Department of Musculoskeletal Biology and Ageing Science, Institute of Life Course and Medical Sciences, University of Liverpool, William Henry Duncan Building, 6 W Derby St, Liverpool L7 8TX, UK.
Centre for Metabolomics Research, Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Biosciences Building, Crown Street, Liverpool, L69 7BE, UK.
Anal Methods. 2022 Sep 29;14(37):3661-3670. doi: 10.1039/d2ay00779g.
Equine osteoarthritis is a chronic degenerative disease of the articular joint, characterised by cartilage degradation resulting in pain and reduced mobility and thus is a prominent equine welfare concern. Diagnosis is usually at a late stage through clinical examination and radiographic imaging, whilst treatment is symptomatic not curative. Extracellular vesicles are nanoparticles that are involved in intercellular communication. The objective of this study was to investigate the feasibility of Raman and Optical Photothermal Infrared Spectroscopies to detect osteoarthritis using plasma-derived extracellular vesicles, specifically differentiating extracellular vesicles in diseased and healthy controls within the parameters of the techniques used. Plasma samples were derived from thoroughbred racehorses. A total of 14 samples were selected (control; = 6 and diseased; = 8). Extracellular vesicles were isolated using differential ultracentrifugation and characterised using nanoparticle tracking analysis, transmission electron microscopy, and human tetraspanin chips. Samples were then analysed using combined Raman and Optical Photothermal Infrared Spectroscopies. Infrared spectra were collected between 950-1800 cm. Raman spectra had bands between the wavelengths of 900-1800 cm analysed. Spectral data for both Raman and Optical Photothermal Infrared Spectroscopy were used to generate clustering principal components analysis and classification models were generated using partial least squared discriminant analysis in order to characterize the techniques' ability to distinguish diseased samples. Optical Photothermal Infrared Spectroscopy could differentiate osteoarthritic extracellular vesicles from healthy with good classification (93.4% correct classification rate) whereas Raman displayed poor classification (correct classification rate = -64.3%). Inspection of the infrared spectra indicated that plasma-derived extracellular vesicles from osteoarthritic horses contained increased signal for proteins, lipids and nucleic acids. For the first time we demonstrated the ability to use optical photothermal infrared spectroscopy combined with Raman spectroscopy to interrogate extracellular vesicles and osteoarthritis-related samples. Optical Photothermal Infrared Spectroscopy was superior to Raman in this study, and could distinguish osteoarthritis samples, suggestive of its potential use diagnostically to identify osteoarthritis in equine patients. This study demonstrates the potential of Raman and Optical Photothermal Infrared Spectroscopy to be used as a future diagnostic tool in clinical practice, with the capacity to detect changes in extracellular vesicles from clinically derived samples.
马的骨关节炎是一种关节的慢性退行性疾病，其特征是软骨降解导致疼痛和运动能力下降，因此是一个突出的马福利问题。诊断通常是通过临床检查和放射影像学在晚期进行的，而治疗是对症的，不是治愈性的。细胞外囊泡是参与细胞间通讯的纳米颗粒。本研究的目的是探讨使用血浆衍生的细胞外囊泡通过拉曼和光热红外光谱检测骨关节炎的可行性，特别是在所用技术的参数范围内区分疾病和健康对照的细胞外囊泡。血浆样品取自纯种赛马。共选择了 14 个样本（对照组；n = 6，病变组；n = 8）。使用差速超速离心法分离细胞外囊泡，并通过纳米颗粒跟踪分析、透射电子显微镜和人类四跨膜蛋白芯片进行表征。然后使用组合拉曼和光热红外光谱对样品进行分析。收集 950-1800 cm 之间的红外光谱。分析了波长在 900-1800 cm 之间的拉曼光谱带。使用拉曼和光热红外光谱的光谱数据生成聚类主成分分析，并使用偏最小二乘判别分析生成分类模型，以表征技术区分病变样本的能力。光热红外光谱能够很好地区分骨关节炎的细胞外囊泡与健康的细胞外囊泡（正确分类率为 93.4%），而拉曼显示出较差的分类（正确分类率=-64.3%）。对红外光谱的检查表明，来自骨关节炎马的血浆衍生的细胞外囊泡含有增加的蛋白质、脂质和核酸信号。我们首次证明了使用光热红外光谱结合拉曼光谱来检测细胞外囊泡和骨关节炎相关样本的能力。在这项研究中，光热红外光谱优于拉曼，能够区分骨关节炎样本，表明其具有潜在的诊断用途，可用于识别马的骨关节炎。本研究表明，拉曼和光热红外光谱具有作为临床实践中未来诊断工具的潜力，能够检测从临床样本中提取的细胞外囊泡的变化。
