Antibacterial effect and biological reaction of calcium phosphate cement impregnated with iodine for use in bone defects

Introduction

Calcium phosphate cement (CPC) is used to address bone defects after the treatment of bone tumors and fractures.^1–4 The main characteristics of CPC are the following: (1) it may be easily molded according to the shape of a bone defect; (2) it has excellent bone conduction and biocompatibility; ⁵ (3) its chemical stability allows the incorporation of various amounts of drugs within its structure; and (4) it presents an excellent sustained-release capacity. ⁶ Hence, CPCs impregnated with antibiotics are used in the treatment of osteomyelitis.^7–9

The antibacterial spectrum of povidone-iodine is particularly broad because it acts not only on general bacteria but also on viruses, tubercle bacilli, and fungi. ¹⁰ Previous studies in our group describe the in vitro antibacterial activity, in vitro cytocompatibility, and in vivo safety characteristics of iodine-supported titanium implants.^11,12 Clinical trials have also demonstrated the safety and effectiveness of these implants when used in patients.^13–16

To treat cases of bone defects that present a high infection risk, developing CPCs that present long-lasting wide-spectrum antibacterial effects is vital. Though there have been some reports of CPC containing antibiotics, no report of CPC with iodine has been described. Therefore, this study describes the antibacterial effect and biological reaction of CPCs impregnated with iodine. The proposed CPCs were impregnated with various amounts of iodine and subjected to in vitro and in vivo experiments to evaluate their efficacy.

Materials and methods

In vitro assessment of iodine contents in CPCs impregnated with iodine

The attenuation of iodine from CPCs with various iodine contents (2.5, 5, and 20%) was analyzed. Each implant was immersed in vials containing 10 mL phosphate-buffered saline (PBS; pH 7.4) and placed inside an incubator maintained at a temperature of 37°C. The PBS in each vial was replaced with new PBS every 24 h. After specified time periods (0, 3, 12, and 24 h, and 1, 2, 3, 5, and 8 weeks), each CPC specimen was removed, and its iodine content was measured using X-ray fluorescence spectroscopy (XFS; Prostec Instruments Company, Sabae, Japan) and averages were obtained for three CPCs.

In vitro antibacterial assessment

Two types of CPC were prepared with different iodine contents: 0 %-iodine CPC, 5 %-iodine CPC; they were analyzed over specified periods (0 h, and 1 and 8 weeks). The antibacterial activity of the CPCs was measured using a method approved by Japanese Industrial Standards, as described in the literature (refer to Figure 1). ¹¹ This procedure was performed in sterility controls. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 700926 were grown overnight in Tryptic Soy Agar (TSA, Bacto^TM, Becton Dickson).^17,18 Approximately 10⁶ colony-forming units were inoculated on a cubic CPC sample placed in a sterile glass Petri dish, which was then covered and incubated at 37°C for 2, 6, or 24 h (n: Number of the samples = 4 per implant type). At each time point, each CPC sample was washed with 5 mL of PBS using a vortex mixer for 1 min. One hundred μL of the washed eluate was diluted to 1 mL of PBS, and 100 μL of the dilute eluate was incubated in TSA at 37°C. The number of bacterial colonies was counted manually using a microscope after 24 h, and the average number of colonies for the triplicate implants at different incubation periods (2–24 h) was used to determine specimen antibacterial activity against S. aureus and E. coli.

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Figure 1.

Antibacterial assessment using a modified version of the Japanese Industrial Standards method.

