dual ag/zno-decorated micro-/nanoporous sulfonated polyetheretherketone with superior...

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1800028 (1 of 12) © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Dual Ag/ZnO-Decorated Micro-/Nanoporous Sulfonated Polyetheretherketone with Superior Antibacterial Capability and Biocompatibility via Layer-by-Layer Self-Assembly Strategy

Yi Deng, Lei Yang, Xiaobing Huang, Junhong Chen, Xiuyuan Shi, Weizhong Yang,* Min Hong, Yuan Wang, Matthew S. Dargusch, and Zhi-Gang Chen*

DOI: 10.1002/mabi.201800028

1. Introduction

Orthopedic and dental implants have been extensively employed for curing irrepa-rable bone defects, resulting from dis-eases, congenital abnormalities, accidents, and military injuries.[1] Polyetheretherk-etone (PEEK), a semicrystalline polymer with ≈30–35% degree crystallinity, has become a popular candidate for replacing typical metallic implants made of cobalt-based alloys, titanium-based alloys, or stainless steels.[2] Unlike metallic bio-materials which display large Young’s modulus over 110 GPa, PEEK has an elastic modulus in the range of 4–5 GPa, comparable to human cortical bone’s (≈18 GPa), that mitigates the risk of bone absorption or osteanabrosis derived from stress shielding as a result of mismatched mechanical properties between bone and the implant.[3] In the view of the clinical processing, PEEK can be prepared using traditional plastic processing production, then disinfected repeatedly and heat-con-toured to fit the shape of bones because of its thermoplasticity. In addition, PEEK

Dental and Orthopaedic Implants

Polyetheretherketone is attractive for dental and orthopedic applications due to its mechanical attributes close to that of human bone; however, the lack of antibacterial capability and bioactivity of polyetheretherketone has substan-tially impeded its clinical applications. Here, a dual therapy implant coating is developed on the 3D micro-/nanoporous sulfonated polyetheretherketone via layer-by-layer self-assembly of Ag ions and Zn ions. Material characterization studies have indicated that nanoparticles consisting of elemental Ag and ZnO are uniformly incorporated on the porous sulfonated polyetheretherketone surface. The antibacterial assays demonstrate that Ag-decorated sulfonated polyetheretherketone and Ag/ZnO-codecorated sulfonated polyetherether-ketone effectively inhibit the reproduction of Gram-negative and Gram-positive bacteria. Owing to the coordination of micro-/nanoscale topological cues and Zn induction, the Ag/ZnO-codecorated sulfonated polyetheretherketone substrates are found to enhance biocompatibility (cell viability, spreading, and proliferation), and hasten osteodifferentiation and -maturation (alkaline phosphate activity (ALP) production, and osteogenesis-related genetic expres-sion), compared with the Ag-decorated sulfonated polyetheretherketone and the ZnO-decorated sulfonated polyetheretherketone counterparts. The dual therapy Ag/ZnO-codecorated sulfonated polyetheretherketone has an appealing bacteriostatic performance and osteogenic differentiation potential, showing great potential for dental and orthopedic implants.

Dr. Y. Deng, X. B. HuangSchool of Chemical Engineering Sichuan University Chengdu 610065, ChinaDr. Y. DengDepartment of Mechanical Engineering The University of Hong Kong 999077, Hong Kong, ChinaProf. L. Yang, J. H. Chen, X. Y. Shi, Prof. W. Z. YangSchool of Materials and Engineering Sichuan University Chengdu 610065, ChinaE-mail: [email protected]

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/mabi.201800028.

Dr. M. Hong, Y. Wang, Prof. Z.-G. ChenCentre for Future Materials University of Southern Queensland Springfield, Queensland 4300, AustraliaE-mail: [email protected]. M. S. Dargusch, Prof. Z.-G. ChenMaterials Engineering The University of Queensland Brisbane, Queensland 4072, AustraliaProf. M. S. DarguschCentre for Advanced Materials Processing and Manufacturing (AMPAM) School of Mechanical and Mining Engineering The University of Queensland Brisbane, Queensland 4072, Australia

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possesses good chemical/environmental resistance, nontox-icity, inherent radiolucency, and magnetic resonance imaging (MRI) compatibility.[4] Nevertheless, the bioinertness and poor antibacterial properties of PEEK have impeded its clinical appli-cations, although it received the approval from American Food and Drug Administration (FDA) in late 1980s.[4a]

Surface engineering is an efficient strategy to modify the biological and mechanical performance of materials while retaining the advantageous bulk properties of the mate-rials.[5] In order to improve osseointegration of the implants, hydroxyapatite (HA) or titanium dioxide (TiO2) have been deposited/coated onto the surface of PEEK via different approaches, such as simulated body fluid immersion,[6] cold spray technology,[7] sol–gel coating,[8] etc. Nonetheless, the inferior physical bonding between PEEK and coated HA or TiO2 commonly leads to some detrimental impact on the mechanical properties and a decreased fatigue limit, which eventually causes implant failure.[9] Another reason for implant failure is implant-associated infections (IAIs).[10] For example, the infection rate for orthopedic implants is as high as 3–5% on average every year.[10] IAIs delay bone reconstruction and repairing, increase patient morbidity, and pose high financial burdens (expenditures projected > $1.63 billion annually, by 2025 for Americans).[11] Once infec-tion occurs, planktonic bacteria first adhere to the implant surface, aggregate in the self-produced extracellular matrix substances, and ultimately evolve into biofilms, which are difficult to destroy using antibacterial agents.[12] Such infec-tions often lead to many undesirable complications such as destruction of the adjacent tissue, implant detachment, second surgery, and amputation, which may be related to extremely huge medical expenditures for patients and cause significant pain and suffer for the patients. Thus, it is urgent to restraint initial attachment of bacteria on the interface of PEEK implants through suitable surface engineering technology.

