ABSTRACT

Osteoarthritis is a common chronic disease of joints characterized by degenerative changes of articular cartilage. An early diagnosis of osteoarthritis may be possible when imaging excised tissue for research in situ at the cellular-molecular scale. Whereas cartilage histopathology is destructive, time-consuming, and limited to 2D views, contrast-enhanced x-ray microscopy (XRM) can image articular cartilage and subchondral bone in 3D. This study evaluates articular cartilage structure ex vivo using both techniques.

Osteochondral plugs were excised from non-diseased bovine knees and stained in phosphotungstic acid for 0 to 32 h. XRM imaging revealed an optimal staining time of 16 h and a saturated staining time of 24 h. Histology sections were cut and analyzed by polarized light microscopy (PLM) and second-harmonic-generation dual-photon (SHG-DP) microscopy. Histology photomicrographs were aligned with matching XRM slices and evaluated for features relevant in histopathological scoring of osteoarthritis cartilage, including the tidemark, collagen architecture and chondrocyte morphology.

The cartilage collagen network and chondrocytes from the 3D contrast-enhanced XRM were correlated with the 2D histology. This technique has two distinct advantages over routine histopathology: (1) the avoidance of dehydration, demineralization, and deformation of histological sectioning, thereby preserving the intact articular cartilage and subchondral bone plate ex vivo, and (2) the ability to evaluate the entire osteochondral volume in 3D. This work explores several diagnostic features of imaging cartilage, including: visualization of the tidemark in XRM and SHG-DP microscopy, validating the morphology of chondrocytes and nuclei with XRM, SHG-DP and PLM, and correlating collagen birefringence with XRM image intensity.

摘要

骨关节炎是一种以关节软骨退行性改变为特征的常见关节慢性疾病。当对切除的组织进行成像以在细胞分子尺度上进行原位研究时，骨关节炎的早期诊断是可能的。尽管软骨组织病理学具有破坏性、耗时且仅限于 2D 视图，但对比增强 X 射线显微镜 (XRM) 可以对关节软骨和软骨下骨进行 3D 成像。本研究使用这两种技术评估离体关节软骨结构。

从未患病的牛膝上切下骨软骨栓，并在磷钨酸中染色 0 至 32 小时。XRM 成像显示最佳染色时间为 16 小时，饱和染色时间为 24 小时。通过偏振光显微镜 (PLM) 和二次谐波产生双光子 (SHG-DP) 显微镜切割和分析组织切片。组织学显微照片与匹配的 XRM 切片对齐，并评估与骨关节炎软骨组织病理学评分相关的特征，包括潮位标记、胶原结构和软骨细胞形态。

来自 3D 对比增强 XRM 的软骨胶原网络和软骨细胞与 2D 组织学相关。与常规组织病理学相比，该技术具有两个明显的优势：(1) 避免组织切片的脱水、脱矿质和变形，从而在体外保留完整的关节软骨和软骨下骨板，以及 (2) 评估整个骨软骨的能力3D 体积。这项工作探讨了成像软骨的几个诊断特征，包括：在 XRM 和 SHG-DP 显微镜下观察潮位标记，使用 XRM、SHG-DP 和 PLM 验证软骨细胞和细胞核的形态，以及将胶原双折射与 XRM 图像强度相关联。