Micro- and nano-patterned elastin-like polypeptide hydrogels for stem cell culture

Abstract

We show that submicron-sized patterns can be imprinted into soft, recombinant-engineered protein hydrogels (here elastin-like proteins, ELP) by transferring wavy patterns from polydimethylsiloxane (PDMS) molds. The high-precision topographical tunability of the relatively stiff PDMS is translated to a bio-responsive, soft material, enabling topographical cell response studies at elastic moduli matching those of tissues. Aligned and unaligned wavy patterns with mold periodicities of 0.24–4.54 µm were imprinted and characterized by coherent anti-Stokes Raman scattering and atomic force microscopy. The pattern was successfully transferred down to 0.37 µm periodicity (width in ELP: 250±50 nm, height: 70±40 nm). The limit was set by inherent protein assemblies (diameter: 124–180 nm) that formed due to lower critical solution temperature behavior of the ELP during molding. The width/height of the ELP ridges depended on the degree of hydration; from complete dehydration to full hydration, ELP ridge width ranged from 79± 9% to 150 ± 40% of the mold width. The surface of the ridged ELP featured densely packed protein aggregates that were larger in size than those observed in bulk/flat ELP. Adipose-derived stem cells (ADSCs) oriented along hydrated aligned patterns with periodicities ≥0.60 µm (height ≥170±100 nm), while random orientation was observed for smaller distances/amplitudes, as well as flat and unaligned wavy ELP surfaces. Hence, micro-molding of ELP is a promising approach to create tissue-mimicking, hierarchical architectures composed of tunable micron-sized structures with nano-sized protein aggregates, which opens the way for orthogonal screening of cell responses to topography and cell-adhesion ligands at relevant elastic moduli.

The effects of surface topography modification on hydrogel properties

“…Robert et al showed that hydrogels with 1.9 μ m of grooves by casting method were effective in inducing cell alignment, but cells aligned poorly on grooves with depth less than 1 μ m. 163 Adipose-derived stem cells were also shown to exhibit alignment when the topography width is larger than 0.60 μ m and height larger than 170 ± 100 nm on elastin-based hydrogel. 164 Fibroblasts were cultured on PAM hydrogels with 5 μ m wide and 1 μ m high lines with stiffness of 13, 37, and 145 kPa. Cell elongation was induced by topography on all substrates.…”

The Use of Microfabrication Techniques for the Design and Manufacture of Artificial Stem Cell Microenvironments for Tissue Regeneration

“…Although hydrogels can be fabricated from synthetic and natural polymers, synthetic polymers have become more relevant due to their long service life, high mechanical strength, and higher water absorption [ 102 ]. Relevant hydrogel materials used to culture stem cells include PEG [ 103 ], poly(2-hydroxyethyl methacrylate) (HEMA) [ 104 ] elastin [ 105 ], collagen [ 106 ], and hyaluronic acid [ 107 ].…”

Surface Topography and Electrical Signaling: Single and Synergistic Effects on Neural Differentiation of Stem Cells

“…[38][39] Patterned topographies with a size range from sub-micrometers to tens of micrometers directly alter cell morphology along the patterns and differentiate stem cells by impact on their alignment and orientation. [40] Nanometer-sized patterns with the dimensions of integrins induce nano-scale integrin clustering, and assembly of focal adhesion proteins such as paxillin, vinculin, and focal adhesion kinase (FAK) in stem cells, which induce cytoskeleton reorganization and alter cytoskeletal tension (Figure 2). [41][42] The mechanical tension activated by specific topographies actuates integrin-mediated intracellular signaling and mechanotransduction pathways via nuclear reorganization and eventually rearranges the centromere through deformation of the nucleus.…”