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Microfabrication of biomimetic hydrogels: bio-inspiration to recreate neural networks or physiological environments onto a novel smart material
G. Dos Reis1, F. Fenili 2, A. Gianfelice3, C. Lenardi4, E. Ranucci2, P. Ferruti2 and P. Milani3
1 C.I.Ma.I.Na. and SEMM, European School of Molecular Medicine, IFOM-IEO campus, Milan, Italy
2 C.I.Ma.I.Na. and Dipartimento di Chimica Organica e Industriale, University of Milan, Italy
3 C.I.Ma.I.Na. and Dipartimento di Fisica, University of Milan, Italy
4 C.I.Ma.I.Na. and Dipartimento di Scienze Molecolari Applicate ai Biosistemi, University of Milan, Italy
Microfabrication of Biomimetic Hydrogels
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Abstract— Development of chemistries for new class of biomimetic materials is major interest in regenerative medicine and materials science. Poly(amidoamine)s are highly hydrophilic synthetic polymers obtained by Michael-type poly-addition of amines to bisacrylamides. When cross-linked in aqueous media, they form optically transparent hydrogels that in many cases proved to be fully biodegradable and biocompatible, warranting potential for biomedical applications. The tendency now, is to move away from old-fashioned substrates to more sophisticated, nature inspired, and finally closer to physiological environments. We report here the possibility of micro-designing PAA hydrogels to enhance and control cell processes such as adhesion, differentiation and proliferation. In particular, we present the development of a direct-writing electron beam lithographic method to pattern the surface of PAA hydrogels. The process is performed at dry state before swelling of hydrogel. The computer assisted methodology enables to control physical and biochemical features, down to a lateral resolution of 500 nm. We demonstrated that surfaces exposed to e-beam could be coated with bioactive proteins or other biomolecules, whose amount can be quantified by using fluorescent labels. The direct-writing approach on biohydrogels can be also used to manufacture larger microstructures, such as embedded microfluidic systems, microelectronic interfaces or other miniaturized systems, easily integrated into cost-competitive biodevices. Finally we interfaced microfabricated hydrogels with cells lines such PC12, able to differentiate into neuronal cells. The cells and neurite recognized the electron beam modified area with strong preference. The microfabrication allowed to precisely controlling the guidance of neurite outgrowth from single cell through microchannels, thus eventually reconstituting neural networks. This technique applied to hydrogels has the potential of creating platforms accurately reproducing the physiological characteristics of cellular microenvironments or neuronal networks.
Keywords— Poly-(amidoamine), hydrogel, neural networks, and microfabrication.
I. INTRODUCTION
Nowadays biomaterials have an essential role in human health, quality of life and environment protection. But a tremendous social-economic impact is still expected from the biomaterials booming time. The current global financial crisis, claims for a readjustment of the domestic economy, by re-launching investment and innovation. Most powerful and innovating materials will make use of biotechnology, promising new capabilities and therapeutic products (1,2). Then the recently available micro- and nanotechnologies applied to existing biomaterials will open new research lines, to find out novel properties and provide cutting-edge tools for construction of clinically helpful micro- and nano devices (2). We foresee that the advent of the coming nanotechnologies will help us to reach a new, more qualitative level, and overcome government difficulties and practical solutions. They will be especially important in social spheres, including health, food and environment protection. The technologies covering the micro and nano-domains shows that biology and materials science needs to rediscover the powerful set of new physical parameters which influences cell behavior. Controlled shape, stiffness, mechanical properties, surface topography, micro and nano features, compartmentalization, specific microenvironments and circulatory systems, are already examples found in nature, which are crucial for biological processes (1). The evolution will occur from simple embedded devices towards more complex and multifunctional materials, bio-inspired devices and materials where physical and chemical properties can be precisely designed (3). At higher scope, we can assume that these properties can be significantly changed and respond in a controlled manner to external stimuli (4,5). These substrates will be the basis to create revolutionary smart materials: able to touch, feel or stimulate biological systems, but also to communicate as organic sensors or circuits. Several microfabrication techniques have been developed to produce miniature components and devices within micro and nano scale, mainly in semiconductor industry. The direct-writing technologies, potentially powerful in fabrication of microelectronics, have also been adopted to manufacture a large variety of new tools and materials for biomedical research and applications. During a typical direct-write approach, patterns or layered structures are built directly without the use of masks, allowing rapid prototyping. Main aspects to be considered are the ease of the technique, and the possibility to precisely control the patterning by computer-assisted design (4). “Top-down” approaches are the common UV lithographic processes or the use of more elaborate lithographic processes such as electron