Effect And Mechanism Of Transcription Factor TTF1 On Chondrogenic Differentiation Of Dental Pulp Stem Cells

Objective: The clinical incidence of articular cartilage defect is high which can be caused by trauma,tumors,and joint degeneration.Long-term unhealed articular cartilage defect can easily lead to the occurrence of osteoarthritis.The surgical methods currently used in clinical include microfracture,autologous chondrocyte transplantation and autologous or allogeneic osteochondral transplantation,which can only relieve symptoms such as pain and swelling in a short period of time.In most cases they can only form fibrous tissue or fibrocartilage with poor strength and don’t have the bearing capacity and durability of healthy articular cartilage,and they cannot achieve the ideal therapeutic effect.Dentalderived stem cells have excellent multi-lineage differentiation ability,which provide a new direction for the treatment of articular cartilage defect.Dental-derived stem cells can be obtained from extracted impacted teeth and orthodontic teeth.The acquisition process is less invasive and has low immunogenicity and immunomodulatory capability.These unique advantages make them better choices for clinical applications.At least seven types of dental-derived mesenchymal stem cells have been isolated and identified in the field of oral research,including dental pulp stem cells,periodontal ligament stem cells,gingival stem cells,dental follicle stem cells,exfoliated deciduous tooth stem cells,apical dental papilla stem cells and alveolar bone marrow mesenchymal stem cells.Dental-derived stem cells isolated from different parts have different multi-lineage differentiation abilities.At present,there is no complete research in vitro and in vivo on the chondrogenic differentiation ability of different dental-derived stem cells.To sum up,we took dental-derived stem cells as the key point of this study by in vitro experiments,subcutaneous implantation experiments in nude mice and the full-thickness cartilage defect model of the knee joint in rats to investigate the cartilage regeneration potential of dental pulp stem cells(DPSCs),periodontal ligament stem cells(PDLSCs)and alveolar bone marrow mesenchymal stem cells(ABMMSCs).The regulation of transcription factor TTF1 and its interaction transcription factor GLIS1 on the chondrogenic differentiation of DPSCs were explored through in vitro and in vivo experiments.This study provided a theoretical basis for the treatment of articular cartilage defect.Methods:1.DPSCs,PDLSCs and ABMMSCs were isolated and cultured from the same individual(n=5),and identified by flow cytometry and three-lineage differentiation experiments.The chondrogenic differentiation ability of them was compared in vitro by q RT-PCR,western blot and immunohistochemical staining.The cartilage repair ability in vivo of the three types of MSCs was further compared through the subcutaneous implantation experiment in nude mice and the repair experiment of full-thickness cartilage defect of rat knee joint.2.We identified the protein expression profile of DPSCs,PDLSCs and ABMMSCs,and the proteomic results were counted and analyzed.Bioinformatics analysis was performed on the differential proteins of the three groups of D＿PL,D＿A,and PL＿A,and the differential proteins related to cartilage differentiation were screened out.Then we predicted transcription factors that bind to them.Through data analysis,TTF1 and GLIS1 were selected as target transcription factors.3.TTF1 and GLIS1 were transfected into DPSCs by lentiviral vector,and DPSCs overexpressing or knocking down TTF1 and GLIS1 were successfully constructed.After that,the functional recovery experiment of chondrogenic differentiation was performed in vitro,and we detected the changes in protein and function level by western blot and immunohistochemistry.Co-immunoprecipitation and immunofluorescence co-localization were used to detect whether TTF1 and GLIS1 were combined with each other.The effect of TTF1 and its interaction gene GLIS1 on the effect of repairability cartilage defect of DPSCs were explored by establishing a full-thickness cartilage defect model of rat knee joint.Results:1.DPSCs,PDLSCs and ABMMSCs all highly expressed mesenchymal stem cell surface markers CD29,CD44,CD73,CD90,CD105 and CD146,and low expressed hematopoietic stem cell markers CD24,CD34 and CD45.After three weeks of osteogenic induction,mineralized nodules were seen by Alizarin Red staining.After three weeks of adipogenic induction,red round lipid droplets were seen by Oil Red O staining.After two weeks of chondrogenic induction,acid