Mechanical Strain Influence On The Expression Of TAZ Of The Periodontal Ligament Stem Cells In Vitro

Objective:Clear and definite the influence of mechanical tensile stress on the biological characteristics and the influence on the expression of TAZ of the periodontal ligament stem cells in vitro.Methods:1. Isolation culture and identify the human periodontal ligament stem cellsCollect healthy and fresh teeth from patients aged 18-30 for the reason of impaction three molars or premolars for orthodontic reasons, separate the periodontal ligament gently from the middle third of the root,and use type I collagenase c’igestion digest the tissue, then culture the tissue explants. Seed single-cell suspension into 96-well plates, achieve the primary PDLSC colonies.3rd generational cells are used for the study of this experiment. Use immunofluorescence assay and flow cytometry to detect the cell surface markers STRO-1、CD146、CD14、CD29、CD44、CD45、 CD90, and use osteogenic and adipogenic induction to detect the differentiation capacity of the PDLSCs.2. Mechanical strain influence on the biological characteristics of the periodontal ligament stem cellsThe experiment adopted a Four-Point bending apparatus to load mechanical strain on PDLSCs, collect and culture 3rd generational cells on the afterburner board, the experimental group set stimulate stress 3000μ. strain and received 0.5HZ mechanic device in 30,60,180,360 and 720min,the control group did not receive mechanical strain stimulation and the other cultural conditions were the same. Use flow cytometry detect the cell cycle,in order to observe the proliferation of the PDLSC after the effect of mechanical strain in a specific point in time, through the change of the expression of Runx2, ALP, PPARy mRNA by real-time PCR to detect the differentiation capacity changes of the PDLSCs.3. Mechanical strain influence on the expression of TAZCollect the PDLSCs after the mechanical strain in a specific point in time, observe the change of the expression of TAZ.Results:1. We obtained the PDLSC by using limiting dilution cloning method, the cells had similar shape, both were fusiform or irregular shape, the cell arrangement were radical or in a spiral shape.The flow cytometry revealed that the cells were positive expression of mesenchymal stem cell makers CD29(91.07%),CD44(90.89%),CD90 (96.16%),and were negative expression of endothelial cells makers CD 14 (4.73%) and CD45 (0.26%).After osteogenic induction, we found calcium deposits by alizarin red staining. After adipogenic induction we found lipid droplets by positive oil red O. These results prove that the PDLSC had ability of proliferation and multi-directional differentiation of stem cells.2. Change of the biological characteristics of the periodontal ligament stem cells after the effect of mechanical strainAfter the effect of the mechanical strain,we found that at the time of 180 min and 360 min can obvious promote the PDLSC proliferation, the expression of the osteogenic related makers ALP mRNA, RUNX2 mRNA and the adipogenic related gene PPARy mRNA were strengthen with the extension of time after 60 minutes later, the expression were significant at 180min.3. Changes of TAZ after the effect of mechanical strainCollect PDLSCs after the effect of mechanical strain, we observe that the expression of TAZ was strengthening with the extension of time after 60 minutes later.Conclusions:1. The PDLSCs that obtained by using limiting dilution cloning method, had the ability of self-renewal and multipotential differentiation.2. The mechanical strain of 3000μstrain can promote the ability of proliferation and multi-directional differentiation of PDLSCs in vitro.3. The mechanical strain of 3000μ strain can promote the expression of TAZ mRNA. It indicated that TAZ participate in the intracellular signal transmission of mechanical tensile stress, TAZ can regulate the differentiation of PDLSCs.