Comparative transcriptomics of fibrocartilage stem cells and knee chondrogenic progenitors identifies a DKK3-PI3K/AKT regulatory axis

Comparative transcriptomics of fibrocartilage stem cells and knee chondrogenic progenitors identifies a DKK3-PI3K/AKT regulatory axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative transcriptomics of fibrocartilage stem cells and knee chondrogenic progenitors identifies a DKK3-PI3K/AKT regulatory axis Qiaoli Dai, Ying Wang, Cunyi Wang, Wenlin Yuan, Yilin Chen, Mengqi Zhu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9282133/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Objective This study aimed to compare fibrocartilage stem cells (FCSCs) and knee chondrogenic progenitor cells (CPCs) through RNA sequencing to identify key mechanisms regulating FCSCs function, and to explore the role of Dickkopf-3 (DKK3) in maintaining FCSCs chondrogenic potential in association with the PI3K/AKT pathway. Design: FCSCs were isolated from temporomandibular joint (TMJ) condylar cartilage and CPCs from knee articular cartilage of 6-week-old male Sprague-Dawley (SD) rats. RNA sequencing analyzed transcriptional differences. DKK3 knockdown in FCSCs was performed using short hairpin RNA (shRNA), followed by chondrogenic differentiation assays. PI3K/AKT pathway activation was assessed via Western blot, immunofluorescence, and rescue experiments with the PI3K/AKT activator Recilisib. Results DKK3 expression was higher in FCSCs than CPCs (p < .05). DKK3 knockdown reduced chondrogenic markers (Sox9, Col2a1, Aggrecan) and PI3K/AKT activity (p < .05), while recombinant DKK3 rescued the IL-1β-induced impairment of FCSCs chondrogenesis (p < .05). Recilisib restored PI3K/AKT signaling and chondrogenic capacity in DKK3-deficient FCSCs (p < .05). Transcriptomics revealed FCSCs’ unique enrichment in extracellular matrix (ECM) remodeling and PI3K/AKT pathways compared to CPCs. Conclusions Our results suggested that DKK3 appears to maintain FCSCs’ chondrogenic potential via the PI3K/AKT pathway activation, highlighting a tissue-specific regulatory mechanism critical for TMJ cartilage homeostasis. Fibrocartilage Stem Cell PI3K/AKT signaling Cartilage RNA Sequence Analyses Temporomandibular joint Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction The incidence of osteoarthritis (OA) is rising, affecting approximately 595 million individuals globally (about 7.6% of the world’s population) with a projected increase of approximately 60.1% by 2050 (Courties et al. 2024 ; Global, regional, and national burden of osteoarthritis, 1990–2020 and projections to 2050: a systematic analysis for the Global Burden of Disease Study 2021 2023). OA is primarily characterized by cartilage degradation, subchondral bone remodeling, and synovitis (Mahmoudian et al. 2021 ). Although knee OA and temporomandibular joint (TMJ) OA are both mechano-inflammatory diseases, they exhibit significant heterogeneity in anatomy, biomechanics and pathological changes. Structurally, TMJ articular surfaces consist of fibrocartilage rich in type I collagen, in contrast to the type II collagen-dominated hyaline cartilage of the knee. This compositional divergence directly dictates biomechanical specialization: TMJ fibrocartilage is optimized for dynamic tensile stresses (e.g., masticatory loading), whereas knee hyaline cartilage primarily resists compressive forces (Lei et al. 2024 ). Pathologically, knee OA typically begins with superficial cartilage fraying and erosion, characterized by surface cartilage fissures. In contrast, TMJ OA is typically characterized by more prevalent deep-layer fissures and milder surface damage. Further differences emerge in inflammatory signaling, hormonal sensitivity, and mechanotransduction pathways, underscoring the site-specific nature of OA pathophysiology (Zhao et al. 2025 ; Tian et al. 2022 ). Articular cartilage, an avascular tissue, relies heavily on resident stem/progenitor cells for injury repair. Fibrocartilage stem cells (FCSCs) localized to the superficial zone (SZ) of the TMJ condylar cartilage (Embree et al. 2016 ) and chondrogenic progenitor cells (CPCs) distributed within knee articular cartilage represent two key reparative populations. However, their spatial distribution and microenvironmental niches differ markedly. FCSCs are restricted to the SZ of TMJ condylar cartilage (Embree et al. 2016 ), directly exposed to dynamic biomechanical stress and synovial biochemical stimuli. In OA, FCSCs exhibit reduced numbers and altered activity, with their main population remaining enriched in the SZ of the condylar cartilage (Wang et al. 2024 ; Tuwatnawanit et al. 2025 ). CPCs reside primarily in the superficial layer of normal knee cartilage but can expand into deeper zones or even migrate from subchondral bone in late‑stage OA (Jiang and Tuan 2015 ; Koelling et al. 2009 ). These microenvironmental differences suggest divergent signaling pathways regulating cartilage homeostasis in FCSCs versus CPCs. However, systematic investigations into their transcriptomic profiles and regulatory networks remain lacking, which limits our understanding of their specific roles in the respective OA conditions. Currently, therapeutic strategies for TMJ OA largely follow those for knee OA (such as anti-inflammatory drugs and intra-articular injections), but their efficacy is controversial (Bielajew et al. 2021 ). Therefore, systematically elucidating the functional differences between FCSCs and CPCs based on the heterogeneity of TMJ and knee OA and the differentiation of stem cell microenvironments is of vital importance. Dickkopf-3 (DKK3), a secreted glycoprotein and Wnt pathway modulator, has emerged as a cartilage-protective factor that inhibits inflammatory matrix degradation (Conde et al. 2021 ) and is highly expressed in the SZ of the TMJ condyle (Utreja et al. 2016 ). This expression pattern suggests a potential role for DKK3 in maintaining FCSCs function, yet studies directly examining this relationship are currently lacking. Here, we compared the cellular characteristics of FCSCs and CPCs, such as proliferation, differentiation, migration, multi-lineage differentiation, and other specific transcriptional features through RNA-seq analysis, in order to identify critical regulatory proteins. Focusing on DKK3, we aimed to elucidate its role in regulating FCSCs function, thereby uncovering novel regulatory mechanisms that could provide clues to the pathogenesis and treatment of TMJ OA. 2 Materials and Methods 2.1 Cell isolation and culture Primary FCSCs and CPCs were isolated and cultured from 6-week-old male SD (Sprague-Dawley) rats (Zhejiang University's experimental animal center) (Hua et al. 2022 ; Embree et al. 2016 ). Only male subjects were selected to eliminate the influence of estrogen (Yue et al. 2025 ). Rats were anesthetized via intraperitoneal injection of 3% pentobarbital sodium (50 mg/kg body weight). Euthanasia was performed by cervical dislocation under deep anesthesia. Primary FCSCs were isolated and cultured from TMJ condylar cartilage. To isolate FCSCs, the condyles were digested by 4 mg/ml type II dispase (Solarbio, D6431) for 15 min before separating the SZ, and then the SZ was digested in 4 mg/ml type II dispase (Solarbio, D6431) and 3 mg/ml type I collagenase (Solarbio, C8140). Single-cell suspensions were cultured in Dulbecco’s Modified Eagles Medium (DMEM with high glucose, Gibco, C11995500BT) containing 10% fetal bovine serum (FBS, Gibco, A5669701), glutamax (Invitrogen, 35050-061) and 1% penicillin-streptomycin (PS, Solarbio, P1400) at 37°C with 5% CO₂. The culture medium was replaced after 48 hours (Embree et al. 2016 ). Once the FCSCs reached 80% confluence, they were digested with 0.25% trypsin containing EDTA (Gibco, 25200056). The cells were then passaged (P1-3) and plated for in vitro experiments. Primary CPCs were isolated and cultured from knee joint cartilage of the same rats. Cartilage tissue from the non-weight-bearing area of the rat knee joint tibial plateau and the lateral femoral condyle was cut into 1 mm pieces and digested in 1.5 mg/ml type II collagenase (Solarbio, C8150) for 2–4 hours. The resulting cells were discarded, and the digested cartilage slices were incubated in DMEM containing 10% FBS and 1% PS at 37°C with 5% CO₂ (Wang et al. 2020 ; Batschkus et al. 2017 ). The culture medium was replaced after 48 hours. Once the migrated CPCs reached 80% confluence, they were digested with 0.25% trypsin containing EDTA, and P1-3 cells were plated for in vitro experiments. Animal experiments were approved by the Ethics Committee of Zhejiang University (Number: ZJU20190007). 2.2 Flow Cytometry CPCs and FCSCs were immunolabeled with fluorescent conjugated antibodies or isotype-matched IgG controls for 30 min at 4°C, and then the cells were washed with Phosphate Buffered Saline (PBS) and resuspended. Cells were subjected to fluorescence activated cell sorting. The fluorescent conjugated antibodies included: anti-CD29-PE (eBioscience,12-0291-82, 1:100), anti-CD44-APC (Abcam, ab81424, 1:200), and anti-CD90-FITC (Abcam, ab226, 1:200) and anti-CD45-PB (BioLegend, 202225, 1:200) (Bi et al. 2020) . Flow cytometry experiments were conducted using FACSAria II and samples were calculated using Flowjo software. 2.3 RNA Sequencing and Bioinformatics RNA sequencing was performed by Biomarker Technologies (Beijing, China). Total RNA was extracted from FCSCs and CPCs (n = 3 biological replicates per group) using TRIzol reagent (Invitrogen, 15596026) (Tosa et al. 2023 ), and RNA integrity was assessed using the Agilent Bioanalyzer 2100. Subsequently, libraries were constructed using the NEBNext Ultra™ RNA Library Prep Kit and sequenced on the Illumina platform. Clean reads were aligned to the reference genome using HISAT2 software, and transcript assembly and expression quantification were performed using StringTie. Differential expression analysis was conducted with DESeq2 software to identify significantly differentially expressed genes, with raw reads aligned to the rat reference genome (rn6) using HISAT2. Differentially expressed genes (DEGs) were identified using DESeq2 software (Fold Change ≥ 2 and FDR < .01) (Tosa et al. 2023 ). Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted to reveal gene functions and pathway changes. 2.4 Colony Forming Assay FCSCs and CPCs at P2 at logarithmic phase were harvested and cultured (40 cells/cm 2 in 6-well plates). After 14 days’ culture, cells were fixed with 4% paraformaldehyde (PFA, Solarbio, P1110) for 15 min, washed in PBS and stained with 0.1% crystal violet (Beyotime, C0121) for 20 min. Cells were next washed with ddH₂O till clarified, and plates were air-dried and photographed. Colonies larger than 50 µm in diameter were blindly counted 3 times by 2 observers respectively. This assay was repeated with 5 biological replicates, and clonogenic efficiency of FCSCs and CPCs was calculated and compared between groups. 2.5 Cell Proliferation Assay Cell proliferation potential of FCSCs and CPCs was evaluated using the Cell Counting Kit-8 (CCK-8, Beyotime, C0042) (Du et al. 2023 ). Cells were seeded in 96-well plates at a density of 2×10³ cells/well and cultured in complete growth medium (DMEM supplemented with 10% FBS and 1% PS). After 24, 48, 72, and 96 hours of incubation at 37°C under 5% CO₂, 10 µL CCK-8 reagent was added to each well. Absorbance at 450 nm was measured using a microplate reader (Bio-Tek Synergy H1) following 2 hours of incubation. Randomization was applied during cell seeding, and operators were blinded to group allocation during absorbance measurement. 2.6 shRNA-Mediated DKK3 Knockdown Lentiviral particles encoding short hairpin RNA (shRNA) targeting DKK3 (shDKK3: 5′- GTGGAGAGAAGATTTAGACCT-3′) and scrambled control (shCtrl) were synthesized by Bioscien (Shanghai, China), a professional supplier of lentiviral vectors and shRNA synthesis services (Moore et al. 2010 ). FCSCs were transfected at 70% confluence with a multiplicity of infection MOI of 100. After 72 hours, puromycin (3 µg/mL, Beyotime, ST551) was added for 5 days to select stable clones. Knockdown efficiency was validated by qRT-PCR and Western blot. 2.7 Multi-lineage differentiation Multi-lineage differentiation protocols were adapted from the study by Embree et al.