Mesenchymal stem cell-derived apoptotic bodies alleviate alveolar bone destruction by regulating osteoclast differentiation and function

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Mesenchymal stem cells (MSCs) are essential for the periodontal regeneration. However, the hypoxic periodontal microenvironment will induce the MSCs apoptosis. Apoptotic bodies (ABs) are the major product of apoptotic cells and are gaining increased attention as potential mediators for periodontitis treatment, thus we investigated the effects of ABs from MSCs on periodontitis. Bone marrow mesenchymal stem cells (BMMSCs) were cultured under hypoxia for 72 h to simulate the periodontal hypoxic microenvironment, after which ABs were isolated using a multi-filtration system from the supernatant of BMMSCs. Transmission electron microscopy, diameter assessment and immunofluorescence were used to characterize ABs. We found that ABs inhibited osteoclast differentiation and alveolar bone resorption, miR-223-3p is highly enriched in ABs and critical for the therapeutic effects of ABs. Targetscan and luciferase activity results confirmed that ITGB1 was targeted by miR-223-3p, which interfered the function of osteoclasts. Additionally, DC-STAMP is one of the key regulators that mediates membrane infusion. ABs and pre-osteoclasts are highly expressed DC-STAMP on the membrane, which mediated the target engulf of ABs by pre-osteoclasts. ABs with knock-down of DC-STAMP (KO-ABs) failed to be engulfed by pre-osteoclasts. Collectively, BMMSC-derived ABs can be targeted engulfed by pre-osteoclast via DC-STAMP, rescued alveolar bone loss by transferring miR-223-3p to osteoclasts, which led to the attenuation of their differentiation and bone resorption. These results suggest that MSCs derived ABs are promising therapeutic agents for the treatment of periodontitis. Health sciences/Diseases Biological sciences/Physiology/Bone mesenchymal stem cells apoptotic bodies osteoclasts miR-223-3p DC-STAMP periodontitis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Periodontitis is a common disease characterized by the destruction of supporting alveolar bone, followed by an inflammatory host response, secondary to infections by periodontal bacteria 1 – 3 .The pathogenesis of periodontitis involves the imbalance between bone formation and resorption. Osteoclasts differentiate from monocytes/macrophages, and macrophage colony stimulating factor (M-CSF) and RANKL produced by osteoblasts mediate the generation of osteoclasts. Osteoclast precursor cells express RANK (RANKL receptor), recognize RANKL expressed by osteoblasts through cell-cell interactions and differentiate into osteoclasts in the presence of M-CSF 4 – 6 . The over-activated osteoclasts are the key factor for periodontal destruction. Mesenchymal stem cells (MSCs) residing in the periodontal tissues are critical for periodontal regeneration. MSCs are undifferentiated cells with self-proliferation and multi‐linage differentiation capabilities that have promising impact for the treatment of bone destruction 7 – 9 . Studies have demonstrated that MSCs rescues the bone destruction by homing to the damaged areas, differentiating into osteoblasts and/or functioning in a paracrine manner 10 , 11 . However, due to the colonization of Gram-negative anaerobic pathogens, damage of surrounding vasculature and respiratory bursts of immune cells in the periodontal tissue, a lower‐oxygen microenvironment become a common feature for periodontitis 12 – 15 . And hypoxia has been shown to induce the MSCs apoptosis. Apoptosis is one type of programmed cell death. During apoptosis, apoptotic cells generate various types of extracellular vesicles, including exosomes, macrovesicles and apoptotic bodies (ABs), the latter being the major product of apoptotic cells. ABs range from 1 to 5 µm in diameter and can be engulfed by macrophages, dendritic cells, epithelial cells, endothelial cells and fibroblasts 16 – 20 . ABs contain cytosolic proteins, lipids and genetic factors, such as mRNAs, miRNAs and lncRNAs 21 – 25 , which are used for immune activation, recruiting apoptotic cells and tissue regeneration. After ABs are engulfed, their contents will be released and regulate downstream receptor cells for intercellular communications. Emerging studies have shown that ABs can be used as promising tools for various treatments, such as Staphylococcus aureus infections, atherosclerosis, hepatic fibrosis, diabetes, wound healing, etc 26 – 32 . However, weather MSCs derived ABs affect osteoclasts and bone destruction remain unknown. In this study, the effects of ABs secreted from MSCs on periodontitis in animal models were investigated. The results show that ABs significantly contribute to the suppression of osteoclast differentiation and the progression of osteoclastogenesis by transferring miRNAs. The mediator for targeted phagocytosis of ABs by osteoclasts was identified. Collectively, MSC-derived ABs are a potential and promising therapeutic agent for the treatment of periodontitis. Results Bone marrow mesenchymal stem cells undergo apoptosis in the periodontal hypoxic microenvironment The periodontitis animal model was established to understand the fate of BMMSCs during periodontitis. The expression level of HIF-1a was significantly induced in periodontitis group compared with control group, which indicated the hypoxia microenvironment in the periodontal tissue (Fig. 1 A-C, ***p < 0.001). Under the hypoxic condition, the number of BMMSCs in the alveolar bone was reduced in periodontitis group, consistently, the Tunel positive BMMSCs was increased (Fig. 1 A, D-F, **p < 0.01, ***p < 0.001). The results revealed that BMMSCs underwent apoptosis under hypoxic microenvironment that induced by periodontitis. ABs inhibits osteoclast differentiation and function In vitro , the BMMSCs were cultured under hypoxia. BMMSCs were cultured under hypoxia for 72 h to induce apoptosis according to the in vitro results (Figure S1 ). The isolated ABs ranged from 1 to 5 µm in diameter assessed by particle size detection and TEM, and were positive for Annexin V and TSP1 antibody staining as well (Fig. 2 A). To investigate the potential regulatory role(s) of ABs, they were co-cultured with pre-OCs. ABs can be engulfed by pre-OCs and accumulated in the cells over time (Fig. 2 B). To understand the effect of ABs on osteoclasts, ABs were co-cultured with pre-osteoclasts. Compared with the osteoclast induction group, ABs significantly inhibited the differentiation of OCs. The number of TRAP-positive cells decreased (Fig. 2 C, **p < 0.01, ***p < 0.001) and the expression level of Cathepsin K (CTSK) and Nfatc1 (Fig. 2 D, F, *p < 0.05, **p < 0.01, ***p < 0.001) was reduced in the AB group. To further examine the effects of ABs on osteoclastogenesis, we evaluated bone resorption activity using the Pit assay. The results showed that the bone resorption ability of osteoclasts was diminished in the presence of ABs (Fig. 2 F). The expression of integrin β1 was induced with osteoclast maturation, however, ABs reduced levels of integrin β1 and interfered with the osteoclastogenesis process (Fig. 2 G, *p < 0.05, **p < 0.01). ABs not only inhibited osteoclast differentiation, but also disturbed the functions of osteoclastogenesis. The application of ABs alleviates alveolar bone destruction In order to examine whether ABs could rescue alveolar bone destruction, we established periodontitis models. ABs were injected into the gingival sulcus and significantly alleviated the bone resorption measured by micro-CT (Fig. 3 A,B, *p < 0.05, **p < 0.01). Further, the differentiation of osteoclasts was highly induced, but the injection of ABs restrained that process and the number of osteoclasts was reduced compared with the periodontitis group as showed by TRAP staining and immunohistochemistry (Fig. 3 C,D, *p < 0.05, **p < 0.01). These results suggested that ABs injection can alleviate bone destruction by inhibiting osteoclasts. miR-223-3p is the key regulator in ABs during osteoclast differentiation and bone resorption Next, we sought to determine how ABs regulate osteoclasts and rescue the bone destruction in the periodontitis. We used microRNA-Seq to detect the cargo in ABs. A total of 147 miRNAs were detected in the ABs and after comparing them with miRNAs reported to be related with osteoclasts, miR-29a-3p, miR-29b-3p, miR-21-5p and miR-223-3p was identified (Fig. 4 A). To identify the key components in ABs, the expression levels of the screened miRNAs were assessed. The results confirmed that miR-223-3p had the highest expression level in ABs (Fig. 4 B, **p < 0.01, ****p < 0.0001). Coincidently, the expression of miR-223-3p was also upregulated in BMMSCs under hypoxia (Fig. 4 C, *p < 0.05). Our previous results showed that ABs are engulfed by OCs, after which miR-223-3p is transferred into osteoclasts since miR-223-3p expression was induced after the co-culture of osteoclasts with ABs (Fig. 4 D, *p < 0.05, **p < 0.01, ***p < 0.001). A miR-223-3p inhibitor was transfected into pre-OCs and confirmed the impact of miR-223-3p on osteoclastogenesis (Figure S2). To verify whether miR-223-3p contributes to the ABs-reduced inhibition of osteoclasts, we used an inhibitor approach to knock-down the expression of miR-223-3p in ABs (Fig. 4 E, **p < 0.01). miR-223-3p silenced ABs (AB-I) were co-cultured with pre-OCs but failed to attenuate osteoclast differentiation. Compared with the ABs group, there was no significant difference between the osteoclast and AB-I groups in the expression of CTSK and Nfatc1 (Fig. 4 F,G, *p < 0.05) or in the number of TRAP-positive cells (Fig. 3 H, **p < 0.01, ***p < 0.001). To further confirm that miR-223-3p is a key factor in