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. 2021 Feb 1;46(17):E911–E915. doi: 10.1097/BRS.0000000000003970

Cobalt–Chromium Alloy Has Superior Antibacterial Effect Than Titanium Alloy

In Vitro and In Vivo Studies

Kota Watanabe a, Satoshi Fukuzaki b, Atsushi Sugino c, Nicholas Benson d, Newt Metcalf d, Masaya Nakamura a, Morio Matsumoto a
PMCID: PMC8357039  PMID: 34384089

Bacterial survival on titanium alloy and cobalt–chromium alloy using in vitro and in vivo experiments was evaluated. Cobalt–chromium alloy suppressed Staphylococcus aureus and Propionibacterium acnes proliferation compared with titanium alloy in vitro and in an in vivo infection model.

Keywords: antibacterial, antibacterial testing, biomaterial, cobalt–chromium alloy, infection model, Propionibacterium acnes, spinal instrumentation, Staphylococcus aureus, surgical site infection

Study Design.

In vitro and in vivo laboratory studies.

Objective.

This study aimed to compare bacterial survival on titanium alloy (Ti) and cobalt–chromium alloy (CC) using in vitro and in vivo experiments.

Summary of Background Data.

Spinal implants are frequently manufactured from Ti and CC. These foreign materials are thought to be susceptible to biofilm formation that contributes to the development of surgical site infections. Certain metals (i.e., silver, cobalt) are known to have antibacterial properties.

Methods.

In the in vitro study, discs made of Ti or CC were incubated with one of two common bacteria: Staphylococcus aureus (S. aureus) and Propionibacterium acnes (P. acnes). After incubation, discs were assessed to determine the number of viable bacterial cells. In the in vivo study, the discs that were made of CC or Ti were implanted into the subcutaneous layer of BALB/c mice. After skin closure, a suspension including either S. aureus or P. acnes was directly inoculated on the implanted discs. The discs were retrieved and analyzed to determine the number of viable bacteria at 0.5, 1, and 3 days after inoculation.

Results.

The number of viable S. aureus cultured from the CC discs was 0.9 ± 0.2 × 103 CFU/disc, which was significantly lower than the cultured Ti discs (114.8 ± 18.3 × 103 CFU/disc). Moreover, a significantly lower mean number of P. acnes were cultured with CC (1.9 ± 1.2 × 103 CFU/disc) compared with the Ti (180.0 ± 72.1 × 103 CFU/disc). The in vivo infection model testing against S. aureus or P. acnes showed a significantly lower number of viable S. aureus or P. acnes on CC discs than Ti discs. The result was seen at all measured time points.

Conclusion.

CC suppressed S. aureus and P. acnes proliferation compared with Ti in vitro and in an in vivo infection model.

Level of Evidence: N/A

Spinal instrumentation, such as pedicle screws or rods made of titanium (Ti) alloy or cobalt–chromium (CC) alloy, is an important tool for correcting adult deformity and adolescent idiopathic scoliosis and for providing stabilization until a definitive fusion for many spinal disorders. Although successful clinical results of instrumented spinal surgery have been extensively published, the incidence of surgical site infection (SSI) remains a concern. This concern is consistent across all orthopedic surgeries including joint arthroplasty and fracture surgery.1,2 The incidence of SSI in spinal surgery is reported to be between 0.6% and 11.9%.3–5 It is well documented that SSI results in prolonged hospitalization, increased morbidity, reduced outcomes, and increased economic costs for patients and hospitals.6

SSI risk factors are multifactorial including patient characteristics and pre-, intra-, and postoperative characteristics.7 Among the intraoperative risk factors, foreign materials, such as suture or spinal instruments at the surgery site, are known to increase the risk of SSI in surgery.8–10 Surface modifications to give an antibacterial effect to reduce the risk of foreign materials have been reported. Coatings including silver are common to provide materials with an antibacterial effect, and some researchers have reported experimental results.11–13 Other promising approaches to add an antibacterial effect are polymer coatings including antibacterial agents,14 such as an iodine-incorporated oxidized layer.15,16 Other materials combined with metallic cations, such as copper, zinc, cobalt, and gallium, and silver ions have also shown an antibacterial effect.17,18 To test the hypothesis that CC is expected to show antibacterial effect according to previous reports, we conducted a comparative study of the antibacterial effect of common materials used for spinal implants.

