ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

Summary

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.

Abstract

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.

Introduction

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 apoptosis^1-3. 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 nanometers^5-7. 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(ε-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 interface^17-19. 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 °C water the PIPAAm remains a solid, but when cooled to 32 °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.

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Protocol

1. Fabrication of Master Mold Using Photolithography

1. 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 μm. Smaller features <10 μm will require a chrome on glass photomask. All of the nanofibers and nanostructures presented here were fabricated using a transparency-based photomask, and thus were nanometer-scale in thickness but not lateral dimensions.
   Note: It is important to distinguish which regions of the photomask will be dark (prevent UV light to pass through) and which will be transparent (allow UV light to pass through) as this, along with the type of photoresist (positive or negative), will dictate the final topography of the master mold.
2. To begin fabrication of the master mold, dehydrate a 4" silicon wafer by placing it on a hotplate set to 150 °C for 15 min.
3. Center the wafer on the vacuum chuck of a spin-coater. Pour SU8-2015 negative photoresist onto the middle of the wafer and continue pouring in concentric circles until about two thirds of the wafer is covered.
   Note: Keep the bottle of SU8 close to the wafer when pouring to minimize the formation of bubbles.
4. Program the spincoater as follows:
   • Spread cycle: 500 rpm with an acceleration of 100 rpm/sec for 10 sec.
   • Spin cycle: 4,000 rpm with an acceleration of 100 rpm/sec for 30 sec.
   Note: This spincoating recipe will form a photoresist layer that is ~10 μm in thickness. By changing the spinning speed or the SU8 formulation, the thickness can be adjusted.
5. Soft bake the wafer by placing it on a hotplate set to 95 °C for 3 min.
6. Expose the wafer with UV light through the photomask for a total dosage of 140 mJ/cm².
   Note: SU8 is a negative photoresist therefore regions where UV light is able to pass through the photomask will remain after developing and become raised features on the master mold.
7. Post exposure bake the wafer by placing it on a hotplate set to 95 °C for 4 min.
8. Develop the wafer by placing it in SU8 developer for 3 min. After 3 min, rinse the wafer with isopropyl alcohol. If a white film is produced during the rinsing, the wafer is not fully developed and it should be placed back in the developer for another 30 sec. Rinse again with isopropyl alcohol. Repeat this process until a white film does not form during the isopropyl alcohol rinsing.
9. Dry the wafer in a stream of nitrogen and place in a 150 mm petri dish to protect from dust.

2. Making the PDMS Stamps

1. Prepare the PDMS prepolymer by combining the elastomer base and curing agent in a 10:1 w/w ratio. Typically 80 g of base and 8 g of curing agent are used to ensure there is sufficient PDMS to cover the master mold in a 1 cm thick layer.
2. Mix and degas the PDMS using a centripetal mixer set to the following:
   • Mix: 2,000 rpm for 2 min
   • Degas: 2,000 rpm for 2 min.
3. If a mixer is unavailable mix the PDMS by hand for 10 min using a 10 ml serological pipette. Degas the mixture by placing it in a vacuum desiccator for 30 min to remove bubbles.
4. Pour enough PDMS prepolymer over the master mold (patterned silicon wafer) to form a 1 cm thick layer. Cure the PDMS by baking at 65 °C for 4 hr or at room temperature for 48 hr.
5. Once cured, The regions containing the patterns can be cut out to form the PDMS stamps. To distinguish the feature side from the backside of the PDMS stamp, cut a notch out of one of the corners on the backside of the stamp.

3. Microcontact Printing of ECM Patterns

1. Clean 25 mm diameter glass coverslips by sonication in 95% ethanol for 1 hr and then dry in a 65 °C oven.
2. Prepare the PIPAAm solution by dissolving PIPAAm in 1-butanol at a concentration of 10% (w/v, typically 1 g in 10 ml).
3. Center a glass coverslip on the vacuum chuck of the spincoater and pipette 200 μl of the PIPAAm solution so that the entire glass surface is covered.
4. Spincoat the coverslip at 6,000 rpm for 1 min.
5. Clean the PDMS stamps by sonication in 50% ethanol for 30 min and then dry under a stream of nitrogen.
   Note: Drying and subsequent steps should be performed in a biosafety cabinet to maintain sterility for applications where the ECM nanostructures will be used with cells.
6. Coat the patterned surface of each PDMS stamp with 200 μl of the protein solution, typically 50 μg/ml in sterile distilled water for FN. Incubate for 1 hr at room temperature.
   Note: This coating volume is for a 1.5 cm² PDMS stamp and will need to be adjusted depending on the size of the PDMS stamp, the ECM protein used and the concentration of the ECM protein in solution.
7. Wash the PDMS stamps in distilled water to remove excess protein and dry thoroughly under a stream of nitrogen.
   Note: Any water left on the stamp will trigger the premature dissolution of the PIPAAm coating on the coverslip and prevent proper protein transfer.
8. For sterile fabrication, place the PIPAAm-coated coverslips inside a closed petri dish and sterilize using UV exposure, 45 min under the UV light in a biosafety cabinet is sufficient. If sterility is not required this step can be omitted.
9. Perform microcontact printing by placing the feature side of the PDMS stamp in contact with the PIPAAm-coated coverslip. If required, use forceps to tap lightly on the back of the stamps to remove any air bubbles and ensure uniform contact.
10. After 5 min, peel off the PDMS stamp from the coverslip.
11. At this stage, additional ECM proteins can be patterned to create more complex and multicomponent structures. Up to 3 printings have been verified to work with this process, and more may be feasible.

4. Release of ECM Nanofibers and Nanostructures

1. Place the patterned PIPAAm coated coverslip in a 35 mm petri dish and inspect the pattern fidelity using phase contrast microscopy. Depending on the pattern, a CCD camera may be necessary to resolve the features of the pattern. Fluorescence microscopy can also be used to inspect the pattern provided the ECM proteins are fluorescently labeled.
2. Add 3 ml of 40 °C distilled water to the petri dish and allow the water to gradually cool.
3. The dissolution of the PIPAAm layer and the release of the ECM protein patterns can be monitored using phase contrast microscopy. If the application does not permit the use of optical techniques, the release can be monitored by measuring the solution temperature. Typically, the water is cooled to room temperature, well below the LCST of PIPAAm (32 °C), to ensure the ECM protein nanostructures have been released.
4. After release, the nanofibers, nanofabrics and other nanostructures are floating in water. To use them for further applications they need to be manipulated. The exact approach will depend on the experimental objective and may include steps such as immobilizing onto another surface, moving with a micromanipulator system or embedding in a hydrogel.

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Results

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 μm 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 ± 0.4...

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Discussion

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...

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Materials

Name	Company	Catalog Number	Comments
Poly(N-isopropylacrylamide) / PIPAAm	Polysciences	21458-10	40,000 Mw
Sylgard 184 Silicone kit (PDMS)	Dow Corning		Mix 10 parts base with 1 part curing agent.
Butanol
Fibronectin	BD biosciences	354008	Human, 1mg
Laminin	BD biosciences	354239	Ultrapure, mouse, 1mg
Negative Photoresist	Microchem	SU8-2015
SU8 Developer	Microchem
Sonicator Branson M3510	Branson Ultrasonic Corporation	CPN-952-318
Thinky ARE-250 Mixer	Thinky Corporation
Spincoater	Specialty Coating Systems	G3P-8
Glass cover 25mm diameter, No 1.5	Fisher Scientific	12-545-86