Fujii Shun, Horie Naoyuki, Ikegami Saki, Imamura Hayata, Zhu Wenliang, Ikegaya Hiroshi, Mazda Osam, Pezzotti Giuseppe, Takahashi Kenji
Department of Orthopaedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kamigyo-ku, 465 Kajii-cho, Kyoto 602-8566, Japan.
Ceramic Physics Laboratory, Kyoto Institute of Technology, Sakyo-ku, Matsugasaki, Kyoto 606-8585, Japan.
Int J Mol Sci. 2025 Sep 3;26(17):8572. doi: 10.3390/ijms26178572.
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 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 (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 , which featured the relative intensity of the 1644 cm signal peculiar to the oxidized molecules with respect to its pyrrole ring-breathing signal at 750 cm, 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.
是引起骨髓炎最常见的病原体，骨髓炎是一种难以恢复的骨感染，给患者带来巨大负担。骨髓炎小鼠模型长期以来成功地用于深入了解发病机制和宿主反应。然而，已发表的文献中仍明显缺乏在细胞水平上对骨微生物发病机制和免疫反应的直接原位监测。在这里，我们更新了Wistar大鼠模型中的标准化脓性骨髓炎，以便通过光谱分辨拉曼光谱进行原位分析，研究大鼠胫骨骨髓炎中的细菌定位和免疫反应。在大鼠胫骨中增加细菌接种的初始剂量后，在感染后1周和5周进行拉曼实验。无标记原位拉曼光谱通过利用特征类胡萝卜素葡萄球菌黄素分子的特殊信号，清楚地揭示了 的存在。数据是作为初始细菌接种剂量和沿胫骨位置的函数收集的。这种与类胡萝卜素碳链主链中的单键和双键相关的强拉曼信号，即使在低感染水平下也可作为有效的细菌标记物。我们还可以检测到来自骨细胞中细胞色素 （及其氧化形式）的强拉曼信号，以响应感染和炎症途径。尽管初始接种仅限于靠近内侧髁的单个位置，但细菌沿整个骨骼扩散至内踝，与初始感染剂量无关。拉曼光谱表征通过与葡萄球菌黄素碳链主链长度相关的特定光谱生物标志物，全面定量地揭示了细菌的代谢状态。此外，真核细胞的生理反应可以通过监测线粒体细胞色素 的氧化水平来量化，根据先前发表的文献，其特征在于氧化分子特有的1644 cm信号相对于其在750 cm处的吡咯环呼吸信号的相对强度。总之，我们在此提出一种新的拉曼光谱方法，用于对骨组织中的细菌浓度和免疫反应进行索引。这种新方法能够对原位骨感染、炎症宿主组织反应以及细菌抗性/适应性进行定位和表征。
