Multi-Species Biofilm Interactions and Their Impact on the Biocorrosion of Copper-Coated CoCr Alloys in Dental Application

Abstract

Biocorrosion of materials used in dental restorations is a complex process involving various bacterial species that coexist as biofilms. Since copper possesses excellent antibacterial properties, it could help minimize this problem. The aim of this study is to assess the antibiofilm characteristics and corrosion resistance of CoCr and copper-coated CoCr alloys in a multispecies biofilm model. CoCr alloys and CoCr coated with copper (CoCr/Cu) using Physical Vapor Deposition (PVD) were investigated. The samples were incubated in media with and without a multispecies biofilm for 24 h and for 15 days. Potentiodynamic Polarization and Electrochemical Impedance Spectroscopy (EIS) were used to assess the corrosion behavior of the samples. Scanning Electron Microscopy (SEM) was utilized to observe the growth of multispecies biofilms and the type of corrosion. The Mann–Whitney U test was employed to examine corrosion results, with significant differences defined as p < 0.05. CoCr/Cu alloys demonstrated superior corrosion resistance at 24 h and 15 days in the presence of biofilm compared to those without coating. No differences were observed in multispecies biofilm formation at 24 h. The study demonstrates that copper-coated CoCr alloys (CoCr/Cu) exhibit a more positive corrosion potential (E_corr) compared to uncoated CoCr alloys, both in the presence and absence of multispecies biofilm (BP) at 24 h and 15 days. After 15 days, the potential of CoCr/Cu with BP was −0.144 V, compared to −0.252 V for uncoated CoCr. These significant differences in E_corr values underscore the protective effect of copper against corrosion in multispecies biofilm environments.

1. Introduction

Cobalt-chromium alloys (CoCr) are extensively used in dentistry to manufacture metallic structures for removable partial dentures, crowns, and bridges, and have recently been employed in creating metallic substructures for implant rehabilitation [1,2]. One of their major disadvantages is the formation of bacterial plaque [2]. Bacteria can biocorrode these alloys by forming biofilms and producing metabolites [3,4,5]. Corrosion of these devices in the mouth can result in the release of metal ions, which may travel to other parts of the body and potentially cause toxic or allergic reactions, as well as certain forms of cancer [4,6,7,8].

More than 700 bacterial species are predicted to inhabit the oral cavity [4,9]. Therefore, the clinical viability of biomaterials in the mouth depends on their antibacterial properties [10]. Copper (Cu) possesses exceptional antibacterial properties [10,11,12,13]. Although CoCr alloys are bioinert and lack antibacterial characteristics, making them prone to biofilm development and biocorrosion [3,10], the tendency for corrosion can be mitigated by applying protective layers to their surfaces. Utilizing thin antibacterial coatings can effectively minimize and control bacterial adhesion on the surface of biomaterials [14]. A copper coating on dental metals may improve corrosion behavior [10].

To date, there has been no research on biocorrosion in CoCr alloys based on copper coatings; similarly, no studies have been conducted to understand the role of multispecies oral biofilms in the biocorrosion of dental metals through electrochemical tests. The objective of this study was to evaluate the biocorrosion behavior of CoCr alloys, with and without copper coating, in a multispecies oral biofilm model (Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, Prevotella melaninogenica, and Porphyromonas gingivalis) after 24 h and after 15 days. The null hypothesis posits that there is no difference in biocorrosion and multispecies biofilm formation between CoCr and CoCr/Cu alloys after these time periods. Despite the prevalent issues of partial edentulism, dental caries, and periodontal diseases, numerous treatment options based on Cr-Co alloys, such as removable, fixed, and implant prosthetics, significantly enhance oral health-related quality of life. To ensure the longevity of dental restorations, preventing the corrosion of dental materials is crucial.

2. Materials and Methods

Table 1. CoCr alloy characterization.

Starloy C CoCr	Alloy Composition (%w)
	Ni	Co	Cr	W	Nb	V	Si	Mo	Fe
	0.0	59.4	24.5	10.0	2.0	2.0	1.0	1.0	0.1

shows the chemical composition of the alloy examined as reported by the manufacturer. To facilitate comparisons with the manufacturer’s data, element distribution was investigated using energy dispersive X-ray (EDS) analysis.

