In vitro assessment of corrosion,antibacterial properties,and degradation behavior of zirconia-coated polyetheretherketone (PEEK)

Sample preparation and coating

The corrosion test samples were prepared using the Pramaan 310 HT global FDM 3D printer machine, Additive Manufacturing Private Ltd., Hyderabad, India. Four different samples were prepared by varying the parameters, such as printing speed and layer height. Table 1 lists the parameters used to prepare the 3D-printed samples. The samples required for coating were polished with 1500 and 2000-grit-size silicon carbide abrasive sheets followed with ultrasonic cleaning for 8 min each with acetone and deionized water in an Ultrasonicator (Labman, New Delhi, India) to improve coating adherence.

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Table 1.

3D printed PEEK FDM parameters.

S. No	Specification	Description
1	Specimen 1 (S1)	L: 0.10 mm; S: 20 mm/s
2	Specimen 2 (S2)	L: 0.10 mm; S: 25 mm/s
3	Specimen 3 (S3)	L: 0.15 mm; S: 20 mm/s
4	Specimen 4 (S4)	L: 0.15 mm; S: 25 mm/s
5	Extruder temperature	420°C
6	Bed temperature	120°C
7	Infill pattern	Grid
8	Infill percentage	100%

L: Layer height; S: Printing speed.

An RF sputtering unit manufactured by Vacutech Systems, Bangalore, India was employed for coating zirconia films over PEEK polymer. Zirconia target of 5 mm diameter and 2 mm thickness with copper backing plate of 99.99% purity from Ultra nanotech Private Limited, Bangalore, India was purchased. The parameters used in the coating process are given in Table 2. Before processing with the deposition, the instrument was maintained in a vacuum until it reached the expected base pressure. Argon gas of 25 sccm was supplied to the working chamber and the working pressure was set for the deposition of the target. The schematic illustration of the RF sputtering process and the equipment used in the study are given in Figure 1.

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Table 2.

Sputtering parameters.

S. No	Specification	Description
1	Target material	Zirconia
2	Substrate	PEEK
3	Base pressure	4 torr × 10–5 torr
4	Work pressure	5 torr × 10–4 torr
5	Substrate temperature	Room temperature
6	Argon gas flow	25 sccm
7	Target substrate distance	20 cm
8	RF power	80 W
9	Deposition time	20 min

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Figure 1.

Schematic illustration: (a) RF magnetron sputtering machine and (b) sputtering process.

The uncoated and coated PEEK samples were immersed in artificial saliva to examine the corrosive behavior, and the samples were immersed in the solution for approximately 30 days. The samples were taken out and weighed at 3 days intervals. Fusayama solution was utilized as artificial saliva in this investigation, and the solution’s composition as well as its concentration, are given in Table 3.

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Table 3.

Artificial saliva composition.

S. No	Components	Concentration (g/l)
1	Potassium chloride	0.4
2	Sodium chloride	0.4
3	Calcium chloride dihydrate	0.906
4	Monosodium phosphate dihydrate	0.69
5	Sodium sulfide nonahydrate	0.005
6	Urea	1

Characterization methods

The phase structure of the specimens immersed in the artificial saliva solution was analyzed using the X-ray diffraction (XRD) technique. The spectra were collected using a Cu-Kα source with applied voltage of 30 kV and current 40 mA over a range of 5°–90° at a scan rate of 0.5 °/s on a Bruker D8 Advance X-ray diffractometer, Germany.

The morphology of the test specimens was examined using a Thermo Fisher FEI QUANTA 250 FEG field emission scanning electron microscope (FESEM), Waltham, Massachusetts.

Furthermore, the passivation of films generated during the corrosion test was analyzed using ULVAC-PHI VersaProbe 4 X-ray photoelectron spectroscopy (XPS), Japan. An Al-Kα X-ray source was employed, and the anode energy was set at 1486.6 eV.

Antimicrobial studies

The antimicrobial test was performed using the broth microdilution method, with gram positive and gram-negative bacteria such as S. aureus and E. coli, respectively. The broth was used to cultivate the appropriate bacteria, and samples were immersed in it for analysis. The microtiter plates are incubated at 37°C for 20 h. Plates were incubated after being filled with 125 l of 0.1% crystal violet solution. Finally, the reading was taken at 550 nm using a microplate reader, BIORAD (USA).

