A multilayered scaffold for regeneration of smooth muscle and connective tissue layers

Supplemental Figure S1 Fiber organization and orientation on opposite sides of the multilayered scaffold. (A) Representative fluorescence confocal maximum intensity projections of the aligned and unaligned sides of the E1001(1k) scaffold. Scale bar = 50 μm. (B) Quantification of the scaffold fiber orientations by Fast Fourier Transform (FFT) analysis of confocal slices is shown. Images were captured at 0, 8, and 26 μm into the scaffold starting from the aligned side (AL, left) and 0, 10, and 26 μm in starting from the unaligned side (UN, right). Peaks between 45 and 90° for AL 0 and AL 8 μm confocal planes indicate that the fibers are oriented in the same direction. The lack of major peaks in the plots taken from the unaligned side as well as for AL 26 μm show that the fibers in these planes and all UN planes have no preferred orientation.

Supplemental Figure S2. Cell organization on different sides of the multilayered scaffold. NIH 3T3 fibroblasts were grown on either the aligned (top) or unaligned (bottom) side of scaffolds. Cells were fixed and stained with DAPI and Texas-Red phalloidin, to detect nuclei (blue) and actin (red), respectively. Horizontal actin filament orientations on the aligned side indicate cell alignment with the scaffold fibers. Scale bar = 50 μm.

Supplemental Figure S3. Cell infiltration into the unaligned and aligned sides of a multilayered electrospun scaffold. The depth of fibroblast infiltration into each side of the multilayered scaffold was visualized by the localization of DAPI staining (blue) in shaded-volume reconstructions showing orthogonal views. Dimensions are indicated on the right. The scaffold is green.

Figure S4: Decellularization of the electrospun scaffold. NIH 3T3 fibroblasts (A and B) or WKY3M22 smooth muscle cells (C) were cultured on either the aligned (A and C) or unaligned (B) side of the E1001(1k) scaffold for 7 days. The scaffolds were decellularized, fixed and stained with R457 anti-fibronectin antiserum. Decellularized cultures of NIH 3T3 and WKY3M22 rat aortic SMCs on the scaffold showed intact FN fibrils oriented in similar directions to scaffold fibers. Representative reflectance (Scaffold) and fluorescence (FN) images of the same fields of view are shown. Scale bar = 50 μm. (D) Orientation of the decellularized FN on the scaffolds was quantified by FFT analysis as in Figure 2. FFT analyses confirmed the orientations of the FN matrix fibrils were similar to the orientation of the underlying scaffold fibers.

Supplemental Figure S5. Delivery of TGF-β1 to smooth muscle cells through an agarose droplet. (A) Schematic depicting experimental setup. (B) A 1% agarose droplet containing 2 ng TGF-β1 was pipetted onto the unaligned side of a transwell scaffold and allowed to solidify. Total release of TGF-β1 into the culture medium will yield a concentration of 2 ng/ml. WKY3M22 SMCs were seeded onto the opposing aligned side of the scaffold and cells were grown for 5 days before fixing and staining with an antibody specific to α-SMA. The fold-change in mean fluorescence intensities of α-SMA in cells with and without TGF-β1 are shown on the images (mean ± S.E.) (n = 3). Scale bar = 50 μm. Note that SMCs grown with the TGF-β1 droplet had prominent α-SMA stress fibers aligned with the underlying scaffold fibers. Mean fluorescence intensity averaged 1.4 ± 0.1-fold higher than control (p < 0.05). This increase was not significantly different (p = 0.37) from the increase in α-SMA intensity when TGF-β1 was added directly to the media.

Supplemental Figure S6. Formation and evaluation of cell growth on a tubular scaffold. (a) Images depicting the rolled scaffold with plastic cylinders to hold the shape during culture (top) and maintenance of a tubular shape after cylinder removal (bottom). (b) 4 x 104 cells/cm² were seeded onto the aligned side of a scaffold sheet and allowed to grow in complete medium for the indicated times. Cells were fixed and stained with DAPI and Texas-Red phalloidin to detect nuclei and actin, respectively. The scaffold fibers were visualized by confocal microscopy in reflectance mode. Difference in cell densities at 8 hr and 7 days demonstrates that cells grew to confluence in the tubular scaffold culture. Scale bar = 50 μm.

Supplemental Figure S7. SEM images of an electrospun tube. (A) Picture of an electrospun bilayer tube that was prepared by depositing, on either a 4 mm or 6 mm diameter rod, a 10% polymer solution at high speed of rotation for 15 min to obtain an aligned layer followed by low speed of rotation for additional 15 min to obtain an unaligned layer. Rods were dipcoated with 100 kDa PEG dissolved in water and dried before electrospinning; this way, the electrospun tube was able to slide off the rods through immersion in water. Scale bar = 1 cm. (B) SEM images showing the fiber orientation for the aligned (inside) and unaligned (outside) scaffold sides of a 6 mm diameter tube. The fibers are ~1 μm in diameter and are densely packed on the aligned side and have larger pores on the unaligned side. Scale bar = 100 μm.