| Objective: Periodontitis is a kind of chronic infectious diseases characterized by gingival inflammation,periodontal pocket formation,attachment loss,and alveolar bone resorption.Periodontal ligament stem cells(PDLSCs)are recognized as the seed cells of choice in periodontal tissue engineering research due to their high tissue homology,self-proliferative viability,and multi-directional differentiation potential.However,PDLSCs in patients with periodontitis are under the inflammatory microenvironment and their regenerative capacity and multi-directional differentiation ability are disrupted.There is an urgent need for researchers to find new approaches to reduce the inflammation and restore the multi-directional differentiation ability of PDLSCs.Gingival mesenchymal stem cells(GMSCs)and gingival mesenchymal stem cells-derived exosomes(GMSC-Exo)have been found to have excellent anti-inflammatory and immunomodulatory effects and to circumvent the drawbacks associated with cellular therapies.The signaling pathways in periodontitis are intricate and complex,NF-κB is an important signaling pathway directly related to inflammation,and Wnt/β-catenin signaling pathway is closely related to the repair and regeneration of periodontal tissues.In this study,we investigated the effect of GMSC-Exo on the proliferation and osteogenic differentiation ability of PDLSCs and further revealed the regulatory role of GMSC-Exo on NF-κB and Wnt/β-catenin signaling pathways in the inflammatory microenvironment.Methods: 1.Isolation,extraction and identification of PDLSCs,GMSCs and GMSC-Exo:We have isolated and cultured PDLSCs and GMSCs by tissue block method and enzyme digestion method.By ultracentrifugation,GMSC-Exo was isolated from the culture supernatant of GMSCs and identified.GMSC-Exo was labeled with fluorescent dye PKH67 and incubated with PDLSCs for 24 h.The uptake effect of PDLSCs on GMSC-Exo was observed by laser confocal microscopy.2.Construction of the internal inflammatory microenvironment of PDLSCs: CCK-8method was used to detect the effects of Porphyromonas gingivalis-lipopolysaccharide(P.g-LPS)at different concentrations on the proliferation of PDLSCs.3.Effects of GMSC-Exo on proliferation differentiation of PDLSCs: The effect of GMSC-Exo on proliferation of PDLSCs was detected by CCK-8 method.4.Effects of GMSC-Exo on osteogenic differentiation of PDLSCs: Fluorescence quantitative polymerase chain reaction(q RT-PCR)was used to detect the effect of different concentrations of GMSC-Exo on the osteogenic differentiation ability of PDLSCs in normal environment.1 μg/m L P.g-LPS was used to construct an inflammatory microenvironment in vitro,and the regulatory effect of 10 μg/m L GMSC-Exo on the osteogenic differentiation of PDLSCs in the inflammatory microenvironment was detected.The experimental groups were as follows: Osteogenic control,P.g-LPS(1μg/m L),P.g-LPS(1 μg/m L)+ GMSC-Exo(10 μg/m L),GMSC-Exo(10 μg/m L).Osteogenic induction solution was added and incubated for 5 and 7 days,respectively.The m RNA expression levels of Runt-related transcription factor 2(Runx2),Alkaline phosphatase(ALP),and β-catenin were detected by q RT-PCR;Western Blot(WB)assay was performed to detect the protein expression levels of Runx2,ALP,nucleu-β-catenin(N-β-catenin),β-catenin,GSK-3β and phosphorylation-GSK-3β(P-GSK-3β).4.Regulatory effects of GMSC-Exo on NF-κB and Wnt/β-catenin signaling pathways in inflammatory microenvironments: Lentivirus transfection of PDLSCs was used to silence the expression of NF-κB/p65 gene in PDLSCs.Following are the experimental groups:Control,P.g-LPS(1 μg/m L),Lenti-NF-κB/p65+P.g-LPS(1 μg/m L),P.g-LPS(1 μg/m L)+GMSC-Exo(10 μg/m L),P.g-LPS(1 μg/m L)+ GMSC-Exo(10 μg/m L)+ DKK1(100nmol/L).Fluorescence microscopy was used to detect the green fluorescence expression in PDLSCs after transfection with lentivirus.The q RT-PCR assay was used to detect the expression of NF-κB/p65 gene in PDLSCs after transfection.The m RNA expression levels of NF-κB/p65,IκBα and β-catenin in PDLSCs of each experimental group were compared by q RT-PCR.Through WB assay,the protein expression levels of NF-κB/p65,phosphorylation-NF-κB/p65(P-NF-κB/p65),IκBα,phosphorylation-IκBα(P-IκBα),β-catenin,GSK-3β and P-GSK-3β within PDLSCs were examined.The nuclear proteins of PDLSCs were extracted,and the expression levels of nucleu-NF-κB/p65(N-NF-κB/p65)and N-β-catenin were detected by WB.Results: 1.Isolation,extraction and identification