Biofilm-Induced Bone Degradation in Osteomyelitis: Insights from a comprehensive ex vivo pathogen interaction study

Abstract

Objectives: Osteomyelitis, characterised by bone inflammation due to microbial infection, presents significant healthcare challenges. While the protective role of biofilm in bacterial immunity and persistence is well-documented, its direct impact on bone degradation in osteomyelitis remains inadequately characterised. This study aimed to comprehensively examine the direct effects of biofilm-forming pathogens on human bone, providing new insights into the mechanisms of bone destruction associated with osteomyelitis.

Methods: Bone sections were collected from patients undergoing total knee replacement surgery at Sultan Qaboos University Hospital, Muscat, Oman, between January 2021 and December 2022. The samples were then inoculated with Staphylococcus aureus to simulate in vitro bone infection. Raman spectroscopy and scanning electron microscopy (SEM) were employed to analyse the bacterial interaction with bone tissue.

Results: Biofilm-forming pathogens directly contributed to bone degradation, as evidenced by SEM images showing marked trabecular destruction. Raman analysis showed a significant increase in the carbonate-to-phosphate ratio in inoculated samples (61.9%) compared to controls (47%). Additionally, mineral content decreased in inoculated samples, and the carbonate-to-amide I ratio reduced by 47% in inoculated samples and by 80% in controls. The inoculated samples exhibited an 82% shift in collagen cross-linking compared to a 72% shift in controls.

Conclusions: This research enhances the comprehension of the mechanisms underlying bone destruction in osteomyelitis and underscores the intricate role of biofilm in disease progression. These findings highlight the importance of biofilm in bone degradation and its potential implications for infection management.

Keywords: Biofilm; Bone and Bones; Infection Control; Oman; Osteomyelitis; Raman Spectroscopy; Scanning Electron Microscopy.

Fig. 1.

A: Typical Raman spectrum of healthy human bone with labelled waves’ peaks of interest. B: Average Raman spectra collected from both control and inoculated samples at 8 weeks of incubation.

Fig. 2.

A: Carbonate to phosphate ratio in inoculated samples compared to controls showing changing bone crystallinity and transformation to a brittle matrix. B & C: Carbonate to amide I ratio in inoculated samples compared to controls showing a reduction in mineral content relative to the organic material. D: Graph showing the collagen cross-linking ratio as a shift of the Raman bands at 1,660 cm^-1 to 1,668 cm^-1 in inoculated samples compared to controls, representing the destruction of collagen cross-linkage.

Fig. 3.

Scanning electron microscopy images showing a divergence at 2 different magnifications (A & B: × 1 mm magnification; C & D: × 200 μm magnification) between the control (A & C) and inoculated groups (B & D).