Bio-Medical Materials and Engineering 26 (2015) 169–181 169 DOI 10.3233/BME-151555 IOS Press 3D cell culture to determine in vitro biocompatibility of bioactive glass in association with chitosan Y. Bédouin a,b,∗ , P. Pellen Mussi a , S. Tricot-Doleux a , D. Chauvel-Lebret a,b , P. Auroy c,d , X. Ravalec b , H. Oudadesse a and F. Pérez a,e a Equipe Chimie du Solide et Matériaux – UMR CNRS 6226 – Sciences Chimiques de Rennes, Université de Rennes 1, Université Européenne de Bretagne, Campus de Beaulieu, 263 avenue du Général Leclerc, 35042 Rennes Cedex, France b CHU de Rennes, Pôle d’Odontologie et de Chirurgie Buccale, 2 place Pasteur, 35000 Rennes, France c Faculté de Chirurgie dentaire, Université d’Auvergne, 11 Boulevard Charles de Gaulle, 63000 Clermont-Ferrand, France d CHU de Clermont-Ferrand, service d’Odontologie, 11 rue Léon Malfreyt, 63000 Clermont-Ferrand, France e CHU de Nantes, service d’Odontologie, 1 Place Alexis Ricordeau, 44000 Nantes, France Received 14 October 2014 Accepted 30 September 2015 Abstract. This study reports the in vitro biocompatibility of a composite biomaterial composed of 46S6 bioactive glass in association with chitosan (CH) by using 3D osteoblast culture of SaOS 2 . The 46S6 and CH composite (46S6–CH) forms small hydroxyapatite crystals on its surface after only three days immersion in the simulated body fluid. For 2D osteoblast culture, a significant increase in cell proliferation was observed after three days of contact with 46S6 or 46S6–CH-immersed media. After six days, 46S6–CH led to a significant increase in cell proliferation (128%) compared with pure 46S6 (113%) and pure CH (122%). For 3D osteoblast culture, after six days of culture, there was an increase in gene expression of markers of the early osteoblastic differentiation (RUNX2, ALP, COL1A1). Geometric structures corresponding to small apatite clusters were observed by SEM on the surface of the spheroids cultivated with 46S6 or 46S6–CH-immersed media. We showed different cellular responses depending on the 2D and 3D cell culture model. The induction of osteoblast differ- entiation in the 3D cell culture explained the differences of cell proliferation in contact with 46S6, CH or 46S6–CH-immersed media. This study confirmed that the 3D cell culture model is a very promising tool for in vitro biological evaluation of bone substitutes’ properties. Keywords: Spheroids, biocompatibility, bioactive glass, chitosan * Corresponding author: Yvan Bédouin, Equipe Chimie du Solide et Matériaux – UMR CNRS 6226 – Sciences Chimiques de Rennes, Université de Rennes 1, Université Européenne de Bretagne, Campus de Beaulieu, 2 avenue du Professeur Léon Bernard, Bâtiment 15, 35043 Rennes Cedex, France. Tel.: +33 2 23 23 43 38; Fax: +33 2 23 23 43 93; E-mail: yvan.bedouin@univ-rennes1.fr. 0959-2989/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved