Abstract
Objectve
We aimed to investigate the cytotoxicity and antibacterial properties of nano-silver-coated polyetheretherketone (PEEK) produced through magnetron sputtering and provide a theoretical basis for its use in clinical applications.
Methods
The surfaces of PEEKs were coated with nano-silver at varying thicknesses (3, 6, 9, and 12 nm) through magnetron sputtering technology. The resulting coated PEEK samples were classified into the following groups according to the thickness of the nano-silver coating: PEEK-3 (3 nm), PEEK-6 (6 nm), PEEK-9 (9 nm), PEEK-12 (12 nm), and PEEK control group. The surface microstructure and composition of each sample were observed by scanning electron microscopy (SEM), atomic force microscopy (AFM), and energy dispersive spectrum (EDS) analysis. The water contact angle of each sample was then measured by contact angle meters. A cell counting kit (CCK-8) was used to analyze the cytotoxicity of the mouse fibroblast cells (L929) in the coated groups (n = 5) and group test samples (n = 6), negative control (polyethylene, PE) (n = 6), and positive control group (phenol) (n = 6). The antibacterial properties of the samples were tested by co-culturing Streptococcus mutans and Straphylococcus aureus . The bacteria that adhered to the surface of samples were observed by SEM. The antibacterial adhesion ability of each sample was then evaluated.
Results
SEM and AFM analysis results showed that the surfaces of control group samples were smooth but compact. Homogeneous silver nano-particles (AgNPs) and nano-silver coating were uniformly distributed on the surface of the coated group samples. Compared with the control samples, the nano-silver coated samples had a significant increase in surface roughness (P < 0.05) as the thickness of their nano-silver coating increased. EDS analysis showed that not only C and O but also Ag were present on the surface of the coated samples. Moreover, the water contact angle of modified samples significantly increased after nano-silver coating modification (P < 0.01). CCK-8 cytotoxicity test results showed that coated samples did not exhibit cytotoxicity. The antibacterial experimental results showed that the nano-silver coating can significantly improve the antibacterial activity and bacterial adhesion ability of the PEEK samples.
Significance
The compact and homogeneous nano-silver coating was successfully prepared on the surface of PEEK through magnetron sputtering. The nano-silver coated PEEKs demonstrated enhanced antibacterial activities and bacterial adhesion abilities and had no cytotoxic effects.
1
Introduction
Owing to the increase in the aging population, the number of patients with dentition defect or loss is increasing annually. In addition, cases of bone defects caused by trauma, tumor resection, or infection has increased recently. Consequently, the demand for oral implant materials increases. At present, titanium and titanium alloys are the most commonly used medical implant materials especially in dental implants and fixation materials for bone defects and fracture splints, because they demonstrate good clinical effect. However, the mismatch between the elastic modulus of the titanium implant materials and human bone tissues produces a “stress shielding effect” after implantation , and thereby leading to implant loosening and even implantation failure. Moreover, when used as a dental implant, titanium metal exhibits changes in its appearance because it is readily exposed and causes allergic reactions in some patients . Therefore, for the resolution of these problems and increase of the success rate of implantation surgery, exploring new implant materials is extremely important.
As a member of the poly (aryl ether ketone) family, polyetherether ketone (PEEK) is a special thermoplastic polymer because it exhibits good mechanical properties, such as high strength, high rigidity, corrosion resistance, hydrolysis resistance, and good biocompatibility . As a medical implant material exhibiting excellent performance, PEEK has been approved by the US Food and Drug Administration (FDA). Compared with titanium, PEEK has an elastic modulus of 3–4 GPa, which is approximate to that of human cortical bone. Furthermore, PEEK can be retained during clinical examination (such as X-ray, CT, MRI) and diagnosis because of its good plastic and semi ray transmissions . During the1990s, PEEK composite materials were extensively used as metal implant replacements in the field of orthopedics and trauma . In 1992, PEEK was first used in dental applications and was mainly used in the manufacture of temporary abutment, implant abutment, and healing cap . Currently, PEEKs are extensively used as implants in various medical fields such as orthopedics, spine surgery, and craniofacial surgery, because of their satisfactory clinical effects .
