Nanofiber Hierarchical Biological Scaffolds

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Suspended Micro/Nanofiber Hierarchical Biological Scaffolds Fabricated using Non-Electrospinning STEP Technique Ji Wang, and Amrinder Singh Nain Langmuir, Just Accepted Manuscript • DOI: 10.1021/la503011u • Publication Date (Web): 13 Oct 2014 Downloaded from http://pubs.acs.org on October 19, 2014

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Suspended Micro/Nanofiber Hierarchical Biological Scaffolds Fabricated using Non-Electrospinning STEP Technique Ji Wang1,3, and Amrinder S. Nain2,1,3* 1

Engineering Science and Mechanics Department, Virginia Tech, Blacksburg, VA, 24061, USA. 2 Mechanical Engineering Department, Virginia Tech, Blacksburg, VA, 24061,USA. 3 Macromolecule and Interface Institute, Blacksburg, VA, 24061, USA. Abstract Extracellular matrix (ECM) is a fibrous natural cell environment, possessing complicated micro-and nano- architectures, which provide extracellular signaling cues and influence cell behaviors. Mimicking this three dimensional micro environment in vitro is a challenge in developmental and disease biology. Here, suspended multilayer hierarchical nanofiber assemblies (diameter from microns to less than 100nm) with accurately controlled fiber orientation and spacing are demonstrated as biological scaffolds fabricated using the nonelectrospinning STEP (Spinneret based Tunable Engineered Parameter) fiber manufacturing technique. Micro/nanofiber arrays were manufactured with high parallelism (relative angles between fibers were maintained less than 6°) and well controlled inter-fiber spacing (< 15%). Using these controls, we demonstrate a bottom up hierarchical assembly of suspended 6 layer structures of progressively reduced diameters and spacing from several polymer systems. We then demonstrate use of STEP scaffolds to study single and multi-cell arrangement at high magnifications. Specifically, using double layer divergent (0° to 90°) suspended nanofibers assemblies, we show precise quantitative control of cell geometry (change in shape index from 0.15 to 0.57 at similar cell areas), and through design of scaffold porosity (80×80 µm2 to 5×5 µm2) quadruple the cell attachment density. Furthermore, using unidirectional or crisscross patterns of sparse and dense fiber arrays, we are able to control the cell spread area from ~400 µm2 to ~700 µm2, while the nucleus shape index increases from 0.75 to 0.99 with cells nearly doubling their focal adhesion cluster lengths (~15 µm) on widely spaced 1

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nanofiber arrays. The platform developed in this study allows a wide parametric investigation of biophysical cues which influence cell behaviors with implications in tissue engineering, developmental and disease biology.

Keywords: ECM (extracellular matrix), nanofiber, hierarchical assembly, bio-scaffolds, cellular interactions, aligned fibers Introduction

Cell behavior is directly affected by extracellular matrix (ECM), which is primarily composed of natural hierarchical assemblies of protein nanofibers.1–3 The hierarchical architecture of the surrounding ECM comprises of individual proteins (30-70nm diameter), which can form bundles (100nm-microns in diameter).4–10 These bundles of fibers are observed to be aligned or seemingly randomly distributed in vivo.11–14 Furthermore, due to imaging limitations in vivo, much of our current understanding in fundamental cell processes is derived from reductionist in vitro platforms, such as flat, gels and fibers, which aim to capture a complete in vivo description replete with simultaneous biophysical and biochemical cues.15–17 Micropatterned flat surface approaches involve micro-fabrication of cell-adhesive patches on cell culture substrates with high precision using soft lithography or photolithography.18,19 The engineered surfaces with the ability to constrain cell spreading to a specific cell-surface area have been shown to dramatically affect cell proliferation, differentiation, migration and apoptosis.20,21 However, the complexity in preparation of the cell-adhering substrates coupled with their long term stability and compatibility in cell-culture medium are typical problems requiring consideration.

