Abstract
Though the three-dimensional (3D) in vitro culture system has received attention as a powerful tool for conducting biological research, in vitro bone formation and osteocyte differentiation studies have mostly been based on results obtained using two-dimensional (2D) culture systems. Here, we introduced a rotatory culture system to fabricate 3D spheroids, using mouse osteoblast precursor cells. These spheroids, incubated for 2 days without chemical induction by osteogenic supplements, exhibited notably up-regulated osteocyte marker levels; osteoblast marker levels were down-regulated, as compared to those of the conventional 2D monolayer model. The cell condensation achieved with the 3D spheroid structure triggered a greater level of differentiation of osteoblast precursor cells into osteocyte-like cells than that observed during chemical induction. Our study might imply that osteoblasts proliferate and become condensed at the targeted bone remodeling site, because of which osteoblasts achieved the capability to differentiate into osteocytes in vivo.
Highlights
Three-dimensional (3D) culture systems known as organoids have recently been in focus, as they have been used to recapitulate the morphogenesis and functioning of various types of organs (Sasai, 2013; Rossi et al, 2018), such as the small intestine (Spence et al, 2011), liver (Takebe et al, 2013), stomach (McCracken et al, 2014), and lungs (Dye et al, 2015)
We examined the changes in the expression of distalless homebox 5 (Dlx5), bone sialoprotein (Bsp), and Ocn
Because we hypothesized that the cell condensation plays a significant role in osteogenesis differentiation, we utilized 3D spheroid models, which enabled us to evoke the cell condensation
Summary
Three-dimensional (3D) culture systems known as organoids have recently been in focus, as they have been used to recapitulate the morphogenesis and functioning of various types of organs (Sasai, 2013; Rossi et al, 2018), such as the small intestine (Spence et al, 2011), liver (Takebe et al, 2013), stomach (McCracken et al, 2014), and lungs (Dye et al, 2015). The 3D structure of cells inducing the appropriate cell-cell interactions during the organ formation process has become known; the demand for the generation of a 3D in vitro culture system by researchers studying human development, disease, and drug screening has increased (Rossi et al, 2018). The structural effects and configurations of cells in the 3D culture system, and especially cellular behavior, including differentiation capability, are not fully understood yet. A conventional two-dimensional (2D) culture system has greatly enabled us to understand cellular behavior, including gene expression and homeostasis, it might alter several intracellular signaling pathways, as compared to those present in vivo, thereby causing distinct biological outcomes. A more detailed study using the 3D culture system would be required to understand cellular behavior in complex native structures. The bone is composed of mineralized collagen fibrils induced via the formation
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