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RANKL-mediated osteoclastic subchondral bone loss at a very early stage precedes subsequent cartilage degeneration and uncoupled bone remodeling in a mouse knee osteoarthritis model

Journal of Orthopaedic Surgery and Research volume 20, Article number: 226 (2025) Cite this article

Abstract

Introduction

Uncoupled bone remodeling in the subchondral bone (SB) has recently been considered as an important process in the progression of knee osteoarthritis (KOA). In this study, we aimed to investigate changes in SB and articular cartilage using a mouse model of destabilization of the medial meniscus (DMM) and determine the effects of bone metabolism on KOA progression.

Methods

DMM or sham surgery was performed on the left knees of 40-week-old male wild-type (WT) mice and Tsukuba hypertensive mice (THM), which exhibit high-turnover bone metabolism. Bone volume/tissue volume (BV/TV) and bone mineral density (BMD) in the medial tibial SB were measured longitudinally in vivo using μCT at 0 (immediately after surgery), 1, 2, 4, 8, and 12 weeks postoperatively. Concurrently, histological evaluations of the articular cartilage in the medial tibial plateau were conducted. Furthermore, the number of endo-periosteal tartrate-resistant acid phosphatase-positive osteoclasts, trabecular RANKL-positive osteocytes, and osteocytes in the trabeculae were measured at 0, 1, 2, and 4 weeks.

Results

In the WT + DMM group, BV/TV and BMD in the SB significantly decreased with time, whereas cartilage degeneration significantly increased. In the THM + DMM group, these changes in BMD and cartilage degeneration were significantly pronounced. Interestingly, in the THM + DMM group, BV/TV significantly decreased up to 4 weeks but then began to increase, although BMD continued to decrease until the 12-week mark. The number of osteoclasts and the percentage of RANKL-positive osteocytes per total number of osteocytes within the total trabecular bone area (%) in the WT + DMM group significantly increased with time, with a significant difference between the WT + DMM and WT + sham groups at 4 weeks. The number of osteocytes in the WT + DMM group significantly decreased with time, and the difference between the WT + DMM and WT + sham groups was significant at 4 weeks postoperatively. These histological changes were significantly enhanced in the THM + DMM group.

Conclusions

The results indicate that early-stage osteocyte death in the SB and RANKL-mediated osteoclastic SB loss precede histological cartilage degeneration and contribute to uncoupled bone remodeling at the later stage. Acceleration of disease processes in the THM + DMM group suggests that high-turnover bone metabolism is a potential risk factor for KOA. Maintaining SB integrity and avoiding continuous SB overload may be key strategies for mitigating disease progression.

Introduction

Osteoarthritis (OA), associated with aging and mechanical overload of joints, is the most prevalent degenerative joint disease. It is characterized by cartilage loss, subchondral bone (SB) sclerosis, and chronic synovitis. The knee is the joint most commonly affected by OA [1]. Knee OA (KOA) affects patients by reducing their quality of life, shortening healthy life expectancy, and placing a substantial financial burden on healthcare systems. In Japan, a country with a super-aging society, approximately 25 million people suffer from KOA, accounting for around 10% of individuals requiring care [2]. Hence, reducing KOA prevalence is highly desirable.

Recent studies have identified uncoupled SB remodeling as an important process in KOA progression [3, 4]. Radin et al., using a bone scan in a rabbit KOA model, first reported that SB remodeling is significantly enhanced in response to mechanical overload of the articular surface and precedes damage to the overlying articular cartilage [5]. Furthermore, they suggested that changes in SB density and architecture influence both the initiation and progression of cartilage degeneration [6, 7]. Additional animal studies have shown that SB loss can occur at the early stage of KOA and that inhibiting bone resorption can suppress KOA progression in experimental models [8,9,10,11,12,13,14,15]. Bellido et al. reported that microstructural impairment in tibial SB associated with increased bone remodeling aggravates cartilage damage in rabbits with previous osteoporosis (OP) [13]. Similarly, Zhu et al. showed that ovariectomy in older rats induces both OP and KOA, and early treatment of OP with bisphosphonates protects both articular cartilage and SB [14]. However, more recent findings by Fang et al. reported no significant SB loss after destabilization of the medial meniscus (DMM) surgery in mice [16]. Therefore, the relationship between SB loss at the early stage of KOA and disease initiation and progression remains unclear.

