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Betaine enhances SCAPs chondrogenic differentiation and promotes cartilage repair in TMJOA through WDR81

Stem Cell Research & Therapy volume 16, Article number: 55 (2025) Cite this article

Abstract

Background

The cartilage tissue regeneration mediated with mesenchymal stem cells (MSCs) is considered as a viable strategy for temporomandibular joint osteoarthritis (TMJOA). Betaine has been confirmed to modulate the multidirectional differentiation of MSCs, while its effect on chondrogenic differentiation of Stem Cells from the Apical Papilla (SCAPs) is unknown. Here, we explored the effects and underlying mechanisms of betaine on chondrogenic differentiation of SCAPs.

Methods

Betaine was added for SCAPs chondrogenic induction. The chondrogenic differentiation potential was assessed using Alcian Blue staining, Sirius Red staining and the main chondrogenic markers. In vivo cartilage regeneration effects were evaluated by the rat TMJOA model. RNA-sequencing and biological analyses were performed to select target genes and biological processes involved. The mechanism betaine acts on chondrogenic differentiation of SCAPs was further explored.

Results

Betain-treated SCAPs demonstrated stronger cartilage regeneration in vitro and promoted cartilage repair of TMJOA in vivo. Betaine enhanced the expression of WDR81 in SCAPs during chondrogenesis. WDR81 overexpression promoted chondrogenic differentiation of SCAPs, while WDR81 depletion inhibited chondrogenic differentiation. In addition, both betaine treatment and WDR81 overexpression reduced intracellular reactive oxygen species levels and increased mitochondrial membrane potential in SCAPs.

Conclusion

Betaine promotes SCAPs chondrogenic differentiation and provided an effective candidate for TMJOA treatment. WDR81 may serve as the potential drug target through mitophagy.

Introduction

Temporomandibular joint osteoarthritis (TMJOA) is characterized by damage to articular cartilage with synovial and subchondral bone changes [1]. It has a prevalence of approximately 8–35% in the population and result in an economic burden of approximately 4 billion dollars annually worldwide, accompanied by an upward trend [2]. Traditional clinical treatment methods for TMJOA include symptomatic drug therapy and surgical intervention. However, medications such as oral anti-inflammatory analgesics, intra-articular injection of lubricants, and bone metabolism regulators mainly provide symptomatic relief, with a significant decrease in efficacy over time [3]. Surgical treatment is commonly used for severe joint damage, but it is associated with significant trauma, high costs, and risks of surgical failure and complications, imposing a substantial socioeconomic burden on patients [4]. Additionally, the limited vascular structure in cartilage hinders the exchange of signaling molecules, migration of progenitor cells, and the supply of nutrients and oxygen, resulting in a low probability of effective cartilage tissue repair [5]. Therefore, evaluation of new strategies to promote cartilage regeneration for the treatment of TMJOA is necessary.

In recent years, mesenchymal stem cells (MSCs)-based therapies have shown great promise in restoring cartilage defects [6, 7]. MSCs derived from bone marrow, cord blood, adipose tissue and odontogenic tissue have demonstrated favorable cartilage differentiation potential and can be assessed as promising alternatives to chondrocytes [8, 9]. Intra-articular injection of MSCs has been shown to enhance cartilage protection and promote further regeneration in TMJOA rabbit models [10]. Moreover, studies have reported the regeneration of full-thickness cartilage defects in the temporomandibular joint using MSCs alone or in combination with carriers such as platelet-rich plasma [11]. Despite their potential, the therapeutic methods utilizing undifferentiated cells for temporomandibular disorders treatment still require further evaluation in humans [12]. Stem cells from apical papilla (SCAPs) derived from the apical papilla of young permanent teeth [13, 14]. SCAPs exhibit higher growth rates, stronger osteo/odontogenic potential, and lower immunogenicity compared to dental pulp stem cells [15]. Numerous studies have also reviewed the strong chondrogenic potential of SCAPs [16]. Therefore, SCAPs may serve as a good cell source for temporomandibular joint cartilage treatment.

Betaine, chemically known as N, N,N-trimethyl glycine, is widely found within plants and animals and regulates various biological processes [17, 18]. Betaine has also been explored to regulate multidirectional differentiation of stem cells. Appropriate concentrations of betaine promote adipogenic differentiation of hUC-MSCs and hAD-MSCs [19]. Through regulating intracellular calcium levels, betaine enhances osteogenic differentiation of immortalised dental pulp stem cells [20]. However, its effect on chondrogenic differentiation of SCAPs is unknown. In addition, betaine treatment has been reported to maintain the microstructure of subchondral bone and mitigate the progression of osteoarthritis in the rat articulatio genus osteoarthritis model [21]. These findings contribute to the exploration of betaine as an adjunct in MSC-based therapies. Therefore, the study investigated the effect of betaine on chondrogenic differentiation of SCAPs and evaluated the feasibility of treating TMJOA.

In this study, we evaluated the role and potential mechanism of betaine on chondrogenic differentiation of SCAPs and validated it in the TMJOA model in vitro. The results suggest that betaine has a significant positive regulatory effect on chondrogenic differentiation. WDR81 may be involved in the above process through mitophagy.

