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1 Harmine Stimulates the Differentiation of Cementoblasts In Harmine stimulates the differentiation of cementoblasts in vitro Thesis Presented in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Graduate School of The Ohio State University By Li Zheng, DDS, PhD The Ohio State University 2020 Thesis Committee Dr. Toru Deguchi, Advisor Dr. Brian L. Foster Dr. Do-Gyoon Kim 1 Copyrighted by Li Zheng 2020 2 Abstract Introduction: One of the major side effects in orthodontic treatment is external apical root resorption (EARR). Recently, a new antiresorptive agent, harmine, was reported to stimulate osteoblast formation, differentiation and function. We hypothesized that harmine can be a potential agent for preventing and even recovering EARR. Therefore, our objective is to examine if harmine stimulates the differentiation of cementoblast in vitro. Methods: A cementoblast cell line (OCCM-30) was obtained for growth assay and scratch assay to investigate the effects of harmine on cementoblast growth. Real-time Polymerase Chain Reaction (RT-PCR) was used to examine transcription factors and differentiation markers such as Runx2, Dlx5, ctnnb1, Msx2, Osterix, Ibsp, Col1a1 and Spp1 in cementoblasts treated with harmine, compared to cells without treatment. Western blot was used to test if the transcription factors and differentiation markers are stimulated or inhibited on the protein level. Differentiation assays such as alizarin red staining and von Kossa staining are used to examine the effects of harmine on cementoblast differentiation. Results: The growth rate of OCCM-30 was inhibited by 10 µm harmine after 2 days treatment and more significant on day 3. Furthermore, scratch assay study showed a dose-dependent inhibition manner. Ten μM harmine decreased healing speed after 12 ii hours, 5 μM harmine slowed down the migration after 24 hours and 1 μM of harmine treatment decreased the speed after 36 hours, compared to control group. RT-PCR and western blot results indicated that harmine stimulates the expression of transcription factors and differentiation markers on both RNA and protein levels. Expression levels of Runx2, Dlx5 and Osterix were stimulated after 14 days treatment. Msx2 expression increased 1.5 fold after treatment on day 4. The expressions of Col1a1, Ibsp and Spp1 increased 3, 1.5 and 10 times, respectively on day 4. Von Kossa and alizarin red staining showed more calcium deposition by OCCM-30 cells after 4 days 10 μM harmine treatment. Conclusions: Harmine stimulates the differentiation of cementoblast in vitro. Therefore, harmine shows potential as a novel candidate for prevention of root resorption during orthodontic treatment and a promote repair cementum after root resorption. iii Acknowledgments This work is supported by the pilot grant from the College of Dentistry, the Ohio State University to Toru Deguchi. iv Vita Year Degree Major Institution 2017 – 2020 Master Dentistry The Ohio State University 2002 – 2006 Ph.D. Dental Sciences Okayama University, Japan 1995 – 2000 D.D.S. Dentistry Tongji University, China Publications Said R, Zheng L, Saunders T, Zeidler M, Papagerakis S, Papagerakis P. Generation of Amelx-iCre Mice Supports Ameloblast-Specific Role for Stim1. J Dent Res. 2019 Aug;98(9):1002-1010 Papagerakis P, Zheng L, Kim D, Said R, Ehlert AA, Chung KKM, Papagerakis S. Saliva and Gingival Crevicular Fluid (GCF) Collection for Biomarker Screening. Methods Mol Biol. 2019;1922:549-562 Hsiao J, Wang Y, Zheng L, Liu R, Said R, Hadjiyski L, Cha H, Botero T, Chatzistavrou X, Dong Q, Papagerakis S, Papagerakis P. In Vivo Rodent Models for Studying Dental Caries and Pulp Disease. Methods Mol Biol. 2019;1922:393-403 Sulaiman Ghandourah B, Lefkelidou A, Said R, Chatzistavrou X, Flannagan S, Gonzáles-Cabezas C, Fenno CJ, Zheng L, Papagerakis S, Papagerakis P. In Vitro Caries Models for the Assessment of Novel Restorative Materials. Methods Mol Biol. 2019;1922:369-377. v Dong Q, Wang Y, Mohabatpour F, Zheng L, Papagerakis S, Chen D, Papagerakis P. Dental Pulp Stem Cells: Isolation, Characterization, Expansion, and Odontoblast Differentiation for Tissue Engineering. Methods Mol Biol. 2019;1922:91-101 Alam MK, Zheng L, Liu R, Papagerakis S, Papagerakis P, Geyer CR. Synthetic antigen-binding fragments (Fabs) against S. mutans and S. sobrinus inhibit caries formation. 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