Abstract
Objective
We aimed to investigate the bioactivity and antibacterial effect of nitrogen plasma immersion ion implantation (PIII) on polyetheretherketone (PEEK).
Methods
According to the different modified parameters, the PEEK specimens were randomly divided into four main groups ( n = 49/group): PEEK-C, PEEK-I, PEEK-L, and PEEK-H. Then, N 2 -PIII surface modification was conducted using the corresponding parameters. The microstructure and composition of the modified PEEK surface was observed by scanning electron microscope (SEM), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The water contact angle of the PEEK surface was also studied by contact angle meters. The bioactive ability of PEEK samples was evaluated by observing the attachment, proliferation, and differentiation of MG63 cells cultured on the PEEK samples. Antibacterial property of the samples against Staphylococcus aureus was detected with the plate colony-counting methods.
Results
SEM and AFM analysis shows that PEEK-C surface is relatively smooth with the Ra value of 50.6 ± 2.52 nm. PEEK-I and PEEK-L surface is rough with the Ra value of 435.9 ± 6.47 nm and 443.23 ± 5.49 nm, respectively, and the PEEK-H surface is the most rough with the Ra value of 608.4 ± 3.14 nm. XPS element analysis demonstrated that nitrogen functional groups were successfully introduced into the surface of PIII-modified PEEK. Biological evaluation and the antibacterial results showed that nitrogen PIII treatment can significantly improve the biological activity of PEEK, and samples showed antibacterial properties against S. aureus .
Significance
PEEK surface subjected to the N 2 -PIII treatment showed better biological activity and antibacterial effect. Therefore, N 2 -PIII-treated PEEK surface is promising in bone tissue engineering and dental applications.
1
Introduction
In recent years, with the continuous increase in the proportion of aged population, dentition defect in patients is increasing year by year. Therefore, the demand for dental implanted materials is also very strong. Due to the increasing number of bone defects caused by trauma, infection, tumor resection, and other conditions such as craniotomy, the patient’s health faces serious threats. Pure titanium, as the most commonly used medical implant material, especially oral implant material, has achieved good clinical effect: it not only successfully helps countless patients with bone defect to receive the ideal morphology, it also objectively promotes the popularization and development of the concept of oral implant. Due to elastic modulus mismatch between titanium implant material and the surrounding bone tissue. When titanium was implanted in vivo, stress peaks at the bone implant interface could occur, causing an overload of peri-implant bone and therefore bone loss. Which is the so-called “stress shielding effect”, eventually leading to loosening of the implants . Some patients using titanium metal undergo allergic reactions , and thin gingival biotype with titanium implants possess easily exposed metal color, thus having aesthetic impact. To overcome these problems and to reduce adverse biological effects after implantation, looking for new alternative materials is very important.
Polyetheretherketone (PEEK), a high-performance metal-free medical implant material, has been used in the clinic with the authorization from the US Food and Drug Administration (FDA). As a semi-crystalline aromatic organic polymer compound, the melting point of PEEK can reach up to 335 °C. Compared with titanium, the biggest advantage of PEEK used as implant material in orthopedics is that it has similar elastic modulus (3–4 GPa) as the cortical bone . And this can reduce the “stress-shielding effect” that can be seen in titanium-based implants . Therefore, PEEK can be considered a promising metal-free prosthesis biomaterial for implantation. In the field of oral medicine, PEEK and its composites are mainly used as temporary abutments of implants, as orthodontic bite bar, in orthodontic aesthetics, and in fixed clamping rings . In the field of orthopedics, PEEK and its composites are mainly used in spinal surgery, joint surgery, wound healing, and other fields, which has achieved satisfactory repairing effect . However, the biological activity of PEEK is very poor because of its inert surface. When used as dental implant or bone defect repair material, it is difficult to obtain optimal osseointegration effect . This is also the reason why PEEK has not been used widely in clinical applications.
Confronted with the critical issue that the PEEK surface does not show biological activity, resulting from its high chemical inertness, current research has focused mainly on two aspects: the preparation of PEEK composites and surface modification. The preparation of PEEK composites could be achieved by the method of blending active substances, such as preparing TiO 2 /PEEK composites by blending TiO 2 , preparing hydroxyapatite (HA)/PEEK composite by blending HA , preparing FA/PEEK composite by blending nano-fluorapatite . Surface modification mainly accomplished by chemical etching , surface coating , plasma surface treatment . The modified strategies mentioned above can improve the biological activity of PEEK to a certain extent. However, chemical etching can result in reduced mechanical strength . The surface coating is easy to be degraded in the body fluid environment because of low crystallinity . There is also the risk of stripping due to thermal expansion coefficient mismatch between coating and matrix . Surface modification layer obtained by plasma surface treatment is thin, which is often unable to meet the properties required to be used as medical implant material .
