bare PEEK increased

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the hardness, elastic modulus as well as UTS similar to those of human cortical bone, indicating that the mechanical properties of the nano-FHA reinforced PEEK could be tailored to mimic human cortical bone avoiding stress shielding (Table 1). Therefore, it was expected that the prepared PEEK/nano-FHA nanocomposites with such mechanical properties could have great potential to be used in biomedicine such as synthetic implant material. 3.3. Bone-like apatite formation Fig. 5 exhibited the surface morphologies of the specimens under SEM after 3 and 14 days static immersion in SBF. There was no apparent observation of CaeP deposition on the surface of bare PEEK group in the first several days, with only a few scattered particle sediments appearing after 14 days immersion (Fig. 5(c)). In contrast, the aggregation of some rounded nodulus started to emerge on the surface of PEEK/nano-FHA composite after 3 days soaking. After 14 days immersion, the number of particles on PEEK/ nano-FHA surfaces was substantially increased and the surface was covered (Fig. 5d). The EDS spectra acquired from the immersed specimens revealed that the formed particle contain calcium and phosphorous and their ratio was about 1.59, which was approximate to that of bone mineral. The XRD pattern affirmed the formation of hydroxyapatite (matches quite well with PDF # 09-0432 of HA) on the surface of the PEEK-based composite. It was obvious that the relative intensity of the diffraction peaks, particularly the three most intense peaks of HA, corresponding to (211), (112), and (300) planes, increased with increasing time, implying improved crystallinity (Fig. S3). Moreover, the XPS Ca 2p and P 2p spectra of the deposited spherical particles on PEEK/nano-FHA immersed in SBF for 14 days were presented in Fig. 5e and f. The Ca 2p spectrum exhibited a doublet at 346.8 and 350.3 eV and the P 2p spectrum showed a single peak at 132.6 eV, which was consistent with the published data for hydroxyapatite (HA). In addition to the hydrophilic enhancement, it is proved that by incorporating the bioactive HA into polymer, the bone-like apatite formation is promptly observed in SBF and it increases with the fraction of HA in the composite [40]. The mechanism can be explained in terms of the electrostatic interaction of the Ca2þ on the surface of FHA with the ions in the SBF. Previous studies show that FHA possess abundant Ca2þ ions on the surface, and can induce heterogeneous nucleation and growth of apatite [40]. Considering the ionic nature, the electrostatic interaction triggers initial nucleation, and the Ca2þ cation presents on the surface may play a pivotal part in anchoring phosphate and hydroxyl ions. Consequently, negatively-charged  ions (HPO2 4 and OH ) in the SBF are incorporated to the surfaces leading to the formation of a hydrated precursor cluster consisting of calcium hydrogen phosphate. Since this phase is metastable, it

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grows spontaneously and transform into stable bone-like apatite  crystals by consuming Ca2þ, HPO2 4 and OH ions from SBF. Our data disclose that the bioactivity on PEEK/nano-FHA is better than that on the bare PEEK control. 3.4. In vitro biocompatibility evaluation It is well-known that PEEK materials have been widely employed in biomedical fields and medical devices particularly in the area of load-bearing orthopedic and degenerative spine diseases applications (such as interbody fusion PEEK cage) [14,15]. FHA crystals, on the other hand, are expected to be favorable for cell proliferation, exhibit excellent cytocompatibility and facilitate osseointegration properties [45,46]. Initial cell adhesion is usually responsible for cellular functions and eventual tissue integration, while cell proliferation is closely correlated with the amount of new bone formation. Better pre-osteoblast adhesion and proliferation probably produce a larger mass of bone tissues around the implants. Hence, in the present study, the cell proliferation and cytotoxicity of the as-prepared PEEK/nano-FHA composite to human osteoblast-like MG-63 cells is an important factor that should be carefully evaluated for use of this composite in biological and biomedical applications, such as dental implant. 3.4.1. Cell attachment and proliferation In this study, the in vitro cell adhesion of the prepared samples was investigated using WST-1 assay on MG-63 cell line. It was interesting to note that, no matter smooth and rough, PEEK groups displayed low OD value among the groups, whereas higher OD value was detected in the group of PEEK/nano-FHA (Fig. 6a), indicating the presence of nano-FHA in the composites could promote the adhesion of MG-63 cells. Analysis of the results from the attachment test also showed that there was a significant positive correlation between surface roughness and the cell attachment, and rough surface significantly induced cell attachment. Therefore, our results suggest that the combined effects of nano-FHA crystal and sand blasting treatment might greatly promote the cell attachment on the surface of PEEK/nano-FHA biocomposite. The results of the indirect cytotoxicity test for PEEK/nano-FHA biocomposite in extraction media for 3, 7 and 14 days, respectively, with bare PEEK as control were shown in Fig. S4. It could be seen that the cells cultured with bare smooth PEEK and rough PEEK extraction media showed no statistical differences in cell viability, but displayed lower cell viability than those of the smooth and rough PEEK/nano-FHA samples after each culture period. This indicated that compared with bare PEEK, the presence of nano-FHA crystals could promote the proliferation of MG-63 cells. Even after a prolonged culturing period, the cell viabilities of studied PEEK/