22,23

On the other hand, hydrogel-cross-linked networks possessing

high water content have been intensively utilized for in vitro cell experiments.24 Cells embedded into hydrogels sense confinement from both mechanical properties of the hydrogels and pericellular accumulation of ECM proteins. Additionally, tuning the mechanical 2


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properties of hydrogels can be difficult as it involves a complex interplay of network properties,

swelling

characteristics,

degradation

rates

and

deposition

of

ECM

macromolecules.25

Recently, ECM as a mesh of nanofiber bundles has been studied using nanofabrication techniques such as nanolithography, micro/nano printing and electrospinning technique.26,27 Nanofiber assemblies are an interesting class of bioassays providing a 3D topology that better mimics the architecture formed by fibrous ECM proteins. It is well known that besides chemical composition and morphology of individual nanofibers, nanofiber orientation and spacing play important roles in guiding cellular responses.28,29 Thus, in the last decade, substantial efforts have been devoted to develop organized nanofiber scaffolds.30 In this regard, Li et al have demonstrated fabrication of aligned electrospun nanofibers using patterned electrodes,31 Katta et al demonstrated aligned electrospun nanofibers on a high speed rotating drum,32 and Zussman et al fabricated nanofiber arrays and crisscross patterns using a wheel-like bobbin collector.33,34 Although a certain degree of fiber alignment has been achieved, these approaches have encountered difficulties in spatially arranging fibers with accurately controlled diameter and spacing due to the inherent electric instabilities. In contrast, direct drawing method using polymer wetted probe tips has recently demonstrated precise micro/nanofibers depositions.35–38 Although well-organized fiber patterns have been demonstrated, it is unclear if these methods can be extended to build large area multi-layer structures at the nanoscale.

Using the Spinneret based Tunable Engineered Parameters (STEP) technique, micro/nano fibers are pulled from a pendent solution droplet and the absence of electrical instability (no electric source in the fabrication process) allows collection of highly aligned fibers of uniform dimension (diameter: sub 100 nm-microns) on the substrate. Using this approach, here we 3


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demonstrate suspended multilayer hierarchical scaffolds with accurately controlled fiber diameter, spacing and orientation for detailed cellular studies at high magnifications.

Experimental Section Solution preparation: All chemicals were used as received without further purification. Polystyrene (PS) of several molecular weights ( M W ) (860K, 1500K, and 2000K g.mol-1, Polymer Scientific, USA) were dissolved at room temperature in p-xylene and tetrahydrofuran (THF) at varying concentrations for one week prior to experiments. Poly (methyl methacrylate) (PMMA, M W =540K g.mol-1, Polymer scientific, USA) was dissolved in chlorobenzene at 134mg.ml-1, and polyurethane (PU5719, Lubrizol Advanced Material, USA) was dissolved in DMF at 292mg.ml-1. Bovine fibrinogen fraction I from plasma (340 kDa, Sigma-Aldrich Chemical Co., USA) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) at 100mg.ml-1. STEP spinning: The viscous polymer solutions were pumped through a 100 micron diameter micropipette spinneret (Jensen Global, USA).The substrate was mounted onto a DC motor, which in turn was mounted onto a motorized three degree of freedom micro-positioning stage (VP-25XA, Newport Inc, USA). No voltage source was used in the experiments to eject polymer solution. Cell culturing: STEP scaffolds were mounted on to glass-bottom six-well plates (MatTek Corp., Ashland, MA). Fiber scaffolds were sterilized by adding 2 ml 70% ethanol for 10 min, then coated in 2µg.ml-1fibronectin (Invitrogen, Carlsbad, CA).8,39 C2C12 mouse myoblasts (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (HyClone Laboratories, Logan, UT). Cells were seeded onto the fiber scaffolds via 30 µl droplets at a concentration of 100,000 cells ml-1 and incubated at 37 °C

4


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and 5% CO2. Cells were then given 2–6 h to attach onto the nanofibers. After 1 hour of seeding, 2 ml of medium was added to each well. Fluorescence microscopy: C2C12 mouse myoblasts were stained for F-actin stress fibers, focal adhesions and the nucleus. Cells were fixed in 4% paraformaldehyde, permeabilized in a 0.1% Triton X100 solution and blocked in 10% goat serum. Primary goat antipaxillin antibodies (Invitrogen) were diluted 1:250 and incubated at 4C° for 1 h. Secondary goat antirabbit antibodies conjugated to Alexa Fluor 488 (Invitrogen) were then added along with a 1:100 dilution of rhodamine phalloidin (Santa Cruz Biotechnology, Santa Cruz, CA) and placed at room temperature for 45 min and protected from light. DAPI counterstaining was performed by adding 300 nM DAPI (Invitrogen) for 5 min. The substrates were then rinsed with PBS and observed using Zeiss microscope. Fiber scaffolds were kept hydrated in 1 ml phosphate buffered saline (PBS) during imaging. SEM characterization: For SEM (Zeiss, Leo 1550) analysis, the samples were placed on goldcoated stubs and sputter coated. Statistical Methods:

Statistical analysis and graphing were performed using GraphPad

software. One way ANOVA testing with Tukey post testing comparing all pairs of data sets was performed. Column graphs are presented with standard error bars.

