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Journal of Orthopaedic Surgery and Research logoLink to Journal of Orthopaedic Surgery and Research
. 2026 Jan 14;21:37. doi: 10.1186/s13018-025-06461-0

Nell-1 regulates fibrocartilage stem cells and alleviates temporomandibular joint osteoarthritis progression by inhibiting hedgehog pathway

Shiyu Hu 1,#, Na Wu 1,#, Cunyi Wang 2, Wen Li 1,✉, Jiejun Shi 1,✉
PMCID: PMC12801752  PMID: 41535869

Abstract

Background

Nel-like molecule-1 (Nell-1) could prevent the damage of cartilage in osteoarthritis (OA), including temporomandibular joint osteoarthritis (TMJOA), though, the definite mechanism of the process is unclear. Fibrocartilage stem cells (FCSCs) were discovered in the temporomandibular joint (TMJ), and were associated with the TMJ health and disease. However, little information is available on the effect of Nell-1 on FCSCs fate in TMJOA progression.

Methods

In this study, TMJOA was induced in rabbits through collagenase injection. 8 weeks after injection, the left and right joints of the rabbits were injected with AAV9-CMV-Nell-1 and AAV9 vector virus supernatant respectively. FCSCs were isolated, cultured, and transfected with recombinant AdNell-1. Histology staining, immunofluorescence staining, micro-CT, qRT-PCR and western blot were applied to detect the condition of FCSCs and condyles in vitro and in vivo.

Results

In the collagenase injection group, we observed histological changes consistent with OA, meanwhile, the expression of Nell-1 was decreased, but Indian hedgehog (Ihh) and Sonic hedgehog (Shh) were significantly elevated in FCSCs and condylar cartilage. Further in vitro and in vivo analyses revealed that Nell-1 markedly reduced Ihh and Shh. In addition, Nell-1 induced the chondrogenic differentiation of FCSCs by stimulating the expression of Collagen II, Aggrecan and Sox-9.

Conclusion

Nell-1 leads to improved chondrogenic capacity of FCSCs in TMJOA and demonstrated an inhibitory effect on Hh signal transduction while concurrently facilitating the process of cartilage remodeling. Collectively, Nell-1 may be a promising drug candidate for the treatment of TMJOA.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13018-025-06461-0.

Keywords: Temporomandibular disorders, Osteoarthritis, Cartilage, Chondrogenic differentiation, Nel-like molecule-1, Hedgehog

Background

The temporomandibular joint (TMJ), a hinge and sliding joint, controls mandibular movement during speech and mastication [1]. Temporomandibular disorders (TMD) are a group of heterogeneous clinical diseases characterized by pain, limitations in jaw function and/or clicking sounds in the TMJ [2]. Temporomandibular joint osteoarthritis (TMJOA), a progressive and irreversible subtype of TMD, is characterized by the degradation of fibrocartilage and subchondral bone sclerosis, which leads to a poor life quality for the patients [3, 4]. Conventional treatments, such as total joint replacement surgery and pharmacological treatments, are not able to target the pathogenesis and do not effectively control TMJOA progression. Apoptosis, inflammation, excessive mechanical stress, and catabolic enzymes are the pathological outcomes of TMJOA [4]. However, the underlying mechanisms of TMJOA have still not been fully clarified.

Injured articular cartilage has a limited regenerative capacity, so stem cell-based regeneration has emerged as a potential approach to treating degenerative joint diseases, including TMJOA [5–8]. While some studies show positive outcomes of stem cells, all interventions are still in the experimental phase, and the potential advantages and disadvantages still need to be balanced by various factors [9, 10]. In addition, fibrocartilage in the TMJ has poorer mechanical and biochemical properties compared with hyaline cartilage in many other joints [11]. The effects of regulating stem cells in TMJ on the progression of TMJOA are still not fully clarified yet. For this reason, understanding the underlying pathological mechanisms of TMJOA and developing stem cell-based regenerative approaches are critical for improving clinical outcomes. Scientists have recently discovered fibrocartilage stem cells (FCSCs), which are resided in the TMJ condyle superficial zone with the potential for regenerating cartilage and bone [12]. FCSCs play an important role in maintaining the fibrocartilage homeostasis by differentiating into chondrocytes. Moreover, a study discovered that transplanted exogenous FCSCs could improve TMJ cartilage repair of the condylar defects in rats [13]. Increasing evidence has revealed the cell features of FCSCs were regulated by some molecular signals, including the canonical Wnt, Notch, NF-κB and IGF1 signaling pathway [12, 14–16], and the microenvironment may contribute to FCSC fate decisions [17]. However, other potential signaling pathways involved in the regulation of the cell fate of FCSCs were largely unclear.