Fig. 8. Cell cycle analysis of osteoblasts (a) grown on smooth PEEK, rough PEEK, smooth PEEK/nano-FHA, and rough PEEK/nano-FHA samples for 3, 7, and 14 days. Percentages of apoptotic cells (b) at 7 days determined from flow cytometric analyses. * represents p < 0.05.

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nano-FHA biocomposite, remaining at the high level of over 97%, were almost the same as that of the negative group, representing a non-toxicity feature. It was reported that the fluoridated hydroxyapatite extraction media containing Ca2þ ions had a significantly higher cell viability than pure cell culture media, indicating its positive effect on the proliferation of osteoblast cells [47]. However, the viabilities of bare PEEK groups showed a decrease in comparison with PEEK/nano-FHA groups after a long culturing period (7 and 14 days). Additionally, the morphologies of cell cultured in extraction media from all PEEK groups displayed healthy spindle-like shape at 7 days, nevertheless, the amount of MG-63 cells cultured with both PEEK/nano-FHA groups was higher than those of both PEEK groups (Fig. S4b), consistent with the results from indirect method. The cell proliferation and cytotoxicity of each PEEK disk was also evaluated by the WST-1 assay at 3, 7 and 14 days through direct methods. Fig. 6b showed that the OD value increased with time when MG-63 cells were co-cultured with different PEEK-based groups, indicating that the PEEK-based composite could affect cell proliferation. It was clear that the viability of cells for short-term (3 and 7 days) displayed no statistical differences among smooth PEEK, rough PEEK and rough PEEK/nano-FHA groups, but smooth PEEK/nano-FHA group showed significantly higher cell proliferation and kept better viability in the short term, indicating that the exposure of nano-FHA might have a positive influence on the cytocompatibility and on improving the bioactivity of PEEK materials. It is reported that nano-FHA shows a positive effect on bone cells (MG-63 and SAOS-2 line cells) adhesion and promotes cell proliferation [46,48], and possesses the potential to induce mesenchymal stromal cells differentiation into the osteoblast lineage [49]. For instant, a flourhydroxyapatite solegel coating on titanium substrate for hard tissue implants was proved by Kim et al. to improve the activity of cellular functions on the substrates [50]. However, after incubating for 14 days (long-term) the proliferation rate revealed no statistical differences among all groups, because the cells became saturated on the disk surface of PEEK groups, and no cytotoxicity was observed in all groups. It could be concluded

from the cell viability results that the incorporation of nano-FHA into the PEEK composite would present superb cytocompatibility and better cell viability than the pristine PEEK, and PEEK/nano-FHA biocomposite has great potential to be used for biomedical applications. 3.4.2. Cell morphology Generally speaking, the cells will undergo their morphological changes to stabilize the cellematerial interface after contacting biomaterials. The whole process of adhesion and spreading consists of cell attachment, filopodial growth, cytoplasmic webbing, flattening of the cell mass and the ruffling of peripheral cytoplasm progressing [51]. And the typical overview of the MG-63 cells morphologies on bare PEEK and PEEK/nano-FHA composite samples for 3, 7 and 14 days were shown in Fig. 7a. The number of cells attached on each sample increased with the extension of culture time. At 3 days, it could be seen that the MG-63 adhered onto the substrates, and spread pseudopodia on the surfaces. The amount of MG-63 grown on the pristine PEEK disc was the least at 3 days because the hydrophobic surface of bare PEEK made it difficult for cells or proteins to attach. On the surfaces of smooth PEEK/nanoFHA biocomposite, the cells spread with ruffling of peripheral cytoplasm, and flattened with a larger attachment area compared to the cells cultured on bare smooth PEEK disc, which indicated that the presence of nano-FHA on PEEK surfaces could promote cell attachment and growth, since the MG-63, which expressed a high surface density of intercellular adhesion molecule, might be bound to Ca2þ ions from FHA, then facilitating its adherence to substrates [52]. And cells cultured on rough surfaces showed a better cell spreading than those on smooth surfaces and exhibited a more highly flattened morphology and numerous cytoplasmic processes as well. It might be because the PEEK substrate after blasting had a relatively rough surface, which provided a larger interfacial contact area. Furthermore, after long-term culture, it was found that no significant difference could be found among all groups, and MG-63 cells covered almost the whole surfaces to form cell layers on the surfaces at 14 days. The multicellular aggregates were strongly

