Printer Friendly

Silver-Containing Hydroxyapatite Coating Reduces Biofilm Formation by Methicillin-Resistant Staphylococcus aureus In Vitro and In Vivo.

1. Introduction

Numerous types of orthopaedic implants are used to repair bone fractures, tumour resections, and artificial joint replacements. However, implants are prone to bacterial infections, which can lead to implant-associated infections at the surgical sites. If a bacterial implant-associated infection occurs, long-term treatment is required, which can affect the patient's quality of life and pose a burden on the surgeon [1, 2].

Several recent articles have described the relationship between biofilms and orthopaedic infections [3-5]. When bacteria form a biofilm on the implant, the biofilm layer protects the bacteria from antibiotic agents [6, 7]. Nishimura et al. demonstrated that sessile Staphylococcus aureus in biofilms were [greater than or equal to] 1,000 times more resistant to antibiotics than planktonic bacteria isolated from infected total hip arthroplasty patients [8]. Biofilm clusters from methicillin-sensitive S. aureus exhibited an antibiotic resistance normally associated with methicillin-resistant S. aureus (MRSA) strains [5]. Moreover, the biofilm structure can inhibit the adaptive and innate immune responses of the host [9]. Treatment becomes even more challenging if the bacteria are resistant to antibacterial drugs. Unfortunately, MRSA causes the majority of implant-associated infections [1, 10].

Multiple approaches to imparting antibacterial properties to implants to prevent infections have been reported [3, 4, 11-13]. Several studies have reported the merits of implants with antibacterial surface coatings such as gentamicin and magnesium [11-13]. Silver has strong, broad-spectrum antibacterial activity and low toxicity. For these reasons, silver has a long history of use as an antimicrobial agent, as in medical device coatings [14]. The mechanism underlying the antibacterial effect of silver was elucidated recently. Silver releases ions from its surface, which can bind to a number of bacterial cell structures, including the peptidoglycan cell wall and plasma membrane, DNA, and proteins [15, 16]. One study reported that ion-binding to the cell wall damaged the outer cell layers, causing loss of cell contents and creating structural abnormalities [17]. These mechanisms of action result in disorganization of surface species such as proteins, of cells, and of their resultant biofilms. Although we previously reported that an excessive dose of silver inhibits bone formation, the dose-dependent influence of silver on osteoconductivity remains to be elucidated [18].

Hydroxyapatite (HA) has good biocompatibility and osteoconductivity. Therefore, it is used to coat implants to improve bone-implant attachment strength [19, 20]. There have been various reports on the use of plasma-sprayed silver-doped HA coatings [21-23]. We developed a silver-containing HA (Ag-HA) coating, with the combined properties of both silver and HA, using a thermal spraying technique [24, 25]. We previously reported that Ag-HA exhibited antibacterial activity against MRSA in vivo. In that study, the number of viable MRSAs on Ag-HA-coated subcutaneous implants was significantly lower than that on HA-coated subcutaneous implants in rats [26]. Akiyama et al. reported similar results in the medullary cavity [27]. Ag-HA can release silver ions [28] and, thus, has antibacterial activity. However, its antibiofilm properties have not been evaluated. We hypothesised that Ag-HA coatings would reduce bacterial biofilm formation and help to manage refractory orthopaedic infections. Therefore, we designed in vitro and in vivo infection models to evaluate the effects of these coatings on biofilm formation.

2. Materials and Methods

2.1. Ag-HA Coating. Pure titanium disks (diameter, 14 mm; thickness, 1 mm) were used as substrates for coating deposition for both the in vitro and in vivo studies. One side of the surface was roughened using a K5 sandblasting machine (TKX Corp., Osaka, Japan) with a 180-grit aluminium oxide medium (Showa Denko K.K., Tokyo, Japan), after which the disks were washed with ethanol for 3 min under ultrasonication. Powdered silver oxide (Kanto Chemical, Tokyo, Japan) was added to powdered HA (KYOCERA Medical Materials, Osaka, Japan) to prepare the specified concentrations (w/w), and the mixtures were stirred for 5 min in plastic bags. HA powders, with or without silver oxide, were thermally sprayed onto the sandblasted surface using a flame-spraying system (Oerlikon Metco Japan Ltd., Tokyo, Japan) to coat the surface of the disks. The temperature of the flame was approximately 2,700[degrees]C. The spraying powder was carried into the flame by a dry air carrier gas during spraying, then melted through the flame, and sprayed onto the disk. The coating process was conducted under normal atmospheric pressure. The physical and chemical properties of Ag-HA, namely, a 40-[micro]m thick layer of Ag-HA containing calcium, phosphorus, and oxygen, in which the amount of calcium/phosphorus is nearly the same as that of HA, have been reported previously [25, 27]. The disks and implants were individually packaged and sterilised using a JS-8500 gamma steriliser (MDS Nordion, Ontario, Canada). All disks were obtained from KYOCERA Medical Corporation (Osaka, Japan).