As a broad-spectrum germicidal drug, silver (Ag) and its ion has been widely employed to enhance the antimicrobial activity of implantable materials.[13] However, the antibacte-rial mechanisms associated with Ag species raises concerns about their biosafety. Excessive release of Ag+ from Ag nano-particles (AgNPs) dampens cell growth and causes serious side effects, such as biotoxicity and internal organ damage.[14] Therefore, some osteogenesis-promoting or cell prolifera-tion–promoting factors are coated/deposited onto implants to alleviate the cytotoxicity derived from Ag, with the purpose of obtaining both an antibacterial and cytocompatible implant interface. It is well-proved that Zn is a key trace element in bone tissue and plays various roles in biofunctions, including nucleic acid metabolism, DNA synthesis, enzymatic activity, hormonal production, and biomineralization[15] with certain antimicrobial feature.[16] Previous reports in the literatures have demonstrated that Zn-doped or Zn-decorated Ti was capable of suppressing the bacterial attachment and dramati-cally promoted the osteogenic commitment of stem cells in vitro as well as stimulated bone development/reconstruc-tion in vivo.[17] Nevertheless, only Zn and ZnO[18] do not have enough ability to kill all the bacteria on the biomaterial surface. Introduction of Ag to Zn surface can significantly

facilitate the bactericidal properties and retain the osteogenic activity of implants. Liu and co-workers fabricated Zn/Ag microgalvanic couples on titanium interfaces through plasma immersion ion implantation and found that the Zn/Ag dual implantation boosts the early adhesion, cell proliferation, osteodifferentiation, and gene expressions of mesenchymal stem cells derived from rat bone marrow (rBMSCs), while Zn/Ag-implanted titanium has good antimicrobial properties both in vitro and in vivo.[17b]

Sulfonation is a facile and effective way to generate a 3D porous and nanostructured network on PEEK, which enhances cell anchoring and osteogenic differentiation.[3a,19] The sul-fonated PEEK (SPEEK) treated by concentrated sulfuric acid has been shown to possess good resistance against Escherichia coli and Staphylococcus aureus.[19] Inspired from these considera-tions mentioned above, a novel type of Ag- and ZnO-codeco-rated hierarchical porous SPEEK implant has been constructed via the typical and simple layer-by-layer self-assembly, where alginate combined with Ag+ as negatively charged poly-electrolyte layer and chitosan with Zn2+ as positively charged polyelectrolyte layer. Polyanion alginate has high electro-static attraction toward Ag+ cations, and the negative groups including carboxyl (COO−) and carbonyl (CO) on chitosan display high chelating affinity toward Zn2+ ions. Alginate and chitosan, two natural polysaccharides, are selected in the pre-sent work because these materials have outstanding nonim-munogenicity, biodegradability, and good biocompatibility.[20] Simultaneously, alginate and chitosan facilitate cell growth and proliferation owing to the structure similar to glycosaminogly-cans in an extracellular matrix.[21] Dopamine (DA), a mussel-inspired structure, undergoes polymerization and adheres onto most materials’ surface without any surface pretreatment.[22] Chitosan can be tightly grafted to polydopamine through the amino reacting with the oxidized catechol groups via Schiff base reactions or Michael addition. To our best knowledge, our work is a pioneering study on the surface engineering of PEEK which combines sulfonation with dual nutrient element modi-fication, which simultaneously bestows both antibacterial prop-erties and osteogenesis-promoting abilities on bioinert PEEK implants. This study settles to: 1) develop and characterize the Ag/ZnO-codecorated porous SPEEK (Ag/Zn–SPEEK); 2) inves-tigate its bactericidal behaviors against Gram-negative bacterial and Gram-positive bacteria; 3) in vitro MG-63 cells’ response to the modified porous structure with different nutrient ele-ments (e.g., Ag, ZnO, and dual Ag/ZnO) regarding attachment, proliferation, alkaline phosphate activity (ALP) activity, and osteogenic gene expression Figure 1.

2. Results and Discussion

2.1. Morphology of Decorated Porous SPEEK

Figure 2a shows the typical scanning electron microscope (SEM) images of different Ag- or/and ZnO-modified and untreated SPEEK samples. As can be seen, PEEK after sulfonation is altered to forming a 3D network structure with intercon-nected micro- and nanopores, and the size of the micropores varies between 0.5 and 1.0 µm. The further cross-sectional




view of the porous SPEEK shows that the thickness of SPEEK is 1.64 ± 0.27 µm (Figure S1, Supporting Information). And, the elemental mapping profiles (Figure S2, Supporting Information) confirm that the coating contains C, O, Ag, and Zn. Although the amount of Ag and Zn is low but they have uniform distribution patterns. Besides, the results from scratch test show that the adhesion strength of Ag/Zn–SPEEK coating is about 36.7 ± 3.2 N, higher than that untreated SPEEK coating with the value of 29.1 ± 2.4 N, indicating that the decorated coat-ings adhere tightly onto the PEEK matrix. But, the layer-by-layer modification does not influence the porous structure of the layer. Homogeneous AgNPs with diameter of about 50–70 nm are distributed on both the surface and the inner wall of the pores in the Ag–SPEEK sample. By contrast, a large number of ZnO particles with sizes of 120–150 nm are observed for the Zn–SPEEK group. After dual decoration, AgNPs and ZnO particles coexist on the porous surface of Ag/Zn–SPEEK sub-strates. The large surface area of the implants and the depth of the pits (as antimicrobials reservoirs) can increase capacity for adsorbing metal ions. Alginate and chitosan macromole-cules adhere uniformly on both the surface and interior porous wall of the SPEEK coatings due to the inherent material-inde-pendent adhesion nature of dopamine, and their negative groups such as carboxyl (COO−) and carbonyl (CO) also have high chelating affinity toward Ag+ and Zn2+ cations. Afterward, the catechols on polydopamine coating reduce most of Ag+ to AgNPs. Nonetheless, the density of AgNPs is not enough. To thoroughly reduce Ag+ ions, especially in the holes, additional UV exposure (1 h) is employed. These cocontribute to densely surface-grafted and pore-embedded AgNPs in the meantime. In terms of Zn2+, part of Zn2+ ions are translated to ZnO in the presence of air and water environments, especially under ozone.