Fig. 9. Hemocompatibility assessment of hemolysis rate (a), and number of adhered platelets (b). The SEM observation of adhered platelets onto the surfaces of bare PEEK (c) and PEEK/nano-FHA (d). The white arrows in (c) and (d) point to the platelets. * represents p < 0.05.

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advocated as a highly useful culture mode for MG-63 instead of the traditional monolayer culture, since its tissue-like structure could promote cell proliferation and differentiation over a long period [53]. The fluorescence images from Fig. 7b indicated on the surface of smooth PEEK/nano-FHA biocomposite, MG-63 with a healthy spindle shape attached and spread more than other PEEK groups at 7 days, indicating that nano-FHA crystals could enhance cell growth, in accordance to previous results. Furthermore, as cell adhesion was enhanced, MG-63 cultured with smooth PEEK/nanoFHA group extended more adhered filopodia and spread more with visible presentation of more mature F-actin intracellular stress fibers. The number of adherent cells with smooth PEEK/nano-FHA group (Fig. S5) was significantly more than that of other groups, indicating that PEEK/nano-FHA biocomposite exhibited an excellent performance in vitro proliferation. 3.4.3. Flow cytometric analysis The cell cycle of the MG-63 cultures on the different samples was further evaluated by measuring the DNA content of nuclei labeled with propidium iodide, and different cytodieresis phases (G0G1: pre-DNA synthesis of resting, S: DNA synthesis phase, G2M: post-DNA synthesis and mitosis) were analyzed. It is reported that F element in FHA coatings prepared by solegel method favors the proliferation process of osteoblast-like cells through increasing the percentage of cell in S period [47]. Fig. 8a showed the total percentage of pre-osteoblast MG-63 cells in the S and G2M phases for the four groups after the various culture periods (3, 7, and 14 days).

Fig. 10. Antibacterial activities (a) of bare PEEK and PEEK/nano-FHA composite samples against S. Mutans bacteria cultured for 1, 4, 12 and 24 h. Live/dead cell staining (b) after 14 days of incubation with S. Mutans: bare PEEK (b-1), PEEK/nano-FHA (b-2). ** represents p < 0.01.

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On the smooth PEEK/nano-FHA surface, the total percentage of cells in S and G2M phases was also significantly higher in early culture period (3 days) with no significant difference found in the other groups. It indicated that in the early culture period, the nano-FA had a more significant effect on stimulating the osteoblast cells to become more active in DNA replication process. With longer culture period, although there was no significant difference found between rough PEEK and rough PEEK/nano-FHA, cells on the rough surfaces had a remarkably greater proportion of cells in S and G2M phases in comparison with those of their smooth counterparts, suggesting that the rough-surfaced PEEK was beneficial to the proliferation of osteoblast cells, and other studies have proved that the rough surface of substrate materials dramatically improves proliferation and differentiation of osteoblasts [54,55]. The effect of surface roughness on osteoblast cells might show up after longer period of interaction between cells and rough surface, which might be due to rough surface offering more space for the cells to proliferate. Apoptosis and death of MG-63 cells were further detected using flow cytometry, with Annexin V-FITC/propidium iodide double-staining of cells. Fig. 8b showed the percentage of apoptotic cells and necrotic cells on bare PEEK and PEEK/nanoFHA groups at 7 days and typical two-parameter figures for the flow cytometric analyses were depicted in Fig. S6. From the images, we could see that more cells were located in B1 and B4 region for smooth PEEK than other PEEK-based groups, implying a number of cells possessed the situation of early apoptosis and death. A significantly higher percentage of apoptotic cells were found on bare PEEK samples at day 7 compared to the PEEK/nanoFHA samples no matter smooth and rough surfaces, indicating that PEEK/nano-FHA samples provided a more favorable environment for cells growth. Moreover, the apoptosis rates of rough PEEK and rough PEEK/nano-FHA samples were measured to be 16.11%  3.1% and 8.57%  1.2% respectively, which were obviously lower than those of their smooth counterparts, in accord with cell cycle results, thus revealing useful effect from the surface treatment. The surface morphology plays an important role in the cytocompatibility of biomaterials, although the relationship between the morphology and biomedical behavior is still unclear [56,57]. These results suggest that the biological activity of PEEK/ nano-FHA biocomposite might be greatly promoted combined with the surface treatment.

