Three-dimensional reconstruction of confocal laser microscopy images to study the behaviour of osteoblastic cells grown on biomaterials.

The adhesion, spreading and cytoskeletal organization of osteoblastic cells seeded onto titanium and titania/hydroxyapatite composite coating (TiO2/HA) were studied using images acquired by confocal laser scanning microscopy. The fluorescence staining technique was employed to visualize actin cytoskeletal organization of cells, 2-D images were exhaustive when the cells were seeded at low density (in the first 24 h of incubation), but they were less clear when the cells proliferated and appeared stacked. Since the shareware software were not satisfactory, a new 3-D image reconstruction was developed using ordinary software and a model was obtained directly from the optical section set, in order to achieve a more realistic and faithful vision of morphological structures and to evaluate the behaviour of bone cells grown on materials. The results showed that the cells grown on titanium conform to the irregular substrate surfaces maximizing the contact between the cell membrane and the substrate and proliferate disposing close to each other. On the contrary, the osteoblasts seeded onto TiO2/HA coating develop clusters where the cells aggregated extending processes in order to establish intercellular connections. Cell aggregation is an early and critical event leading to cell differentiation and mineralization process and could be a first signal of the tendency of TiO2/HA coating to stimulate cell differentiation.