(Embree et al. 2016 ) with minor modifications. We tested adipogenic, osteogenic, and chondrogenic differentiation in vitro using chemically defined media. For adipogenesis, FCSCs and CPCs with a cell density of 2×10⁴ were cultivated in a 24-well plate. Upon reaching 70% confluence, the cells were cultured using commercial adipogenic media (Oricell, RAXMD-90031). After 2 weeks, the cells were fixed in 4% PFA (Solarbio, P1110) and stained with Oil Red O. For osteogenesis, FCSCs were seeded at a density of 2×10⁴ cells per well in a 24-well plate. When the cells reached 70% confluence, they were cultured with commercial osteogenic induction medium (Amizona Scientific, AMK8006-200). After 1 week, the cells were fixed with 4% PFA and stained with alkaline phosphatase (ALP) (Beyotime, C3206). Chondrogenic differentiation was conducted via two culture systems (2D monolayer and 3D pellet). For 2D monolayer chondrogenic induction, FCSCs and CPCs with a cell density of 2×10⁴ were cultivated in a 24-well plate. The cells were cultured in high glucose DMEM medium supplemented with 100 nM dexamethasone (MCE, HY-14648), 1 mM sodium pyruvate (Gibco, 11360070), 50 µg/ml L-ascorbic-2-phosphate (Sigma, A8960), 40 mg/ml L-proline (Aladdin, P108709), 1% insulin-transferrin-selenium (Beyotime, C0341), and 10 ng/ml transforming growth factor-beta 3 (TGF-β3, PeproTech, 100-21C), when they reached 90% confluence. The cells were cultured for 3 weeks and stained with Alcian Blue (OriCell, ALCB-10001) for glycosaminoglycans. For 3D pellet chondrogenic induction, cells (1 × 10 6 per pellet) were pelleted in 15 ml polypropylene tubes by centrifugation and cultured (5% CO 2 , 37°C) for 3 weeks in the above chondrogenic induction medium. The medium was changed every 2–3 days to maintain nutrient supply. After 3 weeks, 3D pellets were fixed with 4% PFA (Solarbio, P1110), cut into 3 µm paraffin sections, and stained with Alcian Blue (OriCell, ALCB-10001) for glycosaminoglycans. Quantification was performed using ImageJ software to calculate the percentage of Alcian Blue-positive area (by color thresholding) relative to total spheroid area, as described by the previous research (Klinder et al. 2020 ). 2.8 RNA extraction and qRT-PCR qRT-PCR was conducted based on a previously published protocol (Bi et al. 2020 ). Total RNA was extracted by SteadyPure Quick RNA Extraction Kit (AG21023, Accurate Biology). cDNA was synthesized by HiScript IV RT SuperMix for qPCR(+gDNA wiper)(R423-01, Vazyme). qRT-PCR was conducted with ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme). The primer sequences were synthesized by Sangon Biotech (Shanghai, China) as listed in Table 1. All genes were analyzed by Thermo Fisher QuantStudio™ 7. The relative content of mRNA was determined by the 2-ΔΔCt method and standardized with the results of GAPDH or β-actin. (Table S1 ) 2.9 Immunofluorescence staining Immunofluorescence staining was conducted based on a previously published protocol (Bi et al. 2020 ). Rat FCSCs and CPCs were seeded in confocal dishes. The cells were then washed with 1×PBS and fixed with 4% PFA at room temperature for 20 minutes. After fixation, the cells were permeabilized with 0.3% Triton X-100 (Beyotime, P0096) for 10 minutes and blocked with 5% bovine serum albumin (BSA, Beyotime, ST023) for 1 hour. Subsequently, the cells were incubated with the primary antibodies against anti-DKK3 (Abcam, ab186409, 1:1000) and anti-p-AKT (Ser473, Cell Signaling, 4060T, 1:2000) overnight at 4°C. Then, the cells were incubated with the corresponding fluorescently labeled secondary antibodies (HABIO, HA1121, HA1122) for 1 hour. After nuclear staining with DAPI, the cells were imaged using a fluorescence microscope. For tissue immunofluorescence, the TMJ and knee joints were harvested from 6-week-old male SD rats and fixed with 4% PFA (Solarbio, P1110). The samples were decalcified for one month, dehydrated, and embedded in paraffin. The paraffin-embedded samples were cut into 4-µm-thick sections. The sections were dewaxed to water and subjected to heat-induced antigen retrieval using citrate buffer (Beyotime, P0081). The subsequent steps were the same as those for the cell staining. 2.10 Western blot analysis Proteins were extracted using RIPA buffer (Beyotime, P0013B) with protease inhibitors. Lysates (30 µg) were separated by 4–12% SDS-PAGE (LABLEAD, P41215) and transferred to PVDF membranes (Millipore, IPFL00010). Membranes were incubated overnight at 4°C with primary antibodies: anti-DKK3 (Abcam, ab186409, 1:1000), anti-p-AKT (Ser473, Cell Signaling, 4060T, 1:2000), anti-AKT (Cell Signaling, 4691T, 1:1000), anti-β-actin (Servicebio,GB15003-100, 1:2000), anti-Collagen Type II (Sigma, SAB4500366, 1:1000), anti-SOX9 (Abcam, ab185966, 1:1000), anti-PI3K (Abmart, T40115, 1:1000). HRP-conjugated secondary antibodies (HABIO, HA1001, 1:10000) were used, and bands were visualized via ECL (Millipore, WBKLS0500). 2.11 Statistical analysis Except for RNA sequencing and flow cytometry (n = 3 biological replicates), all other experiments were performed with 5 biological replicates and 3 technical replicates per group, following randomization and with operators blinded to group allocation. Statistical analyses were conducted using GraphPad Prism version 9 (GraphPad Software, Boston, Massachusetts, USA). Data were expressed as mean ± SD. The statistical difference between two normally distributed groups was compared by Mann-Whitney U test. Multiple groups were compared using one-way ANOVA followed by Tukey’s post hoc test. For multiple comparisons, a two-way ANOVA followed by Tukey’s post hoc or Bonferroni’s post hoc test was used. Differences were considered statistically significant at p < 0.05. 3 Results 3.1 FCSCs and CPCs exhibit different tendencies during multi-lineage differentiation FCSCs and CPCs were isolated from the TMJ condylar cartilage and knee articular cartilage of 6-week-old male SD rats, respectively. Both populations expressed mesenchymal stem cell (MSC) markers (CD29, CD44, CD90) and lacked CD45, and exhibited comparable colony-forming capacity (Figure S1 ) , which confirmed the mesenchymal origin and stem cell phenotype of FCSCs and CPCs. To further clarify the cellular characteristics of FCSCs compared to CPCs, we performed bulk RNA-seq on these well-characterized cells (n = 3 per group). After mapping the raw RNA-seq data to the relevant genomes, we used statistical methods DESeq2 to compare gene expression levels in different groups and identify significantly DEGs. GO enrichment analysis and KEGG pathway analysis of the identified DEGs were also conducted. The transcriptomic profiles of gene expression in FCSCs and CPCs were compared. Cluster analysis showed that each sample in triplicate clustered together within groups, indicating distinct gene expression profiles between FCSCs and CPCs. A total of 1,599 DEGs were identified between the two groups (fold change > 2.0, p < .05), with 819 genes upregulated and 780 genes downregulated in the FCSCs group compared to the CPCs group ( Fig. 2 A ). GO enrichment revealed that genes upregulated in FCSCs were enriched in negative regulation of the Wnt pathway (GO: 0030178), which is consistent with previous literature indicating that the inhibition of the Wnt pathway is crucial for maintaining the FCSCs pool and cartilage stability (Embree et al. 2016 ). Simultaneously, compared to the CPCs group, the FCSCs group showed upregulation in GO terms related to ECM organization (GO: 0030198) and collagen fiber organization (GO: 0030199), suggesting that FCSCs possess a stronger capacity for ECM remodeling and cartilage homeostasis compared to CPCs. Additionally, in the FCSCs group compared to the CPCs group, GO terms related to osteogenesis were upregulated, such as osteoblast differentiation (GO: 0001649) and bone mineralization (GO: 0030282) ( Fig. 2 B ). This indicates that FCSCs may have superior osteogenic and chondrogenic capabilities compared to CPCs. Conversely, downregulated DEGs in FCSCs were associated with inflammatory pathways, such as TNF signaling pathway (KEGG: map04668) and cytokine-cytokine receptor interaction (KEGG: map04060) ( Fig. 2 C ). CPCs showed stronger associations with pathways related to inflammatory response and immune regulation. To validate the results of the transcriptomic analysis, we conducted in vitro verification using primary FCSCs and CPCs. We performed proliferation assays using the CCK8 method, which revealed that CPCs had a higher proliferation rate than FCSCs (p < .0001) ( Fig. 2 D ). Trilineage differentiation assays demonstrated that both cell types possessed the potential for multilineage induction, but with distinct differentiation capacities ( Fig. 2 E ) . CPCs exhibited stronger adipogenic differentiation potential than FCSCs, while FCSCs showed greater osteogenic and chondrogenic differentiation potential than CPCs ( Fig. 2 E-F ) . This suggests that, compared to CPCs, FCSCs possess unique chondrogenic capabilities. We further examined the expression levels of chondrogenic-related genes in FCSCs and CPCs after 14 days of chondrogenic induction. The results showed that the mRNA level of Aggrecan (p < .01) and Col10a1 (p < .01) were significantly higher in FCSCs than in CPCs ( Fig. 2 F ) , which indicated that FCSCs exhibit a stronger chondrogenic potential, particularly in cartilage matrix synthesis and cartilage maturation. 3.2 DKK3 maintains chondrogenic differentiation capacity of FCSCs Through GO enrichment analysis, we found that the negative regulation of the Wnt pathway is more pronounced in FCSCs. Based on this, we compared the expression differences of Wnt pathway-related genes between FCSCs and CPCs and created a heatmap ( Fig. 3 A ) . The results showed that the Wnt pathway-related gene DKK3 was more highly expressed in FCSCs than that in CPCs (5.6-fold, p < .01) ( Fig. 3 A ) . This was validated at the protein level by Western blot and immunofluorescence (p < .05) ( Fig. 3 B-C ) . Our tissue immunofluorescence results suggested that DKK3 expression appeared stronger in the fibrocartilaginous SZ of the condylar cartilage than in the superficial layer of knee cartilage (p < .05), and also appeared more intense compared to deeper zones within the condylar cartilage itself ( Fig. 3 C ) . This finding is consistent with previous studies (Utreja et al. 2016 ; Xu et al. 2022 ). This specific expression pattern suggests DKK3 is a potential marker of the condylar cartilage SZ. Previous research has confirmed that DKK3 can protect articular cartilage by inhibiting the Wnt and NF-κB pathways (Snelling et al. 2016 ; Conde et al. 2021 ). These results suggest that DKK3 may play an important role in the functional regulation of FCSCs. To verify the regulatory effect of DKK3 on the function of FCSCs, we successfully knocked down the DKK3 gene in FCSCs using shRNA technology, resulting in a significant decrease in both mRNA and protein levels ( Fig. 3 D-E ). After 14 days of chondrogenic induction, qPCR results revealed that the expression levels of chondrogenic-related genes (Col2a1, Sox9, Aggrecan and Col10a1, p < .01) in the shDKK3 group were significantly lower than those in the control group ( Fig. 3 E ). Western blotting further confirmed reduced expression of Col2a1 and Sox9 (p < .01) ( Fig. 3 F ) , and Alcian blue staining indicated diminished cartilage matrix synthesis in the shDKK3 group (p < .05) ( Fig. 3 G ) . These findings indicated that DKK3 was required for the efficient chondrogenic differentiation of FCSCs in rats. 3.3 DKK3 activates PI3K/AKT signaling in FCSCs To identify the downstream pathways through which DKK3 regulates the functions of FCSCs, we conducted in-depth analysis of the transcriptomic datasets. KEGG and GSEA analyses consistently revealed significant enrichment of the PI3K/AKT signaling pathway in FCSCs ( Fig. 4 A ). Single-gene enrichment analysis further revealed that genes exhibiting a strong positive correlation with DKK3 expression (Pearson correlation coefficient > 0.8) were predominantly associated with the PI3K/AKT signaling pathway ( Fig. 4 B ) . To verify the results of the transcriptomic analysis, we used Western blot to detect the expression levels of proteins related to the PI3K/AKT pathway. The results showed that the protein expression level of p-AKT (phosphorylated AKT) /AKT in FCSCs was significantly higher than that in CPCs (p < .01), indicating that the PI3K/AKT pathway was more active in FCSCs ( Fig. 4 C ) . Immunofluorescence also showed that the expression of p-AKT in the superficial layer of condylar cartilage is greater than in knee cartilage (p < .001) ( Fig. 4 D ). After knocking down the DKK3 gene in FCSCs using shRNA technology, Western blot quantification indicated a marked decrease in the p-AKT/AKT ratio (p < .001) ( Fig. 4 E ) , and immunofluorescence appeared to show a reduction in p-AKT nuclear translocation (p < .01) ( Fig. 4 F ) , indicating that the knockdown of DKK3 appeared to inhibit the activity of the PI3K/AKT pathway. 