Plasma Immersion Ion Implantation (PIII) is a novel surface modification method, which can change the surface microstructure and chemical composition of polymeric materials without affecting their bulk properties . Moreover, the treatment is not only easy to conduct but also highly efficient. The corresponding elements and chemical groups can be introduced into the surface of PEEK by different plasma injection sources . Previous studies have shown that nitrogen-containing functional groups can be introduced on the surface of polymer materials by nitrogen PIII, and the resulting surface exhibits osteogenic activity and antibacterial properties .
As a linear aromatic macromolecule, the surface structure and composition of PEEK can be changed after being bombarded by high-energy plasma particles. Wang et al. reported that many “ravined structures” and hydroxyl groups are constructed on water-PIII-treated PEEK surfaces, which were favorable for the adhesion and proliferation of osteoblast precursor cells MC3T3-E1 . Similarly, the PEEK surface also can be modified with the formation of hydrophilic nanostructures using titanium PIII. The obtained surface layer promoted the adhesion, proliferation, and osteoblast differentiation of cells cultured on PEEK surface .
The aim of this present study was to investigate the bioactivity and antibacterial effect of nitrogen PIII modification on PEEK. Finally, we lay a foundation for their widespread clinical application of PEEK and its composites in the field of dental implant, bone defect repair and joint replacement.
2
Materials and methods
2.1
Sample preparation
A total of 196 biomedical grade disk PEEK standard samples (Jilin University Super Engineering Plastics Research Co., Ltd., China), with dimensions of 10 mm diameter and 1 mm thickness, were prepared. Prior to surface treatment, all samples were ground with 600-, 800-, 1000-, and 1200-grit silicon carbide abrasive papers, as well as diamond paste to achieve a near mirror finish. Then, they were ultrasonically cleaned using acetone, ethanol, and deionized water in an ultrasonic water bath (Euronda, Italy) for 10 min. After air-drying carefully, all samples were randomly divided into the PEEK-C and the N 2 -PIII-treated group. Then, all samples of the PIII-treated group were modified in the PIII device (Harbin Institute of Technology, Harbin, China). The schematic of N 2 -PIII modification on PEEK is illustrated in Fig. 1 . According to the different treatment parameters, the N 2 -PIII-treated group was assigned to PEEK-I, PEEK-L, and PEEK-H. Table 1 lists the key treatment parameters of PIII-treated group.
| Group | PEEK-C | PEEK-I | PEEK-L | PEEK-H |
|---|---|---|---|---|
| Gas type | – | N 2 | N 2 | N 2 |
| Gas flow rate | – | 20 sccm | 20 sccm | 20 sccm |
| Voltage | – | – | −20 kV | −20 kV |
| Pressure | – | 1 × 10 −2 Pa | 1 × 10 −2 Pa | 1 × 10 −2 Pa |
| Pulse width | – | – | 30 μS | 50 μS |
| Frequency | – | – | 1000 W | 1000 W |
| Time | – | 90 min | 90 min | 90 min |
2.2
Surface characterization
Surface morphology was assessed using scanning electron microscopy (SEM, XL30, SEI, USA). In order to characterize the three-dimensional morphology of the sample surfaces, atomic force microscopy (AFM, Nano Wizard II, JPK Instruments, Germany) was used in contact mode with a scanned area of 20 μm × 20 μm, and the surface roughness was also acquired in duplicate from 5 samples. The chemical composition of samples was detected by X-ray photoelectron spectroscopy (XPS; ESCALAB 250Xi, Thermo Scientific, USA). The water contact angle of samples was measured by contact angle measurement (Harvey, Main, & Co., Ltd., Hong Kong) using sessile distilled water (10 μL per drop) at 20 °C ± 1 °C. Each contact angle was acquired from an average of five measurements on each sample for statistical accountability.