In vitro cytocompatibility assessment

Cytocompatibility was assessed by counting the number of colonies of the V 79 cell line (Chinese hamster fibroblast) provided by the RIKEN BioResource Center Cell Bank (Tsukuba, Japan). The culture medium consisted of an alpha-minimum essential medium (a-MEM) supplemented with 10% fetal calf serum (FBS), 100 U ml⁻¹ penicillin, and 100 μg ml⁻¹ streptomycin sulfate. Experiments were conducted in an incubator at 37°C with a humidified atmosphere of 95% air and 5% CO₂ for 24 h. The extraction liquid was prepared by soaking CPC with various iodine contents (0, 5%) in 10 mL a-MEM for 24 h. A cell suspension of trypsinized subcultured V79 cells was diluted from 10⁶ to 10² cells ml⁻¹. Next, 2 mL of the cell suspension were seeded on 6 mL of the extraction liquid in dishes, to provide 300 cells per dish. Control dishes that did not contain CPCs were also prepared. After seeding, the dishes were gently shaken and cultured in the incubator. After one week, the medium was extracted, and the cells were fixed with 5 mL 10% formalin for 30 min, stained with 8 mL of 0.15% methylene blue for an additional 30 min, washed thoroughly, and dried. Colony formation in the dishes was compared manually using a microscope with that in the control dishes by counting the number of colonies present (n = 3 per each group).^11,19,20

Results

In vitro assessment of iodine content in CPC impregnated with iodine

Of all the specimens, the 5 %-iodine CPC samples presented the highest iodine concentration after one week (p < 0.05) (Figure 2). 48.6% of the iodine content remained in the 5%-iodine CPC samples after one week, and 15% after eight weeks.

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Figure 2.

In vitro temporal profile of iodine content in CPC, showing an initially rapid attenuation, followed by a more gradual attenuation. The 5%-iodine CPC presented the highest iodine concentration after one week (p < 0.05).

In vitro antibacterial assessment

The 5%-iodine CPC inhibited colony formation of both S. aureus and E. coli compared with 0 %-iodine CPC. Fewer colonies formed on the 5%-iodine CPC samples within the specified periods (0 h, 1 and 8 weeks) at all time points (p < 0.05) (Figures 3 and 4). The antibacterial effects of the 5 %-iodine CPC samples were maintained for up to eight weeks.

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Figure 3.

In vitro antibacterial activities of CPCs with different iodine concentrations on Staphylococcus aureus. The antibacterial effect was maintained for up to eight weeks with the 5%-iodine CPC.

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Figure 4.

In vitro antibacterial activities of CPCs with different iodine concentrations on Escherichia. Coli. The antibacterial effect was maintained until eight weeks with the 5%-iodine CPC.

In vitro cytocompatibility assessment

There was no difference in the number of colonies between any of the specimen groups. The 5%-iodine CPC samples exhibited no obvious cytotoxicity (Figure 5).

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Figure 5.

In vitro cytocompatibility assessments of CPCs with different iodine concentrations using V79 cells. Control, 0%-iodine CPC, 5%-iodine CPC. There were significantly no differences between the amounts of colony formation in each group.

In vivo osteoconductivity assessment

There were no significant differences between the bone implant percentages of each group (Figure 6). There was space present between the 5%-iodine CPC and bone at 2 weeks after surgery. However, some contact areas were confirmed at eight weeks after surgery (Figure 7). After HE staining, immature bone tissue around the 5%-iodine CPC was shown at two weeks after surgery. The shape was round because the bone tissue contacted to a few areas of the CPC. Also, mature bone tissue around the 5%-iodine CPC was shown at eight weeks after surgery. The shape was almost square because the bone tissue contacted to about 50% of areas of the CPC. (Figure 8). The 5%-iodine CPC did not suppress bone conductivity.

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Figure 6.

In vivo osteoconductivity assessment using a scanning electron microscope. The bone implant percentages did not differ significantly between each group.

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Figure 7.

In vivo osteoconductivity assessments using an scanning electron microscope. A: 2 weeks after surgery, and B: 8 weeks after surgery. There was space between 5%-iodine CPC and the bone at two weeks after surgery. However, some contact areas were confirmed.

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Figure 8.

In vivo osteoconductivity assessment using HE staining. A: 2 weeks after surgery. Immature bone tissue around the 5%-iodine CPC was shown at two weeks after surgery. The shape was round because the bone tissue contacted to a few areas of the CPC. B: 8 weeks after surgery. Mature bone tissue around the 5%-iodine CPC was shown at eight weeks after surgery. The shape was almost square because the bone tissue contacted to about 50% of areas of the CPC.

