Construction Of Polyelectrolvte Multilayers Gradients And Their Influence On Cell Migration

Cell migration plays a crucial role in a variety of physiological and pathological processes ranging from embryonic development, cancer metastasis, blood vessel formation and remolding, tissue regeneration, immune surveillance and inflammation. Materials-cells interactions, as one of the major challenges in regenerative medicine and tissue engineering, can be used to optimize cell migration in a controlled manner. Among the various surface engineering methods, the layer-by-layer (LBL) assembly can diversely tailor the substrate properties and is suitable to address the materials-cells interactions.In this work, poly(sodium4-styrenesulfonate)(PSS)/poly(diallyldimethyl-ammonium chloride)(PDADMAC) multilayers were treated with1-5M NaCl solutions, resulting in continuous changes in physicochemical properties of the multilayers. For Multilayers-1,2M (multilayers post-treated by the1M and2M NaCl), the original structure and properties of the multilayers are mostly retained, with a PDADMAC dominated, positively charged surface, and a larger swelling ratio in water. The hardly swollen Multilayers-3M film, by contrast, has a PSS dominated and negatively charged surface as a result of larger loss of PDADMAC. For the Multilayers-4,5M films, PSS becomes abundant and the swelling ratio is pretty high, especially for the Multilayers-5M which shows the highest hydration in water.The cell migration on substrate is known to be influenced by many surface physicochemical properties. Chemical composition, surface charge, hydration degree/stiffness, thickness and roughness on salt-treated PSS/PDADMAC thin films are all varied, which are suitable to be applied to study the cell migration behaviors, and to obtain a comprehensive view of multi-stimuli to cells migration on the surface. The surface chemistry and viscoelasticity of the salt-treated multilayers are the major factors governing the cell migration rate. Migration rate of human smooth muscle cells (SMCs) was slowest on the Multilayers-3M, which was comparable with that on tissue culture plate, but was highest on the Multilayers-5M. To elucidate the intrinsic mechanisms, cell adhesion, proliferation, and gene expression of related adhesion and migration was further investigated. The SMCs preferred to attach, spread and proliferate on the PSS-dominated surfaces with well organized focal adhesion and actin fibers, especially on the Multilayers-3M. while kept round and showed low viability on the PDADMAC-dominated surfaces. The relative mRNA expression of adhesion related genes such as laminin and focal adhesion kinase. and migration related genes such as myosin IIA and Cdc42were compared to explain the different cellular behaviors.Cells migrate in response to gradually gradient signals such as dissolved chemoattractants (chemotaxis), or surface-attached molecules (haptotaxis) in vivo. Hence, gradient surface is effective to turn cell migration direction. In the next experiment, gradient multilayers with similar chemical composition but gradually changed mechanical properties were further fabricated in a gradient salt solution of3-5M concentration. The cells of high culture density on the gradient multilayers showed the fast directional migration behaviors to the3M side. After24h migration, large amount of cells gathered at the side of3M region. In contrast, cells of low culture density migrate randomly on the gradient multilayers, demonstrating the pure gradient surface can not regulate cell directional migration. The cell focal adhesions, the interactions between cells and substrate, and the immunostaining of Cdc42, myosin and RhoA were compared with the discrete multilayers, showing that the cell-cell interactions play a significant role in the directional migration.Groove patterns can be an important cue to control the cell alignment, elongation, and migration along the direction of ridges. Hence, to improve the effective directional migration, the surfaces with both gradient and groove patterns were fabricated. The multilayers were directly assembled on the grooved polydimethylsiloxane (PDMS) stamp with a feature of20x20x3.5μm in width, space and depth, respectively. Results showed that cells of low culture density could migrate to the3M side of salt-treated gradient multilayers. Because the groove on the substrate can be used to force cell orientation, limit their migration direction, and increase the cell length in the gradient direction, the gradient multilayers can guide cell to move directionally from5M side to3M side.Finally, a solvent-assisted micro-molding technology was developed to fabricate clear physical patterns on hydrated PSS/PDADMAC multilayers driven by the capillary force. Influences of molding temperature, multilayer thickness, and solvent quality on the pattern formation were elucidated, confirming that the pattern topography and height could be conveniently mediated. The multilayers with a groove feature of nano-width and20μm in space were chosen and post-treated with0,1and2M NaCl solutions. We found this pattern feature with a relatively low height (several hundred nanometers) could better control cell to migrate along the groove direction, and cell migrated more randomly with the decrease of the pattern height.