FIG. 7.

(a) Low- and high-magification images of MSC differentiation into smooth muscle actin (SMA)⁺ (brown) cells. Nuclei appear blue. The boxes in the low-magnification images (upper rows) show the location of the high-magnification images (lower rows). No SMA staining was observed in EC-only modules (data not shown). For static MSC-only modules, SMA⁺ cells were sparsely located throughout the modular construct. With flow, the MSC migrated toward the intermodular region, and by day 21, SMA⁺ cells were found around the surface of modules, as well as within the intermodular spaces. The presence of EC and flow appeared to accelerate the appearance of SMA staining and the MSC migration such that many SMA⁺ cells were observed in the intermodular region at day 21. Scale bars=25 μm. (b) The effect of flow and EC on MSC differentiation into SMA⁺ cells. The graph shows the number of SMA⁺ cells (bars) and the total number of cells (inset table) found within the average 10×field of the constructs' histological sections. The number of SMA⁺ cells in MSC-only cases was similar for both static and flow conditions. When EC were added to the system, static cases showed no changes in the number of SMA⁺ cells, but when flow was added to the system, significantly more SMA⁺ cells were observed, suggesting that the flow-conditioned EC were guiding the differentiation of MSC into SMA⁺ cells. The day 21 increase in SMA⁺ cells under the MSC+EC flow conditions resulted in concomitantly more total cell numbers than in the corresponding day 21 MSC+EC static controls. Differentiation of existing MSC and MSC proliferation followed by differentiation probably caused the greater number of SMA⁺ cells. (c) The effect of flow on the population of SMA⁺ cells. The ratio of SMA⁺ cells to the total number of cells was used to determine the percentage of SMA⁺ MSC. For MSC-only constructs, no statistically significant differences in the population of SMA⁺ cells were observed under static or flow conditions, but for MSC+EC constructs, flow caused an immediate increase in the proportion of SMA⁺ cells at day 7, illustrating the ability of flow and EC to accelerate MSC differentiation into SMA⁺ cells. The SMA⁺ cells also remained the dominant cell type, despite the presence of EC. Connecting lines represent statistically significant differences between conditions (p<0.05, ANOVA); all error bars are±SEM, and number of analyzed remodeling chambers is given in the inset table. F, flow; S, static. Color images available online at www.liebertonline.com/tea