title: Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells.
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.
  
  
  CONCLUSIONS
  3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling.
authors.affiliation: 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.
authors.forename: Mioko
authors.initials: M
authors.lastname: Yamamoto
authors.affiliation: 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.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Kawashima
authors.affiliation: 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.
authors.forename: Nami
authors.initials: N
authors.lastname: Takashino
authors.affiliation: 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.
authors.forename: Yu
authors.initials: Y
authors.lastname: Koizumi
authors.affiliation: 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.
authors.forename: Koyo
authors.initials: K
authors.lastname: Takimoto
authors.affiliation: 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.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Suzuki
authors.affiliation: Division of Operative Dentistry, Department of Restorative Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Saito
authors.affiliation: 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.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Suda
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D015687:Cell Hypoxia; D003771:Dental Papilla / Q000166:cytology*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D016023:Integrins / Q000378:metabolism; D051379:Mice; D009804:Odontoblasts / Q000502:physiology*; D010006:Osteoblasts / Q000502:physiology*; D010641:Phenotype; D016133:Polymerase Chain Reaction; D016596:Vinculin / Q000378:metabolism
chemical_list: D015415:Biomarkers; C513308:Dmp1 protein, mouse; D016326:Extracellular Matrix Proteins; D016023:Integrins; D016596:Vinculin; D000469:Alkaline Phosphatase
keywords: Dental papilla cells; Integrin signalling; Odonto/osteoblastic differentiation; Spheroid culture