Characterizing calcification on the joint surface during OA development. Normal samples and cartilage tissues in early-stage OA (OA-E) and advanced-stage OA (OA-A) were collected and carefully identified through different histologic features for further analysis (Fig. S1). Intact surface and superficial fibrillation were mainly present in OA-E samples, while OA-A samples were largely eroded, a large amount of cartilage had been lost. Next, we examined the joint surface by SEM techniques coupled with energy-dispersive X-ray spectroscopy (EDX) to visualize the topographical characteristics and elemental compositions for each group. The normal joint surface demonstrated collagen fiber alignment without mineral deposition in both SEM and density-dependent color (DDC)-SEM micrographs. In contrast, OA-E surfaces showed some thickened collagen fibrils (Fig. 1b), spherical mineral particles (dimensions ranged from 25 nm to 2.1 µm, n = 841) and calcified fibers (Fig. 1e, Fig. 1k, and Fig. S2). Notably, OA-A surfaces had apparent mineral deposition in three distinct forms: spherites (dimensions of 80 nm ~ 2.4 µm, n = 996), calcified fibers, and compact materials (Fig. 1c, 1f and 1l, Fig. S2). In addition to mineral accumulation on the cartilage surface, mineral deposition in deeper cartilage was detected in OA-A samples (Fig. S3).
Moreover, the presence of calcium (Ca), phosphorus (P), and small amounts of magnesium (Mg) and sodium (Na) were revealed by EDX between the OA-E and OA-A samples. There were no significant variations in Ca and P (Fig. 1h and Fig. 1i), while OA-E appeared to have slightly higher Mg content than OA-A. As Mg is a phase stabilizer for mineral precursors but highly unstable in bulk HAp,18 it is possible that the higher Mg level contributes to the mineral nucleation in OA-E, and lower Mg may be related to spherite growth in OA-A. In addition, we analyzed the Ca/P ratios within minerals with respect to the degree of mineralization.19 The Ca/P ratios in OA-E cartilage (1.59 ± 0.047) were comparable to those of the mature bone matrix.20 Meanwhile, the minerals in the OA-A cartilage were large with a wider distribution than those in OA-E cartilage (Fig. 1k and Fig. 1l), which may correspond to lower Mg concentrations. It appears that a larger mineral particle size correlated with greater OA severity. This phenomenon was also demonstrated in the calcified cardiovascular tissues of humans.21
Calcified matter was previously identified histologically and radiologically in OA cartilage and is considered an important feature of OA progression.2,22 However, mechanisms for the formation and progression of calcified matter in OA have never been extensively investigated.3 We found a large amount of Ca- and P-containing spherites arising at early-stage OA cartilage surfaces. Such spherites appeared to grow larger with different morphologies and even extended to the deeper zones of cartilage in OA-A (Fig. 1h). Such submicron spherical structures have not been examined previously in bone but were discovered in calcified cardiovascular tissues with a diverse range of diseases and in the lacunae micropetrosis in aged bone.23,24 These mineralized spherites seemed to be an intermediate form of calcified minerals, and it was suggested that they were important building blocks in the formation of the calcified region at lesion sites.21 The Mg-rich feature of the spherites deposited on the joint surface may suggest their cellular origin given that Mg2+ ions were important intracellular ions that were largely concentrated in apoptotic bodies and matrix vesicles.25,26 The globular apoptotic bodies and matrix vesicles contained biomolecules such as phosphatidylserine,27 tissue-nonspecific alkaline phosphatase (TNAP)28 and annexins.29 which allow Ca2+ and phosphate (PO43−) accumulation and uptake from the surface26,30. The accumulation of Ca2+ and PO43− was reported to mainly surround the chondrocytes in OA31 and is known to induce chondrocyte hypertrophy that is closely associated with matrix mineralization.2 Together with the higher content of Mg2+, which stabilizes the precursor,18,32 the accumulation of Ca2+ and PO43− regulates spherite formation and growth in OA-E. OA cartilage that was found to increase levels of Ca, P and Mg, supporting mineral deposition.31,33 Thus, cartilage calcification is not a passive deposition of CaP but an early event during OA initiation alongside the phenotypic alterations of chondrocytes.2,5,34
