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Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_17 | Pages 21 - 21
24 Nov 2023
RETHINKING THE INOCULUM USED IN ANIMAL MODELS OF IMPLANT-ASSOCIATED OSTEOMYELITIS: THE FORMATION AND APPLICATION OF BACTERIAL AGGREGATES
Hartmann KT Nielsen RL Mikkelsen F Ingmer H Kvich LA Aalbaek B Odgaard A Jensen HE Lichtenberg M Bjarnsholt T Jensen LK
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Aim

To make an inoculum for induction of Implant-Associated Osteomyelitis (IAO) in pigs based on bacterial aggregates resembling those found on the human skin, i.e. aggregates of 5–15 µm with low metabolic activity. The aggregates were evaluated and compared to a standard planktonic bacterial inoculum.

Method

The porcine Staphylococcus aureus strain S54F9 was cultured in Tryptone Soya Broth for seven days. Subsequently, the culture was filtered through cell strainers with pore sizes of 15 µm and 5 µm, respectively. The fraction of 5–15 µm aggregates in the top of the 5 µm filter was collected as the aggregate-inoculum. The separation of aggregates into different size fractions was evaluated by light microscopy. The metabolism of the aggregate-inoculum and a standard overnight planktonic inoculum was evaluated with isothermal microcalorimetry. In total, six female minipigs were allocated into three groups (n=2), receiving different inoculums. Group A: overnight planktonic inoculum; 104 CFU S. aureus (S54F9), Group B: seven days old 5–15 µm aggregate-inoculum; 104 CFU S. aureus (S54F9), Group C: saline. All inoculums were placed in a pre-drilled implant cavity in the right tibia of the pig and a sterile stainless-steel implant was inserted. The pigs were euthanized seven days after surgery. Postmortem macroscopic pathology, microbiology, computed tomography and histopathology were performed.

Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_10 | Pages 30 - 30
1 Oct 2022
NEW INORGANIC/ORGANIC NANO-STRUCTURED XEROGEL COATING PREVENTS DEVELOPMENT OF OSTEOMYELITIS IN A PORCINE MODEL
Jensen LK Jensen HE Blirup SA Bue M Hanberg P Soto S Aalbaek B Arkas M Vardavoulias M
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Aim

To develop a new system for antibacterial coating of joint prosthesis and osteosynthesis material. The new coating system was designed to release gentamicin immediately after insertion to eradicate surgical contamination.

Method

Steel implants (2×15mm) were coated with a solid nanocomposite xerogel made from silica and the dendritic polymer, hyperbranched polyethyleneimine. The xerogel was anchored inside a porous surface made by pre-coating with titanium microspheres. Finally, gentamicin was encapsulated in the xerogel, i.e. no chemical binding. A total of 50 µg gentamicin was captured into each implant. The efficacy of the new coating was evaluated in a porcine model of implant associated osteomyelitis. In total, 30 female pigs were randomized into 3 study groups (n=10). Group A; plain implants + saline, Group B; plain implants + 104 CFU of Staphylococcus aureus, and Group C; coated implants + 104 CFU of S. aureus. Implant + inoculum was placed into a pre-drilled implant cavity of the right tibia and the pig was euthanized 5 days afterwards. Postmortem microbiology and pathology were performed. Two additional pigs were used in a pharmacokinetic study where microdialysis (MD) catheters were placed alongside coated implants. Extracellular fluid was sampled regularly for 24 hours from the MD catheters and analyzed for gentamicin content.

Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_10 | Pages 90 - 90
1 Oct 2022
HISTOPATHOLOGICAL CHARACTERIZATION OF THE FIRST AND LAST REMOVED BONE TISSUE DURING DEBRIDEMENT OF CHRONIC OSTEOMYELITIS
Jensen LK Jensen HE Gottlieb H
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Aim

To describe the histopathology of the first and last debrided bone tissue in chronic osteomyelitis and answer the following research question; is the last debrided bone tissue viable and without signs of inflammation?

Method

In total, 15 patients with chronic osteomyelitis were allocated to surgical treatment using a one stage protocol including extensive debridement. Suspected infected bone tissue eradicated early in the debridement procedure was collected as a clearly infected sample (S1). Likewise, the last eradicated bone tissue was collected as a suspected non-infected sample (S2), representing the status of the bone void. In all cases, the surgeon debrided the bone until visual confirmation of healthy bleeding bone. The samples were processed for histology, i.e. decalcification and paraffin embedding, followed by cutting and staining with Haematoxylin and Eosin. Immunohistochemistry with MAC-387 antibodies towards the calprotectin of neutrophil granulocytes (NGs) was also performed and used for estimation of a neutrophil granulocyte (NG) score (0, 1, 2 or 3), by the method described for fracture related infections (1).

Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_15 | Pages 47 - 47
1 Dec 2021
DEMONSTRATION OF LOCAL ACUTE PHASE RESPONSE DURING OSTEOMYELITIS
Lüthje FL Skovgaard K Jensen HE Heegaard P Gottlieb H Kirketerp-M⊘ller K Blirup SA Jensen LK
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Aim

The liver is the major source of acute phase proteins (APPs) and serum concentrations of several APPs are widely used as markers of inflammation and infection. The aim of the present study was to explore if a local extra hepatic osseous acute phase response occurs during osteomyelitis.

Method

The systemic (liver tissue and serum) and local (bone tissue) expression of several APPs during osteomyelitis was investigated with qPCR and ELISA in a porcine model of implant associated osteomyelitis (IAO) at 5, 10 and 15 days after inoculation with S. aureus or saline, respectively. Additionally, samples were also collected from normal heathy pigs and pigs with spontaneous, chronic, haematogenous osteomyelitis. Afterwards, immunohistochemistry towards different upregulated APPs was performed on the porcine osteomyelitis lesions and on bone biopsies from human patients with chronic osteomyelitis.

Bone & Joint Research
Vol. 9, Issue 7 | Pages 394 - 401
1 Jul 2020
Pathological and microbiological impact of a gentamicin-loaded biocomposite following limited or extensive debridement in a porcine model of osteomyelitis
Blirup-Plum SA Bjarnsholt T Jensen HE Kragh KN Aalbæk B Gottlieb H Bue M Jensen LK
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Aims

CERAMENT|G is an absorbable gentamicin-loaded biocomposite used as an on-site vehicle of antimicrobials for the treatment of chronic osteomyelitis. The purpose of the present study was to investigate the sole effect of CERAMENT|G, i.e. without additional systemic antimicrobial therapy, in relation to a limited or extensive debridement of osteomyelitis lesions in a porcine model.

Methods

Osteomyelitis was induced in nine pigs by inoculation of 104 colony-forming units (CFUs) of Staphylococcus aureus into a drill hole in the right tibia. After one week, the pigs were allocated into three groups. Group A (n = 3) received no treatment during the study period (19 days). Groups B (n = 3) and C (n = 3) received limited or extensive debridement seven days postinoculation, respectively, followed by injection of CERAMENT|G into the bone voids. The pigs were euthanized ten (Group C) and 12 (Group B) days after the intervention.

Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_14 | Pages 93 - 93
1 Dec 2019
GUIDELINE FOR PRECLINICAL STUDIES OF BONE INFECTIONS
Jensen LK Henriksen NL Blirup SA Jensen HE
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Aim

To conduct a systematic review of non-rodent animal models (rabbit, pig, dog, goat and sheep) of bone infection. In the future, anti-infective technologies aiming to fight bone infections are depending on evaluation in reliable animal models. Therefore, it is highly relevant to evaluate the scientific quality of existing bone infection models.

Method

PubMed and Web of Science were searched systematically. To be included in the systematic review, publications had to deal with bacterial inoculation of non-rodent animals in order to model bone infections in humans. Data was extracted on study design e.g. bacterial inoculation dose and infection time, methodological quality and post-mortem evaluation with respect to registration and quantification of pathology and microbiology.

Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_14 | Pages 77 - 77
1 Dec 2019
EXTENSIVE DEBRIDEMENT IS FUNDAMENTAL FOR THE SUCCESS OF AN ABSORBABLE GENTAMICIN-LOADED BIO-COMPOSITE
Jensen LK Blirup SA Aalbæk B Bjarnsholt T Kragh KN Gottlieb H Bue M Jensen HE
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Aim

To study the antimicrobial effect of a gentamicin loaded bio-composite bone void filler in relation to a limited or extensive debridement of osteomyelitis lesions, respectively.

Methods

Nine pigs were inoculated into the right proximal tibial bone with a high virulent gentamicin sensitive strain of Staphylococcus aureus (104 CFU). Seven days after inoculation, Group A pigs (n=3) were exposed to a limited debridement of the bone lesion, whereas Group B pigs (n=3) were exposed to an extensive debridement. The bone defects of Groups A and B were filled with (2–5 ml) of an absorbable gentamicin (175 mg/10 mL) loaded bio-composite. The animals of Group A and B were euthanized 12 days after revision surgery. Group C animals did not undergo revision surgery and were euthanized seven (n=1) or nineteen (n=2) days post inoculation in order to follow the development of the untreated infection. None of the animals were treated with systemic antimicrobials. All bones were exposed to a post mortem CT scan and rigours pathological examinations. The surrounding bone tissue and the bio-composite were sampled for microbiology.

Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_17 | Pages 64 - 64
1 Dec 2018
EXTREME HIGH LOCAL INTRAOPERATIVE GENTAMICIN CONCENTRATIONS ARE NEEDED TO PREVENT BIOFILM FORMATION IN VIVO
Jensen LK Bjarnsholt T Henriksen NL Kragh KN Jensen HE
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Aim

To investigate the local intra-operative concentration of gentamicin needed to prevent biofilm formation in a porcine model of implant-associated osteomyelitis.

