Mitofusins Regulates Osteogenic Differentiation Of PDLSCs In Inflammatory Microenvironment

Periodontitis is a chronic inflammatory disease characterized by progressive destruction of periodontal support tissues and is the leading cause of premature loss of teeth in adults.Periodontitis has become a major disease that endangers oral and systemic health,thereby affecting the quality of patients' lives.However,the current treatment of periodontal disease can not effectively restore the regenerative capacity of periodontal tissue,and therefore failed to fundamentally solve the important clinical problems of periodontal tissue defects.In recent years,there are more and more researches on the application of stem cells for tissue and organ repair and regeneration.There is a class of mesenchymal stem cells—periodontal ligament stem cells(PDLSCs)existing in periodontal ligament tissues.These cells may become the key cells of periodontal regeneration.Previous studies have found that PDSLCSs derived from periodontitis are characterized by decreased ability to regenerate.Therefore,it is clear that the regulatory mechanism of PDLSCs with reduced proliferative ability is the key to solve the defect of periodontitis tissue repair and regeneration.Mitochondrial fusion proteins exist in the mitochondrial outer membrane and play an important role in mitochondrial fusion and fission.It is noteworthy that the mitochondrial fusion protein also exists in the endoplasmic reticulum membrane,so that the endoplasmic reticulum and mitochondria are closely linked in physical structure.A research showed that the endoplasmic reticulum stress can decrease osteogenic differentiation of PDLSCs in the inflammatory microenvironment.What's more,mitochondria has a crucial role not only in the energy metabolism of cells,but also in the proliferation and differentiation of stem cells.Therefore,mitochondrial fusion protein as an important protein molecule that can regulate both endoplasmic reticulum and mitochondria may be involved in the regulation of the differentiation and regeneration of periodontal ligament stem cells.In addition,previous studies by the research group indicated that inflammatory cytokines can activate the wnt/?-catenin pathway and thus inhibit the osteogenic differentiation of PDLSCs.However,the specific regulatory mechanism is still unclear.In this study,we isolated and cultured normal and inflammatory PDLSCs,and used inflammatory cytokines to mimic the inflammatory microenvironment.First,it was clarified that the inflammatory microenvironment could lead to the decrease of osteogenic differentiation of PDLSCs.Then,the changes of mitochondrial fusion protein expression,mitochondrial and endoplasmic reticulum coupling degree and mitochondrial fusion were observed under electron microscope and confocal microscopy.The effect of mitochondrial fusion protein on osteogenic differentiation ability of PDLSCs was further explored.Finally,Wnt signaling pathway was detected to regulate PDLSCs osteogenetic differentiation via mitochondrial fusion protein.This research can not only provide a new target for periodontitis stem cell therapy,but also enrich the relationship between inflammatory microenvironment and stem cell activity at the level of organelle,which has a important theoretical and clinical significance.Part I The effect of inflammatory microenvironment on osteogenic differentiation of PDLSCs Objective H-PDLSCs derived from healthy tissues and P-PDLSCs derived from inflammatory tissues were isolated and cultured in vitro.Cell biological characteristics were identified and the osteogenic differentiation ability of PDLSCs from two different sources was compared.We use TNF-? to mimic inflammatory microenvironment to detect the osteogenic differentiation of PDLSCs under inflammatory microenvironment Methods(1)PDLSCs were isolated from normal and chronic inflammatory periodontal ligament tissues respectively,and cultured by limiting dilution method.(2)Detect PDLSCs mesenchymal stem cell markers with flow cytometry.(3)The second generation of H-PDLSCs were selected and cultured in the medium supplemented with 10ng/ml of TNF-a mimicking inflammatory microenvironment for 7 days,then passaged to P3,P4 and cultivated in a normal medium.(4)Induce osteogenic differentiation of PDLSCs in normal,inflammatory and mimic inflammatory microenvironments.Osteogenic related genes were detected by qRT-PCR.Alizarin red staining was applied to observe mineralized nodule formation.Results(1)PDLSCs derived from two sources were detected by flow cytometry in this experiment.The CD29 and CD90 markers of MSCs were strongly positive,and the surface markers of CD34 and CD45 derived from hematopoietic system were negative.(2)The osteogenic differentiation induced H-PDLSCs and P-PDLSCs and the ability of osteogenic differentiation was detected.The results showed that PDLSCs from both sources were able to form mineralized nodules.However,the expression of osteogenic genes and mineralized nodules in P-PDSLCs were significantly lower than those of H-PDLSCs.(3)The osteogenic genes of PDLSCs in mimic inflammatory microenvironment were lower than those of the control group,and the ability of mineralized nodules was still decreased.Conclusions(1)PDLSCs applied in this experiment are mesenchymal stem cells,and have osteogenic differentiation ability.