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Staphylococcus aureus is the most common pathogen causing osteomyelitis, a hardly recoverable bone infection that generates significant burden to patients. Osteomyelitis mouse models have long and successfully served to provide phenomenological insights into both pathogenesis and host response. However, direct in situ monitoring of bone microbial pathogenesis and immune response at the cellular level is still conspicuously missing in the published literature. Here, we update a standard pyogenic osteomyelitis in Wistar rat model, in order to investigate bacterial localization and immune response in osteomyelitis of rat tibia upon adding in situ analyses by spectrally resolved Raman spectroscopy. Raman experiments were performed one and five weeks post infections upon increasing the initial dose of bacterial inoculation in rat tibia. Label-free in situ Raman spectroscopy clearly revealed the presence of Staphylococcus aureus through exploiting peculiar signals from characteristic carotenoid staphyloxanthin molecules. Data were collected as a function of both initial bacteria inoculation dose and location along the tibia. Such strong Raman signals, which relate to single and double bonds in the carbon chain backbone of carotenoids, served as efficient bacterial markers even at low levels of infection. We could also detect strong Raman signals from cytochrome c (and its oxidized form) from bone cells in response to infection and inflammatory paths. Although initial inoculation was restricted to a single location close to the medial condyle, bacteria spread along the entire bone down to the medial malleolus, independent of initial infection dose. Raman spectroscopic characterizations comprehensively and quantitatively revealed the metabolic state of bacteria through specific spectroscopic biomarkers linked to the length of staphyloxanthin carbon chain backbone. Moreover, the physiological response of eukaryotic cells could be quantified through monitoring the level of oxidation of mitochondrial cytochrome c, which featured the relative intensity of the 1644 cm⁻¹ signal peculiar to the oxidized molecules with respect to its pyrrole ring-breathing signal at 750 cm⁻¹, according to the previously published literature. In conclusion, we present here a novel Raman spectroscopic approach indexing bacterial concentration and immune response in bone tissue. This new approach enables locating and characterizing in situ bone infections, inflammatory host tissue reactions, and bacterial resistance/adaptation. Full article

Osteomyelitis is an infection of the bone that is often difficult to treat and causes a significant healthcare burden. Staphylococcus aureus is the most common pathogen causing osteomyelitis. Osteomyelitis mouse models have been established to gain further insights into the pathogenesis and host response. Here, we use an established S. aureus hematogenous osteomyelitis mouse model to investigate morphological tissue changes and bacterial localization in chronic osteomyelitis with a focus on the pelvis. X-ray imaging was performed to follow the disease progression. Six weeks post infection, when osteomyelitis had manifested itself with a macroscopically visible bone deformation in the pelvis, we used two orthogonal methods, namely fluorescence imaging and label-free Raman spectroscopy, to characterise tissue changes on a microscopic scale and to localise bacteria in different tissue regions. Hematoxylin and eosin as well as Gram staining were performed as a reference method. We could detect all signs of a chronically florid tissue infection with osseous and soft tissue changes as well as with different inflammatory infiltrate patterns. Large lesions dominated in the investigated tissue samples. Bacteria were found to form abscesses and were distributed in high numbers in the lesion, where they could occasionally also be detected intracellularly. In addition, bacteria were found in lower numbers in surrounding muscle tissue and even in lower numbers in trabecular bone tissue. The Raman spectroscopic imaging revealed a metabolic state of the bacteria with reduced activity in agreement with small cell variants found in other studies. In conclusion, we present novel optical methods to characterise bone infections, including inflammatory host tissue reactions and bacterial adaptation. Full article

Osteomyelitis is a chronic bone infection that is often treated with adjuvant antibiotic-impregnated poly(methyl methacrylate) (PMMA) cement spacers in multi-staged revisions. However, failure rates remain substantial due to recurrence of infection, which is attributed to the poor performance of the PMMA cement as a drug release device. Hence, the objective of this study was to design and evaluate a bioresorbable calcium phosphate scaffold (CaPS) for sustained antimicrobial drug release and investigate its efficacy in a murine model of femoral implant-associated osteomyelitis. Incorporating rifampin and sitafloxacin, which are effective against bacterial phenotypes responsible for bacterial persistence, into 3D-printed CaPS coated with poly(lactic co-glycolic) acid, achieved controlled release for up to two weeks. Implantation into the murine infection model resulted in decreased bacterial colonization rates at 3- and 10-weeks post-revision for the 3D printed CaPS in comparison to gentamicin-laden PMMA. Furthermore, a significant increase in bone formation was observed for 3D printed CaPS incorporated with rifampin at 3 and 10 weeks. The results of this study demonstrate that osteoconductive 3D printed CaPS incorporated with antimicrobials demonstrate more efficacious bacterial colonization outcomes and bone growth in a single-stage revision in comparison to gentamicin-laden PMMA requiring a two-stage revision. Full article