Results and Discussion In a typical STEP fiber fabrication process, polymer solution is extruded from a micropipette and forms a droplet (Figure 1(A)). The droplet is brought in contact with a rotating substrate mounted onto a three degree of freedom micro-positioning stage. Thus, in this set up, the substrate undergoes both rotational (Vr) and translational (Vt) motions. As the rotating substrate translates vertically, a polymeric filament is continuously extracted from the solution droplet and subsequently deposited on the substrate in parallel configurations. Controlled fiber deposition on various shapes (cube, cylinder, sphere, and shape assemblies) 5


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makes STEP an attractive technique for achieving fiber assemblies on curved surfaces of varying geometries (Figure 1(B-E)).

Figure 1. (A) Schematic illustration of STEP, (B-E) 3-D STEP fiber collections on cube, cylinder, sphere and shape assemblies respectively, (F) PS fiber diameters as a function of substrate rotating speed ‘ω’ for 2000K (g.mol-1) PS 65.1mg.ml-1solution in p-xylene. Each fiber diameter was averaged over 50 measurements, (G) PU fiber assembly, and (H) fibrinogen fiber assembly. Inserts in (G) and (H) are higher magnification images of the assemblies with 5 µm scale bars each. STEP fiber formation depends on both material and processing parameters. Fiber diameter scales with rotational speed (ω) as shown in Figure 1(F), and in this study, all fibers were obtained at a constant rotating speed ω=40 RPS (substrate width=8 mm). Smooth and uniform diameter fibers start to form as the solution concentration approaches critical entanglement concentration Ce, which signifies sufficient number of polymer chain entanglements to form a long range elastic network required for fiber formation. Using this technique, we have obtained aligned micro/nanofibers from various polymer species, including polystyrene (PS), poly(methyl methacrylate) (PMMA), poly(ethylene oxide) (PEO), poly(lactic-co-glycolic acid) (PLGA), polyurethane (PU) and fibrinogen (Figure 1(G-H)).40 Fiber diameter was controlled through a combination of material parameters, including solvent volatility (e.g. 6


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boiling point), solution concentration and molecular weight of the polymer. Using these approaches, we have been able to control fiber diameters in a fairly large range from 30 nm to 8 µm (See supplementary Information, Table S1). Note that the diameter of fibrous ECM proteins ranges from 50 nm (e.g. the approximate diameter of a collagen fibril) to microns (e.g. diameters of collagen fiber bundles), which is well within the diameter range of STEP fibers.41 Therefore, it is possible to topographically mimic native ECM at different scales using STEP fibers.

STEP based fiber manufacturing provides a single stable filament from the pendant droplet, leading to uniform diameter fibers which can be deposited precisely in ordered configurations, thus enabling accurate control of fiber diameter, alignment and spacing. As no solution filament spraying or branching occurs during STEP fabrication, the fiber diameter variation is well controlled within 12%. (See supporting Information, Table S1). In addition, since the filament is directly pulled out from the solution droplet by the collecting substrate, the sourceto-target distance, where instability is typically found in electrospinning, is absent. The absence of filament whipping in STEP results in a very high degree of alignment, where relative angles between fibers in an array are maintained less than 3° for microfiber and less than 6° for sub 100 nm diameter fibers (Figure 2(A-E)). Furthermore, STEP technique allows patterning ultrathin nanofibers of 30 nm in diameter (Figure 2(D)) , which is comparable to the lowest diameter that can be aligned with electrospinning.33,42,43 To achieve desired fiber spacing, the required translation speed Vt (mm/s) of the substrate is easily estimated by: Vt = d × RPS + L × (RPS-1), where d is the fiber diameter and L is the desired fiber spacing. (See supporting Information, Table S2). Using this, the average fiber spacing variation was maintained less than 7% for micro and 15% for nanoscale fiber arrays as shown in Figure 2(A-D) and (F). Double-layer structures were obtained by depositing fiber arrays on the previously deposited layer. Angles between two layers can be altered from 0° to 90° by 7


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rotating the substrate as shown in Figure 2(G) i-iii, which, when coupled with the ability to control spacing between consecutive fibers, enables a bottom-up assembly environment with fibers serving as 1D building blocks. As shown in Figure 2(H-K), PS nanofibers were

