SN Composites Integration with PEKK

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Bio-interactions and Biocompatibility

Microporous Coatings of PEKK/SN Composites Integration with PEKK Exhibiting Antibacterial and Osteogenic Activity, and Promotion of Osseointegration for Bone Substitutes Han Wu, Lili Yang, Jun Qian, Deqiang Wang, Yongkang Pan, Xuehong Wang, Saha Nabanita, Tingting Tang, Jun Zhao, and Jie Wei ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01508 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 15, 2019

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ACS Biomaterials Science & Engineering

Microporous Coatings of PEKK/SN Composites Integration with PEKK Exhibiting Antibacterial and Osteogenic Activity, and Promotion of Osseointegration for Bone Substitutes

Han Wua, Lili Yangb, Jun Qiana, Deqing Wanga, Yongkang Pana, Xuehong Wanga, Saha Nabanita*c, Tingting Tang*d, Jun Zhaoe, Jie Wei*a

a

Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of

Science and Technology, Shanghai 200237, China b

Department of Orthopaedic Surgery, Changzheng Hospital, The Second Military Medical

University, Shanghai 200003, China c

Centre of Polymer Systems, University Institute, Tomas Bata University, Tr T Bati 5678, Zlin

76001, Zlin, Czech Republic d

Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedic Surgery,

Shanghai Ninth People’s Hospital, Shanghai Jiaotong University, Shanghai 200011, China e

Shanghai Key Laboratory of Stomatology, Department of Orthodontics, Shanghai Ninth

People’s Hospital, Shanghai Jiaotong University, Shanghai 200011, China ⁎ Corresponding authors: E-mail addresses: [email protected] (Jie Wei), [email protected] (Tingting Tang), [email protected] (Saha Nabanita)

Abstract: To improve the antibacterial and osteogenic activities of polyetherketoneketone (PEKK), concentrated sulfuric acid (H2SO4) suspension with silicon nitride (SN) microparticle was utilized to modify PEKK surface. Through sulfonation reaction, microporous coatings of PEKK/SN composites were created on PEKK surface, which were 1 ACS Paragon Plus Environment

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integrated with PEKK substrate. The results showed that the content of SN in the microporous coatings increased with the increase of SN content in H2SO4 suspension (PKS without SN, PKSC5 and PKSC10 with 5wt% and 10wt% SN content in H2SO4) and the surface roughness and hydrophilicity of microporous coatings on PEKK were significantly improved with the SN content increasing. In addition, the microporous coating of PKSC10 with high SN content exhibited excellent antibacterial activity due to the synergistic action of the presence of amino (-NH2) and sulfonic acid (-SO3H) groups as well as the improvement of protein absorption. Moreover, the microporous coating of PKSC10 obviously stimulated adhesion, proliferation and osteogenic differentiation of rat bone mesenchymal stem cells. Furthermore, histological and push-out load evaluation indicated that the microporous coating of PKSC10 significantly promoted osteogenesis and osseointegration in vivo. The results suggested that the microporous coatings of PKSC10 with high SN content display good biocompatibility, antibacterial and osteogenic activities and osseointegration ability, which would have great potential for bone substitutes.

Keywords: Polyetherketoneketone; Silicon nitride; Microporous coating; Antibacterial activity; Osteogenic activity

1. Introduction Polyaryletherketone (PAEK) is a kind of special functional plastic, which contains two typical representatives: polyetherketoneketone (PEKK) and polyetheretherketone (PEEK).1 Due to good biocompatibility, chemical stability and mechanical strength as well as elastic modules similar to bone of human body, etc., PAEK (such as PEEK) are widely used in surgical treatments for bone fractures, spinal fusions, and joint arthroplasties as well as in craniofacial and dental applications.2,3 However, the poor osteogenic activity of PAEK as bone implants leads to inferior osseointegration, which reduces primary fixation and long-term stability of 2 ACS Paragon Plus Environment

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the implants during prosthetic implantation.4 Moreover, due to the poor osseointegration, the implants may clinically encounter many complications such as displacement, cage subsidence or pseudoarthrosis, loosening or failure, etc.5 Osseointegration of a biomaterial implant is regarded as the criteria for successful bone implantation.6 Thus, how to enhance the osteogenic activity of PAEK implants for local osseointegration has been the challenges in orthopedic applications. Various methods of surface modification have been developed to improve the biological properties of PAEK for accelerating osteogenesis and osseointegration, such as bioactive coating, grafting specific functional groups, physical modifications (e.g. surface sandblasting and plasma treatment), etc.4,7,8 Nevertheless, the bonding strength of the bioactive coating (e.g. hydroxyapatite) is generally weak, which might detach from the PAEK surface.9 Grafting specific functional groups and plasma treatment can improve the biological properties of PAEK; however, the properties are usually unstable.4 Although sandblasting can enhance surface roughness, which might be beneficial to cell responses, the biological inertia of PAEK remains

unsolved.8

Blending

PAEK

with

bioactive

inorganic

materials

(e.g.

