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 cytosk eletal organization of cells. 2-D images were exhaustive when the cells wer e seeded at low density (in the first 24 h of incubation), but they were le ss clear when the cells proliferated and appeared stacked. Since the sharew are software were not satisfactory, a new 3-D image reconstruction was deve loped using ordinary software and a model was obtained directly from the op tical section set, in order to achieve a more realistic and faithful vision of morphological structures and to evaluate the behaviour of bone cells gr own on materials. The results showed that the cells grown on titanium confo rm to the irregular substrate surfaces maximizing the contact between the c ell membrane and the substrate and proliferate disposing close to each othe r. On the contrary, the osteoblasts seeded onto TiO2/HA coating develop clu sters 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 f irst signal of the tendency of Ti02/11A coating to stimulate cell different iation, (C) 2001 Elsevier Science Ltd. All rights reserved.