Immunomodulatory thermosensitive hydroxypropyl chitin hydrogel platform maintains cartilage-synovium homeostasis for enhanced osteoarthritis therapy

Abstract

Osteoarthritis (OA) progression is fueled by a self-perpetuating cycle of synovitis and cartilage degradation. Current clinical therapies exhibit suboptimal efficacy, primarily due to the lack of strategies that target the multifactorial pathogenesis of OA. Herein, we developed an injectable thermosensitive hydroxypropyl chitin hydrogel (HPCH) loaded with an optimized concentration of dimethyloxalylglycine (DMOG), designated as HD₂₅, to disrupt this pathological cycle and achieve multi-modal OA therapy. HD₂₅ reprograms macrophages toward an anti-inflammatory M2 phenotype via the JAK-STAT pathway, thereby attenuating synovitis and inflammation-driven matrix degradation. In parallel, HD₂₅ provides favorable lubrication to ameliorate mechanical stress-induced wear and tear, promotes mesenchymal stem cell recruitment and chondrogenesis, and directly protects cartilage by suppressing chondrocyte hypertrophy, apoptosis, and extracellular matrix catabolism. In surgery-induced OA mice, HD₂₅ treatment effectively preserved cartilage integrity, attenuated synovitis, and restored subchondral bone remodeling. At 10 weeks, HD₂₅ reduced the proportion of MMP13-positive chondrocytes from approximately 35% to 13%. Moreover, it decreased the proportion of pro-inflammatory F4/80⁺iNOS⁺ synovial macrophages from approximately 60% to 20%, while increasing anti-inflammatory F4/80⁺CD206⁺ macrophages from approximately 15% to 30%. Crucially, in situ macrophage-depletion experiments confirmed that the therapeutic efficacy of HD₂₅ largely depends on macrophage reprogramming, underscoring the superiority of immunomodulation over cell clearance. This multifunctional hydrogel platform orchestrates chondroprotection, immunomodulation, and tissue remodeling to break the OA vicious cycle, presenting a promising disease-modifying strategy for OA management.

Graphical abstract

Highlights

• •An injectable HPCH hydrogel loaded with DMOG achieves multi-modal OA therapy.
• •HD₂₅ disrupts OA vicious cycle via coordinated immunomodulation and chondroprotection.
• •HD₂₅ reprograms macrophages toward an M2 phenotype via the JAK-STAT signaling pathway.
• •Immune education via macrophage reprogramming, not ablation, sustains joint homeostasis.
• •HD₂₅ preserves cartilage, synovitis, and subchondral bone homeostasis in vivo.

1. Introduction

Osteoarthritis (OA) is a prevalent degenerative joint disease globally, affecting more than 500 million people. Both its prevalence and the associated years lived with disability have risen substantially since 1990, and this upward trend is projected to continue, with an estimated 1.1 billion individuals affected by 2050 and imposing a substantial and growing global burden of pain, disability, and reduced quality of life [1]. OA is no longer regarded as a simple consequence of cartilage “wear and tear,” but rather a complex whole-joint disorder involving progressive cartilage degradation, chronic synovitis, and aberrant subchondral bone remodeling [[2], [3], [4]]. These pathological processes are tightly interconnected and form a self-perpetuating vicious cycle. Mechanical or biochemical insults to the joint initiate the release of damage-associated molecular patterns (DAMPs), which trigger synovial inflammation, leading to further tissue destruction and the release of additional DAMPs [5,6]. Despite advances in symptomatic management, current therapies fail to halt or reverse structural deterioration, underscoring the urgent need for disease-modifying strategies capable of disrupting this pathogenic feedback loop [7].

Among the interconnected pathological features of OA, synovitis has emerged as a pivotal driver of disease initiation and progression [3]. The presence and severity of synovial inflammation correlate strongly with clinical symptoms and radiographic progression across all disease stages [[8], [9], [10]]. Macrophages, the most abundant innate immune cells in the synovium, are central orchestrators of this inflammatory milieu [11,12]. Their functional polarization into pro-inflammatory (M1) or pro-regenerative (M2) phenotypes critically influences disease trajectory [13,14]. Notably, synovitis alone can initiate OA-like pathological changes, even in the absence of primary cartilage injury, highlighting the causal role of immune dysregulation [15]. An imbalance toward the M1 phenotype fuels cartilage breakdown by driving cytokine release (e.g., IL-1β, TNF-α), creating a feed-forward loop of inflammation and degradation. This sustained inflammatory state drives chondrocyte dysfunction, extracellular matrix (ECM) degradation, and pathological bone remodeling [16]. Therefore, a promising therapeutic strategy is to target both arms of this cycle concurrently, rebalancing the dysregulated immune microenvironment to resolve inflammation while directly protecting cartilage integrity to reduce the initial release of DAMPs. This strategy could effectively disrupt the pathological feedback loop, offering a potent approach to halt OA progression.

To implement such a dual-action strategy, an effective local delivery system is required. Direct intra-articular delivery offers a rational approach to OA therapy by overcoming the avascularity of cartilage and minimizing systemic side effects [17]. Given the multifactorial pathology of OA, combination strategies targeting multiple joint tissues are increasingly emphasized [[18], [19], [20]]. Injectable hydrogels have emerged as particularly attractive biomaterial-based delivery platforms, enabling sustained, localized release of therapeutics while providing biomechanical and biochemical support [5,21]. As soft, three-dimensional networks that retain substantial water, hydrogels offer unique advantages for joint applications, including excellent biocompatibility, inherent lubricating properties, and the capacity for sustained drug release, all of which are critical for managing chronic conditions such as OA [[22], [23], [24]]. Their minimally invasive nature allows for direct administration, conformal filling of the irregular joint space, and localized, prolonged delivery of therapeutics [25]. Among these platforms, thermosensitive hydrogels, which undergo a reversible sol-gel transition at physiological temperature, provide exceptional operational simplicity.

Among the diverse therapeutic agents that could be delivered via such platforms, dimethyloxalylglycine (DMOG), a small-molecule inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylases, exhibits multifaceted potential. DMOG can stabilize HIF-1α, promoting chondrocyte survival and anabolism [26,27]. Furthermore, DMOG favorably directs stem cell fate, promoting chondrogenic differentiation while inhibiting undesired hypertrophy, a critical balance for functional cartilage repair [28,29]. Beyond these anabolic actions, DMOG can skew macrophage polarization toward a reparative phenotype, thereby enhancing matrix deposition [30,31]. However, the clinical translation of free DMOG is hampered by rapid clearance by joint synovial fluid turnover, and its immunomodulatory mechanism remains unclear.

To overcome these limitations, our group previously developed a thermosensitive hydroxypropyl chitin hydrogel (HPCH), which has demonstrated robust performance as a scaffold for cartilage regeneration and as a sustained drug-delivery depot, particularly for hydrophilic small molecules such as DMOG [[32], [33], [34]]. Building on this foundation, we engineered an injectable, thermosensitive HPCH hydrogel loaded with DMOG (denoted HD) as an integrated therapeutic platform for enhanced OA therapy (Scheme 1). In this study, we characterized the key physicochemical properties of HD with an optimized concentration of DMOG (HD₂₅), including its microstructure, degradation, drug release kinetics, and lubricating performance. We then systematically investigated its immunomodulatory capacity, elucidating the central role of the JAK-STAT signaling pathway in HD₂₅-mediated macrophage reprogramming. Using in vitro multicellular 3D cartilage spheroids and ex vivo cartilage explants, we further demonstrated the direct chondroprotective and anabolic effects of HD₂₅. The therapeutic efficacy of HD₂₅ was rigorously evaluated in vivo in a surgically induced murine OA model. Finally, through in vivo macrophage-depletion experiments, we established that the therapeutic benefits of HD₂₅ depend on macrophage reprogramming rather than ablation, thereby validating a paradigm of immunomodulation over simple cell clearance. This study presents a potent, multifunctional therapeutic candidate and provides mechanistic insights into the critical vicious cycle between synovitis and cartilage homeostasis, offering a promising disease-modifying OA strategy.

Scheme 1.

Schematic illustration for the preparation of a hydrogel platform and its application for effective OA alleviation by modulating cartilage-synovium homeostasis.

