Comparison of Actin Cytoskeleton Between 2D and 3D Cultured MC3T3-E1

Objective: Differentiation of osteoblast to osteocyte associates with dramatic change in their shape with reorganization of actin cytoskeleton. The cuboidal-shaped osteoblasts that occupy bone surface switch to star-shaped osteocytes as encased in the bone. In the meanwhile, it is reported that osteoblast-like cell line MC3T3-E1 in collagen gel develop long processes similar to the osteocyte network in bone. Therefore, we examined the actin cytoskeleton in MC3T3-E1 in 2 dimensional (2D) and 3 dimensional (3D) culture. In addition, cytoskeleton of 3D-cultured MC3T3-E1 cell was compared with that of 3D-cultured primary osteocyte. Methods: Cells were incubated on/within the collagen type I gel. Primary osteocytes are isolated from 16-day-old chick embryonic calvaria with serial treatment of collagenase and EDTA. Fluorescence staining was performed for visualizing actin and actin-binding proteins with their antibodies. 3D reconstruction was performed by IMARIS software from optically sliced images taken by confocal microscopy. Results: 2D-MC3T3-E1 displayed a flattened morphology with thick bundles of actin filaments 3h after seeding. On the other hand, 3D-MC3T3-E1 dramatically started to form multiple processes from 1h and had well-developed branches after 3h. The processes and branches were confirmed to be actin rich as seen in 3D-primary osteocytes. Next, we focused on the distribution of actin-binding proteins. In 2D-MC3T3-E1, alpha-actinin, myosin, and tropomyosin were located in the stress fibers. In 3D-MC3T3-E1, those proteins were reorganized in their processes and branches. Fimbrin, which was diffusely distributed in cell body in 2D-MC3T3-E1, reorganized to the cell processes in 3D-gel culture. Interestingly, the distribution of actin-binding proteins in 3D-MC3T3-E1 was similar to that in 3D-primary osteocytes. Conclusions: MC3T3-E1 changes their shape to osteocytic morphology with similar distribution of actin cytoskeleton of primary osteocytes when the cells were cultured in 3D-gel.