bioceramics/bioglasses) to prepare PAEK based composites, which might enhance the bioactivity of PAEK.10 However, the agglomeration of inorganic materials in the polymer matrix is used to be a tricky problem, which might decrease the mechanical strength and biological properties of the composites. Therefore, development of new modification technology to improve surface biological properties of PAEK is still a significant need for bone repair. With the rapid development of biomaterial industry, implant centered infections have become a very knotty clinical problem.11 Implant-related infection is one of the primary causes of orthopedic implant failure, which usually require extensive surgical debridement, implant extraction, and prolonged antibiotic treatment.12 Therefore, resistance to bacterial infection is a very desirable property of biomaterial for the orthopedic implant, and there has 3 ACS Paragon Plus Environment


been a great quest to produce antimicrobial implants. Generally, some efforts have been made to mainly focus on coating existing orthopedic implants with:13-16 (1) metal ions (e.g.silver and copper) which have proven to be limited due to toxicity issues; and (2) antibiotics (e.g., gentamicin and vancomycin) which limit the osteogenic activity of the implant. Thus, a necessary alternative approach is used to develop an implantable biomaterial that is inherently antibacterial and osteogenic, which would likely be multifunctional to simultaneously promote osteogenesis and osseointegration while inhibit bacterial infection. Silicon nitride (Si3N4, SN) is a synthetic nonoxide ceramic that has been investigated and adapted as a biomedical material since 1989.17 More than twenty years had passed before SN was actually introduced to the biomedical market of the United States and European Union as an orthopedic biomaterial for interbody fusion devices. In addition, SN bioceramics have shown encouraging outcomes in the spine and maxillofacial surgery.18 Due to its outstanding compression strength and resistance to brittle fractures, SN was also proved to be useful in developing bearings to improve wear and longevity of hip and knee prostheses.18 SN has good biocompatibility, bioactivity, antibacterial activity and displays excellent bone affinity.19,20 Thus, this ceramic has attracted a lot of attention as a candidate for skeletal prosthetic biomaterial. In this study, to improve the antibacterial and osteogenic activities of PEKK, concentrated sulfuric acid (H2SO4) suspension with SN microparticle was utilized to modify PEKK surface. The objective of the study is to prepare a microporous coatings of PEKK/SN composites on PEKK surface, which can be completely integrated with PEKK substrate (avoiding coating fall off), and the microporous coatings containing SN can enhance the surface biological properties of PEKK while do not decrease its mechanical performances. To test these assumptions, the surface properties, antibacterial activity and rat bone mesenchymal stem cells (rBMSCs) responses to the microporous composite coatings on PEKK in vitro were

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investigated. Furthermore, the in vivo osteogenesis and osseointegration of the microporous composite coatings on PEKK were also evaluated.

2. Materials and methods 2.1 Preparation of microporous coatings on PEKK. SN (β phase, 99.9% metals basis, Aladdin Industrial Co., Ltd., China) was cleaned with ethanol and deionized water, then dried via compressed air. The concentrated H2SO4 suspension with SN particles was prepared by adding dried SN into 98wt% H2SO4 (Lingfeng Chemical Reagent Co., Ltd., China) at ambient temperature. The suspension was put into an ultrasonic cleaner (KQ5200DE, Kun Shan Ultrasonic Instruments Co., Ltd., China) to disperse the SN particles for 10 minutes. PEKK (OXPEKK-C, Oxford Performance Materials, UK) samples were prepared by using molds through cold pressing sintering method. Cylindric and square parallelepiped samples of PEKK (Ф10 mm × 2 mm, 50 mm × 50 mm × 2 mm and Ф5 mm × 5 mm) for different applications were prepared by using different molds. The PEKK samples were ground by 800-grit silicon carbide paper and ultrasonic cleaning with deionized water before surface modification. The PEKK samples were immersed in concentrated H2SO4 suspension with a different concentration of SN particles (0wt%, 5wt%, and 10wt%) for 8 minutes at ambient temperature. The modified PEKK samples (labeled as PKS, PKSC5, and PKSC10, respectively) were then immersed in water at 90℃ for 24 hours. After that, the samples were cleaned in the ultrasonic cleaner (UC-30, Shanghai Titan Scientific Co., Ltd.) for 30 minutes to obtain the final samples. 2.2 Characterization of microporous coatings on PEKK. SN particles were characterized by Fourier transform infrared spectrometry (FTIR, Nicolet 6700, Nicolet, USA), X-ray diffraction (XRD, D/MAX 2550 VB/PC, Rigaku Co., Japan), scanning electron microscopy (SEM, S-4800, Hitachi, Japan) and laser light scattering spectrometer (CGS5022F, ALV Co., Germany). In addition, the samples of PKS, PKSC5, and PKSC10 (Ф10 5 ACS Paragon Plus Environment


mm × 2 mm) were characterized by using SEM, Laser confocal 3D microscope (VK-X 110, Keyence Co., Japan), FTIR, XRD and energy dispersive spectrometry (EDS, S-4800, Hitachi, Japan). The water contact angles of these samples were assessed by a water contact angle measurement (XG-CAMB3, Xuanyichuangxi Industrial Equipment Co., Ltd., China) with about 5 μL of deionized water droplet for each test at ambient temperature. 2.3 In vitro protein adsorption. The properties of protein adsorption on the surfaces of samples (Ф10 mm × 2 mm) were determined by adsorbing protein of bovine serum albumin (BSA). The samples of PKS, PKSC5, and PKSC10 were placed in 10% BSA-PBS solution in a cell incubator for 24 hours at 37℃. Then the samples were taken out and lightly washed with PBS 2 times to remove the unadsorbed proteins. After that, 500 μl of 1% sodium dodecyl sulfate solution was added and shaken for 15 minutes to detach proteins from the surfaces of these samples. The total protein concentrations in the collected sodium dodecyl sulfate solutions were determined by NanoDrop 2000C device (Thermo Scientific, USA) at a wavelength of 280 nm. 2.4 Changes of Si ions concentration and pH values in SBF. The samples of PKS, PKSC5 and PKSC10 (Ф10 mm × 2 mm) were soaked in simulated body fluid (SBF, Beijing Leagene Biotechnology Co., Ltd., China) and kept in a constant temperature oscillator (HZQX300, Yiheng science instruments Co., Ltd., China) at 37℃ to determine ions release behaviors of the samples. The concentration of Si ions in SBF was tested by an inductively coupled plasma atomic emission spectrometer (ICP-AES, Varian 710-ES, Agilent Technologies, USA). Before the ICP analyses, the SBF solution was filtered to remove impurities and diluted 10 times to prevent the solutes from SBF from interfering with the assay. The pH values of the solution were measured by a pH meter (FE20, Mettler Toledo) after the samples soaked into SBF for 1, 3, 5, 7, 10 and 14 days. 2.5 In vitro antibacterial activity. The bacteria counting method was used to evaluate the antibacterial activity of PKS, PKSC5, and PKSC10 (50 mm × 50 mm × 2 mm). All the 6 ACS Paragon Plus Environment

