Effects And Interference Of Different Periodontal Environmental Factors On Osteogenic Differentiation Of Periodontal Ligament Stem Cells

Background and Objectives:Periodontitis is a chronic and progressive disease that involves the destruction of periodontal support tissue,ultimately leading to tooth loss.Periodontitis can severely affect patient' s masticatory function,aesthetic appearance,and physical and mental health.On the basis of controlling infection,the ultimate aim of periodontal treatment is to reconstruct periodontal tissue.With the development and application of stem cells and tissue engineering technology,it provides more effective methods for treatment of periodontitis.Periodontal ligament stem cells(PDLSCs)are the main functional cells of periodontal regeneration and the main seed cells of periodontal tissue engineering.It is found that periodontal ligament stem cells have the ability of self-renewal and multi-differentiation,which can be differentiate into cementum,alveolar bone and periodontal tissue.Because of its low immunogenicity,stable performance after implantation,strong adaptability to environment and little damage tobody,it has become a good seed cell forperiodontal regeneration.The regeneration function of PDLSCs is influenced by different periodontal environmental factors.The periodontal environmental factors include cells,extracellular matrix,cytokines,cell surface molecules,signal molecules,intercellular interactions,inflammatory factors and other factors.First,Periodontal tissue maintains its physiological and metabolic functions derived from a variety of cell interactions,and the absorption and regeneration of alveolar bone is completed by osteoclasts and osteoblasts.PDLSCs,as seed cells,are bound to interact with osteoblasts or osteoclasts.Many scholars at home and abroad have confirmed that Osteoblasts can promote osteogenic differentiation of mesenchymal stem cells through secreted factors.Our previous study also showed that PDLSCs could promote osteogenic differentiation of osteoblasts.However,the effect of osteoblasts on theosteogenic differentiation of PDLSCs is not clear.In addition,the function of PDLSCs is likely to be affected by inflammatory factors.Currently,TNF-a is recognized as one of the most important inflammatory factors causing periodontal tissue destruction.Studies have shown that TNF-a inhibits the osteogenic differentiationof PDLSCs and MC3T3-E1 cells.Therefore,exploring the antagonistic measures against TNF-? and promoting the periodontal regeneration ability of PDLSCs in the inflammatory microenvironment have important clinical significance.PGRN is a growth factor with multiple effects,Some studies have shown PGRN can directly promote the osteogenic differentiation of mesenchymal stem cells.However,whether PGRN can reverse inhibitory effect of TNF-a on osteogenic differentiation of PDLSCs and directly promote the osteogenic differentiation of PDLSCs has not been reported.The purpose of this study was to observe the effect of osteoblasts on osteogenic differentiation of PDLSCs by indirect co-culture experiments,further clarify the effect of TNF-? on osteogenic differentiation of PDLSCs and explore the reversing effect of PGRN on TNF-?-inhibited PDLSCs osteogenic differentiation and the positive effect of PGRN on osteogenic differentiation of PDLSCs.It provides an experimental basis for understanding the mechanism of periodontal regeneration and improving the strategy of periodontal tissue engineering,and also provides new strategies and ideas for the clinical diagnosis and treatment of periodontitis.Materials and Methods1.Isolation and identification of human PDLSCs1.1 Isolation and culture of PDLSCsPDLSCs were obtained from premolars extracted for orthodontic reason or from Third molars without caries and periodontitis extracted for impacted reason.Periodontal membrane were collected from the middle third of the root surface by tissue block and enzyme digestion methods.Clonings of single cells were gotten by the limiting dilution method.Cells with higher activity were used in the following experiments.1.2 Identification of human PDLSCsPDLSCs were incubated respectively in osteogenic induction medium(a-MEM containing 5%FBS,0.1 ?M dexamethasone,10 mM ?