Three-Dimensional Quantitative Morphometric Analysis (QMA) for In Situ Joint and Tissue Assessment of Osteoarthritis in a Preclinical Rabbit Disease Model

Abstract

This work utilises advances in multi-tissue imaging, and incorporates new metrics which define in situ joint changes and individual tissue changes in osteoarthritis (OA). The aims are to (1) demonstrate a protocol for processing intact animal joints for microCT to visualise relevant joint, bone and cartilage structures for understanding OA in a preclinical rabbit model, and (2) introduce a comprehensive three-dimensional (3D) quantitative morphometric analysis (QMA), including an assessment of reproducibility. Sixteen rabbit joints with and without transection of the anterior cruciate ligament were scanned with microCT and contrast agents, and processed for histology. Semi-quantitative evaluation was performed on matching two-dimensional (2D) histology and microCT images. Subsequently, 3D QMA was performed; including measures of cartilage, subchondral cortical and epiphyseal bone, and novel tibio-femoral joint metrics. Reproducibility of the QMA was tested on seven additional joints. A significant correlation was observed in cartilage thickness from matching histology-microCT pairs. The lateral compartment of operated joints had larger joint space width, thicker femoral cartilage and reduced bone volume, while osteophytes could be detected quantitatively. Measures between the in situ tibia and femur indicated an altered loading scenario. High measurement reproducibility was observed for all new parameters; with ICC ranging from 0.754 to 0.998. In conclusion, this study provides a novel 3D QMA to quantify macro and micro tissue measures in the joint of a rabbit OA model. New metrics were established consisting of: an angle to quantitatively measure osteophytes (σ), an angle to indicate erosion between the lateral and medial femoral condyles (ρ), a vector defining altered angulation (λ, α, β, γ) and a twist angle (τ) measuring instability and tissue degeneration between the femur and tibia, a length measure of joint space width (JSW), and a slope and intercept (m, Χ) of joint contact to demonstrate altered loading with disease progression, as well as traditional bone and cartilage and histo-morphometry measures. We demonstrate correlation of microCT and histology, sensitive discrimination of OA change and robust reproducibility.

Fig 1. Overview Schema.

Schema illustrating key aspects of the experimental procedure and parameters of interest.

Fig 2. Comparison of microCT and histology.

(a) Plot of 2D cartilage thickness measured by microCT against histology, R = 0.94, p < 0.001, and (b) a Bland-Altman plot showing relatively even scatter at both low and high values of mean measures, indicating no obvious trend of increasing scatter with Cg.Th. (c) Plot of mean grey value measured with microCT against histology (grouped by staining batch), and (d) a Bland-Altman plot showing the spread of scatter points with a strong proportional bias with increasing mean measures, independent of histology staining batch.

Fig 3. User reproducibility for cartilage segmentation.

Plot of 2D cartilage thickness (Cg.Th) measured by three users segmenting a random selection of matching microCT and histology images; 10 femur and 10 tibia image pairs from both NO and OP joints, demonstrating user independence of the segmentation procedure. ICC (microCT) = 0.996 (0.992, 0.998); ICC (histology) = 0.993 (0.984, 0.997), p < 0.001.

Fig 4. Typical examples of 3D QMA showing differences between OP and NO joints.

(a) Thickness maps of top: femoral, and bottom: tibial cartilage compartments indicating increased Cg.Th in OP medial and lateral femoral compartments, and medial tibial compartment, as well as increased Cg.S and Cg.V in OP medial femoral condyle, and increased Cg.S in lateral tibial plateau. (b) Larger JSW, smaller α angle, and more acute medial σ angles are evident in the OP joint. (c) Upon virtual loading of the femur onto the tibia in a stepwise manner (i) the distance to reach first contact, χ - indicated in red—is significantly higher in lateral OP compartments compared to NO. (ii) and (iii) Once contact is reached, rate of increase, m, rises significantly faster in medial OP compartments.

Fig 5. Bivariate Regressions of QMA measures.

Relationships between QMA measures indicate significant associations between and within tissue measures (Cg.Th, Ct.Th) and whole joint measures (JSW, χ, β, σ). Correlations were observed between Cg.Th and JSW both (a) laterally (R = 0.64, p < 0.01) and (b) medially (R = 0.66, p < 0.01), where Cg.Th is the addition of mean tibial and femoral compartmental values. This was also seen laterally (c) but not medially (d) for JSW and χ (R = 0.88, p < 0.01). The β angle is strongly negatively correlated with both (e) lateral and (f) medial tibial Cg.Th (R = -0.69 and -0.66, respectively, p < 0.01), and Ct.Th is negatively correlated with σ in the (g) lateral and (h) medial tibia (R = -0.52 and -0.55, respectively, p < 0.05).

Fig 6. Visualisation of osteophytes.

Osteophytes (red arrows) are indicated in the femur (a,c,e,g) and tibia (b,d,f,h) as visualised with (a-b) histology, (c-h) microCT: (c-d) the matching microCT image, (e-f) a cut-through the microCT 3D greyscale image showing low attenuating osteophyte masses, and (g-h) a 3D reconstruction of the microCT scan (PRE).

Fig 7. Measurement reproducibility.

Typical HEX1/HEX2/HEX3 scans show excellent measurement reproducibility (ICC > 0.74) for cartilage, bone and in situ joint measures. (a) Bone morphometric ICC values were low for tibial BS/BV (0.077) and Tb.Th (0.403), due to penetration of Hexabrix^® into bone tissue. Scale bar = 5 mm. (b) Femoral and (c) tibial cartilage thickness maps demonstrate good reproducibility in cartilage compartments.