Quantitative three-dimensional collagen orientation analysis of human meniscus posterior horn in health and osteoarthritis using micro-computed tomography.

OBJECTIVE: Knee osteoarthritis (OA) is associated with meniscal degeneration that may involve disorganization of the meniscal collagen fiber network. Our aims were to quantitatively analyze the microstructural organization of human meniscus samples in 3D using micro-computed tomography (µCT), and to compare the local microstructural organization between OA and donor samples. METHOD: We collected posterior horns of both medial and lateral human menisci from 10 end-stage medial compartment knee OA patients undergoing total knee replacement (medial & lateral OA) and 10 deceased donors without knee OA (medial & lateral donor). Posterior horns were dissected and fixed in formalin, dehydrated in ascending ethanol concentrations, treated with hexamethyldisilazane (HMDS), and imaged with µCT. We performed local orientation analysis of collagenous microstructure in 3D by calculating structure tensors from greyscale gradients within selected integration window to determine the polar angle for each voxel. RESULTS: In donor samples, meniscus bundles were aligned circumferentially around the inner border of meniscus. In medial OA menisci, the organized structure of collagen network was lost, and main orientation was shifted away from the circumferential alignment. Quantitatively, medial OA menisci had the lowest mean orientation angle compared to all groups, -24° (95%CI -31 to -18) vs medial donor and -25° (95%CI -34 to -15) vs lateral OA. CONCLUSIONS: HMDS-based µCT imaging enabled quantitative analysis of meniscal collagen fiber bundles and their orientations in 3D. In human medial OA menisci, the collagen disorganization was profound with overall lower orientation angles, suggesting collagenous microstructure disorganization as an important part of meniscus degradation.

Three-dimensional microstructure of human meniscus posterior horn in health and osteoarthritis.

OBJECTIVE: To develop and perform ex vivo 3D imaging of meniscus posterior horn microstructure using micro-computed tomography (µCT), and to compare specimens from healthy references against end-stage osteoarthritis (OA) using conventional section-based histology and qualitative µCT. DESIGN: We retrieved human medial and lateral menisci from 10 deceased donors without knee OA (healthy references) and medial and lateral menisci from 10 patients having total knee replacement for medial compartment OA. Meniscal posterior horns were dissected and fixed in formalin. One subsection underwent hexamethyldisilazane (HMDS) treatment and µCT imaging. Pauli's histopathological scoring was performed for 3 other subsections. The differences in histopathological scores were estimated using mixed linear regression, resulting in fixed effects estimates for within-knee comparisons and adjusted for age and body mass index for between-subjects comparisons. RESULTS: 3D visualization with µCT qualitatively revealed similar microstructural changes in the posterior horns as conventional histology. The mean histopathological score was higher for medial menisci from OA knees vs both medial reference menisci (mean difference [95% CI], 3.9 [2.6,5.3]), and lateral menisci from OA knees (3.9 [2.9,5.0]). The scores were similar between lateral menisci from OA knees and lateral reference menisci (0.8 [-0.6,2.2]), and between medial and lateral reference menisci (0.8 [-0.3,1.9]). CONCLUSIONS: HMDS-based µCT protocol allows unique 3D visualization of meniscus microstructures. Posterior horns of medial menisci from medial compartment OA knees had higher histopathological scores than both the lateral posterior horns from the same OA knees and medial reference menisci, suggesting a strong association between meniscus degradation and unicompartmental knee OA.

3D morphometric analysis of calcified cartilage properties using micro-computed tomography.

OBJECTIVE: Our aim is to establish methods for quantifying morphometric properties of calcified cartilage (CC) from micro-computed tomography (µCT). Furthermore, we evaluated the feasibility of these methods in investigating relationships between osteoarthritis (OA), tidemark surface morphology and open subchondral channels (OSCCs). METHOD: Samples (n = 15) used in this study were harvested from human lateral tibial plateau (n = 8). Conventional roughness and parameters assessing local 3-dimensional (3D) surface variations were used to quantify the surface morphology of the CC. Subchondral channel properties (percentage, density, size) were also calculated. As a reference, histological sections were evaluated using Histopathological osteoarthritis grading (OARSI) and thickness of CC and subchondral bone (SCB) was quantified. RESULTS: OARSI grade correlated with a decrease in local 3D variations of the tidemark surface (amount of different surface patterns (rs = -0.600, P = 0.018), entropy of patterns (EP) (rs = -0.648, P = 0.018), homogeneity index (HI) (rs = 0.555, P = 0.032)) and tidemark roughness (TMR) (rs = -0.579, P = 0.024). Amount of different patterns (ADP) and EP associated with channel area fraction (CAF) (rp = 0.876, P < 0.0001; rp = 0.665, P = 0.007, respectively) and channel density (CD) (rp = 0.680, P = 0.011; rp = 0.582, P = 0.023, respectively). TMR was associated with CAF (rp = 0.926, P < 0.0001) and average channel size (rp = 0.574, P = 0.025). CC topography differed statistically significantly in early OA vs healthy samples. CONCLUSION: We introduced a µ-CT image method to quantify 3D CC topography and perforations through CC. CC topography was associated with OARSI grade and OSCC properties; this suggests that the established methods can detect topographical changes in tidemark and CC perforations associated with OA.

In vitro method for 3D morphometry of human articular cartilage chondrons based on micro-computed tomography.

OBJECTIVE: The aims of this study were: to 1) develop a novel sample processing protocol to visualize human articular cartilage (AC) chondrons using micro-computed tomography (µCT), 2) develop and validate an algorithm to quantify the chondron morphology in 3D, and 3) compare the differences in chondron morphology between intact and osteoarthritic AC. METHOD: The developed protocol is based on the dehydration of samples with hexamethyldisilazane (HMDS), followed by imaging with a desktop µCT. Chondron density and depth, as well as volume and sphericity, were calculated in 3D with a custom-made and validated algorithm employing semi-automatic chondron selection and segmentation. The quantitative parameters were analyzed at three AC depth zones (zone 1: 0-10%; zone 2: 10-40%; zone 3: 40-100%) and grouped by the OARSI histological grades (OARSI grades 0-1.0, n = 6; OARSI grades 3.0-3.5, n = 6). RESULTS: After semi-automatic chondron selection and segmentation, 1510 chondrons were approved for 3D morphometric analyses. The chondrons especially in the deeper tissue (zones 2 and 3) were significantly larger (P < 0.001) and less spherical (P < 0.001), respectively, in the OARSI grade 3-3.5 group compared to the OARSI grade 0-1.0 group. No statistically significant difference in chondron density between the OARSI grade groups was observed at different depths. CONCLUSION: We have developed a novel sample processing protocol for chondron imaging in 3D, as well as a high-throughput algorithm to semi-automatically quantify chondron/chondrocyte 3D morphology in AC. Our results also suggest that 3D chondron morphology is affected by the progression of osteoarthritis (OA).