mucopolysaccharides were seen by Alcian Blue staining,and the expression levels of SOX9,COL2A1 and ACAN in DPSCs at the m RNA and protein levels were higher than those in PDLSCs and ABMMSCs(P<0.05).2.Nude mice subcutaneous ectopic chondrogenesis experiments showed that none of the three types of MSCs formed tumor-like tissue.The tissues formed by DPSCs were smooth and translucent,similar to the color of hyaline cartilage.The tissues formed by PDLSCs were chalky and opaque.The tissues formed by ABMMSCs were partly chalky and translucent,and mixed with yellow-brown bone-like structure.HE staining and immunofluorescence staining further confirmed that DPSCs could form a large amount of type II collagen and a small amount of type I collagen.PDLSCs could only form a small amount of type II collagen,mainly type I collagen which was more similar to fibrous connective tissue.The chondrogenic ability of ABMMSCs was in the middle,and it was more inclined to osteogenic differentiation.The results of HE,safranin O-fast green and immunohistochemical staining in the cartilage defect area of the rat knee joint showed that compared with the pure cartilage defect group and the pure scaffold material group,type II collagen and ACAN in the defect repair area of the DPSCs group increased,while type I collagen was relatively less,and was the closest to the sham-operated group.The PDLSCs group was dominated by type I collagen.The ABMMSCs group had increased type I collagen,type II collagen,and ACAN,among which the proportion of type I collagen was higher,and HE staining showed typical bone-like structures.3.Based on TMT mass spectrometry,a total of 4223 proteins were identified in DPSCs,PDLSCs and ABMMSCs.GO enrichment analysis was performed on the three groups of D＿PL,D＿A,and PL＿A,and the GO function analysis of the top 30 differentially expressed proteins whose differences were greater than 1.5 times and P<0.05 found that these proteins were mainly closely related to cellular components,cell binding,catalytic activation,and biological regulation.We further screened out the differential proteins related to cartilage differentiation and predicted the related transcription factors TTF1 and GLIS1.4.TTF1 or GLIS1 were transfected into DPSCs by constructing a lentiviral vector.The in vitro results showed that overexpression of TTF1 could promote the chondrogenic differentiation of DPSCs,knockdown of TTF1 could inhibit the chondrogenic differentiation of DPSCs.Overexpression of GLIS1 could inhibit the chondrogenic differentiation of DPSCs,knockdown of GLIS1 could promote the chondrogenic differentiation of DPSCs.In vivo experiments we found that DPSCs overexpressing TTF1 or knocking down GLIS1 could repair full-thickness cartilage defect in rat knee joints.The newly repaired area of the defect contained a large amount of type II collagen,and the cells were arranged in an orderly manner.The results of co-immunoprecipitation and immunofluorescence co-localization showed that TTF1 interacted with GLIS1,and GLIS1 decreased after overexpression of TTF1,and overexpression of TTF1 could activate the Smad5 signaling pathway.The results suggested that overexpression of TTF1 may promote chondrogenic differentiation of DPSCs by inhibiting the expression of the gene GLIS1 and then activated the Smad5 signaling pathway to promote the synthesis of cartilage matrix.Conclusions:1.Allogeneic dental-derived mesenchymal stem cell transplantation could promote cartilage repair of articular cartilage defect.Among them,DPSCs had the strongest cartilage repair ability,and the formed cartilage was the closest to normal knee hyaline cartilage.PDLSCs mostly formed fibrous connective tissue.ABMMSCs had the middle chondrogenic ability and were more inclined to osteogenic differentiation.2.A total of 4223 proteins were detected by TMT mass spectrometry on the three types of stem cells.A total of eight differential proteins related to cartilage differentiation were screened by the differences are greater than 1.5 times and P<0.05,and the transcription factors TTF1 and GLIS1 could bind to six of them.3.Overexpression of TTF1 could promote the chondrogenic differentiation of DPSCs,knockdown of TTF1 could inhibit the chondrogenic differentiation of DPSCs.Overexpression of GLIS1 could inhibit the chondrogenic differentiation of DPSCs,and knockdown of GLIS1 could promote the chondrogenic differentiation of DPSCs.Overexpression of TTF1 promoted chondrogenic differentiation of DPSCs possibly by inhibiting the expression of GLIS1 and activating the Smad5 signaling pathway to promote the synthesis of cartilage matrix.