Nel-like molecule-1 (Nell-1) is an osteochondral regulatory factor that was identified through gene screening, highlighting differential expression between normal and prematurely fused cranial sutures [18]. Previous studies have shown that Nell-1 can promote the proliferation of chondrocytes and enhance the expression of chondrogenic markers [19, 20]. In addition, another study has indicated that the application of Nell-1 resulted in a notable enhancement of the healing process within TMJOA [21]. Besides, our lab has constructed a composite system of the tri-block polyethylene glycol injectable hydrogel and Nell-1, and found out that it could reverse the damage caused by TMJOA [22]. However, the detailed mechanisms by which Nell-1 regulates the cell fate of FCSCs and cartilage regeneration in TMJOA remain to be explored.

Numerous signaling pathways contribute to the pathogenesis of OA. For example, interleukin-1 (IL-1) signaling, NF-κB signaling, Wnt/β-catenin signaling, and Hedgehog (Hh) signaling all represent unique pathways in OA [23–25]. The Hh signaling pathway is a significant regulator of metazoan development [26]. Indian hedgehog (Ihh), sonic hedgehog (Shh), and desert hedgehog (Dhh) are members of the Hh family proteins. Among them, Ihh is necessary for maintaining the growth of adjacent proliferative chondrocytes, and Shh plays a crucial role in inducing early chondrogenic differentiation [27, 28]. It has been documented that Ihh signaling in experimental OA cartilage is associated with hypertrophic chondrocyte formation. In this regard, the Ihh signal was activated in OA. Meanwhile, OA can be more severe if the level of Ihh signaling transduction in chondrocytes is higher. Besides, genetic inhibition of Ihh signaling reduces the severity of OA [29]. Ihh signaling is elevated with Nell-1 activation during osteogenic differentiation and endochondral ossification in many studies, which appears to contradict the content about OA described earlier [30, 31]. However, Ihh was downregulated during Nell-1-induced osteogenic differentiation of human adipose-derived stem cells [32]. Given the interplay between Hh signaling and OA, understanding whether and how Nell-1 regulates Hh signaling and FCSC function in the OA environment could contribute to the exploration of TMJOA pathogenesis and therapies.

To understand the pathogenesis of TMJOA, it is valuable to build an animal model. There are many ways to establish animal models, including mechanical, surgical, chemical, and genetic models. An experimental rabbit model of collagenase-induced TMJOA was used in this study [33]. Collagenase is a common drug in animal models of TMJOA, which can cause cartilage matrix degradation with small trauma. Maintaining a sufficient concentration of Nell-1 in the joints over an extended period is a crucial aspect of our research. Gene delivery to the joint cavity using adeno-associated virus (AAV) vectors may offer an efficacious alternative. AAV represents high transduction efficiencies in vivo and can maintain long-term AAV-induced gene expression with low toxicity [34, 35]. AAV9 was found to exhibit high and sustainable gene expression in a rat model of OA [36]. In the present study, to mediate widespread expression of Nell-1 in condylar cartilage, we injected AAV9-CMV-Nell-1 into the joints of rabbits.

This study aims to evaluate the role and mechanism of Nell-1 on characteristics of FCSCs. At the same time, the study endeavors to elucidate the contributions of Nell-1 regulated Hh signaling in the process of cartilage remodeling within the context of TMJOA. The results suggest that Nell-1 induced the chondrogenic differentiation of FCSCs by inhibiting Hh pathway. Such strategy will help to provide new therapeutic targets for the clinical therapy of TMJOA.

Methods

Animals

Female New Zealand white rabbits (2.8–3.0 kg, 9–10 weeks) were obtained from the experimental animal center of Zhejiang Academy of Medical Sciences. They were raised separately and allowed to drink and eat at will. Rabbits were randomly divided into experimental and control groups. The rabbits were intravenously injected with 3% pentobarbital sodium 1 mg/kg. After shaving the zygomatic and joint area, the location of the zygomatic process was determined by palpation. A more detailed morphology was visible in the transverse or sagittal CT slices. In order to confirm the direction of injection, we used cone beam CT. The experimental group (n = 8) was injected with 250 μL of 4 mg/mL collagenase solution (Sigma-Aldrich), and the normal control (NC) group was injected with PBS. For the experimental group, 9 weeks after injection, the left and right joints of the rabbits were injected with 10μL of AAV9-CMV-Nell-1 and AAV9 vector virus supernatant (1012 genome copies/mL) respectively (Fig. 1A). Treatment was not provided to the NC group. The rabbits were euthanized by air embolism under general anesthesia at the end of the study. The animal care and all procedures were conducted following the agency guidelines. The Ethics Committee of Zhejiang University approved the experiment (Number: ZJU20190056).