-glycerophosphate,and 50 mg/ml ascorbate-2-phosphate).The mineralized nodules were characterized by 2%Alizarin red staining four weeks later.Cells were cultured in adipogenic induction medium(?-MEM containing 10%FBS,1 M dexamethasone,10 g/ml insulin,0.5 mM IBMX,0.2 mM indomethacin).Staining of Oil Red O was performed to identify the oil globules three weeks later.2.Effects and mechanism of MC3T3-E1 cells on osteogenic differentiation of PDLSCsIn Transwell system,PDLSCs was placed in the lower chamber,while MC3T3-E1 cells and the control cells(Human gingival fibroblasts,HGFs)were placed in the upper chamber.These cells were treated with osteogenic induction medium.Alkaline phosphatase activity(ALP)and gene/protein expression levels of ALP,runt-related transcription factor-2(Runx2)and osteopontin(OPN)were assessed.Cementum attachment protein(CAP)and cementum protein 23(CP-23)mRNA expressions were also evaluated.The formation of mineralized nodules was observed to indicate the effect of osteoblasts on osteogenic differentiation and mineralization of PDLSCs.To explore the underlying mechanism of the positive effect of osteoblasts on PDLSCs differentiation and mineralization,bone morphogenetic protein 2(BMP-2)secreted by the HGFs/MC3T3-El cells was assessed by ELISA assay.3.Effects of TNF-a on osteogenic differentiation of PDLSCsPDLSCs was treated with 10 ng/ml TNF-ca,ALP activity and gene/protein expressions levels of ALP,Runx2 and OPN were evaluated.4.Effects of PGRN on osteogenic differentiation of PDLSCs in normal and inflammatory environmentCell-counting kit-8(CCK8),ALP activity and gene expressions levels of PDLSCs simulated with different concentrations of PGRN were detected,and the optimum concentration of PGRN in promoting osteogenic differentiation was screened.Then,four groups were set up:Group A:Control group:PDLSCs were cultured alone;Group B:PDLSCs treated with 10 ng/ml TNF-a;Group C:PDLSCs treated with 25 ng/ml PGRN;Group D:PDLSCs treated with 10 ng/ml TNF-? + 25 ng/ml PGRN;ALP activity and gene/protein expressions levels of ALP,Runx2 and OPN were evaluated.Extracellular matrix calcification was measured by Alizarin Red staining to quantify calcium content three weeks later.Results1.Isolation,identification of human PDLSCsPDLSCs was successfully harvested and cultured in a good condition.The mineralized nodules were characterized by 2%Alizarin red staining four weeks later.We can clearly see the red calcium nodules of different sizes formed under the microscope.PDLSCs was cultured in adipogenic inductionmedium for three weeks,lipid droplets with visible red dye were formation.2.MC3T3-E1 cells enhance osteogenic differentiation of PDLSCsPDLSCs co-cultured with MC3T3-E1 cells exhibited a significant increase in ALP activity compared with the HGFs and control groups at day 7 and 14.With peak expression in the P1M2 group at day 7,and in the P1M1 and P1M2 groups at day 14.Secreted BMP-2 from MC3T3-E1-CM was significantly and time-dependently increased compared with that in the HGFs-CM at day 3,7 and 14(p<0.05).ALP,Runx2 and OPN mRNA expression was increased in the majority of OB co-culture groups,ALP mRNA expression in P1M1 group at day 7 and 21 or PI M2 group at day 14 changed most significantly compared to the other experiment groups(p<0.05).Runx2 expression was enhanced to a greater extent in the P1M2 group compared with all other experimental groups(p<0.05).OPN mRNA expression was greatest in the PI M2 and P2M1 groups compared with all other experimental groups(p<0.05).CAP mRNA expression was significantly increased in the PI M1 and PI M2 groups at day 7 and 14,and in all OB co-culture groups at day 21,except for the P1M4 group(p<0.05).CP-23 mRNA expression was enhanced in the majority of OB co-culture groups,with peaking at PI M2 group(p<0.05).OB co-culture groups had significantly