ABs, we constructed recombinant ABs (rABs) to eliminate the effect of other miRNAs in ABs. The components in ABs were excluded by centrifugation to obtain ghost ABs (gABs). The gABs were incubated with the miR-223-3p mimic under ultrasonication after which we harvested the rABs. The expression of miR-223-3p was dramatically induced in rABs (Fig. 4 I, ****p < 0.0001) and the size of rABs was around 1 µm as assessed by TEM (Fig. 4 J). Compared to ABs, rABs inhibited the expression of CTSK, Nfatc1 and Itgb1 more efficiently (Fig. 4 K,L, *p < 0.05, **p < 0.01). TRAP staining also showed that rABs significantly reduced the differentiation of osteoclasts (Fig. 4 M,N, *p < 0.05, **p < 0.01, ****p < 0.0001). These results confirmed the effects of miR-223-3p on osteoclasts and revealed that miR-223-3p is the key regulator in ABs during osteoclast differentiation and bone resorption. miR-223-3p attenuates the osteoclastogenesis process via targeting Itgb1 In the present study, we characterized the interaction between miR-223-3p and Itgb1. Targetscan was used to predict the miR-223-3p binding region in the 3’UTR of Itgb1 and a paired target region was identified (Fig. 5 A left). A mutation of Itgb1 was constructed according to the conserved target sites. We found that miR-223-3p significantly decreased the luciferase activity of Itgb1-WT and that Itgb1-Mu rescued the suppression (Fig. 5 A right, **p < 0.01, ***p < 0.001). FITC-phalloidin staining and Pit formation assay results showed that AB-I didn’t affect the function of osteoclasts (Fig. 5 B) or the expression of integrin β1 (Fig. 5 C, *p < 0.05). In vivo , the injection of AB-I had no benefit on rescuing bone destruction (Fig. 5 D,E, **p < 0.01) or the inability to reduce the number of osteoclasts (Fig. 5 D-F, *p < 0.05, **p < 0.01, ***p < 0.001). DC-STAMP mediates the targeted phagocytosis of ABs by osteoclasts Since the infusion of ABs was able to ameliorate the alveolar bone resorption, we asked what mediated the targeted phagocytosis of ABs by pre-OCs. DC-STAMP is expressed on the cell membrane of osteoclasts and mediates the cell membrane fusion, as well as ATP6v0d2 and CD9. We found that the expression of DC-STAMP in BMMSCs was significantly upregulated by hypoxia (Fig. 6 A,B, ***p < 0.001), however, ATP6v0d2 and CD9 were suppressed under hypoxic conditions (Fig. 6 A, ****p < 0.0001). Further, we detected the expression level of DC-STAMP in pre-OCs and in ABs and confirmed that they both highly expressed DC-STAMP (Fig. 6 C). DC-STAMP was the predicted mediator in the membrane fusion of ABs and osteoclasts. Except for osteoclasts, stem cells and neutrophils also possess the ability to phagocytose ABs. We compared the DC-STAMP expression level and affinity for ABs among the cells. The expression of DC-STAMP was the highest in pre-OCs compared with stem cells and neutrophils (Fig. 6 D,E, ***p < 0.001, ****p < 0.0001). In addition, we co-cultured the same amounts of ABs with three kinds of cells. Only a few ABs were engulfed by stem cells or neutrophils but many ABs were phagocytosed by pre-OCs (Fig. 6 F). These results indicated that DC-STAMP may mediate the targeted phagocytosis of ABs by osteoclasts. To confirm the weather DC-STAMP is the key mediator, DC-STAMP silencing technology (Figure S3) and DC-STAMPKO mice were applied. BMMSCs were isolated from DC-STAMPKO mice and ABs were harvested (KO-ABs) under hypoxia. Consistent with our previous results, KO-ABs were not be engulfed by pre-OCs (Movie 1–2), and KO-ABs didn’t show any significant inhibition of CTSK, Nfatc1 and Itgb1 compared with the ABs group (Fig. 6 G,H, *p < 0.05, **p < 0.01). The number of TRAP-positive cells and the sealing zone showed no difference between the KO-ABs and osteoclast groups (Fig. 6 I-K, **p < 0.01, ***p < 0.001). Collectively, these findings imply that DC-STAMP is necessary for the targeted phagocytosis of ABs by osteoclasts. A DC-STAMP deficiency attenuates the inhibitory effect of ABs on bone destruction We demonstrated that DC-STAMP mediates the phagocytosis of ABs by osteoclasts in vitro . Next, we established periodontitis models to validate the effects of DC-STAMP. The local injection of KO-ABs showed no impact on rescuing the alveolar bone resorption compared with the periodontitis group (Fig. 7 A,B, **p < 0.01). Further, the differentiation of osteoclasts wasn’t inhibited after treated with KO-ABs (Fig. 7 C,D, **p < 0.01). Taken together, DC-STAMP mediates the targeted phagocytosis of ABs by osteoclasts; ABs transfer miR-223-3p into osteoclasts and inhibit the expression of NFIA and Itgb1, which interferes with the differentiation of osteoclasts as well as the function of osteoclastogenesis. Finally, MSC-derived ABs possess the ability to alleviate bone destruction in periodontitis (Fig. 7 E, created with Biorender.com). Discussion MSCs are critical for periodontal tissue regeneration, however, in our study we found that the hypoxic microenvironment was one of the major reasons that induced MSCs apoptosis in periodontitis. During the apoptosis process, the chromatin block (nuclear fragments) formed by the condensation and fragmentation of nuclei, and the membrane vesicles gradually divide into different sizes and form ABs 33 , 34 . ABs can be engulfed by macrophages, fibroblasts or stem cells, which is mediated by specific interactions between phagocytes and ABs 35 , 36 . ABs are the major product of apoptotic cells, which contain large amounts of regulatory molecules that are involved in a wide range of biological functions. Studies have shown that ABs possess different regulatory roles derived from different sources of cells. However, the role of ABs in bone homeostasis remain unknown. Liu et al. found that circulating ABs maintained the homeostasis of MSCs and ameliorated osteopenia 34 . In this study, MSCs-derived ABs effectively alleviated bone loss in periodontitis model. The application of ABs showed a significant recovery of bone destruction. Hence, we found that ABs suppressed the differentiation and function of osteoclasts by transferring miRNAs. There were 147 miRNAs in ABs, in which miR-21a-5p, miR-29a-3p, miR-29a-5p and miR-223-3p were identified that are related with osteoclasts 37 – 39 . miR-223-3p had the highest expression level in ABs and we confirmed that miR-223-3p is the key miRNA in osteoclast regulation. Studies have shown that NFIA and IKK-α are target genes of miR-223-3p. miR-223-3p inhibited the expression of IKK-α and resulted in the suppression of Nfatc1 through the classic and non-classic NF-kB pathways, thereby inhibiting osteoclast differentiation 40 – 42 . Besides, ruffled borders and clear zones are highly polarized cytoplasmic structures of osteoclasts, which are responsible for bone resorption. Integrin and actin form the sealing zone at the mature stage of osteoclast differentiation and the integrity of the sealing zone is crucial to the function of osteoclasts 43 – 46 . Integrins are transmembrane matrix receptors, which have αv, α2, β1 and β3 subunits. Those subunits form heterodimer receptors that are necessary for podosome-related osteoclast adhesion and absorption functions 47 – 49 . Recent studies have shown that a deficiency of integrin β1 disrupts the formation of podosomes and invadopodia on osteoclasts 50 , 51 . Consistently, we found that the bone resorption ability of osteoclasts was weakened by ABs. Luciferase activity revealed that miR-223-3p in ABs bound to the 3’UTR of Itgb1 and inhibited its expression and finally interfered with the function of osteoclasts. ABs regulated the differentiation and function of osteoclasts by transferring miR-223-3p. DC-STAMP is preferentially expressed in myeloid DCs, macrophages and OCs, however, the expression level of DC-STAMP in osteoclasts is the highest among those cells 52 – 54 . DC-STAMP is expressed on the osteoclast membrane as a dimer and plays an essential and vital role in the fusion of mononuclear osteoclasts, thus increases the absorbing activity of osteoclasts 55 . Kukita et al. found that the overexpression of DC-STAMP on L1.2 cells induced membrane interactions with osteoclast precursors 56 . Moreover, many multinucleated cells (≥ 3 nuclei per cell) are observed after transfection of the pCMV6-DC-STAMP plasmid into RAW 264.7 cells within 16 h in the absence of RANKL 57 . Those studies indicated that intercellular membrane fusion is possible only when the cells co-express DC-STAMP, and the presence of DC-STAMP may be the inducer of its ligand. From this study, we found that osteoclast precursors and ABs express high levels of DC-STAMP on their membranes and the expression of DC-STAMP was lower in other types of phagocytes (such as stem cells and neutrophils) than in pre-OCs, which led to a restricted engulfment of ABs in these cells compared with pre-OCs. To further confirm the role of DC-STAMP, we generated DC-STAMPKO mice and harvested KO-ABs from BMMSCs. Surprisingly, a dynamic phagocytosis test showed that KO-ABs can not be engulfed by pre-OCs (Movie 1–2). Accordingly, KO-AB failed to regulate the differentiation and function of osteoclasts and there was no rescue of KO-AB on the bone destruction in the periodontitis models. It appears that DC-STAMP mediates the phagocytosis of ABs by pre-OCs, and the existence of DC-STAMP on the membrane is necessary for the expression of its ligand. However, the putative ligand of DC-STAMP and the underlying interaction between DC-STAMP and its ligand still need further investigation. In summary, our study revealed that MSC derived ABs can effectively reduce bone loss in periodontitis models. ABs inhibited the differentiation and bone resorption ability of osteoclasts by transferring miR-223-3p, which suppressed the expression of NFIA and Itgb1. DC-STAMP mediated the engulfment of ABs by osteoclasts. Although this study focused on osteoclasts, it is possible that ABs may also regulate osteoblasts during bone regeneration, which will be further investigated in the future. Therefore, miR-223-3p are enriched in ABs and synergistically contribute to bone repair. ABs are new and efficient therapeutic agents for the treatment of periodontitis. Material and Methods 1. Cell culture Mouse bone marrow mesenchymal stem cells (BMMSCs) were flushed from the cavities of femurs and tibias with cell culture medium (20% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Invitrogen)) and were incubated at 37 ℃ and 5% CO 2 in a humidified environment. BMMSCs at passage 2 were used in the present study. 