Implant materials must exhibit not only good biological activity but also good antibacterial properties. Presently, research for the improvement of the biological activities of the PEEK implants is gradually maturing and is focused on modification strategies, such as surface coating modification, chemical etching, plasma surface treatment, and plasma immersion ion implantation. For example, nitrogen-containing functional groups were successfully integrated on a PEEK surface through nitrogen ion implantation, and the resulting modified PEEK demonstrated enhanced biological activity . However, PEEK has poor antibacterial ability. In addition, studies on the antibacterial activity of PEEK are currently immature. Meanwhile, implant inflammation instigated by plaque is the most frequent complication of implant surgery and is the leading cause of implant failure . After an in vivo implantation, bacteria can quickly attach to the surface of the implant. Bacteria firmly attach on the implant surface through the specific binding of their cell surfaces with the receptors on the acquired film and formatted plaque biofilm . The biofilm renders the bacteria highly resistant to the host defense response and antimicrobial agents, and thus results in chronic infection and bone resorption around the implants; as such, implant loosening and shedding eventually results in implantation failure . Therefore, preventing the inflammation around the implant by modifying the implant material is an important strategy that can inhibit the adhesion of bacteria and formation of biofilm. This inhibitory effect can improve the antibacterial property of the implant material.
Silver has good anti-inflammatory and bactericidal properties. Notably, the antibacterial properties of silver are already long acknowledged and extensively applied. As a nonspecific broad-spectrum antibacterial agent, silver is used as support in the surface of the implant to enhance the antibacterial properties of the latter. Compared with silver ions, nano-silver exhibits quantum effect, small-size effect, and large specific surface area, presents some distinguishing features, such as durable antibacterial property, broad antibacterial spectrum, and low toxicity and has been applied to a variety of medical materials and equipment, such as wound dressings, catheters, blood vessels, and bone fixation devices, to prevent infection . Therefore, preparation of a nano-silver modified layer on the surface of a material is presently receiving considerable attention because this process can improve the antibacterial properties of the material. Wan et al. and Cao et al. found that implanting silver ions onto the surface of titanium and titanium alloy enhances the antibacterial properties of these materials. Moderate silver on the surface of material can improve antibacterial property without adversely affecting cell proliferation and differentiation.
Magnetron sputtering is a high-speed and low-temperature deposition technology developed in during the 1970s. It can prepare uniform and strong adhesion film on the surface of polymers, composite materials, and ceramics . The basic principle of the magnetron sputtering is as follows: The plasma in Ar and O 2 mixed gas subjected to an electric field and alternating magnetic fields and high-energy particles accelerated the bombardment of the target surface. After the energy exchange, the atoms of the target surface escape from the original atomic lattice, and then transfer to the surface of the substrate, and finally form a film. The entire process is extremely fast, and it requires low temperature and generates low damage. Magnetron sputtering requires simple equipment and thus is easy to control. In addition, it has low substrate temperature, high film forming rate, and strong film adhesion .
Huang et al. prepared silver coating on the surface of tantalum oxide through magnetron sputtering. The resulting silver-cotated tantalum oxide exhibited increase in water contact angle, improved hydrophobicity, and good antibacterial properties to Straphylococcus aureus . Similarly, Gao et al. prepared silver coating on the surface of titanium implants through magnetron sputtering, and their antibacterial experiment showed that the silver-coated titanium implants exhibited long-term antibacterial effects against S. aureus and Escherichia coli (the antibacterial rate was maintained above 97%).
The aim of this present study was to investigate the cytotoxicity and antibacterial effect of nano-silver modification on PEEK. There were three hypotheses as follows: (1) The compact and homogeneous nano-silver modified layer was prepared on the surface of PEEK by magnetron sputtering. (2) The modified PEEK implants have no cytotoxicity. (3) The antibacterial activity and bacterial adhesion ability of the modified PEEK implants are enhanced significantly. So, we provided a solid foundation for the broad clinical application of PEEK and its composites in the fields of joint replacement, bone defect repair, and dental implantation.