2.2. Chemical Composition of Decorated Porous SPEEK Coating

In order to probe the change of chemical constitutes after distinct stages of surface modification, the modified PEEK substrates were characterized using Fourier transform infrared with atten-uated total reflection (ATR-FTIR), contact angle goniometry, and X-ray photoelectron spectroscopy (XPS) spectra. ATR-FTIR analysis of these samples before and after modification is pre-sented in Figure 2b, the peak observed at 1652 cm−1 is ascribed to CO stretching vibration, and obvious peaks centered at 1592 and 1496 cm−1 originate from in-plane vibration of benzene. The absorption bands of CH vibration at about 837 and 768 cm−1 are tentatively corresponding to the divided bands of the out-of-plane bending vibration of benzene. These are all characteristic peaks of PEEK. After layer-by-layer self-assembly, however, the new bands at 1530 cm−1 are apparent, and they are attributed to the deformation vibration of amide II (δNH).[23] The char-acteristic broad bands of the hydroxy group (OH−) are detected at ≈3413 cm−1 corresponding to alginate and chitosan molecules.

The contact angle on a material has been widely employed to track the effectiveness of surface decoration protocols. Ini-tially, the pristine SPEEK substrate is relatively hydrophobic (83.75° ± 0.75°), in line with previous work.[3a] The contact angle on the porous SPEEK substrate after layer-by-layer assembly dramatically decrease by about 53°, as witnessed in Figure 2c, because of the hydrophilic groups (OH, NH2, NHCO) of chitosan and alginate on the surface of SPEEK. Although, there is no significant difference in contact angle between Ag–SPEEK and Zn–SPEEK, a slight increase in hydro-philicity has been observed for the Ag/ZnO-codecorated SPEEK substrate. It is widely accepted that the hydrophilic implant surface enables to promote the cell attachment and prolifera-tion through improving the adsorption of proteins.[24]


Figure 1. Schematic drawing of the preparation of Ag/ZnO dual-decorated micro-/nanoporous SPEEK, and its bactericidal effect and osteogenic activity.



To cross-check the results obtained from FTIR and con-tact angle, we conducted XPS technology to offer more proofs of chemical constitutes and states (Figure 3 and Table S2 and Figure S3 (Supporting Information)). In the wide scan spectrum of the bare SPEEK substrate, C and O elements are the domi-nating components with a handful of nitrogen and sulfur ele-ments. Successful anchoring of chitosan and alginate is revealed by an enhancement in O 1s and N 1s amount and a corre-sponding reduction in the C 1s amount, as shown in Table S2 (Supporting Information). Meanwhile, the weak peak of Na 1s is caused by the contamination from sodium chloride (NaCl) solu-tion used in the sample preparation process. After modification, in the wide-scan spectra, Ag or Zn signal appears on the surface of Ag–SPEEK or Zn–SPEEK, respectively. Notably, sliver and zinc peaks coexist for Ag/Zn–SPEEK samples, indicating the successful immobilization of Ag and ZnO on the hierarchically porous SPEEK coating, although the contents of the two metal elements are lower than those in Ag–SPEEK and Zn–SPEEK samples. Figure 3b depicts XPS deconvolution spectra of Ag 3d and Zn 2p for Ag/Zn–SPEEK substrate surface. The 6 eV gap in

the binding energies of Ag 3d3/2 and Ag 3d5/2 appear at 373.9 and 367.7 eV, respectively, which are between the binding energy of Ag metal (374.2 eV for Ag 3d3/2, and 368.2 eV for Ag 3d5/2) and that of Ag(I) oxide (373.5 eV for Ag 3d3/2, and 367.5 eV for Ag 3d3/2).[25] The fraction of silver oxide incorporated in the coatings is about 8.2%. The generation of AgNPs from the reduction of Ag+ to Ag0 is verified. The Zn 2p peaks are well-fitted at 1021.9 and 1045.2 eV, and assigned to the Zn 2p in ZnO groups, sug-gesting the presence of ZnO on the surface.[26] Moreover, an obvious alteration in the C bond composition found in the high-resolution C 1s supports the conclusion. The C 1s spectrum of pristine SPEEK is deconvoluted into three curves. The binding energies at 284.8, 286.3, and 291.8 eV can be associated with the CC/CH, COH, and C(O)O bonds. After layer-by-layer self-assembly, two bands of CN and CO at about 285.1 and 288.2 eV are observed on functionalized substrates, demonstrating the presence of chitosan and alginate. Besides, as shown in Figure 3c, the intensity of the CC/CH decreases significantly accompanied by an increase of the peaks of the CO and COH groups, compared with the pristine


Figure 2. a) SEM images of SPEEK, Ag-SPEEK, Zn-SPEEK, and Ag/Zn-SPEEK. b) ATR-FTIR spectra of the pure SPEEK and Ag/Zn-SPEEK substrates. c) Contact angle and corresponding photos of droplets on various porous SPEEK.



SPEEK substrate. There are similar contents of CC/CH, COCOH, and CN bands for three modified SPEEK sub-strates, suggesting that the same amounts of macromolecules are contained on the sur-face of the Ag–SPEEK, Zn–SPEEK, and Ag/Zn–SPEEK samples. Additionally, through fitting the distribution of the high-resolution O 1s spectra of various samples, two peaks are found for the pristine SPEEK, and three peaks are resolved for the modified SPEEK substrates after layer-by-layer self-assembly (Figure S3, Supporting Information). The peaks centered at 531.4 and 533.3 eV are attributed to the bond of CO, COH, or COC of bio-macromolecule coatings, respectively. Other peaks in the modified SPEEK at 532.1–532.4 eV should be ascribed to the metal–O interaction including Ag–O and Zn–O. Hence, the interaction between oxygen and Ag or Zn have been further demonstrated.