Materials and methods

Cell culture

The immature apical papilla tissue from the third molars was gently isolated at Beijing Stomatological Hospital, Capital Medical University. Informed consent was signed prior to the study (Ethical Review NO. CMUSH-IRB-KJ-PJ-2023-06). Initially, the apical papilla tissue was incubated in Phosphate Buffer Saline (PBS) containing type I collagenase (Invitrogen) and Dispase (Invitrogen) for 1 h at 37 °C. Subsequently, suspensions were cultured in DMEMα-Eagle’s Medium (Invitrogen, Carlsbad) containing 20% FBS (Invitrogen) and 10% penicillin-streptomycin (Invitrogen) at 37 °C and 5% CO2. Stem cells were identified, and the results have been previously reported in our work [22]. The cells of 3–4 generations were used for further experiments.

Cell viability assay kit

Cell counting kit-8 (CCK-8 kit) (Dojindo, Japan, Lot.GB707) was used to detect the viability of SCAPs with a density of 2000 cells/well were added to the 96-well plate and different concentrations of betaine (0, 1, 5, 10, 20, 50 mm) was added. The culture was continued for 24 and 48 h, respectively. The medium was removed, and every well was treated with αMEM containing 10 µL CCK-8 solution for 2 h at 37 °C. 0 mM betaine-treated cells served as the control and cell-free wells served as the blank. Absorbance of the sample at 450 nm. Each group contains three replicates. Relative cell viability (%) = (Abs test-Abs blank)/(Abs control-Abs blank) × 100%.

Alcian blue staining

SCAPs were seeded in a 12-well plate with chondrogenic differentiation medium at a density of 1.0 × 105 cells/well. The cells were cultured in StemPro chondrogenic differentiation medium for 21 days. Fixed with 4% paraformaldehyde and washed using PBS, the cells were stained with alcian blue solution (Solarbio, G1563) for 40 min. 0.1 M hydrochloric acid removed unstained areas and be neutralized by water.

Pellet culture system and histological examination

Briefly, samples containing 2.0 × 105 cells per milliliter were centrifuged at 1100 rpm for 6 min in 15-milliliter polypropylene conical centrifuge tubes. The aggregates were then cultured in StemPro chondrogenic for 3 weeks. The cell aggregates were fixed and embedded. The samples were stained with 1% Alcian Blue (Solarbio, G1563) for 1 h, and counterstained with Nuclear Fast Red. For Picro Sirius Red staining followed as the instruction (Solarbio, G1470).

Real-time reverse transcriptase PCR (RT-qPCR)

Total RNA of SCAPs was obtained by TRIzol Reagent. Then following procedures were performed by the instructions (Invitrogen). Data analysis: The 2−△△Ct method was employed for normalization, with mRNA using GAPDH as the reference gene. The primer sequences involved are provided in Table 1.

Table 1 The sequences of RT-PCR primers
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Immunofluorescence staining

Immunofluorescence staining was performed according to the instructions. Briefly, the samples were fixed with paraformaldehyde, treated with 0.1% Triton X-100 and sealed, and incubated overnight with primary antibody at 4 °C. Then the samples were incubated with the corresponding secondary antibody. Relevant primary antibodies included: COL2 (Proteintech, cat no. 28459-1-AP), COL5 (Proteintech, cat no. 67604-1-Ig), and SOX9 (Proteintech, cat no. 67439-1-Ig).

Western blot analysis

Follow the instructions. Briefly, the cells were lysed with lysate buffer and protease inhibitor to extract protein. Subsequent procedures were as previously described. The primary antibodies used in this study were as follows: WDR81 (Proteintech, cat no. 24874-1-AP) and GAPDH (Proteintech, cat no. 60004-1-Ig).

RNA-Sequencing (RNA-seq)

RNA samples were extracted from SCAPs in the control and Betaine groups. Transcriptome high-throughput sequencing was performed by Shanghai Bioprofile Technology Company Ltd. (Shanghai, China).

Reactive oxygen species (ROS) detection

ROS levels were measure by DCFH-DA assay and DHE assay. For DCFH-DA assay, SCAPs was inoculated in a culture plate for three days with 1.8 × 105 cells per well. DCFH-DA was diluted according to the instructions (Beyotime Biotechnology, S0033S) and added into the plate. The cells were washed after incubated at incubator for 20 min. Fluorescence images were observed at 488 nm excitation wavelength and 525 nm emission wavelength.

For DHE assay, SCAPs was inoculated in a culture plate for three days with 1.8 × 105 cells per well. DHE was diluted according to the instructions (Beyotime Biotechnology, S0064S) and added into the plate. The cells were washed after incubated at incubator for 20 min. Fluorescence images were observed at 535 nm excitation wavelength and 610 nm emission wavelength.

Mitochondrial membrane potential detection

TMRE cationic fluorescent probe (MedChemExpress, HY-D0985A) was choose. Briefly, SCAPs was inoculated in a culture plate for one day with 1.8 × 105 cells per well. Add the prepared working liquid and treat at room temperature for 30 min. After washing with PBS, fluorescence microscopy was used to observe and capture images.

