Abstract

Background

   Synovitis is a key factor in temporomandibular joint osteoarthritis
   (TMJOA) and could be an early sign of the disease. Notably, synovitis
   and its macrophage component represent a target of interest for
   developing treatments. Dental pulp stem cells (DPSCs) are derived from
   the neural crest, coincidentally with maxillofacial tissues, thus
   attracting significant interest in situ maxillofacial regenerative
   medicine. However, there is a relative scarcity of studies
   investigating the role of DPSCs in the temporomandibular joint (TMJ)
   synovial macrophage.

Objective

   This study aimed to evaluate the regulatory and reparative capabilities
   of DPSCs on synovitis in TMJOA rat models and to elucidate their
   specific mechanisms of action on synovial macrophages.

Method

   In vivo, three groups were established: the control group, which had
   neither induction nor treatment; the OA group, consisting of rats with
   OA but without any treatment; and the DPSCs treatment group, where rats
   with OA received DPSCs therapy. Progressive TMJOA was induced in rats
   via intra-articular injection of complete Freund’s adjuvant and sodium
   iodoacetate. After 2 weeks, DPSCs were injected into the joint cavity
   as a therapeutic intervention. Meanwhile, normal saline was injected
   into the joints of rats in both the control and OA groups. Four weeks
   after treatment, histological analysis was performed to evaluate the
   repair of synovitis. In addition, an in vitro co-culture system,
   consisting of macrophages and DPSCs. Each group was assessed using
   techniques, including mRFP-GFP-LC3 lentivirus transfection,
   Mito-Tracker Red and Lyso-Tracker Green staining, transmission electron
   microscopy, and Western blot analysis. Furthermore, apoptotic primary
   neutrophils were co-cultured with polarized macrophages to observe the
   phagocytic ability of macrophages towards apoptotic primary neutrophils
   under different treatments in vitro.

Results

   In the rat model, compared with the OA group, the DPSCs group exhibited
   reduced cell infiltration and collagen deposition, along with elevated
   levels of anti-inflammatory CD206 and decreased levels of inflammatory
   CD86, and enhanced the ability of macrophages to phagocytize apoptotic
   cells in vivo and in vitro. Notably, DPSCs exhibited enhanced efficacy
   in upregulating autophagosome expression, promoting co-localization
   with mitochondria and lysosomes, and modulating the expression of
   mitochondrial mitophagy proteins. Furthermore, inhibition of mitophagy
   in M1 macrophages partially attenuated the M2 polarization effect and
   phagocytosis induced by DPSCs.

Conclusion

   DPSCs significantly mitigate synovitis in TMJOA rats by enhancing M2
   polarization and efferocytic functions of macrophages. This study
   underscores the potential of DPSCs as a therapeutic strategy for TMJOA
   by modulating synovial macrophage functions through the regulation of
   mitophagy.

Graphical Abstract

   [41]graphic file with name 13287_2025_4468_Figa_HTML.jpg

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s13287-025-04468-2.

Introduction

   Synovitis could potentially precede temporomandibular joint
   osteoarthritis (TMJOA) and is crucial in its progression [[42]1–[43]4].
   The healthy synovial membrane comprises two layers: the lining
   surrounding the joint cavity and the sublining layer within the tissue.
   These layers predominantly comprise fibroblasts and tissue-resident
   macrophages, which are rich in blood vessels and facilitate the
   recruitment of cells during inflammatory responses [[44]5]. The balance
   within the joint depends on the joint’s microenvironment. In the
   temporomandibular joint (TMJ), synovial macrophages are the predominant
   cell type in the synovial membrane [[45]6]. In inflammatory conditions,
   there is a notable increase in the number of macrophages. The
   dysregulation in apoptotic processes of inflammatory macrophages and
   synovial fibroblasts underlies the pathogenesis of osteoarthritis.
   Professional phagocytes, including macrophages, are crucial in clearing
   apoptotic cells. The presence of dying cells in tissues is rarely
   noticeable, and apoptotic materials typically do not cause inflammation
   due to the remarkably efficient clearance process known as
   “efferocytosis“ [[46]7]. Efferocytosis eliminates cellular debris with
   inflammatory potential and initiates intracellular reprogramming,
   promoting pro-resolution signaling pathways [[47]8]. Depending on the
   immune context, macrophages can polarize into classically activated
   pro-inflammatory M1 macrophages or activated anti-inflammatory M2
   macrophages. Studies have shown a significant rise in M1 macrophages
   within the inflamed synovial membrane [[48]9–[49]11].

   Mesenchymal stem cells (MSCs) regenerative therapy holds promising
   potential for treating osteoarthritis. These cells interact with
   different innate immune cells, including macrophages and neutrophils
   [[50]12, [51]13]. Through direct contact or paracrine effects, MSCs can
   exert regulatory influence on macrophage maturation, migration, and
   proliferation [[52]4]. They possess the ability to induce the
   production of transforming growth factor-beta (TGF-ß) and arginase-1
   (Arg-1) in macrophages, thereby promoting their polarization from an M1
   phenotype towards an M2-like phenotype through secretion of
   cyclooxygenase-1 (COX-1) and prostaglandin E2 (PGE2) [[53]14]. Dental
   pulp stem cells (DPSCs) play a role in systemic immune regulation and
   suppression of inflammation. Their homology to craniofacial tissue
   makes them particularly promising for repairing craniofacial tissue.
   DPSCs have demonstrated safety and efficacy in treating stroke,
   COVID-19, and diabetes [[54]15–[55]17]. Their low immunogenicity
   enables their utilization by individuals and family members, while
   their storage value and therapeutic potential garner increasing
   attention. Consequently, DPSCs are poised to offer a safer and more
   regenerative alternative to traditional medicines [[56]18]. In a mouse
   model, DPSCs transfusion has been shown to manage local rheumatoid
   arthritis synovitis and autoimmune status effectively [[57]19].
   However, there is a relative scarcity of studies investigating the role
   of DPSCs in TMJOA synovitis.