A total of 36 sheets (7 mm × 7 mm × 1 mm) were obtained using a Struers Accutom-100 cutting machine (Struers, Santiago, Chile) (n = 12 controls, n = 12 for biocorrosion, and n = 12 for biofilm studies). The slides were polished with optical paper, cleaned in an ultrasonic bath for 5 min using ethanol and distilled water, stored in a desiccator for 24 h, and then autoclaved at 121 °C for 15 min before electrochemical pickling. Physical Vapor Deposition (PVD) was used to coat 18 of the 36 sheets with copper. This process involved an evaporator (Ladd Research Industries, Essex Junction, VT, USA) supplying a current of 30–40 A for 20 s at a temperature of 1084 °C. The coatings were characterized using Scanning Electron Microscopy (SEM) and X-ray Energy Dispersion Spectrometry (EDS) with a JEOL JSM 6010 PLUS microscope (JEOL Ltd., Tokyo, Japan).

Static measurements of the water contact angle were conducted using 3µL water droplets and a KRUSS goniometer (SCA SOFTWARE OCA-20, Data Physics Instruments, Filderstadt, Germany) at room temperature. The average value was calculated based on measurements from three samples each of CoCr and CoCr/Cu. All samples were autoclaved at 121 °C for 15 min.

Strains of S. mutans 25175, S. sanguinis 10556, V. parvula 10790, P. gingivalis 33277, and P. melaninogenica 25,845 from the American Type Culture Collection (ATCC) were used to create the multispecies biofilm. Each strain was independently cultured on blood agar plates (TSA II 5% SB, BD & Co., Franklin Lakes, NJ, USA). S. mutans, S. sanguinis, and V. parvula were grown on 5% TSA II plates, while separate cultures of P. melaninogenica and P. gingivalis were grown on 5% TSA II plates supplemented with 5 mg/mL hemin and 1 mg/mL vitamin K1. All strains were incubated at 37 °C for 48 h under anaerobic conditions. Bacterial cells were then transferred to sterile tubes containing 2.5 mL of brain–heart infusion (BHI) media and cultured for another 12 h at 37 °C under anaerobic conditions. The bacterial suspensions were vortexed for 10 s. The optical density (OD600nm) of each bacterial culture was adjusted to 10⁸ cells/mL, and, after correcting for bacterial cell size, a final concentration of 10⁵ cells/150 µL of each bacterium was achieved. The cultures of all five bacterial species were then combined in equal parts. In total, 150 µL of the mixed suspension was added to each of the twenty-four wells containing the slides. The medium was supplemented with 1 µg/mL hemin, 1 µg/mL vitamin K, 3 µg/mL yeast extract, and 10 µg/mL sucrose.

Of the 24 sheets used in the multispecies biofilm development, 12 were allocated for the biocorrosion study. Of these, 6 sheets were incubated for 24 h (3 CoCr and 3 CoCr/Cu) and another 6 for 15 days (3 CoCr and 3 CoCr/Cu). For the biofilm study samples (n = 12), 6 were incubated for 24 h and another 6 for 15 days, respectively, at 37 °C under anaerobic conditions. The medium for the samples incubated for 15 days was changed twice weekly. The controls (n = 12) were divided equally, with 6 incubated in sterile BHI at 37 °C for 24 h and the remaining 6 for 15 days.

Electrochemical tests were conducted on both the controls and the biocorrosion study samples after 24 h and 15 days. Before measurements, the multispecies biofilm was removed using ethanol and distilled water for 5 min in an ultrasonic cleaner.

The specimens were placed in an Avesta type cell, with the test pieces serving as the working electrode (WE), a platinum counter electrode, and a saturated calomel reference electrode (SCE). The electrolytic solution used was artificial saliva at 37 °C; its composition is detailed in

Table 2. Chemical composition of artificial saliva.

Composite	Concentration (g/L)
NaCl	0.40
KCl	0.40
CaCl2	0.60
NaH2PO4·12H2O	0.54
Urea	1.00
Destiller water	Up to 1 L

. The pH of this solution was controlled and maintained within a range close to neutrality, specifically between 6.8 and 7.2. This range is representative of the typical oral environment and was chosen to simulate physiological conditions relevant to dental applications.

Electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization studies were conducted on the samples. The EIS parameters included a frequency range of 100 kHz to 50 Hz, 10 measurements per decade, and an amplitude of 10 mV. All data were analyzed using VersaStudio 2.49.2 (AMETEK, Inc., Berwyn, PA, USA) and ZsimpWin 3.60 (AMETEK, Inc., Berwyn, PA, USA) software (version v3.60).

