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. Sci Rep. 2018 Jul 5;8(1):10173 Zheng L, Zinn V, Lefkelidou A, Taqi N, Chatzistavrou X, Balam T, Nervina J, Papagerakis S, Papagerakis P. Orai1 expression pattern in tooth and craniofacial ectodermal tissues and potential functions during ameloblast differentiation. Dev Dyn. 2015 Oct;244(10):1249-58. Wang YY, Chatzistavrou X, Faulk D, Badylak S, Zheng L, Papagerakis S, Ge L, Liu H, Papagerakis P. Biological and bactericidal properties of Ag-doped bioactive glass in a natural extracellular matrix hydrogel with potential application in dentistry. Eur Cell Mater. 2015 Jun 20;29:342-55. Papagerakis P, Pannone G, Zheng L, Athanassiou-Papaefthymiou M, Yamakoshi Y, McGuff HS, Shkeir O, Ghirtis K, Papagerakis S. Clinical significance of kallikrein- related peptidase-4 in oral cancer. Anticancer Res. 2015 Apr;35(4):1861-6. Matossian M, Vangelderen C, Papagerakis P, Zheng L, Wolf GT, Papagerakis S. In silico modeling of the molecular interactions of antacid medication with the endothelium: novel therapeutic implications in head and neck carcinomas. Int J Immunopathol Pharmacol. 2014 Oct-Dec;27(4):573-83. Desiderio V, Papagerakis P, Tirino V, Zheng L, Matossian1 M, Prince M, Paino F, Mele L, Montella R, Papaccio G, Papagerakis S. Increased fucosylation h as a pivotal role in invasive and metastatic properties of head and neck cancer stem cells. (2015) Oncotarget. Jan 1;6(1):71-84 Papagerakis S, Pannone G, Zheng L, About I, Taqi N, Nguyen NP, Matossian M, McAlpin B, Santoro A, McHugh J, Prince ME, Papagerakis P. Oral epithelial stem cells-Implications in normal development and cancer metastasis. (2014) Exp Cell Res. Jul ;325(2):111-129 Zheng L, Ehardt L, McAlpin B, About I, Kim D, Papagerakis S, Papagerakis P. The tick tock of odontogenesis. (2014) Exp Cell Res. Jul ;325(2):83-89 Papagerakis S, Zheng L, Schnell S, Sartor MA, Somers E, Marder W, McAlpin B, Kim D, McHugh J, Papagerakis P. The circadian clock in oral health and diseases. (2014) J Dent Res. Jan;93(1):27-35 vi Czerwinski MJ, Desiderio V, Shkeir O, Papagerakis P, Lapadatescu MC, Owen JH, Athanassiou-Papaefthymiou M, Zheng L, Papaccio G, Prince ME, Papagerakis S. In vitro evaluation of sialyl Lewis X relationship with head and neck cancer stem cells. Otolaryngol Head Neck Surg. 2013 Jul;149(1):97-104. Zheng L, Seon YJ, Mourão MA, Schnell S, Kim D, Harada H, Papagerakis S, Papagerakis P. Circadian Rhythms Control Ameloblasts Differentiation. (2013) Bone. Jul;55(1):158-65 Desiderio V, Papagerakis P, Zheng L, Prince M, Czerwinski MJ, Papaccio G, Owen J, Cezar IM, Papagerakis P. In Vitro Evaluation of Sialyl Lewis X Relationship with Head and Neck Cancer Stem Cells. (2013) Otolaryngol Head Neck Surg. Jul;149(1):97-104 Zheng L, Seon YJ, McHugh J, Papagerakis S, Papagerakis P. Clock genes show circadian rhythms in salivary glands. (2012) J Dent Res. Aug;91(8):783-8. Selected for the journal’s cover. Zheng L, Papagerakis S, Schnellc SD, Hoogerwerfd WA, Papagerakis P. Expression of Clock Proteins in Developing Tooth. (2011) Gene Expr Patterns. Mar-Apr;11(3- 4):202-6. Simmer JP, Papagerakis P, Smith CE, Fisher DC, Rountrey AN, Zheng L, and Hu J.C.-C. Regulation of Dental Enamel Shape and Hardness. (2010) J Dent Res. Oct;89(10):1024-38. Zheng L, Amano K, Iohara K, Ito M, Into T, Matsushita K, Nakamura H, Nakashima M. Matrix Metalloproteinase 3 Accelerates Wound Healing Events after Dental Pulp Injury. (2009) Am J Patho. 175(5):1905-14 Iohara K, Zheng L, Ito M, Ishizaka R, Nakamura H, Into T, Matsushita K, Nakashima M. Regeneration of dental pulp after pulpotomy by transplantation of CD31(-)/CD146(-) side population cells from a canine tooth. (2009) Regen Med. 4(3):377-85. Amano K, Nakashima M, Zheng L, Iohara K, Matsui H, Yamashaki M, Matsushita K, Nakamura H. MMP-3 Regulates Wound Healing Process in Rat Dental Pulp. (2008) The Japanese Society of Conservative Dentistry. 51(6), 602-613 (paper in Japanese) Iohara K, Zheng L, Wake H, Ito M, Nabekura J, Wakita H, Nakamura H, Into T, Matsushita K, Nakashima M. A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp. (2008) Stem Cells. 26(9):2408-18. vii Zheng L, Iohara K, Ishikawa M, Into T, Takano-Yamamoto T, Matsushita K, Nakashima M. Runx3 negatively regulates Osterix expression in dental pulp cells. (2007) Biochem J. 405(1):69-75. Yamashiro T, Zheng L, Shitaku Y, Saito M, Tsubakimoto T, Takada K, Takano- Yamamoto T, Thesleff I. Wnt10a regulates dentin sialophosphoprotein mRNA expression and possibly links odontoblast differentiation and tooth morphogenesis. (2007) Differentiation. 75(5):452-62. Nakashima M, Iohara K, Zheng L. Gene therapy for dentin regeneration with bone morphogenetic proteins. (2006) Curr Gene Ther. 6(5):551-60. Iohara K, Zheng L, Ito M, Tomokiyo A, Matsushita K, Nakashima M. Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis. (2006) Stem Cells. 24(11):2493-503. Zheng L, Yamashiro T, Fukunaga T, Balam TA, Takano-Yamamoto T. Bone morphogenetic protein 3 expression pattern in rat condylar cartilage, femoral cartilage and mandibular fracture callus. (2005) Eur J Oral Sci. 113(4):318-25. Balam TA, Yamashiro T, Zheng L, Murshid Ahmed S, Fujiyoshi Y, Takano- Yamamoto T. Experimental tooth movement upregulates preproenkephalin mRNA in the rat trigeminal nucleus caudalis and oralis. (2005) Brain Res. 1036(1-2):196-201. Fujiyama K, Yamashiro T, Fukunaga T, Balam TA, Zheng L, Takano-Yamamoto T.