2
Materials and methods
2.1
Sample preparation
Two sets of biomedical grade standard PEEK disks were obtained from Jilin University Super Engineering Plastics Research Co., Ltd., China. One set contained the PEEK disks with diameters of 10 mm and thickness of 1 mm, while the other set contained those with diameters of 40 mm and thickness of 2 mm. Prior to surface treatment, all the disks were ground with 600-, 800-, 1000-, 1200-, and 1500-grit silicon carbide abrasive papers. Then they were ultrasonically cleaned using acetone, ethanol, and deionized water in an ultrasonic water bath (Euronda, Italy) for 30 min. After air-drying carefully, they were randomly divided into the control group (PEEK group) and the nano-silver coating groups. All the PEEKs of the nano-silver coating groups were modified using a magnetron sputtering apparatus (SBC-12, Beijing KYKY technology CO., Ltd., China). The schematic of nano-silver coating through magnetron sputtering on PEEK is illustrated in Fig. 1 . During the process, the distance between the 99.99% silver target and the standard test piece was 5 cm, and the room temperature vacuum was adjusted to 3 × 10 3 Pa. Approximately 40 sccm of argon was then subjected to a work pressure of 0.5 Pa. The nano-silver coatings at thicknesses of 3, 6, 9, and 12 nm were deposited onto the PEEKs through magnetron sputtering under 80 W. The modified PEEKs were divided into the following groups according to coating thickness: PEEK-3 (3 nm), PEEK-6 (6 nm), PEEK-9 (9 nm), and PEEK-12 (12 nm).
2.2
Surface characterization
The surface morphologies of the modified PEEK samples were assessed using scanning electron microscopy (SEM, S-4800, Hitachi, Japan). The chemical compositions of the samples were detected by energy dispersive spectrometer (EDS, QUANTAX 400, Luke AXS Co., Germany). The three-dimensional morphologies of the sample surfaces were characterized through atomic force microscopy (AFM, Veeco Instruments, USA). The surface roughness of each sample was also acquired. Furthermore, the water contact angle of each sample was measured by contact angle measurement (DSA20, MK2 KR SS Edward Keller company, Germany) using the hanging drop method using deionized water on the surface of the samples at different positions. For statistical accountability, the average of the five contact angle measurements of each sample was obtained.
2.3
Cytotoxicity test in vitro
2.3.1
Preparation of disinfection and extraction solution of the samples
The samples were wiped with 75% alcohol and rinsed with deionized water thrice. After drying, the samples were transferred to a clean bench (BLB-1300, Suzhou Sujing baishen Technology Co., Ltd.) and subjected to UV irradiation for 2 h and then turned over after 1 h. A high glucose culture medium (Nanjing Jiancheng biological Co., China) was added to the culture medium containing the sterile test piece. The resulting culture medium was then placed in a CO 2 incubator (MCO-20IL, Sanyo, Japan) at 37 °C for 72 h to prepare the extraction solution.
2.3.2
CCK-8 cytotoxicity test
The logarithmic growth phase mouse fibroblast cell suspension (L929, Cells Resource Center, Shanghai Institutes of Biological Science, China) with a concentration of 2 × 10 4 /ml was added to 96-well plates (Costar, USA). Each hole contained 2000 cells. The following groups were then used for subsequent experiment: blank control (pure high glucose medium), negative control (PE), positive control (phenol), PEEK, PEEK-3, PEEK-6, PEEK-9 and PEEK-12. The marked 96-wellplates were placed at a cell incubator (5%CO 2 , 95% humidity). After incubation for 24 h at 37 °C, the supernatant was removed and 100 μl extraction solution was added into each hole. After incubation for 1, 3, 5, and 7 days, 10 μlCCK-8 agentia (CCK-8, Sigma, USA) was added into each hole in darkness and then incubated for 2 h at 37 °C in a CO 2 cell incubator. The absorbance (A) of the supernatant was measured using a microplate reader (BL340, Biotech, USA) at 450 nm wave length. The relative growth rate (RGR) of the cells was calculated using the following formula: RGR = experimental group A value/blank control group A value × 100%. Furthermore, the toxicity levels of the samples and the safety standards were determined according to Table 1 .