Plasmid construction and viral infection

When 70% confluency was reached in 10 mm culture dishes, SCAPs were infected with lentiviruses using a lentivirus infection system and polybrene for transduction enhancement. After 12 h of infection, fresh culture medium was replaced, and stable transfection of SCAPs was achieved by selecting with puromycin for 3 days. The sequences for WDR81 shRNA1 (WDR81sh1) and WDR81 shRNA2 (WDR81sh2) were 5′-GCAGTGGATGAGAAGCTTTGC-3′ and 5′-GCAACTTCCACTACCTCATGC-3′, respectively. The control shRNA (Consh), WDR81 and negative control (Vector) lentiviruses were obtained from Genechem Company.

TMJOA model induction and treatments

As reported, male Sprague-Dawley (SD) rats (aged 8 weeks, body weight 250–280 g, n = 40) were purchased from the Beijing Xinuoyin Biotechnology Co., Ltd (Beijing, China), which is known for extensive disease modeling [23]. They were housed in groups and were allowed a period to acclimatize to the laboratory environment. The work has been reported in line with the ARRIVE guidelines 2.0. and approved by the Animal Ethic Committee of School of Stomatology Capital Medical University (Approved project: Ethical Approval Document for Biological Research, Approval No. CMUSH-IRB-KJ-PJ-2023-06, Date of approval: Feb 13, 2023). Animals were housed under a controlled environment with a 12 / 12 h light / dark cycle and provided with food and water. In the first animal experiment, rats were randomly assigned into four groups (n = 5): Control group, monosodium iodoacetate (MIA)-induced TMJOA model group (MIA group), SCAPs treatment group (MIA/SCAPs group) and SCAPs/Betaine treatment group (MIA/SCAPs/Betaine group). In the second animal experiment, rats were randomly assigned into four groups (n = 5): Control group, MIA group, MIA/SCAPs group and SCAPs overexpressing WDR81 treatment group (MIA/SCAPs/WDR81 group). To create the animal model, all rats were induced by inhalation of isofurane (concentration as 3–3.5%) through a mask for 2–3 min, and then the concentration was changed to 2–2.5% for the maintenance of anesthesia. Buprenorphine was injected subcutaneously 30 min before surgery (0.05 mg/kg) for effective analgesia. The hair in the temporomandibular joint (TMJ) area of the rats was shaved and the area was sterilized with iodine. 50 µL of 4 mg/kg MIA was injected into the TMJ cavity using a microsyringe to establish the TMJOA model. The control group was deal with isopyknic PBS. In the MIA/SCAPs group, 50 µL of 4 × 106/mL SCAPs suspension was injected into the TMJ after 7 days of MIA injection. The MIA/SCAPs/Betaine group received an additional 5 mM betaine along with SCAPs treatment. In the MIA/SCAPs/WDR81 group, SCAPs were replaced with SCAPs overexpressing WDR81, and the injection method and dosage were the same as in the MIA/SCAPs group. As for the MIA group and Control group, equal doses of PBS was injected in subsequent treatments. Rats were euthanized by cervical dislocation at 5 weeks after initial injection, and samples were collected for morphological and molecular biological analysis. Rats appeared healthy and exhibited normal activity levels prior to the start of the study. Rats exhibiting signs of illness or abnormal behavior were excluded from the study initially. Animals were excluded from analysis only if they experienced complications unrelated to the TMJOA model or experimental procedures, such as infections or physical abnormalities unrelated to the induced TMJOA. Rats were experimented with little heterogeneity under inclusion and exclusion criteria.

Micro-CT

Micro-CT imaging was performed using a Micro-CT system (SkyScan 1176; Bruker Corp) with a current of 385 µA and voltage of 65 kV. DataViewer, and CTAn software were used to reconstruct and analyze relevant parameters, including bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N). The data processor was blinded of the animal groups.

Histological examination

About histological examination, Harvested specimens were fixed, decalcified and sliced. Histological staining includes hematoxylin and eosin (HE), Alcian blue staining and Safranin O staining (Solarbio). The protein stained by immunofluorescence was COL2. Quantitative data were calculated by Image J software. Mankin score was performed to evaluate TMJOA as previous study [24]. The data processor was blinded of the animal groups.

Statistical analyses

All data are expressed as mean ± SD. Experiments were repeated three times. An independent sample t-test was performed to determine statistical differences using SPSS 22.0. *p < 0.05, **p < 0.01, ***p < 0.001.

Results

Betaine promotes chondrogenic differentiation of SCAPs

First, the concentrations of betaine required for effective treatment were screened. The results, as shown in Fig. 1A, demonstrated that after 24 h incubation with betaine, the viability of SCAPs increased to varying degrees. After 48 h of culture, differences in cell viability were observed among the different concentrations of betaine, with the 5 mM betaine group showing the highest cell viability at 107.90%. Therefore, we selected 5 mM betaine for subsequent experiments.

