Surface patterning is a key feature of materials science that requires the design of complex structures. Surface topographical features have often been generated through ‘top-down’ strategies using micro-contact printing or by electron beam-, photo- or dip pen lithography. 1,2
These techniques provide control over size and arrangement in the micro- and nano- scales with remarkable reproducibility. However, the need for patterned surfaces has extended its niche from the electronics industry to surface chemistry, protein biology, biosensors, and even cell biomechanics.
For biological applications in particular, surface engineering has dominated recent biomaterials design and shown how specific surface functionalities are cell adhesive, e.g. carboxyl, amine, or hydroxide groups 3
. The topological arrangement of such chemistries has an equally important effect; the order of nanoscale surface roughness can control and direct cell functions 2,4
. This is particularly critical for stem cell engineering where there is strong requirement to control cell phenotype 4
or direct differentiation 2
. Clustering cell controlling moieties into a more mimetic and well-spaced arrangement can alter cell adhesion and spreading 5
but only when sites are adequately spaced and with sufficient heterogeneity to generate forces6
. Key biomaterial design aspects must therefore mimic native extracellular matrix (ECM) of the body; they must provide both structural support and intrinsic properties to the cell to influence its behavior 7
, e.g. topography 2
, stiffness 8
, and cell binding site spacing 9
and more recently ECM tethering10
. These cues show exquisite micro- and nano-scopic organization in vivo
, and in the absence of traditional growth factor cocktails, their spatiotemporal presentation alone can regulate cellular behavior, e.g. adhesion, proliferation, differentiation, and apoptosis 11
While these efforts highlight how nano-scale topological properties influence cells, most of these studies have been limited to two-dimensional (2D) systems. Three-dimensional (3D) scaffolds often present uniform surface chemistry via surface immobilization or direct crosslinking of a binding motif to the scaffold, yielding either homogeneous or protein polymer hydrogels 7
. Often however, these materials have very little control over their surface topology, which can be substantially different from native ECM 9
. This suggests the need for rationally designed materials that present cues in a way that reflects ECM’s complexity.
Herein we propose the synthesis of a new class of three-dimensional matrices using a ‘bottom-up’ approach to better mimic the adhesive heterogeneity of matrix and thus control cell adhesion in vitro
. We propose the combination of high internal phase emulsion (HIPE) templating with interface confined block copolymer self-assembly to engineer 3D porous nano-functionalized materials as scaffolds for cell culture 12,13
(). To date, the most utilized polyHIPE systems are surfactant-stabilized water-in-oil emulsions, where the oil phase consists of polymerizable monomers, and the controlled combination of polymerization and internal phase destabilization gives rise to highly porous materials 14
. However such an approach is intrinsically limited by matrix hydrophobicity and the difficulty of introducing surface functionality. One way to introduce hydrophilicity is by preparing ‘inverse’ oil-in-water polyHIPEs, copolymerizing hydrophilic monomers such as acrylic acid 15
and hydrophilic monomers such as poly(ethylene glycol) methacrylate in water-in-oil emulsions 16
. Surface functionalization of these materials has been achieved with plasma polymerization 17
or polymer grafting through azide-alkyne Huisgen cyclo-addition, which is also known as click chemistry 18
. However, multi-step surface modifications often lack control over efficiency. Alternatively, surfactant free Pickering polyHIPEs that utilizes colloidal particles to stabilize the oil-water interface have also been synthesized. For example, PMMA nanoparticles 19
trapped at the oil-water interface upon polymerization, may offer control over surface topography in 3D. On the other hand, block copolymers can form nanostructured materials in bulk and in solution by exploiting controlled micro-phase separation 20
. These nanomaterials have now been translated successfully to control cell adhesion in 2D 21
, but they cannot form structured 3D microenvironments.
Figure 1 HIPE Polymerisation Scheme. a) Schematic of high internal phase emulsion templating to form surface cell adhesive and inert domains through amphiphilic block copolymer phase separation at the oil-water interface. b) Macro- and micro-porosity of 3D foams (more ...)
Herein we combine the HIPE process with amphiphilic block copolymers polystyrene-b-poly(ethylene oxide) (PS-PEO) and/or polystyrene-b-poly(acrylic acid) (PS-PAA). What results is a polystyrene (PS)-based foam with high affinity between the amphiphilic copolymer and the PS matrix, anchoring the copolymer at the scaffold surface (). By mixing different amphiphilic copolymers, we can drive the formation of patchy interfaces 22,23
, now with domain size ranging from tens to hundreds of nanometers. Thus we can design interfaces where cell active motifs (PAA) can be confined on clusters surrounded by inert motifs (PEO) matrix and vice-versa (). Furthermore, these foams exhibit architectural features ranging from porosity in the 100μm range to surface topography in the 10 nm range. We finally demonstrate that stem cells grown on these foams adhere in a block copolymer dependent manner indicating the complexity of adhesive heterogeneity as a cue for stem cell adhesion.