Micronano Bubble Containing Ozone is Efficient for Bacterial Biofilms on Orthopaedic Material.+1Morimoto, Y; 1Yoshida, A; 2Murakami, S; 1Tsuji, H; 3Yoshitaka, T; 1Ozaki, T

+1Science of Functional Recovery and Reconstruction, Okayama University, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, JAPAN, 2KYOWAKISETSU Co.,Ltd., Fukuyama, JAPAN, 3Department of Regenerative Medicine, National Research Institute for Child Health and Development, Tokyo, JAPAN

yumorimo@md.okayama-u.ac.jpINTRODUCTION: Chronic osteomyelitis and infection after orthopaedic surgery using implants are often difficult to cure, and the cost of treatment and patient’s disability are increased. The infection is resistant to host defence mecha-nisms and antimicrobial therapy, because of not only multiple-drug-resistant bacteria, but also the bacterial biofilms was formed. The biofilm is an aggregate of microorganisms and is adherent to a solid surface. In the biofilm, bacteria is embedded within a self-produced extracellular polysaccharide matrix which allows for the bacteria to become antibiotic resistant and difficult to wash even if radical debridement is performed. If the control of the infection is failure, infected implants must be removed or amputation can not avoid. In this study, we investigated a novel method against biofilm-related infections. The technology is already applied to wash silicon wafers and industrial pipe with a minute gas bubble in liquid. Gas bubbles which diameter between 10 to several 10µm are called micro bubble. Gas bub-bles which diameter several 100 nm or smaller called nano bubble. We call “ Micronano bubble (MNB)” which contains both micro and nano bubbles. The bubbles stay in water several months. The type of gas and liquid are alterable such as room air, oxygen, and ozone. We evaluated the effectiveness of MNB against biofilm in vitro.METHODS: We used Staphylococcus aureus (S. aureus: Seattle1945 strain) ,which have bacterial biofilm formation ability. After the S. aureus strain had grown in trypticase soy broth (TSB), it was incubated at 37°C for 8 hrs until the exponential growth phase had been reached. We created the bacteria-containing TSB (2x109 cfu/ml) for exposure. We designed two infection models with stainless washer and NEOBONE® (Covalent Mate-rial, Japan), which is hydroxyapatite ceramic with an interconnected po-rous structure and is clinically used for bone graft. The materials were suspended in 48 well-plate filled with 700µl TSB containing 100µl bacte-rial suspension kept at 37°C. TSB was changed 24 hrs later, and the incu-bation was continued for another 24 hrs. And then, we rinse the samples in saline thoroughly. The method to generate MNB was shearing air-water mixed liquid. Distilled water at 4°C were used for liquid, and room air or ozone were for gas. Ozone was generated with ozone-generating device provided by OHNIT (JAPAN). Three kinds of wash water were prepared: (1) water without MNB (control), (2) water with room air MNB (RA-MNB), and (3) water with ozonated MNB (O-MNB). The MNB generator, BUVI-TAS® and its prototype model, was provided by KYOWAKISETSU (JA-PAN). The operation time was 10 min at the flow gas volume of 0.7 l/min. The size and volume distributions of MNB was measured by MultisizerTM

3 / Coulter Counter® and LS 13 320 Laser Diffraction Particle Size Ana-lyzer (Beckman Coulter, USA). The ozone concentration in O-MNB was measured with O3-3F (Kasahara Chemical Instruments, JAPAN). We resuspended in the each wash liquid of interest at a 1:10 dilution from the bacterial suspension. To investigate the ability of MNB to kill bacteria, bacterial cell numbers in culture were measured, using assay for colony forming unit on tryptic soy agar after 1 hr and 12 hrs cultivation at room temperature. Next, we washed the samples of infection model with 1L liquid at the constant flow rate (130ml/ min). To investigate the biofilms attached to surface, samples were examined with a scanning electron microscope (SEM: S-4200, HITACHI, Japan) For SEM, samples prepared with stan-dard methods. The samples were also stained with a LIVE/DEAD kit (Invitrogen Molecular Probes, USA) following the manufacturer's instruc-tions. This stain contains SYTO 9 and propidium iodide (PI), both binding to DNA. The SYTO 9 generally stains all bacteria in a population. In contrast, PI stains only bacteria with damaged membranes. We examined treated and untreated biofilms with a Zeiss LSM 510 confocal laser scan-ning microscope. Image analyses and export were performed in a Zeiss LSM Image Browser ver 4.2.0.RESULTS: The ozone solubility in O-MNB were assessed changes over time (Fig 1). In case of ozonated water, it is commonly known that ozone disappear in several minutes. However, O-MNB kept effective ozone solubility 0.5 ppm during about 6 hrs. Compared with control, RA-MNB has little abil-

ity to kill bacteria (data not shown). In contrast, O-MNB indicate good sterilizing ability (Fig 2). In SEM imaging, the surface of samples without wash was fully cov-ered with bacteria and biofilms (Fig3-a). In the group of washing with control, bacteria were removed roughly, but we could observed remaining biofilms (Fig3-b). In the group of washing with RA-MNB, the size and number of remaining biofilms were smaller than with water alone. How-ever, the appearance of remaining bacteria was the same (Fig3-c). In the group of O-MNB washing, remaining biofilms were more decreasing than of RA-MNB. Furthermore, the bacteria shrinking and flattening were observed (Fig3-d). For LIVE/DEAD staining, washing with control did not damage to cell membrane and remaining bacteria and biofilms were still alive. In wash-ing with RA-MNB, biofilms were not damaged, but a part of remaining bacteria was damaged. O-MNB had a damaging effect on almost of all remaining bacteria and biofilms. DISCUSSION: Our generated MNB persists for more than several months. Ozone is known to have a strong bacteriocidal capacity, but to have low solubility in water. In addition, ozonated water shows very short-lived effect. Our O-MNB combine the best properties of MNB and ozone. O-MNB has a high solubility in water for a relatively-long time. Additionally, O-MNB had an antibacterial effect on S. aureus cells and those embedded in biofilms. These results suggest the possibility of useful effect for infection after orthopaedic surgery. Kill ratio of O-MNB in this study is not enough, then more volume and flow is necessary to kill any remaining bacteria cells. And we must clarify the response of body to O-MNB to prevent adverse side effects.

Fig 1. The solubility of ozone in ozonated MNB water.

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Fig 2. The activity of ozonated MNB to kill bacteria.

Fig 3. The surface of washers in SEM imaging.

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Poster No. 2147 • 56th Annual Meeting of the Orthopaedic Research Society