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Controlling Cell Behavior on Silk Nanofiber Hydrogels with Tunable Anisotropic Structures Lili Wang, Guozhong Lu, Qiang Lu, and David L Kaplan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00969 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 2018

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ACS Biomaterials Science & Engineering

Controlling Cell Behavior on Silk Nanofiber Hydrogels with Tunable Anisotropic Structures Lili Wanga, b, Guozhong Luc, Qiang Lua, b,*, David L. Kapland

a

National Engineering Laboratory for Modern Silk & Collaborative Innovation Center of Suzhou

Nano Science and Technology, Soochow University, Suzhou 215123, People’s Republic of China b

Key Laboratory of Stem Cells and Biomedical Materials of Jiangsu Province and Chinese

Ministry of Science and Technology, Soochow University, Suzhou 215123, People’s Republic of China c

Department of Burns and Plastic Surgery, The Third Affiliated Hospital of Nantong University,

Wuxi 214041, People's Republic of China d

Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA

Corresponding author: *

Qiang Lu, Tel: (+86)-512-67061649; E-mail: [email protected]

ACS Paragon Plus Environment

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Abstract Understanding the influence of various physical cues on cell behavior is critical for the design of bioactive biomaterials optimized for tissue regeneration. Hydrogels provide microenvironments suitable for cells and tissues, but the introduction of tunable physical cues in hydrogels is often challenging. Here, silk nanofiber hydrogels with tunable, aligned structures were prepared under electric fields and used to influence cell behavior. Cell responses to the aligned structures were qualitatively and quantitatively evaluated by investigating cell proliferation, morphology and migration behavior, when compared to cells cultured on silk nanofiber hydrogels with homogeneous structures as controls. The cells seeded on anisotropic hydrogels showed oriented morphology, suggesting the link between aligned physical cues on cell morphology. The results indicated that tunable, aligned structures in hydrogels are effective for the design of bioactive biomaterials with preferred functions.

Keywords: Silk; Hydrogel; Alignment; Cell Behavior; Physical Cues


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1. Introduction The surface morphology of biomaterials provides critical physical cues to influence the behavior of cells, including morphology, migration, growth and differentiation.1-10 Various techniques including photochemistry,11 soft lithography12 and inkjet printing13 have been developed to fabricate surfaces with designed topographies, but challenges remain to introduce topographies in three-dimensional (3D) biomaterials to mimic the features of extracellular structures. Electrospinning methods have evolved to control the surface topography of 3D nanofibrous biomaterials to improve cell adhesion, proliferation and differentiation towards tissue regeneration.14-16 However, significant differences remain for these biomaterial systems when compared to the microenvironments found in native tissues. Due to the similar biophysical properties exhibited by hydrogels to native extracellular matrices, these materials provide appealing applications in tissue engineering.17, 18 Different chemical and physical cues, such as composition, the presence of bioactive growth factors and nanofibrous structure morphology can be introduced into this class of materials, providing useful microenvironments for tissue engineering.19-22 Considering that many complex tissues (e.g., nerves, bone, skin, muscles) retain an anisotropic architecture for normal biological functions and efficient cell-cell communication,23-26 hydrogels with oriented microstructures have been prepared and have achieved improved tissue regeneration.27, 28 While a few processes have been developed to form anisotropic hydrogels,29-32 limitations with these methods resulted in challenges in the formation of aligned structures. The influence of hierarchically aligned structures in hydrogels and their impact on cell behavior and tissue regeneration remains to be elucidated.


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Silk fibroin (hereafter referred to as silk), derived from Bombyx mori, has been widely used in biomaterial matrices for soft and hard tissue engineering due to its tunable mechanical properties, degradability in vivo, biocompatibility and facile fabrication into different material formats.33, 34 Diverse physical and chemical crosslinking methods have been applied to form silk hydrogels with various microstructures, mechanical properties and degradation behavior.35-37 Although these hydrogels exhibited preferred performance in tissue regeneration, the introduction of anisotropic structures remains elusive, yet would be a useful feature to explore towards enhanced tissue outcomes. Silk hydrogels easily formed under electrical fields, providing a mild and feasible way of designing microenvironments for cell culture and tissue regeneration.38-40 These hydrogels were termed as e-gels and have been used in tissue repair and drug delivery. Recently, we assembled silk nanofibers in aqueous solutions and formed hydrogels with aligned structures under an electric field.41 The anisotropic structures could be tuned by changing the concentrations of silk, providing a suitable platform to study anisotropic cues in hydrogels on cell behavior. Here, silk nanofiber hydrogels with tunable anisotropic structures were used to culture cells and investigate the influence of various aligned cues on cell proliferation, morphology and migration. Significantly different cell behaviors, especially migration, were observed. The study emphasizes the critical effects of physical cues on controlling cell behavior and reveals the possibility of exploiting bioactive silk hydrogels towards directed control of cell and tissue outcomes. 2. Experimental Section Preparation of silk solution: Silk solution was prepared based on previous published processes.42 Simply, Bombyx mori silks were degummed in boiling 0.02 M sodium carbonate


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solution for 20 min followed by washing in deionized water. The degummed fibers were dried in air and dissolved in 9.3 M LiBr solution at 60oC, obtaining a blend solution. After dialysis against distilled water for 72 h, the solution was centrifuged at 9,000 rpm for 20 min at 4oC to remove tiny of aggregates, finally achieving transparent aqueous solution with concentration of about 6 wt%. Fabrication of silk nanofiber hydrogels: The silk nanofibers were prepared via a concentration-dilution method.43 Fresh solution (6 wt%) was slowly concentrated to above 20 wt% over 24 h at 60oC to form unstable nanoparticles by covering a lid with holes on the solution. The concentrated solution was diluted to 0.5 wt%, 1 wt%, and 2 wt% with distilled water, respectively. Then the diluted silk fibroin solution was treated at 60oC in the sealed beaker to induce the transformation until the formation of nanofiber hydrogels. The homogeneous nanofiber hydrogels with a concentration of 0.5 wt% were used as control and termed as RSH0.5. All the nanofiber hydrogels with different concentrations were then used to form aligned silk hydrogels under electrical field. Fabrication of aligned silk hydrogels: Electrodes were immersed in an aqueous hydrogel of silk nanofibers with concentrations of 0.5 wt%, 1 wt% and 2 wt%, respectively and 50 V DC was applied over via a pair of conductive electrodes. Within 30 min, a visible aligned hydrogel formed at the positive electrode.41 According to the concentration of the used silk nanofiber hydrogels, the aligned silk nanofiber hydrogels were termed ASH-0.5, ASH-1 and ASH-2, respectively. SEM: The microstructure of the hydrogels was evaluated by scanning electron microscopy (SEM, Hitachi S-4800, Hitachi, Tokyo, Japan) at 3 kV. Gold sputter-coated treatment was