Fig. 1.

Fig. 1

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Characterization of control and OA rabbit TMJ condyles. A Schematic illustrations for the methods of whole research. An intra-articular injection of collagenase solution (experimental group) and PBS (control group) was performed. 8 weeks after injection, primary cells were isolated for in vitro studies. As for in vivo studies, after 9 weeks, the left and right joints of the rabbits (experimental group) were injected with 10μL of AAV9-CMV-Nell-1 and AAV9 vector virus supernatant respectively. B The condyles became markedly swollen. 1: control, 2: OA. C The OARSI scores were significantly higher in the TMJOA group. D–F Histomorphology of Safranin O staining and H&E staining of condyle surface and subchondral bone, respectively. G 3D reconstructions of representative rabbit TMJ condyles. H The bone structure analysis results of condyles. Means ± SD, n = 5 per group (*p < 0.05, **p < 0.01, ***p < 0.001)

Histology and immunofluorescence staining

The rabbits were sacrificed 12 weeks after collagenase treatment. After being fixed with 4% paraformaldehyde (PFA) for 24 h, the TMJ condylar samples were decalcified with 20% EDTA for 8 weeks, dehydrated and embedded in paraffin. The standard lateral sections at 5 μm were stained with Safranin O staining and H&E.

To assess the extent of joint injury histologically, the OARSI scale was adopted [37, 38]. Two qualitative trained observers (unknown sample sources) were identified according to H&E staining analysis of cartilage fragments. The average score of each condylar sample was then statistically analyzed for OA severity assessment.

For immunostaining paraffin-embedded sagittal sections of rabbit condyles were deparaffinized and rehydrated. After being incubated with 3% hydrogen peroxide for 15 min, the slides were blocked with 5% BSA at room temperature for 2 h and incubated with primary antibodies against Ihh, Shh, Nell-1, COL2A1, Aggrecan, Sox-9, COL1A1, Ki67, and CD90 in blocking buffer at 4 °C overnight. On the second day, the sections were washed with 1× PBS three times, and incubated using secondary antibody conjugated with fluorescence for 1 h. The nuclei were stained by DAPI.

Micro-CT

A high-resolution microcomputed tomography scanner U-CT-XUHR (Milabs, Houten, Netherlands) was used to scan the bilateral condyles under aseptic conditions. Scans were performed without a filter, employing the following parameters: 50 kV voltage, 0.24 μA current, and 15 ms exposure time. X-ray projections were reconstructed using Imalytics Preclinical 2.1 software, and subchondral cancellous bone was defined as the cancellous bone region 0.5 mm beneath the calcified cartilage–bone junction. For trabecular bone parameter analysis, a cylinder with a radius of 1.5 mm and length of 1 mm was chosen from each specimen. Parameters assessed included the percentage of bone volume (BV/TV, %), trabecular separation (Tb.Sp, cm) and bone mineral density (BMD, g/cm3).

Cell isolation and culture

Primary mandibular condyle chondrocytes (MCCs) and FCSCs were isolated and cultured from TMJ condylar cartilage. To isolate MCCs, the TMJ was washed, sliced, and digested by 0.25% trypsin for 0.5 h and 0.2% type II collagenase for 1 h. As for FCSCs, the condyles were digested by 4 mg/mL type II dispase for 15 min before separating the superficial zone (SZ), and then the SZ was digested in 4 mg/mL type II dispase and 3 mg/mL type I collagenase. Single-cell suspensions were cultured in Dulbecco’s Modified Eagles Medium (DMEM) containing 20% fetal bovine serum, glutamax and 1% PS. The growth of MCCs and FCSCs was observed under the microscope. Throughout the study, in vitro experiments were performed in passage 1 and 2.

Flow cytometry

The immunophenotypes of FCSCs were identified using flow cytometry. FCSCs were incubated with fluorescent conjugated antibodies for 20 min. The following antibodies were used: anti-CD29-PE, anti-CD44-APC, and anti-CD90-FITC (all dilutions, 1:200) (Biolegend). Flow cytometry experiments were conducted using BD Fortessa and samples were calculated using Flojo software.

