1. Introduction
Metals and alloys are widely used in the production of medical implants for use in dentistry, cardiology, orthopedic fractures, etc. Titanium alloys, austenitic stainless steel and chromium–cobalt–nickel alloys are mainly used for implants. First of all, these materials must have adequate biocompatibility, i.e., they must not be irritating, toxic, allergic, inflammatory, mutagenic or carcinogenic to surrounding tissues [
1,
2]. For example, in the case of dental and orthodontic implants, some studies show that biofilms caused by attachment of bacteria are responsible for about 65% of infections such as periodontal disease and peri-implant diseases [
3]. To prevent these adverse effects, surface treatments are used, among which the deposition of coatings by PVD and CVD methods are one of the ways to improve their mechanical and biological properties [
4,
5,
6,
7,
8]. In particular, the coatings should fulfill a number of functions, such as bacterial resistance, i.e., resistance to adhesion and colonization of bacteria [
9,
10]. They should be biocompatible [
11], have adequate thrombogenicity [
12,
13] in cardiac surgery applications and create a suitable diffusion barrier for elements causing allergic reactions in the case of deposition of coatings on medical steels [
4,
14]. Simultaneously, the coatings should have pitting corrosion resistance, a low coefficient of friction and wear resistance, high hardness and low Young’s modulus [
15,
16,
17]. The aforementioned range of properties by which coatings should be characterized is a function of many factors, the most important of which is the chemical composition of coatings and their structure (monolayers, multilayers, nanocomposites, gradient coatings, etc. [
18]). In the case of antibacterial properties, due to the fact that the interaction of the material with bacteria is adhesive, the chemical activity is important (contact angle, free surface energy, etc.) and depends primarily on the chemical composition and type of bonds in the surface layer of coatings deposited on implants. The roughness of the surface is also taken into account.
Promising candidates meeting these requirements are thin coatings made of transition metal carbides. Among them, zirconium-carbide-based coatings are interesting due to their chemical, mechanical and electrical properties [
19,
20,
21,
22]. The microstructure and phase composition of these coatings are mainly influenced by the atomic concentration of carbon as well as the parameters of the coating deposition process [
23,
24]. Relatively significant possibilities of controlling properties are obtained due to the formation of free carbon in the amorphous form surrounding the grains of nanocrystalline carbide [
25], although comparing to coatings based on transition metal nitrides, carbide coatings with higher carbon concentrations are characterized with lower hardness, however, production of coatings with a lower coefficient of friction is possible. Hence, by appropriately changing the phase composition and using the potential of carbon to combine into various structures, there are wide possibilities of shaping the properties of coatings based on Zr–C, which also inspire research on the perspectives of their application on medical implants [
4,
26,
27]. In particular, in the studies presented in [
4], ZrC coatings were deposited at different substrate temperatures (25, 200 and 400 °C) on substrates of 316L steel. It has been shown that the surface energy, hydrophobicity and surface roughness have the greatest influence on the hemocompatibility of these coatings. The authors of [
28] present research on the analysis of antibacterial activity and cell proliferation activity of Zr–C–N coating with different C concentration deposited on titanium (Ti). Coatings with the highest C concentration (22 at.%) have been shown to increase the antibacterial ability against
Staphylococcus aureus, but also meet the requirement for HGF (human gingival fibroblasts) biocompatibility. Tests of CN
x-doped W, Ti and Zr coatings for antibacterial activity and wear are presented in [
6]. It was shown that the CN
x-Zr coating, whose surface had the highest hydrophilicity, provided the best antibacterial effect. However, the CN
x-Zr coating showed lower wear resistance than the CN
x-W and CNx-Ti coatings.
The research presented in this article concerns Zr–C coatings which were deposited on 304L steel by reactive magnetron sputtering from the Zr target in an Ar-C2H2 atmosphere at various mass acetylene flow rates conditioning a specific carbon concentration in the coating. The evolution of phase composition of coatings was studied with increasing carbon concentration and the composition correlated with mechanical, antibacterial and corrosive properties. For comparison, these properties were also tested for 304L steel without a coating. In particular, the correspondence between chemical and phase composition of coatings and biological properties was studied. Corrosion properties and contact angle were tested in an environment that simulates the oral cavity, i.e., artificial saliva, taking into account the possible application of Zr–C coatings on orthodontic wires.
2. Materials and Methods
2.1. Coating Deposition
Zr–C coatings deposited by pulsed, reactive magnetron sputtering on high-speed steel substrates were the object of research previously described by the authors in [
17]. Coatings considered in this paper were deposited on 304L medical steel substrates in the form of discs with a diameter of 32 mm and a thickness of 4 mm in the same deposition processes, methodology which has been described in detail in previous work [
17]. The objects of research in this work are Zr–C coatings with different carbon concentrations obtained at various acetylene mass flow rates during the deposition process. In addition, zirconium coatings deposited without carbon-carrier gas in the working chamber were tested for comparative purposes.
2.2. Structural and Compositional Characterisation
The surface morphology and the elemental distribution in individual coatings were characterized using field emission scanning electron microscopy (FESEM, JEOL, JSM-7100F) with an energy-dispersive X-ray analytical system (Thermo Fisher SEM-EDX).