3.4 PI3K/AKT activation rescues DKK3 knockdown-induced chondrogenic impairment in FCSCs To validate the causal role of the DKK3-PI3K/AKT axis, the shDKK3 FCSCs were treated with the PI3K/AKT activator Recilisib (25µM, 24 h), followed by chondrogenic induction for 14 days. Recilisib (25µM, 24h) significantly restored PI3K/AKT pathway activation in shDKK3 FCSCs (p < .0001) ( Fig. 5 A-B ) . Chondrogenic induction assays revealed upregulation of chondrogenic markers (Col2a1, Sox9, Aggrecan and Col10a1, p < .01) at mRNA levels in the shDKK3 + Recilisib group compared to shDKK3 alone ( Fig. 5 C ) . Western blot results showed that, compared to the shDKK3 group alone, the shDKK3 + Recilisib group exhibited a significant upregulation of Sox9 (p < .001) ( Fig. 5 D ) . Alcian Blue staining further confirmed that Recilisib treatment partially rescued the proteoglycan synthesis deficiency in shDKK3 cells (p < .001) ( Fig. 5 E ) . These findings demonstrated that PI3K/AKT pathway activation can partially counteract the chondrogenic suppression caused by DKK3 knockdown, indicating that PI3K/AKT signaling was involved in mediating DKK3’s effects on FCSCs chondrogenesis. 3.5 DKK3 alleviates IL-1β-induced impairment of chondrogenic capacity in FCSCs To further evaluate the role of DKK3 in sustaining chondrogenic capacity under pathological stress, FCSCs were pretreated with IL‑1β (10 ng/mL, 24 h). Recombinant DKK3 protein (125 or 250 ng/mL) was then added for 12 h, followed by chondrogenic induction for 7 days. qPCR and Western blot analyses revealed that IL‑1β stimulation markedly impaired the chondrogenic capacity of FCSCs (p < .05) ( Fig. 6 A ). The addition of recombinant DKK3 (125 or 250 ng/ml) restored the mRNA expression of Sox9, Col2a1, and Aggrecan, as well as the protein levels of Sox9 and Aggrecan (p < .05) ( Fig. 6 A-B ) . These findings suggested that DKK3 may help mitigate inflammation‑induced impairment of chondrogenic differentiation in FCSCs. 4 Discussion Knee OA and TMJ OA share similar pathological changes and symptoms, but TMJ OA exhibits distinct characteristics. TMJ cartilage is fibrocartilaginous and primarily resists tensile and shear forces, whereas knee cartilage is hyaline and mainly bears compressive loads. These biomechanical differences partly explain why current therapeutic strategies for knee OA often show limited efficacy in TMJ OA, highlighting the necessity of exploring TMJ-specific cellular and molecular mechanisms. Given the critical roles of FCSCs and CPCs in TMJ and knee cartilage repair, elucidating functional differences between FCSCs and CPCs may provide valuable insights for TMJ OA treatment. In this study, we isolated FCSCs and CPCs from TMJ and knee cartilage, respectively. Both cell types expressed classic mesenchymal stem cell markers such as CD29, CD44, and CD90, while lacking the hematopoietic marker CD45, and demonstrated comparable colony-forming capacity. These phenotypic characteristics are consistent with previous reports on FCSCs(Embree et al. 2016 ; Gong et al. 2025 ) and CPCs(Koelling et al. 2009 ; Jayasuriya et al. 2019 ), confirming their mesenchymal progenitor identity. Subsequently, we systematically compared FCSCs and CPCs using transcriptomic profiling and functional assays. Transcriptomic data showed FCSCs exhibited upregulated activity in Wnt pathway negative regulation, ECM organization, collagen fibril organization, and osteogenic pathways compared to CPCs, indicating superior ECM remodeling, cartilage homeostasis, and osteogenic differentiation capabilities. Conversely, CPCs showed stronger associations with inflammatory response and immune regulation pathways. Functional assays further demonstrated that CPCs displayed higher proliferative capacity. In terms of tri-lineage differentiation potential, FCSCs have weaker adipogenic capacity than CPCs, while FCSCs exhibit stronger osteogenic and chondrogenic potential. These findings align with previous observations that human FCSCs (hFCSCs) possess greater chondrogenic capacity than human orofacial mesenchymal stem cells (hOFMSCs) (Bi et al. 2020 ), enriching the comparative characterization of region-specific stem cell populations. Based on the significant potential of FCSCs in chondrogenesis, exploring the regulatory mechanisms of chondrogenic differentiation of FCSCs may provide a unique perspective for TMJ disease treatment. Among the differentially expressed genes, DKK3 emerged as a molecule of interest. Although classified within the Dickkopf family, DKK3 does not function solely as a canonical Wnt inhibitor (Hu et al. 2024 ; Cooney et al. 2023 ; Mourtada et al. 2023 ; Sadeghi et al. 2019 ), and its role in bone and cartilage development has gained growing recognition. DKK3 exhibits unique expression patterns in mouse cranial bones distinct from DKK1 and DKK2 (Nie et al. 2005 ). During limb development, DKK3 shows spatiotemporal specificity, highly expressed in joint cells and surrounding perichondrium (Witte et al. 2009 ), implicating it in joint formation and cartilage differentiation. Additionally, DKK3 marks quiescent periosteal cells in the fibrous layer, co-expressed with SMAA and Col3.6, suggesting a role in mesenchymal progenitor differentiation into fibrocartilage cells (Mori et al. 2016 ). In ATDC5 cells, DKK3 expression increases during the early stages of chondrogenesis and sharply decreases during the chondrocyte hypertrophy stage, indicating its role in early cartilage development. (Snelling et al. 2016 ; Conde et al. 2021 ). DKK3 has been found to protect cartilage by inhibiting Wnt signaling, enhancing TGF-β signaling, and suppressing NF-κB signaling pathways, thereby reducing the production of MMP-13 and protecting cartilage matrix from inflammatory factors (Conde et al. 2021 ). Dynamic DKK3 modulation during cartilage injury repair was observed in murine hip dislocation models (Snelling et al. 2016 ). Within the TMJ, DKK3 exhibits a strikingly specific distribution pattern. Previous studies localize DKK3 predominantly to the condylar cartilage superficial zone, where dynamic mechanical loading upregulates its expression and is associated with cartilage thickening (Utreja et al. 2016 ), suggesting a role in tissue adaptation. Consistent with this, our transcriptomics revealed significantly higher DKK3 expression in TMJ FCSCs versus knee CPCs, establishing DKK3 as a potential marker for the TMJ cartilage superficial zone. However, its precise function within the TMJ remains poorly understood. Using shRNA knockdown, we found reduced DKK3 expression significantly impaired FCSCs chondrogenic differentiation, evidenced by downregulated Sox9, Col2a1, and Aggrecan. These findings align with the documented protective role of DKK3 in cartilage homeostasis (Conde et al. 2021 ), yet the underlying mechanisms remain incompletely defined. The PI3K/AKT pathway is a central regulator of cell survival, proliferation, and differentiation (Jafari et al. 2019 ), and plays a crucial dual role in cartilage biology. In TMJ, the activation of the PI3K/AKT pathway can protect condylar chondrocytes from IL-1β-induced apoptosis and promote the expression of ECM (Chen et al. 2016 ; Shen et al. 2021 ). Additionally, exosomes derived from bone marrow mesenchymal stem cells (BMSCs) alleviate chondrocyte apoptosis in the TMJ disc of TMJOA via the PI3K/AKT pathway (Chen et al. 2025 ). Conversely, pathological PI3K/AKT activation mediates endoplasmic reticulum stress-induced apoptosis in TMJ chondrocytes (Zhou et al. 2024 ) and may exacerbate degeneration by disrupting FCSCs function (Yin et al. 2023 ), highlighting the need for precise regulation. Our study reveals tissue-specific PI3K/AKT activation in FCSCs compared to CPCs, with pathway activity strongly correlated to DKK3 expression. Previous studies demonstrate that DKK3 activates PI3K/AKT signaling to promote cell proliferation and migration in head and neck squamous cell carcinoma (HNSCC) and benign prostatic hyperplasia models (Katase et al. 2019 ; Xia et al. 2024 ), and confers neuroprotection in post-cardiac arrest brain injury (PCABI) models (Xu et al. 2025 ). Consistently, our transcriptomic analysis linked DKK3 to PI3K/AKT pathway in FCSCs, and DKK3 knockdown reduced AKT phosphorylation, confirming pathway regulation. Critically, PI3K/AKT activators can reverse the chondrogenic defects caused by DKK3 knockdown, thereby establishing a unique DKK3-PI3K/AKT axis in FCSCs. These findings reveal DKK3’s tissue-specific role in fine-tuning PI3K/AKT activity to balance chondrogenesis and stem cell maintenance—advancing FCSC biology in TMJ OA and revealing novel therapeutic targets. However, it is noteworthy that the PI3K/AKT activator did not fully restore the chondrogenic differentiation level of FCSCs, suggesting that the regulatory network governed by DKK3 may extend beyond a single pathway. It is known that DKK3 can interact with multiple signaling cascades, including the canonical/non-canonical Wnt and NF-κB pathways (Mourtada et al. 2023 ) (Conde et al. 2021 ), which are crucial for cartilage homeostasis. In addition, AKT can stabilize β-catenin and inhibit GSK-3β to enhance Wnt signaling, and PI3K-dependent IKK activation can modulate NF-κB activity (Rogers et al. 2008 ; Li et al. 2018 ). Reactivating PI3K/AKT alone may therefore be insufficient to compensate for the loss of DKK3’s simultaneous modulation of other axes. Future studies are needed to further elucidate these crosstalk mechanisms. Inflammatory cytokines are key mediators of cartilage degeneration in TMJ OA. Inflammatory cytokines such as IL-1β are elevated in TMJ OA. In human OA cartilage, IL-1β treatment reduces DKK3 expression (Conde et al. 2021 ).In our study, IL-1β impaired FCSC chondrogenesis, while exogenous DKK3 restored chondrogenic marker expression, suggesting that DKK3 acts as a protective regulator under inflammatory conditions, preserving the chondrogenic potential of FCSCs. This aligns with reports that exogenous DKK3 can inhibit IL-1β-mediated proteoglycan loss by suppressing NF-κB signaling (Conde et al. 2021 ). This study has several limitations. First, 6-week-old male rats were selected because adolescent rats provide a cartilage environment with high progenitor cell activity, and the use of males eliminates the confounding influence of cyclic estrogen fluctuations, thereby focusing on core mechanisms. Consequently, it does not recapitulate the pathophysiology of aged or osteoarthritic joints, and the potential influence of estrogen on the identified axis remains unexplored. Second, our study primarily employed in vitro experiments to elucidate the regulation of the DKK3-PI3K/AKT axis on FCSC chondrogenesis under both physiological and inflammatory conditions. In vivo validation is still lacking. Future research should therefore focus on confirming the protective role of this axis in TMJ OA animal models and exploring whether its targeted modulation can delay or reverse disease progression. 