   Autophagy is a cellular degradation and recycling mechanism that plays
   a crucial role in maintaining the stability of the intracellular
   environment and resisting external stress. It regulates macrophage
   phagocytosis, antigen presentation function, and polarization direction
   to modulate their involvement in inflammation development and
   resolution [[58]20]. Artificial mitochondrial transplantation (AMT) can
   reverse the aging of mesenchymal stromal cells and improve their
   immunomodulatory properties in lipopolysaccharide (LPS)-induced
   synovial cell inflammation by upregulating autophagy [[59]21].
   Autophagy plays an essential role in the occurrence and development of
   synovitis, and the regulation of autophagy may become a potential
   target for future treatment of synovitis [[60]22]. Mitophagy is a
   selective autophagy that removes damaged mitochondria [[61]23].
   Previous studies have demonstrated that TNF-α induces mitophagy in
   synovial fibroblasts of rheumatoid arthritis, and inhibition of this
   process can ameliorate arthritic synovitis [[62]24]. However, a dearth
   of research investigating mitophagy in macrophages associated with TMJ
   synovitis exists. Therefore, further investigation is imperative to
   comprehensively elucidate the autophagy mechanisms underlying synovitis
   in TMJOA.

   Given the crucial role of synovitis in the progression of TMJOA, and
   the current insufficient understanding of the potential of DPSCs in the
   treatment of TMJOA synovitis, this study aims to explore the hypothesis
   that DPSCs alleviate TMJOA synovitis by regulating macrophage
   polarization and efferocytosis. Specifically, we hypothesize that DPSCs
   can alleviate synovial inflammation by regulating autophagy, promoting
   the polarization of synovial macrophages from the pro-inflammatory M1
   phenotype to the anti-inflammatory M2 phenotype, and enhancing their
   efferocytosis. This study aims to clarify the effects of DPSCs on
   macrophage polarization and efferocytosis in synovitis of TMJOA, and to
   explore the potential therapeutic mechanism by which DPSCs regulate the
   inflammatory microenvironment of TMJ.

Methods

Animal experiments in vivo

Animal model

   The Sprague-Dawley (SD) rats utilized in this study were procured from
   the Laboratory Animal Research Center at Zhejiang Chinese Medical
   University. This study received approval from the Ethics Committee of
   Zhejiang Chinese Medical University on Feb 6, 2023 (Project name:
   Investigation into the Mechanism of Cartilage Repair by Dental Pulp
   Mesenchymal Stem Cells in Temporomandibular Arthritis. Ethics Approval
   Number: IACUC-20221031-14). A total of 18 SD rats were randomly
   assigned to three groups: the Control group (n = 6), the OA group
   (n = 6), and the DPSCs group (n = 6). To induce TMJOA, a mixture of 50
   µL of monosodium iodoacetate (MIA) at a concentration of 0.5 mg
   (Sigma-Aldrich, USA), 25 µL of normal saline (NS), and 25 µL of
   complete Freund’s adjuvant (CFA) (Sigma-Aldrich, USA) was administered
   intra-articularly once into both TMJs and induced for 2 weeks
   (Fig.[63]S1). The control group did not receive any induction during
   this phase. Following the 2-week MIA/CFA induction period, the OA and
   DPSCs groups underwent weekly intra-articular injections into bilateral
   TMJs for 4 consecutive weeks. Specifically, the OA group received 50 µL
   of normal saline, while the DPSCs group received 50 µL of DPSCs
   suspension. Meanwhile, in the control group, 50 µL of normal saline was
   injected into the bilateral temporomandibular joints once a week for 4
   consecutive weeks. These injections were administered using the 30G
   insulin needle (Becton, Dickinson and Company, USA). The rats exhibited
   no obvious signs of suffering throughout the experimental period. At
   the end of the 4-week treatment period, the rats were euthanized via
   intraperitoneal injection of Shutai 50 solution (BIOFIVEN, China) at a
   dose of 20 mg/kg body weight, with anesthesia being achieved within
   3–5 min. This study adhered to the ARRIVE guidelines 2.0 for the
   reporting of in vivo experiments (Fig. [64]1).

Fig. 1.

   Fig. 1
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   Injection time point diagram

Histology and immunohistochemical/immunofluorescence staining

   For the histological examination, samples from the temporomandibular
   joints were initially fixed in 4% paraformaldehyde for 3 days.
   Subsequently, they were decalcified in a 14% Ethylene Diamine
   Tetraacetic Acid (EDTA) solution at room temperature for 90 days. The
   samples were then systematically dehydrated using an automatic
   dehydrator and embedded in paraffin. Sagittal sections, each 3 μm in
   thickness, were cut from the paraffin blocks. After deparaffinization
   and rehydration, the sections were stained with Hematoxylin-Eosin (HE)
   and Masson staining for morphological observation. The assessment of
   the synovial membranes was performed on H&E-stained slides, focusing on
   three key features: the synovial lining cell layer, stromal cell
   density, and inflammatory infiltrate. The above indicators were
   semiquantitatively evaluated (from 0, absent to 3, strong), and each
   feature was graded separately. The scores for these parameters were
   then combined and interpreted as follows: a total score of 0–1
   indicated no synovitis, 2–4 indicated low-grade synovitis, and 5–9
   indicated high-grade synovitis [[66]25].

