Denosumab attenuates knee osteoarthritis progression by inhibiting synovial inflammation via the RANK/TRAF6/FSTL1 signalling

Domain: biology
Published: 2025-12-17
Editorial outcome: minor_revision

Abstract
Synovitis has recently been shown to be a critical early stage in development of osteoarthritis (OA), and inhibiting synovitis significantly alleviates OA symptoms. Denosumab, a monoclonal antibody targeting RANKL, previously developed for osteoporosis. Here, we report the effect of denosumab on fibroblast-like synoviocytes (FLSs), attenuating synovitis and consequently OA progression. We first demonstrate that RANKL is highly expressed in the knee synovium of OA mice, as well as in patients. Next, we show that denosumab, injected systemically, accumulates in the synovium and effectively reduces synovitis through RANK/TRAF6/FSTL1 signalling in post-traumatic, inflammatory, and aged OA murine models and a beagle dog OA model, delaying OA progression. Finally, a single-arm clinical trial with primary endpoints of VAS and OKS scores, and secondary endpoints of WOMAC score and adverse events rate, shows that denosumab alleviates synovitis and pain, improving joint function in knee OA patients. These findings provide a translational basis for using denosumab to treat knee OA in the clinic.

Introduction
Knee osteoarthritis (OA) is one of the most common degenerative joint diseases and a major cause of pain and disability1, affecting an estimated 250 million people worldwide2. Knee OA is a whole-joint disorder characterized by articular cartilage destruction, subchondral bone sclerosis, osteophyte formation and inflammation of the synovial membrane3–6. Despite extensive research, the complex pathological mechanisms underlying the onset and development of OA remain unknown. Consequently, no disease-modifying drugs are available, and current treatments are limited to painkillers such as non-steroidal anti-inflammatory drugs (NSAIDs) until the joint deteriorates to the stage where surgery is necessary. However, NSAIDs can cause gastrointestinal and cardiovascular side effects7, while surgery carries risks of infection and other complications. Therefore, new drugs are urgently needed to treat knee OA in the early stages.

Recently, we and others have found that synovial inflammation (synovitis), characterized by synovial lining hyperplasia, infiltration of macrophages and lymphocytes, neoangiogenesis and fibrosis, is strongly associated with cartilage degeneration, abnormal subchondral bone remodelling and osteophyte formation8. Many studies have demonstrated that synovitis is associated with pain and poor function, and it is thus considered to play a critical role in radiographic OA onset and structural progression4,9,10. Both mechanical and non-mechanical factors can transform FLSs to an activated, pathological states during OA, where upon they secrete various pro-inflammatory and chondrocyte catabolic cytokines, such as tumour necrosis factor (TNF), interleukins (ILs), receptor activator of nuclear factor κB ligand (RANKL), matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family members. These factors contribute to joint damage, pain, and OA progression11,12. Therefore, targeting synovitis is a promising therapeutic strategy for OA.

Denosumab is used to treat osteoporosis (OP) and giant cell tumour of bone, acting by downregulating the RANKL/RANK signalling pathway thereby inhibiting the maturation of monocyte–macrophage–osteoclast lineage cells13, which play a significant role in synovial inflammation and potentially OA progression. Although denosumab has been shown to attenuate OA progression in mouse models of post-traumatic OA by modifying subchondral bone remodelling14, the detailed mechanisms remain largely uncharacterized. Furthermore, its efficacy in OA patients requires further validation, as rodent joint biomechanics and physiology differ significantly from those of humans15.

In this work, we use three different murine OA models–post-traumatic, inflammatory and aged OA, and a beagle dog knee OA model to validate the effects of denosumab on synovitis and OA progression. Following this, we conduct a single-arm clinical trial, and find that denosumab attenuates synovitis, reduces joint pain and improves joint function in OA patients. These effects are likely mediated by the RANK/TRAF6/FSTL1 signalling pathway in FLSs.

Results
Expression of RANK and RANKL in healthy and pathological joint tissues
We first investigated the expression levels of RANK and RANKL in OA progression in a mouse model at 4, 8, and 12 weeks post-destabilisation of the medial meniscus (DMM) in mice. Immunohistochemical staining revealed that RANK and RANKL expression in articular cartilage (AC) were not significantly different from the sham groups, but both were markedly increased in synovial membrane (SM) of DMM-induced mice at 4 weeks. Furthermore, expression levels of RANK and RANKL in AC and SM gradually increased over 8 and 12 weeks post-DMM and were more enriched in SM than AC (Fig. 1A–C). Next, we assessed the expression level of RANKL in human healthy and OA joint fluid using ELISA. Results showed that RANKL expression was significantly increased in OA joint fluid compared to healthy joint fluid (Fig. 1D). We then obtained normal human AC and SM tissues from thigh amputation or hemipelvectomy and OA-affected AC and SM tissues from knee arthroplasty of patients at the Union Hospital, Tongji Medical College (Wuhan, China), for western blot and PCR analyses. The results confirmed that, compared with normal AC and SM, the expression levels of RANK and RANKL were significantly increased in OA AC and SM, particularly in the OA SM (Fig. 1E, F). To further consolidate our conclusion, mouse chondrocytes, FLSs and macrophages were isolated from AC or SM and incubated with IL-1β for 24 h to induce inflammation. In line with the previous data, the expression of RANK and RANKL was markedly higher in IL-1β-induced chondrocytes and macrophages and especially in IL-1β-induced FLSs (Fig. 1G). PCR assay of RANK and RANKL in IL-1β-induced chondrocytes, the macrophages and FLSs showed the same result (Fig. 1H). Previous studies have identified Col VI as a marker for FLSs11,16. Therefore, we performed co-immunofluorescence staining of RANK or RANKL with Col VI to mark FLSs. As shown in Fig. 1I and Supplementary Fig. 1A, RANK+ cells, RANKL+ cells and Col VI+ cells were all obviously increased in OA synovium (Fig. 1J and Supplementary Fig. 1B). Strikingly, assessment of the proportions of the cell population revealed a change in percentage from 40% to 65% of RANKL+ Col VI+/Col VI+ (Fig. 1K) and 33% to 55% of RANK+ Col VI+/Col VI+ (Supplementary Fig. 1C) at 8 weeks after DMM surgery, indicating that FLSs expressing RANK and RANKL greatly expanded under OA conditions. In summary, these data suggest that FLSs with higher expression of RANK and RANKL are strongly associated with OA initiation and progression.

Denosumab ameliorated OA progression in post-traumatic, inflammatory and aged OA models
Denosumab is a monoclonal antibody targeting RANKL, so we first sought to study its effects in a DMM-induced mouse post-traumatic OA model. Safranin-O/Fast Green staining revealed that cartilage erosion was mild in both femoral condyle and tibial plateau of DMM knees at 4 weeks post-surgery but aggravated at 8 and 12 weeks post-surgery. However, in denosumab-treated DMM mice, there was a significant retention of proteoglycan and a marked reduction in the thickness of calcified cartilage areas at 8 and 12 weeks compared to DMM controls (Fig. 2A). Further analysis demonstrated that denosumab reduced the Osteoarthritis Research Society International (OARSI) score to 28.6% and 29.5% at 8 and 12 weeks, respectively, in OA progression compared with DMM controls (Fig. 2A). We next performed haematoxylin and eosin (H&E) staining of synovium and found abundant inflammatory cell infiltration and synovial hyperplasia at 4, 8 and 12 weeks post-DMM. Treatment with denosumab significantly inhibited these inflammatory effects and reduced the synovitis score (Fig. 2B–E). Micro-CT analysis to evaluate subchondral bone remodelling revealed that DMM caused significant subchondral bone resorption. However, this detrimental effect was effectively reversed by denosumab treatment (Fig. 2G). The results were further confirmed by quantitative analysis of structural parameters, including BV/TV and thickness of subchondral bone plates (SBP. Th) (Fig. 2H). Furthermore, tartrate-resistant acid phosphatase (TRAP) staining revealed hyperactivity of osteoclasts in the DMM group, which was inhibited after denosumab treatment (Supplementary Fig. 2A). Osteophytes, one of the main radiographic features of OA, have significant clinical implications and can be a cause of pain and loss of function1,17. At 8 weeks post-DMM, we observed osteophyte formation and radiological focal defects in both the medial tibial plateau and femoral condyle of mice in the DMM group. Denosumab treatment significantly reduced both osteophyte formation and OA grades compared to the DMM group (Fig. 2F, H and Supplementary Fig. 2B). Since osteophytes are possibly derived from GDF5-lineage cells in synovium18,19, we performed IHC staining of GDF5. Consistent with previous studies, we found a large number of GDF5+ cells in the region where the synovium attached to the edge of the AC in the 8-week post-DMM mice. Intriguingly, GDF5+ cells were markedly reduced in the denosumab-treated mice compared with DMM mice (Supplementary Fig. 2C, D), indicating that denosumab inhibited osteophyte formation by reducing the numbers of osteophyte-derived cells. Markers of cartilage matrix degradation, MMP-13 and ADAMTS5, were also significantly reduced, whereas Col2a1 and Aggrecan were correspondingly increased in denosumab-treated DMM knees (Supplementary Fig. 2E, F). Moreover, the von Frey assay revealed that the paws of DMM mice were sensitive to the stimulus, whereas denosumab improved the paw withdrawal threshold (PWT) compared with control DMM mice (Fig. 2I), suggesting that denosumab heightens mechanical pain sensitivity after DMM.

There are several types of OA, which may be the main reason for the heterogeneous response to drugs20. We further evaluated the therapeutic effects of denosumab on a mono-iodoacetate (MIA)-induced inflammatory OA rat model. Histology showed that intra-articular injection of MIA resulted in an irregular surface and a focal area of cartilage loss and degeneration, while denosumab greatly attenuated this phenotype (Fig. 3A), due to reduced levels of ADAMTS5 and MMP-13 (Supplementary Fig. 3A, B). H&E staining showed that obvious inflammatory cell infiltration and hyperplasia in MIA synovium were significantly alleviated by denosumab treatment (Fig. 3B). Denosumab also produced an elevation in the PWT in MIA-treated rats compared with vehicle-treated MIA rats, consistent with the DMM model (Supplementary Fig. 3C).

Moreover, we also investigated the effect of denosumab in an age-induced OA model. In line with other studies, mice displayed moderate cartilage degradation at 18 months of age, while administration of denosumab caused a partial but significant reduction in cartilage degeneration and OARSI scores (Fig. 3C). Synovial hyperplasia analysed by H&E staining was observed in 18-month-old mice and was attenuated after treatment with denosumab (Fig. 3D). IHC results further confirmed that the expression of MMP-13 and ADAMTS5 was upregulated in old mice, but both were downregulated in denosumab-treated old mice (Supplementary Fig. 3C, D).

Finally, denosumab treatment did not alter the body weight, mechanical pain sensitivity, microscopic morphology of the main organs (heart, liver, spleen, lung, kidney and knee joint), or liver and kidney function, spontaneous locomotor activity, nor did it induce an anxiety-like behaviours in mice (Supplementary Fig. 4). Taken together, all these results demonstrated the therapeutic effect of denosumab in delaying synovitis and OA progression, and confirmed that this treatment was harmless in vivo.

Denosumab alleviated RANKL/RANK-mediated synovitis in FLSs
As OA is a complex disease involving multiple tissues of the whole joint, we next tried to identify the main tissues and specific cell type responding to denosumab. We first investigated the distribution of denosumab in vivo. Mice were injected subcutaneously with denosumab, fluorescently labelled with CY5.5 dye (red), and this revealed that denosumab primarily distributed in the synovium, compared to other tissues of the joint (Supplementary Fig. 5A, B).

We next harvested cells from the whole joint of sham, DMM and DMM+denosumab groups at 4 weeks after DMM surgery for single-cell RNA sequencing (scRNA-seq), including cartilage, subchondral bone, meniscus, and synovium (Fig. 4A). Based on the expression of marker genes (Supplementary Table 5), uniform manifold approximation and projection (UMAP) identified 14 distinct single-cell clusters: fibroblast-like synoviocytes (FLSs), chondrocytes, seven groups of immune cells (neutrophils, basophils, macrophages, monocytes, dendritic cells, T cells, and B cells), haematopoietic stem cells (HSCs), endothelial cells, Schwann cells, pericytes and erythrocytes (Fig. 4B). Interestingly, among all the clusters with increased cell proportions after DMM, FLSs showed the most dramatic decrease in cell proportion after denosumab treatment (Fig. 4C). Consequently, we explored the intercellular communication among FLSs, chondrocytes and macrophages, contributing to cartilage erosion and joint inflammation. Interestingly, we identified that FLSs, as the dominant communication “hub”, dramatically increased the number and strength of communications with the other two cell types during OA. However, these effects were strongly inhibited by denosumab treatment (Fig. 4D), indicating that FLSs were the main denosumab-responsive cells. Next, we analysed the detailed changes in the outgoing and incoming signals among FLSs, chondrocytes and macrophages. We found that in the DMM group, FLSs were the dominant contributors to the outgoing inflammatory signals, including TNF, IL6, CCL, CSF and MIF (Supplementary Fig. 6A). On the other hand, the communication patterns of incoming signals revealed that chondrocytes were the main receivers of the inflammatory signals secreted by FLSs, both of which were significantly inhibited by denosumab treatment (Supplementary Fig. 6B). These data suggested that in the early stages of OA, activated OA FLSs are the main contributors to the propagation of inflammation and destruction of the cartilage matrix, acting by secreting pro-inflammatory cytokines and proteolytic enzymes. Denosumab markedly blocked the pro-inflammation mediators secreted by FLSs, thereby alleviating the structural progression of OA.

