Periodontal ligament cells-derived exosomes promote osteoclast differentiation via modulating macrophage polarization

www.nature.com/scientificreports OPEN Periodontal ligament cells‑derived exosomes promote osteoclast differentiation via modulating macrophage polarization Xinyi Bai 1,2,7, Yingxue Wang 6,7, Xinyuan Ma 2,3, Yingying Yang 5, Cong Deng 1,2, Mengling Sun 2,3, Chen Lin 2,4* & Linkun Zhang 2,4* Several studies have demonstrated that exosomes (Exos) are involved in the regulation of macrophage polarization and osteoclast differentiation. However, the characteristics as well as roles of exosomes from human periodontal ligament cells (hPDLCs-Exos) in M1/M2 macrophage polarization and osteoclast differentiation remain unclear. Here, periodontal ligament cells were successfully extracted by method of improved Type-I collagen enzyme digestion. hPDLCs-Exos were extracted by ultracentrifugation. hPDLCs-Exos were identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting (WB). Osteoclast differentiation was evaluated by real-time quantitative polymerase chain reaction (RT-qPCR), WB and tartrateresistant acid phosphatase (TRAP) staining. M1/M2 macrophage polarization were evaluated by RT-qPCR and WB. The results showed hPDLCs-Exos promoted osteoclast differentiation and M2 macrophage polarization, but inhibited M1 macrophage polarization. Moreover, M1 macrophages inhibited osteoclast differentiation, whereas M2 macrophages promoted osteoclast differentiation. It has shown that hPDLCs-Exos promoted osteoclast differentiation by inhibiting M1 and promoting M2 macrophage polarization. Orthodontic tooth movement (OTM) occurs in the periodontal steady-state microenvironment through bone remodeling1. Periodontal ligament cells (PDLCs) are the most common cells in the periodontal t issues2,3. The periodontal ligament is one of fibrous structures that connects the cementum on the tooth root surface to the alveolar bone, aiming anchoring the teeth in the alveolar s ocket4. Exosomes (Exos) are nanovesicles derived from various types of cells. With diameters ranging from 40 to 200 nm5, Exos play an important role in intercellular communication6. Exos derived from PDLCs, gingival cells and dental pulp cells can play an important role in enhancing the function of recipient cells (proliferation and differentiation, etc.)7. PDLCs, which is exposed to OTM for a long time, can perceive mechanical signals and convert them into chemical s ignals8,9. Exosomes from human periodontal ligament cells (hPDLCs-Exos) can be transported to nearby or distant cells, delivering a series of signals that affect the microenvironmental stability of bone r emodeling10. OTM is widely recognized as a good reflection of bone immunology because it involves both immune responses and bone r emodeling11. The interaction between immunity and osteoclasts is of great significance during OTM1,12. Macrophages, derived from monocytes13, are important members of the immune system14 and play crucial roles in tissue repair and inflammation suppression15. Macrophages can exhibit different functional phenotypes based on local changes in the m icroenvironment16, and are generally classified into two types: proinflammatory M1 macrophages and anti-inflammatory M2 m acrophages17. Classically activated M1 macrophages are induced by cytokines such as interferon-gamma (IFN-γ) or lipopolysaccharide (LPS), and promote inflammation in the context of innate immunity by producing inflammatory cytokines such as tumor necrosis factoralpha (TNF-α) and interleukin-6 (IL-6)18. In contrast, alternatively activated M2 macrophages are induced by interleukin-4 (IL-4) or interleukin-13 (IL-13), and produce anti-inflammatory c ytokines19. Osteoclasts, which 1 School of Medical, NanKai University, Tianjin 300071, China. 2Department of Orthodontics, Tianjin Stomatological Hospital, Tianjin 300041, China. 3School of Clinical Stomatology, Tianjin Medical University, Tianjin 300070, China. 4Tianjin Key Laboratory of Oral and Maxillofacial Function Reconstruction, 75 Dagu Road, Heping District, Tianjin 300041, China. 5Fuzhou Second Hospital, Fuzhou 350007, China. 6Tianjin Kanghui Hospital, Tianjin, Tianjin 300385, China. 7These authors contributed equally: Xinyi Bai and Yingxue Wang. *email: linchen@ tmu.edu.cn; Scientific Reports | (2024) 14:1465 | https://doi.org/10.1038/s41598-024-52073-9 1 Vol.:(0123456789) www.nature.com/scientificreports/ are bone-resorbing multinucleated cells, also orgin from the monocyte lineage20. Macrophage colony-stimulating factor (M-CSF) activation of its receptor c-Fms and receptor activator of the NF-κB ligand (RANKL) activation of receptor activator of the NF-κB (RANK) are important signaling events that prompt osteoclasts (OC) precursors proliferation and d ifferentiation21. Although macrophages and osteoclasts share the same precursor 22 cells , the potential impact of macrophages on osteoclastogenesis, particularly the differences between M1 and M2 macrophages, is mainly unknown. In recent years, Exos have been found in periodontal tissues and have been increasingly studied in relation to orthodontic tooth movement-related bone remodeling23. However, there is limited research on the macrophage polarization by hPDLCs-Exos, as well as the potential impact of different types of macrophages on osteoclast differentiation. Here, we hypothesize that hPDLCs-Exos can influence macrophage polarization, thereby participate in osteoclast differentiation. Result Uptake of hPDLCs‑Exos by RAW264.7 macrophages The isolation and identification of hPDLCs-Exos were conducted following the protocols described in previous article24. Ultracentrifugation was employed for exosome isolation, while TEM, NTA and WB were utilized for exosome characterization. Confocal microscopy showed that hPDLCs-Exos labeled by PKH-26 were taken up by RAW264.7 macrophages (Fig. 1a). The CCK-8 results demonstrated that exosomes of different concentrations exhibited the promoting effect on cell proliferation. Meanwhile the most significant promotion was observed at a concentration of 25 µg/ml. The differences were found to be statistically significant (Fig. 1b). Therefore, 25 µg/ ml exosomes were selected for induction in subsequent experiments. hPDLCs‑Exos promote osteoclast differentiation of RAW264.7 macrophages Osteoclasts were induced successfully using M-CSF and RANKL (Appendix Fig. 1). RAW264.7 macrophages treated with hPDLCs-Exos (Exos-OC group) had a significantly higher osteoclast differentiation potential than OC group, as demonstrated by TRAP staining (Fig. 2a). The RT-qPCR results showed a statistically significant increase in the expression of osteoclast marker genes Acp5, Mmp9, and Ctsk in Exos-OC group, compared with OC group (Fig. 2b). The expression of osteoclast marker proteins was significantly higher in Exos-OC group compared to OC group, as demonstrated by WB analysis. The difference was statistically significant (Fig. 2c). Figure 1.  Uptake of hPDLCs-Exos and Osteoclasts. (a) RAW (...truncated)