2. Materials and methods

2.1. Materials

Chitin was supplied by Golden Shell Biochemical Co., Ltd. (China). The viscosity-average molecular weight (Mη) of chitin is 3.75 × 10⁵ Da, and the degree of acetylation is 0.97 according to our previous work [34,35]. Dimethyloxalylglycine (DMOG) was obtained from MedChemExpress (China). Hyaluronic acid (HA, prepared by microbial fermentation, weight-average molecular weight (Mw) = 1.4 × 10⁶ Da) was obtained from Bloomage Biopharm (China). Cell culture reagents, including Dulbecco's Modified Eagle Medium (DMEM), phosphate-buffered saline (PBS), 0.25% trypsin-EDTA, fetal bovine serum (FBS), penicillin-streptomycin (P/S), L-glutamine, and sodium pyruvate, were purchased from Gibco (USA). Cell culture plates, tubes, and Transwell plates were sourced from Corning (USA). Staining kits (Safranin O/Fast Green, Alcian Blue) and a bicinchoninic acid (BCA) protein assay kit were provided by Solarbio (China). Recombinant cytokines (M-CSF, IL-4, IL-13, and IFN-γ) were obtained from Peprotech (USA). The CCK-8 assay kit was purchased from Dojindo (Japan). The total RNA extraction kit was obtained from Omega Bio-Tek (USA), while the cDNA synthesis and quantitative PCR kits were from TransGen Biotech (China). Fluorescently conjugated antibodies for flow cytometry (FCM) were procured from BioLegend (USA) and Invitrogen (USA). ELISA kits were purchased from Jiangsu Meimian Industrial Co., Ltd (China). Primary and secondary antibodies for western blotting and immunofluorescence (IF) were supplied by Abcam (USA). Lipopolysaccharide (LPS), ITS (insulin, transferrin, sodium selenite) supplement, ascorbic acid, dexamethasone, β-glycerol phosphate, 4',6-diamidino-2-phenylindole (DAPI), and general chemicals were acquired from Sigma-Aldrich (USA).

2.2. Synthesis and characterization of the HD composite

HPCH was synthesized according to our established homogeneous NaOH/urea aqueous method, and its degree of substitution and acetylation were determined by ¹H NMR in D₂O at 25 °C as previously described [33,35]. Briefly, chitin dissolved in NaOH/urea was reacted with 1,2-propylene oxide at 15 °C for 6 h. The product was neutralized, then subjected to extensive dialysis, followed by lyophilization. For hydrogel formation, the lyophilized HPCH was dissolved in PBS to a final concentration of 2.5% (w/v). To prepare the drug-loaded composite, a concentrated DMOG stock solution was thoroughly mixed with the HPCH precursor solution, yielding final concentrations of 2% (w/v) HPCH and 25 μg/mL DMOG (HD₂₅). These concentrations were derived from our previous work [32,36] and corroborated by the preliminary dose-response screening results detailed in Section 3.2. The surface morphology of the hydrogel system was examined by a field-emission scanning electron microscope (FESEM, Sirion 200, Holland). HPCH degradation was assessed by weight loss in a 3 mg/mL lysozyme solution at 37 °C for 18 days, with PBS as the control. At each time point, the samples were removed from the solution and weighed. The weight-loss ratio was normalized to the samples' initial weight. Temperature-dependent rheological properties were investigated using a rheometer (Anton Paar MCR301, Austria) equipped with a parallel-plate geometry and a Peltier temperature control system. The lubricating properties of the hydrogel (2% w/v) were evaluated using a tribometer (Anton Paar TRP3, Switzerland) in reciprocating mode. Experiments were performed at room temperature (RT) using a polytetrafluoroethylene (PTFE) ball against a stainless-steel disk under a 5 N load, with a sliding amplitude of 4 mm and a frequency of 1 Hz for 600 cycles. The coefficient of friction (COF) was recorded throughout the test.

2.3. In vitro drug release study

The release profile of DMOG from the HPCH hydrogel was investigated using a conventional sample-and-separate method. Briefly, the DMOG-loaded hydrogel samples were placed in 15-mL centrifuge tubes containing 5 mL of phosphate-buffered saline (PBS, pH 7.4) and incubated at 37 °C under constant orbital shaking at 100 rpm. At predetermined time intervals (0.5, 1, 2, 6, 10, 20, 36, 72, and 144 h), an aliquot of 4 mL (80% of the total release medium) was withdrawn and replaced with an equal volume of fresh pre-warmed PBS to maintain sink conditions. The concentration of released DMOG in the aliquots was quantified using reverse-phase high-performance liquid chromatography (HPLC, UltiMate 3000 system, USA). A calibration curve was constructed using serial dilutions of DMOG standard in PBS. The cumulative percentage of DMOG released was calculated based on the measured concentrations, corrected for the volume replacement. All experiments were performed in triplicate. Quantification was based on a validated calibration curve.

2.4. Cell isolation and culture

Bone marrow-derived macrophages (BMDMs) were isolated from the femurs and tibiae of 6 to 8 weeks old C57BL/6 mice [37]. BMDMs were generated by culturing bone marrow progenitors for 5-7 days in medium containing 30 ng/mL macrophage colony-stimulating factor (M-CSF). Primary chondrocytes were isolated from the articular cartilage of 1-week-old mice and cultured in DMEM/F12 medium with 10% FBS. The C3H10T1/2 cell line was obtained from ATCC as a mouse stem cell. All cells were cultured under standard conditions at 37 °C in a humidified atmosphere of 5% CO₂.

2.5. Biocompatibility assessment

The biocompatibility of the hydrogel system was evaluated using a CCK-8 assay [38]. MSCs were seeded in 96-well plates and cultured with various hydrogel systems for 1 and 3 days, after which cell viability was quantified according to the manufacturer's protocol. For 3D biocompatibility assessment, primary chondrocytes were encapsulated within the hydrogel, cultured for 3 days, and assessed using Live/Dead staining visualized by confocal laser scanning microscopy (CLSM).

2.6. Flow cytometry (FCM)

Following treatment, BMDMs were harvested for flow cytometric analysis of surface marker expression. Cells were gently detached using Accutase™ (Invitrogen, USA) and collected by centrifugation at 300 × g for 5 min at 4 °C. The cell pellet was washed twice with ice-cold PBS and resuspended in FACS buffer (PBS supplemented with 1% bovine serum albumin). Cell density was adjusted to approximately 1 × 10⁶ cells/mL, and 1 mL of the cell suspension was aliquoted into each polystyrene flow cytometry tube for unstained controls, fluorescence-minus-one (FMO) controls, and fully stained samples. For surface marker analysis, cells were first incubated with FVS780 for 30 min at 4 °C in the dark to exclude dead cells from subsequent analysis. After viability staining, cells were washed with FACS buffer and centrifuged at 300 × g for 5 min at 4 °C. Subsequently, cells were stained with the following fluorochrome-conjugated anti-mouse antibodies for 30 min at 4 °C in the dark: CD11b-FITC, F4/80-PE, CD86-PE-Cy7, and CD206-AF647. All antibodies were titrated prior to use to determine the optimal working concentrations. Following staining, cells were washed twice with FACS buffer and centrifuged to remove unbound antibodies, and finally resuspended in FACS buffer for acquisition.

For phospho-protein analysis, after the indicated interventions, BMDMs were fixed and permeabilized using Cytofix fixation buffer and Phosflow Perm buffer III (BD, USA), respectively. Phosphorylated STAT1 (p-STAT1) and STAT6 (p-STAT6) were used to stain phosphorylated signaling molecules. FCM was performed on a Beckman CytoFLEX instrument, and data were analyzed using FlowJo software. Positive controls for M1 (LPS/IFN-γ) and M2 (IL-4/IL-13) polarization were included in the experiments.

