FIG. 4.

Proteoglycan staining of modular constructs. At day 14, large differences in blue proteoglycan staining (Alcian blue, pH 2.5) were observed between the EC, MSC, and MSC+EC conditions. Nuclei were counterstained in red. Low- and high-magnification images are shown, with the boxes in the low-magnification images showing the location of the higher-magnification image. These differences were not seen at earlier times. For EC-only modules under flow, neither cell nor proteoglycan staining was observed, whereas static controls showed cells (on the module periphery (red)) but no proteglycan staining. In MSC-only modules, a small cloud of proteoglycan surrounded embedded MSC with flow (visible in the higher-magnification images), which was not seen in static controls. For MSC+EC modules, more cell and proteoglycan staining was observed within the intermodular regions (on the periphery of the modules) with flow. This contrasted with the lack of staining in static controls, which was similar to that of the MSC-only static controls. Thus, it appeared that flow enhanced the amount of proteoglycan synthesis for flow-conditioned constructs, illustrating changes in the overal remodeling process. Scale bars=25 μm. Color images available online at www.liebertonline.com/tea