2.2. Bacteria and Culture Conditions. The MRSA strain used in this study was UOEH6 (University of Occupational and Environmental Health Hospital, Fukuoka, Japan). This strain had been isolated from a blood sample of a septic patient and was characterised as a biofilm-producing strain. Bacteria were cultured overnight in Tryptic Soy Broth (Eiken Chemical, Tokyo, Japan) at 37[degrees]C. Immediately after inoculation, serial dilutions of the residual suspension were incubated on agar plates for 48 h at 37[degrees]C, and colony-forming units (CFU)/mL were calculated.

2.3. In Vitro Experiment. Four types of implants were prepared: Ti with HA coating (HA), Ti with 0.5% Ag-HA coating (0.5% Ag-HA), Ti with 1.0% Ag-HA coating (1% Ag-HA), and Ti with 3.0% Ag-HA coating (3% Ag-HA). One implant was aseptically placed into each well of 24-well sterile polystyrene tissue culture plates (Corning, Corning, NY, USA), and 500 [micro]L of an MRSA suspension containing 4 x [10.sup.5] CFU/mL was inoculated onto each implant. The disks were incubated at 37[degrees]C for 1h and then rinsed twice with 500 [micro]L sterile phosphate buffered saline (PBS) to eliminate nonadherent bacteria. The disks were transferred to Petri dishes containing 20 mL heat-inactivated 100% foetal bovine serum (Thermo Fisher Scientific, Wilmington, DE, USA). The dishes were incubated at 37[degrees]C with continuous slow stirring on magnetic stirrers for 7 or 14 days. The stir bar was spun at 60 revolutions per minute. The medium was changed every 3 days. In the 7-day experiment, 7 disks with HA coating, including 2 disks of 0.5% Ag-HA, 2 disks of 1% Ag-HA, and 3 disks of 3% Ag-HA, were used. In the 14-day experiment, 9 disks with HA and 3 disks of each group of Ag-HA were used.

2.4. In Vivo Experiment

2.4.1. Animals. We used ten 8-week-old male Sprague-Dawley rats, weighing on average 331.6 g (range 316.5-355.1 g), from Kyudo (Kumamoto, Japan). The rats were housed in pairs with 12-h light-dark cycles and acclimated for 5 days prior to use in a room in which a suitable environment was maintained. All animal procedures were conducted with the approval of the Animal Research Ethics Committee at Saga University (Approval Number 27-018-0). According to their recommendation, the number of experimental animals was minimised.

2.4.2. Surgical Technique. All rats were anaesthetised using a mixture of anaesthetic agents (0.375 mg/kg medetomidine, 2 mg/kg midazolam, and 2.5 mg/kg butorphanol) administered by intraperitoneal injection [28]. The infection model, using the back of the rat, was previously described by Shimazaki et al. [26] and was generated with slight modifications in our study.

Disks coated with HA or 3% Ag-HA were prepared. The back of the rat was shaved, cleaned with povidone-iodine, and dried. Four sagittal 1-cm incisions were made on the dorsum; two were at the level of the scapula and two were at the level of the lower rib cage, each 2 cm lateral to midline, and pockets were made not to connect to another pocket. One disk was aseptically implanted into each subcutaneous pocket; two HA-coated disks were implanted on the right side of the rats and two Ag-HA-coated disks on the left side. MRSA suspension (8.8-10.0 x [10.sup.8] CFU, 20 [micro]L per disk) was inoculated onto the disks in the pockets. Incisions were closed using interrupted 3-0 nylon suture. After the surgery, atipamezole (0.75 mg/kg) was used to induce recovery of animals from the anaesthesia. No analgesia was used after the operation. After 1 (3 rats), 3 (3 rats), or 7 days (4 rats), the animals were euthanised and the disks removed.

