| Background and PurposesPeriodontitis is a chronic infectious disease characterized by periodontal support tissue destruction.Plaques,as the initiating factor,activate the host immune system.As an essential part of the immune response,the characteristics and functions of macrophages also change with the local microenvironment.In the local inflammatory microenvironment,the ratio of macrophages of M1/M2 was unbalanced.M1 macrophages release many kinds of inflammatory factors,such as IL-6 and TNF-α,which not only destroys periodontal tissue,but also affects the process of periodontal tissue regeneration.At present,the main purpose of the treatment of periodontitis is to control inflammation by eliminating or reducing plaque biofilm but will not in order to prevent the further destruction of periodontal tissue.Therefore,we hypothesis whether the local environmental supplementation of M2 macrophages can inhibit the inflammatory process and thus provide a favorable environment for the osteogenic differentiation of mesenchymal stem cells(MSCs).Human Gingival mesenchymal stem cells(GMSCs)can secret a large number of growth factors and cytokines,which have anti-inflammatory and immunomodulatory effects on inflammatory diseases to a certain extent.Compared with stem cell transplantation,the conditioned culture medium of stem cells is safer and more convenient to use.Our previous studies have shown that GMSCs-CM can promote periodontal tissue regeneration by regulating local immune factors,but its further mechanism has not yet been clarified.Growing researches document that MSCs can promote the polarization of macrophage(Mφ)to M2 and reduce the inflammatory response.The polarization effect of MSCs on macrophages may be an important part of the function of mesenchymal stem cells.Human acute mononuclear leukemia(THP-1)cells,in morphology and function,are similar to primary monocytes and macrophages,and their homogeneous genetic background can minimize the degree of phenotypic variation,which is conducive to reproducing the results of research.Therefore,In this study,THP-1 cell line was pretreated with GMSCs-CM in vitro and implanted into the periodontal tissue defect of rats with experimental periodontitis to observe the effect of THP-1 cell line on periodontal tissue regeneration.Our study may not only provide a new method for periodontal tissue regeneration but also further clarify the mechanism of conditional culture medium of gingival stem cells on promoting periodontal tissue regeneration.Materials and MethodsIn vitro experiment:1.Acquirement of GMSCs-CM:human gingival fibroblasts were obtained by tissue block culture method,then GMSCs were culture medium was concentrated by ultrafiltration centrifugation to get the concentrated condition medium(GMSCs-CM)and stored for later use.2.M0-Mφ/M1-Mφ macrophages induction:THP-1 cells were induced into M0-Mφ by 100 ng/mL Phorbol Ester(PMA),and different concentrations of LPS were added to screen out the concentration under which M0-Mφpolarized into M1-Mφ.In the experimental group,GMSCs-CM was added and cultured for 72 h.Total RNA was extracted for real-time quantitative polymerase chain reaction(qRT-PCR)to detect the mRNA expression of Arg-1,CD206,IL-10,TNF-α and IL-6 in each group.3.Neutral red phagocytosis experiment:DMEM and GMSCs-CM were used to induce M0-Mφ for 48 h,respectively.Neutral red staining solution was added,and the absorbance OD value of each group was detected by enzyme plate instrument,so as to detect the effect of GMSCs-CM on the phagocytic function of macrophages.In vivo experiment:1.Rat model of experimental periodontitis was established by local ligation combined with LPS injection.2.Establishment of periodontal defect and the following treatment:Forty male Wistar rats were randomly divided into four groups:(1)PBS group(as blank control group);(2)M0 macrophages group;(3)DMEM group:The macrophages were treated with DMEM;(4)GMSCs-CM group:The macrophages were treated with GMSCs-CM.The periodontal defect was established in the buccal side of left mandibular first molar of rats by surgically removing the alveolar bone periodontal ligament and the cementum on the root surface.Then the collagen membrane loaded with macrophages from each group was transplanted into the periodontal defect and the wound was closed tightly.3.Specimen progressing procedure:Three weeks after surgery,all animals were sacrified.The left mandibular first molars and its surrounding tissues were separated,fixed in 4%paraformaldehyde,and then decalcified.Paraffin slices were procedured to HE stain,modified Masson staining and immunohistochemical staining of TNF-α,IL-6,IL-10,CD163 and iNOS.Periodontal tissue regeneration were histologically observed and the newly formed alveolar bone were histolometrically observed and the newly formed alveolar bone were histologically analysed.Immunohistochemical stained density of TNF-α,IL-6,IL-10,CD 163 and iNOS were acquired by imaging software.All the data were statically analysed by GraphPad Prism software and difference between the groups was analysed by one-way analysis of varianceResults:In vitro experiment:1.In the absence of LPS,mRNA expressions of Arg-1 and IL-10 were significantly increased compared with blank group and DMEM group after GMSCs-CM induction of M0-Mcp,and mRNA expressions of TNF-α and IL-6 were decreased compared with DMEM group.2.After the addition of LPS,the macrophages were polarized into M1-Mφ at a concentration of 1 μg/mL.Compared with the blank group and DMEM group,after adding GMSCs-CM,the mRNA expression of IL-10 and CD206 increased significantly,and the expression of TNF-α and IL-6 decreased markedly.3.The OD value of GMSCs-CM group was significantly higher than that of control group in neutral erythrocytosis experiment(P<0.0001).In vivo experiment:1.The results of HE and Masson staining showed that at 3 weeks,the defect area was almost full of new bone in GMSCs-CM group,and the connective tissue between collagen membrane and root surface was more orderly and dense.Statistical analysis of histological measurements showed that the height of new alveolar bone in the GMSCs-CM group was significantly higher than that in the PBS group、the DMEM group and the M0 group.2.Cells with positive expression were mainly concentrated in the extracellular matrix,and distributed throughout the defect area.CD163 immunohistochemical staining results showed that the expression of CD 163 in the GMSCs-CM group was slightly higher than that of the other three groups(P<0.05),and there was no statistical difference in the other three groups.The results of iNOS immunohistochemical staining showed that iNOS in GMSCs-CM and M0 group was higher than that in DMEM and PBS group.The expression of IL-10 in M0 group was significantly greater than that of GMSCs-CM group,DMEM group and PBS group,while the expression of IL-6 and TNF-α in GMSCs-CM and M0 group was statistically lower than that of DMEM group and PBS group(P<0.05).Expression of IL-10 and IL-6 were slightly higher in the DMEM group than in the PBS group,and there was no visible difference in the expression of TNF-α between the two groups;Conclusion:The above results show that the conditioned culture medium of gingival mesenchymal stem cells may promote the polarization of M0-Mφ toward M2-Mφ,inhibit the differentiation to M1-Mφ under the action of LPS,and promote the reverse polarization of M1-Mφ into M2-Mφ.Conditioned culture medium of gingival mesenchymal stem cells could significantly enhance the phagocytic function of macrophages.M0-Mφtreated with conditioned medium from gingival mesenchymal stem cells may promote the regeneration of periodontal tissue in an inflammatory environment,by inhibiting the expression of inflammatory factors in the invasion area,while promoting the secretion of anti-inflammatory factors.Our research may provide a new treatment method for periodontal regeneration. |
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