beam lithography (5). Electron microscopy as a microfabrication tool presents as one of its advantage, the three-dimensional control gained by a motorized stage and programmable parameters (including x, y, and z planes, intensity or exposure dose). Here we present the physical and biochemical microfabrication based on electron beam lithography relevant for single cell biology, on recent and innovator poly-amidoamine (PAA) hydrogels (6,7,8). These hydrogels are synthetic cross-linked polymers, highly hydrophilic and obtained by Michael-type poly-addition of amines to bisacrylamides. The hydrogels are optically transparent, biocompatible and biodegradable. Short peptides, complex biomolecules (such as growth factors, extracellular matrix components, and drugs) can participate during the hydrogel synthesis. The synthesis of PAA polymers that will degrade in predictable ways makes them potential candidates for smart drug delivery systems (9). In conclusion PAA hydrogels itself results as an excellent advanced material for a wide range of “in vitro” or “in vivo” applications, such as scaffolds for cell culture, tissue engineering and regenerative medicine (7). With the increasing demand for last generation of biomaterials to evolve from the present substrates to highly physiologic 3D environments adapted to live complexity, PAA hydrogels represents an opportunistic tool for the coming stem cells and micro and nanotechnologies advents (2). We can envisage new body implantable theranostics devices, able to deliver precise treatment at the same time is able to exchange information with external environment, being minimally invasive and naturally eliminated by human body, cost effective, providing less traumatic treatment for patient, and improved patient outcomes. We can also foresee the development of fast, inexpensive, and high throughput drug screening, sensing and biosystems to recreate human body “in vitro”.
II. MICROFABRICATION OF paa HYDROGELS
A. Electron Beam Lithography for direct writing on PAA hydrogels
Using a Scanning Electron Microscope (SEM), we described a new electron beam (e-beam) lithography method for direct writing on hydrogel surface. The novelty of the lithographic process occurs by physical ablation and chemical modification of irradiated hydrogel surface. These modifications are relevant for cell biology. The microfabrication process consists of exposing dry hydrogel films to e-beam (computer-assisted) in high vacuum chamber (figure 1). After the lithographic process, the hydrogel is hydrated and swelling of hydrogel and patterns occurs (fig 1).
Fig.1 Schema describing the electron beam lithography process on hydrogel surface. (b) The process occurs in a scanning electron microscope (SEM) chamber. Computer assisted software (CAD) controls the patterning process. (c) Hydration leads to the proportional swelling of the hydrogel and patterns.
Then as exemplified in Fig. 2, the directly-writing method on hydrogel, allows creating complex or biologically relevant microstructures on PAA hydrogels.
Fig. 2 a) University of Milan logo, b) electron beam lithography is able to precisely pattern complex bitmap images onto hydrogel surface, c) during swelling patterned is proportionally maintained.
The approach meets the requirement to reproduce sterile samples compatible for cell systems. We developed the method, and find out and characterized the basic set of parameters (such as dose exposure, and e-beam energy) to have full control of hydrogel surface topography. Accurate tests and further characterizations of PAA hydrogels were performed at nanoscale by Atomic Force Microscopy (Data not shown). AFM data showed that lithographic process occurs by physical ablation. The removed depth is dependent of exposure dose. By controlling exposure dose we control the depth of removed material and other effects of irradiated area, such as changing the cross-linking degree or the chemical structure of the surface. The e-beam irradiated hydrogel characterized by AFM, allowed finding out basic set of parameters to make three-dimensional designs on PAA hydrogel surface.
B. From simple hydrogel designs to wet microdevices
The microfabricated hydrogels with specific mechanical robustness and stiffness (easily controlled by the cross-linking degree or e-beam exposure doses) combined with properties such as responsiveness (also cross-linking degree dependent), conductivity and actuation would be useful as multifunctional materials. We can expect that these materials will be the basis for wet devices, that combine their ability to change shape and respond to chemical, physical and biological environments (4). Micromanufacturing and downscaling of systems will allow a better understanding of the micro domain that are not yet known, and the creation of devices and applications with lower sizes, smaller energy, volumes, cost consumptions, and finally an increasing environment protection. The full control of our lithographic technique allows manipulating the topographic surface in a three-dimensional manner (fig. 3), where a smooth dose gradient, creates and sculpture surface topologies and patterns with a precise variable depth (as exemplified in fig 3a), and sizes (100, 50, 10, and 1µm) (fig. 3b), where the maximum writing lateral resolution of 500 nm (figure 3c) attained with our instrumentation, goes far beyond the scale of a single cell (fig. 3c, indicated by ***). We can envisage new commercial applications using a new biocompatible and biodegradable material for fast, inexpensive, and high throughput drug screenings devices, sensing or drug delivery biosystems that can be directly implanted in human body (fig. 3f-g).
