Pre-osteoblast MC3T3-E1 cells were cultured in hyaluronic acid-modified chitosan/collagen/nano-hydroxyapatite (HA-CS/Col/nHAP) composite scaffolds and treated with phytoestrogen α-zearalanol (α-ZAL) to improve bone tissue formation for bone tissue engineering. Perfusion and dynamic strain were applied to three-dimensional (3D) cultured cells, which simulates mechanical microenvironment in bone tissue and solves mass transfer issues. The morphology of cell-scaffold constructs in vitro was then examined and markers of osteogenesis were assessed by immunohistochemistry staining and western blotting. The results showed that cells expanded their pseudopodia in an irregular manner and dispersed along the walls in 3D-dynamic culture. Osteogenic phenotype was increased or maintained by enhanced collagen I (COLI) levels, decreased osteopontin expression and having little effect on osteocalcin expression during the 12 days of in vitro culture. In response to α-ZAL, the cell-scaffold constructs showed inhibited cellular proliferation, enhanced the alkaline phosphatase (ALP) activity and increased ratio of osteoprotegerin to receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL). Application of perfusion and dynamic strain to cells-scaffold constructs treated with α-ZAL represents a promising approach in the studies of osteogenesis stimulation of bone tissue engineering.