2. Isolation and characterization of ABs BMMSCs were incubated in an AnaeroPack system (Mitsubishi Gas Chemical Co., Inc.) for 72 h to induce apoptosis under hypoxia. The supernatant was collected and centrifuged for 10 min at 300 g to remove cell debris. The supernatant was subsequently filtered with 5 µm and 1 µm filters to collect extracellular vesicles between 1 and 5 µm in diameter. Next, the supernatant was centrifuged at 2,000 × g for 20 min to pellet the ABs. The diameters of ABs were assessed using a DelsaMax Pro. The collected ABs were stained with 1 µg FITC-Annexin V (1:100, 55567, BD Bioscience) and PE-TSP1 (1:100, sc-59886, Santa Cruz). ABs were defined as Annexin V and TSP1 positive vesicles. 3. Transmission electron microscopy ABs were fixed with 1% glutaraldehyde solution for 15 min and washed 3 times with distilled water. The fixed ABs were centrifuged at 2,000 × g for 20 min and the supernatant was removed. Next, the samples were placed on formvar-carbon-coated copper grids (Ted Pella, Inc.) for 15 min. The liquid was removed, the ABs were fixed with acetic acid dioxygen glaze for 2 min and then washed 3 times with distilled water. The grids were dried and then observed using a JEM-2100F field-emission electron microscope (JEOL Ltd.). Images were captured on a Tecnai F20 Twin TEM operated at 120 kV. 4. Animal models Five-week-old female C57BL/6J mice were purchased from Vital River and DC-STAMP knockout (DC-STAMPKO) mice were obtained from Cyagen Biosciences. All mice were housed in separate pathogen-free animal facilities with a 12:12-h light:dark cycle. DC-STAMPKO mice were used to obtain DCSTAMPKO ABs (KO-ABs). Twenty C57BL/6J mice were used for the periodontitis model and were divided into a control group (n = 5), a periodontitis group (n = 5), an ABs injection group (n = 5) and a KO-ABs injection group (n = 5). Mice were anesthetized with 1% chloral hydrate. The neck of the second molar of each mouse was ligated with 5 − 0 silk thread for 2 weeks to establish the periodontitis model. The mice were sacrificed by anesthesia followed by cervical dislocation. Alveolar bone resorption was assessed by micro-computed tomography (micro-CT, Bruker; tube voltage: 80kV, tube current: 90µA, time: 430ms). All animal experiments were performed after approval by the Institute’s Ethics Committee of Beijing Stomatological Hospital, Capital Medical University (KQYY-202206-007). 5. Immunohistochemistry and immunofluorescence staining Sections of alveolar bones were subjected to antigen unmasking with sodium citrate buffer (pH 6.0), after which they were incubated with 10% normal blocking serum for 30 min at 37℃. The primary antibodies (anti-CTSK: 1:200, ab19027, Abcam; anti-Integrin β1, ab179471, Abcam) were placed on the slides at 4°C overnight. The specimens were then incubated with secondary antibodies conjugated to peroxidase for 30 min at room temperature. Images were captured using a microscope (Olympus), and the numbers of immunostaining positive cells were calculated using ImageJ software. 6. Osteoclastogenesis detection Pre-osteoclasts (pre-OCs) were flushed and harvested from the bone cavity and cultured with 10% FBS, 2 mM L-glutamine (Invitrogen), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen) and 30 ng/ml M-CSF in a-MEM (Gibco). Pre-OCs were seeded at 1 × 10 5 cells in 12-well plates and were induced for 4 days in osteoclast differentiation medium (30 ng/ml M-CSF, 100 ng/ml RANKL and/or AB). Tartrate-resistant acid phosphate (TRAP) staining (Kamiya Biomedical Company) was used to detect osteoclast differentiation. The Pit assay (Corning) and Phalloidin staining (Abcam) were used to detect the bone resorption ability of osteoclasts. Images were captured using a microscope (Olympus) and TRAP-positive osteoclasts were counted. 7. Manipulation of ABs The ABs were subjected to Hypotonic Lysis Buffer (Chromatrap, WXM) at 4°C for 1 h, and then sonicated for 5 s (VCX 130 PB, Sonics). After centrifugation at 100 g for 10 min to remove debris, the supernatant was centrifuged at 10,000 g for 10 min to concentrate ghost ABs (gABs). The gABs were incubated with miR-223-3p mimic and sonicated for 2 min in a bath sonicator (SY25­12, Shengyuan Supersonic), then the recombinant ABs (rABs) were centrifuged at 5,000 g for 10 min. The morphology of rABs was determined by TEM (JEOL Ltd., Japan). 8. Quantitative reverse transcription-PCR (qRT-PCR) Total RNA was extracted from BMMSCs using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Two µg aliquots of RNA were synthesized using random hexamers or oligo (dT) and reverse transcriptase for real-time PCR. The reactions were performed using a QuantiTect SYBR Green PCR kit (Qiagen) and an IcycleriQ Multi-color Real-time PCR Detection System. 9. Western blot analysis Lysis Buffer was used to extract total proteins and 20 µg protein was used for each Western blot using standard protocols. The primary antibodies were listed in Table S1 . The membranes were incubated with horseradish peroxidase-conjugated anti-mouse/rabbit IgG (1:2000; 98164, Cell Signaling Technology) at room temperature for 1 h. An ECL Plus Western Blotting Detection System (GE Healthcare) was used to visualize and capture images of bound antibodies. 10. microRNA array For miRNA analysis in ABs, 1.5 mg of each sample was subjected to SurePrint G3 mouse miRNA 8 x 60 K arrays (Rel. 21.0, Agilent) and the data were analyzed using Agilent GeneSpring GX software. 11. Luciferase activity TargetScan and miRDB were used to predict binding sites between miR-223-3p and Itgb1. A mutant vector of the Itgb1 3’-UTR in the seeding sequence was constructed according to specified base-pairing rules. HEK 293T cells (1.0 × 10 4 cells/well) were treated in 96-well plates with Lipofectamine™ 6000 (Thermo Fisher Scientific) following the manufacturer's instructions for transient transfection. The cells were co-transfected with the non-target control or the miR-223-3p mimics. Reporter assays used a Dual-Glo Luciferase Reporter Assay system (E1910; Promega Corp.) at 48 h post-transfection. All experiments were performed in triplicate and means and standard deviations were calculated. 12. Statistical analysis Statistical analyses were performed using SPSS 19.0 software. Statistical significance (p-value < 0.05) was assessed by an independent two-tailed Student's t-test or analysis of variance (ANOVA). Declarations Acknowledgements This work was supported by grants from National Key R&D Program of China (Grant NO. 2022YFC2504200), the National Nature Science Foundation of China (81991504 and 81974149 to YL; 82201052 to XYL), the Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX202121 to YL), the Innovation Research Team Project of Beijing Stomatological Hospital, Capital Medical University (CXTD202202), the Beijing Municipal Administration of Hospitals’ Ascent Plan (DFL20181501 to YL), the Beijing Municipal Administration of Hospitals’ Youth Programme (QML20181501 to LJG.; QML20231505 to XYL), the Beijing Stomatological Hospital, Capital Medical University Young Scientist Program (NO. YSP202103 to XYL.),the Innovation Foundation of Beijing Stomatological Hospital, Capital Medical University (21-09-18 to LJG). Conflict of Interest The authors declared no conflict of interest. Author Contributions XYL, YYJ and XL performed the experiments; XYL and LG wrote the manuscript; XL, JFF and JD conceived the study and analyzed the data; ZHL, YYJ and JFF designed the animal experiments; JJX, UKB, YL and LG supervised interpretation of the data and critical review of the manuscript. All authors read and approved the final manuscript. Data Availability Statement The datasets used and analyzed in this study are available from the corresponding authors ( [email protected] or [email protected] ) on reasonable request. References Trindade, F. et al. Uncovering the molecular networks in periodontitis. Proteomics Clin. Appl. 8 , 748-761, doi:10.1002/prca.201400028 (2014). Kinane, D. Causation and pathogenesis of periodontal disease. Periodontol. 2000 25 , 8-20, doi:10.1034/j.1600-0757.2001.22250102.x (2001). Lourenço, T. et al. Microbial signature profiles of periodontally healthy and diseased patients. J. Clin. Periodontol. 41 , 1027-1036, doi:10.1111/jcpe.12302 (2014). 