Method

In total 24 pigs were allocated to six groups. Group A (n=6) was inoculated with saline. Groups B (n=6), C (n=3), D (n=3), E (n=3) and F (n=4) were inoculated with 10 μL saline containing 104 CFU of Staphylococcus aureus, however, different minimal inhibitory concentrations (MIC) of gentamicin were added to the inoculum of Groups C(160xMIC), D(1600xMIC), E(16000xMIC) and F(160000xMIC). The inoculums were injected into a pre-drilled implant cavity proximally in the right tibial bone. Following inoculation, a steel implant (2 × 15 mm) was placed in the cavity. The pigs were euthanized after five days. The implants were sonicated and swabs were taken from the implant cavity for microbiological evaluation. The peri-implant tissue was analyzed by histopathology including estimation of neutrophil infiltration.

Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_17 | Pages 45 - 45
1 Dec 2018
SINGLE-DOSE BONE PHARMACOKINETICS OF VANCOMYCIN IN A PORCINE IMPLANT-ASSOCIATED OSTEOMYELITIS MODEL
Bue M Hanberg P Koch J Jensen LK Lundorff M Aalbæk B Jensen HE Søballe K Tøttrup M
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Aim

The increasing incidence of orthopaedic methicillin-resistant Staphylococcus aureus (MRSA) infections represents a significant therapeutic challenge. Being effective against MRSA, the role of vancomycin may become more important in the orthopaedic setting in the years to come. Nonetheless, vancomycin bone and soft tissue penetration during infection remains unclear. We assessed the effect of a traumatically induced, implant-associated acute osteomyelitis on vancomycin bone penetration in a porcine model.

Method

In eight pigs, implant-associated osteomyelitis was induced on day 0, using a Staphylococcus aureus strain. Following administration of 1,000 mg of vancomycin on day 5, vancomycin concentrations were obtained with microdialysis for eight hours in the implant bone cavity, in cancellous bone adjacent to the implant cavity, in subcutaneous adipose tissue (SCT) adjacent to the implant cavity, and in healthy cancellous bone and healthy SCT in the contralateral leg. Venous blood samples were also obtained. The extent of infection and inflammation was evaluated by post-mortem computed tomography scans, C-reactive protein serum levels and cultures of blood and swabs.

Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_22 | Pages 60 - 60
1 Dec 2017
HISTOPATHOLOGICAL DIAGNOSIS OF BIOFILM
Jensen LK Henriksen NL Jensen HE
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Aim

Despite the expanding research focusing on bacterial biofilm formation, specific histochemical biofilm stains have not been developed for light microscopy. Therefore, pathologists are often not aware of the presence of biofilm formation when examining slides for diagnosing bacterial infections, including orthopaedic infections. The aim of the present study was to develop a combined histochemical and immunohistochemical biofilm stain for simultaneous visualization of Staphylococcus aureus bacteria and extracellular matrix in different colours using light microscopy.

Methods

Infected bone tissue was collected from two different porcine models of osteomyelitis inoculated with the biofilm forming S. aureus strain S54F9. The infection time was 5 and 15 days, respectively. First, 25 common histochemical protocols were used in order to find stains that could identify extracellular biofilm matrix. Hereafter, the histochemical protocols for Alcian Blue pH3, Luna and Methyl-pyronin green were combined with an immunohistochemical protocol based on a specific antibody against S. aureus. Finally, the three new combined protocols were applied to infected bone tissue from a child suffering from chronic staphylococcal osteomyelitis for more than a year. For all combined protocols applied on all types of tissue (porcine and human) the number of double stained bacterial aggregates were counted. On the same sections the percentage of extracellular matrix of representative bacterial aggregates was calculated by image analysis.

Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_23 | Pages 77 - 77
1 Dec 2016
INFECTED BONE TISSUE DECREASES THE PENETRATION OF CEFUROXIME
Tøttrup M Bue M Koch J Jensen LK Hanberg P Aalbæk B Fuursted K Jensen HE Søballe K
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Aim

A reason for treatment failure, in cases of periprosthetic bone infections and osteomyelitis, may be incomplete or heterogeneous tissue distribution of antimicrobials to the affected bone. Decreased bioavailability has been demonstrated in healthy bones but never in pathological bone tissue. Therefore, the aim was to obtain pharmacokinetic parameters of cefuroxime in infected bone tissue by means of microdialysis in a porcine model of implant associated osteomyelitis

Method

An implant cavity of 4 mm in diameter was drilled 25 mm into the right tibial bone of ten pigs (30 kg/BW). Subsequently, a small steel implant (K-wire 2 × 2 mm) and 104 CFU of Staphylococcus aureus was inserted and injected into the implant cavity. Five days after inoculation, two additional drill holes of 2 × 25 mm were drilled into the trabecular bone tissue adjacent to the implant cavity and into the left uninfected tibia. After intravenous administration of 1500 mg of cefuroxime, the concentration was measured in plasma and in the three tibial drill holes for 8 hours. All measurements were performed with microdialysis. Post mortem, the presence of bone infection was assessed by computed tomography (CT) scans and cultures of swabs.