Multi-lineage differentiation

We tested adipogenic, osteogenic and chondrogenic differentiation in vitro using chemically defined media. For adipogenesis, FCSCs with a cell density of 1.8 × 104 were cultivated in a six-well plate. Upon reaching 70% confluence, the cells were cultured using commercial adipogenic media (Gibco). After 1 week, the cells were fixed in 4% PFA and stained with Oil Red O. For osteogenesis, FCSCs were cultured at a density of 1.8 × 104 in a six-well plate in the osteogenic differentiation medium consisting of αMEM, 10% FBS, 100 nM dexamethasone, 100 μM l-ascorbic acid, and 2 mM β-glycerophosphate for 3 weeks. Cells were fixed with 4% PFA and stained with alkaline phosphatase (ALP). For chondrogenesis, three dimensional (3D) pellets consisting of 3 × 106 cells were made by centrifugation and cultured (5% CO2, 37 °C) for 3 weeks in high glucose DMEM medium supplemented with 1% insulin, transferrin, selenium (ITS), 5% FBS, 40 mg/mL l-proline, 100 nM dexamethasone, 100 μM l-ascorbic acid, 1 mM pyruvate, and 10 ng/mL TGF-β1. After 3 weeks, 3D pellets were fixed with 4% PFA, cut into 12 μm cryosections, and stained with Alcian Blue for glycosaminoglycans.

Recombinant AdNell-1 and transfection

We constructed the adenovirus vectors harboring Nell-1 (from CELL-LAND company). Upon reaching 70–80% confluence, rabbit FCSCs were transfected with recombinant AdNell-1 at 200 pfu/cell of active virus particles. After FCSCs were transfected with the Nell-1 for 48–72 h, the cells were observed by fluorescence, and the Nell-1 mRNA expression level was detected by qRT-PCR.

RNA extraction and qRT-PCR

Total RNA was extracted by Trizol method (Qiagen). cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit (Thermo). qRT-PCR was conducted with SsoAdvance Universal SYBR Green Supermix PCR Kit (Bio-Rad). The primer sequences were synthesized by Sunny Company as listed in Table 1. All genes were analyzed by CFX connect qRT-PCR System (Bio-Rad). The relative content of mRNA was determined by the 2−ΔΔCt method and standardized with the results of GAPDH.

Table 1.

Primer sequences for the genes measured in this experiment

Gene Sequence (5′–3′)
Shh F-CAGCCTCTCTCACAAGCTCTCTAG
R-TCGCAGGAAGTCTCTGTAATTTC
Ihh F-GGCGAGCCGGGCAGCTCAG
R-TCTACCAACTTAAATAATTTTTATTAC
Nell-1 F-GCAAGTTTGGCCGCTCC
R-TGGGACAAAATCACAACAGGCAAC
Sox-9 F-TGTATCTATGGAGGGAAAG
R-CACAGTAGGCAGGGTCT
Col2A1 F-CTGTCCTGTGCGACGACATA
R-TCCTTTCTGCCCCTTTGGTC
IL-1β F-TTGAAGAAGAACCCGTCCTCTG
R-CTCATACGTGCCAGACAACACC
Aggrecan F-GCTACGGAGACAAGGATGAGTTC
R-CGTAAAAGACCTCACCCTCCAT
GAPDH F-GGTGAAGGTCGGAGTGA
R-TTGATGTTGGCGGGAT
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Cellular immunofluorescence staining

Rabbit MCCs were seeded on the cover glass of the 6-well plate. Then, the cells were washed with 1× PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. The fixed cells were then permeabilized with 0.3% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Subsequently, the cells were incubated with the primary antibodies against COL2A1 and Aggrecan at 4 °C overnight. Then, the cells were incubated using secondary antibody conjugated with fluorescence for 1 h. After labeling the cell nuclei with DAPI, the treated MCCs were imaged using a fluorescence microscope.

Western blotting

Protein concentration was quantified by the BCA method (Beyotime Biotechnology). The protein aliquots (20 μg) were separated and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk and incubated overnight with primary antibodies at 4 °C, respectively, including anti-Ihh, anti-Shh, anti-Nell-1, anti-Sox-9, and anti-COL2A1 (all dilutions, 1:1000) (Bioss). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, and the signals were examined with an enhanced ECL kit: Clarity™ Western (Bio-Rad). In three repeated experiments, the band strength of each imprinted protein was quantified by scanning densitometer. The results were scanned using ChemiDoc Touch Imaging System (Bio-Rad) and densitometry measurements were performed with Image Lab version.

Statistical analysis

The data were expressed by mean ± SD and analyzed by SPSS software (version 19.0). The statistical difference between two normally distributed groups was compared by unpaired or paired Student’s t-test. Multiple groups were compared using one-way ANOVA followed by Tukey’s post hoc test. p value less than 0.05 was considered to be statistically significant.

