Female Sprague-Dawley rats (6 and 4 weeks of age) were purchased from the Guangdong Provincial Medical Experimental Animal Center (Guangzhou, China). The experimental procedure was approved by the Animal Care and Use Committee of Southern Medical University. The animals were subjected to light/dark cycles of 12:12 h and a constant temperature of 25°C. Ad libitum water and standard chow were provided.

A discectomy-induced TMJOA rat model was employed in the current study. Twenty-four female Sprague-Dawley rats (6 weeks old) were subjected to surgery and divided randomly into two groups: the surgery group (16 rats) and the sham-operated group (8 rats). The partial TMJ discectomy surgery was performed as previously described (Liu et al, 2021; Hua et al, 2022). Briefly, for the surgery group, following anesthesia with chloral hydrate (10% mL/kg), the joint capsule of the temporomandibular joint was incised, a small T-shaped incision was made in the rat articular disc with a scalpel, and the lateral third of the articular disc was excised to expose the condylar surface. The capsule was incised without exposing or destroying the joint disc in the sham-operated group.

A week post-surgery, the rats were once more randomly divided into two groups (vehicle-treated group and KGN-treated group) and administered intra-articular injections of 50 μL vehicle and KGN (100 nmol·L^-1) for 8 weeks, respectively. The TMJ condyles of all rats were collected 4 weeks after the last injection for histological and immunohistochemical (IHC) analysis.

The TMJ condyles were dissected and isolated. Three independent observers evaluated the macroscopic alterations of the TMJ condyle cartilage using the modified OARSI scoring system (Table 1). The TMJ condyles were then fixed in 4% paraformaldehyde solution for 24 h at room temperature, decalcified for 6 weeks with 10% ethylenediaminetetraacetic acid (EDTA) solution (E1171, Solarbio, Beijing, China) at 37°C, dehydrated in graded ethanol solutions, and embedded in paraffin. The sections were cut using a hard tissue slicer (Leica HistoCore MULTICUT, Germany) along the sagittal plane with a thickness of 4 μm. Hematoxylin and eosin (H&E) and Safranin O/fast green staining were used to stain cartilage tissue sections of the condyles. Three independent observers assessed osteoarthritis severity based on OARSI scoring (Table 2).

To examine osteoclasts and osteoblasts in the subchondral bone of the TMJ, TRAP staining was performed in accordance with the manufacturer’s protocol (Servicebio, G1050-50T, Wuhan, China). Briefly, paraffin sections of rat TMJ condyles were dewaxed in water, incubated with pure water for 2 h, and then stained with TRAP reagent (37°C, 30 min). Three independent observers counted the number of TRAP-positive osteoclasts at three randomly chosen sites in each sample, and the mean of these three sites was used for statistical analysis.

Sections of rat TMJ condyles (4 μm thickness) were deparaffinized with xylene, rehydrated with graded alcohol, and then incubated with rabbit primary antibodies against Runx2 (1:500; ab192256; Abcam), Aggrecan (1:400; ab216965; Abcam), Collagen II (1:400; ab34712; Abcam), Sox9 (1:200; ab185966; Abcam), and Ki67(1:300; ab236639; Abcam) overnight at 4°C. After washing with PBS, the sections were incubated with a horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (Goldbridge, Beijing, China) at 37°C for 20 min, and then stained with diaminobenzidine (Goldbridge, Beijing, China). Images were obtained at ×20 magnification with an Aperio Scan Scope slide scanner (Aperio VERSA; Leica Biosystems, United States). The number of positively stained cells at three randomly chosen sites from each sample was counted using the ImageJ software. The average number of positive cells per site was calculated, and the difference between groups was statistically analyzed.

FCSCs were isolated from rats according to a previous protocol (Hua et al, 2022). Briefly, the superficial zone tissues of the condyles from 10 rats were carefully separated under a stereomicroscope, and the obtained tissue was cut into small pieces of 1 mm³ in a cell culture dish containing PBS, followed by digestion with collagenase I and dispase II (3 mg·mL^-1 and 4 mg·mL^-1) at 37°C for 3 h. Single-cell suspensions of FCSCs were cultured (5% CO₂, 37°C) in basal medium consisting of Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 20% fetal bovine serum (FBS, Hyclone), GlutaMAX (Gibco), 1% penicillin/streptomycin (Gibco), and 100 mM 2-mercaptoethanol (Macklin) for 4–6 days. FCSCs were digested with trypsin-EDTA (Gibco) and plated at P1 for in vitro experiments.