   Histology sections were utilized for immunohistochemical (IHC) and
   immunofluorescent (IF) staining. The slices were initially subjected to
   dewaxing, followed by rehydration and subsequent washing in phosphate
   buffer (PBS) for three cycles of 5 min each. Subsequently, the slices
   were soaked overnight in a citric acid solution in a water bath at 60℃
   to facilitate antigenic repair. After repair, the slices were washed
   three times with PBS and then treated with endogenous peroxidase
   blocker at room temperature for 10 min before being rewashed with PBS
   three times. The sections were then sealed with 5% goat serum at room
   temperature for 1 h (IHC staining only). Next, the sections were
   incubated overnight at 4℃ with the primary antibody. Then, the
   secondary antibody was applied using IHC or IF staining techniques and
   incubated at room temperature for 1 h. For IHC-stained sections,
   diaminobenzidine and hematoxylin stains were used before dehydration
   and sealing took place. For IF-stained slices, DAPI solution was
   applied followed by sealing using glass slides. Antibodies used for
   IHC/IF staining were as follows: P62 (HUABio, R1309-8, China), Beclin1
   (ABclone, A7353, China), LC3B (Novusbio, NB100-2220, USA), CD206
   (Proteintech, 18704-1-AP, China), CD86 (Proteintech, 13395-1-AP,
   China), Col2 (Abcam, ab39012, UK), MMP13 (Abcam, ab39012, UK).

TUNEL staining

   The TUNEL assay was conducted following the protocol provided by the
   manufacturer (TUNEL BrightGreen Apoptosis Detection Kit, Vazyme
   Biotech, China) to identify cell death within the synovial membrane.
   This assay leveraged the green channel at a wavelength of 488 nm for
   detection. DAPI was employed as a counterstain to visualize the nuclei
   detected in the blue channel at 460 nm. The imaging process was carried
   out using a fluorescent microscope (Olympus BX43, Japan).

Access to gene expression omnibus (GEO) datasets

   The dataset of relevant studies was retrieved from the GEO database,
   and the [67]GSE49604 dataset was downloaded. The gene expression
   profile, [68]GSE49604 on the [69]GPL8432 platform, was found for
   further analysis. The experimental group comprised primary human
   synovial macrophages from patients with arthritis, while the control
   group consisted of primary human peripheral blood mononuclear cells
   from healthy individuals. Utilize GEO2R for differentially expressed
   genes (DEGs) between macrophages of human synovitis and healthy
   macrophages, followed by subsequent visualization and statistical
   analysis of the DEGs. The threshold was set to obtain DEGs as absolute
   log fold change|log[2]FC|>1 and P < 0.05. The heatmap was plotted by
   [70]https://www.bioinformatics.com.cn, an online platform for data
   analysis and visualization.

Cell culture in vitro

Cell culture

   The RAW264.7 macrophages were sourced from Punosai Life & Technology
   Co., LTD. These cells were cultured in Dulbecco’s modified Eagle medium
   supplemented with 10% Fetal Bovine Serum (FBS), 100 U/ml penicillin,
   and 100 µg/ml streptomycin and incubated in a humidified incubator with
   5% CO[2] at 37 °C. After three passages, the cells were harvested for
   subsequent experiments. Additionally, 100 ng/mL LPS (Sigma-Aldrich,
   USA) and 10 ng/mL IFN-γ (PeproTech, USA) were used to differentiate
   RAW264.7 cells into M1-type macrophages. The DPSCs were generously
   provided by the Institute of Medicine, Chinese Academy of Sciences &
   Tongce Biological Dental Stem Cell Bank and Research and Development
   Center and the donors had signed informed consent [[71]26]. The DPSCs
   were cultured with alpha modification of Eagle’s medium (containing 10%
   fetal bovine serum) at 37 °C with 5% CO[2]. DPSCs were identified using
   a previously described method, and cells with three passages were used
   [[72]26] (Fig.[73]S2).

RAW264.7 cells and DPSCs co-culture system

   The study was divided into four groups: the control group, the LPS
   group, the LPS + DPSCs group, and the LPS + DPSCs + Mdivi-1 group. In
   the control group, RAW264.7 macrophages received no treatment. For the
   LPS group, RAW264.7 macrophages were differentiated into M1-type
   macrophages using 100 ng/mL LPS (Sigma-Aldrich, USA) and 10 ng/mL IFN-γ
   (PeproTech, USA). A co-culture model was established with DPSCs
   (2 × 10⁶/well) in the upper layer and RAW264.7 macrophages
   (2 × 10⁵/well) in the lower layer. In the LPS + DPSCs group, the cells
   were stimulated with LPS and IFN-γ for 24 h before being co-cultured
   with DPSCs for 24 h. In the LPS + DPSCs + Mdivi-1 group, RAW264.7
   macrophages were pre-treated with the mitophagy inhibitor Mdivi-1 (25
   µmol/L, Abcam, UK) for 3 h before LPS/IFN-γ treatment and subsequent
   co-culture with DPSCs [[74]23]. After a 24-h co-culture period between
   DPSCs and RAW264.7 cells, RAW264.7 cells were systematically harvested
   for further experimental analysis (Fig. [75]2).

Fig. 2.

   Fig. 2
   [76]Open in a new tab

   In vitro model diagram of cell co-culture. DPSCs were placed in the
   upper chamber, and RAW264.7 cells were cultured in the lower chamber

Western blot

   The RAW264.7 cells of the above groups were lysed in RIPA buffer
   (Beyotime, Shanghai, China) for 20 min, followed by centrifugation at
   12,000 g for 4 min. The primary antibodies of Beclin1 (Proteintech,
   11306-1-AP, China), P62 (Abcam, ab211324), LC3B (Novusbio, NB100-2220,
   USA), Parkin (Proteintech, 14060-1-AP, China), PINK1 (Proteintech,
   23274-1-AP, China), Arginase-1 (Proteintech, 16001-1-AP, China) and
   GAPDH (Abcam, ab181602, UK) were utilized. The membranes underwent
   incubation with primary antibodies, followed by the application of
   horseradish peroxidase-conjugated secondary antibodies to facilitate
   the detection process. The relative gray level of the protein bands was
   determined using an enhanced chemiluminescence system (ASPEN, AS1059,
   China). The AlphaEaseFC software processing system ensured accurate
   protein expression level quantification.