(2)The ability of osteogenic differentiation of PDLSCs decreased under the inflammatory microenvironment in vitro and in vivo,and the descending characteristics of osteogenic differentiation were still retained after being separated from the inflammatory microenvironment for many passages.Part II Effect of Inflammatory Microenvironment on the Expression of Mitofusins PDLSCs and the related Change of Organelle Activity ObjectiveThe expression levels of Mfn1 and Mfn2 were examined and mitochondrial fusion and endoplasmic reticulum-mitochondrial coupling activities were further explored both in the normal and inflammatory microenvironment.Methods(1)The expression of Mfn1 and Mfn2 in PDLSCs of each group was detected by qRT-PCR.(2)The mitochondrial length and endoplasmic reticulum-mitochondrial coupling structure in H-PDLSCs,P-PDLSCs and H-PDLSCs+TNF-? group were observed under transmission electron microscope and quantified with Image Pro Plus.(3)Endoplasmic reticulum and mitochondria were stained with specific organelles probe respectively.The endoplasmic reticulum and mitochondria of H-PDLSCs and P-PDLSCs,H-PDLSCs and H-PDLSCs + TNF-? were observed under confocal microscope.We use Image Pro Plus to measure and quantify the degree of coupling.Results(1)Compared with H-PDLSCs group,the expression of Mfn1 and Mfn2 in P-PDSLCs group and H-PDLSCs + TNF-? group were significantly increased.(2)The results of transmission electron microscopy showed that the mitochondrial length,the degree of endoplasmic reticulum and mitochondrial coupling in P-PDSLCs group and H-PDLSCs + TNF-? group were significantly increased than those in H-PDSLCs group.(3)Compared with H-PDLSCs,the endoplasmic reticulum and mitochondria overlap in P-PDSLCs group and H-PDLSCs+TNF-? increased.The statistical results showed that the colocalization coefficients of the two kinds of organelles were significantly increased in the inflammatory microenvironment.Conclusions(1)In the inflammation microenvironment,the expression of Mfn1/2 increased.(2)Endoplasmic reticulum and mitochondrial coupling and mitochondrial fusion activity increased in the inflammatory microenvironment.Part III Mfn1/2 Regulates Osteogenic Differentiation of PDLSCs Objective To investigate the effect of Mfn1 and Mfn2 on osteogenic differentiation of PDLSCs under inflammatory microenvironment.Methods(1)The expression of Mfn1 and Mfn2 in P-PDLSCs were down-regulated by siMfn1 and siMfn2 respectively.Osteogenic related genes were detected by qRT-PCR.Mineralized nodules were quantified by alizarin red staining and cetylpyridinium chloride.(2)Mfn1 and Mfn2 expression in H-PDLSCs were overexpressed by plasmids.Osteogenic related genes were detected by qRT-PCR.Mineralized nodules were quantified by alizarin red staining and cetylpyridinium chloride.Results(1)After down-regulation of Mfn1 and Mfn2 expression in P-PDLSCs,the expression of osteogenic factor increased compared with the control group,and alizarin red staining showed that the mineralization ability was enhanced.(2)After up-regulating the expression of Mfn1 and Mfn2 in H-PDLSCs respectively,the expression of osteogenesis-related genes decreased.Alizarin red staining showed that the mineralized nodules decreased.ConclusionsIncreased expression of Mitofusins can inhibit osteogenic differentiation of PDLSCs.Part IV Role of Wnt/?-catenin pathway in the regulation of osteogenic differentiation of PDLSCs via mitochondrial fusion protein Objective To explore the effect of Wnt/?-catenin pathway on Mfn1/2,endoplasmic reticulum-mitochondrial coupling structure and mitochondrial fusion activity.To detect the role of Mfn1/2 during the process of Wnt/?-catenin pathway regulating PDLSCs osteogenic differentiation.Methods(1)LiCl is used to activate Wnt/?-catenin pathway in H-PDLSCs.After induced by osteogenic medium,the expression of osteogenic factor was detected by qRT-PCR.Alizarin red staining was used to detect the formation of mineralized nodules.(2)The expression of Mfn1 and Mfn2 in PDLSCs after LiCl stimulation was detected by qRT-PCR.(3)We use transmission electron microscopy and confocal microscopy to observe the endoplasmic reticulum-mitochondrial coupling structure and analyze the colocalization degree.(4)After activation of Wnt pathway,siMfn1 and siMfn2 were respectively used to downregulate the expression of Mfn1 and Mfn2 in PDLSCs.The osteogenic differentiation of H-PDLSCs+LiCl,H-PDLSCs+LiCl+siMfn1,H-PDLSCs+LiCl+siMfn2 groups were simultaneously induced and detected Results(1)Compared with H-PDLSCs group,the expression of osteogenic genes and the mineralized nodules in H-PDLSCs + LiCl group decreased.(2)The expression of Mfn1 and Mfn2 in PDLSCs increased after stimulation with LiCl.The results of transmission electron microscopy and confocal microscopy showed that the colocalization of endoplasmic reticulum and mitochondria decreased.(3)The down-regulation of Mfn1 and Mfn2 expression in H-PDLSCs + LiCl enhanced the osteogenic differentiation ability.Conclusions(1)The activation of Wnt/?-catenin pathway decreased the osteogenic differentiation ability in H-PDLSCs.(2)After activation of Wnt/?-catenin pathway,the expression of Mfn1/2 in PDLSCs increased,and the degree of ER-mitochondrial coupling increased.(3)The down-regulation of Mfn1/2 expression could reverse the osteogenic differentiation of PDLSCs induced by activation of Wnt pathway.