Fig. 11. ALP activity of MG-63 cells after cultivating for 3, 7 and 14 days with different PEEK-based composites. *represents p < 0.05.

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Fig. 12. The production of mineralized extracellular matrix of hMSCs cultured in different PEEK-based composite groups was stained with Alizarin Red S on days 14 (a) and 21 (b). * represents p < 0.05, ** represents p < 0.01 and # represents p < 0.05 compared with other groups.

3.4.4. Blood compatibility of PEEK/nano-FHA composite For potential blood-contacting dental biomaterials, the adhesion of platelets to the biointerfaces and the interplay between blood and implant affect bone formation and healing. Platelet adhesion can be mediated by the integrin on the surface of the platelet that binds to the adsorbed proteins, especially fibrinogen, and causes platelet activation [51]. The activated platelets then accelerate thrombosis and lead to further coagulations. Thus, in this study, the morphologies of the adherent platelets and the adhered platelet number were investigated to evaluate the blood compatibility. Fig. 9c showed typical SEM of human platelets adhering on bare PEEK and PEEK/nano-FHA smooth surfaces after incubation in PRP for 1 h. A higher amount of platelets was detected on bare PEEK sample compared to PEEK/nano-FHA samples (Fig. 9b), which indicated that thrombus formation might be more likely to occur on the surface of bare PEEK. From the enlarged images, it could be seen that platelets adhering to both PEEK groups surfaces were nearly round with only one or two short pseudopodia, implying a negative activation. No significant difference in hemolysis percentage could be observed from both treated and untreated PEEK samples. It was noteworthy that both the hemolysis rates for bare PEEK and PEEK/nano-FHA samples stayed much lower than 5%, which is regarded as a biosafety threshold and stands for well-behaved hemocompatibility as illustrated in Fig. 9a. Endosseous implants initially come into contact with blood. The interactions between blood and endosseous implants have a tremendous influence on subsequent bone healing events in the peri-implant healing compartment. Our findings indicate that PEEK/nano-FHA biocomposite with

good blood compatibility have great potential to be used for endosseous dental implants. 3.5. Antibacterial activities against bacteria The primary goal of this study is to endow PEEK implant with antibacterial activity for dental applications, thus, it is essential to examine the antibacterial properties of nano-FHA/PEEK composite. The semi-quantification of viable bacterial cell on the surfaces of substrates was done via Microbial Viability Assay Kit, which is a widely used method to determine bacterial cell

Fig. 13. Micro-CT 3D reconstruction models showing the regenerated bone of about 0.5 mm width ring around PEEK and PEEK/nano-FHA implants surface at 8 weeks.

Please cite this article in press as: Wang L, et al., Polyetheretherketone/nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.085

L. Wang et al. / Biomaterials xxx (2014) 1e18

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Table 2 Micro-CT results after 4 and 8 weeks of implantation (n ¼ 6 per group). Groups 4 weeks 8 weeks

BV/TV (%) PEEK PEEK/nano-FHA PEEK PEEK/nano-FHA

47.69 58.53 56.21 70.46

   

0.56 0.72* 3.40 4.81*

Tb.Th (mm) 92.26 175.96 177.41 317.50

   

9.81 7.64** 13.05 10.77**

Tb.N (1/mm) 1.72 2.44 2.25 3.53

   

0.35 0.10* 0.26 0.19*

Tb.Sp (mm) 390.73 352.28 315.23 287.97

   

18.04** 19.86 15.29* 25.05

* represents p < 0.05. ** represents p < 0.01.

