The development of a label-free, non-destructive and safe analytical method such as Raman spectroscopy for assessing cartilage degradation is highly desirable. Compared to non-optical imaging modalities, Raman mapping offers a more sensitive means of directly assessing the chemical composition of cartilage in three-dimensional space and the potential to monitor cartilage degeneration to inform intervention and treatment strategies. Herein, we report the application of Raman spectroscopic methods ex vivo and at arthroscopy to identify molecular alterations in cartilage specimens containing minor focal lesions characteristic of the early disease phase. Our initial ex vivo analysis, obtained by single-point Raman spectroscopy of cartilage samples, supports previous findings based on S-O stretching vibration bands associated with sulphated glycosaminoglycans (sGAGs). We extended the analyses to the high-wavenumber region where we observed that vibrational bands assigned to C-H and O-H stretching modes discriminated early cartilage alterations from healthy cartilage samples. Furthermore, we performed a proof-of-concept in-clinic study using a custom-built optical probe to acquire Raman spectral measurements for the first time in patients undergoing arthroscopy of knee joints. Spectra were obtained with adequate signal-to-noise ratios that similarly discriminated between lesion and adjacent cartilage sites and identified reductions in sGAGs in apparently healthy cartilage. Building on this, we present initial results from Raman mapping to spatially resolve the molecular constituents of cartilage through its depth and across a lesion. Mapping revealed a non-uniform and reduced sGAG distribution within the lesion and peripheral cartilage that was otherwise visually normal, similar to the in-clinic observations, showing that the degradative influence of the lesion extended beyond its border. This was accompanied by a decreased fluorescence signal intensity, which suggests that fluorescence may provide valuable information as an adjunct to the Raman signal in discriminating normal and degenerating cartilage. This work demonstrates the value of Raman mapping over single-point Raman measurements for the analysis of the anisotropy of articular cartilage and highlights the potential of the technology for in vivo articular joint arthroscopy applications.

开发一种无标记、非破坏性和安全的分析方法，如用于评估软骨退化的拉曼光谱是非常需要的。与非光学成像模式相比，拉曼映射提供了一种更敏感的方法，可以直接评估三维空间中软骨的化学成分，并有可能监测软骨退化，为干预和治疗策略提供信息。在此，我们报告了拉曼光谱方法在体外和关节镜下的应用，以识别含有早期疾病阶段特征的轻微局灶性病变的软骨标本中的分子改变。我们最初的体外通过软骨样品的单点拉曼光谱获得的分析支持先前基于与硫酸化糖胺聚糖 (sGAG) 相关的 SO 伸缩振动带的发现。我们将分析扩展到高波数区域，我们观察到分配给 CH 和 OH 拉伸模式的振动带将早期软骨改变与健康软骨样本区分开来。此外，我们使用定制的光学探头进行了概念验证临床研究，首次在接受膝关节关节镜检查的患者中获取拉曼光谱测量值。获得的光谱具有足够的信噪比，类似地区分病变和相邻的软骨部位，并确定明显健康的软骨中 sGAG 的减少。在此基础上，我们展示了拉曼映射的初步结果，以通过其深度和跨病变在空间上解析软骨的分子成分。映射显示病变和外周软骨内的 sGAG 分布不均匀且减少，否则视觉上是正常的，类似于临床观察，表明病变的降解影响超出了其边界。这伴随着荧光信号强度的降低，这表明荧光可以作为拉曼信号的辅助手段提供有价值的信息，以区分正常和退化的软骨。这项工作证明了拉曼映射相对于单点拉曼测量在分析关节软骨各向异性方面的价值，并强调了该技术在以下方面的潜力活体关节关节镜应用。