To assess the effect of KGN on cartilage anabolic factors and catabolic factors, cells were starved overnight in α-MEM containing 1% FBS, then treated for 24 h with 10 ng·mL^-1 rhTNF-α (peprotech 315-01A) or KGN (100 nmol·L^-1) alone or in combination.

Multi-lineage differentiation potential of rat FCSCs was tested in vitro using chemically defined media for chondrogenesis, osteogenesis, and adipogenesis as previously described (Hua et al, 2022). For chondrogenesis, cells (1× 10⁶ per pellet) were pelleted in 15 mL polypropylene tubes by centrifugation and cultured (5% CO2, 37 °C) for 3 weeks in high glucose Dulbecco’s Modified Eagle medium (Gibco) supplemented with 10^–7 M dexamethasone, 100 μM L-ascorbic acid, 1% insulin, transferrin, selenium (ITS), 1 mM pyruvate, and 10 ng/mL TGF-β1 (Novoprotein). After 3 weeks, pellets were processed for histology. To induce osteogenesis, FCSCs (5 × 10⁴) were cultured in a 12-well plate for 4–5 weeks in media containing αMEM supplemented with 20% FBS, dexamethasone (10^–8 M), 100 μM L-ascorbic acid, and 2 mM β-glycerophosphate (Coolaber). To induce adipogenesis, cells (5 × 10⁴) were cultured in a 12-well plate using commercial adipogenic media (BGsciences BGM-1131) for 1 week. Calcium nodules and fat were visualized by staining with alizarin red and Oil Red O, respectively.

Cells were incubated for 30 min with or without conjugated specific antibodies at recommended concentrations at 4°C. Then cells were washed, resuspended in PBS, and analyzed on a Flow Cytometer (DxFLEX, Beckman). Unlabeled cells were used as blank control. The antibodies included anti-CD29 (FITC) (102,205, BioLegend), anti-CD45 (Alexa Fluor 647) (202,211, BioLegend), and anti-CD90 (PE) (202,523, BioLegend).

Following the protocol of cell counting kit-8 (CCK-8) assay (APExBIO, K1081, Houston, United States), the growth of FCSCs in 96-well plates was assessed at 1, 3, 5, 7, and 9 days. FCSCs were seeded into 96-well plates at 2000 cells/well and incubated for 24 h for attachment. KGN (100 nmol·L^-1) and PBS were added to the media of the experimental and control groups, respectively, and the solution was changed every 2 days. OD values (450 nm) of cell proliferation on days 1, 3, 5, 7, and 9 were measured using a microplate reader.

A total of 1 × 10⁶ FCSCs were pelleted in a 15-mL polypropylene tube via centrifugation at 2000 rpm for 5 min and then cultured in DMEM supplemented with 10⁻⁷ mol·L^-1 dexamethasone, 100 μmol·L^-1 ascorbic acid, 1% insulin, transferrin, selenium (ITS), 1 mmol·L^-1 pyruvate, and 10 ng·mL^-1 TGF-β1 (Novoprotein) for 3 weeks, with medium changes every 2 days.

TRIzol Reagent (AG, AG21102, Hunan, China) was used to extract total RNA from cultured rat FCSCs according to the manufacturer’s instructions. Total RNA (1,000 ng) was reverse-transcribed to cDNA using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (AG, AG11728, Hunan, China). Real-time PCR was performed on a PCR instrument (Roche, Lightcycle 96, Switzerland) using SYBR Green Premix Pro Taq HS qPCR Kit II (AG, AG11702, Hunan, China) to analyze SOX9, Aggrecan, Col2a1, and GAPDH mRNA expression, following the manufacturer’s instructions. The relative mRNA expression levels of target genes were normalized to those of GAPDH and then calculated using the 2^−ΔΔCT method. The primer sequences used for quantitative real-time PCR are listed in Table 3.

