WDR63 enhances the chondrogenic differentiation and regenerative potential of stem cell from apical papilla by facilitating vimentin function to promote mitochondrial fission

Abstract

Background

Research on cartilage repair in the knee joint is crucial for treating knee arthritis or injuries. The application of mesenchymal stem cells (MSCs) for cartilage tissue regeneration represents a promising therapeutic approach. Among the critical aspects in cartilage formation, the enhancement of MSC chondrogenic differentiation stands as a pivotal challenge. WDR63 is a cytoplasmic dynein that plays a significant role in promoting stem cell differentiation and is closely associated with the cytoskeleton and energy metabolism processes. In the current study, our objective is to elucidate the phenotypic manifestations and mechanisms of WDR63 in relation to its chondrogenic differentiation function in MSCs.

Methods

Stem cells from apical papilla (SCAP) were used. The Alcian Blue staining technique, pellet culture system, and cell transplantation in rabbit knee cartilage defects were employed to assess the chondrogenic differentiation capabilities of MSCs. Western blot and real-time RT-PCR were utilized to investigate the molecular mechanisms involved.

Results

In vitro, WDR63 overexpression in SCAPs enhanced chondrogenic differentiation, as evidenced by upregulating collagen type II (COL2), collagen type V (COL5), and sex-determining region Y box protein 9 (SOX9), and robust pellet formation, whereas WDR63 knockdown produced opposite effects. In vivo, implantation of WDR63-overexpressing SCAP promoted cartilage repair in a rabbit osteochondral defect model, showing improved hyaline cartilage matrix deposition, higher COL2 expression, reduced collagen type X(COLX) expression, and increased collagen type Ι (COL1) expression in the subchondral bone. Mechanistically, WDR63 interacted and co-localized with vimentin (VIM), and its overexpression enhanced VIM expression and WDR63–VIM binding. WDR63 upregulates DRP1 expression, and rescues the Mdi-suppressed mitochondrial fission.

Conclusions

WDR63 may promote chondrogenic differentiation of SCAPs by interacting with VIM and enhancing its expression, potentially through facilitating mitochondrial fission.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-026-04959-w.

Introduction

Knee arthritis and knee injuries are common and troublesome health issues that many people face nowadays. As people age, the wear and degeneration of joints can lead to the onset of knee arthritis, while sports and trauma can also cause cartilage damage, resulting in joint pain, swelling, and limited mobility, severely affecting patients’ daily lives. The cartilage tissue in joints has a low content of cells and blood vessels, consisting of sparse cartilage cells enveloped by extracellular matrix. Damage caused by natural degeneration or trauma may result in permanent structural and functional damage, therefore, treatment for the injury and repair of knee cartilage is crucial for alleviating patients’ conditions. However, current treatment methods have limited effects on restoring the normal state of articular cartilage and lack significant efficacy [1]. As a result, there is a pressing necessity to explore novel and efficient approaches for cartilage regeneration. In recent years, MSC-assisted regeneration of articular cartilage technology has attracted much attention and is considered a promising treatment method. MSCs can give rise to a range of distinct cell types, including bone/dentin, cartilage, and adipose tissue. Most studies on cartilage differentiation and regeneration focus on bone marrow mesenchymal stem cells (BMSCs) or cartilage-sourced stem or progenitor cells [2, 3]. Evidence is mounting that bone marrow-derived MSCs are capable of differentiating into multiple cell lineages. However, the relatively low efficiency of BMSC-mediated chondrogenesis and the uncertain maintenance time of cartilage differentiation are problems that need to be solved. Cartilage-derived stem cells are also challenging to isolate, and the rejection reaction of allogeneic stem cell re-implantation is also difficult to solve [4, 5]. Recent research has found that MSCs derived from dental tissues hold great promise for cartilage repair due to their strong chondrogenic differentiation capacity. In addition, they exhibit superior proliferative ability and broader multipotent potential compared to both bone marrow-derived and cartilage-derived mesenchymal stem cells [6]. Furthermore, evidence indicates that MSCs sourced from dental pulp outperform their bone marrow counterparts in terms of cartilage differentiation capacity under both in vivo and in vitro conditions [7]. Among many tooth-derived stem cells, SCAP stand out due to their easy accessibility, minimal damage, high proliferation and differentiation efficiency, and good cartilage repair effect. Since apical papilla stem cells originate from autologous tissue, they have lower immunogenicity in cartilage regeneration applications, which can reduce the occurrence of immune rejection reactions. Recent advances have highlighted the growing potential of apical papilla stem cells in cartilage repair and regeneration [8, 9]. Consequently, dental-derived stem cells have gradually become a focal point of interest for researchers. Researchers have delved deeply regarding the chondrogenic properties of dental stem cells. These cells have shown significant differentiation potential and self-renewal capabilities [10]. Among the various populations of dental-origin stem cells, SCAP have garnered attention due to their faster growth rate and greater potential for bone/dentin generation [11]. Given its superior differentiation ability, SCAP holds significant promise as a regenerative cell source [12]. Moreover, SCAP have been reported to show considerable chondrogenic potential in various experimental settings [13]. Nevertheless, a clear understanding of the differentiation mechanisms in BMSCs is fundamental to harnessing the full potential of induced pluripotent stem cells for lineage-specific differentiation. Furthermore, fully identifying cell lineages and the decision of cell fate is a necessary condition for generating effective directed differentiation.

In the investigation of cartilage repair processes, epigenetic regulation emerges as a key contributing factor. Previous studies have identified KDM2B as a negative regulator of chondrogenic differentiation in SCAP, and its inhibition may enhance cartilage regeneration mediated by MSCs [8]. Moreover, in the process of osteogenic differentiation, KDM2B has been recognized as an inhibitor of WDR63 in SCAP, while WDR63 plays a positive role in promoting its differentiation ability [9]. Therefore, we will focus our research on the functional characteristics of WDR63.

WDR63, as a cytoplasmic dynein, has been shown to interact with Arp2/3, thereby affecting cell migration behavior, indicating that WDR63 has the ability to bind with cytoskeletal components [14]. Furthermore, WDR63 is closely related to the movement of sperm flagella, and its absence can lead to infertility [15]. At the same time, WDR63 has also been linked to neurodevelopment and the mechanisms underlying nerve repair [16]. These studies consistently indicate that there is a correlation between WDR63 and the cytoskeleton as well as cellular movement. Based on this, we hypothesize that WDR63 may have some connection with mitochondria, the site of cellular movement energy production. It is likely that this interaction plays a functional role in cartilage development and regeneration.

