Organic di-selenide hydrogel microspheres for multimodal treatment of osteoarthritis

Domain: neuroscience
Published: 2026-02-03
Editorial outcome: minor_revision

Abstract
Osteoarthritis (OA) involves multiple pathological processes and presents significant clinical challenges in treatment. Traditional therapies focus on individual factors in cartilage, synovium, or subchondral bone, limiting their ability to comprehensively address OA pathogenesis. In this study, a ROS/MMP13 dual-responsive organic selenium hydrogel microsphere (HSPHR) is developed to trigger a localized microenvironmental response specific to early OA by exploiting the disease’s pathological features. Simultaneously, the organic selenium component effectively enhances selenoprotein levels in cartilage, synovium, and subchondral bone, enabling multimodal treatment for osteoarthritis. HSPHR injections into joints reduce cartilage damage, synovial hyperplasia, and bone sclerosis in post-traumatic OA, while promoting new cartilage in defect models. It enhances selenoprotein synthesis and activates the PI3K-AKT-mTOR pathway in key cells, improving mitochondrial function and antioxidant capacity, thus reversing OA-related changes. Here, we present a multimodal therapeutic strategy for OA lesions and reveal shared regulatory pathways among different cell types. This approach offers distinct insights for the multimodal treatment of degenerative joint diseases.

Introduction
Osteoarthritis (OA) is one of the most prevalent degenerative joint disorders, with increasing incidence and prevalence, particularly among the elderly population. The Global Burden of Disease Study 2021 reports a substantial rise in the global burden of OA from 1990 to 2021, with ~466.3 million new cases and a total prevalence of about 606.9 million cases in 20211,2. The pathogenesis of OA involves alterations across multiple joint structures. Cartilage matrix degradation, a hallmark of OA, is often driven by abnormal chondrocyte metabolism and overexpression of matrix-degrading enzymes, such as matrix metalloproteinases (MMPs) and members of the ADAMTS family3. Additionally, subchondral bone hyperplasia and sclerosis are central to OA progression. Changes in the subchondral bone microenvironment, including bone marrow edema and angiogenesis, have been shown to precede cartilage degeneration and are strongly linked to OA pain and progression4. Synovial hyperplasia, typically accompanied by low-grade inflammation, is another critical pathological feature of OA. Proliferation and fibrosis in the synovial tissue may be driven by various inflammatory factors and signaling pathways, with the role of CEMIP (KIAA1199) in synovial inflammation and fibrosis well-documented5. Moreover, chronic synovial inflammation can impact cartilage and subchondral bone health through the release of inflammatory mediators6. The pathomechanisms of OA are multifaceted, involving complex interactions among cartilage, subchondral bone, and synovium. A deeper understanding of these mechanisms is crucial for developing effective therapeutic approaches7,8.

Mitochondrial dysfunction and oxidative stress are key features of OA. Mitochondria, as the primary source of cellular energy, generate ATP via oxidative phosphorylation (OXPHOS). However, mitochondrial impairment leads to reduced ATP production and excessive reactive oxygen species (ROS) generation. This is associated with increased mitochondrial DNA (mtDNA) damage in chondrocytes from patients with OA compared to healthy individuals9. Additionally, mitochondrial dysfunction in synovial tissue correlates with an imbalance between M1 and M2 macrophages in OA. M1 macrophages, in particular, produce excessive ROS, contributing to mitochondrial dysfunction and sustained inflammation10. ROS also serve as secondary messengers, triggering the differentiation of hematopoietic stem cells or monocyte/macrophage progenitor cells into osteoclasts. Osteoclast activation and abnormal bone remodeling are central to OA pathogenesis11. Therefore, regulating ROS production and clearance while maintaining intracellular redox balance holds significant clinical potential for simultaneously addressing OA across multiple tissue types.

Selenoproteins, crucial antioxidants, are essential for maintaining the body’s redox balance12. Glutathione peroxidase (GPX) and thioredoxin reductase (TrxR) are two key selenoproteins that play a pivotal role in scavenging hydrogen peroxide and lipid peroxides, thereby protecting cells from oxidative damage13. In recent years, there has been growing interest in the use of selenium nanoparticles in orthopedics and OA. Exploiting their antioxidant properties, bioactive materials derived from selenium have been shown to enhance selenoprotein expression, reduce excess ROS in OA cartilage, and improve the local microenvironment, thereby promoting cartilage regeneration14–16. Despite these promising properties, the application of selenium nanoparticles in OA treatment remains limited, as their mechanisms of action are not fully understood, and effective strategies for coordinated management of multiple pathological tissues in OA are still lacking.

This study chemically conjugated selenocysteine using a thio-alkene click reaction to create a ROS/MMP13 dual-responsive organic bis-selenium hydrogel microsphere (HSPHR). This approach, designed to leverage the pathological characteristics of OA, enables early-stage, localized microenvironmental responses to the disease. At the same time, the organic selenium component replenishes selenoprotein levels in cartilage, synovium, and subperiosteal tissues. This not only provides multidimensional precision therapy for joint lesions but also offers deeper insights into the critical role of selenoproteins in OA pathogenesis. Intra-articular injection of HSPHR alleviated cartilage degeneration, improved synovial hyperplasia, and delayed subperiosteal bone sclerosis in traumatic OA models, while accelerating new cartilage formation in cartilage defect models. Compared to conventional selenium nanoparticles, this microsphere demonstrated enhanced antioxidant activity and prolonged retention in the joint cavity. Through multidimensional targeted intervention, HSPHR promotes cartilage repair and functional restoration. Mechanistically, HSPHR significantly upregulates TXNRD1 expression, activates the PI3K-AKT-mTOR signaling pathway, and shifts cellular energy metabolism, thereby restoring chondrocyte metabolic homeostasis. Notably, activation of the PI3K-AKT-mTOR pathway was also observed in synovial macrophages and osteoclasts treated with HSPHR. In summary, this study proposes a therapeutic strategy based on OA’s pathophysiological features to achieve synergistic treatment of multiple joint lesions, providing distinct insights for precision therapy in degenerative joint diseases.

Results
Role of oxidative stress imbalance mediated by selenoprotein in osteoarthritic cartilage damage
The selenoprotein family consists of proteins containing selenocysteine (Sec) residues, which play vital roles in antioxidant defense, immunomodulation, and toxin antagonism in vivo. Knee tissues from six patients, including both normal individuals and patients with OA, were analyzed for cartilage appearance and imaging. Patients with OA exhibited significant cartilage degradation and prominent osteophyte growth around the knee joint (Fig. 1A, B). Selenoprotein gene expression was measured in both normal and damaged cartilage via PCR, revealing the presence of Gpx1, Txnrd1, Txnrd2, Txnrd3, Sellnow, Sellnot, and Msrb1 in human chondrocytes. Notably, Gpx1 and Txnrd1 exhibited the most significant expression differences between normal and OA cartilage (Fig. 1C). Similar trends were observed in protein tissue levels from both normal and OA cartilage (Fig. 1D–F). Immunofluorescence analysis further demonstrated significantly lower protein levels of GPX1 and TXNRD1 in normal chondrocytes compared to OA chondrocytes, with a negative correlation to the pathological markers COL2 and MMP13 in OA (Fig. 1G, H). Histological assessments of patients with OA identified regions of relatively normal and damaged cartilage. In damaged regions, there was considerable loss of the cartilage matrix, and immunohistochemical analyses showed a marked decrease in GPX1 and TXNRD1 expression at these damaged sites. Similar observations were made in 14-week-old DMM rats (Fig. 1I–N). These results highlight a significant reduction in selenoproteins, particularly antioxidant proteins like GPX1 and TXNRD1, in OA-associated chondrocytes. The antioxidant function of selenoproteins is particularly crucial, as they maintain cellular redox balance by participating in redox reactions, thereby protecting cells from oxidative stress-induced damage. Moreover, selenoproteins play a key role in immune regulation, enhancing immune responses. A deficiency in selenoproteins is closely linked to the development of various metabolic disorders17,18. GPX1 serves as an antioxidant enzyme, protecting articular cartilage and slowing the progression of OA by scavenging ROS. TXNRD1, involved in cellular redox homeostasis and antioxidant defense, contributes to OA progression. Inhibition of TXNRD1 has been shown to prevent cartilage extracellular matrix degradation by activating the Nrf2 signaling pathway, thereby decelerating OA progression. The synthesis of selenoproteins in the body relies on selenium participation.

SeNPs counteract OA cartilage degeneration by reactivating selenoprotein-driven antioxidant pathways
The imbalance in the antioxidant capacity of chondrocytes is a key mechanism in the pathogenesis of OA. Selenoproteins, such as GPX and TXNRD1, play essential roles in cellular antioxidant pathways. A clear quantitative and qualitative association has been established between selenoprotein deficiency in cartilage tissues and OA progression. Selenium nanoparticles (SeNPs), developed based on prior studies19, were designed to directly replenish selenium in chondrocytes. Transmission electron microscopy revealed the structural morphology of SeNPs (Fig. 2A). The particle size was approximately 250 nm, consistent with dynamic light scattering measurements (Fig. 2C). Energy-dispersive X-ray (EDX) confirmed the uniform distribution of selenium on the SeNPs surface (Fig. 2B). SeNPs exhibited a Zeta potential of −11.7 mV (Fig. 2D). Cytotoxicity evaluations revealed that rat chondrocytes exposed to SeNPs at concentrations of 20 ng/mL and 50 ng/mL maintained over 97% viability, as indicated by live-dead cell staining (Fig. 2E). SeNPs at these concentrations were then administered to IL-1β-treated rat chondrocytes, resulting in upregulation of cartilage synthesis-related genes and modulation of chondrocyte degradation-related genes. This effect appeared to be linked to the concentration-dependent upregulation of Gpx1, Txnrd1, and Sephs1 genes in response to SeNPs (Fig. 2G). Immunofluorescence analysis further revealed a significant downregulation of selenoproteins in IL-1β-treated osteoarthritic chondrocytes, which was reversed by SeNPs treatment, restoring cartilage matrix metabolism (Fig. 2F, H, Supplementary Fig. 1). Additionally, SeNPs treatment increased the expression of phosphorylated selenoprotein synthase, with a concomitant dose-dependent increase in the expression of its downstream targets, GPX1 and TXNRD1 (Fig. 2I–K).

To further assess whether SeNPs exert therapeutic effects in OA by restoring intra-articular microenvironmental homeostasis, the impact of SeNPs on DMM was evaluated following intra-articular injection (Supplementary Fig. 2A). Micro-CT analysis revealed that SeNPs treatment increased the number of trabecular bones in the coronal plane and expanded the volume of subchondral bone in the tibial region in the sagittal plane. Trabecular parameters, including BV/TV, Tb/Th, and Tb/Sp, indicated improved subchondral bone heterotopia after the restoration of selenoprotein synthesis (Supplementary Fig. 2B–D). H&E staining showed that the inhomogeneous articular surface was improved in the SeNPs-treated group compared to the saline group. Furthermore, the number of arthroplasts was reduced, as demonstrated by S.O. staining, and the superficial cartilage layer was thicker, with a corresponding decrease in OARSI scores in the SeNPs-treated group (Supplementary Fig. 2E–H). Further analysis of cartilage matrix composition revealed that COL2 levels were restored to normal after SeNPs treatment, while the cartilage degradation marker MMP13 was significantly downregulated (Supplementary Fig. 2I–K). Additionally, this study determined whether SeNPs rescued cartilage loss by restoring selenoprotein synthesis in chondrocytes. The number of GPX1 and TXNRD1 positive cells in joint tissues treated with SeNPs was higher than in saline-treated DMM rats. This suggests that SeNPs supplementation effectively upregulated the selenoprotein levels in chondrocytes Supplementary Fig. 2L–N). SeNPs were employed for the treatment of DMM mice to further evaluate their impact on post-traumatic OA locomotor function and willingness to move (Supplementary Fig. 3). Spontaneous activity levels were recorded during a 3-min open field test (OFT). Compared to the sham-operated group, DMM operated mice exhibited significantly reduced activity levels, active time, active distance, and average speed. Treatment with 50 ng/mL SeNPs improved the mice’s willingness to move (Supplementary Fig. 3A–E). Furthermore, pain and gait status were assessed through footprint experiments, showing that SeNPs alleviated pain in DMM mice, improving stride and step length while shortening forefoot and hindfoot footprint lengths (Supplementary Fig. 3F–H). These results suggest that SeNPs may reverse the OA process by enhancing selenoprotein synthesis in OA chondrocytes, restoring metabolic homeostasis in the intra-articular microenvironment, slowing cartilage matrix degeneration, and ultimately restoring joint function. SeNPs have demonstrated efficacy in treating lumbar disc herniation, where SeNP supplementation promoted GPX1 protein synthesis in the nucleus pulposus cells (NPCs), maintained ECM homeostasis in NPCs, and significantly enhanced mitochondrial activity19. Despite the promising attributes of SeNPs, their long-term safety and biometabolism pose certain limitations20. Although these nanoparticles exhibit low toxicity in vivo, their narrow therapeutic window and precise toxicity thresholds limit their clinical applicability. As a result, several researchers have explored combining SeNPs with cartilage tissue engineering. For example, Hu W et al. developed OHA/HA-ADH@SeNPs hydrogels for the sustained release of SeNPs, targeting GPX1 activation15. Gao W et al. created Se/PRP-OGel hydrogels, utilizing zero-valent selenium nanoenzymes for radical scavenging while activating cartilage regeneration through PRP growth factors14. While these studies have broadened the scope of SeNPs applications, most have focused on the radical scavenging effects of the nanoparticles themselves, with limited exploration of the mechanisms through which selenoproteins regulate disease progression.

HSPHR facilitates the release of organic selenium in response to ROS/MMP13 stimuli
To enhance the in vivo bioavailability of elemental selenium, selenocysteine was grafted onto the hyaluronic acid (HA) chain via EDC/NHS-activated HA using thio-mercapto-ene click chemistry, resulting in the synthesis of a selenocysteine-rich selenium di-selenium bonded HA-Se-Se (HS) hydrogel (Fig. 3A). To improve the environmental responsiveness of HA-Se-Se, a MMP13 responsive short peptide (MRP, KCGPQG) was incorporated into the HS via an amidation reaction, forming the HSM hydrogel. FTIR analysis revealed a broad peak at 3287 cm–1, corresponding to the NH stretching vibration in HSM. By integrating methacryloyl glycoside (MA) modified HA (HAMA) with RGD peptide (RGDMA) and utilizing classical microfluidic techniques, complex hydrogel microspheres (HSPHR) were efficiently synthesized (Supplementary Fig. 4A), exhibiting uniform porosity with diameters ranging from 100 to 120 µm (Supplementary Fig. 4B). Scanning electron microscopy analysis revealed that the microspheres retained a loose porous structure during lyophilization, with a significant reduction in pore size compared to single-phase HAMA. The surface also exhibited modifications resembling short peptide-like structures (Fig. 3C). 77Se NMR analysis identified a single Se-Se peak at 290.23 ppm (Fig. 3B). Raman spectroscopy revealed a characteristic Se-Se metallic-like vibrational peak at 254 cm–1 (Fig. 3D), while FTIR analysis confirmed Se-Se peaks (Fig. 3E). XPS further detected characteristic Se peaks (Fig. 3F, G). Based on the distinct characteristics of Se-Se, which responds specifically to the ROS environment, and MRP, which is responsive to MMP13, the in vitro degradation and release of Se elements from HSPHR were evaluated in the presence of H2O2, the MMP13 enzyme, and a combination of both. The results indicated that HSPHR exhibited accelerated degradation when exposed to both H2O2 and the MMP13 enzyme, with the degradation process further expedited when both agents were present simultaneously (Fig. 3I, Supplementary Fig. 4C). These results suggest the presence of two distinct degradation mechanisms responsive to the microenvironment. The release efficiency of elemental selenium was significantly enhanced within the first 24 h upon the introduction of H2O2 and the MMP13 enzyme. This enhancement is likely due to the combined effects of ROS-induced oxidation, which disrupts the Se-Se bond, and MMP13-mediated cleavage of the MRP peptide (Fig. 3H). The environment-responsive burst release of organic selenium not only boosts its antioxidant capacity early on but also contributes to the depletion of MMP13 in the early OA environment, thereby fostering an optimal protochondral microenvironment during the maintenance period. To track the retention of HSPHR microspheres within the joint cavity, Cy5.5-labeled HSPHR microspheres were injected into the joint cavities of both normal and OA-affected rats. A sustained release over a two-week period was achieved in vivo, with significantly high fluorescence intensities confirming the environment-responsive performance of HSPHR (Fig. 3J, K). Fluorescence intensity measurements in various organs suggested that partially absorbed nanoparticles were metabolized through the liver (Supplementary Fig. 4D). In vitro antioxidant capacity testing revealed that HSPHR exhibited superior antioxidant efficacy compared to 50 ng/mL SeNPs alone (Fig. 3L–N). The onset and progression of OA are intricately linked to the dynamics of pathological factors, such as ROS, MMPs, and other components of the joint microenvironment. While moderate selenium supplementation can effectively restore intracellular protein metabolism and intra-articular redox homeostasis, it is important to note that the therapeutic window for selenium is quite narrow21. Se-Se bonds have been extensively reported to efficiently respond to ROS and exhibit distinct functional properties22–25. Building on this, a structure of selenium-diselenylated HA grafted with an MMP13-responsive peptide was developed, capable of achieving a focal dual response to ROS and MMP13, thereby intelligently facilitating selenium release. The release mechanism was achieved through a feedback loop of “environmental signaling-structural dissociation”, enabling adaptive regulation within the biological system. HSPHR demonstrated superior biosafety and enhanced in vitro antioxidant activity compared to conventional selenium supplementation methods.

