Fig 2: Characterization and properties of THG hydrogel. (A) Schematic of hyaluronic acid (HA)-gallic acid (GA) polymer synthesis, a precursor of THG hydrogel. (B) Schematic of N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1, N1, N3, N3-tetramethylpropane-1,3-diaminium (TSPBA) polymer synthesis, a precursor of THG hydrogel. (C) 1H NMR spectra of HA, HA–NH2, and HA–GA in D2O. (D) 1H NMR spectrum of TSPBA. (E) Schematic illustration of gel formation: HA–GA mixed with TSPBA at a 2:1 vol ratio forms an opaque white gel. (F) Scanning electron microscopy shows the formation of a porous network in THG hydrogel (scale bars: 50 μm and 10 μm). (G) Rheological evaluation of the storage (G′) and loss (G″) moduli of THG hydrogel at 37 °C. (H) Release profile of FGF18 from reactive oxygen species (ROS)-sensitive THG hydrogels immersed in 10 μM H2O2 or phosphate-buffered saline (PBS) solutions. (I) Degradation pattern of ROS-sensitive THG hydrogels immersed in 10 μM H2O2 or PBS solutions. Values are presented as the mean ± standard error of the mean (SEM) of three independent experiments. Significance was determined by Tukey's multiple comparison test following two-way analysis of variance (ANOVA). ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P < 0.0001.

Fig 3: Schematic illustration of the mechanism by which ROS-sensitive THG hydrogel encapsulating FGF18 active protein (FGF18/THG) treats temporomandibular joint (TMJ) osteoarthritis (TMJOA). A ROS-responsive, biodegradable anti-inflammatory hydrogel (THG) is designed by combining HA–GA with N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1, N1, N3, N3-tetramethylpropane-1,3-diaminium (TSPBA). The hydrogel encapsulates FGF18 (FGF18/THG), which is injected into the TMJ cavity. Upon exposure to the reactive oxygen species (ROS)-enriched TMJOA microenvironment, the boronic ester groups in FGF18/THG degrade, leading to ROS depletion. Simultaneously, the released FGF18 and anti-inflammatory molecules (e.g., GA) further neutralize ROS and inhibit the abnormally activated NF-κB pathway in osteoarthritic condylar chondrocytes, upregulating cartilage-specific genes (Col2, Acan) and downregulating pro-inflammatory genes (e.g., Adamts5, Mmp13). This synergistic process promotes cartilage regeneration, reduces inflammation, and prevents extracellular matrix (ECM) degradation. Additionally, in milder cases of TMJOA, the therapeutic effects of FGF18 are further enhanced, slowing disease progression in vivo.

Fig 4: Macroscopic evaluation and histopathological assessment of the temporomandibular joint (TMJ) in SD rats with experimental TMJ osteoarthritis (TMJOA) after two weeks of intra-joint injection treatment with FGF18/THG. (A) Schematic diagram of the SD rat TMJOA experimental model, indicating the time points for partial discectomy, intra-joint injection of FGF18, THG, or FGF18/THG, and sacrifice. (B) Representative macroscopic images of the TMJ condyles in sham-operated and PDE-operated rats after eight weeks of treatment with FGF18/HA–GA or control. (C) Representative histological images stained with H&E and Safranin O/fast green showing histological changes in cartilage sections from different groups of SD rats eight weeks post-PDE surgery. (D) Representative immunohistochemical images of COL2, ACAN, ADAMTS5, and MMP13 staining in the TMJ condyles of rats two weeks post-sham surgery or PDE surgery, treated with FGF18/THG or control. The dashed box highlights the repair area, and arrows indicate the inflammatory regions. (E) Macroscopic semi-quantitative scoring of the TMJ condylar cartilage in SD rats. (F) Semi-quantitative scoring of Safranin O/fast green staining in the TMJ condylar cartilage of different groups of SD rats. (G) Semi-quantitative analysis of COL2, ACAN, ADAMTS5, and MMP13 immunohistochemical staining in TMJ cartilage of different groups of SD rats. Values are presented as the mean ± standard error of the mean (SEM) of four independent experiments. Significance was determined by one-way analysis of variance (ANOVA): ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P < 0.0001.

Fig 5: In vitro anti-inflammatory and cytotoxicity evaluation of FGF18/THG. (A) Representative fluorescence images of the reactive oxygen species (ROS) levels in condylar chondrocytes co-cultured with FGF18, THG, and FGF18/THG using ROS fluorescence staining. (B) Immunofluorescence staining of catalase (CAT) and superoxide dismutase 1 (SOD1) expression in IL-1β-induced condylar chondrocytes treated with FGF18/THG. (C) Quantitative evaluation of ROS levels in condylar chondrocytes using ROS fluorescence staining. (D) mRNA expression of Cat and Sod1 in IL-1β-induced condylar chondrocytes treated with FGF18/THG. (E) CCK-8 assay results showing the cytotoxicity of FGF18/THG on condylar chondrocytes based on cell proliferation. Values are presented as the mean ± standard error of the mean (SEM) of three independent experiments. Significance was determined by one-way analysis of variance (ANOVA): ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P < 0.0001.