Mitochondria, as the main producer of ATP in cells, their dynamic characteristics in the process of cartilage development and regeneration are receiving extensive attention [17]. Studies have confirmed that the growth and development of chondrocytes are highly dependent on energy supply, and mitochondria are responsible for this critical role [17–19]. Mitochondria can flexibly adjust their morphology and quantity through their dynamic processes, including division, fusion, and mitochondrial autophagy, to meet the high energy demand of chondrocytes [20]. In the case of cartilage damage, mitochondrial dynamics also demonstrate their importance by adjusting mitochondrial morphology and quantity, promoting the proliferation and motility of chondrocytes, contributing to an accelerated cartilage regeneration process [21]. Additionally, mitochondria can release signaling molecules such as growth factors, which have a positive effect on the synthesis and deposition of cartilage matrix [22].

In order to further explore this issue, we used SCAP as a research model to conduct a detailed study on the role of WDR63 in cartilage formation and differentiation. The study revealed that WDR63 drives SCAP differentiation toward chondrocytes via upregulation of mitochondrial fission, providing new ideas and strategies for cartilage regeneration. This finding not only helps us better understand the mechanism of cartilage differentiation but also identifies a potential new target for cartilage repair interventions.

Materials and methods

Cell culture

Human SCAP were purchased from Shanghai Anwei Biotechnology Co. Ltd. (Shanghai, China). The cell suspensions were cultured in α-MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS; Invitrogen), 200 mM L-glutamine, and 10,000 U/mL penicillin-streptomycin (Invitrogen) at 37 °C and 5% carbon dioxide atmosphere. Stem cell identification was performed using flow cytometry, with results previously reported in our earlier publication [23].

Western blot analysis

The procedures for protein extraction and gel electrophoresis followed methods described in our earlier work [23]. The primary antibodies were as follows: WDR63 (Cat No. ab216126, abcam, England), COL2 (Cat No. 28459-1-AP, Proteintech, Wuhan, China), SOX9 (Cat No. bs-4177R, Bioss, Beijing, China), VIM (Cat No. 22031-1-AP, Proteintech, Wuhan, China), β-actin (Cat No. AC038, ABclonal, Wuhan, China).

Plasmid construction and viral infection

The cDNA of WDR63 and short hairpin RNAs (shRNAs) were inserted into the pQCXIN lentiviral vector (Beijing Hesheng, China) and the plKO.1 lentiviral vector (Beijing Hesheng, China), respectively, for the construction of SCAP with WDR63 overexpression or WDR63 knockout. The gene target sequences of the shRNAs are as follows: Vector group shRNA: 5′-AAACGTGACACGTTCGGAGAACGAATTCTCCGAACGTGTCACGTTT-3′; WDR63 shRNA: 5′-GGACATAAGAAAGTAATTACACAGACTGTAATTACTTTCTTATGTCC-3′. Cells were allocated into four groups according to the type of viral vector used for transduction: knockdown control group (Scramsh) and stable WDR63 knockdown group (WDR63sh); overexpression control group (Vector) and stable WDR63 overexpression group (HA-WDR63). For lentiviral infection, SCAP were seeded at 8 × 10⁵ cells/dish in 10 cm culture dishes and cultured until they reached 50%~60% confluency. The medium was replaced 12 h after lentiviral infection, and cells were selected with 1 µg/mL puromycin 48 h post-infection.

Reverse transcriptase-polymerase chain reaction (RT-PCR) and real‐time RT-PCR

Total RNA was extracted from SCAP using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. cDNA was synthesized from 2 µg of total RNA using oligo(dT) or random primers and reverse transcriptase (Invitrogen). Quantitative real-time PCR was conducted using the QuantiTect SYBR Green PCR Kit (Qiagen, Hilden, Germany) on an iCycler iQ Multicolor Real-Time PCR Detection System. Each sample was analyzed in technical triplicates, and the entire experiment was independently repeated three times. Primer sequences are listed in Table S1.

Alcian blue staining

Chondrogenic differentiation of SCAP was induced using the StemPro Chondrogenesis Differentiation Kit (Invitrogen, Carlsbad, CA, USA). Cells were plated in 6-well plates at a density of 2 × 10⁵ cells per well and cultured in chondrogenic induction medium to evaluate their chondrogenic potential. The medium was refreshed every 3 days, and the induction period lasted for 2 weeks. For Alcian blue staining, cells were fixed with 4% formaldehyde at room temperature for 1 h, followed by thorough washing with PBS. After fixation, cells were incubated with 1% Alcian blue solution for 30 min. Excess stain was removed by rinsing three times with 0.1 N HCl, and the pH was subsequently neutralized with distilled water. Stained samples were visualized using an inverted biological microscope, and representative images were captured for further analysis.

Pellet culture system and histological examination

To generate cell aggregates, 2 × 10⁵cells/mL were centrifuged at 1100 rpm for 6 min in 15 mL polypropylene conical tubes. The resulting aggregates were maintained in StemPro chondrogenic differentiation medium at 37 °C in a 5% CO₂ atmosphere for 14 days, with medium changes every 3 days. After induction, the primary aggregates were fixed in 4% paraformaldehyde (PFA) for 24 h and subsequently embedded in paraffin. Paraffin sections were prepared and subjected to hematoxylin and eosin (H&E), Alcian Blue (G2541, Solarbio, Beijing, China), and Picrosirius Red (G1470, Solarbio, Beijing, China) staining. For Alcian Blue staining, sections were pre-treated with 3% acetic acid (pH 2.5) for 5 min, followed by staining with 1% Alcian Blue solution for 1 h, and counterstained with nuclear fast red for 5 min. For Picrosirius Red staining, tissue sections were incubated in a saturated aqueous solution of picric acid containing 0.1% Sirius Red for 1 h, then rinsed in 0.01 M HCl for 2 min.

Cartilage defect established in rabbit knee and SCAP transplant experiment

The care and use of all animals were conducted in accordance with the animal welfare guidelines stipulated by the Beijing Stomatological Hospital, Capital Medical University (Ethical Review KQYY-202307-007). All procedures complied with the guidelines outlined in the Experimental Animal Management Ordinance. A total of 12 two-month-old male New Zealand white rabbits were used in this study, weighing between 3 and 3.5 kg to create knee cartilage defects. These rabbits were subsequently randomized into four distinct groups: Sham, Matrigel, SCAP/Vector, and SCAP/HA-WDR63, with each group consisting of six rabbits, each with a knee cartilage defect. After the conclusion of the three-month experiment, animals were euthanized under deep anesthesia by intravenous injection of an overdose of sodium pentobarbital (Product code: MYM_MP0787, Biopike, Beijing, China).

In the Sham group, the rabbits underwent a procedure where only the joint capsule was opened and then sutured back together. Anesthesia for the rabbits was induced intravenously using 3% pentobarbital sodium at a dosage of 35 mg/kg. Rabbit knee joint exposure was performed using a medial parapatellar technique. Utilizing a standard 5-mm hollow drill, we created a cylindrical full-thickness osteochondral defect measuring 5 mm in diameter and 3 mm in depth in the medial femoral condyle. Subsequently, a total of 1 × 10⁶ cells suspended in 50 µL of Matrigel^® Matrix (Cat No. 356234, Corning, USA) was introduced into the defect area.