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samples were sterilized by ethylene oxide. 0.2 mL of E. coli (ATCC 25922) and S. aureus (ATCC 25923) (1 × 106 CFU/mL) suspension were inoculated on each sample and incubated for 24 hours at 37℃. A vortex mixer (VORTEX-5, Beijing Jinye Dexiang Technology Co., Ltd. China) was used to dissociate bacteria from the porous surfaces of the samples. After that, the dissociated bacteria suspension was diluted and introduced to agar culture Petri dish. After incubated for 24 hours at 37℃, the active bacteria were calculated according to National Standard of China GB/T 4789.2 protocol (PEKK was used as a control). The bacteriostatic rate was calculated using the formula (1):

Xs 

A B 100% A

(1)

Where Xs represents the bacteriostatic rate, A represents the average number of colonies for PEKK, B represents the average number of colonies for PKS, PKSC5, and PKSC10. 2.6 In vitro cell responses. 2.6.1 Cell culture. The samples of PKS, PKSC5, and PKSC10 (Ф10 mm × 2 mm) were sterilized by ethylene oxide before cell experiments. The rBMSCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific Inc., USA) with the supplement of fetal bovine serum (FBS, 10%, GibcoBRL, USA), streptomycin (100 μg/mL) and penicillin (100 U/mL) at 37℃ in an atmosphere of 95% air and 5% CO2. The medium for cell culture was changed every 3 days. 2.6.2 Cell adhesion and morphology. Firstly, 1 mL of culture medium with 5 × 104 cells was seeded on each sample, which was placed in a 24-well plate and cultured for 24 hours. Then the specimens were gently washed with phosphate buffered saline (PBS, pH=7.4) for 3 times, after that, the cells were fixed with 0.25% glutaraldehyde solution (Lingfeng Chemical Reagent Co., Ltd., China) for 1 hour. Then the fixed cells were washed with PBS for 3 times. FITC-phalloidin isothiocyanate (FITC, Beyotime Biotech, Jiangsu, China) was utilized to stain actin cell filaments for 30 minutes. After that, the FITC was washed away by PBS and 2(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Beyotime Biotech, Jiangsu, 7 ACS Paragon Plus Environment


China) was added on samples to stain the nuclei of the cell for 10 minutes. The stained rBMSCs were observed by a confocal laser scanning microscope (CLSM, Nikon AIR, Nikon, Japan). To determine the cell attachment ratio on the samples, the rBMSCs were cultured with the density of 5 × 104 cells/well on the samples in 24-well culture plate. After cultivated for 6, 12 and 24 hours, the culture medium with unattached cells was carefully removed from the wells by pipette; then the samples were washed with PBS for three times. After that, the cells remaining on the samples were collected and counted by CyQUANT® assay kits (Life Technologies, Carlsbad, USA) (tissue culture plate without sample was used as a control) and the cell attachment ratio was presented as the value of cell numbers on the samples divided by that on tissue culture plate). 2.6.3 Cell proliferation. The methylthiazol tetrazolium test (MTT) was used to investigate the proliferation of rBMSCs on the samples after the cells were cultured for 1, 3, and 5 days. 1 mL of the cell suspension was seeded on samples in a 24-well plate with a cell density of 5 × 104 cells/mL and kept at 37℃ in a humidified incubator with 5% CO2. After that, 100 μL of the MTT solution (0.5 mg/mL) was added, and the samples were incubated for 4 hours at 37℃ after the specimens were placed into new 24-well plates. After incubation, 100 μL dimethyl sulfoxide (DMSO, Sinopharm, Shanghai, China) was added into each well. Then, the solution was incubated for 15 minutes at 37℃. An automated plate reader (Synergy HT Multi-detection Microplate) was used to measure the optical density (O.D.) values at a wavelength of 570 nm. 2.6.4 Alkaline phosphatase activity. Alkaline phosphatase (ALP) activity of the cells was measured to evaluate cell differentiation ability. The rBMSCs were cultured on the three samples with the density of 5×104 cells/mL, and cultured for 7, 10 and 14 days. Then the culture medium was removed, and the samples were washed for 3 times with PBS. The cell 8 ACS Paragon Plus Environment