Epidemiological studies have associated KOA development to high systemic bone mineral density (BMD). Several studies have shown that patients with KOA tend to have higher BMD than those without KOA [17,18,19]. Recently, both systemic and subchondral BMD have also been positively associated with increased cartilage thickness in patients with radiographic OA [20]. Contrarily, several studies have reported a relationship between low BMD and KOA [21]. KOA prevalence, which is higher in women than in men, increases sharply with age, particularly after menopause [22, 23]. Postmenopausal OP, which results from estrogen deficiency, is the most common type of OP and rapidly progresses with age due to high-turnover bone metabolism [24]. However, the causal relationship between high-turnover OP and increased KOA prevalence has not been determined owing to the cross-sectional design of these epidemiological studies. The objective of this study was to investigate early changes in SB and articular cartilage in detail using a mouse model of DMM and to determine the effects of high-turnover metabolism on KOA progression.

Materials and methods

Animals

All animal experiments were performed in accordance with the guidelines of the National Institutes of Health and the institutional rules for the use and care of laboratory animals at Kindai University. All procedures were approved by the Experimental Animal Welfare Committee of Kindai University (approval number: KAME-2022-008).

Forty-week-old C57/BL6 male wild-type (WT) mice were used as controls. To investigate whether high-turnover bone metabolism affects SB loss and uncoupled bone metabolism in the SB, 40-week-old male Tsukuba hypertensive mice (THM) were used. THM mice are double transgenic mice on a C57BL/6 genetic background, created by crossing a transgenic mouse expressing the human angiotensinogen gene with a transgenic mouse expressing the human renin gene, which were supplied by the RIKEN Biolease Center (RIKEN, Tsukuba, Japan; with permission from Dr. Fukamizu A). In THM, serum concentrations of angiotensin II (Ang II) are four to five times higher than in WT mice, as human angiotensinogen is cleaved by human renin to Ang I, which is then converted to Ang II by angiotensin-converting enzyme [25]. Furthermore, Ang II has been demonstrated to accelerate OP by activating osteoclasts via RANKL expression in osteoblasts [26], and activation of the renin-angiotensin system in aged male THM induces high-turnover OP independent of hypertension [27].

All mice were kept in a standard laboratory environment (room temperature: 23 ± 2 °C, 12-h light/dark cycle, 2–3 mice per cage) with free access to food and water. To account for weight as a potential confounding factor affecting KOA, mice weights were assessed before knee surgery. Although the mean weight of THM mice was lower than that of WT mice, the difference was not statistically significant (WT + DMM: 23.58 ± 1.07 g, THM + DMM: 22.71 ± 1.21 g, p > 0.05; n = 7 per group). Forty-week-old male WT and THM mice were divided into two groups: DMM and sham. Thus, four groups (WT + DMM, THM + DMM, WT + sham, and THM + sham; n = 7 per group) were established for bone parameter measurements using μCT. For histological evaluation of cartilage degeneration using the Osteoarthritis Research Society International (OARSI) score in mice, four groups (WT + DMM, THM + DMM, WT + sham, and THM + sham; n = 7 per group at 0 (immediately after surgery), 1, 2, 4, 8, and 12 weeks postoperatively,) were also established. Furthermore, four groups (WT + DMM, THM + DMM, WT + sham, and THM + sham; n = 7 per group at 0, 1, 2, and 4 weeks postoperatively) were set up for measuring the number of osteoclasts (endo-periosteal tartrate-resistant acid phosphatase [TRAP]-positive cells), trabecular RANKL-positive osteocytes, and the number of osteocytes and empty lacunae.

Surgically induced KOA model in mice

The DMM model was used to induce KOA in mice. This model is often used as a surgical method for KOA induction due to its reproducibility and is particularly suited for studying the early stage of KOA because of the slower disease progression it produces [28]. In this study, DMM or sham surgery was performed on the left knee of 40-week-old male WT and THM mice. In the DMM surgery, a 3-mm longitudinal skin incision was made from the proximal patella to the proximal tibial plateau, followed by an arthrotomy just medial to the patellar tendon. Subsequently, the medial meniscotibial ligament was transected, and medial displacement of the medial meniscus was confirmed. In the sham surgery, only the skin incision and arthrotomy were performed before closure. After surgery, all animals were reared in the standard environment described previously.

