Influence of polydimethylsiloxane substrate stiffness on corneal epithelial cells

Abstract

Many cell types are known to modulate their behaviour in response to changes in material stiffness; however, little is known about how stiffness affects corneal epithelial cells. This study aims to investigate the response of a corneal epithelial cell line to polydimethylsiloxane (PDMS) substrates with a range of Young's moduli from 10 to 1500 kPa. Cellular morphology, proliferation, differentiation and mechanobiology were examined. Cells grown on PDMS adopted the typical cobblestone morphology exhibited by the corneal epithelium. Proliferative markers pERK and Ki67 were higher in cells cultured on stiffer substrates compared with those on softer substrates. Material stiffness was also found to influence the cell phenotype with cells on stiffer substrates having higher cytokeratin 3 gene expression, a mature epithelial marker, while cells on softer substrates expressed more cytokeratin 14, a basal epithelial marker. Cells grown on softer substrates also displayed higher levels of focal adhesions and intermediate filaments compared with cells on stiff substrates. This research will aid in designing novel biomaterials for the culture and transplantation of corneal epithelial cells.

Keywords: cornea; epithelium; mechanobiology; substrate stiffness.

Figure 1.

Characterization of PDMS substrates. (a) Thickness of spin-coated PDMS; (b) Young's modulus of PDMS; (c) contact angle of PDMS and TCP; (d) percentage of cell adhered to substrates after 4 h seeding. Data are presented as mean + s.d., significance calculated via one-way ANOVA with post-Tukey test, N = 4–6, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 2.

Metabolic activity and proliferation of cells in response to different substrate stiffness: (a) metabolic activity of cells after 7 days in culture; (b) western blot of cells grown on different substrates for pERK, TERK and GAPDH proteins at day 7; (c) densitometry analysis of western blot data at day 7, all data normalized to TERK and GAPDH; (d) fluorescence intensity of Ki67 staining at day 3 (i) and day 7 (ii); (e) Ki67 staining of cells grown on different substrates at day 3 (i)–(v) and day 7 (vi)–(x) (scale bar, 10 µm). Data are presented as the mean (±s.d.), significance calculated via one-way ANOVA with the post-Tukey test, N = 3–6, *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

Figure 3.

Real-time PCR to determine relative expression after 3 and 7 days: (a) CK3 gene expression at day 7; there was no detection of CK3 at day 3 in any groups; (b) CK14 gene expression; (c) ABCG2 gene expression; (d) ΔNP63 gene expression. Data are presented as the mean (±s.d.), significance calculated via one-way ANOVA for CK3 and two-way ANOVA for CK14, ΔNP63 and ABCG2 with the post-Tukey test, N = 3, *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

Figure 4.

Western blot of CK14 and isoforms of ΔNP63 at day 7: (a)(i) Sample western blot and (ii) densitometry analysis for CK14 and GAPDH; (b)(i) sample western blot and (ii) densitometry analysis of each NP63 isoforms. Data are presented as the mean (±s.d.), significance calculated via one-way ANOVA with the post-Tukey test, N = 3–4, ***p ≤ 0.001.

Figure 5.

Immunocytochemical staining for stem cell markers after 7 days. (i) Fluorescence intensity of ABCG2 staining and (ii) NP63 staining. Top row (a–e) stained in green for ABCG2 protein; second row (f–j) stained in green for NP63. All cells were counterstained with f-actin (red) (scale bar, 10 µm). Data are presented as the mean (±s.d.), significance calculated via one-way ANOVA with the post-Tukey test, N = 3.

Figure 6.

Immunocytochemical staining for vinculin. (i) Fluorescence intensity of vinculin at day 3 and (ii) day 7. (a–e) Cells stained in green for vinculin at day 3. (f–j) Cells at day 7. All cells were counterstained with f-actin (red) and DAPI (blue) (scale bar, 10 µm). Data are presented as mean (±s.d.), significance calculated via one-way ANOVA with post-Tukey test, N = 3, *p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001.

Figure 7.

Immunocytochemical staining for vimentin. (i) Fluorescence intensity of vimentin at day 3 and (ii) day 7. (a–e) Cells stained in green for vimentin at day 3. (f–j) Cells at day 7. All cells were counterstained with f-actin (red) and DAPI (blue) (scale bar, 10 µm).

Figure 8.

Immunocytochemical staining for phosphorylated YAP (pYAP) protein. (i) Fluorescence intensity of pYAP at day 3 and (ii) day 7. (a–e) Cells stained in green for pYAP at day 3. (f–j) Cells at day 7. All cells were counterstained with f-actin (red) (scale bar = 10 µm). Data are presented as the mean (±s.d.), significance calculated via one-way ANOVA with the post-Tukey test, N = 3, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.