Early alterations of the cartilage–subchondral bone unit in spontaneous osteoarthritis

To the Editor: The cartilage–subchondral bone (SB) unit that comprises the articular cartilage (AC) layer and subchondral bone bears the most severe impact force of the knee joint. The mechanical integrity of cartilage and its resistance to injury depend not only on the biochemical composition of its extracellular matrix but also on its interaction with the underlying SB. Both structures act in concert with complementary mechanical functions, the former as a bearer and the latter as a structural girder and shock absorber.[1] The SB absorbs most of the mechanical stimuli transmitted by joints.[1] SB comprises two distinct anatomic entities: the subchondral bone plate (SP) and the subchondral trabecular (ST) bone, which differ greatly in compositional and mechanical properties. Bone is a nanocomposite material in which hard inorganic (hydroxyapatite crystallites) and organic (collagen fibrils) components are hierarchically arranged on the nanometer scale.[2] Bone quality is dependent on the spatial distributions of the shape, size, and composition of bone constituents (minerals, collagen, and water).[3,4] Previous studies have indicated that an initial decrease in the elastic modulus of SB occurred during the osteoarthritis (OA) pathological process.[5] AC degeneration might be directly affected by the mechanical environmental changes induced by changes in the SB.[5] However, to the best of our knowledge, the understanding of nanoscale mechanical properties across the osteochondral region is limited, which restricts the ability to understand the disease progression and to engineer replacement materials. Few studies have investigated the relationship between changes in the nanoscale surface topography and the mechanical properties of SB and those of AC. Therefore, one objective of our study was to evaluate changes in the topographical and mechanical properties of AC in Dunkin Hartley (DH) guinea pig knees relative to the stage of OA, with a specific focus on surface roughness—a key determinant of tissue lubrication. Another aim was to correlate observed changes in AC surface topography and mechanical properties with those of SB, which is crucial for understanding the functional interplay within the cartilage–subchondral bone unit in OA pathogenesis. We hypothesized that AC surface roughness and the indentation moduli of ST and SP increase with OA progression and play more significant roles in OA pathogenesis than their bone matrix topographical features. This study was performed in strict accordance with the recommendations of the Laboratory Animal Standardization Committee and was approved by the Science and Ethics Committee of the School of Biological Science and Medical Engineering in Beihang University, China (Approval ID: BM201900064). Forty male DH strain guinea pigs at the ages of 1 month, 3 months, 6 months, and 9 months (with 10 in each age group) were procured from Beijing Xinglong Experimental Animal Center. These guinea pigs were sacrificed via intraperitoneal injection of 100 mg/kg pentobarbital after 3 days of feeding. The left knee joints were fixed in 4% paraformaldehyde (PFA) for 3 days and prepared for histological and immunofluorescence analyses. The right knee joints were stored at −20°C prior to atomic force microscopy (AFM) and nanoindentation tests [Figure 1A].Figure 1: Synopsis of site-specific measurements performed in the tibial AC and SB regions, a sketch map of indentation areas and sites in the sagittal plane of knee joints, macroscopic images of the cartilage and modified Mankin score (n = 40) from 1-, 3-, 6-, and 9-month-old guinea pigs. (A) Synopsis of site-specific measurements performed in the tibial AC and SB regions. (B) Typical images of tibial plateau oriented (medial on left, lateral on right) with macroscopic lesions of increasing severity from no lesions, (C) to moderate surface fissuring, and (D,E) severe erosion to bone. The diseased areas in panels (C–E) are identified by the dotted demarcated region. (F) Modified Mankin score variations with animal age (this result was published in the previous study, and permission has been obtained from Springer Nature[ 9 ]). * P <0.05, compared with the 1-month group; † P <0.05, compared with the 3-month group; ‡ P <0.05, compared with the 6-month group. AC: Articular cartilage; SB: Subchondral bone.From a macroscopic perspective, the AC surface of 1-month-old guinea pigs appeared smooth without any signs of degeneration [Figure 1B]. Occasionally, mild lesions became apparent at 3 months of age [Figure 1C]. At 6 months of age, moderate fissures and evident loss of matrix were observed on the cartilage surface [Figure 1D]; by 9 months of age, there was a noticeable increase in the proportion of cartilage areas exposed to the bone surface [Figure 1E]. Significant differences were observed in the modified Mankin scores among the groups at 1 month, 3 months, 6 months, and 9 months [Figure 1F]. The presence of collagen type I fibers (orange) and type II fibers (yellow and brown) was noted in each group, whereas type III fibers (green) were observed only in 6- and 9-month-old guinea pigs [Supplementary Figure 1A–D, https://links.lww.com/CM9/C386]. Immunofluorescence imaging revealed a layered, nonhomogeneous distribution of collagen type II and zonal stratification of AC cells in AC [Supplementary Figure 1E–H, https://links.lww.com/CM9/C386]. The indentation moduli and hardnesses of the SP and ST in the tibial plateaus of 1-, 3-, 6-, and 9-month-old guinea pigs were measured using nanoindentation testing [Supplementary Figure 2, https://links.lww.com/CM9/C386]. The indentation moduli and hardnesses of the SP and ST in the tibial plateau of 3-, 6-, and 