Bone parameter measurements with μCT

Anesthesia was induced with 5% isoflurane using an inhalation anesthesia device suitable for small animals (MARCOBIT-E; Natsume Seisakusho, Tokyo, Japan), and the absence of reflexes to tactile stimulation was confirmed. While under 3% isoflurane anesthesia, mice were positioned supine on the CT table with the knee extended and the patella oriented upward. Subsequently, 10-μm sections were scanned from the upper end of the patella to the tibial tubercle using a μCT device (Cosmo Scan GXII; Rigaku Co., Tokyo, Japan) at a tube voltage of 90 kV, tube current of 88 μA, imaging time of 4 min, and an isotropic voxel size of 10 μm. Longitudinal measurements were taken in the same animals at 0, 1, 2, 4, 8, and 12 weeks postoperatively.

Trabecular bone parameters, including bone volume/tissue volume (BV/TV, %), trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm), and BMD (g/cm3) were measured longitudinally in the same individuals at 0, 1, 2, 4, 8, and 12 weeks after surgery (n = 7 per group) using software preinstalled on the μCT system (Bone Microarchitecture Analysis ver. 14.0; Rigaku Co.). To begin, a cuboidal region of interest containing the entire SB of the medial tibial condyle was defined. This region extended: (1) mediolaterally, from the medial edge of the SB to the medial tibial eminence; (2) anteroposteriorly, from the anterior edge to the posterior edge of the SB; and (3) superiorly to inferiorly, from the upper edge to the lower edge of the SB. Approximately 40 axial sections, each of 10 μm thickness, were extracted for each subject, and the software automatically segmented trabecular bone from cortical bone based on CT values (trabecular bone, 200–500; cortical bone, 500–1000). Manual corrections were then performed on each axial section to rectify any segmentation errors between the subchondral trabecular and cortical bones before measuring the bone parameters. Trabecular bone parameters for the medial tibial SB were calculated as the mean values derived from approximately 40 sections.

Histological evaluation

The left knee joints were excised after euthanasia using carbon dioxide gas at 0, 1, 2, 4, 8, and 12 weeks (n = 7 per group per time point). The excised joints were fixed in a 10% neutral-buffered formalin solution for 48 h and subsequently demineralized with ethylenediaminetetraacetic acid for 2 weeks. Fixed and demineralized samples were embedded in paraffin in a frontal orientation and then sliced into serial frontal sections of 6-µm thickness. From these sections, samples containing the anterior cruciate ligament were selected, deparaffinized, and hydrophilized with alcohol. All histological evaluations were performed under an optical microscope (BIOREVO, BZ-9000; Keyence, Osaka, Japan).

Articular cartilage degeneration

Safranin O and fast green staining were used to evaluate articular cartilage at 0, 1, 2, 4, 8, and 12 weeks after DMM surgery (n = 7 per group). Sections were soaked in Weigert’s iron hematoxylin solution for 2 min and then rinsed under running water for 5 min. Next, sections were stained with a 0.02% fast green solution for 10 min, soaked in 1% acetic acid solution 10 times, and subsequently stained with a 0.1% safranin O solution for 10 min. Articular cartilage degeneration on the medial tibial plateau was evaluated using the OARSI scoring system [29], which assesses degeneration depth and classifies it into six grades, with normal cartilage being grade 0. Two independent observers, blinded to the groups, performed the scoring, and the mean of their scores was considered as the true value.