C. Designing bioactive patterns on hydrogels
We found that e-beam irradiated hydrogel surface, is not only physically modified in terms of topography, cross-linking degree, material ablation, swelling and other mechanical properties. It also changes the chemical structure of the polymer. The modifications create an optimum subtract to directly attach proteins or other biomolecules. We demonstrated that the surfaces exposed to the e-beam can be coated with proteins or other biomolecules whose amount can be quantified by using fluorescent labels (Fluorescein Isothiocyanate FITC, or Tetramethyl Rhodamine Iso- Thiocyanate TRITC) (fig 4). We tested a reference protein BSA – FITC (Bovine Serum Albumin, 66 kDa), the hormone EGF – FITC (6 kDa Epidermal Growth Factor which induce cell proliferation), the biomolecule Phalloidin - TRITC (MW 789.2 from the phallotoxin group, used as an imaging tool), and fibrinogen – Alexa (not shown) (340 kDa glycoprotein precursor of fibrin). They all attach selectively to e-beam irradiated surface in a dose dependent manner (fig. 4a,b and c). Higher exposure doses, leads to a higher protein or biomolecules attachment.
Fig.3 Patterns created on hydrogel surface by electron beam lithography. White bar: 100 µm. (a) dose exposure gradient (* lower dose to ** higher dose) leads to a removed depth gradient. (b-c) maximum writing lateral resolution of 500 nm (point indicated by ***). The patterns can be used to incorporate smart devices and responsive systems on hydrogel. Some designs exemplify possible applications for microfluidic (d-e), micro valves (f) or pumps and microarrays (g) on hydrogels.
Fig.4 Confocal microscopy shows that electron beam creates on hydrogel a surface optimal for protein or biomolecules adsorption. Increasing dose exposure leads to higher protein adsorption: (a) BSA-FITC, (b) Phalloidin-TRITC and (c) EGF-FITC). White bar: 100 µm. (d) examples of a micropatterns and its negative, coated with BSA-FITC.
Parameters induced by irradiation, affects the polymer chemistry, the nano and micro features of surface (such topology, roughness, and polymer network porosity).Then the protein intrinsic properties (such size, shape and chemistry) might be crucial in explaining the protein-electron beam substrate interactions. The microlithographic approach on PAA hydrogels, gives the opportunity to create systems that can be functionalized and designed with biomolecules relevant for biological studies. These represent an inherent potential to create a bioactive materials controlled at microscale to interface and mimic living systems.
D. Microfabricated hydrogels interfacing cells
Cells lines such MDCK (Madin-Darby canine kidney) and PC12 (rat pheochromocytoma) were cultivated on patterned surface. MDCK growth was observed along all surfaces, independently of any modification, while PC12 presented a strong preference for e-beam modified substrate (not shown). The fact cells are able to sense the surrounding environments is well known, but how its machinery interacts with interfaces is far away of being full understood. The deep insight of processes involved in different cells and substrate interactions, are vital to design enhanced substrates and reveals the importance of custom materials, prepared for tissue complexity. With our microlithographic process and hydrogel, we are able to control PC12 adhesion and growth into desired areas by creating microwells or chambers (from single cell size to a container of several cells). PC12 when induced by the hormone Nerve Growth Factor (NGF) is able to differentiate into neural cells. By treating PC12 cell culture with NGF, through this chambers or single cells, we are able to guide neurite outgrowth using microchannels (1 µm thickness) interconnecting microwells (10 µm) (fig 5). With this technique and material we promote in a controlled manner neurite outgrowth. It represents a technological tool to assemble neural networks on a last generation biomaterial.
Fig.5 Neural networks on a microfabricated PAA hydrogel: Immune-staining (Phalloidin-TRITC) of PC12 cells treated with NGF. By creating microchambers interconnected by microchannels we are able to control neurite outgrowth or assemble neural networks on hydrogels.
III. CONCLUSIONS
We demonstrated that the versatile PAA hydrogel can be designed and functionalized in a microscale precision manner on the surface, with bioactive properties using proteins. They allow the creation of platforms to study cell-substrate interactions, or to control the guidance of neurite outgrowth throw microchannels. Thus represents an inherent potential to create low-cost biocompatible systems, in such way to reproduce very accurately the physiological characteristics of cellular microenvironments on hydrogels. Several challenges remain, including developing drug delivery systems, or materials that can sense biochemical signals in the body. Understanding more about extracellular matrix biology, immunology and cell membrane will help in predict, how the body will respond to specific materials. Analogously, advances in micro and nanomaterials will create new opportunities to mimic body entities. An increasing need already exists, and solutions become limited to the new technical, economic or ecological demands. The subject of bio-inspired materials approach is promising and it is at frontier between biological and material sciences, chemistry, physics together with biotechnology and computing.