9-month-old guinea pigs were found to be higher than those of 1-month-old guinea pigs (P <0.001) [Supplementary Figure 2, https://links.lww.com/CM9/C386]. The mean roughness of the SP in the tibial plateaus of 3-, 6-, and 9-month-old guinea pigs was found to significantly increase compared with that of 1-month-old guinea pigs (P <0.001) [Supplementary Figure 3, https://links.lww.com/CM9/C386]. There was no significant difference between the 1- and 3-month groups in the mean roughness of the ST. In addition, the mean roughness of the ST in the tibial plateau of 6- and 9-month-old guinea pigs was found to be higher than that of 1-month-old guinea pigs (P <0.001) [Supplementary Figure 3, https://links.lww.com/CM9/C386]. The roughness of AC varied with animal age, and the mean roughness of AC on the tibial plateau increased significantly from 3-month-old to 6-month-old guinea pigs, whereas there was no significant change in the roughness of AC of the 9-month group compared with that of the 3-month group [Supplementary Figure 4, https://links.lww.com/CM9/C386] (P <0.001). The moduli and hardness of SP had significant correlations with OA score (r = 0.6610, r = 0.3870, respectively; P <0.001). The increased OA score was significantly correlated with the modulus of ST (r = 0.7390, P <0.001). The roughness of SP, ST, and AC was significantly correlated with the OA score (r = 0.5200, r = 0.5210, r = 0.8800, respectively; P <0.001) [Supplementary Table 1, https://links.lww.com/CM9/C386]. The degeneration of the cartilage surface during the progression of OA has always been thought to be one of the most important pathological characteristics. OA may lead to nanoscale topographical variations in AC. To clarify this speculation, AFM analyses were performed in this study. The results showed that cartilage roughness increased in the early stage of OA. The indentation moduli and hardness of SP and ST bone showed a significant increase in the 3-month-old group compared with the 1-month-old group, which was consistent with the findings of a previous investigation.[6] SB in OA undergoes an altered mineralization process, which results in nanostructural changes in apatite crystals that are likely to account for the compromised mechanical properties of SB in OA. Additionally, the increase in mechanical properties with OA may directly correspond to the increase in bone metabolism and roughness of the bone matrix.[7] The indentation moduli of SP and ST exhibited significant positive correlations with the OA score [Supplementary Table 1, https://links.lww.com/CM9/C386]. This suggests that the mechanical properties of SB could serve as crucial biomarkers in OA diagnosis. AC and SB form a functional unit and interact with each other in the knee joint. The content of collagen II in AC decreased considerably with the increased indentation moduli of SP and ST during the progression of spontaneous OA. These results were consistent with those of a previous study.[5] The ST is believed to deliver nutrients to the adjacent cartilage through its capillaries, which need to pass through the SP. This function seems to be limited by SP sclerosis, which further changes the content of collagen II and sGAG in AC. Moreover, the indentation moduli of SP and ST had significantly positive correlations with AC surface roughness [Supplementary Table 1, https://links.lww.com/CM9/C386]. This indicated that AC degeneration might be directly affected by changes in the mechanical environment, which were induced by alterations in the mechanical properties of SB during OA progression. One recently published research has suggested that subchondral bone properties in OA are affected at the compositional, mechanical, and cellular levels in hip OA, which were closely related to cartilage degeneration.[8] Thus, for clinical application, SB might be a new treatment target for this type of OA. Furthermore, compared with the changes in the roughness of the bone matrix of SB, the biomechanical property of SB was more highly correlated with the variations in the roughness of AC [Supplementary Table 1, https://links.lww.com/CM9/C386]. This suggests that the contribution of altered biomechanical changes may be more significant than the contributions of the increased bone matrix roughness of SP and ST in AC degeneration. Furthermore, SB became stiffer because of the increase in its indentation modulus, which disrupted the mechanical equilibrium between AC and SB. This study has a few limitations. First, only the primary OA models of the 1-, 3- 6-, and 9-month-old groups were investigated in this study. Although significant changes at the early stage of OA were observed in our study, future studies using 2-month-old guinea pigs have been designed to assess the initial changes in AC and SB of OA. Second, the sample size is relatively small. A larger sample size may improve the ability to obtain more relationships among the topographical and mechanical characterizations of AC and SB. In conclusion, this study illuminated the AC roughening in early OA, characterized by the more non-homogeneous distribution of collagen type II and the increase of modulus of subchondral bone plate. Notably, these nanoscopic deficiencies occurred in macroscopically intact regions of AC, potentially linked to processes that result in the reduced modulus of AC. Moreover, the moduli of the SP and ST showed significant correlations with the OA score. Based on the interactions between the increased modulus of subchondral bone and AC degeneration, future therapeutic OA approach may target compositional and microstructural properties of the subchondral bone. Funding This work was supported by grants from the National Natural Science Foundation of China (Nos 12102278 and 12272029). Conflicts of interest None.