Osteoclast activation

Osteoclast activation was evaluated by quantifying TRAP- and RANKL-positive osteocytes at 0, 1, 2, and 4 weeks after DMM surgery. TRAP staining was used to determine osteoclast numbers (n = 7 per group) at these time points with a commercially available kit (TRAP/ALP Stain Kit, 294-67001; Fujifilm Wako, Tokyo, Japan) [30]. For each sample, the entire medial tibial SB was observed using a 4 × objective lens, then divided into a 3 × 2 grid, and each section was magnified 40 × . Osteoclasts (TRAP-positive cells containing three or more nuclei) were counted in each field, and the total across six fields was recorded [31]. The length of the endo-periosteal membrane (trabecular bone surface) of the medial tibial SB was measured in each field using image processing software (ImageJ ver. 1.8.0; NIH, Bethesda, ML), following previously established methods [32, 33]. The number of osteoclasts was determined by dividing the total number of osteoclasts by the total length of the endo-periosteal membrane (n/mm), using a previously described method [34, 35].

Furthermore, immunohistochemical staining for RANKL was performed in sections at 0, 1, 2, and 4 weeks postoperatively (n = 7 per group) [36]. For staining, antigen activation was achieved using the proteolytic enzyme proteinase K. Endogenous peroxidase was inactivated with 0.3% hydrogen peroxide in methanol for 30 min. After blocking with normal goat serum for 20 min, the sections were incubated with a primary antibody (anti-RANKL polyclonal antibody, 1:100 in PBS; #bs-0747R; Bioss Inc., Woburn, Massachusetts, USA) for 90 min at 23 °C. They were then incubated with a secondary antibody for 30 min (Histofine Simple Stain Mouse MAX-PO(R), #414341; Nichirei Biosciences Inc., Tokyo, Japan). Staining was visualized using a diaminobenzidine substrate kit (#425011; Nichirei Biosciences Inc.) and Meyer’s hematoxylin solution. The number of trabecular RANKL-positive osteocytes in each field was counted, and their total number across six fields was recorded. The total area of subchondral trabecular bone (in square millimeters per millimeter of standard length) was measured from digitized images using image processing software (ImageJ ver. 1.8.0), following previously described methods [32, 33]. The number of RANKL positive osteocytes in the SB was quantified by dividing the total number of RANKL positive osteocytes by the total trabecular bone area (n/mm2), and the percentage of RANKL-positive osteocytes per total number of osteocytes within the total trabecular bone area (%) was measured [34].

Measurements of the numbers of osteocytes and empty lacunae

To evaluate trabecular SB viability, the numbers of osteocytes and empty lacunae were assessed at 0, 1, 2, and 4 weeks after DMM surgery (n = 7 per group) using the same TRAP-stained sections as mentioned above. An osteocyte was defined as a trabecular mononuclear spindle-shaped cell within a hematoxylin-stained lacuna. An empty lacuna was defined as a small, oval space within the trabecular bone without a nucleus. The numbers of osteocytes and empty lacunae were obtained as described above.

Statistical analysis

The results are presented as mean ± standard deviation and were analyzed using Microsoft Excel 2016 (Microsoft Corp., Redmond, WA). Statistical differences between the DMM and sham groups were evaluated using the Tukey–Kramer multiple comparison test. Williams’ multiple comparison test was performed with the Excel Statistics Add-in software (Social Survey Research Information Co., Ltd., Tokyo, Japan) to identify the point at which significant changes after surgery became evident during longitudinal observation. Statistical significance was set at p < 0.05.

Results

Changes in bone parameters

Representative μCT images were shown in Fig. 1A. No significant differences in parameters were observed between the THM + DMM and WT + DMM groups at week 0 (Fig. 1B–E). Similarly, in the THM + sham and WT + sham groups, none of the bone parameters showed significant changes during the postoperative observation period from 0 to 12 weeks. In the WT + DMM group, BV/TV decreased with time after surgery, with significant differences from time-0　and WT + sham group at 4 weeks postoperatively (Fig. 1B). However, BV/TV continued to decrease only until week 8, with no further changes thereafter. Tb.Th exhibited a similar temporal pattern to BV/TV in this group (Fig. 1C). TB.Sp increased with time, reaching significant differences from time-0 and the WT + sham group at 4 weeks postoperatively (Fig. 1D). BMD also decreased with time after surgery, with significant differences from time-0 and the WT + sham group observed at 8 weeks postoperatively (Fig. 1E).

