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The ability to promote osteoblast differentiation is a desirable property of root-end filling materials. Several in vitro studies compare the cytotoxicity and physical properties between mineral trioxide aggregate (MTA) and Endosequence root repair material (ERRM), but not their osteogenic potential. Three-dimensional cultures allow cells to better maintain their physiological morphology and better resemble in vivo cellular response than 2-dimensional cultures. Here we examined the osteogenic potential of MTA and ERRM by using a commercially available 3-dimensional Alvetex scaffold.Mandibular osteoblasts were derived from 3-week-old male transgenic reporter mice where mature osteoblasts express green fluorescent protein (GFP) driven by a 2.3-kilobase type I collagen promoter (Col(I)-2.3). Mandibular osteoblasts were grown on Alvetex in direct contact with MTA, ERRM, or no material (negative control) for 14 days. Osteoblast differentiation was evaluated by expression levels of osteogenic genes by using quantitative polymerase chain reaction and by the spatial dynamics of Col(I)-2.3 GFP-positive mature osteoblasts within the Alvetex scaffolds by using 2-photon microscopy.ERRM significantly increased alkaline phosphatase (Alp) and bone sialoprotein (Bsp) expression compared with MTA and negative control groups. Both MTA and ERRM increased osterix (Osx) mRNA significantly compared with the negative control group. The percentage of Col(I)-2.3 GFP-positive cells over total cells within Alvetex was the highest in the ERRM group, followed by MTA and by negative controls.ERRM promotes osteoblast differentiation better than MTA and controls with no material in a 3-dimensional culture system. Alvetex scaffolds can be used to test endodontic materials.Alvetex supports growth of mouse mandibular osteoblasts.Alvetex significantly induces greater expression of mouse osteoblastic markers than 2D cultures.Alvetex can be used to test the osteogenic potential of root-end filling materials. The results are the following:Stimulation of mouse osteoblast marker genes: ERRM > MTA > polystyrene.Number of 2.3 GFP positive cells (mature mOBs): ERRM > MTA > polystyrene.Number of total cells on Alvetex: polystyrene > ERRM > MTA.