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lysates were obtained by adding 1 mL of 0.2% Nonidet P-40 solution. After that, the lysates were transferred into another 24-well plate and incubated with 0.100 L Tris-HCl solution (1 mol/L), 0.025 L MgCl2 solution (4 mmol/L) and 0.025 L p-nitrophenyl phosphate (4 mmol/L) solution for half an hour at 37℃. The conversion of p-nitrophenyl phosphate to p-nitrophenol was stopped by adding 1×10-4 L NaOH solution (0.5 mol/L). The O.D. values of pnitrophenol were detected by a spectrophotometer (UV-vis 8500, Shanghai) at 405 nm. 2.7 Implantation in vivo. The osteogenesis and osseointegration of the samples (PKS, PKSC5, and PKSC10) in vivo were evaluated using the model of rabbit femur cavity defects. In this study, 36 New Zealand white rabbits (18 for histological and 18 for biomechanical analysis) with 3-month age and average weight of 2.5 kg were used. The procedure was approved by the Animal Care and Experiment Committee of Shanghai Ninth People’s Hospital affiliated to Shanghai Jiao Tong University (Shanghai, China). All implants were sterilized by γ-ray (10 kGv) radiation before surgery procedure. The rabbits were anesthetized with intramuscular injection of sodium pentobarbital (80 mg/kg, Sigma, USA). Then the defects (5 mm in diameter and 5 mm in depth) were created in the right shaved knee. After that, the samples of PKS, PKSC5, and PKSC10 (Ф5 mm × 5 mm) were implanted into the defects. After implanted for 1 and 3 months, the rabbits were sacrificed and the femurs were removed to obtain experimental animal samples. The push-out loads of implants with bone were determined by a universal materials testing system (Instron, HighWycombe, UK) with a load rate of 5 mm/min. The load-displacement curve was recorded during the pushing period, and the failure load was defined as the peak value of the load-displacement curves. The femur specimens were dehydrated by ascending concentrations of ethanol and then embedded in polymethylmethacrylate. After that, the embedded specimens were cut into slices and polished till the thickness was 50 μm. Van Gieson's picro fuchsin was used to stain the slices. The stained slices were observed by an inverted microscope (TE2000-U, Nikon, 9 ACS Paragon Plus Environment


Japan), and the Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, USA) was used to calculate the percentage of bone-implant contact (BIC). 2.8 Statistical analysis. All experiments in this study were independently carried out in triplicates, and each data point represents three replicate measurements. The data were expressed as averages standard deviations. The results were statistically analyzed using the one-way analysis of variance (ANOVA) with Tukey’s post hoc test and all the statistical analyses were determined by the GraphPad Prism 5 statistical software package. p < 0.05 was considered to be statistically significant.

3. Results 3.1 Characterization of SN. Figure 1a and 1b are FTIR and XRD patterns of SN. For the FTIR pattern of SN, the broad peak around 918 cm-1 was assigned to asymmetrical telescopic vibration of Si-N.21 The peaks at 1610 cm-1 and 3250 cm-1 were attributed to the flexural vibration and telescopic vibration of N-H, indicating that the amino (-NH2) groups presented on the SN surface.22 In addition, the peak at 3400 cm-1 was stretching vibration of O-H, indicating that hydroxyl (-OH) groups appeared on the SN surface. For the XRD pattern of SN, the narrow peaks at 13.4° (100), 23.4° (110), 27.0° (200), 33.5° (101), 36.1° (210), 52.2° (301), and 69.9° (321) could be observed. These peaks were typical characteristic peaks of β phase SN with high crystallization.23 Figure 1c and 1d are SEM images and particle size distribution analysis of SN. It could be seen that the size of SN particles were 2-4 μm, and the particle size distribution of SN was around 3 μm.


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Figure 1 FTIR pattern (a), XRD pattern (b), SEM image (c) and particle size distribution (d) of SN

3.2 SEM images of PKS, PKSC5, and PKSC10. The SEM images of surface morphology of PKS, PKSC5 and PKSC10 and cross-section of PKSC10 are shown in Figure 2. It could be seen that micropores ranging from 3 μm to 5 μm were found on surfaces for all the samples. In addition, the SN particles with the size around 3 μm were presented in the micropores after PEKK was modified by concentrated H2SO4 suspension with SN particles. The results indicated that microporous coatings of SN/PEKK composites were prepared on PEKK surface. A small SEM image of cross-section of PKSC10 was inserted in Figure 2e. From the small image, it was difficult to distinguish the interface between coating and PEKK, indicating that the microporous coatings of SN/PEKK composites were wholly integrated with PEKK substrate, which can avoid coating shedding.



Figure 2 SEM images of PKS (a, b), PKSC5 (c, d), PKSC10 (e, f) under different magnification, and small SEM image of cross-section of PKSC10 was inserted in “Figure 2e”

3.3 FTIR and XRD of PKS, PKSC5, and PKSC10. Figure 3a is the FTIR spectra of PKS, PKSC5, and PKSC10. All the characteristic bands of PEKK were presented in PKS, PKSC5, and PKSC10. The peaks at 1153 cm-1 and 1233 cm-1 were assigned to asymmetric stretching vibrations of C-O-C bond in the diaryl groups, and the peak at 1584 cm-1 was attributed to CC stretching vibrations in the aromatic ring. Moreover, the peak at 1051 cm-1 was assigned to 12 ACS Paragon Plus Environment

