Construct thickness and cellularity.

<p>(A) Shows a graph of construct thickness at 4 weeks. TGF-ß3 treatment led to a significant increase in construct thickness for all conditions (*p<0.05). (<b>B</b>)_Graph of construct's total cells calculated from counts in optical sections of fixed cultures at 4 weeks incubation. Both cell types showed similar numbers ± TGF-ß3 when serum was present. CSSCs without serum had a significantly lower cell density (*p<0.05). Error bars = SD (n = 3).</p

Expression of keratocyte genes by HCF and CSSC.

<p>The absolute mRNA abundance in cells before culture (yellow) and after 4 weeks of culture without TGF-ß3 (red) or with TGF-ß3 (blue) was determined for 6 genes associated with keratocyte phenotype (ALDH3A1, AQP1, B3GNT7, CHST6, KERA, PTGDS) as described in Methods. Error bars show standard deviation of triplicate assays. Some error bars are too small to be visualized on the plot.</p

Collagen Type III.

<p>Confocal micrographs demonstrating immunolocalization of type III collagen at 4 weeks in (A, B) HCF, (C, D) CSSC in serum-containing media, and (E, F) CSSC in serum-free media. Constructs treated without TGF-ß3 are on the left (A, C, E) and with TGF-ß3 are on the right (B, D, F). Bar = 50 microns.</p

Collagen Type I in constructs.

<p>Confocal micrographs demonstrating immunolocalization of type I collagen at 4 weeks in (A, B) HCF, (C, D) CSSC in serum-containing media, and (E, F) CSSC in serum-free media. Constructs treated without TGF-ß3 are on the left (A, C, E) and with TGF-ß3 are on the right (B, D, F). Bar = 50 microns.</p

Ultrastructural Features of Polycarbonate Transwell Membranes.

<p>(A) Scanning electron micrograph (5000×) shows the 0.45 µm pores in the membranes. Scale Bar is 10 µm. (B) A 20,000× view shows parallel features on the membranes. Bar is 1 µm. (C) A portion of the image in (B) (0.5 µm×5 µm) was transformed using the “Surface Plot” algorithm of FIJI image analysis software to illustrate the regularity and spacing of the parallel features. Physical depth of the apparent grooves was not measured directly, but approximately 23 ridges are seen in the 5 µm section indicating an average spacing of 218 nm. (D) Electrospun PEUU nanofibers used as substratum for previous studies of CSSC average 165 nm in diameter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086260#pone.0086260-Wu1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086260#pone.0086260-Wu2" target="_blank">[19]</a> and the spacing in this sample averaged 227 nm. Bar = 1 µm.</p

Second harmonic generation images of cell constructs.

<p>Images generated by fibrillar collagen were collected by excitation at 830–4×2 µm optical sections from a series of images through the entire construct. Below each image is an image representing a 100 µm×100 µm area of the full thickness image stack rotated on the X-axis. The height of these images provides representation of the thickness of the constructs. Cell distribution is visualized by Sytox Green staining of nuclei appearing green. (A, B) HCF (C, D) CSSC+Serum and (E, F) CSSC–serum free. Cultures were carried out without TGF-ß3 (A, C, E) or with TGF-ß3 (B, D, F).</p