viability. S. Mutans is one of the early colonized bacteria in oral biofilm and the major pathogen responsible for dental caries in humans. As shown in Fig. 10a, at the stage of initial adhesion at 4 h, compared with the pristine PEEK, few bacteria cells were detected on PEEK/nano-FHA biocomposite surface, though no significant differences were observed between PEEK and PEEK/ nano-FHA at 1 h, suggesting PEEK/nano-FHA displayed good antifouling properties. At the proliferation stage, as for PEEK, it showed that OD value increased with time when bacteria were cultured on PEEK substrate surfaces, indicating that the pristine PEEK implant were prone to bacterial cells proliferation, which would lead to subsequent infection. Conversely, there was a clear reduction in the number of adherent cells on PEEK/nano-FHA biocomposite surfaces. For instance, at 24 h, when challenged with S. Mutans PEEK/nano-FHA substrates showed more than about 86.5% reduction in bacterial adhesion compared with bare PEEK surface. More importantly, the number of S. Mutans on PEEK/nano-FHA surfaces decreased with time went on, which was significantly different from that of pristine PEEK surfaces, indicating that PEEK/nano-FHA surface significantly inhibited bacterial adhesion and proliferation. After initial bacterial attachment onto the implant surface, there is a prolonged accumulation phase involving cell proliferation and intercellular interaction followed by biofilm formation. Consequently, cells inside biofilm have a much higher antibiotic tolerance compared to their planktonic counterparts which makes them very difficult to eradicate [58]. In addition to in situ biofilm formation, pathogens with high motility such as S. Mutans can migrate along the dental implant surface, adhere and grow on new sites to form new colonies, and subsequently result in whole implant infection, eventually implant failure and bone loss [59]. Therefore, an effective way to prevent infection is to rupture bacterial when they are forming biofilm on PEEKbased implant surface. To examine the long-term antibacterial function of PEEK/nano-FHA biocomposite, mature biofilm formation within 14 days of exposure to S. Mutans bacteria solution was observed and monitored by CLSM technique. Fig. 10b showed CLSM images of 14 days bacterial biofilms on bare PEEK and PEEK/nano-FHA composite surfaces with LIVE/DEAD BacLight bacterial viability kit. For the pristine PEEK surface, extensive bacterial biofilm was formed and reached to about 90 mm thickness with visible live bacteria. In sharp contrast, more dead bacterial cells were evident on PEEK/nano-FHA biocomposite surface with red color (Fig. 10b2), suggesting that antimicrobial activity of PEEK/nano-FHA could be preserved for an extended period of time. The mechanisms by which fluoride may interfere with bacterial metabolism and dental plaque acidogenicity include the inhibition of the glycolytic enzyme enolase and the proton-extruding ATPase as well as the bacterial colonization and competition [32]. Furthermore, intracellular or plaque-associated enzymes such as acid phosphatase, pyrophosphatase, peroxidase and catalase may be affected by fluoride ions, thus resulting in disintegrating bacterial cells [32,60]. Meanwhile, as verified above, the addition of n-FHA can promote

the cell adhesion on the PEEK/nano-FHA composite surface. Upon implantation, the fate of a biomaterial could be described as a race between cell adhesion and bacterial adhesion to its surface [61]. For a successful implantation, tissue integration should occur prior to bacterial adhesion, thereby preventing bacterial colonization of the implant. The chemical element F and better cell adhesion on the surface by improving the “run for the surface” towards the cells and against the bacterial may cocontribute to the antimicrobial activity of the PEEK/nano-FHA. Overall, our developed PEEK/nano-FHA biocomposite performs excellent antibacterial activity and is able to confer the enhanced short-term and long-term antimicrobial activity to bare PEEK substrate which may be momentous in reducing bacterial contamination during implantation.