This study aimed to compare the Staphylococcus aureus (S. aureus) and Propionibacterium acnes (P. acnes) survival on Ti and CC used as common spinal implant materials in in vitro and in vivo experiments. S. aureus and P. acnes are common SSI-causing pathogens and have been linked to late-onset screw loosening.19–21

MATERIALS AND METHODS

Specimen

For the in vitro antibacterial testing, discs (15 mm in diameter and 3 mm in thickness) made of either Ti (TI-6Al-4 V, ASTM F136) or CC (Co-28Cr-6Mo, ASTM F1537) were used. A disc (10 mm in diameter) made of polyethylene (Organo Corp, Tokyo, Japan) was used as a control. For in vivo testing, discs (7 mm in diameter and 1 mm in thickness) made from either from Ti or CC were used. To reproduce and validate the widely used implant materials, discs were provided from Medtronic Sofamor Danek Co, Ltd, in similar alloys to currently available spinal implants.

In Vitro Antibacterial Testing

Two types of bacteria, S. aureus (NBRC 12732, National Institute of Technology and Evaluation, Tokyo, Japan) and P. acnes (NBRC 107605, National Institute of Technology and Evaluation, Tokyo, Japan), were used in the in vitro experiments. S. aureus was cultured in a liquid nutrient broth (NB) medium (Nissui Pharmaceutical Co, Ltd, Tokyo, Japan) at 35°C for 48 hours under aerobic conditions. P. acnes was cultured in a liquid Gifu Anaerobic Medium (GAM) broth (Nissui Pharmaceutical Co, Ltd, Tokyo, Japan) at 35°C for 5 days under anaerobic conditions. The cells harvested by centrifugation were washed three times with 0.9% NaCl solution and then suspended in 0.9% NaCl solution. Cell suspensions were diluted with 1/100 NB medium or 1/100 GAM at appropriate dilution and used as the test inoculums.

The in vitro antibacterial testing was conducted according to the Japanese Industrial Method Z 2801:2010 “Antibacterial products–Test for antibacterial activity and efficacy” with minor modifications. To spread the inoculum on the surface and ensure close contact with the material surface, 40 μL of cell suspension was injected onto the discs and then covered with polyethylene film. The discs contacted with S. aureus and P. acnes were incubated at 35°C for 24 hours and then washed out with 4 mL of 0.9% NaCl solution containing 0.7% Tween 80 (Kanto Chemical Co, Inc, Tokyo, Japan). To determine the number of viable cells, serial decimal dilutions of 0.9% NaCl solution were prepared, and 0.1 mL was uniformly spread on an agar plate including appropriate culture mediums. The plates for S. aureus and P. acnes were incubated at 35°C for 24 hours and 5 days under aerobic and anaerobic conditions, respectively. The data of viable cell numbers were counted as colony-forming units per disc (CFU/disc) and calculated antibacterial activity as a log reduction by comparing the number of viable cells on polyethylene with that on each alloy. Mann–Whitney U test with a 5% significance threshold was used to determine the difference of viable cells between CC and Ti. A sample size of 5 was used in the experiments.

In Vivo Infection Model

Two types of bacteria, S. aureus (ATCC 25923, American Type Culture Collection, VA) and P. acnes (NBRC 107605), were used in the in vivo experiments. S. aureus was cultured on Staphylococcus agar (Becton, Dickinson and Company, NJ) at 37°C for 2 days under aerobic condition. P. acnes was cultured on GAM agar (Nissui Pharmaceutical Co, Ltd, Tokyo, Japan) at 37°C for 3 days under the anaerobic condition. Then, the prepared cell suspensions were diluted with a concentration of 2 × 107 CFU/mL by using physiological saline (Otsuka Pharmaceutical Factory, Inc, Tokyo, Japan).

