Background
Osteoarthritis (OA) is a leading cause of disability and joint dysfunction in adults. Since the predominant feature of OA is degeneration of articular cartilage, most studies into the pathogenesis of OA have tended to focus on the mechanisms involved in the destruction of the articular cartilage. However, subchondral bone sclerosis is also a well-characterized manifestation in OA and many studies have emphasized the importance of subchondral bone changes, such as composition, architecture, quality, and regulation as important distinguishing features of OA [
1‐
3]. Studies have demonstrated abnormal biochemistry in subchondral bone in OA compared to normal controls, with increased bone formation and relatively high bone mineral density (BMD) [
3,
4]. It is well known that changes to the composition of the subchondral bone matrix in OA are associated with alterations in bone microarchitecture. During the end stage of OA, microarchitectural characteristics of the subchondral bone are (i) thickening of subchondral bone plate and trabecular bone, (ii) increased bone volume fraction, (iii) decrease of trabecular separation and bone marrow spacing, (iv) and transformation of the trabeculae from a rod-like to a plate-like configuration [
5]. Disordered microarchitecture within the subchondral bone causes it to become relatively stiffer and denser in OA affected bone and leads to a disruption of the equilibrium of the mechanical loading between cartilage and subchondral bone. Although sclerotic bone is less well mineralized, it suffers greater absorption of local stresses, reducing load transmission to the deeper subarticular region and resulting in OA progression [
6].
Despite the growing body of research into the biophysical and mechanical properties of OA bone [
7,
8] there are few studies that have analysed the structure of the subchondral sclerosis at the nanoscale. It is therefore not well understood how the hypomineralized subchondral sclerosis region responds to the increased mechanical strains. The hierarchical structure of bone, from nano scale to the organ level, ultimately determines its mechanical strength and properties. At the nano-scale, bone is a composite with a quasiperiodic structure, consisting of carbonated hydroxyapatite (HA) crystals, which are embedded into collagen fibrils. An exact match of collagen fibrils and mineral crystal organization provides bone with its capacity to withstand mechanical loads. Until now, the evaluation of OA includes an assessment of a patient’s bone mineral density (BMD), using techniques such as computer tomography (CT, or micro-CT) and magnetic resonance imaging (MRI). Changes seen using micro-CT (μCT) are morphometric parameters, such as bone volume fraction and trabecular number, thickness, and separation. However, these techniques do not provide an understanding of mechanical properties such as the hardness, modulus, and toughness of the tissue and the quality of mineral and fibres at nano-scale, all of which are independent of bone mass or micro-architecture.
In this study we evaluated the subchondral bone structure at various length-scales in two representative Polar Regions, with and without sclerosis (grade IV OA and grade I OA), in patient matched samples with an aim of providing a better understanding of the structural and compositional determinants of bone strength. For this purpose we have used advanced imaging techniques to characterize the material quality of the OA bone and its mechanical strength at the nano-scale level. Nanoindentation was used to determine hardness and elastic modulus at defined local positions of sub-micrometer sizes in various subchondral bone and trabecular areas. Transmission electron microscopy (TEM) imaging, Electron Diffraction, and Elemental Analysis techniques were used to explore bone fibrils banding patterns, the nature of the mineral phase and the orientation of crystal lattices. Furthermore, subchondral bone nano-hydroxyapatite powders were prepared and characterised using high-resolution transmission electron microscopy (HR-TEM) and Fourier transform infrared spectroscopy (FTIR). Applying these techniques, we found that severe OA-affected bone had altered nano-structural and mechanical properties.
Discussion
We hypothesized that OA bone changes were related to changes to the physicochemical properties of bone materials and not simply changes to overall bone mass. To test this hypothesis we analysed subchondral bone from the superior and inferior sectors of tibial sections from OA patients. The results from this study demonstrated a relationship between the pathological changes in OA bone and changes to mineral phase of the bone at the nano-structural level.
It is widely accepted that bone stiffness and ductility are strongly influenced by the collagen fibers and the physiochemical property of carbonated HA, respectively [
23,
24]. In this study, we observed that the fibrilar skeleton lost its well-organized appearance in the severely affected subchondral bone plate and sclerotic trabeculae (Figs.