2.7. Western blotting

Total protein extracts were obtained via the lysis of macrophages or MSCs treated in cold RIPA buffer supplemented with a protease inhibitor cocktail (Thermo, USA). After incubation on ice for 30 min, lysates were centrifuged at 12,000 × g for 15 min at 4 °C to remove insoluble debris. Protein concentrations in the supernatants were determined using a BCA protein assay kit. Equal amounts of protein (20–30 μg per lane) were mixed with 4× Laemmli loading buffer and denatured by boiling for 10 min. Subsequently, equal concentrations of the samples were separated by 10% SDS-PAGE on a precast gel (Epizyme, China) and then transferred to polyvinylidene fluoride membranes (Millipore, USA). Membranes were blocked with 5% non-fat milk or 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature to prevent non-specific binding. Following blocking, the membranes were incubated with specific antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. GAPDH was used as a loading control. Image Lab 3.0 software (Bio-Rad) was used to quantify protein band intensities, which were normalized to GAPDH.

2.8. Enzyme-linked immunosorbent assay (ELISA)

According to a standardized protocol, the concentrations of secreted cytokines (IL-1β, TNF-α, IL-10, and Arg-1) in cell culture supernatants were quantified using commercial ELISA kits. Absorbance measurements at 450 nm were obtained using a microplate reader (BioTek, USA).

2.9. qRT-PCR

Total RNA was extracted from treated cells using a commercial RNA extraction kit according to the manufacturer's instructions. RNA concentration and purity were determined spectrophotometrically using a NanoDrop 2000c (Thermo Fisher Scientific, USA). First-strand cDNA was synthesized from 1 μg of total RNA, followed by quantitative PCR [39]. Melting curve analysis was conducted to confirm primer specificity. The relative expression of target genes was calculated using the 2^−ΔΔCt method, with Gapdh serving as the endogenous reference. Primer sequences are listed in Supplementary Table 1.

2.10. Cellular immunofluorescence

BMDMs were seeded in confocal dishes at a density of 1 × 10⁴ cells per well and treated as specified. Following treatment, cells were processed for immunostaining using a standard protocol: fixation with 4% paraformaldehyde (PFA, 10 min), permeabilization with 0.2% Triton X-100 (10 min), and blocking with 5% BSA (30 min at RT). Cells were then incubated overnight at 4 °C with primary antibodies against CD206 (1:200) or iNOS (1:200), followed by incubation with Alexa Fluor-conjugated secondary antibodies (1:500) for 1 h at RT. Nuclei were counterstained with DAPI for 5 min. Stained samples were imaged using CLSM. All experiments were performed in triplicate to ensure reproducibility.

2.11. Bulk-RNA sequencing

HD₂₅, HPCH, or PBS-treated macrophages were processed for transcriptomic profiling following the manufacturer's instructions (Lianchuan Bio, China). Visualization and functional enrichment analyses (e.g., GO, KEGG, and GSEA) were conducted using the OmicStudio tools (https://www.omicstudio.cn) to further explore the biological pathways modulated by the treatments. |log₂FC| > 1 and adjusted P < 0.05 were used to define differentially expressed genes.

2.12. Induction of chondrogenesis

To assess the direct chondrogenic potential of the hydrogel system, C3H10T1/2 cells were seeded at a density of 1 × 10⁵ cells per well in a 24-well plate and cultured in chondrogenic differentiation medium. The medium consisted of DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 1× ITS, 50 μM ascorbic acid, 100 nM dexamethasone, 1% sodium pyruvate, and 10 ng/mL recombinant TGF-β1. Cells were cultured for 14 days with medium changes every 2-3 days. Chondrogenesis was evaluated using multiple endpoints, including qRT-PCR, Alcian blue staining, the dimethylmethylene blue (DMMB) assay, and Western blotting (WB). For the DMMB assay, cell layers were digested with papain, and the digested lysate was reacted with DMMB dye. Absorbance was measured at 525 nm and compared to a chondroitin sulfate standard curve. Results were normalized to total protein content, as determined by a BCA assay.

2.13. Multicellular cartilage spheroids

Chondrospheroids were generated using primary chondrocytes as previously described. Briefly, chondrocytes were resuspended in DMEM/F12 medium at a density of 1 × 10⁶ cells/mL. 500 μL of the cell suspension was transferred to conical tubes and centrifuged (300 × g, 5 min) to form a pellet. The supernatant was carefully removed, and the cell pellet was gently resuspended in chondrogenic induction medium and centrifuged again to promote aggregate formation. After 48 h of stabilization, the resulting spheroids were transferred to 24-well plates and cultured with or without the hydrogel composite. The culture medium was changed every two days during the 21-day differentiation culture. On day 21, spheroids were harvested, fixed in 4% PFA, and cryosectioned for subsequent histological and molecular analyses.

2.14. Evaluation of cell apoptosis

To evaluate cellular apoptosis, chondrocytes exposed to IL-1β (10 ng/mL) treated with or without HD₂₅ were first detached using Accutase™. The harvested cell suspensions were collected by centrifugation at 800g for 4 min. After three washes with PBS and a subsequent rinse with binding buffer, apoptotic cells were identified via the Annexin V-FITC/PI dual-staining kit (Dojindo, Japan), strictly adhering to the provider's instructions. Fluorescence signals were acquired on a Beckman flow cytometer controlled by Cytexpert software under identical voltage settings for all runs, and the resulting data were processed using FlowJo 10.9.0.

2.15. Transwell assay

Cell migration was evaluated using a Transwell system with 8.0 μm pore inserts [40]. MSCs, 2 × 10⁴ cells/insert in serum-free medium, were placed above HD-containing chemoattractant medium. After a 12-h migration (37 °C, 5% CO₂), transmigrated cells were fixed with PFA and stained with 0.5% crystal violet. Migrated cells from five random fields per insert were counted using ImageJ software.

2.16. Animal experiments

All animal procedures were performed in accordance with protocols approved by the Ethics Committee of Guangdong Provincial People's Hospital (No. KY-Z-2021-544-01) and complied with the National Research Council's “Guide for the Care and Use of Laboratory Animals”. OA was surgically induced in the right knee joint of 12-week-old male C57BL/6 mice via destabilization of the medial meniscus (DMM). Age-matched sham-operated mice (incision without meniscal destabilization) served as controls. The joint capsule and skin incisions were closed in layers using sutures. One week post-surgery, mice were randomly allocated into four treatment groups (n = 5): Sham, Saline, HPCH, and HD₂₅. Weekly intra-articular injections (10 μL) commenced at this time point and continued throughout the study. For macrophage depletion within the articular cavity, animals received weekly intra-articular injections of 10 μL clodronate-encapsulated liposomes at 5 mg/mL, beginning at one week post-surgery. An equal volume of PBS liposomes served as the control. Depletion efficiency was subsequently evaluated by immunofluorescence staining for macrophage marker F4/80. Mice were euthanized at 5 and 10 weeks post-surgery. Harvested knee joints were fixed in 4% PFA and then imaged using a high-resolution micro-computed tomography (micro-CT) system (Bruker Skyscan, UK). Following micro-CT scanning, the samples were decalcified, dehydrated, embedded in paraffin, and sectioned into 5 μm-thick slices. The sections were subsequently processed for histological analysis (H&E, Safranin O/Fast Green, Toluidine Blue, TRAP staining), immunofluorescence, and immunohistochemistry (as detailed in Supporting Information).

2.17. Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0 (GraphPad Software Inc, USA). Data are presented as mean ± standard deviation (SD). Differences between the two groups were analyzed by an unpaired Student's t-test. For comparisons among three or more groups, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used. Before ANOVA, normality was assessed using the Shapiro-Wilk test and homogeneity of variance was assessed using Levene's test. When assumptions were not met, the Kruskal-Wallis non-parametric test was applied. A p-value <0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001). All experiments were performed with at least three independent replicates.