2.3. Ion Release from Scaffolds

Because the released ion content from scaf-folds is important for subsequent cell and bacteria responses, the liberation of Ag+ and Zn2+ ions was investigated. The total loading amounts of ions for different deco-rated scaffolds are shown in Figure S4a (Supporting Information). The total amount of Ag in Ag–SPEEK and the total amount of Zn is Zn–SPEEK are slightly higher than those in Ag/Zn–SPEEK substrates (Ag: 42.82 ± 7.08 µg per scaffold; Zn: 29.38 ± 3.22 µg per scaffold). With respect to Ag ion, about 27.04% of Ag is leaked into phosphate buffered saline (PBS) (3.86 ± 0.09 µg mL−1) in the initial 24 h. The value gradually grows to 11.25 ± 0.31 µg mL−1 (78.82% of total) upon 5 days immersion. The liberation rate slows further from 5 to 10 days, but there is still 3.41% of remnant. As for Zn ion, about 62.36% (9.16 ± 0.26 µg mL−1) of Zn is leached at 5th day of immer-sion and finally still 7.76% of remnant after 10 days. The profiles also show that the delivery of Ag from Ag–SPEEK and the delivery of Zn from Zn–SPEEK are always lower than those from Ag/Zn–SPEEK, revealing better durability.

2.4. Antibacterial Activity

The attachment of bacteria to implant surfaces is the initial and prerequisite step for biofilm formation, that plays


Figure 3. XPS spectra analysis: a) XPS wide spectra; b) high-resolution spectra of Ag 3d and Zn 2p; c) high-resolution spectra of carbon peaks (C 1s) for pristine (c1) SPEEK, (c2) Ag–SPEEK, (c3) Zn–SPEEK, and (c4) Ag/Zn–SPEEK.



a vital role in the pathogenesis of infection.[27] The pre-vention of bacterial attachment and growth to implant-able materials, during the early postimplantation period, is effective to long-term success of an implant.[28] Thus, the wide range of antibacterial action of the modified SPEEK surfaces toward Gram-negative E. coli and Gram-positive S. aureus, the common bacteria cells during implant infection, was assessed on the samples for 24 h in 3 h intervals. As shown in Figure 4a,b, untreated SPEEK has no negative effect on the reproduction of E. coli and S. aureus, and the amount of bac-teria increases with the extension of time. Nevertheless, there is a little decline in the amount of adherent bacteria on the Zn–SPEEK surfaces for two bacteria, indicating that ZnO does not have a notably positive antimicrobial performance toward Gram-positive bacteria and Gram-negative bacteria. Noticeably, both the Ag–SPEEK and dual Ag/Zn–SPEEK substrates inhibit the proliferation of E. coli and S. aureus. For example, after 24 h contact with E. coli, Ag–SPEEK and Ag/Zn–SPEEK groups show more than a 99.2% decrease in bacterial attachment than the bare SPEEK. In the case of S. aureus, Ag/Zn–SPEEK sam-ples display similar efficacy (around 99.3%) in reducing the amount of bacteria. These demonstrate that Ag/Zn–SPEEK samples exhibit prominent antibacterial effects because of the combined effect of the liberation of Ag+ and the existence of ZnO.

In order to investigate whether the diffusion of the ions sterilizes the bacteria, the agar diffusion assay was utilized. As shown in Figure 4c,d and Table S3 (Supporting Information), as for E. coli, Ag–SPEEK, Zn–SPEEK, and Ag/Zn–SPEEK sam-ples have an inhibition zone, but the sizes of the inhibition zones around Zn–SPEEK (5.36 ± 0.22 µm) and Ag/Zn–SPEEK (5.91 ± 0.19 µm) are significantly smaller than that around the Ag–SPEEK (8.42 ± 0.34 µm). This suggests that compared with Ag/Zn–SPEEK, the Ag–SPEEK substrate is much supe-rior in antibacterial properties on E. coli. Differing from the phenomena of E. coli, only Ag-containing SPEEK shows inhi-bition zone toward S. aureus, and there are no distinct bacte-rial growth inhibition zones around the pristine SPEEK and Zn–SPEEK disks, providing further evidence for the absence of obvious antibacterial effects on S. aureus, consistent with the results of the bactericidal curves presented in Figure 4b. This is possibly because that the Zn2+ ions have hardly liber-ated from ZnO surface, resulting in inferior antibacteria. The size of the inhibition zone around Ag/Zn–SPEEK substrate for S. aureus is 10.37 ± 0.38 µm, similar to that around Ag–SPEEK (10.43 ± 0.21 µm), indicating that the incorporation of Zn has little negative impact on germicidal action of Ag ions.

The morphology and membrane integrity of S. aureus and E. coli are presented in Figure 5. Clearly, the E. coli on pure SPEEK has an integrated rod shape, whereas the number of


Figure 4. Bactericidal curves against a) E. coli and b) S. aureus on SPEEK, Ag–SPEEK, Zn–SPEEK, and Ag/Zn–SPEEK substrates. Inhibition zones around the different SPEEK samples against c) E. coli and d) S. aureus with red circles showing the inhibition zones.



bacteria on Zn–SPEEK is lower than that on SPEEK. A mass of bacterial cell debris are detected on Ag–SPEEK sample, and bacteria with a normal shape are difficult to find. Intrigu-ingly, the bacteria cells on Zn/Ag–SPEEK appear severed and distorted, demonstrating that Zn/Ag–SPEEK display good antibacterial property toward E. coli. Similarly, the S. aureus possesses a spherical shape with a smooth and intact surface on the pristine SPEEK, but irregular shape and shrinking/corrugated membrane on the Zn–SPEEK groups. Though the shapes of a part of bacteria are found on Zn–SPEEK, the membrane is not as smooth as that on the bare SPEEK, and small cells are detected, indicating that ZnO can suppress the growth of bacteria via contact killing mode. However, no micro-bial cells are detected for Ag–SPEEK. The high magnification images provide more details about the bacterial morphology and membrane. Completely lysed bacteria and cell debris are observed on the surface of Zn/Ag–SPEEK substrates. Unlike E. coli, SEM reveals that there are many S. aureus cells trapped in the 3D micropores. S. aureus is spherical with a size of about 0.5 mm, but E. coli is a lanky rod with a size of about 1 mm.[17b] The elongated shape impedes the capability of the 3D structure to trap E. coli, but the spherical shape of S. aureus enables them to be more easily trapped. Our results reveal that the death of S. aureus and E. coli is partly because of the disruption of the membrane integrity and lysis of bacteria cells.