Advancement of calcified matter from calcified cartilage into deep cartilage during OA processes. Significant changes in the composition and structure of cartilage and SB have been thoroughly investigated in OA,11 but those of the connective layer that cause the breakdown of cartilage to predispose OA are unknown. 35 As the invasion of calcified cartilage into the overlying cartilage and duplicated tidemarks are hallmarks of OA,11 we next focused on the detailed mineral assembly patterns and structural organization involved in pathological calcification in different OA stages. Distinct microstructural units at the osteochondral interface were revealed by histological staining in different OA stages (Fig. 2a). Further study by DDC-SEM was conducted to image and examine the microstructures associated with OA status (Figs. 2b). As a result, normal calcified cartilage showed a gradient of spherical minerals proximal to cartilage (zone-1, active calcified matter formation sites) and densely packed minerals adjacent to SB (zone-2) (Fig. 2c). In OA-E cartilage, we found an obvious 100–200 µm-wide calcified layer invading the overlying original calcified cartilage, forming a sandwich structure at the osteochondral transitional zone (Fig. 2b, 2f). The mineral distribution patterns in OA-E cartilage were largely identical to those of the normal calcified cartilage (Fig. 2c). In contrast, the microstructures of calcified cartilage were completely disrupted in OA-A samples (Figs. 2c). A large number of chondrocytes residing in partially calcified lacunae were localized within the mineralization front region of calcified cartilage tissues in both OA-E and OA-A samples, suggesting that these chondrocytes underwent hypertrophy or apoptosis to contribute to calcifying invasion into cartilage (Fig. S4). The progressive mineralized region (zone-1) at the mineralization front of the interface was the active region for calcified invasion. The subsequent EDX line scan indicated an increased thickness of zone-1 that was predominant in both OA-E and OA-A cartilage, compared with healthy samples (Fig. 2d). This suggests the intrusion of calcification into the overlying cartilage to some extent when OA develops (Fig. S4-S5). The elemental compositions of minerals differed between zone-1 and zone 2 for all samples, accompanied by an increased Ca/P ratio from zone-1 to zone-2 (Fig. 2e). The OA-E samples had a Ca/P ratio similar to that of the healthy control in both zone-1 (1.52 ± 0.23 vs. 1.56 ± 0.12) and zone-2 (1.80 ± 0.10 vs. 1.76 ± 0.08), whereas that of OA-A was slightly lower in both zones compared with the control (1.41 ± 0.12 for zone-1, 1.66 ± 0.16 for zone-2). As the Ca/P ratio is indicative of the mineralization degree,19 it was suggested that minerals in calcified cartilage exhibited local variations in their assembly patterns during the evolution of OA pathology.
To understand the changes in mineral assemblies that occurred in the OA calcified cartilage, FIB-SEM was applied to map the nanoscale structural details. In contrast to the normal control, OA-E and OA-A cartilage showed thickened gradient mineralized regions (Fig. 2g), consistent with the EDX line scan results (Fig. 2d) and bright field TEM images (Fig. S5). From the mineralization front to the highly mineralized region of calcified cartilage, three typical mineral morphologies within the matrix were visualized for normal samples. The morphologies included isolated spherites with diverse sizes, fibers assembled within collagen, and well-organized compact materials with uniformly distributed voids (Figs. 2g-2i). As predicted, the OA-E samples showed different morphologies in the calcified cartilage. For instance, in the mineralization front region, larger and denser spherites with clear outlines as well as larger spherites in needle-shaped crystals were observed (Fig. 2h). Such spherites were rarely associated with the collagen matrix and were absent from the normal control. In the highly mineralized region, the densely packed particles and compact materials were shown to have more, larger voids (yellow arrowheads). Such voids were predominantly embedded or located at the peripheries of the particles (Figs. 2h and 2i). These mineral particles, which varied in size and morphology, were discretely distributed at the front of the mineralization region of OA-E (Figs. 2h and 2i). In the OA-A samples, larger spherites with fuzzy peripheries were prominent at the mineralization front, where more numerous and larger voids formed in the surrounding matrix (Fig. 2h). The compact materials possessed smaller but more uniformly distributed voids within the structures in the highly mineralized region (Fig. 2h and 2i). The increasing sizes of voids were associated with OA progression (Figs. 2h-2j). The 3D rendering of typical mineral spherites at the mineralization front of calcified cartilage further illustrated the nanoscale variations in mineral structures in OA (Fig. 2j). The OA-E and OA-A samples lost the fiber morphologies of minerals (Figs. 2h and 2i), which was important to the mineral-extracellular matrix (ECM) assemblies during the mineralization process in bone or other soft-to-hard interfaces.17,36 Meanwhile, considerable variations in mineral morphologies were found at the mineralization front in OA-E and OA-A compared with normal controls (Fig. 2h-2j), possibly indicating different assembly patterns of mineral formation that subsequently promoted the invasion of calcified matter into cartilage or the thickening of calcified