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Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 27 , 79-92, doi:10.1002/jbmr.531 (2012). Additional Declarations (Not answered) Supplementary Files Mov1AB.mp4 Movie-1 Mov2KOAB.mp4 Movie-1 Supplementaryfile.docx Supplementary file FigureS1.jpg FigureS2.jpg FigureS3.jpg Cite Share Download PDF Status: Published Journal Publication published 01 Dec, 2023 Read the published version in International Journal of Oral Science → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3231435","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":227132216,"identity":"b37d0f61-8cb9-4d89-99c7-29f4700a0309","order_by":0,"name":"Yi Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYBACPmbmhgMPDBgY2NgbGx9+IEYLGzNjw4EEkBaew83GEkRpYWBsYEgAsSTS2wR4iNLCzth4IKHgcGKf5MM2BgkGOzndBuIcdjixTTqx7UEBQ7Kx2QEStLQbSDAcSNxGvBbJg20SPKRpkWAkTUu6cRtPIjCQDYjwCz//4cMfPvyxlp3ffvzhww8VdnIEtcCAYwOYMiBSOQjYk6B2FIyCUTAKRhoAAADzQOM0J9K3AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4998-5547","institution":"Capital Medical University School of Stomatology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Liu","suffix":""},{"id":227132217,"identity":"39a3c842-9fa5-4947-909e-81105c2855ec","order_by":1,"name":"Xiaoyan Li","email":"","orcid":"","institution":"Capital Medical University School of Stomatology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Li","suffix":""},{"id":227132218,"identity":"229e7a4a-b614-43b1-822f-55fd4e8304ec","order_by":2,"name":"Yiyang Jiang","email":"","orcid":"","institution":"Capital Medical University School of Stomatology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yiyang","middleName":"","lastName":"Jiang","suffix":""},{"id":227132219,"identity":"9f18f416-4102-438f-b215-153028675402","order_by":3,"name":"Liu Xu","email":"","orcid":"","institution":"Capital Medical University School of Stomatology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liu","middleName":"","lastName":"Xu","suffix":""},{"id":227132220,"identity":"17ca79aa-6b6f-4bff-9151-ccac62f59443","order_by":4,"name":"Jingfei Fu","email":"","orcid":"","institution":"Capital Medical University School of Stomatology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingfei","middleName":"","lastName":"Fu","suffix":""},{"id":227132221,"identity":"7159311a-ff7a-4394-87f8-38dd45f231d3","order_by":5,"name":"Juan Du","email":"","orcid":"","institution":"Capital Medical University School of Stomatology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Du","suffix":""},{"id":227132222,"identity":"685d0e2e-160a-44e8-9dfd-606d402471de","order_by":6,"name":"Zhenhua Luo","email":"","orcid":"","institution":"Capital Medical University School of Stomatology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhenhua","middleName":"","lastName":"Luo","suffix":""},{"id":227132223,"identity":"d9b8fa48-33e8-410a-add6-1bc9cae74efb","order_by":7,"name":"Junji Xu","email":"","orcid":"","institution":"Capital Medical University School of Stomatology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Junji","middleName":"","lastName":"Xu","suffix":""},{"id":227132224,"identity":"5ca4b62d-f076-4964-ba31-7d3d0d370f61","order_by":8,"name":"Ujjal Bhawal","email":"","orcid":"","institution":"Nihon University School of Dentistry at Matsudo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ujjal","middleName":"","lastName":"Bhawal","suffix":""},{"id":227132225,"identity":"e6562099-611b-471d-87b2-e8bbe1fc5a0d","order_by":9,"name":"Lijia Guo","email":"","orcid":"","institution":"Capital Medical University School of Stomatology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lijia","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2023-08-03 13:02:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3231435/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3231435/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41368-023-00255-y","type":"published","date":"2023-12-01T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":42431060,"identity":"db5d0660-4009-4d31-a760-38ed6802ccea","added_by":"auto","created_at":"2023-08-31 14:34:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4035058,"visible":true,"origin":"","legend":"\u003cp\u003eBone marrow mesenchymal stem cells undergo apoptosis in the periodontal hypoxic microenvironment. (A-C) The expression level of HIF-1a was significantly induced in periodontitis group compared with control group, which indicated the hypoxia microenvironment in the periodontal tissue. (D-F) Under the hypoxic condition, the number of BMMSCs in the alveolar bone was reduced in periodontitis group, consistently, the Tunel positive BMMSCs was increased. Scale bars: 50 μm.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/d81f8b5f8b8057f49a1247a8.jpg"},{"id":42433636,"identity":"b69a8f00-a57a-4503-bdda-0ee615e0004f","added_by":"auto","created_at":"2023-08-31 14:42:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3722534,"visible":true,"origin":"","legend":"\u003cp\u003eABs inhibits osteoclast differentiation and function. \u003cstrong\u003e(A) \u003c/strong\u003eThe isolated ABs ranged from 1 to 5 μm assessed by particle size detection and TEM, and were positive for Annexin V and TSP1 antibody staining. \u003cstrong\u003e(B)\u003c/strong\u003e ABs can be engulfed by pre-OCs and accumulated in those cells over time. \u003cstrong\u003e(C)\u003c/strong\u003e Compared with the osteoclast induction group, ABs significantly inhibited the differentiation of TRAP-positive osteoclasts. \u003cstrong\u003e(D, E) \u003c/strong\u003eThe expression levels of Cathepsin K (CTSK) and Nfatc1 were reduced in the AB group. \u003cstrong\u003e(F) \u003c/strong\u003eThe sealing zone formation and bone resorption ability of osteoclasts was diminished in the presence of ABs. \u003cstrong\u003e(G)\u003c/strong\u003e ABs had reduced levels of integrin β1 and interfered with the osteoclastogenesis process. Scale bars: 20 μm. Data are reported as means ± SD (n = 3). *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/e9088e43971daf2d6807b148.jpg"},{"id":42431058,"identity":"5e768a69-c47e-4ada-98cc-e8ccaed909fe","added_by":"auto","created_at":"2023-08-31 14:34:22","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3512214,"visible":true,"origin":"","legend":"\u003cp\u003eThe application of ABs alleviates alveolar bone destruction. \u003cstrong\u003e(A, B)\u003c/strong\u003e ABs significantly alleviated the bone resorption measured by micro-CT.\u003cstrong\u003e (C, D)\u003c/strong\u003e The injection of ABs restrained the differentiation of osteoclasts as showed by immunohistochemistry and Trap staining. Scale bars: 20 μm. Data are reported as means ± SD (n = 5). *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/9ae2e0e8527a2d747b429dab.jpg"},{"id":42433634,"identity":"596d6039-7ca2-4df5-967d-45f65315b2f2","added_by":"auto","created_at":"2023-08-31 14:42:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4003497,"visible":true,"origin":"","legend":"\u003cp\u003emiR-223-3p is the key regulator in ABs during osteoclast differentiation and bone resorption.\u003cstrong\u003e (A)\u003c/strong\u003e147 miRNAs were detected in ABs, and after comparing with miRNAs reported to be related with osteoclasts, miR-29a-3p, miR-29b-3p, miR-21-5p and miR-223-3p were identified. \u003cstrong\u003e(B)\u003c/strong\u003e miR-223-3p showed the highest expression level in ABs. \u003cstrong\u003e(C)\u003c/strong\u003eThe expression of miR-223-3p was also upregulated in BMMSCs under hypoxia. \u003cstrong\u003e(D) \u003c/strong\u003emiR-223-3p expression was induced after co-culture of osteoclasts with ABs. \u003cstrong\u003e(E) \u003c/strong\u003eAn inhibitor approach was used to knock-down the expression of miR-223-3p in ABs. \u003cstrong\u003e(F-H)\u003c/strong\u003e Compared with the ABs group, there was no significant difference between the osteoclast and AB-I groups in the expression of CTSK and Nfatc1, as well as the number of TRAP-positive cells. \u003cstrong\u003e(I,J)\u003c/strong\u003e The expression of miR-223-3p was dramatically induced in rABs and the size of rABs was around 1 μm as assessed by TEM. \u003cstrong\u003e(K,L) \u003c/strong\u003eCompared to ABs, rABs inhibited the expression of CTSK, Nfatc1 and Itgb1 more efficiently. \u003cstrong\u003e(M,N) \u003c/strong\u003eTRAP staining showed that rABs significantly reduce the differentiation of osteoclasts. Scale bars: 20 μm. Data are reported as means ± SD (n = 3). *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/cabd79a8049c1cd804a55d67.jpg"},{"id":42433639,"identity":"f9be2939-7cbc-4c43-9320-b56f917f1eb4","added_by":"auto","created_at":"2023-08-31 14:42:22","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4266180,"visible":true,"origin":"","legend":"\u003cp\u003emiR-223-3p attenuates the osteoclastogenesis process via targeting Itgb1.\u003cstrong\u003e (A)\u003c/strong\u003e Left, a paired target region identified between miR-223-3p and the 3’UTR of Itgb1 as predicted by Targetscan. Right, miR-223-3p significantly decreased the luciferase activity of Itgb1-WT and Itgb1-Mu rescued the suppression. \u003cstrong\u003e(B, C) \u003c/strong\u003eFITC-phalloidin staining and Pit assays showed that AB-I didn’t affect the function of osteoclasts or the expression of integrin β1.