The Vector group received SCAP/Vector combined with Matrigel, while the HA-WDR63 group was treated with SCAP/HA-WDR63 also mixed with Matrigel. The Sham-operated group functioned as the untreated control. The mixture was allowed to solidify before implantation. Upon completion of the procedure, the femur and patella were restored to their original positions and sutured in layers using absorbable materialz. To prevent infection, each rabbit was administered an intramuscular injection of penicillin at a dosage of 100,000 U/kg for seven consecutive days. All animals were humanely euthanized twelve weeks post-surgery.

Micro-CT

Place the freshly collected femoral joints into a sample tube and perform a micro-CT scan (SkyScan1276). The samples were scanned at 100 kV, 110µA, and an integration time of 600 ms, with a slice thickness of 13 μm and a pixel size of 1024 × 1024. Then, use CTvox software to perform a 3D reconstruction of the subchondral bone, and calculate bone-related data using CTan software.

Histological examination of the repaired cartilage

After completing the Micro-CT scan, we conducted macroscopic observations of the femur. Subsequently, all specimens were harvested, fixed in 10% neutral-buffered formalin, and decalcified with 10% EDTA. Tissue samples were then embedded, sectioned at 5 μm thickness, and stained with H&E. Cartilage and matrix formation were evaluated using Safranin O staining kits (Cat No. G1470, Solarbio, Beijing, China) and Sirius Red staining (Cat No. G1472, Solarbio, Beijing, China). Immunohistochemistry staining with Anti-Human Nuclear Antigen antibody[235-1](Cat No. Ab191181, abcam, England), Immunofluorescence staining with COL1(67288-1-lg, Proteintech, Wuhan, China), COL2, COLⅩ(26984-1-AP, Proteintech, Wuhan, China) antibodies was performed to assess their expression within the regenerated tissue. Histological scoring of cartilage repair was independently conducted by five blinded evaluators using both the International Cartilage Repair Society (ICRS) scoring system and the Modified O’Driscoll scale, as described in previous studies [13, 24].

Tissue section staining

Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed in sodium citrate buffer at 95 ℃ for 15 min. Sections were rinsed in deionized water and permeabilized with 0.5% Triton X-100 for 15 min.

For immunohistochemistry, endogenous peroxidase activity was quenched with 3% H₂O₂ for 10 min, followed by blocking with 10% donkey serum for 1 h at room temperature. Sections were incubated with primary antibody overnight at 4℃. After washing with PBS, sections were incubated with HRP-conjugated secondary antibody for 30 min at room temperature. Following PBS washes, signals were developed using DAB substrate and nuclei were counterstained with hematoxylin.

For immunofluorescence staining, sections were blocked with 10% donkey serum for 1 h after permeabilization and incubated with primary antibody overnight at 4℃. After PBS washes, sections were incubated with fluorophore-conjugated secondary antibody for 30 min at room temperature. Nuclei were counterstained with DAPI after PBS washes, and images were captured using a fluorescence microscope.

Immunofluorescence staining of cells

SCAP were seeded onto cell slides placed in 24-well plates and subjected to chondrogenic induction for 72 h. Cells were washed with PBS and fixed in 4% paraformaldehyde for 30 min at room temperature, followed by permeabilization with 0.5% Triton X-100 and blocking with 10% donkey serum. Samples were incubated overnight at 4℃ with primary antibodies against WDR63 and VIM. After PBS washes, fluorophore-conjugated secondary antibodies were applied for 2 h at room temperature, and nuclei were counterstained with DAPI. Fluorescent images were captured using a fluorescence microscope (Olympus, Japan). Then the colocalization rate of WDR63 and VIM was calculated: Colocalization ratio % = pixels (colocalization)/pixels (total).

Protein-protein molecular docking

Protein sequences of WDR63 and VIM were obtained from UniProt. Because no suitable experimental structures were available, their 3D models were predicted using ProteinX (an open-source framework reproducing AlphaFold3-level performance) and refined with Rosetta Relax. After structural preparation (dehydration, hydrogen addition) in PyMOL, rigid docking was first performed using HDOCK to generate initial complex conformations, followed by RosettaDock for flexible docking refinement. The optimal models were selected based on Rosetta scoring functions. Protein–protein interaction interfaces and binding energies were analyzed using LigPlot and the FoldX Interface_Analyzer module. PyMOL was used for final visualization.

Co-immunoprecipitation (Co-IP) assay

A total of 2 × 10⁶ cells were collected and lysed in IP lysis buffer (Invitrogen, USA) containing a protease inhibitor cocktail (MCE, USA). The lysates were clarified by centrifugation at 14,000 rpm for 10 min at 4℃. The supernatants were then incubated with the appropriate primary antibody for 4 h at 4℃, followed by the addition of protein A/G Sepharose beads (MCE, USA) for overnight incubation at the same temperature. After incubation, the beads were washed three times with lysis buffer and resuspended in SDS-PAGE loading buffer. Samples were subsequently subjected to Western blot analysis using specific antibodies.

Mitochondrial morphology analysis

Cells were cultured on 14 mm cell slides (BS-14-RC, Biosharp, China) placed in 24-well plates and stained with 200 nM MitoTracker™ Deep Red at 37℃ for 30 min. Imaging was performed using a Nikon Eclipse Ti2 microscope equipped with a 100×/1.4 NA oil immersion objective at 3× or 5× magnification. Fluorescence images were acquired with an Andor Zyla sCMOS camera, and all acquisition parameters were managed using NIS-Elements software. Line averaging was set to 2, and dual-channel signals were captured in continuous scanning mode. To visualize the red staining of cells by Mitotracker™, we utilized the Mitochondria Analyzer plugin and FIJI software. The perimeter and aspect ratio (AR) of mitochondria are key parameters for analyzing mitochondrial morphological changes.

Transmission electron microscope(TEM)

Cells were scraped from culture dishes and fixed with 2.5% glutaraldehyde (EM grade; Cat. No. P1126, Solarbio, China). After resin embedding, 60–80 nm sections were prepared and stained with uranyl acetate and lead citrate. Images were obtained using a HITACHI HT7700 transmission electron microscope at 80 kV.

CCK-8

SCAP cell viability under various concentrations of Mdivi was assessed using the Cell Counting Kit-8 (CCK-8) assay. Cells were seeded into 96-well plates and exposed to Mdivi at concentrations of 0, 5, 10, 15, 20, 25, and 30 µM for 24 h. Following treatment, CCK-8 solution was added to each well and incubated for 1 to 4 h. Absorbance at 450 nm was then measured using a microplate reader. Cell viability was expressed as a percentage relative to the untreated control group.