Fig. 14. Histotomy of bone contact immunostained by toluidine blue-fuchsine at 8 weeks of bare PEEK (aeb) and PEEK/nano-FHA implants (ced) postoperatively. (b) and (d) refer to the higher-magnification images of (a) and (c). Dark red area represents the newly formed bone, and dark black area represents the PEEK-based implant. White scale: 200 mm, black scale: 100 mm (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

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Ti substrate, indicating its positive effect on the differentiation of human osteoblast cells [63]. Also, surface roughness modification of implants is reported to dramatically improve the osseointegration properties [64]. In the present work, the good osteogenic property of rough PEEK/nano-FHA suggests that the combined effects of nano-FHA and surface roughness could improve the osteogenic differentiation activity of bioinert PEEK materials. 3.7. In vivo study

Fig. 15. In histological analysis, new bone formation around bare PEEK (aeb) and PEEK/nano-FHA implants (ced) were detected by bone labeling (calcein, calcein blue, and tetracycline). (b) and (d) refer to the higher-magnification images of (a) and (c). S: sample; NgB: newly grown bone deposition and remodeling zone; PeB: pre-existing bone tissue zone.

3.6. Osteogenic differentiation in vitro The osteogenic differentiation activity of cell to the biointerfaces, as far as bone-repair biomaterials concerned, is a key event in bone formation. Among the major osteogenic hallmarks, the upregulation of ALP activity is a central event occurring during the early time points of osteogenesis [62]. In vitro ALP activities of MG-63 cells cultivating with PEEK/nano-FHA biocomposite were evaluated at 3, 7 and 14 days. As shown in Fig. 11, ALP value increased with time went on when MG-63 cells were co-cultured with different PEEK-based groups. It could be seen that at 3 and 7 days, there were no significant differences between all PEEK-based groups. Whereas, at 14 days higher ALP activities were obtained for MG-63 cells cultured with rough PEEK/nano-FHA group than other groups, indicating that rough PEEK with nano-FHA particles could trigger an upregulation of ALP, correlated with the first check-point for osteogenic differentiation. Concomitantly, we analyzed the efficiency of the mineralization stage by using Alizarin Red S staining as a marker for the inorganic calcium, a characteristic common to bone-like structures. Fig. 12 showed the effect of addition of nano-FHA and surface roughness of PEEK matrix on the formation of mineralized matrix in osteoinductive media on days 14 and 21. The hMSC cells cultured on rough surfaces clearly showed more positive and brighter red staining (the typical calcium deposition) than smooth groups. These findings reinforced the statement that surface roughness could act as a promoter of osteogenic differentiation. On the other hand, a significant increase in the number of nodules and the amount of mineralized matrix were observed due to addition of nano-FHA underlining the role of nano-FHA crystals in the enhancement of osteogenic differentiation efficiency. As mentioned above, nanoFHA crystals have been accepted to greatly up-regulate biological markers during osteogenesis and enhance osteogenic differentiation of bone cells, therefore facilitating the formation of bone [46,49]. Montanaro et al. suggested that nano-FHA coating on titanium surface had a substantial higher osteogenic activity than pure

3.7.1. Micro-CT results In vitro evaluation indicated that the roughness surfaces were beneficial to the bioactivity of both pure PEEK and PEEK/nano-FHA composite. Thereby, all of the cylindrical implants were blasted by the same process before in vivo tests. Micro-CT has been utilized extensively in the study of trabecular architecture with X-ray radiation as a penetrating probe. This technology not only offers detailed microstructural information from almost any material, but also enables to generate high-resolution images/data to quantify the changes in percent of bone volume/tissue volume (BV/TV) histologically and then to validate the micro-CT data [39,65]. In Fig. 13, the implanted PEEK/nano-FHA was thickly surrounded by natural bone, which showed the trabeculae about 0.5 mm thickness vertical to the longitudinal axis of cylindrical implants, 2 mm length along the longitudinal axis of implants. However, discontinuous parts of the adjacent bone were found on the implanted bare PEEK groups. Owing to the radiolucency of PEEK, the bone recovery around the implant can be readily probed by X-ray examination. The bone resorption and looseness of implants after recovery were not seen showing good bind to adjacent bone tissue, however, dark shadow was evident in Ti implant due to the radiopacity of metal alloys as shown in Fig. S7. As shown in Table 2, higher bone volume/ tissue volume (BT/TV) was maintained in PEEK/nano-FHA cylindrical implant group than in bare PEEK implant group (p ¼ 0.05) at 4 and 8 weeks after surgery. The trabecular numbers (Tb.N) for PEEK/nano-FHA implant group after implantation were dramatically higher than those of bare PEEK implant group (p ¼ 0.05). Moreover, the trabecular thickness (Tb.Th) in PEEK/nano-FHA implant group was also significantly higher than that in bare PEEK implant group at 4 and 8 weeks after implantation (p ¼ 0.01). Nevertheless, at 4 and 8 weeks of recovery, the trabecular separation (Tb.Sp) for PEEK/nano-FHA implant group at 4 and 8 weeks after implantation were significantly lower than those of bare PEEK group (p ¼ 0.01 and p ¼ 0.05, respectively). These results showed that the quantity of newly formed bone in contact with the implants of PEEK/nano-FHA biocomposite group was drastically higher than that for bare PEEK group. It is proved that fluoride modification of titanium implants surfaces shows a significant increase in gene expression levels of osteocalcin and tartrateresistant acid phosphatase (TRAP) and also exhibit higher degrees both in the newly woven bone and in older Haversian bone reconstruction [66]. When PEEK/nano-FHA is in contact with bone marrow after implantation, the exposure of nano-FHA inevitably promotes the growth of osteoblasts and gives rise to bone formation. The results are closely correlated with osteoblast proliferation and differentiation in vitro as well. Thus, the PEEK polymer after blending nano-FHA crystals not only positively affects the osseointeration between implant and bone but also increases bone formation surrounding the implant. Our results provide unequivocal proof that PEEK/nano-FHA composite benefits the enhancement of in vivo bioactivity and osteogenic activity. 3.7.2. Histological analysis Fig. 14 showed the tissue response to bare PEEK and PEEK/ nano-FHA implants after 8 weeks with immunohistochemical