2.3
In vitro biological evaluation
2.3.1
Cell culture
Human osteoblast-like MG63 cells (Cells Resource Center, Shanghai Institutes of Biological Science, China) were cultured in Hepes-buffered Dulbecco’s modified Eagle’s medium (H-DMEM; Gibco, USA) with 10% (v/v) fetal calf serum (Gibco, USA), in a humidified air incubator at 37 °C. And 15 samples were evaluated for each group. After incubation for 1, 2, and 3 days, the cell culture medium was sucked out and the samples were rinsed with sterile PBS three times. The MG-63 cells were seeded on the samples at a density of 2 × 10 5 cells/cm 2 in 24-well plates (Costar, USA). The culture medium was refreshed at 3-day intervals.
2.3.2
Cells attachment
After incubation for 2 h, the cell culture medium was sucked out and the samples were rinsed with sterile PBS three times. The cells adhesive on samples were fixed with 4% (v/v) paraformaldehyde (Sigma, USA) for 30 min at 4 °C. Then 4, 6-diamidino-2-phenylindole (DAPI, Roche, Switzerland) was added into 24-well plates. After stained for 20 min, the samples were rinsed three times with PBS. Finally, the cells adhesion number was observed by inverted fluorescence microscope (OLYMPUS U-RFL-T, Japan).
2.3.3
Cell viability assay
The proliferation viability of MG-63 cells cultured on the surface of samples was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, USA) assay. A total of 45 samples ( n = 15/group) were evaluated for cell viability assay. After incubation for 1, 2, and 3 days, the cell culture medium was sucked out and the samples were rinsed with sterile PBS three times. Then, 200 μL 5 mg/mL MTT solution and 800 μL medium without serum and phenol were into new 24-well plates (Costar, USA). After incubation for 2 h at 37 °C, formazan was formed, then 1 mL of dimethyl sulfoxide (Sigma–Aldrich, USA) was added into the culture plate to dissolve the formazan. Finally, 200 μL of the above medium was transferred to another 96-well plate for measurement. Absorbance of the supernatant was measured using a microplate reader (BL340, Biotech, USA) at the wavelength of 490 nm.
2.3.4
Cell morphology
After incubation for 12 h, the cell culture was terminated and cells were rinsed with PBS three times. A total of 20 samples ( n = 5/group) were used to observe SEM morphology of cells adsorbed on samples surface. The MG-63 cells cultured on the surface of samples were fixed with volume fraction of 4% paraformaldehyde (Sigma, USA) for 24 h at room temperature. The samples were removed and rinsed three times with PBS, followed by dehydration with 30%, 50%, 70%, 80%, 90%, 95%, and 100% (v/v) graded ethanol, with incubation at each concentration for 15 min. The samples were dried by a vacuum dryer at the critical point of CO 2 and sprayed with gold coating before observing by SEM.
2.3.5
Alkaline phosphatase (ALP) activity assay
ALP activity of MG-63 grown on the sample surface was detected using ALP reagent (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). A total of 20 samples ( n = 5/group) were used. After culturing for 14 days, the samples were rinsed with PBS three times and immersed in 1 mL 0.25% trypsin for 2 min. Then, the lysate was transferred to the centrifugal tube, the supernatant was discarded, and 200 μL of PBS was added per tube to dissolve the cell pellet. The resulting solutions were lysed with 100 μL cell lysate containing 0.1% Triton X-100 overnight at 4 °C. After no cell structure was observed under the optical microscope, the absorbance value was measured at 562 nm wavelength and the resulting nitrophenol was calculated following the instructions of the ALP Kit. Referring to the normal standard, total intracellular protein content was detected using BCA Protein Assay Kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). The relative activity of ALP was calculated by dividing the amount of nitrophenol into the corresponding total protein.
2.4
Bacterial culture
To evaluate the antibacterial properties of the samples by colony-counting method, Staphylococcus aureus (American Type Culture Collection 25923) was used as the experimental strain. First, samples were placed in 75% ethanol for 60 min and dried in sterilized clean bench; both sides of the sample were irradiated with ultraviolet light for 30 min and then put into a 24-well cell culture plate. The concentration of S. aureus was adjusted to 1 × 10 7 colony-forming units/mL using a visible light spectrophotometer (LAMBDA 25, Perkin Elmer, CT, USA). And 60 μL of the bacterial suspension was added on the sample surface in 24-well plates. After culturing at 37 °C and 90% humidity in a constant temperature environment for 24 h, samples were removed and washed three times with PBS to remove non-adherent bacteria. Then, they were placed in a sterile test tube containing 5 mL PBS. The test tube was placed on an ultrasonic oscillator (Euronda, Italy) for 15 min to remove the bacteria adhered to the material’s surface, the method has been reported in many studies . The number of bacteria contained in the liquid was detected by the plate-counting method. According to the above results, the number of adhesive bacteria on the surface of the specimen was calculated.
