Rina Muraoka

Immunohistochemical expression of heat shock proteins in the mouse periodontal tissues due to orthodontic mechanical stress

The histopathology of periodontal ligament of the mouse subjected to mechanical stress was studied. Immunohistochemical expressions of HSP27 and pHSP27 were examined. Experimental animals using the maxillary molars of ddY mouse by Waldo method were used in the study. A separator was inserted to induce mechanical stress. After 10 minutes, 20 minutes, 1 hour, 3 hours, 9 hours and 24 hours, the regional tissues were extracted, fixed in 4% paraformaldehyde and 0.05 M phosphate-buffered fixative solution. Paraffin sections were made for immunohistochemistry using HSP27 and p-HSP27. In the control group, the periodontal ligament fibroblasts expressed low HSP27 and p-HSP27. However, in the experimental group, periodontal ligament fibroblasts expressed HSP27 10 minutes after mechanical load application in the tension side. The strongest expression was detected 9 hours after inducing mechanical load. p-HSP27 was also expressed in a time-dependent manner though weaker than HSP27. The findings suggest that HSP27 and p-HSP27 were expressed for the maintenance of homeostasis of periodontal ligament by the activation of periodontal ligament fibroblasts on the tension side. It also suggests that these proteins act as molecular chaperones for osteoblast activation and maintenance of homeostasis.

Promotion of transplanted bone marrow-derived cell migration into the periodontal tissues due to orthodontic mechanical stress.

Background: Bone marrow-derived cells (BMCs) have abilities of cell migration and differentiation into tissues/organs in the body and related with the differentiation of teeth or periodontal tissue including fibroblasts. Then, we examined the effect of orthodontic mechanical stress to the transplanted BMC migration into periodontal tissues using BMC transplantation model. Material and Method: BMC from green fluorescence protein (GFP) transgenic mice were transplanted into 8-week-old female C57BL/6 immunocompromised recipient mice, which had undergone 10 Gy of lethal whole-body-irradiation. Five mice as experimental group were received orthodontic mechanical stress using separator between first molar (M1) and second molar (M2) 1 time per week for 5 weeks and 5 mice as control group were not received mechanical stress. The maxilla with M1 and M2 was removed and was immunohistochemically analyzed using a Dako Envision + Kit-K4006 and a primary anti-GFP-polyclonal rabbit antibody. Immunohistochemically stained was defined as positive area and the pixel number of positive area in the periodontal tissue was compared with the previously calculated total pixel number of the periodontal tissue. Results: The immunohistochemistry revealed that GFP positive cells were detected in the periodontal tissues, both in the experimental and control specimens. The ratio of pixel number in the examination group showed 5.77 ± 3.24 % (mean ± SD); and that in the control group, 0.71±0.45 % (mean ± SD). The examination group was significantly greater than that of control group (Mann-Whitney U test: p<0.001). Conclusion: These results suggest that orthodontic mechanical stress accelerates transplanted BMC migration into periodontal tissues.

Histological Evaluation of Periodontal Ligament in Response to Orthodontic Mechanical Stress in Mice.

The purpose of the study was to determine the cell dynamics in periodontal ligament in response to mechanical stress during orthodontic movement. Following Waldos method, a square sheet of rubber dam was inserted in between the first and second maxillary molars in 10 ddY mice leaving the stress load for 3 hours. After 3 days and at 1 week, cell count on pressure and tension sides of the periodontal ligament was determined. Furthermore, the type of cell present after mechanical stress was identified using GFP bone marrow transplantation mouse model. Immunohistochemistry was carried out at 0 min (immediately after mechanical stress), 24 hours, 1 week, 2 weeks and 6 months. Temporal changes in the expression of GFP-positive bone marrow derived cells were examined. Moreover, double immunofluorescent staining was performed to determine the type of cell in the periodontal ligament. Cell count on the tension side tremendously increased 3 days after mechanical stress. At 1 week, spindle and round cell count increased compared to the control group. These changes were observed on both tension and pressure sides. Cell count on pressure side at 3 days (22.11+/-13.98) and at 1 week (33.23+/-11.39) was higher compared to the control group (15.26+/-8.29). On the tension side, there was a significantly increased at 3 days (35.46+/-11.85), but decreased at 1 week (29.23+/-13.89) although it is still higher compared to the control group (AD+/-SD: 10.37+/-8.69). Using GFP bone marrow transplantation mouse model, GFP positive cell count increased gradually over time in 6 months. GFP positive cells were also positive to CD31, CD68 and Runx2 suggesting that fibroblasts differentiated into osteoclasts and tissue macrophages. In conclusion, mechanical stress during orthodontic movement promoted the increase in the number of cells in the periodontal ligament on both tension and pressure sides. The increase in the number of cells in the periodontal ligament is believed to be due to the migration and cell division of undifferentiated mesenchymal cells.

Functional Role of HSP47 in the Periodontal Ligament Subjected to Occlusal Overload in Mice

We carried out an experiment to induce traumatic occlusion in mice periodontal tissue and analyzed the expression of HSP47. Continuous traumatic occlusion resulted to damage and remodeling of periodontal ligament as well as increase in osteoclasts and bone resorption. Four days after traumatic occlusion, osteoclasts did not increase but Howships lacunae became enlarged. That is, the persistent occlusal overload can destroy collagen fibers in the periodontal ligament. This was evident by the increased in HSP47 expression with the occlusal overload. HSP47 is maintained in fibroblasts for repair of damaged collagen fibers. On the other hand, osteoclasts continue to increase although the load was released. The osteoclasts that appeared on the alveolar bone surface were likely due to sustained activity. The increase in osteoclasts was estimated to occur after load application at day 4. HSP47 continued to increase until day 6 in experiment 2 but then reduced at day 10. Therefore, HSP47 appears after a period of certain activities to repair damaged collagen fibers, and the activity was returned to a state of equilibrium at day 30 with significantly diminished expression. Thus, the results suggest that HSP47 is actively involved in homeostasis of periodontal tissue subjected to occlusal overload.