BACKGROUND:Cytoskeleton plays an important role in the transduction of mechanical signal, and intermittent tensile stress can promote osteogenic differentiation. However, there is no relevant study about the change of cytoskeleton in osteoporosis rat bone marrow mesenchymal stem cells under intermittent tensile stress. OBJECTIVE:To investigate the effects of intermittent tensile stress on the cytoskeleton of osteoporosis rat bone marrow mesenchymal stem cells during osteogenic differentiation. METHODS:Bone marrow mesenchymal stem cells were obtained from osteoporosis rats and cultured in vitro. The 5%, 10%and 15%tensile stress were strained on the bone marrow mesenchymal stem cells through FX-4000T Flexcell. No stress was in the control group. Osteogenic differentiation of bone marrow mesenchymal stem cells was observed through alkaline phosphatase staining, while the change of cytoskeleton was observed by confocal laser scanning microscopy with figures col ected for analysis by Image-ProPlus 6.0 software. The area of cells, ratio of length to width and integrated fluorescence intensity of cytoskeleton protein F-actin were measured. RESULTS AND CONCLUSION:Under tensile stress, bone marrow mesenchymal stem cells from osteoporosis rats arranged in the direction vertical to mechanical stimulation. cells under different tensile stress differentiated towards osteoblasts. The result of alkaline phosphatase staining showed the most significant difference in 10%group, and quite an amount of cells lining lost succession in the 15%group. Under stress, the F-actin filaments were rearranged in paral el accordingly, which showed a reconstruction of cytoskeleton. Imaging analysis indicated that the area of bone marrow mesenchymal stem cells was decreased in 10%and 15%groups (P<0.05) with the increased ratio of length to width (P<0.05), and expression of F-actin increased in5%, 10%, 15%groups (P<0.05) after tensile stress. Under mechanical stimulation, the cytoskeleton of bone marrow mesenchymal stem cells from osteoporosis rats is shown to have corresponding alterations during osteogenic differentiation.

OBJECTIVETo test the hypothesis that the CCL2/CCR2 signaling pathway plays an important role in pain induced by experimental tooth movement.

METHODSMale Sprague- Dawley rats weighing between 200 and 300 g were used in this study. Expression of CCL2/CCR2 in the trigeminal ganglion (TG) was determined by Western blotting 0 h, 4 h, 1 d, 3 d, 5 d, 7 d after tooth movement. Localization of the CCL2 was revealed by immunohistochemistry. Changes in body weight, nocifensive behaviors, and the effects of CCL2/CCR2 antagonists on these changes in pain behaviors were evaluated. Exogenous CCL2 was injected into periodontal tissues and added to TG neurons in culture and the resulting c-fos expression and pain responses were detected. In addition, the expression and cellular localization of CCL2 in the medullary dorsal horn (MDH) was determined by immunohistochemistry 3 d and 14 d after tooth movement.

RESULTSExperimental tooth movement led to a statistically significant increase in CCL2/CCR2 expression at the protein level from day 3 to 7 after application of force initiating tooth movement.When compared with control group (1.000 ± 0.000), CCL2 increased to (2.620 ± 0.128), (3.300 ± 0.197) and (1.740 ± 1.290) at day 3, 5 and 7 respectively, which were statistically significant (P < 0.05). CCR2 expression levels were (1.636 ± 0.061) and (1.766 ± 0.126) compared with that in control group (1.000 ± 0.000) at day 3 and 5 respectively with statistical significance (P < 0.05). Both of them peaked on day 5 (3.3 and 1.8 time compared to control group). Application of recombinant CCL2 led to the up-regulation of c-fos expression in vivo and in vitro, and triggered a corresponding nocifensive behavior in rats. The magnitude of the nocifensive behavior could be reduced by a CCR2 antagonist, and by CCL2 neutralizing antibody. Furthermore, we found a significant increase in the expression of CCL2, corresponding well to the up-regulation of the time spent on nocifensive behaviors after ETM. In addition, CCL2 was up-regulated in TG neurons and astrocytes in Vc.

CONCLUSIONSThe CCL2/CCR2 axis was modulated by experimental tooth movement and involved in the development of tooth movement pain, and thus palyed an important role in orthodontic pain mechanism.

Animals ; Chemokine CCL2 ; physiology ; Immunohistochemistry ; Male ; Neurons ; Rats ; Rats, Sprague-Dawley ; Receptors, CCR2 ; physiology ; Signal Transduction ; Tooth Movement Techniques ; adverse effects ; Toothache ; etiology ; Trigeminal Ganglion ; Up-Regulation