ATC rescues osteogenic differentiation of hMSCs on soft substrates. (A) Bright field images showing hMSCs stained for ALP after 6 d of culture in osteogenic differentiation medium on PDMS micropost arrays (PMAs) with different rigidities as indicated. hMSCs were loaded with microbubbles (MBs) before ultrasound (US) stimulation (+US +MB). Negative control of hMSCs without MBs and US stimulation (−US −MB) was included for comparison. Scale bar, 250 μm. (B) Bar plot showing percentage of hMSC osteogenesis as a function of substrate rigidity under different conditions as indicated. hMSCs without MBs but stimulated with US (+US −MB) were included for comparison. (C) Relative expression of osteogenic markers in hMSCs cultured on PMAs with different rigidities. Expression levels were normalized to the value of TBP gene. For +US +MB group, hMSCs were stimulated using US each day for 5 min, with US parameters being frequency 1 MHz, pulse repetition frequency 10 Hz, pulse duration 50 ms, treatment duration 5 min, and acoustic pressure 0.08 MPa. Each day before US stimulation, cells were re-coated with MBs with the average MB number per cell kept as constant. Error bar denotes SEM, with n ≥ 3. *, P < 0.05; **, P < 0.01.