3. Results

3.1. Synthesis and characterization of the DMOG-loaded hydrogel (HD)

The molar substitution (MS) of hydroxypropyl groups and the degree of acetylation (DA) for the synthesized HPCH hydrogel were determined from its ¹H NMR spectrum to be 0.74 and 0.89, respectively (Fig. S1). HPCH exhibited a reversible thermosensitive sol-gel transition, remaining fluid at 4 °C and forming a stable gel at 37 °C (Fig. 1A and B). This property enabled easy injection through a fine needle at low temperature, followed by in situ gelation at body temperature to form stable constructs of various shapes, confirming its suitability for minimally invasive intra-articular delivery (Fig. 1C). Scanning electron microscopy (SEM) revealed that both HPCH and HD₂₅ possessed a highly porous, three-dimensional network structure with interconnected pores and a uniform pore-size distribution (Fig. 1D), an architecture favorable for nutrient diffusion and controlled drug release. In vitro degradation studies showed that the hydrogel maintained structural stability in PBS with minimal degradation over 18 days, while undergoing lysozyme-mediated hydrolysis, losing approximately 50% of its mass within 5 days in a 3 mg/mL lysozyme solution (Fig. 1E). The release profile of DMOG was characterized by an initial rapid release within the first 12 h, followed by a sustained release lasting over 6 days (Fig. 1F). Rheological analysis of 2% HPCH further confirmed the thermosensitive behavior, showing a sharp increase in storage modulus (G′) and loss modulus (G″) upon heating from 5 °C to 40 °C, with the gelation temperature determined to be approximately 24.2 °C, which falls well below physiological temperature, confirming its suitability as an injectable hydrogel system that rapidly forms a stable gel upon in vivo administration (Fig. 1G). Given the critical role of lubrication in joint function, the tribological performance of HPCH was evaluated using a ball-on-disk configuration under simulated physiological load (5 N) and frequency (1 Hz). The COF of HPCH (2% w/v ) was comparable to the standard intra-articular lubricant, hyaluronic acid (HA, 1 mg/mL), demonstrating its favorable lubricating properties (Fig. 1H and I; Fig. S1). The biocompatibility of the system was assessed with key joint cell types. A CCK-8 assay indicated that both HPCH and HD₂₅ supported high viability of MSCs over 3 days (Fig. 1J). Furthermore, primary chondrocytes encapsulated within the HD₂₅ hydrogel maintained excellent viability with minimal cell death after 3 days in 3D culture, as visualized by Live/Dead staining (Fig. 1K).

Fig. 1.

Synthesis and characterization of the HD composite. (A) Schematic diagram showing the synthesis of the hydrogel system. (B) Photographs illustrating the reversible thermosensitive sol-gel transition of the hydrogel. (C) Photograph demonstrating the injectability of the HPCH precursor solution. (D) Representative SEM images of the porous structures of HPCH hydrogel and HD. Scale bar: 200 μm. (E) In vitro degradation profiles of HPCH hydrogels incubated in PBS or in 3 mg/mL lysozyme solution at 37 °C. (F) Cumulative release profile of DMOG from HD₂₅ in PBS at 37 °C (n = 3). (G) Temperature-dependent rheological properties of 2% HPCH: storage modulus (G′) and loss modulus (G″). (H) COF versus time curves and (I) the corresponding average COF values of hyaluronic acid and HPCH hydrogel. (J) Viability of C3H10T1/2 cells cultured with the hydrogel systems for 1 and 3 days, tested by CCK-8 assay (n = 4). (K) Live/Dead staining of primary chondrocytes encapsulated within the HPCH hydrogel after 3 days of culture. ns indicates no significant difference.

3.2. HD reprograms macrophage polarization to an anti-inflammatory phenotype in vitro

Given the pivotal role of macrophage polarization in OA pathogenesis, we investigated the immunomodulatory capacity of the HD hydrogel system on bone marrow-derived macrophages (BMDMs). LPS/IFN-γ or IL-4/IL-13 intervention served as positive controls for M1 and M2 polarization, respectively. To determine the optimal DMOG concentration for immunomodulation, we first screened a range of DMOG concentrations (0, 10, 25, 50, and 100 μg/mL) in BMDMs. PCR analysis revealed that HD₂₅ (25 μg/mL) most effectively upregulated M2-associated genes (Mrc1, Arg1, and Il10) while suppressing M1 markers (Il1β and Tnf). Notably, higher concentrations (50 and 100 μg/mL) exhibited diminished or even reversed effects, suggesting a dose-dependent biphasic response (Fig. S2). Based on this profile, HD₂₅ was selected as the optimal formulation for subsequent experiments, with HD₅₀ included for comparison in key assays [32]. We first examined the phenotypic modulation of BMDMs under basal conditions (cultured in complete medium without exogenous cytokines). Western blot analysis revealed that HD treatment significantly upregulated CD206 protein expression while downregulating CD86 and TNF-α compared with the control group, as shown by the representative blots and corresponding densitometric quantification, with HD₂₅ showing the best regulatory effects (Fig. 2A and B; Fig. S3 and S4A). IF staining further confirmed the enhanced CD206 signal in HD-treated BMDMs (Fig. 2C), whereas the iNOS signal was decreased in HD-treated BMDMs compared to the HPCH group (Fig. S4B). Flow cytometric analysis demonstrated that HD₂₅ treatment significantly elevated the proportion of CD206⁺ cells (Fig. 2D and E). Notably, HPCH alone increased CD86 expression, whereas the incorporation of DMOG effectively shifted the phenotype toward CD206 upregulation while concurrently suppressing CD86, highlighting the prominent immunomodulatory role of DMOG (Fig. 2D and E). Consistent with this phenotypic shift, HD₂₅ treatment significantly upregulated the mRNA levels of M2-associated genes Il10 and Arg1 (Fig. 2F) and elevated the secretion of Arg-1 and IL-10 proteins, as determined by ELISA, with approximately 2-fold increases over the Ctrl group (Fig. 2G).

Fig. 2.

Immunomodulatory effects of HD on macrophages in vitro. (A) Representative Western blot bands and (B) semi-quantitative analysis of CD86 and CD206 expression in BMDMs following various treatments. (C) IF staining of CD206 (green) in BMDMs after different treatments. Nuclei were counterstained with DAPI (blue). Scale bar: 25 μm. (D) Representative FCM plots and (E) the corresponding quantitative analysis of the expression of M1 marker (CD86), M2 marker (CD206) and the CD206/CD86 ratio, reflecting the phenotypic shift. (F) Relative mRNA expression of M2-associated genes (Il10 and Arg1) assessed by qRT-PCR (n = 3). (G) Secretion of M2-associated factors Arg-1 and IL-10, measured by ELISA (n = 3–4). (H) qRT-PCR analysis of both M1-and M2-associated gene expression profiles in treated BMDMs (n = 3). (I) Representative FCM histograms and quantitative fluorescence intensity of CD86 in M1 macrophages post-treatment (n = 3). (J) Levels of IL-1β and TNF-α in the supernatant of M1 macrophages after treatment with different hydrogel systems were assessed by ELISA (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001.

We next challenged BMDMs with LPS/IFN-γ to induce a pro-inflammatory M1 phenotype and assessed the capacity of HD to reverse this activated state. HPCH alone did not exacerbate inflammation and showed a modest capacity to reduce Il1β and Cd86, accompanied by a slight increase in Arg1. Notably, HD₂₅ treatment robustly suppressed the expression of key M1 markers (Il1β, Tnf, Nos2, and Cd86) at the transcriptional level, while simultaneously enhancing the expression of M2-related genes including Arg1, Pparg, and Il10 (Fig. 2H). Flow cytometric analysis further confirmed that HD₂₅ treatment significantly reduced CD86 mean fluorescence intensity (MFI) in M1 macrophages (Fig. 2I). Consistently, ELISA results demonstrated that HD₂₅ markedly decreased the secretion of pro-inflammatory cytokines IL-1β and TNF-α from M1 macrophages compared with the HPCH and Ctrl groups.

Collectively, these data demonstrate that the HD composite effectively reprograms macrophages toward an anti-inflammatory phenotype, with DMOG providing the principal immunomodulatory stimulus. This modulation establishes a key mechanism through which HD may alleviate synovial inflammation and contribute to the restoration of cartilage homeostasis.