Statistical analyses

All statistical analyses were conducted using SPSS software (version 22) and GraphPad Prism 9. In vitro experiments were independently repeated three times, while in vivo analyses included five biological replicates per group. Data were analyzed using either Student’s t-test or one-way ANOVA, depending on the experimental design. A p-value less than 0.05 was considered statistically significan. And the work has been reported in line with the ARRIVE guidelines 2.0.

Results

Overexpression of WDR63 enhances the chondrogenic differentiation potential of SCAP

To gain insight into the involvement of WDR63 in SCAP chondrogenesis, its temporal expression was profiled during the differentiation timeline. Western Blot results showed that WDR63 significantly increased after 3 days of chondrogenic differentiation induction and was maintained until day 7 (Fig. 1A). Following transduction with WDR63 or control vectors, SCAP were subjected to puromycin selection (2 µg/mL) for 3 days to eliminate non-transduced cells. The Western blot results confirmed the WDR63 overexpression (Fig. 1B). Genetically modified SCAP were maintained in chondrogenic induction medium to evaluate their capacity for chondrogenic differentiation. Two weeks post-induction, WDR63-transduced SCAP exhibited significantly greater glycosaminoglycan accumulation than controls, as shown by Alcian Blue staining (Fig. 1C). Quantitative real-time RT-PCR analysis revealed upregulation of chondrogenic markers, including COL2, COL5, and SOX9, in WDR63-transduced SCAP after two weeks of chondrogenic induction, in comparison with the control group (Fig. 1D). Besides, the results of Western blot confirmed an upregulation of COL2 and SOX9 protein levels (Fig. 1E, F). Furthermore, to explore the impact of WDR63 on chondrogenesis, SCAP were subjected to in vitro culture conditions to generate cartilage pellets. The outcomes of Two weeks after induction, pellets formed from SCAP transduced with WDR63 showed stronger chondrogenic characteristics than those from the control group, as evidenced by Alcian Blue and Picro Sirius Red staining (Fig. 1G).

Fig. 1.

WDR63 over-expression enhanced the chondrogenic differentiation in SCAP. A Western blot results showed the WDR63 expression in SCAP after chondrogenic induction. B Western blot results showed the WDR63 expression in different groups of SCAP. C Alcian Blue staining show that WDR63 over-expression enhanced chondrogenic differentiation in SCAP. Scale bar, 100 μm. D Real-time RT-PCR results showed that WDR63 over-expression upregulated the expression of COL2, COL5, and SOX9 in SCAP after 14d. E, F Western blot results showed the COL2 (E) and SOX9 (F) expression in SCAP. The relative protein levels quantified by densitometry and normalized to β-actin. G H&E, Alcian Blue and Picro Sirius Red staining results of chondrogenesis induced pellet. Scale bar, 100 μm. β-actin and GAPDH was used as an internal control. Student’s t test analysis was performed to determine the statistical significance. All error bars represent s.d. (n = 3). *P<0.05

Knockdown of WDR63 inhibits the chondrogenic differentiation ability of SCAP

To further clarify the function of WDR63 in SCAP chondrogenesis, its expression was silenced via lentiviral delivery of WDR63-targeting shRNA. Following transduction, SCAP were subjected to puromycin (2 µg/mL) treatment for three days to enrich for infected cells. The Western blot analysis indicated that WDR63 was effectively silenced in the SCAP (Fig. 2A). At two weeks after induction, diminished glycosaminoglycan deposition was observed in WDR63-silenced SCAP, as demonstrated by Alcian Blue staining (Fig. 2B). At the two-week time point, COL2, COL5, and SOX9 transcript levels were found to be downregulated, as determined by real-time RT-PCR. Correspondingly, Western blot analysis indicated that SOX9 expression diminished in WDR63-depleted SCAP following 2 weeks of chondrogenic induction, as compared to the control group (Fig. 2C–E). Following two weeks of cartilage pellet cultivation, histological staining with Alcian Blue and Picro Sirius Red indicated that WDR63-deficient SCAP generated pellets with compromised chondrogenic differentiation (Fig. 2F).

Fig. 2.

WDR63 knock-down inhibited the chondrogenic differentiation in SCAP. A Western blot results showed the WDR63 expression in different groups of SCAP. B Alcian Blue staining showed that WDR63 knock-down downregulated chondrogenic differentiation in SCAP. C Real-time RT-PCR results showed that WDR63 knock-down downregulated the expression of COL2, COL5, and SOX9 in SCAP after 14d. D, E Western blot results showed the COL2 (D) and SOX9 (E) expression in SCAP. The relative protein levels quantified by densitometry and normalized to β-actin. F H&E, Alcian Blue and Picro Sirius Red staining results of chondrogenesis induced pellet. Scale bar, 100 μm. β-actin and GAPDH was used as an internal control. Student’s t test analysis was performed to determine the statistical significance. All error bars represent s.d. (n = 3). *P<0.05. **P<0.01

WDR63 enhances SCAP-mediated cartilage regeneration in rabbit knee cartilage defect model

To study whether WDR63 promotes cartilage repair and regeneration in vivo, we constructed full-thickness cartilage defects in the femoral trochlea of rabbit knees, and hydrogels, or hydrogels mixed with SCAP, were re-implanted into the defect areas. At 12 weeks following transplantation, a rigorous examination of the regeneration of cartilage tissue was conducted to assess and compare the chondrogenic efficacy of the genes under investigation.

Subchondral bone serves as a crucial support for cartilage, and cartilage layers with superior subchondral bone structure and density demonstrate enhanced repair capabilities. The three-dimensional reconstruction results from Micro-CT, as well as the BMD and BV/TV values of the injured regions, indicate that overexpression of WDR63 can effectively enhance the repair of subchondral bone injuries, thereby facilitating cartilage recovery (Fig. 3A, C, D).

Fig. 3.

WDR63 enhanced SCAP-mediated cartilage regeneration in rabbit knee cartilage defect models. A representative Micro-CT images of different groups. Scale bar, 1 mm. B Macroscopic observation and sagittal images of cartilage defect healing. Scale bars, 1 mm. C, D Quantitative analysis of the BMD (C) and BV/TV (D) at the defect site. E Quantitative assessment of macroscopic results. One-way ANOVA analysis was performed to determine the statistical significance. All error bars represent s.d. (n = 5). *P<0.05. **P<0.01

In the SCAP/Matrigel group, which is the treatment group with direct implantation of hydrogel into the defect area, it can be clearly observed that the surface is irregular, with obvious fibrosis and even epidermal stratification in the gross observation of cartilage repair. The SCAP/Vector group was similar to the Matrigel group, with surface fibrosis accompanied by cracks and significant concavities. Compared with the SCAP/HA-WDR63 group, the smoothness of the tissue surface was reduced. In contrast, the SCAP/HA-WDR63 group had more normal morphological characteristics. Specifically, in terms of color, the tone of the SCAP/HA-WDR63 group was translucent and milky white, similar to the color of natural joint cartilage, and seamlessly covered the joint end of the femoral condyle, resembling the morphological characteristics of healthy joint cartilage. Observing from the profile of the defect area, the thickness and area of cartilage repair tissue in the SCAP/HA-WDR63 group were larger, and the boundary with surrounding tissue was not obvious (Fig. 3B). After our careful assessment, it is consistent with the previous expectations. The ICRS score (cartilage repair assessment of international cartilage repair society) data from macro-observation showed that the score of SCAP/HA-WDR63 group was significantly higher and its value was close to the non-surgical group (Fig. 3E). This result further confirmed our expectations.