Because of the porous structure (natural reservoir) and the metal-binding carboxyl groups (regulated release), Ag+ amounts both on the surface and in the micro-/nanopores should be much more than that for the planar counterparts. Most planktonic pioneers are first dispelled and eliminated via surrounding ionized Ag+ (called “release killing”) on the Ag-incorporated and Ag/ZnO-incorporated substrates. Some settled cells touch the nano-Ag-containing interface directly, and underwent membrane injury through Ag per se through multiple antibacterial actions, including 1) protein denatura-tion, 2) disruption of protons circulation and metabolism, 3) reactive oxygen species (ROS) overproduction, 4) charge transfer, and 5) inhibition of DNA synthesis (i.e., “contact killing”).[13b,29] Besides, chitosan is a natural bio-macromolecule which displays good antibacterial action against Gram-negative bacteria and Gram-positive bacteria.[30] The main antibacterial mechanism of chitosan is that positively charged polyelectrolyte chitosan interacts with anionic groups on bacterial surface, thus leading to enhancing membrane permeability and probably its disruption and subsequent leakage of cell proteins.[31] Chitosan toxicity against bacteria has been proved to be dependent on the molecular weight and degree of deacetylation. Here, these anti-bacterial modes from the chitosan in conjunction with silver synergistically work together to affect the antibacterial proper-ties of the coatings, ultimately killing bacteria. The presence of Zn elements enhances the bactericidal ability through long-range interactions, because the liberation of Zn2+ and locally ionized Ag+ occur simultaneously on the Zn/Ag-codecorated SPEEK surface. Moreover, the presence of Ag ion boosts the germicidal properties of Zn ions due to much stronger steri-lization capability for Ag ions than that for Zn ions. The Zn2+ ions delivered to the surrounding play a long-range role in antibacterial ability, while these embedded AgNPs play a short-range role synergistically. Therefore, the synergistic effect of the

long-range and short-range interplays boosts the antibacterial action of dual Ag/Zn-codecorated SPEEK substrates.

2.5. In Vitro Cytocompatibility Evaluation

The toxicity of the dual nutrient element–modified biomaterials to human osteoblast-like MG-63 cells should be assessed when the novel PEEK is considered for dental/bone implants. Figure 6 shows the time-dependent proliferation of MG-63 on the micro-nanoporous SPEEK substrate before and after layer-by-layer self-assembly via cell counting kit-8 (CCK-8) kit. The optical density (OD) value increases with the extension of time when MG-63 cells are cultured on pure PEEK, Zn–SPEEK, and Ag/Zn–SPEEK groups. However, the cells on Ag–SPEEK show the lowest prolif-eration among groups, and OD value decreases after 5 days cul-ture, indicating that Ag ions are adverse to cell proliferation. It is


Figure 5. SEM observation of the amount and morphology of the a) E. coli and b) S. aureus after culturing on the bare SEEK and modifed SPEEK surfaces. Red arrows point to the cut and distorted E. coli cells. Green arrows point to the disintegrating S. aureus cell debris.



apparent that more cells are detected on Zn–SPEEK substrate than that on Ag–SPEEK and Ag/Zn–SPEEK groups at 1 and 3 days. It is reported that zinc (Zn), an important trace element required for bone healing, can significantly simulate cell prolif-eration at low zinc concentration (1 × 10−6–3 × 10−6 m) in vitro.[32] Interestingly, Ag/Zn–SPEEK substrate displays higher cell via-bility than Zn–SPEEK, indicating that dually decorated substrates are beneficial to the proliferation/growth of MG-63 cells with no biotoxicity. With regard to Ag nanomaterials, an emerging topic is their potential cytotoxicity to eukaryotic cells. In a large extent, it is based on the fact that mobile particles can be internalized.[33] Freestanding AgNPs are very reactive, and they are free from oxi-dization and stable when being exposed to water, thus generating persistent, high Ag+ environment that is toxic to cells. Yet on-the-surface AgNPs (in appropriate content) with confined movability on surfaces or immobilized within nanostructures display tai-lored, slow liberation profiles, which might be biocompatible in vitro and in vivo.[13b,34] Besides, the presence of Zn on implants can alleviate the cytotoxicity from Ag, rendering implant interface both antibacterial and cytocompatible properties.

An overview of the cellular morphologies of MG-63 on the various functionalized SPEEK substrates for 5 days is presented in Figure 7. Cells on pristine SPEEKs are sparsely distributed but spread well. But, cells on Ag–SPEEKs substrates are less spread out and display a round shape, indicating that Ag-containing surfaces are negative for cell spreading. In particular, cells on dual Ag/ZnO-decorated surfaces proliferate well and spread out with a healthy fusiform osteoblastic shape. Cells also display many elongated and overlapped lamellipodia on whole surface, covered with ruffling peripheral cytoplasm. Cells are already confluent at 3 days, and they adhere to the micro-/nanoporous surfaces tightly. On the contrary, those on the Zn–SPEEK sub-strate are poor in terms of morphology and quantity. Then, the ability of MG-63 cells to develop cytoskeleton was examined, as shown in Figure 8. After 3 days culture, nearly no stress fibers are formed through F-actin for the Ag–SPEEK samples, com-pared with other substrates. Fluorescence images indicate that the MG-63 cells display filamentous morphology on both the

bare SPEEK and Zn–SPEEK substrates with limited spread, and F-actin is poorly developed. Nevertheless, many MG-63 cells adhere and spread onto Ag/Zn–SPEEK samples. Besides, with improved cell adhesion, the cells cultivated on the Ag/Zn–SPEEK group are associated with well spread out, more adhered filopodia, and more visible presentation of F-actin intracellular stress nanofibers, implying that the Ag/Zn–SPEEK has a good in vitro cytocompatibility. These results confirm that the coincor-poration of Ag and Zn ions on the porous surfaces can enhance cell growth and spreading, and highly improve the bioactivity of PEEK-based materials.

