Abstract: Abstract : Objective To observe the effect of mechanical stimulation on the differentiation of periodontal ligament stem cells in vitro. Methods Isolation and culture of human periodontal ligament cells by enzymatic digestion. Monoclonal method was used to separate hPDLSCs. The surface marker of hPDLSCs was detected by flow cytometry. Crystal violet staining was used to observe the cell clones of hPDLSCs, and then induced into osteoblast and adipose cells. The potential differentiation was identified by Alizarin red and oil red O staining. Cyclic hydrostatic pressure was performed on hPDLSCs using independently developed mechanical loading device, the pressure is 0~120kPa and the frequency of load is 0.1Hz, and cells were treated with pressure 1h/d for consecutive 7 days. The mRNA expression levels of peroxisome proliferators-activated receptor-γ ( PPAR-γ), runt-related transcription factor 2 (Runx2), Scleraxis and Cementum Protein 1( CEMP1) were measured by Real-time PCR. Results The hPDLSC obtained by the limited dilution were positive for CD90 (95.2%), CD105 (95.5%) and negative for CD45 (1.3%), CD34 (1.36%). Single colonies were observed after crystal violet staining. Alizarin red-positive mineral deposits and Oil Red O-positive cells indicated the cultured cells have osteogenic and adipogenic potential. After mechanical stimulation, the expression of Scleraxis was significantly higher than that in control group, whereas the expression of PPAR-γ, Runx2 and CEMP1 had no significant difference compared with the control group. Conclusion Cyclic hydrostatic pressure could maintain the potentials of hPDLSCs for periodontal ligament fibroblasts differentiation.

Key words: Hydrostatic pressure, human Periodontal Ligament Stem Cells (hPDLSCs), Differentiation