| Toxicity levels | RGR | Safety standards |
|---|---|---|
| 0 | >100% | Safe |
| I | 75%–100% | Safe |
| II | 50%–75% | Insecurity |
| III | 25%–50% | Insecurity |
| IV | 1%–25% | Insecurity |
| V | <1% | Insecurity |
2.4
Antibacterial test in vitro
2.4.1
Cultivation of strains and preparation of bacterial suspensions
The freeze-dried strains were recovered, and bacterial suspension at a concentration of 1 × 10 9 CFU L −1 was prepared using the third generation strain. Streptococcus mutans (UA159) was cultured anaerobically with BHI culture medium for 48 h, while S. aureus (ATCC6538) was cultured aerobically with an LB culture medium for 24 h.
2.4.2
Antibacterial experiment
Approximately 50 ml of bacteria suspension (1 × 10 9 CFU L −1 ) was dispersed onto the surface of each sterile sample, which was then covered with a sterile polyethylene film. The resulting samples were then incubated in a bacteria incubator (SLI-1200, SANYO, Japan) for 48 h at a relative humidity (RH) of higher than 90% and temperature of 37 °C. Afterward, the samples were thoroughly washed with 10 ml PBS buffer 10 times. Meanwhile, 50 μl of washing solution was then uniformly coated on a BHI/LB agar plate after the elution solution was diluted to a certain factor. After incubation for 48 h under similar conditions, the active bacteria were counted, and the antibacterial effect was calculated using the following formula: R(%) = (B − A)/B × 100, where R is antibacterial effect (%), A is the mean number of bacteria on the modified samples (CFU/sample), and B is the mean number of bacteria on the control samples (CFU/sample).
(The experimental results must meet the following conditions: first, the above experimental operation was repeated thrice and the average result was obtained. Second, the same group with three viable parallel specimen numbers was consistent with the maximum–minimum value or average value of ≤0.3.)
2.4.3
Antibacterial adhesion test
2.4.3.1
Bacterial adhesion plate colony count
Approximately 2 ml of bacterial suspension (1 × 10 9 CFU L −1 ) was co-cultured with sterile samples under anaerobic environment for 48 h. All the samples were then taken out sequentially from the bacterial suspension and rinsed thrice with sterile PBS buffer for 10 s to remove non-adherent bacteria. The adherent bacteria were detached ultrasonically from the samples in10 ml of the same PBS buffer for 5 min. After the solution was diluted to a certain factor, 50 ml of washing solution was uniformly coated on the BHI/LB agar plate. After the solution was cultured anaerobically for 48 h, the bacterial colonies were counted.
2.4.3.2
SEM observation of the surface adhesion of bacteria
The sterile samples were co-cultured with 2 ml bacterial suspension (1 × 10 9 CFU L −1 ) under an anaerobic environment for 24 h. All samples were then extracted sequentially from the bacterial suspension and rinsed thrice with sterile PBS buffer solution to remove the non-adherent bacteria. The bacteria cultured on the surface of the samples were fixed with a volume fraction of 2.5% glutaraldehyde (Sigma, USA) for 24 h at4 °C. The samples were removed and rinsed with sterile PBS buffer thrice, and then dehydrated with 30%, 50%, 70%, 80%, 90%, 100%, and 100% (v/v) graded ethanol, successively, with 15 min incubation at each concentration. The samples were dried by a vacuum dryer at a critical point of CO 2 and sprayed with gold coating before the SEM observation.
2.5
Statistical analysis
Data were described as mean ± SD and statistically analyzed by one-way ANOVA using SPSS 22 software (SPSS Inc., Chicago, IL, USA). The difference was considered to be statistical significance and significant statistical significance when P < 0.05 and 0.01.