HSPHR improve cartilage metabolic homeostasis in OA by restoring mitochondrial function regulation through TXNRD1-mediated PI3K-AKT-mTOR pathway
Following a 3-day incubation period, chondrocytes cultured on HSPHR exhibited significantly elevated levels of COL2 and notably reduced expression of MMP13 compared to those cultured on HAMA hydrogel microspheres (Supplementary Fig. 5A, B, E, F). Furthermore, the expression of GPX1 and TXNRD1 was significantly upregulated (Supplementary Fig. 5C, D, G, H). Following pretreatment with IL-1β, a comparative analysis of HR microspheres, 50 ng/mL SeNPs, and HSPHR microspheres was conducted to evaluate their efficacy in enhancing selenoprotein synthesis and reversing OA in vitro. Consistent with previous results, SeNPs effectively regulated selenoprotein synthesis and maintained chondrogenic metabolic homeostasis. However, chondrocytes co-cultured with HSPHR showed more pronounced improvements, with significantly increased expression of GPX1, TXNRD1, SEPHS1, and COL2 proteins, and marked downregulation of MMP13 protein synthesis (Supplementary Fig. 5I–N). To explore the molecular mechanisms underlying the protective effects of HSPHR on cartilage extracellular matrix homeostasis, RNA sequencing was performed on chondrocytes treated with HSPHR. Batch analysis identified 343 differentially expressed genes (DEGs), with 114 genes upregulated and 229 genes downregulated in response to HSPHR treatment (Supplementary Fig. 6A). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed alterations in chondrocyte matrix metabolism and the enrichment of osteoblast-associated pathways, which were strongly correlated with the PI3K-AKT signaling pathway. Additionally, pathway enrichment analysis suggested an inflammatory-protective role of HSPHR (Supplementary Fig. 5O, P). Proteomic analysis further confirmed the upregulation of TXNRD1 and GPX1 protein expression after HSPHR treatment (Supplementary Fig. 5Q). Gene Set Enrichment Analysis (GSEA) revealed significant enrichment of glycolysis-related genes in chondrocytes (Supplementary Fig. 6B).

We investigated the roles of the TXNRD1 and GPX1 proteins in cartilage biology. In the ATDC5 chondrocyte cell line, TXNRD1 and GPX1 were individually overexpressed via transfection with overexpression plasmids or silenced using small interfering RNA (siRNA). Chondrogenic induction assays revealed that TXNRD1 overexpression significantly enhanced chondrogenic efficiency in the presence of IL-1β (Supplementary Fig. 7A–D). Conversely, TXNRD1 silencing substantially impaired chondrogenic progression (Supplementary Fig. 7E–H). We propose that this observation is likely attributable to TXNRD1 overexpression promoting the scavenging of ROS in OA chondrocytes (Supplementary Fig. 7I–L). Therefore, TXNRD1 may play a more crucial role in cartilage protection. Consequently, this study examined the restoration of mitochondrial function through TXNRD1, a key protein that enhances cellular antioxidant capacity and regulates the PI3K-AKT-mTOR pathway26. To further investigate the downstream signaling events triggered by TXNRD1, TRi-1, a chemical inhibitor of TXNRD127, was employed. Under HSPHR treatment, TXNRD1 expression was significantly elevated, leading to the activation of the PI3K-AKT-mTOR pathway and increased phosphorylation of associated proteins. IL-1β treatment, however, significantly downregulated TXNRD1 expression and inhibited the phosphorylation of PI3K, AKT, and mTOR. Following HSPHR treatment, TXNRD1 expression returned to normal, restoring proper regulation of the PI3K-AKT-mTOR pathway. The addition of TRi-1 again suppressed this pathway (Supplementary Fig. 8A), indicating that HSPHR activates TXNRD1, which in turn initiates downstream PI3K-AKT-mTOR signaling. Further analysis of mitochondrial structural proteins, morphology, and function (Supplementary Fig. 8B, G–J) revealed that IL-1β significantly downregulated intrinsic mitochondrial proteins ATP5A, MT-ND4, COX 4, and TOMM20. HSPHR treatment restored these proteins to normal levels, while TRi-1 application reduced their expression. Similar trends were observed for mitochondrial membrane potential (MMP) (Supplementary Fig. 8D) and mitochondrial staining (Supplementary Fig. 8E). Transmission electron microscopy showed that IL-1β induced mitochondrial swelling and cristae loss, which was reversed by HSPHR treatment, while TRi-1 blocked this effect. Additionally, HSPHR restored the decreases in matrix synthetic proteins and reduced matrix degradative proteins induced by IL-1β, while enhancing the production of matrix synthetic proteins in chondrocytes. These results collectively demonstrate that HSPHR exerts its effect by upregulating TXNRD.

The regulatory role of HSPHR in the PI3K-AKT-mTOR signaling pathway was further explored using LY294002 (a PI3K phosphorylation inhibitor28) and 740Y-P (a PI3K phosphorylation activator29) (Fig. 4 and Supplementary Fig. 10). HSPHR-mediated activation of the PI3K-AKT-mTOR pathway restored the expression of mitochondrial-specific proteins in osteoarthritic chondrocytes (Fig. 4A, B, F–I, Supplementary Fig. 9A–F, K, L), reestablished mitochondrial morphology (Fig. 4D), and reversed the abnormal MMP (Fig. 4E; Supplementary Fig. 9M). These protective effects were similarly validated following 740Y-P administration (Supplementary Fig. 10A, B, D–R), with HSPHR demonstrating equivalent activation potential to 740Y-P. LY294002 treatment reversed these effects. Additionally, Seahorse extracellular flux analysis was employed to investigate the metabolic response of inflammatory chondrocytes to HSPHR, evaluating glycolytic stress states by real-time monitoring of the extracellular acidification rate (ECAR) following sequential glucose and 2-deoxy-d-glucose (2-DG) injections. Compared to controls, IL-1β-induced inflammatory chondrocytes exhibited significantly enhanced glycolytic activity and glycolytic reserve. However, in the HSPHR group, chondrocyte glycolytic parameters showed a progressive decline. Intervention with the PI3K inhibitor LY294002 significantly increased glycolytic reserve and accelerated mitochondrial damage (Fig. 4J, Supplementary Fig. 9Q, R). Similarly, sequential treatment effects of oligonucleotides, cyanocarbonamide, trifluoromethylbenzohydrazide (FCCP), and rotenone were recorded via real-time oxygen consumption rate (OCR) measurement. Cartilage cell OXPHOS parameters were assessed based on cellular mitochondrial stress detection (Fig. 4K, Supplementary Fig. 9N–P). Compared to the control group, the IL-1β group exhibited markedly reduced basal respiration, ATP production, and maximal respiration, while OXPHOS parameters progressively increased in the HSPHR group. Correspondingly, cellular OXPHOS significantly decreased after LY294002 intervention. These findings suggest that HSPHR incubation induces metabolic reprogramming in inflammatory chondrocytes, shifting energy production from glycolysis to OXPHOS, potentially linked to the activation of the chondrocyte PI3K-AKT-mTOR signaling pathway. Subsequent analysis of antioxidant capacity revealed that HSPHR treatment enhanced the clearance of excessive O2–, •OH, and other free radicals (Fig. 4L–O). Collectively, these results demonstrate that HSPHR therapy restores mitochondrial protein synthesis pathways, reestablishes mitochondrial OXPHOS, and exerts intracellular antioxidant effects, thereby mitigating cartilage damage.

HSPHR modulate the joint microenvironment, restoring bone metabolic homeostasis and macrophage polarization
In OA pathogenesis, the imbalance in the synovial microenvironment and abnormal bone metabolism are central factors. Synovial macrophages directly induce chondrocyte catabolism by secreting pro-inflammatory factors such as IL-1β and TNF-α, which not only upregulate MMP-13 and ADAMTS-5 expression but also activate osteoclast precursors, promoting their differentiation into mature osteoclasts. Overactivation of osteoclasts through the RANKL/RANK signaling pathway leads to abnormal subchondral bone resorption, disrupting the mechanical balance of the bone-cartilage unit and releasing bone-derived factors, thereby establishing a vicious cycle of “inflammation-bone destruction-bone remodeling.” Our study demonstrated that HSPHR treatment significantly impacted the inflammatory signaling pathways in chondrocytes and bone mineralization. To further investigate the regulatory effects of HSPHR on these two cell types, 50 ng/mL RANKL was initially used to induce osteoclast differentiation in RAW264.7 cells. HSPHR treatment effectively inhibited osteoclast activity and prevented further osteoblastic fusion of the cells (Supplementary Fig. 11A, B). Additionally, markers associated with osteoblastic activity, including CTSK, MMP9, and CD40L, were significantly reduced (Supplementary Fig. 11C, D). Evaluation of subchondral bone lesions in rats two weeks after DMM revealed excessive osteoclast activation in the DMM group at the two-week postoperative time point. HSPHR treatment effectively suppressed this process, restoring the bone density that was initially reduced (Supplementary Fig. 11E, F, I–L). The mechanism may involve the upregulation of selenoproteins within osteoclast precursor cells, accelerating the clearance of ROS generated by cartilage tissue, thereby inhibiting osteoclast activation in subchondral bone (Supplementary Fig. 11C, G, H). An alternative explanation is a direct effect of HSPHR on osteoclasts. Studies suggest that increased selenoprotein synthesis can directly influence osteoclast activity and reverse their activation process36. These findings highlight that HSPHR treatment significantly impacts inflammatory signaling pathways, bone mineralization, and metabolism, indicating its potential to restore bone metabolic homeostasis by inhibiting osteoclast formation.

Transcriptome sequencing was performed on osteoclasts treated with HSPHR to elucidate the mechanism by which HSPHR protects against abnormal subchondral bone ossification. The analysis identified 3202 DEGs. Notably, upregulation of the Pik3cg gene indicated activation of the PI3K-AKT-mTOR signaling pathway (Fig. 5A). KEGG and GSEA enrichment analyses further highlighted the critical role of the PI3K-AKT pathway (Fig. 5B, C). Selective activation and inhibition of the PI3K-AKT-mTOR pathway in osteoclasts using 740Y-P and LY294002 demonstrated that pathway inactivation effectively promoted osteoclast activation (Fig. 5D–G, I–L), while pathway activation inhibited osteoclast activation (Fig. 5H, M, Supplementary Fig. 12). Additionally, the regulatory effect of HSPHR on synovial macrophages was investigated. Flow cytometry (FCA) revealed significant changes in macrophage polarization following HSPHR treatment, with a shift from the M1 to the M2 phenotype (Supplementary Fig. 13A–D). This was further confirmed by immunofluorescence co-staining (Supplementary Fig. 13E). Transcriptome sequencing of HSPHR-treated M1 macrophages revealed significant enrichment of the PI3K-AKT pathway as well (Supplementary Fig. 14A–C). Selective activation and inhibition of the PI3K-AKT-mTOR pathway in macrophages using 740Y-P and LY294002, respectively, showed that inactivation of this pathway exacerbated the inflammatory state of macrophages (Supplementary Fig. 14D–Q), while pathway activation promoted anti-inflammatory differentiation (Supplementary Fig. 15A–N).

HSPHR enables full-cycle treatment of OA pathology, promotes cartilage regeneration, and slows the progression of OA
To evaluate the effect of HSPHR on delaying the progression of OA in vivo, a medial meniscus DMM-induced OA model was established (Fig. 6A). Histological analysis showed cartilage erosion and destruction in the saline-treated DMM group eight weeks post-surgery. Injection of HR microspheres alone partially restored glycosaminoglycan levels. The therapeutic efficacy was further enhanced by the precise and efficient delivery of HSPHR (Fig. 6B–E). HSPHR exhibited dual responsiveness to ROS and MMP13 in the early stages of OA. This intelligent function promoted COL2 synthesis while effectively inhibiting MMP13 levels (Fig. 6F–H). Additionally, HSPHR enhanced the endogenous synthesis and expression of GPX1 and TXNRD1 proteins (Fig. 6I–K), which was associated with downstream activation of the PI3K-AKT pathway and induction of mitochondrial autophagy (Fig. 6L–N). The impact of HSPHR on synovial inflammation following DMM surgery was also assessed. HSPHR treatment significantly reduced synovial inflammation, downregulated the immune-regulation marker INOS, and increased IL-10 secretion in DMM rats (Supplementary Fig. 13F–H, K, L). These findings may be attributed to the upregulation of selenoproteins in inflamed synovial tissue (Supplementary Fig. 13I, J, M, N). Motor function in DMM mice treated with HSPHR was evaluated next. The results showed significantly reduced relative activity levels, active time, active distance, and average velocity in DMM mice compared to the sham-operated group. HSPHR treatment reversed these trends (Supplementary Fig. 16A–E), improving stride and step length while reducing forefoot and hindfoot footprint dimensions (Supplementary Fig. 16J, K). Micro-CT analysis demonstrated that HSPHR effectively prevented the development of subchondral bone sclerosis (Supplementary Fig. 16F–H). These findings indicate that HSPHR has the capacity to address OA through a multidimensional approach, restoring metabolic homeostasis within cartilage mechanisms.

To further investigate the beneficial effects of HSPHR on cartilage regeneration, an in vivo model of full-thickness cartilage defects was developed. Chondrogenic progenitor cells (CPCs) were incorporated into HSPHR microspheres, following our established treatment protocol for advanced cartilage defects41. Gross examination revealed limited regenerative capacity in both the 4-week and 8-week post-operative saline-treated groups, as evidenced by the presence of cavities and irregular margins (Supplementary Fig. 17B, H). The enhanced protochondral formation potential of HSPHR facilitated accelerated cartilage formation, attributed to its unique cell-loading properties, which allowed controlled selenium release in response to injury localization. This process resulted in optimal tissue integration and the development of hyaline-like cartilage morphology (Supplementary Fig. 17C, I). In contrast, saline treatment failed to regenerate collagen for the extracellular matrix components of the newly formed cartilage. However, HSPHR treatment successfully restored the natural hyaline cartilage-like ECM, as demonstrated by increased expression of COL2 and suppression of the fibrocartilage marker COL1 (Supplementary Fig. 17E–G, K–M). Mechanistically, HSPHR effectively modulates chondrocyte energy metabolism through the PI3K-AKT-mTOR pathway, restoring mitochondrial function and promoting cartilage regeneration. Following HSPHR treatment, no significant abnormalities were observed in vital organs (heart, liver, spleen, lungs, and kidneys) (Supplementary Fig. 18A) or blood biochemical parameters (Supplementary Fig. 18B, C). In the early stages of OA, inflammatory dysregulation leads to an imbalance in the joint microenvironment and excessive osteoclast activation. In advanced stages, irreversible cartilage destruction, periosteal bone remodeling, and abnormal osteoblast differentiation occur.

Methods
SeNPs preparation
SeNPs were synthesized via microbial reduction using sodium selenite (Na2SeO3) as the precursor46. Saccharomyces cerevisiae was first inoculated into a yeast extract-peptone-dextrose (YPD) medium, containing 1% yeast extract, 2% peptone, and 2% glucose, and incubated in a shaker at 30°C for 12 h to activate the culture. Following this, 10% of the culture was transferred to a shaker containing 30 mL of medium supplemented with 100 g/L mother liquor and incubated for another 12 h. Sodium selenite was then added to achieve a final concentration of 2 g/L in the medium. Distilled water was added, and the resulting precipitate was homogenized. The mixture was incubated at 30°C for 48 h, followed by autolysis at 40°C for 24 h. Finally, the SeNPs were isolated using ultrasonic disruption and subsequent centrifugation.

Preparation of HSP
To prepare a solution of HA, 100 mg of HA was dissolved in an appropriate volume of water and sonicated for 30 min. The solution was then dialyzed in 0.001 M dilute hydrochloric acid for 4 h to achieve acidification. The acidified HA was subsequently dissolved in dimethyl sulfoxide (DMSO), with the addition of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for activation, allowing the reaction to proceed for 6 h. Selenocystamine and an MMP13-responsive peptide (KCGPQG) were then dissolved in the activated HA solution, maintaining a weight ratio of selenocystamine, HA, and short peptides at 2:2:1. The mixture was sonicated for 30 min and allowed to react for 12 h to facilitate the formation of diselenide bonds. The resulting DMSO solution was placed in a dialysis bag with an appropriate molecular weight cutoff and subjected to aqueous dialysis, followed by lyophilization for 48 h to obtain the hyaluronan monomer containing diselenide bonds.

Preparation of HAMA
The synthesis of HAMA was conducted following previously established protocols. Briefly, 4 mL of methacrylic anhydride (MA) was gradually added to 100 mL of a 2 wt.% HA solution. Subsequently, 5 M NaOH was incrementally introduced up to 4 mL using a micro-syringe pump, and the reaction was allowed to proceed overnight under ice bath conditions. After completion, the reaction mixture was dialyzed against ultrapure water for three days using a dialysis membrane with a molecular weight cutoff of 3500 Da to remove residual reactants. The purified product was then freeze-dried and stored at −20°C for future use.

Preparation of microspheres
The three-way connector was interfaced with two independent microfluidic syringe pumps via hoses. The external phase, designated as the continuous phase, was used to carry the internal phase, which consisted of a mixture of HAMA, HSP, and RGD-MA (Engineering For Life, China) solution, including a photoinitiator, serving as the dispersed phase. Two types of microspheres were synthesized: HAMA microspheres and composite HSR microspheres, with the latter comprising HAMA, HSP, and RGD-MA in a mass ratio of 5:5:1. The aqueous and oil phases were sequentially introduced into the microfluidic device inlet at a flow rate ratio of 15:500, facilitating a photo-crosslinking reaction under UV irradiation to solidify the microsphere structure. The collected microspheres were then washed repeatedly with 75% ethanol to remove any residues of the continuous phase. Following this, the microspheres were transferred to a freshly prepared PBS buffer for 4 h, a process repeated three times to ensure thorough removal of residual additives. To create a porous microsphere structure, the purified microspheres were first subjected to freeze treatment at −80°C for 4 h, followed by lyophilization in a freeze-dryer for 48 h. After drying, the microspheres were stored in a freeze-dryer.

