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Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_10 | Pages 63 - 63
1 Oct 2022
ULTRASTRUCTURAL ANALYSIS OF MITOCHONDRIA FROM OSTEOBLASTS AND OSTEOCYTES FROM PATIENTS WITH OSTEOMYELITIS
Mendelsohn DH Walter N Niedermair T Alt V Brochhausen C Rupp M
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Aim

Osteomyelitis is a difficult-to-treat disease with high chronification rates. The surgical amputation of the afflicted limb sometimes remains as the patients’ last resort. Several studies suggest an increase in mitochondrial fission as a possible contributor to the accumulation of intracellular reactive oxygen species and thereby to cell death of infectious bone cells. The aim of this study is to analyze the ultrastructural impact of bacterial infection and its accompanying microenvironmental tissue hypoxia on osteocytic and osteoblastic mitochondria.

Method

19 Human bone tissue samples from patients with osteomyelitis were visualized via light microscopy and transmission electron microscopy. Osteoblasts, osteocytes and their respective mitochondria were histomorphometrically analyzed. The results were compared to the control group of 5 non-infectious human bone tissue samples.

Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_15 | Pages 5 - 5
1 Dec 2021
RIFAMPICIN RESTORES THE METABOLIC AND BONE FORMATION ACTIVITIES OF OSTEOBLASTS AFTER INTRACELLULAR STAPHYLOCOCCUS AUREUS INFECTION
Alagboso F Mannala G Steinmann S Docheva D Rupp M Brochhausen C Alt V
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Aim

Bone regeneration following the treatment of Staphylococcal bone infection or osteomyelitis is challenging due to the ability of Staphylococcus aureus to invade and persist within bone cells, which could possibly lead to antimicrobial tolerance and incessant bone destruction.

Here, we investigated the influence of Staphylococcal bone infection on osteoblasts metabolism and function, with the underlying goal of determining whether Staphylococcus aureus-infected osteoblasts retain their ability to produce extracellular mineralized organic matrix after antibiotic treatment.

Method

Using our in vitro infection model, human osteoblasts-like Saos-2 cells were infected with high-grade Staphylococcus aureus EDCC 5055 strain, and then treated with 8 µg/ml rifampicin and osteogenic stimulators up to 21-days.

Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_15 | Pages 12 - 12
1 Dec 2021
CAN NECROTIC BONE BE OBJECTIVELY IDENTIFIED IN CHRONIC FRACTURE-RELATED INFECTIONS? FIRST CLINICAL EXPERIENCE WITH AN INTRAOPERATIVE FLUORESCENCE IMAGING TECHNIQUE
Rupp M Henssler L Brochhausen C Zustin J Geis S Pfeifer C Alt V Kerschbaum M
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Aim

Adequate debridement of necrotic bone is of paramount importance for eradication of infection in chronic osteomyelitis. Currently, no tools are available to detect the exact amount of necrotic bone in order to optimize surgical resection. The aim of the present study was to evaluate the feasibility of an intraoperative illumination method (VELscope®) and the correlation between intraoperative and pathohistological findings in surgically treated chronic fracture related infection patients.

Method

Ten consecutive patients with chronic fracture related infections of the lower extremity were included into this prospectively performed case series. All patients had to be treated surgically for fracture related infections requiring bony debridement. An intraoperative illumination method (VELscope®) was used to intraoperatively differentiate between viable and necrotic bone. Tissue samples from the identified viable and necrotic bone areas were histopathologically examined and compared to intraoperative findings.

Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_15 | Pages 87 - 87
1 Dec 2021
MICROBIOLOGICAL AND ULTRASTRUCTURAL EVALUATION OF THE 191219 S. AUREUS BACTERIOPHAGE ACTIVITY AGAINST PLANKTONIC, INTRACELLULAR, AND BIOFILM INFECTION WITH STAPHYLOCOCCUS AUREUS
Mannala G Rupp M Walter N Brunotte M Alagboso F Docheva D Brochhausen C Alt V
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Aim

Here, we are aimed to evaluate bacteriophage (191219) to treat S. aureus implant-associated bone infections by means of testing against S. aureus during its planktonic, biofilm and intracellular growth phases and finally assessing antimicrobial effect on in vivo biofilm formed on metal K-wire in an alternative insect model Galleria mellonella.

Method

The bacteriophages (191219) were provided from D&D Pharma GmbH. These bacteriophages were tested against S. aureus EDCC 5055 (MSSA) and S. aureus DSM 21979 (MRSA) strains. To assess the activity of bacteriophages against planktonic growth phase, bacteriophages, and S. aureus EDCC 5055(1×107 CFU/ml) were co-cultured in LB media as multiplicity of infection (MOI) of 10, 1, 0.1, and 0.01 for 24 hours at 37oC and finally plated out on the LB agar plates to estimate the bacterial growth. The antimicrobial activity of bacteriophages on biofilms in vitro was measured by analysing the incubating the several fold dilutions of bacteriophages in LB media with biofilms formed on 96-well plate. The eradication of biofilm was analysed with crystal violet as well as CFU analysis methods. Later, the effect of bacteriophages on intracellular growth of S. aureus in side osteoblast was tested by treating the S. aureus infected osteoblasts at 2h, 4h and 24h time points of post treatment. In addition, we have analysed synergistic effect with gentamicin and rifampicin antibiotics to clear intracellular S. aureus. Finally, experiments are performed to prove the effect of bacteriophages to clear in vivo biofilm using alternative insect model G. mellonella as well as to detect the presence of bacteriophages inside the osteoblasts through transmission electron microscopy (TEM) analysis.

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