Preliminary Study On The Marker And Differentiation Mechanism Of Human Periodontal Ligament Cells

Periodontal ligament cells(PDLCs) comprise a heterogeneous cell population and possess stem cells which are believed to be responsible for maintaining homeostasis in periodontal ligament. These stem cells can differentiate into cementoblast, osteoblast and periodontal fibroblast. However, the precise mechanisms of periodontal ligament stem cells differentiation are unclear.During the regeneration of periodontium, there are reports about the successfully regenerate tissues by periodontal ligament cells and bone marrow stromal cells(BMSCs). But there's a few report about the relationship between these two cells.Scleraxis is a basic helix-loop-helix (bHLH) transcription factor and express highly in mesenchymal progenitors which form connective tissues (including tendon, ligament, cartilage cells) terminally. Scleraxis is required for mesoderm formation and play an important role in chondrogenic and/or osteogenic differentiation. Periodontal ligament stem cells expressed a higher level of Scleraxis than did bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs).To find the relationship between human bone marrow cells (hBMSCs) and periodontal ligament cells (hPDLCs), and to investigate the involvement of Scleraxis in human periodontal ligament cells during differentiation in vitro. HPDLCs, hBMSCs and gingival fibroblasts (hGFs) were cultured in vitro. Immunochemistry was used to detect the difference of surface markers on hPDLCs and hBMSCs. The expression of Scleraxis in hPDLCs from different passages was detected by RT-PCR. Furthermore, these three cells were induced to differentiate and the changes of Scleraxis expression levels were observed during this process.Experiment 1 Comparison of surface character on hPDLCs and hBMCsAim: To compare the surface character on hPDLCs and hBMCs.Methods: HPDLCs and hBMCs were cultured in vitro. The expression of CD14, CD44, CD105, CD34 on these cells were detected by immunocyto-chemistry and analyzed by image analysis software.Results: Similar to hBMCs, hPDLCs expressed CD44 and CD105, the expression of CD14 and CD34 were negative.Conclusions: HPDLCs and hBMCs had some similar surface antigen charcter.Experiment 2 Expression of Scleraxis in hPDLCs from different passagesAim: To detect if Scleraxis can express in hPDLCs in vitro, and to study if the expression levels change with the increased passages.Methods: HPDLCs were isolated from healthy periodontal ligament of premolars extracted for orthodontic reasons. HPDLCs were cultured primarily by explant method and passaged serially. The expression of Scleraxis in hPDLCs from 3rd, 5th, 7th, 9th passages was analyzed by RT-PCR.Results: Scleraxis were detectable in hPDLCs from different passages and the expression levels were decreased with the increased passage numbers.Conclusions: Scleraixis can express in hPDLCs. Maybe the expressions of Scleraxis have relations with the differentiation potential of hPDLCs.Experiment 3 The role of Scleraxis during hPDLCs differentiation in vitroAim: To investigate the expression of Scleraxis in hPDLCs during osteogenic differentiation.Methods: HPDLCs, hBMCs and hGFs were cultured in vitro and each cell was divided into two groups. The induction groups were cultured in osteogenic induction conditions, and the uninduction groups were culutred in normal conditions. The expression of Scleraxis in these cells was detected by RT-PCR.Results: No matter in uninduction groups or the induction groups, the expression of Scleraxis was highest in hPDLCs and lowest in hGFs. The Scleraxis mRNA expression level increased in hPDLCs in the induction group compared with the uninduction group. On the contrary, hBMCs and hGFs in the induction group express lower Scleraxis mRNA than the uninduction groups.Conclusions: Scleraxis take part in the differentiation of hPDLCs in vitro. And Scleraxis might be a specific marker of periodontal ligament cells.