2
Materials and methods
2.1
Sample preparation
Two sets of biomedical grade standard PEEK disks were obtained from Jilin University Super Engineering Plastics Research Co., Ltd., China. One set contained the PEEK disks with diameters of 10 mm and thickness of 1 mm, while the other set contained those with diameters of 40 mm and thickness of 2 mm. Prior to surface treatment, all the disks were ground with 600-, 800-, 1000-, 1200-, and 1500-grit silicon carbide abrasive papers. Then they were ultrasonically cleaned using acetone, ethanol, and deionized water in an ultrasonic water bath (Euronda, Italy) for 30 min. After air-drying carefully, they were randomly divided into the control group (PEEK group) and the nano-silver coating groups. All the PEEKs of the nano-silver coating groups were modified using a magnetron sputtering apparatus (SBC-12, Beijing KYKY technology CO., Ltd., China). The schematic of nano-silver coating through magnetron sputtering on PEEK is illustrated in Fig. 1 . During the process, the distance between the 99.99% silver target and the standard test piece was 5 cm, and the room temperature vacuum was adjusted to 3 × 10 3 Pa. Approximately 40 sccm of argon was then subjected to a work pressure of 0.5 Pa. The nano-silver coatings at thicknesses of 3, 6, 9, and 12 nm were deposited onto the PEEKs through magnetron sputtering under 80 W. The modified PEEKs were divided into the following groups according to coating thickness: PEEK-3 (3 nm), PEEK-6 (6 nm), PEEK-9 (9 nm), and PEEK-12 (12 nm).
2.2
Surface characterization
The surface morphologies of the modified PEEK samples were assessed using scanning electron microscopy (SEM, S-4800, Hitachi, Japan). The chemical compositions of the samples were detected by energy dispersive spectrometer (EDS, QUANTAX 400, Luke AXS Co., Germany). The three-dimensional morphologies of the sample surfaces were characterized through atomic force microscopy (AFM, Veeco Instruments, USA). The surface roughness of each sample was also acquired. Furthermore, the water contact angle of each sample was measured by contact angle measurement (DSA20, MK2 KR SS Edward Keller company, Germany) using the hanging drop method using deionized water on the surface of the samples at different positions. For statistical accountability, the average of the five contact angle measurements of each sample was obtained.
2.3
Cytotoxicity test in vitro
2.3.1
Preparation of disinfection and extraction solution of the samples
The samples were wiped with 75% alcohol and rinsed with deionized water thrice. After drying, the samples were transferred to a clean bench (BLB-1300, Suzhou Sujing baishen Technology Co., Ltd.) and subjected to UV irradiation for 2 h and then turned over after 1 h. A high glucose culture medium (Nanjing Jiancheng biological Co., China) was added to the culture medium containing the sterile test piece. The resulting culture medium was then placed in a CO 2 incubator (MCO-20IL, Sanyo, Japan) at 37 °C for 72 h to prepare the extraction solution.
2.3.2
CCK-8 cytotoxicity test
The logarithmic growth phase mouse fibroblast cell suspension (L929, Cells Resource Center, Shanghai Institutes of Biological Science, China) with a concentration of 2 × 10 4 /ml was added to 96-well plates (Costar, USA). Each hole contained 2000 cells. The following groups were then used for subsequent experiment: blank control (pure high glucose medium), negative control (PE), positive control (phenol), PEEK, PEEK-3, PEEK-6, PEEK-9 and PEEK-12. The marked 96-wellplates were placed at a cell incubator (5%CO 2 , 95% humidity). After incubation for 24 h at 37 °C, the supernatant was removed and 100 μl extraction solution was added into each hole. After incubation for 1, 3, 5, and 7 days, 10 μlCCK-8 agentia (CCK-8, Sigma, USA) was added into each hole in darkness and then incubated for 2 h at 37 °C in a CO 2 cell incubator. The absorbance (A) of the supernatant was measured using a microplate reader (BL340, Biotech, USA) at 450 nm wave length. The relative growth rate (RGR) of the cells was calculated using the following formula: RGR = experimental group A value/blank control group A value × 100%. Furthermore, the toxicity levels of the samples and the safety standards were determined according to Table 1 .
| Toxicity levels | RGR | Safety standards |
|---|---|---|
| 0 | >100% | Safe |
| I | 75%–100% | Safe |
| II | 50%–75% | Insecurity |
| III | 25%–50% | Insecurity |
| IV | 1%–25% | Insecurity |
| V | <1% | Insecurity |
2.4
Antibacterial test in vitro
2.4.1
Cultivation of strains and preparation of bacterial suspensions
The freeze-dried strains were recovered, and bacterial suspension at a concentration of 1 × 10 9 CFU L −1 was prepared using the third generation strain. Streptococcus mutans (UA159) was cultured anaerobically with BHI culture medium for 48 h, while S. aureus (ATCC6538) was cultured aerobically with an LB culture medium for 24 h.