Discussion

Osteomyelitis is defined as inflammation of the bone and marrow cavity. ²² The prevalence of osteomyelitis in the human population is increasing along with the prevalence of diseases such as diabetes. Kremers et al. ²³ reported that rates of osteomyelitis have increased with each calendar year, from 11.4 cases per 100,000 person-years in the period from 1969 to 1979 to 24.4 per 100,000 person-years in the period from 2000 to 2009. Chronic osteomyelitis is one of the most severe problems addressed by orthopedic surgeons, increasing both the risks of morbidity and long-term mortality.^24,25 Despite long-term antibiotic treatment and temporary healing with remission of osteomyelitis, there is a high relapse rate of approximately 30%, and in cases involving Pseudomonas aeruginosa, the recurrence rate is as high as 50%. ²⁶ High recurrence rates or the poor control of osteomyelitis result in prolonged times required for second-stage bone grafting, and an excessively long treatment cycle that increases the suffering and financial costs of patients. ²⁷ Even in current practice, intractable severe osteomyelitis is still treated with amputation.^23,28

The insufficiency of general antibiotics used to combat resistant bacteria in infections such as osteomyelitis is well known.^29,30 Povidone-iodine has a wide antibacterial spectrum and does not produce resistant bacteria.^10,31 Osteomyelitis is a largely drug-resistant bacterial infection such as methicillin-resistant S. aureus that produces polysaccharide protein complexes and forms a biofilm to protect bacteria inside the film. ³² Once a biofilm is formed, the effectiveness of antibiotics is significantly reduced. Iodine-supported implants have the potential to reduce or inhibit bacterial attachment and, therefore, biofilm development. ¹⁷ In addition, they retain their iodine content and antibacterial activity against methicillin-sensitive S. aureus, methicillin-resistant S. aureus, E. coli, and P. aeruginosa for up to eight weeks.^11,12,18 Iodine is also a trace metal and an essential component of the thyroid hormone. It can be excreted by the kidneys and often used for contrast-enhanced CT. In a clinical study performed on iodine-supported implants, thyroid-stimulating hormone (TSH), free triiodothyronine (FT3) and free thyroxine (FT4) levels were within the normal range during the study period.^10,33 Therefore, povidone-iodine is a key tool that may be used in the treatment of infection.

In general, antibiotic therapy for 4−8 weeks is required to treat osteomyelitis.^30,34 The results of this study show that 5%-iodine CPC had an antibacterial effect on S. aureus and E. coli for up to eight weeks. The management of antibiotic toxicity and dead space is key to the effective treatment of osteomyelitis.^35,36 In this study, 5%-iodine CPC had showed no obvious cytotoxicity to V79 cells and presented good bone affinity. In addition, povidone-iodine has a wide antibacterial spectrum and does not produce resistant bacteria.^10,31 Therefore, CPC impregnated with iodine may have the potential to be applied to bone defect cases with a high risk of infection.

This study presented certain limitations. First, the long-term safety and efficacy of the proposed CPC implant impregnated with iodine after more than eight weeks was not evaluated. Second, biofilm study was not performed. In the future, we will perform it refer to past our study. ¹⁷ Third, its mechanical strength was not assessed. Among this research of In vitro assessment of iodine content in CPC impregnated with iodine, 5%-iodine CPC had a higher concentration of iodine at one week compared to 20%-iodine CPC. The strength of 20%-iodine CPC is considered weaker than 5%-iodine CPC. Fourth, the infection model used in this study was not previously validated and tested. However, 5%-iodine CPC presented antibacterial activity in contact with S. aureus and E. coli for up to eight weeks. In addition, it presented showed no obvious cytotoxicity to V79 cells and revealed good bone affinity.

Conclusion

The proposed 5%-iodine CPC presented antibacterial activity when in contact with S. aureus and E. coli for up to eight weeks. In addition, it presented no obvious cytotoxicity and revealed a good bone affinity. Though further research will be required before it can be used in patients, CPC impregnated with iodine may be considered an effective option in the management of bone defects in cases with a high risk of infection.