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applied to the freeze-dried samples before examination.44 To analyze the space width and lamella thickness of aligned hydrogels prepared from different silk nanofiber concentrations, at least 300 samples for each study group were counted using the Image-Pro Plus software based on the SEM images. CD: The secondary structures of the silk hydrogels were investigated using a Jasco-815 CD spectrophotometer (Jasco Co., Japan).45,

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The CD spectra were collected in the wavelength

range of 190-250 nm at a scanning rate of 100 nm min-1. Five scans were accumulated for every measurement and the average data was obtained from three samples. FTIR: FTIR spectra of silk hydrogels were obtained with a Nicolet FTIR 5700 spectrometer (Thermo Scientific, FL, USA) in the range of 400-4000 cm-1.44 Fourier self-deconvolution (FSD) of the amide I region (1595-1705 cm-1) was achieved by PeakFit 4.12 software to analyze silk secondary structures.47, 48 Cell culture: Bone marrow mesenchymal stem cells (BMSCs) derived from Sprague-Dawley (SD) rats were used to investigate the effect of hydrogels on cell attachment, morphology and migration. The use of the SD rats was approved by the animal ethics committee of Soochow University. The derived BMSCs were cultured in Dulbecco's modified Eagle medium (DMEM, low glucose) supplemented with 10% fetal bovine serum, 100 U/mL penicillin-streptomycin (all from Invitrogen, Carlsbad, CA). When the confluence was above 90%, cells were detached and seeded in the 96-well plates with sterilized hydrogels (60Co γ-irradiation 25 kGy) at a density of 1.0 ×105 cells / hydrogel. Cell morphology and proliferation: The cell morphology on the hydrogels was examined by confocal laser scanning microscopy (CLSM, Olympus FV10 inverted microscope, Nagano,


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Japan).49 After culturing for 1, 3, 6, and 12 d, the cell-seeded hydrogels were stained with phalloidin-FITC (Sigma-Aldrich, St. Louis, MO, USA) and DAPI (Sigma-Aldrich, St. Louis, MO, USA) according to the protocol of the manufacturer. CLSM was used to photograph fluorescence images of the samples under excitation/emission at 358/462 nm and 494/518 nm. PicoGreen™ DNA assay (Invitrogen, Carlsbad, CA, USA) was used to evaluate cell proliferation on the hydrogels. At indicated time points (from 1 to 12 d), the samples were immersed in proteinase K solution overnight at 56oC to digest the hydrogel.50 The digested samples (n=5) were measured with a spectrofluorometer (BioTeK Synergy 4, Winooski, VT, USA) at an excitation wavelength of 480 nm and emission wavelength of 530 nm. Then the DNA content was calculated based on the standard curve. Quantitation of cell cytoskeleton alignment: 2-D FFT image analysis was applied to quantify cell cytoskeleton alignment on the hydrogels.51-53 Images of the cells stained with phalloidinFITC were characterized to determine the extent of alignment of the cell cytoskeleton. A CLSM image was cropped to a square of 512 × 512 px and then overlaid with a black square mask with a concentric transparent circle (512 px in diameter) to avoid edge/corner effects. The masked image was computed using the FFT function in Image J. Pixel intensity along each radius (from 0° to 359° with 1° increment) in the FFT plot was summed using the Image J plugin ‘‘Oval Profile’’. The pixel intensity of each radius was normalized by the minimum intensity value. Subsequently, the baseline was shifted to 0 by subtracting 1 from each value. Quantitation of cell nucleus alignment: To quantify cell nucleus alignment of DAPI-stained samples, the nucleus angle for each cell was determined using Image J. The cell nucleus orientation angle was the angle between the hydrogel alignment direction and the major axis of


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the cell nucleus. At least 100 cells for each study group were analyzed to calculate the orientation angle of cell nucleus. Cell migration: Lower initial density of 3×104 cells/cm2 was used to cell aggregates. After 12 hrs culture, the cell migration was monitored using a live cell station equipped with an incubation chamber (37oC and 5% CO2 humidified atmosphere) and a time-lapse digital camera (CELL R, Olympus, Japan).54-56 Images were taken every 15 min within 24 h to assess migration behaviors of the cells. The migration trajectories of BMSC were reconstructed according to the center positions of individual cells over the observation period. The information of twenty cells such as trajectories, migration rate and net displacement was collected for every sample. Statistical methods: SPSS v.16.0 software was used to perform statistical analyses. Comparison of the mean values of the data sets was performed using one-way AVOVA. P < 0.05 was considered statistically significant unless otherwise specified. 3. Results and Discussion 3.1. Characterization of silk nanofiber hydrogels. Silk hydrogels are suitable matrices for simulating the influence of different niche features on cell behavior due to their tunability in terms of mechanical properties and microstructures. Various processes have been exploited to form silk hydrogels with tunable structures and performances. Although plenty of studies revealed the influence of critical factors such as silk concentration, molecular weight and conformation on nucleation rates of gelation and hydrogel morphology,57-59 it remains a challenge to introduce nano-topography and anisotropic cues into these systems. Recently, we prepared silk nanofibers in aqueous solution and formed aligned hydrogels under electric fields, providing an improved material system for revealing cell


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behavior under ECM-biomimetic microenvironments. Different to previous silk hydrogel systems, stable silk nanofibers containing rich beta-sheet structures were assembled in aqueous solutions before gelation. The silk nanofibers had high negative charge density, making them move and aggregate near positive electrodes under electrical field. Although the gelation process and mechanism were consistent with that of previous silk e-gels derived from traditional silk solution,38,

