| BackgroundDental pulp stem cells(DPSCs)have the potential of self-renewal and multi-directional differentiation,participating in the repair of dental pulp tissue.It is one of the ideal seed cells in regeneration tissue engineering.However,with increasing age,the regenerative potential of stem cells decreases,which may be related to the decrease in the number of stem cells,the changes of microenvironment and the decline of stem cells intrinsic function.When the dental pulp tissue is stimulated by inflammation,the DPSCs can proliferate and differentiate into odontoblasts and participate in the repair process of the pulp tissue.Studies have shown that the inflammatory microenvironment can alter the biological properties of dental pulp stem cells.Lipopolysaccharide(LPS)as the main virulence factor of the cell wall of Gram-negative bacteria,plays an important role in the pathogenesis of dental pulp infection and is currently used to simulate inflammatory microenvironment.The biological characteristics of dental pulp stem cells are effected by the age factor.We suspect that their ability to respond to inflammatory stimuli and their ability to repair are also different,and there is few relative reports at present.Therefore,this experiment intends to explore the changes of proliferation and mineralization of DPSCs isolated form different age rat in the inflammatory microenvironment stimulated by LPS.And to provide basic research for contribution of inflammatory DPSCs in tissue regeneration engineering and pulp repair.Materials and Methods1.Isolation,culture and identification of dental pulp stem cells from juvenile and adult ratsThe dental pulp stem cells were isolated and purified from the dental pulp tissue of juvenile and adult rats incisor by the method named "modified tissue block enzymatic digestion".The juvenile rat pulp stem cells were called as JDPSCs and adult rat dental pulp stem cells were called ADPSCs.Adipogenic and osteogenic differentiation Induction were used to detect multi-directional differentiation potential;flow cytometry was used to detect cell surface markers of JDPSCs and ADPSCs.2.Comparison of age-related changes in dental pulp tissue and DPSCs of juvenile and adult ratsHE staining of Dental pulp tissue and senescence-associated ?-galactosidase staining of JDPSCs and ADPSCs and tissue to compare age-related changes.CCK8 experiment was taken to compared the proliferation ability of JDPSCs and ADPSCs.Flow cytometry was adopted to detect the Cell state of JDPSCs and ADPSCs3.Effects of different concentrations of LPS on proliferation and mineralization of JDPSCs and ADPSCsThe effects of LPS on the proliferation of JDPSCs and ADPSCs were examined by CCK8 assay and flow cytometry.Under the condition of mineralization induction,the effects of LPS on osteogenesis/dentinogenesis differentiation of JDPSCs and ADPSCs were detected by alizarin red staining,alkaline phosphatase staining,qRT-PCR and Western Blot decting the expression of mineralization related genes or protein.Result1.Age-related changes of biological characteristics of rat DPSCs and dental pulp tissueIn this experiment,JDPSCs and ADPSCs were successfully isolated and purified.Both cells can differentiate into osteoblasts and adipocytes and positively expressed mesenchymal stem cell markers CD90 and CD29?and negatively expression of hematopoietic stem cell marker CD45.The ability of proliferation of ADPSCs was lower than JDPSCs(P<0.01),and the expression of senescence-associated p-galactosidase was increased((P<0.01).In addition,the vascular content and senescence-associated p-galactosidase of dental pulp tissue in Adult rats was higher than juvenile one(P<0.01).2.Effect of LPS on cell proliferation and mineralization of JDPSCs and ADPSCsCCK8 results showed LPS at the concentration of 0.01-1?g/mL,the experimental group both JDPSCs and ADPSCs had higher OD value(P<0.01)than the control group(without LPS)at the same time point from the first day to the fifth day.The change trend of JDPSCs group was obviously than that of ADPSCs group.The OD value of JDPSCs and ADPSCs were increased in the early stage(24h)stimulated with LPS at concentration of 10?g/mL or 10?g/mLat the same time point.However,With the prolongation of stimulation time,the OD value of the experimental group was lower than that of the control group.The change of JDPSCs with LPS stimulation was more obviously than that of ADPSCs.Under the condition of mineralization induction,low concentration of LPS(<1?g/mL)can promote the formation of mineralized nodules of JDPSCs and ADPSCs.The increase of JDPSCs was more obvious than that of ADPSCs.The results of PCR and Western Blot indicated that lower concentrations of LPS promoted expression of mineralization related genes or protein,and JDPSCs were promoted significantly higher than ADPSCs.Contclusion1.Dental pulp stem cells can be isolated and cultured in the pulp tissue of juvenile and adult rats.JDPSCs and ADPSCs are derived from mesenchyme with multi-directional differentiation potential.JDPSCs have higher proliferative capacity and lower p-galactosidase expression levels than ADPSCs.2.Low concentration LPS(0.1-1?g/mL)stimulation can promote the proliferation of JDPSCs and ADPSCs,and the promotion of JDPSCs is significantly higher than that of ADPSCs.Short-term stimulation of higher concentrations of LPS can promote the proliferation of JDPSCs and ADPSCs,and inhabitation of proliferation with prolonged stimulation time.3.Low concentrations of LPS can promote the expression of genes and proteins related to mineralization of JDPSCs and ADPSCs.The increase trend of juvenile group is more obvious than that of adult group.There may be a stronger repair ability for JDPSCs in the inflammatory microenvironment. |
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