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symmetric stretching of S=O, and the peak at 1200 cm-1 was the antisymmetric stretching vibration of S=O.24 It could be deduced that sulfonic acid (-SO3H) groups were grafted on PEKK surface after sulfonation reaction between PEKK and concentrated H2SO4. With the increasing of SN concentration in concentrated H2SO4 suspension, the intensity of characteristic peaks of SN was significantly enhanced (around 918 cm-1) in PKSC10 (compared with PKSC5). Moreover, the -NH2 and -OH groups were found on the surfaces of both PKSC5 and PKSC10. Figure 3b is the XRD patterns of PKS, PKSC5, and PKSC10. The peaks at 2θ=15.6° (020), 18.5° (021), 22.9° (110) and 28.5° (102) in all samples were attributed to the characteristic peaks of PEKK. Compared with PKS, the peaks at 13.4° (100), 23.4° (110), 27.0° (200), 33.5° (101), 36.1° (210), 52.2° (301), and 69.9° (321) were attributed to the characteristic peaks of SN in both PKSC5 and PKSC10. 3.4 EDS analysis of PKS, PKSC5, and PKSC10. Figure 3c-3k are the results of EDS analysis of PKS, PKSC5, and PKSC10. From the element distribution maps (Figure 3c, 3d, 3f, 3g, 3i, and 3j), no Si signal distribution was found on PKS surface while a lot of Si signal dots were found to be evenly dispersed on the surfaces of PKSC5 and PKSC10, indicating that SN was evenly coated on PEKK surface. In addition, the number of Si signal dots on PKSC10 was more than on PKSC5 surface, indicating more SN particles were coated on PKSC10 surface. Moreover, a few S signals were found in all samples. Therefore, combining Figure 4a (FTIR), it could be confirmed that, after sulfonation modification, -SO3H groups were introduced on PEKK surface. Figure 3e, 3h, and, 3k show the element energy spectrum of PKS, PKSC5, and PKSC10, respectively. The peak of S element was found on all the samples while the peak of Si element was only found on the surfaces of PKSC5 and PKSC10. The results of SEM, FTIR, XRD and EDS analysis showed that the microporous coatings of SN/PEKK composites were prepared on PEKK surface after modified by H2SO4 suspension with SN. In addition, the 13 ACS Paragon Plus Environment


higher concentration of SN in concentrated H2SO4, the more SN particles were presented in microporous coatings on PEKK.

Figure 3 FTIR (a) and XRD (b, “ · ” represents characteristic peak of PEKK and “*” represents characteristic peak of SN) patterns of PKS, PKSC5 and PKSC10. Element distribution maps (c, d, f, g, i and j) and element energy spectrum (e, h and k) of PKS (c, d and e), PKSC5 (f, g and h) and PKSC10 (i, j and k), in which red dots represent Si signal, yellow dots represent S signal


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3.5 Roughness, hydrophilicity and protein adsorption. The laser microscope 3D images of the surface morphology of PKS, PKSC5 and PKSC10 are shown in Figure 4a, 4b, and 4c. As shown in Figure 4d, it was found that all the samples exhibited rough surface after modification, and the roughness (Ra: arithmetical mean deviation of the profile) of PKS, PKSC5, and PKSC10 was 5.34 ± 0.30 μm, 7.78 ± 0.24 μm and 8.61 ± 0.32 μm, respectively. The results revealed that the roughness of the samples increased with the increase of SN content in concentrated H2SO4 (PKSC10 > PKSC5 > PKS).

Figure 4 Laser microscope 3D images of surface morphology of PKS (a), PKSC5 (b), PKSC10 (c), and roughness (d) of the samples (*p < 0.05, vs PKS)

The hydrophilicity of the samples was characterized by measuring the water contact angle, and the results are shown in Figure 5a. The water contact angles of PKS, PKSC5, and PKSC10 were 90.0°, 64.1° and 53.8°, respectively. The results indicated that the hydrophilicity of the samples was enhanced with the increase of SN content in the microporous coatings (PKSC10 > PKSC5 > PKS). 15 ACS Paragon Plus Environment


Figure 5b shows the property of protein adsorption on the surfaces of samples soaking for 24 hours. The protein adsorption amount on PKS, PKSC5, and PKSC10 was 14.1 ± 1.0 μg/mL, 30.3 ± 2.1 μg/mL and 50.6 ± 3.3 μg/mL, respectively. The results indicated that the protein adsorption of the samples significantly increased with the increase of SN content in the microporous coatings. 3.6 Changes of Si ions concentration and pH values in SBF. Figure 5c shows the change of concentration of Si ions in solution after PKS, PKSC5 and PKSC10 were immersed into SBF for 1, 3, 5, 7, 10 and 14 days. It could be found that no change of concentration of Si ions for PKS in SBF, indicating that no Si ions released from PKS. However, the concentration of Si ions increased with time for both PKSC5 and PKSC10, and the concentration of Si ions for PKSC10 was higher than PKSC5 at all time points, indicating that the release of Si from PKSC10 into SBF was faster than PKSC5. Figure 5d is the change of pH values in solution after PKS, PKSC5, and PKSC10 were immersed into SBF for 1, 3, 5, 7, 10 and 14 days. For PKSC10, the pH values decreased in the first day and then increased with time. For PKSC5, the pH values decreased in the first three days, and then increased with time. For PKSC, the pH values also decreased in the first three days, and then showed no visible change. On day 5, the pH values for PKSC5 and PKSC10 were 7.37 and 7.53 while the pH value for PKS was 7.03. Furthermore, on day 14, the pH values for PKSC5 and PKSC10 were 7.57 and 7.81 while the pH value for PKS was 7.03.


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Figure 5 Water contact angles (a) and adsorption of total protein (b) of PKS, PKSC5 and PKSC10 (*p < 0.05, vs PKS) and the function curves of Si ions concentration (c) and pH values (d) with time after PKS, PKSC5 and PKSC10 were immersed in SBF for 1, 3, 5, 7, 10 and 14 days

3.7 In vitro antibacterial activity. Figure 6 is the photos of cultivated E. coli and S. aureus colonies after cultured on agar for 24 hours, which were dissociated from PEKK, PKS, PKSC5, and PKSC10. It was found that, for PEKK, a large number of colonies were found for both E. coli and S. aureus, indicating no antibacterial activity. Compared with PEKK, the colonies of E. coli and S. aureus were obviously reduced for PKS, indicating that PKS had antibacterial activity. Furthermore, almost no bacteria was found for PKSC5 and PKSC10, indicating that both PKSC5 and PKSC10 exhibited better antibacterial activity against E. coli and S. aureus than PKS.