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staining. In the dyeing mage of histotomy, dark red area represented the newly formed bone and dark black area represented the PEEK-based implant. All implants showed direct contact with the newly formed bones. Direct fusion of bone to PEEK/nano-FHA biocomposite surface was demonstrated, and there was no sign of a fibrous layer which implied a loosening of the implants. Moreover, no inflammation or necrosis was observed on both PEEK/nano-FHA and PEEK samples, suggesting that the implants did not produce observable toxic effects in the surrounding tissues although a longer time point was necessary prior to clinical acceptance and fathom the healing process. There was no obvious increase in bone fusion in bare PEEK implant group at 8 weeks after surgery (Fig. 14aeb). In particularly, more bones were found around PEEK/nano-FHA implants than PEEK control. Fig. S8 showed the percentage of contact (BIC) on bare PEEK and PEEK/nano-FHA implants after week 8. There was more BIC on PEEK/nano-FHA implant in comparison with PEEK control, and the results were consistent with those obtained by the micro-CT analysis. Further, fluorochrome bone marker labels (calcein, calcein blue, and tetracycline) were clearly observed for bone tissue bonding to the cylindrical implants (Fig. 15). More bone deposition and remodeling were found around PEEK/nano-FHA implants, suggesting a greater degree of bone regeneration than pure PEEK implants. Moreover, the ingrowth of new-formed lamellar bone or trabecular alignment were dramatically distinguished without fibrous tissue encapsulation on the interface between bone and the PEEK/nano-FHA implant at 8 weeks, which showcased phenomenal osteoinductive integration. As a consequence, in vivo tests clearly indicate our PEEK/nano-FHA biocompose implant possess the superior ability to bond with host bones and significantly promote osseointegration thereby boding well application to dental implants. 4. Conclusions We show here that, compared with bare PEEK, PEEK/nano-FHA biocomposite, after surface treatment, is cytocompatible, possesses good antibacterial activity, and triggers a set of events in vitro that follow the temporal pattern of osteogenesis (ALP activity and cell mineralization). More importantly, the rough PEEK/nano-FHA implant exhibits improved bioactivity, osseointegration, and bone-implant contact in vivo due to the co-effects of rough structure and nano-FHA crystals. These results have paved the way for the PEEK-based composite to be used as dental implant material in many challenging applications with combined improvement in antibacterial activity and osseointegration. Acknowledgments This work was supported by National Natural Science Foundation of China (Grant 30973317), Natural Science Foundation of Hainan Province (813191), Key Science and Technology Plan Projects of Hainan Province (ZDXM20110051), and Peking University’s 985 Grant. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.04.085. References [1] Feng Y-F, Wang L, Zhang Y, Li X, Ma Z-S, Zou J-W, et al. Effect of reactive oxygen species overproduction on osteogenesis of porous titanium implant in the present of diabetes mellitus. Biomaterials 2013;34:2234e43.

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Please cite this article in press as: Wang L, et al., Polyetheretherketone/nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.085