Autophagy detection through transmission electron microscopy

   The RAW264.7 cells of the above groups were first gently washed twice
   with phosphate-buffered saline (PBS) to remove any residual substances,
   followed by fixation in 2.5% glutaraldehyde (Sinopharm, China) for
   24 h. Subsequently, the samples were post-fixed with 1% osmic acid
   (Sinopharm, China) for 2 h, dehydrated using graded ethanol (Sinopharm,
   China), and then infiltrated and embedded with epon 812 (Sinopharm,
   China). Ultrathin slices of the samples were sectioned using an
   ultramicrotome (Leica, Germany) and examined under Transmission
   Electron Microscopy (TEM) (Thermo Fisher Scientific, USA).

Autophagy detection through mRFP-GFP-LC3 adenovirus transfection

   The RAW264.7 cells in each group were transfected with the
   Ad-mRFP-GFP-LC3 adenoviral vector to detect the autophagic flux levels.
   Before transfection, the Ad-mRFP-GFP-LC3 adenoviral vector (Hanbio
   Biotechnology, China) was retrieved from the − 80℃ freezer and thawed
   slowly on ice. Based on the number of cells, the adenovirus was diluted
   to the working concentration to achieve a multiplicity of infection
   (MOI) of 50. The diluted adenoviral solution was added to the cell
   culture wells to ensure adequate contact with the cells. The culture
   plate was then incubated at 37℃ in a 5% CO₂ incubator for 4 h. After
   incubation, the adenovirus culture medium was aspirated and replaced
   with fresh complete culture medium (DMEM containing 10% FBS). After the
   cells were further cultured for 72 h, the fluorescence signals of
   mRFP-GFP-LC3 in the cells were observed and imaged using confocal laser
   scanning microscopy (Zeiss, Germany), and the autophagy-related changes
   were analyzed.

Autophagy detection through lysosome and mitochondria colocalization

   The distribution relationship between mitochondria and lysosomes in the
   above groups of RAW264.7 macrophages was explored through fluorescence
   staining and colocalization analysis. The mitochondria of live cells
   were stained with a working solution of Mito-Tracker Red (Beyotime,
   China) at a concentration of 100 nM and incubated at 37℃ for 20 min.
   Subsequently, the cells were further stained with a working solution of
   Lyso-Tracker Green (Beyotime, China) at a concentration of 50 nM and
   incubated at 37℃ for 15 min. Cell nuclei were counterstained with
   Hoechst 33,342. The co-localization analysis of lysosomes and
   mitochondria was performed using ImageJ software.

Efferocytosis assay

   Primary neutrophils were isolated from C57BL/6 mice and then cultured
   in RPMI-1640 medium (Hyclone, USA) for 48 h without serum to induce
   apoptosis. The primary neutrophils were subsequently labeled with PKH67
   dye (Sigma-Aldrich, USA) following the manufacturer’s instructions.
   Then, PKH67-labeled apoptotic primary neutrophils were added in the
   Control, LPS, LPS + DPSCs, and LPS + DPSCs + Midiv1 groups at a ratio
   of 10:1, and incubated for another 48 h. Subsequently, the cells were
   thoroughly washed three times with PBS to eliminate unengulfed primary
   neutrophils. The cells were subsequently labeled with Dil (Beyotime,
   China) solution according to the manufacturer’s instructions. The
   engulfment ability of macrophages towards apoptotic primary neutrophils
   was visualized using immunofluorescence microscopy. The efferocytotic
   index was calculated as follows: (number of macrophages containing
   apoptotic bodies)/(total macrophages) × 100%, then normalized to the
   control group set as 100%.

Statistical analysis

   All experiments were conducted with a minimum of three repetitions to
   ensure the reliability of the results. The data obtained were presented
   as the means ± standard deviation. Data was analyzed using GraphPad
   Prism 8.0 software (GraphPad Software, USA). To discern differences
   between the two groups, Student’s t-test was employed. When comparing
   three or more groups, a one-way analysis of variance (ANOVA) was
   utilized to assess the variability among the groups. Subsequently,
   Tukey’s test was applied to determine the specific between-group
   differences. For this study, statistical significance was defined as
   P < 0.05.

Result

DPSCs reduce synovial inflammation and fibrosis

   Firstly, we sought to assess the protective impact of DPSCs on
   synovitis in vivo. The OA rat model was induced by injecting complete
   Freund’s adjuvant (CFA) and sodium iodoacetate (MIA). The animals were
   divided into Control, OA, and DPSCs groups, which received weekly
   injections of normal saline (NS) and DPSCs suspension for 4 weeks.
   After 4 weeks of DPSCs injections into the joint cavity, TMJ joint
   specimens of rats were collected for Masson and HE staining. The
   morphology of the TMJ synovium in the control group was normal.
   However, following the induction of arthritis via MIA/CFA, OA rats
   exhibited pronounced pathological alterations in TMJ synovium,
   characterized by synovial collagen fibrohyperplasia accompanied by
   vascular opacity (as evidenced by Masson staining) and infiltration of
   inflammatory cells (demonstrated through HE staining). After four weeks
   of treatment with DPSCs, the DPSCs group showed a marked reduction in
   the number of synovial lining cell layers and blood vessels, alongside
   a decrease in both the density and quantity of infiltrating
   inflammatory cells and collagen fibers within the synovial tissue when
   compared to the OA group (Fig. [77]3a-b). The synovitis scores of the
   OA groups were significantly higher than those of the control and DPSCs
   groups (Fig. [78]3c). The findings suggest that applying DPSCs
   significantly attenuates synovial inflammation and fibrosis in OA
   synovitis.

Fig. 3.

