| Background and purposesPeriodontitis is an inflammatory disease caused by plaque biofilm and is characterized by the destruction of periodontal tissue.Its hazard is irreversible alveolar bone resorption,which can cause teeth to loosen or even fall out in severe cases.Therefore,the main treatment goal for patients with periodontitis is to repair tooth supporting alveolar bone defects.Mesenchymal stem cells(MSCs)have the potential for multidirectional differentiation,which can promote tissue repair and regeneration,and are an ideal source of seed cells in tissue engineering.However,its function often fails to achieve the expected biological effects due to the intervention of inflammatory factors in the inflammatory microenvironment.Thus,if we can improve the inflammatory microenvironment of stem cells and at the same time promote osteogenic differentiation,it is bound to better promote periodontal tissue regeneration.Sema3 A was originally discovered as a neuro-directing molecule.Further studies have found that Sema3 A has the unique biological characteristics of not only promoting the differentiation of osteoblasts,but also inhibiting the differentiation of osteoclasts,avoiding traditional osteogenic factors(such as BMPs,IGF.etc.)Due to the side effects caused by osteogenesis and osteoclast coupling regulation,it can more stably promote bone regeneration.Our previous study found that Sema3 A can promote the osteogenic differentiation of rBMSCs and has the potential to promote periodontal tissue regeneration,but It does not have the ability to improve the inflammatory microenvironment.Interleukin 10(IL-10)is a classic anti-inflammatory cytokine that plays a key role in preventing inflammation and autoimmune diseases.It also has an inhibitory effect on periodontal tissue inflammation.Therefore,in this study,IL-10,which has a significant inhibitory effect on periodontal inflammation,and Sema3A,which has a unique osteogenesis effect,were combined to study the effect of the combined application of IL-10/Sema3A on the osteogenic differentiation of rBMSCs on the premise of improving the microenvironment of stem cells,and further explored the potential mechanism of IL-10/Sema3A combined application on the osteogenic differentiation of rBMSCs in an inflammatory environment.Materials and methods1.Isolation,culture and identification of rBMSCsThe whole bone marrow adherence method was used to isolate and culture rBMSCs,the clone formation experiment was used to determine the clone formation ability,the surface markers of stem cells were detected by flow cytometry,and the osteogenic and adipogenic induction were used to detect the multidirectional differentiation potential.2.Establishment and detection of Sema3A and IL-10/Sema3A overexpression cell models mediated by lentiviral vectorLentiviral transfection technology was used to construct rBMSCs overexpressing Sema3A gene(LV-Sema3A),rBMSCs overexpressing IL-10/Sema3A gene(LV-IL-10/Sema3A)and control empty vector rBMSCs(LV-NC),and observe under a fluorescence microscope GFP green fluorescent protein was expressed and the expression levels of Sema3A and IL-10 genes and proteins were detected by qRT-PCR and Western Blot.CCK-8 and clone formation test were used to detect the effect of lentiviral transfection on the proliferation ability of rBMSCs.3.The effect of Sema3A and IL-10/Sema3A overexpression on the osteogenic differentiation of rBMSCs stimulated by LPSThe periodontitis cell model was constructed by E.coli LPS stimulation,and the osteogenic differentiation and mineralization ability of the LV-NC group,LV-NC+LPS group,Sema3A+LPS group and LV-IL-10/Sema3A group cells were detected by qRT-PCR,Western bolt,ALP activity detection,alkaline phosphatase staining,and alizarin red staining.4.The effect of Sema3A and IL-10/Sema3A overexpression on the expression of inflammatory factors in rBMSCs stimulated by LPSCells in LV-NC group,LV-NC+LPS group,LV-Sema3A+LPS group and LV-IL-10/Sema3A group were treated for 2h,6h,12h,and 24h after the supernatant and RNA were extracted,and then ELISA and qRT-PCR were used to detect Inflammatory factors expression.5.The combined application of IL-10/Sema3A reduces the expression of inflammatory factors in rBMSCs and promotes their osteogenic differentiation pathwaysWestern blot was used to detect whether the combined application of IL-10/Sema3A reduces the expression of inflammatory factors in rBMSCs and promotes their osteogenic differentiation through the NF-κB signaling pathway.Results1.The rBMSCs are isolated and obtained by the whole bone marrow adherence method and purified by subculture.The rBMSCs are spindle-shaped and grow radially.Wright-Giemsa staining observed that the third-generation cells were spindle-shaped and star-shaped.The cell body is hypertrophy,rich in cytoplasm,loose cytoplasm,lighter coloration,larger nucleus,darker coloration,and most cells have 2-4 nucleoli.The clone formation test showed that it has a strong proliferation ability.Flow cytometry analysis of rBMSCs surface antigens CD-44,CD-90,and CD-105 are high expression,CD-34 and CD-45 are low expression,indicating that rBMSCs have mesenchymal stem cell characteristics.Mesenchymal stem cell characteristics.Alizarin red staining and oil red O staining indicate that rBMSCs have multi-directional differentiation potential.2.According to the cell status and transfection efficiency of rBMSCs transfected with Sema3A and IL-10/Sema3A lentivirus,the optimal MOI values were determined to be 75 and 25,respectively.The fluorescence intensity of the cells transfected by the lentivirus was observed under an inverted fluorescence microscope for 72 hours,and the fluorescence intensity would not decrease after the cells were passaged.The results of qRT-PCR and Western Blot showed that the expression level of Sema3A gene and protein in LV-Sema3A group was significantly higher than that in LV-NC group(P<0.05),and the expression of IL-10 and Sema3A gene and protein in LV-IL-10/Sema3A group was significantly higher than that of the LV-NC group(P<0.05).The expression levels of Sema3A mRNA and protein in the LV-IL-10/Sema3A group were not statistically different from those in the LV-Sema3A group.CCK-8 and clone formation experiments showed that the overexpression of IL-10/Sema3A enhanced the proliferation of rBMSC.3.Seven days after osteogenic induction,the ALP activity of the LV-IL-10/Sema3 A+LPS group was significantly higher than that of the LV-Sema3 A+LPS group.The alkaline phosphatase staining showed that the LV-IL-10/Sema3A+LPS group was compared with other inflammatory stimulation groups,ALP staining was the strongest,while the LV-NC+LPS group was the weakest.21 days after osteoinduction,Alizarin Red staining showed that the LV-IL-10/Sema3A+LPS group had more mineralized nodes than the LV-Sema3A+LPS group Section formed.On the 7th and 14th days after osteogenic induction,the results of qRT-PCR and Western Blot indicated that the combined application of IL-10/Sema3A could improve the osteogenic differentiation of rBMSCs under inflammation more than Sema3 A alone.4.LPS significantly increased the expression of IL-6 and TNF-α at the gene level,and the combined application of IL-10/Sema3A inhibited the expression of inflammatory cytokines IL-6 and TNF-α stimulated by LPS at different times5.Compared with the LV-NC group,LPS significantly increased the phosphorylation of P65,IκBα and the nuclear translocation of P65,while the expression of these proteins in the LV-IL-10/Sema3A+LPS group was significantly lower than that of the LV-NC+LPS or LV-Sema3A+LPS group.ConclusionThe combined application of IL-10/Sema3A can inhibit inflammation and promote the osteogenic differentiation of rBMSCs.This study provides new ideas for periodontal regeneration treatment. |