3.3. HD₂₅ reprograms macrophage polarization via the JAK-STAT signaling pathway

To elucidate the molecular mechanism underlying HD-mediated immunomodulation, we performed bulk RNA sequencing on BMDMs treated with PBS, HPCH, or HD₂₅ (Fig. 3A), as HD₂₅ exhibited the optimal in vitro immunomodulatory effects. Volcano plot analysis revealed 880 genes significantly upregulated (red) and 642 downregulated (blue) in the HD₂₅ group relative to the PBS control. A focused comparison between HD₂₅ and HPCH identified 71 differentially expressed genes (31 upregulated, 40 down-regulated), highlighting the specific transcriptional reprogramming induced by DMOG (Fig. 3B). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes (DEGs) demonstrated that the HD₂₅ versus Ctrl comparison was significantly enriched in pathways critical for immune regulation, including the cytokine-cytokine receptor interaction, TNF signaling pathway, PI3K-Akt signaling pathway, and, notably, the JAK-STAT signaling pathway (Fig. 3C and D). Enrichment of the JAK-STAT and TNF signaling pathways was also prominent when comparing HD₂₅ and HPCH. Furthermore, Gene Ontology (GO) analysis confirmed that inflammatory response, immune system processes, and cytokine activity were prominently associated with the regulatory mechanisms of HD₂₅ (Fig. S5A and B). Gene Set Enrichment Analysis (GSEA) revealed suppression of the TNF signaling pathway, IL-1 family signaling, IL-6 family signaling, and tyrosine phosphorylation of STAT proteins in HD₂₅-treated BMDMs, alongside an upregulation of the PPAR signaling pathway (Fig. 3E). In addition, the overall downregulation of inflammatory and immune responses in BMDMs after HD₂₅ treatment was evident in GSEA (Fig. S5C). Among the pathways identified by the transcriptomic analysis, we prioritized the JAK-STAT axis for subsequent validation based on both the enrichment results and the known biological properties of HPCH. HPCH is a water-soluble hydroxypropylated derivative of chitin. Chitin and its derivatives can interact with macrophages through pattern-recognition receptors, thereby regulating innate immune responses [41]. Previous studies have also linked the immunomodulatory activities of chitin-based materials to JAK-STAT signaling, including STAT1 and STAT6 [[42], [43], [44]]. These findings, together with the enrichment of JAK-STAT-related pathways in our RNA-seq dataset, provided the rationale for prioritizing this signaling axis for further investigation.

Fig. 3.

Mechanistic investigation of HD₂₅-mediated regulation of macrophage polarization. (A) Schematic illustration of the experimental workflow for RNA-seq and bioinformatics analysis from BMDMs treated under different conditions (Created in BioRender). (B) Volcano plots showing transcriptomic alterations in BMDMs induced by HD₂₅ relative to control and HPCH. (C–D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for comparisons (C) HD₂₅ vs. Ctrl and (D) HD₂₅ vs. HPCH. (E) Gene Set Enrichment Analysis (GSEA) of hallmark signaling pathways altered in HD₂₅-treated BMDMs. (F) Representative histograms and (G) quantified MFI of phosphorylated STAT1 (p-STAT1) and STAT6 (p-STAT6) in BMDMs following various treatments (n = 3). (H) Representative FCM plots and (I) quantification of CD86 and CD206 expression in BMDMs treated with HD₂₅ with or without the JAK inhibitor Ruxolitinib (n = 3–4). (J) Quantified MFI of p-STAT1 and p-STAT6 in BMDMs (n = 3–4). *P < 0.05, **P < 0.01, ***P < 0.001.

The JAK-STAT signaling pathway is a well-established master regulator of macrophage polarization, in which distinct STAT isoforms drive divergent functional phenotypes [45]. To decipher the mechanism underlying HD₂₅-mediated immunomodulation, we analyzed the activation of key STAT proteins. Intracellular FCM revealed that HPCH alone treatment increased the phosphorylation of STAT1 (p-STAT1), with a modest increase in p-STAT6. Strikingly, HD₂₅ treatment fundamentally altered this signaling profile. HD₂₅ markedly attenuated p-STAT1 levels compared to HPCH alone while simultaneously enhancing p-STAT6 phosphorylation (Fig. 3F and G). This shift mirrors the phenotypic switch from a mixed state to a definitive M2-polarizing signature (HD₂₅) and aligns with the pathway enrichment predicted by transcriptomic analysis. These data pinpoint the specific modulation of the JAK-STAT axis, suppressing STAT1 while potentiating STAT6, as a central mechanism through which the HD₂₅ composite reprograms macrophages toward a therapeutic, anti-inflammatory phenotype.

To establish the causal link between JAK-STAT signaling and the immunomodulatory function of HD₂₅, we employed the pharmacological JAK inhibitor Ruxolitinib [46]. Co-treatment with Ruxolitinib effectively abolished HD₂₅-induced macrophage polarization, as evidenced by a significant reduction in CD206 and a restoration of CD86 expression (Fig. 3H and I). Furthermore, Ruxolitinib effectively attenuated the HD₂₅-induced phosphorylation of both STAT1 and STAT6 (Fig. 3J and Fig. S6). Taken together, these inhibitor studies confirm that the activation and differential modulation of the JAK-STAT pathway are functionally necessary for HD₂₅ to reprogram macrophages. These data demonstrate that DMOG plays an indispensable role within the composite, actively steering STAT phosphorylation away from a pro-inflammatory (STAT1) toward a pro-regenerative (STAT6) profile, which is essential for achieving the desired M2-polarizing outcome.

3.4. HD maintains cartilage homeostasis and protects chondrocytes from inflammatory stress in vitro

To evaluate the direct chondroprotective effects of HD, we used a three-dimensional chondrocyte pellet culture model that recapitulates key aspects of the native cartilage microenvironment, including hypoxia [47]. After 3 weeks of culture, histological analysis revealed that pellets treated with HD exhibited enhanced glycosaminoglycan (GAG) deposition, as evidenced by stronger SO/FG staining, and a more hyaline-like morphology with reduced central hypertrophy compared to control or HPCH-treated pellets (Fig. 4A and Fig. S7). Molecular analysis confirmed that HD treatment significantly suppressed the expression of hypertrophic markers (Col10a1 and Runx2) while upregulating the chondrogenic transcription factor Sox9 (Fig. 4B). IF results corroborated these findings, revealing that HD treatment markedly increased SOX9 protein level, approximately 3-fold and 1.7-fold higher than those in the Ctrl and HPCH groups, respectively, while simultaneously reducing COL10A1 expression (Fig. 4C–F), with HD₂₅ showing the optimal effects.

Fig. 4.

HD maintains cartilage homeostasis in vitro. (A) Representative images of Safranin O-stained sections of primary chondrocyte pellets cultured under different conditions. (B) Relative mRNA expression levels of chondrogenic and hypertrophy-related markers. (C) Representative IF images and (D) semi-quantitative analysis of SOX9 (green) in chondrocyte pellet sections. (E) Representative IF images and (F) semi-quantitative analysis of COL10A1 (red) in chondrocyte pellet sections. (G) Toluidine Blue staining images of primary chondrocytes treated with PBS, IL-1β alone, or IL-1β plus HD₂₅ for 2 days. (H) FCM plots of Annexin V/PI staining and (I) quantitative analysis of the total apoptotic cell rate after 2 days of treatment with different interventions. (J) SO/FG staining of cartilage explants following the indicated treatments. (K) Corresponding quantification of proteoglycan loss (n = 3). (L) TUNEL staining of cartilage explant sections. (M) Quantification of the percentage of TUNEL-positive cells (n = 3). Scale bars: 200 μm *P < 0.05, **P < 0.01, ***P < 0.001.

Having established that the HD₂₅ formulation exhibited the most pronounced chondroprotective effects under normal conditions, we next sought to evaluate its therapeutic potential under inflammatory conditions mimicking the OA microenvironment. To this end, chondrocytes and cartilage explants were stimulated with IL-1β, a standard pro-inflammatory cytokine widely used to model OA in vitro, and treated with the HD₂₅ formulation to assess its ability to counteract IL-1β-induced catabolic and inflammatory responses. In monolayer cultures, IL-1β (10 ng/mL) induced a catabolic state characterized by ECM loss, oxidative stress, and apoptosis. Co-treatment with HD₂₅ effectively reversed these effects (Fig. 4G–I; Fig. S8). To validate these findings in a more physiologically relevant context, we employed an ex vivo cartilage explant model. IL-1β stimulation caused significant GAG loss, which was reduced by approximately 50% with HD₂₅ treatment (Fig. 4J and K). Furthermore, HD₂₅ significantly attenuated IL-1β-induced chondrocyte apoptosis in explants, as shown by TUNEL staining (Fig. 4L and M).