After the repair of the tissue, further histological examination and analysis were carried out. Considering that SCAP are of human origin, we performed Immunohistochemistry staining using an anti-human nuclear antibody. The results showed detectable DAB-positive signals were detectable in both the SCAP/Vector group and the SCAP/HA-WDR63 group (Fig. S1A). The H&E staining results, including panoramic and detailed images, showed that there were cracks and defects in the surface tissue of the regenerative cartilage in the SCAP/Matrigel group, and the cartilage tissue was disordered, mostly composed of fibrous tissue without obvious characteristic cells of cartilage; the SCAP/Vector group was superior to the SCAP/Matrigel group in terms of cell quantity, but the surface layer was still composed of fibrous tissue. Only a few typical cartilage tissue structures formed in the deep part of the defect, and a few columnar cells were observed. Extensive regeneration of cartilage-like tissue was observed in the defect area of the SCAP/HA-WDR63 group, characterized by a compact structure and color similar to that of the surrounding native cartilage. The thickness is slightly thicker than normal tissue. Moreover, numerous columnar chondrocyte-like cells were identified in the deeper zones of the regenerated tissue, and the subchondral bone structure closely resembled that of native tissue (Fig. 4A). The results of Safranin O-Fast Green staining showed that cartilaginous tissue in the SCAP/Matrigel group only existed in the middle tissue, while the cartilaginous matrix in the SCAP/Vector group was primarily confined to the deeper layers of the defect. In the SCAP/HA-WDR63 group, cartilaginous tissue occupied half of the thickness in the defect area, demonstrating excellent integration with the adjacent cartilaginous tissue. The amount of regenerated cartilaginous matrix was marginally reduced compared to native cartilage (Fig. 4B). The quantitative analysis of transparent cartilage percentage showed that the cartilaginous matrix in the SCAP/HA-WDR63 group was significantly more than that in the SCAP/Matrigel group and SCAP/Vector group, and basically the same as that in the SCAP/sham group (Fig. 4C).

Fig. 4.

WDR63 enhanced SCAP-mediated cartilage regeneration in rabbit knee cartilage defect models. A H&E staining results. The area within the frame represents a typical region for cartilage damage repair. Scale bars, 500 μm. B Safranin-O-fast green staining results.The area within the frame represents a typical region for cartilage damage repair. Scale bars, 500 μm. C Immunofluorescence staining results of COL2. Scale bars, 500 μm. D Quantitative detection results of transparent cartilage percentage. E Quantitative statistics of average fluorescence intensity by immunofluorescence staining of COL2. F Total score based on modified O’Driscoll scores of repaired tissues after 2 months. One-way ANOVA analysis was performed to determine the statistical significance. All error bars represent s.d. (n = 5). **P<0.01

According to immunofluorescence analysis, COL2 expression was faint on the surface of rabbit knee cartilage in the SCAP/Matrigel group, contrasting with more pronounced COL2 presence in the central and deep zones of regenerated tissue in the SCAP/Vector group. However, the SCAP/HA-WDR63 group had a uniform and strong COL2 expression in the regenerative cartilaginous tissue sites (Fig. 4C). Quantitative analysis of the average fluorescence intensity of the four sets of immunofluorescent-stained tissue images also confirmed this (Fig. 4F). Based on the above results, histological scores were evaluated using the Modified O’ Driscore. The results showed that, as in our experimental results, the scores for the SCAP/Matrigel, SCAP/Vector, and SCAP/HA-WDR63 groups increased sequentially (Fig. 4D). Additionally, immunofluorescence staining of the hypertrophic cartilage marker COLX showed the highest expression in the Matrigel group, moderate expression at the bone–cartilage interface in the Vector group, and markedly reduced expression in the HA-WDR63 group (Fig. S1B). In contrast, COL1 revealed that, compared with the Vector group, the HA-WDR63 group exhibited higher COL1 expression in the subchondral bone and lower expression within the cartilage region, closely resembling the pattern observed in the Sham group. (Fig. S1C). Although the re-implantation of SCAP with overexpressed WDR63 showed stronger ability in tissue recovery, matrix generation and collagen expression, it can be found from the histological score that there is still a certain gap between the cartilage score after repair and the intact cartilage.

WDR63 combines with VIM in the process of chondrogenesis

To explore the mechanism by which WDR63 regulates chondrogenic differentiation of SCAP, we conducted an IP experiment using WDR63 on SCAP, and performed protein mass spectrometry on the 45-55kd band with the highest binding amount. As shown in Supplementary Table 2, the top three proteins are VIM, RBMX, and B4DY09. RBMX is a binding protein associated with mRNA and nuclear transcription [25]; B4DY09 is the coding gene for interleukin enhancer binding factor 2 (ILF2), primarily related to inflammation [26]; and VIM is a necessary cytoskeletal component for chondrocytes to maintain their shape [27]. Considering both the binding amount and relevance to chondrogenesis, we ultimately chose VIM for further experimental validation.

In the STRING protein–protein interaction network, we identified a potential association between WDR63 and VIM (Fig. S2A). Analysis using the InterfaceAnalyzer module of FoldX revealed that the protein-protein docking binding free energy between WDR63 and VIM proteins is -37.63 kcal/mol, indicating a strong binding propensity (Fig. S2B). To explore the relationship between VIM and WDR63 in protein expression, we first used Western blot to detect the VIM protein content of cells with overexpressed and knocked-down WDR63. The results showed that the expression level of VIM in SCAP overexpressing WDR63 increased, while it decreased in the opposite case (Fig. 5A). This indicates a positive relationship between the expression of WDR63 and VIM.

Fig. 5.

