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Growth and differentiation of osteoblasts are often studied in cell cultures. In vivo, however, osteoblasts are embedded within a complex three-dimensional (3D) microenvironment, which bears little relation to standard culture flasks. Our study characterizes osteoblast-like cells cultured in 3D collagen gels and compares them with cells in two-dimensional (2D) cultures. Primary rat osteoblasts and MC3T3-E1 cells were seeded within type I collagen gels, and differentiation was determined by mineral staining and gene expression analysis. Cells growing in 3D gels showed positive mineral staining and induction of osteoblast marker genes earlier than cells growing in 2D. A number of genes, including osteocalcin, bone sialoprotein, alkaline phosphatase and dentin matrix protein 1, were already highly upregulated in 3D cultures 24 h after seeding. The early expression of osteoblast genes was dependent on the 3D structure and was not induced in cells growing on collagen-coated dishes in 2D. Comparison of thymidine incorporation between cells in 3D and 2D cultures treated with agents that induce proliferation-transforming growth factor β, platelet-derived growth factor and lactoferrin-showed a much greater response in 3D gels. Cells in 3D cultures were also much more sensitive to inhibition of proliferation by the protein kinase inhibitor imatinib mesylate. The 3D collagen gels better represent the physiological bone environment and offer a number of technical advantages for the study of osteoblasts in vitro. These studies have additional practical implications as 3D collagen gels are considered as a scaffold material in regenerative medicine for the repair of bone defects.

Original publication




Journal article


Bonekey Rep

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