Physical characterization of the microspheres
The morphology and dimensions of the microspheres were examined and documented using optical microscopy (CX23, Olympus, Japan). Particle size was measured using ImageJ-win64 software, followed by statistical analysis. The lyophilized microspheres underwent gold sputter coating, and their structures were analyzed using scanning electron microscopy (SEM, SU8100, Hitachi, Japan). Pore sizes on the microsphere surfaces were quantified using ImageJ-win64. Comprehensive analysis of the structural properties of the synthesized microspheres and nanoparticles was conducted using Fourier Transform Infrared Spectroscopy (FTIR, Thermo Fisher Scientific, USA). Samples were prepared with potassium bromide pellets and scanned 128 times at a resolution of 4 cm–1 across a spectral range of 400–4000 cm–1. The surface elemental composition of the microspheres was analyzed using Energy Dispersive Spectroscopy (EDS, EDAX, USA). Changes in the surface characteristics of the microspheres and nanoparticles were assessed using X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Thermo Fisher Scientific, USA). The experimental parameters were carefully configured: the vacuum within the analysis chamber was maintained at 10–9 mBar, with Al Kα radiation (hv=1486.68 eV) used as the excitation source. The X-ray spot diameter was set to 500 μm, and the operational voltage was set at 15 kV, with a filament current of 10 mA. The signal accumulation spanned 5 to 10 cycles, and the energy pass band was set to 30 eV with an incremental step width of 0.05 eV. Data analysis was performed through peak fitting using Avantage software, with the C1s binding energy standard value of 284.80 eV as the reference point for charge correction.

Antioxidant capacity of the microsphere
The antioxidant capacity of the microspheres was evaluated by assessing the scavenging activity against DPPH radicals, O2-• radicals, and •OH radicals. Microspheres from each experimental group were homogenized using a tissue grinder. A 100 μM DPPH solution was prepared, and various samples were mixed and dispersed in 2.0 mL of ethanol, with PBS as the blank control. The mixture was incubated for 30 min at 37°C in the absence of light. Absorbance was measured at 515 nm using a UV-Vis spectrophotometer.
Next, 100 μL of either HSRHR microsphere or SeNPs solution was combined with 100 μL of a 100 μM ABTS solution. After thorough mixing, absorbance was measured at 734 nm to evaluate ABTS radical scavenging activity.
The scavenging ability of the microspheres against hydroxyl radicals (•OH) generated through the Fenton reaction was assessed by mixing 100 mg of the sample with 500 μL of a 1 mM FeSO4 solution and 500 μL of a 100 mM H2O2 solution. The mixture was incubated at 37°C for 1 h. After cooling to 25°C, 100 μL of the supernatant was combined with 100 μL of a 10 mM 3,3′,5,5′-tetramethylbenzidine (TMB) solution (dissolved in DMSO). Absorbance was measured at 536 nm using a microplate reader.

Degradation of microspheres
For microsphere degradation, microspheres containing an equivalent mass were weighed and placed into a 6-well plate. These were incubated with 3 mL of a composite solution consisting of MMP13 protein (5 units/mL), 0.1% H2O2, and a combination of MMP13 protein with 0.1% H2O2. The mixture was incubated at 37°C with shaking at 120 rpm. At predetermined time intervals, the microspheres were collected, examined, and photographed to assess their morphology using an optical microscope. After observation, the microspheres were washed multiple times with sterile deionized water, lyophilized, and weighed. The degradation rate was calculated using the following formula:
DP (%) = (W0 − Wt) / W0 × 100%
Where W0 is the weight of the initial lyophilized sample and Wt is the weight of the lyophilized sample at time t.

Drug release experiments
Microspheres containing an equivalent mass of the drug were weighed and placed into a 6-well plate. These were incubated with 3 mL of a composite solution consisting of MMP13 protein (5 units/mL), 0.1% H2O2, and a combination of MMP13 protein with 0.1% H2O2. The concentration of organic valence selenium was measured at predetermined intervals (6, 12, 18, and 24 h, as well as 1, 3, 5, 7, 14, and 21 days) by extracting 1.5 mL of the sample solution, which was then supplemented with an equal volume of buffer and added back to the plate. Supernatants were analyzed spectrophotometrically at 490 nm using a Bio-Tek spectrophotometer (Winooski, VT, USA). A standard curve for selenium radical concentration was generated under identical conditions, and the drug concentration was determined from the obtained absorbance values.

Human cartilage samples assay
Articular cartilage specimens were obtained from patients undergoing total knee arthroplasty, with informed consent. The study was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University (Approval No. 2025066). A total of six patients participated, with detailed information available in Supplementary Table 1. OA was diagnosed based on radiographic evaluation of the lower limbs. Cartilage samples were categorized into relatively normal (N) and OA sections according to the extent of tissue damage. For subsequent protein and RNA extraction, the cartilage samples were rapidly frozen in liquid nitrogen and stored at −80°C.

Histological analysis
Human cartilage tissue and rat knee joints were fixed in 10% neutral formalin for 24 h and decalcified in an EDTA solution (pH 7.4) for 4 weeks. After decalcification, the tissues were embedded in paraffin, and the resulting wax blocks were sectioned into 5 µm serial sections for Safranin O/fast green staining. Sections were collected starting from the point where the medial meniscus began to detach, continuing until the meniscus was no longer visible, yielding approximately 50 sections per sample. Cartilage destruction was evaluated by two independent observers under blinded conditions using the Osteoarthritis Research Society International (OARSI) scoring system (grades 0–6) on Safranin O/fast green-stained sections.

Live/Dead cell staining assay
Chondrocytes were treated with SeNPs at concentrations of 25 and 50 ng/mL in 24-well plates. Following treatment, Live/Dead cell staining was performed using reagents from Thermo Fisher Scientific, USA (Waltham, MA, USA) for 20 min. Viable cells were stained green, while non-viable cells were stained red. Fluorescence microscopy (Zeiss, Oberkochen, Germany) was employed to capture images of the stained cells.

Fluorescence measurement of mitochondrial morphology
Mitochondrial detection in chondrocytes was performed using Mito-Tracker Red (Beyotime, China). Prior to analysis, cells were stained with 200 nM MitoTracker Red for 15 min. Mitochondrial morphology was then observed using an orthogonal fluorescence microscope.

Mitochondrial membrane potential (MMP) level
The MMP was assessed using the Mitochondrial Membrane Potential Assay Kit with JC-1, obtained from Beyotime, China. Chondrocytes were exposed to 0.5 µM JC-1 working solution for 30 min at 37°C, in the dark, followed by incubation with cold JC-1 staining buffer. Fluorescent images were captured using a fluorescence microscope, and the MMP value was determined by calculating the ratio of red fluorescence intensity (representing JC-1 aggregates) to green fluorescence intensity (representing JC-1 monomers) using ImageJ-win64 software.

Metabolic assays using the XFe96 extracellular flux analyzer
ECAR and OCR measurements were conducted using a Seahorse XFe96 Extracellular Flux Analyzer and a Seahorse XF Glycolytic Rate Assay Kit (Agilent Technologies), following the manufacturer’s instructions47. Briefly, 2 × 105 cells per well were plated in XF96 Cell Culture Microplates coated with Cell-Tak. The cells were then incubated in XF RPMI medium (without phenol red) supplemented with 2 mM glutamine, 10 mM glucose, 1 mM pyruvate, and 5 mM HEPES for 45 min at 37°C (non-CO2 incubator) prior to the assay. ECAR and OCR were measured at the basal stage (basal glycolysis + mitochondrial acidification), in response to Rot/AA (mitochondrial electron transport chain inhibitors; compensatory glycolysis) and 2-deoxy-D-glucose (a glucose analog; post-2-DG acidification). The basal and compensatory glycolytic rates were calculated using the Seahorse Glycolytic Rate Assay.

TEM images of mitochondria in chondrocytes
Cell samples were fixed with a 2.5% glutaraldehyde solution (Sigma-Aldrich, USA) in phosphate buffer for 4 h. After rinsing with phosphate buffer, the samples were treated with 1% osmium tetroxide solution (OsO4, Sigma-Aldrich, USA) in phosphate buffer for 2 h. The samples were then immersed in ethanol at varying concentrations for 15 min. Following this, the samples underwent treatment with a mixture of embedding agent and acetone (Sigma-Aldrich, USA) for 3 h. The permeated samples were embedded and subjected to overnight heating at 70°C to facilitate embedding. The embedded samples were sectioned using an ultra-thin microtome, and the sections were stained with uranyl acetate and alkaline lead citrate (Sigma-Aldrich, USA) for 10 min each. Finally, the sections were examined using an H-7650 transmission electron microscope (TEM) (Hitachi, Japan).

Cell culture
To isolate and culture articular cartilage cells, rats were euthanized and sterilized by immersion in 75% ethanol. The skin over the knee joint was incised using sterile instruments to aseptically extract the articular cartilage tissue. The tissue blocks were minced and repeatedly rinsed with a 10% penicillin-streptomycin solution (Gibco, USA). Subsequently, the tissue was washed with sterile phosphate-buffered saline (PBS, RG-CE-10, KETU, China) and digested overnight with 0.2% type II collagenase (Sigma-Aldrich, USA) at 37°C in an incubator. The next day, undigested tissue was removed by filtration through a cell sieve, and the digested solution was centrifuged, resuspended, and cultured in 100 mm cell culture dishes (704202, NEST Biotechnology, China) to obtain P0 generation rat chondrocytes. These cells were cultured in complete medium (DMEM/F-12, Keygen BioTECH, China) supplemented with 10% fetal bovine serum (FBS, F103, Vazyme Biotech Co., Ltd, China), penicillin (100 units/mL), and streptomycin (100 units/mL) at 37°C in a 5% CO2 incubator. Subsequent experiments were conducted using P1 generation rat chondrocytes. For the isolation and culture of bone marrow-derived macrophages (BMMs), six-week-old male SD rats were euthanized by cervical dislocation following anesthesia. After euthanasia, the rats were sterilized with 75% ethanol and placed on a sterile glass plate. The femur and tibia were meticulously isolated, and the bones, immersed in PBS, were transferred to a sterile workbench. The metaphyses of the femur and tibia were excised to expose the medullary cavities. The diaphyseal cavities were then flushed with a complete medium containing 30 ng/mL macrophage colony-stimulating factor (M-CSF, CB34, Novoprotein, China) using a 10 mL sterile syringe and a 20-gauge needle. This process was repeated until the medullary cavities appeared white. The resulting cell suspension was treated with red blood cell lysate, followed by cell lysis and centrifugation. The isolated cells were cultured in a 10 cm petri dish for 16 h. The supernatant was then transferred and treated with M-CSF (30 ng/mL) for three days. For further experimentation, BMMs were cultured with M-CSF (30 ng/mL) and receptor activator of nuclear factor kappa-B ligand (RANKL, 50 ng/mL).

Cellular intervention
Chondrocytes were inoculated at an initial density of 3000 cells/cm². To simulate the arthritic microenvironment in vitro, interleukin-1 beta (IL-1β) at a concentration of 10 ng/mL was used for co-culturing with the cells. Each group of microspheres was thoroughly washed and sterilized using 75% ethanol before being co-cultured with the chondrocytes. A control group was also established, with the culture medium replaced every two days for a total of seven days. To inhibit TXNRD1 activity, TRi-1 is present at a concentration of 12 nM in the cell culture medium48. To inhibit the phosphorylation of PI3K, LY294002 was maintained at a concentration of 0.5 μM in the culture media of chondrocytes, macrophages, and osteoclasts49. To activate the phosphorylation process of PI3K, the concentration of 740Y-P in the culture medium for chondrocytes, macrophages, and osteoclasts was set to 3 μM29.

Cell transfection
ATDC-5 cells were seeded into 6-well plates (5 × 10⁵ cells/well) and cultured until 80–90% confluency. Interventions were performed as follows: TXNRD1 overexpression, GPX1 overexpression, si-TXNRD1, and si-GPX1 (Anzhenbio, China) corresponding plasmids and siRNA sequences are listed in Supplementary Tables 4, 5. For overexpression groups, following Lipofectamine 3000 instructions, dissolve 2 μg of corresponding overexpression plasmid and 5 μL reagent in serum-free medium, incubate, and mix to form a complex. For knockdown groups, transfect 50 nM corresponding siRNA using Lipofectamine RNAiMAX. The complex was added dropwise to the cell culture system and incubated at 37°C with 5% CO2. Transfection efficiency was validated by Western blot analysis 48 h post-transfection.

Induction of Cartilage Formation
ATDC-5 mouse chondrocytes (CL-0856, Pronase Life Technology, China). A 100 μL suspension containing 400,000 cells was pipetted into the center of each well of a 24-well cell culture plate. Cells were cultured at 37°C in a 5% CO2 environment for 2 h, then transferred to medium containing 1% insulin-transferrin-selenium, 10 ng/ml transforming growth factor-β3, 100 nM dexamethasone, 50 μg/ml vitamin C, 1 mM sodium pyruvate, and 40 μg/ml proline. Cultures continued for 7 days with concurrent IL-1β and HSPHR treatment. The culture supernatant was discarded, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 20 min, stained with A.B. and S.O., observed under an optical microscope, and images were captured. Quantitative analysis was performed using ImageJ-win64 software.

Trap stain assay
To facilitate in vitro osteoclastogenesis, Raw264.7 cells were seeded at a density of 2 × 104 cells per well in 48-well plates. The cells were then incubated for five days in the presence or absence of macrophage colony-stimulating factor (M-CSF) at a concentration of 30 ng/mL and receptor activator of nuclear factor kappa-B ligand (RANKL) at 50 ng/mL. Tartrate-resistant acid phosphatase (TRAP) staining was performed following the manufacturer’s protocol. After fixation with 4% paraformaldehyde for 20 min, staining solution I was applied, and the cells were incubated at 37°C for 30 min in the dark. Images were captured and stored using an inverted microscope (Carl Zeiss, Germany).

Macrophage polarization detection
Raw264.7 cells (CL-0190, Pronase Life Technology, China), at a concentration of 2 × 104 cells/mL, were cultured in the presence of microspheres. Following a 24-h treatment with lipopolysaccharide (LPS) at 10 ng/mL and interferon-gamma (IFN-γ) at 20 ng/mL, the cells were harvested. The cells were then stained with antibodies specific to F4/80, CD86, and the mannose receptor (CD206), sourced from eBioscience, USA. After washing with PBS, the expression levels of F4/80, CD86, and CD206 were analyzed using FCA with the Guava® easyCyte system (Merck Millipore, Germany).

Western blot assay
For protein extraction, cells treated with the respective interventions were lysed using RIPA buffer (Beyotime, China). The supernatant was collected after ultracentrifugation to yield the protein sample, and the concentration was determined using a BCA kit (Beyotime, China). Protein samples were mixed with protein loading buffer (Beyotime, China) at a 4:1 ratio and boiled at 100°C for 5 min. SDS-PAGE electrophoresis was performed using a 4–12% gel (GenScript, China), and the transferred nitrocellulose membrane was incubated in blocking solution (Beyotime, China) for 30 min at room temperature. After blocking, the membrane was incubated overnight at 4°C with the appropriate primary antibodies (diluted 1:2000). The antibodies used included anti-COL2 (ab188570, Abcam, USA), anti-ACAN (A11691, Abclonal, China), anti-MMP13 (ab39012, Abcam, USA), anti-ADAMTS5 (A2836, Abclonal, China), anti-SEPHS1 (A11435, Abclonal, China), anti-GPX1 (A0968, Abclonal, China), anti-TXNRD1 (A20805, Abclonal, China), anti-ATP5A (A0207, Abclonal, China), anti-TOMM20 (A0208, Abclonal, China), anti-LC3b (A19664, Abclonal, China), anti-MT-ND4 (A11247, Abclonal, China), anti-CTSK (A1782, Abclonal, China), anti-MMP9 (A0289, Abclonal, China), anti-CD40L (A0327, Abclonal, China), anti-CD86 (A1199, Abclonal, China), anti-CD206 (A26948, Abclonal, China), anti-ARG1 (A1847, Abclonal, China), anti-NOS2 (A3774, Abclonal, China), anti-PI3K (A22730, Abclonal, China), anti-AKT (A18675, Abclonal, China), anti-P-PI3K (4228, Cell Signaling Technology, USA), anti-P-AKT (4060, Cell Signaling Technology, USA), anti-P-mTOR (5536, Cell Signaling Technology, USA) and anti-β-Actin (AC038, Abclonal, China). The next day, primary antibodies were removed, and the membrane was incubated with an HRP-conjugated secondary antibody solution (diluted 1:10,000, Abclonal, China) specific to the corresponding species for 1 h at room temperature. The membrane was washed three times for 5 min each with the washing solution. Ultrasensitive Enhanced Chemiluminescent (ECL) substrate (NCM Biological Technology Co. Ltd, China) was prepared and incubated with the membrane for 5 min at room temperature, followed by exposure using a VersaDoc™ imaging system (Bio-Rad, USA). The intensities of the bands were quantified using ImageJ-win64 software for statistical analysis.