cartilage. The larger and increasing number of pores that emerged in calcified cartilage in OA-E may establish communicating channels to facilitate the transfer of molecules to promote crosstalk between cartilage and SB; the largest ones in OA-A may even allow the extension of vessels from SB to the cartilage region.9,37,38 Certain physicochemical metastability at the osteochondral interfaces of healthy samples was hypothesized to maintain the ultrastructural organization of mineral deposits associated with the surrounding ECM.39 It could be altered by activating cellular or molecular processes involved in initiating downstream mineralization events at the mineralization front of the calcified cartilage, resulting in calcified matter invasion from calcified cartilage to the overlying cartilage in OA.11,37 Such a process commonly raises the possibility for concurrent compositional changes in minerals at the osteochondral transitional zone during OA.10
Hypermineralized and hypomineralized HAp minerals in calcified cartilage in early-stage and advanced-stage OA. Variations in the assembly patterns of minerals in calcified cartilage prompted us to analyze their detailed compositions at multiple scales during OA development. Using XRD, FTIR and Raman spectroscopy analysis, carbonate-substituted hydroxyapatite (HAp)10 was detected as the main phase of calcified cartilage for all samples (Fig. S6). HAp was also identified as the main mineral phase of the duplicated “calcified region” in OA-E (Fig. S7). This specific region was formed adjacent to the duplicated tidemarks (Fig. 2, Fig. S7). Acidic components (-COOH) of tidemarks could chelate the supersaturated Ca2+ and PO43− ions in OA cartilage to form crystals.40,41 The spatial distribution of HAp across the calcified cartilage was further assessed using Raman spectroscopy. The characteristic bands in the Raman spectra at 961 cm− 1 (PO43− v1 symmetric stretch) and 1071 cm− 1 (CO32− v1 in-plane vibrations) were used to visualize the spatial distribution of PO43− and CO32− of minerals (Fig. 3a and 3b). The ionic substitution ratios (CO32−/PO43−) and crystallinity (full-width at half-maximum (FWHM) of the peak at 961 cm− 1) of HAp across calcified cartilage were also mapped (Fig. 3c and 3d). A depth-dependent increase in PO43− and CO32− contents, as well as a concomitant decrease in CO32− substitution ratios and FWHM, which inversely indicated mineral crystallinity, created a graded 30 µm-wide region at the mineralization front of calcified cartilage for normal cartilage. In contrast, OA-E samples have higher PO43− and CO32− contents, thus creating a sharp boundary at the mineralization front. Within the same region, CO32− substitution ratios were slightly decreased, while mineral crystallinities were markedly improved compared with normal controls. This indicated a hypermineralized feature of HAp minerals in the calcified cartilage in OA-E.42 OA-A samples have greatly reduced PO43− and CO32− contents in calcified cartilage, where significantly higher CO32− substitution ratios and lower crystallinities were more predominant, suggesting a hypomineralization phenomenon that is completely inverse to OA-E. A previous study also revealed this trend of HAp in calcified cartilage in OA.10
Next, we examined the underlying internal nanoscale compositions and organizations of the minerals. Crystalline domains measuring 2–5 nm progressively enlarged and elongated across the interface until they were completely fused at calcified cartilage of normal samples (Fig. 3e). In OA-E samples, the minerals contained larger crystal size, higher aspect ratio, and higher crystallinity near cartilage. The crystalline aggregates fused precociously before reaching the highly mineralized region, revealing more crystalline-like features of minerals at the mineralization front of OA-E. In contrast, OA-A contained crystalline domains with smaller sizes and lower crystallinity from the mineralized front to the highly mineralized region. These results were in good agreement with the Raman results (Fig. 3a-3d) and further supported the morphological variations of minerals in FIB-SEM. Moreover, minerals in OA-E contained higher contents of Ca and P, whereas those in OA-A were significantly decreased when compared with normal samples (Fig. 3f). As the Ca content determines the Ca/P ratios of minerals, this could explain the higher Ca/P ratios of minerals in OA-E and lower in OA-A (Fig. 3g). These results further revealed the nanoscale hypermineralized and hypomineralized phenomena of minerals in calcified cartilage in OA-E and OA-A cartilage.10 Combining Raman spectroscopy with electron microscopy, we unveiled the compositional variations of minerals in calcified cartilage on a multilevel scale in OA development. Compared with normal cartilage, the substantial deviations in the spatial compositions of HAp at the active mineralization front of calcified cartilage in OA-E and OA-A were closely associated with the morphological changes (Fig. 2).