Fig. 1
figure 1

Betaine concentration screening and its effect on chondrogenic differentiation of SCAPs. (A) Cell viability on SCAPs after 24 h and 48 h cultured with different concentrations of betaine. (B) RT-qPCR results for COL2, COL5 and SOX9 of chondrogenic induction for 2 weeks treated with betaine (5 mM). (C) Alcian Blue staining results of chondrogenic induction for 3 weeks treated with betaine (5 mM) and corresponding quantification (D). (E-G) Immunofluorescence images of anti-COL2, anti-COL5 and anti-SOX9 in SCAPs of chondrogenic induction treated with betaine (5 mM) for 1 week. (H) Fluorescence intensity of COL2, COL5 and SOX9 expression. (I) Alcian Blue and Sirius Red staining results of chondrogenic induced pellet and corresponding quantification treated with betaine (5 mM) for 3 weeks (J and K). n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: mean ± SD

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The influence of betaine on chondrogenesis of SCAPs was investigated by adding betaine to the chondrogenic medium. The selected cartilage markers were COL2, COL5, and SOX9. COL2 is a cartilage-specific major structural protein that is significantly expressed during cartilage differentiation. COL5 contributes to the formation of the cartilage matrix in conjunction with COL2. SOX9, a transcription factor, activates a series of genes associated with cartilage formation, and its expression is a crucial step in chondrocyte differentiation. RT-qPCR results demonstrated that mRNA expression of COL2, COL5 and SOX9 were significantly up-regulated after 2 weeks treated by betaine of chondrogenic induction (Fig. 1B). Alcian Blue staining indicated SCAPs betaine-treated produced increased production of glycosaminoglycans (Fig. 1C and D). Immunofluorescence staining confirmed the increased fluorescence intensity of COL2, COL5, and SOX9 after betaine treatment from the protein level (Fig. 1E-G), as supported by quantitative analysis (Fig. 1H).

The Pellet culture system provides a more suitable environment for cell differentiation in three-dimensional state. The shade of Sirius Red staining reflects the collagen fiber content within the cell or tissue. According to Fig. 1I, SCAPs treated with betaine exhibited darker Alcian Blue stain and Sirius Red stain than untreated group after 3 weeks of induction, supported by quantitative analysis (Fig. 1J and K). This indicates a higher potential for cartilage formation. Above findings illustrate betaine can promote chondrogenic differentiation of SCAPs.

Betaine enhances the therapeutic effect of SCAPs in alleviating rat experimental TMJOA cartilage degeneration

Based on Micro-CT examination (Fig. 2A), at 4 weeks post-surgery, the condyle of MIA group exhibited serious bone damage and subchondral discontinuity. The lesions in the SCAPs group were reduced but still a certain degree of cartilage and subchondral bone destruction was still visible. By comparison, the MIA/SCAPs/betaine group showed a significant reduction in lesions and improved appearance. Micro-CT parameters, including BV/TV, Tb.Th and Tb.Sp, were consistent with the morphological evaluation, but no differences were observed in Tb.N (Fig. 2B).

Fig. 2
figure 2

Betaine enhances the therapeutic effect of SCAPs in alleviating rat experimental TMJOA subchondral bone destruction. (A) Representative sagittal view and 3D reconstructed images for four groups of TMJ condyles by Micro-CT. (B) Statistical analysis of relative bone deterioration parameters: BV/TV, Tb. Th, Tb. N and Tb. Sp. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: mean ± SD

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Safranin O staining revealed unclear boundaries between the condylar cartilage layers, disordered cellular arrangement and degenerative manifestations: fibrosis of the superficial cartilage, proliferation of the superficial cells, and cleavage of the superficial layers of the cartilage in the MIA group (Fig. 3B, E). In contrast, the control group maintained abundant and regular proteoglycan staining in the cartilage. It should be noted that SCAPs exhibited improved reduction of MIA-induced cartilage proteoglycan loss, and the addition of betaine further enhanced cartilage restoration. HE staining also revealed improvements in the structure and cellular components of cartilage after betaine administration (Fig. 3A). The Mankin scoring system, a widely accepted standard for evaluating the degree of cartilage degeneration in osteoarthritis, was used to assess the TMJOA repair effects in different treatment groups (Fig. 3D). There was significant differences in the scores among the four groups. The MIA group had the highest scores than others (9.00 ± 1.00). The addition of betaine to SCAPs treatment showed the best improvement in terms of structural integrity and cartilage matrix, with the score of 3.33 ± 0.58. Immunohistochemical staining and quantitative analysis indicated lowest number of COL2-positive cells appeared in the MIA group, while the control and betaine groups exhibited the highest expression of COL2 (Fig. 3C, F). These results collectively indicate that SCAPs can protect cartilage from degradation to some extent, and betaine can enhance this protective effect.

