The aim of this study was to investigate the effects of perfusion co-culture on bone tissue regeneration in vitro. Human mesenchymal stem cell (hMSC)-derived osteoblasts and THP-1 human acute monocytic leukemia cell line-derived osteoclasts were dynamically co-cultured on the chitosan-hydroxyapatite (chitosan-HA) superporous hydrogel. In the perfusion bioreactor set-up, bidirectional recycled perfusion with 6 mL/h flow rate was applied and cell seeding was realized in two-steps with a preculture time of 12 days. Outcomes were compared to static cultures. Two-step cell seeding and long preculture ensured good adhesion of cells on the scaffold surface and minimized cell loss during perfusion. The perfusion bioreactor enhanced mass transfer throughout the scaffolds, thus increased cellularity and provided flow-induced mechanical stimulation for osteoblastic and osteoclastogenic differentiation. The results indicated that osteoblast and osteoclaft co-cultures in perfusion bioreactors provide a one-step approach to in vitro bone tissue engineering and emphasized the significance of enhanced mass transfer and mechanical stimulation on cellular activity and differentiation. (C) 2016 Elsevier Ltd. All rights reserved.