Study for the induction of the periodontal ligament cell apoptosis by mechanical stress

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In the orthodontic treatment, the tooth moves in the alveolar bone by the mechanical stress induced by various devices. However, it still remains unclear that how the physical mechanical stress is replaced into the various biological events. To find a clue to clarify this mechanism, we tried to identify the genes expressed by mechanical stress in the human periodontal ligament(PDL) primary culture cells, using the cDNA array method.The patient, who was diagnosed as tooth extraction from orthodontic reasons, was thoroughly informed about the objective of the experiments, and their consent was obtained. PDL tissue derived from a premolar of the patient was excised, and was placed onto flexible bottom plate. One month later, the heterogeneous cells reached confluence, and continuous mechanical stress was loaded onto the cells for 1, 6, 24 hours. Then, total RNA was extracted and purified. The total RNA was reverse transcribed into cDNA, and amplified by PCR method. Afterwards, it was labe … More led by ^<32>P and hybridized to the human array membrane. As a result, among 1176 genes studied, many genes were up-regulated or down-regulated within a wide range after 1 hour of stretching ; however, almost all of them returned to the control level by 6 or 24 h. The up-regulated or down-regulated genes at 1h stretching were related to cell cycle arrest and apoptosis, as well as cytokines, hormones, extracellular matrixes, adhesion moleclues, protein kinases, and others. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) for selected genes confirmed the results of the cDNA array analysis. We also demonstrated that the gene most markedly induced by continuous stretching and related to the cell cycle or apoptosis was in part activated via the p38 mitogen-activated protein kinase (MAPK) pathway, by Western blotting and the inhibitor experiment. This work has established an important baseline for clarifying the mechanisms whereby the PDL responds to mechanical stresses, using cDNA array analysis. Less