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Updated: Nov 23, 2025
Combined In vivo Optical and µCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice
Published on: October 16, 2014
Emerging electron microscopy and 3D methodologies to interrogate Staphylococcus aureus osteomyelitis in murine models
Karen L de Mesy Bentley1,2,3, Chad A Galloway2, Gowrishankar Muthukrishnan1,3
1Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, USA.
Abstract:
Recent breakthroughs in our understanding of orthopaedic infections have come from advances in transmission electron microscopy (TEM) imaging of murine models of bone infection, most notably Staphylococcus aureus invasion and colonization of osteocyte-lacuno canalicular networks of live cortical bone during the establishment of chronic osteomyelitis. To further elucidate this microbial pathogenesis and evaluate the mechanism of action of novel interventions, additional advances in TEM imaging are needed. Here we present detailed protocols for fixation, decalcification, and epoxy embedment of bone tissue for standard TEM imaging studies, as well as the application of immunoelectron microscopy to confirm S. aureus occupation within sub-micron canaliculi. We also describe the first application of the novel Automated-Tape-UltraMicrotome system with three-dimensional reconstruction and volumetric analyses to quantify S. aureus occupation within the osteocyte-lacuno canalicular networks. Reconstruction of the three-dimensional volume broadened our perspective of S. aureus colonization of the canalicular network and, surprisingly, revealed adjacent noninfected canaliculi. This observation has led us to hypothesize that viable osteocytes of the osteocyte-lacuno canalicular networks respond and resist infection, opening future research directions to explain the paradox of adjacent uninfected canaliculi and life-long deep bone infection in patients with chronic osteomyelitis.
Insights
Advanced transmission electron microscopy reveals Staphylococcus aureus colonization within bone's microscopic networks. Surprisingly, some networks resist infection, suggesting osteocyte defense mechanisms against chronic osteomyelitis.
Area of Science:
- Orthopaedic research
- Microbial pathogenesis
- Advanced imaging techniques
Background:
- Chronic osteomyelitis is a persistent bone infection.
- Staphylococcus aureus invades osteocyte-lacuno canalicular networks.
- Understanding microbial invasion mechanisms is crucial for developing new treatments.
Purpose of the Study:
- To detail protocols for preparing bone tissue for transmission electron microscopy (TEM).
- To apply immunoelectron microscopy and 3D reconstruction to study Staphylococcus aureus in bone.
- To investigate Staphylococcus aureus colonization within the osteocyte-lacuno canalicular network.
Main Methods:
- Detailed protocols for bone tissue fixation, decalcification, and epoxy embedment for TEM.
- Immunoelectron microscopy to confirm bacterial presence in canaliculi.
- Automated-Tape-UltraMicrotome system for 3D reconstruction and volumetric analysis.
Main Results:
- Established protocols for high-resolution TEM imaging of bone infections.
- Confirmed Staphylococcus aureus presence within sub-micron canaliculi.
- 3D reconstruction revealed Staphylococcus aureus colonization patterns and adjacent uninfected canaliculi.
Conclusions:
- Osteocyte-lacuno canalicular networks are sites of Staphylococcus aureus colonization in chronic osteomyelitis.
- The presence of uninfected canaliculi suggests potential osteocyte resistance mechanisms.
- Further research is needed to understand osteocyte responses and the persistence of bone infections.