Fig. 1
figure 1

Changes in bone parameters in the four groups. A Representative axial and sagittal micro-CT images at 0, 1, 4, and 12 weeks after surgery in the THM + DMM and WT + DMM groups. Upper row: axial images. Lower row: sagittal images. B Changes in BV/TV in the four groups. C Changes in Tb.Th. D Changes in Tb.Sp. E Changes in BMD. Significant differences: BV/TV, Tb.Th, and TB.Sp for WT + DMM vs. WT + sham at > 4 weeks (p = 0.0013 at 4 weeks); BMD for WT + DMM vs. WT + sham at > 8 weeks; BV/TV for WT + DMM at time-0 vs. > 4 weeks (p = 0.0024 at 4 weeks); BMD for WT + DMM at time-0 vs. > 8 weeks; BV/TV, Tb.Th, and TB.Sp for THM + DMM vs. THM + sham at > 1 week (p < 0.001 at 1 week); BMD for THM + DMM at time-0 vs. > 2 weeks (p = 0.0080 at 2 weeks); BMD for THM + DMM vs. THM + sham at > 2 weeks (p = 0.049 at 2 weeks)

Full size image

In the THM + DMM group, BV/TV decreased more rapidly with time after surgery than that in the WT + DMM group, with significant differences from time-0 and the THM + sham group at 1 week postoperatively (Fig. 1B). However, BV/TV began to increase again by 4 weeks after surgery. Tb.Th exhibited a similar temporal change to BV/TV in this group (Fig. 1C). Tb.Sp increased with time, with significant differences from time-0 and the THM + sham group at 1 week postoperatively (Fig. 1D). BMD decreased more rapidly with time compared with that in the WT + DMM group, with significant differences from time-0 and the THM + sham group at 2 weeks postoperatively (Fig. 1E).

Histological evaluation

Cartilage degeneration

Representative histological images of Safranin O and fast green staining were shown in Fig. 2A. No significant difference in OARSI scores was found among the groups at week 0 (Fig. 2B). The OARSI scores in the THM + sham and WT + sham groups did not show any significant changes during the postoperative observation from 0 to 12 weeks. In the WT + DMM group, the OARSI score increased with time after surgery, with significant differences from time-0 and the WT + sham group at 8 weeks postoperatively. In the THM + DMM group, the score increased more rapidly with time, with significant differences from time-0 and the THM + sham group at 2 weeks postoperatively.

Fig. 2
figure 2

Histopathological evaluation of medial tibial articular cartilage with the OARSI scoring system. A Representative histological samples at 0, 2, 4, and 8 weeks after surgery in the THM + DMM and WT + DMM groups. B Changes in OARSI scores. Significant differences: WT + DMM vs. WT + sham at > 8 weeks (p < 0.001 at 8 weeks); WT + DMM at time-0 vs. > 8 weeks (p < 0.001 at 8 weeks); THM + DMM vs. THM + sham at > 2 weeks (p < 0.001 at 2 weeks); and THM + DMM at time-0 vs. > 2 weeks (p < 0.001 at 2 weeks)

Full size image

Osteoclast activation

Representative histological images of TRAP staining and immunohistochemical staining for RANKL were shown in Figs. 3A, and 4A, respectively. In the WT + DMM group, the number of osteoclasts significantly increased at 4 weeks after surgery, with significant differences observed from the WT + sham group as well at this time point (Fig. 3B). In the THM + DMM group, the number of osteoclasts increased more rapidly with time after surgery than that in the WT + DMM group, reaching significant differences from time-0 and the THM + sham group at 1 week postoperatively (Fig. 3B).