Quantitative real-time PCR analysis
Chondrocytes cultured in six-well plates were subjected to intervention treatment, and RNA was extracted by lysing the cells with Trizol reagent (Sigma-Aldrich, USA). The RNA concentration and purity were assessed using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, USA). Complementary DNA (cDNA) was then synthesized using a reverse transcription kit (Thermo Fisher Scientific, USA). The synthesized cDNA was mixed with SYBR Green PCR Master Mix (TaKaRa, Kusatsu, Japan), specific primers, and DEPC-treated water to establish the reaction system. Gene expression analysis was performed using the CFX96 real-time PCR system (Bio-Rad, USA), with Gapdh as the reference control. Primer sequences are detailed in Supplementary Tables 2 and 3. The relative expression levels of target genes were calculated using the 2−ΔΔCt method.

Immunofluorescence staining
Chondrocytes were co-cultured with microsphere groups and treated with IL-1β. After treatment, the samples were fixed with 4% paraformaldehyde (Servicebio, China) for 30 min, followed by a 30-min incubation with an immunostaining blocking solution (Beyotime, China). The primary antibody working solution (1:200) was prepared, and incubation with the primary antibody occurred overnight at 4°C. The next day, the primary antibody was removed, and the cells were incubated with the Alexa 488 or Alexa 594 fluorescent secondary antibody (1:500, Abcam, USA) at room temperature for 1 h. After washing with PBS, the nuclei were stained with DAPI (Sigma, USA). Imaging was performed using a Zeiss Axiovert 40 CFL microscope, and all steps were conducted under light-protected conditions.

RNA sequencing
To evaluate differential gene expression, rat chondrocytes were exposed to IL-1β (10 ng/mL) alone or in combination with HSPHR microspheres. RAW264.7 cells were exposed to RANKL (50 ng/mL) or co-exposed with HSPHR microspheres. Alternatively, RAW264.7 cells were exposed to LPS (100 ng/mL) + IFN-γ (20 ng/mL) or co-exposed with HSPHR microspheres. The resulting samples were submitted to Wekemo Tech Group Co., Ltd. (Shenzhen, China) for comprehensive RNA sequencing analysis using the Affymetrix GeneChip microarray platform (Affymetrix, Santa Clara, CA, USA). RNA expression levels were also assessed with an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Differential gene expression was analyzed with Microarray software, and genes with a fold change greater than 2 or less than 0.05 were identified as DEGs. Differential expression analysis between three 30°C and two 4°C samples was performed using the R (v. 4.0.3) package DESeq2 (v. 1.30.0). GSEA was performed with GSEA (v. 4.1.0) using the GSEAPreranked tool, whereby genes were preranked on the basis of their P values and fold changes.

Animals and Ethics
The experimental subjects comprised SPF-grade SD rats and C57BL/6J mice. All husbandry practices and experimental procedures were conducted in strict accordance with the guidelines for laboratory animal welfare and ethics, receiving approval from the Animal Experiment Management Committee of Soochow University (SUDA20241210A8). The SD rats and C57BL/6J mice were housed within a SPF barrier environment, maintained at a temperature of 22±2°C, with 50±10% relative humidity, and subjected to a 12-h light-dark cycle. The animals were accommodated in individually ventilated cages, with 3–5 animals per cage, and were provided with sterile bedding, SPF-grade feed, and acidified sterile water available ad libitum. Health assessments were performed daily to ensure the well-being of the animals.

Gait analysis
Prior to tissue collection, gait analysis was performed on all mice. The forepaws were marked with red pigment, and the hind paws with green pigment. A white recording sheet was placed along a 50 cm long, 10 cm wide, and 10 cm high runway. A black enclosure, with an opening facing the recording sheet, was positioned at the end of the runway to encourage the mice to enter. Each mouse was placed individually at the starting point of the runway and allowed to traverse the path freely, without any external interference. Upon completion of the experiment, consecutive, clearly distinguishable footprints were selected from the recording sheet for subsequent statistical analysis.

Open field test
After the DMM model was successfully developed up to week 8, spontaneous activity and exploratory behavior were evaluated using the VisuTrack system (Shanghai XinRuan Information Technology, China). Mice were placed in a 50 × 3 × 50 cm enclosed arena under dim lighting conditions. A video camera recorded their movement for 3 min, allowing assessment of relative mobility, activity duration, distance traveled, and average speed.

In vivo imaging systems (IVIS) imaging
The degradation of glycopeptide hydrogels within the joint cavity of mice was assessed using the IVIS in vivo imaging system (Spectrum, PerkinElmer). Mice were anesthetized with isopentane before the injection of 100 μL of Cy5.5-labeled microspheres (10 mg/mL) into the joint cavity. Imaging sessions were conducted on days 1, 3, 5, 7, and 14, with anesthesia maintained using the same protocol. The rats were carefully positioned on the imaging chamber stage, and the testing temperature was kept at 37°C during each exposure. Exposure duration and imaging parameters were optimized for visualization and applied consistently across all sessions. Kinetic imaging curves were generated after all imaging sessions. The region of interest (ROI) was delineated as the joint cavity area in each mouse. The IVIS imaging system (Xenogen) was used to integrate bioluminescence signals, and quantification of these signals determined the average radiance. The resulting ROI photon flux was used as an indicator of hydrogel retention within the joint cavity.

Micro-CT
At eight weeks postoperatively, the rats were euthanized, and knee specimens were collected for further analysis. These specimens were fixed in a 4% paraformaldehyde solution (Servicebio, China) for 48 h. Following fixation, the specimens were scanned and analyzed using a high-resolution Micro-CT system (SkyScan 1176, Aartselaar, Belgium). Initial scans were performed at 60 kV, 170 mA, and 0.7° rotation with a resolution of 18 μm, maintaining consistent scanning parameters across all samples. The scanned data were reconstructed and subjected to three-dimensional quantitative analysis using NRecon, Data Viewer, CTAn, and Mimics software. Key metrics, including bone volume to tissue volume ratio (BV/TV), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th), were evaluated to assess the extent of OA.

Histological staining
After fixation, specimens were decalcified using 10% EDTA (pH 7.4, Sigma-Aldrich, USA) over approximately two months. Post-decalcification, the samples underwent dehydration and were embedded in paraffin (Leica, Germany). Tissue blocks were sectioned into 6 μm slices. These sections were deparaffinized in xylene, dehydrated through a graded ethanol series, and stained with hematoxylin and eosin, Safranin O/fast green, and toluidine blue. Microscopic images were captured using a light microscope (CX23). Cartilage degeneration was evaluated using the OARSI scoring system and the ratio of hyaline cartilage (HC) to calcified cartilage (CC). Cartilage defect repair was assessed using the International Cartilage Repair Society (ICRS) and the Modified O’Driscoll histological (MODS) scoring systems. Additionally, sections of embedded rat visceral specimens were prepared to evaluate the in vivo toxicity of the materials, with hematoxylin and eosin staining used for assessment.

Immunohistological staining and immunohistofluorescence
Sections underwent deparaffinization in xylene and dehydration through a graded ethanol series. They were then incubated with 3% hydrogen peroxide for 15 min and subjected to antigen retrieval using 2 mg/mL hyaluronidase (Sigma-Aldrich, USA) at 37°C. The sections were blocked with 1.5% goat serum and incubated overnight with the primary antibody at a dilution of 1:200 at 4°C. The next day, the primary antibodies were removed, and the sections were incubated with HRP-conjugated secondary antibodies at a dilution of 1:500 for 1 h at room temperature. The sections were then stained using the 3,3′-diaminobenzidine (DAB) working solution (Vector Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin. For immunofluorescence staining, the sections were incubated with Alexa 488 or Alexa 594 fluorescent secondary antibodies at a dilution of 1:500 for 1 h at room temperature, protected from light, and counterstained with DAPI. Imaging was performed using a Zeiss Axiovert 40 CFL microscope. The percentage of positive cells was quantified using ImageJ-win64 software.

DMM model
Eight-week-old male SD rats were used as the experimental model. All microsurgical instruments were rigorously sterilized prior to the start of the experiment. Anesthesia was induced via intraperitoneal injection of 1% sodium pentobarbital at a dose of 400 μL per 100 g of body weight. Following anesthesia, the patellar region was shaved and disinfected, and a sterile surgical drape was applied. A longitudinal incision was made along the medial side of the patella, followed by sequential dissection of the skin, subcutaneous tissue, and joint capsule. The patella was then displaced laterally to expose the medial condyles of the femur and tibia, as well as the meniscal structures. The adipose tissue beneath the patella was carefully blunt-dissected, and the medial anterior tibial ligament was transected cautiously to avoid damaging the cartilage. Subsequently, the meniscus was dislocated from its original stable position, and the surrounding tissues were sutured at multiple layers. In the control group, the joint capsule was incised and then closed without further manipulation. Postoperatively, the surgical site was thoroughly cleaned and disinfected, followed by intramuscular administration of penicillin to prevent infection. One week after arthroplasty, the animals were randomly assigned to experimental groups and received intra-articular injections according to the treatment protocol. At 8 weeks post-surgery, the rats were euthanized, and their knee joints were harvested for subsequent analysis.

Cartilage defect model
Eight-week-old male SD rats were used for the experimental procedures. All microsurgical instruments were strictly sterilized prior to surgery. Anesthesia was induced via intraperitoneal injection of 1% sodium pentobarbital at a dose of 0.4 mL per 100 g of body weight. After confirming anesthesia and ensuring stabilization, the rats were placed in a supine position. The surgical site was shaved, with particular attention to the knee joint, followed by local disinfection and sterile draping. A longitudinal incision was made along the medial side of the patella using a sterile scalpel, and sequential incisions were performed through the subcutaneous tissue and joint capsule. The patella was laterally dislocated using hemostatic forceps to expose the femoral condyle and trochanter. Thereafter, a full-thickness cartilage defect measuring 1.5 mm in diameter and 1.5 mm in depth was created on the femoral trochlear groove using an electric drill. The surgical site was irrigated with saline to remove debris, after which the patella was repositioned, and the joint cavity was closed by suturing the tissue layers sequentially. In the sham-operated group, only exposure and closure of the joint cavity were performed without creating a cartilage defect. After completing the surgical procedures, local disinfection was carried out, and penicillin (200,000 U/kg) was administered intramuscularly to prevent infection. Once the rats had recovered from anesthesia, they were returned to a controlled environment for postoperative care. One week after surgery, rats with full-thickness articular cartilage defects were randomly assigned to one of four treatment groups (excluding the sham group) and received intra-articular injections of 200 μL PBS, HAMA (2 mg/mL), or HSPHR (2 mg/mL). At 8 weeks post-surgery, the rats were euthanized, and their knee joints were harvested for further analysis.

In vivo toxicity
To assess the safety profile of HAMA and HSR microspheres, rat serum samples were collected following intra-articular injection. Total protein (TP), albumin (ALB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine (Cre) concentrations were measured using standard assay kits according to the manufacturers’ instructions. Histological examination of cardiac, hepatic, splenic, pulmonary, and renal tissues was then conducted using H&E staining.