To verify the effects of RANKL and denosumab on FLSs, mouse FLSs were incubated with RANKL or denosumab for 24 h before RNA was extracted for mRNA sequencing. We identified differentially expressed genes (DEGs) that were upregulated in RANKL-treated FLSs but were significantly inhibited after denosumab treatment, including MMP-13, MMP-9, ADAMTS5, ADAMTS4 and TNF-α, shown as a heatmap (Supplementary Fig. 6C). KEGG pathway analysis revealed that these DEGs were enriched in the TNF signalling pathway, the NF-κB signalling pathway and the IL-17 signalling pathway (Supplementary Fig. 6D). Furthermore, GO analysis revealed that these DEGs were strongly associated with inflammatory response, osteoclast differentiation, cellular response to interleukin-1 and extracellular matrix catabolic enzymes (Supplementary Fig. 6E), indicating that denosumab significantly attenuated RANKL-mediated inflammatory reaction in FLSs. Further western blot and PCR analyses also confirmed that MMP-13 and TNF-α expression levels in mouse FLSs were significantly increased by RANKL, while these changes were abolished in denosumab-treated FLSs (Fig. 4E and Supplementary Fig. 7A). Interestingly, when we treated chondrocytes directly with RANKL, we found that RANKL induced modest but not significant catabolic effects on chondrocytes (Supplementary Fig. 7B–D). Therefore, we speculated that RANKL, and consequently denosumab, are more likely to influence chondrocytes in an indirect manner through FLSs. We harvested conditioned medium (CM) from FLSs after RANKL treatment (RANKL-CM) without or with denosumab (RANKL+denosumab-CM) treatment. Western blotting and PCR demonstrated that RANKL-CM markedly reduced anabolic gene expression (Col2a1) and increased catabolic gene expression (Col10a1) in chondrocytes, while RANKL+denosumab-CM significantly reversed these changes (Fig. 4F and Supplementary Fig. 7E). In addition, RANKL-CM significantly increased CD86 expression and reduced Arg1 expression to induce M1 macrophage polarisation. Similarly, these effects were markedly reversed by RANKL+denosumab-CM, as demonstrated by western blotting and immunofluorescence staining (Fig. 4G and Supplementary Fig. 7F). Meanwhile, a transwell assay also showed that RANKL-CM induced the migration ability of macrophages, which was substantially inhibited by RANKL + denosumab-CM (Supplementary Fig. 7G). To further consolidate our conclusion in vivo, we injected RANKL with or without denosumab into the mouse knee joints once a week for 4 weeks. Safranin-O/Fast Green staining showed mild cartilage surface erosion in the RANKL-treated knees, a typical sign of early OA, but this was attenuated by denosumab treatment, in line with changes in the OARSI score (Fig. 4H, I). H&E staining showed that the synovial lining layer was thickened and had higher synovitis scores in RANKL-treated knees but not in denosumab-treated knees compared to vehicle (Fig. 4J, K).

In conclusion, we validated the proinflammatory function of RANKL on FLSs, which possibly indirectly induces chondrocyte catabolism and M1 macrophage polarisation, thereby promoting OA progression. These effects were significantly attenuated by denosumab.

FSTL1 expression in FLSs was strongly associated with DMM-induced OA progression but was significantly down-regulated by denosumab treatment
Next, we focused on the DEGs in the FLS cluster. We identified DEGs that were upregulated in the DMM group but were significantly inhibited after denosumab treatment. GO analysis showed that these DEGs were inflammation-related functional genes, such as regulation of inflammatory response, leucocyte migration involved in inflammatory response, leucocyte aggregation, chronic inflammation response and leucocyte cell−cell adhesion (Fig. 5A). Among them, follistatin-like protein 1 (FSTL1) was the most significantly expressed gene in the FLS cluster (Fig. 5B–D). IHC staining confirmed the presence of higher amounts of FSTL1 on the SM of DMM-induced mice compared to the sham group, while this increase was significantly attenuated by denosumab treatment (Supplementary Fig. 8). Furthermore, co-immunofluorescence staining of FSTL1 with the FLS marker Col VI revealed that the percentage of FSTL1+ Col VI+/Col VI+ was significantly reduced in the DMM+denosumab group compared to DMM controls (Fig. 5E). Previous studies demonstrated that FSTL1 is a biomarker of joint damage in OA patients21, and the levels of this protein correlate with OA severity (assessed by Kellgren–Lawrence [KL] and Western Ontario and McMaster Universities arthritis index [WOMAC] scores)22. In our work, we found that FSTL1 markedly upregulated proinflammatory factors (TNF-α, IL-1β, MMP-13, ADAMTS5) in FLSs and cartilage catabolic factors (Col10a1, MMP-13, ADAMTS5) in chondrocytes, but these were both significantly attenuated by denosumab (Supplementary Fig. 9A–D). Moreover, FSTL1 significantly enhanced macrophage migration, which was substantially blocked by denosumab (Supplementary Fig. 9E). In summary, these data suggest that FSTL1 expression in FLSs is positively correlated with OA progression and confirmed that denosumab rescues OA cartilage degeneration by targeted downregulation of FSTL1 in FLSs.

Denosumab inhibited activation of RANKL-induced TRAF6/NF-κB pathways in FLSs
To further clarify the mechanism by which denosumab regulates FSTL1 expression in FLSs, KEGG pathway enrichment was performed. Results showed that DEGs in FLSs were highly enriched in the TNF and NF-κB signalling pathways (Fig. 6A), which play important roles in synovitis and OA pathobiology23,24. Therefore, we focused on the expressions of these key factors involved in the NF-κB and TNF signalling pathways after RANKL or RANKL+denosumab treatment. Notably, the expression of FSTL1, TRAF6 and phosphorylated IκBα were significantly increased after stimulation with RANKL (Fig. 6B). Additionally, RANKL increased the expression of p50 and p65 in the SM (Supplementary Fig. 10A). Moreover, incubation of FLSs with RANKL in vitro upregulated the expression of total p50 and p65 and their nuclear co-localisation (Fig. 6B, C). However, denosumab treatment reversed all these effects of RANKL induction. To demonstrate whether denosumab participated in TRAF6 interaction with RANK after stimulation with RANKL, Co-IP experiments were performed. As shown in Fig. 6D, TRAF6 was clearly bound to RANK after stimulation of FLSs with RANKL; however, this effect was abolished by denosumab treatment. Thus, we speculated that denosumab prevents RANKL from binding to the RANK–TRAF6 receptor complex, thereby inhibiting FSTL1 expression in FLSs via the NF-κB pathway.

In order to verify the function of this signalling pathway, TRAF6 siRNA and BAY11-7082, an NF-κB inhibitor, were introduced to FLSs. We found that the expression of TRAF6 was significantly downregulated by TRAF6 siRNA compared with NC siRNA (Supplementary Fig. 10B, C) and either TRAF6 siRNA or BAY11-7082 markedly suppressed the expression of FSTL1 and phosphorylated IκBα induced by RANKL (Fig. 6E). Moreover, RANKL-induced total p50 and p65 expression and nuclear translocation were also substantially inhibited by TRAF6 siRNA and BAY11-7082 (Fig. 6E, F, H and Supplementary Fig. 10D). These results indicated that RANKL-regulated FSTL1 expression depended exclusively on RANK/TRAF6/NF-κB signalling, which was abolished by denosumab (Fig. 6G).

To further clarify the pro-inflammatory and chondrocyte catabolic effects of the TRAF6/FSTL1 axis, we knocked down the expression of TRAF6 and FSTL1 in FLSs, which were subsequently incubated with RANKL (Supplementary Fig. 11A, B). The results revealed that downregulation of TRAF6 or FSTL1 strongly attenuated the expression of chondrocyte catabolic (MMPs and ADAMTSs) and inflammatory (TNF-α) cytokines induced by RANKL in FLSs (Fig. 6I and Supplementary Fig. 11C).

Together, these results led to the conclusion that the effects of RANKL are mediated via the RANK/TRAF6/NF-κB axis, which upregulates FSTL1, promoting synovitis and chondrocyte catabolism, and thereby inducing OA progression. Denosumab downregulates this signalling axis, thereby attenuating synovial inflammation and subsequent OA development.

Denosumab alleviated synovitis in a larger animal (Beagle) OA model
Based on the therapeutic efficacy of denosumab observed in vitro and in vivo in mice, we used surgical anterior cruciate ligament transection (ACLT)-induced OA in the beagle dog knee as a large animal model (Fig. 7A), which is more comparable to humans25. According to the OARSI canine OA scoring system26, OA-like lesions were grossly observed in the femoral condyle at 2 months after ACLT surgery in a macroscopic view. In contrast, administration of denosumab inhibited AC damage in beagle dogs after ACLT surgery, as shown by a smooth surface and a reduced cartilage score (Fig. 7B). MRI analysis showed that treatment with denosumab protected against cartilage loss as well as the formation of articular hydrops, demonstrated by higher cartilage thickness and reduced effusion-synovitis scores observed in denosumab-treated beagle dogs at 2 months after ACLT surgery (Fig. 7C, D). Safranin-O/Fast Green staining demonstrated that compared with the sham group, the tibial plateau exhibited obvious cartilage damage and proteoglycan loss in the ACLT group. However, the denosumab-treated tibial plateau displayed only minor signs of degeneration and preserved cartilage integrity, effectively alleviating cartilage wear (Fig. 7E, F). H&E staining further showed that denosumab treatment alleviated synovitis, demonstrated by decreased synovial hyperplasia and infiltration of inflammatory cells, when analysed by synovitis scoring in denosumab-treated beagle dogs (Fig. 7E, F). Similarly, we did not observe any obvious morphologic changes in heart, liver, spleen, lung or kidney in denosumab-treated beagle dogs (Supplementary Fig. 12). Because denosumab attenuates OA progression in larger animals, these findings suggest that denosumab has the potential to be used clinically to treat patients with OA.

Denosumab alleviated synovitis in OA patients
A randomised placebo-controlled phase 2 atrial published in Nature Medicine by Wittoek et al. demonstrated that denosumab delayed hand OA progression27. However, the anatomy of the knee joint is different from that of the hand, so we explored whether denosumab exerted any effects on synovitis by a single-arm clinical trial on knee OA patients. In line with Wittoek et al., we found that OKS, WOMAC and VAS scores decreased gradually after injection compared with baseline, with the most significant improvement observed at the final follow-up (Supplementary Fig. 13A). Moreover, MRI showed that, at baseline, the synovium was obviously thickened and there was a large amount of effusion in the joint cavity and suprapatellar bursa, whereas the manifestations of synovitis were significantly improved at 6 months after systemic injection of denosumab, with significant improvements in Hoffa-synovitis and effusion-synovitis scores (Supplementary Fig. 13B, C). In addition, OA patients who needed total knee arthroplasty were injected with denosumab three weeks before surgery. The synovium near the femoral condyle was then harvested for H&E, IHC staining and western blot analysis (Supplementary Fig. 14A). Notably, H&E staining demonstrated that the layers of lining cells were remarkably increased, showing villous hyperplasia in OA synovium, whereas only a slight increase of lining cells was observed in denosumab-treated synovium (Supplementary Fig. 14B). Further IHC staining and western blotting confirmed that the release profiles of RANKL, FSTL1, proinflammatory cytokines (IL-1β, TNF-α) and collagenases (MMP-13, ADAMTS5) within OA synovium were higher than those in OA + denosumab synovium (Supplementary Fig. 14C, D). These data further confirmed our findings in animal models, indicating an important role of FLS-mediated synovitis in OA treatment by denosumab.

Methods
Animals
This study does not involve sex-based analysis and sex was not considered in study design, Therefore, in order to control variables, we used only male mice in this study. Approximately one hundred male C57BL/6J mice at 12 weeks old, twelve male C57BL/6J mice at 12 months old and eighteen male Sprague-Dawley (SD) rats (weight 200–225 g) were used as experimental animals, purchased from Beijing SPF Biotechnology Co. Ltd. Animals were housed under standard animal feeding conditions (22 ± 2 °C, 50 ± 10% humidity, 12-hour light/dark cycle) with free access to a conventional rodent chow and autoclaved water. For aging OA mouse models, 12-month-old male C57BL/6J mice were kept until 18 months of age. Twelve male dogs (beagles), with a mean body weight of 10.0 ± 1.5 kg, were purchased from Wuhan Xinzhou Wanqianjiahe Experimental Animal Breeding Farm Co., Ltd. and housed on the first floor of the Huazhong University of Science and Technology Tongji Medical College Laboratory Animal Centre in an open environment.

