AbstractBioreactor systems are very precious tools to generate living bone grafts in vitro. The aim of this study was to compare the effectiveness of rotating and perfusion bioreactor in the production of a living bone construct. Human bone marrow-derived mesenchymal stem cells (BMDSCs) were seeded on the surfaces of hydroxyapatite-based scaffolds and cultured for 21 days in three different conditions: (1) static 3D culture, (2) 3D culture in a perfusion bioreactor, and (3) dynamic 3D culture in a rotating bioreactor. Quantitative evaluation of cell number showed that cultivation in the perfusion bioreactor significantly reduced cell proliferation compared to the rotating bioreactor and static culture. Osteogenic differentiation test demonstrated that BMDSCs cultured in the rotating bioreactor produced significantly greater amount of osteopontin compared to the cells cultured in the perfusion bioreactor. Moreover, Raman spectroscopy showed that cultivation of BMDSCs in the rotating bioreactor enhanced extracellular matrix (ECM) mineralization that was characterized by B-type carbonated substitution of hydroxyapatite (associated with PO43− groups) and higher mineral-to-matrix ratio compared to the ECM of cells cultured in the perfusion system. Thus, it was concluded that the rotating bioreactor was much more effective than the perfusion one in the generation of bone tissue construct in vitro.