Statistical analysis
Data are presented as mean ± standard deviation (SD). To evaluate differences between groups, Student’s t test was used for comparisons between two groups, and one-way ANOVA was used for multiple group comparisons. A p value < 0.05 was considered statistically significant. All data analysis was performed using GraphPad Prism 10.1.2 software.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The technology reported here is interesting, although the novelty is difficult to evaluate. In this manuscript, the authors constructed a ROS/MMP13 dual-responsive organoselenium hydrogel microsphere (HSPHR), which can significantly upregulate the expression of TXNRD1, activate the PI3K-AKT-mTOR signaling pathway, and enhance mitophagy and mitochondrial function. The aim is to promote cartilage repair and functional reconstruction through multidimensional targeted intervention. We appreciate the authors' efforts; however, there are some questions and concerns that arose during the review of the manuscript, which are listed as follows:\n1. The authors have not provided evidence to confirm the successful preparation of Se NPs as opposed to other possible substances. Notably, they conducted both in vitro and in vivo studies using these materials without first performing any material characterization experiments to verify the identity and properties of the Se NPs.\n2. The font size in Figures 4I-J is too small, which hinders effective extraction of information from the images. It is recommended that the authors increase the font size to improve readability and clarity.\n3. On page 18, line 7, the authors state that \"HSPHR exhibited accelerated degradation when exposed to both H₂O₂ and the MMP13 enzyme.\" However, in DMM mice, the degradation rate of HSPHR appears to be slower.\n4. In Figure 6A, the specificity of the PI3K protein bands appears to be suboptimal. Furthermore, the consistency of the AKT and P-AKT results raises doubts as to whether these proteins were detected on the same membrane. Please provide the Western data with whole membrane and shown with marker ladder.\n5. In Figures 5–6, a group without IL-1β treatment should be included as a blank control to verify the successful induction of inflammation by IL-1β. The absence of such a control group makes it difficult to determine whether the observed effects are specifically due to IL-1β-induced inflammation.\n6. Several aspects of Figure 7 require clarification:\na. The S.O. staining of cartilage in the DMM group appears to be more intense, whereas the staining in the HSPHR group is unexpectedly lighter. The authors should explain the reasons for these observations.\nb. The immunohistochemical staining results for TXNRD1 and GPX1 in the DMM group are inconsistent with those of other groups, with marked differences in nuclear staining intensity. This raises concerns about the experimental procedures and comparability among groups.\nc. Previous studies (Bone Res. 2019 Aug 5;7:23. doi: 10.1038/s41413-019-0062-y) have demonstrated that AKT signaling is activated during OA. However, in this study, the level of p-AKT in DMM mice is significantly decreased. The authors should provide a rationale for this discrepancy.\nd. The number of biological replicates per group (n=4) is insufficient to provide robust evidence that HSPHR alleviates OA progression.\n7. The naming of the control groups in Figure 6D is inconsistent between the representative images and the corresponding quantitative data.\n8. On page 21, line 8, the authors mention that \"HSPHR hydrogel microspheres improve cartilage metabolic...\". However, the manuscript does not provide sufficient evidence to clarify whether TXNRD1 restores mitochondrial function via the PI3K-AKT-mTOR pathway, and the causality between these factors is ambiguous. To strengthen the mechanistic interpretation, the authors should consider assessing mitochondrial function and/or the expression of relevant proteins following activation or inhibition of the PI3K-AKT-mTOR pathway. This would provide more direct evidence for the involvement of this signaling axis in the observed effects.", "reviewer_2": "The manuscript presents a novel synthesis of a ROS/MMP13 dual-responsive organic double selenium hydrogel microsphere to realize early OA local microenvironmental response by utilizing the pathological characteristics of osteoarthritis, and at the same time, the organic selenium can effectively supplement the selenoprotein level in the cartilage, synovium and subchondral bone. Intra-articular injection of HSPHR not only reduced cartilage degeneration, improved synovial hyperplasia, and delayed sclerosis of the subchondral bone in the post-traumatic OA model, but also accelerated the formation of new cartilage in the cartilage defect model. While the study is innovative and holds significant potential, I would like to provide the following constructive suggestions to enhance the scientific rigor and presentation clarity of this manuscript before it can be considered for publication.\n1.The \"Introduction\" section is too long. Authors should focus only on the most important aspects for a good understanding of the developed work.\n2. The authors propose that their \"three birds with one stone\" modified organic diselenide hydrogel microspheres can simultaneously treat multiple aspects of osteoarthritis. However, this conceptual framework requires clarification for several reasons:\nWe recommend either:\na) Providing direct experimental evidence for triple-targeting efficacy, or\nb) Modifying the conceptual framework to better reflect the actual therapeutic mechanisms observed.\nThis would significantly strengthen the manuscript's mechanistic claims while maintaining scientific rigor. The current presentation risks overinterpretation of the technology's targeting capabilities.\n3. While the abstract states that organic selenium supplementation restores selenoprotein levels across multiple joint tissues (cartilage, synovium, and subchondral bone), the study primarily demonstrates mechanistic effects in cartilage without providing selenium quantification data for synovium or subchondral bone. This limitation should be acknowledged, as it affects the interpretation of the compound's proposed multi-tissue targeting capability.\n4. While the authors showed HSPHR's inhibitory effect on osteoclast formation in vitro in Supplementary Figure 6, the in vivo study only evaluated late-stage OA pathology (8 weeks post-operation). Since osteoclasts are known to mediate early-stage OA pathogenesis, could the authors address why osteoclast activity was not monitored at earlier time points? This information would strengthen the mechanistic connection between HSPHR's cellular effects and its therapeutic outcomes.\n5. The Graphic Abstract does not clearly convey the \"three-in-one\" concept, nor does it show the microspheres' impact on other cells.\n6. To further explore the advantageous effects of HSPHR on cartilage regeneration, the authors developed an in vivo model featuring a full-thickness cartilage defect. Chondrogenic progenitor cells were incorporated into microspheres of HSPHR in order to enhance the therapeutic outcomes for cartilage repair. The study primarily elucidates the role of selenoproteins (particularly GPX1 and TXNRD1) in pathological cartilage, as demonstrated in both the main text and schematic diagrams. However, the relationship between selenoproteins and chondrocyte differentiation remains unclear. Several critical questions need to be addressed:\na) Molecular Mechanisms: Are GPX1 and TXNRD1 similarly involved in chondrocyte differentiation and cartilage homeostasis? What specific selenoproteins regulate chondrogenic differentiation pathways?\nb) Experimental Evidence: The study would benefit from in vitro experiments using:\n1) Chondrocyte differentiation models;\n2) Selenoprotein-specific knockdown/overexpression systems;\n3) Comprehensive analysis of redox regulation during differentiation.\n7. In Figure 4, the authors report solid-state NMR characterization of HA-Se-Se, but the presented spectrum shows only a single peak with no observable selenium signal. This raises several technical and interpretational concerns that need to be addressed.\n8. While the manuscript investigates selenium-based therapy for OA, several critical concerns regarding its novelty and mechanistic depth need to be addressed:\nInnovation Gap:\nThe therapeutic use of selenium nanoparticles in OA has been extensively reported (e.g., studies by DOI: 10.1016/j.mtbio.2023.100864; DOI: 10.1016/j.biomaterials.2025.123195; DOI: 10.1016/j.jcis.2025.137612).\nThe current work must better differentiate itself by:\nSelenoprotein Analysis Limitations:\nThe cartilage selenoprotein expression profiling GPX1/TXNRD1 largely replicates prior work without advancing the field.\nMechanistic Shortcomings:\nWhich specific selenoproteins mediate the therapeutic effects in cartilage;\nHow their regulation differs across joint tissues (synovium vs. cartilage vs. bone);\nThe downstream pathways linking selenoproteins to chondroprotection.", "reviewer_3": "This study developed ROS/MMP13-responsive selenium microspheres to target OA pathology. HSPHR restores selenoproteins and activates PI3K-AKT-mTOR-mediated mitophagy, mitigating cartilage degeneration, synovitis, and subchondral sclerosis in post-traumatic OA models while enhancing regeneration in cartilage defects. While the work addresses an important clinical issue, it falls short in several critical areas, particularly regarding novelty and experimental depth. As such, the current version of the manuscript is not suitable for publication in Nature Communications.\n1. The abstact and the introduction should be rewritten, the novelty of the work is not highlighted. What does \"Three birds with one stone\" mean in the title? It needs to be clearly stated in the introduction.\n2. The graphical abstract fails to effectively convey the \"Three birds with one stone\" concept and requires redesign to explicitly highlight the innovative therapeutic strategy of the HSPHR system.\n3. The article uses a great many abbreviations, some of which are not explained at all (e.g. CEMIP, HSR, DMM, Ser, PTEN, WB, etc.). The abbreviations of all text are very confusing. Full names should be given when an abbreviation first appears in the text. Additionally, the manuscript contains redundant abbreviation definitions (e.g., osteoarthritis (OA) appears with parenthetical clarification multiple times) and inconsistent terminology for key abbreviations. Specifically, the term HSPHR is ambiguously defined as both:\n“dual-responsive organicdouble selenium hydrogel microsphere (HSPHR)”\n“dual-responsive selenium nanoparticle hydrogel microspheres (HSPHR) ”\n4. Given that most prior studies refer to “SeNPs” as inorganic selenium nanoparticles, while this work focuses on organic selenium, what specific form does “SeNPs” denote in this context? Please discuss the advantages of organic selenium compared to inorganic selenium.\n5. The authors claim SeNPs reverse OA cartilage degeneration by restoring antioxidant pathways. However, given the significant influence of nanoparticle morphology and size on bioactivity, the current characterization of these parameters is insufficient and requires enhancement. For instance, comprehensive characterization techniques such as TEM (Transmission Electron Microscopy) for morphology visualization, DLS (Dynamic Light Scattering) for size distribution analysis, and zeta potential measurement for surface charge evaluation should be employed to provide a more accurate and detailed understanding of the SeNPs' physicochemical properties. Additionally, justification is needed for selecting 20 nM and 50 nM concentrations in validating SeNPs' effects on chondrocyte matrix metabolic homeostasis.\n6. Please standardize the capitalization of the first letters of the x- and y-axis labels, as well as the font style and size in all figures, and define the abbreviations for each group.\n7. The presentation of many data in the manuscript is not substandard. All morphological images must include scale bars, such as Figures 2A,3E-M, 5A-D, 7B-M, S7E-I, and S8. All fluorescently stained images should provide information on the fluorescent probes used, such as Figures 5A-D, 7L-M, S6B, and S7E-F, etc.\n8. Selenium plays a critical role in maintaining redox homeostasis by participating in antioxidant processes and regulating selenoprotein synthesis. However, this study lacks experimental evidence demonstrating how SeNPs modulate redox homeostasis.\n9. The western blot results in Figure 1D, and 6B lack reliability due to inconsistent loading controls.\n10. The therapeutic efficacy evaluation of HSPHR microspheres in early-stage osteoarthritis lacks critical validation through imaging modalities (e.g., Micro-CT, MRI) and behavioral assessments (e.g., gait analysis), undermining the clinical relevance of reported histological improvements.\n11. Figures 7L-M, S2, S5, S6A-B, S7E, and S9 exhibit poor quality requiring enhanced resolution and annotation. Critically, Figure S5 lacks organ labeling in HSPHR biodistribution images. The significant hepatic retention (at 14 days) raise toxicity concerns, demanding expanded safety studies (hepatorenal function, long-term clearance). These revisions are essential to validate therapeutic biosafety.\n12. The discussion section is superficial and lacks depth. The authors should provide a more thorough analysis of their results, comparing their findings with existing literature and highlighting the implications of their work for clinical applications.\n13. This manuscript needs a revision. The writing of this paper is too rough. The manuscript requires thorough language revision due to frequent formatting errors (e.g., incorrect subscript/superscript notation in chemical formulas like Na2SeO3 and H2O2), spelling mistakes (such as ofor, and SeNPss, etc.), and grammatical inconsistencies. Comprehensive English editing is recommended to ensure scientific rigor and clarity."}, "author_response": "ResponsetoReviewers'Comment\nReviewer#1(RemarkstotheAuthor):\nThe technology reported here is interesting, although the novelty is difficult to evaluate. In\nthis manuscript, the authors constructed a ROS/MMP13 dual-responsive organoselenium\nhydrogel microsphere (HSPHR), which can significantly upregulate the expression of\nTXNRD1, activate the PI3K-AKT-mTOR signaling pathway, and enhance mitophagy and\nmitochondrial function. The aim is to promote cartilage repair and functional reconstruction\nthrough multidimensional targeted intervention. We appreciate the authors' efforts; however,\nthere are some questions and concerns that arose during the review of the manuscript, which\narelistedasfollows:\nResponse：We sincerely appreciate the reviewer's constructive suggestions, which have\nsignificantlyenhancedthequalityofourmanuscript(seedetailsbelow).\nComment1.The authorshavenot providedevidence toconfirmthesuccessful preparationof\nSeNPs as opposed to other possible substances. Notably, they conducted both in vitro and in\nvivo studies using these materials without first performing any material characterization\nexperimentstoverifytheidentityandpropertiesoftheSeNPs.\nResponse:Wesincerelyappreciatethereviewers'valuablecomments.Thecharacterizationof\nbiomaterials constitutes a critical aspect of our research. SeNPs were comprehensively\ncharacterized in our previous studies (J Nanobiotechnology 2024;22(1):634), and we\napologize foranyconfusionthatmayhave arisenfromtheomissionofthisinformationinthe\nmanuscript. Following the reviewers' suggestions, we have provided a detailed\ncharacterizationoftheSeNPspreparedinthisstudy.Transmissionelectronmicroscopy(TEM)\nwas employed to examine the morphological structure of the SeNPs (Figure R1A). The\nparticlesize was determinedtobe approximately250nm, whichisconsistentwiththe results\nfrom dynamic light scattering analysis (Figure R1C). Energy-dispersive X-ray spectroscopy\n(EDX) confirmed the uniform surface distribution of selenium on the SeNPs (Figure R1B).\nAdditionally,thezetapotentialoftheSeNPswasmeasuredat-11.7mV(FigureR1D).\nSeNPs,(B)SurfaceEDXspectrumofSeNPs,(C)ParticlesizedistributionofSeNPs,(D)Zeta\npotentialofSeNPs.\nComment 2. The font size in Figures 4I-J is too small, which hinders effective extraction of\ninformation from the images. It is recommended that the authors increase the font size to\nimprovereadabilityandclarity.\nResponse：We sincerely appreciate your valuable feedback. We have carefully corrected all\ninstances of non-compliant font usage throughout the manuscript, including in the main text,\nandhavemadecomprehensiveadjustmentsinothersectionstoenhanceoverallreadability.\nComment 3. On page 18, line 7, the authors state that \"HSPHR exhibited accelerated\ndegradation when exposed to both H₂O₂ and the MMP13 enzyme.\" However, in DMM mice,\nthedegradationrateofHSPHRappearstobeslower.\nResponse:Wesincerelyappreciateyour valuablefeedback.Throughinvitroexperiments, we\nhave confirmed that HSPHR microspheres exhibit responsiveness to both MMP13 enzyme\nand reactive oxygen species (ROS), characterized by increased selenium release. To observe\nthis phenomenon in rats, Cy5.5 dye labeling combined with infrared imaging is necessary.\nNotably, HSPHR contains a selenium-selenium double bond cross-linking structure that\nfacilitates three-dimensional network formation, thereby enhancing drug encapsulation\ncapacity. For instance, previous studies have utilized this selenium-selenium structure to\ndeliver NGF-responsive drugs to brain injury sites for the treatment ofAlzheimer's disease (J\nMater Chem B 2021;9(37):7835-7847). When Cy5.5 is encapsulated within HSPHR, the\nfluorophore may remain shielded under non-inflammatory conditions due to the stability of\n2\nthe Se-Se crosslinks. Therefore, fluorescence intensitymust be interpretedacross two distinct\ntime windows: during the early phase (Day 1–7), abundant fluorophores allow gradual Cy5.5\nrelease, resulting in strong fluorescence in both Sham and DMM groups. During the late\nphase(Day8–14),the DMMgroupexhibits sustainedinflammatory-like responses, including\nelevated ROS and MMP13 levels, which trigger the cleavage of Se-Se bonds and subsequent\nre-exposure of the Cy5.5 fluorescent moiety. Consequently, the DMM group demonstrates\nsignificantly higher fluorescence intensity compared to the Sham group. Similar results have\nbeen reported in ROS-responsive delivery systems used in models of middle cerebral artery\nocclusion (Acta Pharm Sin B 2023;13(12):5107-5120) and liver fibrosis (Biomaterials\n2025;314:122887).\nComment4.InFigure6A,thespecificityofthePI3Kproteinbandsappearstobesuboptimal.\nFurthermore,theconsistency of theAKTandP-AKTresults raises doubtsas towhetherthese\nproteins were detected on the same membrane. Please provide the Western data with whole\nmembraneandshownwithmarkerladder.\nResponse:Wesincerelyappreciatethereviewer'scomment.TheoriginalblotsforallWestern\nblotproteinbandshavebeenprovidedinSupplementaryFile1.\nComment 5. In Figures 5–6, a group without IL-1β treatment should be included as a blank\ncontrol to verify the successful induction of inflammation by IL-1β. The absence of such a\ncontrolgroup makes it difficult todetermine whether the observed effects arespecifically due\ntoIL-1β-inducedinflammation.\nResponse: We sincerely appreciate your insightful suggestions. Implementing blank controls\nas a quality control measure is essential to ensure the reliability and validity of our\nexperimental results. In response, we have revised the experiments shown in Figures 5–6\n(Figures R2–R3) by incorporating blank controls. Our results consistently show that IL-1β\ntreatmentinducesanarthritis-associatedphenotypeinchondrocytes,characterizedbyreduced\nCOL2 protein synthesis and increased MMP13 expression. Administration of SeNPs and\nHSPHR effectively mitigates this pathological phenotype (Figures R2A–C), with HSPHR\ndemonstrating a more significant protective effect than SeNPs. Further analysis of\n3\nselenoprotein expression revealed that IL-1β treatment markedly decreases the levels of\nselenoprotein synthase, GPX1, and TXNRD1. However, treatment with both SeNPs and\nHSPHR successfully restores the expression of these selenoproteins, with HSPHR showing\nsuperiortherapeuticpotential(FiguresR2A,D–F).