Enzyme-Linked Immunosorbent Assay (ELISA)
The levels of RANKL in the joint fluid were measured using a Human TRANCE/TNFSF11/RANKL ELISA Kit (Abclonal, RK00341) according to the manufacturer’s protocol.

OA induction and treatment
DMM surgery was performed to create an OA mouse model in C57BL/6J male mice at 12 weeks of age48. Briefly, mice were anesthetized with pentobarbital sodium (50 mg/kg), and a longitudinal skin incision was made on the medial side of the right knee. The medial meniscal tibial ligament was transected under a surgical microscope. Mice which underwent the incision without meniscal tibial ligament transection formed the sham group.

Rats were subjected to mono-iodoacetate (MIA; Sigma, MO, USA) to induce OA49. Briefly, rats were anesthetized with pentobarbital sodium and received a single intra-articular injection of 3.0 mg/50 μL MIA (in PBS) into the right knee joint using a 31G insulin syringe. The control group received an equal volume of 0.9% sterile saline into the left knee.

For the canine model, OA was induced by surgical anterior cruciate ligament transection (ACLT)50. Under standard anaesthetic conditions, the anterior cruciate ligament of the right knee joint was transected using a pair of curved scissors and a sham operation was performed on the left knee joint. Bleeding and soft tissue damage were minimised as much as possible. After surgery, the synovium, fasciae, and skin were each sutured in sequence. The contralateral knee served as an internal control. The animals received analgesics and antibiotics during the first 3 days after surgery. Starting 2 days after surgery, the dogs were allowed out every day for 10 min on the patio for daily exercise.

Animals in the treatment group were injected subcutaneously with denosumab (10 mg/kg/week). Meanwhile, animals in the RANKL group were injected subcutaneously with sRANKL (1 mg/kg). The sham group received an equivalent volume of vehicle. All experimental mice were euthanised 4, 8 or 12 weeks after surgery for further analysis.

OA pain analysis
Mouse knee pain was measured using von Frey fibres at 0, 2, 4, and 8 weeks after DMM surgery. Before the test, each mouse was acclimatised on an individual 4 × 3 × 7 cm metal grid (Shanghai Yuyan Instrument Co., LTD., Shanghai, China) for 20 min. Von Frey fibres (Shanghai Yuyan Instrument Co., LTD.) in the range 0.008 to 300 g were used for this assay. According to the “up-down method“51, the plantar surface of the hind paw of each mouse was stimulated with each fibre until the fibre bent for 3 s. X indicates a withdrawal response and O indicates no response. The formula shows that the threshold force causing paw contraction is expressed as the 50% paw contraction threshold: (10[Xr+kδ])/10,000 (Xr = value of the last von Frey fibre used in the sequence (expressed in logarithmic units), k = table value, δ = mean difference of forces between fibres).

Open-field test
Mice were tested in the open field to measure their spontaneous locomotor activity and anxiety-like behaviours. Briefly, each mouse was gently put into an open field box (50 × 50 × 50 cm3), the bottom of the box was mapped by the system. The mice were placed in the central area, habituated to the experimental environment for 30 min before the test and then allowed to move freely for 5 min. Afterwards, the total distance travelled, walking speed, and time duration in the central area were recorded and calculated.

Micro-CT analysis
Mouse knee joints were harvested at 8 weeks after DMM surgery and fixed in 4% paraformaldehyde (PFA) for 48 h. Micro-CT analyses were performed using a high-resolution micro-CT scanner (Bruker SkyScan 1176, Karlsruhe, Germany) at 9 μm resolution, with a 1 mm aluminium filter, 90 kV voltage, and 273 μA current. NRecon reconstruction software and CT-VOX 3D reconstruction analysis software provided by SkyScan were used for volumetric reconstruction and 3D image generation. The grade of OA progression was analysed based on the micro-CT images52.

Histology and immunohistochemistry (IHC)
After performing micro-CT scanning, knee joints were immersed in 4% PFA overnight, decalcified with 10% EDTA for 4 weeks, and then embedded in paraffin. After embedding, the specimens were sectioned at a thickness of 7 μm from the medial side to the cruciate ligament junction. Safranin O/fast green, H&E and TRAP staining were performed. The Mankin Score or OARSI score was used to evaluate the severity and progression of OA after safranin-O staining53. Factors affecting synovitis score included thickening of the entire synovial membrane, activation of resident cells/synovial stroma and aggregation of inflammatory cells, revealed by H&E staining54. For IHC staining, slides were incubated with primary antibodies: anti-RANK (1:100, Abclonal, A13382), anti-RANKL (1:100, Proteintech, 23408-1-AP), anti-Col2a1 (1:100, Abcam, ab34712), anti-Aggrecan (1:100, Abcam, ab315486), anti-MMP-13 (1:100, Proteintech, 18165-1-AP), anti-ADAMTS-5 (1:100, Boster, A02802-1), anti-FSTL1 (1:100, Proteintech, 20182-1-AP), anti-GDF5 (1:100, Abclonal, A13167). After rinsing twice, sections were incubated with appropriate secondary antibodies and photographed under an optical microscope (Olympus, Tokyo, Japan; IX73P1F).

Immunofluorescence
For immunofluorescent staining, frozen sections were rewarmed to room temperature, permeabilized with 3‰ Triton X-100 at room temperature for 15 minutes, then blocked in 5% bovine serum. After rinsing, slides were incubated with primary antibodies against RANKL (1:100, Proteintech, 23408-1-AP), RANK (1:100, Abclonal, A13382), or Col VI (1:100, Proteintech, 17023-1-AP) overnight at 4 °C, then sections were labelled with secondary antibody for 1 h in the dark, and counterstained with DAPI for 10 min and then imaged under a confocal microscope (Nikon A1).

Cells cultured on slides were fixed with 4% PFA for 20 min at room temperature then incubated with a primary antibody against p50 (1:100, Proteintech, 14220-1-AP) or p65 (1:100, Proteintech, 10745-1-AP), CD86 (1:100, Proteintech, 13395-1-AP) or Arg1 (1:100, Affinity Biosciences, DF6657), overnight at 4 °C. Then the next day, the cells were incubated with a fluorophore-conjugated secondary antibody for 1 h. Cell nuclei were counterstained with DAPI for 10 min in the dark. Images were acquired under a confocal microscope (Nikon A1).

Single-cell RNA-sequencing (scRNA-seq)
Articular tissues of mice in the sham, DMM and DMM+denosumab treatment groups were isolated and dissociated into single cell suspensions with 100 μg/mL of the enzyme mixture Liberase™ (Sigma-Aldrich) and 100 μg/mL DNase I (New England Biolabs, Ipswich, MA, USA)55. Briefly, a single-cell RNA-Seq library kit v3 was used for cDNA amplification and chromium library construction (10× Genomics, Pleasanton, CA, USA). cDNA libraries were sequenced on an Illumina NovaSeq 6,000 (Illumina Inc., San Diego, CA, USA) with a reading length of 150 bp by the Wuhan Biobank Co. Ltd. The R package Seurat (Version 3.0.2) was used to perform data analysis56. Briefly, a single-cell suspension of 1.8 × 104 cells was examined and filtered (nFeature 6,000, nCount 10,000, percent_mt 0.25) for downstream analysis. Cells were visualised using the Uniform Manifold Approximation and Projection (UMAP). Differentially expressed genes (DEGs) were defined by a P value threshold < 0.05 and logFC > 0.25.

mRNA sequencing (RNA-seq)
Total RNA was isolated from mouse FLSs of Veh, RANKL and RANKL+denosumab treatment groups using Trizol (TaKaRa Bio) and evaluated using an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). An RNA sequencing library was prepared and sequenced by Capital Bio Technology (Beijing, China). The DESeq2 package was used to identify DEGs between groups, and the threshold of adjusted P value was 0.05. Gene ontology (GO) analysis, and Kyoto encyclopaedia of genes and genomes (KEGG) enrichment analysis for DEGs were visualized and analysed using the DAVID Bioinformatics Resources 6.8.

Cell isolation, treatment and transfection
FLSs were harvested from 8-week-old mouse knee joints. After euthanising mice, the synovium tissue was dissected and digested in 0.25% trypsin for 30 min, followed by digestion with 0.2% collagenase Type 2 for 6 h57. Then cells were centrifuged and filtered, plated into T25 flasks and cultured with Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Carlsbad, CA, USA) containing 10% foetal bovine serum (FBS) (Gibco), and supplemented with penicillin (100 U/ mL) and streptomycin (100 g/mL).

Chondrocytes were harvested from the knee joint articular cartilage of 3-day-old mice. Cartilage samples were harvested, cut up into pieces using a sterile scalpel under a dissection microscope and digested in 0.25% trypsin for 30 min. Then the pieces of cartilage were further digested with 900 U/mL collagenase Type I for 2 h. Dissociated cells were centrifuged and resuspended in DMEM containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 g/mL).

For chondrogenesis, 4.0 × 105 chondrocytes were centrifuged into pellets and then cultured in chondrogenic medium (high-glucose DMEM containing 40 mg/mL L-proline, 0.1 mM dexamethasone, 1% penicillin/ streptomycin, 100 mg/mL sodium pyruvate, 50 mg/mL ascorbate-2-phosphate, 1% ITS+ Premix). Before chondrogenic induction, 10 ng/mL TGF-β1 (PeproTech, Rocky Hill, NJ, USA) was freshly added to the differentiation medium, which was changed every 3 days. After culturing for 2 weeks, the cell pellets were fixed in 4% PFA and then embedded in paraffin. To evaluate collagen deposition, paraffin sections were stained with alcian blue.

FLSs were cultured in a 6-well plate at a density of 1 × 104 cells/well, then transfected with TRAF6 siRNA (siTRAF6) or FSTL1 siRNA (siFSTL1) sequences (GenePharma Co., Ltd., Shanghai, China) using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA). After transfection for 36 h, the culture medium was changed, and the cells were treated with RANKL (50 ng/mL) or denosumab (1 μg/mL) for 24 h.

Conditioned medium
FLSs were seeded into a 6-well plate and incubated with RANKL or RANKL + denosumab for 24 h. The next day, the cells were washed three times with PBS, and the culture medium was changed to serum-free DMEM. After another 24 h, the conditioned medium was collected for further experiments.

Migration assay
To evaluate migration, 1 × 104 RAW 264.7 macrophages (American Type Culture Collection, TIB-71, Manassas, VA, USA) in 150 μL growth medium were plated into the upper chamber of a 24-well Transwell™ plate. RANKL-CM or RANKL+denosumab-CM was added to the lower chamber. After incubating for 12 h, cells in the upper chamber were removed using a swab and cells on the bottom surface of each Transwell™ were fixed with 4% PFA and stained with crystal violet. The number of macrophages that had migrated was observed and quantified under a microscope.

Real-time polymerase chain reaction (RT-PCR)
Total RNA was isolated from cells or tissues using Trizol reagent (Invitrogen) according to the manufacturer’s protocol, and cDNA was reverse transcribed using a reverse transcription kit (TaKaRa Bio). TB Green Premix Ex Taq II (TaKaRa Bio) was used for RT-PCR detection. Expression levels were normalised to GAPDH which was used as the internal reference. All primer sequences for RT-PCR are listed in Supplementary Table 1.

Western blotting and co-immunoprecipitation (Co-IP)
Total protein was harvested from cells or tissues using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with phosphatase and proteinase inhibitors, and the concentration was determined by the BCA assay (Beyotime Institute of Biotechnology). Proteins were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a 0.45 μm polyvinylidene difluoride membrane. The membrane was then incubated overnight at 4 °C with the following primary antibodies against the following proteins: RANK (1:1,000, Abclonal, A13382), RANKL (1:1,000, Proteintech, 23408-1-AP), MMP-13 (1:1,000, Proteintech, 18165-1-AP), ADAMTS-5 (1:1,000, Boster, A02802-1), Col2a1 (1:1,000, Bioss, bs-0709R), Col10a1 (1:1,000, Abcam, ab182563), TNF-α (1:1,000, Abclonal, A11534), IL-1β (1:1,000, Affinity Biosciences, AF5103), CD86 (1:1,000, Proteintech, 13395-1-AP), Arg1 (1:1,000, Affinity Biosciences, DF6657), FSTL1 (1:1,000, Proteintech, 20182-1-AP), TRAF6 (1:1,000, Santa Cruz, sc8409), IκB-α (1:2,000, Proteintech, 10268-1-AP), p-IκB-α (1:1000, CST, 2859T), p-65 (1:1,000, Proteintech, 10745-1-AP), p-50 (1:1,000, Proteintech, 14220-1-AP) and H3 (1:1,000, ab32356, Abcam); GAPDH (1:3,000, Proteintech, 60004-1-Ig) was used as the internal reference. After washing with TBS containing 0.01% Tween 20 (TBST) three times, HRP-conjugated secondary antibodies were added and incubated for 1 h at room temperature. Signals on the membrane were visualized using ECL chemiluminescence.