\u003cstrong\u003e (D-F)\u003c/strong\u003e \u003cem\u003eIn vivo\u003c/em\u003e, the injection of AB-I had no effect on the rescue of bone destruction or the inability to reduce the number of osteoclasts. Scale bars: 20 μm. Data are reported as means ± SD (n = 3). *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/8b599bf17826a5b832d5fe06.jpg"},{"id":42431061,"identity":"aa835c9e-08d3-4c3c-aed3-2ae897a2d2a7","added_by":"auto","created_at":"2023-08-31 14:34:22","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3911417,"visible":true,"origin":"","legend":"\u003cp\u003eDC-STAMP mediates the targeted phagocytosis of ABs by osteoclasts. \u003cstrong\u003e(A,B)\u003c/strong\u003e The expression of DC-STAMP in BMMSCs is significantly upregulated in hypoxia while ATP6v0d2 and CD9 are suppressed under hypoxic conditions. \u003cstrong\u003e(C)\u003c/strong\u003e Pre-OCs and ABs both express high levels of DC-STAMP. \u003cstrong\u003e(D,E)\u003c/strong\u003e The expression of DC-STAMP was the highest in pre-OCs compared with BMMSCs and neutrophils. \u003cstrong\u003e(F)\u003c/strong\u003e ABs were co-cultured with these 3 cell lines, only a few ABs were engulfed by stem cells or neutrophils, but many ABs were phagocytosed by pre-OCs. \u003cstrong\u003e(G,H)\u003c/strong\u003e KO-ABs didn’t significantly inhibit CTSK, Nfatc1 or Itgb1 compared with the ABs group. \u003cstrong\u003e(I-K)\u003c/strong\u003e The number of TRAP-positive cells and the sealing zone showed no difference between the KO-ABs and osteoclast groups. Scale bars: 20 μm. Data are reported as means ± SD (n = 3). *\u003cem\u003e p\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/9e32ed81d03c8579c34b18f6.jpg"},{"id":42435137,"identity":"997c8b18-c7e9-4fd1-a2ec-f02cb9e22dad","added_by":"auto","created_at":"2023-08-31 14:50:23","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3922198,"visible":true,"origin":"","legend":"\u003cp\u003eA deficiency of DC-STAMP attenuates the inhibitory effect of ABs on bone destruction. \u003cstrong\u003e(A,B)\u003c/strong\u003e The local injection of KO-ABs showed no impact on rescuing the alveolar bone resorption compared with the periodontitis group. \u003cstrong\u003e(C,D)\u003c/strong\u003e The differentiation of osteoclasts wasn’t inhibited after treated with KO-ABs \u003cstrong\u003e(E) \u003c/strong\u003eDC-STAMP mediates the targeted phagocytosis of ABs by osteoclasts; ABs transferred miR-223-3p to osteoclasts and inhibited the expression of NFIA and Itgb1, interfering with the differentiation of osteoclasts as well as the function of osteoclastogenesis. Finally, MSC-derived ABs possess the ability to alleviate bone destruction in periodontitis. Scale bars: 20 μm. Data are reported as means ± SD (n = 5). **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/a8f95d224bd62cade453e1e2.jpg"},{"id":47477526,"identity":"77b44471-93c5-4ac0-b711-3282db5f122f","added_by":"auto","created_at":"2023-12-02 08:32:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1830627,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/c028418b-aea4-4223-9fb6-f92868c2d8be.pdf"},{"id":42435138,"identity":"d403ebe5-4b7d-4e8d-ba8c-952b3390c005","added_by":"auto","created_at":"2023-08-31 14:50:23","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1614701,"visible":true,"origin":"","legend":"Movie-1","description":"","filename":"Mov1AB.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/051036442291c921ce1e0777.mp4"},{"id":42431057,"identity":"15aa02c9-d39c-4490-85f9-e38b80fe46b8","added_by":"auto","created_at":"2023-08-31 14:34:22","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1520117,"visible":true,"origin":"","legend":"Movie-1","description":"","filename":"Mov2KOAB.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/7d8054d62367648204383ed2.mp4"},{"id":42433637,"identity":"eb645c0b-cb65-4dcf-be41-c877493c990c","added_by":"auto","created_at":"2023-08-31 14:42:22","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20714,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary file\u003c/p\u003e","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/f0ad50fd3bc6bcb210319d45.docx"},{"id":42431067,"identity":"9c6c48e0-d0b2-49e0-abd7-1a44b5cc1a4c","added_by":"auto","created_at":"2023-08-31 14:34:22","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3162977,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/4456780217963d5a9dab720a.jpg"},{"id":42431071,"identity":"8fa33710-99db-4eb6-8db7-3e519152bb77","added_by":"auto","created_at":"2023-08-31 14:34:23","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3927217,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/076052bc8efc5a631ffcc381.jpg"},{"id":42431064,"identity":"bd302c03-ae62-4ad2-8ee4-2be8816b8d4e","added_by":"auto","created_at":"2023-08-31 14:34:22","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2174984,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3231435/v1/7138084e246004af84de4c0e.jpg"}],"financialInterests":"(Not answered)","formattedTitle":"Mesenchymal stem cell-derived apoptotic bodies alleviate alveolar bone destruction by regulating osteoclast differentiation and function","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePeriodontitis is a common disease characterized by the destruction of supporting alveolar bone, followed by an inflammatory host response, secondary to infections by periodontal bacteria \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.The pathogenesis of periodontitis involves the imbalance between bone formation and resorption. Osteoclasts differentiate from monocytes/macrophages, and macrophage colony stimulating factor (M-CSF) and RANKL produced by osteoblasts mediate the generation of osteoclasts. Osteoclast precursor cells express RANK (RANKL receptor), recognize RANKL expressed by osteoblasts through cell-cell interactions and differentiate into osteoclasts in the presence of M-CSF \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The over-activated osteoclasts are the key factor for periodontal destruction.\u003c/p\u003e \u003cp\u003eMesenchymal stem cells (MSCs) residing in the periodontal tissues are critical for periodontal regeneration. MSCs are undifferentiated cells with self-proliferation and multi‐linage differentiation capabilities that have promising impact for the treatment of bone destruction \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Studies have demonstrated that MSCs rescues the bone destruction by homing to the damaged areas, differentiating into osteoblasts and/or functioning in a paracrine manner \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, due to the colonization of Gram-negative anaerobic pathogens, damage of surrounding vasculature and respiratory bursts of immune cells in the periodontal tissue, a lower‐oxygen microenvironment become a common feature for periodontitis \u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. And hypoxia has been shown to induce the MSCs apoptosis.\u003c/p\u003e \u003cp\u003eApoptosis is one type of programmed cell death. During apoptosis, apoptotic cells generate various types of extracellular vesicles, including exosomes, macrovesicles and apoptotic bodies (ABs), the latter being the major product of apoptotic cells. ABs range from 1 to 5 \u0026micro;m in diameter and can be engulfed by macrophages, dendritic cells, epithelial cells, endothelial cells and fibroblasts \u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. ABs contain cytosolic proteins, lipids and genetic factors, such as mRNAs, miRNAs and lncRNAs \u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, which are used for immune activation, recruiting apoptotic cells and tissue regeneration. After ABs are engulfed, their contents will be released and regulate downstream receptor cells for intercellular communications. Emerging studies have shown that ABs can be used as promising tools for various treatments, such as Staphylococcus aureus infections, atherosclerosis, hepatic fibrosis, diabetes, wound healing, etc \u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30 CR31\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, weather MSCs derived ABs affect osteoclasts and bone destruction remain unknown.\u003c/p\u003e \u003cp\u003eIn this study, the effects of ABs secreted from MSCs on periodontitis in animal models were investigated. The results show that ABs significantly contribute to the suppression of osteoclast differentiation and the progression of osteoclastogenesis by transferring miRNAs. The mediator for targeted phagocytosis of ABs by osteoclasts was identified. Collectively, MSC-derived ABs are a potential and promising therapeutic agent for the treatment of periodontitis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eBone marrow mesenchymal stem cells undergo apoptosis in the periodontal hypoxic microenvironment\u003c/h2\u003e\n\u003cp\u003eThe periodontitis animal model was established to understand the fate of BMMSCs during periodontitis. The expression level of HIF-1a was significantly induced in periodontitis group compared with control group, which indicated the hypoxia microenvironment in the periodontal tissue (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-C, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Under the hypoxic condition, the number of BMMSCs in the alveolar bone was reduced in periodontitis group, consistently, the Tunel positive BMMSCs was increased (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, D-F, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The results revealed that BMMSCs underwent apoptosis under hypoxic microenvironment that induced by periodontitis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eABs inhibits osteoclast differentiation and function\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e, the BMMSCs were cultured under hypoxia. BMMSCs were cultured under hypoxia for 72 h to induce apoptosis according to the \u003cem\u003ein vitro\u003c/em\u003e results (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). The isolated ABs ranged from 1 to 5 \u0026micro;m in diameter assessed by particle size detection and TEM, and were positive for Annexin V and TSP1 antibody staining as well (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). To investigate the potential regulatory role(s) of ABs, they were co-cultured with pre-OCs. ABs can be engulfed by pre-OCs and accumulated in the cells over time (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eTo understand the effect of ABs on osteoclasts, ABs were co-cultured with pre-osteoclasts. Compared with the osteoclast induction group, ABs significantly inhibited the differentiation of OCs. The number of TRAP-positive cells decreased (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and the expression level of Cathepsin K (CTSK) and Nfatc1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD, F, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was