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the repulsive interaction between silk nanofibers resulted in aligned structures

rather than homogenous porous morphology. Unlike previous silk hydrogel systems, bigger porous spaces appeared for aligned silk hydrogels derived from higher concentrated solutions, also due to their higher charge repulsion. As shown in Figure 1, the hydrogels showed aligned lamellar structures at the micrometer scale. The lamella was further composed of oriented silk nanofibers, suggesting hierarchical anisotropic structures within the hydrogels. Through controlling the concentration of silk nanofibers, the hydrogels exhibited tunable spacing intervals in the 15-85 µm range, and changeable lamella thickness from 2 µm to 24 µm. Further detailed microstructures of the aligned hydrogels were revealed in our recent study.41 All the results showed that silk nanofiber hydrogels with homogeneously aligned structures were prepared under the electric field where the structures and thickness of the hydrogels were regulated by changing the concentration of silk nanofiber, providing them tunability. Both CD and FTIR analyses indicated that the same conformations were present (Figure 2), with high beta-sheet content of above 45% (Table 1). All the aligned hydrogels showed high swelling capacity with swelling ratios of above 10 (Figure S1). The swelling ratios decreased gradually from 16.3 to 13.5 and 10.8 for ASH-0.5, ASH-1 and ASH-2 hydrogels, respectively. Due to high beta-sheet structure of the hydrogels, all the hydogels were stable without significant destruction when cultured for above 28 days in PBS solution. Similar to our previous results, the aligned hydrogels


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also exhibited tunable and anisotropic mechanical properties (Figure S2). When the compressive force was parallel to the silk layers, the compressive moduli were 0.29, 1.04 and 1.53 kPa for ASH-0.5, ASH-1 and ASH-2 hydrogels. The moduli were then decreased to 0.18, 0.63 and 0.89 kPa, respectively after the compressive force was orthogonal to the layers. Considering the tunability, these anisotropic hydrogels can provide useful physical cues to influence cell behavior. 3.2. Controllable cell behavior on the hydrogels. The influence of anisotropic cues on in vitro cell compatibility was evaluated with BMSC attachment and proliferation. The silk nanofiber hydrogels without electric field treatment were used as controls to assess the influence of the aligned structures. The cells grew well on all the hydrogels without reaching a plateau after 12 d. DNA content indicated significantly better cell proliferation on the anisotropic hydrogels (Figure 3A), suggesting that the aligned structures formed in the hydrogels facilitated cell proliferation. Further improvement of proliferation behavior following increased nanofiber concentration implied that the in vitro cell control could be optimized by tuning the anisotropic topography. Confocal microscopy confirmed favorable cell growth on the silk nanofiber hydrogels (Figure 3B). At day 12, dense cell aggregates appeared on all of the hydrogels with and without aligned structures. Besides the improved proliferation, the cells cultured on the anisotropic hydrogels showed various morphologies. An elongated morphology along the aligned orientation of the gels was present for the cells cultured on all of the anisotropic hydrogels. This response was different than that on the isotropic hydrogels where random entangled morphology was observed. Unlike on the isotropic hydrogels with a random morphology, the cells showed preferential anisotropic alignment along the orientational cues at 1, 3, 6, 12 days of incubation, finally forming confluent cell layers with


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highly aligned spindle shaped. Similar aligned morphology was achieved for the cells cultured on different anisotropic hydrogels, suggesting that all the orientated layers with different spacings provided strong topographical cues to modulate cell behavior. The alignment of BMSCs on the anisotropic hydrogels was quantified with 2-D FFT image analysis (Figure 4). Although both the aligned cues of lamella and nanofibers could influence the behaviors of the cells, only the cues derived from the lamella were used in our present study since the two interfaces of aligned lamella and nanofibers were orthogonal. The cells stained with phalloidin-FITC were characterized to determine the extent of alignment of the cell cytoskeletons. The representative FFT results of the cells cultured on the isotropic hydrogels showed symmetrically and circular pixels while the FFT curves for the cells on the aligned hydrogels exhibited non-random and elliptical pixels. The shape and height of the FFT curves indicated the degree of alignment of the cell cytoskeleton. There was no distinct peak for the cells cultured on homogeneous hydrogels, indicating random distributions. Two strong peaks appeared for the cells cultured on the aligned hydrogels, confirming the ordered orientation of the cytoskeleton. Cell alignment was further quantified by measuring the angle of the cell nucleus (Figure 5). A narrow distribution of cell nucleus angles represents a high degree of alignment. When cells were cultured on the aligned hydrogels, more than 80% of the cells were aligned with orientation angles in the range from 0° to 40°, while the nuclear angles of the cells cultured on isotropic hydrogels were randomly distributed between 0° and 90°. These results indicated that the aligned cues provided by the hydrogels with different special intervals enhanced cell orientation and elongation, an outcome similar to that which occurs on electrospun fibers and films with aligned topographies.14, 60, 61


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Live cell staining was used to investigate cell migration on the silk nanofiber hydrogels (Figure 6). Many studies suggest that cells migrate preferentially along aligned cues.62-64 Therefore, we compared the migration behavior of stem cells on homogeneous silk nanofiber hydrogels and anisotropic nanofiber hydrogels with various aligned spaced intervals. When the cell migration speed was divided into two sections that were perpendicular to, or parallel to, the aligned direction, significantly higher velocities appeared along the parallel direction. This response was further improved at the more widely spaced intervals. The parallel/perpendicular ratios increased from 1.29 to 1.61 and 1.82 for the cells cultured on ASH-0.5, ASH-1 and ASH-2 hydrogels, respectively. The results indicated the influence of aligned cues on rates of cell migration. The cells migrated preferentially along the aligned morphologies which is consistent with several previous studies.2,

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The preferred migration was further regulated by tuning the spaces

between aligned intervals, suggesting that fine fabrication of aligned cues was effective way of optimizing bioactive biomaterials used in various tissue regenerations such as muscle and nerves. Interestingly, the different aligned spaced intervals also resulted in different average cell speeds. After the same culture time, the rate of cell migration on the homogeneous hydrogels (RSH-0.5) was 3.24 ± 1.6 µm/h, and then decreased to 2.69 ± 1.3, 2.52 ± 0.7 and 2.16 ± 0.4 µm/h on the ASH-0.5, ASH-1 and ASH-2 gels, respectively, possibly since the larger lamellar gaps of the hydrogels restrained the cells to cross. Net cell displacement results that are usually used to assess cell movement further indicated various cell movement capacities for the different silk nanofiber hydrogels. Similar to the migration results, a higher net displacement was observed on the RSH-0.5 hydrogels while lowest net displacement occurred on the ASH-2 hydrogels. Several studies suggested that the adhesion of cells was influenced by the topography of silk hydrogels.20, 21