Fig. 3
figure 3

Changes in osteoclasts, osteocytes, and empty lacunae in the four groups. A Representative TRAP-positive cell samples at 0 and 4 weeks after surgery in the THM + DMM and WT + DMM groups. Red triangles indicate TRAP-positive cells, black triangles indicate osteocytes, and asterisks indicate empty lacunae. B Changes in TRAP-positive cells in the four groups. Significant differences: WT + DMM vs. WT + sham at 4 weeks (p < 0.001 at 4 weeks); WT + DMM at time-0 vs. 4 weeks (p < 0.001 at 4 weeks); THM + DMM vs. THM + sham at > 1 week (p < 0.001 at 1 week); and THM + DMM at time-0 vs. > 1 week (p < 0.001 at 1 week). C Changes in the number of osteocytes. Significant differences: WT + DMM vs. WT + sham at 4 weeks (p = 0.0033 at 4 weeks); WT + DMM at time-0 vs. 4 weeks (p = 0.0096 at 4 weeks); THM + DMM vs. THM + sham at > 2 weeks (p = 0.0020 at 2 weeks); and THM + DMM at time-0 vs. > 2 weeks (p = 0.0028 at 2 weeks). D Changes in the number of empty lacunae. Significant differences: WT + DMM vs. WT + sham at 4 weeks (p = 0.029 at 4 weeks); WT + DMM at time-0 vs. 4 weeks (p < 0.001 at 4 weeks); THM + sham at > 2 weeks (p = 0.0013 at 2 weeks); and THM + DMM at time-0 vs. > 2 weeks (p = 0.0021 at 2 weeks)

Full size image
Fig. 4
figure 4

Changes in RANKL-positive osteocytes. A Representative RANKL-positive cell samples at 0 and 4 weeks after surgery in the THM + DMM and WT + DMM groups. Red triangles indicate RANKL-positive cells. B Changes in percentage of RANKL-positive osteocytes per total number of osteocytes in the total trabecular bone area in the four groups. Significant differences: WT + DMM vs. WT + sham at > 2 weeks (p = 0.034 at 2 weeks); WT + DMM at time-0 vs. > 2 weeks (p = 0.0023at 2 weeks); THM + DMM vs. THM + sham at > 1 week (p < 0.001 at 1 week); and THM + DMM at time-0 vs. > 1 week (p < 0.001 at 1 week)

Full size image

Number of osteocytes and empty lacunae

In the WT + DMM group, the number of osteocytes decreased with time after surgery, with significant differences from time-0 and the WT + sham group at 4 weeks postoperatively (Fig. 3C). The number of empty lacunae increased with time, with significant differences from time-0and the WT + sham group at 4 weeks postoperatively (Fig. 3D). In the THM + DMM group, the number of osteocytes decreased more rapidly with time, with significant differences from time-0 and the THM + sham group at 2 weeks postoperatively (Fig. 3C). The number of empty lacunae increased more rapidly with time, with significant differences from time-0 and the THM + sham group at 2 weeks postoperatively (Fig. 3D). The percentage of RANKL-positive osteocytes per total number of osteocytes in the WT + DMM group increased with time, with significant differences from time-0 and at 2 weeks postoperatively (Fig. 4B). Similarly, the percentage of RANKL-positive osteocytes per total number of osteocytes in the THM + DMM group increased with time, with significant differences from time-0 and at 1 week postoperatively (Fig. 4B).

Discussion

The results of the present study on longitudinal changes in bone parameters indicate that SB loss in the mouse DMM model occurs very early after surgery in both the WT + DMM and THM + DMM groups, preceding histological changes in the articular cartilage. Furthermore, SB loss after surgery was significantly greater in the THM + DMM group than in the WT + DMM group. In a previous study by Fang et al. [16], BMD in the tibial SB of WT mice was measured after DMM surgery using μCT at 2, 5, and 10 weeks. BMD did not show statistically significant differences between the DMM and sham groups at 2 weeks after surgery but significantly increased at both 5 and 10 weeks after surgery. Several factors may account for the differences between their results and ours. In their study, running wheels were provided in the colony cage after surgery to encourage exercise, whereas we reared the mice in a standard, natural environment. Thus, SB remodeling and cartilage degeneration in WT + DMM mice in our study may have progressed more slowly due to reduced mechanical overload on the joint than those in their study. To observe changes at the very early stage of KOA in the DMM mouse model, housing the mice in a calm environment may be advantageous. Furthermore, we conducted in vivo longitudinal measurements of bone parameters from time-0 to 12 weeks after surgery, whereas they conducted ex vivo measurements at 2, 5, and 10 weeks after surgery, which could have influenced the outcomes. In addition, the age of the mice used in each study varied: they used 12-week-old mice, whereas we used 40-week-old mice. Age differences may have affected the biological responses of the SB and cartilage after DMM surgery.