WDR63 associated with VIM and formed protein complex in SCAP. A Co-IP results showed the relationship between WDR63 and the combined amount of WDR63 and VIM in SCAP. B Co-IP results showed more WDR63-VIM complex formation in SCAP after chondrogenic differentiation for 7 days. C, D Immunofluorescence staining results of WDR63 (C) and VIM (D) in chondrogenic differentiation of 0 and 3 days SCAP. Scale bars, 10 μm. E Co-location ratio of immunofluorescence staining. F Western blot results showed WDR63 and VIM expression after chondrogenic differentiation for 0d, 3d, and 7d. G Western blot results showed VIM expression in different groups. β-actin served as the internal control. Student’s t test analysis was performed to determine the statistical significance. All error bars represent s.d. (n = 5). *P<0.05

In order to explore the changes in the endogenous binding quantity of WDR63 and VIM during the process of chondrogenesis, proteins were extracted from 0 day and 7 day after chondrogenic induction for co-IP experiments. The experiment showed that the binding quantity of WDR63 and VIM was very low in the initial state; after 7 days of chondrogenic differentiation, the binding quantity of WDR63 and VIM increased significantly (Fig. 5B). Meanwhile, SCAP were subjected to immunofluorescence experiments to detect the co-location of WDR63 and VIM. The results showed that WDR63 and VIM co-localized in the cytoplasm (Fig. 5C). Data analysis revealed that the co-location ratio increased after 3 days of chondrogenic differentiation induction (Fig. 5D, E).

Changes in VIM during chondrogenesis were also detected. Compared with that in undifferentiated SCAP, the protein expression of VIM was significantly upregulated at 3 and 7 days after chondrogenic differentiation (Fig. 5F). To further clarify the effect of WDR63 expression on VIM, the expression of VIM in SCAP with overexpressed and knocked-down WDR63 was detected. The results show that high expression of WDR63 promotes the increase of VIM expression, while conversely, it inhibits VIM expression. (Fig. 5G).

WDR63 promotes mitochondrial fission in SCAP and inhibits mitochondrial fusion

Moreover, during chondrogenic differentiation, mitochondrial fission increases while fusion decreases [28]. Therefore, the mRNA expression levels of OPA1 (mitochondrial fusion marker) and DRP1 (mitochondrial fission marker) in SCAP overexpressing or knocking down WDR63 were first detected. The results showed that overexpressing WDR63 increased the expression of DRP1 and inhibited the expression of OPA1 (Fig. 6A, B); More intuitively, transmission electron microscopy is used as a method to show the specific shapes of mitochondria. The typical shapes indicated by arrows in the figure are representative. As shown in the figure, the mitochondria in the Vector group were mostly short and rod-shaped, while the number of mitochondria in the HA-WDR63 group increased, showing a splitting state with more circular or spindle-shaped mitochondria (Fig. 6C). A quantitative analysis of the electron microscope results revealed that the mitochondrial perimeter and aspect ratio (AR) in the HA-WDR63 group were lower than in the control group. When the perimeter of mitochondria is shorter and the AR is closer to 1, it indicates that the mitochondrial morphology tends to be rounder, with more pronounced fission; whereas as the perimeter and AR increase, the mitochondrial morphology tends to be more linear, indicating a more pronounced fusion phenomenon. So, the result described above indicates that overexpressing WDR63 makes mitochondria smaller and closer to circular shapes (Fig. 6D). Immunofluorescence images showed that overexpressing WDR63 enhances mitochondrial fission and reduces mitochondrial length (Fig. 6E, F).

Fig. 6.

WDR63 regulates mitochondrial fission in SCAP. A, B, G, H RT-PCR results showed the DRP1 and OPA1 expression in SCAP. C, I Transmission electron microscopy (TEM) photomicrographs of SCAP with WDR63 overexpression (C) or knockdown (I). Scale bars, 500 nm. D, J Quantification of perimeter and area using ImageJ software. Mitochondria are highlighted in red arrows. (n = 35). E, K Representative immunofluorescence images of the mitochondrial network in SCAP with WDR63 overexpression (E) or knockdown (K). Red: Mitotracker, Blue: DAPI. Scale bars, 5 μm. F, L The length of mitochondria was quantified by Image J. GAPDH served as the internal control. Student’s t test analysis was performed to determine the statistical significance. All error bars represent s.d. (n = 3). *P<0.05, ** P<0.01

Conversely, knocking down WDR63 can inhibit DRP1 and increase the expression of OPA1 (Fig. 6G, H). The WDR63sh group showed increased mitochondrial fusion and less clear mitochondrial cristae (Fig. 6I). Both the mitochondrial perimeter and aspect ratio of the WDR63sh group were higher than those of the control group, indicating that knocking down WDR63 leads to an increase in mitochondrial volume and a more elongated shape (Fig. 6J). Relatively, knocking down WDR63 causes the mitochondrial dynamic balance to shift towards fusion and results in an increase in mitochondrial length (Fig. 6K, L). Based on the experimental results above, it is possible that WDR63 facilitates mitochondrial fission through VIM, thereby enhancing the chondrogenic differentiation of SCAP both in vivo and in vitro.

WDR63 overexpression rescues Mdivi-suppressed mitochondrial fission and enhances chondrogenic differentiation in SCAP

To deeply explore the mechanism of WDR63 in regulating mitochondrial fission, we conducted rescue experiments using the specific inhibitor of DRP1 protein, Mdivi. In the experiments, Mdivi was first diluted to a working concentration range of 0–25 µg/mL and co-incubated with SCAP cells for 4 h. Subsequently, RNA was extracted and processed with CCK-8 experiments. The results indicated that with the increase in Mdivi concentration, the proliferation ability of SCAP cells exhibited a significant downward trend (Fig. 7A). qRT-PCR analysis demonstrated that Mdivi treatment significantly altered mitochondrial dynamics-related gene expression, including downregulation of DRP1 and upregulation of OPA1 (Fig. 7B, C). Based on the results of CCK-8 and PCR, we used a drug concentration of 20 µg/mL of Mdivi for further experiments. SCAP were divided into four experimental groups: SCAP, SCAP + Mdi, SCAP + Mdi+Vector, SCAP + Mdi+HA-WDR63, to induce differentiation into chondrocytes for 14 days. Alcian Blue staining confirmed the restorative effect of WDR63 overexpression on chondrogenic differentiation capacity under Mdivi treatment (Fig. 7D). qRT-PCR results further revealed that the overexpression of WDR63 can rescue the inhibition of DRP1 expression by Mdivi (Fig. 7E). At the same time, the expression of chondrogenic differentiation markers COL5, COL2, and SOX9 in the SCAP + Mdi+HA-WDR63 group was significantly higher compared to the SCAP + Mdi group and the control virus group (Fig. 7F–H). Furthermore, Western blot results indicated that the overexpression of WDR63 effectively rescued the reduction in COL2 and SOX9 expression caused by DRP1 inhibition (Fig. 7I–K). The above results confirm that the overexpression of WDR63 effectively rescued the reduction in mitochondrial fission and inhibition of SCAP chondrogenic potential caused by Mdivi inhibition. In other words, WDR63 can indeed promote SCAP chondrogenic differentiation by facilitating mitochondrial fission.