As shown in Figure 7, the cells adhered on silk nanofibers and formed different morphologies


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due to the diverse topographies of the hydrogels. The different morphologies usually affect cell movement towards tunable migration behavior. Different studies have revealed that cells with inferior movement capacity on biomaterials may achieve higher rates of proliferation.65-69 Therefore, it is reasonable to conclude a relationship between proliferation and migration of cells on different hydrogels. All of these results suggest that tunable aligned structures of the hydrogels are effective cues to regulate cell behavior, including proliferation, morphology and migration. Different with previous silk biomaterials, the silk nanofiber hydrogel system could provide tunable aligned physical cues, making it possible to investigate the elaborate influence of the cues on cell behaviors and possess promising application in soft tissue regeneration such as nerve. Multiple cell behaviors such as proliferation, morphology and migration were significantly tuned by simply changing the aligned structures, which suggested the possibility of developing bioactive biomaterials through optimizing aligned structures. It emphasized the function of physical factors in regulating cell behavior and tissue regeneration. 4. Conclusions Silk nanofiber hydrogels with tunable aligned structures were developed to regulate cell behavior. Besides aligned morphology, the cells showed different proliferation and migration behaviors on the hydrogels depending on the different anisotropic structures, suggesting that physical alignment cues with the hydrogels were effective factors in controlling complex cell behavior. Therefore, the present study revealed that introducing tunable alignment cues to the hydrogels provides a strategy to optimize bioactive control of silk-based biomaterials. The system described here also provides a suitable platform to clarify relationships between cell behavior and various physical factors.


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Supporting Information The results of swelling and mechanical properties of the hydrogels were supplied in support information. This material is available free of charge. ACKNOWLEDGMENTS The authors thank the National Key Research and Development Program of China (2016YFE0204400), the NIH (R01NS094218, R01AR070975) and the AFOSR. We also appreciate the second affiliated hospital of Soochow University preponderant clinic discipline group project funding (NO.XKQ2015010) for support of this work.


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REFERENCES (1) Lavenus, S.; Poxson, D. J.; Ogievetsky, N.; Dordick, J. S.; Siegel, R. W. Stem Cell Behavior on Tailored Porous Oxide Surface Coatings. Biomaterials 2015, 55, 96-109, DOI: 10.1016/j.biomaterials.2015.03.033. (2) Deng, J.; Zhao, C. S.; Spatz, J. P.; Wei, Q. Nanopatterned Adhesive, Stretchable Hydrogel to Control Ligand Spacing and Regulate Cell Spreading and Migration. ACS Nano 2017, 11, 82828291, DOI: 10.1021/acsnano.7b03449. (3) Huang, C. Y.; Hu, H.; Wei, Z. H. Comparison of Cell Behavior on PVA/PVA-Gelatin Electrospun Nanofibers with Random and Aligned Configuration. Sci. Rep. 2016, 6, 37960, DOI: 10.1038/srep37960. (4) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Micropatterned Surfaces for Control of Cell Shape, Position, and Function. Biotechnol. Prog. 1998, 14, 356-363, DOI: 10.1021/bp980031m. (5) Mpoyi, E. N.; Cantini, M.; Reynolds, P. M.; Gadegaard, N.; Dalby, M. J.; Salmeron-Sanchez, M. Protein Adsorption as a Key Mediator in the Nanotopographical Control of Cell Behavior. ACS Nano 2016, 10, 6638-6647, DOI: 10.1021/acsnano.6b01649. (6) Yao, X.; Peng, R.; Ding, J. D. Cell–Material Interactions Revealed via Material Techniques of Surface Patterning. Adv. Mater. 2013, 25, 5257-5286, DOI: 10.1002/adma.201301762. (7) Chen, W. Q.; Villa-Diaz, L. G.; Sun, Y. B.; Weng, S. N.; Kim, J. K.; Lam, R. H. W.; Han, L.; Fan, R.; Krebsbach, P. H.; Fu, J. P. Nanotopography Influences Adhesion, Spreading, and SelfRenewal of Human Embryonic Stem Cells. ACS Nano 2012, 6, 4094-4103, DOI: 10.1021/nn3004923.


15


Page 16 of 33

(8) Jeon, H.; Koo, S.; Reese, W. M.; Loskill, P.; Grigoropoulos, C. P.; Healy, K. E. Directing Cell Migration and Organization via Nanocrater-Patterned Cell-Repellent Interfaces. Nat. Mater. 2015, 14, 918-923, DOI: 10.1038/NMAT4342. (9) Richert, L.; Vetrone, F.; Yi, J. H.; Zalzal, S. F.; Wuest, J. D.; Rosei, F.; Nanci, A. Surface Nanopatterning to Control Cell Growth. Adv. Mater. 2008, 20, 1488-1492, DOI: 10.1002/adma.200701428. (10) Abagnale, G.; Steger, M.; Nguyen, V. H.; Hersch, N.; Sechi, A.; Joussen, S.; Denecke, B.; Merkel, R.; Hoffmann, B.; Dreser, A.; Schnakenberg, U.; Gillner, A.; Wagner, W. Surface Topography Enhances Differentiation of Mesenchymal Stem Cells Towards Osteogenic and Adipogenic

Lineages.

