Creating Biomimetic Anisotropic Architectures with Co-Aligned

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Creating Biomimetic Anisotropic Architectures with Co-Aligned Nanofibers and Macrochannels by Manipulating Ice Crystallization Linpeng Fan, Jing-Liang Li, Zengxiao Cai, and Xun-Gai Wang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01648 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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Creating Biomimetic Anisotropic Architectures with Co-Aligned Nanofibers and Macrochannels by Manipulating Ice Crystallization Linpeng Fan, Jing-Liang Li*, Zengxiao Cai, and Xungai Wang* Institute for Frontier Materials, Deakin University, Geelong, VIC 3216, Australia KEYWORDS anisotropic 3D scaffolds, aligned nanofibers, aligned macrochannels and macropores, silk fibroin nanofibers, alginate nanofibers, gelatin nanofibers, neurites and vascularization

ABSTRACT Continuous evolution of tissue engineering scaffolds has been driven by the desire to recapitulate structural features and functions of the natural extracellular matrix (ECM). However, it is still an extreme challenge to create a three-dimensional (3D) scaffold with both aligned nanofibers and aligned interconnected macrochannels to mimic the ECM of anisotropic tissues. Here, we develop a facile strategy to create such a scaffold composed of oriented nanofibers and interconnected macrochannels in the same direction, with various natural polymers typically used for tissue regeneration. The orientation of nanofibers and interconnected macrochannels can be easily tuned by manipulating ice crystallization. The scaffold

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demonstrates both structural and functional features similar to the natural ECM of anisotropic tissues. Taking silk fibroin as an example, the scaffold with radially orientated both nanofibers and interconnected macrochannels is more efficient to capture cells and promote growth of both non-adherent embryonic dorsal root ganglion neurons (DRGs) and adherent human umbilical vein endothelial cells (HUVECs) compared to the widely-used scaffold types. Interestingly, DRGs and neurites on the SF scaffold demonstrate a 3D growth mode similar to that of natural nerve tissues. Furthermore, the co-aligned nanofibers and macrochannels of the scaffold can direct HUVECs to assemble into blood vessel-like structures and deposit collagen matrix in their arrangement direction. The strategy could inspire the design and development of multifunctional 3D scaffolds with desirable structural features for engineering different tissues.

Scaffolds formed by natural polymers such as proteins and polysaccharides play crucial roles in tissue engineering due to their excellent biocompatibility and biodegradability.1-4 An ideal scaffold should not only provide a three-dimensional (3D) environment and support, but also direct cell behaviors and functions by interacting with cells and mediating the complex multicellular interactions spatially and temporally.5-7 To this end, it is important for a scaffold to mimic the structural features and functions of natural extracellular matrix (ECM). A natural ECM has a 3D porous and intricate architecture of collagen nanofibers with diameter between 50 and 500 nm.8 In many tissues with anisotropic structural characteristics (such as dural, tendon, ligament, tympanic and muscle tissues), cells and ECM nanofibers are highly aligned.9-14 These specific alignments support the specific physiological functions of tissues and organs. For example, radially aligned nanofibrous matrices of the dural and tympanic tissues are essential for carrying blood and conducting sound, respectively.9,13 In skeletal muscle, tendon


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and ligament tissues, longitudinally aligned nanofibers play a key role in supporting movement and mechanical load.11,14 Apart from aligned nanofibers, in anisotropic tissues such as nerve tissues, interconnecting macrochannels along the long axis of tissues provide space for the orientated growth of nerve fibers and facilitate the exchange and transport of oxygen, nutrients and waste.15-17 Different techniques, especially electrospinning,2,18 3D printing19,20 and lyophilization,15,21,22 have been widely used to produce scaffolds for tissue engineering applications. However, none of them has been capable of coupling aligned nanofibers with aligned macrochannels which always go in the opposite direction. Although electrospinning is able to produce 2D aligned nanofibrous films, the films have very small pores and low porosity due to the mechanical stretching or high-speed drawing during fabrication. As a result, cells cannot infiltrate into the films and grow only on their surface.23-25 Meanwhile, these 2D films cannot mimic the extracellular matrix of native anisotropic tissues to provide support to cells and tissues in a 3D space.26-28 3D printing is an emerging technique that offers greater flexibility in tailored design of scaffolds, whereas the resolution of the printed structures is generally on micrometer or submillimetre scales.19,20 Creating hierarchical nanostructures presents enormous challenge to this technique. Lyophilisation is widely used to fabricate 3D porous scaffolds. Typically, a solution of polymer is frozen in a freezer. Upon sublimation of ice crystals, a porous polymer scaffold forms. However, anisotropic scaffolds with oriented nanofibers and macrochannels also have not been achieved with this technique to date. This is due to uncontrolled ice nucleation and growth, which leads to formation of random pores and structures. The pore characteristics of a scaffold depend on how ice nucleates and grows in the polymer matrix. The size and morphology of ice crystals in turn determine the structure of the solid part of a scaffold. Neglecting the interplays