Fig. 7.

WDR63 over-expression reverses the decreased chondrogenic differentiation potential of SCAP caused by Mdivi. A CCK-8 assay demonstrates SCAP viability under varying Mdivi concentrations. B, C Real-time RT-PCR results showed the DRP1 (B) and OPA1 (C) expression in SCAP. D Alcian Blue staining results show that WDR63 over-expression reverses the decreased chondrogenic differentiation potential of SCAP caused by Mdivi. Scale bar, 100 μm. E Real-time RT-PCR results show that WDR63 over-expression rescued the expression of DRP1 in SCAP. F–H Real-time RT-PCR results show that WDR63 over-expression rescued the expression of COL5 (F), COL2 (G), and SOX9 (H) in SCAP. I–K Western blot results showed the COL2 (I) and SOX9 (J) expression in SCAP. The relative protein levels quantified by densitometry and normalized to β-actin (K). β-actin and GAPDH was used as an internal control. Student’s t test analysis was performed to determine the statistical significance. All error bars represent s.d. (n = 3). *P<0.05

Discussion

The repair process of cartilage tissue after injury involves multiple biological mechanisms. Specifically, this process involves cell differentiation, extracellular matrix synthesis and deposition, as well as the formation of both fibrous and hyaline cartilage. Among these steps, the generation of hyaline cartilage is particularly important, as it is decisive in assessing the degree of cartilage morphology and functional recovery. To elucidate the involvement of WDR63, we systematically performed experiments targeting its role in the aforementioned process [29]. This work focused on evaluating the function of WDR63 in promoting chondrogenic differentiation in SCAP. The experimental results show that WDR63 is up-regulated during the chondrogenic differentiation of SCAP. WDR63 has also been shown to support cartilage differentiation, accompanied by elevated levels of chondrogenic genes like COL2, COL5, and SOX9. In vivo, human cells were observed in the repaired tissue following transplantation of human SCAP. This suggests that the implanted SCAP may have functioned in the defect region.

As the predominant component of the extracellular matrix in cartilage, COL2 plays a critical role in maintaining cartilage integrity by providing structural support and biomechanical flexibility. The fibrous structure of COL2 protein provides attachment sites for chondrocytes, which helps with the proliferation, differentiation, and maintenance of chondrocyte function. In the case of cartilage injury or pathology, the supplementation of COL2 protein can promote the proliferation and differentiation of chondrocytes, accelerate the synthesis and repair process of cartilage matrix [30]. These findings are highly relevant to the development of treatments for osteoarthritis and cartilage defect-related disorders. In our study, it was observed that the overexpression of WDR63 promoted the expression of COL2 mRNA and protein at the cellular level, and an increase in COL2 expression was also observed in the regenerative tissue in a cartilage injury repair model. This indicates that WDR63 accelerates the in vitro chondrogenic differentiation of SCAP and in vivo cartilaginous formation. Moreover, the natural repair of cartilage in an injured state often results in fibrocartilage. Our experimental results found that the reimplantation of SCAP overexpressing WDR63 can effectively increase the proportion of hyaline cartilage and the deposition of cartilage matrix in the repair area. COLX is a hallmark of immature or pathological hypertrophic cartilage. Lower COLX expression indicates that the regenerated tissue is closer to normal hyaline cartilage, reflecting higher-quality cartilage repair [31]. The decreased expression of COLX upon WDR63 overexpression further supports the conclusion that WDR63 facilitates cartilage regeneration. In addition, during cartilage repair, restoration of the subchondral bone is also critical. Moreover, COL1 is a major component of the subchondral bone matrix, where it provides structural support for the formation and maintenance of hyaline cartilage and facilitates bone regeneration within the defect area [32]. Previous studies have shown that WDR63 promotes the osteogenic differentiation of SCAP, which is also reflected in the present study that WDR63 enhances COL1 expression in the subchondral bone region and promotes bone mineralization within the defect area, thereby contributing to the formation of a supportive subchondral bone scaffold essential for effective cartilage repair. The precise mode and underlying mechanisms of this repair process warrant further investigation. In summary, we found that the effect of implanting SCAP in the defect area of rabbit knee joint cartilage is better than the cartilage regeneration effect of only implanting Matrigel, and implanting SCAP with overexpressed WDR63 can effectively improve the level of cartilage regeneration.

To clarify the modulatory function of WDR63 in SCAP-mediated cartilage differentiation, its associated binding proteins were investigated. By performing mass spectrometry on the protein bands with the strongest binding to WDR63, we identified proteins as shown in Supplementary Table 2, among which VIM, RBMX, and B4DY09 had higher proportions. RNA-binding motif protein X-linked (RBMX) is an RNA-binding factor involved in the regulation of both pre- and post-transcriptional events. RBMX also produces hnRNP G, a nuclear protein known to influence RNA splicing, ensure proper sister chromatid cohesion, and support genomic integrity [25]. Multiple studies have shown that RBMX binds to DNA promoters or pre-mRNA, promoting or inhibiting transcription [33–35]. However, there is no clear evidence to suggest a correlation between RBMX and genes related to the development and regeneration of cartilage or bone. The B4DY09 code encodes ILF2, which is a transcription factor required for T cells to express the interleukin 2 (IL-2) gene. In the innate immune system, ILF2 contributes to immune regulation by influencing T cell functionality and enhancing M2-type macrophage polarization. Additionally, aberrant expression of ILF2 is associated with various malignant tumors, such as non-small cell lung cancer (NSCLC), liver cancer, small cell lung cancer, pancreatic cancer, and stomach cancer. It may promote tumor cell proliferation by regulating mitochondrial homeostasis [36–39]. In other words, ILF2 may have a potential correlation with cartilage inflammation in terms of immunity and promoting macrophage polarization, but our main focus is on the ability of cartilage to repair and regenerate under conditions of damage [26, 40].

However, VIM, as a key member of the intermediate filament protein family, plays an indispensable role in various cell types, including chondrocytes [27]. In chondrocytes, VIM constitutes the core part of the cytoskeleton, which is crucial for maintaining cell morphology, preserving cell integrity, and ensuring smooth intercellular interactions [41]. The dynamic nature of VIM is particularly critical for cellular flexibility, as it provides necessary elasticity and stability when the cell is subjected to external mechanical forces to ensure normal cell function [42]. Furthermore, VIM not only participates in maintaining cell morphology but also regulates intracellular signaling pathways and material transportation through synergistic interactions with other cytoskeletal proteins [43]. Chondrocyte function, including proliferation, lineage commitment, and cartilage matrix production, is significantly influenced by the physiological levels of VIM expression, thereby ensuring the normal development and function maintenance of cartilage tissue [44]. In this study, we found that the interaction between WDR63 and VIM exhibits an increasing trend during chondrogenic differentiation, and the extent of this binding is positively correlated with the expression level of WDR63. Concomitantly, the expression level of VIM is upregulated during chondrogenic induction, and WDR63 can positively regulate VIM expression. Therefore, we hypothesize that there is a interaction between WDR63 and VIM, which may be involved in the regulation of chondrogenic differentiation. Therefore, considering the binding quantity with WDR63 and the correlation with cartilage, we have chosen VIM as the subject of our study.

