In orthopaedic surgery the tissues damaged by injury or disease could be replaced using constructs based on biocompatible materials, cells and growth factors. Scaffold design, porosity and early colonization are key components for the implant success. From biological point of view, attention may be also given to the number, type and size of seeded cells, as well as the seeding technique and cell morphological and volumetric alterations. This paper describes the use of the microCT approach (to date used principally for mineralized matrix quantification) to observe construct colonization in terms of cell localization, and make a direct comparison of the microtomographic sections with scanning electron microscopy images and confocal laser scanning microscope analysis. Briefly, polycaprolactone scaffolds were seeded at different cell densities with MG63 osteoblastic-like cells. Two different endpoints, 1 and 2 weeks, were selected for the three-dimensional colonization and proliferation analysis of the cells. By observing all images obtained, in addition to a more extensive distribution of cells on scaffolds surfaces than in the deeper layers, cell volume increased at 2 weeks compared to 1 week after seeding. Combining the cell number quantification by deoxyribonucleic acid analysis and the single cell volume changes by confocal laser scanning microscope, we validated the microCT segmentation method by finding no statistical differences in the evaluation of the cell volume fraction of the scaffold. Furthermore, the morphological results of this study suggest that an effective scaffold colonization requires a precise balance between different factors, such as number, type and size of seeded cells in addition to scaffold porosity.