Exosomes Derived From Human Gingival Mesenchymal Stem Cells Attenuate The Inflammatory Response In Periodontal Ligament Stem Cells

ObjectivePeriodontitis is a chronic infectious disease which occurs in periodontal supporting tissue.Inflammation and destruction of periodontium are the main clinical features of periodontitis.One treatment strategy is utilizing the human periodontal ligament stem cells(PDLSCs)isolated from periodontal ligaments,which can generate cementum/PDL-like tissue.PDLSCs in the inflammatory environment show significantly attenuated osteogenic differentiation potential,hindering the regeneration of periodontal tissues.Therefore,it is necessary to diminish the inflammatory response and restore the therapeutic potential of PDLSCs.Gingival mesenchymal stem cells(GMSCs)have attracted increasing attention in cellular therapy for their excellent anti-inflammatory and immunomodulatory effects.As a novel cell-free therapy,exosomes derived from GMSCs(GMSC-Exo)not only have immuno-inflammatory and modulatory capabilities similar to those of GMSCs but also overcome numerous disadvantages of cellular therapy.In addition,accumulating evidence suggests that NF-κB signaling pathway and Wnt family member 5a(Wnt5a)are significant for the development and disease of periodontal tissues.The cross-talk between them is of great significance to the process of inflammatory response.Therefore,we speculate that GMSC-Exo might play an important role in regulating the inflammation of PDLSCs which might contribute to facilitating periodontal tissue regeneration,and GMSC-Exo might exert its effects by regulating NF-κB signaling and Wnt5 a.This study provides a novel therapeutic approach against periodontitis and other inflammatory disorders by exploring the role of GMSC-Exo on the inflammatory response of PDLSCs in inflammatory microenvironment and its potential mechanism.MethodsPDLSCs and GMSCs were isolated and cultured from human periodontal ligament and gingival connective tissue by tissue enzymatic digestion and had been identified.GMSC-Exo was isolated from the culture supernatant of GMSCs by ultracentrifugation and had been identified.To identify the concentration of Lipopolysaccharide(LPS)for the investigation,the role of LPS on PDLSCs proliferation was evaluated using the CCK-8 assay.The experimental groups were as follows: Control,GMSC-Exo(30μg/m L),LPS(10μg/m L),LPS(10μg/m L)+ GMSC-Exo(30μg/m L).CCK-8 assay was used to detect the proliferation of PDLSCs on the 4th day.The quantities of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-10(IL-10)in the supernatant of PDLSCs were compared using an enzyme-linked immunosorbent assay(ELISA).TNF-α,IL-1β,IL-10,p65,and Wnt5 a mRNA expression in PDLSCs were detected via Real-time quantitative PCR(qRT-PCR).The protein expression of p65,P-p65,and Wnt5 a in PDLSCs was detected by Western blotting(WB).ResultsBy using tissue enzymatic digestion,PDLSCs and GMSCs were effectively identified and cultivated from human periodontal ligament and gingival connective tissue.The cells had a spindle-shaped or fibroblast-like morphology with the ability to form Colony Forming Unit-Fibroblasts in vitro and the potential to differentiate into osteoblasts and adipocytes.Flow cytometry assay revealed positive expression of the surface markers CD73,CD90,and CD105 and negative expression of CD45.GMSC-Exo showed the typical cup-shaped appearance of exosomes and were enriched with exosomal positive markers ALIX,TSG101,CD9,and CD81.The majority of particles were distributed in approximately 100 nm.CCK-8 assay showed that the proliferation of PDLSCs was promoted by LPS in a concentration-dependent manner,and the promotion effect was more pronounced with the increase of LPS concentration.But the promotion effect of 100μg/m L LPS was reduced(P<0.05).LPS at 10μg/m L was selected to simulate the inflammatory microenvironment.GMSC-Exo had no significant effect on the proliferative capacity of PDLSCs cells either under normal conditions or in the inflammatory microenvironment.The expression of pro-inflammatory cytokines TNF-α and IL-1β in the supernatant of GMSC-Exo group was decreased(P<0.05)compared to the control group,while the expression of IL-10 in the supernatant and the mRNA expression of TNF-α,IL-1β and IL-10 were unchanged.Compared to the control group,the expression of TNF-α and IL-1β in supernatant was meaningfully induced in the LPS group,whereas the expression of anti-inflammatory cytokines IL-10 was significantly declined(P<0.05);TNF-α and IL-1β mRNA expression was dramatically reduced,whereas IL-10 mRNA expression was obviously lessened(P<0.05).The expression of TNF-α and IL-1β in the supernatant of LPS+GMSC-Exo group was significantly reduced than LPS group,while the expression of IL-10 was significantly improved(P<0.05);similarly,TNF-α and IL-1βwas significantly declined and the expression of IL-10 mRNA was clearly improved than LPS group(P<0.05).Compared with control group,the expression of p65 and Wnt5 a mRNA and the expression of P-p65/p65 and Wnt5 a protein in LPS group were suggestively increased(P<0.05).In contrast with LPS group,the expression of p65 and Wnt5 a in LPS+GMSC-Exo group was decreased,and the protein expression of P-p65/p65 and Wnt5 a showed similarly reduction(P<0.05).In addition,the expression of Wnt5 a mRNA and protein in GMSC-Exo group was higher than that in control group(P<0.05).ConclusionGMSC-Exo attenuates the inflammatory response of PDLSCs in the inflammatory microenvironment,which is achieved by regulating the expression of NF-κB signaling pathway and Wnt5 a.