Six-week-old, specific-pathogen-free, female BALB/c mice (Japan SLC Inc, Shizuoka, Japan) were used. A sample size of 10 animals was measured at each time period and study arms. The mice were anesthetized by an intramuscular injection of the mixture of 80 mg/kg ketamine (Daiichi Sankyo Propharma Co, Ltd, Tokyo, Japan) and 10 mg/kg xylazine (Bayer Yakuhin, Ltd, Osaka, Japan). After the back of each animal was shaved and disinfected with alcohol, an approximately 10-mm skin incision was made at the midline of the back, followed by the creation of a subcutaneous pocket for the discs. The discs made of CC and Ti were implanted onto the pocket, and then the surgical site was sutured. Then, 0.05 mL of each of cell suspensions (1 × 106 CFU) was directly injected on the surface of implanted disc. The mice were sacrificed at 0.5, 1, or 3 days with direct inoculation of excessive isoflurane anesthesia of isoflurane. Each removed disc was transferred to a test tube containing 1 mL of Hank's balanced salt solution (Thermo Fisher Scientific, MA), sonicated, and vigorously mixed to collect the S. aureus or P. acnes. The serial diluted suspension of S. aureus was then cultured on Staphylococcus agar plates at 37°C for 2 days under aerobic condition. The suspension of P. acnes was cultured on GAM agar plates at 37°C for 3 days under anaerobic conditions. The number of colonies on the plates was measured. Wilcoxon rank-sum test with a 1% significance threshold was used to determine the difference of viable cells between CC and Ti. A sample size of 10 was examined at each time point for the examined alloys. All experiments were approved by the Committee for the Ethical Treatment of Animals of Nihon Bioresearch Inc (Gifu, Japan) and were conducted in accordance with the guidelines published from the Ministry of Health, Labor and Welfare and Nihon Bioresearch Inc.

RESULTS

In Vitro Antibacterial Efficacy

The results of the in vitro antibacterial testing against S. aureus and P. acnes on Ti and CC are presented in Table 1. The number of viable S. aureus on CC (0.9 ± 0.2 × 103 CFU/disc) was significantly lower than that on Ti (114.8 ± 18.3 × 103 CFU/disc) 24 hours after inoculation (P < 0.05). The reduction ratio of CC alloy and Ti alloy (vs. polyethylene) against S. aureus was 99.9% and 82.6% respectively. The antibacterial activity of CC against S. aureus was calculated using a log reduction comparison polyethylene. The log reduction was measured at 2.9, which means a 99.9% reduction in viable S. aureus on CC surface. The numbers of viable P. acnes on CC and Ti after inoculation for 24 hours were 1.9 ± 1.2 × 103 CFU/disc and 180.0 ± 72.1 × 103 CFU/disc, respectively. Statistical difference in the number of viable P. acnes between Ti and CC was observed (P < 0.05). The reduction ratio of CC alloy and Ti alloy (vs. polyethylene) against P. acnes was 99.4% and 43.4% respectively. The antibacterial activity of CC against P. acnes was 2.2, which is able to reduce to approximately 100th of viable P. acnes in comparison with Ti.

TABLE 1.

Results of the In Vitro Antibacterial Testing Against Staphylococcus aureus and Propionibacterium acnes on Titanium Alloy and Cobalt–Chromium Alloy

Material Staphylococcus aureus Propionibacterium acnes
Viable Cell / × 103 CFU/Disc (Mean ± SD) Antibacterial Activity Viable Cell / × 103 CFU/Disc (Mean ± SD) Antibacterial Activity
Cobalt–chromium alloy 0.9 ± 0.2∗ 2.9 1.9 ± 1.2∗ 2.2
Titanium alloy 114.8 ± 18.3 0.8 180.0 ± 72.1 0.2
Polyethylene 660.0 ± 143.4 N/A 318.0 ± 67.6 N/A
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∗

P < 0.05 (Mann–Whitney U test). A sample size of 5 was used in the experiments. The number of viable S. aureus cultured from the cobalt–chromium (CC) discs was 0.9 ± 0.2 × 103 CFU/disc, which was significantly lower than the cultured Ti discs (114.8 ± 18.3 × 103 CFU/disc). In addition, a significantly lower mean number of P. acnes were cultured with CC (1.9 ± 1.2 × 103 CFU/disc) compared with the Ti (180.0 ± 72.1 × 103 CFU/disc).

In Vivo Antibacterial Efficacy

The number of viable S. aureus on Ti and CC in the in vivo infection model is presented in Figure 1. The number of viable S. aureus on the Ti was increased with extended implantation periods. In contrast, the number of viable S. aureus on the CC was significantly decreased from the 0.5 days after inoculation. The significant reduction of the viable S. aureus on the CC was kept for all implantation periods (P < 0.01).

Figure 1.

Figure 1

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The number of viable Staphylococcus aureus on titanium alloy and cobalt–chromium alloy in the in vivo infection model (data shown as mean ± 2SE).