   [79]Fig. 3
   [80]Open in a new tab

   DPSCs ameliorate synovium inflammation and synovial fibrosis in TMJOA
   synovitis. (a-b) The synovium thickness, inflammatory cell
   infiltration, and collagen fiber deposition in the Control, OA and
   DPSCs groups were assessed by Masson’s trichrome and HE staining,
   respectively. Scale bar: 20 μm. Amplification region: 10 μm. (c)
   Statistical analysis of synovitis scores in HE-stained sections. Data
   are presented as mean ± SD. Statistical significance was determined
   using one-way ANOVA with Tukey’s post hoc test. ***p < 0.001,
   ****P < 0.0001

DPSCs promote synovial macrophage M2 polarization and efferocytosis

   Subsequently, we employed immunofluorescence to assess the shift in
   polarization of TMJ synovial macrophages. Notably, after the
   administration of MIA and CFA, there was a significant increase in both
   the M1 macrophage-associated markers (CD86) and the M2
   macrophage-associated markers (CD206) within the synovial membrane of
   the OA group. In comparison, the group treated with DPSCs displayed a
   significant upregulation of the M2 macrophage marker CD206 and a
   concurrent downregulation of the pro-inflammatory M1 macrophage marker
   CD86 when contrasted with the OA group (Fig. [81]4a-d). Efferocytosis
   is an indispensable process through which dead and dying cells are
   removed by phagocytic cells. In this study, immunofluorescent TUNEL
   staining showed that the number of apoptotic cells in synovial tissue
   increased with the progression of inflammation, and the number of
   apoptotic cells in the DPSCs group was down-regulated.
   Immunofluorescence staining of CD206 and TUNEL revealed that the
   injection of DPSCs augmented the co-localization between CD206^+
   macrophages and apoptotic cells labeled with TUNEL in vivo, thereby
   further substantiating the capacity of DPSCs to induce polarization of
   synovial macrophages towards an M2 phenotype and enhance their
   phagocytic activity towards apoptotic cells in OA synovium
   (Fig. [82]4e-f). To investigate the effects of DPSCs on macrophage
   phagocytosis and function in vitro, we stimulated RAW264.7 cells with
   LPS/γ-IFN to simulate an inflammatory environment and co-cultured these
   LPS-stimulated cells with DPSCs for 24 h. Subsequently, the macrophages
   were stained with Dil and co-cultured with apoptotic neutrophils
   labeled with PKH67 for 48 h to assess the phagocytosis level. As
   anticipated, it was also confirmed in vitro that the efferocytosis
   function of macrophages was impaired in the LPS microenvironment,
   resulting in a reduced ability of macrophages (Dil-labeled) to
   effectively clear fluorescent apoptotic neutrophils (PKH67-labeled)
   compared with the control group. In addition, co-culture with DPSCs
   enhanced the phagocytosis activity of macrophages (Fig. [83]4g-h).
   Based on these findings, we propose that DPSCs can potentially promote
   the polarization of M1 synovial macrophages toward the M2 phenotype and
   enhance their efferocytosis.

Fig. 4.

   [84]Fig. 4
   [85]Open in a new tab

   DPSCs promote the polarization of synovial macrophages towards the M2
   phenotype and enhance their phagocytosis in TMJOA synovitis. (a-b)
   Immunohistochemistry staining was performed to visualize the expression
   and localization of CD86 and CD206. Scale bar: 50 μm. (c-d)
   Quantitative analysis of CD86 and CD206 protein expression. (e) The
   expression and co-localization of CD206 and TUNEL were observed in
   synovial tissue by immunofluorescent staining. Scale bar: 50 μm. (f)
   Quantification of CD206^+ macrophages engulfing apoptotic cells as a
   proportion of total CD206^+ cells. (g) Fluorescent staining for Dil
   (red) in RAW264.7 cells and PKH67 (green) in apoptotic neutrophils
   after co-culture for 48 h. Scale bar: 20 μm. (h) Quantification of
   Dil-PKH67-positive macrophages (yellow) as a proportion of total
   macrophages. Data are presented as mean ± SD. Statistical significance
   was determined using one-way ANOVA with Tukey’s post hoc test.
   *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001

Autophagy is identified as the key signal in macrophages of synovitis

   To elucidate the mechanisms underlying the therapeutic effects of DPSCs
   on macrophages, we searched the National Center for Biotechnology
   Information (NCBI) GEO database. The differentially expressed genes
   (DEGs) in the gene expression profiles ([86]GSE49604) were identified
   using GEO2R, an online data analysis tool. Subsequently, the resulting
   dataset was analyzed using volcano plots to visualize the DEGs. We
   identified 507 DEGs between macrophages of human synovitis and healthy
   macrophages (Fig. [87]5a). Gene Ontology (GO)/Kyoto Encyclopedia of
   Genes and Genomes (KEGG) heatmap was plotted by
   [88]https://www.bioinformatics.com.cn, an online data analysis and
   visualization platform. KEGG pathway enrichment analysis showed that
   these genes were involved in many signaling pathways, mainly related to
   lysosomal, autophagy, and apoptosis activity (Fig. [89]5b, d). As shown
   in Fig. [90]5c, the top two enriched biological process (BP) terms were
   the neutrophil activation involved in immune response and neutrophil
   degranulation. Taken together, autophagy activity and the immune
   response involved in neutrophils may play an essential role in
   macrophages of synovitis.

Fig. 5.