For immunoprecipitation, cell lysate was precipitated with 5 μL of anti-TRAF6 antibody (Santa Cruz, sc8409) or control IgG for 24 h using magnetic beads. Then the immune complexes were separated by magnetic adsorption and washed twice with PBS. Finally, the immune complexes were boiled for further western blot assays to detect the expression of RANK.

Clinical study design and collection of human synovium samples
The clinical study was performed between August 2021 and December 2025 in Wuhan Union Hospital. The protocol was registered on the Chinese Clinical Trial Registry (www.chictr.org.cn, number ChiCTR2200060412), and ClinicalTrials.gov (identifier: NCT06357741) and was approved prior to patient recruitment by the Institutional Review Board according to the tenets of the Declaration of Helsinki. All patients included in this study signed a written informed consent form and volunteered for the collection and use of their tissue samples (cartilage, synovium, and synovial fluid) in scientific research. In addition, publication of any potentially identifiable images or data, as approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Ethical approval number: 2025-0423). It was designed as a 3+3 single-arm Phase I study to evaluate the safety and efficacy of denosumab injection in knee OA patients with OP. This study does not involve sex-based analysis and sex was not considered in the study design. Therefore, for human participants, we did not assign a particular sex. Nine patients were enroled and followed up over three distinct study visits of 1, 3 and 6 months after systemic denosumab injection. The demographic characteristics and clinical status of these patients are listed in Supplementary Table 2. Fifty-six percent of the patients had a high BMI.

Human synovium samples were collected from patients with knee OA who received total knee replacement at the Union Hospital, Tongji Medical College (Wuhan, China) (Supplementary Table 3). We obtained informed consent from all participants in this study, and the tenets of the Declaration of Helsinki were followed throughout.

Outcomes
The primary outcome was the 10-point visual analogue scale (VAS) for knee pain and the Oxford Knee Score (OKS) for knee function. The secondary outcome was the WOMAC for knee function and the rate of adverse events (AEs). WOMAC consists of three sub scores, pain, comprising five items; stiffness, with two items; and functionality, with 17 items. AEs were recorded at each follow-up and described in terms of symptoms, incidence, severity, and relatedness according to the World Health Organization-Uppsala Monitoring Centre causality assessment system58. The severity of AEs was divided into five levels according to the National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE)59. No serious adverse events (AEs), or cases of permanent disability, septic arthritis, or neoplasia were reported at any of the follow-up visits. All AEs were mild and no patients discontinued the trial due to AEs (Supplementary Table 4).

Radiologic outcomes were analysed by magnetic resonance imaging (MRI) according to the MRI Osteoarthritis Knee Score (MOAKS), which is a validated semi-quantitative tool to describe OA progression over time60. Thus, the knee is divided into 14 subregional divisions (tibia–medial and lateral, anterior/central/posterior; femoral–medial and lateral, trochlea/central/posterior; patella–medial and lateral) with each region achieving a corresponding score for features of OA. The features assessed in this study were articular cartilage, synovitis and bone marrow lesions (BMLs). All data were collected at intervals of baseline, 1, 3 and 6 months follow-up by an orthopaedic physician with no knowledge of the treatment group.

Statistical analysis
Each experiment was repeated at least three times and only data from a representative experiment are presented. Data are represented as the median with interquartile range and all statistical analyses and mapping were performed using GraphPad Prism 8 software (GraphPad Software Inc., La Jolla, CA, USA). We employed unpaired two-tailed t-test or two-tailed Mann–Whitney U test for statistical analysis between two groups and one-way ANOVA with Tukey’s post hoc test or Kruskal–Wallis test with Tukey’s post hoc test for multiple comparisons. n indicates the number of biologically independent samples, animals per group, or human specimens. Values of P<0.05 were considered significant.

Ethics approval
All animal study designs were reviewed and approved by the Animal Care and Ethics Committee of Huazhong University of Science and Technology (Ethical approval number: 3852).

