Microfabrication of poly (glycerol–sebacate) for contact guidance applications
Introduction
Extracellular matrix proteins are known to contain a rich three-dimensional surface topography that presents biophysical cues to cells [1]. The topographic features within basement membranes have been shown to contain sub-micron length-scales [2]. This discovery led to the well-tested hypothesis that cellular structure and function can be influenced by culturing cells on a variety of substrates modified with micron and sub-micron sized features [3]. Inducing cellular responses using synthetic micro- and nanofabricated substrates [4], termed contact guidance [5], has been observed in a variety of cell types [6] including, but not limited to epithelial cells [7], [8], [9], fibroblasts [10], [11], [12], [13], oligodendrocytes [14], and astrocytes [14]. Contact guidance has been shown to induce a variety of cellular responses such as the up regulation of fibronectin mRNA in human fibroblasts [15], increased adhesion of epithelial cells [8], and increased mineralization and alkaline phosphatase activity in rat bone marrow cells [16]. However, if substrates are fabricated with sub-micron features, the cellular response varies across cell type and depends heavily on other factors such as feature size, geometry [17] and cell–cell interactions [18].
One of the most promising applications of contact guidance is in the field of tissue engineering. The ability to control cell morphology and orientation using mechanical cues alone is a potentially useful technique. However, one limiting factor in previous work in contact guidance has been the choice of material. Microfabricated silicon and replica molded poly(dimethyl siloxane) (PDMS), although ubiquitous and inexpensive, are not biodegradable, have limited biocompatibility, and therefore are not suitable biomaterials for tissue engineering systems. Poly(l-lactic–glycolic acid) (PLGA), while biodegradable, exhibits sub-optimal properties for an implant material such as rigid mechanical properties [19], bulk degradation kinetics [20], and questionable biocompatibility in some cases [21]. High concentrations of PLGA by-products has also been shown to be cytotoxic [22], which is a major limitation in the prospect of fabricating large, organ-size scaffolds using PLGA. Poly(glycerol–sebacate) (PGS), a recently synthesized biocompatible and biodegradable elastomer with superior mechanical properties, serves as a promising alternative material for fabricating contact guidance substrates [20]. PGS is a tough, biodegradable, elastomer that is biocompatible, inexpensive, and easy to synthesize. Polymers containing sebacic acid and glycerol, the monomers used in PGS synthesis, have both been approved for use in medical applications by the FDA. Both in vitro and in vivo biocompatibility studies [20], [21] suggest improved cellular response and morphology of PGS when compared to PLGA, which eliminates the need for surface treatment through chemical modification. PGS is also a suitable material from a processing perspective. PGS pre-polymer can be replica molded and cured on silicon masters to form layers as thin as 100 μm in a process that is analogous to replica molding of PDMS [23]. PGS degrades via a surface erosion mechanism [21], which allows the substrate to retain feature fidelity after implantation thus continually providing contact guidance cues throughout the lifetime of the material.
The corpus of work in contact guidance has focused primarily on fabricating substrates with well-defined microstructures of various feature sizes to study the response of cellular morphology. Obtaining well-defined topographical features with tight distributions in feature size is characteristic of work that utilizes traditional microfabrication processes. However, one unfortunate consequence of traditionally microfabricated substrates is the creation of sharp features with abrupt salient corners. While it has been shown that the cellular responses often vary as a function of the length-scale of basic ridge–groove features or randomly oriented, nanometer scale posts [24], [25], [26], the sharp edges of these features are not biologically relevant. There are, however, reports of mammalian cells aligning and elongating in response to rounded geometries, namely along the longitudinal axis of cylindrical substrates [27] and in the direction of continuous wavy features with micron-scale periodicities [28].
In this study, we developed a method for fabricating flexible, biodegradable substrates with micron-scale features for contact guidance applications. We have developed a fabrication technique for achieving sub-micron scale features down to 500 nm in a flexible biodegradable material platform. This technique is flexible, allowing for the application of this process to microfabricate rounded features on other types of replica-molded biomaterials. Using bovine aortic endothelial cells (bAECs) as a model cell system, we studied the morphology of bAECs grown on substrates with periods of approximately 2.5 and 4.5 μm with constant feature height of 0.45 μm. Cells cultured on substrates with smaller periods exhibited stronger alignment and reduced circularity relative to substrates with larger features. A hypothesis that describes a possible mechanism for contact guidance is proposed to rationalize the results, which focuses on the role of filipodia in the detection of local topographic cues.
Section snippets
Microfabrication of silicon masters
Standard photolithographic and plasma etching techniques were utilized to produce “negative mold” silicon masters for use in replica molding just as PDMS is used to microfabricate polymeric layers quickly and easily [23]. Briefly, patterned chrome-on-glass masks were used in the photolithography step. Single-crystal silicon, 100 mm diameter wafers were patterned with photoresist using one photolithography cycle. Features were etched using an STS etcher (Surface Technology Systems, Newport, UK)
Microfabricated substrates
Three microfabricated surfaces with varying feature sizes were used in contact guidance studies. SEM images of the silicon masters for patterns G2 and G3 reveal some degree of non-uniformities in the feature sizes and spacing (Fig. 2C and E). This can be most likely attributed to the inaccuracies that accompany the microfabrication of sub-micron features using near-UV wavelength photolithography. SEM and AFM imaging studies suggest that the surfaces of the replica-molded PGS substrates were
Adaptable process for developing smoothed microfeatures for biopolymers
The microfabrication process demonstrated in this work has potential applications for engineering surfaces of implants and other tissue engineering systems. No solvents are used in this generalized replica-molding process, which can be expanded to produce biomaterials with rounded features of micron and sub-micron scale. This process can be expanded to fabricate any large range of materials that can be microfabricated using solvent casting, embossing, or similar processes that employ a mold.
Conclusions
We have fabricated flexible, biodegradable, biocompatible substrates with rounded features as small as 500 nm. Substrates containing smooth features with periods on the order of microns can be used to align and elongate bovine aortic endothelial cells. The superior mechanical and biocompatibility properties of PGS coupled with the observed strong contact guidance responses of cells cultured on microfabricated PGS renders this system appropriate for in vivo applications. This could lead to the
Acknowledgments
The authors would like to thank Yadong Wang for technical consultation and Connie Cardoso as well as the entire MEMS group at the Draper Laboratory. This work was supported by the National Institutes of Health (Grants HL060435 and DE013023). The views expressed are not endorsed by the sponsor. This work was supported in part by Draper Laboratory, under Award no. DL-H-550154 and by the MRSEC Program of the National Science Foundation under Award no. DMR 02-13282.
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