2d and
3d) in grade IV bone (Figs.
2e and
3e). Moreover, grade IV bone adjacent to the severely affected lesion also displayed non-hierarchical intra-fibrillar mineralization; however, the Ca/P ratios showed some kind of crosscurrent distribution in subchondral bone plate and trabeculae. Increasing electron density in grade IV bone was suggestive of mineral aggregation which could result in the fibrils having less ductility and being subjected to greater compressive stress [
25]. The subchondral bone plate lies immediately beneath the calcified zone of the articular cartilage. Due to its anatomical position, the subchondral bone plate carries most of the load passing through the joint. It is therefore under constant stress and consequently has a high rate of metabolism [
26], which could alter the distribution of calcium ion and the form of bony salts. Consistent with our observations, Buchwald et al. [
27] observed the ratio of carbonate apatite to hydroxyapatite is higher in the subchondral bone plate from OA patients, which indicated deficient mineralization and had an impact on mineral crystal growth [
28]. In another study, it has been reported that bone from the iliac crest have higher mineral contents by density fractionation of cortical bone and back scattered electron microscopy. These data indicate that site and bone type may be important factors governing the changes caused by OA. The high frequency of bone turnover in OA subchondral bone plate leads to an unstable environment for lesion recovery and normal mineralization, and failure to form crystalline and correctly oriented mineral crystals. Deterioration of subchondral bone plate structure could expose subjacent trabeculae to abnormal mechanical stresses and thus caused pathological and adaptive changes in trabeculae [
29]. Changes in collagen could also affect the mineralization process [
30]. The arrangement of fibrils could alter the way that collagen molecules interact with each other and with surrounding macromolecules and would, therefore, ultimately affect the morphology and arrangement of minerals formed in the collagen matrix [
30]. The diffused 002 planes in the grade IV subchondral bone plate suggest changes to the orientation and phase of mineral crystals (Fig.
2g and h). In the severely affected lesion of grade IV bone (Fig.
3h), non-parallel arrangement of fibrils and mineral crystals could cause an abnormal load transmission and makes it hard for fibers to dissipate the deformation energy and thus promote the micro-damage. Nano-sized mineral crystals showed a highly ductile behavior, but at the macro-scale were increasingly brittle [
31]. We made the observation by TEM that fibrils in sclerotic subchondral bone plates and trabeculae undergo changes to mineralization and rearrangements, something which highlights the complex pathological mechanism of OA disease. The amorphous profile of mineral crystals in sclerotic trabeculae is indicative of a coalescence of mineral particles that is reported to lead increased bone brittleness.
Disordered arrangement of organic and inorganic composition had a negative effect on load transmission [
32]. Distinctly heterogeneous distribution of Ca/P ratios in subchondral bone plate is adverse to form a proper mineral phase that could absorb load stresses, conversely, more abnormal stresses were transmit to subjacent trabeculae. Bone strength and stiffness increased with increasing mineral crystallinity [
31]. As a compensatory reaction, increased mineral crystallinity in trabeculae could be a possible explanation to the increment of localized stress absorption. Varying Ca/P ratio affected mineral crystals in the physical, mineralogical and mechanical characteristics [
28,
33]. Compared with the grade I tabeculae, Ca/P ratio in grade IV trabeculae showed an uneven distribution (Fig.
3). The ratio could be affected by organic phosphate in tissue. To eliminate this possibility, we extracted the mineral ingredients from the fresh trabeculea. The plate-like structure in OA trabeculae has been reported [
8,
34], which reflected high mechanical stress and was associated with similar morphology of mineral crystals as shown in this study (Fig.