Biomaterials

2015,

61,

316-326,

DOI:

10.1016/j.biomaterials.2015.05.030. (11) Nakayama, Y.; Miyamura, M.; Hirano, Y.; Goto, K.; Matsuda, T. Preparation of Poly (Ethylene Glycol)–Polystyrene Block Copolymers Using Photochemistry of Dithiocarbamate as a Reduced Cell-Adhesive Coating Material. Biomaterials 1999, 20, 963-970, DOI: 10.1016/S0142-9612(98)00252-X. (12) Qin, D.; Xia, Y. N.; Whitesides, G. M. Soft Lithography for Micro-and Nanoscale Patterning. Nat. Protoc. 2010, 5, 491-502, DOI: 10.1038/nprot.2009.234. (13) Sheets, K.; Wunsch, S.; Ng, C.; Nain, A. S. Shape-Dependent Cell Migration and Focal Adhesion Organization on Suspended and Aligned Nanofiber Scaffolds. Acta Biomater. 2013, 9, 7169-7177, DOI: 10.1016/j.actbio.2013.03.042. (14) Choi, J. S.; Lee, S. J.; Christ, G. J.; Atala, A.; Yoo, J. J. The Influence of Electrospun Aligned Poly(Epsilon-Caprolactone)/Collagen Nanofiber Meshes on the Formation of Self-


16

Page 17 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Aligned

Skeletal

Muscle

Myotubes.

Biomaterials

2008,

29,

2899-2906,

DOI:

10.1016/j.biomaterials.2008.03.031. (15) Zhou, Q. H.; Xie, J.; Bao, M.; Yuan, H. H.; Ye, Z. Y.; Lou, X. X.; Zhang, Y. Z. Engineering Aligned Electrospun PLLA Microfibers with Nano-Porous Surface Nanotopography for Modulating the Responses of Vascular Smooth Muscle Cells. J. Mater. Chem. B 2015, 3, 44394450, DOI: 10.1039/c5tb00051c. (16) Wang, Y. M.; Shi, H. G.; Qiao, J.; Tian, Y.; Wu, M.; Zhang, W.; Lin, Y.; Niu, Z. W.; Huang, Y. Electrospun Tubular Scaffold with Circumferentially Aligned Nanofibers for Regulating Smooth Muscle Cell Growth. ACS Appl. Mater. Interfaces 2014, 6, 2958-2962, DOI: 10.1021/am405556x. (17) Khademhosseini, A.; Vacanti, J. P.; Langer, R. Progress in Tissue. Sci. Am. 2009, 300, 6471, DOI: 10.1038/scientificamerican0509-64. (18) Hoare, T. R.; Kohane, D. S. Hydrogels in Drug Delivery: Progress and Challenges. Polymer 2008, 49, 1993-2007, DOI: 10.1016/j.polymer.2008.01.027. (19) Chau, M.; De France, K. J.; Kopera, B.; Machado, V. R.; Rosenfeldt, S.; Reyes, L.; Chan, K. J. W.; Forster, S.; Cranston, E. D.; Hoare, T.; Kumacheva, E. Composite Hydrogels with Tunable Anisotropic Morphologies and Mechanical Properties. Chem. Mater. 2016, 28, 34063415, DOI: 10.1021/acs.chemmater.6b00792. (20) Aubin, H.; Nichol, J. W.; Hutson, C. B.; Bae, H.; Sieminski, A. L.; Cropek, D. M.; Akhyari, P.; Khademhosseini, A. Directed 3D Cell Alignment and Elongation in Microengineered Hydrogels. Biomaterials 2010, 31, 6941-6951, DOI: 10.1016/j.biomaterials.2010.05.056.


17


Page 18 of 33

(21) Yamada, M.; Sugaya, S.; Naganuma, Y.; Seki, M. Microfluidic Synthesis of Chemically and Physically Anisotropic Hydrogel Microfibers for Guided Cell Growth and Networking. Soft Matter 2012, 8, 3122-3130, DOI: 10.1039/c2sm07263g. (22) Tseng, H.; Puperi, D.S.; Kim, E. J.; Ayoub, S.; Shah, J. V.; Cuchiara, M. L.; West, J. L.; Grande-Allen, K. J. Anisotropic Poly (Ethylene Glycol)/Polycaprolactone Hydrogel–Fiber Composites for Heart Valve Tissue Engineering. Tissue Eng., Part A 2014, 20, 2634-2645, DOI: 10.1089/ten.tea.2013.0397. (23) Georgiou, M.; Bunting, S. C. J.; Davies, H. A.; Loughlin, A. J.; Golding, J. P.; Phillips, J. B. Engineered Neural Tissue for Peripheral Nerve Repair. Biomaterials 2013, 34, 7335-7343, DOI: 10.1016/j.biomaterials.2013.06.025. (24) Place, E. S.; Evans, N. D.; Stevens, M. M. Complexity in Biomaterials for Tissue Engineering. Nat. Mater. 2009, 8, 457-470, DOI: 10.1038/NMAT2441. (25) Seliktar D. Designing Cell-Compatible Hydrogels for Biomedical Applications. Science 2012, 336, 1124-1128, DOI: 10.1126/science.1214804. (26) Rivron, N. C.; Rouwkema, J.; Truckenmuller, R.; Karperien, M.; De Boer, J.; Van Blitterswijk, C. A. Tissue Assembly and Organization: Developmental Mechanisms in Microfabricated

Tissues.

Biomaterials

2009,

30,

4851-4858,

DOI:

10.1016/j.biomaterials.2009.06.037. (27) Takahashi, H.; Shimizu, T.; Nakayama, M.; Yamato, M.; Okano, T. The Use of Anisotropic Cell Sheets to Control Orientation During the Self-Organization of 3D Muscle Tissue. Biomaterials 2013, 34, 7372-7380, DOI: 10.1016/j.biomaterials.2013.06.033.