Collectively, the HD system directly promotes a stable chondrocyte phenotype by enhancing anabolic activity, inhibiting hypertrophy, and providing potent protection against inflammation-driven ECM degradation and apoptosis.

3.5. HD promotes MSC recruitment and chondrogenic differentiation

Given the importance of endogenous repair in OA, we assessed HD's ability to recruit MSCs and direct their differentiation. HD significantly enhanced MSC migration in a Transwell assay, with approximately a two-fold increase compared to control medium (Fig. 5A and B). This was accompanied by the upregulation of key chemotaxis-related genes (Mif, Ccl2, and Ccl3) in MSCs exposed to HD (Fig. 5C).

Fig. 5.

HD promotes MSC recruitment and chondrogenesis by remodeling the niche. (A) Representative images of MSC migration in a Transwell assay after 12-h stimulation with hydrogel systems. Scale bar = 200 μm. (B) Quantification of migrated cells (n = 3). (C) Relative mRNA expression levels of migration-related genes (Mif, Ccl2, and Ccl3) in MSCs after 12-h stimulation. (D) Relative gene expressions of Col2a1, Sox9, Acan, and Hif1a in MSCs after 14 days of chondrogenic induction in the presence of hydrogel systems. (E) Alcian blue staining of MSCs after chondrogenic induction and (F) semi-quantification of the positive area (n = 3). (G) Quantification of GAG content in MSCs cultured under different interventions after chondrogenic induction. (n = 4). (H–I) Representative Western blot images and corresponding semi-quantitative analysis of SOX9 and COL2A1 protein expression in MSCs. *P < 0.05, **P < 0.01, ***P < 0.001.

To evaluate direct chondrogenic effects, MSCs were treated with HPCH or the HD system for 14 days. While HPCH intervention moderately upregulated chondrogenic genes, the incorporation of DMOG in the HD system further potentiated this response, significantly increasing the expression of Sox9, Acan, Col2a1, and Hif1a, with Sox9 elevation being particularly pronounced (Fig. 5D). Among the tested formulations, HD₂₅ exhibited the most pronounced pro-chondrogenic activity (Fig. S9). Alcian blue staining revealed more intense proteoglycan deposition (Fig. 5E and F), and a dimethylmethylene blue (DMMB) assay quantified a ∼1.8-fold increase in GAG content compared to the control (Fig. 5G). Western blotting further corroborated these findings, showing the highest protein expression levels of the key chondrogenic factors SOX9 and type II collagen (COL2A1) in the HD-treated groups (Fig. 5H and I). These findings indicate that HD promotes chondrogenic differentiation of MSCs in vitro.

Since macrophage-derived factors are known to influence tissue regeneration, we investigated whether HD's immunomodulatory effect could indirectly promote chondrogenesis. Conditioned medium (CM) from BMDMs pretreated with HD was collected and used to supplement MSC chondrogenic cultures. Remarkably, this macrophage CM significantly enhanced proteoglycan deposition in MSC pellets, an effect comparable to that induced by medium from classical M2-polarized macrophages (Fig. S11).

In summary, HD not only directly recruits MSCs and potently enhances their chondrogenic differentiation, but also, through its modification of the macrophage secretome, creates a pro-regenerative paracrine environment that further supports cartilage repair.

3.6. HD₂₅ ameliorates OA progression in mice

Based on the in vitro immunomodulatory capacity and pro-chondrogenic effects of HD, we next evaluated its therapeutic efficacy in vivo using a destabilization of the medial meniscus (DMM) mouse model. HD₂₅ was selected for the in vivo study due to its optimal in vitro efficacy. Prior to the efficacy study, we first assessed the in vivo retention behavior of the HD₂₅ system to inform the dosing regimen. The results showed that Cy5.5-encapsulated HPCH exhibited significantly prolonged fluorescence signal within the joint cavity following intra-articular injection, with detectable signal persisting for more than 6 days. In contrast, free Cy5.5 was rapidly cleared within 48 h (Fig. S10). This extended retention supports a weekly injection schedule, which was therefore adopted for the subsequent therapeutic evaluation. OA mice were treated with different materials by weekly intra-articular injection 1 week after constructing the OA model, and at 5 and 10 weeks post-surgery, mice were sacrificed to collect samples for subsequent experiments (Fig. 6A). Subchondral bone remodeling is a hallmark of OA. Micro-CT analysis at 5 weeks revealed that DMM surgery induced significant early-stage subchondral bone loss, as quantified by a reduction in bone volume fraction (BV/TV). Treatment with HD₂₅ effectively rescued the bone loss (Fig. 6B and C). Consistent with the structural preservation, histomorphometric analysis showed a rapid increase in TRAP⁺ osteoclasts in the DMM group, which was significantly suppressed by HD₂₅ treatment, returning osteoclast numbers to near-normal levels (Fig. 6D and E). This anti-osteoclastic effect aligned with our in vitro findings, where HD₂₅ inhibited osteoclast differentiation (Fig. S12), and was corroborated by KEGG pathway enrichment, which highlighted an impact on osteoclast differentiation (Fig. 3C). By the 10-week time point, saline-treated DMM mice exhibited advanced OA pathology, including prominent periarticular osteophyte formation, disorganized joint architecture, and substantial subchondral bone sclerosis. While HPCH treatment alone provided limited benefit, HD₂₅ treatment significantly ameliorated these late-stage pathological changes. The HD₂₅ group displayed a more orderly trabecular bone structure and a notable reduction in osteophyte formation, closely resembling the sham group (Fig. 6F and G). These results demonstrate that intra-articular delivery of HD₂₅ effectively attenuates OA-associated subchondral bone pathology, mitigating early bone loss and inhibiting aberrant bone formation at later stages, thereby confirming successful drug delivery and potent therapeutic action on deeper joint tissues.

Fig. 6.

Therapeutic efficacy of HD₂₅ in a mouse OA model. (A) Schematic illustration of the experimental design for in vivo intervention and evaluation (Created in BioRender). (B–C) Representative three-dimensional (3D) reconstructed micro-computed tomography (micro-CT) images of the mouse joints and tibial subchondral bone microstructure at (B) 5 weeks post-surgery and (C) microarchitectural quantitative assessment of subchondral bone (n = 5). (D) Representative images of TRAP staining at 5 weeks post-surgery. (E) Quantification of TRAP-positive multinucleated cells (n = 4). (F–G) Representative micro-CT images of the mouse joints and tibial subchondral bone microstructure at (F) 10 weeks post-surgery and (G) the quantitative results of BV/TV (n = 5). Scale bars: 200 μm *P < 0.05, **P < 0.01, ***P < 0.001.

3.7. HD₂₅ attenuates cartilage degeneration and maintains synovial homeostasis

We next evaluated the impact of HD₂₅ on cartilage integrity and synovial inflammation. Histological assessment at 5 and 10 weeks post-surgery revealed progressive cartilage degeneration in the saline group, characterized by surface fibrillation, erosion, and full-thickness loss (Fig. 7A and B; Fig. S13). While HPCH treatment alone provided moderate protection, HD₂₅ treatment significantly preserved cartilage structure at both time points, as evidenced by markedly lower OARSI scores (Fig. 7C). Interestingly, free DMOG alone, despite containing the same drug dosage as HD₂₅, failed to achieve significant cartilage preservation at 5 weeks post-treatment, with OARSI scores and subchondral bone parameters showing no statistically significant differences compared to the saline group (Fig. S14). This confirms that the HPCH-mediated sustained release is essential for achieving meaningful therapeutic efficacy. Toluidine blue staining confirmed enhanced proteoglycan retention in the HD₂₅ group compared to all other treatment groups (Fig. 7B).

Fig. 7.

