Effect Of Compressive Force On The Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells

Objective:To investigate the effect of compressive force on the osteogenic differentiation of human periodontal ligament stem cells,in order to provide experimental evidence for further research on hPDLSCs signal network under mechanical stimulation.Methods:1.hPDLCs were cultured by the method of tissue adhering,and the limiting dilution method was adopted to separate hPDLSCs.2.The characterizations of hPDLSCs were identified by morphological observation,cloning formation experiment,cell phenotype identification,cell cycle analysis,immunofluorescence staining,osteogenic induction experiment and fat induction experiment.3.The mRNA and protein expression levels of ALP,RUNX2 and OCN were investigated by RT-qPCR and Western blot after loading different magnitude of continuous static compressive force(1,2,3g/cm~#)for different time(12,24,72h),respectively.Result:1.hPDLSCs were successfully cultured in vitro.The cells at 4 to 6passages with stable shape were employed for the next experiment.2.The acquired cells had clonality and low proliferation.Most of the cells were in phase G_%/G_&(93.87%)and there were high expression of CD90(98.26%)and CD146(61.02%),low expression of CD34(0.02%).Immunofluorescence staining showed that the cells were positive for vimentin protein and negative for keratin.The cells could be induce to differentiate into osteoblast and steatoblast in vitro.The acquired cells showed the characteristics of stem cells.3.1g/cm~#compressive force could up-regulate the mRNA expression of ALP and RUNX2after loading for 12h,then the mRNA expression of ALP,RUNX2 and OCN decreased gradually over time,and began to lower than the control group at 24h.The protein expression of RUNX2 were increased at 12h,then the protein expression of ALP,RUNX2 and OCN decreased gradually over time.4.When2g/cm~#compressive force was applied to hPDLSCs,the mRNA expression of ALP were increased in 24h,and then less than the control group.The mRNA expression of RUNX2 and OCN were lower than the control group after loading for 24h.The expression trend of the three protein were similar to the mRNA.5.When 3g/cm~#compressive force was applied to hPDLSCs,only the expression of ALP mRNA was increased at 12h,and the mRNA expression levels of the other groups were decreased over time.The expression trend of the three protein were similar to the mRNA.Conclusions:hPDLSCs were successfully cultured in vitro,and the acquired cells showed the characteristics of stem cells.The expression of osteogenic makers were up-regulated after loading compressive force for 12h,so it could promoted the osteogenic differentiation of hPDLSCs at early loading time.With the increase of loading force and loading time,the loading of compressive force had inhibitory effect on the osteogenic differentiation of hPDLSCs.