2.5
Statistical analysis
The data were statistically analyzed by SPSS 21 software (SPSS Inc., Chicago, IL, USA). The results of the experiment are shown as mean ± standard deviation. Two independent samples were compared with the t -test and the comparison between multiple groups was done using single-factor analysis of variance. P < 0.05 indicated that the difference has statistical significance. All biological tests were independently performed in quintuplicate ( n = 5/group) and each data point represents five replicate measurements.
2
Materials and methods
2.1
Sample preparation
A total of 196 biomedical grade disk PEEK standard samples (Jilin University Super Engineering Plastics Research Co., Ltd., China), with dimensions of 10 mm diameter and 1 mm thickness, were prepared. Prior to surface treatment, all samples were ground with 600-, 800-, 1000-, and 1200-grit silicon carbide abrasive papers, as well as diamond paste to achieve a near mirror finish. Then, they were ultrasonically cleaned using acetone, ethanol, and deionized water in an ultrasonic water bath (Euronda, Italy) for 10 min. After air-drying carefully, all samples were randomly divided into the PEEK-C and the N 2 -PIII-treated group. Then, all samples of the PIII-treated group were modified in the PIII device (Harbin Institute of Technology, Harbin, China). The schematic of N 2 -PIII modification on PEEK is illustrated in Fig. 1 . According to the different treatment parameters, the N 2 -PIII-treated group was assigned to PEEK-I, PEEK-L, and PEEK-H. Table 1 lists the key treatment parameters of PIII-treated group.
| Group | PEEK-C | PEEK-I | PEEK-L | PEEK-H |
|---|---|---|---|---|
| Gas type | – | N 2 | N 2 | N 2 |
| Gas flow rate | – | 20 sccm | 20 sccm | 20 sccm |
| Voltage | – | – | −20 kV | −20 kV |
| Pressure | – | 1 × 10 −2 Pa | 1 × 10 −2 Pa | 1 × 10 −2 Pa |
| Pulse width | – | – | 30 μS | 50 μS |
| Frequency | – | – | 1000 W | 1000 W |
| Time | – | 90 min | 90 min | 90 min |
2.2
Surface characterization
Surface morphology was assessed using scanning electron microscopy (SEM, XL30, SEI, USA). In order to characterize the three-dimensional morphology of the sample surfaces, atomic force microscopy (AFM, Nano Wizard II, JPK Instruments, Germany) was used in contact mode with a scanned area of 20 μm × 20 μm, and the surface roughness was also acquired in duplicate from 5 samples. The chemical composition of samples was detected by X-ray photoelectron spectroscopy (XPS; ESCALAB 250Xi, Thermo Scientific, USA). The water contact angle of samples was measured by contact angle measurement (Harvey, Main, & Co., Ltd., Hong Kong) using sessile distilled water (10 μL per drop) at 20 °C ± 1 °C. Each contact angle was acquired from an average of five measurements on each sample for statistical accountability.
2.3
In vitro biological evaluation
2.3.1
Cell culture
Human osteoblast-like MG63 cells (Cells Resource Center, Shanghai Institutes of Biological Science, China) were cultured in Hepes-buffered Dulbecco’s modified Eagle’s medium (H-DMEM; Gibco, USA) with 10% (v/v) fetal calf serum (Gibco, USA), in a humidified air incubator at 37 °C. And 15 samples were evaluated for each group. After incubation for 1, 2, and 3 days, the cell culture medium was sucked out and the samples were rinsed with sterile PBS three times. The MG-63 cells were seeded on the samples at a density of 2 × 10 5 cells/cm 2 in 24-well plates (Costar, USA). The culture medium was refreshed at 3-day intervals.