Figure 6i and 6j show the bacteriostatic rate of the samples against E. coli and S. aureus. The results revealed that the bacteriostatic rate of PKS against E. coli was 83.92% and S. aureus was 82.16%, the bacteriostatic rates of PKSC5 against E. coli and S. aureus were 94.33% and 92.72%, and the bacteriostatic rates of PKSC10 against E. coli and S. aureus were 99%.

Figure 6 Photos of cultivated E. coli and S. aureus colonies on agar for 24 hours, which were dissociated from PEKK(a, e), PKS (b, f), PKSC5 (c, g) and PKSC10 (d, h), and bacteriostatic rates of E. coli (i) and S. aureus (j) (*p < 0.05, vs PKS)

3.8 In vitro cell responses. 3.8.1 Cell adhesion and morphology. Figure 7a, 7b, and 7c show the CLSM images of rBMSCs after the cells were cultured on PKS, PKSC5 and PKSC10 for 24 hours. The number of cells on PKSC10 was more than PKSC5, and PKSC5 was more than PKS. Moreover, the cell spread better on PKSC10 than PKSC5, and PKSC5 18 ACS Paragon Plus Environment

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was better than PKS. In particular, the cells on PKSC10 exhibited plenty of prominent filopodia and unidirectional lamellipodia compared with PKSC5 and PKS. Figure 7d is the attachment ratio of cells on the samples at the different time. It could be observed that the attachment ratio of cells on the samples increased over culture time. At 6 hours, the attachment ratios of cells on PKSC5 and PKSC10 were higher than PKS. At 12 and 24 hours, the attachment ratio of cells on the samples obviously increased with the increase of SN content in the microporous coatings (PKSC10 > PKSC5 > PKS), indicating good cytocompatibility of the samples. 3.8.2 Cell proliferation and ALP activity. As shown in Figure 7e, the O.D. values (indicating cell proliferation) of rBMSCs on all samples increased over culture time. On day 1, no noticeable difference of O.D. value was found for the three samples. However, on day 3 and day 5, the O.D. values for PKSC10 and PKSC5 were significantly higher than PKS, and PKSC10 was higher than PKSC5. Figure 7f is the ALP activity (an early osteogenic marker, indicating osteogenic differentiation) of rBMSCS cultivated on PKS, PKSC5, and PKSC10 for 7, 10 and 14 days. The ALP activity of cells on all the samples increased over culture time. On day 7, no obvious difference was found for all samples. On day 10, the ALP activity for PKSC10 was significantly higher than PKSC5 and PKS. On day 14, the ALP activity for the samples obviously increased with the increase of the SN content in the microporous coatings (PKSC10 > PKSC5 > PKS). The proliferation and differentiation of rBMSCs on the three samples increased with time, indicating good cytocompatibility.



Figure 7 CLSM images of rBMSCs cultured on PKS (a), PKSC5 (b), PKSC10 (c) for 24 hours, and attachment ratio (d), O.D. values (e) and ALP activity (f) of rBMSCs cultured on PKS, PKSC5 and PKSC10 for different time (*p < 0.05, vs PKS)

3.9 In vivo osteogenesis and osseointegration. The images of the histological sections stained with Van Gieson's picro fuchsin are shown from Figure 8a to 8f. Both the amount of new bone tissues (NB) and bone-implant contact (BIC) increased with time (3 months > 1 20 ACS Paragon Plus Environment

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month). Moreover, at both 1 and 3 months, the amount of NB and BIC increased with SN content in the microporous coatings on PEKK (PKSC10 > PKSC5 > PKS). Notably, at 3 months, obvious gap appeared at the interface between NB and PKS, and only a few NB appeared around PKS. However, almost no gap presented at the interface between NB and PKSC10, and NB directly formed on PKSC10 surface. Clearly, PKSC10 obtained the best contact with NB. Figure 8g shows the quantitative analysis of BIC at both 1 and 3 months. It was found that the BIC of the samples not only increased with time but also increased with SN content in the microporous coatings. At 3 months, the BIC of PKSC10 was the highest (82.1%) compared with PKS (21.8%) and PKSC5 (62.5%). The push-out loads of the implants are shown in Figure 8h, the push-out loads of PKSC5 and PKSC10 significantly increased with the extension of implantation time while no significant change was found for PKS. In addition, at 1 month, there were no obvious differences of push-out loads for PKS and PKSC5 while the push-out load for PKSC10 was much higher than that of both PKS and PKSC5. Moreover, at 3 months, the push-out loads for the samples increased with the increase of SN content in the microporous coatings, and PKSC10 also had the highest push-out load (501.3 N).



Figure 8 Photos of histological sections by V&G staining after PKS (a, b), PKSC5 (c, d) and PKSC10 (e, f) implanted in vivo for 1 (a, c and e) and 3 months (b, d and f); M: implant material, B: newly formed bone tissues. Quantitative analysis of bone-implant contact (g) and push-out loads (h) of the samples after implanted in vivo for 1 and 3 months (*p < 0.05, vs PKS)