5 Conclusion This study elucidates that DKK3 maintains the chondrogenic capacity of FCSCs via the PI3K/AKT signaling pathway. Compared with CPCs, FCSCs exhibit a unique differentiation bias and rely on the DKK3-mediated pathway. Targeting the DKK3-PI3K/AKT axis offers new perspectives for understanding TMJ cartilage biology and stem cell regulation. Further exploration of the in vivo efficacy of DKK3-based interventions is needed in the future. CRediT authorship contribution statement Qiaoli Dai : Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization Ying Wang : Writing – review & editing, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Cunyi Wang : Writing – review & editing, Project administration, Methodology, Investigation. Wenlin Yuan : Writing – review & editing, Project administration, Methodology, Funding acquisition. Yilin Chen : Writing – review & editing, Data curation. Mengqi Zhu : Writing – review & editing, Data curation. Weilin Zhang : Writing – review & editing, Data curation. Jiejun Shi : Writing – review & editing, Funding acquisition, Conceptualization. Zuping Wu : Writing – review & editing, Funding acquisition, Conceptualization. All authors critically revised the manuscript and approved the final version. Abbreviations Abbreviations: TMJ temporomandibular joint hFCSCs human fibrocartilage stem cells OA osteoarthritis hOMSCs human orofacial mesenchymal stem cells FCSCs fibrocartilage stem cells ECM extracellular matrix CPCs chondrogenic progenitor cells shRNA short hairpin RNA RNA-seq RNA sequencing RUNX2 Runt-related transcription factor 2 DKK3 Dickkopf-3 GO gene ontology DMEM Dulbecco’s Modified Eagles Medium SZ superficial zone DEGs differentially expressed genes p-AKT phosphorylated AKT PCABI post-cardiac arrest brain injury BMSCs bone marrow mesenchymal stem cells HNSCC head and neck squamous cell carcinoma MSC mesenchymal stem cell FBS fetal bovine serum PS penicillin-streptomycin PFA paraformaldehyde ALP alkaline phosphatase Declarations 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. Acknowledgments We want to express our gratitude for the drawing materials provided by BioRender. Funding This work was supported by the grants from the National Natural Science Foundation of China (82170984), the National Natural Science Foundation of China (No. 82501200), the Joint TCM Science & Technology Projects of National Demonstration Zones for Comprehensive TCM Reform (No. GZY-KJS-ZJ-2025-094), and Zhejiang Provincial Natural Science Foundation of China under Grant No. Q24H140004. Author Contribution **Qiaoli Dai:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization **Ying Wang:** Writing – review & editing, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Cunyi Wang:** Writing – review & editing, Project administration, Methodology, Investigation. **Wenlin Yuan:** Writing – review & editing, Project administration, Methodology, Funding acquisition. **Yilin Chen:** Writing – review & editing, Data curation. **Mengqi Zhu:** Writing – review & editing, Data curation. **Weilin Zhang:** Writing – review & editing, Data curation. **Jiejun Shi:** Writing – review & editing, Funding acquisition, Conceptualization. **Zuping Wu:** Writing – review & editing, Funding acquisition, Conceptualization. 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(molecular function) and KEGG pathway terms of downregulated DEGs in FCSCs versus CPCs. \u003cstrong\u003e(D)\u003c/strong\u003e CCK8 proliferation assay of FCSCs and CPCs. \u003cstrong\u003e(E)\u003c/strong\u003eOil red O staining in FCSCs and CPCs after 14 days of adipogenic differentiation (left), ALP staining in FCSCs and CPCs after 7 days of osteogenic differentiation (middle), Alcian Blue staining in FCSCs and CPCs after 21 days of chondrogenic differentiation (right). \u003cstrong\u003e(F)\u003c/strong\u003e The relative mRNA levels of Pparg, Dagt, Adipoq and Plinh in FCSCs and CPCs after 14 days of adipogenic differentiation (left), the relative mRNA levels of Osx, Alp, Ocn and Col1a1 in FCSCs and CPCs after 14 days of osteogenic differentiation (middle), the relative mRNA levels of Aggrecan, Col10a1, Sox9 and Col2a1 in FCSCs and CPCs after 7 days of chondrogenic differentiation (right). Except for RNA‑seq (n = 3 biological replicates), other experiments were performed with n = 5 biological replicates, all data were expressed as the Means ± SD (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9282133/v1/6578cadc570803ee38c056ab.png"},{"id":106323263,"identity":"a3deaa60-0d8a-4120-9890-646286e8c0b8","added_by":"auto","created_at":"2026-04-07 12:41:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2078401,"visible":true,"origin":"","legend":"\u003cp\u003eDKK3 knockdown impairs chondrogenic differentiation of FCSCs. \u003cstrong\u003e(A)\u003c/strong\u003e The heatmap of expression differences of Wnt pathway-related genes between FCSCs and CPCs (left). Gene expression levels of DKK3 in FCSCs and CPCs (right). \u003cstrong\u003e(B)\u003c/strong\u003eWestern blotting of DKK3 in FCSCs and CPCs (left). Protein expressions of DKK3 in FCSCs and CPCs (right). \u003cstrong\u003e(C)\u003c/strong\u003e Immunofluorescence of DKK3 in the two groups. DKK3: green fluorescence, DAPI: blue fluorescence. \u003cstrong\u003e(D)\u003c/strong\u003e Western blotting and protein expressions of DKK3 in shC and shDKK3 group. \u003cstrong\u003e(E) \u003c/strong\u003eRelative mRNA levels of DKK3, Sox9, Aggrecan, Col2a1 and Col10a1 in shC and shDKK3 group after 14 days of chondrogenic differentiation. \u003cstrong\u003e(F)\u003c/strong\u003e Protein expressions of DKK3, Sox9 and Col2a1 in shC and shDKK3 group after 14 days of chondrogenic differentiation. \u003cstrong\u003e(G)\u003c/strong\u003e Alcian Blue staining in shC and shDKK3 group after 21 days of chondrogenic differentiation. n = 5 biological replicates, all data were expressed as the Means ± SD (*p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9282133/v1/fa689721bd63f4e49da09a78.png"},{"id":106403905,"identity":"68cf9d19-59dc-4f99-b0d1-b7bec3cdf668","added_by":"auto","created_at":"2026-04-08 09:15:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2759429,"visible":true,"origin":"","legend":"\u003cp\u003eDKK3 knockdown inhibits the activity of the PI3K/AKT pathway in FCSCs. \u003cstrong\u003e(A) \u003c/strong\u003eThe top 16 significant KEGG pathway terms of upregulated DEGs in FCSCs versus CPCs. GSEA of PI3K/AKT signaling pathway gene sets in FCSCs group and CPCs group. \u003cstrong\u003e(B)\u003c/strong\u003e single-gene enrichment analysis of DKK3 in FCSCs group. \u003cstrong\u003e(C) \u003c/strong\u003eWestern blotting and protein expressions of PI3K/AKT pathway in CPCs and FCSCs. \u003cstrong\u003e(D)\u003c/strong\u003e Immunofluorescence of p-AKT in the TMJ and knee. p-AKT: green fluorescence, DAPI: blue fluorescence. \u003cstrong\u003e(E)\u003c/strong\u003eWestern blotting and protein expressions of PI3K/AKT pathway in Control, shC and shDKK3 group. \u003cstrong\u003e(F)\u003c/strong\u003e Immunofluorescence of DKK3 and p-AKT in shC and shDKK3 group. DKK3 and p-AKT: red fluorescence, DAPI: blue fluorescence. n = 5 biological replicates, all data were expressed as the Means ± SD (**p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9282133/v1/77cd5e591702e5617481d20f.png"},{"id":106323449,"identity":"d7b06dbd-b0ac-450b-9777-3de2cd167b05","added_by":"auto","created_at":"2026-04-07 12:42:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1932948,"visible":true,"origin":"","legend":"\u003cp\u003ePI3K/AKT activation rescues DKK3 knockdown-induced chondrogenic impairment in FCSCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Western blotting of p-AKT in shC, shDKK3 and shDKK3+Recilisib group. \u003cstrong\u003e(B)\u003c/strong\u003e Immunofluorescence of p-AKT in shC, shDKK3 and shDKK3+Recilisib group. p-AKT: red fluorescence, DAPI: blue fluorescence. \u003cstrong\u003e(C)\u003c/strong\u003eRelative mRNA levels of DKK3, Aggrecan, Sox9, Col2a1 and Col10a1 in shC, shDKK3 and shDKK3+Recilisib group after 14 days of chondrogenic differentiation. \u003cstrong\u003e(D)\u003c/strong\u003eWestern blotting and protein expressions of Sox9 in shC, shDKK3 and shDKK3+Recilisib group. \u003cstrong\u003e(E)\u003c/strong\u003e Alcian Blue staining in shC, shDKK3 and shDKK3+Recilisib group after 21 days of chondrogenic differentiation. n = 5 biological replicates, all data were expressed as the Means ± SD (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9282133/v1/e3c26cb6cf8848e3a05205aa.png"},{"id":106404451,"identity":"8cd68c0d-b1ce-4f4a-8976-30a53529633e","added_by":"auto","created_at":"2026-04-08 09:16:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":505418,"visible":true,"origin":"","legend":"\u003cp\u003eRecombinant DKK3 mitigates IL-1β-induced impairment of chondrogenic differentiation in FCSCs. \u003cstrong\u003e(A) \u003c/strong\u003eRelative mRNA levels of Sox9, Aggrecan, Col2a1 and Col1a1 in FCSCs, FCSCs+IL-1β, FCSCs+IL-1β+125ng/ml DKK3 and FCSCs+IL-1β+250ng/ml DKK3 groups after 7 days of chondrogenic differentiation. \u003cstrong\u003e(B)\u003c/strong\u003e Western blotting and protein expressions of Sox9 and Aggrecan in FCSCs, FCSCs+IL-1β, FCSCs+IL-1β+125ng/ml DKK3 and FCSCs+IL-1β+250ng/ml DKK3 groups after 7 days of chondrogenic differentiation. n = 5 biological replicates, all data were expressed as the Means ± SD (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9282133/v1/fea214cd2884e536e23001bb.png"},{"id":106404089,"identity":"c05d8507-88c7-490c-98cc-e032eed04e59","added_by":"auto","created_at":"2026-04-08 09:15:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":648132,"visible":true,"origin":"","legend":"\u003cp\u003eThe DKK3-PI3K/AKT axis in FCSCs. (Image created with BioRender.com, with permission).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9282133/v1/f9b3e35f560d942905321a49.png"},{"id":106406021,"identity":"2e1d2dd6-23fb-4737-8fa8-8ded845d4b2f","added_by":"auto","created_at":"2026-04-08 09:29:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11117343,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9282133/v1/bc6d6eee-ea3a-4a24-90be-ece212370d44.pdf"},{"id":106323274,"identity":"66f9726d-8435-46f7-bb44-c892f036e955","added_by":"auto","created_at":"2026-04-07 12:41:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16087,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1caption.docx","url":"https://assets-eu.researchsquare.com/files/rs-9282133/v1/838a391a3d73c1255c702a98.docx"},{"id":106404443,"identity":"ab08016c-992a-4c78-bfc2-c7209b66a9d2","added_by":"auto","created_at":"2026-04-08 09:16:01","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":39441892,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-9282133/v1/970b02f41cae9130aae792a8.tif"},{"id":106323448,"identity":"464d1686-125c-46c1-a245-3f311afec888","added_by":"auto","created_at":"2026-04-07 12:42:09","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18511,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9282133/v1/f413302cfe7f8c0e536e5425.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative transcriptomics of fibrocartilage stem cells and knee chondrogenic progenitors identifies a DKK3-PI3K/AKT regulatory axis","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe incidence of osteoarthritis (OA) is rising, affecting approximately 595\u0026nbsp;million individuals globally (about 7.6% of the world\u0026rsquo;s population) with a projected increase of approximately 60.1% by 2050 (Courties et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Global, regional, and national burden of osteoarthritis, 1990\u0026ndash;2020 and projections to 2050: a systematic analysis for the Global Burden of Disease Study 2021 2023). OA is primarily characterized by cartilage degradation, subchondral bone remodeling, and synovitis (Mahmoudian et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although knee OA and temporomandibular joint (TMJ) OA are both mechano-inflammatory diseases, they exhibit significant heterogeneity in anatomy, biomechanics and pathological changes. Structurally, TMJ articular surfaces consist of fibrocartilage rich in type I collagen, in contrast to the type II collagen-dominated hyaline cartilage of the knee. This compositional divergence directly dictates biomechanical specialization: TMJ fibrocartilage is optimized for dynamic tensile stresses (e.g., masticatory loading), whereas knee hyaline cartilage primarily resists compressive forces (Lei et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Pathologically, knee OA typically begins with superficial cartilage fraying and erosion, characterized by surface cartilage fissures. In contrast, TMJ OA is typically characterized by more prevalent deep-layer fissures and milder surface damage. Further differences emerge in inflammatory signaling, hormonal sensitivity, and mechanotransduction pathways, underscoring the site-specific nature of OA pathophysiology (Zhao et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Tian et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eArticular cartilage, an avascular tissue, relies heavily on resident stem/progenitor cells for injury repair. Fibrocartilage stem cells (FCSCs) localized to the superficial zone (SZ) of the TMJ condylar cartilage (Embree et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and chondrogenic progenitor cells (CPCs) distributed within knee articular cartilage represent two key reparative populations. However, their spatial distribution and microenvironmental niches differ markedly. FCSCs are restricted to the SZ of TMJ condylar cartilage (Embree et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), directly exposed to dynamic biomechanical stress and synovial biochemical stimuli. In OA, FCSCs exhibit reduced numbers and altered activity, with their main population remaining enriched in the SZ of the condylar cartilage (Wang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Tuwatnawanit et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). CPCs reside primarily in the superficial layer of normal knee cartilage but can expand into deeper zones or even migrate from subchondral bone in late‑stage OA (Jiang and Tuan \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Koelling et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). These microenvironmental differences suggest divergent signaling pathways regulating cartilage homeostasis in FCSCs versus CPCs. However, systematic investigations into their transcriptomic profiles and regulatory networks remain lacking, which limits our understanding of their specific roles in the respective OA conditions.