Salivary gland cell differentiation and organization on micropatterned PLGA nanofiber craters

Abstract

There is a need for an artificial salivary gland as a long-term remedy for patients suffering from salivary hypofunction, a leading cause of chronic xerostomia (dry mouth). Current salivary gland tissue engineering approaches are limited in that they either lack sufficient physical cues and surface area needed to facilitate epithelial cell differentiation, or they fail to provide a mechanism for assembling an interconnected branched network of cells. We have developed highly-ordered arrays of curved hemispherical "craters" in polydimethylsiloxane (PDMS) using wafer-level integrated circuit (IC) fabrication processes, and lined them with electrospun poly-lactic-co-glycolic acid (PLGA) nanofibers, designed to mimic the three-dimensional (3-D) in vivo architecture of the basement membrane surrounding spherical acini of salivary gland epithelial cells. These micropatterned scaffolds provide a method for engineering increased surface area and were additionally investigated for their ability to promote cell polarization. Two immortalized salivary gland cell lines (SIMS, ductal and Par-C10, acinar) were cultured on fibrous crater arrays of various radii and compared with those grown on flat PLGA nanofiber substrates, and in 3-D Matrigel. It was found that by increasing crater curvature, the average height of the cell monolayer of SIMS cells and to a lesser extent, Par-C10 cells, increased to a maximum similar to that seen in cells grown in 3-D Matrigel. Increasing curvature resulted in higher expression levels of tight junction protein occludin in both cell lines, but did not induce a change in expression of adherens junction protein E-cadherin. Additionally, increasing curvature promoted polarity of both cell lines, as a greater apical localization of occludin was seen in cells on substrates of higher curvature. Lastly, substrate curvature increased expression of the water channel protein aquaporin-5 (Aqp-5) in Par-C10 cells, suggesting that curved nanofiber substrates are more suitable for promoting differentiation of salivary gland cells.

Fig. 1

Creation and characterization of nanofibrous crater arrays. (A, B) After thermal reflow, photoresist mound arrays are highly ordered and uniform. (C) Plot shows a decrease in degree of curvature as feature radius increases. “Crater radius” refers to the radius of the circle used in the photolithography mask and is independent from the height of the mounds or the depth of the craters. Due to the nearly identical mound size within each array, standard deviations are negligible. (D) After transferring the arrays to PDMS, craters are formed which exhibit the negative shape and same order as the mounds. (E) Once PLGA nanofibers have been deposited onto craters and incubated in PBS for 3 days and cell media for 1 day, fibers confirm to the crater curvature and samples are ready for cell seeding. As a result of fiber stretching during conformation, pore size of fibers deposited on the flat areas (Ei) are smaller than those within the crater (Eii).

Fig. 2

SIMS mouse submandibular cell line conforms to the shape of 30 μm radius nanofibrous craters after 96 h of growth. SEM images show cell conformity from (A) top-down and (B) angled views. (C) A tilted view of a fluorescence confocal Z-stack 3-D projection with SIMS stained for F-actin (green, phalloidin), and nuclei (blue, DAPI), conforming to a crater lined with nanofibers (red). The Z-plane is denoted by the arrow. The arrangement of nuclei on (D) 30 μm, (E) 80 μm radius craters, and (F) flat nanofibers, reveals that nuclei preferentially localize within the craters, more obviously on 30 μm radius craters. Dotted circles outline a portion of the craters in each sample. On 80 μm radius craters, some regions exhibited cell overlapping (red arrows), whereas on 30 μm radius craters and flat nanofibers, cells grew largely as one monolayer over the entirety of the samples.

Fig. 3

Confocal top-down and cross-sectional views illustrating differences in height of SIMS cell monolayer on a (A) 30 μm radius crater, (B) flat nanofibers, and in (C) 3-D Matrigel. Cells on fibers are stained for F-actin (phalloidin, green) and nuclei (DAPI, blue). Nanofibers are stained red. Cells in Matrigel were stained for F-actin (phalloidin, green), and nuclei (DAPI, blue). (D) Measured heights of SIMS cell monolayers on craters of various sizes, flat nanofibers, and Matrigel. Measurements in craters were acquired at the center (bottom) of the craters. Thin Matrigel is a layer of Matrigel deposited such that it is too thin for cells to penetrate into and grow in full 3-D, whereas 3-D Matrigel is deep enough for cells to grow within the gel. *** indicates data point is significantly different from others (ANOVA, p < 0.001).

