Mineral Crystal Thickness in Calcified Cartilage and Subchondral Bone in Healthy and Osteoarthritic Human Knees

Abstract

Osteoarthritis (OA) is the most common joint disease, where articular cartilage degradation is often accompanied with sclerosis of the subchondral bone. However, the association between OA and tissue mineralization at the nanostructural level is currently not understood. In particular, it is technically challenging to study calcified cartilage, where relevant but poorly understood pathological processes such as tidemark multiplication and advancement occur. Here, we used state-of-the-art microfocus small-angle X-ray scattering with a 5-μm spatial resolution to determine the size and organization of the mineral crystals at the nanostructural level in human subchondral bone and calcified cartilage. Specimens with a wide spectrum of OA severities were acquired from both medial and lateral compartments of medial compartment knee OA patients (n = 15) and cadaver knees (n = 10). Opposing the common notion, we found that calcified cartilage has thicker and more mutually aligned mineral crystals than adjoining bone. In addition, we, for the first time, identified a well-defined layer of calcified cartilage associated with pathological tidemark multiplication, containing 0.32 nm thicker crystals compared to the rest of calcified cartilage. Finally, we found 0.2 nm thicker mineral crystals in both tissues of the lateral compartment in OA compared with healthy knees, indicating a loading-related disease process because the lateral compartment is typically less loaded in medial compartment knee OA. In summary, we report novel changes in mineral crystal thickness during OA. Our data suggest that unloading in the knee might be involved with the growth of mineral crystals, which is especially evident in the calcified cartilage. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Keywords: ANALYSIS/QUANTITATION OF BONE; BONE MODELING AND REMODELING; COLLAGEN; MATRIX MINERALIZATION; OSTEOARTHRITIS.

Fig. 1

Schematic representation of osteochondral sample collection and μSAXS measurement of healthy and osteoarthritic knee joints. (A) Osteochondral plugs were collected from both lateral and medial femoral condyles. (B) Schematic cross‐sectional representation of samples taken from healthy and osteoarthritic knee joints. The white dotted line represents the 500 μm wide μSAXS measurement area extending from the deep articular cartilage to the subchondral bone (500–1000 μm).

Fig. 2

Histopathological assessment of the osteochondral samples. (A) Box plot with jitter showing the OARSI grades (with subgrades) in the lateral and medial condyles of cadaveric donors and total knee replacement (TKR) patients. Each grade represents the following key feature. Grade 0: Fully intact cartilage; grade 1: Intact surface with cellular changes and/or edema; grade 2: Surface discontinuity; grade 3: Vertical fissures; grade 4: Cartilage erosion; grade 5: Denudation (articular cartilage matrix loss to calcified cartilage); and grade 6: Deformation. (B) Box plot with a pairwise comparison showing the number of tidemarks, which were counted from the histopathological images. There was complete erosion of calcified cartilage in three samples from the Medial^TKR group and these patients were excluded from the pairwise comparison.

Fig. 3

Tissue segmentation from 2D μSAXS images. Step 1: The mineral crystal thickness maps were binarized using an absolute threshold on scattering intensity. Step 2: K‐means cluster analysis of the I(q) scattering curves. Step 3: Eight clusters were found to be optimal for most of the samples when comparing the cluster images to the cSAXS visualizations of crystal orientation and asymmetric intensity. The cluster assignments were further confirmed by comparing the cluster images to the histopathological images of adjacent sections. Step 4: Final tissue‐specific binary masks for CC and SB were generated after de‐speckling. CC = calcified cartilage; SB = subchondral bone.

Fig. 4

SAXS parameters in the two layers of CC of the samples with tidemark multiplication. Box plots showing the pairwise comparison of the mineral crystal thickness and degree of orientation between the superior and inferior layers of calcified cartilage in the osteochondral samples with multiple tidemarks, from both lateral (15 samples) and medial (8 samples) compartments, respectively. CC = calcified cartilage.

Fig. 5

Compartment‐specific comparison of the mineral crystal thickness between cadaveric donors (donor) and TKR patients. Differences in mineral crystal thickness are displayed with a 95% confidence interval and p value in the calcified cartilage and the subchondral bone, respectively. The model was adjusted for age, and then for age and BMI. The comparison between medial and lateral compartments from the same knee is adjusted for all individual‐ and knee‐level confounding through the design and use of a mixed‐effects model. TKR = total knee replacement.

Fig. 6

Compartment‐specific comparison of the degree of orientation of mineral crystals in the calcified cartilage and the subchondral bone between cadaveric donors (donor) and TKR patients. Differences in the degree of orientation are displayed with a 95% confidence interval and p value. The mixed‐effects model was adjusted for age, and then for age and BMI. TKR = total knee replacement.