The concentration of the individual elements in the deposited coatings was determined by wavelength-dispersive X-ray spectroscopy (WDX) using a Thermo Scientific’s Magnaray system with an X-ray microprobe working in a wave mode.
The microstructure of Zr–C coatings was analyzed by means of X-ray diffraction (XRD) using the conventional symmetrical Bragg–Brentano configuration (θ/2θ) with the Co-Kα radiation source on a DRON device. Further data processing was performed by HighScore Plus with ICDD PDF-4+ database software.
The analysis of the structure (type of bonds) of the carbon phase in Zr–C coatings was carried out by Raman spectroscopy using Renishaw in Via with laser excitation of the 534 nm wavelength.
Chemical and phase composition of the coatings was determined by means of X-ray photoelectron spectroscopy (XPS) using a Kratos Axis Supra system with monochromatic Al Kα1,2 radiation of energy 1486.6 eV. The calibration of the photoelectron spectrum was carried out with use of carbon C 1s (284.8 eV). The deconvolution of the XPS spectrum was performed through the fitting of peaks with the GL(30) function (Gauss and Lorenz functions at a ratio 30:70) and sigmoidal asymmetric function.
2.3. Mechanical Evaluation
Hardness measurements were performed by nanoindentation using the Fisherscope HM2000 nanoindenter equipped with the diamond Berkovich indenter. The load–depth indentation curves were obtained in the mode of the linear load increase up to 50 mN. The values of the hardness and the Young’s modulus were determined based on the indentation curves using the Oliver–Pharr model [
29].
2.4. Microbiological Tests
Four different species of microorganisms from the American Pure Culture Collection (Candida albicans ATCC 2091, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Streptococcus mutans ATCC 35668) were used. The antimicrobial activity of the coatings was assessed by direct method based on the (BPR-ECHA-EU) regulation criteria (SN 195920–ASTM E2922), while the evaluation of antifungal activity was performed based on the SN 195921 standard. The fungus culture was carried out on Sabouraud medium, while the bacteria culture was on TSA medium. Test microorganisms were inoculated on the surface of the medium with a suspension concentration of 108 cfu/mL (0.5 Mc Farland); the test samples faced with coatings were placed into the medium after 20 min and incubated at 37 °C for 48 h. After incubation, the growth inhibition zone of the tested microorganisms was evaluated. The criterion for the assessment of fungicidal and bactericidal properties was the presence and size of the zone inhibiting the growth of the tested microorganisms. The tests were carried out in three repetitions for each tested sample and each tested microorganism.
To test the susceptibility of the surface of the coatings to microbial adhesion, a bacterial suspension with a density of 0.5 McFarland was applied to the test specimens and incubated at 37 °C for 24 h. After culturing, the samples were washed three times with distilled water to remove nonadherent cells and then shaken for 15 min in distilled water. Afterwards, the coatings were dried and the 0.5 cm3 of benzidine bis (for observation of live cells) was applied to the surface and incubated for 120 s. After rinsing in distilled water, 0.3 cm3 of propidium iodide (dead cell observation) was placed on the surface of the coatings for 120 s and again washed with water. After drying, the surfaces of the samples were observed using a fluorescence microscope (Motic BA 410 E M, MOTIC ASIA, Hong Kong). For each coating and the observed microorganism, two series of measurements were made and the final total number of microorganisms was the average of ten 1 cm2 fields of view calculated using the Motic Live Imaging (MOTIC ASIA, Hong Kong) module.
The results of microbial adhesion to the coatings were calculated in relation to the control sample (304L sample without coating) using the formula:
where N represents the number of living/dead cells on the sample in cfu/cm
2 and N
0 represents the number of living/dead cells on the uncoated 304L reference sample.
2.5. The Wettability of Coating Surface and Corrosion Behavior
The wettability of the coating surface was determined by measuring the static contact angles through the sessile drop method using F4 Goniometer by Rame-Hart Instrumental Co. Drops with a volume of approximately 4 μL were deposited onto the investigated surfaces using a microsyringe. Fusayama-Meyer’s artificial saliva, with the composition given in
Table 1, was used as a test liquid. The contact angles were measured at 3–5 different points at the surface of each sample. The images were recorded over 300 s and analyzed using the DROPimage CA (Rame-Hart Instrumental Co) program.
The corrosion behavior of substrate/coating systems was evaluated by potentiodynamic polarization tests, which were carried out using an ATLAS 0531 Electrochemical Unit (Atlas-Sollich, Poland). Conventional electrochemical three-electrode cell was used with a sample with the active area of 0.292 cm
2 as the working electrode, saturated calomel electrode (SCE, Hg/Hg
2Cl
2/KCl) as the reference electrode and a platinum sheet as counter electrode. The electrolyte was Fusayama-Meyer artificial saliva. Polarization tests were carried out in a temperature of 25 ± 1 °C at the scan rate of 0.001 V/s. Prior to polarization, the samples were kept in contact with the electrolyte for 1 h at open circuit conditions. Measurements were repeated until obtaining three similar results (polarization curves). Corrosion potential (E
corr), corrosion current density (i
corr) and polarization resistance (R
pol—a value inversely proportional to i
corr) were estimated by the Tafel extrapolation method. Since the anodic curves were influenced by passivation reactions, only the cathodic curves were used to determine i
corr, according to [
30].