Cytotoxic studies

Human Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) were cultured in Minimum Essential Medium Alpha (MEM α, Sigma) at 37°C in a humidified environment with 5% CO₂. Before conducting in vitro tests, samples were cleaned with ethyl alcohol and then sterilized in an autoclave. To test cell viability, 25,000 cm⁻² of WJ-MSCs were seeded on sample surfaces in triplicate. After 48 h of incubation, the MTT [3-(4,5-Dimethylthiazol-2-yl)−2,5-Diphenyltetrazolium Bromide] assay was performed and the colorimetric readings were taken at 595 nm using an automatic cell counter, BIORAD (USA).

Characterization of ZrO₂ coated PEEK

Zirconia films were deposited over the PEEK surface using an RF sputtering method within the chamber at a base pressure and working pressure of 4 × 10^-5 Torr and 5 × 10^-4 Torr respectively to achieve a high-quality, uniform coating. ZrO₂ plasma developed around the target begins to deposit evenly on top of the PEEK samples placed on the sample holder. The X-ray diffraction pattern of zirconia coated PEEK sample is shown in Figure 2(a). The diffraction pattern displays well-defined and scattered peaks in the 2θ range of 10° to 70°, with the most prominent peaks observed between 25° and 42°. The diffraction pattern revealed that zirconia has a polycrystalline characteristic, namely with a monoclinic phase featuring lattice planes (011), (111), (201), (022), (131), and (023). ²¹ In addition, the 2θ spacing of the samples precisely aligned with the standards provided by the Joint Committee on Powder Diffraction Standards (JCPDS), which gives information about their relative intensity.

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Figure 2.

Characteristics of ZrO₂ coated PEEK: (a) XRD, (b) micrograph at 5 µm, (c) micrograph at 500 nm, and (d) EDS image and elemental mapping.

Figure 2(b)–(d) shows the surface morphology of the zirconia-coated PEEK sample by FESEM analysis along with the EDS mapping, which exhibits crack-free and uniform morphology. The elemental map reveals the homogeneous distribution of the zirconia particles over the sample surface, showing the efficiency of the RF sputtering process. Figure 3 shows the cross-sectional image of zirconia coated PEEK sample using scanning electron microscopy and the thickness of the zirconia coating deposited over the PEEK surface was measured to be approximately 1 micron. This thickness range was chosen as it is beneficial to the stability of coating and reduces crack force and delamination. ²⁶ In addition, increasing the coating thickness increases surface roughness, which promotes bacterial adherence. ²⁷

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Figure 3.

Coating characterization: (a) cross-section FESEM of zirconia coated PEEK specimen and (b) EDS and elemental mapping.

Characterization of PEEK immersed in artificial saliva

The PEEK samples printed with four different parameters, S1, S2, S3, and S4 each, were immersed in a 10 ml beaker containing artificial saliva solution, for a total immersion period of 30 days. The pH of the prepared artificial saliva solution was found to be 6.7. The samples were weighed before being immersed in the solution. After immersion, the samples were removed, dried, and weighed at 72-h intervals for 30 days. The pH of the saliva solution was monitored regularly and the new solution was changed every 3 days. Following the conclusion of the 30-day immersion period, the samples were collected and subjected to drying in a vacuum oven before commencing the process of sample characterization. Figure 4 shows the X-ray diffraction analysis of the four specimens. The peak analysis revealed the presence of the following compounds: potassium chloride (KCl), sodium chloride (NaCl), potassium bisulfite (KHSO₃), sodium phosphate (Na₃PO₄), and calcium sulfate (CaSO₄). The presence of these compounds on the surfaces was confirmed by matching the 2θ spacing with JCPDS standards along with the elemental mapping using FESEM microscopy. The JCPDS card numbers, together with their corresponding relative intensity values for the various chemical compounds, are presented in Figure 4. Sodium phosphate was found to exhibit a cubic phase with lattice planes (111) and (220) with 2θ spacing of 20.67° and 34.02°, respectively. Potassium bisulfite showed an orthorhombic structure with lattice planes (022), (240), and (222) at 2θ angles of 23.14°, 26.11°, and 29.48°, respectively. Both potassium chloride and sodium chloride exhibited cubic structure with a lattice plane (200) at 2θ spacing of 28.30° and 31.69°, respectively.

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Figure 4.

XRD characteristics of PEEK samples immersed in artificial saliva solution: (a) S1, (b) S2, (c) S3, and (d) S4.