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between these two components, i.e. polymer and ice, is the reason for failing to fabricate scaffolds with all the structural features. Furthermore, the interconnectivity of the pores in the scaffolds, which generally have a wall-like (non-fibrous) structure, is not sufficient for infiltration, migration and growth of cells and tissues.15,29 It also limits the transport of oxygen, nutrients and wastes, which is a general cause of cell necrosis and failure of tissue growth.15,30,31 In this work, we show that 3D scaffolds with aligned both nanofibers and interconnected macrochannels can be created with various biomacromolecules including silk fibroin (SF), using a facile guided ice-crystal growth and nanofiber assembly strategy. Firstly, oriented fine ice crystals were created to guide the assembly of oriented nanofibers. In brief, a solution of silk fibroin was immersed into a medium with a very low temperature (e.g. liquid nitrogen, Scheme 1a). Such a low temperature created a high temperature gradient in the solution, leading to formation of radially aligned fine ice crystals, among which biomacromolecules were assembled into nanofibers (Scheme 1, top view of (a)). Following removal of the fine ice crystals by sublimation (Scheme 1(i)), 3D scaffolds (AF, A denotes ‘aligned’ and F denotes ‘nanofibers’) composed of radially aligned nanofibers were obtained (Scheme 1b). Secondly, the orientated nanofibers guided orientated growth of large ice crystals along the fiber direction into the scaffold, which in turn led to further assembly of the nanofibers to form aligned macrochannels in their long-axis direction. In short, after post treatment in ethanol (Scheme 1(ii)), the AF scaffold with radially aligned nanofibers was immersed in water and frozen at -20 oC (Scheme 1c). The post treatment was used to fix the protein structure of the nanofibers to make them insoluble in water. In this step, the relatively low temperature gradient and cooling rate led to formation of large ice crystals. The radially aligned nanofibers of the AF scaffold guided the growth of large ice crystals into the scaffold in the alignment direction of nanofibers (Scheme 1c


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and its top view). Removal of the large ice crystals by lyophilization led to formation of macrochannels co-aligned with the nanofibers, where the aligned nanofibers assembled to form walls of the macrochannels (Scheme 1d and its top view). Due to squeezing and further assembly of nanofibers during macrochannel formation, pores on the nanofibrous channel walls were created, making the macrochannels interconnected (Scheme 1d, top view). Meanwhile, a central macrochannel from the top to the bottom of the scaffold was formed to interconnect with all radially aligned macrochannels (Scheme 1d). The scaffold with radially co-aligned nanofibers and macrochannels was indicated as A(F&C), where A represents ‘aligned’, F represents ‘nanofibers’ and C represents ‘channels’. Effects of the resulting silk fibroin (SF) scaffold on cell behavior and fate were investigated using the classic non-adherent embryonic dorsal root ganglion neurons (DRGs) and adherent human umbilical vein endothelial cells (HUVECs), respectively. In comparison with traditional 3D porous SF scaffolds, our 3D SF scaffolds with co-aligned nanofibers and interconnected macrochannels were more efficient in capturing both adherent cells and non-adherent cells. Furthermore, the scaffold not only significantly promoted cell proliferation, but also promoted 3D growth of DRGs and neurites, which is similar to the growth mode of natural nerve tissues. More interestingly, HUVECs in the scaffold were directed to assemble into blood vessel-like structures and deposit collagen matrix in the alignment direction of nanofibers and macrochannels.


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Scheme 1. Fabricating 3D scaffolds with radially co-aligned nanofibers and interconnected macrochannels (A(F&C) scaffolds) by a facile guided ice-crystal growth and nanofiber assembly strategy, with silk fibroin (SF) as an example. The bottom pictures in (b) and (d) are digital photos of the AF scaffold and the A(F&C) scaffold, respectively.


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RESULTS AND DISCUSSION

Figure 1. 3D aligned nanofibrous scaffolds with various geometries, diameters and thicknesses as well as different fiber orientations. (a) Representative SEM images of 3D aligned silk fibroin (SF) nanofibrous scaffolds (AF scaffolds in Scheme 1b). Fast Fourier transform (FFT) pattern (the inset on the bottom left of (AF) image in (a)) suggests the nanofibers are well aligned along the radial direction of cylinder scaffolds (the red arrows indicate the alignment direction). Scale bars: from left to right 2, 1 and 10 µm, respectively. (b) Directionally freezing SF solutions in liquid nitrogen by self-made devices produces 3D SF nanofibrous scaffolds with controllable nanofiber orientations indicated by red arrows as well as various geometries (including cylinders, tubes and particles), diameters and thicknesses.