Fig. 3
figure 3

Betaine enhances the therapeutic effect of SCAPs in alleviating rat experimental TMJOA cartilage degeneration. Representative HE staining (A) and safranin O staining (B) images for four groups of TMJ condyles. (C) Immunohistochemistry staining of COL2 protein expression in TMJ cartilage. (D) Mankin scores obtained according to safranin O and HE staining. (E) The percentage of proteoglycan for four groups obtained by safranin O staining. (F) Quantification of the COL2 positive staining. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: mean ± SD

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Betaine upregulates the expression of WDR81 during chondrogenic differentiation of SCAPs

Considering the mechanism of betaine-induced chondrogenesis, RNA-seq was performed to present the gene expression of SCAPs in the betaine and control groups. Compared with the group without betaine treatment, the betaine group appeared 1240 upregulated genes (including FTH1, ARPC2, IMMT, TCP1, PPP1CB, etc.) and 1153 downregulated genes (including TNFAIP3, TPT1, MGLL, MTHFD1, DNM1L, etc.) (Supplementary excel 1). Figure 4A presents 10 representative upregulated and downregulated genes. Gene Ontology (GO) enrichment analysis (Fig. 4B) revealed that betaine could affect establishment of protein localization to mitochondrial membrane and Phosphatidylinositol 3-kinase binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated the involvement of betaine in multiple signaling pathways (Fig. 4C). Mitochondrial membrane proteins participate in‌ mitophagy and regulate the differentiation direction and function of stem cells through energy metabolism,‌ antioxidant pathway, etc. [25]. As presented in Fig. 4D-G, Betaine treatment reduced DCF fluorescence intensity, indicating a decrease in intracellular ROS levels. Similarly, the decrease in DHE fluorescence level indicated the decrease of superoxide anion after betaine treatment. TMRE staining, a cationic fluorescent probe, revealed an increase in orange fluorescence intensity in SCAPs treated with betaine, indicating an elevation in mitochondrial membrane potential.

Fig. 4
figure 4

Results of RNA-seq and related validation. (A) Heat map of differentially expressed genes between control cells and betaine-treated cells. The bubble charts of GO enrichment analysis (B) and KEGG enrichment pathways (C). (D) DCFH-DA, DHE and TMRE staining images treated with betaine. (E-G) Relative intensity of DCFH-DA, DHE and TMRE staining. (H) RT-qPCR for WDR81 at 7 days of chondrogenic induction treated with betaine. (I and J) Western blot results and protein quantitative analysis for WDR81 at 7 days of chondrogenic induction treated with betaine. Full-length blots are presented in Figure S1. (K) RT-qPCR results for WDR81 at 0 and 7 days of chondrogenic induction. (L and M) Western blot results and protein quantitative analysis for WDR81 at 0 and 7 days of chondrogenic induction. Full-length blots are presented in Figure S2. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: mean ± SD

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WDR81 has been reported to localize to mitochondria as one of the genes presented by sequencing to be highly expressed and up-regulated after betaine treatment (approximately 10-fold) (Fig. 4A) [26]. It can promote the mitophagy process by binding ubiquitinated substrates and autophagosomes through the structural domain of mitochondrial autophagy bridging protein [27]. Therefore, WDR81 is probably involved in betaine regulation of SCAPs differentiation through the mitochondrial pathway. We chose WDR81 for the following experiments.

The RNA-seq results were validated at both the RNA and protein levels. As illustrated in the Fig. 4H-J, when betaine was added to the culture medium during chondrogenic induction of SCAPs, the expression of WDR81 was also upregulated. Additionally, significant increase of WDR81 expressed during chondrogenic induction of SCAPs (Fig. 4K-M). This suggests that WDR81 is likely to be critical in SCAPs chondrogenesis. All of the above suggested WDR81 is a downstream target of betaine in the process of chondrogenic differentiation.

Overexpression of WDR81 enhances the chondrogenic differentiation of SCAPs

Stable cell lines were established by transfecting lentiviral vectors containing the full-length WDR81 sequence to evaluate its influence on the chondrogenic process. As expected, RT-qPCR and Western blot data demonstrated effective overexpression of WDR81 (Fig. 5A-C). RT-qPCR results demonstrated significant upregulation of COL2, COL5 and SOX9 expression in the WDR81 overexpression group (Fig. 5D). Alcian Blue staining showed increased production of glycosaminoglycans in SCAPs overexpressing WDR81 (Fig. 5E and F). Furthermore, immunofluorescence staining confirmed enhanced production of COL2, COL5, and SOX9 in the WDR81 overexpression group (Fig. 5G-J). Similarly, in the pellet system, overexpressing WDR81 exhibited more quantitatively glycosaminoglycan and collagen fiber stained by Alcian Blue and Sirius Red (Fig. 5K-M).