   [91]Fig. 5
   [92]Open in a new tab

   Functional enrichment based on DEGs. (a) Volcano plot depicting the
   distribution of DEGs between human arthritis macrophages and healthy
   controls from the [93]GSE49604 dataset. Red, blue, and gray dots
   represent gene expression levels corresponding to upregulated,
   downregulated, and insignificant expression. DEGs, differentially
   expressed genes. (b) KEGG pathway enrichment analysis of DEGs in human
   arthritis macrophages and healthy controls. KEGG, Kyoto Encyclopedia of
   Genes and Genomes. (c) GO-BP analysis for DEGs shows significant terms
   through (c) a bubble plot. GO, Gene Ontology; BP, biological process;
   (d) Gene expression in the adherens junction pathway was plotted by
   PATHVIEW

DPSCs enhance macrophage M2 polarization and efferocytosis by enhancing
mitophagy

   LC3B, P62, and Beclin1 are the most commonly used markers to indicate
   autophagy activity. Consistent with our expectations, we observed a
   significant upregulation of Beclin1 and LC3B expression and a
   significant downregulation of P62 expression in the synovium of the OA
   group. These findings suggest that MIA/CFA-induced mitophagy can be
   activated, and DPSCs further enhance mitophagy to suppress
   MIA/CFA-induced inflammation in vivo (Fig. [94]6a-d). To elucidate the
   mechanism by which DPSCs regulate macrophages, we established an in
   vitro co-culture system of DPSCs and macrophages by stimulating
   RAW264.7 cells with LPS and γ-IFN. To confirm the regulatory effect of
   DPSCs on macrophage polarization in vitro, macrophages were
   pre-stimulated with LPS and then co-cultured with or without DPSCs.
   Immunofluorescence staining of macrophages showed that DPSCs reduced
   the expression of CD86 ^+ cells and promoted polarization toward the M2
   phenotype (CD206 ^+ cells) (Fig. [95]6e-f). These results demonstrate
   that DPSCs inhibit LPS-stimulated macrophage M1 polarization and
   reverse LPS-suppressed macrophage M2 polarization. These findings
   indicate that DPSCs may promote the M2 polarization of macrophages by
   autophagy, thereby mitigating synovitis.

Fig. 6.

   [96]Fig. 6
   [97]Open in a new tab

   DPSCs promote autophagy and M2 polarization in macrophages. (a)
   Immunohistochemical analysis of autophagy-associated proteins P62,
   Beclin1 and LC3 in synovial macrophages of various treatment groups.
   Scale bar: 10 μm. (b-d) Quantifying the histomorphometric analysis of
   the expression of proteins P62, Beclin1 and LC3. (e-f)
   Immunofluorescence staining of CD86 and CD206 in RAW264.7 macrophages.
   Scale bar: 10 μm. Data are presented as the mean ± SD. Statistical
   significance was determined using one-way ANOVA with Tukey’s post hoc
   test. *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001

   Furthermore, transmission electron microscopy illustrated that LPS and
   γ-IFN stimulation escalated the number of autophagosomes within
   macrophages. TEM analysis revealed an increase in the number of
   autophagosomes following treatment with DPSCs in M1 macrophages
   (Fig. [98]7a). We utilized an RFP-GFP-LC3 reporter assay to investigate
   autophagic flux in cells treated with LPS and DPSCs. This assay reveals
   autophagosome formation through the visibility of both RFP and GFP
   signals, presenting as yellow puncta. Upon the fusion of autophagosomes
   with lysosomes, the acidic environment quenches GFP signals, leaving
   only RFP signals and, thus red autolysosomes visible. Our findings
   indicated that DPSCs prompted an increase in autophagosome formation
   (yellow) (Fig. [99]7b, f). Next, we evaluated the expression of
   mitophagy-associated proteins in M1-polarized macrophages. We found
   that P62 expression was decreased, while Becin1 and LC3 II/I ratio
   protein expression levels were significantly increased in LPS-induced
   RAW264.7 cells. The expression of P62 protein in RAW264.7 cells was
   further downregulated. In contrast, Becin1 and LC3 II/I ratio protein
   expression were further upregulated upon LPS-induced co-culture of
   RAW264.7 cells with DPSCs (Fig. [100]7c, d). Full-length blots/gels are
   presented in Supplementary Figure [101]S3. The mitophagy pathway
   encompasses various mechanisms, with the most classical and
   comprehensively understood being the Pink1/Parkin pathway. To elucidate
   the impact of DPSCs on mitophagy, we evaluated the activation level of
   the PINK1/Parkin pathway. Western blot analysis results demonstrated
   that LPS stimulation upregulated the expression of PINK1 and Parkin in
   M1 macrophages, and treatment with DPSCs further augmented PINK1 and
   Parkin expression (Fig. [102]7e, g). Full-length blots/gels are
   presented in Supplementary Figure [103]S4.

Fig. 7.

   [104]Fig. 7
   [105]Open in a new tab

   DPSCs enhance macrophage mitochondrial autophagy in vitro. (a) The
   visualization of the ultrastructure of macrophages via transmission
   electron microscope (TEM). In the images, membrane-like vesicles in
   macrophages were observed. The distribution of autophagosomes (green
   circle) in macrophages was visualized using the TEM. Scale bar: 1 μm.
   Amplification region: 500 nm. (b, f) Autophagocytic flux in macrophages
   was analyzed by mRFP-GFP-LC3 (yellow puncta). Scale bar: 10 μm. (c-d)
   Western blot showing P62, Beclin1 and LC3-II/I expression in
   macrophages after various treatments. (e, g) Western blot showing the
   expression of PINK1 and Parkin in macrophages after various treatments.
   Data are presented as the mean ± SD. Statistical significance was
   determined using one-way ANOVA with Tukey’s post hoc test. *p < 0.05,
   **p < 0.01, ***p < 0.001, ****P < 0.0001

   To investigate the interplay between macrophage mitophagy and M2
   polarization, we implemented a simultaneous intervention involving LPS,
   DPSCs, and Mdivi-1 to impede mitochondrial mitophagy. Furthermore,
   fluorescence microscopy revealed that LPS induction significantly
   increased the co-localization of mitochondria and lysosomes in
   macrophages. The overlapping signals of MitoTracker and LysoTracker
   were significantly improved in M1 macrophages following co-culture with
   DPSCs, indicating that DPSCs could regulate mitochondrial autophagy in
   M1 macrophages. The application of the mitochondrial autophagy
   inhibitor, Midiv-1, markedly reduced the colocalization signals between
   MitoTracker and LysoTracker (Fig. [106]8a-b). The Western blot analysis
   revealed an upregulation of Arg-1 (M2 macrophage-associated markers)
   expression in M1 macrophages upon co-culture with DPSCs. Furthermore,
   treatment with Mdivi-1 significantly attenuated the expression of Arg-1
   compared to the LPS + DPSCs group (Fig. [107]8c-d). Full-length
   blots/gels are presented in Supplementary Figure [108]S5. The above
   assessments encompassed alterations in M2 polarization among
   macrophages across varied groups, revealing that mitophagy suppression
   partially annulled the M2 polarization enhancement of DPSCs. Moreover,
   the ratio of phagocytic apoptotic neutrophils in the Midiv-1 treatment
   group decreased significantly (Fig. [109]8e, f). In conclusion, the
   data indicated that mitophagy may promote the phagocytosis and
   polarization of macrophages.