reduced in the AB group. To further examine the effects of ABs on osteoclastogenesis, we evaluated bone resorption activity using the Pit assay. The results showed that the bone resorption ability of osteoclasts was diminished in the presence of ABs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). The expression of integrin \u0026beta;1 was induced with osteoclast maturation, however, ABs reduced levels of integrin \u0026beta;1 and interfered with the osteoclastogenesis process (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). ABs not only inhibited osteoclast differentiation, but also disturbed the functions of osteoclastogenesis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eThe application of ABs alleviates alveolar bone destruction\u003c/h2\u003e\n\u003cp\u003eIn order to examine whether ABs could rescue alveolar bone destruction, we established periodontitis models. ABs were injected into the gingival sulcus and significantly alleviated the bone resorption measured by micro-CT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA,B, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Further, the differentiation of osteoclasts was highly induced, but the injection of ABs restrained that process and the number of osteoclasts was reduced compared with the periodontitis group as showed by TRAP staining and immunohistochemistry (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC,D, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results suggested that ABs injection can alleviate bone destruction by inhibiting osteoclasts.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003emiR-223-3p is the key regulator in ABs during osteoclast differentiation and bone resorption\u003c/h2\u003e\n\u003cp\u003eNext, we sought to determine how ABs regulate osteoclasts and rescue the bone destruction in the periodontitis. We used microRNA-Seq to detect the cargo in ABs. A total of 147 miRNAs were detected in the ABs and after comparing them with miRNAs reported to be related with osteoclasts, miR-29a-3p, miR-29b-3p, miR-21-5p and miR-223-3p was identified (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). To identify the key components in ABs, the expression levels of the screened miRNAs were assessed. The results confirmed that miR-223-3p had the highest expression level in ABs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Coincidently, the expression of miR-223-3p was also upregulated in BMMSCs under hypoxia (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Our previous results showed that ABs are engulfed by OCs, after which miR-223-3p is transferred into osteoclasts since miR-223-3p expression was induced after the co-culture of osteoclasts with ABs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n\u003cp\u003eA miR-223-3p inhibitor was transfected into pre-OCs and confirmed the impact of miR-223-3p on osteoclastogenesis (Figure S2). To verify whether miR-223-3p contributes to the ABs-reduced inhibition of osteoclasts, we used an inhibitor approach to knock-down the expression of miR-223-3p in ABs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). miR-223-3p silenced ABs (AB-I) were co-cultured with pre-OCs but failed to attenuate osteoclast differentiation. Compared with the ABs group, there was no significant difference between the osteoclast and AB-I groups in the expression of CTSK and Nfatc1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF,G, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) or in the number of TRAP-positive cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n\u003cp\u003eTo further confirm that miR-223-3p is a key factor in ABs, we constructed recombinant ABs (rABs) to eliminate the effect of other miRNAs in ABs. The components in ABs were excluded by centrifugation to obtain ghost ABs (gABs). The gABs were incubated with the miR-223-3p mimic under ultrasonication after which we harvested the rABs. The expression of miR-223-3p was dramatically induced in rABs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and the size of rABs was around 1 \u0026micro;m as assessed by TEM (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Compared to ABs, rABs inhibited the expression of CTSK, Nfatc1 and Itgb1 more efficiently (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eK,L, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). TRAP staining also showed that rABs significantly reduced the differentiation of osteoclasts (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eM,N, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). These results confirmed the effects of miR-223-3p on osteoclasts and revealed that miR-223-3p is the key regulator in ABs during osteoclast differentiation and bone resorption.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-223-3p attenuates the osteoclastogenesis process\u003c/strong\u003e \u003cstrong\u003evia\u003c/strong\u003e \u003cstrong\u003etargeting Itgb1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the present study, we characterized the interaction between miR-223-3p and Itgb1. Targetscan was used to predict the miR-223-3p binding region in the 3\u0026rsquo;UTR of Itgb1 and a paired target region was identified (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA left). A mutation of Itgb1 was constructed according to the conserved target sites. We found that miR-223-3p significantly decreased the luciferase activity of Itgb1-WT and that Itgb1-Mu rescued the suppression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA right, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). FITC-phalloidin staining and Pit formation assay results showed that AB-I didn\u0026rsquo;t affect the function of osteoclasts (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB) or the expression of integrin \u0026beta;1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eIn vivo\u003c/em\u003e, the injection of AB-I had no benefit on rescuing bone destruction (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD,E, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) or the inability to reduce the number of osteoclasts (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD-F, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eDC-STAMP mediates the targeted phagocytosis of ABs by osteoclasts\u003c/h2\u003e\n\u003cp\u003eSince the infusion of ABs was able to ameliorate the alveolar bone resorption, we asked what mediated the targeted phagocytosis of ABs by pre-OCs. DC-STAMP is expressed on the cell membrane of osteoclasts and mediates the cell membrane fusion, as well as ATP6v0d2 and CD9. We found that the expression of DC-STAMP in BMMSCs was significantly upregulated by hypoxia (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA,B, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), however, ATP6v0d2 and CD9 were suppressed under hypoxic conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Further, we detected the expression level of DC-STAMP in pre-OCs and in ABs and confirmed that they both highly expressed DC-STAMP (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). DC-STAMP was the predicted mediator in the membrane fusion of ABs and osteoclasts. Except for osteoclasts, stem cells and neutrophils also possess the ability to phagocytose ABs. We compared the DC-STAMP expression level and affinity for ABs among the cells. The expression of DC-STAMP was the highest in pre-OCs compared with stem cells and neutrophils (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD,E, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In addition, we co-cultured the same amounts of ABs with three kinds of cells. Only a few ABs were engulfed by stem cells or neutrophils but many ABs were phagocytosed by pre-OCs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF). These results indicated that DC-STAMP may mediate the targeted phagocytosis of ABs by osteoclasts.\u003c/p\u003e\n\u003cp\u003eTo confirm the weather DC-STAMP is the key mediator, DC-STAMP silencing technology (Figure S3) and DC-STAMPKO mice were applied. BMMSCs were isolated from DC-STAMPKO mice and ABs were harvested (KO-ABs) under hypoxia. Consistent with our previous results, KO-ABs were not be engulfed by pre-OCs (Movie 1\u0026ndash;2), and KO-ABs didn\u0026rsquo;t show any significant inhibition of CTSK, Nfatc1 and Itgb1 compared with the ABs group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG,H, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The number of TRAP-positive cells and the sealing zone showed no difference between the KO-ABs and osteoclast groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eI-K, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Collectively, these findings imply that DC-STAMP is necessary for the targeted phagocytosis of ABs by osteoclasts.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eA DC-STAMP deficiency attenuates the inhibitory effect of ABs on bone destruction\u003c/h2\u003e\n\u003cp\u003eWe demonstrated that DC-STAMP mediates the phagocytosis of ABs by osteoclasts \u003cem\u003ein vitro\u003c/em\u003e. Next, we established periodontitis models to validate the effects of DC-STAMP. The local injection of KO-ABs showed no impact on rescuing the alveolar bone resorption compared with the periodontitis group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA,B, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Further, the differentiation of osteoclasts wasn\u0026rsquo;t inhibited after treated with KO-ABs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC,D, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Taken together, DC-STAMP mediates the targeted phagocytosis of ABs by osteoclasts; ABs transfer miR-223-3p into osteoclasts and inhibit the expression of NFIA and Itgb1, which interferes with the differentiation of osteoclasts as well as the function of osteoclastogenesis. Finally, MSC-derived ABs possess the ability to alleviate bone destruction in periodontitis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE, created with Biorender.com).