Interestingly, in the THM + DMM group, BV/TV and Tb.Th decreased up to the 4-week mark but then increased, whereas BMD continued to decrease until 12 weeks. This biphasic change in BV/TV and Tb.Th was also observed at the later stage in the WT + DMM group. The increase in BV/TV and the decrease in BMD at the later stage after surgery indicate that uncoupled SB remodeling, characterized by increased bone formation but reduced bone mineralization, occurred in the SB. Decreased mineralization in the SB can lead to decreased bone strength, which could promote the accumulation of microdamage under continuous mechanical overload after DMM. SB sclerosis with a low mineralization pattern has been reported in human hip OA [37] and KOA [38].

The OARSI scores in the WT + DMM group increased with time after surgery, with a significant difference from the WT + sham group at 8 weeks. This time-dependent progression of cartilage degeneration was also enhanced in the THM + DMM group, with significant differences from the THM + sham group observed at 2 weeks postoperatively. These findings in the THM + DMM group align with previous observations using a forced-running KOA model in THM [35]. Increased serum concentrations of Ang II in THM may affect cartilage metabolism in KOA, as hypertrophic chondrocytes express angiotensin type 1 receptors, which can further stimulate chondrocyte differentiation [36, 39]. However, hypertrophic chondrocytes were not observed histologically at the early 1-week mark after surgery in the THM + DMM group (data not shown), despite a significant decrease in BV/TV being evident. Therefore, the effects of increased serum Ang II concentration in THM on early cartilage degeneration are considered limited after surgery.

To demonstrate osteoclastic activity after DMM, we histologically evaluated postoperative changes in the number of osteoclasts in the endo-periosteal membranes and in the percentage of RANKL-positive osteocytes per total number of osteocytes within the subchondral trabeculae. Our results indicate that osteoclast activation in the SB occurs very early after surgery in both the WT + DMM and THM + DMM groups, suggesting that this activation is mediated, at least in part, by RANKL expression in osteocytes. Mechanical overload-induced fatigue in trabecular bone may cause microcracks or microdamage in the SB, potentially resulting in apoptosis of subchondral osteocytes and subsequent osteoclast activation driven by RANKL expression in nearby non-apoptotic osteocytes [32, 33, 40]. Furthermore, continuous mechanical overload on bone has been reported to induce bone resorption with increased osteoclastic activity due to RANKL expression in osteocytes [41]. To determine the effects of continuous mechanical overload after DMM on subchondral trabecular bone viability, we evaluated the number of osteocytes and empty lacunae in the SB trabeculae. We observed a significant decrease in osteocyte numbers very early after surgery, suggesting that DMM-induced mechanical overload on the SB led to bone fatigue and osteocyte death. In the THM + DMM group, RANKL-mediated osteoclast activation was enhanced by the upregulation of RANKL expression in osteoblasts, stimulated by high serum concentrations of Ang II [25, 42]. Asaba et al. demonstrated by RT-PCR that RANKL expression in bone was increased in THM mice [27], though we were unable to evaluate RANKL expression in osteoblasts due to excessive staining of RANKL in bone marrow cells. It was a limitation of our study and alternatively we evaluated RANKL-positive osteocytes within the subchondral trabeculae, and demonstrated that the percentage of RANKL-positive osteocytes per total number of osteocytes in the THM + DMM group increased with time more rapidly than the WT + DMM group.