2.4.3. Calculation of Biofilm Coverage Rates (BCRs). All disks (from in vitro and in vivo experiments) were rinsed twice with 500 [micro]L of sterile PBS and stained with biofilm stain (FilmTracer calcein red-orange biofilm stain, Thermo Fisher Scientific) for 1 h. After staining, disks were washed twice with 500 [micro]L of sterile PBS. Thereafter, biofilm formation on each disk was observed under a fluorescence microscope (Axioplan 2; ZEISS, Jena, Germany). All quantifications were performed at a magnification of 50x. Random areas (seven per disk) were recorded as digital images. The BCRs on the disk surfaces were calculated using the image analysis software program ImageJ (National Institutes of Health, Bethesda, MD, USA) [29].

2.4.4. Statistical Analysis. All data are expressed as the median (range). For the in vitro study, groups were compared using the Kruskal-Wallis test. For the in vivo study, groups were compared using the Mann-Whitney U test. All statistical analyses were performed using the SPSS software program version 23 for Mac (IBM Corp., Armonk, NY, USA). A p value of <0.05 was considered significant.

3. Results

3.1. In Vitro Effects of the Ag-HA Coating. Figure 1 shows representative fluorescence microscopic images after bacterial culture for 7 and 14 days. The median BCRs on HA, 0.5% Ag-HA, 1% Ag-HA, and 3% Ag-HA after 7 days were 19.1% (2.8%-34.9%; 49 images), 8.0% (2.2%-28.8%; 14 images), 5.6% (3.0%-11.7%; 14 images), and 3.9% (1.8%-6.9%; 21 images), respectively. The median BCRs after 14 days were 38.6% (13.9%-55.7%; 63 images), 27.3% (9.8%-46.1%; 21 images), 23.2% (8.6%-40.0%; 21 images), and 6.6% (3.0%-19.7%; 21 images), respectively. The BCRs on all the Ag-HA-coated disks were significantly lower than those on the HA-coated disks (7 days: 0.5%, p = 0.011; 1%, p < 0.001; 3%, p < 0.001; 14 days: 0.5%, p = 0.024; 1%, p < 0.001; and 3%, p < 0.001). At 14 days the BCRs on 3% Ag-HA-coated disks were significantly lower than those on 0.5% (p < 0.001) and 1% Ag-HA-coated disks (p = 0.013; Figure 2).

3.2. In Vivo Effects of the Ag-HA Coating. None of the rats in any of the groups died during the experiment. Figure 3 shows the skin of a representative rat at 7 days after implantation. None of the rats exhibited any skin disorders or poor wound healing, indicating no observable toxic effects of the Ag-HA. Figure 4 shows representative fluorescence microscopic images. The BCRs of Ag-HA-coated disks were significantly lower (p < 0.001) than those of the HA-coated disks at all time points (Figure 5).

4. Discussion

In this study, we performed a systematic evaluation of the effects of Ag-HA coating on biofilm formation by MRSA. For evaluation of the biofilm coverage rates, the MRSA biofilm was stained with calcein red-orange. Methylene blue and SYTO 9 green fluorescent nucleic acid stain (Live/Dead staining) were not used, because these stains react not only with the MRSA biofilm but also with the debris of rat inflammatory tissues. To our knowledge, this study is the first to demonstrate that silver-containing HA reduces biofilm formation in vivo.

In our study, 3% Ag-HA reduced, but did not fully prevent, MRSA biofilm formation. We did not use any antibiotics in in vivo experiment. However, we usually use antibiotics to prevent surgical site infection at the time of orthopaedic surgery in humans. Morones-Ramirez et al. reported that silver ions enhanced the activity of antibiotics and broadened the spectrum of vancomycin [30]. Thus, synergism between Ag-HA and antibiotics could be expected to increase their effectiveness in treating implant-associated infection. Moreover, because silver has broad-spectrum antibacterial activity, Ag-HA can affect other bacteria as well.

In addition, we evaluated the potential toxic effects of the silver in Ag-HA in vivo. Argyria, a grey-blue tissue discoloration that can be observed in humans exposed to silver or using silver-containing medications, is the most common toxic effect of silver [31, 32]. In this study, none of the animals exhibited any signs of skin disorders or poor wound healing caused by silver toxicity, despite implantation of the disks just under the skin. Silver levels in the blood below 200 ppb are considered normal, whereas levels higher than 310 ppb are reported to cause argyria, argyrosis, and liver and kidney damage [33]. Tsukamoto et al. previously demonstrated that, in a rat tibia model with Ag-HA-coated implants, serum silver levels were sufficiently low to avoid harmful effects, and no degeneration was observed in the brain, liver, kidneys, or spleen. The amount of silver required for Ag-HA coating of femoral replacements in humans is low enough to avoid argyria [34]. In fact, in a 1-year follow-up study, we found that none of the patients developed any adverse reactions to silver from Ag-HA-coated implants in total hip arthroplasties [35].