Formation, Structure and Features of 3D Scaffolds. Figure 1a demonstrates the nanofibrous structure of the AF scaffold with radially aligned nanofibers, obtained after removal of the radially aligned fine ice crystals created during directional freezing of a SF solution in liquid


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nitrogen. The nanofibers have a smooth morphology with an average diameter 276 ± 78 nm. Fast Fourier transform (FFT) pattern (the inset on the bottom left of (AF) image in Figure 1a) shows the nanofibers are well aligned along the radial direction of cylinder scaffolds (the red arrows indicate the alignment direction of scaffold nanofibers). Interestingly, the nanofibers are decorated with nanoparticles. The formation of nanoparticles could be due to further nucleation of free SF molecules during the process of freeze drying for ice sublimation. In a solution of macromolecules, microscopically, there is always a concentration heterogeneity. Nucleation of nanoparticles is thermodynamically favored at the locations where the concentration of SF is higher. However, since the overall concentration of free SF after nanofiber formation is low, isolated nanoparticles rather than continuous structures (such as nanofibers) formed on the existing nanofibers. Quenching of a SF solution is facile and allows the fabrication of samples with various geometries (such as cylinders, tubes and particles), diameters and thicknesses (Figure 1b). In addition, the orientation of nanofibers can be easily varied by controlling the direction of temperature gradient. For example, apart from cylinders and tubes with radially aligned nanofibers, cylinders and tubes with vertically aligned nanofibers can also be fabricated by slowly lowering the SF solution-containing tube into liquid nitrogen to induce a vertical temperature gradient (the red arrows indicate the alignment direction of nanofibers) (Figure 1b(i),(ii)). Furthermore, by directly dropping SF solution into liquid nitrogen, particles with nanofibers radially aligned towards the center of the particle were obtained (Figure 1b(iii)). We observed that fast freezing with a high temperature gradient is beneficial to the formation of nanofibers and directional structures. Instead of using liquid nitrogen, SF solutions contained in the same glass tubes were frozen in freezers at -80 oC and -20 oC, respectively, followed by removing ice crystals with a freeze drier (i.e., the conventional lyophilization method). SF


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scaffolds from freezing at -80 oC have a hybrid structure consisting of random short channels/pores/nanofibers, but the short channels and pores are not interconnected (Supporting Information, Figure S1a). In the following study, the hybrid 3D SF scaffold from freezing at -80 o

C is presented as W&F, where W denotes ‘walls of the channels and pores’, F denotes

‘nanofibers’. In comparison, only random pores were observed in the SF scaffolds from freezing at -20 oC and the pores are also not well connected to form a network (Supporting Information, Figure S1b). Hence, when temperature gradient and cooling rate are not high enough, large ice crystals generally form, facilitating the formation of large but not interconnected pores and the scaffold has a wall-like structure. In the following study, the porous wall-like 3D scaffold from freezing at -20 oC is represented by W which denotes ‘wall-like structures’.


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Figure 2. Hierarchical structure of 3D SF scaffolds. (a) Micro-CT images of aligned macrochannels in the A(F&C) SF scaffold. Scale bars: 1000 µm. White arrows indicate that the


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direction of radially aligned macrochannels in cylinder scaffolds. (b) SEM images of the macrochannel wall of the A(F&C) SF scaffold, at various magnifications, revealing the aligned SF nanofibers decorated with nanoparticles and pores along the long-axis direction of the aligned macrochannel. Yellow arrows indicate the direction of radially aligned nanofibers along the long-axis direction of the aligned macrochannel. The green, blue and red arrows indicate the aligned nanofiber, the nanoparticle and the pore, respectively. Scale bars: from left to right 10, 2, and 1 µm, respectively. (c) A schematic dimension presentation of the relevant structures of the A(F&C) scaffold. (d) Micro-CT and SEM images of the W&F scaffold showing a hybrid structure of random short channels/pores/nanofibers and the W scaffold revealing a wall-like structure. Scale bars: 1000 and 100 µm, respectively, for Micro-CT and SEM images.

As described in the Scheme 1, the target 3D A(F&C) scaffold with radially co-aligned nanofibers and macrochannels was obtained after removal of the radially aligned large ice crystals in the 3D AF scaffold (Scheme 1c-d). 3D micro-CT images (Figure 2a) and the movie (Supporting Information, Movie S1) demonstrate that each radially aligned macrochannel (diameter, 100-1000 µm) connects the side surface and center of the A(F&C) scaffold (SEM profiles of macrochannels in the A(F&C) scaffold were presented in Supporting Information, Figure S2). As shown in Figure 2b, the channel walls are composed of SF nanoparticles and nanofibers (diameter, 50-600 nm) that are well aligned along the channel direction (indicated by yellow arrows). Many pores with diameters in the range of 50-1000 nm can be identified on the channel wall and these pores make the channels interconnected. The pores are also aligned along the same direction as nanofibers. The formation of pores could be due to contraction of nanofibers during the drying process. More interestingly, a central channel (diameter, 0.4-2 mm)