\u003c/p\u003e \u003cp\u003eCurrently, therapeutic strategies for TMJ OA largely follow those for knee OA (such as anti-inflammatory drugs and intra-articular injections), but their efficacy is controversial (Bielajew et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, systematically elucidating the functional differences between FCSCs and CPCs based on the heterogeneity of TMJ and knee OA and the differentiation of stem cell microenvironments is of vital importance. Dickkopf-3 (DKK3), a secreted glycoprotein and Wnt pathway modulator, has emerged as a cartilage-protective factor that inhibits inflammatory matrix degradation (Conde et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and is highly expressed in the SZ of the TMJ condyle (Utreja et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This expression pattern suggests a potential role for DKK3 in maintaining FCSCs function, yet studies directly examining this relationship are currently lacking. Here, we compared the cellular characteristics of FCSCs and CPCs, such as proliferation, differentiation, migration, multi-lineage differentiation, and other specific transcriptional features through RNA-seq analysis, in order to identify critical regulatory proteins. Focusing on DKK3, we aimed to elucidate its role in regulating FCSCs function, thereby uncovering novel regulatory mechanisms that could provide clues to the pathogenesis and treatment of TMJ OA.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell isolation and culture\u003c/h2\u003e \u003cp\u003ePrimary FCSCs and CPCs were isolated and cultured from 6-week-old male SD (Sprague-Dawley) rats (Zhejiang University's experimental animal center) (Hua et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Embree et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Only male subjects were selected to eliminate the influence of estrogen (Yue et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Rats were anesthetized via intraperitoneal injection of 3% pentobarbital sodium (50 mg/kg body weight). Euthanasia was performed by cervical dislocation under deep anesthesia. Primary FCSCs were isolated and cultured from TMJ condylar cartilage. To isolate FCSCs, the condyles were digested by 4 mg/ml type II dispase (Solarbio, D6431) for 15 min before separating the SZ, and then the SZ was digested in 4 mg/ml type II dispase (Solarbio, D6431) and 3 mg/ml type I collagenase (Solarbio, C8140). Single-cell suspensions were cultured in Dulbecco\u0026rsquo;s Modified Eagles Medium (DMEM with high glucose, Gibco, C11995500BT) containing 10% fetal bovine serum (FBS, Gibco, A5669701), glutamax (Invitrogen, 35050-061) and 1% penicillin-streptomycin (PS, Solarbio, P1400) at 37\u0026deg;C with 5% CO₂. The culture medium was replaced after 48 hours (Embree et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Once the FCSCs reached 80% confluence, they were digested with 0.25% trypsin containing EDTA (Gibco, 25200056). The cells were then passaged (P1-3) and plated for in vitro experiments. Primary CPCs were isolated and cultured from knee joint cartilage of the same rats. Cartilage tissue from the non-weight-bearing area of the rat knee joint tibial plateau and the lateral femoral condyle was cut into 1 mm pieces and digested in 1.5 mg/ml type II collagenase (Solarbio, C8150) for 2\u0026ndash;4 hours. The resulting cells were discarded, and the digested cartilage slices were incubated in DMEM containing 10% FBS and 1% PS at 37\u0026deg;C with 5% CO₂ (Wang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Batschkus et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The culture medium was replaced after 48 hours. Once the migrated CPCs reached 80% confluence, they were digested with 0.25% trypsin containing EDTA, and P1-3 cells were plated for in vitro experiments. Animal experiments were approved by the Ethics Committee of Zhejiang University (Number: ZJU20190007).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Flow Cytometry\u003c/h2\u003e \u003cp\u003eCPCs and FCSCs were immunolabeled with fluorescent conjugated antibodies or isotype-matched IgG controls for 30 min at 4\u0026deg;C, and then the cells were washed with Phosphate Buffered Saline (PBS) and resuspended. Cells were subjected to fluorescence activated cell sorting. The fluorescent conjugated antibodies included: anti-CD29-PE (eBioscience,12-0291-82, 1:100), anti-CD44-APC (Abcam, ab81424, 1:200), and anti-CD90-FITC (Abcam, ab226, 1:200) and anti-CD45-PB (BioLegend, 202225, 1:200) \u003csup\u003e(Bi et al. 2020)\u003c/sup\u003e. Flow cytometry experiments were conducted using FACSAria II and samples were calculated using Flowjo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 RNA Sequencing and Bioinformatics\u003c/h2\u003e \u003cp\u003eRNA sequencing was performed by Biomarker Technologies (Beijing, China). Total RNA was extracted from FCSCs and CPCs (n\u0026thinsp;=\u0026thinsp;3 biological replicates per group) using TRIzol reagent (Invitrogen, 15596026) (Tosa et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and RNA integrity was assessed using the Agilent Bioanalyzer 2100. Subsequently, libraries were constructed using the NEBNext Ultra\u0026trade; RNA Library Prep Kit and sequenced on the Illumina platform. Clean reads were aligned to the reference genome using HISAT2 software, and transcript assembly and expression quantification were performed using StringTie. Differential expression analysis was conducted with DESeq2 software to identify significantly differentially expressed genes, with raw reads aligned to the rat reference genome (rn6) using HISAT2. Differentially expressed genes (DEGs) were identified using DESeq2 software (Fold Change\u0026thinsp;\u0026ge;\u0026thinsp;2 and FDR \u0026lt; .01) (Tosa et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted to reveal gene functions and pathway changes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Colony Forming Assay\u003c/h2\u003e \u003cp\u003eFCSCs and CPCs at P2 at logarithmic phase were harvested and cultured (40 cells/cm\u003csup\u003e2\u003c/sup\u003e in 6-well plates). After 14 days\u0026rsquo; culture, cells were fixed with 4% paraformaldehyde (PFA, Solarbio, P1110) for 15 min, washed in PBS and stained with 0.1% crystal violet (Beyotime, C0121) for 20 min. Cells were next washed with ddH₂O till clarified, and plates were air-dried and photographed. Colonies larger than 50 \u0026micro;m in diameter were blindly counted 3 times by 2 observers respectively. This assay was repeated with 5 biological replicates, and clonogenic efficiency of FCSCs and CPCs was calculated and compared between groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell Proliferation Assay\u003c/h2\u003e \u003cp\u003eCell proliferation potential of FCSCs and CPCs was evaluated using the Cell Counting Kit-8 (CCK-8, Beyotime, C0042) (Du et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Cells were seeded in 96-well plates at a density of 2\u0026times;10\u0026sup3; cells/well and cultured in complete growth medium (DMEM supplemented with 10% FBS and 1% PS). After 24, 48, 72, and 96 hours of incubation at 37\u0026deg;C under 5% CO₂, 10 \u0026micro;L CCK-8 reagent was added to each well. Absorbance at 450 nm was measured using a microplate reader (Bio-Tek Synergy H1) following 2 hours of incubation. Randomization was applied during cell seeding, and operators were blinded to group allocation during absorbance measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 shRNA-Mediated DKK3 Knockdown\u003c/h2\u003e \u003cp\u003eLentiviral particles encoding short hairpin RNA (shRNA) targeting DKK3 (shDKK3: 5\u0026prime;- GTGGAGAGAAGATTTAGACCT-3\u0026prime;) and scrambled control (shCtrl) were synthesized by Bioscien (Shanghai, China), a professional supplier of lentiviral vectors and shRNA synthesis services (Moore et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). FCSCs were transfected at 70% confluence with a multiplicity of infection MOI of 100. After 72 hours, puromycin (3 \u0026micro;g/mL, Beyotime, ST551) was added for 5 days to select stable clones. Knockdown efficiency was validated by qRT-PCR and Western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Multi-lineage differentiation\u003c/h2\u003e \u003cp\u003eMulti-lineage differentiation protocols were adapted from the study by Embree et al.(Embree et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) with minor modifications. We tested adipogenic, osteogenic, and chondrogenic differentiation in vitro using chemically defined media. For adipogenesis, FCSCs and CPCs with a cell density of 2\u0026times;10⁴ were cultivated in a 24-well plate. Upon reaching 70% confluence, the cells were cultured using commercial adipogenic media (Oricell, RAXMD-90031). After 2 weeks, the cells were fixed in 4% PFA (Solarbio, P1110) and stained with Oil Red O. For osteogenesis, FCSCs were seeded at a density of 2\u0026times;10⁴ cells per well in a 24-well plate. When the cells reached 70% confluence, they were cultured with commercial osteogenic induction medium (Amizona Scientific, AMK8006-200). After 1 week, the cells were fixed with 4% PFA and stained with alkaline phosphatase (ALP) (Beyotime, C3206). Chondrogenic differentiation was conducted via two culture systems (2D monolayer and 3D pellet). For 2D monolayer chondrogenic induction, FCSCs and CPCs with a cell density of 2\u0026times;10⁴ were cultivated in a 24-well plate. The cells were cultured in high glucose DMEM medium supplemented with 100 nM dexamethasone (MCE, HY-14648), 1 mM sodium pyruvate (Gibco, 11360070), 50 \u0026micro;g/ml L-ascorbic-2-phosphate (Sigma, A8960), 40 mg/ml L-proline (Aladdin, P108709), 1% insulin-transferrin-selenium (Beyotime, C0341), and 10 ng/ml transforming growth factor-beta 3 (TGF-β3, PeproTech, 100-21C), when they reached 90% confluence. The cells were cultured for 3 weeks and stained with Alcian Blue (OriCell, ALCB-10001) for glycosaminoglycans. For 3D pellet chondrogenic induction, cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e per pellet) were pelleted in 15 ml polypropylene tubes by centrifugation and cultured (5% CO\u003csub\u003e2\u003c/sub\u003e, 37\u0026deg;C) for 3 weeks in the above chondrogenic induction medium. The medium was changed every 2\u0026ndash;3 days to maintain nutrient supply. After 3 weeks, 3D pellets were fixed with 4% PFA (Solarbio, P1110), cut into 3 \u0026micro;m paraffin sections, and stained with Alcian Blue (OriCell, ALCB-10001) for glycosaminoglycans. Quantification was performed using ImageJ software to calculate the percentage of Alcian Blue-positive area (by color thresholding) relative to total spheroid area, as described by the previous research (Klinder et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 RNA extraction and qRT-PCR\u003c/h2\u003e \u003cp\u003eqRT-PCR was conducted based on a previously published protocol (Bi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Total RNA was extracted by SteadyPure Quick RNA Extraction Kit (AG21023, Accurate Biology). cDNA was synthesized by HiScript IV RT SuperMix for qPCR(+gDNA wiper)(R423-01, Vazyme). qRT-PCR was conducted with ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme). The primer sequences were synthesized by Sangon Biotech (Shanghai, China) as listed in Table\u0026nbsp;1. All genes were analyzed by Thermo Fisher QuantStudio\u0026trade; 7. The relative content of mRNA was determined by the 2-ΔΔCt method and standardized with the results of GAPDH or β-actin. \u003cb\u003e(Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was conducted based on a previously published protocol (Bi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Rat FCSCs and CPCs were seeded in confocal dishes. The cells were then washed with 1\u0026times;PBS and fixed with 4% PFA at room temperature for 20 minutes. After fixation, the cells were permeabilized with 0.3% Triton X-100 (Beyotime, P0096) for 10 minutes and blocked with 5% bovine serum albumin (BSA, Beyotime, ST023) for 1 hour. Subsequently, the cells were incubated with the primary antibodies against anti-DKK3 (Abcam, ab186409, 1:1000) and anti-p-AKT (Ser473, Cell Signaling, 4060T, 1:2000) overnight at 4\u0026deg;C. Then, the cells were incubated with the corresponding fluorescently labeled secondary antibodies (HABIO, HA1121, HA1122) for 1 hour. After nuclear staining with DAPI, the cells were imaged using a fluorescence microscope.