18

Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(28) Pawar, K.; Prang, P.; Willer, R.; Caioni, M.; Bogdahn, U.; Kunz, W.; Weidner, N. Intrinsic and Extrinsic Determinants of Central Nervous System Axon Outgrowth into Alginate-Based Anisotropic Hydrogels. Acta Biomater. 2015, 27, 131-139, DOI: 10.1016/j.actbio.2015.08.032. (29) Liu, M. J.; Ishida, Y.; Ebina, Y.; Sasaki, T.; Hikima, T.; Takata, M.; Aida, T. An Anisotropic Hydrogel with Electrostatic Repulsion Between Cofacially Aligned Nanosheets. Nature 2015, 517, 68-72, DOI: 10.1038/nature14060. (30) Barrow, M.; Zhang, H. F. Aligned Porous Stimuli-Responsive Hydrogels via Directional Freezing and Frozen UV Initiated Polymerization. Soft Matter 2013, 9, 2723-2729, DOI: 10.1039/c2sm27722k. (31) Bai, H.; Polini, A.; Delattre, B.; Tomsia, A. P. Thermoresponsive Composite Hydrogels with Aligned Macroporous Structure by Ice-Templated Assembly. Chem. Mater. 2013, 25, 45514556, DOI: 10.1021/cm4025827. (32) Zhang, S. M.; Liu, X.; Barreto-Ortiz, S. F.; Yu, Y. X.; Ginn, B. P.; DeSantis, N. A.; Hutton, D. L.; Grayson, W. L.; Cui, F. Z.; Korgel, B. A.; Gerecht, S.; Mao, H. Q. Creating Polymer Hydrogel Microfibres with Internal Alignment via Electrical and Mechanical Stretching. Biomaterials 2014, 35, 3243-3251, DOI: 10.1016/j.biomaterials.2013.12.081. (33) Vepari, C.; Kaplan, D. L. Silk as a Biomaterial. Prog. Polym. Sci. 2007, 32(8), 991-1007, DOI: 10.1016/j.progpolymsci.2007.05.013. (34) Rockwood, D. N.; Preda, R. C.; Yucel, T.; Wang, X. Q.; Lovett, M. L.; Kaplan, D. L. Materials Fabrication from Bombyx Mori Silk Fibroin. Nat. Protoc. 2011, 6, 1612-1631, DOI: 10.1038/nprot.2011.379.


19


Page 20 of 33

(35) Luo, K. Y.; Yang, Y. H.; Shao, Z. Z. Physically Crosslinked Biocompatible Silk-FibroinBased Hydrogels with High Mechanical Performance. Adv. Funct. Mater. 2016, 26, 872-880, DOI: 10.1002/adfm.201503450. (36) Whittaker, J. L.; Dutta, N. K.; Elvin, C. M.; Choudhury, N. R. Fabrication of Highly Elastic Resilin/Silk Fibroin Based Hydrogel by Rapid Photo-Crosslinking Reaction. J. Mater. Chem. B 2015, 3, 6576-6579, DOI: 10.1039/c5tb00970g. (37) Kim, M. H.; Park, W. H. Chemically Cross-Linked Silk Fibroin Hydrogel with Enhanced Elastic Properties, Biodegradability, and Biocompatibility. Int. J. Nanomed. 2016, 11, 29672978, DOI: 10.2147/IJN.S106467. (38) Leisk, G. G.; Lo, T. J.; Yucel, T.; Lu, Q.; Kaplan, D. L. Electrogelation for Protein Adhesives. Adv. Mater. 2010, 22, 711-715, DOI: 10.1002/adma.200902643. (39) Lu, Q.; Huang, Y. L.; Li, M. Z.; Zuo, B. Q.; Lu, S. Z.; Wang, J. N.; Zhu, H. S.; Kaplan, D. L. Silk Fibroin Electrogelation Mechanisms. Acta Biomater. 2011, 7, 2394-2400, DOI: 10.1016/j.actbio.2011.02.032. (40) Kojic, N.; Panzer, M. J.; Leisk, G. G.; Raja, W. K.; Kojic, M.; Kaplan, D. L. Ion electrodiffusion governs silk electrogelation. Soft matter 2012, 8, 6897-6905, DOI: 10.1039/c2sm25783a. (41) Lu, Q.; Bai, S. M.; Ding, Z. Z.; Guo, H.; Shao, Z. Z.; Zhu, H. S.; Kaplan, D. L. Hydrogel Assembly with Hierarchical Alignment by Balancing Electrostatic Forces. Adv. Mater. Interfaces 2016, 3, 1500687, DOI: 10.1002/admi.201500687. (42) Han, F.; Liu, S.; Liu, X.; Pei, Y.; Bai, S.; Zhao, H.; Lu, Q.; Ma, F.; Kaplan, D. L.; Zhu, H. Woven Silk Fabric-Reinforced Silk Nanofibrous Scaffolds for Regenerating Load-Bearing Soft Tissues. Acta Biomater. 2014, 10, 921-930, DOI: 10.1016/j.actbio.2013.09.026.


20

Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(43) Bai, S. M.; Zhang, X. L.; Lu, Q.; Sheng, W. Q.; Liu, L. J.; Dong, B. J.; Kaplan, D. L.; Zhu, H. S. Reversible Hydrogel–Solution System of Silk with High Beta-Sheet Content. Biomacromolecules 2014, 15, 3044-3051, DOI: 10.1021/bm500662z. (44) Dong, X. D.; Zhao, Q.; Xiao, L. Y.; Lu, Q.; Kaplan, D. L. Amorphous Silk Nanofiber Solutions for Fabricating Silk-Based Functional Materials. Biomacromolecules 2016, 17, 30003006, DOI: 10.1021/acs.biomac.6b00863. (45) Bai, S.; Liu, S.; Zhang, C.; Xu, W.; Lu, Q.; Han, H.; Kaplan, D. L.; Zhu, H. Controllable Transition of Silk Fibroin Nanostructures: An Insight into in Vitro Silk Self-Assembly Process. Acta Biomater. 2013, 9, 7806-7813, DOI: 10.1016/j.actbio.2013.04.033. (46) Yin, Z. P.; Wu, F.; Zheng, Z. Z.; Kaplan, D. L.; Kundu, S. C.; Lu, S. Z. Self-Assembling Silk-Based Nanofibers with Hierarchical Structures. ACS Biomater. Sci. Eng. 2017, 3, 26172627, DOI: 10.1021/acsbiomaterials.7b00442. (47) Chen, X.; Shao, Z. Z.; Knight, D. P.; Vollrath, F. Conformation Transition Kinetics of Bombyx Mori Silk Protein. Proteins: Struct., Funct., Bioinf. 2007, 68, 223-231, DOI: 10.1002/prot.21414. (48) Ling, S. J.; Qi, Z. M.; Knight, D. P.; Shao, Z. Z.; Chen, X. Synchrotron FTIR Microspectroscopy of Single Natural Silk Fibers. Biomacromolecules 2011, 12, 3344-3349, DOI: 10.1021/bm2006032. (49) Bai, S. M.; Han, H. Y.; Huang, X. W.; Xu, W. A.; Kaplan, D. L.; Zhu, H. S; Lu, Q. Silk Scaffolds with Tunable Mechanical Capability for Cell Differentiation. Acta Biomater. 2015, 20, 22-31, DOI: 10.1016/j.actbio.2015.04.004.