4. Discussions


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SN is biocompatible, antimicrobial and osteoconductive, making it a very attractive biomaterial for orthopedic surgery.20 SN has shown satisfactory performances as an implantable biomaterial, and implants of intervertebral discs and spinal fusion devices made of SN bioceramic have been applied in the clinic.25 PEKK, as one of PAEK polymers with good biocompatibility, high strength and a low modulus of elasticity compared with metal, has been investigated as an orthopedic biomaterial.2 However, PEKK lacks antibacterial activity and osteogenic activity. In this study, to enhance antibacterial and osteogenic activities of PEKK, the microporous coatings of PEKK/SN composites on PEKK surface were fabricated by sulfonation reaction through immersing PEKK in concentrated H2SO4 suspension with SN particles. The results revealed that the microporous coatings of PEKK/SN composites were wholly integrated with PEKK substrates. Therefore, this microporous coatings containing SN on PEKK could avoid the coatings fall off while maintain the mechanical properties of PEKK, and improve the biological properties of PEKK. Moreover, -SO3H groups were introduced onto the PEKK surfaces (including PKSC10, PKSC5, and PKS) through sulfonation reaction. Many studies have shown that the rough surface of the biomaterial can promote cell adhesion, and the effect of roughness on cell adhesion has been mainly attributed to an increased surface-to-volume ratio that may provide more sites for cell attachment.26 In this study, PKSC10, PKSC5, and PKS exhibited rough surface, and the roughness of the samples increased with the SN content increasing in the microporous coatings. It can be suggested that the SN content in the microporous coating played an essential role in the improvement of the roughness of the samples. In addition, the hydrophilic surface of the biomaterial is not only suitable for cell adhesion/growth but also beneficial to new bone tissue growth when implanted in vivo.27 SN is an excellent hydrophilic biomaterial that has complex hydrolytic reactions in water, and hydrophilic groups such as -OH and -NH2 can be naturally formed on its surface.20 In this study, the hydrophilicity of the samples was obviously improved with the 23 ACS Paragon Plus Environment


increase of SN content in the microporous coatings, indicating that the SN played key roles in the enhancement of the hydrophilicity of the samples. Therefore, the improvements of the roughness and hydrophilicity of PKSC10 were attributed to the presence of SN in the microporous coatings. The adsorption of protein on biomaterial surface is beneficial to cell adhesion, which promotes cell membrane in contact with biomaterial and the extension of pseudopodia.28 The previous study has shown that the increase of protein adsorption on biomaterial surface can reduce bacterial adhesion and then inhibit the biofilm formation of bacteria, and thus display antibacterial activity.29,30 In this study, the protein adsorption of the samples increased with the increase of SN content. It can be suggested that incorporating SN into the microporous coatings on PEKK could improve their surface roughness, hydrophilicity, and protein adsorption of the samples. After soaked into SBF, Si ions were found to slow release from PKSC10 and PKSC5 into the solution due to the slight dissolution of SN, and PKSC10 containing more SN released more Si ions than PKSC5. Moreover, the pH values in the solution slightly decreased for PKS and formed a weak acid micro-environment. However, the pH values increased with time for PKSC5 and PKSC10, and weak alkaline micro-environment was formed. The decrease of pH values for PKS was attributed to the presence H+ ions, which were ionized from -SO3H groups on PEKK in the microporous coatings while the increase of pH values for both PKSC10 and PKSC5 were ascribed to the presence of -NH2 groups on SN (-NH2 groups reacted with H+ ions and formed -NH3+ groups).31 When the H+ ions ionized from -SO3H groups completely reacted, the excess -NH2 groups on SN ionized water and produced -OH-; thus the pH value increased later and caused a weak alkaline micro-environment. Therefore, the presence of SN could regulate the acid-base properties of the microporous coatings on PEKK in the physiological environment.


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Bacterial infection of the implant is a severe risk, which causes loosening and abscission of the implant, and eventually leads to failure of implantation in vivo.32 Biomaterials with antibacterial activity can inhibit bacterial infection and guarantee the long-term stability of the implant in vivo.33 The -SO3H groups on the biomaterials have been proved to have antibacterial activity.34 In addition, previous studies have shown that SN surface was inherently resistant to bacteria and inhibited biofilm formation due to the presence of -NH2 groups, which can form -NH3+ groups that are positively charged in acid environment while the bacteria is used to be negatively charged, thus the interaction of positively charged -NH3+ groups with negatively charged bacteria led to bacterial membrane disruption and lysis.17 In this study, PKS showed antibacterial activity due to the presence of -SO3H groups. However, PKSC5 and PKSC10 displayed excellent antibacterial activity because of the presence of both -NH2 and -SO3H groups on the microporous coatings. Therefore, the enhancements of antibacterial activity of both PKSC5 and PKSC10 were ascribed to the effects of both -NH2 and -SO3H groups on bacteria, in which PKSC10 containing more SN exhibited the highest antibacterial activity. One of the reasons for SN surface with antibacterial activity is because of its fast protein absorption, which reduced bacterial adhesion and then prevented bacteria from biofilm formation.35 In this study, the protein absorption of the samples increased with the increase of SN content in the microporous coatings, and PKSC10 showed the highest protein absorption. Therefore, the excellent antibacterial activity of PKSC10 was ascribed to the synergistic effects of the presence of functional groups and improvement of protein absorption. Osteoblasts interact with the substrates via integrins, which cluster and associate with cytoskeletal elements forming focal adhesions, and the rough and hydrophilic surface of the biomaterials could augment the density of focal adhesions.36 As a result of the extracellular cues, the more numerous focal adhesions present on the substrates increase cell complexity and enable the formation of ﬁlopodia-like structures, through the cytoskeleton arrangement.37 25 ACS Paragon Plus Environment