Fig. 8.

   [110]Fig. 8
   [111]Open in a new tab

   DPSCs facilitate M2 polarization and effercytosis of macrophages by
   inducing mitophagy. (a-b) The autolysosomes, mitochondria, and nucleus
   were labeled by Lyso-Tracker Green and Mito Tracker Red, respectively.
   Scale bar: 20 μm. (c-d) Representative Western blot image of Arg1 in
   macrophages after treatment of LPS, DPSCs, and Midivi-1. (e-f)
   Fluorescent staining for Dil (red) in RAW264.7 cells and PKH67 (green)
   in apoptotic neutrophils after co-culture for 48 h. Scale bar: 20 μm.
   Phagocytosis was observed under the fluorescence microscope. Data are
   presented as the mean ± SD. Statistical significance was determined
   using one-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01,
   ***p < 0.001, ****P < 0.0001

Discussion

   Synovial inflammation, also known as synovitis, is a crucial factor in
   the progression of osteoarthritis from its earliest clinical stages and
   is a key driver of pain initiation [[112]27, [113]28]. Current research
   indicates that DPSCs have some potential in controlling symptoms of
   synovitis, but most studies have focused on their effects on
   fibroblast-like synoviocytes (FLSs). These studies have revealed that
   DPSCs can mitigate the pathological course of synovitis by influencing
   FLSs and the secretion of inflammatory factors [[114]19, [115]29].
   However, the pathogenesis of synovitis is complex, involving not only
   FLSs but also the interaction of various immune cells, especially the
   polarization state of macrophages, which plays a key role in
   inflammatory responses. In this study, we aim to investigate the impact
   of DPSCs on macrophages in TMJOA.

   Based on our previous findings and a comprehensive literature review,
   we propose that DPSCs play a crucial role in synovial inflammation and
   macrophage polarization. The homing ability of stem cells facilitates
   their targeting of inflamed and damaged areas, potentially expediting
   the repair process [[116]30]. Moreover, stem cell homing has the
   potential to modulate macrophage polarization and thereby regulate the
   inflammatory response [[117]4, [118]31]. DPSCs have garnered attention
   for their potential to combat inflammation and prevent fibrotic tissue
   formation, positioning them as viable options for addressing various
   conditions related to the immune system, inflammation, and fibrosis
   [[119]32]. The preliminary histological evaluation demonstrated that
   DPSCs effectively mitigated synovitis, as evidenced by reduced in
   collagen fibroplasia and inflammatory cell infiltration. These findings
   support our initial hypothesis and highlight the potential therapeutic
   role of DPSCs in modulating synovial inflammation.

   Synovial macrophages play a role in regulating joint inflammation and
   OA severity [[120]33, [121]34]. M1 macrophages release proinflammatory
   cytokines, while M2 macrophages release anti-inflammatory cytokines
   that contribute to the resolution of inflammation [[122]35]. The new
   study found that electrical signals improve DPSC-mediated paracrine
   patterns by up-regulating macrophage M2 polarization [[123]17,
   [124]36]. In addition, the conditioned medium (CM) of stem cells from
   human exfoliated deciduous teeth (SHEDs) can exhibit significant
   anti-inflammatory effects on synovitis by inducing the polarization of
   M2 macrophages. However, it remains unclear which specific secretory
   factors or signaling pathways play a dominant role in inducing the
   polarization of M2 macrophages [[125]37]. Consistent with these
   findings, we discovered that DPSCs induced M2 polarization of TMJ
   synovial macrophages in vivo and in vitro, but the specific mechanism
   remained unclear.

   Through further bioinformatics analysis, we speculate that macrophage
   autophagy and immune activities may play a crucial role in the
   pathogenesis of synovitis. However, the impact of DPSCs on macrophage
   autophagy remains to be elucidated. Additionally, the intricate
   relationship between autophagy and macrophage polarization is still not
   fully understood. In inflammation, efferocytosis by macrophages is a
   key process for clearing apoptotic cells, crucial for maintaining
   inflammatory balance and tissue repair. When large numbers of
   neutrophils are recruited to the site of injury in the early stages of
   inflammation and complete their phagocytic and bactericidal tasks, they
   rapidly enter apoptosis. If these apoptotic neutrophils are not cleared
   promptly, they may release pro-inflammatory contents, further
   exacerbating the inflammatory response [[126]38]. A previous study
   indicated that individuals with knee osteoarthritis exhibit a higher
   prevalence of apoptotic synovial cells than healthy controls. The
   fraction of synovial tissue macrophages participating in efferocytosis
   and the amount of material engulfed by individual macrophages
   significantly decreased in patients with osteoarthritis [[127]39].
   Consistent with these findings, a notable decrease in the proportion of
   synovial tissue macrophages engaged in efferocytosis was observed in
   TMJOA rats. In light of these observations, we further investigated the
   effects of DPSCs on macrophage function. Our results demonstrate that
   the administration of DPSCs significantly enhances the phagocytic
   activity of macrophages, both in vivo and in vitro. This finding is
   novel and has not been previously reported, suggesting that DPSCs may
   play a unique role in modulating macrophage function and promoting the
   clearance of apoptotic cells in inflammatory conditions.