Fig. 5
figure 5

WDR81 overexpression enhanced the chondrogenic differentiation of SCAPs. (A, B) RT-qPCR and western blot results show WDR81 is overexpressed. Full-length blots are presented in Figure S3. (C) Protein quantitative analysis. (D) RT-qPCR results for COL2, COL5 and SOX9 of chondrogenic induction for 2 weeks in SCAPs. (E) Alcian Blue staining results of chondrogenic induction for 3 weeks and corresponding quantification (F). (G-I) Immunofluorescence images of anti-COL2, anti-COL5 and anti-SOX9 in SCAPs of chondrogenic induction for 1 week. (J) Fluorescence intensity of COL2, COL5 and SOX9 expression. (K) Alcian Blue and Sirius Red staining results of chondrogenesis induced pellet and corresponding quantification for 3 weeks (L and M). (N) DCFH-DA, DHE and TMRE staining images of SCAPs. (O -Q) Relative intensity of DCFH-DA, DHE and TMRE staining. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: mean ± SD

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We investigated the intracellular ROS levels by DCFH-DA and DHE fluorescent probe. As displayed in Fig. 5N, both the significant decrease in fluorescence intensity in SCAPs overexpressing WDR81 compared to the Vector group, indicating a reduction in ROS levels. TMRE staining results were similar to those observed after betaine treatment, with an increase in red fluorescence intensity in SCAPs overexpressing WDR81, indicating an elevation in mitochondrial membrane potential. Quantitative results also support the above (Fig. 5O-Q).

Knockdown of WDR81 inhibits the chondrogenic differentiation of SCAPs

To investigate WDR81’s contribution in SCAPs chondrogenic differentiation, WDR81 knockdown SCAPs were induced under chondrogenic culture environment. WDR81 was effectively knocked down at both the mRNA and protein levels (Fig. 6A-C). The mRNA levels of COL2, COL5, and SOX9 were significantly reduced in WDR81-knockdown SCAPs after 2 weeks of chondrogenic induction compared to the control group (Fig. 6D). Alcian Blue staining exhibited a decrease in glycosaminoglycan production in WDR81-knockdown SCAPs (Fig. 6E and F). Furthermore, Immunofluorescence staining (Fig. 6G-I) and quantitative analysis (Fig. 6J) also performed that Knockdown of WDR81 downregulated fluorescence intensity of cartilage-forming markers in varying degrees. Additionally, in the pellet system, WDR81-knockdown SCAPs exhibited lower glycosaminoglycan and collagen fiber production compared to control (Fig. 6K-M).

Fig. 6
figure 6

WDR81 knockdown inhibited the chondrogenic differentiation in SCAPs. (A, B) RT-qPCR and western blot results show efficient knockdown of WDR81. Full-length blots are presented in Figure S4. (C) Protein quantitative analysis. (D) RT-qPCR results for COL2, COL5 and SOX9 of chondrogenic induction for 2 weeks in SCAPs. (E) Alcian Blue staining results of chondrogenic induction for 3 weeks and corresponding quantification (F). (G-I) Immunofluorescence images of anti-COL2, anti-COL5 and anti-SOX9 in SCAPs of chondrogenic induction for 1 week. (J) Fluorescence intensity of COL2, COL5 and SOX9 expression. (K) Alcian Blue and Sirius Red staining results of chondrogenesis induced pellet and corresponding quantification for 3 weeks (L and M). (N) DCFH-DA, DHE and TMRE staining images of SCAPs (O-Q) Relative intensity of DCFH-DA, DHE and TMRE staining. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: mean ± SD

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As expected, ROS levels in SCAPs increased significantly after WDR81 knockdown. The fluorescence intensity of TMRE staining also decreased significantly (Fig. 6N-Q). These results indicate that WDR81 possesses anti-oxidative stress ability and protective effect on mitochondrial membrane potential.

Overexpression of WDR81 enhances the therapeutic effect of SCAPs in alleviating rat experimental TMJOA cartilage degeneration

The rat TMJOA model was performed to evaluate the therapeutic effect of SCAPs overexpressing WDR81. Similar to the betaine treatment results, Micro-CT analysis showed the most pronounced cartilage discontinuity and subchondral bone destruction in the MIA group, while the SCAPs group exhibited some improvement but still showed rough and uneven cartilage and subchondral bone surfaces (Fig. 7A). In comparison to the MIA and MIA/SCAPs groups, the MIA/SCAPs/WDR81 group demonstrated the strongest cartilage and subchondral bone repair ability, with a smoother cartilage surface observed. Micro-CT parameters, including BV/TV, Tb.N and Tb.Sp, were consistent with the morphological evaluation (Fig. 7B).

Fig. 7
figure 7

Overexpression of WDR81 enhances the therapeutic effect of SCAPs in alleviating rat experimental TMJOA subchondral bone destruction. (A) Representative sagittal view and 3D reconstructed images for four groups of TMJ condyles by Micro-CT. (B) Statistical analysis of relative bone deterioration parameters: BV/TV, Tb. Th, Tb. N and Tb. Sp. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: mean ± SD

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Significant differences in Mankin scores were observed in the control, MIA, MIA/SCAPs and MIA/SCAPs/WDR81 groups (Fig. 8D). The MIA group had the highest scores (8.67 ± 0.58), indicating the greatest degree of cartilage degeneration. The control group exhibited the lowest scores (0.67 ± 0.58), considering the unavoidable trauma of the surgical procedure itself. The addition of WDR81 to SCAPs treatment showed the most significant improvement in terms of cartilage structural integrity (3.67 ± 0.58). HE and Safranin O staining also revealed increased proteoglycan restoration in cartilage after WDR81 overexpression (Fig. 8A, B and E), which was more pronounced than in the SCAPs group. Immunohistochemical staining and quantitative analysis showed that the MIA group had the fewest COL2-positive cells, whereas the control and WDR81 overexpression groups exhibited the highest COL2 expression (Fig. 8C and F). These results collectively indicate that SCAPs can protect cartilage from degradation to some extent, and overexpression of WDR81 can enhance this protective effect.