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMSCs are critical for periodontal tissue regeneration, however, in our study we found that the hypoxic microenvironment was one of the major reasons that induced MSCs apoptosis in periodontitis. During the apoptosis process, the chromatin block (nuclear fragments) formed by the condensation and fragmentation of nuclei, and the membrane vesicles gradually divide into different sizes and form ABs \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. ABs can be engulfed by macrophages, fibroblasts or stem cells, which is mediated by specific interactions between phagocytes and ABs \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eABs are the major product of apoptotic cells, which contain large amounts of regulatory molecules that are involved in a wide range of biological functions. Studies have shown that ABs possess different regulatory roles derived from different sources of cells. However, the role of ABs in bone homeostasis remain unknown. Liu et al. found that circulating ABs maintained the homeostasis of MSCs and ameliorated osteopenia \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, MSCs-derived ABs effectively alleviated bone loss in periodontitis model. The application of ABs showed a significant recovery of bone destruction. Hence, we found that ABs suppressed the differentiation and function of osteoclasts by transferring miRNAs. There were 147 miRNAs in ABs, in which miR-21a-5p, miR-29a-3p, miR-29a-5p and miR-223-3p were identified that are related with osteoclasts \u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. miR-223-3p had the highest expression level in ABs and we confirmed that miR-223-3p is the key miRNA in osteoclast regulation.\u003c/p\u003e \u003cp\u003eStudies have shown that NFIA and IKK-α are target genes of miR-223-3p. miR-223-3p inhibited the expression of IKK-α and resulted in the suppression of Nfatc1 through the classic and non-classic NF-kB pathways, thereby inhibiting osteoclast differentiation \u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Besides, ruffled borders and clear zones are highly polarized cytoplasmic structures of osteoclasts, which are responsible for bone resorption. Integrin and actin form the sealing zone at the mature stage of osteoclast differentiation and the integrity of the sealing zone is crucial to the function of osteoclasts \u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Integrins are transmembrane matrix receptors, which have αv, α2, β1 and β3 subunits. Those subunits form heterodimer receptors that are necessary for podosome-related osteoclast adhesion and absorption functions \u003csup\u003e\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Recent studies have shown that a deficiency of integrin β1 disrupts the formation of podosomes and invadopodia on osteoclasts \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Consistently, we found that the bone resorption ability of osteoclasts was weakened by ABs. Luciferase activity revealed that miR-223-3p in ABs bound to the 3\u0026rsquo;UTR of Itgb1 and inhibited its expression and finally interfered with the function of osteoclasts. ABs regulated the differentiation and function of osteoclasts by transferring miR-223-3p.\u003c/p\u003e \u003cp\u003eDC-STAMP is preferentially expressed in myeloid DCs, macrophages and OCs, however, the expression level of DC-STAMP in osteoclasts is the highest among those cells \u003csup\u003e\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. DC-STAMP is expressed on the osteoclast membrane as a dimer and plays an essential and vital role in the fusion of mononuclear osteoclasts, thus increases the absorbing activity of osteoclasts \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Kukita et al. found that the overexpression of DC-STAMP on L1.2 cells induced membrane interactions with osteoclast precursors \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Moreover, many multinucleated cells (\u0026ge;\u0026thinsp;3 nuclei per cell) are observed after transfection of the pCMV6-DC-STAMP plasmid into RAW 264.7 cells within 16 h in the absence of RANKL \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Those studies indicated that intercellular membrane fusion is possible only when the cells co-express DC-STAMP, and the presence of DC-STAMP may be the inducer of its ligand. From this study, we found that osteoclast precursors and ABs express high levels of DC-STAMP on their membranes and the expression of DC-STAMP was lower in other types of phagocytes (such as stem cells and neutrophils) than in pre-OCs, which led to a restricted engulfment of ABs in these cells compared with pre-OCs. To further confirm the role of DC-STAMP, we generated DC-STAMPKO mice and harvested KO-ABs from BMMSCs. Surprisingly, a dynamic phagocytosis test showed that KO-ABs can not be engulfed by pre-OCs (Movie 1\u0026ndash;2). Accordingly, KO-AB failed to regulate the differentiation and function of osteoclasts and there was no rescue of KO-AB on the bone destruction in the periodontitis models. It appears that DC-STAMP mediates the phagocytosis of ABs by pre-OCs, and the existence of DC-STAMP on the membrane is necessary for the expression of its ligand. However, the putative ligand of DC-STAMP and the underlying interaction between DC-STAMP and its ligand still need further investigation.\u003c/p\u003e \u003cp\u003eIn summary, our study revealed that MSC derived ABs can effectively reduce bone loss in periodontitis models. ABs inhibited the differentiation and bone resorption ability of osteoclasts by transferring miR-223-3p, which suppressed the expression of NFIA and Itgb1. DC-STAMP mediated the engulfment of ABs by osteoclasts. Although this study focused on osteoclasts, it is possible that ABs may also regulate osteoblasts during bone regeneration, which will be further investigated in the future. Therefore, miR-223-3p are enriched in ABs and synergistically contribute to bone repair. ABs are new and efficient therapeutic agents for the treatment of periodontitis.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e1. Cell culture\u003c/h2\u003e \u003cp\u003eMouse bone marrow mesenchymal stem cells (BMMSCs) were flushed from the cavities of femurs and tibias with cell culture medium (20% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Invitrogen)) and were incubated at 37 ℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified environment. BMMSCs at passage 2 were used in the present study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2. Isolation and characterization of ABs\u003c/h2\u003e \u003cp\u003eBMMSCs were incubated in an AnaeroPack system (Mitsubishi Gas Chemical Co., Inc.) for 72 h to induce apoptosis under hypoxia. The supernatant was collected and centrifuged for 10 min at 300 g to remove cell debris. The supernatant was subsequently filtered with 5 \u0026micro;m and 1 \u0026micro;m filters to collect extracellular vesicles between 1 and 5 \u0026micro;m in diameter. Next, the supernatant was centrifuged at 2,000 \u0026times; g for 20 min to pellet the ABs. The diameters of ABs were assessed using a DelsaMax Pro. The collected ABs were stained with 1 \u0026micro;g FITC-Annexin V (1:100, 55567, BD Bioscience) and PE-TSP1 (1:100, sc-59886, Santa Cruz). ABs were defined as Annexin V and TSP1 positive vesicles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3. Transmission electron microscopy\u003c/h2\u003e \u003cp\u003eABs were fixed with 1% glutaraldehyde solution for 15 min and washed 3 times with distilled water. The fixed ABs were centrifuged at 2,000 \u0026times; g for 20 min and the supernatant was removed. Next, the samples were placed on formvar-carbon-coated copper grids (Ted Pella, Inc.) for 15 min. The liquid was removed, the ABs were fixed with acetic acid dioxygen glaze for 2 min and then washed 3 times with distilled water. The grids were dried and then observed using a JEM-2100F field-emission electron microscope (JEOL Ltd.). Images were captured on a Tecnai F20 Twin TEM operated at 120 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4. Animal models\u003c/h2\u003e \u003cp\u003eFive-week-old female C57BL/6J mice were purchased from Vital River and DC-STAMP knockout (DC-STAMPKO) mice were obtained from Cyagen Biosciences. All mice were housed in separate pathogen-free animal facilities with a 12:12-h light:dark cycle. DC-STAMPKO mice were used to obtain DCSTAMPKO ABs (KO-ABs). Twenty C57BL/6J mice were used for the periodontitis model and were divided into a control group (n\u0026thinsp;=\u0026thinsp;5), a periodontitis group (n\u0026thinsp;=\u0026thinsp;5), an ABs injection group (n\u0026thinsp;=\u0026thinsp;5) and a KO-ABs injection group (n\u0026thinsp;=\u0026thinsp;5). Mice were anesthetized with 1% chloral hydrate. The neck of the second molar of each mouse was ligated with 5\u0026thinsp;\u0026minus;\u0026thinsp;0 silk thread for 2 weeks to establish the periodontitis model. The mice were sacrificed by anesthesia followed by cervical dislocation. Alveolar bone resorption was assessed by micro-computed tomography (micro-CT, Bruker; tube voltage: 80kV, tube current: 90\u0026micro;A, time: 430ms). All animal experiments were performed after approval by the Institute\u0026rsquo;s Ethics Committee of Beijing Stomatological Hospital, Capital Medical University (KQYY-202206-007).