Cyclic tensile force stimulates BMP9 synthesis and in vitro mineralization by human periodontal ligament cells.

Periodontal ligament (PDL) cells are mechanosensitive and have the potential to differentiate into osteoblast-like cells under the influence of cyclic tensile force (CTF). CTF modulates the expression of regulatory proteins including bone morphogenetic proteins (BMPs), which are essential for the homeostasis of the periodontium. Among the BMPs, BMP9 is one of the most potent osteogenic BMPs. It is yet unknown whether CTF affects the expression of BMP9 and mineralization. Here, we demonstrated that continuously applied CTF for only the first 6 hr stimulated the synthesis of BMP9 and induced mineral deposition within 14 days by human PDL cells. Stimulation of BMP9 expression depended on ATP and P2Y 1 receptors. Apyrase, an ecto-ATPase, inhibited CTF-mediated ATP-induced BMP9 expression. The addition of ATP increased the expression of BMP9. Loss of function experiments using suramin (a broad-spectrum P2Y antagonist), MRS2179 (a specific P2Y 1 receptor antagonist), MRS 2365 (a specific P2Y 1 agonist), U-73122 (a phospholipase C [PLC] inhibitor), and thapsigargin (enhancer of intracytosolic calcium) revealed the participation of P2Y 1 in regulating the expression of BMP9. This was mediated by an increased level of intracellular Ca 2+ through the PLC pathway. A neutralizing anti-BMP9 antibody decreased mineral deposition, which was stimulated by CTF for almost 45% indicating a role of BMP9 in an in vitro mineralization. Collectively, our findings suggest an essential modulatory role of CTF in the homeostasis and regeneration of the periodontium.

The effect of cyclic tensile force on the actin cytoskeleton organization and morphology of human periodontal ligament cells.

To explore Girdin/Akt pathway protein expression and morphology change by cyclic tension in the periodontal ligament cells. Human periodontal ligament cells were exposed to cyclic tension force at 4000 µstrain and 0.5 Hz for 6 h though a four-point bending system. Cyclic tension force upregulated F-actin, Girdin and Akt expression in hPDL. In transmission electron microscope assay showed that there are more and bigger mitochondria, more and longer cynapses, more cellular organisms after tension force stimulation than control. The actin filament was changed to be regular lines and pointed to poles of cells. However, we found that the Girdin-depleted cells are small and there are more micro-organisms including more lysosomes and matrix vesicles than control. These finding suggest that the STAT3/Girdin/Akt pathway in PDL to response to mechanical stimulation as well, and Girdin may play a significant role in triggering cell proliferation and migration during orthodontic treatment. It provided an insight into the molecular basis for development of a vitro cell model in studying orthodontic treatment.

Cyclic tensile force up-regulates BMP-2 expression through MAP kinase and COX-2/PGE2 signaling pathways in human periodontal ligament cells.

Periodontal ligament cells play important roles in the homeostasis of periodontal tissue by mechanical stress derived from mastication, such as tension, compression, fluid shear, and hydrostatic force. In the present study, we showed that cyclic tensile force increased the gene expression level of bone morphogenetic protein (BMP)-2, a crucial regulator of mineralization, in human periodontal ligament cells using real-time PCR. Signaling inhibitors, PD98059/U0126 (extracellular signal-regulated kinase (ERK) inhibitors) and SB203580/SB202190 (p38 inhibitors), revealed that tensile force-mediated BMP-2 expression was dependent on activation of the ERK1/2 and p38 mitogen-activated protein (MAP) kinase pathways. Cyclic tensile force also induced cyclooxygenase-2 (COX-2) gene expression in a manner dependent on ERK1/2 and p38 MAP kinase pathways, and induced prostaglandin E2 (PGE2) biosynthesis. NS-398, a COX-2 inhibitor, significantly reduced tensile force-mediated BMP-2 expression, indicating that PGE2 synthesized by COX-2 may be involved in the BMP-2 induction. The inhibitory effect of NS-398 was completely restored by the addition of exogenous PGE2. However, stimulation with PGE2 alone in the absence of tensile force had no effect on the BMP-2 induction, indicating that some critical molecule(s) other than COX-2/PGE2 may be required for cyclic tensile force-mediated BMP-2 induction. Collectively, the results indicate that cyclic tensile force activates ERK1/2 and p38 MAP kinase signaling pathways, and induces COX-2 expression, which is responsible for the sequential PGE2 biosynthesis and release, and furthermore, mediates the increase in BMP-2 expression at the transcriptional level.

Cyclic tensile force-upregulated IL6 increases MMP3 expression by human periodontal ligament cells.

OBJECTIVE: Cyclic tensile force (CTF) modulates physiological responses of periodontal ligament (PDL) cells. PDL cells are mechanosensitive and are able to maintain tissue homeostasis; a process mediated by the expression of particular cytokines including interleukin 6 (IL6). It is unknown whether CTF-induced IL6 regulates the expression of MMPs, enzymes needed for tissue remodeling. DESIGN: Human PDL cells were subjected to 10% elongation strain of CTF at a frequency of 60 rpm continuously for 6 h. RNA and proteins were extracted and analyzed for IL6 and MMP expression by quantitative real-time PCR and ELISA, respectively. Using a neutralizing anti-IL6 antibody and addition of recombinant human IL6 at concentrations of 0.1, 1, 10 ng.mL-1 were performed to clarify whether CTF-upregulated IL6 increased MMP expression. Inhibitors of intracellular signaling molecules were employed to reveal possible pathway(s) of IL6-induced MMP expression. RESULTS: CTF-induced IL6 expression coincided with an increased MMP3 expression. A neutralizing anti-IL6 antibody attenuated the CTF-increased MMP3 expression, whereas stimulating the cells with recombinant human IL6 increased MMP3 expression. Both PI3K and MAPK pathways were essential in the IL6 induced expression of MMP3. CONCLUSION: Our findings suggest a role of CTF in the modulation of expression of IL6 and MMP3 and thus in the regulation of homeostasis and remodeling of the periodontal ligament.