Next, the distinct local chemical environment of minerals at the calcified region of different cartilages was further studied using EELS. Four typical mineral particles across the calcified cartilage were selected for detailed analysis. Carbon K edge signals (284 eV − 302 eV) revealed the organic matrix for all samples, and the calcium peak at 348 eV − 352 eV confirmed the mineral compounds (Fig. 3h). We generated maps of Ca signals to visualize their distribution within mineral particles across the calcified cartilage (Fig. 3i). The Ca intensity of minerals gradually increased throughout the calcified cartilage region for all samples. Among them, the Ca intensity of minerals in OA-E was greater in the core. In contrast, OA-A gave a more diffuse, cloudy pattern of Ca distribution within minerals that contained a fairly low amount of Ca. This result corresponded with the EDX mapping in Fig. 3f. Higher Ca intensity contributed to a higher level of crystal perfection in OA-E; Ca loss, in turn, induced imperfection of crystalline aggregates in OA-A.
The striking variations in carbonate content have been reported to cause mineral formation and growth in physiological bone and pathological calcification in kidney and cardiovascular tissues15,16,19. Hence, we further studied the fine structures of the carbon K edge. After performing the multi-Gaussian fitting, a small amount of carbonate (peak C at 290 eV)16,19 was noticeable in the cores of minerals in calcified cartilage for all samples (Fig. 3h, Fig. S8, Table S2). Consistent with the Raman results, the intensity of carbonate was greater within spherical particles in OA-A and less in OA-E. Recently, the detection of carbonate in the core of bone mineral precursors19 has led to the postulation that carbonated calcium may act as bioseeds for calcium phosphate formation at the mineralization front of calcified cartilage in OA-A. Moreover, the reduction in mineral crystallinity by carbonate substitution into the lattice43 explains how the graded crystallinity of minerals in normal samples was altered to undergo hypermineralization and hypomineralization in OA-E and OA-A samples, respectively.
We also detected a small number of carbonyl signals (peak B at 287 eV) from mineral structures in OA-E and OA-A samples (Fig. 3h, Fig. S8). These carbonyl signals seemed to be more intense in minerals in OA-E. Their presence should be ascribed to organic compounds such as osteopontin and TNAP (not limited), which were reported to be highly expressed in OA cartilage and played a role in mediating mineral nucleation and growth.3,44 Such organic compounds assemble onto the membranes of matrix vesicles to entrap ions (Ca2+ and PO43−) for nucleation and crystal growth.45 A similar process may occur at the mineralization front of calcified cartilage in OA-E.
Overall, these observations suggested that great variations in mineral particles in calcified cartilage, in terms of morphology and composition for OA-E and OA-A tissues, may be ascribed to different nucleation mechanisms: organic compounds (matrix vesicles) for OA-E and calcium carbonate for OA-A.
Calcium-containing matrix vesicles secreted by hypertrophic chondrocytes drive two kinds of nucleation in OA development. To ascertain the abovementioned nucleation process, hypertrophic chondrocytes in lacunae in calcified cartilage were further analyzed using DDC-SEM and FIB-SEM (Fig. 4). DDC-SEM micrographs showed the presence of many dense materials (minerals) around the hypertrophic chondrocytes in OA cartilage (Fig. 4a, Fig. S4 and S9). FIB-SEM images validated their submicron structures and surroundings (Fig. 4b). The hypertrophic chondrocytes in OA-E resided in a confined lacunar region located at a distance from the surrounding mineralized islands, while those in OA-A were closely surrounded by minerals. Hypertrophic chondrocytes in OA-E behaved like osteoblasts by secreting inhibitors (e.g., osteopontin) to keep the closest pericellular matrix free of minerals and release promoters (e.g., TNAP) to remove the inhibitors.46 Without the inhibition of nucleation, crystallization occurred in the ECM.20,47 Matrix vesicles containing disordered precursors of CaP were closely associated with this mineralization process.20,48,49 Thus, we further examined their presence and features in normal and OA samples.
