Comparison Of Three Types Of Dental Tissue-derived Stem Cells And The Effects Of LPS On Human Periodontal Ligament Stem Cells

Backgrounds and objectivesMedical technologies,such as tissue engineering,often incite enthusiasm regarding their potential to treat diseases that cause soft and hard tissue damage.A key aspect of tissue engineering is identifying a type of multipotent stem cell that has excellent proliferation,self-renewal and differentiation abilities,which determines the success or failure of the regeneration process.In recent years,tissue regeneration has taken a key step forward with the isolation and identification of human dental tissue-derived mesenchymal stem cells(MSCs).To date,a variety of dental tissue-derived MSCs have been isolated and characterized,including dental pulp stem cells(DPSCs),stem cells from human exfoliated deciduous teeth(SHED),periodontal ligament stem cells(PDLSCs),dental follicle progenitor cells(DFPCs),stem cells from apical papilla(SCAP)and gingival MSCs(GMSCs).Among all these types of cells,DPSCs,PDLSCs and GMSCs are readily available and can be easily obtained from one donor,so these three types of dental tissue-derived MSCs were chosen for further exploration to systemically investigate the differences in biological performance among these cells in vitro,and thus provide theoretical support for the selection of optimal seed cells for tissue engineering and offer specific and targeted strategies for performance optimization of different dental stem cells.Furthermore,considering that bone tissue engineering is the most active field in research of tissue engineering,the type of cells with the strongest osteogenesis ability are further evaluated in inflammatory microenvironment to find out whether the osteogenic ability is affected and the potential mechanism.Lipopolysaccharide(LPS),the major component of the outer membrane of gram-negative bacteria,is a pertinent deleterious factor in the oral microenvironment.The aim of this part was to investigate the effect of LPS on the proliferation and osteogenic differentiation of PDLSCs,as well as the mechanisms involved.Materials and methods1.Primary DPSCs,PDLSCs and GMSCs were isolated and cultured by using enzyme-digested culture methods.The immunophenotypes of cells were analyzed by flow cytometry.Osteogenic,adipogenic and chondrogenic di□erentiation induction were used to detect the multiple differentiation ability of cells.Cell passage,cell cryopreservation and recovery were also operated to obtain P4 and P11 of these three types of cells.2.Cell counting kit-8(CCK-8),EdU detection kit were used to detect proliferation;β-galactosidase staining was used to detect senescence,Annexin V-APC staining was used to detect apoptosis;ALP staining,ALP activity assay,Alizarin red staining were used to detect the osteogenic differentiation;the adipogenic differentiation of three types of cells was detected by the oil red O staining.The sternness related mRNA(C-MYC,NANOG,KLF4,OCT4)and osteogenic differentiation related mRNA(ALP,RUNX2,Col I,etc.)were detected by qRT-PCR;expression of proteins related with cell cycle,senescence,apoptosis,osteogenic differentiation,adipogenic differentiation,and core elements in several signaling pathways were evaluated by Western Blot.3.To determine the role of TAZ on LPS-induced changes of osteogenesis in PDLSCs,siRNA and lentivirus packaging and transfection assay were used to knockdown the TAZ gene;Wnt/β-catenin inhibitor,DKK1,was used to explore the role of canonical Wnt signaling pathways in this environment.Results1.Primary DPSCs,PDLSCs and GMSCs from the same donor were successfully isolated and stably passaged to P4 and P11.The results of flow cytometry indicated that GMSCs,DPSCs and PDLSCs were all positive for MSC-specific surface markers(CD90,CD44,CD 105,CD73,STRO-1)but negative for haematopoietic and endothelial cell-specific markers(CD34,CD11b,CD19,CD45,HLA-DR).All three types of stem cells have multi-potentiality(osteoblast,adipocyte,chondrocyte).2.The three cells in P4 and P11 were evaluated in terms of their proliferation,senescence,apoptosis,multi-lineage differentiation,and sternness maintenance.GMSCs showed superior proliferation capability,whereas patient-matched PDLSCs exhibited excellent osteogenic and adipogenic differentiation ability;DPSCs achieved mediocre results in both aspects.In addition,GMSCs were the least susceptible to senescence,while PDLSCs were the most prone to aging.Furthermore,the biological properties of these three types of cells were all affected after long-term in vitro culture.3.LPS(0.5μg/ml)did not affect the proliferation of PDLSCs but promoted osteogenesis.The promotion of osteogenic differentiation was related to fortification of TAZ activity.The activation of TAZ was mostly mediated by the Wnt/β-catenin pathway.ConclusionsThree types of dental tissue-derived stem cells all have self-renewal and multiple differentiation potentials in vitro.PDLSCs are the best candidate cells for bone regeneration,but the application of PDLSCs might be limited to certain number of passages.Improving the differentiation of GMSCs remains the key issue regarding their application in tissue engineering.PDLSCs-governed bone tissue regeneration was not necessarily reduced under the stimulation of LPS and could be modulated by Wnt and TAZ.