Furthermore, it is important to consider conglutination of implants to the bone. Yonekura et al. reported that 50% Ag-HA coating inhibited bone formation, while 3% Ag-HA coating showed good osteoconductivity [18]. Accordingly, Eto et al. reported that 3% Ag-HA supported viability and function of osteoblasts, as well as anchorage strength. In pull-out tests using rat femurs, there were no significant differences between 3% Ag-HA and HA, whereas 50% Ag-HA required less force [36]. Therefore, we speculate that orthopaedic implants coated with 3% Ag-HA will have low silver toxicity while maintaining antibiofilm activity of silver, combined with the good osteoconductivity of HA. In our clinical trial with Ag-HA-coated implants, there were no implant failures [35].

Despite our promising results, this study had some limitations. The experiments were performed for only 7 (in vivo) or 14 (in vitro) days, which reflect acute infection. Such a short duration is not suitable for the evaluation of antibiofilm activity in chronic infections. In a previous study using a rat tibial model, antibacterial activity was demonstrated 4 weeks after implantation, suggesting that Ag-HA could sufficiently prevent acute and subacute infections [27]. To evaluate potential resistance to chronic infection, other models are needed.

In our in vivo study, we observed differences between HA-and Ag-HA-coated disks implanted in the same rat. Thus, the released silver ions affected only the local surgical site. Usually, only the part of the implant surface that is in contact with the bone, and not the entire orthopaedic implant, is coated. However, because the released silver ions can spread, the effectiveness of the antibiofilm activities of Ag-HA on the uncoated part of the implant surface remains to be clarified.

5. Conclusion

Ag-HA coating reduced biofilm formation by MRSA in vitro and in vivo, indicating that Ag-HA coatings could help manage refractory orthopaedic infections. Coating of orthopaedic implants with Ag-HA might be expected to decrease the incidence of postoperative implant-associated infections, improve the quality of life of patients, and promote favourable outcomes in orthopaedic surgery.

http://dx.doi.org/10.1155/2016/8070597

Competing Interests

The authors declare no conflict of interests.

Acknowledgments

This research was supported by a Grant-in-Aid for Young Scientists (B) (no. 26861199) from the Japan Society for the Promotion of Science.

References

[1] B. A. Rogers and N. J. Little, "Surgical site infection with methicillin-resistant Staphylococcus aureus after primary total hip replacement," The Journal of Bone & Joint Surgery--British Volume, vol. 90, no. 11, pp. 1537-1538, 2008.

[2] S. M. Kurtz, E. Lau, J. Schmier, K. L. Ong, K. Zhao, and J. Parvizi, "Infection burden for hip and knee arthroplasty in the United States," Journal of Arthroplasty, vol. 23, no. 7, pp. 984-991, 2008.

[3] S. J. McConoughey, R. Howlin, J. F. Granger et al., "Biofilms in periprosthetic orthopedic infections," Future Microbiology, vol. 9, no. 8, pp. 987-1007, 2014.

[4] K. D. Secinti, H. Ozalp, A. Attar, and M. F. Sargon, "Nanoparticle silver ion coatings inhibit biofilm formation on titanium implants," Journal of Clinical Neuroscience, vol. 18, no. 3, pp. 391-395, 2011.

[5] C. A. Fux, S. Wilson, and P. Stoodley, "Detachment characteristics and oxacillin resistance of Staphyloccocus aureus biofilm emboli in an in vitro catheter infection model," Journal of Bacteriology, vol. 186, no. 14, pp. 4486-4491, 2004.

[6] L. Hall-Stoodley, J. W. Costerton, and P. Stoodley, "Bacterial biofilms: from the natural environment to infectious diseases," Nature Reviews Microbiology, vol. 2, no. 2, pp. 95-108, 2004.

[7] H. Anwar, M. K. Dasgupta, and J. W. Costerton, "Testing the susceptibility of bacteria in biofilms to antibacterial agents," Antimicrobial Agents and Chemotherapy, vol. 34, no. 11, pp. 2043-2046, 1990.

