Abstract
Introduction
Histology remains the gold standard in morphometric and pathological analyses of osteochondral tissues in human and experimental bone and joint disease. However, histological tissue processing is laborious, destructive and only provides a two-dimensional image in a single anatomical plane. Micro computed tomography (μCT) enables non-destructive three-dimensional visualization and morphometry of mineralized tissues and, with the aid of contrast agents, soft tissues. In this study, we evaluated phosphotungstic acid-enhanced (PTA) μCT to visualize joint pathology in spine osteoarthritis.
Methods
Lumbar facet joint specimens were acquired from six patients (5 female, age range 31–78) undergoing decompression surgery. Fresh osteochondral specimens were immediately fixed in formalin and scanned in a benchtop μCT scanner (65 kV, 153 mA, 25 μm resolution). Subsequently, samples were completely decalcified in 5% formic acid, equilibrated in 70% ethanol and stained up to ten days in 1% PTA (w/v) in 70% ethanol. PTA-stained specimens were scanned at 70 kV, 140 mA, 15 μm resolution. Depth-dependent analysis of X-ray attenuation in cartilage tissues was performed using ImageJ. Bone structural parameters of undecalcified and PTA-stained specimens were determined using CT Analyser and methods were compared using correlation and Bland-Altman analysis.
Results
The maximal penetration depth of PTA in decalcified facet joint was 5 mm. Bone tissue showed strong and uniformly distributed X-ray attenuation, while mild to moderate and differentially distributed attenuation was observed in articular cartilage and subchondral marrow spaces. Measurements of bone volume (r=0.90, p=0.01) and bone surface (r=0.95, p=0.004) were strongly correlated between undecalcified and PTA-stained samples. Compared with PTA-stained samples, measurements in undecalcified specimens were consistently higher (∼14%). PTA-enhanced μCT visualization of cartilage tissues enabled the identification of individual chondrocytes and their pericellular microenvironment (chondrons). Owing to loss of collagen lower X-ray attenuation was observed in the middle and deep cartilage layers at the central, but not peripheral, regions of the degenerated facet joint specimens. Depth-dependent analysis of PTA-staining intensity suggested that the extent of collagen loss in articular cartilage might correlate with the thickness of the subchondral cortical plate.
Conclusion
PTA-enhanced μCT is a low-cost, non-toxic and highly feasible method for ex vivo 3D-visualization of osteochondral pathology in human osteoarthritis. The method enables bone morphometric analysis, as well as collagen distribution in all anatomical planes. Contrast enhanced μCT has several applications in bone and osteoarthritis research including 3D histopathological grading, tissue stratification, and imaging and analysis of aberrant collagen metabolism in osteochondral disease.