\u003c/p\u003e \u003cp\u003eFor tissue immunofluorescence, the TMJ and knee joints were harvested from 6-week-old male SD rats and fixed with 4% PFA (Solarbio, P1110). The samples were decalcified for one month, dehydrated, and embedded in paraffin. The paraffin-embedded samples were cut into 4-\u0026micro;m-thick sections. The sections were dewaxed to water and subjected to heat-induced antigen retrieval using citrate buffer (Beyotime, P0081). The subsequent steps were the same as those for the cell staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western blot analysis\u003c/h2\u003e \u003cp\u003eProteins were extracted using RIPA buffer (Beyotime, P0013B) with protease inhibitors. Lysates (30 \u0026micro;g) were separated by 4\u0026ndash;12% SDS-PAGE (LABLEAD, P41215) and transferred to PVDF membranes (Millipore, IPFL00010). Membranes were incubated overnight at 4\u0026deg;C with primary antibodies: anti-DKK3 (Abcam, ab186409, 1:1000), anti-p-AKT (Ser473, Cell Signaling, 4060T, 1:2000), anti-AKT (Cell Signaling, 4691T, 1:1000), anti-β-actin (Servicebio,GB15003-100, 1:2000), anti-Collagen Type II (Sigma, SAB4500366, 1:1000), anti-SOX9 (Abcam, ab185966, 1:1000), anti-PI3K (Abmart, T40115, 1:1000). HRP-conjugated secondary antibodies (HABIO, HA1001, 1:10000) were used, and bands were visualized via ECL (Millipore, WBKLS0500).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e \u003cp\u003eExcept for RNA sequencing and flow cytometry (n\u0026thinsp;=\u0026thinsp;3 biological replicates), all other experiments were performed with 5 biological replicates and 3 technical replicates per group, following randomization and with operators blinded to group allocation. Statistical analyses were conducted using GraphPad Prism version 9 (GraphPad Software, Boston, Massachusetts, USA). Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The statistical difference between two normally distributed groups was compared by Mann-Whitney U test. Multiple groups were compared using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. For multiple comparisons, a two-way ANOVA followed by Tukey\u0026rsquo;s post hoc or Bonferroni\u0026rsquo;s post hoc test was used. Differences were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 FCSCs and CPCs exhibit different tendencies during multi-lineage differentiation\u003c/h2\u003e \u003cp\u003eFCSCs and CPCs were isolated from the TMJ condylar cartilage and knee articular cartilage of 6-week-old male SD rats, respectively. Both populations expressed mesenchymal stem cell (MSC) markers (CD29, CD44, CD90) and lacked CD45, and exhibited comparable colony-forming capacity \u003cb\u003e(Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e, which confirmed the mesenchymal origin and stem cell phenotype of FCSCs and CPCs.\u003c/p\u003e \u003cp\u003eTo further clarify the cellular characteristics of FCSCs compared to CPCs, we performed bulk RNA-seq on these well-characterized cells (n\u0026thinsp;=\u0026thinsp;3 per group). After mapping the raw RNA-seq data to the relevant genomes, we used statistical methods DESeq2 to compare gene expression levels in different groups and identify significantly DEGs. GO enrichment analysis and KEGG pathway analysis of the identified DEGs were also conducted. The transcriptomic profiles of gene expression in FCSCs and CPCs were compared. Cluster analysis showed that each sample in triplicate clustered together within groups, indicating distinct gene expression profiles between FCSCs and CPCs. A total of 1,599 DEGs were identified between the two groups (fold change\u0026thinsp;\u0026gt;\u0026thinsp;2.0, p \u0026lt; .05), with 819 genes upregulated and 780 genes downregulated in the FCSCs group compared to the CPCs group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGO enrichment revealed that genes upregulated in FCSCs were enriched in negative regulation of the Wnt pathway (GO: 0030178), which is consistent with previous literature indicating that the inhibition of the Wnt pathway is crucial for maintaining the FCSCs pool and cartilage stability (Embree et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Simultaneously, compared to the CPCs group, the FCSCs group showed upregulation in GO terms related to ECM organization (GO: 0030198) and collagen fiber organization (GO: 0030199), suggesting that FCSCs possess a stronger capacity for ECM remodeling and cartilage homeostasis compared to CPCs. Additionally, in the FCSCs group compared to the CPCs group, GO terms related to osteogenesis were upregulated, such as osteoblast differentiation (GO: 0001649) and bone mineralization (GO: 0030282) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e This indicates that FCSCs may have superior osteogenic and chondrogenic capabilities compared to CPCs. Conversely, downregulated DEGs in FCSCs were associated with inflammatory pathways, such as TNF signaling pathway (KEGG: map04668) and cytokine-cytokine receptor interaction (KEGG: map04060) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e).\u003c/b\u003e CPCs showed stronger associations with pathways related to inflammatory response and immune regulation.\u003c/p\u003e \u003cp\u003eTo validate the results of the transcriptomic analysis, we conducted in vitro verification using primary FCSCs and CPCs. We performed proliferation assays using the CCK8 method, which revealed that CPCs had a higher proliferation rate than FCSCs (p \u0026lt; .0001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e).\u003c/b\u003e Trilineage differentiation assays demonstrated that both cell types possessed the potential for multilineage induction, but with distinct differentiation capacities \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. CPCs exhibited stronger adipogenic differentiation potential than FCSCs, while FCSCs showed greater osteogenic and chondrogenic differentiation potential than CPCs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F\u003cb\u003e)\u003c/b\u003e. This suggests that, compared to CPCs, FCSCs possess unique chondrogenic capabilities. We further examined the expression levels of chondrogenic-related genes in FCSCs and CPCs after 14 days of chondrogenic induction. The results showed that the mRNA level of Aggrecan (p \u0026lt; .01) and Col10a1 (p \u0026lt; .01) were significantly higher in FCSCs than in CPCs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e, which indicated that FCSCs exhibit a stronger chondrogenic potential, particularly in cartilage matrix synthesis and cartilage maturation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 DKK3 maintains chondrogenic differentiation capacity of FCSCs\u003c/h2\u003e \u003cp\u003eThrough GO enrichment analysis, we found that the negative regulation of the Wnt pathway is more pronounced in FCSCs. Based on this, we compared the expression differences of Wnt pathway-related genes between FCSCs and CPCs and created a heatmap \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The results showed that the Wnt pathway-related gene DKK3 was more highly expressed in FCSCs than that in CPCs (5.6-fold, p \u0026lt; .01) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. This was validated at the protein level by Western blot and immunofluorescence (p \u0026lt; .05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C\u003cb\u003e)\u003c/b\u003e. Our tissue immunofluorescence results suggested that DKK3 expression appeared stronger in the fibrocartilaginous SZ of the condylar cartilage than in the superficial layer of knee cartilage (p \u0026lt; .05), and also appeared more intense compared to deeper zones within the condylar cartilage itself \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. This finding is consistent with previous studies (Utreja et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This specific expression pattern suggests DKK3 is a potential marker of the condylar cartilage SZ. Previous research has confirmed that DKK3 can protect articular cartilage by inhibiting the Wnt and NF-κB pathways (Snelling et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Conde et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These results suggest that DKK3 may play an important role in the functional regulation of FCSCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify the regulatory effect of DKK3 on the function of FCSCs, we successfully knocked down the DKK3 gene in FCSCs using shRNA technology, resulting in a significant decrease in both mRNA and protein levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E\u003cb\u003e).\u003c/b\u003e After 14 days of chondrogenic induction, qPCR results revealed that the expression levels of chondrogenic-related genes (Col2a1, Sox9, Aggrecan and Col10a1, p \u0026lt; .01) in the shDKK3 group were significantly lower than those in the control group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u003cb\u003e).\u003c/b\u003e Western blotting further confirmed reduced expression of Col2a1 and Sox9 (p \u0026lt; .01) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e, and Alcian blue staining indicated diminished cartilage matrix synthesis in the shDKK3 group (p \u0026lt; .05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. These findings indicated that DKK3 was required for the efficient chondrogenic differentiation of FCSCs in rats.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 DKK3 activates PI3K/AKT signaling in FCSCs\u003c/h2\u003e \u003cp\u003eTo identify the downstream pathways through which DKK3 regulates the functions of FCSCs, we conducted in-depth analysis of the transcriptomic datasets. KEGG and GSEA analyses consistently revealed significant enrichment of the PI3K/AKT signaling pathway in FCSCs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e Single-gene enrichment analysis further revealed that genes exhibiting a strong positive correlation with DKK3 expression (Pearson correlation coefficient\u0026thinsp;\u0026gt;\u0026thinsp;0.8) were predominantly associated with the PI3K/AKT signaling pathway \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify the results of the transcriptomic analysis, we used Western blot to detect the expression levels of proteins related to the PI3K/AKT pathway. The results showed that the protein expression level of p-AKT (phosphorylated AKT) /AKT in FCSCs was significantly higher than that in CPCs (p \u0026lt; .01), indicating that the PI3K/AKT pathway was more active in FCSCs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Immunofluorescence also showed that the expression of p-AKT in the superficial layer of condylar cartilage is greater than in knee cartilage (p \u0026lt; .001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e).