FIG. 8.

(a) Low- and high-magnification images of MSC differentiation into desmin⁺ (brown) cells. Nuclei appear blue. The boxes in the low-magnification images (upper rows) show the location of the high-magnification images (lower rows). The migration and organization of desmin⁺ cells appeared to parallel those of the SMA⁺ cells in Figure 7a. No desmin staining was observed in EC-only modules (data not shown). Scale bars=25 μm. (b) The effect of flow and EC on MSC differentiation into desmin⁺ cells. The graph shows the number of desmin⁺ cells (bars) and the total number of cells (inset table) found within an average 10×field of the remodeling chambers' histological sections. For static cases (white bars), almost none of the cells were desmin⁺, independent of the presence of EC. This contrasts with flow cases (grey bars), in which EC showed a clear effect. For MSC-only flow cases, the number of desmin⁺ cells increased modestly over time, although this increase was not statistically significant. This modest increase contrasted with the patent increase in desmin⁺ cells for MSC+EC flow cases such that day 14 and 21 showed significantly more desmin⁺ cells than at day 7 or the corresponding static controls (p<0.05, ANOVA). Thus, flow and EC increased the number of desmin⁺ cells within the constructs. An increase in the total cell number accompanied the greater number of desmin⁺ cells for MSC+EC flow cases at day 21. The total number of cells was significantly higher than with day 21 MSC+EC static controls and MSC-only flow cases. Moreover, there was a rise in the total cell number between days 7 and 21 for MSC+EC flow cases. Similar to the SMA results, the increased number of desmin⁺ cells was likely caused by a combination of MSC differentiation as well as MSC proliferation followed by differentiation. (c) The effect of flow on the population of desmin⁺ cells. The percentage of desmin⁺ cells was determined as the ratio of the number of desmin⁺ cells to the total number of cells. For MSC-only flow cases, no statistically significant changes in the proportion of desmin⁺ cells were found, and desmin⁺ cells were in the minority. For MSC+EC constructs, there was almost no desmin⁺ MSC population in the static cases. With flow, the population of desmin⁺ cells was significantly higher than in static controls at all time points, and by day 14, the majority of cells within the remodeling chambers were desmin⁺. This illustrates the ability of flow and EC to guide the differentiation of MSC toward a desmin⁺ phenotype. Connecting lines represent a statistically significant difference between conditions (p<0.05, ANOVA), all error bars are±SEM, and the number of analyzed remodeling chambers is given in the inserted table. F, flow; S, static. Color images available online at www.liebertonline.com/tea