This study has several limitations. First, we were unable to evaluate the effects of hypertension on SB metabolism in the THM + DMM group. Hypertension in THM may affect SB remodeling after DMM because bone ischemia due to hypertension has been suggested to cause osteocyte apoptosis, potentially upregulating RANKL expression in adjacent living osteocytes [43]. However, we could not evaluate the effects of bone ischemia due to hypertension on osteocyte death in THM mice. Second, at time-0, the BV/TV in the medial tibial SB of THM mice did not significantly differ from that of WT mice in this study. Previous research by Asaba et al. reported a decrease in BV/TV owing to high-turnover bone metabolism in the proximal tibia of 40-week-old THM mice [27]. Differences in the effects of renin-angiotensin system activation on bone parameters may be due to variations in anatomical locations, specifically the epiphyseal SB evaluated in this study versus the entire metaphyseal SB assessed in the prior study. Third, we could not perform histological evaluation of osteoclast activation and trabecular bone viability beyond 4 weeks after surgery. This limitation is due to SB remodeling at 8 and 12 weeks, which altered the trabecular structures. Fourth, the increase in the number of empty lacunae was unexpectedly small relative to the decrease in the number of osteocytes. However, the reason for this discrepancy remains unclear. Empty oval lacunae in the trabecular bone may disappear after osteocyte death owing to continuous compressive overload after DMM. Fifth, we did not evaluate RANKL-VEGF gene expression in the subchondral bone of THM, while it was revealed in the previous study that Ang II acted on osteoblasts and increased RANKL and VEGF, thereby stimulating the formation of osteoclasts in transgenic THM [27]. In addition, the effect of ACE inhibitor for the prevention of the promotion of osteoarthritis in THM + DMM model was also not evaluated in this study, though the ACE inhibitor enalapril was demonstrated to suppress osteoporosis [27]. It should be a future research topic to investigate whether ACE inhibitor treatment suppresses the promotion of osteoarthritis in THM.

Conclusion

Osteocyte death in the SB and RANKL-mediated osteoclastic resorption of SB occurred at the very early stage after DMM surgery, preceding histologically detectable cartilage degeneration at the early stage and uncoupled bone remodeling at the later stage. The acceleration of this disease process in THM + DMM mice suggests that high-turnover bone metabolism is a risk factor for KOA. Maintaining SB integrity and avoiding continuous mechanical overload on the joint surface may be basic strategies for preventing disease onset and slowing progression.

Availability of data and materials

The datasets generated and/or analyzed in the current study are available from the corresponding author upon reasonable request.

Abbreviations

Ang II:

Angiotensin II

BMD:

Bone mineral density

BV/TV:

Bone volume/tissue volume

DMM:

Destabilization of the medial meniscus

KOA:

Knee osteoarthritis

MPR:

Multiplanar reconstruction

OARSI:

Osteoarthritis Research Society International

TRAP:

Tartrate-resistant acid phosphatase

OP:

Osteoporosis

RANKL:

Receptor activator of nuclear factor-B ligand

SB:

Subchondral bone

Tb.Sp:

Trabecular separation

Tb.Th:

Trabecular thickness

THM:

Tsukuba hypertensive mice

WT:

Wild type

µCT:

Microcomputed tomography

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Acknowledgements

We gratefully acknowledge Prof. Hiroshi Kaji and Dr. Naoyuki Kawao of the Department of Physiology and Regenerative Medicine, Kindai University Faculty of Medicine for their critical and constructive comments on interpreting the results of this study.

Funding

This work was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (JSPS KAKENHI, grant number: 20K09443).

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Authors and Affiliations

  1. Department of Orthopaedic Surgery, Kindai University Hospital, 377-2 Ohno-Higashi, Osaka-Sayama City, Osaka, 589-8511, Japan

    Teruaki Hashimoto, Masao Akagi, Ichiro Tsukamoto, Kazuhiko Hashimoto, Takafumi Morishita, Tomohiko Ito & Koji Goto

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  1. Teruaki Hashimoto
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Contributions

TH, MA, and IT designed and conceived the study. TH, TM, and TI acquired the data. TH, KH, MA, and KG analyzed and interpreted the data. MA, KG, and TH drafted the manuscript. MA had full access to all data in the study and takes responsibility for the integrity of the data and accuracy of the data analysis.

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Correspondence to Teruaki Hashimoto.

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All animal experimental procedures were performed in accordance with the guidelines of the National Institutes of Health and the institutional rules for the use and care of laboratory animals at Kindai University. All procedures were approved by the Experimental Animal Welfare Committee of Kindai University (approval number: KAME-2022-008). Consent to participate was not required.

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Hashimoto, T., Akagi, M., Tsukamoto, I. et al. RANKL-mediated osteoclastic subchondral bone loss at a very early stage precedes subsequent cartilage degeneration and uncoupled bone remodeling in a mouse knee osteoarthritis model. J Orthop Surg Res 20, 226 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13018-025-05578-6

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Journal of Orthopaedic Surgery and Research

ISSN: 1749-799X