[8] S. Nishimura, T. Tsurumoto, A. Yonekura, K. Adachi, and H. Shindo, "Antimicrobial susceptibility of Staphylococcus aureus and Staphylococcus epidermidis biofilms isolated from infected total hip arthroplasty cases," Journal of Orthopaedic Science, vol. 11, no. 1, pp. 46-50, 2006.

[9] P. 0. Jensen, M. Givskov, T. Bjarnsholt, and C. Moser, "The immune system vs. Pseudomonas aeruginosa biofilms," FEMS Immunology and Medical Microbiology, vol. 59, no. 3, pp. 292-305, 2010.

[10] J. L. Del Pozo and R. Patel, "Clinical practice. Infection associated with prosthetic joints," The New England Journal of Medicine, vol. 361, no. 8, pp. 787-794, 2009.

[11] V. Alt, A. Bitschnau, F. Bohner et al., "Effects of gentamicin and gentamicin-RGD coatings on bone ingrowth and biocompatibility of cementless joint prostheses: An experimental study in rabbits," Acta Biomaterialia, vol. 7, no. 3, pp. 1274-1280, 2011.

[12] H. Vester, B. Wildemann, G. Schmidmaier, U. Stockle, and M. Lucke, "Gentamycin delivered from a PDLLA coating of metallic implants: in vivo and in vitro characterisation for local prophylaxis of implant-related osteomyelitis," Injury, vol. 41, no. 10, pp. 1053-1059, 2010.

[13] Y. Li, G. Liu, Z. Zhai et al., "Antibacterial properties of magnesium in vitro and in an in vivo model of implant-associated methicillin-resistant Staphylococcus aureus infection," Antimicrobial Agents and Chemotherapy, vol. 58, no. 12, pp. 7586-7591, 2014.

[14] J. L. Clement and P. S. Jarrett, "Antibacterial silver," Metal-Based Drugs, vol. 1, no. 5-6, pp. 467-482, 1994.

[15] K. Chaloupka, Y. Malam, and A. M. Seifalian, "Nanosilver as a new generation of nanoproduct in biomedical applications," Trends in Biotechnology, vol. 28, no. 11, pp. 580-588, 2010.

[16] S. A. Brennan, C. N1 Fhoghlu, B. M. Devitt, F. J. O'Mahony, D. Brabazon, and A. Walsh, "Silver nanoparticles and their orthopaedic applications," The Bone & Joint Journal, vol. 97, no. 5, pp. 582-589, 2015.

[17] M. Yamanaka, K. Hara, and J. Kudo, "Bactericidal actions of a silver ion solution on Escherichia coli, studied by energy-filtering transmission electron microscopy and proteomic analysis," Applied and Environmental Microbiology, vol. 71, no. 11, pp. 7589-7593, 2005.

[18] Y. Yonekura, H. Miyamoto, T. Shimazaki et al., "Osteoconductivity of thermal-sprayed silver-containing hydroxyapatite coating in the rat tibia," The Journal of Bone and Joint Surgery-British Volume, vol. 93, no. 5, pp. 644-649, 2011.

[19] T. Hara, K. Hayashi, Y. Nakashima, T. Kanemaru, and Y. Iwamoto, "The effect of hydroxyapatite coating on the bonding of bone to titanium implants in the femora of ovariectomised rats," The Journal of Bone & Joint Surgery--British Volume, vol. 81, no. 4, pp. 705-709, 1999.

[20] Y. Nakashima, K. Hayashi, T. Inadome et al., "Hydroxyapatite-coating on titanium arc sprayed titanium implants," Journal of Biomedical Materials Research, vol. 35, no. 3, pp. 287-298, 1997.

[21] M. Roy, G. A. Fielding, H. Beyenal, A. Bandyopadhyay, and S. Bose, "Mechanical, in vitro antimicrobial, and biological properties of plasma-sprayed silver-doped hydroxyapatite coating," ACS Applied Materials and Interfaces, vol. 4, no. 3, pp. 1341-1349, 2012.

[22] O. Braissant, P. Chavanne, M. de Wild et al., "Novel microcalorimetric assay for antibacterial activity of implant coatings: the cases of silver-doped hydroxyapatite and calcium hydroxide," Journal of Biomedical Materials Research, Part B: Applied Biomaterials, vol. 103, no. 6, pp. 1161-1167, 2015.

