M2 macrophage-derived exosomes enable osteogenic differentiation and inhibit inflammation in human periodontal ligament stem cells through promotion of CXCL12 expression

Abstract

Background: Periodontitis is a dental disease characterized by inflammation of periodontal tissues and loss of the periodontal ligaments and alveolar bone. Exosomes are a class of extracellular vesicles that are involved in a variety of diseases by releasing active substances. In this study, we aimed to investigate the effect and mechanism of exosomes from M2 polarized macrophages (M2-exos) on osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs).

Methods: M2-exos were isolated from IL-4-induced RAW264.7 cells (M2 macrophages) and then treated on hPDLSCs. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, measurement of osteogenic differentiation-related genes and proteins, and inflammation was evaluated by measuring the levels of inflammatory factors. The mechanism of M2-exo was confirmed through qPCR, western blot, ALP and ARS staining.

Results: Results suggested that M2-exo improved osteogenic differentiation and inhibited inflammation in LPS-induced hPDLSCs. CXCL12 expression was elevated in M2 macrophages, but decreased in LPS-induced hPDLSCs. Moreover, the effect of M2-exo on osteogenic differentiation and inflammation in LPS-induced hPDLSCs was reversed by CXCL12 knockdown.

Conclusion: We demonstrated that M2-exo facilitated osteogenic differentiation and suppressed inflammation in LPS-induced hPDLSCs through promotion of CXCL12 expression. These results suggested the potential of M2-exo in the treatment of periodontitis, which may provide a new theoretical basis for M2-exo treatment of periodontitis.

Keywords: CXCL12; Exosome; Macrophage; Osteogenic differentiation; Periodontitis.

Fig. 1

Identification of M2 macrophages, M2-exo and hPDLSCs (A) Markers of M2 macrophage CD163 and CD206 was identified by flow cytometry. (B) The protein levels of Arg1 and CD206 were detected by western blot. (C) The characteristic of M2-exos was identified by TEM. (D) The size of M2-exos was measured by NTA. (E) The PKH67 labeled ADSC-exos taken up by the hPDLSCs was identified by PKH67 staining. (F) Western blot was performed to detect the exosome labeled protein of M2-exos. (G-H) ALP staining, ARS staining and Oil red O staining were performed to identify hPDLSCs

Fig. 2

M2-exo restored osteogenic differentiation in hPDLSCs inhibited by LPS. (A) Osteogenic differentiation was assessed by ALP and ARS staining. (B) ALP activity in hPDLSCs was measured using an ALP activity kit. (C) The absorbance of ARS staining was measured at 562 nm. (D-F) The expression of osteogenic differentiation-related genes ALP, OCN and Runx2 was measured by qPCR. (G) The protein levels of ALP, OCN and Runx2 were identified by western blot. (H-J) The levels of inflammatory factors IL-1β, IL-6 and TNF-α were measured by ELISA assay

Fig. 3

Expression of CXCL12 in macrophages and hPDLSCs (A-C) qPCR was performed to measure the expression of CXCL12 in macrophages and hPDLSCs

Fig. 4

M2-exo restored osteogenic differentiation in LPS-induced hPDLSCs is suppressed by CXCL12 knockdown (A) The expression of CXCL12 was measured by qPCR. (B) Osteogenic differentiation was assessed by ALP and ARS staining. (C) ALP activity in hPDLSCs was measured using an ALP activity kit. (D) The absorbance of ARS staining was measured at 562 nm. (E-G) The expression of osteogenic differentiation-related genes ALP, OCN and Runx2 was measured by qPCR. (H) The protein levels of ALP, OCN and Runx2 were identified by western blot. (I-K) The levels of inflammatory factors IL-1β, IL-6 and TNF-α were measured by ELISA assay