Financial support was provided to J.M.S. from the NIH Biomechanics in Regenerative Medicine T32 Training Program (2T32EB003392), to Q.J. from the Dowd-ICES Fellowship and to A.W.F. from the NIH Director's New Innovator Award (1DP2HL117750).

1. Fabrication of Master Mold Using Photolithography
<ol>
	<li>The ECM protein nanofibers, nanofabrics and nanostructures to be fabricated are first designed using Computer Aided Design (CAD) software. This CAD file is then transferred to a photomask. The type of photomask will depend on the resolution of the features; with a transparency-based photomask adequate for feature sizes down to ~10 &#956;m. Smaller features &#60;10 &#956;m will require a chrome on glass photomask. All of the nanofibers and nanostructures pre...

<ol>
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E., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+electrospinning+design+and+nanofibre+assemblies.">A review on electrospinning design and nanofibre assemblies.</a> Nanotechnology. 17, (2006).</li><li>Reneker, D. H., Chun, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometre+diameter+fibres+of+polymer,+produced+by+electrospinning.">Nanometre diameter fibres of polymer, produced by electrospinning.</a> Nanotechnology. 7, 216-21 (1996).</li><li>Ma, P. 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A., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+chitosan-based+nanofibers+and+their+cellular+compatibility.">Electrospun chitosan-based nanofibers and their cellular compatibility.</a> Biomaterials. 26, 6176-6184 (2005).</li><li>Jin, H. -. J., Chen, J., Karageorgiou, V., Altman, G. H., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bone+marrow+stromal+cell+responses+on+electrospun+silk+fibroin+mats.">Human bone marrow stromal cell responses on electrospun silk fibroin mats.</a> Biomaterials. 25, 1039-1047 (2004).</li><li>Wierzbicka-Patynowski, I., Schwarzbauer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+fibronectin+matrix+assembly.">The ins and outs of fibronectin matrix assembly.</a> Journal of Cell Science. 116, 3269-3276 (2003).</li><li>Mosher, D. F., Johnson, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+formation+of+disulfide-bonded+fibronectin+multimers.">In vitro formation of disulfide-bonded fibronectin multimers.</a> Journal of Biological Chemistry. 258, 6595-6601 (1983).</li><li>Little, W. C., Smith, M. 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The authors declare that they have no competing financial interests.

The SIA method presented here mimics cell-mediated matrix assembly and enables the engineering of ECM protein nanofibers and nanostructures with tunable size, organization and composition. While not identical to cell-generated ECM, SIA creates ECM composed of nanoscale protein fibrils20 that undergo reversible folding/unfolding during mechanical strain21 and can bind cells20. This provides a unique capability to build ECM protein materials that recapitulate many properties of the ECM foun...

The extracellular matrix (ECM) in tissues is composed of multifunctional proteins involved in physical and chemical regulation of multiple cell processes including adhesion, proliferation, differentiation, and apoptosis1-3. The ECM is synthesized, assembled, and organized by cells and the constituent protein fibrils have unique compositions, fiber size, geometries and interconnected architectures that vary with tissue type and developmental stage. Recent work has demonstrated that the ECM can provide instructive cues to guide cells to form engineered tissues4, suggesting that recapitulating the ECM in terms of composition and structure might enable the development of biomimetic materials for tissue engineering and biotechnology applications.
A number of fabrication methods have been developed to engineer polymeric scaffolds that can mimic aspects of the ECM in tissues. For example, electrospinning and phase separation have both demonstrated the ability to form porous matrices of fibers with diameters ranging from tens of micrometers down to tens of nanometers5-7. Both techniques have also shown that highly porous matrices of nanofibers can support cell adhesion and infiltration into the scaffold8. However, these approaches are limited in the possible fiber geometries, orientations and 3D architectures that can be created. Electrospinning typically produces scaffolds with either randomly oriented or highly aligned fibers whereas phase separation produces scaffolds with randomly oriented fibers. There are also limitations on the materials, with researchers typically using synthetic polymers, such as poly(&#949;-caprolactone)8 and poly(lactic-co-glycolic acid)9, that are subsequently coated with ECM proteins to promote cell adhesion. Natural biopolymers are also used, including collagen type I10, gelatin11, fibrinogen12, chitosan13, and silk14, but represent only a small subset of the proteins found in native tissue. Most tissues contain a larger milieu of ECM proteins and polysaccharides including fibronectin (FN), laminin (LN), collagen type IV and hyaluronic acid that are difficult or impossible to fabricate nanofibers using existing methods.
To address this challenge, we have focused our research efforts on mimicking the way cells synthesize, assemble and organize ECM protein fibrils in their surroundings. While the specific fibrillogenesis process varies for different ECM proteins, typically a conformational change in the ECM protein molecule is triggered by an enzymatic or receptor-mediated interaction, which exposes cryptic self-assembly sites. Here we use FN as a model system to better understand the fibrillogenesis process. Briefly, FN homodimers bind to integrin receptors on the cell surface via the RGD amino acid sequence in the 10th type III repeat unit. Once bound, the integrins move apart via actomyosin contraction and unfold the FN dimers to expose cryptic self-assembly sites. The exposure of these FN-FN binding sites enables the FN dimers to assemble into an insoluble fibril right on the cell surface15. Work in cell-free systems has demonstrated that cryptic FN-FN binding sites can be revealed through unfolding using denaturants16 or surface tension at an air-liquid-solid interface17-19. However, the FN fibers created by these techniques are restricted to specific fiber sizes and geometries and are typically bound to a surface.
Here we describe an approach termed surface-initiated assembly (SIA) 20 that overcomes these limitations by utilizing protein-surface interactions to create free-standing insoluble nanofibers, nanofabrics (2D sheets) and other nanostructures composed of single or multiple ECM proteins (Figure 1). In this process, ECM proteins are adsorbed from a compact, globular conformation in solution and partially denatured (unfolded) onto a patterned, hydrophobic polydimethylsiloxane (PDMS) stamp. The ECM proteins are then transferred in this state onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface through microcontact printing22. When hydrated with 40 &#176;C water the PIPAAm remains a solid, but when cooled to 32 &#176;C it passes through a lower critical solution temperature (LCST) where it becomes hydrophilic, swells with water and then dissolves, releasing the assembled ECM nanostructures off of the surface. The SIA method provides control over the dimensions with nanometer-scale precision. By controlling key parameters such as composition, fiber geometry, and architecture, it is possible to recapitulate many properties of the ECM found in vivo and to develop advanced scaffolds for tissue engineering and biotechnology applications.