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from the top to the bottom of the scaffold was created spontaneously (Scheme 1d). This special structure could be produced by contraction of the scaffold during the lyophilization for ice sublimation. All the relevant sizes of the structures within the hierarchical 3D A(F&C) scaffold were summarized in Figure 2c. The macrochannels with porous nanofibrous walls in the A(F&C) scaffold and the central channel are very important for cell and tissue infiltration, as well as their growth by providing space and faciliating the transport of oxygen, nutrients and wastes. The aligned nanofibers on channel walls can have an important role in promoting cell capturing and proliferation as well as directing cell migration and mediating the complex multicellular interactions. Furthermore, nanofibers and nanoparticles can be good carriers for the delivery of growth factors or drugs. The A(F&C) scaffolds have also been achieved from mixtures of SF and gelatin as well as other biomacromolecules such as sodium alginate (Supporting Information, Figure S3). Without guidance of the aligned nanofibers, macrochannels cannot be made in randomly porous W and W&F scaffolds under the same conditions (Figure 2d; Supporting Information, Figure S4 and S5; Supporting Information, Movie S2 and S3 for W&F and W scaffolds, respectively). The random stiff wall-like structures in the W and W&F scaffolds made the growth of ice crystals be confined in the free space of the scaffolds and thus no long channels formed. Secondary Structure and Mechanical Characteristics of 3D Scaffolds. To make the scaffolds insoluble in water, an ethanol post-treatment (Scheme 1(ii)) was used to transform the random coil structure of SF to the β-sheet crystalline structure. Silk fibroin is water-insoluble when β-sheets are dominant.18,32 The secondary-structure change of SF can be indicated by the shift of its characteristic absorption peaks (1600–1500 cm−1 for amide II and 1700–1600 cm−1 for amide I) in ATR-FTIR spectra.18,32,33 Before post treatment with ethanol, all three types of SF


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scaffolds showed one of the main characteristic peaks at around 1644 cm−1 (which suggests random coils) (Supporting Information, Figure S6a).18 Noteworthily, the scaffolds respectively formed at -20 oC and -80 oC (W and W&F) presented another main characteristic peak at 1517 cm-1, which indicates dominant β-sheets.18 However, the scaffold (AF) formed by freezing in liquid nitrogen (about -196 oC) showed another main characteristic peak at 1533 cm-1, which suggests dominant random coils.18 These results suggest that the freezing treatment in liquid nitrogen could be beneficial to formation of random coils in SF scaffolds (Supporting Information, Figure S6a). After post treatment with ethanol, all three types of SF scaffolds presented main characteristic peaks at around 1700, 1622 and 1517 cm-1, suggesting the ethanoltreated scaffolds mainly consist of β-sheets (Supporting Information, Figure S6b).18,32


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Figure 3. Mechanical characteristics of scaffolds and representative scaffold morphologies after compressing. (a) Compressive modulus of scaffolds and their corresponding morphologies after the mechanical test: porous wall-like 3D silk fibroin (SF) scaffolds (W), 3D SF scaffolds with short channels/pores/nanofibers (W&F) and 3D SF scaffolds with radially co-aligned nanofibers and macrochannels (A(F&C)). (b) Stress-strain plots of 3D SF scaffolds. (c) Hysteresis loops


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during the first loading cycle for 3D SF scaffolds. (d) Peak stresses of 3D SF scaffolds during the first loading cycle.

Mechanical characteristics of different scaffolds were given in Figure 3. The A(F&C) scaffold (with co-aligned nanofibers and macrochannels) has a compressive modulus of around 80 kPa, which is lower than that of the porous wall-like scaffold (W) and the scaffold with short channels/pores/nanofibers (W&F) (around 100 and 140 kPa, respectively) (Figure 3a). The lower modulus of the A(F&C) scaffold could be due to its large channel-based structure with nanofibers. Noteworthily, the A(F&C) scaffold did not fracture even if it was compressed to approximately 85% of its original height (Figure 3b). Furthermore, after being compressed in the mechanical test, the A(F&C) scaffold still maintained a well radially aligned morphology and structure, with just some minor collapses seen on its surface, probably due to damage of some macrochannels (Figure 3a). Although the A(F&C) scaffold has a relatively low peak stress (Figure 3d), its stress-stain curve under cyclic compression shows a hysteresis loop (Figure 3c), confirming its viscoelastic nature similar to that of the natural ECMs.34-36


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Figure 4. Co-aligned nanofibers and macrochannels of 3D A(F&C) scaffolds facilitate capture of embryonic dorsal root ganglion neuron cells (DRGs), and direct 3D growth of DRG neurites. (a) Viability (MTS absorbance index) of DRGs captured by radially aligned 3D SF nanofibrous scaffolds without channels (AF), porous wall-like 3D SF scaffolds (W), 3D SF scaffolds with short channels/pores/nanofibers (W&F) and 3D SF scaffolds with radially co-aligned nanofibers and macrochannels (A(F&C)). (b) Confocal fluorescence microscopic images reveal that the structures of W, W&F and AF scaffolds limited DRGs and DRG neurites to grow on surface of


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the scaffolds only. Scale bars: 100 µm for the W and W&F scaffolds; 50 µm for the AF scaffold. (c) Co-aligned nanofibers and macrochannels directed 3D growth of DRG neurites in the 3D A(F&C) scaffold. Scale bars: from left to right 75, 25 and 25 µm, respectively.