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
Source data of all figures and RNA-seq data of this study are provided in the Source Data File. The scRNA-seq data of this study are provided and accessible through National Genomics Data Center (https://ngdc.cncb.ac.cn/omix/release/OMIX011832). The study protocol and the information of the volunteers enrolled in the clinical trial are available within the article and its supplementary files. Any additional requests for information can be directed to, and will be fulfilled by the corresponding authors. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript shows that Denosumab - a monoclonal antibody targeting RANKL and currently used in osteoporosis - is effective in treating pain and cartilage integrity in osteoarthritis and identifies important aspects of the mechanism of action. The mechanism of action, relies on interference of Denosumab in the binding of RANKL to RANK and TRAF6, mostly in the synovial membrane, inhibiting NF-kB and suppressing the expression of FSTL1, which in turn causes release of inflammatory cytokines in FLS and catabolic enzymes in cartilage. The efficacy is demonstrated in three different models in rodents, in instability-induced osteoarthritis in dogs and, albeit less convincingly, in humans.\nIn terms of novelty, the efficacy of Denosumab in osteoarthritis was already shown by Shangguan et al. 2024 (doi: 10.1016/j.reth.2024.03.019) in mice and by Wittoek et al. Nat Med 2024 (doi: 10.1038/s41591-024-02822-0) in humans in a randomised controlled clinical trial. The former is acknowledged in both introduction and discussion, the second, however, a high-profile randomised, placebo controlled trial, only in discussion. This should be corrected. I believe that, in addition to replication, which is per se important, what this manuscript brings in terms of novelty, is the mode of action, which is novel. RANKL/RANK function is well known for its role in osteoclast differentiation and therefore for bone remodelling. The anti-inflammatory function in this context is novel. Of particular value are the experiments with conditioned medium of FLS treated with RANKL +/- denosumab and the FSTL1 loss of function experiments clearly showing that the mechanism on cartilage can be mediated by synovial inflammation, albeit - in my opinion, it does not show that this is the only mechanism.\nCriticisms.\n1. In osteoarthritis clinical trials there is usually a placebo effect + regression to the means around 30%. This is a major problem in the interpretation of the single arm clinical trial. I think that the best way to keep this part is to present it as a replication of the the Wittoek paper in a different form of osteoarthritis (the Wittoek paper was in hand osteoarthritis) and to emphasize the histological aspects of synovitis in the experiments performed in patients undergoing knee replacement and acknowledging the limitations of a single arm study in osteoarthritis.\n2. While it is true that the experiments show that the effects of RANKL and Denosumab are more intense in the FLS than in cartilage and that the supernatant of RANKL treated FLS induce catabolic effects in cartilage, a direct effect on cartilage cannot be excluded. For instance, in Fig. S6 there is some degree of increase of Col2a1 when Denosumab is applied on chondrocytes. N=3 is really small and these experiments, in chondrocytes, may be underpowered particularly in monolayer cultures which, compared to micromass/pellet cultures are much less responsive. Cartilage is a slow-reacting tissue and slow but persistent changes are likely to have a sizable effect in the context of osteoarthritis. I think that emphasizing the effect on synovitis is well-justified, but the authors have not convincingly excluded a direct effect on cartilage itself.\n3. The “elephant in the room” here is the bone. Denosumab clearly limits bone remodeling and bone remodeling is a well-established driver of osteoarthritis. Consequently, a better characterization of bone remodeling is required it at least DMM, including bone density, BV/TV, thickness of the subchondral plate, osteoclast count, the size of the epiphysis, and ideally also rate of bone remodeling. This is imperative. In addition, a discussion point should be added, depending on the results, that albeit there are clear anti-inflammatory effects, there are also effects on bone remodeling (and direct effects on cartilage, although less intense) and that the interplay fo these components is likely to result on the efficacy of denosumab.\n4. The Mankin score is ill-suited for murine OA because murine cartilage is much more cellular than human cartilage and most of the parameters of cellularity and cell shape do not really apply. For these reasons, people developed the OARSI score, which is the standard scoring system in the mouse. It would be best to rescore using the OARSI score to facilitate comparison with other studies of this type. Also, the picture of the Denosumab group at 8 weeks does not seem to be representative of the score in the graph. Please chose a representative picture i.e. one close to the median of the group. In fact, the picture shown is essentially normal cartilage, whereas in the graph, in the denosumab group, there is no data point that is normal. Is this correct?\n5. Fig. 2F the microCT of the DMM+denosumab group seems to be taken from the contralateral knee, judging from the shape of the tibia, unless it is a projection effect. Please check if correct. In addition, the ossicle that normally is present in the anterior horn of the medial meniscus is not present and also the entire patellar grove is not visible. Please check. Were the soft tissues dissected before microCT? Was the thresholding the same as the other images?\n6. Fig 2B-D and all figures about scoring of synovitis: the part behind the meniscus is the joint capsule. The synovium is limited to the tissue lining the joint where there is no cartilage (and no menisci). I do not think that the scores will change but please correct.\n7. Fig 3C middle panel, there is a cutting artifact with rolling of the cartilage of the femoral condyle which makes it look thinner. Please chose a different section.\n8. Fig. 7. Cartilage histology and histological score of the dog model should be shown.\nMinor points and suggestions\n- P5. all the refs proposed to support a role of inflammation in osteoarthritis progression, in reality only show association rather than causation. Synovial inflammation is a well-known driver of symptoms in osteoarthritis, but its role in cartilage breakdown is still debated.\n- ln 137-138. OA cartilage and SF was obtained from patients undergoing knee replacement. Presumably not the healthy cartilage and synovial membrane. Please state the origin of the healthy tissue.\nFigure 1 E;F, please state if the values are normalised by GAPDH. Also please make the legend more legible.\n- I would suggest stating the statistical test used for every panel in the figure legends.\n- Fig S4 distribution of denosumab in vivo. This was achieved by injecting systemically Cy5-conjugated denosumab and looking at epifluorecence in the slides. It is difficult to assess the specificity of the fluorescence because cartilage and synovial membrane are highly autofluorescent. An appropriate negative control is essential. The staining, particularly in cartilage, appears to be patchy, which, in the absence of a convincing control, is suspicious.\n- In figure S6 and S10 are important and convincing and in my opinion would deserve a place in the main figures.\n- Fig 4. The labeling of the panels is wrong in the legend. There is no panel I and I believe F is western blot of TNF and MMP13, G is PCR, H is the representative images and I the synovitis score.\n- Fig 4H-I and related text. The text mentions mild cartilage erosions in the mice treated with RANKL but not in Denosumab. I would suggest showing some images of that and quantify using OARSI score.\n- Li 465-466 “synovial inflammation is an initial event in the development of OA\": I would replace “initial” with “critical”. This is because, in most patients, biomechanical factors on cartilage prime the pathological process, including synovial inflammation. The same applies for “initiation” in li 94.\n- Li 502-503. I would recommend care when making statements such as “rodents are very different from humans” because they may be easily misconstrued as “models in rodents are not predictive of human disease” and used to argue against the use of rodents in science. After all this manuscript beautifully shows how instead very predictive they are! I would rephrase on the lines of “for confirmation in larger animals with a joint structure and load more similar to the human one….”", "reviewer_2": "In this manuscript, the authors investigated the role of denosumab, a monoclonal antibody targeting RANKL, in three different murine models and in a dog model of osteoarthritis (OA). After confirming that RANKL and RANK were highly expressed in both murine and human joint tissues, particularly in the synovium (and mainly in fibroblast-like synoviocytes (FLSs)), denosumab was injected systemically in the murine and dog models. This resulted in a significant inhibition of synovitis. To uncover the underlying mechanisms, the authors performed scRNA-seq demonstrating that FLSs were the most significantly altered cell cluster having the most intense cell–cell interaction with chondrocytes. These phenomena were reversed by denosumab treatment. Further immunoprecipitation assay revealed that RANK–TRAF6 binding that activates NF-κB signaling led to the upregulation of catabolic and pro-inflammatory factors in FLSs. In subsequent analyes, the follistatin-like protein 1 (FSTL1) was identified as the most upregulated cytokine in FLSs, highly-likely being responsible for the catabolic and pro-inflammatory effects in chondrocytes. In addition, the authors performed a single-arm clinical trial using denosumab that similar to animal models reduced synovitis and OA-associated pain via the same FLSs/FSTL1 mechanism.\nThe topic is of course of great interest and highly relevant because still no disease-modifying drugs exist that effectively and causally slow down OA progression. The authors offer a large amount of information and have conducted relevant experiments at different levels. However, I have one substantial concern regarding the missing sham+denosumab control as explained in more detail below. Another major weakness in my opinion is the small sample size in the majority of experiments, see also below. In addition, I have a few minor comments, some of which concern the content or the scientific writing/symbols.\nMAJOR\n1. Missing sham+denosumab control: Without this control group, the OA-independent effects of denosumab can not be detected. Accordingly, no side effects are visible. The authors state in the manuscript (line 84) ‘NSAIDs can cause gastrointestinal and cardiovascular side effects’… denosumab has also side effects (e.g. https://www.prolia.com/), some of them are quite serious (cardiovascular, fatigue, bone remodeling, pain! among others).\nTherefore a further question arises: why was denosumab applied systemically? Did the authors check body weight of the animals, or their activity – e.g. denosumab might cause lethargy and lack of energy and consequently decreased activity that might be OA protective…\n2. Small sample size: Although the results seem to be heterogeneous – which surprised me anyway, especially in the animals models, I never obtained such low variability in my murine studies – a sample/group size of n=3 is very low when performing multiple statistical comparisons. This would be even more difficult when the missing sham+denosumab mice were included….\n3. The authors talk about the MIA model in mice in the sections abstract, results (figure legends), and discussion…Then, in the section methods, the reader is surprised by the fact that rats were used for the MIA model...that is very confusing and therefore, I am not able to evaluate this part of the results.\n4. Can the denosumab effect be sex-specific? Since almost only female patients were included (and females deal differently with pain…)?\n5. What about a denosumab effect on monocytes/macrophages, since they and their interaction with FLS were/was also strongly influenced, s. Fig.4. Moreover, RANKL induces macrophage polarization to the pro-inflammatory M1 phenotype. Wouldn´t it be worth/necessary to investigate this?\nMINOR\n6. Line 61 – redundancy with line 57-58\n7. Line 90: there will never be ONE central mechanism because OA pathogenesis is multifactorial, extremely complex\n8. Line 114 – 2x three\n9. Lines 138-139: healthy donors underwent arthroplasty???\n10. Line 145 and the entire manuscript: IL-1β is the correct spelling\n11. Line 147: ‘inflammatory chondrocytes’??? Chondrocytes were treated with IL-1β and are therefore in an inflammatory microenvironment, but they are not inflammatory per se…\n12. Gene names should be italic – ist RANK/RANKL correct, or should TNFRSF11A/ TNFSF11 be used?\n13. Lines 200-201 and Fig S2: no, there is no Col2a1 increase after denosumab treatment. There is just no difference to Ctrl. Type II does not disappear anyway in DMM! Collagens are very stable. First, proteoglycans are degraded, so a proteoglycan loss is visible in OA cartilage. Maybe type II collagen also decreases in the defect area but not in the entire cartilage. I can not believe Fig, S2D Col2.\n14. Lines 202-206 and Fig. 2G: Frey assay is an indirect measurement of pain sensitivity and does not directly evaluate OA-associated joint pain. So, the effect might be some ‘side effect’ of denosumab – which could be clarified using the sham+denosumab mice, see my major comment 1.\n15. The MIA model is often preferred when studying pain (quick and harsh). Why was pain not assessed in the MIA model?\n16. Lines 241-242: ‘suspension of 1.8 × 104 cells’ – such details are not needed in section results, it is a methodological detail.\n17. General to MMP and ADAMTS - WB and staining does not tell anything about enzyme activity - be careful with interpretation.\n18. Fig. 7 I: Huffa synovitis score – typo (Hoffa is correct)\n19. The dogs are missing in the Methods/Animals section (Age, sex, number…).\n20. The order of methods is not logical – does not follow the results order.\n21. How was the synovitis scoring performed? There are different protocols published with measuring synovial thickness, quantifying infiltrating immune cells etc. The authors do not provide information on that.", "reviewer_3": "This review only concerns the statistical part of the submitted work.\nThere is no mention of how many mice or rats were used in the study. There were no mention of dogs in the Materials and Methods section but results were presented for them (line 400). 8 human patient were used in the study. Although a formal sample size calculation is not needed in a Phase I clinical trial, a justification of the numbers used is desirable.\nIn the Statistical Analysis section it is mentioned that 'groups were compared by t test. One-way ANOVA and two-way ANOVA were used for multiple comparisons'. The Results section should clearly present the results of group comparisons showing the test statistics, p-value and the degrees of freedom as appropriate.\nFootnotes of Figures 1, 2, 3, 4, 6 & 7 says that 'Data presented as mean +/- SD'. It was not clear what are these values in those Figures.\nAs the study sample sizes, be it animal or human, are very small, parametric tests such as t test and ANOVAs are not appropriate. Instead the authors should use non-parametric test. Also for descriptive statistics it is desirable to use median and range (or Inter Quartile Range, IQR) should be used instead of mean and SD.\nAs the data were collected at baseline, 4, 8 and 12 weeks, it is desirable to use longitudinal analysis to see how the variables change over time. Instead comparisons were made with baseline values or between two time points. It is understandable that longitudinal analysis may not be feasible due to small sample size and therefore an acknowledgement of this would make the analyses, with baseline or between two time points, acceptable.\nWherever a statement like 'did not differ significantly' such as in Line 129, 136 etc., please quote the p-value to justify the statement.\nIn my opinion, the Methods and Materials section should be presented before the Results section."