[23] S. Guimond-Lischer, Q. Ren, O. Braissant, P. Gruner, B. Wampfler, and K. Maniura-Weber, "Vacuum plasma sprayed coatings using ionic silver doped hydroxyapatite powder to prevent bacterial infection of bone implants," Biointerphases, vol. 11, no. 2, Article ID 011012, 2016.

[24] Y. Ando, H. Miyamoto, I. Noda et al., "Calcium phosphate coating containing silver shows high antibacterial activity and low cytotoxicity and inhibits bacterial adhesion," Materials Science and Engineering C, vol. 30, no. 1, pp. 175-180, 2010.

[25] I. Noda, F. Miyaji, Y. Ando et al., "Development of novel thermal sprayed antibacterial coating and evaluation of release properties of silver ions," Journal of Biomedical Materials Research, Part B: Applied Biomaterials, vol. 89, no. 2, pp. 456-465, 2009.

[26] T. Shimazaki, H. Miyamoto, Y. Ando et al., "In vivo antibacterial and silver-releasing properties of novel thermal sprayed silver containing hydroxyapatite coating," Journal of Biomedical Materials Research, Part B: Applied Biomaterials, vol. 92, no. 2, pp. 386-389, 2010.

[27] T. Akiyama, H. Miyamoto, Y. Yonekura et al., "Silver oxide-containing hydroxyapatite coating has in vivoantibacterial activity in the rat tibia," Journal of Orthopaedic Research, vol. 31, no. 8, pp. 1195-1200, 2013.

[28] S. Kawai, Y. Takagi, S. Kaneko, and T. Kurosawa, "Effect of three types of mixed anesthetic agents alternate to ketamine in mice," Experimental Animals, vol. 60, no. 5, pp. 481-487, 2011.

[29] M. D. Abramoff, P. J. Magelhaes, and S. J. Ram, "Image processing with Image J," Biophotonics International, vol. 11, no. 7, pp. 36-42, 2004.

[30] J. R. Morones-Ramirez, J. A. Winkler, C. S. Spina, and J. J. Collins, "Silver enhances antibiotic activity against gram-negative bacteria," Science Translational Medicine, vol. 5, no. 190, Article ID 190ra81, 2013.

[31] J. M. L. White, A. M. Powell, K. Brady, and R. Russell-Jones, "Severe generalized argyria secondary to ingestion of colloidal silver protein," Clinical and Experimental Dermatology, vol. 28, no. 3, pp. 254-256, 2003.

[32] M. A. Hollinger, "Toxicological aspects of topical silver pharmaceuticals," Critical Reviews in Toxicology, vol. 26, no. 3, pp. 255-260, 1996.

[33] A. T. Wan, R. A. J. Conyers, C. J. Coombs, and J. P. Masterton, "Determination of silver in blood, urine, and tissues of volunteers and burn patients," Clinical Chemistry, vol. 37, no. 10, part 1, pp. 1683-1687, 1991.

[34] M. Tsukamoto, H. Miyamoto, Y. Ando et al., "Acute and sub-acute toxicity in vivo of thermal-sprayed silver containing hydroxyapatite coating in rat tibia," BioMed Research International, vol. 2014, Article ID 902343, 8 pages, 2014.

[35] S. Eto, S. Kawano, S. Someya, H. Miyamoto, M. Sonohata, and M. Mawatari, "First clinical experience with thermal-sprayed silver oxide-containing hydroxyapatite coating implant," Journal of Arthroplasty, vol. 31, no. 7, pp. 1498-1503, 2016.

[36] S. Eto, H. Miyamoto, T. Shobuike et al., "Silver oxide-containing hydroxyapatite coating supports osteoblast function and enhances implant anchorage strength in rat femur," Journal of Orthopaedic Research, vol. 33, no. 9, pp. 1391-1397, 2015.