Scaffolds with Co-aligned Nanofibers and Macrochannels Facilitating DRGs Capturing and 3D Growth of Neurites. To understand effects of the co-aligned nanofibers and macrochannels on cells, the ability of the scaffolds to capture cells and promote their growth was investigated using embryonic dorsal root ganglion neurons (DRGs). As shown in Figure 4a, the A(F&C) scaffold (with co-aligned nanofibers and macrochannels) demonstrated superior DRG capturing capacity. The AF scaffold (with aligned nanofibers, but without channels) showed the lowest DRG capturing. Figure 4b illustrates the areas of scaffolds that were scanned and the corresponding images. Obviously, affluent neurites were aligned in the direction of nanofibers on the surface of the AF scaffold, but they were not clearly observed in the inner of this scaffold, as shown by the image of AF cross-section. In the porous W and W&F scaffolds, neurites were also mainly aggregated on the surface. In the inner of the porous wall-like W scaffold, the neurite infiltration of aggregated DRGs happened along the pore walls only (Supporting Information, Figure S7). In the inner of the W&F scaffold (with short channels/pores/nanofibers), the pore walls also led to the aggregation of DRGs and limited the neurite outgrowth (Supporting Information, Figure S7). These results indicate that in the absence of the natural nerve conduitlike channels, it’s difficult for DGRs to grow in the inner of these scaffolds during 21 days of culture, and thus the neurite outgrowth and extension of DRGs were suppressed. In comparison with the structures of the scaffold AF, W and W&F, the radially aligned macrochannels towards the center of scaffolds provided enough space similar to the natural nerve conduit for migration,


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and 3D growth of the DRGs and neurites. Figure 4c illustrates the scanned areas of the A(F&C) scaffold and the corresponding images. In the scaffold A(F&C), DRGs can be clearly seen, and lots of long neurites had grown through the macrochannels (the channels, channel walls and neurites were indicated by white arrows). Interestingly, zooming in on the channels revealed that DRGs and neurites mainly grew along the channels, suggesting a 3D growth mode of neurites similar to that of natural nerve fibers.17 This is totally different from the 2D growth of DRGs and neurites along the aligned nanofibers on the surface of the AF scaffold (Figure 4b). From the last image (inside of A(F&C)) in Figure 4c, neurites in bundles are obvious, which is very important for the formation of nerve tissues.17 These observations demonstrate that the co-aligned nanofibers and macrochannels can not only promote the adhesion of non-adherent DRGs, but also direct them to grow, migrate and interact in the 3D space similar to the natural ECM.


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Figure 5. 3D A(F&C) scaffolds with radially co-aligned nanofibers and macrochannels enhance capturing and proliferation of adherent Human Umbilical Vein Endothelial Cells (HUVECs), and direct cell migration and growth. (a) Viability (MTS absorbance index) of HUVECs captured by radially aligned 3D silk fibroin (SF) nanofibrous scaffolds without macrochannels (AF), porous wall-like 3D SF scaffolds (W), 3D SF scaffolds with short channels/pores/nanofibers (W&F) and 3D SF scaffolds with radially co-aligned nanofibers and macrochannels (A(F&C)). (b) Viability (MTS absorbance index) of HUVECs in 3D AF, W, W&F and A(F&C) scaffolds after different periods of culture. (c) Scheme illustrating how to read the images presented in (d). (d) Growth of HUVECs in 3D AF, W, W&F and A(F&C) scaffolds after three days of culture. All the scaffolds were stained by two fluorescent dyes. To clearly show the morphology of cells and scaffolds,


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blue color was used for the W and W&F scaffolds and red color was used for the AF and A(F&C) scaffolds. Scale bars: 25 µm in W, W&F, AF and Inset 1; 75 µm in A(F&C).

