Influences of surface chemistry and swelling of salt-treated polyelectrolyte multilayers on migration of smooth muscle cells

Abstract

The cell migration plays a crucial role in a variety of physiological and pathological processes and can be regulated by the cell-substrate interactions. We found previously that the poly(sodium 4-styrenesulphonate) (PSS)/poly(diallyldimethylammonium) chloride (PDADMAC) multilayers post-treated in 1-5 M NaCl solutions result in continuous changes of their physico-chemical properties such as thickness, chemical composition, surface charge, swelling ratio and wettability. In this study, the responses of human smooth muscle cells (SMCs) on these salt-treated multilayers, particularly the governing factors of cellular migration that offer principles for designing therapeutics and implants, were disclosed. The cell migration rate was slowest on the 3 M NaCl-treated multilayers, which was comparable with that on tissue culture plates, but it was highest on 5 M NaCl-treated multilayers. To elucidate the intrinsic mechanisms, cell adhesion, proliferation, adhesion and related gene expressions were further investigated. The SMCs preferred to attach, spread and proliferate on the PSS-dominated surfaces with well-organized focal adhesion and actin fibres, especially on the 3 M NaCl-treated multilayers, while were kept round and showed low viability on the PDADMAC-dominated surfaces. The relative mRNA expression levels of adhesion-related genes such as fibronectin, laminin and focal adhesion kinase, and migration-related genes such as myosin IIA and Cdc42 were compared to explain the different cellular behaviours. These results reveal that the surface chemistry and the swelling of the salt-treated multilayers govern the cell migration behaviours.

Figure 1.

Cell migration on the salt-treated (PSS/PDADMAC)₇ multilayers. (a) Random cell migration trajectories on the multilayers post-treated with 1, 3 and 5 M NaCl solutions, respectively. (b) Cell migration rates averaged from greater than or equal to 15 cells on TCPS and the salt-treated multilayers. (c) The relative migration ability of cells on TCPS and the salt-treated multilayers. The relative migration ability is defined as the ratio of cell migration distance to its characteristic length. Asterisks indicate significant difference, which is determined by one-way analysis of variance (ANOVA) in the Origin software. The statistical significance of asterisks is set as p < 0.05 and the Tukey means comparison method is performed.

Figure 2.

SMCs adhesion and proliferation on the multilayers post-treated with different NaCl solutions. (a) The ratio of living SMCs cultured on the multilayers for 4 h with and without FBS, respectively. The live and dead cells were distinguished by FDA and PI staining. (b) Cell spreading area, (c) cell number and (d) OD value of the SMCs on the multilayers after being cultured in DMEM/10 per cent FBS for different periods of time. (e) Cell cycle analysis of G0/G1, S and G2/M phases of SMCs incubated for 24 h on TCPS and the multilayers post-treated with 1, 2, 3, 4 and 5 M salt solutions, respectively. Asterisks indicate significant difference, which is determined by one-way analysis of variance (ANOVA) in the Origin software. The statistical significance of asterisks is set as p < 0.05 and the Tukey means comparison method is performed.

Figure 3.

Morphology and focal contact formation of the SMCs cultured on the salt-treated multilayers for 24 h. Representative CLSM images of SMCs on the multilayers treated with (a) 1, (b) 2, (c) 3, (d) 4 and (e) 5 M NaCl solutions, respectively. (a(i)–e(i)) Merge fluorescence images of vinculin (green), actin (red) and nucleus (blue). (a(ii)–e(ii)) and (a(iii)–e(iii)) vinculin and actin in a single cell, respectively. Arrows in (c(ii)), (d(ii)) and (e(ii)) indicate areas of large focal adhesion plaques. Two-head arrows indicate the polarity of the SMCs, and the texts illustrate the cell front (cell leading edge) and rear.

Figure 4.

Gene expression analysis of SMCs cultured on TCPS and the salt-treated multilayers for 24 h. (a) Cell adhesion-related genes of fibronectin, laminin and FAK, and (b) migration-related genes of myosin IIA and Cdc42 detected by real-time quantitative RT-PCR. The data were normalized to those control cells cultured on TCPS. Asterisks indicate significant difference at p < 0.05.

Figure 5.

Scheme of the SMCs migration behaviours on the multilayers with different surface charge and swelling properties. (a) The SMCs have a moderate migration rate on the positively charged and hydrated surface (the multilayers-1 M). (b) The SMCs migrate slowest on the negatively charged and dehydrated surface (the multilayers-3 M). (c) The SMCs migrate fastest on the negatively charged and highly hydrated surface (the multilayers-5 M).