Fig. 8
figure 8

Overexpression of WDR81 enhances the therapeutic effect of SCAPs in alleviating rat experimental TMJOA cartilage degeneration. Representative HE staining (A) and safranin O staining (B) image for four groups of TMJ condyles. (C) Immunohistochemistry staining of COL2 protein expression in TMJ cartilage. (D) Mankin scores obtained according to safranin O and HE staining. (E) The percentage of proteoglycan for four groups obtained by safranin O staining. (F) Quantification of the COL2 positive staining. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: mean ± SD

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Discussion

Most current first-line treatments for TMJOA are palliative and cannot replace degraded cartilage and subchondral bone. Stem cell therapy, as a promising treatment method, has been shown to be as effective as chondrocytes in joint cartilage repair, with the additional benefits of cost reduction and decreased morbidity at donor sites. Clinical trials have demonstrated the ability of stem cell injections to improve clinical symptoms such as knee osteoarthritis pain, joint stiffness, and physical functional impairment. Therefore, enhancing the chondrogenic capacity of MSCs is a promising future direction for TMJOA therapy.

Betaine, an important methyl donor for methylation modifications, plays a crucial role in various physiological activities [28]. The role of betaine on MSCs chondrogenesis was explored first in this study. As we expected, the expression of markers of chondrogenesis and glycosaminoglycans in SCAPs was significantly elevated by the addition of betaine during chondrogenesis induction. MIA intracapsular injection is the most widely used method of chemical induction in animal TMJOA models. MIA destroys chondrocytes glycolysis, leading to histological and morphological changes in condylar cartilage similar to those seen in OA patients [29]. In this study, SCAP injection attenuated MIA-induced TMJOA manifestations, reduced cartilage destruction, and promoted condylar cartilage recovery. As a supplement to stem cell therapy, betaine-treated SCAPs exhibited enhanced recovery capability. In this part, we also noted the protective effect of betaine on subchondral bone. Absorption of subchondral bone was observed in early OA [30]. Subchondral bone destruction is not only an important manifestation of OA, but also one of the key factors to promote the progression of OA. Early repair can slow down cartilage degeneration, maintain joint stability, and protect joint function ‌.‌ Studies have reported that intra articular injection of nanoparticles containing betaine can effectively protect cartilage and subchondral bone from damage [31]. Our results support this view.

In addition, according to Mankin’s score, OA stages were divided into: early stage (0–6); middle stage (7–9); late stage (10–13) [32]. Early OA usually occurs 1–7 days after MIA injection, characterized by the beginning of slight damage to the articular cartilage, but the overall structure is intact [33]‌. With the extension of molding time, the cartilage damage was aggravated, and obvious fissures appeared, possibly reaching the middle layer, with the reduction of cartilage matrix. The late stage OA usually occurs 4–8 weeks or more after injection [34]. Notably, once OA reaches late stage, the cartilage is almost completely degraded and the only option is the replacement prosthesis. Thus, early management of OA could provide option for maintaining or even regenerating cartilage while the pathology is reversible. Our works indicated that betaine could stimulate the synthesis of cartilage matrix and proteoglycans at the early OA stages, suggesting the prospective ability to relieve TMJOA.

To elucidate how betaine regulates cartilage differentiation, we performed RNA-seq. GO enrichment analyses indicated that betaine affects the “establishment of protein localization to mitochondrial membrane”. Mitochondrial membrane proteins, especially outer membrane proteins, perform critical functions in mitophagy. They are directly involved in the signaling transmission of autophagy process and the recognition of damaged mitochondria. For example, SYNJ2BP and SYNJ2 localize PTEN induced putative kinase 1 (Pink1) to mitochondria by binding to Pink1 mRNA [35]. In addition, BNIP3 and NIX directly initiate the non-ubiquitination-dependent mitophagy pathway by binding to microtubule-associated proteins light chain 3 (LC3) [36]. ‌Previous studies have reported the positive role of mitochondrial autophagy in MSCs differentiation [37]. For example, the expression of BNIP3L was elevated during chondrogenic induction of BMSCs [38]. The expression of Col2A increased during chondrogenic differentiation with the addition of the autophagy inducer rapamycin. Betaine, as the additive, has been demonstrated to significantly increase the expression of hepatic autophagy marker proteins LC3II/LC3I and Atg7 in mice [39]. Thus, betaine is probably affecting chondrogenic differentiation of SCAPs through mitophagy. This was also demonstrated by our ROS and TMRE staining: reduced ROS levels and increased mitochondrial membrane potential within betaine-treated SCAPs.