4. Discussion
On the basis of hydrophobicity and microbiological properties, i.e., bacteriostatic tests and resistance to bacterial adhesion and colonization, it is clear that the antibacterial properties of the Zr–C coatings studied grow in accordance with the sequence:
As demonstrated in the assessment of the antibacterial properties of the Zr–C coatings tested, their hydrophobicity plays an important role by determining the contact angle. All tested coatings with C concentration above 50 at.% showed a higher contact angle than uncoated 304L steel. Considering the contact angle as a measure of hydrophobicity and at the same time as an assessment of potential antibacterial properties, it can be generally observed that surfaces with Zr–C coatings have better antibacterial properties than surfaces of uncoated 304L steel and that coated with Zr. In particular, a rapid increase in antibacterial properties is observed when the carbon concentration in Zr–C coatings exceeds 50 at.%, while practically no significant differences occur in the bacteriostatic tests as well as resistance to bacterial adhesion and colonization. In this context, it is interesting to compare the results of the chemical and phase composition of the coatings carried out using the XPS method and the participation in coatings sp2 and sp3 bonds determined by Raman spectroscopy. First of all, the results of these tests indicate low or no participation of phases with graphite and diamond carbon bonds in coatings with a carbon concentration below 50 at.%. In turn, in coatings with a carbon concentration above 50 at.%, based on the analysis of Raman spectra it can be concluded that the carbon has an amorphous form with a dominance of sp2 bonds.
In [
40], hydrogenated amorphous carbon (α-C:H) coatings were produced on PET in an acetylene atmosphere at different working pressures. Coatings differing in the share of sp
3/sp
2 bonds were obtained. On the basis of the conducted research it was shown that the adhesion of bacteria to the coatings depends on the structure of the coatings and decreases with decreasing sp
3/sp
2 ratio, i.e., an increase in the share of sp
2 bonds. Additionally, in [
41] it has been proved that the sp
3/sp
2 ratio is an important factor affecting the biological response of carbon coatings. As already mentioned, in the test results presented in this article, the coatings produced above 50 at.% C have the observed advantage of sp
2 bonds over sp
3 bonds, and these relationships, i.e., the ratio of sp
3/sp
2 bonds, do not change with increasing carbon concentration; the antibacterial properties also do not change. These results correspond to the results presented in the cited papers [
40,
41].
In the context of the obtained results characterizing antibacterial properties, it should also be taken into account that in the tested coatings there is a specific hydrogen concentration due to the fact that Zr–C coatings are produced in the C
2H
2 atmosphere. In [
5], it is stated that the presence of hydrogen in carbon coatings deteriorates their antibacterial activity compared to a coating containing no hydrogen. Bacterial adhesion is an interfacial process and therefore electrostatic and van der Waals forces are most likely responsible for the interaction between material and bacterial surfaces. Due to the presence of hydrogen in the carbon layer, polarized C-H bonds are formed, which are promoters of Lifshitz van de Waals forces between the microorganism and the polarized surface of the material [
42]. Thus, on the one hand, the advantage of sp
2 bonds in the coating promotes its antibacterial effect, while on the other hand, the presence of hydrogen may slightly worsen this effect.
The antibacterial properties of the tested coatings were also examined by analyzing the number of cells attached to the coatings. Both live and dead cells were considered. In these studies, it was found that the number of adsorbed cells, both dead and living, decreases significantly after exceeding 50 at.% C compared to uncoated 304L steel and that coated with Zr, i.e., as in other tests, the antibacterial properties of coatings increase significantly after exceeding 50 at.%. In the case of dead cells, studies described in [
43,
44,
45] suggest that the main mechanism for the death of bacteria is the physical interaction with carbon-based coatings. Carbon aggregates can cause irreversible destruction of the outer cell membrane of bacteria that results in the release of cell contents [
43]. The smallest wettability of the coating with a carbon concentration of ~63% and the smallest roughness observed in SEM images may be the reason for the weakest adhesion, i.e., the highest antibacterial effectiveness, of this coating in relation to some bacterial strains.
In summary, due to biological properties, the tested coatings after exceeding 50 at.% C are characterized by higher antibacterial properties both compared to uncoated 304L steel and to coatings with a lower carbon concentration. Taking into account the possibility of shaping a wide range of mechanical properties of these coatings, especially for coatings with a carbon concentration above 50 at.%, it can be concluded that these coatings are a good candidate for orthodontic applications. An additional argument strengthening this conclusion is the result of corrosion tests. In particular, increasing the carbon concentration in the coating above the stoichiometry results in hindered oxidation of the metallic components of the systems. For these coatings, there is also no clear pitting initiation area on the polarization curves (no breakdown potential was determined). This indicates a high protective potential of these coatings against pitting in the environment of artificial saliva.