   The reprogramming of macrophage polarization is closely associated with
   autophagy [[128]40, [129]41], a process that elucidates MSC-mediated
   immunomodulation [[130]42]. Research suggests that the absence of
   essential autophagy genes or specific autophagy-related components can
   lead to increased production of TNF-αand IL-6, key proinflammatory
   cytokines. This deficiency also promotes the development of
   proinflammatory cell phenotypes typical of M1 macrophages. These
   effects have been observed in both in vivo animal models and in vitro
   cell culture systems, highlighting the critical role of autophagy in
   regulating inflammatory responses and the balance between M1 and M2
   macrophage polarization [[131]43]. Low-intensity pulsed ultrasound
   attenuated mature IL-1β production primarily by augmenting
   autophagy-mediated degradation of pyruvate kinase, muscle, and
   sequestosome1 in macrophages, thereby ameliorating synovitis [[132]44].
   Some researchers synthesized ROS reactive polymer, which self-assembled
   with astaxanthin and autophagy activator rapamycin to form
   nanoparticles. These nanoparticles could restore mitochondrial membrane
   potential, induce intracellular autophagy, and effectively repolarize
   M1 macrophages into M2 phenotype, thus significantly slowing down the
   progression of synovitis [[133]45]. These findings indicate a potential
   modulation of synovial macrophage polarization by autophagy, yet
   definitive evidence is currently lacking.

   Mitophagy has recently garnered increasing attention in the field. It
   is a crucial intracellular clearance mechanism for maintaining
   mitochondrial function and quality, effectively modulating cellular
   metabolism, survival, and programmed cell death pathways [[134]46]. The
   novel functionalized nanoparticles have modulated synovial macrophage
   pyroptosis and mitophagy interactions to mitigate osteoarthritis
   progression [[135]34]. However, research on TMJOA synovitis mitophagy
   remains scarce. In this research, we investigated the impact of LPS and
   DPSCs on mitophagy levels in macrophages. The findings suggest that
   co-culture with DPSCs increases mitophagy in macrophages, which is
   associated with M2 polarization and reduced inflammation. Additionally,
   DPSCs induce mitophagy in a PINK1/Parkin pathway-dependent manner. The
   PINK1/Parkin pathway is a well-researched mechanism that triggers
   mitophagy, a process where damaged mitochondria are targeted for
   degradation. When mitochondria are damaged, their membrane potential
   decreases, preventing PINK1 from moving to the inner membrane. Instead,
   PINK1 accumulates on the outer mitochondrial membrane. Here, it
   interacts with the translocase of the outer mitochondrial membrane,
   leading to its phosphorylation at Ser65. This phosphorylation activates
   Parkin, a ubiquitin ligase, which then ubiquitinates mitochondrial
   proteins, marking the damaged mitochondria for degradation [[136]47,
   [137]48]. The ubiquitin chains are recognized by p62, an autophagy
   adaptor protein. p62 connects LC3 to polyubiquitinated proteins during
   autophagosome formation, targeting them for degradation. Finally, p62
   is degraded in the autolysosome, completing mitophagy [[138]48].
   Consistent with the classical theory of mitophagy, our study showed
   increased mitophagy in the OA model, further enhanced by DPSCs
   treatments. This enhancement is evidenced by the upregulation of
   mitophagy-associated proteins, including LC3, PINK1, Parkin, and
   Beclin1, coupled with the downregulation of the mitophagy-inhibitory
   protein P62. It has been reported that IFN-γ induced autophagy of
   cervical cancer cells, further promoting macrophage phagocytosis
   [[139]49]. To demonstrate the correlation between mitophagy and
   macrophage polarization and phagocytosis, we employed Mdivi-1 to
   inhibit mitophagy and observed a decrease in the M2 polarization index
   and phagocytosis capacity of macrophages. These findings suggest that
   DPSCs promote mitophagy in macrophages, thereby modulating M2
   polarization and efferocytosis, consequently alleviating synovial
   inflammation. This study is groundbreaking as it unveils the role and
   mechanism of DPSCs in TMJ synovitis. Furthermore, it demonstrates the
   correlation between mitochondrial autophagy and synovial macrophage
   polarization and function.

   Nevertheless, the study has certain limitations. Currently,
   investigating the interplay between stem cells and macrophages has
   emerged as a prominent research area. The novel study found that
   “superactivated” macrophages release the lipid metabolite 11,12-EET
   through Gasdermin D, mediating the metabolic communication between
   macrophages and stem cells and thus promoting the repair of tissue
   damage [[140]50]. However, in our study, a detailed exploration of the
   impact of DPSC on macrophage mitophagy mechanisms was lacking. Although
   our study evidenced the participation of PINK1/Parkin in mitophagy
   initiation, the precise processes through which DPSCs trigger the
   PINK1/Parkin pathway and how mitophagy induces M2 polarization and
   macrophage efferocytosis remain ambiguous, demanding further
   investigation.

Conclusion

   In conclusion, our comprehensive investigation has elucidated the
   pivotal role of DPSCs in modulating macrophage polarization both in
   vitro and in vivo. Through intra-articular injection, DPSCs have
   demonstrated a remarkable capacity to mitigate the infiltration of
   M1-polarized macrophages while concurrently augmenting the presence of
   M2-polarized macrophages within rat synovial tissue. Additionally, the
   enhancement of macrophage efferocytosis function further underscores
   the therapeutic potential of DPSCs. Notably, our study has unraveled
   the intricate involvement of mitophagy in these processes, revealing
   that DPSCs facilitate macrophage M2 polarization and efferocytosis by
   promoting mitophagy. These findings collectively underscore the immense
   significance of DPSCs in the treatment of TMJOA. Future research should
   explore its potential mechanism and optimize its application in
   clinical practice.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [141]Supplementary Material 1^ (2.2MB, docx)

Acknowledgements