Mechanism Of Noggin Interfering With The Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells

Periodontitis is a chronic inflammatory disease involving the periodontal support tissues.It is the chief culprit in the loosening of adult teeth.At present,the conventional treatments for periodontitis,e.g.scaling and root planning(SRP),periodontal surgery,and photodynamic therapy,can block the progression of periodontal inflammation,but the recovery effect is still unrecognizable for the defective periodontal supporting tissues,especially the defective alveolar bone.Periodontal ligament stem cells(PDLSCs)are mesenchymal stem cells isolated from the periodontal ligament.They have strong self-proliferation and multi-directional differentiation ability,and become a hopeful seed cell for tissue regeneration engineering.Therefore,it is very important to study the mechanism and application of osteogenic differentiation ability of PDLSCs.In the previous experiment,the differently expressed gene NOG was screened out of PDLSCs under the condition of simulated high altitude periodontitis.At present,the research on Noggin mostly involves the nervous system.Besides,Noggin can specifically inhibit the BMP(bone morphogenetic protein,BMP)signaling pathway,and plays an important role in the formation of bone,cartilage and joints.In addition,Noggin also plays an important role in tooth morphology and periodontal tissue alteration.Therefore,this experiment is to investigate the effect of Noggin on the osteogenic differentiation of human PDLSCs and its related molecular mechanisms,and provide a theoretical basis for the application of human PDLSCs in tissue engineering to repair the missing alveolar bone in periodontitis.Methods:1.Isolation,culture and identification of PDLSCs: periodontal ligament cells were isolated from human periodontal ligament by tissue block method combined enzymatic digestion method,the ability of clonal formation was detected by limiting dilution method,the multi-directional differentiation ability was detected by osteogenic,adipogenic,and chondrogenic differentiation,and the mesenchymal stem cell surface markers were detected by flow cytometry.2.Teeth from patients with periodontitis and teeth extracted by orthodontic treatment from patients without periodontal problems were removed and collected.The quantitative polymerase chain reaction(qPCR)and Western blot were used to detect the expression of Noggin mRNA and protein in the periodontal tissues of periodontitis,and the expressions of osteogenic related genes,e.g.alkaline phosphatase(ALP),collagen type 1(COL1),osteocalcin(OCN),and runt-related transcription factor 2(RUNX2),in the periodontal tissues of periodontitis,were also detected by qPCR.3.Western blot and cellular immunofluorescence and other methods were used to detect the expression of Noggin protein in human PDLSCs.And the expression of Noggin protein was also detected when human PDLSCs were treated by lipopolysaccharide from Gram-negative Porphyromonas gingivalis(LPS-PG)to simulate the inflammatory conditions in periodontitis.4.Human PDLSCs were induced for the osteogenic differentiation when treated by exogenous recombinant Noggin protein.The mineralization of human PDLSCs,gene and protein expression of osteogenic related factors ALP,COL1,OCN and RUNX2,were examined.5.siRNA NOG was transfected into human PDLSCs,and the expression of BMP2 was detected by Western blot.Human PDLSCs were then treated by the exogenous recombinant Noggin protein,and Western blot was used to detect the expression of RUNX2.Results:1.Human periodontal ligament cells isolated from the periodontal ligament can form cell clones and have the ability to differentiate into bone,fat,and cartilage in vitro.They positively express surface markers of mesenchymal stem cells,such as CD29 and CD44,and negatively express the surface of hematopoietic stem cells,such as CD34 and CD45.2.The expression of osteogenic related genes ALP,COL1,OCN and RUNX2 in periodontal tissues of periodontitis is decreased,and the expression of Noggin mRNA and protein is significantly increased.3.Noggin protein is expressed in human PDLSCs,and the expression of Noggin protein is increased after human PDLSCs were stimulated by LPS-PG to simulate the inflammatory conditions of periodontitis.4.Exogenous recombinant Noggin protein can interfere with the osteogenic differentiation of human PDLSCs,resulting in decreased mineralized nodules,and decreased expressions of the genes and proteins of osteogenesis-related factors ALP,COL1,OCN and RUNX2.5.Western blot confirmed that BMP2 expression was decreased after siRNA NOG was transfected into human PDLSCs.After human PDLSCs were stimulated by exogenous recombinant Noggin protein,Western blot confirmed that the expression of RUNX2 was decreased.Conclusions:1.This study first found that Noggin gene and protein expression in inflammatory periodontal tissues was higher than that in healthy periodontal tissues,suggesting that Noggin may be related to the course of periodontitis.2.In this study,Noggin was found to reduce mRNA and protein expression of osteogenic factors ALP,COL1,OCN and RUNX2 during the osteogenic differentiation of PDLSCs,as well as the formation of calcification nodules.3.The results of this experiment suggest that Noggin may contribute to the stable expression of BMPs,and that Noggin may interfere with the osteogenic differentiation of human PDLSCs by interfering with the signaling of the BMP signaling pathway and inhibiting the expression of RUNX2.