}, "author_response": "Thank you very much for your insightful comments and professional suggestions. As you rightly pointed out, the therapeutic effects of denosumab in hand OA have been well documented by Wittoek et al. In light of this, we have relocated the results of the single-arm study (including VAS, WOMAC and OKS scores) to the Supplementary Fig 13, and emphasized the effects of denosumab on synovitis in the revised Results section as follows:\nA randomized placebo-controlled phase 2a trial published in Nature Medicine by Wittoek et al. demonstrated that denosumab delayed progression hand OA. However, the anatomy of the knee joint is different from that of the hand, so we explored whether denosumab exerted any effects on synovitis by a single-arm clinical trial on knee OA patients. In line with Wittoek et al., we found that OKS, WOMAC and VAS scores decreased gradually after injection compared with baseline, with the most significant improvement observed at the final follow-up (Supplementary Fig. 13A). Moreover, MRI showed that, at baseline, the synovium was obviously thickened and there was a large amount of effusion in the joint cavity and suprapatellar bursa, whereas the manifestations of synovitis were significantly improved at 6 months after systemic injection of denosumab, with significant improvements in Hoffa-synovitis and Effusion-synovitis scores (Supplementary Fig. 13B, C). In addition, OA patients who needed total knee arthroplasty were injected with denosumab three weeks before surgery. The synovium near femoral condyle was then harvested for H&E, IHC staining and western blot analysis (Supplementary Fig. 14A). Notably, H&E staining demonstrated that the layers of lining cells were remarkably increased, showing villous hyperplasia in OA synovium, whereas only a slight increase of lining cells was observed in denosumab-treated synovium (Supplementary Fig. 14B). Further IHC staining and western blotting confirmed that the release profiles of RANKL, FSTL1, proinflammatory cytokines (IL-1β, TNF-α) and collagenases (MMP-13, ADAMTS5) within OA synovium were higher than those in OA + denosumab synovium (Supplementary Fig. 14C, D). These data further confirmed our findings in animal models, indicating an important role of FLS-mediated synovitis in OA treatment by denosumab.\nAdditionally, we have incorporated a brief discussion on the limitations of this single-arm study in the Discussion part at lines 865-884.\nSupplementary Fig 13. Denosumab treatment attenuated pain and improved joint function in OA patients. (A) Quantification of OKS Score, VAS Score and WOMAC Score at 1, 3 and 6 months post-injection of denosumab in OA patients, compared with baseline. n = 9. (OKS Score: 1 month: P = 0.0012. 3 month: P = 0.0004. 6 month: P < 0.0001; VAS Score: 1 month: P = 0.0294. 3 month: P = 0.0090. 6 month: P = 0.0004; WOMAC Score: 1 month: P = 0.0002. 3 month: P = 0.0001. 6 month: P < 0.0001). (B) Representative MRI images of knee joints at 6 months after denosumab injection in OA patients. Red arrows indicate the effusion of joint cavity and suprapatellar bursa. (C) Hoffa-synovitis score and Effusion-synovitis score based on MRI images, quantified at 6 months after denosumab injection in OA patients, compared with baseline. n = 9. (Hoffa-synovitis Score, P = 0.0004; Effusion-synovitis Score, P = 0.0023). Data are presented as median with interquartile range. Statistical analysis was performed using Mann–Whitney U test (A, C). n indicates the number of biologically independent samples, mice per group, or human specimens. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.\nSupplementary Fig 14. Denosumab treatment alleviated synovial inflammation in OA patients. (A) The boxed area shows where the synovium was harvested for analysis during surgery. (B) Representative H&E staining of synovial tissues of OA patients after 3 weeks of denosumab treatment. Magnified images of the boxed areas are shown in the panel below. Scale bar = 200 μm (upper image); 100 μm (lower image). Synovitis scores were quantified. OA-synovium, n = 6; OA + denosumab-synovium, n = 3. (P = 0.0119). (C) Immunostaining of RANK, RANKL, FSTL1, MMP-13, ADAMTS5 and TNF-α in the synovial membrane of OA patients after 3 weeks of denosumab treatment before surgery. Scale bar = 100 μm. (D) Western blotting analysis of RANK, RANKL, FSTL1, ADAMTS5, MMP-13 and TNF-α in the indicated synovium. Data are presented as median with interquartile range. Statistical analysis was performed using Mann–Whitney U test (B). n indicates the number of biologically independent samples, mice per group, or human specimens. *P < 0.05.\n2. While it is true that the experiments show that the effects of RANKL and Denosumab are more intense in the FLS than in cartilage and that the supernatant of RANKL treated FLS induce catabolic effects in cartilage, a direct effect on cartilage cannot be excluded. For instance, in Fig. S6 there is some degree of increase of Col2a1 when Denosumab is applied on chondrocytes. N=3 is really small and these experiments, in chondrocytes, may be underpowered particularly in monolayer cultures which, compared to micromass/ pellet cultures are much less responsive. Cartilage is a slow-reacting tissue and slow but persistent changes are likely to have a sizable effect in the context of osteoarthritis. I think that emphasizing the effect on synovitis is well-justified, but the authors have not convincingly excluded a direct effect on cartilage itself.\nAuthor response: We thank the reviewer for raising this professional point. However, we obviously did not make our explanation clear; the n value in our in vitro experiments was 3, but we repeated the experiment at least three times. We are very sorry for missing this important information from our initial manuscript, and we have now added this clarification at lines 386-387 in our revised manuscript.\nPellet cultures indeed provide a better model to study chondrogenesis. As recommended, we performed 3D-cultured chondrocyte pellet cultures and conducted Alcian blue staining on the sections. The results aligned with the reviewer’s expectations, revealing that RANKL exerted a modest but not dramatic influence on the pellets (Supplementary Fig. 7C). To further investigate this observation, we extended the duration of RANKL treatment of chondrocytes (48 h and 72 h) and performed western blot and PCR analyses. However, the data demonstrated no significant metabolic effects of RANKL under these conditions.\nOverall, we agree with the reviewer’s perspective that the direct effect of RANKL on chondrocytes is indeed subtle and slow-acting, in contrast to its more pronounced indirect effects. Therefore, we have maintained our focus on the indirect mechanisms in this study. As suggested, we have added a clarification at lines 579-583 of the revised manuscript and showed the results as follows:\nInterestingly, when we treated chondrocytes directly with RANKL and we found that RANKL induced modest but not significant catabolic effects on chondrocytes (Supplementary Fig. 7B–D). Therefore, we speculated that RANKL, and consequently denosumab, are more likely to influence chondrocytes in an indirect manner through FLSs.\nSupplementary Fig 7. Part C:\n(C) Alcian blue staining of sections from 3D-cultured chondrogenic cell pellets after culture in chondrogenic medium for 2 weeks with RANKL or denosumab. Scale bar = 100 µm.\n3. The “elephant in the room” here is the bone. Denosumab clearly limits bone remodeling and bone remodeling is a well-established driver of osteoarthritis. Consequently, a better characterization of bone remodeling is required it at least DMM, including bone density, BV/TV, thickness of the subchondral plate, osteoclast count, the size of the epiphysis, and ideally also rate of bone remodeling. This is imperative. In addition, a discussion point should be added, depending on the results, that albeit there are clear anti-inflammatory effects, there are also effects on bone remodeling (and direct effects on cartilage, although less intense) and that the interplay fo these components is likely to result on the efficacy of denosumab.\nAuthor response: We thank the reviewer for raising this professional point and making the conclusions of our paper more accurate. The pathology of OA is very complicated, our groups previously found that the whole joint is involved including cartilage1,2, subchondral bone3, and synovium4,5. As a result, there is a great deal of interplay between cells within those different tissues, which together cause OA progression. Basically, denosumab is mainly used to treat osteoporosis, and thus its effects on subchondral bone remodelling definitely cannot be ignored. We did find some changes in osteoclast function, revealed by micro-CT and TRAP staining, but focused on synovium according to our sequencing data.\nIn response, we utilized micro-CT to construct three-dimensional images of the subchondral bone in mice at 8 weeks post-DMM surgery. Based on these images, we performed quantitative analyses of bone volume fraction (BV/TV) and thickness of subchondral bone plates (SBP.Th). Additionally, we further conducted tartrate-resistant acid phosphatase (TRAP) staining to quantify osteoclast numbers, which allowed us to assess bone resorption activity and bone turnover rates. These results have been added to Fig. 2F–H and Supplementary Fig. 2A, B of the revised manuscript and also shown as below.\nIndeed, as you noted, denosumab treatment effectively suppressed osteoclast activation and abnormal subchondral bone remodelling in DMM models. However, the primary mechanistic focus of our study was on synovium, which is a novel therapeutic mechanism. While we did not perform an in-depth investigation into subchondral bone changes, we acknowledge that an overemphasis on synovium might lead to potential misunderstandings. We have now included a brief discussion at lines 802-810 and checked throughout the manuscript to better reflect that denosumab influences multiple joint tissues, with synovial modulation being the central focus of our mechanistic exploration.\nFig. 2F–H\n(F) Representative views of the anterior of the knee joints in the indicated mouse groups derived from micro-CT analysis at 8 weeks after DMM surgery. Red arrowheads indicate osteophyte formation. (G) Three-dimensional μCT images of the sagittal view of medial tibial subchondral bone at 8 weeks after DMM surgery. (H) Quantification of the grade of OA change and tibial subchondral bone: BV/TV, thickness of subchondral bone plate (SBP. Th). n = 6. (Grade of OA change: Sham versus DMM, P < 0.0001. DMM versus DMM + denosumab, P = 0.0044; BV/TV: Sham versus DMM, P < 0.0001. DMM versus DMM + denosumab, P = 0.0001; SBP. Th: Sham versus DMM, P < 0.0001. DMM versus DMM + denosumab, P = 0.0003).\nSupplementary Fig. 2A, B\n(A) Representative images showing TRAP staining of tibial subchondral bone at 8 weeks after DMM surgery. TRAP-positive cells are indicated by black-colored arrows. Scale bar = 100 µm. (B) Quantification of the number of osteoclasts in subchondral bone of mice indicated. n = 6. (Sham versus DMM, P < 0.0001. DMM versus DMM + denosumab, P = 0.0003).\n4. The Mankin score is ill-suited for murine OA because murine cartilage is much more cellular than human cartilage and most of the parameters of cellularity and cell shape do not really apply. For these reasons, people developed the OARSI score, which is the standard scoring system in the mouse. It would be best to rescore using the OARSI score to facilitate comparison with other studies of this type. Also, the picture of the Denosumab group at 8 weeks does not seem to be representative of the score in the graph. Please chose a representative picture i.e. one close to the median of the group. In fact, the picture shown is essentially normal cartilage, whereas in the graph, in the denosumab group, there is no data point that is normal. Is this correct?\nAuthor response: Thank you for your professional suggestions, we have re-scored all murine models using the OARSI score system throughout the revised manuscript. These results have been graphed and relevant content has been added to the MATERIALS AND METHOD section at lines 201-203.\nWe have replaced the safranin-O staining of the denosumab group at 8 weeks and re-scored using OARSI score. These results have been graphed and are also shown as below.\nFigure 2A\n(A) Safranin O/Fast green staining of knee joints in the indicated mouse groups at 4, 8 and 12 weeks after DMM surgery. Scale bar = 200 μm. The OA severity of knee joints was evaluated using the OARSI Score. n = 6. (4 W: Sham versus DMM, P = 0.0023. DMM versus DMM + denosumab, P = 0.4894; 8 W: Sham versus DMM, P = 0.0001. DMM versus DMM + denosumab, P = 0.0028; 12 W: Sham versus DMM, P < 0.0001. DMM versus DMM + denosumab, P = 0.0003).\n5. Fig. 2F the microCT of the DMM+denosumab group seems to be taken from the contralateral knee, judging from the shape of the tibia, unless it is a projection effect. Please check if correct. In addition, the ossicle that normally is present in the anterior horn of the medial meniscus is not present and also the entire patellar grove is not visible. Please check. Were the soft tissues dissected before microCT? Was the thresholding the same as the other images?\nAuthor response: Thank you for pointing out our mistakes. Corrections have been made in Fig. 2F in the revised manuscript and also shown as below. We did indeed remove some of the periarticular soft tissues prior to CT scanning. This step was taken to enhance the clarity of the joint structures in the subsequent CT reconstructions. Furthermore, in our comparative analysis of the sham, DMM, and DMM + denosumab groups, we applied identical parameter thresholds across all groups. This standardization was implemented to minimize the influence of residual soft tissue artefacts, ensuring that our findings are not confounded by unnecessary soft tissue shadows.\nFig. 2F–H\n(F) Representative views of the anterior of the knee joints in the indicated mouse groups derived from micro-CT analysis at 8 weeks after DMM surgery. Red arrowheads indicate osteophyte formation. (G) Three-dimensional μCT images of the sagittal view of medial tibial subchondral bone at 8 weeks after DMM surgery. (H) Quantification of the grade of OA change and tibial subchondral bone: BV/TV, thickness of subchondral bone plate (SBP. Th). n = 6. (Grade of OA change: Sham versus DMM, P < 0.0001. DMM versus DMM + denosumab, P = 0.0044; BV/TV: Sham versus DMM, P < 0.0001. DMM versus DMM + denosumab, P = 0.0001; SBP. Th: Sham versus DMM, P < 0.0001. DMM versus DMM + denosumab, P = 0.0003).\n6. Fig 2B-D and all figures about scoring of synovitis: the part behind the meniscus is the joint capsule. The synovium is limited to the tissue lining the joint where there is no cartilage (and no menisci). I do not think that the scores will change but please correct.\nAuthor response: Thank you for pointing out our mistakes. We have meticulously re-annotated the synovial tissue with dashed lines and subsequently re-assessed the scores.\n7. Fig 3C middle panel, there is a cutting artifact with rolling of the cartilage of the femoral condyle which makes it look thinner. Please chose a different section.\nAuthor response: Thank you for pointing out our mistakes. The apparent thinning of the femoral condyle cartilage in the middle panel of Fig. 3C is indeed due to a sectioning artefact, as noted. To address this concern, we have replaced the image and re-sored using OARSI score. These results have been graphed and also shown as below:\nFigure 3C\n(C) Safranin O/Fast green staining of knee joints in young (3-month-old), old (18-month-old) and old + denosumab mice. Scale bar = 200 μm. The OA severity of the knee joints was evaluated using the OARSI Score. n = 6. (OARSI Score: Young versus Old, P = 0.0001; Old versus Old + denosumab, P = 0.0034).\n8. Fig. 7. Cartilage histology and histological score of the dog model should be shown.\nAuthor response: We thank the reviewer for kindly pointing this out. We initially encountered some difficulties in decalcifying the canine joints due to their substantial size, which precluded us from performing paraffin sectioning at that time. Consequently, we only provided macroscopic images of the articular cartilage. However, we have since addressed this limitation by cutting hard tissue sections of the canine joints. Using these sections, we performed safranin-O staining to strengthen our conclusion, and these results have been graphed and added to Figure 7E, F of the revised manuscript. We also show the results below:\nFigure 7E–F\n(E) Representative Safranin O/Fast green staining of tibial plateau and H&E staining of synovium among Sham, ACLT and ACLT + denosumab dog groups. Magnified images of the boxed areas are shown in the panel below. Scale bar = 500 μm (upper image); 100 μm (lower image). (F) Mankin score, OARSI score and Synovitis score were quantified. n = 6. (Mankin score: Sham versus ACLT, P < 0.0001; ACLT versus ACLT + denosumab, P = 0.0167; OARSI score: Sham versus ACLT, P < 0.0001; ACLT versus ACLT + denosumab, P = 0.0026; Synovitis scores: Sham versus ACLT, P = 0.0003; ACLT versus ACLT + denosumab, P = 0.0014).\nMinor points and suggestions\n9. P5. all the refs proposed to support a role of inflammation in osteoarthritis progression, in reality only show association rather than causation. Synovial inflammation is a well-known driver of symptoms in osteoarthritis, but its role in cartilage breakdown is still debated.\nAuthor response: Thank you for your professional suggestions. We fully agree that OA is indeed a highly complex disease, and the causal relationship between synovial inflammation and cartilage damage has long been a subject of debate. While our in vitro experiments have demonstrated that inflammatory FLSs can induce catabolic effects in chondrocytes, the in vivo scenario is considerably more intricate. We checked the whole manuscript, and we now use a more accurate interpretation of the data, especially regarding the cause–effect interpretation which has been changed to a strong association. Moreover, as discussed in our manuscript, we plan to further investigate whether synovial inflammation can lead to cartilage damage by utilizing ColVI-Cre RANKLflox/flox or ColVI-Cre FSTL1flox/flox mice to conditionally knock out RANKL or FSTL1 in FLSs in a future study.