\u003c/b\u003e After knocking down the DKK3 gene in FCSCs using shRNA technology, Western blot quantification indicated a marked decrease in the p-AKT/AKT ratio (p \u0026lt; .001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e, and immunofluorescence appeared to show a reduction in p-AKT nuclear translocation (p \u0026lt; .01) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e, indicating that the knockdown of DKK3 appeared to inhibit the activity of the PI3K/AKT pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 PI3K/AKT activation rescues DKK3 knockdown-induced chondrogenic impairment in FCSCs\u003c/h2\u003e \u003cp\u003eTo validate the causal role of the DKK3-PI3K/AKT axis, the shDKK3 FCSCs were treated with the PI3K/AKT activator Recilisib (25\u0026micro;M, 24 h), followed by chondrogenic induction for 14 days. Recilisib (25\u0026micro;M, 24h) significantly restored PI3K/AKT pathway activation in shDKK3 FCSCs (p \u0026lt; .0001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e. Chondrogenic induction assays revealed upregulation of chondrogenic markers (Col2a1, Sox9, Aggrecan and Col10a1, p \u0026lt; .01) at mRNA levels in the shDKK3\u0026thinsp;+\u0026thinsp;Recilisib group compared to shDKK3 alone \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Western blot results showed that, compared to the shDKK3 group alone, the shDKK3\u0026thinsp;+\u0026thinsp;Recilisib group exhibited a significant upregulation of Sox9 (p \u0026lt; .001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Alcian Blue staining further confirmed that Recilisib treatment partially rescued the proteoglycan synthesis deficiency in shDKK3 cells (p \u0026lt; .001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. These findings demonstrated that PI3K/AKT pathway activation can partially counteract the chondrogenic suppression caused by DKK3 knockdown, indicating that PI3K/AKT signaling was involved in mediating DKK3\u0026rsquo;s effects on FCSCs chondrogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 DKK3 alleviates IL-1β-induced impairment of chondrogenic capacity in FCSCs\u003c/h2\u003e \u003cp\u003eTo further evaluate the role of DKK3 in sustaining chondrogenic capacity under pathological stress, FCSCs were pretreated with IL‑1β (10 ng/mL, 24 h). Recombinant DKK3 protein (125 or 250 ng/mL) was then added for 12 h, followed by chondrogenic induction for 7 days. qPCR and Western blot analyses revealed that IL‑1β stimulation markedly impaired the chondrogenic capacity of FCSCs (p \u0026lt; .05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e The addition of recombinant DKK3 (125 or 250 ng/ml) restored the mRNA expression of Sox9, Col2a1, and Aggrecan, as well as the protein levels of Sox9 and Aggrecan (p \u0026lt; .05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e. These findings suggested that DKK3 may help mitigate inflammation‑induced impairment of chondrogenic differentiation in FCSCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eKnee OA and TMJ OA share similar pathological changes and symptoms, but TMJ OA exhibits distinct characteristics. TMJ cartilage is fibrocartilaginous and primarily resists tensile and shear forces, whereas knee cartilage is hyaline and mainly bears compressive loads. These biomechanical differences partly explain why current therapeutic strategies for knee OA often show limited efficacy in TMJ OA, highlighting the necessity of exploring TMJ-specific cellular and molecular mechanisms. Given the critical roles of FCSCs and CPCs in TMJ and knee cartilage repair, elucidating functional differences between FCSCs and CPCs may provide valuable insights for TMJ OA treatment.\u003c/p\u003e \u003cp\u003eIn this study, we isolated FCSCs and CPCs from TMJ and knee cartilage, respectively. Both cell types expressed classic mesenchymal stem cell markers such as CD29, CD44, and CD90, while lacking the hematopoietic marker CD45, and demonstrated comparable colony-forming capacity. These phenotypic characteristics are consistent with previous reports on FCSCs(Embree et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Gong et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and CPCs(Koelling et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Jayasuriya et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), confirming their mesenchymal progenitor identity. Subsequently, we systematically compared FCSCs and CPCs using transcriptomic profiling and functional assays. Transcriptomic data showed FCSCs exhibited upregulated activity in Wnt pathway negative regulation, ECM organization, collagen fibril organization, and osteogenic pathways compared to CPCs, indicating superior ECM remodeling, cartilage homeostasis, and osteogenic differentiation capabilities. Conversely, CPCs showed stronger associations with inflammatory response and immune regulation pathways. Functional assays further demonstrated that CPCs displayed higher proliferative capacity. In terms of tri-lineage differentiation potential, FCSCs have weaker adipogenic capacity than CPCs, while FCSCs exhibit stronger osteogenic and chondrogenic potential. These findings align with previous observations that human FCSCs (hFCSCs) possess greater chondrogenic capacity than human orofacial mesenchymal stem cells (hOFMSCs) (Bi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), enriching the comparative characterization of region-specific stem cell populations.\u003c/p\u003e \u003cp\u003eBased on the significant potential of FCSCs in chondrogenesis, exploring the regulatory mechanisms of chondrogenic differentiation of FCSCs may provide a unique perspective for TMJ disease treatment. Among the differentially expressed genes, DKK3 emerged as a molecule of interest. Although classified within the Dickkopf family, DKK3 does not function solely as a canonical Wnt inhibitor (Hu et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Cooney et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mourtada et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sadeghi et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and its role in bone and cartilage development has gained growing recognition. DKK3 exhibits unique expression patterns in mouse cranial bones distinct from DKK1 and DKK2 (Nie et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). During limb development, DKK3 shows spatiotemporal specificity, highly expressed in joint cells and surrounding perichondrium (Witte et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), implicating it in joint formation and cartilage differentiation. Additionally, DKK3 marks quiescent periosteal cells in the fibrous layer, co-expressed with SMAA and Col3.6, suggesting a role in mesenchymal progenitor differentiation into fibrocartilage cells (Mori et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In ATDC5 cells, DKK3 expression increases during the early stages of chondrogenesis and sharply decreases during the chondrocyte hypertrophy stage, indicating its role in early cartilage development. (Snelling et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Conde et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). DKK3 has been found to protect cartilage by inhibiting Wnt signaling, enhancing TGF-β signaling, and suppressing NF-κB signaling pathways, thereby reducing the production of MMP-13 and protecting cartilage matrix from inflammatory factors (Conde et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Dynamic DKK3 modulation during cartilage injury repair was observed in murine hip dislocation models (Snelling et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWithin the TMJ, DKK3 exhibits a strikingly specific distribution pattern. Previous studies localize DKK3 predominantly to the condylar cartilage superficial zone, where dynamic mechanical loading upregulates its expression and is associated with cartilage thickening (Utreja et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), suggesting a role in tissue adaptation. Consistent with this, our transcriptomics revealed significantly higher DKK3 expression in TMJ FCSCs versus knee CPCs, establishing DKK3 as a potential marker for the TMJ cartilage superficial zone. However, its precise function within the TMJ remains poorly understood. Using shRNA knockdown, we found reduced DKK3 expression significantly impaired FCSCs chondrogenic differentiation, evidenced by downregulated Sox9, Col2a1, and Aggrecan. These findings align with the documented protective role of DKK3 in cartilage homeostasis (Conde et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), yet the underlying mechanisms remain incompletely defined.\u003c/p\u003e \u003cp\u003eThe PI3K/AKT pathway is a central regulator of cell survival, proliferation, and differentiation (Jafari et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and plays a crucial dual role in cartilage biology. In TMJ, the activation of the PI3K/AKT pathway can protect condylar chondrocytes from IL-1β-induced apoptosis and promote the expression of ECM (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, exosomes derived from bone marrow mesenchymal stem cells (BMSCs) alleviate chondrocyte apoptosis in the TMJ disc of TMJOA via the PI3K/AKT pathway (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Conversely, pathological PI3K/AKT activation mediates endoplasmic reticulum stress-induced apoptosis in TMJ chondrocytes (Zhou et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and may exacerbate degeneration by disrupting FCSCs function (Yin et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), highlighting the need for precise regulation. Our study reveals tissue-specific PI3K/AKT activation in FCSCs compared to CPCs, with pathway activity strongly correlated to DKK3 expression. Previous studies demonstrate that DKK3 activates PI3K/AKT signaling to promote cell proliferation and migration in head and neck squamous cell carcinoma (HNSCC) and benign prostatic hyperplasia models (Katase et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Xia et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and confers neuroprotection in post-cardiac arrest brain injury (PCABI) models (Xu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Consistently, our transcriptomic analysis linked DKK3 to PI3K/AKT pathway in FCSCs, and DKK3 knockdown reduced AKT phosphorylation, confirming pathway regulation. Critically, PI3K/AKT activators can reverse the chondrogenic defects caused by DKK3 knockdown, thereby establishing a unique DKK3-PI3K/AKT axis in FCSCs. These findings reveal DKK3\u0026rsquo;s tissue-specific role in fine-tuning PI3K/AKT activity to balance chondrogenesis and stem cell maintenance\u0026mdash;advancing FCSC biology in TMJ OA and revealing novel therapeutic targets.\u003c/p\u003e \u003cp\u003eHowever, it is noteworthy that the PI3K/AKT activator did not fully restore the chondrogenic differentiation level of FCSCs, suggesting that the regulatory network governed by DKK3 may extend beyond a single pathway. It is known that DKK3 can interact with multiple signaling cascades, including the canonical/non-canonical Wnt and NF-κB pathways (Mourtada et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) (Conde et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which are crucial for cartilage homeostasis. In addition, AKT can stabilize β-catenin and inhibit GSK-3β to enhance Wnt signaling, and PI3K-dependent IKK activation can modulate NF-κB activity (Rogers et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Reactivating PI3K/AKT alone may therefore be insufficient to compensate for the loss of DKK3\u0026rsquo;s simultaneous modulation of other axes. Future studies are needed to further elucidate these crosstalk mechanisms.\u003c/p\u003e \u003cp\u003eInflammatory cytokines are key mediators of cartilage degeneration in TMJ OA. Inflammatory cytokines such as IL-1β are elevated in TMJ OA. In human OA cartilage, IL-1β treatment reduces DKK3 expression (Conde et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).In our study, IL-1β impaired FCSC chondrogenesis, while exogenous DKK3 restored chondrogenic marker expression, suggesting that DKK3 acts as a protective regulator under inflammatory conditions, preserving the chondrogenic potential of FCSCs. This aligns with reports that exogenous DKK3 can inhibit IL-1β-mediated proteoglycan loss by suppressing NF-κB signaling (Conde et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, 6-week-old male rats were selected because adolescent rats provide a cartilage environment with high progenitor cell activity, and the use of males eliminates the confounding influence of cyclic estrogen fluctuations, thereby focusing on core mechanisms. Consequently, it does not recapitulate the pathophysiology of aged or osteoarthritic joints, and the potential influence of estrogen on the identified axis remains unexplored. Second, our study primarily employed in vitro experiments to elucidate the regulation of the DKK3-PI3K/AKT axis on FCSC chondrogenesis under both physiological and inflammatory conditions. In vivo validation is still lacking. Future research should therefore focus on confirming the protective role of this axis in TMJ OA animal models and exploring whether its targeted modulation can delay or reverse disease progression.