A scanning electron microscope (SEM) linked to an EDS unit (JEOL JSM 6010 PLUS, JEOL Ltd., Tokyo, Japan) was used to analyze the surface and morphology of the biofilm on the samples, with an acceleration voltage of 20 kV and magnifications of 500× and 2500×. The samples were cleaned in phosphate-buffered saline (PBS) and fixed in 2% glutaraldehyde for 30 min prior to microscopic examination. They were then rinsed in PBS and dehydrated for 10 min in a series of graded ethanol solutions (30, 50, 70, and 90%). Finally, the samples were dried with anhydrous ethanol and coated with a gold–platinum (Au-Pt) layer before SEM analysis. Statistical analyses were conducted using SPSS v16.0 software (version v16.0) (IBM, Armonk, NY, USA). The Mann–Whitney U test was employed, and significant differences were considered at p < 0.05.

3. Results

Figure 1 and Figure 2 illustrate the morphological characteristics of the surface and the EDS spectra of CoCr and CoCr/Cu. The EDS analyses enabled the determination of the element distribution in the samples. Figure 3 summarizes the water contact angle measurements on CoCr and CoCr/Cu samples. The average contact angle for the copper-coated samples was 73.4°, while it was 85.2° for the uncoated samples, indicating a tendency towards hydrophilicity in both cases.

There were no significant differences between CoCr and CoCr/Cu after 24 h of exposure to the multispecies biofilm (Figure 4a,b). However, after 15 days, significant changes were observed; CoCr/Cu exhibited minimal biofilm growth, reduced bacterial size, and localized corrosion compared to CoCr (Figure 4c–e). When compared to uncoated samples, CoCr/Cu alloys with multispecies biofilm demonstrated superior corrosion potential (E_corr) at both 24 h and 15 days. A similar result was obtained when comparing samples without biofilm over the same periods (see Figure 5).

Table 3. Corrosion potential of the studied samples.

Sample	Potential/Ecorr (V)	p
CoCr (BP-15D)	−0.252	0.025 *
CoCr/Cu (BP-15D)	−0.144
CoCr (BP-24H)	−0.199	0.025 *
CoCr/Cu (BP-24H)	−0.084
CoCr (SBP-24H)	−0.126	0.025 *
CoCr/Cu (SBP-24H)	−0.065
CoCr (SBP-15D)	−0.156	0.025 *
CoCr/Cu (SBP-15D)	−0.085

* Statistically significant differences (p < 0.05). (BP: Biofilm with bacteria, SBP: Biofilm without bacteria).

shows that the E_corr value for CoCr migrated towards more negative values compared to the copper-coated alloys. The difference in corrosion potential between the coated and uncoated samples was statistically significant (p < 0.05), with all copper-coated samples performing better.

Significant changes in corrosion current density (i_corr) were discovered depending on the presence or absence of Cu coating (p < 0.05) (

Table 4. Corrosion current density.

Sample	Corrosion Current Density/Icorr (mA)	p
CoCr (BP-15D)	4.61 × 10−9	0.025 *
CoCr/Cu (BP-15D)	2.76 × 10−9
CoCr (BP-24H)	3.58 × 10−9	0.025 *
CoCr/Cu (BP-24H)	9.24 × 10−9
CoCr (SBP-24H)	3.95 × 10−9	0.025 *
CoCr/Cu (SBP-24H)	1.69 × 10−9
CoCr (SBP-15D)	5.36 × 10−9	0.025 *
CoCr/Cu (SBP-15D)	8.93 × 10−9

* Statistically significant differences (p < 0.05). BP: Biofilm with bacteria, SBP: Biofilm without bacteria).

).

Except for the CoCr/Cu samples with biofilm at 24 h and without biofilm at 15 days, whose corrosion rate was 1.6 times higher than those without coating, EDS analyses revealed a lack of copper and an uneven distribution of the element (Figure 6 and Figure 7). Impedance measurements on CoCr/Cu samples showed values that were 1 to 2 times higher than those without coating at both 24 h and 15 days, in both the presence and absence of biofilm, with the exceptions of CoCr/Cu with biofilm at 24 h and CoCr/Cu without biofilm at 15 days (

Table 5. EIS data of the studied samples.

Samples
|Z|0.1 Hz	|Z|0.1 Hz	p
CoCr (SBP-24H)	CoCr/Cu (SBP-24H)	0.513
3.07 × 10−7	5.57 × 10−7
CoCr (BP-24H)	CoCr/Cu (BP-24H)	0.513
3.32 × 10−7	1.50 × 10−7
CoCr (SBP-15D)	CoCr/Cu (SBP-15D)	0.827
1.72 × 10−7	1.18 × 10−6
CoCr (BP-15D)	CoCr/Cu (BP-15D)	0.050
1.79 × 10−7	4.57 × 10−7

). Figure 8 presents the Bode diagrams obtained at 24 h and 15 days for both CoCr and CoCr/Cu samples.