Scaffolds with Co-aligned Nanofibers and Macrochannels Promoting HUVECs Capturing and Growth. Effects of the co-aligned nanofibers and macrochannels on cells were further confirmed using adherent human umbilical vein endothelial cells (HUVECs). At all-time points, the A(F&C) scaffold demonstrated significantly higher capacity of cell capturing and proliferation than the porous wall-like 3D silk fibroin (SF) scaffold (W) and the 3D SF scaffold with short channels/pores/nanofibers (W&F) (Figure 5a,b). This indicates that the aligned macrochannel and nanofibrous structures of the A(F&C) scaffold are beneficial to cell capturing and proliferation. Compared with the W scaffold, the W&F scaffold demonstrated higher cell adhesion and proliferation viabilities, after 8 hours and 6 days, respectively, which is probably due to the presence of nanofibers in the W&F scaffold. These findings are consistent with the observations from other researchers that nanofibers can promote cell adhesion and proliferation, probably by providing more cues and binding sites.5 To further identify the effect of macrochannels, the AF scaffold (with radially aligned nanofibers, but without channels) was also used as cell culture substrates in this study (Figure 5a,b). Obviously, the A(F&C) scaffold demonstrated significantly higher cell viability than the AF scaffold at all time-points, confirming the advantages of macrochannels in cell capturing and proliferation. Furthermore, the W and W&F scaffolds also showed higher cell viability in comparison with the AF scaffold. This is probably due to the fact that the W and W&F scaffolds have more space for cell adhesion and proliferation with their larger pores.


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To gain more insight into effect of the co-aligned nanofibers and macrochannels, cells grown in the scaffolds for 3 days were imaged using confocal fluorescence microscopy (Figure 5d). To date, it remains a problem that cell behaviors including cell spreading, migration, elongation and interaction are often hindered, due to the small pores and their low interconnectivity of scaffolds as well as the absence of binding and guiding cues in a scaffold.15,29 This is also true to both the W and W&F scaffolds. As shown in Figure 5d, cell spreading was significantly limited by pore walls (indicated by yellow arrows in (W)) or presented with blunt edges (indicated by white arrows in (W&F)) as if cells were cultured on surface of a flat material. Although cells were also observed in the AF scaffold, it was difficult to find them during scanning under confocal microscopy due to the small number of cells inside this scaffold. Cells in the AF scaffold were not well aligned and elongated in the direction of nanofibers, exhibiting a relatively flat and polygonous morphology. This is probably due to that the loosely aligned nanofibers provided cells with many surrounding signals from different directions.5 Obviously, cells on the channel wall of the A(F&C) scaffold were well elongated and aligned along with nanofibers, and they seemed to be in their dynamic migrating modes. The presence of large 3D channels reduced space in the scaffold so that nanofibers were compacted on walls of the channels, providing cells with more signals in the long-axis direction of nanofibers (the directions of channels and nanofibers were indicated by white arrows, respectively). This could explain the cell growth and morphologies observed in the A(F&C) scaffold.


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Figure 6. Co-aligned nanofibers and macrochannels in 3D scaffolds direct formation of the CD31-positive vessel-like structures and deposition of the collagen matrix from HUVECs by regulating the growth, migration and interaction of cells (Figure 5c above illustrates how to read the images presented in this Figure). (a) Growth and interaction of HUVECs in the 3D silk fibroin (SF) scaffold with radially co-aligned nanofibers and macrochannels (A(F&C)), the radially aligned 3D SF nanofibrous scaffold without channels (AF), the 3D SF scaffold with short channels/pores/nanofibers (W&F) and the porous wall-like 3D SF scaffold (W). Scale bars: 50 µm in A(F&C), W&F and W; 25 µm in AF. (b) Sequential confocal slices of the A(F&C)


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channel shown in (a) demonstrating many vessel-like structures formed in the long-axis direction of the channel. Scale bars: 50 µm. (c) Deposition of collagen matrix from HUVECs in the A (F&C), AF, W&F and W scaffolds. For the AF scaffold, collagen matrix only on the surface of this scaffold was stained and imaged (it was difficult to observe collagen matrix in the inside of this scaffold due to a small number of cells). The blue arrows indicate the collagen matrix stained in bright red. The black arrow in AF indicates the direction of aligned nanofibers and the black arrow in A(F&C) indicates the direction of the channel. Scale bars: 100 µm in W, W&F and AF; 200 µm in A(F&C).

Scaffolds with Co-aligned Nanofibers and Macrochannels Directing Vascularisation and Collagen Matrix Deposition. Proliferation, migration and interaction of endothelial cells are very important for formation of the tubal structures in both vasculogenesis and angiogenesis.37 HUVECs are a classic endothelial cell model for studying vascularization.38,39 As observed above (Figure 5), the A(F&C) scaffold can promote the proliferation of HUVECs. Hence, it was anticipated that the cell migration and elongation induced by the co-aligned nanofibers and macrochannels would enhance intercellular interactions to facilitate formation of vessel-like structures. To prove this, we cultured HUVECs up to 21 days to observe vascularization behaviours of cells in the different scaffolds (Figure 6a,b; Figure 5c illustrates how to read the images in Figure 6). Clearly, all cells are CD31-positive (CD31 is a glycoprotein expressed on endothelial cells), where the CD31-positive cells are in bright green, with their cell nuclei in bright blue (Figure 6a,b and Supporting Information, Figure S8). This suggests they still maintained the characteristics of HUVECs in the scaffolds after a long term of culture. In the W and W&F scaffolds, many cells showed a round morphology with just a few nuclei elongated