Fig. 4

(A) Confocal top-down images of nuclei (DAPI, blue) and expression of adherens junction protein E-cadherin (green) in SIMS cells grown on nanofibrous substrates of varying curvature. The protein is expressed and localized mostly to cell membranes on all substrate types. (B) A representative Western blot shows no definitive trend in E-cadherin expression as substrate curvature increases. Normalized band intensities are plotted beside blot image. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

(A) Confocal top-down images of nuclei (DAPI, blue) and expression of tight junction protein occludin (green) in SIMS cells grown on nanofibrous substrates of varying curvature. The protein is expressed and localized to cell membranes on all substrate types. (B) A representative Western blot shows an increase in occludin expression as substrate curvature increases. Normalized band intensities are plotted beside blot image. (C) Cross-sectional views of confocal z-stacks stained for nuclei (DAPI, blue), occludin (green), and fibers (red) show that occludin is more apically-localized (top of the cells) in SIMS on 30 μm radius craters as compared to those grown on flat nanofibers, where the protein fluorescence extends from the apical to the basal side of the cells. (D) Profiles of occludin fluorescence intensity for lines drawn though the apical (blue) and basal (red) side of SIMS cell monolayers on 30 μm radius craters and flat nanofibers, respectively. Cells on the crater show markedly larger peaks for a line drawn though the apical region of the cell monolayer as compared to the basal profile, whereas cells on flat nanofibers have similarly-sized peaks for both apical and basal regions. Areas of locally-intense staining in (C) and their corresponding graphical peaks in (D) are numbered.

Fig. 6

Par-C10 cell organization on nanofibrous substrates of varying curvature. The arrangement of nuclei in (A) 30, (B) 80 μm radius craters, and (C) flat nanofibers reveals that Par-C10 cells are more spread than SIMS, and nuclei do not preferentially localize within craters. Dotted circles outline a portion of the craters in each sample. Confocal top-down and cross-sectional views showing height of Par-C10 cell monolayer on (D) 30 μm radius crater (E) flat nanofibers, and (F) 3-D Matrigel respectively. The cross-sectional views reveal that cell monolayer layer heights for both nanofibrous substrates are distinctly lower than cells grown in 3-D Matrigel. Cells are stained for F-actin (phalloidin, green) and nuclei (DAPI, blue). Nanofibers are stained red. When Par-C10 heights were quantified (G), cells grown on 30 mm radius craters had significantly higher monolayer heights than cells on substrates of lesser curvature, and those growing in 3-D Matrigel exhibited strikingly higher cell layer heights than any of the other samples (ANOVA **p < 0.01, ***p < 0.001).

Fig. 7

(A) Confocal top-down images of nuclei (DAPI, blue) and expression of adherens junction protein E-cadherin (green) in Par-C10 cells grown on nanofibrous substrates of varying curvature. The protein is expressed and marginally localized to cell membranes on all substrate types. (B) A representative Western blot shows no definitive trend in E-cadherin expression as substrate curvature increases. Normalized band intensities are plotted beside blot image. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

(A) Confocal top-down images of nuclei (DAPI, blue) and expression of tight junction protein occludin (green) in Par-C10 cells grown on nanofibrous substrates of varying curvature. The protein is expressed and localized to cell membranes on all substrate types. (B) A representative Western blot shows that occludin expression increases as substrate curvature increases, especially on 30 μm radius craters. Normalized band intensities are plotted beside blot image. (C) Cross-sectional views of confocal z-stacks stained for nuclei (DAPI, blue), occludin (green), and fibers (red) show that occludin is more apically-localized (top of the cells) in Par-C10s on 30 μm radius craters as compared to those grown on flat nanofibers, where the protein fluorescence extends from the apical to the basal side of the cells. (D) Profiles of occludin fluorescence intensity for lines drawn though the apical (blue) and basal (red) side of Par-C10 cell monolayers on 30 μm radius craters and flat nanofibers, respectively. Cells on the crater show markedly larger peaks for a line drawn though the apical region of the cell monolayer as compared to the basal profile, whereas cells on flat nanofibers have similarly-sized peaks for both apical and basal regions. Areas of locally-intense staining in (C) and their corresponding graphical peaks in (D) are numbered.

Fig. 9

Water channel aquaporin-5 (Aqp-5) expression in Par-C10 cells on engineered substrates. A representative Western blot and corresponding normalized band intensity plot showing that expression of Aqp-5 increases when cells are grown on 30 μm craters as compared to substrates of lesser curvature. An additional intermediate crater size of 50 μm radius was analyzed in these experiments.