Results

An TMJOA model was obtained in the rabbit

To determine whether TMJOA models were obtained successfully in the rabbits, we performed histology staining and micro-CT scanning using TMJ condyle samples from experimental group and control group at 8 weeks after intra-articular injection (Fig. 1A). The condyles of TMJ became significantly swollen in the TMJOA group (Fig. 1B). Besides, compared with the control group, OARSI score of condylar cartilage in the TMJOA group was significantly higher (Fig. 1C).

In the control group, the surface of the condylar joint was complete and smooth with a clear structure and good continuity (Fig. 1E). In the TMJOA group, the fibrous layer of the surface layer became thinner. We also found the loss of Safranin O staining in the condyles, which exhibited significant ECM deficiency (Fig. 1D). Some bone trabeculae were discontinuously broken, and the subchondral bone marrow cavity showed fibrosis-like structures (Fig. 1F).

Condylar samples were scanned with micro-CT, and the bone structure analysis revealed that BV and BV/TV were significantly reduced in the TMJOA model, while Tb.Sp was significantly improved compared to the control group (Fig. 1G, H). Our results showed articular cartilage damage and subchondral bone remodeling, indicating a successful TMJOA model was created in rabbits.

Characteristics of MCCs in TMJOA rabbit

We then isolated the primary MCCs from articular cartilage of TMJOA and control group. Normal chondrocytes had a triangular, polygonal, or irregular shape, while TMJOA chondrocytes were larger than those in the normal group (Fig. 2A). To determine whether the characteristics of cells were influenced in TMJOA group and to verify the success of animal modeling, we used toluidine blue staining and collagen type II immunohistochemistry to character the primary MCCs (Fig. 2B, C). The results showed the expression of proteoglycans and collagen type II was reduced in TMJOA group.

Fig. 2.

Fig. 2

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The condylar chondrocytes identification. A Morphological observation under inverted phase contrast microscope. B MCCs were stained with toluidine blue. C Immunohistochemistry revealed cytoplasmic staining for type II collagen. D The relative mRNA levels of IL-1β and Sox-9. E Cellular immunofluorescence staining for Aggrecan and COL2A1. Means ± SD, n = 5 per group (*p < 0.05, **p < 0.01)

To clarify the biological effects of TMJOA MCCs, the levels of genes and protein related to inflammation and chondrocyte extracellular matrix metabolism were determined by qRT-PCR and immunofluorescence staining. As shown in Fig. 2D, the mRNA level of IL-1β expression in TMJOA MCCs were significantly increased compared with those in the control group, and the mRNA level of Sox-9 were significantly decreased in the experimental group. Immunofluorescence staining for Aggrecan and COL2A1 from isolated rabbit MCCs was performed, and found that the expression of Aggrecan and COL2A1 was decreased in TMJOA MCCs (Fig. 2E).

Increased Ihh and Shh levels in TMJOA

To investigate the function of Nell-1 and Hh signaling in the progression of TMJOA, we examined the expression levels of Nell-1, Ihh and Shh. FCSCs play an important role in maintaining the fibrocartilage homeostasis, so we investigated the characteristics of the cells. qRT-PCR (Fig. 3A) and western blot analyses (Fig. 3B, C) demonstrated a significant increase in the expression levels of Ihh and Shh, but a decreased expression level of Nell-1 in FCSCs within the TMJOA group. Immunofluorescence revealed that the expression of Ihh and Shh was markedly increased in TMJOA condyles, but the expression of Sox-9, Aggrecan, COL2A1 and COL1A1 was reduced (Fig. 3D, E). These results suggest that Hh signaling is aberrantly activated in the context of TMJOA. However, the expression of Nell-1 in the TMJOA group was significantly lower compared to the normal group, implying Hh signaling might be regulated by Nell-1.

Fig. 3.

Fig. 3

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Expression of Hh, Nell-1 and chondrogenic markers in rabbit TMJs. A The relative mRNA expressions of Shh, Ihh and Nell-1. B Western blotting of Shh, Ihh and Nell-1. C Protein expressions of Shh, Ihh and Nell-1. D Immunofluorescence of Shh, Ihh, Nell-1, COL2A1, Sox-9, Aggrecan and COL1A1 in rabbit healthy and OA condylar cartilage. E Semi-quantitative immunofluorescence determination of Ihh, Shh, Nell-1, Sox-9, COL2A1, Aggrecan and COL1A1 protein. Means ± SD, n = 5 per group (*p < 0.05, **p < 0.01, ***p < 0.001)