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(Figure 6a,b and Supporting Information, Figure S8). Obviously, the spreading, migration and elongation of cells were limited by scaffold walls, leading to local aggregation and interaction of some cells. In the AF scaffold, although some cell nuclei were elongated, most of cells were not significantly aligned and elongated, presenting a polygonous morphology (Figure 6a,b and Supporting Information, Figure S8). Interestingly, in the A(F&C) scaffold, all cells and cell nuclei were elongated and aligned on the wall of channels where they interacted and assembled into CD31-positive vessel-like structures (the channel, channel walls, vessel-like structures as well as aligned and elongated cell nuclei were indicated by white arrows) (Figure 6a,b and Supporting Information, Figure S8). A comparison between Figures 6 and 4 indicates that the adherent HUVECs were mainly directed by the aligned nanofibers on the channel wall of the A(F&C) scaffold, and the non-adherent DRGs and neurites preferred to grow along the 3D channel space. Fourteen sequential confocal slices of the A(F&C) macrochannel in Figure 6a were presented in Figure 6b. Clearly, there are many vessel-like structures aligned on the wall of channel in the inner of the A(F&C) scaffold. These findings demonstrate the co-aligned nanofibers and macrochannels facilitated the spreading, migration, elongation and interaction of HUVECs to assemble into the vessel-like structures. More interestingly, the structure of the A(F&C) scaffold not only directed the behaviors of cells, but also directed deposition of the collagen matrix of cells in the scaffold. As shown in Figure 6c, in the W and W&F scaffolds, the collagen matrix from HUVECs was in a random arrangement (after staining, the collagen matrix was in bright red, as indicated by the blue arrows in Figure 6c). For the AF scaffold, the collagen matrix from HUVECs only on the surface of the scaffold was stained and imaged (it was difficult to observe the collagen matrix in the inside of


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this scaffold due to the lack of cells). Although the collagen matrix was deposited along the nanofiber direction (indicated by the black arrow in Figure 6c) of the AF scaffold, the matrix was not well aligned, which may be due to the flat and polygonous arrangement of HUVECs on the loosely aligned nanofibers, as shown in Figure 5d and 6a. Obviously, in the A(F&C) scaffold, collagen matrix on the channel wall was well deposited along the aligned nanofibers, which is consistent with the good alignment and elongation behaviors of HUVECs on the channel wall of this scaffold, as indicated in Figure 5d and 6a,b. Stability of 3D Scaffolds. In order to investigate the in vitro degradation of 3D SF scaffolds, the scaffolds were incubated in PBS and protease/PBS solution, respectively, for 1, 5, 10, 15, 21 and 28 days (Supporting Information, Figure S9a). After being incubated in PBS for up to 28 days, all the three types of scaffolds experienced only a small loss in weight (≤5%). In the presence of protease, the weight loss of the A(F&C) scaffold is about 54%, while those of W and W&F scaffolds are around 38% and 33%, respectively. The higher weight loss of the A(F&C) scaffold at all time-points may be due to the fact that its nanofibrous structure provides a higher surface area to interact with protease molecules. To further provide insight into the degradation of the scaffolds, morphologies of the scaffolds after incubation in the solutions for 10 days were examined using SEM (Supporting Information, Figure S9b and S10). In the presence of protease, some small pores (indicated by red arrows) on the surface of nanofibers or walls of the scaffolds were observed. Meanwhile, some cracks on the relatively thin nanofibers in the A(F&C) scaffold were seen, as indicated by the blue arrows in Figure S9b (Supporting Information). Despite these, all three types of scaffolds maintained a good morphology and integrity even in the presence of protease, as shown by SEM images taken at low magnifications (Supporting Information, Figure S9b and S10). The excellent stability of the scaffolds was also demonstrated


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in the in vitro cell-culture studies, in which the channels still showed a good morphology and structure after 21 days of cell culture (Figure 4c, 5d and 6a,b). With the pure PBS solution, no obvious changes on the surface of nanofibers of the A(F&C) scaffold and walls of the W and W&F scaffolds were identified. These results suggest that although the scaffolds are biodegradable, their morphology and integrity can be maintained for a long time.