2.6. Osteogenic Potential

2.6.1. ALP Activity

It is well-established that the chemical and physical cues from materials’ composition and morphology enable to regulate and steer cell fates.[35] Hence, cell differentiation (ALP activity) was investigated on different porous SPEEK substrates after 7 days. Because alkaline phosphate is expressed in large con-tents in the differentiated phase during bone cell development, the assay enables to show early osteogenic phenotypic expres-sions.[36] From Figure 9, higher ALP activities are detected for MG-63 cultured on the Zn–SPEEK than the pristine SPEEK, suggesting that Zn elements can induce the osteogenic activity of cells, consistent with Cai and co-workers’ research in which Zn-incorporated coatings with the best Zn contents could sig-nificantly improve osteoblasts’ proliferation/differentiation and the in vivo osseointegration.[17a] Besides, Ag–SPEEK shows the lowest ALP activity among the groups, since high Ag ion con-centration liberated from Ag–SPEEK inhibits cell growth and causes the death of cells, as mentioned in CCK-8 results. Ag/Zn–SPEEK substrate has the largest ALP expression, and the order of obtained porous SPEEK surface promoting differ-entiation of MG-63 cells is as follows: Ag/Zn–SPEEK > Zn–SPEEK > SPEEK > Ag–SPEEK. These results indicate that both


Figure 6. Proliferation of MG-63 on various SPEEK substrates surfaces for 1, 3, 5 days. * represents p < 0.05, # represents p < 0.05 compared with other groups, and ## represents p < 0.01 compared with other groups.

Figure 7. Typical SEM images of MG-63 on the pure SPEEK, Ag–SPEEK, Zn–SPEEK, Ag/Zn–SPEEK substrate surfaces after 5 days culturing. Red arrows point to pseudopodia.



micro-/nanoporous structure and Zn ions cocontribute to the enhanced osteogenic differentiation of the SPEEK implant.

2.6.2. Real-Time Polymerase Chain Reaction

We further evaluated ALP, runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and collagen type I alpha 1 (Col1a1) expressions on different porous SPEEK samples at day 14. The fold change in ALP production in cells on Zn–SPEEK samples is greater than that in the pristine SPEEK group (Figure 10a). Clearly, the cells on Ag/Zn–SPEEK show stronger messenger ribonucleic acid (mRNA) expression of Runx2 compared with that on Ag–SPEEK substrates. Runx2 is a tran-scription factor that regulates the transcription of osteo-related genes.[37] OCN and Col1a1 are markers of the mature osteoblast and production of the organic bone matrix, respectively, both of which play key roles in the process of bone mineralization.[1b,38] Runx2 and OCN expressions are closely related to that of ALP in the bare SPEEK, Ag–SPEEK, Zn–SPEEK, Ag/Zn–SPEEK groups, although the Col1a1 expression shows no statistical difference between SPEEK and Zn–SPEEK substrates. Cells on the Ag–SPEEK group express the lowest osteogenesis-related genes due to the fewest cell numbers. While, the Ag/Zn–SPEEK surface displays the highest ALP, Runx-2, Col1a1, and

OCN genes among the groups, demonstrating that the Ag and Zn ions have a synergism in accelerating the osteogenic differ-entiation potential of PEEK biomaterials at a molecular level. Previous studies have reported that the hierarchically porous structure and Zn ions in low appropriate concentration (such as <10 ppm) have been found to be factors that trigger osteoblast functions and bone reconstruction. Though high concentration of Ag+ ions leads to toxicity,[39] Ag is safe and cytocompatible at low concentration.[35] Therefore, the enhanced MG-63 cell functions of the Ag/ZnO-incorporated coatings might originate from the synergetic benefits of released Zn ions and micro-/nanoporous topology on the surfaces.

3. Conclusion

In this study, three types of nutrient element–incorporated SPEEK coatings (Ag–SPEEK, Zn–SPEEK, and Ag/Zn–SPEEK) were prepared by a combination of sulfonation processes and layer-by-layer self-assembly strategy. The porous and nanostruc-tured SPEEK coatings containing Ag and/or ZnO have been formed on a PEEK substrate surface. The results from these antibacterial experiments indicate that the Ag-incorporated and Ag/ZnO-incorporated SPEEK coatings can greatly suppress the growth of E. coli and S. aureus with a bactericide rate up to 99%, while Ag–SPEEK presents serious cytotoxicity. In vitro biocom-patibility assessment demonstrates that the adhesion, prolifera-tion, and spreading of MG-63 on the Ag/Zn–SPEEK surface are significantly enhanced compared with single ion immobilized SPEEK (Ag–SPEEK and Zn–SPEEK). Moreover, osteoblasts express higher level of ALP activity and osteogenesis-related genes on the Ag/ZnO dual-incorporated SPEEK coatings. The better antibacterial activity, cytocompatibility, and the capa-bility to boost osteoblastic differentiation of the Ag/Zn–SPEEK implant might be ascribed to the synergistic effect of ion induc-tion and a 3D porous topological structure. Our study indicates that the innovative Ag/ZnO-incorporated SPEEK is a promising


Figure 8. Attached morphologies and F-actin cytoskeletal organization (rhodamine-labeled phalloidin, counterstained with DAPI for nuclei) of cells on different porous SPEEK substrates.