WDR81 is a transmembrane protein localized in mitochondria [26]. In this study, WDR81 expression was detected to elevate in the betaine group during chondrogenic induction. We first confirmed the positive function of WDR81 as the target of betaine in chondrogenic differentiation of SCAPs. Results in the animal TMJOA model were similar to those after betaine treatment: overexpression of WDR81 also functioned in the early stages of OA to restore cartilage and subchondral bone destruction. In addition, WDR81 is also associated closely with mitophagy. WDR81 interacts with p62 and promotes the binding of ubiquitinated proteins to p62 [27]. p62, as an essential selective autophagy bridging protein, binds ubiquitinated substrates and autophagosomes through its structural domains and transports substrates to be degraded to the autophagic lysosomal system. Meanwhile, WDR81 also specifically binds to LC3C through LIR in its BEACH structural domain and promotes the recruitment of ubiquitinated proteins to LC3C, thus further promoting the autophagy process [40]. Furthermore, enrichment analyses indicated that betaine was significantly associated with PI3K. PI3Kα catalytic subunit inhibits cellular autophagy [41]. Inhibition of PI3K activity, particularly of the PI3Kα catalytic subunit, contributes to the promotion of cellular autophagy, including mitophagy, which maintains intracellular homeostasis and responds to stressful situations. ‌WDR81 could inhibit assembly of the PI3K-III complex [42]. Thus, WDR81 is most probably involved as an intermediate target in the promotion of chondrogenic differentiation by betaine through mitophagy.‌.

Conclusion

In summary, we demonstrated that betaine promotes SCAPs chondrogenic differentiation and betaine-treated SCAPs promotes cartilage recovery in the TMJOA model via WDR81. Our study expands the potential clinical applications of mesenchymal stem cells and suggests that betaine provides an effective candidate for TMJOA treatment, while WDR81 could serve as the potential drug target through mitophagy.

Data availability

All experimental protocols and data obtained in this study were available upon request to the corresponding author. All the data supporting the conclusions of this study including the RNA sequencing data are included within the article and supplementary data.

Abbreviations

MSCs:

Mesenchymal stem cells

TMJOA:

Temporomandibular joint osteoarthritis

SCAPs:

Stem cells from the apical papilla

RNA-seq:

RNA-sequencing

ROS:

Reactive oxygen species

PBS:

Phosphate buffer saline

CCK-8:

Cell counting kit-8

RT-qPCR:

Real-time reverse transcriptase PCR

MIA:

Monosodium iodoacetate

TMJ:

Temporomandibular joint

BV/TV:

Bone volume fraction

Tb.Th:

Trabecular thickness

Tb.Sp:

Trabecular separation

Tb.N:

Trabecular number

HE:

Hematoxylin and eosin

GO:

Gene ontology

KEGG:

Kyoto encyclopedia of genes and genomes

Pink1:

PTEN induced putative kinase 1

LC3:

Microtubule-associated proteins light chain 3

PI3K:

Phosphatidylinositol 3-Kinase

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Acknowledgements

The authors thank Beijing Laboratory of Oral Health for the completion of the work. The authors declare that they have not use AI-generated work in this manuscript.

Funding

This study was supported by National Key Research and Development Program of China (No. 2022YFA1104401), National Natural Science Foundation of China (No. 82201010 and No. 81771026).

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Authors and Affiliations

  1. Department of Stomatology, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China

    Meiyue Wang, Zejie Wu, Xiaoyu Zheng & Runtao Gao

  2. Beijing Laboratory of Oral Health, Capital Medical University, Beijing, 100050, China

    Yishu Huang, Yizhou Jin, Jiaxin Song, Wanzhen Lei, Hua Liu & Haoqing Yang

  3. Department of Stomatology, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100050, China

    Riyue Yu

  4. Research Unit of Tooth Development and Regeneration, Chinese Academy of Medical Sciences, Beijing, 100050, China

    Haoqing Yang

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Contributions

WMY: Methodology and writing original draft preparation; WZJ: Software; ZXY and HYS: Investigation; JYZ and SJX: Formal analysis; LWZ and LH: Animal experiment performance; YRY: Validation and visualization; YHQ: Supervision and funding acquisition; GRT: Review and editing.

Corresponding authors

Correspondence to Riyue Yu, Haoqing Yang or Runtao Gao.

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Ethics approval and consent to participate

This research does not contain clinical experiments. The procedure for obtaining human cells from hospitals was approved by the ethics committee of School of Stomatology Capital Medical University (Approval Number: CMUSH-IRB-KJ-PJ-2023-06; Title: m6A methylation regulates the regeneration of apical papilla stem cells; Date of approval: 02/13/2023). Informed consent was signed prior to the study. All the animal experiments are performed at Beijing Laboratory of Oral Health and approved under the project of “m6A methylation regulates the regeneration of apical papilla stem cells” by the Ethic Committee of School of Stomatology Capital Medical University. (Approval Number: CMUSH-IRB-KJ-PJ-2023-06; Date of approval: 02/13/2023).

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Wang, M., Wu, Z., Zheng, X. et al. Betaine enhances SCAPs chondrogenic differentiation and promotes cartilage repair in TMJOA through WDR81. Stem Cell Res Ther 16, 55 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04161-4

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512