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

Abstract

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.

Keywords: Collagen organization; Contrast agent free micro-computed tomography; Meniscus microstructure; Osteoarthritis; Structure tensors.

Figure 1

Schematic representation of 3D local orientation analysis. The principal orientation direction is calculated from the image by storing the original μCT images as 3D arrays of grey values using structure tensors. Eigenvalue decomposition of this structure tensor matrix, three eigenvalues and corresponding eigenvectors are produced. The smallest eigenvalue represents the principal direction of local fiber orientation. In the equation, W(p) is the integration window, vector p is position of integration window, and vector r with the components (x, y, z) is the single point in image I relative to the integration window. More details can be found in in ref.

Figure 2

A) Representative μCT 3D reconstructions of tissue pieces acquired from posterior horns of meniscus from a healthy donor on the top row and a total knee replacement (TKR) patient on the bottom row. B) A cubic volume-of-interest (VOI) was selected from the middle of the meniscus piece for local orientation analysis. C) VOIs with calculated orientations show mean angles for each voxel with dominantly high angles in the donor sample and dominantly low angles in the TKR sample. D) Graphical illustration how polar angle theta (⊖) is calculated in the 3D space. Color bar shows the representative mean orientation angle in each voxel.

Figure 3

A) Representative μCT 3D volumes from medial OA, medial donor, lateral OA and lateral donor groups. B) The selected volume-of-interest (VOI) with size of 1800 μm x 1800 μm x 1800 μm from the meniscus middle layer. C) The analyzed VOIs representing orientation angles of the meniscus microstructure.

Figure 4

The mean percentage of voxels as a function of local orientation angle (0-90 degrees) in the studied sample groups (medial OA, lateral OA, medial donor, and lateral donor) with 95% confidence intervals, as predicted from the Poisson regression model (see statistical methods section for details). Medial OA group had the highest amount of low angle orientations indicating most disorganization in the tissue when compared to all other groups. Medial donor and lateral OA groups have similar low and high angle distributions. Lateral donor group had the smallest amount of low angle orientations indicating the healthiest tissue group.

Figure 5

The percentage of voxels at each angle for each individual sample for OA samples (A) and donor samples (B).