4b) [
35]. The crystals from the grade IV OA trabeculae produced sharper diffraction rings by electron diffraction plus Ca/P ratio values approaching the theoretical value of HA, suggesting a possibly higher crystallinity. This notion was supported by the changes in the infrared splitting factor, the value of which showed a numerical increase in the mineral crystals from sclerotic trabeculae. Increased mineral crystallinity and non-hierarchical intra-fibrillar mineralization in subchondral sclerosis would further enhance the localized stiffness of bone material and lead to a corresponding absorption of local stresses, the non-affected region near the lesion could suffer from the atrophy of disuse and thus display localized stress shielding, evidenced by lower Ca/P ratio in grade I bone [
36]. This opens up to the possibility of a “mineralization adaptation zone” between the lesion area and non-affected area, which would assist the localized load transmission and lead to increased subchondral sclerosis [
37]. Excessive intra-fibrillar mineralization not only increases the intra-fibrillar density of mineral content but also contains minerals with higher crystallinity. However, at some point high carbonated HA crystallinity is associated with bone brittleness, which implies that the more crystalline the bone the more liable it is to form critical sized cracks, since it is less able to withstand deformation [
38]. It is worth noting that technical limitations make it impossible to extract the mineral crystals from the subchondral bone plate.
Previous studies have revealed that structural changes to OA bone is the result of mechano-regulated bone adaptation [
39,
40]. In the sclerotic trabeculae, pathological remodeling of the bone results in disordered fibrils and mineral crystal arrangements. In the present study, the grade IV OA trabeculae obtained a higher
E
r and
H values as OA progresses, whereas disordered structure and high crystalline mineral content made grade IV OA bone less tolerant to micro-cracks of the order of several hundred micro-meters, a size which may be essential for normal bone remodeling. During the active OA stages, the mineral deposition is attenuated in the lesion region by high bone turnover, resulting in hypomineralization of the bone [
41]. This is associated with less stiffness and causes the bone structure to collapse more readily under load. Micro-cracks were generated and healed to form a thicker and denser subchondral bone structure for mechanical adaptation. However, the healing progresses were depressed due to low bone turnover at the late OA stage and thus produced more micro-cracks in the sclerotic lesion [
41,
42].
Altered anisotropic mechanical properties were found between the grade I and grade IV OA regions, which may increase the bone brittleness, thus leading to macroscopic failure of the tissue and the risk of catastrophic bone fractures. When the mechano-regulatory pathway of bone is activated [
39], the continued deposition of minerals may lead to a localized hyper-mineralized phase of the subchondral bone during the OA stationary stage, and low bone turnover in the lesion region results in an abnormal aggregation of mineral crystals in the sclerotic region. This creates a stable micro-environment for mineral crystallization and an increased
E
r
value, which, is in turn, compels the bony stiffness to deal with more force. Paradoxically, the ductility of bone is suppressed by non-hierarchical intra-fibrillar mineralization and high crystalline mineral crystal, showing a higher
H value, which is indicative of high bone brittleness in OA. Both increments of the
E
r and
H values in grade IV trabeculae indicated that both osteons and lamellae were subject to significant changes in mechanical properties during OA disease progression. A higher modulus increases resistance to elastic deformation and an increased hardness accounted for the stiff but brittle properties of bone. This supports the notion that sclerotic trabecular bone had a denser structure and stiffer property than the grade I OA trabecular bone that had suffered osteoporosis [
43]. Furthermore, increased crystallinity of the mineral phase increases the chemical stability of the crystals [
44] and leads to reduced rates of bone turnover in sclerosis and, therefore, results in stiffer bone material property than the grade I trabeculae. Using micro-indentation testing and electron probe microanalysis of the hip, Coats et al. has shown a reduced hardness and elastic modulus in OA bone when compared to osteoporotic bone [
45]. However, in our study we found an increase in the hardness and elastic modulus compared to mild OA bone. These differences could be attributed to the location of the sample site differences between hip and knee. In another study, Li et al., showed an altered mechanical and material properties of the subchondral bone plate from the femoral head of patients with either OA or osteoporosis [
46,
47]. Our data add a novel perspective to the general understanding of the bone stiffening mechanism in subchondral sclerosis. It is well-documented that sclerotic bone has less mineralization in the lesion region [
6] but absorbs the most stress of the bone [
43], and in this study we observed that hypomineralized subchondral sclerosis displayed a disordered mineralization distribution and that hypermineralized parts in trabeculae could assist with localized stress absorption. Increased intra-fibrillar mineral density also results in the fibrils having less ductility and being subjected to greater compressive stress. Furthermore, increased crystallinity of the mineral phase renders higher stiffness to bone, and increased chemical stability of the crystals leads to reduced rates of bone turnover in sclerosis [
44].