Figure 9. Alkaline phosphatase activity of MG-63 cells on these different micro-/nanoporous SPEEK for 7 days. * represents p < 0.05 between groups, ## represents p < 0.01 compared with other groups.




candidate for dental and orthopedic implants with excellent bactericidal capability and biocompatibility.

4. Experimental Section

4.1. Materials

Medical grade PEEK discs (450G) were obtained from Victrex (Thornton Cleveleys, UK). Chitosan (molecular weight = 310–375 kDa with a degree of deacetylation greater than 90%) and sodium alginate (viscosity = 200 ± 30 mPa s) were obtained from Aladdin (Shanghai, China), and dopamine hydrochloride was supplied by Sigma-Aldrich (China). Tris[hydroxymethyl]-aminomethane (Tris-HCl), NaCl, concentrated sulfuric acid (H2SO4), acetic acid, silver nitrate (AgNO3), and zinc nitrate (Zn(NO3)2) were all provided from Chengdu KeLong Reagent (Chengdu, China).

4.2. Preparation of Porous SPEEK

PEEK disks with dimensions of Φ 8.5 × 2 mm3 were fabricated for material characterization, and bacteria/cell experiments in 48-well cell culture dishes. All samples were polished with a series of abrasive SiC papers. They then were rinsed in D.I.: double distilled water, and dried at 50 °C for 24 h. Sulfonation was conducted in concentrated sulfuric acid (96–98 wt%) at ambient temperature for 5 min. To attain the uniform porous structure, supersonic stirring was used throughout the entire process. The samples were subsequently taken out, and rinsed in D.I. water and acetone repeatedly at least three times using ultrasonic treatment to remove the redundant sulfuric acid. The porous SPEEK dishes were dried at room temperature for subsequent modification.

4.3. Preparation of Ag/ZnO-Codecorated Porous SPEEK

The biocompatible and bactericidal multilayers were built on the porous SPEEK using electrostatic interactions between the chitosan and

alginate polyelectrolytes via the layer-by-layer self-assembly approach. Zn ion–containing chitosan solution was prepared through dissolving 20 mg chitosan in 8 mL of 10 × 10−3 m Zn(NO3)2 solution, and Ag ion–containing sodium alginate solution was prepared through dissolving 10 mg sodium alginate in 8 mL of 10 × 10−3 m AgNO3 solution. The porous SPEEK samples were soaked in a dopamine solution (2 × 10−3 m in 10 × 10−3 m Tris-HCl, pH = 8.5) overnight. After being rinsed with D.I. water for three times, the samples were immersed in Zn ion–containing chitosan solution for 30 min, and then rinsed for three times in 0.15 mol L−1 NaCl solution. Next, the substrate was immersed in Ag ion–containing sodium alginate solution for 30 min and then also rinsed with NaCl solution. These dipping cycles corresponded to the deposition of one bilayer, and 10 cycles were repeated. The resulting dually decorated samples after multilayer assembly were dried under a gentle stream of nitrogen gas, before exposure to 1 h UV/ozone (λ = 254 nm, 3.5 W m−2) to further reduce metallic nanoparticles, which were denoted to Ag/Zn–SPEEK. Figure 1 shows the preparation of dual Ag/ZnO-codecorated porous SPEEK coating. Ag-modified porous SPEEK substrates (Ag–SPEEK) were prepared through assembling pure chitosan with Ag ion–containing sodium alginate, and ZnO-modified porous SPEEK substrates (Zn–SPEEK) were also prepared by assembling Zn ion–containing chitosan with pure sodium alginate using the same approach as control groups in this work.

4.4. Material Characterization

Surface morphologies of different SPEEK samples were tested using the field emission SEM (FE-SEM, JSM-6701F, JEOL, Japan). Before SEM observation, samples were precoated with Au for 60 s. ATR-FTIR spectra (Magna-IR 750, Nicolet, USA) were recorded to analyze the functional groups of the pristine SPEEK and Ag/Zn–SPEEK from 670 to 4000 cm−1.

The hydrophilicity on surfaces was investigated using a contact angle goniometry apparatus (SL200B, Kino, USA) with 10 µL of droplets. Six samples were done to show the average value. The variation of chemical constituents and elemental states of these samples were probed using XPS (Kratos Analytical Ltd., UK) for wide and high-resolution spectra.

Figure 10. RT-PCR analysis of osteogenic gene expressions including(a) ALP, b) Runx2, c) Col1a1, d) OCN of MG-63 incubated with pristine and func-tionalized SPEEK substrates.




4.5. Adhesion Strength Tests of Coatings

The adhesion force between the decorated coatings and PEEK samples was assessed using the Auto Scratch Coating Tester according to the earlier description.[40] The critical load was defined as the smallest load at which a recognizable failure occurs. The failure force was determined from the load versus acoustic output characteristics.

4.6. Ion Release Measurement

In order to study the liberation behavior of Ag and Zn ions from the coatings, substrates (n = 3) were soaked in PBS solution at 37 °C under static conditions for 1, 3, 5, 7, 10 days. The leaching medium was collected at the expected time, and new PBS was refilled accordingly. Analysis was carried out through an inductively coupled plasma mass spectrometry (ICP-MS, Leeman, USA). Besides, the total Ag and Zn contents were also tested by immersing in HNO3 (n = 3).

4.7. Antibacterial Kinetic Tests

The antibacterial capability of these substrates was evaluated using E. coli (ATCC, 25923) and S. aureus (ATCC, 25922), which were cultivated in liquid Luria-Bertani (LB) medium (containing 10 g L−1 sodium chloride, 10 g L−1 peptone, 5 g L−1 yeast extract). After 24 h of culture at 37 °C, respective colony-forming unit (CFU) counts were determined from 600 nm wavelength absorbance comparing with the previously established standard curve, and the concentrations of E. coli and S. aureus in broth were adjusted to a concentration of 106 CFU mL−1. For the bacterial adhesion and reproduction test, various decorated PEEK samples were disinfected under UV irradiation for 0.5 h each side, then put into a 48-well cell culture dishes (Corning) and covered with 20 µL bacterial suspension and 1 mL LB medium. All samples were incubated at 37 °C. After culturing for desired time periods, the absorbance value (OD value) was recorded at 600 nm through a microplate reader (SAF-680T, BAIIU, Shanghai). Six specimens were done for each sample at each incubation time in order to improve statistics associated with the collected data.

