IRUCAA@TDC : FUNCTIONAL EXPRESSION OF RANKL AND OPG IN RAT PERIODONTAL LIGAMENT CELLS IN RESPONSE TO MECHANICAL FORCE IN VIVO AND IN VITRO

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Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College, Available from http://ir.tdc.ac.jp/

Title

FUNCTIONAL EXPRESSION OF RANKL AND OPG IN RAT

PERIODONTAL LIGAMENT CELLS IN RESPONSE TO

MECHANICAL FORCE IN VIVO AND IN VITRO

Author(s)

Ana, Tricia, Moreira

Journal

歯科学報, 114(4): 388-389

URL

http://hdl.handle.net/10130/3380

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論文内容の要旨１．Purpose：

The aim of this study was to evaluate the expression of receptor activator of nuclear factor kappa-B ligand（RANKL）and osteoprotegerin（OPG）in rat periodontal ligament（PDL）cells in response to mechanical force bothin vivo and in vitro.

２．Materials and Methods：

In this experiment Sprague-Dawley rats were used. In thein vivo study, a traction force of approximately 200 g was applied in the bucco-palatal direction by first placing a stainless steel wire around the crown of each of the rats（n＝２０）first maxillary molars and then connecting them with an elastic rubber band. The animals were sacrificed at 3, 7, or 14 days after fixation of the orthodontic appliance. In the in vivo experi-ment, RANKL and OPG immunoreactivity was observed during orthodontic tooth movement. In thein vitro study, PDL cells were obtained from other rats（n＝５）incisors and cultured 3-dimensionally in type-1 colla-gen gel. A closed glass receptacle containing water weighing a total of 25 g was placed on the collacolla-gen gel culture to apply approximately 5 g/cm２

of pressure for 24 hr to simulate application of mechanical force. Periodontal ligament cells in collagen gel with no application of force were observed as a control group. The cells were then observed at the molecular level after 1, 3, and 7 days. HE, immunohistochemical and im-munofluorescence staining methods were used for evaluation. Primary anti-bodies of RANKL and OPG were used for immunohistochemical staining evaluation. Phalloidin and DAPI were used for actin filaments staining evaluation of the cell behavior in 3-D scaffold, under confocal microscope.

３．Results：

At 7 days, PDL cells were positive for RANKL and OPG in the central area of the PDL and close to cementum. However, OPG positive cells gradually increased, whereas RANKL decreased. After the loss of the elastic rubber band s elasticity, OPG positive cells began to increase due to initiation of regeneration and subsequent recovery of PDL cells. In thein vitro study, mRNA expression of RANKL showed small amount

氏名（本籍）

アナトリシアモレイラ

Ana Tricia Moreira

（ブラジル）

学位の種類博士（歯学）

学位記番号第１９７８号（甲第１２１９号）

学位授与の日付平成２５年３月３１日

学位授与の要件学位規則第４条第１項該当

学位論文題目 FUNCTIONAL EXPRESSION OF RANKL AND OPG IN RAT PERIODONTAL LIGAMENT CELLS IN RESPONSE TO MECHANICAL FORCE IN VIVO AND IN VITRO

掲載雑誌名 CLINICAL DENTISTRY AND RESEARCH 第３７巻２号３−１３頁２０１３年１０月論文審査委員（主査）井上孝教授（副査）末石研二教授新谷誠康教授齋藤淳教授東俊文教授３８８歯科学報 Vol．１１４，No．４（２０１４） ― ８４ ―

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and decreased with time and there was no significant difference at any of the time period comparing to the control group. Expression of OPG, however, showed an increase after 1 day due to cellular response to pres-sure.

４．Conclusion：

These results suggest that RANKL and OPG are regulators of regeneration of PDL and its surrounding tissues during orthodontic tooth movement.

論文審査の要旨

Tuesday, July 24, 2012, thesis examination of Ana Trícia Moreira was held. The defense, Professor Inoue Takashi requested the offence professors, Prof. Shintani Seikou, Prof. Saito Atsushi and Prof. Azuma Toshi-fumi, and the defense professor, Prof. Sueishi Kenji to share their comments about the thesis and ask the questions.

１）Are there many investigators studying about RANKL and OPG？ Answer : Yes, the majority of the researchers are inducing resorption using closed-coil springs for continuous force, but I was using elastic rubber band, which the forces decrease with the time. ２）Were the cells used in the in vitro study collected from the rats of thein vivo study？ Answer : No, the cells used in the in vitro study were collected from different rats. ３）About the material and methods in thein vitro study, what was the density of the

cells mixed in the collagen gel？ Answer : １×１０５

periodontal ligament cells were mixed with 2 ml collagen gel to make a cell suspension at a final concentration of 5×10４

cells/cm３

and placed in 35-mm polystyrene dishes in for incubation in 3-dimensional（3-D）culture. ４）Even the force decreased with the time of the ex-periment, the remaining force was still heavy for the rats. How was the force measured？ Answer : The force was measured with a dynamometer, but it was not possible to remove the appliance to measure it perfectly and also the teeth were already moved, suggesting that the force was weaker than what was showing in the graphic. It was the elastic force, but now the actual force for the rats.

After the presentation, the professors suggested to change the nature of the force used in this study （intermittent force）should be changed to continuous- decreasing force, because the magnitude of the force

is still decreasing with the time. And the intermittent force , as we used, needs a rest period.

Finally the entire external examiner Professors were agreed to submit this thesis paper for the PhD thesis.

歯科学報 Vol．１１４，No．４（２０１４）３８９