Quantitative mouse model of implant-associated osteomyelitis and the kinetics of microbial growth, osteolysis, and humoral immunity.

The Center for Musculoskeletal Research, University of Rochester Medical Center, 601 Elmwood Avenue, Box 665, Rochester, New York 14642, USA.
Journal of Orthopaedic Research (Impact Factor: 2.97). 01/2008; 26(1):96-105. DOI: 10.1002/jor.20452
Source: PubMed

ABSTRACT Although osteomyelitis (OM) remains a serious problem in orthopedics, progress has been limited by the absence of an in vivo model that can quantify the bacterial load, metabolic activity of the bacteria over time, immunity, and osteolysis. To overcome these obstacles, we developed a murine model of implant-associated OM in which a stainless steel pin is coated with Staphylococcus aureus and implanted transcortically through the tibial metaphysis. X-ray and micro-CT demonstrated concomitant osteolysis and reactive bone formation, which was evident by day 7. Histology confirmed all the hallmarks of implant-associated OM, namely: osteolysis, sequestrum formation, and involucrum of Gram-positive bacteria inside a biofilm within necrotic bone. Serology revealed that mice mount a protective humoral response that commences with an IgM response after 1 week, and converts to a specific IgG2b response against specific S. aureus proteins by day 11 postinfection. Real-time quantitative PCR (RTQ-PCR) for the S. aureus specific nuc gene determined that the peak bacterial load occurs 11 days postinfection. This coincidence of decreasing bacterial load with the generation of specific antibodies is suggestive of protective humoral immunity. Longitudinal in vivo bioluminescent imaging (BLI) of luxA-E transformed S. aureus (Xen29) combined with nuc RTQ-PCR demonstrated the exponential growth phase of the bacteria immediately following infection that peaks on day 4, and is followed by the biofilm growth phase at a significantly lower metabolic rate (p < 0.05). Collectively, these studies demonstrate the first quantitative model of implant-associated OM that defines the kinetics of microbial growth, osteolysis, and humoral immunity following infection.

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    ABSTRACT: Background Implant-related osteomyelitis (IRO) is recently controlled with local antibiotic delivery systems to overcome conventional therapy disadvantages. In vivo evaluation of such systems is however too little.Questions/purposesWe asked whether vancomycin (V)-containing poly-l-lactic acid/ß-tricalcium phosphate (PLLA/ß-TCP) composites control experimental IRO and promote bone healing in vivo.Methods Fifty-six rats were distributed to five groups in this longitudinal controlled study. Experimental IRO was established at tibiae by injecting methicillin-resistant Staphylococcus aureus (MRSA) suspensions with titanium particles in 32 rats. Vancomycin-free PLLA/ß-TCP composites were implanted into the normal and infected tibiae, whereas V-PLLA/ß-TCP composites and coated (C)-V-PLLA/ß-TCP composites were implanted into IRO sites. Sham-operated tibiae established the control group. Radiological and histological scores were quantified with microbiological findings on weeks 1 and 6.ResultsIRO is resolved in the CV- and the V-PLLA/ß-TCP groups but not in the PLLA/ß-TCP group. MRSA was not isolated in the CV- and the V-PLLA/ß-TCP groups at all times whereas the bacteria were present in the PLLA/ß-TCP group. Radiological signs secondary to infection are improved from 10.9¿±¿0.9 to 3.0¿±¿0.3 in the V-PLLA/ß-TCP group but remained constant in the PLLA/ß-TCP group. Histology scores are improved from 24.7¿±¿6.5 to 17.6¿±¿4.8 and from 27.6¿±¿7.9 to 32.4¿±¿8.9 in the CV-PLLA/ß-TCP and the V-PLLA/ß-TCP groups, respectively. New bone was formed in all the PLLA/ß-TCP group at weeks 1 and 6.ConclusionsCV- and V-PLLA/ß-TCP composites controlled experimental IRO and promoted bone healing.Clinical relevanceCV- and V-PLLA/ß-TCP composites have the potential of controlling experimental IRO and promoting bone healing.
    Journal of Orthopaedic Surgery and Research 11/2014; 9(1):114. DOI:10.1186/s13018-014-0114-3 · 1.58 Impact Factor
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    ABSTRACT: Bacterial biofilms causing implant-associated osteomyelitis is a severe complication with limited antimicrobial therapy options. We designed an animal model, in which implant associated osteomyelitis arise from a Staphylococcus aureus biofilm on a tibia implant. Two bioluminescently engineered (luxA-E transformed), strains of S. aureus were utilized, Xen29 and Xen31. Biofilm formation was assessed with epifluorescence microscopy. Quantitative measurements were performed day 4, 6, 8, 11 and 15 post-surgery. Bacteria were extracted from the biofilm by sonication and the bacterial load quantified by culturing. Biofilm formation was evident from day 6 post-implantation. Mean bacterial load from implants was ∼1×104 CFU/implant, while mean bacterial load from infected tibias were 1×106 CFU/bone. Bioluminesence imaging revealed decreasing activity throughout the 15-day observation period, with signal intensity for both strains reaching that of the negative control by day 15 while there was no significant reduction in bacterial load. The model is suitable for testing antimicrobial treatment options for implant associated OM, as treatment efficacy on both biofilm and viable counts can be assessed.
    PLoS ONE 10/2014; 9(10):e103688. DOI:10.1371/journal.pone.0103688 · 3.53 Impact Factor
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    ABSTRACT: Infections associated with deep wounds require extensive surgical and medical care. New adjunctive treatments are required to aid in the eradication of the bacterial biofilms found on infected wounds and, in particular, any underlying hardware. Ozone has been used as a safe and efficient disinfectant in water treatment plants for many years. The purpose of this study is to investigate the anti-biofilm potential of ozonated saline against biofilms of Staphylococcus aureus, a microorganism commonly implicated in wound infections. A custom-made bacterial biofilm bioreactor was used to grow S. aureus biofilms on discs of medical grade titanium alloy. An ozone generator was connected in-line and biofilms and planktonic bacteria were exposed to ozone in saline. Cytotoxicity was assessed against primary ovine osteoblasts in the same system. In tests against planktonic S. aureus, a 99% reduction in bacterial numbers was detected within 15 minutes of exposure. S. aureus biofilms were significantly more resistant to ozone, although complete eradication of the biofilm was eventually achieved within 5 hours. Ozonated saline was not found to be cytotoxic to primary ovine osteoblasts. Ozonated saline may be suitable as an adjuvant therapy to treat patients as an instillation fluid for wound irrigation and sterilisation. © 2015 Inc and John Wiley & Sons Ltd.
    International Wound Journal 01/2015; DOI:10.1111/iwj.12412 · 2.02 Impact Factor

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