higher mRNA expression of ALP,Runx2 and OPN than HGFs group at day 7(p<0.05).ALP protein expression peaked in the P1 M2 group at day 7 and 14,and in the P1M1 and P1M2 groups at day 21 compared with the other OB groups(p<0.05).Runx2 expression peaked in the P1M2 and P2M1 groups at day 7 and in the P1M1 group at day 21(p<0.05).OPN expression was superior in the P1 M2 group at all three time points compared with all other groups(p<0.05).PDLSCs co-cultured with MC3T3-E1 cells had significantly increased the mineralization and protein expression of ALP,Runx2 and OPN and than those co-cultured with HGFs at day 7(p<0.05).3.TNF-a inhibits osteogenic differentiation of PDLSCsAt day 7 and 14,When PDLSCs was treated with 10 ng/ml TNF-a,ALP activity and ALP,Runx2,OPN gene/protein expression was significantly lower than control group(p<0.05).4.PGRN promotes osteogenic differentiation of PDLSCs4.1 The proper concentration of PGRN promotes proliferation and osteogenesis differentiation of PDLSCsAfter cultured for 24 h,the proliferation of PDLSCs treated with 25,50 ng/ml PGRN was significantly higher than control group(p<0.05).At 48 and 72 h,the proliferation of PDLSCs treated with 5,25 ng/ml PGRN was remarkably higher than control group(p<0.05).At day 7 and 14,when compared with the control group,5,25 ng/ml PGRN was significantly increased in ALP activity(p<0.05),cell proliferation and ALP activity peaked at the concentration of 25 ng/ml(p<0.05).However,when the concentration of PGRN increased to 100 ng/ml,inhibited the proliferation and ALP activity of PDLSCs(p<0.05).ALP and Runx2 mRNA expression of PDLSCs treated with 5,25,50 ng/ml PGRN had significantly promoted than control group(p<0.05),With peak expression in 25 ng/ml PGRN at day 3 and 7.100 ng/ml PGRN inhibited ALP and Runx2 mRNA expression compared with the control group(p<0.05).4.2 PGRN enhances osteogenic differentiation of PDLSCsAt day 7 and 14,compared with the control group,10 ng/ml TNF-a group had significantly inhibited ALP activity in PDLSCs(p<0.05),but 25 ng/ml PGRN grouphad significantly promoted ALP activity in PDLSCs(p<0.05).10 ng/ml TNF-? + 25 ng/ml PGRN group had no statistical significance compared with the control group(p>0.05).At day 7,14,21,compared with the control group,ALP,Runx2 and OPN mRNA/protein expression in 10 ng/ml TNF-? group was significantly down-regulated,whereas significantly up-regulated in 25 ng/ml PGRN group(p<0.05).Runx2 mRNA expression in 10 ng/ml TNF-? + 25 ng/ml PGRN group for 7 and 14 days was remarkably higher than control group(p<0.05).Runx2 protein expression in 10 ng/ml TNF-? + 25 ng/ml PGRN group for 7 days was significantly higher than that in the control group(p<0.05).At the three time points,mRNA/protein expression levels were highest at 25 ng/ml PGRN group,followed by 10 ng/ml TNF-?+ 25 ng/ml PGRN group.After cultured for 3 w,10 ng/ml TNF-? group inhibited the mineralization of PDLSCs,while 25 ng/ml PGRN group promoted the mineralization of PDLSCs(p<0.05),10 ng/ml TNF-? + 25 ng/ml PGRN group had no statistical significance compared with the control group(p>0.05).Conclusions1.Osteogenic differentiation and mineralization of PDLSCs are positively affected by osteoblasts:The ability of osteogenic/cementoblastic differentiation and mineralization of PDLSCs was enhanced with an optimal cell ratio in the range of 2:1 to 1:1.The mechanism of osteoblasts enhance osteoblastic/cementoblastic differentiation and mineralization of PDLSCs is related to paracrine BMP-2.2.Osteogenic differentiation and mineralization of PDLSCs are negatively affected by inflammatory factors such as TNF-?:Osteogenic differentiation was inhibited in PDLSCs treated with TNF-? at the concentration of 10 ng/ml.3.PGRN can directly promote proliferation,osteogenic differentiation and mineralization of PDLSCs,and can reverse the inhibitory effect of TNF-? on osteogenic differentiation of PDLSCs;PGRN at the concentration of 5,25,50 ng/ml can promote the proliferation and differentiation of PDLSCs,and the optimal concentration is 25 ng/ml(p<0.05).