HFOb cell count and differentiation assay for the four groups tested. (A) cell count for the Flat, 500, 250 and 100 mc throughout four time points: 6, 12, 18, and 24 days. No proliferation is seen throughout the four weeks. There is a significant decrease in cells found on the 100 group at the last time point. Also seen are the differentiation markers RUNX2 (B), ALP (C), OPN (D). The RUNX2 groups do not show any significant difference between the groups although we can see a trend in which the 250 μm diameter microchannels have lower levels than the rest of the groups. The ALP activity shows two different peaks at day 12 and at day 24. At the last time point the 500 and 250 μm diameter microchannels showed a significant higher ALP activity than the Flat control group. The OPN levels do show significant difference on day 18 between the 100 μm diameter microchannels and the control group. At day 24 the significant difference was seen between the 500 μm and the Flat. All data shown as mean ± standard error. * P < 0.05; ^ P < 0.01; # P < 0.001.