\n10. ln 137-138. OA cartilage and SF was obtained from patients undergoing knee replacement. Presumably not the healthy cartilage and synovial membrane. Please state the origin of the healthy tissue.\nFigure 1 E;F, please state if the values are normalised by GAPDH. Also please make the legend more legible.\nAuthor response: Thank you for pointing out this omission. In our study, normal articular cartilage (AC) and synovial membrane (SM) samples were obtained from patients undergoing either thigh amputation or hemipelvectomy, while OA-affected AC and SM were collected from patients undergoing knee arthroplasty. We have now clarified this point in the revised manuscript at lines 411-414 to ensure accuracy.\nIn this study, all WB and PCR results were normalized to GAPDH as an internal control. We have added a detailed description of this at lines 309 and 329 in the MATERIALS AND METHOD section. Additionally, we have revised Figure 1E, F to improve the clarity of the figure legend and ensure that the data presentation is more intuitive and accessible.\n11. I would suggest stating the statistical test used for every panel in the figure legends.\nAuthor response: We thank the reviewer for kindly pointing this out. Related content has now been added in the figure legends.\n12. Fig S4 distribution of denosumab in vivo. This was achieved by injecting systemically Cy5-conjugated denosumab and looking at epifluorecence in the slides. It is difficult to assess the specificity of the fluorescence because cartilage and synovial membrane are highly auto-fluorescent. An appropriate negative control is essential. The staining, particularly in cartilage, appears to be patchy, which, in the absence of a convincing control, is suspicious.\nAuthor response: Thank you for your comments. To address your concerns, we have included the negative controls, which confirmed that neither synovium nor cartilage exhibited autofluorescence. Furthermore, we repeated systemic administration of Cy5-conjugated denosumab and observed that its distribution in cartilage remained patchy (show as below). We attribute this phenomenon primarily to the unique physicochemical properties of the cartilage extracellular matrix (ECM). Cartilage ECM is densely packed with highly negatively charged proteoglycans, which likely hinder homogeneous penetration of the antibody–drug conjugate6. Denosumab, as a large monoclonal antibody (~150 kDa), faces additional diffusion barriers due to size exclusion effects and electrostatic repulsion from the anionic glycosaminoglycan (GAG) chains in the ECM.\nSupplementary Fig 5\n(A) CY5.5 dye-labeled denosumab was subcutaneously injected into mice. After 1, 4 or 7 days, knee sections were scanned by confocal microscopy and synovium, cartilage area were emphatically observed as regions of interest.\n13. In figure S6 and S10 are important and convincing and in my opinion would deserve a place in the main figures.\nAuthor response: Thank you again for your professional suggestions. Accordingly, we have incorporated some of the results from Supplementary Fig. 6 and 10 into the Fig. 4 and 6.\n14. Fig 4. The labeling of the panels is wrong in the legend. There is no panel I and I believe F is western blot of TNF and MMP13, G is PCR, H is the representative images and I the synovitis score.\nAuthor response: Thank you for pointing out our mistakes. Accordingly, corrections have been made in the legend of Fig 4.\n15. Fig 4H-I and related text. The text mentions mild cartilage erosions in the mice treated with RANKL but not in Denosumab. I would suggest showing some images of that and quantify using OARSI score.\nAuthor response: We initially put the relevant images in Fig. S6F, but now we do think these images are important and convincing, as suggested by the reviewer. Therefore, we have added them into Fig. 4H–I and re-scored using OARSI score. They are also shown below.\nFig. 4H and I\n(H) Safranin O/Fast green staining of knee joints in all groups at 4 weeks after RANKL or RANKL + denosumab injection. Scale bar = 200 μm. (I) The OA severity of the knee joints was evaluated using the OARSI Score. n = 6. (Veh versus RANKL, P < 0.0001. RANKL versus RANKL + denosumab, P = 0.0012).\n16. Li 465-466 “synovial inflammation is an initial event in the development of OA\": I would replace “initial” with “critical”. This is because, in most patients, biomechanical factors on cartilage prime the pathological process, including synovial inflammation. The same applies for “initiation” in li 94.\nAuthor response: Thank you for your professional suggestions, we agree that the reviewer’s suggestions are more accurate and precise, so we have corrected the sentences at line 86 and 769, and we checked through the manuscript and made corresponding revisions.\n17. Li 502-503. I would recommend care when making statements such as “rodents are very different from humans” because they may be easily misconstrued as “models in rodents are not predictive of human disease” and used to argue against the use of rodents in science. After all this manuscript beautifully shows how instead very predictive they are! I would rephrase on the lines of “for confirmation in larger animals with a joint structure and load more similar to the human one….”\nAuthor response: Thank you for your professional suggestions, we totally agree that your proposed wording is more precise and logically rigorous. In response to your comment, we have corrected this sentence in the revised manuscript at lines 810-814 accordingly, incorporating your recommended changes to improve the clarity and accuracy of our presentation, as follows:\nHowever, different OA subtypes share different pathology and respond to treatments differently. Additionally, confirmation in larger animals is necessary, since their joint structures and loading conditions are more similar to humans.\nReviewer #2 (Remarks to the Author):\nIn this manuscript, the authors investigated the role of denosumab, a monoclonal antibody targeting RANKL, in three different murine models and in a dog model of osteoarthritis (OA). After confirming that RANKL and RANK were highly expressed in both murine and human joint tissues, particularly in the synovium (and mainly in fibroblast-like synoviocytes (FLSs)), denosumab was injected systemically in the murine and dog models. This resulted in a significant inhibition of synovitis. To uncover the underlying mechanisms, the authors performed scRNA-seq demonstrating that FLSs were the most significantly altered cell cluster having the most intense cell–cell interaction with chondrocytes. These phenomena were reversed by denosumab treatment. Further immunoprecipitation assay revealed that RANK–TRAF6 binding that activates NF-κB signaling led to the upregulation of catabolic and pro-inflammatory factors in FLSs. In subsequent analyes, the follistatin-like protein 1 (FSTL1) was identified as the most upregulated cytokine in FLSs, highly-likely being responsible for the catabolic and pro-inflammatory effects in chondrocytes. In addition, the authors performed a single-arm clinical trial using denosumab that similar to animal models reduced synovitis and OA-associated pain via the same FLSs/FSTL1 mechanism.\nThe topic is of course of great interest and highly relevant because still no disease-modifying drugs exist that effectively and causally slow down OA progression. The authors offer a large amount of information and have conducted relevant experiments at different levels. However, I have one substantial concern regarding the missing sham+denosumab control as explained in more detail below. Another major weakness in my opinion is the small sample size in the majority of experiments, see also below. In addition, I have a few minor comments, some of which concern the content or the scientific writing/symbols.\nMAJOR\n1. Missing sham+denosumab control: Without this control group, the OA-independent effects of denosumab can not be detected. Accordingly, no side effects are visible. The authors state in the manuscript (line 84) ‘NSAIDs can cause gastrointestinal and cardiovascular side effects’… denosumab has also side effects (e.g. https://www.prolia.com/), some of them are quite serious (cardiovascular, fatigue, bone remodeling, pain! among others).\nTherefore a further question arises: why was denosumab applied systemically? Did the authors check body weight of the animals, or their activity – e.g. denosumab might cause lethargy and lack of energy and consequently decreased activity that might be OA protective…\nAuthor response: We thank the reviewer for raising this professional point. We realize that this is a very important issue and always overlooked by us. We have dedicated a specific supplementary figure to comprehensively evaluate whether denosumab induced any adverse effects or behavioral alterations in mice. Given the 3-month revision timeline, we focused on monitoring body weight and mechanical pain sensitivity over the 8-week period following denosumab administration. In this study, we opted for subcutaneous administration of denosumab, as this is the recommended method according to the prescribing information, and local injection might lead to adverse reactions such as pain. Our results demonstrate that subcutaneous injection of denosumab did not elicit significant pain responses or notable changes in body weight. Furthermore, the open field test revealed that denosumab treatment did not affect spontaneous locomotor activity, nor did it induce anxiety-like behaviors in mice. To further assess potential systemic effects, we collected and histologically examined major organs (heart, liver, spleen, lungs, and kidneys) as well as knee joints after the 8-week treatment period. Results revealed no significant morphological changes in any of these tissues. Additionally, we have supplemented our findings with histological data from beagle dogs treated with denosumab for 2 months, which similarly showed no remarkable alterations in the examined organs.\nWe have included the relevant content in the revised manuscript at lines 176-184, 512-519 and 715-717. These results have been graphed and added to Supplementary Fig. 4 and Supplementary Fig. 12 of the revised manuscript, as below:\nSupplementary Fig 4. Physiological function analysis after denosumab administration in mice. (A) Body weight was measured at 0, 2, 4, 6 and 8 weeks after denosumab injection. n = 6. (Sham versus Sham + denosumab: 2 W: P = 0.3312; 4 W: P = 0.8296; 6 W: P = 0.5931; 8 W: P = 0.9957). (B) Pain analysis was performed using Von Frey assay according to paw withdrawal threshold (PWT) at 0, 2, 4, 6 and 8 weeks after denosumab injection. n = 6. (Sham versus Sham + denosumab: 2 W: P = 0.4069; 4 W: P = 0.8966; 6 W: P = 0.8160; 8 W: P = 0.7078). (C, D) Representative locomotion trajectory of different mice in the open-field at 4 and 8 weeks after denosumab injection. (E) Total distance travelled, walking speed and time duration in the central area were analyzed. n = 6. (Total distance travelled: 4 W, P = 0.4848. 8 W, P = 0.6991; walking speed: 4 W, P = 0.3939. 8 W, P = 0.8182; time duration in the central area: 4 W, P = 0.5887. 8 W, P = 0.8182). (F) Representative H&E staining images of heart, liver, spleen, lung and kidney isolated from mice after subcutaneous injection of vehicle or denosumab for 8 weeks. Scale bar = 100 μm. (G, H) Representative Safranin O/Fast green and H&E staining of knee joints after subcutaneous injection of vehicle or denosumab for 8 weeks. Scale bar = 100 μm. (I) OARSI scores and synovitis scores were measured. n = 6. (OARSI scores: P = 0.7560; Synovitis scores: P = 0.5490). (J) Hematology tests of liver and kidney functions of the mice following subcutaneous injection of vehicle or denosumab for 8 weeks. n = 6 per group. (ALT: P = 0.8984; AST: P = 0.5761; Urea: P = 0.8009; Crea: P = 0.6524). ALT = alanine transaminase; AST = aspartate transaminase; Urea = blood urea nitrogen; Crea = creatinine; Data are presented as median with interquartile range. Statistical analysis was performed using unpaired two-tailed t-test (A, B, E, I, J). n indicates the number of biologically independent samples, mice per group, or human specimens. ns = not significant.\nSupplementary Fig 12. Denosumab treatment does not affect vital internal organs. (A) Representative H&E staining images of heart, liver, spleen, lung and kidney isolated from beagle dogs after administration of denosumab for 2 months. Scale bar = 100 μm.\n2. Small sample size: Although the results seem to be heterogeneous – which surprised me anyway, especially in the animals models, I never obtained such low variability in my murine studies – a sample/group size of n=3 is very low when performing multiple statistical comparisons. This would be even more difficult when the missing sham+denosumab mice were included….\nAuthor response: Thank you for your insightful comment regarding the sample sizes in our figures. In our study, we consistently used 6 animals for each experimental group. However, in the immunohistochemical staining analysis, while we initially had 6 mice, we randomly selected 3 samples for immunohistochemical analysis, which is why the reported n-value was 3 in the original manuscript. To address this, we have now included the remaining mice in our analysis and updated the corresponding n-values in the revised manuscript. Additionally, in Figure 1D, we have incorporated three groups of synovial fluid samples from both normal individuals and OA patients. We have re-analyzed these samples using ELISA to ensure comprehensive and robust data representation.\n3. The authors talk about the MIA model in mice in the sections abstract, results (figure legends), and discussion…Then, in the section methods, the reader is surprised by the fact that rats were used for the MIA model...that is very confusing and therefore, I am not able to evaluate this part of the results.\nAuthor response: Thank you for pointing out our mistake and inconsistencies. In this study, we indeed used rats instead of mice for the MIA model, as the joint cavity volume in mice is too small, which could lead to instability in the modelling process. We sincerely apologize for the incorrect description in the manuscript, where we mistakenly referred to the animals as mice. This was an oversight on our part, and we have now corrected this error in the revised manuscript to accurately reflect the use of rats.\n4. Can the denosumab effect be sex-specific? Since almost only female patients were included (and females deal differently with pain…)?\nAuthor response: Thank you for your question. OA is more prevalent in the female gender than in males7. Women are known to be more susceptible to OA onset and development than men are8,9. Several studies suggest that this disparity may be linked to the plunge in sex hormone levels in menopausal women10,11. Additionally, compared to male OA patients, female patients often exhibit higher levels of joint inflammation and clinical pain, thinner articular cartilage, and more severe joint mobility problems8. While denosumab has historically been used for the treatment of osteoporosis in postmenopausal women, it was approved for use in male osteoporosis patients in 2023. Our study also indicates that denosumab has therapeutic effects in male OA patients, albeit in a small sample size. Consequently, we do not believe the treatment effects of denosumab to be sex-specific. However, large-scale clinical studies are necessary to substantiate these findings as the reviewer kindly pointed out, which is our plan for further research regarding this project.\n5. What about a denosumab effect on monocytes/macrophages, since they and their interaction with FLS were/was also strongly influenced, s. Fig.4. Moreover, RANKL induces macrophage polarization to the pro-inflammatory M1 phenotype. Wouldn´t it be worth/necessary to investigate this?\nAuthor response: We are very grateful to the reviewer for this incisive question. As you point out, denosumab is classically known for its role in inhibiting the monocyte/macrophage/osteoclast lineage, thereby treating osteoporosis12. In this study, we found that among all the clusters with increased cell proportions after DMM, FLSs showed the most dramatic decrease in cell proportion after denosumab treatment. In addition, the cellular interactions between FLSs and chondrocytes or macrophages are most prominent under OA conditions, which can be significantly inhibited by denosumab. Therefore, we focused on investigating the effects of denosumab on FLSs to alleviate synovial inflammation. As the reviewer rightly pointed out, the interaction between macrophages and FLSs is also influenced by denosumab, and macrophage polarization plays a significant role in synovial inflammation. We appreciate this insightful observation. Our preliminary data also suggest that denosumab directly inhibits macrophage senescence (as shown below). This will be the focus of our next study. However, in this work, we primarily aimed to elucidate the crosstalk between FLSs and macrophages or chondrocytes. Therefore, to further support our findings, as kindly suggested by the reviewer, we examined the effect of RANKL-CM on macrophage polarization. We treated macrophages with RANKL-CM and assessed the expression of M1 and M2 markers. By WB and immunofluorescence staining, we demonstrated that RANKL-CM significantly increased the expression of CD86, a marker of M1 macrophages, while suppressing the expression of ARG1, a marker of M2 macrophages. Importantly, these effects were also effectively reversed by denosumab. These findings further corroborate our hypothesis that RANKL-mediated FLS–macrophage crosstalk promotes a pro-inflammatory microenvironment in OA, which can be therapeutically targeted by denosumab. These results have been graphed and added to Fig. 4G and Supplementary Fig. 7F of the revised manuscript.