Figure 5 shows accumulation of corrosion particles on the PEEK surface and elemental mapping of sample S1. The presence of potassium and sulfite layers and a minimal weight percentage of sodium and chlorine were observed. The presence of potassium and sulfur compounds leads to rupture, resulting in the breakdown of the polymer when the film peels off externally. The microstructure is accompanied by elemental distributions represented with corresponding color maps. The morphological and elemental map distributions of samples S2 and S3 are shown in Figures 6 and 7. Sample 2 indicates the presence of alkali such as Na, K and Cl, with Na and Cl elements constituting a higher weight proportion. The presence of sodium and chlorine shows the deterioration of the polymer surface, as their concentration is higher in the saliva solution. In sample 3, a small amount of sulfite compound was observed, significantly less than the other samples, indicating a low level of degradation. Furthermore, a significant quantity of phosphate compound was detected, likely indicating the presence of sodium phosphate complex. Phosphates have lower porosity, inhibiting the penetration of invasive particles onto the surface and limiting their deterioration. ²⁸ Figure 8 displays the morphology and EDS mapping of sample S4. The presence of elemental traces like Na, K, Cl, Ca, S, and a trace of phosphate compounds were observed on the sample. The sample S2, which was printed with a thickness of 0.10 mm and a print speed of 25 mm/s, exhibited more degradation compared to the other samples due to the presence of both alkali chlorides and sulfite compound. The field emission secondary electron images reveal that film deterioration was evident wherever potassium and sulfite compound was present. Sample S3, printed at a layer thickness of 0.15 mm and a print speed of 20 mm/s, has better degradation resistance than other samples, indicating that the layers have good bonding strength and thus minimize degradation. This shows the effect of layer thickness and printing speed parameters on sample fabrication.

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Figure 5.

Micrograph of S1 PEEK sample immersed in artificial saliva: (a) micrograph at 50 µm, (b) micrograph at 10 µm, and (c) EDS image and elemental mapping.

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Figure 6.

Micrograph of S2 PEEK sample immersed in artificial saliva: (a) micrograph at 50 µm, (b) micrograph at 10 µm, and (c) EDS image and elemental mapping.

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Figure 7.

Micrograph of S3 PEEK sample immersed in artificial saliva: (a) micrograph at 50 µm, (b) micrograph at 10 µm, and (c) EDS image and elemental mapping.

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Figure 8.

Micrograph of S4 PEEK sample immersed in artificial saliva: (a) micrograph at 50 µm, (b) micrograph at 10 µm, and (c) EDS image and elemental mapping.

The XPS results of all four PEEK samples immersed in artificial saliva for 30 days are shown in Figure 9, with all binding energies calibrated by the C1s peak at 284.8 eV. ²⁹ The survey scan encompassed the range of binding energy from 1100 eV to 0 eV, and the intensity was quantified. The high-resolution spectrum provides the specific elements, their corresponding peak intensity, and the position of the electron orbitals. Since the kinetic energy varies from one element to another, the spectrum is derived by plotting the number of detected electrons per energy interval against their kinetic energy. The C1s analysis revealed a prominent peak at 287.3 eV, indicating the presence of the ketone (C = O) functional group, specifically the PEEK family. An intense phosphorous signal at 131 eV, corresponding to the 2p_1/2 configuration, was detected in sample S3. The detection of phosphate compounds in sample S3, as evidenced by the intense peaks in the O1s and P2p_1/2 spectra, implies that these compounds contribute to the polymer’s resistance to degradation than the other three samples which is in line with the findings from the EDS analysis. The high-energy wave spectra showed the presence of calcium at binding energies of 351 eV and 348.6 eV, corresponding to the 2p_1/2 and 2p_3/2 configurations suggesting the potential existence of CaSO₄ and CaCl₂ in all four samples. At a binding energy of 199.2 eV, a discrepancy in the wave spectrum suggests the likely presence of alkali chlorides, specifically NaCl and KCl. Sample 2 demonstrates the existence of a peak spectrum of salt, chlorine, and calcium elements, which results in increased polymer breakdown, suggesting the influence of rising print speed with low layer thickness. The results of the possible degradation products from the immersion test corresponded with each characterization performed.

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Figure 9.

X-ray photoelectron spectroscopy of PEEK samples immersed in artificial saliva solution.