In this study, the cells adhesion and spreading on the samples were improved with the increase of SN content in the microporous coatings, demonstrating that SN played important roles in promoting cells adhesion and spreading. Cells proliferation and differentiation are the most key stages of cell/biomaterial interactions, which further influence the tissues formation.38 In this study, the proliferation and differentiation of rBMSCs for PKSC10 were obviously higher than PKSC5, and PKSC5 were higher than PKS, indicating that the SN content in the microporous coatings played important roles in promoting cells proliferation and differentiation. The surface characteristics (such as chemical composition, morphology, roughness, and hydrophilicity, etc.) of the biomaterials have obvious influences on the cell responses (such as cell adhesion, proliferation and osteogenic differentiation).27,39 In this study, compared with PKSC5 and PKS, PKSC10 exhibited the highest surface roughness and hydrophilicity. Therefore, the improvement of cells adhesion and spreading on PKSC10 surface was attributed to the enhancements of the surface roughness and hydrophilicity. Previous studies confirmed that a certain amount concentration of Si ions released from Si-based biomaterials could promote cell proliferation and differentiation.40 Moreover, silicon assists with the synthesis of glycosaminoglycans and proteoglycans,41 which can promote the proliferation and differentiation of osteoblasts. Furthermore, weak alkaline micro-environment has been proved to induce positive cell response (e.g. cells proliferation and differentiation).42 In this study, both PKSC5 and PKSC10 not only slowly released Si ions but also caused a weak alkaline micro-environment in SBF. Therefore, it could be suggested that the enhancement of cells proliferation and differentiation on PKSC10 were attributed to the release of Si ions and creation of weak alkaline micro-environment. In short, the cell responses to PKSC10 were the functions of surface roughness, hydrophilicity, release of Si ions and formation of weak alkaline micro-environment.


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The osteogenesis and osseointegration of the samples in vivo were determined by histological and push-out load evaluation.43 The images of histological sections showed that the NB not only increased with time but also improved with SN content in the microporous coatings. At 3 months, only a few NB appeared on PKS surface, and obvious gap was found at the interface between the NB and PKS. However, a large number of NB directly formed on PKSC10 and no gap was found at the interface between the NB and PKSC10. Clearly, compared with PKSC5 and PKS, PKSC10 exhibited the highest osteogenesis and obtained excellent osseointegration. The BIC, defined as the percentage of implant surface covered by NB, is one of the critical measures used to quantify the degree of osseointegration.44 In this study, the BIC of the samples not only increased with time but also increased with SN content increasing in the microporous coatings. At 3 months, compared with PKS and PKSC5, PKSC10 got the highest BIC. The results demonstrated that the microporous coating of PKSC10 containing high SN content displayed the highest osteogenic activity and obtained excellent osseointegration. The push-out loads of the implants with new bone tissues are also regarded as one of the critical determination used to quantify the degree of osseointegration.45 In this study, the push-out loads for the samples increased with the increase of SN content in the microporous coatings, and PKSC10 obtained the highest push-out loads. Therefore, SN in the microporous coating of PKSC10 played key roles in promoting osteogenesis and osseointegration. The finding in this study indicated that the rough and hydrophilic surface of PKSC10 was more favored osteoblasts adhesion, proliferation, and osteogenic differentiation as well as new bone tissue formation due to the presence of SN in microporous coatings integration with PEKK both in vitro and in vivo. In addition, the presence of functional groups of -NH2 and SO3H as well as the improvement of protein adsorption of PKSC10, making it an antibacterial biomaterial. Moreover, the release of Si ions from SN in the microporous coating of PKSC10 might stimulate new bone tissues formation in vivo. Furthermore, successful osseointegration 27 ACS Paragon Plus Environment


of PKSC10 was achieved in vivo, indicating that SN content in the microporous coating of PKSC10 played key roles in osteogenesis and osseointegration. In summary, the microporous coatings of PEKK/SN composite integration with PEKK with good biocompatibility, antibacterial activity and osteogenic activity significantly promoted osteogenesis and osseointegration, which would have a great potential for bone repair.

5. Conclusions PEKK surface was modified by concentrated H2SO4 suspension with SN microparticles, and microporous coatings of PEKK/SN composites wholly integrated PEKK were fabricated by sulfonation reaction. The results showed that roughness and hydrophilicity of the microporous coatings on PEKK were significantly improved with SN content increasing. In addition, the microporous coatings on PEKK obviously promoted rBMSCs adhesion, proliferation, and differentiation, which depended on the SN content. Moreover, the microporous coatings on PEKK exhibited excellent antibacterial activity due to the synergistic action of the presence of -NH2 and -SO3H groups as well as improvement of protein absorption. Furthermore, the osteogenesis of microporous coatings on PEKK was obviously enhanced with SN content increasing, and successful osseointegration was achieved for PKSC10 in vivo. In short, microporous coating of PKSC10 exhibited excellent biocompatibility, antibacterial and osteogenic activities, which might be a promising candidate for bone repair.

Conflicts of interest The authors declare no conflict of interest.

Acknowledgements The grants were from the National Natural Science Foundation of China (81572194 and 81771990), Key Medical Program of Science and Technology Development of 28 ACS Paragon Plus Environment

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Shanghai (17441900600 and 17441902000) as well as by the Ministry of Education, Youth and Sports of the Czech Republic-Program NPU I (LO1504).

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For Table of Contents Use Only Microporous Coatings of PEKK/SN Composites Integration with PEKK Exhibiting Antibacterial and Osteogenic Activity, and Promotion of Osseointegration for Bone Substitutes Han Wua, Lili Yangb, Jun Qiana, Deqing Wanga, Yongkang Pana, Xuehong Wanga, Saha Nabanita*c, Tingting Tang*d, Jun Zhaoe, Jie Wei*a

Microporous coatings of PEKK/SN composites were created on PEKK surface through sulfonation reaction. The surface roughness, hydrophilicity, protein adsorption, cell responses and osteogenesis of the samples increased with the increase of SN content in the coatings. The microporous coating of PKSC10 with high SN content exhibited excellent biocompatibility, antibacterial and osteogenic activities.


SN Composites Integration with PEKK

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