\nFigure R2. Differences in HAMA, 50 ng/mL SeNPs, and HSPHR protein expression\nfollowing IL-1β intervention. (A-F) Western blot bands for MMP13, COL2A, GPX1,\nTXNRD1, and SEPHS1 and their quantitative statistical analysis. (n = 3 per group, *: *P <\n0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001 by one-way ANOVA with Tukey’s\nmultiplecomparisontest.)\nIn Figure R3, blank controls were incorporated, and all experiments—including Western\nblotting,immunofluorescence, andtransmissionelectronmicroscopy—werereorganized.The\naddition of IL-1β significantly downregulated TXNRD1 expression and inhibited\nphosphorylation of PI3K, AKT, and mTOR. Following HSPHR treatment, TXNRD1\nexpression was restored to normal levels, thereby reactivating the downstream\nPI3K-AKT-mTOR signaling pathway. However, administration of the TXNRD1 chemical\ninhibitorTRi-1onceagainsuppressedthePI3K-AKT-mTORsignalingpathway(FigureR3A),\nsuggestingthat HSPHR exertsits effects through activationofTXNRD1,whichsubsequently\nactivates the downstream PI3K-AKT-mTOR signaling pathway. Further analysis of\nmitochondrialstructuralproteinexpression,morphology,andfunctionalchanges(FigureR3B,\nG-J) revealed that IL-1β significantly reduced the expression of mitochondrial intrinsic\nproteinsATP5A, MT-ND4, COX 4, and TOMM20. HSPHR treatment restored these proteins\nto baseline levels, whereas TRi-1 administration led to a subsequent decrease in their\nexpression. Similar trends were observed in mitochondrial membrane potential (Figure R3D)\n4\nand mitoTracker staining intensity (Figure R3E). Transmission electron microscopy\ndemonstrated that IL-1β induced mitochondrial swelling and loss of cristae, alterations that\nwere reversed by HSPHR treatment but blocked by TRi-1. Moreover, HSPHR restored\nIL-1β-induced reductions in matrix synthesis proteins and suppressed matrix degradation\nproteins in chondrocytes. Our findings indicate that HSPHR exerts its protective effects by\nupregulating TXNRD1, thereby activating the PI3K-AKT-mTOR signaling pathway and\nrestoringmitochondrialfunctionandchondrocytemetabolism.\nsynthesistoameliorate osteoarthritisprogression. (A)WBbands ofTXNRD1, PI3K,P-PI3K,\nAKT, P-AKT, and P-mTOR. (B) WB bands of ATP5A, MT-ND4, COX 4, TOMM20, and\nLC3B. (C) WB bands of ACAN, COL2, MMP13, and ADAMTS5. (D) JC-1 staining. (E)\nMitotracker staining and skeletal analysis. (F) TEM of mitochondria. (G) TOMM20\nimmunofluorescence staining. (H) ATP5A immunofluorescence staining (I) COX IV\n6\nimmunofluorescence staining, (J) MT-ND4 immunofluorescence staining, (K-V) quantitative\nstatistical analysis of TXNRD1, PI3K, P-PI3K, AKT, P-AKT, P-mTOR, ATP5A, MT-ND4,\nCOX4,TOMM20,LC3B,ACAN, COL2, MMP13,andADAMTS5proteinlevels. (n=4per\ngroup, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001 by one-way ANOVA with\nTukey’smultiplecomparisontest.)\nComment6.SeveralaspectsofFigure7requireclarification:\na. The S.O. staining of cartilage in the DMM group appears to be more intense, whereas the\nstaining in the HSPHR group is unexpectedly lighter. The authors should explain the reasons\nfortheseobservations.\nResponse: We sincerely appreciate your valuable feedback and deeply regret the unexpected\nstaining outcomes observed in our study. We fully recognize that the hallmark pathological\nfeatures of DMM involve degeneration of the cartilage matrix and morphological alterations\nin cartilage structure. In the DMM group, pronounced changes in joint architecture were\nevident,includingthecompletelossofjointspace.This observationmaybecloselyrelatedto\nthe thickness and uniformity of the tissue sections. Structural disruptions in the joints likely\ncontributedtoslight variationsinsectionthicknessacrosscertainspecimens,whichmayhave\nled to localized dye over-absorption. For this, we offer our sincere apologies. In response to\nthis concern, we have repeated the experiment using an expanded sample size of eight\nanimals, and the findings were consistent with those presented in the original manuscript.\nFurthermore,wehavesystematicallyreviewedallcomparableinstancesinthemanuscriptand\nmadeappropriaterevisionsaccordingly(FigureR4B-D).\n(A)Schematicillustrationdepictingtheintra-articularinjectionofhydrogelmicrospheresinto\nthe knee joint cavity. (B-E) Images of S.O, toluidine blue, hematoxylin, and eosin-stained\nsections of rat knee joint sections from different treatment subgroups and their quantitative\nstatistical analysis. (F-K) Representative images of COL2, MMP13, TXNRD1, GPX1\n8\nimmunohistochemical staining of knee joint sections from different treatment subgroups of\nrats and their quantitative statistical analysis (L-N) Representative images of p-mTOR,\np-AKT immunofluorescence staining of knee joint sections from different treatment\nsubgroupsof ratsandtheir quantitative statistical analysis. (n= 8 rats per group,*P< 0.05,\n**P < 0.01, ***P < 0.001, and ****P < 0.001 by one-way ANOVA with Tukey’s multiple\ncomparisontest.)\nb. The immunohistochemical staining results for TXNRD1 and GPX1 in the DMM group are\ninconsistent with those of other groups, with marked differences in nuclear staining intensity.\nThisraisesconcernsabouttheexperimentalproceduresandcomparabilityamonggroups.\nResponse: We sincerely appreciate your insightful feedback. As previously mentioned, we\nused the same specimen, and it is possible that variations in section thickness within this\nbatchledtounevendye absorptionincertainregions.However,itiscrucialtoemphasizethat\nimmunohistochemical staining relies on antigen-antibody binding reactions, which are not\naffected by excessive dye absorption and should not compromise the consistency of the\nresults. Nevertheless, we acknowledge the experimental inconsistencies and sincerely\napologize for them. To address this issue, we have conducted additional experiments,\nincreasing the sample size to eight animals per group. Moreover, we have carefully reviewed\nall findings that might have caused confusion in the manuscript and have made appropriate\nrevisions,asshownin(FigureR4I–J).\nc. Previous studies (Bone Res. 2019 Aug 5;7:23. doi: 10.1038/s41413-019-0062-y) have\ndemonstrated that AKT signaling is activated during OA. However, in this study, the level of\np-AKT in DMM mice is significantly decreased. The authors should provide a rationale for\nthisdiscrepancy.\nResponse: Thank you for your insightful comments. The precise role of AKT signaling in\nmaintaining articular cartilage homeostasis and its involvement in the progression of\nosteoarthritis remain largely unclear. Numerous in vitro studies have reported conflicting\nfindings regarding the function of AKT signaling in cartilage homeostasis. While persistent\nactivation of AKT signaling may compromise articular cartilage integrity, other studies\n9\nsuggest that AKT activation exerts protective effects on chondrocytes (Nat Commun\n2023;14(1):3159; Sci Adv 2024;10(10):eadk6084). Moreover, activation of this pathway has\nbeenshowntoincreaseintracellularATPlevelsandpromotechondrogenicdifferentiation(Sci\nAdv 2024;10(42):eadp7872). The PI3K/AKT signaling pathway has been demonstrated to\nexert chondroprotective effects by regulating chondrocyte survival, proliferation, and\nextracellularmatrixsynthesis.Furthermore,downstreamactivationofmTORhasbeenshown\nto protect chondrocytes by promoting CoQ10 production through the mTORC1-HMGCR\nsignaling pathway (Nat Aging 2025;5(7):1295-1316). This study demonstrates that HSPHR\ntreatment activates the mTOR signaling pathway, thereby modulating cellular energy\nmetabolism. Specifically, it shifts cellular energy production from anaerobic glycolysis to\noxidative phosphorylation (OXPHOS), enhances mitochondrial antioxidant capacity, and\nthereby provides protection to chondrocytes. This protective effect is likely attributed to the\nmoderateandtransientactivationofphosphorylatedAKT(P-AKT).\nd. The number of biological replicates per group (n=4) is insufficient to provide robust\nevidencethatHSPHRalleviatesOAprogression.\nResponse: We sincerely appreciate your valuable suggestions. In response to your concern\nregarding the previously inadequate sample size, we have implemented specific measures to\naddress this issue. Specifically, we have conducted additional in vivo experiments, thereby\nincreasingthetotalsamplesizepergrouptoeight.Allrelevantinvivoexperimentshavebeen\nrepeated, and thorough quantitative statistical analyses have been carried out. The results\nconsistently demonstrate that HSPHR treatment significantly enhances the expression of\nselenium-related proteins, namely TXNRD1 and GPX1, in articular cartilage. Moreover, it\nactivates the PI3K-AKT-mTOR signaling pathway in chondrocytes and effectively delays the\nprogressionofosteoarthritisinDMMrats,asshowninFigureR4.\nComment 7. The naming of the control groups in Figure 6D is inconsistent between the\nrepresentativeimagesandthecorrespondingquantitativedata.\nResponse: Thank you for your valuable suggestions. We have carefully reviewed and\ncorrectedtheinconsistenciesingroupnamesthroughoutthemanuscript.\nimprovecartilage metabolic...\". However,themanuscriptdoes notprovide sufficient evidence\nto clarify whether TXNRD1 restores mitochondrial function via the PI3K-AKT-mTOR\npathway,andthecausalitybetweenthesefactors isambiguous. Tostrengthenthemechanistic\ninterpretation, the authors should consider assessing mitochondrial function and/or the\nexpression of relevant proteins following activation or inhibition of the PI3K-AKT-mTOR\npathway.Thiswouldprovidemoredirectevidencefortheinvolvementofthissignalingaxisin\ntheobservedeffects.\nResponse：We sincerely appreciate your valuable feedback. In response to your concern\nregarding the insufficient direct evidence for the regulation of the PI3K-AKT-mTOR\nsignaling pathway, we have carefully reconsidered this issue and conducted additional\ncomprehensive experiments. By utilizing LY294002, a specific inhibitor of PI3K\nphosphorylation, we further investigated the regulatory effect of HSPHR on the\nPI3K-AKT-mTOR signaling pathway, as illustrated in Figure R5. The administration of\nLY294002 significantly attenuated the activation of the PI3K-AKT-mTOR pathway induced\nby HSPHR (Figure R5A). Further analysis of mitochondrial intrinsic proteins demonstrated\nthat HSPHR effectively restored their expression levels in inflammatory chondrocytes;\nhowever, this effect was inhibited by LY294002 treatment (Figure R5B, F–I). Transmission\nelectron microscopy revealed that HSPHR administration promoted the formation of\nmitochondrial autophagosomes and facilitated the restoration of normal mitochondrial\nmorphology (Figure R5D). Additionally, mitochondrial membrane potential measurements\nindicated that HSPHR intervention effectively counteracted the IL-1β-induced disruption of\nmitochondrial membrane potential (Figure R5E). Moreover, Seahorse extracellular flux\nanalysiswasemployedtoassessthemetabolicstatusofinflammatorychondrocytesfollowing\nHSPHR treatment. Glycolytic stress testing was performed by measuring the real-time\nextracellular acidification rate (ECAR) after sequential injections of glucose and\n2-deoxy-D-glucose (2-DG), thereby enabling the evaluation of chondrocyte glycolytic\nparameters.Comparedtothe Ctrl group,IL-1β-inducedinflammatorychondrocytes exhibited\nsignificantlyelevatedglycolysis andglycolytic reserve.However,inthe HSPHR group,these\n11\nglycolytic parameters showed a progressive decline. Following treatment with the PI3K\ninhibitor LY294002, glycolytic reserve was significantly increased, which was accompanied\nbyacceleratedmitochondrialdamage(FigureR5J).Inparallel,real-timeoxygenconsumption\nrate (OCR) was measured after sequential administration of oligomycin, carbonyl\ncyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone, allowing for the\nevaluation of chondrocyte oxidative phosphorylation (OXPHOS) parameters based on\nmitochondrial stress assays (Figure R5K). Basal respiration, ATP production, and maximal\nrespiration were notably reduced in the IL-1β group compared to the control group, whereas\nOXPHOSparametersgraduallyincreasedintheHSPHRgroup.Notably,LY294002treatment\nledto a significant decrease incellularOXPHOS capacity.These results suggest that HSPHR\ntreatment promotes metabolic reprogramming in inflammatory chondrocytes, shifting energy\nmetabolism from glycolysis toward oxidative phosphorylation, potentially mediated through\nactivation of the PI3K-AKT-mTOR signaling pathway. Subsequently, cells from each group\nwere collected to assess antioxidant capacity. HSPHR treatment enhanced the clearance of\nexcessive reactive oxygen species, including O₂⁻, •OH, and other free radicals (Figure\nR5L–O). Collectively, these findings indicate that HSPHR therapy restores mitochondrial\nprotein synthesis pathways and reestablishes mitochondrial oxidative phosphorylation,\ntherebyexertingintracellularantioxidanteffectsandalleviatingcartilagedamage.\noxidativephosphorylationandmitigatechondrocytedamage.(A)WBbandsofPI3K,P-PI3K,\nAKT, P-AKT, and P-mTOR WB bands. (B) WB bands of ATP5A, MT-ND4, COX 4,\nTOMM20,andLC3BWB.(C)WBbandsofACAN,COL2, MMP13,andADAMTS5bands.\n(D)TEMofmitochondria.(E)JC-1staining.(F)TOMM20immunofluorescencestaining.(G)\nATP5A immunofluorescence staining . (H) COX IV immunofluorescence staining. (I)\nMT-ND4 immunofluorescence staining. (J) Real-time ECARs of chondrocytes during\nglycolytic stress testing, (K) Real-time OCR in chondrocytes during mitochondrial stress\ntesting.(L–O)ScavengingefficiencyofchondrocytesagainstthreetypicalfreeradicalsPTIO,\n***P < 0.001, and ****P < 0.001 by one-way ANOVA with Tukey’s multiple comparison\ntest.)\nFurthermore, when the experiment was repeated using 740Y-P (a phospho-activated\nPI3K activator), we observed that the therapeutic effect of HSPHR was comparable tothat of\ndirectactivationby740Y-P.Thistreatmenteffectivelyrestoredmitochondrialintrinsicprotein\nexpression levels and normalized mitochondrial morphology and function (Figure R6). In\nsummary, our findings clearly indicate that the mechanism of action of HSPHR is closely\nlinkedtotheactivationofthePI3K-AKT-mTORsignalingpathway.\noxidativephosphorylationandmitigatechondrocytedamage.(A)WBbandsofPI3K,P-PI3K,\nAKT, P-AKT, and P-mTOR. (B) WB bands of ATP5A, MT-ND4, COX 4, TOMM20, and\nLC3B. (C) WB bands of ACAN, COL2, MMP13, and ADAMTS5. (D–P) Quantitative\nanalysisof theaforementionedWBbands.(Q)TEMof mitochondria. (R–S) JC1stainingand\nits statistical analysis. (T) Real-time OCR during mitochondrial stress testing and\n15\nsemi-quantitative analysis of basal respiration, maximal respiration, and ATP production in\nchondrocytes. (n = 4 per group, *: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001\nbyone-wayANOVAwithTukey’smultiplecomparisontest.)\nThe manuscript presents a novel synthesis of a ROS/MMP13 dual-responsive organic\ndouble selenium hydrogel microsphere to realize early OA local microenvironmental response by\nutilizing the pathological characteristics of osteoarthritis, and at the same time, the organic\nselenium can effectively supplement the selenoprotein level in the cartilage, synovium and\nsubchondral bone. Intra-articular injection of HSPHR not only reduced cartilage\ndegeneration, improved synovial hyperplasia, and delayed sclerosis of the subchondral bone\nin the post-traumatic OA model, but also accelerated the formation of new cartilage in the\ncartilage defect model. While the study is innovative and holds significant potential, I would\nlike to provide the following constructive suggestions to enhance the scientific rigor and\npresentationclarityofthismanuscriptbeforeitcanbeconsideredforpublication.\nResponse: We are grateful to the Reviewer for the constructive Comment, which have\nimprovedourmanuscript(detailsbelow).\nComment 1. The \"Introduction\" section is too long. Authors should focus only on the most\nimportantaspectsforagoodunderstandingofthedevelopedwork.\nResponse: Thank you for your valuable suggestions. We have carefully revised the\nintroduction section of the manuscript to enhance its clarity and focus on the key aspects of\ntheresearch.Thissectionhasbeenmarkedintherevisedmanuscript.\nComment 2. The authors propose that their \"three birds with one stone\" modified organic\ndiselenide hydrogel microspheres can simultaneously treat multiple aspects of osteoarthritis.\nHowever,thisconceptualframeworkrequiresclarificationforseveralreasons:\nWerecommendeither:\na)Providingdirectexperimentalevidencefortriple-targetingefficacy,or\nb) Modifying the conceptual framework to better reflect the actual therapeutic mechanisms\nobserved.\nThis would significantly strengthen the manuscript's mechanistic claims while maintaining\nscientificrigor.The current presentationrisks overinterpretation ofthe technology's targeting\ncapabilities.\na) Your suggestions are highly insightful. HSPHR can only exert therapeutic effects in\nresponseto elevatedlevels of ROS andMMP13 withinthe joint. Substantial ROS production\nwithintissuesmayleadtobothmacrophage inflammationandexcessive osteoclastactivation.\nTherefore,HSPHR demonstrates synergistictherapeutic effectsacrossmultiple tissues,which\nis fully consistent with our original objective. Our initial aim was to develop a therapeutic\nstrategy targeting three distinct intra-articular tissues. By leveraging its unique\nROS-responsive properties, HSPHR enhances intracellular selenoprotein expression, thereby\neffectivelymodulatingallthreedistinctpathologicaltissuesassociatedwithOA.\nb) Our research primarily focused on the therapeutic effects of selenoproteins on\nchondrocytes, with limited exploration of synovial macrophages and osteoclasts. Given the\npremisethatHSPHRexertstherapeuticeffectsonbothsynovialmacrophagesandosteoclasts,\nwe further investigated the potential mechanisms underlying HSPHR treatment in these two\ncell types. This study aimed to determine whether HSPHR alleviates synovial inflammation,\nsuppressesosteoclastactivation,andrestoreschondrocyteactivitythroughasharedregulatory\nmechanism. Notably, transcriptomic sequencing revealed that HSPHR's therapeutic effects\nsimultaneously activate the PI3K-AKT-mTOR signaling pathway in chondrocytes, synovial\nmacrophages,andsubchondralosteoclasts(FigureR7B,R9B).Tovalidatethishypothesis,we\nconducted follow-up experiments. By selectively activating and inhibiting the\nPI3K-AKT-mTOR pathway in osteoclasts and synovial macrophages, respectively, we found\nthat HSPHR therapy activates this pathway to promote M2 polarization in synovial\nmacrophages, and that pathway inhibition counteracts the therapeutic effects of HSPHR\n(Figure R7D-Q, R8A-N). Furthermore, activation of the PI3K-AKT-mTOR pathway\neffectively mitigates early excessive osteoclast activity in subchondral bone (Figure R9D-Q).\nThese findings are consistent with our research hypothesis: HSPHR intervention exerts\ntherapeutic effects on three cell types simultaneously, akin to achieving a \"triple-target\"\noutcomewithasingleintervention.\nsignaling pathway. (A) Volcano plot of differentially expressed genes between HSPHR and\nCTRL. (B) KEGG pathways enriched by differentially expressed genes between HSPHR and\nCTRL. (C) ESGA diagram of KEGG pathways enriched. (D–I) CD68/CD86, CD68/CD206\nimmunofluorescence,representativeflowcytometrychannels,andquantitative analysis.(J-Q)\nP-PI3K, P-AKT, and P-mTOR after HSPHR treatment of synovial macrophages. (n = 4 per\ngroup,*:*P< 0.05,**P< 0.01, ***P< 0.001,and****P< 0.001byone-wayANOVAwith\nTukey’smultiplecomparisontest.)\nFigure R8. The PI3K-AKT-mTOR pathway activation induces M2 macrophage polarization.