Previous studies have clearly pointed out that VIM is a key component of the cytoskeleton, offering necessary support and anchoring function for mitochondria, and is crucial for maintaining the spatial positioning and morphological stability of mitochondria. In addition, VIM has been proved to have an auxiliary motor relationship with mitochondria [45]. By sustaining the structural stability of the cytoskeleton, VIM exerts an indirect influence on mitochondrial shape and activity. The knock-out of VIM will lead to abnormal changes of mitochondrial fragmentation, swelling and disorder, which may further affect the metabolism and viability of cells [46]. The dynamic adjustment of mitochondria relies on the flexibility and stability characteristics exhibited by the cytoskeleton, in which the elasticity and stability provided by VIM play an indispensable role [47]. It is worth noting that under pathological conditions such as cancer, VIM and mitochondrial function may experience significant changes. Elevated VIM levels in tumor cells may represent an adaptive response supporting accelerated growth and metastasis; concurrently, mitochondrial behavior is closely associated with the energy metabolism and invasiveness of these cells [48]. The dynamic changes of mitochondria (such as fission and fusion) require flexibility and stability of the cytoskeleton, and the elasticity and stability provided by VIM play an important role in this. Moreover, during chondrogenic differentiation, mitochondrial fission increases while fusion decreases [28]. These findings indicate that VIM regulates mitochondrial dynamics, but the role of WDR63-VIM binding in mitochondrial fusion and fission requires more rigorous validation.

For the growth and development of chondrocytes, sufficient energy supply is indispensable, and mitochondria are the main sites for ATP generation in cells. Through a series of complex mitochondrial dynamic processes, including division, fusion, and mitochondrial autophagy, mitochondria can flexibly adjust their morphology and quantity to precisely match the high energy demand of chondrocytes. Importantly, timely adjustment of mitochondrial dynamics can promote the proliferation and migration of chondrocytes after cartilage tissue damage, thereby accelerating the process of cartilage repair [17]. Additionally, existing studies have also revealed that regulating the expression levels of mitochondrial dynamics-related proteins (such as DRP1, OPA1, etc.) can effectively affect the biological characteristics of chondrocytes, thereby implementing precise regulation of cartilage development and regeneration [49, 50]. Our research further confirms that the overexpression of WDR63 can significantly promote mitochondrial fission, while reduced expression of WDR63 inhibits mitochondrial fission and promotes mitochondrial fusion instead. This study further discovered that WDR63 binds to and positively regulates VIM, while WDR63 positively regulates mitochondrial fission and negatively regulates mitochondrial fusion. Given the connection between WDR63 and chondrogenesis, this study hypothesizes that WDR63 may regulate mitochondrial dynamics through VIM, thereby affecting the chondrogenic differentiation process of SCAP.

A limitation of the present study is that, although we demonstrate the interaction between WDR63 and VIM and observe its correlation with chondrogenic outcomes and mitochondrial morphology, we did not directly perturb the WDR63–VIM interaction to establish its necessity in regulating mitochondrial dynamics and chondrocyte differentiation. Another limitation is biological variability among SCAP. Previous work using multiple donor-derived SCAP strains showed highly consistent chondrogenic differentiation behaviors, supporting the generalizability of the findings, but more cell line should be examined to confirm the discoveries in the future. In further studies, we will validate the role and mechanisms of the WDR63–VIM complex in chondrogenic differentiation and explore its effect in other tooth-derived stem cell populations.

Conclusion

In summary, we have found that the key protein WDR63 can significantly enhance the chondrogenic differentiation potential of SCAP. And WDR63 binds to VIM and enhances its expression, and also promotes mitochondrial fission. Given the established roles of VIM in chondrogenesis and mitochondrial dynamics, we propose that WDR63 may regulate chondrogenic differentiation by interacting with VIM, upregulating its expression, and promoting mitochondrial fission while suppressing fusion (Fig. 8). This discovery reveals the important targets in chondrogenic differentiation and provides a new perspective for understanding of the potential mechanisms and for promoting the differentiation of tooth-derived mesenchymal stem cells into specific directions and for cartilage tissue regeneration.

Fig. 8.

The potential mechanism of WDR63 in regulating SCAP chondrogenic differentiation. WDR63 can significantly enhance the chondrogenic differentiation potential of SCAP. WDR63 binds to VIM protein, promotes VIM expression, facilitates mitochondrial fission and inhibits mitochondrial fusion, thereby regulating the cartilage differentiation process. Created in BioRender. Chou, J. (2026) https://BioRender.com/ss3eubk

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AR

Aspect ratio

BMSCs

Bone marrow mesenchymal stem cells

COL1

Collagen type Ι

COL2

Collagen type II

COL5

Collagen type V

COLⅩ

Collagen type Ⅹ

DRP1

Dynamin-related protein 1

hnRNP G

Heterogeneous nuclear ribonucleoprotein G

ICRS

Cartilage repair assessment of international cartilage repair society

IL-2

Interleukin 2

ILF2

Interleukin enhancer binding factor 2

KDM2B

Lysine (K) demethylase 2B

MSCs

Mesenchymal stem cells

OPA1

Optic atrophy1

RBMX

RNA-binding motif protein X-linked

SCAP

Stem cell from apical papilla

SOX9

Sex-determining region Y box protein 9

VIM

Vimentin

WDR63

WD repeat domain 63

Author contributions

Jiawei Zhou was responsible for collection, data analysis and manuscript writing, and final approval of the manuscript. Yangyang Cao contributed to collecting data. Ziyan Sun and Yishu Huang contributed to picture production.Zhipeng Fan and Mengyuan Zhu were responsible for conception and design, manuscript revising, financial support and final approval of the manuscript. All authors have read and approved the final version of the manuscript.

Funding

This work was supported by grants from the National Key Research and Development Program (2022YFA1104401), and Young Scientist Program of Beijing Stomatological Hospital, Capital Medical University, NO. 23-09-02.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

SCAP was purchased from Shanghai Anwei Biological Company, which ensured the ethical approval of the original samples and obtained informed consent from all donors. Ethical Committee of Beijing Stomatological Hospital, Capital Medical University (Ethical Review NO: KQYY-202307-007). Title of the approved project: The repair effect of WDR63 overexpression on rabbit cartilage defect was investigated by animal experiments. Date of approval: 2023.8.25.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.