Mioko Yamamoto1, Nobuyuki Kawashima2, Nami Takashino1, Yu Koizumi1, Koyo Takimoto1, Noriyuki Suzuki1, Masahiro Saito3, Hideaki Suda1. 1. Pulp Biology and Endodontics, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. 2. Pulp Biology and Endodontics, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Electronic address: kawashima.n.endo@tmd.ac.jp. 3. Division of Operative Dentistry, Department of Restorative Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan.
Abstract
OBJECTIVE: Three-dimensional (3D) spheroid culture is a method for creating 3D aggregations of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into odontoblasts. METHODS: We cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture. Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signalling-related proteins were detected immunohistochemically. RESULTS: Odonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs (p<0.05). Histologically, 3D spheroid colonies consisted of two compartments: a cell-dense peripheral zone and cell-sparse core zone. Proliferating cells with high ALP activity and DMP1 expression were found mainly in the peripheral zone that also showed strong expression of vinculin and integrin signalling-related proteins. In contrast, apoptotic and hypoxic cells were detected in the core zone. CONCLUSION: 3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling.
OBJECTIVE: Three-dimensional (3D) spheroid culture is a method for creating 3D aggregations of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into odontoblasts. METHODS: We cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture. Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signalling-related proteins were detected immunohistochemically. RESULTS: Odonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs (p<0.05). Histologically, 3D spheroid colonies consisted of two compartments: a cell-dense peripheral zone and cell-sparse core zone. Proliferating cells with high ALP activity and DMP1 expression were found mainly in the peripheral zone that also showed strong expression of vinculin and integrin signalling-related proteins. In contrast, apoptotic and hypoxic cells were detected in the core zone. CONCLUSION: 3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling.
Authors: M Widbiller; S R Lindner; W Buchalla; A Eidt; K-A Hiller; G Schmalz; K M Galler Journal: Clin Oral Investig Date: 2015-07-01 Impact factor: 3.573
Authors: Carolina Cucco; Zhaocheng Zhang; Tatiana M Botero; Daniel J Chiego; Rogerio M Castilho; Jacques E Nör Journal: J Endod Date: 2020-09 Impact factor: 4.171
Authors: Sarah Hani Shoushrah; Janis Lisa Transfeld; Christian Horst Tonk; Dominik Büchner; Steffen Witzleben; Martin A Sieber; Margit Schulze; Edda Tobiasch Journal: Int J Mol Sci Date: 2021-06-15 Impact factor: 5.923
Authors: Jörg Neunzehn; Sandra Pötschke; Christian Hannig; Hans-Peter Wiesmann; Marie-Theres Weber Journal: Head Face Med Date: 2017-12-08 Impact factor: 2.151