2.4.2
Antibacterial experiment
Approximately 50 ml of bacteria suspension (1 × 10 9 CFU L −1 ) was dispersed onto the surface of each sterile sample, which was then covered with a sterile polyethylene film. The resulting samples were then incubated in a bacteria incubator (SLI-1200, SANYO, Japan) for 48 h at a relative humidity (RH) of higher than 90% and temperature of 37 °C. Afterward, the samples were thoroughly washed with 10 ml PBS buffer 10 times. Meanwhile, 50 μl of washing solution was then uniformly coated on a BHI/LB agar plate after the elution solution was diluted to a certain factor. After incubation for 48 h under similar conditions, the active bacteria were counted, and the antibacterial effect was calculated using the following formula: R(%) = (B − A)/B × 100, where R is antibacterial effect (%), A is the mean number of bacteria on the modified samples (CFU/sample), and B is the mean number of bacteria on the control samples (CFU/sample).
(The experimental results must meet the following conditions: first, the above experimental operation was repeated thrice and the average result was obtained. Second, the same group with three viable parallel specimen numbers was consistent with the maximum–minimum value or average value of ≤0.3.)
2.4.3
Antibacterial adhesion test
2.4.3.1
Bacterial adhesion plate colony count
Approximately 2 ml of bacterial suspension (1 × 10 9 CFU L −1 ) was co-cultured with sterile samples under anaerobic environment for 48 h. All the samples were then taken out sequentially from the bacterial suspension and rinsed thrice with sterile PBS buffer for 10 s to remove non-adherent bacteria. The adherent bacteria were detached ultrasonically from the samples in10 ml of the same PBS buffer for 5 min. After the solution was diluted to a certain factor, 50 ml of washing solution was uniformly coated on the BHI/LB agar plate. After the solution was cultured anaerobically for 48 h, the bacterial colonies were counted.
2.4.3.2
SEM observation of the surface adhesion of bacteria
The sterile samples were co-cultured with 2 ml bacterial suspension (1 × 10 9 CFU L −1 ) under an anaerobic environment for 24 h. All samples were then extracted sequentially from the bacterial suspension and rinsed thrice with sterile PBS buffer solution to remove the non-adherent bacteria. The bacteria cultured on the surface of the samples were fixed with a volume fraction of 2.5% glutaraldehyde (Sigma, USA) for 24 h at4 °C. The samples were removed and rinsed with sterile PBS buffer thrice, and then dehydrated with 30%, 50%, 70%, 80%, 90%, 100%, and 100% (v/v) graded ethanol, successively, with 15 min incubation at each concentration. The samples were dried by a vacuum dryer at a critical point of CO 2 and sprayed with gold coating before the SEM observation.
2.5
Statistical analysis
Data were described as mean ± SD and statistically analyzed by one-way ANOVA using SPSS 22 software (SPSS Inc., Chicago, IL, USA). The difference was considered to be statistical significance and significant statistical significance when P < 0.05 and 0.01.
3
Results
3.1
Characterization of the sample surface
Compared with the PEEK group, the surface of the nano-silver coating groups was slightly gray. With the increase of the coating thickness, their colors did not significantly change.
Fig. 2 shows the SEM topography of the surfaces of all the samples. The surfaces of the PEEK samples in the control group were relatively smooth without the nano-particles. After magnetron sputtering, uniformly distributed Ag nano-particles (AgNPs) were visible on the surfaces of the samples in PEEK-3, PEEK-6, PEEK-9, and PEEK-12. The silver particles were round and compact, and when the silver content increased, the arrangement of the silver particles became compact.
Fig. 3 displays the results of the surface element analysis on the PEEKs and nano-silver coated PEEKs. EDS observation results showed that the surfaces of the modified PEEKs had silver elements, unlike the surfaces of the pure PEEKs. Furthermore, when the thickness of the nano-silver coating increased, the peak of the silver element gradually increased.