Calcium phosphate-containing matrix vesicles were observed in the ECM within the lacuna of OA-E (Figs. 4b-4d, Fig. S10). Fewer matrix vesicles were found in the same region in OA-A and were rarely detected in normal samples (Figs. 4b-4d). To examine their phase details, the Ca/P ratios of these vesicles were further calculated, and the values for OA-E and OA-A were 0.53 ± 0.21 and 1.34 ± 0.10, respectively (Fig. 4e). In a previous study, a value of 0.75 was identified in intracellular mineral-containing matrix vesicles to be the precursor for HAp.20 The polyphosphate-Ca complex at new bone formation sites is known to have a Ca/P ratio of 0.5.50 Other studies similarly revealed precursors within the ECM to have Ca/P ratios of 1.3.19,51 In our present study, despite the difference in Ca/P ratios between globular vesicles in OA-E and OA-A, the lower Ca/P ratios indicated their disordered features as precursors. The matrix vesicles also contained multiple organic components, which showed various affinities to trap Ca2+ or PO43−. For example, higher TNAP activity allowed the influx of PO43−, and annexins, and phosphatidylserine enabled the influx of Ca2+.26,52 49,53 The matrix vesicles in OA-E and OA-A may have different biomolecule compositions and result in different Ca/P ratios. In the extracellular mineralization process, the remnants of these matrix vesicles may support the carbonyl signal detected within minerals by EELS in OA-E (Fig. 3h). In addition to the small amount of carbonyl signal in minerals, the carbonate signals in OA-A were more intense in the cores of minerals due to the carbonate calcium that acts as nucleation sites (Fig. 3h). Furthermore, the enzyme carbonic anhydrase attached to matrix vesicles may help mediate the formation of such bioseeds.54,55 The concentrations of Ca2+ and PO43− in OA cartilage tissues increased locally for the supersaturation of crystal growth.31 Thus, the calcium phosphate-containing matrix vesicles contained different enzymes to dictate two kinds of nucleation processes at calcified cartilage in various OA stages (Fig. 4f).
The hypertrophic chondrocytes at calcified cartilage were activated in OA to initiate mineralization by secreting calcium-containing matrix vesicles to the ECM. In the ECM, nucleation and growth occurred to form carbonated HAp, leading to calcified cartilage thickening. Matrix vesicles equipped with different components drive two kinds of nucleation mechanisms in OA-E and OA-A. Higher carbonate substitutions at phosphate and hydroxide sites mean more crystal defects and fewer crystalline features of minerals in OA-A (Fig. 3). The size of the minerals should depend on the balance between nucleation and growth. More matrix vesicles favor more nuclei formation and induce smaller particle size in OA-E, whereas in OA-A, fewer matrix vesicles favor less nuclei formation, which leads to larger crystal growth due to a local depletion of ions (Fig. 2 and Fig. 4). The surrounding ECM alterations in OA, such as a higher expression of collagen (type I and X) and carboxymethylation of collagens at the calcified cartilage front, further favored crystal deposition and growth in OA development.3,56
Interestingly, these two kinds of nucleation mechanisms were found in the early event of mineralization in the human kidney.16 Several studies have also suggested that matrix vesicles are also associated with pathological calcification, such as atherosclerosis, arteriosclerosis, and tumors.4,15,57 Chen et al. revealed that matrix vesicles enriched in human body fluids may initiate ectopic calcifications.58 Thus, ectopic calcification in human bodies may share a similarity in the early formation events of OA.59 Concerning cartilage calcifications in OA, we further traced the programed development and the material mechanism at the upper zone of cartilage and the underlying calcified cartilage. Although the pathological calcifications at both sites were likely associated with the transdifferentiated chondrocytes, their surrounding environments were significantly different. The joint surface was closely buffered by synovial fluids that contained inflammatory factors, ions and growth factors, while calcified cartilage may undergo uncoupled crosstalk between the overlying cartilage and underlying SB,38 implying their potentially different mineralization process.