Lower corrosion currents (I_corr) are observed in CrCo samples with biofilm compared to other conditions, which could be due to several factors. The biofilm can act as a physical barrier, reducing the direct exposure of the material to the corrosive environment and, consequently, the corrosion rate. Additionally, the microorganisms in the biofilm can consume oxygen, reducing its availability at the metal surface, which decreases the rate of cathodic reactions and, consequently, the corrosion current. They can also alter the local pH, creating conditions less favorable for corrosion, and produce compounds that inhibit corrosion by adsorbing onto the metal surface. In the specific context of the study, copper-coated CrCo (CrCo/Cu) samples demonstrated greater corrosion resistance compared to uncoated samples. However, it was observed that, in certain cases, samples with biofilm showed a lower corrosion current, possibly due to the factors mentioned above, highlighting the complexity of biocorrosion processes and the variable effect of biofilms on the corrosion of metallic materials.

4. Discussion

Single-species biofilm models are a widely used global research tool to simulate real oral biofilms. However, due to the diversity of species found in the oral cavity, they still lack sufficient precision [15]. Unlike previous studies that demonstrated the effectiveness of copper in inhibiting the formation of monospecies biofilms at 24 h, either as an element incorporated in the production of new alloys or as a coating, this study reveals the ineffectiveness of copper in inhibiting the formation of multispecies biofilms at the same timeframe [10,11,13]. There were no significant differences in the formation of multispecies biofilms at 24 hours between CoCr and CoCr/Cu in this study (Figure 4a,b). This discrepancy in the results observed in the formation of multispecies biofilms in the CoCr/Cu samples at 24 h, compared to earlier studies on monospecies biofilms [10,11,13], could be attributed to the fact that biofilms composed of two or more species, as in the current study, produce more extracellular matrix. This results in limited diffusion of antimicrobial substances into microbial cells; additionally, they are more resistant to antibacterial agents, as bacterial interactions affect the formation, function, and structure of biofilms [15]. Five types of bacteria were used in this study, which could explain these results at 24 hours. However, SEM analysis indicated that at 15 days, CoCr/Cu exhibited minimal biofilm formation and altered bacterial morphology compared to CoCr (Figure 4c,d). Taking into account copper’s suppressive influence on multispecies oral biofilm growth over time, Cu⁺² ions are effective antibacterial and biofilm inhibitors. The findings of this study are consistent with those of Liang T. et al. [12], who developed copper coatings on Ti-6Al-4V alloys through microarc oxidation (MAO), demonstrating good long-term antibacterial capabilities against S. aureus and E. coli strains.

The contact angle of water on a hydrophilic surface typically ranges between 10° and 90°, whereas on a hydrophobic surface, it is between 90° and 150° [16]. Bacterial adhesion is more pronounced on hydrophobic surfaces [17]. Prior to biofilm formation, water contact angle measurements indicated that both CoCr and CoCr/Cu specimens are hydrophilic (Figure 3). Consequently, the effectiveness of the coating in preventing biofilm formation can be attributed to the presence of Cu⁺² ions [11,18,19]. Biofilm formation is considered the primary cause of microbiologically induced corrosion. Therefore, selecting appropriate materials to mitigate biofilm formation is crucial in material design [19]. The results from potentiodynamic polarization curves show that CoCr/Cu alloys demonstrated significantly more positive corrosion potential (E_corr) compared to uncoated specimens; specifically, CoCr/Cu exhibited enhanced corrosion resistance over periods of 24 h and 15 days, indicating that copper positively affects corrosion potential (Table 3, Figure 5). Additionally, significant differences in corrosion current density (I_corr) were observed between CoCr and CoCr/Cu samples in the presence of a multispecies biofilm (Table 4). I_corr, which correlates with corrosion rate [6], was lower in the CoCr/Cu samples, suggesting that they corrode more slowly than uncoated CoCr at 15 days. This finding underscores the complex nature of corrosion, influenced by interactions among anaerobic and aerobic microorganisms [14]. This study aligns with previous research on monospecies biofilms [6,19]. Similar findings were reported by Ameer et al. [6] for NiCr alloys coated with chitosan, hydroxyapatite, and TiO₂ nanocoatings (CS/TiO₂/HA), which displayed superior E_corr and I_corr values in the presence of S. mutans at 24 h and 6 days compared to uncoated samples. Additionally, Zhao et al. [19] found comparable results when testing 304 SS stainless steel and copper-alloyed 304 SS stainless steel in the presence of S. mutans over 14 days in electrochemical tests.