4.8. Bacterial Growth Inhibition Zone Tests

The agar diffusion tests were used to identify whether ion diffusion causes bacteria sterilization. Four types of samples (SPEEK, Ag–SPEEK, Zn–SPEEK, and Ag/Zn–SPEEK) were placed at the center surface of the standard agar LB medium, which uniformly coated 100 µL of E. coli or S. aureus suspension liquid at a concentration of 106 CFU mL−1. After culturing for 1 day, we observed the bacterial growth inhibition zones, and then the size of the inhibition zone was evaluated.

4.9. SEM Characterizations of Bacteria

Samples for morphological observation of bacterial were put in 500 µL suspension with two kinds of bacteria (E. coli and S. aureus) at a concentration of 106 CFU mL−1 and cultivated at 37 °C for 1 day. Afterward, substrates were washed using PBS three times and fixed using 2.5% glutaraldehyde solution for 2 h. The samples were serially dehydrated using ethanol solutions with different concentrations (20%, 60%, 80%, 90%, 95%, and 100%) for 15 min each and dried. The morphologies of the bacteria cells on the treated substrates were analyzed by FE-SEM (JSM-6701F).

4.10. Cell Culture

Human osteoblast-like MG-63 cell line obtained from ATCC was employed for cytocompatibility tests. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (KeyGEN BioTECH, Nanjing, China) supplemented with 10% fetal bovine serum (Gibco, USA), 1% penicillin–streptomycin (KeyGEN BioTECH), and then incubated under a standard cell culture atmosphere of 5% CO2 at 37 °C. The growth medium was refreshed every 2–3 days.

4.11. Cell Viability Tests

Viability of MG-63 was tested by a CCK-8 assay (Dojindo, Japan). After counting, cells were reseeded onto distinct modified PEEK substrates at a density of 2 × 104 per well. After incubating for 1, 3, and 5 days, CCK-8 kit was introduced into each well at a ratio of 1:10 for 2 h incubation in the dark place. Then, 100 µL of supernatant of each sample was transferred to fresh 96-well dishes. The OD value was measured at 450 nm with the microplate reader (SAF-680T). Six substrates were done for each incubation period to obtain the statistic.

4.12. SEM and Cytoskeletal Characterizations of Cells

The cellular morphologies cultured on materials were observed by a FE-SEM. After culturing of 3 days, all samples with cells were taken out, washed with PBS, and fixed in 2.5% w/v glutaraldehyde solution, followed by dehydration with ethanol solutions (15 min for each step). Dehydrated samples were dried before sputter coating with Au SEM observation (JSM-6701F).

After 48 h incubation on different substrates, MG-63 cells were rinsed with PBS and fixed with 4% w/v paraformaldehyde for 15 min. Then, cells were permeabilized using 0.1% Triton X-100 (Sigma-Aldrich) for 5 min, before 0.5% bovine serum albumin (BSA)/PBS incubation at 37 °C for 0.5 h to block nonspecific binding, followed by adding 5 µg mL−1 fluorescein isothiocyanate (FITC)-labeled phalloidin to stain MG-63 for 0.5 h at ambient temperature. After washing with PBS, specimens were reacted for 10 min with 10 µg mL−1 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). The stained cells on substrates were thoroughly washed and captured under a confocal laser scanning microscopy (CLSM, LSM510, Carl Zeiss, Germany).

4.13. Alkaline Phosphate Activity Assay

Intracellular release of ALP activity was determined at the 7th day through an ALP assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) based on the manufacture’s instruction. To normalize, the total protein content was measured by a Bicinchoninic Acid (BCA) protein assay kit (KeyGEN BioTECH). At last, ALP activity was expressed as the total protein content (U g−1 prot). Six samples were repeated for every group and tested time.

4.14. RNA Extraction and RT-PCR

At 14 days, the total mRNA was isolated by TRIzol (Invitrogen, USA) treatment and converted into complementary deoxyribonucleic acid (cDNA) by a RevertAid First Strand cDNA Synthesis Kit (Thermo, USA) following the manufacturer’s protocol. RT-PCR detection was conducted on an ABI 7500 Real-time PCR machine (Applied Biosystems, USA) with SYBR Green (Roche, USA). Each sample was run in triplicate and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was assigned as the endogenous control. Primers (5′–3′) are provided in Table S1 (Supporting Information). The thermal process of the RT-PCR was 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The mRNA folds were calculated through the standard ΔΔCt method.

4.15. Statistical Analysis

All experiments were performed in triplicate, and the data were presented as mean ± standard deviation. Statistical analysis was conducted using SPSS 17.0 software.

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.




AcknowledgementsThe work was financially supported by the Sichuan Science and Technology Program (2017FZ0046, 2018JZ2006), the China Postdoctoral Science foundation (2017M610600), the Fundamental Research Funds for the Central Universities (Full-time Postdoctoral Research Funding of SCU, 2017SCU12016), the Hong Kong Scholars Program, and the Australian Research Council. Z.-G.C. sincerely thanks the USQ Start-up Grant and Strategic Research Funds. M.S.D. would like to acknowledge the support of the ARC Research Hub for Advanced Manufacturing of Medical Devices. The authors would also like to thank Chenghui Li and Hui Wang (Analytical & Testing Center, SCU) for their help in CLSM and SEM observations, respectively.

Conflict of InterestThe authors declare no conflict of interest.

KeywordsAg, antibacterial, cytocompatibility, dental and orthopedic implants, polyetheretherketone, ZnO

Received: January 19, 2018Revised: April 18, 2018

Published online:

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