Nell-1 led to the inhibition of Hh signaling and enhancememt of chondrogenesis of FCSCs

We analyzed the expression of cell surface markers CD29, CD44 and CD90 in FCSCs by flow cytometry. Similar to previous studies, flow cytometric analysis showed the cells expressed these surface markers [6]. Moreover, we confirmed the osteogenic, adipogenic and chondrogenic differentiation potential of FCSCs via ALP, Oil Red O and Alcian Blue staining (Fig. 4A). Two days after 200 pfu/cell AdNell-1 transduction, the morphology of FCSCs did not change (Fig. 4B). The expression levels of Nell-1 in AdNell-1-transduced FCSCs were significantly elevated compared to the control group (Fig. 4C), confirming the successful transfection of the FCSCs with the Nell-1 gene via adenoviral vectors. To investigate the relationship between Nell-1 and Hh signaling, Ihh and Shh expression was also examined in both AdNell-1-transduced FCSCs and control group. qRT-PCR (Fig. 4D) and western blot analysis (Fig. 4E, F) revealed that the expression levels of Ihh and Shh were significantly reduced in the AdNell-1 transduction group compared to the control. Specific Hh signaling antagonist-cyclopamine showed similar results to those observed with Nell-1, which indicated that Hh signaling was inhibited by overexpression of Nell-1. To elucidate the differentiation characteristics of FCSCs following AdNell-1 transduction, we employed qRT-PCR (Fig. 4D) and western blot analysis (Fig. 4E, F) to show the expression levels of COL2A1 and Sox-9 in AdNell-1-transduced FCSCs were significantly higher. These results suggested that Nell-1 facilitates the chondrogenic differentiation of FCSCs and contributes to the process of cartilage remodeling.

Fig. 4.

Fig. 4

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AdNell-1 transduction inhibited Hh signaling. A Flow cytometric analysis of cell markers of FCSCs. B Multipotential differentiation of FCSCs. For adipogenesis, Oil Red O staining in lipid droplets (left row, scale bar: 50 μm). For chondrogenesis, staining of glycosaminoglycans with Alcian Blue (middle row, scale bar: 100 μm). For osteogenesis, ALP staining demonstrates calcium deposition (right row, scale bar: 200 μm). C FCSCs morphology after viral transfection, as observed via fluorescence microscopy. Scale bars: 20 μm. D Relative mRNA expressions of Nell-1 in normal and Nell-1+ group. E Relative mRNA expression of Shh, Ihh, Sox-9 and COL2A1 of FCSCs in Nell-1+, blank and cyclopamine groups. F Protein expressions of Shh, Ihh, Sox-9 and COL2A1 of FCSCs in three groups. G Western blotting of Shh, Ihh, Sox-9 and COL2A1 of FCSCs in three groups. Means ± SD, n = 5 per group (*p < 0.05, **p < 0.01, ***p < 0.001)

Nell-1 treatment alleviated the damage of cartilage and subchondral bone in the rabbit OA models through maintaining FCSC pool

To further confirm the effect of Nell-1 treatment in TMJOA, we then overexpressed Nell-1 in the TMJOA rabbit model (Fig. 1A). Condylar samples were extracted, and micro-CT scanning was performed (Fig. 5A, B). The micro-CT results indicated a notable restoration of bone mass on the AAV9-CMV-Nell-1 sides. Further quantitative analysis showed that, compared with the AAV9 vector virus supernatant treated sides, trabecular bone parameters showed a tendency to improve in the AAV9-CMV-Nell-1 treated sides, as the percentage of bone volume (BV/TV) was increased in the experimental group. Additionally, a significant reduction in Tb.Sp was observed on the AAV9-CMV-Nell-1 treated sides, which might be associated with the restoration of subchondral bone.

Fig. 5.

Fig. 5

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Nell-1 alleviated the progression of TMJOA by inhibiting expression of Ihh and Shh. A 3D reconstructions of representative rabbit TMJ condyles. B The bone structure analysis results of condyles. C and D Histomorphology of Safranin O staining and H&E staining of condyles, respectively. E The OARSI scores were significantly higher on the AAV9 vector virus supernatant treated sides. F Representative images for Ihh, Shh, Nell-1, Sox-9, COL2A1 and Aggrecan immunofluorescent staining of condylar sections on AAV9-CMV-Nell-1 and AAV9 vector virus supernatant treated sides. G Semi-quantitative immunofluorescence determination of Ihh, Shh, Nell-1, Sox-9, COL2A1 and Aggrecan protein. Means ± SD, n = 5 per group (*p < 0.05, **p < 0.01)