The number of viable P. acnes on Ti and CC in the in vivo infection model is presented in Figure 2. The number of viable P. acnes on Ti and CC for 0.5 days after inoculation was 2.8 ± 1.1 × 101 CFU/mm2 and 0.5 ± 0.1 × 101 CFU/mm2, respectively. Significant differences were observed in the number of viable P. acnes on CC in comparison with Ti for 0.5 days after inoculation (P < 0.01). The proliferation of P. acnes on the Ti was continued for all implantation time points, but not on CC. Significant difference was also observed in the number of the viable P. acnes on 1 and 3 days after inoculation on the CC in comparison with the Ti.

Figure 2.

Figure 2

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The number of viable Propionibacterium acnes on titanium alloy and cobalt–chromium alloy in the in vivo infection model (data shown as mean ± 2SE).

DISCUSSION

Because CC alloys has high corrosion resistance, wear resistance, fatigue strength, and biocompatibility, they have been widely used for currently available spinal implants. In this study, CC alloy significantly reduced S. aureus and P. acnes proliferation compared with Ti alloy in the both in vitro and in vivo experiments.

The experiments were conducted with two common SSI pathogens, S. aureus and P. acnes. S. aureus, which has been reported as the most common pathogen, is an aerobic bacterium that causes superficial SSI (64%)19 and deep incisional SSI (63%)4 in orthopedic and spinal surgeries, respectively, whereas P. acnes is an anaerobic bacterium that has been found in delayed postoperative spinal infections and late-onset screw loosening for spinal instrumentation.19,20 Shiono et al. reported that P. acnes and P. species are the most frequent pathogens from intraoperative specimens collected during posterior correction surgery.22 They also reported that delayed infection of P. acnes occurred only in the presence of implants in the in vivo experiments.23 Bacteria are initially attached to the material surface in a reversible adhesion. After changes to the irreversible adhesion on the material surface, the bacteria start to proliferate and produce extracellular polysaccharides as biofilms. The formed biofilms prevent the penetration of antimicrobial agents and disable their efficacy. The antibacterial effect of the CC alloy might be expected to suppress the S. aureus and P. acnes proliferation on the surface during initial phases of SSI development and may reduce one of the risks of SSI and delayed postoperative spinal infection and late-onset screw loosening in the spinal instrumentation. Only one clinical report can be found that assessed the effect of implant materials on SSI in pediatric scoliosis surgery, and there are no significant differences in SSI with stainless steel, Ti, or CC alloy/Ti alloy instrumentation.24

Similar studies have been reported with different antibacterial testing methods and/or different species. Patel et al.25 reported that the biofilm formation rate on CC against S. aureus was significantly higher than that on Ti. The testing methodology used small cut rods made of CC or Ti, and the rods were soaked in a culture medium with S. aureus for 24 hours. Optical density values and CFUs were measured to find the results. Conversely, Koseki et al.26 found a lower biofilm coverage rate of S. epidermidis on CC than Ti after culturing for 6 hours. In their experimental method, the bacteria suspension was directly poured on the discs made from various materials. There was no statistical difference between CC and Ti after 2 and 4 hours of culture. Although the experimental methods of these two papers and our in vitro testing methods seem similar, the testing methods were quite different. Direct comparisons are difficult to make leading to the need for an agreed-upon testing standard.

In conclusion, this study demonstrates that CC alloy suppresses S. aureus and P. acnes growth and survival compared with Ti alloy in both standardized in vitro antibacterial testing and in an in vivo infection model. This study has many limitations, such as a small sample size, the use of a single small animal type, the use of CFUs as a proxy for infection strength, and an absence of clinical data. Adequately powered clinical studies are required to investigate whether differences in SSI incidence and screw loosening related to the examined pathogens exist.

Key Points

  • The antibacterial effects of CC alloy and Ti alloy against Staphylococcus aureus and Propionibacterium acnes were measured.

  • CC alloy significantly suppressed Staphylococcus aureus and Propionibacterium acnes proliferation and inhibited survival compared with Ti alloy using both in vitro and in vivo experiments.

  • Clinical studies are required to determine whether differences in surgical site infections exist between Ti and CC implant materials.

Footnotes

The manuscript submitted does not contain information about medical device(s)/drug(s).

Medtronic Sofamor Danek Co, Ltd research funds were received in support of this work.

Relevant financial activities outside the submitted work: employment.

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