HD₂₅ maintains cartilage and synovial homeostasis in mouse OA joints. (A) Representative histological images of knee joint sections stained with H&E and SO/FG from sham-operated or DMM-operated mice at 5 weeks post-surgery. Scale bars: 200 μm. (B) Representative images of joint sections after 10 weeks of operation, stained with SO/FG, Toluidine Blue, and IHC of MMP13. Scale bars: 200 μm. (C) Quantitative assessment of cartilage degradation severity at 5- and 10-week post-surgery using the Osteoarthritis Research Society International (OARSI) scoring system (n = 5). (D) Quantification of the percentage of MMP13-positive chondrocytes in cartilage (n = 4). ROI: The entire hyaline cartilage layer, excluding the calcified cartilage zone. (E) Representative H&E-stained images of synovial tissue at 10 weeks post-surgery. Scale bar: 200 μm. (F) Quantitative scores of synovial hyperplasia and inflammatory cell infiltration (n = 5). (G–H) IF of macrophage polarization in the synovium at week 10. (G) Co-staining for iNOS (red) and the pan-macrophage marker F4/80 (green). (H) Co-staining for CD206 (red) and F4/80 (green). Scale bars: 200 μm. (I–J) Quantitative analysis of macrophage phenotypes within the synovium. (I) Percentage of F4/80⁺iNOS⁺ cells and (J) Percentage of F4/80⁺CD206⁺ cells among total F4/80⁺ macrophages (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.

To investigate the mechanism underlying cartilage preservation, we analyzed the expression of MMP13 and ADAMTS5, two key extracellular matrix-degrading enzymes driving OA progression [48]. Immunohistochemical (IHC) analysis at 10 weeks revealed that HD₂₅ treatment dramatically suppressed these catabolic markers. The percentage of MMP13-positive chondrocytes in the hyaline cartilage layer was reduced from ∼35% in the saline group to ∼13% in the HD₂₅ group, and ADAMTS5 expression was similarly inhibited (Fig. 7B and D; Fig. S15). This indicates that HD₂₅ stabilizes the cartilage matrix by directly inhibiting catabolic activity.

Concomitantly, HD₂₅ potently mitigated synovitis. Histological analysis of synovial tissue at 10 weeks showed that HD₂₅ treatment significantly reduced synovial hyperplasia and inflammatory cell infiltration compared to the saline and HPCH groups (Fig. 7E and F). IF results revealed that OA induction expanded F4/80⁺ synovial macrophages, predominantly of the iNOS⁺ (M1) phenotype, with few CD206⁺ macrophages observed (Fig. 7G and H). HD₂₅ treatment effectively reversed this imbalance, decreasing the proportion of F4/80⁺iNOS⁺ M1 macrophages from ∼60% to ∼20% while increasing the proportion of F4/80⁺CD206⁺ M2 macrophages from ∼15% to ∼30% (Fig. 7G–J).

Collectively, intra-articular HD₂₅ delivery ameliorates OA pathology by decelerating cartilage degradation through suppression of catabolic enzymes and by reprogramming synovial macrophages toward an anti-inflammatory phenotype, thereby resolving synovitis and restoring joint homeostasis.

3.8. HD₂₅ maintains cartilage-synovium homeostasis in vivo in a macrophage-dependent manner

Macrophage infiltration in the synovium plays an essential role in triggering the inflammation in OA [24]. To determine whether the therapeutic action of HD₂₅ depends on its modulation of synovial macrophages, we performed local macrophage depletion using clodronate liposomes in the DMM model (Fig. 8A). Mice were divided into four groups: DMM with control liposomes (Ctrl), DMM with clodronate liposomes (MφD), DMM treated with HD₂₅ and control liposomes (HD₂₅), and DMM treated with HD₂₅ alongside clodronate liposomes (HD₂₅-MφD). Successful depletion was confirmed by a drastic reduction of F4/80⁺ cells in the synovium of clodronate-treated groups (Fig. S16).

Fig. 8.

HD₂₅ maintains cartilage-synovium homeostasis in vivo in a macrophage-dependent manner. (A) Schematic of the experimental design for macrophage depletion using clodronate liposomes (CLs) in conjunction with hydrogel treatments in the DMM-induced OA model. (B) Representative SO/FG-stained images of articular cartilage at 5- and 10-week post-surgery from mice treated with or without CLs. Scale bar: 200 μm. (C–D) Quantitative assessment of cartilage degeneration using the OARSI scoring system at each time point (n = 5). (E) H&E-stained images of synovial tissue at 10 weeks post-surgery across different treatment groups. Scale bar: 200 μm. (F) Quantitative scores of synovitis for each treatment group (n = 5). (G) Histological assessment of major organs (heart, liver, spleen, lung, kidney) at the study endpoint. Scale bars: 200 μm *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no significant difference.

Histological evaluation revealed that macrophage depletion alone (MφD) afforded only transient protection of cartilage at 5 weeks, with no sustained benefit at 10 weeks compared with the Ctrl group (Fig. 8B–D; Fig. S17). This suggests that the mere elimination of inflammatory macrophages provides only temporary relief and does not alter the long-term disease trajectory. In contrast, when macrophages were depleted in HD₂₅-treated mice, the therapeutic efficacy of HD₂₅ was significantly compromised at both time points, exhibiting worse cartilage integrity and higher OARSI scores than the HD₂₅ group (Fig. 8B–D). Notably, the HD₂₅-MφD group still performed better than the Ctrl group, indicating a residual direct protective effect from the hydrogel or DMOG. These results demonstrate that the optimal cartilage protection by HD₂₅ requires the presence and reprogramming of synovial macrophages. Furthermore, macrophage depletion exacerbated synovitis in both Ctrl and HD₂₅ contexts at the late stage (Fig. 8E and F), suggesting that prolonged absence of macrophages disrupts joint immune homeostasis. Finally, histopathological assessment of major organs showed no signs of systemic toxicity in HD₂₅-treated mice (Fig. 8G).

4. Discussion

OA is characterized by a vicious cycle of synovitis and cartilage degradation, in which dysregulated synovial macrophages play a central, orchestrating role. In this study, we developed a thermosensitive injectable hydrogel loaded with DMOG to disrupt this cycle through coordinated immunomodulation and chondroprotection. We demonstrate that intra-articular HD₂₅ delivery effectively halted OA progression in a murine model by simultaneously reprogramming synovial macrophages toward an anti-inflammatory M2 phenotype largely via modulation of the JAK-STAT signaling pathway, and directly augmenting cartilage anabolism. Crucially, macrophage-depletion experiments demonstrated that immunomodulation is a superior strategy to simple immune cell ablation for achieving sustained cartilage protection. Our multifunctional HD₂₅ system, which combines immunomodulatory and chondroprotective activities, could serve as a promising therapeutic approach for OA treatment.

The success of this strategy stems from a synergistic design integrating a multifunctional material platform with a pleiotropic drug. Injectable, thermosensitive hydrogels, such as HPCH, are well suited for intra-articular therapy, enabling minimally invasive administration, conformal filling of the joint space, and sustained, localized drug release [49]. These advantages could directly address the pharmacokinetic limitations of systemic drug delivery [50]. Beyond its role as a depot for DMOG, the HPCH matrix also plays a critical role in joint homeostasis. First, its favorable lubricating properties, evidenced by a COF comparable to that of clinical HA, help reduce articular surface wear, thereby mitigating a key biomechanical driver of OA [51]. Second, as a derivative of the natural polysaccharide chitin, HPCH and its degradation products are not only highly biocompatible but can also exert inherent chondroprotective effects, supporting proteoglycan synthesis and attenuating inflammatory chondrocyte apoptosis [52,53]. Thus, HPCH serves not merely as a passive carrier but as an active, multifunctional component that establishes a protective and pro-regenerative niche within the joint.