Characterization of ZrO₂ coated PEEK immersed in artificial saliva

The characterization of zirconia coated PEEK samples immersed in the artificial saliva solution was carried out like the uncoated samples. Figure 10 shows the diffraction pattern obtained from XRD analysis of all four samples. Sample S1 exhibited peaks corresponding to CaSO₄, KHSO₃, KCl, and NaCl, indicating that the smaller layer thickness does not effectively adhere to the coating surface, hence permitting the entry of chemicals into the sample. However, samples S3 and S4, which were printed with a layer thickness of 0.15 mm, do not exhibit any peaks indicative of sulfite compounds. This suggests that the ideal thickness parameter, when combined with a zirconia coating, enhances the resistance of the PEEK polymer to chemical penetration or reaction. Zirconia was detected on the samples at various 2θ intervals, specifically on the lattice planes (011) and (111). Sample S3 and S4 exhibit an extra phase identification of PEEK at a 2θ spacing of 21.5°, with a lattice plane (212), in comparison to samples S1 and S2. This indicates that the immersion in the artificial saliva solution does not significantly influence the basic polymer material. In addition, there were minimal amounts of alkali chloride peaks detected, possibly resulting from their high concentration in the saliva solution.

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Figure 10.

XRD characteristics of ZrO₂ coated PEEK samples immersed in artificial saliva solution: (a) S1, (b) S2, (c) S3, and (d) S4.

Figure 11 shows the micrography analysis of the sample S1 coated with zirconia after immersion in the saliva solution. The analysis revealed additional precipitated layers because of the presence of sulfur, potassium, sodium, and calcium in high concentrations on the sample surface. The microstructure and EDS mapping of the sample S2 are shown in Figure 12. Sodium and chlorine agglomeration and accumulation of P, K, Ca, and Zr elements were observed on the sample surface. The EDS map revealed that Zr was scattered and aggregated in some areas, inhibiting chemical reactions with the polymer, and resulting in a high percentage of carbon weight in the sample. The high percentage of carbon and oxygen shows the presence of base PEEK polymer with no adverse effect on their basic performance. Figures 13 and 14 show the surface micrograph and EDS mapping of sample S3 and S4 respectively. The elemental distribution analysis of samples S3 and S4 revealed a large quantity of carbon weight percentage of around 75.3% and 72.1%, respectively. Furthermore, sample S3 showed a minimal accumulation of precipitated particles with very low concentration and no evidence of chlorine whereas sample S4 showed alkali chloride precipitates probably NaCl and KCl. The energy dispersive spectroscopy analysis of sample S3 and S4 revealed that the dispersion of ZrO₂ on their surface was equivalent which prevents the degradation reaction toward the chemical solution.

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Figure 11.

Micrograph of S1 ZrO₂ coated PEEK sample immersed in artificial saliva: (a) micrograph at 50 µm, (b) micrograph at 10 µm, and (c) EDS image and elemental mapping.

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Figure 12.

Micrograph of S2 ZrO₂ coated PEEK sample immersed in artificial saliva: (a) micrograph at 50 µm, (b) micrograph at 10 µm, and (c) EDS image and elemental mapping.

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Figure 13.

Micrograph of S3 ZrO₂ coated PEEK sample immersed in artificial saliva: (a) micrograph at 50 µm, (b) micrograph at 10 µm, and (c) EDS image and elemental mapping.

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Figure 14.

Micrograph of S4 ZrO₂ coated PEEK sample immersed in artificial saliva: (a) micrograph at 50 µm, (b) micrograph at 10 µm, and (c) EDS image and elemental mapping.

Figure 15 shows the XPS analysis of zirconia-coated PEEK samples immersed in the artificial saliva solution. Like the uncoated samples, coated samples were characterized in the high energy spectrum analysis. There was no identification of the presence of alkali salts in sample S3. However, alkali salts were found in samples S1 and S2, indicating unfavorable printing conditions characterized by a layer thickness of 0.10 mm and a printing speed of 25 mm/s. The high-energy wave spectra revealed the existence of ZrO₂ with a binding energy of 182.5 eV, which corresponds to the electron configuration of 3d_3/2. Furthermore, the analysis revealed the detection of sulfur in sample S1, namely in the 2p_1/2 configuration with a binding energy of 165.6 eV. The S3 sample, coated with zirconia, exhibits a superior spectrum characterized by the absence of irregular peaks, indicating excellent resistance to degradation. This reveals the effect of coating and good printing conditions on deterioration resistance, with improved bonding between each layer opposing foreign particle penetration.

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Figure 15.

X-ray photoelectron spectroscopy of ZrO₂ coated PEEK samples immersed in artificial saliva solution.