21


Page 22 of 33

(50) Han, H. Y.; Ning, H. Y.; Liu, S. S.; Lu, Q.; Fan, Z. H.; Lu, H. J .; Lu, G. Z.; Kaplan, D. L . Silk Biomaterials with Vascularization Capacity. Adv. Funct. Mater. 2016, 26, 421-432, DOI: 10.1002/adfm.201504160. (51) Ayres, C. E.; Jha, B. S.; Meredith, H.; Bowman, J. R.; Bowlin, G. L.; Henderson, S. C.; Simpson, D. G. Measuring Fiber Alignment in Electrospun Scaffolds: a User's Guide to the 2D Fast Fourier Transform Approach. J. Biomater. Sci., Polym. Ed. 2008, 19, 603-621, DOI: 10.1163/156856208784089643. (52) Ayres, C.; Bowlin, G. L.; Henderson, S. C.; Taylor, L.; Shultz, J.; Alexander, J.; Telemeco, T. A.; Simpson, D. G. Modulation of Anisotropy in Electrospun Tissue-Engineering Scaffolds: Analysis of Fiber Alignment by the Fast Fourier Transform. Biomaterials 2006, 27, 5524-5534, DOI: 10.1016/j.biomaterials.2006.06.014. (53) Tseng, L. F.; Mather, P. T.; Henderson, J. H. Shape-Memory-Actuated Change in Scaffold Fiber Alignment Directs Stem Cell Morphology. Acta Biomater. 2013, 9, 8790-8801, DOI: 10.1016/j.actbio.2013.06.043. (54) Wu, P.; Fu, Y.; Cai, K. Y. Regulation of the Migration of Endothelial Cells by a Gradient Density of Vascular Endothelial Growth Factor. Colloids Surf., B 2014, 123, 181-190, DOI: 10.1016/j.colsurfb.2014.09.013. (55) Mi, H. Y.; Salick, M. R.; Jing, X.; Crone, W. C.; Peng, X. F.; Turng, L. S. Electrospinning of Unidirectionally and Orthogonally Aligned Thermoplastic Polyurethane Nanofibers: Fiber Orientation and Cell Migration. J. Biomed. Mater. Res., Part A 2015, 103, 593-603, DOI: 10.1002/jbm.a.35208. (56) Ren, T. C.; Yu, S.; Mao, Z. W.; Gao, C. Y. A Complementary Density Gradient of Zwitterionic Polymer Brushes and NCAM Peptides for Selectively Controlling Directional


22

Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Migration

of

Schwann

Cells.

Biomaterials

2015,

56,

58-67,

DOI:

10.1016/j.biomaterials.2015.03.052. (57) Floren, M. L.; Spilimbergo, S.; Motta, A.; Migliaresi, C. Carbon Dioxide Induced Silk Protein Gelation for Biomedical Application. Biomacromolecules 2012, 13, 2060-2072, DOI: 10.1021/bm300450a. (58) Floren M.; Migliaresi C.; Motta A. Processing Techniques and Applications of Silk Hydrogels in Bioengineering. J. Funct. Biomater. 2016, 7, 26, DOI:10.3390/jfb7030026. (59) Elliott, W. H.; Bonani, W.; Maniglio, D.; Motta, A.; Tan, W.; Migliaresi, C. Silk Hydrogels of Tunable Structure and Viscoelastic Properties Using Different Chronological Orders of Genipin and Physical Cross-Linking. ACS Appl. Mater. Interfaces 2015, 7, 12099-12108, DOI: 10.1021/acsami.5b02308. (60) Lu, L. X.; Wang, Y. Y.; Mao, X.; Xiao, Z. D.; Huang, N. P. The Effects of PHBV Electrospun Fibers with Different Diameters and Orientations on Growth Behavior of BoneMarrow-Derived

Mesenchymal

Stem

Cells.

Biomed.

Mater.

2012,

7,

015002,

DOI: 10.1088/1748-6041/7/1/015002. (61) Bashur, C. A.; Dahlgren, L. A.; Goldstein, A. S. Effect of Fiber Diameter and Orientation on Fibroblast Morphology and Proliferation on Electrospun Poly(D, L-lactic-co-glycolic acid) Meshes. Biomaterials 2006, 27, 5681-5688, DOI: 10.1016/j.biomaterials.2006.07.005. (62) Wang, H. B.; Mullins, M. E.; Cregg, J. M.; McCarthy, C. W.; Gilbert, R. J. Varying the Diameter of Aligned Electrospun Fibers Alters Neurite Outgrowth and Schwann Cell Migration. Acta Biomater. 2010, 6, 2970-2978, DOI: 10.1016/j.actbio.2010.02.020.


23


Page 24 of 33

(63) Berns, E. J.; Sur, S.; Pan, L. L.; Goldberger, J. E.; Suresh, S.; Zhang, S. M.; Kessler, J. A.; Stupp, S. I. Aligned Neurite Outgrowth and Directed Cell Migration in Self-Assembled Monodomain Gels. Biomaterials 2014, 35, 185-195, DOI: 10.1016/j.biomaterials.2013.09.077. (64) Sheets, K.; Wunsch, S.; Ng, C.; Nain, A. S. Shape-Dependent Cell Migration and Focal Adhesion Organization on Suspended and Aligned Nanofiber Scaffolds. Acta Biomater. 2013, 9, 7169-7177, DOI: 10.1016/j.actbio.2013.03.042. (65) Kim, D. H.; Han, K.; Gupta, K.; Kwon, K. W.; Suh, K. Y.; Levchenko, A. Mechanosensitivity of Fibroblast Cell Shape and Movement to Anisotropic Substratum Topography

Gradients.