Notably, the HPCH hydrogel provides a platform for sustained drug delivery. We leveraged this capability to encapsulate DMOG, thereby augmenting the system's therapeutic potential against OA. The selection of an optimal DMOG concentration (25 μg/mL, D₂₅) was informed by prior optimization and confirmed in this study, in which HD₂₅ demonstrated superior efficacy in preserving cartilage ECM and reprogramming macrophages compared with a higher dose (50 μg/mL) [32]. This may reflect a nuanced, dose-dependent role of HIF-1α stabilization. While moderate activation supports chondroprotection and M2 polarization, excessive HIF-1α signaling has been linked to heightened glycolytic metabolism and a pro-inflammatory state [54], which could undermine therapeutic outcomes. From an immunomodulatory perspective, DMOG potently shifts the immune microenvironment by skewing macrophage polarization toward an M2 phenotype, thereby indirectly enhancing cartilage matrix synthesis and suppressing the recruitment of inflammatory cells. Our data corroborate that DMOG effectively inhibits M1 polarization both in vitro and in vivo. Although the immunomodulatory effects of DMOG and thermosensitive hydrogels have been reported separately, the underlying mechanisms remain elusive. Macrophage polarization is governed by a complex network of signaling pathways. We identified the JAK-STAT pathway as a critical and specific target of the HD₂₅ system. Most cytokines activate more than one STAT [55]. We found that HPCH increased STAT1 phosphorylation, accompanied by a comparatively modest increase in STAT6 phosphorylation. The upstream basis of this response remains unresolved. Recognition of the chitin-derived polymer or its degradation products by pattern-recognition receptors, including TLR2, Dectin-1, and the mannose receptor, represents a plausible mechanism [41,56]. However, potential contributions from physicochemical parameters, such as osmolarity, pH, and mechanical cues, cannot be excluded. Future studies incorporating matched physicochemical controls, targeted receptor analysis, and receptor-blocking experiments will be required to define the materials-related origin of HPCH-induced STAT1 activation. The incorporation of DMOG fundamentally altered this signaling profile, attenuating STAT1 activation while significantly enhancing STAT6 phosphorylation. Previous studies have shown that STAT6 and PPARγ physically interact and co-occupy target gene promoters, with STAT6 facilitating PPARγ recruitment and transcriptional activity to synergistically drive M2 polarization [57]. This shift toward STAT6 activation, coupled with the observed upregulation of the PPAR-γ pathway, a known driver of M2 polarization [58], explains the composite system's ability to reprogram macrophages toward an anti-inflammatory phenotype. Although the full regulatory network is undoubtedly complex, our work delineates a central mechanism through which the HD₂₅ system coordinates immune repair. Future material designs could build on this insight by incorporating more precise STAT pathway modulators to refine the immune response further and achieve optimal anti-inflammatory and regenerative outcomes.

Beyond its immunomodulatory effects, DMOG exerts direct, potent effects on cartilage homeostasis by stabilizing HIF-1α, a master regulator of chondrocyte anabolism and survival under stress. HIF-1α is essential for maintaining the hyaline phenotype, promoting the synthesis of matrix components such as type II collagen and aggrecan, and supporting cell survival under stress [59]. Its depletion accelerates cartilage degeneration [60]. DMOG has been utilized for cartilage repair and OA treatment, with studies confirming its ability to enhance stem cell chondrogenesis while inhibiting hypertrophy [28,61]. However, its clinical translation has been hindered by poor pharmacokinetics, as a small hydrophilic molecule, free DMOG undergoes rapid systemic clearance, leading to inconsistent local bioavailability and variable therapeutic outcomes, which may explain conflicting reports in the literature [27,32,62]. Our HD₂₅ platform directly addresses this limitation. Encapsulation of DMOG within the HPCH hydrogel was designed to prolong its local availability while limiting systemic exposure. Consequently, the therapeutic advantage of the HD₂₅ system arises not from chemical conjugation or complex formulation optimization, but from the rational integration of these two components. DMOG not only directly enhances cartilage matrix synthesis and cell viability but, in concert with the hydrogel, synergistically reprograms the inflammatory microenvironment. Importantly, a known small-molecule drug is delivered with a hydrogel carrier that addresses its pharmacokinetic limitations. Together, these integrated actions, including sustained drug delivery, immune reprogramming, lubrication, and stem cell recruitment, distinguish our system from existing approaches. While previous injectable hydrogels for OA have focused on a single function, either as passive depots, lubricants, or cell scaffolds, our HPCH-based system cohesively integrates four therapeutically relevant actions into one injectable platform, enabling coordinated disruption of the OA vicious cycle rather than isolated symptomatic mitigation. In vivo macrophage-depletion studies provide critical insights into the therapeutic paradigm for HD₂₅. While transient macrophage ablation early after DMM injury provided moderate cartilage protection by removing the primary inflammatory drivers, sustained depletion failed to halt long-term OA progression and even exacerbated synovitis. This may be attributed to two factors. Firstly, the persistent inflammation in advanced OA is mediated by a more complex cellular repertoire beyond macrophages. Secondly, the absence of macrophages might trigger compensatory activation of other inflammatory cells, potentially worsening certain pathological features [3]. Furthermore, in late-stage OA, the joint engages in compensatory mechanisms, such as chondrocyte hypertrophy and fibrocartilage formation, to maintain function. Interestingly, inflammatory macrophages themselves can secrete factors that may paradoxically support these adaptive, albeit pathological, responses [63]. Therefore, their complete removal might inadvertently hinder these late-stage compensatory processes. In contrast, HD₂₅ achieved superior, sustained outcomes by reprogramming rather than eliminating synovial macrophages. The observation that macrophage depletion abolished the therapeutic benefit of HD₂₅ confirms that its primary efficacy is macrophage-dependent. Importantly, the residual protection in the “HD₂₅ + depletion” group (compared to depletion alone) underscores an additional, direct chondroprotective effect of the hydrogel matrix and DMOG. Collectively, these depletion experiments provide compelling evidence that strategically reprogramming the synovial macrophage phenotype is a more effective and sustainable therapeutic strategy than their physical ablation for mitigating OA progression, a conclusion consistent with other studies [14,64]. This validates the core premise of our work and suggests that future therapies should focus on precise immunomodulation of specific macrophage subsets rather than on their broad clearance [65]. The findings also advocate for active immune education as a more durable and physiologically compatible approach, providing better guidance for the treatment of arthritis and other inflammatory disorders.

While promising, our study has limitations. The tribological assessment was performed using a PTFE ball-on-steel disk configuration as a simplified model system, and native cartilage-on-cartilage articulation involves more complex tribological conditions, including poroelastic behavior and interstitial fluid pressurization. Future studies using native cartilage-on-cartilage models are warranted. The requirement for repeated injections highlights a translational challenge. Future work should focus on engineering the delivery system to achieve longer-term, zero-order release kinetics of DMOG, thereby enhancing clinical compliance and efficacy. Strategies such as pre-encapsulating DMOG in nanoparticles, microspheres, or liposomes prior to hydrogel incorporation could enable multistage release profiles, thereby optimizing drug bioavailability and duration of action. Additionally, the immunomodulatory performance of the material platform holds potential for refinement. Tuning the physical, chemical, and biological properties of HPCH, such as its stiffness and degradation rate, and incorporating additional bioactive cues could enable more precise modulation of the innate immune response, thereby fostering a more robust and sustained pro-regenerative microenvironment.

5. Conclusion

We have developed and validated an injectable, thermosensitive hydrogel platform that effectively mitigates OA progression through a synergistic mechanism that couples direct chondroprotection with macrophage reprogramming via the JAK-STAT pathway. By demonstrating that immune modulation surpasses cell depletion, our work advocates for a paradigm shift in OA therapeutics. This multifunctional, immunomodulatory hydrogel platform represents a significant step toward a disease-modifying intra-articular therapy, with a design philosophy amenable to further refinement for future clinical translation.

Data availability

Data will be made available on request.

CRediT authorship contribution statement

Hongwei Shao: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Writing – original draft, Writing – review & editing. Zehua Lei: Data curation, Investigation, Methodology, Writing – review & editing. Wenjie Yang: Data curation, Formal analysis, Investigation, Writing – review & editing. Jiaqi Zhou: Data curation, Formal analysis, Investigation. Qian Li: Investigation, Methodology, Software. Shunxiang Xu: Formal analysis, Methodology. Wenjie Shi: Methodology, Software. Ziyang Xu: Formal analysis, Software. Guowen Luo: Methodology, Software. Xin Qian: Investigation, Methodology. Zhiyang Zhou: Methodology. Hongyi Liao: Investigation, Resources, Writing – review & editing. Xulin Jiang: Resources, Validation, Writing – review & editing. Yu Zhang: Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing. Xiongfa Ji: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.

Ethics approval and consent to participate

All animal procedures were performed in accordance with protocols approved by the Ethics Committee of Guangdong Provincial People's Hospital (No. KY-Z-2021-544-01) and complied with the National Research Council's “Guide for the Care and Use of Laboratory Animals”.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.