Figure 2. STEP capability in achieving control in single and double layer structures. (A-D) Arrays of 8000, 500, 150 and 30nm diameter PS fibers, (E) relative angles between adjacent fibers, (F) fiber spacing variation. Each data point in (E) and (F) is averaged over 30 measurements, (G) (i-iii) schematic illustrations of achieving interlayer angle (α12) in double layer structures, (H-J) SEM images of PS double layer structures with interlayer angle 15°,30°and 45° respectively, (K-M) orthogonally deposited PS double layer structures with unit cell size of 16×16 µm2, 5×2 µm2 and alternating 5×2 µm2 and 5×10 µm2. 8


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deposited in double layer configurations and the angle between two layers (α12) varied from 15° to 30°, 45° and 90°. By adjusting Vt in both directions, unit cell size was varied from 16×16 µm2(Figure 2(K)) to 5×2 µm2 (Figure 2(L)) and alternating 5×2 µm2 and 5×10 µm2 unit cells within the same architecture (Figure 2(M)).

Using STEP fibers as 1D building blocks, we then developed suspended 6 layer scaffold (thickness ~12 µm and Figure 3) in which large diameter PS fibers (diameter~5µm) were first deposited in two layers at unit cell area of 100×100µm2. The third and fourth layers of PMMA fiber (diameter~1µm) were deposited at 50×50µm2 unit cell size on top of PS fibers PU unit cell 10×10 µm2 III

PU

6 5

PS unit cell 100×100 µm2 I 4

PMMA unit cell 50×50 µm2 II

PMMA

3 3

1

PS V

IV

2

6th PU

6 5

6

4

5 4

3

3

2 1

5th PU 3rd

PMMA 4th PMMA

Figure 3. Schematic and SEM images of partial section of six layer bottom up hierarchical assembly with numbers denoting the bottom up sequence of deposition. Highlighted frames in inserts I-III are PS, PMMA and PU unit cells respectively. Insert IV and V demonstrates the bottom up deposition sequence. Scale bars represent 10 µm in inserts I-V. 9


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and finally, fifth and sixth layer of PU fibers (300nm in diameter) were deposited on top of PMMA fibers at 10×10 µm2 unit cell size. To maintain fiber conformations (fiber spacing and orientation), each new layer was fused to the previously deposited layers at the intersections using an in-house solvent vapor treatment method. Using STEP, there is potentially no limitation on the numbers of layers that can be stacked on top of each other and the architectures of each layer (fiber material, diameter and conformation) can be customized as required. (See Supporting Information, Figure S1) However, not unique to only STEP, but also to other fiber manufacturing platforms, challenges in deposition of fragile nanoscale fibers of substantial lengths at sub-micron spacing will need to be overcome to realize a complete hierarchical assembly design space.

A large number of studies using flat or gel culture systems have highlighted the role of extrinsic biophysical cues from the surrounding extra-cellular matrix (ECM) on cellular interactions.44 Topographic reactions of cells to micro-meter range features such as wells, islands, and pillars have been intensively investigated based on micro-patterned approaches.45 It has been shown that cell shape, position and function can be regulated by attachment of cells on engineered surfaces coated with different density of ECM.46 Recently, electrospun nanofibrous scaffolds have been successfully produced from both synthetic and natural polymers with diameter ranging from nanometer to micrometers.27 Cell studies on these scaffolds have revealed that cell attachment and spreading is affected by nanofiber diameter and scaffolds porosity.47,48 However, the role of suspended fiber spacing and orientation in influencing cell behavior has yet to be demonstrated. Cells on suspended fibers react to mechanical cues by wrapping around the suspended fibers, which has been shown by us to elicit different cell behaviors.8,39,49–52 Suspended cell-fiber culture systems afford a wide parametric investigation of the role of topographical cues on cellular interactions. In this study, we present the development of hierarchical nanofiber assemblies as a platform technology to 10


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control cell geometry with the aim of studying the arrangement of cellular cytoskeleton on suspended fiber networks.

Figure 4. (A)-(D) Representative fluorescent images of C2C12 mouse myoblasts on divergent fibers with varying angles (A) 0°, (B) 30°, (C) 60°, (D) 90°, (E-F) cell shape index and cell spread area as a function of divergent angles ±SE. * shows statistical significance (P

Nanofiber Hierarchical Biological Scaffolds

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