FCSCs were lysed in 1x RIPA buffer (Beyotime, P0013B, Shanghai, China) containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail (Beyotime, P1005, P1096, Shanghai, China). The BCA Protein Assay Kit (Beyotime, P0012, Shanghai, China) was used to measure the protein concentration in the lysates. Ten micrograms of total protein per sample were loaded in each lane, separated using 10% Precast SDS-PAGE Gel (ACE, Nanjing, China), and transferred to a nitrocellulose membrane (BioRad, United States). After blocking with 1x QuickBlock™ Blocking Buffer for Western blot (Beyotime, P0252, Shanghai, China) for 15 min at room temperature, the membranes were incubated with primary antibodies (SOX9: 1:2000; ab185966; Abcam; Runx2: 1:2000; ab236639; Abcam; GAPDH: 1:5,000; ab37168; Abcam) overnight. The membrane was then washed with TBS-T buffer and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000; ab6721; Abcam). After washing, the blots were developed using an enhanced chemiluminescence system (ECL, Merck Millipore, United States). Band density was analyzed using Image Lab Software.

Statistical analyses were performed using GraphPad Prism 8 statistical software (GraphPad Inc., La Jolla, CA, United States) or SPSS 22.0 statistical software (IBM, Chicago, IL, United States). Quantitative data are presented as the mean ± SD. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used to determine the statistical significance of differences between three or more groups. At least three repetitions of each experiment were conducted, and p-values <0.05 were considered statistically significant. Statistically significant differences are indicated by asterisks (*, p < 0.05).

We first established a partial discectomy-induced TMJOA rat model. A week post-surgery, the vehicle-treated and KGN-treated rats received intra-articular injections of 50 μL vehicle or KGN (100 nmol·L^-1), respectively, for 8 weeks (Figure 1A). At 12 weeks post-operation, condylar surface injury was more severe in the vehicle-treated group than in the sham-operated group. However, the condylar surface of KGN-treated group rats was smoother than that of vehicle-treated rats, exhibiting no evident damage or irregularities (Figure 1B). The condylar surface morphology of each group was evaluated using a macroscopic semi-quantitative scoring system. The KGN-treated group scored substantially higher than the sham-operated group and significantly lower than the vehicle-treated group (Figure 1E). The sections stained with Safranin O/fast green and TMJ condylar cartilage areas were further assessed using the OARSI histological scoring system. The vehicle- and KGN-treated groups scored considerably higher than the sham-operated group, indicating that the condylar cartilage exhibited evident pathological degeneration at 12 weeks after surgery. However, the KGN-treated group exhibited significantly improved condylar morphology, as indicated by the significantly lower score compared to the vehicle-treated group (Figures 1C,F). TRAP staining was used to examine histopathological alterations in the TMJ condylar subchondral bone of rats. According to our findings, the subchondral bone of the vehicle-treated group contained significantly more TRAP-positive osteoclasts than the sham-operated group, whereas KGN treatment significantly inhibited osteoclast formation 12 weeks post-surgery (Figures 1D,G). These results indicated that intra-articular KGN injection can improve condylar cartilage health and attenuate the abnormal remodeling of the subchondral bone in the partial discectomy-induced TMJOA rat model.

Next, we investigated the effect of KGN treatment on the proliferation of condylar chondrocytes by counting the number of nuclei in the superficial zone (SZ), polymorphic, and chondrocyte layers of condylar cartilage via HE staining (Figure 2A). Although the number of nuclei in the SZ layers of vehicle-treated rats was slightly lower than that in the sham-operated group, the number of nuclei in the polymorphic and chondrocyte layers was significantly decreased. Meanwhile, the numbers of nuclei in the SZ, polymorphic, and chondrocyte layers of KGN-treated rats were significantly higher than those in the sham-operated and vehicle-treated groups (Figure 2B). The hypertrophic chondrocyte layer in the KGN-treated group was clearly thicker than that in the vehicle-treated and sham-operated groups when viewed under a microscope. Based on these findings, KGN treatment may promote FCSC proliferation and chondrogenic differentiation in the SZ layer under TMJOA, which in turn increases the number of cells in the polymorphic and chondrocyte layers, thereby promoting the thickening of articular cartilage. IHC was used to analyze the expression of Ki67 in TMJ condylar cartilage so as to determine whether KGN treatment promoted cell proliferation in vivo (Figure 2C). Our results showed that, while the expression of proliferation marker Ki67 was slightly reduced in the vehicle-treated group compared to that in the sham-operated group, it was significantly upregulated in the KGN-treated group compared to both the sham-operated and vehicle-treated groups (Figure 2D). These findings implied that KGN therapy promoted condylar chondrocyte proliferation.