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e5. Immunohistochemistry and immunofluorescence staining\u003c/h2\u003e \u003cp\u003eSections of alveolar bones were subjected to antigen unmasking with sodium citrate buffer (pH 6.0), after which they were incubated with 10% normal blocking serum for 30 min at 37℃. The primary antibodies (anti-CTSK: 1:200, ab19027, Abcam; anti-Integrin β1, ab179471, Abcam) were placed on the slides at 4\u0026deg;C overnight. The specimens were then incubated with secondary antibodies conjugated to peroxidase for 30 min at room temperature. Images were captured using a microscope (Olympus), and the numbers of immunostaining positive cells were calculated using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e6. Osteoclastogenesis detection\u003c/h2\u003e \u003cp\u003ePre-osteoclasts (pre-OCs) were flushed and harvested from the bone cavity and cultured with 10% FBS, 2 mM L-glutamine (Invitrogen), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen) and 30 ng/ml M-CSF in a-MEM (Gibco). Pre-OCs were seeded at 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells in 12-well plates and were induced for 4 days in osteoclast differentiation medium (30 ng/ml M-CSF, 100 ng/ml RANKL and/or AB). Tartrate-resistant acid phosphate (TRAP) staining (Kamiya Biomedical Company) was used to detect osteoclast differentiation. The Pit assay (Corning) and Phalloidin staining (Abcam) were used to detect the bone resorption ability of osteoclasts. Images were captured using a microscope (Olympus) and TRAP-positive osteoclasts were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e7. Manipulation of ABs\u003c/h2\u003e \u003cp\u003eThe ABs were subjected to Hypotonic Lysis Buffer (Chromatrap, WXM) at 4\u0026deg;C for 1 h, and then sonicated for 5 s (VCX 130 PB, Sonics). After centrifugation at 100 g for 10 min to remove debris, the supernatant was centrifuged at 10,000 g for 10 min to concentrate ghost ABs (gABs). The gABs were incubated with miR-223-3p mimic and sonicated for 2 min in a bath sonicator (SY25\u0026shy;12, Shengyuan Supersonic), then the recombinant ABs (rABs) were centrifuged at 5,000 g for 10 min. The morphology of rABs was determined by TEM (JEOL Ltd., Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e8. Quantitative reverse transcription-PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from BMMSCs using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Two \u0026micro;g aliquots of RNA were synthesized using random hexamers or oligo (dT) and reverse transcriptase for real-time PCR. The reactions were performed using a QuantiTect SYBR Green PCR kit (Qiagen) and an IcycleriQ Multi-color Real-time PCR Detection System.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e9. Western blot analysis\u003c/h2\u003e \u003cp\u003eLysis Buffer was used to extract total proteins and 20 \u0026micro;g protein was used for each Western blot using standard protocols. The primary antibodies were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The membranes were incubated with horseradish peroxidase-conjugated anti-mouse/rabbit IgG (1:2000; 98164, Cell Signaling Technology) at room temperature for 1 h. An ECL Plus Western Blotting Detection System (GE Healthcare) was used to visualize and capture images of bound antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e10. microRNA array\u003c/h2\u003e \u003cp\u003eFor miRNA analysis in ABs, 1.5 mg of each sample was subjected to SurePrint G3 mouse miRNA 8 x 60 K arrays (Rel. 21.0, Agilent) and the data were analyzed using Agilent GeneSpring GX software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e11. Luciferase activity\u003c/h2\u003e \u003cp\u003eTargetScan and miRDB were used to predict binding sites between miR-223-3p and Itgb1. A mutant vector of the Itgb1 3\u0026rsquo;-UTR in the seeding sequence was constructed according to specified base-pairing rules. HEK 293T cells (1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were treated in 96-well plates with Lipofectamine\u0026trade; 6000 (Thermo Fisher Scientific) following the manufacturer's instructions for transient transfection. The cells were co-transfected with the non-target control or the miR-223-3p mimics. Reporter assays used a Dual-Glo Luciferase Reporter Assay system (E1910; Promega Corp.) at 48 h post-transfection. All experiments were performed in triplicate and means and standard deviations were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e12. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS 19.0 software. Statistical significance (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was assessed by an independent two-tailed Student's t-test or analysis of variance (ANOVA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from National Key R\u0026amp;D Program of China (Grant NO. 2022YFC2504200), the National Nature Science Foundation of China (81991504 and 81974149 to YL; 82201052 to XYL), the Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX202121 to YL), the Innovation Research Team Project of Beijing Stomatological Hospital, Capital Medical University (CXTD202202), the Beijing Municipal Administration of Hospitals\u0026rsquo; Ascent Plan (DFL20181501 to YL), the Beijing Municipal Administration of Hospitals\u0026rsquo; Youth Programme (QML20181501 to LJG.; QML20231505 to XYL), the Beijing Stomatological Hospital, Capital Medical University Young Scientist Program (NO. YSP202103 to XYL.),the Innovation Foundation of Beijing Stomatological Hospital, Capital Medical University (21-09-18 to LJG).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXYL, YYJ and XL performed the experiments; XYL and LG wrote the manuscript; XL, JFF and JD conceived the study and analyzed the data; ZHL, YYJ and JFF designed the animal experiments; JJX, UKB, YL and LG supervised interpretation of the data and critical review of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed in this study are available from the corresponding authors ([email protected] or [email protected]) on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTrindade, F.\u003cem\u003e et al.\u003c/em\u003e Uncovering the molecular networks in periodontitis. \u003cem\u003eProteomics Clin. Appl.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 748-761, doi:10.1002/prca.201400028 (2014).\u003c/li\u003e\n\u003cli\u003eKinane, D. 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Mesenchymal stem cells (MSCs) are essential for the periodontal regeneration. However, the hypoxic periodontal microenvironment will induce the MSCs apoptosis. Apoptotic bodies (ABs) are the major product of apoptotic cells and are gaining increased attention as potential mediators for periodontitis treatment, thus we investigated the effects of ABs from MSCs on periodontitis. Bone marrow mesenchymal stem cells (BMMSCs) were cultured under hypoxia for 72 h to simulate the periodontal hypoxic microenvironment, after which ABs were isolated using a multi-filtration system from the supernatant of BMMSCs. Transmission electron microscopy, diameter assessment and immunofluorescence were used to characterize ABs. We found that ABs inhibited osteoclast differentiation and alveolar bone resorption, miR-223-3p is highly enriched in ABs and critical for the therapeutic effects of ABs. Targetscan and luciferase activity results confirmed that ITGB1 was targeted by miR-223-3p, which interfered the function of osteoclasts. Additionally, DC-STAMP is one of the key regulators that mediates membrane infusion. ABs and pre-osteoclasts are highly expressed DC-STAMP on the membrane, which mediated the target engulf of ABs by pre-osteoclasts. ABs with knock-down of DC-STAMP (KO-ABs) failed to be engulfed by pre-osteoclasts. Collectively, BMMSC-derived ABs can be targeted engulfed by pre-osteoclast \u003cem\u003evia\u003c/em\u003e DC-STAMP, rescued alveolar bone loss by transferring miR-223-3p to osteoclasts, which led to the attenuation of their differentiation and bone resorption. These results suggest that MSCs derived ABs are promising therapeutic agents for the treatment of periodontitis.\u003c/p\u003e","manuscriptTitle":"Mesenchymal stem cell-derived apoptotic bodies alleviate alveolar bone destruction by regulating osteoclast differentiation and function","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-31 14:34:17","doi":"10.21203/rs.3.rs-3231435/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-oral-science","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ijos2","sideBox":"Learn more about [International Journal of Oral Science](http://www.nature.com/ijos/)","snPcode":"41368","submissionUrl":"https://mts-ijos.nature.com/cgi-bin/main.plex","title":"International Journal of Oral Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5a47de48-9681-47af-bdd2-be485f90be08","owner":[],"postedDate":"August 31st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":24116515,"name":"Health sciences/Diseases"},{"id":24116516,"name":"Biological sciences/Physiology/Bone"}],"tags":[],"updatedAt":"2023-12-02T08:32:10+00:00","versionOfRecord":{"articleIdentity":"rs-3231435","link":"https://doi.org/10.1038/s41368-023-00255-y","journal":{"identity":"international-journal-of-oral-science","isVorOnly":false,"title":"International Journal of Oral Science"},"publishedOn":"2023-12-01 05:00:00","publishedOnDateReadable":"December 1st, 2023"},"versionCreatedAt":"2023-08-31 14:34:17","video":"","vorDoi":"10.1038/s41368-023-00255-y","vorDoiUrl":"https://doi.org/10.1038/s41368-023-00255-y","workflowStages":[]},"version":"v1","identity":"rs-3231435","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3231435","identity":"rs-3231435","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}