A model for in vitro three dimensional cultures of osteoblast-like cells

Bone is dynamic tissue built by osteoblasts. When implanting exogenous matherial to correct bone defects, osteointegration by osteoblasts is desirable. However, in vitro analysis approaches for testing new implant matherial are at present insufficient. We set up a novel three-dimensional model of osteoblast cell culture, that we believe is most suitable for investigating the mechanism of osteointegration. The osteoblast-like cell line ROS.SMER = 14 was grown into a rotating (16rpm) wall vessel bio-reactor (RWV) for 48 hr, then cells were withdrawn and evaluated for their phenotype. Under this culture condition, osteoblasts formed three-dimnensional multicellular aggregates of variable size, termed organoids, in which cells survived in suspension without contacts with the vessel wall. The organoids were disaggregated and the osteoblasts harvested. These cells expressed specific osteoblast markers. They were positive for the alkaline phosphatase activity, and expressed the bone-matrix proteins osteopontin, osteonectin, and bone sialoprotein II. They also expressed the bone morphogenetic proteins (BMP)-2 and –4, and the cytokine interleukin-6, thus confirming the maintenance of the osteoblast phenotype. We believe that the three-dimensional organoids obtained by culturing osteoblasts in the RWV device may provide a novel model for testing the osteointegration ability of implant materials prior to in vivo applications.