In the evaluation of E_corr and I_corr in CoCr and CoCr/Cu samples, both in the absence of a multispecies biofilm, the CoCr/Cu demonstrated superior performance at both 24 h and 15 days (Table 3 and Table 4). Ameer et al. [6] found similar results in their study comparing NiCr samples with and without a chitosan, hydroxyapatite, and TiO₂ nanocoating (CS/TiO₂/HA), in the absence of S. mutans at 24 h and 6 days. Liang et al. [2] assessed the corrosion resistance of CoCr alloys with and without silver coating, noting slightly improved outcomes in the coated samples in the absence of biofilm. Similarly, Zhao et al. [19] observed comparable results in 304 SS stainless steel and copper-alloyed 304 SS stainless steel without the presence of S. mutans during 14-day electrochemical tests. Additionally, electrochemical impedance spectroscopy (EIS) measurements in CoCr/Cu samples showed higher values compared to plain CoCr both in the absence and presence of biofilm at 24 h and 15 days (Table 5), suggesting enhanced performance of the coating [6,20]. Ameer et al. [6] reported similar findings in NiCr samples with and without (CS/TiO₂/HA) nanocoating, both in the absence and presence of S. mutans at 24 h and 6 days. Zhao et al. [19] also found analogous outcomes with 304 SS stainless steel with and without copper in both scenarios over 14 days.

Pitting corrosion was noted in the CoCr and CoCr/Cu samples immersed in a multispecies biofilm at 15 days; however, the extent of pitting was significantly reduced in samples with a copper coating (Figure 4d,e). Previous studies have shown that bacteria like S. mutans and S. sanguinis lower the pH of the environment, leading to pitting corrosion in alloys such as NiTi and Ti [9,21]. The addition of copper not only reduces biofilm coverage, but also mitigates the tendency towards pitting corrosion, thanks to the continuous release of Cu ²⁺ ions [11,19]. Exceptions were noted in CoCr/Cu samples with a biofilm at 24 h and without a biofilm at 15 days, which exhibited significant pitting due to the uneven distribution of copper (Figure 6 and Figure 7).

One limitation of the study was the sample type, which did not closely resemble actual Cr-Co restorations. Further research involving real Cr-Co restorations, such as removable partial dentures, is necessary to explore the role of copper as an adjunct in the long-term management of multispecies oral biofilms.

It is true that a more positive corrosion potential generally indicates a lower tendency to corrode at that potential. However, the corrosion current (I_corr) is a more direct and precise measure of the actual corrosion rate. In our study, although copper-coated CrCo (CrCo/Cu) samples showed a more positive corrosion potential, it is possible that the specific conditions of the biofilm affected the observed corrosion current. The presence of biofilms can influence ion distribution and the electrochemical behavior of surfaces, which could explain the discrepancies in the I_corr values. We will continue investigating these effects to better understand the relationship between corrosion potential and corrosion current in the presence of multispecies biofilms.

It is also important to mention that, during our corrosion experiments, the concentration of leached copper in the test medium, which consisted of artificial saliva, was evaluated. Measurements were taken using atomic absorption spectroscopy to determine the concentration of copper ions released into the solution. The results showed that the concentration of leached copper was consistently low, remaining below the maximum contaminant level (MCL) allowed in drinking water, which is 1.3 mg/L according to the United States Environmental Protection Agency (EPA). Specifically, the measured copper concentrations ranged from 0.05 to 0.15 mg/L for both samples incubated for 24 hours and those incubated for 15 days. These values indicate that the copper leaching from the coated CoCr alloys.

5. Conclusions

This study evaluated the interaction of multispecies biofilms and their impact on the biocorrosion of copper-coated CoCr alloys for dental applications. It was observed that CoCr/Cu alloys exhibited a more positive corrosion potential (E_corr) and a lower corrosion rate (I_corr) compared to uncoated alloys, both in the presence and absence of biofilm, demonstrating greater corrosion resistance. However, in some cases, the presence of biofilm reduced the corrosion current due to the formation of a protective layer and the consumption of oxygen by the microorganisms. The copper coating also inhibited the formation of biofilms and decreased the incidence of pitting corrosion. These findings suggest that copper enhances the durability and corrosion resistance of CoCr alloys in oral environments, providing additional benefits by reducing the formation of biofilms and the release of metal ions.