Through histological analyses of the condyles, we observed that the intra-articular administration of AAV9-CMV-Nell-1 resulted in enhanced expression of the cartilage matrix, as evidenced by improved Safranin-O staining (Fig. 5C). Additionally, the fibrocartilage surface of the condyles was continuous and smooth with injection of AAV9-CMV-Nell-1 (Fig. 5D). On the AAV9-CMV-Nell-1 treated sides, the TMJ cartilage demonstrated a well-organized multilayer structure with an increase in cartilage thickness (Fig. 5D). Compared with the experimental group, OARSI score of condylar cartilage on the AAV9 vector virus supernatant treated sides was significantly higher (Fig. 5E).

Subsequently, we further detected Ihh, Shh, Nell-1, Sox-9, Aggrecan and COL2A1 expression in the cartilage. Immunofluorescence staining indicated that Nell-1 diminished the expression of Hh and protected against ECM degradation (Fig. 5F, G). In addition, we used immunofluorescence staining of stem cell surface marker CD90 and proliferating cell associated antigen Ki67 to determine the localization of FCSCs. The results showed that FCSCs were clustering in the superficial zone of cartilage with AAV9-CMV-Nell-1 injection, but the amount of FCSCs was largely decrease in cartilage on the AAV9 vector virus supernatant treated sides (Fig. 6). Immunofluorescence staining revealed that Nell-1 treatment maintained FCSC pool and enhanced the repair of condylar cartilage.

Fig. 6.

Fig. 6

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Nell-1 treatment maintained FCSC pool. A Representative images for Ki67 and CD90 immunofluorescent dual staining of condylar sections on AAV9-CMV-Nell-1 and AAV9 vector virus supernatant treated sides. White arrows represent a FCSC. B Nell-1 stimulation elevated CD90 + Ki67 cell populations. Means ± SD, n = 3 per group (*p < 0.05, **p < 0.01)

Discussion

Previous studies have demonstrated that Nell-1 exerts a beneficial influence on cartilage repair [39, 40]. Furthermore, our lab and another group have confirmed that Nell-1 could heal the cartilage damage caused by TMJOA [21, 41]. Nevertheless, the molecular mechanisms underlying this process remain inadequately characterized. This study highlights the role of Nell-1 in regulating the fate of FCSCs through inhibiting Hh signaling in TMJOA.

Previous studies have shown that Hh signaling plays a critical role in tissue development, repair and homeostasis, influencing the differentiation and proliferation of chondrocytes [42, 43]. Accumulating evidence indicated that Hh signaling was abnormally activated in OA [44]. Furthermore, it has been observed that elevated levels of Ihh signaling transduction in chondrocytes correlate with more severe tissue destruction [29, 45]. Our study was the first to detect the impact of Hh signaling on TMJOA in a collagenase-induced experimental model in the rabbit, and our results showed that the expression levels of Ihh and Shh were increased similar to previous studies, thereby indicating that Hh signaling is activated abnormally in TMJOA. Numerous studies have highlighted the significant role of Ihh protein in the progression of TMJOA, while Shh has been implicated in chondrocyte differentiation [46]. Notably, Shh has been found to be highly expressed in rheumatoid arthritis (RA) [47]. Despite the distinct etiology and pathology of OA and RA, both diseases exhibit cartilage damage. Our study was the first to demonstrate the pathological role of Shh in TMJOA.

Inhibition of Hh signaling has been shown to mitigate the extent of cartilage tissue damage in murine models of OA in vivo, as well as in human in vitro studies [29]. Hh signaling stimulated chondrocytes maturation and mineralization, whereas the application of Hh inhibitors prevented such maturation process [48]. These observations suggest that Hh signaling inhibition may serve as a therapeutic strategy for the degeneration of articular cartilage. However, toxicity and side effects associated with Hh pharmacological inhibitors are what people concerned about. Prior research has indicated that Nell-1 exerts a beneficial effect on cartilage repair [39, 40]. In this study, we performed qRT-PCR, western blot and immunofluorescence to assess the expression of Nell-1, Ihh and Shh, and we observed that Nell-1 reversed the increased expression of Ihh and Shh in TMJOA, which suggested a role of Nell-1 as a Hh-inhibiting agent (Fig. 7). While a previous study indicated an alternative Ihh-activation through Nell-1 mediated by Runx3 in chondrogenic differentiation and maturation, this report contrasts with the OA circumstances in our study [40]. Collectively, these findings demonstrated the complex crosstalk between the Nell-1 and Hh signaling pathways.