Masaya Ueno, (1) Hiroshi Miyamoto, (2) Masatsugu Tsukamoto, (1) Shuichi Eto, (1) Iwao Noda, (2,3) Takeo Shobuike, (2) Tomoki Kobatake, (1) Motoki Sonohata, (1) and Masaaki Mawatari (1)

(1) Department of Orthopaedic Surgery, Faculty of Medicine, Saga University, Saga 849-8501, Japan

(2) Department of Pathology and Microbiology, Faculty of Medicine, Saga University, Saga 849-8501, Japan

(3) Research Department, KYOCERA Medical Corporation, Osaka 532-0003, Japan

Correspondence should be addressed to Masaya Ueno; 13624003@edu.cc.saga-u.ac.jp

Received 7 November 2016; Revised 1 December 2016; Accepted 7 December 2016

Academic Editor: Paul M. Tulkens

Caption: Figure 1: Representative fluorescence microscopic images of implants after bacterial culture for 7 and 14 days in vitro. The bar indicates 100 [micro]m. (a) Hydroxyapatite (HA) coating at 7 days, (b) 0.5% silver- containing HA (Ag-HA) coating at 7 days, (c) 1% Ag-HA coating at 7 days, (d) 3% Ag-HA coating at 7 days, (e) HA coating at 14 days, (f) 0.5% Ag-HA coating at 14 days, (g) 1% Ag-HA coating at 14 days, and (h) 3% Ag-HA coating at 14 days. Calculated BCRs: (a) 19.8%, (b) 9.7%, (c) 6.1%, (d) 3.3%, (e) 43.7%, (f) (36.3%), (g) 29.1%, and (h) 8.4%.

Caption: Figure 2: Box-and-whisker plots of biofilm coverage rates (BCRs) of implants after bacterial culture for 7 days and 14 days in vitro. In the 7-day experiment, 49 images of HA coating, including 14 images of 0.5% Ag-HA, 14 images of 1% Ag-HA, and 21 images of 3% Ag- HA, were used. In the 14-day experiment, 63 images of HA and 21 images of each group of Ag-HA were used. * denotes significant differences between the BCRs of HA and each concentration of Ag-HA. The significance levels are as follows: 7 days: 0.5% Ag-HA, p = 0.011; 1% Ag-HA, p < 0.001; and 3% Ag-HA, p < 0.001; 14 days: 0.5% Ag-HA, p = 0.024; 1% Ag-HA, p < 0.001; and 3% Ag-HA, p < 0.001. ([dagger]) denotes significant differences between the BCRs of 3% Ag-HA and other concentrations (0.5%, 1%) of Ag-HA. The significance levels are 0.5% Ag-HA, p < 0.001, and 1% Ag-HA, p = 0.013.

Caption: Figure 3: Representative photograph of rat dorsal skin at 7 days after implantation. None of the animals exhibited any signs of skin disorders or poor wound healing.

Caption: Figure 4: Representative fluorescence microscopic images of implants (50x objective) after bacterial infection in vivo. The bar indicates 100 [micro]m. The red coloured areas in the picture correspond to the locations covered by biofilms of methicillin- resistant Staphylococcus aureus (MRSA). (a) HA coating at 1 day, (b) HA coating at 3 days, (c) HA coating at 7 days, (d) 3% Ag-HA coating at 1 day, (e) 3% Ag-HA coating at 3 days, and (f) 3% Ag-HA coating at 7 days. Calculated BCRs: (a) 16.0%, (b) 26.6%, (c) 30.1%, (d) 8.9%, (e) 14.6%, and (f) 13.7%.

Caption: Figure 5: Box-and-whisker plots of BCRs of implants at 1, 3, and 7 days after bacterial infection. * denotes significant differences (p < 0.001) between the BCRs of HA and Ag-HA. Calculated BCRs after 1 day: HA 14.7% (5.4%-54.7%; 42 images) and 3% Ag-HA 7.6% (0.8%-28.2%; 42 images); after 3 days: HA 27.2% (10.4%-53.3%; 42 images) and 3% Ag-HA 13.6% (2.8%-33.0%; 42 images); and after 7 days: HA 28.8% (12.0%-61.3%; 56 images) and 3% Ag-HA 11.0% (2.3%-32.2%; 56 images).
COPYRIGHT 2017 Hindawi Limited
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2017 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Research Article
Author:Ueno, Masaya; Miyamoto, Hiroshi; Tsukamoto, Masatsugu; Eto, Shuichi; Noda, Iwao; Shobuike, Takeo; Ko
Publication:BioMed Research International
Article Type:Report
Date:Jan 1, 2017
Words:4425
Previous Article:Identification of Genes Involved in the Responses of Tangor (C. reticulata x C. sinensis) to Drought Stress.
Next Article:The Role of Three-Dimensional Scaffolds in Treating Long Bone Defects: Evidence from Preclinical and Clinical Literature--A Systematic Review.
Topics:

Terms of use | Privacy policy | Copyright © 2022 Farlex, Inc. | Feedback | For webmasters |