<table border="0" cellpadding="0" cellspacing="0" width="651">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Poly(N-isopropylacrylamide) / PIPAAm</td>
			<td style="width:145px;">Polysciences</td>
			<td style="width:123px;">21458-10</td>
			<td style="width:167px;">40,000 Mw</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sylgard 184 Silicone kit (PDMS)</td>
			<td style="width:145px;">Dow Corning</td>
			<td style="width:123px;"></td>
			<td style="width:167px;">Mix 10 parts base with 1 part curing agent.&#160;</td>
		</tr>
		<tr height="21">
			<td height="42" style="height:42px;width:216px;">Butanol</td>
			<td style="width:145px;"></td>
			<td style="width:123px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Fibronectin</td>
			<td style="width:145px;">BD biosciences</td>
			<td style="width:123px;">354008</td>
			<td style="width:167px;">Human, 1mg</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Laminin</td>
			<td style="width:145px;">BD biosciences</td>
			<td style="width:123px;">354239</td>
			<td style="width:167px;">Ultrapure, mouse, 1mg</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Negative Photoresist</td>
			<td style="width:145px;">Microchem</td>
			<td style="width:123px;">SU8-2015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">SU8 Developer</td>
			<td style="width:145px;">Microchem</td>
			<td style="width:123px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:216px;">Sonicator Branson M3510</td>
			<td style="width:145px;">Branson Ultrasonic Corporation</td>
			<td style="width:123px;">CPN-952-318</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Thinky ARE-250 Mixer</td>
			<td style="width:145px;">Thinky Corporation</td>
			<td style="width:123px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Spincoater</td>
			<td style="width:145px;">Specialty Coating Systems</td>
			<td style="width:123px;">G3P-8</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Glass cover 25mm diameter, No 1.5</td>
			<td style="width:145px;">Fisher Scientific</td>
			<td style="width:123px;">12-545-86</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

ecm protein nanofibers and nanostructures engineered using surface-initiated assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

SIA is capable of engineering ECM protein nanofibers with precise control over fiber dimensions. To demonstrate this, arrays of FN nanofibers with planar dimensions of 50 x 20 &#956;m were patterned onto a PIPAAm coated coverslip (Figure 2A). Upon release, the fibers contracted because they were under an inherent pre-stress when patterned on the PIPAAm surface (Figure 2B). Analysis of the FN nanofibers revealed they were monodisperse pre-release with an average length of 50.19 &#177; 0.4...

Watch this Scientific Journal Video about ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly at JoVE.com

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

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JoVE Journal

Bioengineering

12.4K vistas.  Carnegie Mellon University.  Un método para obtener nanofibras y nanoestructuras complejas de proteínas de la matriz extracelular individuales o múltiples se describe. Este método utiliza las interacciones proteína-superficie para la creación de materiales a base de proteínas exentas con la composición sintonizable y la arquitectura para el uso en una variedad de aplicaciones de ingeniería de tejidos y de biotecnología. 