Fig. 7.

Fig. 7

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Mechanism diagram. During the TMJOA process, Hh signaling is abnormally activated, and FCSC populations in the superficial zone of cartilage decreased. Nell-1 stimulation inhibits the Hh signaling and maintain the FCSC pool. At the same time, Nell-1 contributes to the chondrogenic differentiation of FCSCs, thus promoting TMJ cartilage repair

Furthermore, qRT-PCR and western blot analyses revealed that Ihh and Shh were downregulated after Nell-1 stimulation in FCSCs. In contrast, Nell-1 increased the expression of Collagen II and Sox-9, which are recognized as chondrogenic specific markers. Thus, we speculate that Nell-1 contributes to the chondrogenic differentiation of FCSCs by inhibiting Hh signaling (Fig. 7). As previously demonstrated, Nell-1 treatment slowed the progression of TMJOA pathology in a rabbit model by preventing ECM degradation and reversing subchondral bone remodeling. Our findings are in line with other studies on Nell-1 and the Hh pathway in various joint tissues. Specifically, the Hh signaling pathway is aberrantly activated in OA [29], while Nell-1 has been shown to alleviate OA progression [39, 40]. Therefore, the results of the present study may hold potential implications for the treatment of other joint disorders, such as knee osteoarthritis and rheumatoid arthritis. Taken together, we show that Nell-1 can regulate the fate decisions of FCSCs and has the potential to be a therapeutic agent for TMJOA through the inhibition of Hh signaling pathways.

There were, however, a number of potential limitations of this work. Due to considerations of the simplicity of operation, controllability of damage, and good repeatability, our animal model was established by collagenase intra-articular injection. Intra-articular injection degenerated the rabbit condyle cartilage, but its pathophysiology is different from that of progressively occurring clinical TMJOA. Furthermore, the TMJs are paired joints that function in tandem. Administering different substances into the bilateral joints of the same rabbit might lead to inaccurate effects of a single injected substance, owing to mechanical interference caused by the contralateral side. Thus, our future studies are warranted to explore a more accurate and physiologically relevant animal model that closely mimics the natural progression of TMJOA.

Conclusions

In conclusion, the utilization of a TMJOA rabbit model reveals that Hh signaling is aberrantly activated during the process of TMJOA. Notably, Nell-1 has the capacity to counteract the elevated expression of Ihh and Shh, while simultaneously enhancing the expression of chondrogenic markers in TMJOA. Furthermore, Nell-1 promotes the chondrogenic differentiation of FCSCs via the inhibition of Hh signaling. Taken together, these results suggest potential novel therapeutic targets for cartilage repair in the context of TMJ disorders.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (3MB, docx)

Acknowledgements

Not applicable.

Abbreviations

Nell-1

Nel-like molecule-1

OA

Osteoarthritis

TMJOA

Temporomandibular joint osteoarthritis

FCSCs

Fibrocartilage stem cells

TMJ

Temporomandibular joint

Ihh

Indian hedgehog

Shh

Sonic hedgehog

TMD

Temporomandibular disorders

IL-1

Interleukin-1

Hh

Hedgehog

Dhh

Desert hedgehog

AAV

Adeno-associated virus

NC

Normal control

PFA

Paraformaldehyde

SZ

Superficial zone

DMEM

Dulbecco’s modified eagles medium

MCC

Mandibular condyle chondrocyte

ALP

Alkaline phosphatase

3D

Three dimensional

ITS

Insulin, transferrin, selenium

RA

Rheumatoid arthritis

Author contributions

SH, NW, and JS designed the study. SH and NW performed the experiments. CW prepared figures and/or table. WL evaluated the data. SH and NW wrote the paper. All authors read and approved the final manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82170984, 82471004) and the National Traditional Chinese Medicine Comprehensive Reform Demonstration Zone Science and Technology Cooperation Project (Project Number: GZY-KJS-ZJ-2025-094).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All experimental designs and protocols involving animals were approved by the Laboratory Animal Welfare and Ethics Review Committee of Zhejiang University (Title of the approved project: Research on the mechanism of Nell-1 regulating cartilage remodeling through the Hedgehog pathway in TMJOA; Approval No: ZJU20190056; Date of approval: 2019/5/24). The care and use of animals were performed strictly following the regulations on the management of experimental animals.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Shiyu Hu and Na Wu contributed equally to this work.

Contributor Information

Wen Li, Email: ewen74@zju.edu.cn.

Jiejun Shi, Email: sjiejun@zju.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (3MB, docx)

Data Availability Statement

No datasets were generated or analysed during the current study.

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