Antibacterial Activity in Iodine-coated Implants Under Conditions of Iodine Loss: Study in a Rat Model Plus In Vitro Analysis

Abstract

Background We developed iodine-coated titanium implants to suppress microbial activity and prevent periprosthetic joint infection (PJI); their efficacy was demonstrated in animal and in vitro models. The iodine content in iodine-coated implants naturally decreases in vivo. However, to our knowledge, the effect of reduced iodine content on the implant’s antimicrobial activity has not been evaluated to date. Questions/purposes (1) How much does the iodine content on the implant surface decrease after 4 and 8 weeks in vivo in a rat model? (2) What effect does the reduced iodine content have on the antimicrobial effect of the implant against multiple bacteria in an in vitro model? Methods This experiment was performed in two parts: an in vivo experiment to determine attenuation of iodine levels over time in rats, and an in vitro experiment in which we sought to assess whether the reduced iodine content observed in the in vivo experiment was still sufficient to deliver antimicrobial activity against common pathogens seen in PJI. For the in vivo experiment, three types of titanium alloy washers were implanted in rats: untreated (Ti), surface-anodized to produce an oxide film (Ti-O), and with an iodine layer on the oxidation film (Ti-I). The attenuation of iodine levels in rats was measured over time using inductively coupled plasma-mass spectrometry. Herein, only the Ti-I washer was used, with five implanted in each rat that were removed after 4 or 8 weeks. For the 4- and 8-week models, two rats and 15 washers were used. For the in vitro study, to determine the antibacterial effect, three types of washers (Ti, Ti-O, and Ti-I) (nine washers in total) were implanted in each rat. Then, the washers were removed and the antibacterial effect of each washer was examined on multiple bacterial species using the spread plate method and fluorescence microscopy. For the spread plate method, six rats were used, and five rats were used for the observation using fluorescence microscopy; further, 4- and 8-week models were made for each method. Thus, a total of 22 rats and 198 washers were used. Live and dead bacteria in the biofilm were stained, and the biofilm coverage percentage for quantitative analysis was determined using fluorescence microscopy in a nonblinded manner. Ti-I was used as the experimental group, and Ti and Ti-O were used as control groups. The total number of rats and washers used throughout this study was 24 and 213, respectively. Results Iodine content in rats implanted with Ti-I samples decreased to 72% and 65% after the in vivo period of 4 and 8 weeks, respectively (p = 0.001 and p < 0.001, respectively). In the in vitro experiment, the Ti-I implants demonstrated a stronger antimicrobial activity than Ti and Ti-O implants in the 4- and 8-week models. Both the median number of bacterial colonies and the median biofilm coverage percentage with live bacteria on Ti-I were lower than those on Ti or Ti-O implants for each bacterial species in the 4- and 8-week models. There was no difference in the median biofilm coverage percentage of dead bacteria. In the 8-week model, the antibacterial activity using the spread plate method had median (interquartile range) numbers of bacteria on the Ti, Ti-O, and Ti-I implants of 112 (104 to 165) × 105, 147 (111 to 162) × 105, and 55 (37 to 67) × 105 of methicillin-sensitive Staphylococcus aureus (Ti-I versus Ti, p = 0.026; Ti-I versus Ti-O, p = 0.009); 71 (39 to 111) × 105, 50 (44 to 62) × 105, and 26 (9 to 31)× 105 CFU of methicillin-resistant S. aureus (Ti-I versus Ti, p = 0.026; Ti-I versus Ti-O, p = 0.034); and 77 (74 to 83) × 106, 111 (95 to 117) × 106, and 30 (21 to 45) × 106 CFU of Pseudomonas aeruginosa (Ti-I versus Ti, p = 0.004; Ti-I versus Ti-O, p = 0.009). Despite the decrease in the iodine content of Ti-I after 8 weeks, it demonstrated better antibacterial activity against all tested bacteria than the Ti and Ti-O implants. Conclusion Iodine-coated implants retained their iodine content and antibacterial activity against methicillin-sensitive S. aureus, methicillin-resistant S. aureus, and P. aeruginosa for 8 weeks in vivo in rats. To evaluate the longer-lasting antibacterial efficacy, further research using larger infected animal PJI models with implants in the joints of both males and females is desirable. Clinical Relevance Iodine-coated titanium implants displayed an antibacterial activity for 8 weeks in rats in vivo. Although the findings in a rat model do not guarantee efficacy in humans, they represent an important step toward clinical application.

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“…When applied to orthopedic implants, it is desirable to have antimicrobial performance even after the installation. Therefore, antimicrobial activity can be imparted in addition to the photocatalytic activity of the anodized TiNbSn alloys in the application of TiNbSn alloys to hip and knee joint replacement prostheses by loading an active antimicrobial substance onto a porous anodic oxide film [ 42 , 43 ]. The development of anodic oxidation techniques for the TiNbSn alloys is expected to lead toward developing biomedical materials with antimicrobial activities.…”

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

“…Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 700926 were grown overnight in Tryptic Soy Agar (TSA, Bacto TM , Becton Dickson). 17,18 Approximately 10 6 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.…”

CORR Insights®: Antibacterial Activity in Iodine-coated Implants Under Conditions of Iodine Loss: Study in a Rat Model Plus In Vitro Analysis

“…The study by Ueoka and colleagues [6] in this month's Clinical Orthopaedics and Related Research ® evaluated the antibacterial activity of iodine-coated titanium implants using a rat model with additional in vitro analyses. Specifically, the authors sought to answer two important questions regarding the use of antibacterial implants: How much does the iodine content decrease over time, and what is the antimicrobial effect of the reduced iodine content?…”

“…For these implants, treatments designed to be bacteriostatic or bactericidal must not interfere with osseointegration. Ueoka and colleagues [6] have provided valuable information in the understanding of the antimicrobial properties of iodine-supported implants over time. But further questions will need to be addressed before this technology can be used in clinical practice.…”

“…A study to compare the push-out strength of porous-coated titanium implants with iodine coating to those without iodine will be necessary before one of these implants could be used in humans. A study like the one described by Ueoka et al [6] could be performed to help determine the length of the antimicrobial effect of this type of iodine coating. Animal models will also help researchers determine the efficacy of this type of implant in treating an established infection versus as a prophylactic agent.…”