CONCLUSIONS In summary, we have developed a facile strategy for guiding growth of ice crystals and assembly of nanofibers to create biomimetic anisotropic 3D scaffolds (the A(F&C)) with coaligned nanofibers and macrochannels using various natural polymers such as silk fibroin (SF). As a model platform for cell culture and study in vitro, the 3D SF A(F&C) scaffold showed significantly higher cell capture and growth-promoting capability than the widely-used porous 3D SF scaffolds and the 3D aligned SF nanofibrous scaffold without macrochannels for both non-adherent DRGs and adherent HUVECs. More importantly, the co-aligned nanofibers and macrochannels of the A(F&C) scaffold can not only direct neurite growth of DRGs in the 3D space similar to the natural nerve conduit, but also regulate growth, migration, alignment, elongation and interaction of HUVECs to assemble into blood vessel-like structures in the scaffold in vitro. It is interesting to observe that the adherent HUVECs were mainly directed by the aligned nanofibers on the wall of the A(F&C) scaffold and the non-adherent DRGs and neurites preferred to grow along the 3D space of macrochannels. More interestingly, the HUVECs were also directed to deposit their collagen matrix along the co-aligned nanofibers and macrochannels. In brief, the scaffold developed in this work served as an excellent model platform for proof-of-concept that creation of the ECM-mimicking 3D structure is very


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important for providing insight into cell behaviors and functions. Considering the facile fabrication technology, the discovery in this work will provide an inspiration for developing biomimetic functional 3D scaffolds based on aligned nanofibers and macrochannels for use in tissue engineering. For example, the tube scaffold with radially co-aligned nanofibers and macrochannels could be beneficial to a multi-layer cell seeding for constructing blood vessel tissues. Likewise, with a similar structure to the natural nerve conduit, the column scaffold containing co-aligned nanofibers and macrochannels in the scaffold long-axis direction could provide a better support for nerve regeneration than the widely-used hollow tube scaffolds with thin walls.

EXPERIMENTAL SECTION Silk Fibroin (SF) Solution Generation. SF solution (2%) was obtained by dissolving 2 g of regenerated SF sponge (see the fabrication detail in Supporting Information) in 100 mL ultrapure water for further use. 3D SF Scaffold Preparation. (1) Scaffolds with radially aligned nanofibers (AF). SF solution in a glass tube (12 mm diameter × 45 mm height) was directly immersed into liquid nitrogen. Radially aligned nanofibrous scaffolds were produced by removing ice crystal using a freeze dryer. The fabrication scheme was shown in Scheme 1. To make the scaffolds insoluble in water, the scaffolds were treated by immersing in ethanol at ambient temperature for 12 h, and then thoroughly rinsed with ultrapure water to obtain water-resistant AF scaffolds. (2) Scaffolds with co-aligned nanofibers and macrochannels (A(F&C)). The AF scaffolds were immersed in


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ultrapure water and then frozen at -20 oC for 72 h. After removing ice crystals with a freeze drier, A(F&C) scaffolds were obtained.

ASSOCIATED CONTENT Supporting Information. Additional experimental details, figures and movies.

AUTHOR INFORMATION Corresponding Authors *[email protected] (J. L. Li) *[email protected] (X. Wang) Author Contributions L. Fan, J. L. Li and X. Wang conceived the project. L. Fan, Z. Cai, J. L. Li and X. Wang designed the experiments. L. Fan and Z. Cai performed the experiments. L. Fan wrote the draft of manuscript. All authors contributed to analysis and discussions of the data, and revision of the manuscript. All authors have given approval to the final version of the manuscript. Notes The authors declare no conflict of interest.


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ACKNOWLEDGMENTS X. Wang and J. L. Li acknowledge the Australian Research Council World Class Future Fiber Industry Transformation Research Hub (IH140100018). J. L. Li also acknowledges the ARC Future Fellowship project (FT130100057). REFERENCES (1) Sayyar, S.; Murray, E.; Thompson, B.; Chung, J.; Officer, D. L.; Gambhir, S.; Spinks, G. M.; Wallace, G. G., Processable Conducting Graphene/Chitosan Hydrogels for Tissue Engineering. J. Mater. Chem. B 2015, 3, 481-490. (2) Fan, L.; Cai, Z.; Geng, X.; Wang, H.; Li, J.; He, C.; Mo, X.; Wang, X., Fabrication and Characterization of Compound Vitamin B/Silk Fibroin Nanofibrous Matrices. J. Controlled Release 2017, 259, e85-e86. (3) Smith, B. D.; Grande, D. A., The Current State of Scaffolds for Musculoskeletal Regenerative Applications. Nat. Rev. Rheumatol. 2015, 11, 213-222. (4) Porter, D.; Vollrath, F., Silk as a Biomimetic Ideal for Structural Polymers. Adv. Mater. 2009, 21, 487-492. (5) Stevens, M. M.; George, J. H., Exploring and Engineering the Cell Surface Interface. Science 2005, 310, 1135-1138. (6)

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The scaffold with radially co-aligned nanofibers and macrochannels can not only significantly capture cells, but also direct cell behaviors and functions by interacting with cells and mediating the complex multicellular interactions spatially and temporally. 41x39mm (300 x 300 DPI)


Creating Biomimetic Anisotropic Architectures with Co-Aligned

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