\nWe also added the related contents at lines 594-598 in the revised manuscript.\nFig, 4G\n(G) Western blotting of Arg1 and CD86 in macrophages incubated with conditioned medium collected from FLSs treated with RANKL or RANKL + denosumab. n = 3. (Arg1: Veh versus RANKL-CM, P = 0.0332. RANKL-CM versus RANKL + denosumab-CM, P = 0.0160; CD86: Veh versus RANKL-CM, P < 0.0001. RANKL-CM versus RANKL + denosumab-CM, P = 0.0037).\nSupplementary Fig. 7F\n(F) Representative immunofluorescence images of Arg1 (red) and CD86 (green) in the indicated macrophages. Blue: DAPI showing total macrophages. Scale bar = 200 μm.\nMINOR\n6. Line 61 – redundancy with line 57-58\nAuthor response: Thank you for your comments. Accordingly, we have corrected this sentence at lines 56-59 as follows:\nMechanistically, cell–cell interaction analysis indicates enhanced FLS–chondrocyte crosstalk in OA, which is markedly reduced following denosumab treatment, suggesting FLSs as the primary responsive cells to denosumab in OA joints.\n7. Line 90: there will never be ONE central mechanism because OA pathogenesis is multifactorial, extremely complex.\nAuthor response: Thank you for your professional suggestion. Correction has been made at lines 82-83 as follows:\nDespite extensive research, the complex pathological mechanisms underlying the onset and development of OA remain unknown.\n8. Line 114 – 2x three\nAuthor response: Thank you for pointing out our mistake. Correction has been made at line 106 as follows:\nIn this work, we use three different murine OA models–post-traumatic, inflammatory and aged OA, and a beagle dog knee OA model to validate the effects of denosumab on synovitis and OA progression.\n9. Lines 138-139: healthy donors underwent arthroplasty???\nAuthor response: Thank you for pointing out our confusing statement. We sincerely apologize for the inaccuracies in our description. Normal articular cartilage (AC) and synovial membrane (SM) samples were obtained from patients undergoing either thigh amputation or hemipelvectomy, while OA-affected AC and SM were collected from patients undergoing knee arthroplasty. Accordingly, we have revised this sentence at lines 411-414 in the revised manuscript.\n10. Line 145 and the entire manuscript: IL-1β is the correct spelling\nAuthor response: Thank you for pointing out our mistakes and inconsistencies. Accordingly, corrections have been made throughout the revised manuscript.\n11. Line 147: ‘inflammatory chondrocytes’??? Chondrocytes were treated with IL-1β and are therefore in an inflammatory microenvironment, but they are not inflammatory per se…\nAuthor response: Thank you very much for your knowledgeable comment. We have corrected this sentence, which is highlighted in red in the revised manuscript at lines 421-424 as follows:\nIn line with the previous data, the expression of RANK and RANKL was markedly higher in IL-1β-induced chondrocytes and macrophages and especially in IL-1β-induced FLSs (Fig. 1G). PCR assay of RANK and RANKL in IL-1β-induced chondrocytes, macrophages and FLSs showed the same result (Fig. 1H).\n12. Gene names should be italic – ist RANK/RANKL correct, or should TNFRSF11A/ TNFSF11 be used?\nAuthor response: We thank the reviewer for kindly pointing this out. Accordingly, corrections have been made throughout the revised manuscript.\nWe have carefully reviewed the GeneCards database and acknowledge that both terminologies are indeed correct. However, considering that RANK/RANKL is more commonly used and widely recognized, we opted to use this terminology in our manuscript to ensure clarity and consistency with the majority of existing studies.\n13. Lines 200-201 and Fig S2: no, there is no Col2a1 increase after denosumab treatment. There is just no difference to Ctrl. Type II does not disappear anyway in DMM! Collagens are very stable. First, proteoglycans are degraded, so a proteoglycan loss is visible in OA cartilage. Maybe type II collagen also decreases in the defect area but not in the entire cartilage. I can not believe Fig, S2D Col2.\nAuthor response: Thank you for pointing out our mistakes. Accordingly, we have replaced the Col2a1 antibody (1:100, Abcam, ab34712) and performed the immunohistochemical staining again. Additionally, we have further supplemented the results with Aggrecan immunohistochemistry to provide a more comprehensive analysis. The revised results are presented in Supplementary Fig. 2F–G, and the corresponding antibody has been added to the MATERIALS AND METHOD section at line 209.\nSupplementary Fig. 2F–G\n(F) Immunostaining of Col2a1, Aggrecan, MMP-13 and ADAMTS5 in the articular cartilage of mice indicated. Scale bar = 50 μm. (G) The percentages of Col2a1+, Aggrecan+, MMP-13+ and ADAMTS5+ cells within the articular cartilage were quantified. n = 6. (Col2a1: Sham versus DMM, P < 0.0001. DMM versus DMM + denosumab, P = 0.0001; Aggrecan: Sham versus DMM, P = 0.0001. DMM versus DMM + denosumab, P = 0.0001; MMP-13: Sham versus DMM, P < 0.0001. DMM versus DMM + denosumab, P = 0.0001; ADAMTS5: Sham versus DMM, P = 0.0001. DMM versus DMM + denosumab, P = 0.0003).\n14. Lines 202-206 and Fig. 2G: Frey assay is an indirect measurement of pain sensitivity and does not directly evaluate OA-associated joint pain. So, the effect might be some ‘side effect’ of denosumab – which could be clarified using the sham+denosumab mice, see my major comment 1.\nAuthor response: We are very sorry that the way we expressed this finding is not accurate. We have corrected this sentence in the manuscript at line 488-489 as follows:\nMoreover, the von Frey assay revealed that the paws of DMM mice were extremely sensitive to the stimulus, whereas denosumab improved the paw withdrawal threshold (PWT) in the OA procession compared with DMM mice (Fig. 2I), suggesting that denosumab heightens mechanical pain sensitivity after DMM.\n15. The MIA model is often preferred when studying pain (quick and harsh). Why was pain not assessed in the MIA model?\nAuthor response: We thank the reviewer for raising this professional point. We are very sorry for the omission of these critical experiments in our initial submission. Accordingly, we have now conducted the experiments as suggested and included the relevant content in the revised manuscript at lines 499-501. These results have been graphed and added to Supplementary Fig. 3C, and are also shown below:\nSupplementary Fig. 3C\n(C) Pain analysis was performed using Von Frey assay according to paw withdrawal threshold (PWT) at 0, 1, 2, 3 and 4 weeks after MIA injection. n = 6.\n16. Lines 241-242: ‘suspension of 1.8 × 104 cells’ – such details are not needed in section results, it is a methodological detail.\nAuthor response: Thank you for your professional suggestions. We have removed this content and added it to the MATERIALS AND METHOD section at line 243.\n17. General to MMP and ADAMTS - WB and staining does not tell anything about enzyme activity - be careful with interpretation.\nAuthor response: Thank you again for your professional suggestions. We have carefully checked through the manuscript and made relevant corrections.\n18. Fig. 7 I: Huffa synovitis score – typo (Hoffa is correct)\nAuthor response: Thank you for pointing out the mistake. Correction has been made in the revised manuscript.\n19. The dogs are missing in the Methods/Animals section (Age, sex, number…).\nAuthor response: Thank you for pointing out this omission. Correction has been made in the revised manuscript at lines 122-127 in the MATERIALS AND METHOD section as follows:\nTwelve male dogs (beagle), with a mean body weight of 10.0 ± 1.5 kg, were purchased from Wuhan Xinzhou Wanqianjiahe Experimental Animal Breeding Farm Co. Ltd. and housed on the first floor of the Huazhong University of Science and Technology Tongji Medical College Laboratory Animal Centre in an open environment.\n20. The order of methods is not logical – does not follow the results order.\nAuthor response: Thank you for pointing this out. We have reorganized the MATERIALS AND METHODS section to match the sequence of the Results section.\n21. How was the synovitis scoring performed? There are different protocols published with measuring synovial thickness, quantifying infiltrating immune cells etc. The authors do not provide information on that.\nAuthor response: We are very sorry for our negligence in omitting this information. We performed synovitis scoring based on a histopathological classification system including hyperplasia/enlargement of synovial lining cell layer, activation of resident cells/synovial stroma and inflammatory infiltration13. Accordingly, we added the reference at line 206.\nReviewer #3 (Remarks to the Author):\nThis review only concerns the statistical part of the submitted work.\n1. There is no mention of how many mice or rats were used in the study. There were no mention of dogs in the Materials and Methods section but results were presented for them (line 400). 8 human patient were used in the study. Although a formal sample size calculation is not needed in a Phase I clinical trial, a justification of the numbers used is desirable.\nAuthor response: Thank you for pointing out our omissions. Corrections have been made in the revised manuscript at lines 116-127 in the MATERIALS AND METHOD section as follows:\nApproximately one hundred male C57BL/6J mice at 12 weeks old, twelve male C57BL/6J mice at 12 months old and eighteen male Sprague-Dawley (SD) rats (weight 200–225 g) were used as experimental animals which were purchased from Beijing SPF Biotechnology Co. Ltd. Twelve male dogs (beagle), with a mean body weight of 10.0 ± 1.5 kg, were purchased from Wuhan Xinzhou Wanqianjiahe Experimental Animal Breeding Farm Co. Ltd. and housed on the first floor of the Huazhong University of Science and Technology Tongji Medical College Laboratory Animal Centre in an open environment.\nInitially, we planned to recruit 30 patients (15 receiving placebo and 15 receiving denosumab) for this study. However, due to the impact of the COVID-19 pandemic in Wuhan and other factors, patient recruitment proved to be extremely challenging. Additionally, given that a randomized placebo-controlled phase 2a trial on denosumab for hand OA has already been reported14. So, we revised the study design to prioritize feasibility while maintaining scientific rigor. After enrolling 8 patients, we have now added one additional participant and transferred to a single-arm Phase I study using a 3+3 dose-escalation design. This approach aligns with established methodologies for early-phase clinical trials, balancing safety assessments with practical constraints. We have clarified these points in the manuscript at lines 346-348.\n2. In the Statistical Analysis section it is mentioned that 'groups were compared by t test. One-way ANOVA and two-way ANOVA were used for multiple comparisons'. The Results section should clearly present the results of group comparisons showing the test statistics, p-value and the degrees of freedom as appropriate.\nAuthor response: Thank you for your professional suggestions. We have annotated the corresponding data for all the Figures in the Figure legends.\n3. Footnotes of Figures 1, 2, 3, 4, 6 & 7 says that 'Data presented as mean +/- SD'. It was not clear what are these values in those Figures.\nAuthor response: Thank you for your comments. In our Figures, the mean values are represented by the bar height, and the standard deviations (±SD) are shown by the error bars.\n4. As the study sample sizes, be it animal or human, are very small, parametric tests such as t test and ANOVAs are not appropriate. Instead the authors should use non-parametric test. Also for descriptive statistics it is desirable to use median and range (or Inter Quartile Range, IQR) should be used instead of mean and SD.\nAuthor response: Thank you for your professional suggestions. In our animal experiments, each experiment was repeated at least three times and only data from a representative experiment are presented. Besides, all animals were housed under strictly standardized conditions (a 12-hour light/dark cycle, with free access to food and water), which significantly reduces inter-individual variability. We respectfully maintain that this approach aligns with widely-accepted practices in preclinical research, where n = 6 is commonly deemed sufficient for achieving statistical power when environmental and genetic variability are controlled. Additionally, our sample size is consistent with prior published studies in this field15-18. As rightly noted by the reviewer, our clinical trial is a small-scale single-arm Phase I study. Based on your suggestions, we have now represented the data as median with interquartile range (IQR) instead of mean ± SD and re-analyzed all the human data under the guidance of an expert statistician, using the Mann–Whitney U test or Kruskal–Wallis test with Tukey’s post hoc test for multiple comparisons and made corresponding revisions in the Statistical analysis section at lines 386-395.\n5. As the data were collected at baseline, 4, 8 and 12 weeks, it is desirable to use longitudinal analysis to see how the variables change over time. Instead comparisons were made with baseline values or between two time points. It is understandable that longitudinal analysis may not be feasible due to small sample size and therefore an acknowledgement of this would make the analyses, with baseline or between two time points, acceptable.\nAuthor response: Thank you for your professional suggestions. As you rightly noted, our sample size (n = 6) limited the statistical power required for robust longitudinal analysis. Our primary aim was to assess whether the intervention elicited significant changes relative to baseline, which could be adequately addressed through pairwise comparisons. While we acknowledge that this approach does not capture temporal dynamics, it provides a conservative and interpretable estimate of the effect of the intervention under the constraints of this study. As suggested, we have explicitly acknowledged this limitation in the revised manuscript at lines 878-884 as follows:\nAnother limitation of this study is the relatively small sample size, so we opted for pairwise comparisons between time points rather than longitudinal analysis, which introduces certain limitations and may lead to potential biases. Therefore, a larger, multicenter randomized controlled trial is needed to strengthen the evidence supporting the beneficial effect of denosumab in knee OA patents.\n6. Wherever a statement like 'did not differ significantly' such as in Line 129, 136 etc., please quote the p-value to justify the statement.\nAuthor response: Thank you for your professional suggestions. We have annotated the analyzed P-values for all the Figures in the Figure legends.\n7. In my opinion, the Methods and Materials section should be presented before the Results section.\nAuthor response: Thank you for pointing this out. Correction has been made in the revised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my comments satisfactorily.", "reviewer_2": "In general, the authors have put a great deal of effort into revising the manuscript and have successfully improved it. My main points of criticism were taken seriously and addressed appropriately; in most cases, new data were generated to support their responses.\nI would still like to request a minor revision: The authors did address the issue of sex specificity in the rebuttal letter. However, I would appreciate it if this point were briefly addressed in the discussion section as well. In contrast to the animal experiments — which included only male mice, rats, and Beagles — the clinical study involved exclusively female patients. This point should be addressed in 2-3 sentences (or limitation?).\nCongratulations to this manuscript.", "reviewer_3": "I am happy to say that I am satisfied with the authors' response to all my comments on the previous version of the manuscript. I am also satisfied with the revised manuscript. I have no further comments or issues with the revised manuscript."}, "author_response": "Thank you for your thorough review and constructive feedback. We appreciate your recognition of our work and the strengths of our study. In response, we have included a brief discussion at lines 598-609 as follows:\nAnd the study population was mainly female (89%), this is because that women are known to be more susceptible to OA onset and development than men are1. Additionally, compared to male OA patients, female patients often exhibit higher levels of joint inflammation and clinical pain, and more severe joint mobility problems2. While denosumab has historically been used for the treatment of osteoporosis in postmenopausal women, it was approved for use in male osteoporosis patients in 2023. Combined with our animal data, which included only male mice, rats, and beagles, we conclude that the efficacy of denosumab is not sex-specific. However, a large-scale clinical trial recruiting more male patients is needed to strengthen the evidence supporting the beneficial and sex-specific effects of denosumab in knee OA patents."}]

Decision letter