2.3.2
Cells attachment
After incubation for 2 h, the cell culture medium was sucked out and the samples were rinsed with sterile PBS three times. The cells adhesive on samples were fixed with 4% (v/v) paraformaldehyde (Sigma, USA) for 30 min at 4 °C. Then 4, 6-diamidino-2-phenylindole (DAPI, Roche, Switzerland) was added into 24-well plates. After stained for 20 min, the samples were rinsed three times with PBS. Finally, the cells adhesion number was observed by inverted fluorescence microscope (OLYMPUS U-RFL-T, Japan).
2.3.3
Cell viability assay
The proliferation viability of MG-63 cells cultured on the surface of samples was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, USA) assay. A total of 45 samples ( n = 15/group) were evaluated for cell viability assay. After incubation for 1, 2, and 3 days, the cell culture medium was sucked out and the samples were rinsed with sterile PBS three times. Then, 200 μL 5 mg/mL MTT solution and 800 μL medium without serum and phenol were into new 24-well plates (Costar, USA). After incubation for 2 h at 37 °C, formazan was formed, then 1 mL of dimethyl sulfoxide (Sigma–Aldrich, USA) was added into the culture plate to dissolve the formazan. Finally, 200 μL of the above medium was transferred to another 96-well plate for measurement. Absorbance of the supernatant was measured using a microplate reader (BL340, Biotech, USA) at the wavelength of 490 nm.
2.3.4
Cell morphology
After incubation for 12 h, the cell culture was terminated and cells were rinsed with PBS three times. A total of 20 samples ( n = 5/group) were used to observe SEM morphology of cells adsorbed on samples surface. The MG-63 cells cultured on the surface of samples were fixed with volume fraction of 4% paraformaldehyde (Sigma, USA) for 24 h at room temperature. The samples were removed and rinsed three times with PBS, followed by dehydration with 30%, 50%, 70%, 80%, 90%, 95%, and 100% (v/v) graded ethanol, with incubation at each concentration for 15 min. The samples were dried by a vacuum dryer at the critical point of CO 2 and sprayed with gold coating before observing by SEM.
2.3.5
Alkaline phosphatase (ALP) activity assay
ALP activity of MG-63 grown on the sample surface was detected using ALP reagent (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). A total of 20 samples ( n = 5/group) were used. After culturing for 14 days, the samples were rinsed with PBS three times and immersed in 1 mL 0.25% trypsin for 2 min. Then, the lysate was transferred to the centrifugal tube, the supernatant was discarded, and 200 μL of PBS was added per tube to dissolve the cell pellet. The resulting solutions were lysed with 100 μL cell lysate containing 0.1% Triton X-100 overnight at 4 °C. After no cell structure was observed under the optical microscope, the absorbance value was measured at 562 nm wavelength and the resulting nitrophenol was calculated following the instructions of the ALP Kit. Referring to the normal standard, total intracellular protein content was detected using BCA Protein Assay Kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). The relative activity of ALP was calculated by dividing the amount of nitrophenol into the corresponding total protein.
2.4
Bacterial culture
To evaluate the antibacterial properties of the samples by colony-counting method, Staphylococcus aureus (American Type Culture Collection 25923) was used as the experimental strain. First, samples were placed in 75% ethanol for 60 min and dried in sterilized clean bench; both sides of the sample were irradiated with ultraviolet light for 30 min and then put into a 24-well cell culture plate. The concentration of S. aureus was adjusted to 1 × 10 7 colony-forming units/mL using a visible light spectrophotometer (LAMBDA 25, Perkin Elmer, CT, USA). And 60 μL of the bacterial suspension was added on the sample surface in 24-well plates. After culturing at 37 °C and 90% humidity in a constant temperature environment for 24 h, samples were removed and washed three times with PBS to remove non-adherent bacteria. Then, they were placed in a sterile test tube containing 5 mL PBS. The test tube was placed on an ultrasonic oscillator (Euronda, Italy) for 15 min to remove the bacteria adhered to the material’s surface, the method has been reported in many studies . The number of bacteria contained in the liquid was detected by the plate-counting method. According to the above results, the number of adhesive bacteria on the surface of the specimen was calculated.
2.5
Statistical analysis
The data were statistically analyzed by SPSS 21 software (SPSS Inc., Chicago, IL, USA). The results of the experiment are shown as mean ± standard deviation. Two independent samples were compared with the t -test and the comparison between multiple groups was done using single-factor analysis of variance. P < 0.05 indicated that the difference has statistical significance. All biological tests were independently performed in quintuplicate ( n = 5/group) and each data point represents five replicate measurements.
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