Bone disorders represent a significant global burden. Currently, animal models are used to develop and screen novel treatments. However, interspecies variations and ethical concerns highlight the need for a more complex 3D bone model. In this study, we developed a simplified in vitro bone-like model using a U-CUP perfusion-based bioreactor system, designed to provide continuous nutrient flow and mechanostimulation through 3D cultures. An immortalized human fetal osteoblastic cell line was seeded on collagen scaffolds and cultured for 21 days in both a perfusion bioreactor system and in static cultures. PrestoBlue™ assay, scanning electron microscopy, and proteomics allowed monitoring of metabolic activity and compared morphological and proteome differences between both conditions. Results indicated an altered cellular morphology in the bioreactor compared to the static cultures and identified a total of 3494 proteins. Of these, 105 proteins exhibited significant upregulation in the static culture, while 86 proteins displayed significant downregulation. Enrichment analyses of these proteins revealed ten significant pathways including epithelial-mesenchymal transition, TNF-alpha signaling via NF-kB, and KRAS pathway. The current data indicated of osteogenic differentiation enhancement within the bioreactor on day 21 compared to static cultures. In conclusion, the U-CUP perfusion bioreactor is beneficial for facilitating osteogenic differentiation in 3D cultures.