\n(A, D-N) Western blots and analysis show CD206, CD86, ARG1, NOS2, P-PI3K, P-AKT,\nand P-mTOR levels in synovial macrophages post-HSPHR treatment. (B-C)\nImmunofluorescence andflow cytometry display CD68/CD86, CD68/CD206 markers. (n = 4\nper group, *: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001 by one-wayANOVA\nwithTukey’smultiplecomparisontest.)\npathway. (A) Volcano plot of differentially expressed genes between HSPHR and CTRL. (B)\nKEGG pathways enriched by differentially expressed genes between HSPHR and CTRL. (C)\nESGA plot of KEGG pathways enriched. (D–F) Representative images and quantitative\nanalysis of Trap staining and β-actin staining after HSPHR treatment of osteoclasts. (E, I-L)\nRepresentative WB images and quantitative analysis of CTSK, CD40L, P-PI3K, P-AKT, and\nanalysis. (H, M) Trap staining and representative Western blot images of CD206, CD86,\nARG1, NOS2, P-PI3K, P-AKT, and P-mTOR following HSPHR and 740Y-P treatment of\nosteoclasts. (n = 4 per group, *: *P < 0.05, **P< 0.01, ***P < 0.001, and****P < 0.001 by\none-wayANOVAwithTukey’smultiplecomparisontest.)\nComment 3. While the abstract states that organic selenium supplementation restores\nselenoproteinlevelsacrossmultiplejointtissues(cartilage,synovium,andsubchondralbone),\nthe study primarily demonstrates mechanistic effects in cartilage without providing selenium\nquantification data for synovium or subchondral bone. This limitation should be\nacknowledged, as it affects the interpretation of the compound's proposed multi-tissue\ntargetingcapability.\nResponse: Wesincerelyappreciate your valuable suggestions.Wesincerelyapologize for the\nissue concerning the manuscript. To further support our findings, we have added detailed\nexperimental data in the relevant section, with the aim of elucidating and refining the\ntherapeutic mechanisms mediated by HSPHR in cartilage, synovium, and subchondral\nbone.We separately measured selenoprotein levels in cartilage and synovial tissues after\nDMM 8 weeks . Results demonstrated that HSPHR treatment effectively restored GPX1 and\nTXNRD1 protein expression in both cartilage and synovial tissue (Figure R10A-B, E-F).\nIdentical outcomes were observed in subchondral bone tissue at 2 weeks after DMM (Figure\nR10C-D). These findings indicate that HSPHR can upregulate selenoproteins across different\ntissues.Subsequently,weanalyzedtheunderlyingmechanismsindetail(FiguresR6-9).These\nregulatoryeffectsallpointtoacommonregulatorypathwayinvolvingPI3K-AKT-mTOR.\nSubchondral Bone Following DMM. (A-B) Representative histochemical staining and\nquantitative analysis of GPX1andTXNRD1 incartilage tissueat 8weeks after DMM. (C-D)\nRepresentative immunohistochemical staining and quantitative analysis of GPX1 and\nTXNRD1 in subchondral bone at 2 weeks after DMM surgery. (E-F) Representative\nimmunohistochemical stainingand quantitative analysis of GPX1 andTXNRD1 in synovium\nat 8 weeks after DMM surgery. (n = 3-8 per group, *: *P < 0.05, **P < 0.01, ***P < 0.001,\nand****P<0.001byone-wayANOVAwithTukey’smultiplecomparisontest.)\nComment 4. While the authors showed HSPHR's inhibitory effect on osteoclast formation in\nvitro in Supplementary Figure 6, the in vivo study only evaluated late-stage OA pathology (8\n23\nweeks post-operation). Since osteoclasts are known to mediate early-stage OA pathogenesis,\ncould the authors address why osteoclast activity was not monitored at earlier time points?\nThis information would strengthen the mechanistic connection between HSPHR's cellular\neffectsanditstherapeuticoutcomes.\nResponse: We sincerely appreciate your valuable suggestions and fully align with your\nperspective. In the early stages of osteoarthritis, subchondral bone may exhibit hyperactive\nosteoclast function. To further investigate this phenomenon, we conducted a new series of\nanimal experiments using Sham, DMM, HAMA, and HSPHR groups. Subchondral bone\nmeasurements and bone density analyses were performed on rats two weeks after DMM\nsurgery.The results demonstrated that rats in the DMM group exhibited excessive osteoclast\nactivation at two weeks post-surgery, whereas HSPHR treatment significantly attenuated this\nactivation and restored the initially reduced bone density (Figure R11A, C–E). Further\nimmunohistochemical staining of knee joint tissues from two-week-old rats revealed\nmarkedly elevated MMP9 expression in the DMM group at this time point. Following\nHSPHR treatment, osteoclast activation was significantly reduced, and MMP9 expression\nreturned to baseline levels (Figure R11B, F). These findings suggest that HSPHR therapy\neffectively modulates osteoclast activation in subchondral bone during the early stages of\narthritis. The underlying mechanism may involve the scavenging of ROS produced by\ncartilage tissue, thereby limiting osteoclast activation in subchondral bone. Alternatively,\nHSPHR may exertdirect effects onosteoclasts. Evidence from previous studies indicates that\nincreased selenoprotein synthesis can directly regulate osteoclast activity and reverse the\nprocessofosteoclastactivation(AdvMater2024;36(27):e2401620).\nweeksafterDMMsurgery,representativeimagesandquantitativeanalysisof(A)subchondral\nbone remodeling, (B, F) MMP9 immunohistochemical staining in the Sham, DMM, HAMA,\nandHSPHRgroups,and(C-F) subchondralbonetrabecularseparation,bonevolume fraction,\nandtrabeculardensity.(n=8pergroup,*:*P<0.05,**P<0.01,***P<0.001,and****P<\n0.001byone-wayANOVAwithTukey’smultiplecomparisontest.)\nComment 5. The Graphic Abstract does not clearly convey the \"three-in-one\" concept, nor\ndoesitshowthemicrospheres'impactonothercells.\nResponse: Thank you very much for your insightful suggestions. Our research mainly\ninvestigates the degenerative process of osteoarthritis. Notably, our experimental results\nrevealed that HSPHR treatment exerts beneficial regulatory effects on both osteoclasts and\nsynovial macrophages. We sincerely apologize for the insufficient presentation of these\nfindings in the original figure abstract. Accordingly, we have revised the figure abstract to\nmoreclearlydepictthemechanismthroughwhichHSPHRregulatesselenoproteinexpression\ntorestorearticularjointhomeostasis.\nthe authors developed an in vivo model featuring a full-thickness cartilage defect.\nChondrogenic progenitor cells were incorporated into microspheres of HSPHR in order to\nenhancethetherapeuticoutcomesforcartilagerepair.The studyprimarilyelucidates the role\nof selenoproteins (particularly GPX1 and TXNRD1) in pathological cartilage, as\ndemonstrated in both the main text and schematic diagrams. However, the relationship\nbetween selenoproteins and chondrocyte differentiation remains unclear. Several critical\nquestionsneedtobeaddressed:\na) Molecular Mechanisms: Are GPX1 and TXNRD1 similarly involved in chondrocyte\n26\ndifferentiation and cartilage homeostasis? What specific selenoproteins regulate\nchondrogenicdifferentiationpathways?\nb)ExperimentalEvidence:Thestudywouldbenefitfrominvitroexperimentsusing:\n1)Chondrocytedifferentiationmodels;\n2)Selenoprotein-specificknockdown/overexpressionsystems;\n3)Comprehensiveanalysisofredoxregulationduringdifferentiation.\nResponse: a) We sincerely appreciate your valuable insights, which have enabled us to\nfurther explore the mechanisms through which selenoproteins promote chondrogenic\ndifferentiation.Accumulating evidence suggests that the selenoprotein family plays a crucial\nrole in early cartilage development, involving the coordinated actions of multiple\nselenoproteins. Recent studies indicate that GPX1 actively contributes to chondrogenic\ndifferentiation, primarily by alleviating oxidative stress in chondrogenic precursor cells,\nenhancingcartilage matrixsynthesis,andfacilitatingthe differentiationprocess (Mater Today\nBio 2023;23:100864; Arthritis Rheumatol 2014;66(12):3349-58). As a homologous member\nof the selenoprotein family, TXNRD1 also participates in cellular redox metabolism across\nvarious tissues (Nat Aging 2024;4(2):185-197). Our findings demonstrate that TXNRD1\nexerts a more pronounced protective effect on cartilage and exhibits enhanced chondrogenic\ndifferentiation capacity(Figure R12).This effect may be associated with the activation of the\nPI3K-AKT-mTOR signaling pathway downstream of TXNRD1, as discussed in our\nmanuscript. Activation of this pathway restores mitochondrial function impaired by\ninflammatory injury, shifting cellular metabolism from glycolysis to oxidative\nphosphorylation,therebymoreeffectivelypromotingchondrogenicdifferentiation.\nb) Thank you for your advice. To investigate whether GPX1 and TXNRD1 can promote\nchondrogenesis, we designed experiments involving the transfection of overexpression\nplasmids of GPX1 and TXNRD1 into ATDC5 chondrogenic precursor cells. Following the\ninduction of chondrocyte pellet formation, Alcian blue and eosin staining were performed.\nThe results demonstrate that overexpression of both GPX1 and TXNRD1 enhances\nchondrogenic differentiation under inflammatory conditions, with TXNRD1 overexpression\nshowing a more pronounced promotion of cartilage matrix secretion (Figure R12A-D). To\n27\nfurther validate these findings, we conducted siRNA-mediated knockdown of GPX1 and\nTXNRD1 in ATDC5 cells, followed by induction of chondrogenic differentiation. Both\nsiGPX1 and siTXNRD1 led to a reduction in cartilage matrix production, with TXNRD1\nknockdown resulting in a significantly greater decrease, thereby severely impairing\nchondrogenic differentiation (Figure R12E-H). Collectively, these findings indicate that both\nGPX1 and TXNRD1 contribute to the promotion of chondrogenic differentiation under\nosteoarthritic conditions. Notably, our study reveals that TXNRD1 exerts a more pronounced\nprotective effect, representing the first observation of its kind during chondrogenic\ndifferentiation. Subsequently, we analyzed changes in ATDC5 cells' antioxidant capacity\nduring chondrogenic differentiation, assessing scavenging abilities following TXNRD1\noverexpression and HSPHR treatment. Results indicate that both HSPHR intervention and\nTXNRD1 overexpression enhance cellular antioxidant capacity, improving chondrocyte\nscavengingefficiencyagainstthreerepresentativefreeradicalsPTIO,ABTS,•OH,andDPPH\n(FigureR12I-L).\nFigure R12. GPX1 and TXNRD1 restore cellular antioxidant capacity and promote in vitro\n28\nchondrogenic differentiation. (A-D) Representative images and quantitative statistics of S.O.\nand A.B. staining in GPX1 and TXNRD1-overexpressing chondrocyte chips under IL-1β\n(E-H) IL-1β conditions. (E–H) Representative images and quantitative analysis of S.O. and\nA.B. stainingin cartilage chips with GPX1 andTXNRD1 knockdown under IL-1β treatment.\n(I–L) Radical scavenging efficiency of cartilage chips after TXNRD1 overexpression against\nthree typical free radicals and DPPH. (n = 3 per group, *: *P < 0.05, **P < 0.01, ***P <\n0.001,and****P<0.001byone-wayANOVAwithTukey’smultiplecomparisontest.)\nComment 7. Figure 4, the authors report solid-state NMR characterization of HA-Se-Se, but\nthe presented spectrum shows only a single peak with no observable selenium signal. This\nraisesseveraltechnicalandinterpretationalconcernsthatneedtobeaddressed.\nResponse: We sincerely apologize for any confusion concerning our results. In Figure 4,\nsolid-state NMR was employed to analyze the selenium spectrum of HSPHR. The sample\npreparationprocess,whichinvolvedrepeatedgrindingandimpurityremoval,mayhaveledto\npartial oxidation of the test samples. Moreover, due to the inherent limitations of solid-state\nNMR, broad spectral peaks were obtained, thereby reducing measurement precision. To\naddress this, we supplemented our analysis with liquid NMR and Raman spectroscopy to\nmore accurately characterize the specific Se-Se bonds in HSPHR. The 77Se NMR spectrum\nrevealed a distinct Se-Se peak at 290.23 ppm (Figure R13A). Likewise, Raman spectroscopy\nidentified a characteristic Se-Se metallic-like vibrational peak at 254 cm-1 (Figure R12B).\nFurtherFTIRanalysisalsodetectedcharacteristicSe-Se absorptionbands(FigureR13C),and\nXPS analysis confirmed the presence of Se 3d signals (Figure R13D–E). Collectively, these\nfindings provide strongevidencethat the synthesizedHSPHR microspheres contain abundant\nSe-Sebonds.\nspectrum comparison between HSPHR and HAMA, (C) FTIR spectrum comparison between\nHSPHRandHAMA,(D–E)XPSspectrumcomparisonbetweenHSPHRandHAMA.\nComment 8. While the manuscript investigates selenium-based therapy for OA, several\ncriticalconcernsregardingitsnoveltyandmechanisticdepthneedtobeaddressed:\nInnovationGap:\nThe therapeutic use of selenium nanoparticles in OA has been extensively reported (e.g.,\nstudies by DOI: 10.1016/j.mtbio.2023.100864; DOI: 10.1016/j.biomaterials.2025.123195;\nDOI:10.1016/j.jcis.2025.137612).\nThecurrentworkmustbetterdifferentiateitselfby:\nResponse: Thank you for your insightful comments. Current selenium-based studies on\narticular cartilage repair mainly emphasize ROS scavenging and the modulation of the local\nmicroenvironment. The OHA/HA-ADH@SeNPs system reported in the Materials Today Bio\npresents notable innovations, including the sustained release of SeNPs and the targeted\nactivation of GPX1. Nevertheless, the underlying mechanisms of selenoprotein-based\ntherapies in OA remain insufficiently explored. The Se/PRP-OGel system introduced in the\nJournal of Colloid and Interface Science employs zero-valent selenium nanoenzymes to\neliminate ROS and utilizes PRP-derived growth factors to promote cartilage regeneration.\nHowever, it does not clarify the specific selenoprotein pathways involved in regulating\nmitochondrialqualitycontrolinOAchondrocytes, nordoesitprovideasystematicevaluation\nof the synergistic pathological interactions between synovial macrophages and subchondral\n30\nosteoclasts. In the Biomaterials study, HASeNPs@AHAMAHMs were developed as\n\"cascade-targeted\" microspheres, highlighting CD44-mediated uptake and enhanced\nmitochondrial oxidative phosphorylation.Yet, similar to previous studies, this work primarily\nfocuses on SeNPs release and antioxidant effects, without offering direct evidence regarding\nwhich selenoproteins are restored or how their upstream signaling pathways contribute to\nmitochondrial homeostasis. Moreover, it lacks a comprehensive intervention strategy\ntargetingthetriadofpathologicalunits—synovium,cartilage,andsubchondralbone.\nThisstudyproposesandvalidatesaninnovativemodificationoforganicselenium(Se-Se)\ndual-responsive microspheres, HSPHR. The modification achieves three key innovations: (1)\ndual ROS/MMP13 responsiveness that aligns with osteoarthritis (OA) pathology, enabling\non-demanddrugrelease;(2)enhancedseleniumabsorptionefficiencyandimprovedbiosafety.\nIt is demonstrated that TXNRD1 activates the PI3K-AKT-mTOR signaling pathway, shifting\ncellular metabolism from glycolysis to OXPHOS. This metabolic reprogramming alters\ncellularenergymetabolismandfacilitatestherecoveryofimpairedmitochondrialfunction.(3)\nThe regulatory role of the PI3K–AKT–mTOR pathway is shown to operate across three\ncellular dimensions, simultaneously alleviating cartilage degeneration, suppressing synovial\nmacrophageinflammation,andinhibitingearlyosteoclastactivation.Thisapproachrepresents\nanoveltherapeuticstrategyforOAthathasnotbeenpreviouslyreportedintheliterature.\nSelenoproteinAnalysisLimitations:\nThecartilageselenoproteinexpressionprofilingGPX1/TXNRD1largelyreplicatespriorwork\nwithoutadvancingthefield.\nResponse: Thank you for your valuable feedback. It is well established that GPX1 and\nTXNRD1havebeenextensivelystudiedandareknowntoexertsignificantchondroprotective\neffects, primarily by enhancing cellular antioxidant capacity. In this study, we show that\nHSPHR microspheres not only effectively upregulate the expression of GPX1 andTXNRD1,\nbutalsodemonstrate thatTXNRD1restores mitochondrialoxidative phosphorylationthrough\nthe activation of the PI3K-AKT-mTOR signaling pathway, thereby reversing the altered\nenergy metabolism in OA chondrocytes. Moreover, we further confirm that the\nPI3K-AKT-mTOR signaling pathway plays a regulatory role in two additional cell types\n31\nassociatedwithosteoarthritis—synovialmacrophagesandosteoclasts.\nMechanisticShortcomings:\nWhichspecificselenoproteinsmediatethetherapeuticeffectsincartilage;\nHowtheirregulationdiffersacrossjointtissues(synoviumvs.cartilagevs.bone);\nThedownstreampathwayslinkingselenoproteinstochondroprotection.\nResponse:Wesincerelyappreciateyourvaluablesuggestions.Ourresearchprimarilyfocused\non the therapeutic effects of selenoproteins on chondrocytes, with a limited investigation into\ntheir impact on synovial macrophages and osteoclasts.As previously outlined, by examining\nthe broad therapeutic potential of selenoproteins across multiple tissue types, combined with\nour transcriptomic sequencing data, we further explored the effects of HSPHR on synovial\nmacrophagesandosteoclasts.Notably,transcriptomic analysisrevealedthat HSPHRactivates\nthe PI3K-AKT-mTOR signaling pathway simultaneously in chondrocytes, synovial cells, and\nsubchondral osteoclasts (Figures R7B, R9B). To validate this finding, we conducted\nfollow-up experiments. By selectively activating and inhibiting the PI3K-AKT-mTOR\npathway in osteoclasts and synovial macrophages, respectively, our results indicate that\nHSPHRtherapypromotesM2polarizationofsynovialmacrophagesthroughactivationofthis\npathway, and that pathway inhibition attenuates the therapeutic efficacy of HSPHR\n(Figures R7D-Q, R8A-N). Concurrent activation of the PI3K-AKT-mTOR pathway effectively\nsuppresses early excessive osteoclast activity in subchondral bone (Figure R9D-Q),\nthereby supporting our hypothesis that HSPHR exerts therapeutic effects on three distinct cell types,\nakintoamulti-targetinterventionstrategy.OurstudycentersontheselenoproteinsGPX1and\nTXNRD1 to identify a novel therapeutic mechanism with potential for comprehensive joint\ntissueprotection.\nNotably, the PI3K-AKT-mTOR signaling pathway demonstrates a range of functional\nroles across different diseases and cell lines. Our novel strategy is centered on the\ndevelopment of an organic selenium-containing hydrogel microsphere capable of\nsimultaneously activating the PI3K-AKT-mTOR pathway in three distinct cell types. This\nmultifunctional approach exerts a comprehensive therapeutic effect by promoting\nchondrocyte regeneration, modulating macrophage-mediated immunity, and suppressing\n32\nosteoclast activation. By integrating these mechanisms, our approach effectively targets\nmultiple pathological processes in osteoarthritis, representing a meaningful step forward in\ntheadvancementofclinicaltherapies.\nComment:\nThisstudydevelopedROS/MMP13-responsiveseleniummicrospherestotargetOApathology.\nHSPHR restores selenoproteins and activates PI3K-AKT-mTOR-mediated mitophagy,\nmitigating cartilage degeneration, synovitis, and subchondral sclerosis in post-traumatic OA\nmodels while enhancing regeneration in cartilage defects. While the work addresses an\nimportant clinical issue, it falls short in several critical areas, particularly regarding novelty\nand experimental depth. As such, the current version of the manuscript is not suitable for\npublicationinNatureCommunications.\nResponse: We sincerely appreciate the reviewer's constructive comments, which have\ncontributedtotheimprovementofourmanuscript(seedetailsbelow).\nComment 1. The abstract and the introduction should be rewritten, the novelty of the work is\nnot highlighted. What does \"Three birds with one stone\" mean in the title? It needs to be\nclearlystatedintheintroduction.\nResponse: We sincerely appreciate your valuable feedback concerning the identified\nshortcomings. In response, we have thoroughly revised the abstract and introduction to more\nclearly emphasize the role of selenium-containing hydrogels in activating the\nPI3K-AKT-mTOR signaling pathway. This activation enables a synergistic therapeutic\nstrategy that addresses three key pathological aspects of osteoarthritis through a triple-action\nmechanism.