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThis study elucidates that DKK3 maintains the chondrogenic capacity of FCSCs via the PI3K/AKT signaling pathway. Compared with CPCs, FCSCs exhibit a unique differentiation bias and rely on the DKK3-mediated pathway. Targeting the DKK3-PI3K/AKT axis offers new perspectives for understanding TMJ cartilage biology and stem cell regulation. Further exploration of the in vivo efficacy of DKK3-based interventions is needed in the future.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCRediT authorship contribution statement\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eQiaoli Dai\u003c/b\u003e: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization \u003cb\u003eYing Wang\u003c/b\u003e: Writing \u0026ndash; review \u0026amp; editing, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. \u003cb\u003eCunyi Wang\u003c/b\u003e: Writing \u0026ndash; review \u0026amp; editing, Project administration, Methodology, Investigation. \u003cb\u003eWenlin Yuan\u003c/b\u003e: Writing \u0026ndash; review \u0026amp; editing, Project administration, Methodology, Funding acquisition. \u003cb\u003eYilin Chen\u003c/b\u003e: Writing \u0026ndash; review \u0026amp; editing, Data curation. \u003cb\u003eMengqi Zhu\u003c/b\u003e: Writing \u0026ndash; review \u0026amp; editing, Data curation. \u003cb\u003eWeilin Zhang\u003c/b\u003e: Writing \u0026ndash; review \u0026amp; editing, Data curation. \u003cb\u003eJiejun Shi\u003c/b\u003e: Writing \u0026ndash; review \u0026amp; editing, Funding acquisition, Conceptualization. \u003cb\u003eZuping Wu\u003c/b\u003e: Writing \u0026ndash; review \u0026amp; editing, Funding acquisition, Conceptualization. All authors critically revised the manuscript and approved the final version.\u003c/p\u003e"},{"header":"Abbreviations","content":" \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAbbreviations:\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eTMJ\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003etemporomandibular joint\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003ehFCSCs\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003ehuman fibrocartilage stem cells\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOA\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eosteoarthritis\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003ehOMSCs\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003ehuman orofacial mesenchymal stem cells\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eFCSCs\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003efibrocartilage stem cells\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eECM\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003eextracellular matrix\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eCPCs\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003echondrogenic progenitor cells\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eshRNA\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003eshort hairpin RNA\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eRNA-seq\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eRNA sequencing\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eRUNX2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003eRunt-related transcription factor 2\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eDKK3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eDickkopf-3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eGO\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003egene ontology\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eDMEM\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eDulbecco\u0026rsquo;s Modified Eagles Medium\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eSZ\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003esuperficial zone\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eDEGs\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003edifferentially expressed genes\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003ep-AKT\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003ephosphorylated AKT\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePCABI\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003epost-cardiac arrest brain injury\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eBMSCs\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003ebone marrow mesenchymal stem cells\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eHNSCC\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ehead and neck squamous cell carcinoma\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eMSC\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003emesenchymal stem cell\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eFBS\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003efetal bovine serum\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003ePS\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003epenicillin-streptomycin\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePFA\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eparaformaldehyde\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eALP\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003ealkaline phosphatase\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003cbr/\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe 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.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to express our gratitude for the drawing materials provided by BioRender.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the grants from the National Natural Science Foundation of China (82170984), the National Natural Science Foundation of China (No. 82501200), the Joint TCM Science \u0026amp; Technology Projects of National Demonstration Zones for Comprehensive TCM Reform (No. GZY-KJS-ZJ-2025-094), and Zhejiang Provincial Natural Science Foundation of China under Grant No. Q24H140004.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e**Qiaoli Dai:** Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization **Ying Wang:** Writing \u0026ndash; review \u0026amp; editing, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Cunyi Wang:** Writing \u0026ndash; review \u0026amp; editing, Project administration, Methodology, Investigation. **Wenlin Yuan:** Writing \u0026ndash; review \u0026amp; editing, Project administration, Methodology, Funding acquisition. **Yilin Chen:** Writing \u0026ndash; review \u0026amp; editing, Data curation. **Mengqi Zhu:** Writing \u0026ndash; review \u0026amp; editing, Data curation. **Weilin Zhang:** Writing \u0026ndash; review \u0026amp; editing, Data curation. **Jiejun Shi:** Writing \u0026ndash; review \u0026amp; editing, Funding acquisition, Conceptualization. **Zuping Wu:** Writing \u0026ndash; review \u0026amp; editing, Funding acquisition, Conceptualization. All authors critically revised the manuscript and approved the final version.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data presented in this study are available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBatschkus S, Atanassov I, Lenz C, Meyer-Marcotty P, Cing\u0026ouml;z G, Kirschneck C, Urlaub H, Miosge N (2017) Mapping the secretome of human chondrogenic progenitor cells with mass spectrometry. 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Tissue Cell 87:102340. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tice.2024.102340\u003c/span\u003e\u003cspan address=\"10.1016/j.tice.2024.102340\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"histochemistry-and-cell-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hacb","sideBox":"Learn more about [Histochemistry and Cell Biology](http://link.springer.com/journal/418)","snPcode":"418","submissionUrl":"https://submission.nature.com/new-submission/418/3","title":"Histochemistry and Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fibrocartilage, Stem Cell, PI3K/AKT signaling, Cartilage, RNA Sequence Analyses, Temporomandibular joint","lastPublishedDoi":"10.21203/rs.3.rs-9282133/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9282133/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study aimed to compare fibrocartilage stem cells (FCSCs) and knee chondrogenic progenitor cells (CPCs) through RNA sequencing to identify key mechanisms regulating FCSCs function, and to explore the role of Dickkopf-3 (DKK3) in maintaining FCSCs chondrogenic potential in association with the PI3K/AKT pathway.\u003c/p\u003e\u003ch2\u003eDesign:\u003c/h2\u003e \u003cp\u003eFCSCs were isolated from temporomandibular joint (TMJ) condylar cartilage and CPCs from knee articular cartilage of 6-week-old male Sprague-Dawley (SD) rats. RNA sequencing analyzed transcriptional differences. DKK3 knockdown in FCSCs was performed using short hairpin RNA (shRNA), followed by chondrogenic differentiation assays. PI3K/AKT pathway activation was assessed via Western blot, immunofluorescence, and rescue experiments with the PI3K/AKT activator Recilisib.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDKK3 expression was higher in FCSCs than CPCs (p \u0026lt; .05). DKK3 knockdown reduced chondrogenic markers (Sox9, Col2a1, Aggrecan) and PI3K/AKT activity (p \u0026lt; .05), while recombinant DKK3 rescued the IL-1β-induced impairment of FCSCs chondrogenesis (p \u0026lt; .05). Recilisib restored PI3K/AKT signaling and chondrogenic capacity in DKK3-deficient FCSCs (p \u0026lt; .05). Transcriptomics revealed FCSCs\u0026rsquo; unique enrichment in extracellular matrix (ECM) remodeling and PI3K/AKT pathways compared to CPCs.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur results suggested that DKK3 appears to maintain FCSCs\u0026rsquo; chondrogenic potential via the PI3K/AKT pathway activation, highlighting a tissue-specific regulatory mechanism critical for TMJ cartilage homeostasis.\u003c/p\u003e","manuscriptTitle":"Comparative transcriptomics of fibrocartilage stem cells and knee chondrogenic progenitors identifies a DKK3-PI3K/AKT regulatory axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-07 12:39:08","doi":"10.21203/rs.3.rs-9282133/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-26T10:53:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-23T03:16:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52509374262113582862671742764478290547","date":"2026-04-07T10:03:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144817257377561152194073007094253290725","date":"2026-04-02T08:57:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102550419054669923482758537968915630487","date":"2026-04-02T00:22:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-01T23:52:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-01T20:36:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-01T11:44:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Histochemistry and Cell Biology","date":"2026-03-31T15:25:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"histochemistry-and-cell-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hacb","sideBox":"Learn more about [Histochemistry and Cell Biology](http://link.springer.com/journal/418)","snPcode":"418","submissionUrl":"https://submission.nature.com/new-submission/418/3","title":"Histochemistry and Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6aab1422-10b2-4ffd-8544-1335e25ecde2","owner":[],"postedDate":"April 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T12:39:09+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-07 12:39:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9282133","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9282133","identity":"rs-9282133","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}