Hypermineralized and hypomineralized HAp minerals drive improved and reduced mechanical responses of calcified cartilage tissues in early- and advanced-stage OA. Fundamental disparities in mineral assembly patterns in calcified cartilage in OA-E and OA-A could further induce varied mechanical responses. We first utilized indentation loading to map the stiffness of the calcified cartilage tissues at the microscale (Fig. 5a-5b). A continuously increased tissue modulus in the range from approximately 2 kPa to 2 GPa was observed over a region of ~ 30 µm in calcified cartilage of normal samples (black arrow), which correlated well with the gradually mineralized features (Fig. 5b).60 OA-E showed a stiffer region that was over ~ 100 µm in width (white arrow) overlying the original calcified cartilage (black arrow) (Fig. 5b). The stiffnesses of both calcified regions in OA-E samples ranged from 4.9 ± 1.26 kPa to 5 ± 0.95 GPa, which were much higher than those in normal samples. This tissue modulus map at the calcified cartilage region was quite consistent with the microstructures observed in Fig. 2 and Fig. S7 and should be driven by the hypermineralized structures (Fig. 3).10,61 Conversely, OA-A exhibited decreased tissue moduli ranging from 0.6 ± 0.27 kPa to 0.75 ± 0.31 GPa at a broader region of calcified cartilage, which was largely ascribed to the hypomineralization of minerals (Fig. 3).10
AFM analyses were further employed to elucidate the nanoscale mechanical profiles of the less mineralized region (i), intermediate mineralized region (ii) and highly mineralized region (iii) in calcified cartilage (Figs. 5c-5e). As a result, these mineralized regions of normal samples showed increasing tissue stiffness with peaks at 1.07 ± 0.21 GPa, 4.13 ± 2.83 GPa and 5.81 ± 0.33 GPa. In each region, OA-E showed improved tissue stiffness with peaks at 4.68 ± 0.37 GPa, 5.1 ± 0.17 GPa, and 6.5 ± 0.67 GPa, respectively. The broadening in the distribution ranged from 16.87 GPa to 48.23 GPa at both the intermediate and the highly mineralized regions indicated a marked mechanical increment in OA-E, implying their stiffer mineral features. Expectedly, the tissue stiffness of these mineralized regions in OA-A peaked at 1.78 ± 0.75 GPa, 2.17 ± 0.98 GPa, and 2.28 ± 0.12 GPa, was lower than that of normal samples. In addition to the peak values, broader and narrower stiffness distributions were predominant in OA-E and OA-A, respectively, and they were largely distinct from the heterogeneity of normal samples that changed from a narrow peak to a broad peak.
The nanoscale mechanical profiles (Figs. 5c-5e) were consistent with the microscale mechanical response of calcified tissues in different OA stages (Figs. 5a-5b). Given that the degree of mineralization and tissue stiffness are intercorrelated,42 their considerable increase and decrease in tissue stiffness were due to the hypermineralized and hypomineralized features of minerals in OA-E and OA-A, respectively.10 A recent study reported that ossification of calcified cartilage could induce a stiffened interface in early-stage OA.61 Calcified cartilage with stiffer features lacks mechanical nanoheterogeneity in the longitudinal direction for normal joints. This is thought to result in a higher localized stress concentration that leads to a deficiency in force transfer and, consequently, microcracks as an early event in OA-E.62,63 Such microcracks subsequently propagated into SB plates and elevated cartilage-bone crosstalk.37 In this process, inflammatory cytokines or growth factors from SB that are detrimental to healthy chondrocytes may diffuse into the overlying cartilage and disrupt cartilage homeostasis.12 Great number of voids generating channels at the calcified cartilage in OA-E further favored this process (Fig. 2).
As skeletal tissue, cartilage is challenged by mechanical loading from the diarthrodial joints and counterforces from the underlying SB. Anomalous loading commonly leads to cartilage degeneration in OA.64,65 SB tissues undergo coordinated remodeling to adapt to mechanical stress and in turn exert dynamic effects on the overlying cartilage.38 In this context, as their connective layer, calcified cartilage should play a role in affecting SB remodeling during OA pathogenesis. In OA-E, a higher mechanical response of calcified cartilage could reduce excessive mechanical loads on adjacent SB, which activates osteoclastic bone resorption of SB. In OA-A, the lower mechanical properties of calcified cartilage induced higher mechanical stress in SB, where osteoblastic bone formation was largely improved.38 Thus, SB tissues showed bone loss in OA-E and bone sclerosis in OA-A through remodeling.66 Our nanoscale dissection of the intrusion of calcified cartilage has provided novel insights into the macroscale features in OA and further understanding of the pathogenesis of OA from the perspective of materials science.