We further determined whether KGN treatment could promote the proliferation and differentiation of FCSCs in vitro. FCSCs were isolated from the condylar surface of rats, and their capacity for multilineage differentiation was examined in vitro. Expression of stem cell surface markers in FCSCs was determined by flow cytometry analysis. The result shown that FCSCs were positive for CD29 and CD90, while negative for CD45 (Figure 3A). This result was consistent with previous finding in rat FCSCs (Embree et al, 2016). Adipogenesis (Oil Red O-positive), mineralization (Alizarin red-positive), and chondrogenesis (Safranin O-positive) were observed in pellet cultures, which indicates that isolated FCSCs can differentiate into multiple lineages (Figure 3B). The CCK8 assay was then used to assess FCSCs proliferation following KGN treatment. Our findings showed that KGN treatment for 1, 3, 5, 7, and 9 days considerably enhanced the proliferation of FCSCs (Figure 3C). A pellet culture system was used to evaluate whether KGN treatment induced FCSC chondrogenesis. Histological analysis using Safranin O staining revealed that the KGN-treated group had greater staining intensity than the control group. Further IHC evaluation also revealed the KGN-treated group had increased type II collagen expression (Figure 3D). These findings demonstrate that KGN treatment promotes FCSCs proliferation and cartilaginous-like tissue formation in vitro.

To elucidate the effect of KGN treatment on the expression of condylar cartilage synthesis- and degradation-related factors in rats, we performed IHC and analyzed the expression of cartilage anabolic factors (Aggrecan, Collagen II, and Sox9) (Figures 4A–C). Our data showed that the percentage of Aggrecan-positive cells and the Collagen II-positive area within the condylar cartilage layer of the vehicle-treated group were significantly lower than in the sham-operated group (Figures 4E–G). However, the expression of these chondrogenesis-associated markers was markedly upregulated in the KGN-treated group relative to the vehicle-treated group. Further, the percentage of Sox9-positive cells was significantly higher than that in the sham-operated group, demonstrating that KGN promotes chondrogenic differentiation and protects condylar cartilage against deterioration. Cartilage catabolic factor Runx2 was significantly downregulated in the KGN-treated compared to the vehicle-treated group while remaining slightly upregulated relative to the sham-operated group (Figures 4D,H). These findings suggest that KGN treatment inhibits chondrocyte hypertrophy in TMJOA.

In osteoarthritis, TNF-α is regarded as the predominant pro-inflammatory cytokine (Alshenibr et al, 2017). We constructed an in vitro osteoarthritis model by adding TNF-α to the culture medium (Hua et al, 2022). In order to further investigate the mechanisms by which KGN induces protective effects in TMJ condylar cartilage, we also treated FCSCs with TNF-α in the presence or absence of KGN.

We first examined the effect of KGN treatment on the mRNA expression of genes linked to cartilage synthesis (Aggrecan, Col2a1, and SOX9) in FCSCs (Figures 5A–C). Our findings demonstrated that, in normal conditions, KGN treatment greatly upregulated the mRNA expression of Col2a1, Aggrecan, and SOX9. However, when FCSCs were maintained in inflammatory conditions induced by TNF-α, we discovered that KGN treatment mitigated the TNF-α-induced inhibition of these genes and even upregulated their expression relative to that under normal culture conditions. Subsequently, we performed Western blot analysis for SOX9 and Runx2. Western blot results for SOX9 were in agreement with gene expression data (Figures 5D,E). Runx2 expression was significantly higher in the TNF-α-induced group than in the DMSO group, whereas KGN treatment effectively decreased Runx2 expression under both normal culture and TNF-α-induced inflammatory conditions (Figures 5F,G). These findings suggest that KGN treatment increased the expression of cartilage synthesis-related proteins while protecting FCSCs from TNF-α-induced inflammatory responses.