\nComment 2. The graphical abstract fails to effectively convey the \"Three birds with one\nstone\" concept and requires redesign to explicitly highlight the innovative therapeutic\nstrategyoftheHSPHRsystem.\nResponse:Wesincerelyappreciateyourvaluablesuggestions.Ourresearchprimarilyfocuses\nonthe degenerative processesaffectingarticular cartilage. Notably,our experimental findings\ndemonstrate that HSPHR treatment exerts beneficial regulatory effects on both osteoclasts\nandsynovial macrophages.Weapologize for the inadequate illustrationof these results inthe\noriginal figure abstract.Accordingly, we have revised the figure abstract to more clearly and\n34\naccurately depict the mechanism by which HSPHR modulates selenoprotein expression to\nrestorehomeostasisinarticularcartilage.\nComment3. The article uses a great many abbreviations, some of whicharenot explainedat\nall (e.g. CEMIP, HSR, DMM, Ser, PTEN, WB, etc.). The abbreviations of all text are very\nconfusing. Full names should be given when an abbreviation first appears in the text.\nAdditionally, the manuscript contains redundant abbreviation definitions (e.g., osteoarthritis\n(OA)appearswithparentheticalclarificationmultipletimes)andinconsistentterminologyfor\nkeyabbreviations.Specifically,thetermHSPHRisambiguouslydefinedasboth:\n“dual-responsiveorganicdoubleseleniumhydrogelmicrosphere(HSPHR)”\n35\n“dual-responsiveseleniumnanoparticlehydrogelmicrospheres(HSPHR)”\nResponse:Wesincerelyapologize forthe use of ambiguous terminology andtheinclusion of\ninadequately defined abbreviations. We have carefully revised the manuscript to improve\nclarityandtobettersupportitsevaluationbyfutureresearchers.\nComment 4. Given that most prior studies refer to “SeNPs” as inorganic selenium\nnanoparticles,whilethiswork focuses onorganicselenium,whatspecificformdoes“SeNPs”\ndenote in this context? Please discuss the advantages of organic selenium compared to\ninorganicselenium.\nResponse: Selenium is an essential trace element for the human body. Inorganic selenium\ntypically lacks C-Se covalent bonds and is commonly found in the form of sodium selenite\n(Na₂SeO₃). In this study, selenocysteine (Se-Cys), which contains C-Se covalent bonds, was\nsynthesized. Following cellular uptake, Se-Cys can be directly incorporated into\nselenium-containing proteins in the body, participating in processes such as biosynthesis and\nredox balance. Organic selenium has a wider safety margin andis generallyregarded as safer\nthan its inorganic counterpart. Research investigating the effects of different selenium\ncompounds on human cells has shown that inorganic selenium is more likely to induce cell\ngrowth inhibition and cell death compared to organic selenium (Bioinorg Chem Appl\n2014;2014:923834). In contrast to inorganic selenium, which exhibits low bioavailability and\na higher risk of carcinogenicity, organically synthesized selenium demonstrates broader\npotential in the development of bioactive materials. Our findings suggest that organically\nbound selenium exhibits superior antioxidant activity in vitro and high direct conversion\nefficiency within cells. It facilitates the more efficient synthesis of antioxidant proteins such\nas GPX1 and TXNRD1, thereby effectively slowing the progression of osteoarthritis and\npromotingcartilageregeneration.\nComment 5. The authors claim SeNPs reverse OA cartilage degeneration by restoring\nantioxidant pathways. However, given the significant influence of nanoparticle morphology\nand size on bioactivity, the current characterization of these parameters is insufficient and\nrequiresenhancement.Forinstance,comprehensivecharacterizationtechniquessuchasTEM\n36\n(Transmission Electron Microscopy) for morphology visualization, DLS (Dynamic Light\nScattering) for size distribution analysis, and zeta potential measurement for surface charge\nevaluationshould be employedto provide a more accurate and detailed understanding of the\nSeNPs' physicochemical properties. Additionally, justification is needed for selecting 20 nM\nand 50 nM concentrations in validating SeNPs' effects on chondrocyte matrix metabolic\nhomeostasis.\nResponse:Wesincerelyappreciatethereviewers'valuablecomments.Thecharacterizationof\nbiomaterials constitutes a critical component of our research. SeNPs were thoroughly\ncharacterized in our previous studies (J Nanobiotechnology 2024;22(1):634), and we\napologize for any confusion caused by the omission of this information in the current\nmanuscript. In response to the reviewers' suggestions, we have now included a detailed\ncharacterization of the SeNPs synthesized in this study. Transmission electron microscopy\nwas employed to analyze the morphological structure of SeNPs (Figure R1A). The particle\nsize of SeNPs was determined to be approximately 250 nm, which is consistent with the\nresults obtained from dynamic light scattering measurements (Figure R1C). To confirm the\nelementalcompositionofthenanoparticles,energy-dispersiveX-rayspectroscopy(EDX)was\nconducted, revealing a uniform distribution of selenium on the surface of SeNPs (Figure\nR1B). As illustrated in Figure R1D, SeNPs exhibit a negative zeta potential of 11.7 mV,\nindicatingfavorablesurfacechargestability.\nRegarding the selection of SeNPs concentration, our prior study investigated the\nbiosafety profile of SeNPs across a range of concentrations (J Nanobiotechnology\n2024;22(1):634). The findings demonstrated that concentrations exceeding 50 ng/mL\nsignificantly inhibited cell proliferation and reduced cell viability. However, this\nconcentration also yielded the most favorable therapeutic outcomes. Consequently, the\noptimal therapeutic concentration of SeNPs employed in this study was established as 50\nng/mL. We apologize for the imprecise unit notations previously used in the figure captions\nandhaverevisedallambiguousannotationsforclarity.\nComment6.Pleasestandardizethecapitalizationofthefirstlettersofthex-andy-axislabels,\naswellasthefontstyleandsizeinallfigures,anddefinetheabbreviationsforeachgroup.\nWe have carefully revised all fonts and formatting elements to better support the subsequent\nreviewprocessbytheexperts.\nComment 7. The presentation of many data in the manuscript is not substandard. All\nmorphologicalimagesmustincludescalebars,suchasFigures2A,3E-M,5A-D,7B-M,S7E-I,\nandS8.Allfluorescentlystainedimagesshouldprovide informationonthefluorescent probes\nused,suchasFigures5A-D,7L-M,S6B,andS7E-F,etc.\nResponse: Thank you for your valuable feedback. We have relabeled all the images\nmentionedaboveandconductedathoroughreviewofallotherimages.\nComment8. Seleniumplays a criticalrole inmaintainingredox homeostasis by participating\nin antioxidant processes and regulating selenoprotein synthesis. However, this study lacks\nexperimentalevidencedemonstratinghowSeNPsmodulateredoxhomeostasis.\nResponse: We sincerely appreciate your insightful comments. Our study further investigated\nthe mechanisms through which HSPHR enhances cellular antioxidant capacity. Experimental\nfindings demonstrated that the in vitro antioxidant capacity of SeNPs is significantly inferior\nto that of HSPHR. In alignment with previous studies, we confirmed that HSPHR activates\nthe PI3K-AKT-mTOR signaling pathway, as evidenced by the use of both PI3K\nphosphorylation inhibitors and activators. Furthermore, we comprehensively analyzed the\nintracellular antioxidant mechanisms of HSPHR, with particular focus on mitochondrial\nprotein expression, morphological changes, shifts in energy metabolism, and the rate of\nsuperoxide anion clearance (Figures R5–R6). Collectively, HSPHR activation promotes the\nrestoration of mitochondrial protein synthesis pathways and the re-establishment of\nmitochondrial oxidative phosphorylation, thereby exerting potent intracellular antioxidant\neffectsandmitigatingcartilagedamage.\nComment9.The westernblotresults inFigure1D,and6Black reliabilityduetoinconsistent\nloadingcontrols.\nResponse: We sincerely apologize for the lack of a blank control in the original study. To\n38\naddress this limitation, we have redesigned the experiments presented in Figure 6 by\nintroducing a Ctrl group without IL-1β as a blank control. All experiments—including\nWestern blotting, immunofluorescence, and transmission electron microscopy—have been\nrepeated with this revised setup (Figure R3). The results remain consistent with our previous\nfindings: HSPHR exerts its effects by upregulating the selenoprotein TXNRD1, which\nsubsequently activates the PI3K-AKT-mTOR signaling pathway. This activation leads to the\nrestoration of normal mitochondrial metabolism and, consequently, the normalization of\nchondrocytemetabolism.\nComment 10. The therapeutic efficacy evaluation of HSPHR microspheres in early-stage\nosteoarthritis lacks critical validation through imaging modalities (e.g., Micro-CT, MRI) and\nbehavioral assessments (e.g., gait analysis), undermining the clinical relevance of reported\nhistologicalimprovements.\nResponse: Thank you for your valuable suggestions. As an exercise-related degenerative\ndisease, osteoarthritis requires early behavioral assessment and imaging observation for\ntimely intervention. Considering the challenges associated with conducting behavioral\nexperimentsinrats,we utilized6-week-oldmiceforDMMmodelingandassessedbehavioral\nchanges eight weeks after HSPHR knee joint injection therapy. Spontaneous activity levels\nwere recorded over a 3-minute period using the open field test (OFT). Observations showed\nthat, compared to the sham-operated group, DMM-operated mice exhibited significantly\nreduced relative activity levels, active time, total activity distance, and average velocity.\nThese impairments were markedly reversed following HSPHR treatment (Figure R14A–E).\nMoreover, we evaluated pain and gait status using footprint analysis. The results indicated\nthat HSPHR treatment significantly alleviated pain in DMM-induced mice, leading to\nimprovements in stride and step length, as well as reductions in fore- and hindlimb print\nlengths (Figure R14J–K). Additionally, we conducted micro-CT scans of the subchondral\nbone at2 weeks (Figure R10A, C–E) and8weeks (Figure R14F–H) after DMM surgery.The\nfindings revealed that HSPHR effectively suppressed the abnormal activation of subchondral\nosteoclasts during the early stages of osteoarthritis, thereby protecting against pathological\nprogression in subchondral bone and preventing subchondral bone sclerosis. In summary,\n39\ncomparedtotheDMM group,the HSPHR-treatedgroupdemonstrated enhancedspontaneous\nlocomotoractivityandsignificantlyimprovedphysicalperformance.\nFigure R14. HSPHR can alleviate subchondral bone sclerosis and enhance physical mobility\nin OA. (A) Representative trajectory plots displayed by activity intensity thresholds, (B–E)\nChanges in spontaneous activity, including relative activity level, activity duration, distance\ntraveled, and average speed, (F) Subchondral bone micro-CT reconstruction, (G–H)\nSubchondral bone bone volume fractionand trabecular thickness, (I) Representative footprint\nimages across groups, (J–K) Changes in relative stride length and stride frequency in\nHSPHR-treatedmice afterDMM surgery.(n= 8pergroup, *: *P < 0.05,**P< 0.01,***P <\n0.001,and****P<0.001byone-wayANOVAwithTukey’smultiplecomparisontest.)\nComment 11. Figures 7L-M, S2, S5, S6A-B, S7E, and S9 exhibit poor quality requiring\nenhanced resolution and annotation. Critically, Figure S5 lacks organ labeling in HSPHR\nbiodistributionimages.\nResponse: We sincerely appreciate the suggestions provided. All images previously\nmentioned have been re-uploaded in high-definition format.We extend our apologies for any\ninconvenience this may have caused during your review. Furthermore, concerning the\npotential concern regarding drug retention in the liver, we conducted a comprehensive\nbiosafety evaluation. Serum samples collected from rats at two and four weeks post-DMM\nadministration were analyzed to assess the long-term safety of microsphere injection (Figure\nR15). The findings demonstrate that HSPHR injection therapy does not induce visceral\ndamage.\nFigure R15. In vivo biological safety of HSPHR. (A) Blood biochemical parameters in the\nHSPHR group versus the Sham group 2 weeks after DMM surgery. (B) Blood biochemical\nparameters in the HSPHR group versus the Sham group 4 weeks after DMM surgery. (n =\n6pergroup, *:*P< 0.05,**P< 0.01,***P< 0.001,and****P< 0.001byone-wayANOVA\nwithTukey’smultiplecomparisontest.)\nprovide a more thorough analysis of their results, comparing their findings with existing\nliteratureandhighlightingtheimplicationsoftheirworkforclinicalapplications.\nResponse: We sincerely appreciate your valuable suggestions. Accordingly, we have\nexpanded the manuscript to incorporate a more comprehensive discussion, highlighting the\ndistinctionsbetweenexistingresearchandthepresentstudy,aswellasexploringthepotential\nclinicalapplicationsofthisworkinthefuture.\nComment 13. This manuscript needs a revision. The writing of this paper is too rough. The\nmanuscript requires thorough language revision due to frequent formatting errors (e.g.,\nincorrect subscript/superscript notation in chemical formulas like Na2SeO3 and H2O2),\nspelling mistakes (such as ofor, and SeNPss, etc.), and grammatical inconsistencies.\nComprehensiveEnglisheditingisrecommendedtoensurescientificrigorandclarity.\nResponse: We sincerely apologize for any difficulties you may have encountered while\nreading the manuscript. We have carefully revised and polished the document to enhance its\nclarity, thereby facilitating a more effective expert review and enabling the provision of\nvaluablefeedback.\n42"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have satisfactorily addressed most of my concerns. However, the term “three birds with one stone”, while catchy, may still be somewhat overstated without direct comparative efficacy data against other multi-target therapies. The authors should tone down the phrasing unless they can provide comparative evidence.", "reviewer_3": "Comments: The authors have satisfactorily addressed the majority of my previous concerns through extensive revisions. The manuscript has been significantly improved in terms of experimental justification, data presentation, and clarity. In my view, the paper now meets the scientific standards of Nature Communications. I recommend acceptance pending minor revisions, primarily focused on further polishing of the language to enhance readability and precision.", "reviewer_4": "The authors have made substantial improvements in response to the previous reviewers' comments. The manuscript now presents a more complete and coherent study, with clarified methodologies and strengthened data interpretation. The conceptual framework of a ROS/MMP13 dual-responsive organic selenium hydrogel microsphere (HSPHR) for multi-tissue synergistic therapy in osteoarthritis is both innovative and well-supported by experimental evidence. The mechanistic insights into TXNRD1 as a key regulator across cartilage, synovium, and subchondral bone pathways significantly enhance the scientific value of this work.\nHowever, a few minor revisions are still required to meet the journal’s publication standards.\nMinor Comments:\n1.Some subheadings are quite long, for example, “HSPHR hydrogel microspheres improve cartilage metabolic homeostasis in osteoarthritis by restoring mitochondrial function…”. It is suggested to refine them appropriately to conform to the standard scientific writing format, which will facilitate readers in quickly locating the key points of the research.\n2.Please provide the full form when an abbreviation appears for the first time (e.g., MRP), and unify the font and line spacing.\n3.Please check whether the label “Glucose” on the ECAR experiment graph in the article should be replaced with “Rot/AA” and make the necessary changes.\n4.The discussion section is overly lengthy and should be streamlined, such as the discussion on SeNPs in Fig. 3.\nOnce these minor issues are addressed, the manuscript should be suitable for publication."}, "author_response": "Response to Reviewers' Comment\nReviewer #1 (Remarks to the Author):\nThe authors have satisfactorily addressed most of my concerns. However, the term “three birds\nwith one stone”, while catchy, may still be somewhat overstated without direct comparative\nefficacy data against other multi-target therapies. The authors should tone down the phrasing\nunless they can provide comparative evidence.\nResponse：We sincerely appreciate the reviewers' constructive suggestions. We have revised\nthe manuscript title to Organic di-selenide hydrogel microspheres for multimodal treatment of\nosteoarthritis in accordance with your recommendations and the journal's guidelines. The\ncorresponding section within the manuscript has also been amended accordingly.\nReviewer #3 (Remarks to the Author):\nComments: The authors have satisfactorily addressed the majority of my previous concerns\nthrough extensive revisions. The manuscript has been significantly improved in terms of\nexperimental justification, data presentation, and clarity. In my view, the paper now meets the\nscientific standards of Nature Communications. I recommend acceptance pending minor\nrevisions, primarily focused on further polishing of the language to enhance readability and\nprecision.\nResponse: We are extremely grateful for your positive comments. We have polished the\nmanuscript to enhance its readability.\nReviewer #4 (Remarks to the Author):\nThe authors have made substantial improvements in response to the previous reviewers'\ncomments. The manuscript now presents a more complete and coherent study, with clarified\nmethodologies and strengthened data interpretation. The conceptual framework of a\nROS/MMP13 dual-responsive organic selenium hydrogel microsphere (HSPHR) for multi-\ntissue synergistic therapy in osteoarthritis is both innovative and well-supported by\nexperimental evidence. The mechanistic insights into TXNRD1 as a key regulator across\ncartilage, synovium, and subchondral bone pathways significantly enhance the scientific value\n1\nof this work.\nHowever, a few minor revisions are still required to meet the journal’s publication standards.\nMinor Comments:\n1. Some subheadings are quite long, for example, “HSPHR hydrogel microspheres improve\ncartilage metabolic homeostasis in osteoarthritis by restoring mitochondrial function…”. It is\nsuggested to refine them appropriately to conform to the standard scientific writing format,\nwhich will facilitate readers in quickly locating the key points of the research.\nResponse: We express our sincere gratitude for your valuable suggestion. In response, we have\nrefined certain excessively lengthy subheadings in the revised manuscript, thereby improving\nboth the precision of the research and the overall readability of the document.\n2. Please provide the full form when an abbreviation appears for the first time (e.g., MRP),\nand unify the font and line spacing.\nResponse: We express our gratitude for your insightful suggestion. We have meticulously\nreviewed all abbreviations appearing for the first time in the text to ensure they are accompanied\nby comprehensive explanations at their initial occurrence.\n3. Please check whether the label “Glucose” on the ECAR experiment graph in the article\nshould be replaced with “Rot/AA” and make the necessary changes.\nResponse: We express our sincere gratitude for your constructive suggestion and offer our\ndeepest apologies for the inaccuracies in labeling presented in the ECAR results. This issue has\nbeen comprehensively addressed and clarified in the Methods section, and we have\nsubsequently corrected the labeling of the experimental components of ECAR in Figure 6.\n4.The discussion section is overly lengthy and should be streamlined, such as the discussion on\nSeNPs in Fig. 3.\nResponse: We sincerely appreciate your constructive suggestion. We have refined all sections\nof the discussion in the manuscript and streamlined the discussion in Figure 3.\n2"}]

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