Mineral crystal thickness in calcified cartilage and subchondral bone in healthy and osteoarthritic knees

Abstract

Osteoarthritis (OA) is the most common joint disease globally. In OA, 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. Especially, it is technically challenging to identify calcified cartilage, where relevant but poorly understood pathological processes like tidemark multiplication and advancement occur. Here, we used state-of-the art micro-focus small-angle X-ray scattering with high 5μm spatial resolution to determine mineral crystal thickness in human subchondral bone and calcified cartilage. Specimens with a wide spectrum of OA severities were acquired from the medial and lateral compartments of medial compartment knee OA patients (n=15) and cadaver knees (n=10). For the first time, we identified a well-defined layer of calcified cartilage associated with pathological tidemark multiplication, containing 0.32nm thicker crystals compared to the rest of calcified cartilage. In addition, we found 0.2nm thicker mineral crystals in both tissues of the lateral compartment in OA compared with healthy knees, indicating a loading-related disease process since the lateral compartment is typically less loaded in medial compartment knee OA. Furthermore, the crystal thickness of the subchondral bone was lower with increasing histopathological OA severity. In summary, we report novel changes in mineral crystal thickness during OA. Our data suggest that unloading in the knee is associated with the growth of mineral crystals, which is especially evident in the calcified cartilage. In the subchondral bone, mineral crystals become thinner with increasing OA severity, which indicates new bone formation with sclerosis.

Collagen pre-strain discontinuity at the bone—Cartilage interface

“…On one side, there is a layer of articular cartilage (AC) with depth-dependent gradients in fibre orientation (first described by the Benninghof architecture [ 7 ]), type II collagen/proteoglycan content ratios, and hydration [ 8 ], and (more recently identified) nanoscale collagen fibrillar pre-strain and intramolecular disorder [ 9 ]. As shown in Fig 1 , collagen fibrils in the AC traverse a tidemark into a layer of calcified cartilage (CC) [ 10 , 11 ], enabling a firm anchorage of the AC cartilage to the underlying subchondral bone (SCB). Below the SCB is the more open spongy or trabecular bone (TB) network.…”

“…Below the SCB is the more open spongy or trabecular bone (TB) network. Scanning X-ray scattering and electron microscopy measurements on the calcified mineral phase in the calcified plate (CP) (the combined calcified cartilage and subchondral bone), have revealed gradients in mineral composition [ 12 ], crystallinity, and size [ 10 ], and recently, differences in the mineral crystallite thickness between healthy and osteoarthritic patients [ 11 , 13 ]. The gradient in compressive properties from cartilage (1-10MPa) to subchondral bone (GPa) reduces high interfacial shear stresses which could otherwise lead to cracks and delamination between the softer cartilage and the stiffer bone.…”

Collagen Pre-strain Discontinuity at the Cartilage - Bone Interface

“…Thirdly, we have limited our sampling to a specific condylar anatomical site in the metacarpophalangeal joint and given that in-vivo strains vary across the joint, it would be interesting to test if nanomechanical parameters like pre-strain shows a correlation with varying physiological force levels across the joint. Lastly, we have not yet considered changes in OA and ageing, to establish a baseline characterisation of the normal BCI in an animal model; a recent study [11] has shown that nanoscale mineral particle thickness is significantly different in OA versus normal human patients and investigating in situ mechanical response in aged human cartilage would no doubt be of importance.…”

“…Below the subchondral bone is the more open spongy or trabecular bone network. Scanning X-ray scattering and electron microscopy measurements on the calcified mineral phase in the calcified plate (the combined calcified cartilage and subchondral bone) have revealed gradients in mineral composition [12], crystallinity, and size [10], and recently, differences in the mineral crystallite thickness between healthy and osteoarthritic patients [11,13]. The gradient in compressive properties from cartilage (1-10MPa) to subchondral bone (GPa) reduces high interfacial shear stresses which could otherwise lead to cracks and delamination between the softer cartilage and the stiffer bone.…”

“…On one side, there is a layer of AC with depth-dependent gradients in fibre orientation (first described by the Benninghof architecture [7]), type II collagen/proteoglycan content ratios, and hydration [8], and (more recently identified) nanoscale collagen fibrillar pre-strain and intramolecular disorder [9]. As shown in Figure 1 , collagen fibrils in the AC traverse a tidemark into a layer of calcified cartilage of [10,11], enabling a firm anchorage of the AC cartilage to the underlying subchondral bone. Below the subchondral bone is the more open spongy or trabecular bone network.…”