Weight measurement analysis

The weight measurement analysis of the coated and uncoated PEEK samples immersed in the artificial saliva solution at different time intervals is shown in Figure 16. Figure 16(a) and (b) illustrate the mass reduction and the deterioration rate for all four samples when immersed in the solution. It was noted that there was an increase in weight over the first 15 days, followed by a decrease in mass observed on day 18. Sample S2 exhibited an increased mass loss of roughly 0.06 g, but sample S3 showed a decreased mass loss of 0.04 g. Although there was little variation in mass loss in response to the initial mass of the sample, the degradation rate was affected. The degradation rate was calculated using the formula ³⁰

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Figure 16.

Weight measurement analysis: (a) mass loss of PEEK samples, (b) degradation rate of PEEK samples, (c) mass loss of ZrO₂ coated PEEK samples, and (d) degradation rate of ZrO₂ coated PEEK samples.

where M_o—initial mass of the sample, M_f—final mass of the sample after immersion over time interval (t).

At regular time intervals of 3 days of up to 30 days, the samples were taken out and dried for a few hours. The resulting mass of each sample was then measured, and the degradation rate was subsequently calculated. From Figure 16(b) it was observed that the PEEK sample S2 exhibited a significant degradation rate of 60%, whereas samples S1, S4, and S3 showed degradation rates of around 54.5%, 45.6%, and 30.8% respectively. The high deterioration rate of sample S2 is due to the accumulation of alkali chloride and potassium bisulfite, which causes the degradation compared to the other samples as evidenced previously in Figure 12. The passive deterioration rate of sample S3 demonstrates the influence of sodium phosphate in preventing degradation. Figure 16(c) and (d) shows the mass change and degradation rate of zirconia coated PEEK samples immersed in the artificial saliva solution. The coated samples showed a mass loss after 27 days and their respective degradation rate was calculated after weight measurement. The results indicated that the sample S3, printed with 0.15 mm layer thickness and 20 mm/s print speed coated with zirconia showed a better degradation rate of 9.01% whereas the other three samples S1, S2 and S4 coated with zirconia showed deterioration rates of 36.4%, 33% and 16% respectively. This low deterioration rate in sample S3 is related to the absence of chlorine, which was previously detected in EDS analysis. The addition of zirconia coating has inhibited the chemical actions on the sample surface. Hence, the degradation rate of PEEK samples was improved with the addition of zirconia coating.

Antibacterial analysis

Figure 17 shows the optical density (OD) values of both PEEK and zirconia coated PEEK samples after the microdilution with E. coli and S. aureus bacteria. The investigation revealed no significant difference in optical density values across the samples. The type of biofilm formation can be determined using the formulation given in Table 4. For uncoated PEEK samples, the optical density control (ODC) values of blank gram negative and gram-positive bacteria were taken as control. By applying the formulation using the given table, the uncoated PEEK samples resulted in weak biofilm formations. For the coated PEEK samples, the optical density values of uncoated PEEK were used as control values. In coated samples there was a reduction in the biofilm formations. In response to the formulation suggested, it could be taken as the coated samples shown better antibacterial property.

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Figure 17.

Antibacterial analysis of PEEK and zirconia coated PEEK samples against E. coli and S. aureus.

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Table 4.

Formulation of biofilm analysis. ³¹

S. No	Formulation	Description
1	OD ⩽ ODC	No formation
2	ODC < OD ⩽ (2 × ODC)	Weak formation
3	(2 × ODC) < OD ⩽ (4 × ODC)	Moderate formation
4	(4 × ODC) < OD	Strong formation

OD: optical density; ODC: optical density control.

Cytotoxic analysis

The assessment of the compatibility of the coated and uncoated samples in a controlled laboratory environment is a necessary procedure to determine the biological effectiveness of the samples. Figure 18 shows the cell viability of the human mesenchymal stem cells on ZrO₂ coated PEEK and uncoated PEEK samples were determined by MTT assay after 48 h of incubation. In comparison to the uncoated PEEK samples, the ZrO₂ coated PEEK samples showed better viability of cells. The increase in cell viability percentage observed in the coated PEEK samples is likely attributed to the presence of surface oxide layers and the generation of oxygen vacancies upon cell contact. ²¹ These factors promote a strong affinity between the functional groups of mesenchymal protein cells, leading to improved cell adhesion and differentiation. Overall, it is evident that even the uncoated PEEK samples demonstrate a cell viability of over 50%, exhibiting that they do not have a toxic effect on human mesenchymal stem cells. Zirconia coated PEEK sample S3 showed a better cell viability of about 79% whereas sample S1, S2 and S4 showed a cell viability of about 61%, 66%, and 78% respectively.

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Figure 18.

Cell viability results of PEEK and ZrO₂ coated PEEK using human cell lines.