Figure 5.

Indirect immunofluorescence of fibrotic markers—SMA (A, B), type III collagen (C, D), and EDA-Fn (E, F)—on full-thickness constructs at 8 weeks that were treated with TGF-β1 for various times: T1–1w: TGF-β1 treatment for the first week, then construct medium for the remaining 7 weeks; and T1–4w: construct medium for the first 4 weeks, then TGF-β1 treatment for the remaining 4 weeks. Single plane-of-focus confocal images were selected from the brightest area of immunofluorescence. Four- and 8-week T1–1w constructs appeared to be similar; therefore, since T1–4w was collected only at 8 weeks, we compared T1–1w at 8 weeks. With the addition of TGF-β1 for only 1 week (T1–1w), there appeared to be a slight increase in the fibrotic markers (A, C, E). As the time of TGF-β1 exposure increased (T1–4w), so did the amount of myofibroblasts (B), type III collagen (D), and EDA-Fn (F). Of interest, the point at which TGF-β1 was added to the system (first week or last four) did not seem to be important; however, the length of time TGF-β1 was added to the system appeared to affect the expression of the fibrotic markers. A 1-week treatment appeared to increase the markers, but not to the same extent as the 4-week treatment. Bar, 50 μm.