Biomaterials

2009,

30,

5433-5444,

DOI:

10.1016/j.biomaterials.2009.06.042. (66) Mitchel, J. A.; Hoffman-Kim, D. Cellular Scale Anisotropic Topography Guides Schwann Cell Motility. PLoS One 2011, 6, e24316, DOI: 10.1371/journal.pone.0024316. (67) Lawrence, B. D.; Pan, Z.; Rosenblatt, M. I. Silk Film Topography Directs Collective Epithelial Cell Migration. PLoS One 2012, 7, e50190, DOI: 10.1371/journal.pone.0050190. (68) Mandal, B. B.; Kundu, S. C. Cell Proliferation and Migration in Silk Fibroin 3D Scaffolds. Biomaterials 2009, 30, 2956-2965, DOI: 10.1016/j.biomaterials.2009.02.006. (69) Tremel, A.; Cai, A.; Tirtaatmadja, N.; Hughes, B. D.; Stevens, G. W.; Landman, K. A.; O'Connor, A. J. Cell Migration and Proliferation During Monolayer Formation and Wound Healing. Chem. Eng. Sci. 2009, 64, 247-253, DOI: 10.1016/j.ces.2008.10.008.


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Table 1. FTIR determination of secondary structures of different silk nanofiber hydrogels through FSD of the amide I region. samplesa

a

Conformation content of silk fibroin β-sheet

Random

Turns

NSH-0.5

44.59±1.67

34.88±1.74

15.75±0.97

ASH-0.5

45.41±0.95

31.89±1.52

17.86±1.58

ASH-1

45.42±1.54

34.46±0.85

16.05±1.46

ASH-2

45.83±1.18

31.13±1.91

18.93±1.35

Ten measurements per condition were obtained


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Figure 1. Hierarchical micromorpholioies of silk nanofiber hydrogels with random and aligned structures: (A) SEM images of hydrogels with homogeneous structure derived from silk nanofibers with 0.5 wt% concentration (RSH-0.5); (B) SEM images of aligned hydrogels derived from silk nanofibers with 0.5 wt% concentration (ASH-0.5); (C) SEM images of aligned hydrogels derived from silk nanofibers with 1 wt% concentration (ASH-1); (D) SEM images of aligned hydrogels derived from silk nanofibers with 2 wt% concentration (ASH-2); (E) Space width of aligned hydrogels prepared from different silk nanofiber concentrations; (F) Lamella thickness of aligned hydrogels prepared from different silk nanofiber concentrations. The inserts (A-D) are the magnified surface morphologies of the porous walls.


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Figure 2. CD curves (A) and FTIR spectra (B) of different silk nanofiber hydrogels. The samples were as follows: RSH-0.5, homogeneous hydrogels derived from silk nanofibers with 0.5 wt% concentration; ASH-0.5, aligned hydrogels derived from silk nanofibers with 0.5 wt% concentration; ASH-1, aligned hydrogels derived from silk nanofibers with 1 wt% concentration; ASH-2, aligned hydrogels derived from silk nanofibers with 2 wt% concentration.


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Figure 3. Cytocompatibility of the hydrogels in vitro. (A) BMSC proliferation on different silk hydrogels measured with DNA analysis. * Statistically significant P < 0.05. (B) Confocal microscopy images of BMSCs cultivated on day 1, 3, 6 and 12 on the different hydrogels. The samples were as follows: RSH-0.5, homogeneous hydrogels derived from silk nanofibers with 0.5 wt% concentration; ASH-0.5, aligned hydrogels derived from silk nanofibers with 0.5 wt% concentration; ASH-1, aligned hydrogels derived from silk nanofibers with 1 wt% concentration; ASH-2, aligned hydrogels derived from silk nanofibers with 2 wt% concentration. The blue color (DAPI) represents silk hydrogels and cell nucleus, while the green color (FITC labeled phalloidin) represents actin cytoskeleton.


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Figure 4. Cell cytoskeleton alignment. (A1, B1, C1 and D1) CLSM images of BMSCs on different silk nanofiber hydrogels; (A2, B2, C2 and D2) FFT output images; (A3, B3, C3 and D3) Pixel intensity plots against the angle of acquisition for the random and aligned cell cytoskeleton. The samples were as follows: RSH-0.5, homogeneous hydrogels derived from silk nanofibers with 0.5 wt% concentration; ASH-0.5, aligned hydrogels derived from silk nanofibers with 0.5 wt% concentration; ASH-1, aligned hydrogels derived from silk nanofibers with 1 wt% concentration; ASH-2, aligned hydrogels derived from silk nanofibers with 2 wt% concentration. Cells were stained with FITC labeled phalloidin (green) to visualize actin cytoskeleton. Double arrows indicate the aligned silk hydrogels direction.


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Figure 5. Distribution of cell nucleus orientation angles on different silk nanofiber hydrogels. The samples were as follows: (A) RSH-0.5, homogeneous hydrogels derived from silk nanofibers with 0.5 wt% concentration; (B)ASH-0.5, aligned hydrogels derived from silk nanofibers with 0.5 wt% concentration; (C)ASH-1, aligned hydrogels derived from silk nanofibers with 1 wt% concentration; (D) ASH-2, aligned hydrogels derived from silk nanofibers with 2 wt% concentration. The 90° angle represents the angle perpendicular to the aligned silk hydrogels direction.


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Figure 6. Cell migration results. (A-D) Representative migration trajectories of BMSCs cultured onto: (A) RSH-0.5, homogeneous hydrogels derived from silk nanofibers with 0.5 wt% concentration; (B)ASH-0.5, aligned hydrogels derived from silk nanofibers with 0.5 wt% concentration; (C)ASH-1, aligned hydrogels derived from silk nanofibers with 1 wt% concentration; (D) ASH-2, aligned hydrogels derived from silk nanofibers with 2 wt% concentration. (E) Cell migration rate. (F) Net displacement. * Statistically significant P < 0.05.


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Figure 7. The initial images of cell migration. (A) RSH-0.5, homogeneous hydrogels derived from silk nanofibers with 0.5 wt% concentration; (B)ASH-0.5, aligned hydrogels derived from silk nanofibers with 0.5 wt% concentration; (C)ASH-1, aligned hydrogels derived from silk nanofibers with 1 wt% concentration; (D) ASH-2, aligned hydrogels derived from silk nanofibers with 2 wt% concentration.


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For Table of Contents Use Only

Controlling Cell Behavior on Silk Nanofiber Hydrogels with Tunable Anisotropic Structures Lili Wanga, b, Guozhong Luc, Qiang Lua, b,*, David L. Kapland


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Controlling Cell Behavior on Silk Nanofiber ... - ACS Publications

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