of PDLSCs,Gingival mesenchymal stem cells(GMSCs)and GMSC-Exo: PDLSCs and GMSCs were successfully isolated and obtained.They were all clonogenic and had the potential to differentiate into osteoblasts and adipocytes.There is little or no expression of CD45 on the surfaces of PDLSCs and GMSCs,while MSC surface markers CD73,CD90,and CD105 are highly expressed.GMSC-Exo obtained from the culture supernatant of GMSCs showed the typical cup-shaped morphology of exosomes,with a particle size around 100 nm,and positively expressed the exosome marker proteins CD9,CD63,ALIX and TSG101.Laser confocal microscopy showed that PKH67 labeled(green fluorescence)GMSC-Exo was co-cultured with PDLSCs for 24 h,and GMSC-Exo gathered around the nuclei of PDLSCs,indicating that GMSC-Exo was successfully taken up by PDLSCs.2.Construction of the internal inflammatory microenvironment of PDLSCs: The CCK-8results showed that P.g-LPS treatment was able to promote the proliferation of PDLSCs in a concentration-dependent manner,but the proliferation-promoting effect of 10 μg/m L P.g-LPS was diminished,and 1 μg/m L P.g-LPS was chosen to simulate the inflammatory microenvironment in this study.3.Effects of GMSC-Exo on proliferation differentiation of PDLSCs: The results showed that different concentrations of GMSC-Exo were able to promote the proliferative ability of PDLSCs cells.And the most effective concentration of GMSC-Exo was 10 μg/m L.4.Effects of GMSC-Exo on osteogenic differentiation of PDLSCs: The results of q RT-PCR showed that GMSC-Exo could significantly enhance the osteogenic differentiation of PDLSCs in normal environment in a concentration-dependent manner.10μg/m L was the optimal concentration for GMSC-Exo to promote the osteogenic differentiation of PDLSCs.The P.g-LPS+GMSC-Exo group exhibited significantly higher m RNA expression of Runx2,ALP,and β-catenin than the P.g-LPS group at 5 and 7 days following osteogenic induction under inflammation environment.As shown in the WB results,the Runx2,ALP,β-catenin,N-β-catenin and P-GSK-3β expressions were significantly higher in the P.g-LPS+GMSC-Exo group compared to the P.g-LPS group.But the protein expression of GSK-3β was not significantly different.5.Regulatory effects of GMSC-Exo on NF-κB and Wnt/β-catenin signaling pathways in inflammatory microenvironments: PDLSCs expressing green fluorescent protein were detected 72 hours after transfection using fluorescent microscopy.The q RT-PCR results showed that the m RNA expression of NF-κB/p65 was significantly lower in the transfected PDLSCs compared with the untransfected group.Compared with the control group,the m RNA expression of NF-κB/p65 and IκBα was significantly higher and the m RNA expression of β-catenin was decreased in the P.g-LPS group.It was observed that the P.g-LPS + GMSC-Exo group had significantly lower expression of NF-κB/p65 and IκBα and higher expression of β-catenin relative to the P.g-LPS group,whereas the Lenti-NF-κB/p65 group had significantly higher expression of β-catenin.Based on WB results,the P.g-LPS group showed an increase in NF-κB/p65,P-NF-κB/p65,N-NF-κB/p65 and P-IκBα protein expression and a decrease in β-catenin,N-β-catenin and P-GSK-3β protein expression compared with the control group.As compared to the P.g-LPS group,the protein expression of NF-κB/p65,P-NF-κB/p65,N-NF-κB/p65 and P-IκBα were significantly decreased in the P.g-LPS+GMSC-Exo group,whereasβ-catenin,N-β-catenin and P-GSK-3β protein expression were increased.In contrast with the P.g-LPS group,the protein expression of NF-κB/p65,P-NF-κB/p65,N-NF-κB/p65 and P-IκBα were significantly decreased in the P.g-LPS + GMSC-Exo group,whereas the protein expression of β-catenin,N-β-catenin and P-GSK-3β were elevated,and the protein expression of β-catenin,N-β-catenin and P-GSK-3β were most evident in the Lenti-NF-κB/p65 group.As compared with the P.g-LPS + GMSC-Exo group,the P.g-LPS+ GMSC-Exo + DKK1 group showed higher levels of m RNA expression for NF-κB/p65 and IκBα,as well as increased expressions of protein for NF-κB/p65,P-NF-κB/p65,N-NF-κB/p65 and P-IκBα.Protein expression of IκBα and GSK-3β between the groups did not differ significantly.Conclusion: GMSC-Exo enhanced the proliferation of PDLSCs in a concentration-dependent manner.In the inflammatory microenvironment,GMSC-Exo reduces the inflammatory response in PDLSCs and promotes bone differentiation by regulating Wnt/β-catenin and NF-κB signaling pathways. |
PDF Full Text Request