Hydraulic pressure has recently been introduced as an effective stimulation in the field of tissue engineering. In this study, a polymer scaffold consisting of polyurethane (PU)-based 1, 4-butanediisocyanate was fabricated. A self-designed bioreactor was employed to produce perfusion and hydrodynamic pressure stimulations. The viability, proliferation and osteogenic differentiation of the rat bone mesenchymal stromal cell (rBMSC) growing in the polymer scaffold were investigated after hydrodynamic pressure stimulation. Additionally, the mechanical properties of the cell-laden constructs were also evaluated. Our findings suggested that the perfusion rate (10 mL/min) and low hydrodynamic pressure stimulation (60 mmHg, 0.5 Hz) maintained the viability of rBMSC during 2 weeks cultivation. The cell proliferation was promoted by 60 mmHg stimulation in the first week. The synthesis of alkaline phosphates and osteocalcin was enhanced after 2 weeks stimulation. Meanwhile, the equilibrium modulus of scaffold was increased by 1.85-fold using 60 mmHg hydrodynamic pressure stimulation. Additionally, type I and III procollagen produced by rBMSC was increased 4.92- and 3.02-fold, respectively. However, no encouraging results were detected in 120 mmHg hydrodynamic pressure group. Our study suggests that the 60 mmHg hydrodynamic pressure is a promising approach to enhance the functional properties of the rBMSC-laden PU-based bone scaffold. (c) 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3445-3455, 2017.