Supporting Information

Transcription

1 Supporting Information 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 or 1

2 Supporting Information Includes: Methods: (1) Fabrication of Regenerated Silk Fibroin (SF). (2) Preparation of 3D SF Porous Wall-like Scaffolds (W) and 3D SF Scaffolds with Short Channels/pores/nanofibers (W&F). (3) 3D SF/gelatin Composite A(F&C) Scaffolds. (4) 3D Sodium Alginate A(F&C) Scaffolds. (5) Characterization. (6) Cell Capturing and Neurite Outgrowth of Rat Embryonic Dorsal Root Ganglion Neurons (DRGs) in 3D SF Scaffolds. (7) Cell Capturing, Growth, in vitro Vascularization and Collagen Deposition of Human Umbilical Vein Endothelial Cells (HUVECs) in 3D SF Scaffolds. (8) In vitro Degradation of 3D SF Scaffolds. (9) Statistical Analysis. Results: (1) Figure S1: SEM Images of 3D SF W and W&F Scaffolds before Ethanol Treatment. (2) Figure S2: SEM Profiles of Macrochannels of 3D SF A(F&C) Scaffolds. (3) Figure S3: SEM Profiles of Macrochannels of 3D A(F&C) Scaffolds from SF/gelatin Mixture and Sodium Alginate, respectively. (4) Figure S4: Micro-CT Images of 3D SF W and W&F Scaffolds after Ethanol Treatment. (5) Figure S5: SEM Images of 3D SF W and W&F Scaffolds after Ethanol Treatment. (6) Figure S6: ATR-FTIR Spectra of 3D SF Scaffolds. (7) Figure S7: Growth of DRGs in SF W and W&F Scaffolds. (8) Figure S8: Formation of Blood Vessel-like Structures in 3D SF Scaffolds. (9) Figure S9 and Figure S10: In vitro Degradation Behaviors of 3D SF Scaffolds and Morphologies of 3D SF Scaffolds after Degradation. (10) Movie S1: Illustration of Inner Structure Characteristics of 3D A(F&C) Scaffolds. (11) Movie S2: Illustration of Inner Structure Characteristics of 3D W&F Scaffolds. (12) Movie S3: Illustration of Inner Structure Characteristics of 3D W Scaffolds. 2

3 METHODS Fabrication of Regenerated Silk Fibroin (SF). Silk cocoons (Jiaxing Silk Co., China) were boiled 4 times (20 min/time) in an aqueous 0.5% (w/v) Na2CO3 solution to remove sericin protein. The degummed silk fibers were rinsed with ultrapure water thoroughly to remove the residual of sercin. Following drying, they were dissolved in a mixture of CaCl2, H2O and CH3CH2OH (in a molar ratio of 1:8:2) at 65 to get a clear solution. Subsequently, the resulting solution was dialyzed against ultrapure water (18.2 mω cm) using cellulose dialysis tubes (molecular weight cut-off: 14 kda; Sigma Aldrich, Australia) at ambient temperature for 4 days. The impurities were removed by filtering and centrifuging at 5000 rpm for 20 min. Finally, regenerated SF sponge was obtained by lyophilizing the centrifuged solution using a freeze dryer (FreeZone 2.5 Liter Benchtop Freeze Dryer; Labconco, Kansas City, MO, USA). Preparation of 3D Porous Wall-like SF Scaffolds (W) and 3D SF Scaffolds with Short Channels/pores/nanofibers (W&F). For comparison, scaffolds were also formed in freezers at -20 and -80, respectively, rather than by instant freezing with liquid nitrogen. For W scaffolds, SF solution in the glass tube was frozen at -20 for 53 h. For W&F scaffolds, SF solution in the glass tube was frozen at -80 for the same time. Following removal of ice crystals with a freeze drier, W and W&F scaffolds were respectively obtained. To make the scaffolds insoluble in water, the W and W&F scaffolds above were further processed with the same procedures used for obtaining A(F&C) scaffolds, i.e., the scaffolds were treated by immersing in ethanol at ambient temperature for 12 h. After removing ethanol and thoroughly rinsing with ultrapure water, the scaffolds in the ultrapure water were frozen at -20 for 72 h. After removing ice crystals using a freeze drier, water-resistant W and W&F scaffolds were obtained, respectively. 3D SF/gelatin Composite A(F&C) Scaffolds. SF/gelatin (Sigma-Aldrich, Australia) solution (2%) was obtained by dissolving 2 g of regenerated SF/gelatin mixture (in a weight ratio of 95:5) in 100 ml ultrapure water. Then the SF/gelatin composite A(F&C) scaffolds were fabricated by the same protocol used for producing SF A(F&C) scaffolds in the manuscript. 3D Sodium Alginate A(F&C) Scaffolds. Sodium alginate (Sigma-Aldrich, Australia) solution (0.3%) was prepared by dissolving 0.3 g of sodium alginate in 100 ml ultrapure water at 50 under stirring. The sodium alginate A(F&C) scaffolds were prepared by the same procedures used for fabricating SF A(F&C) scaffolds, except that the 3D sodium 3

4 alginate scaffolds with aligned nanofibers after the first step of freeze-drying were treated using an aqueous CaCl2 solution instead of ethanol to obtain water-resistant scaffolds. Characterization. The morphology of materials was observed using a scanning electron microscopy (SEM) (Zeiss Supra 55VP), and fiber diameter was determined from representative SEM images by an image processing software (Image-J 1.34). 3D structures of SF scaffolds were imaged using Micro X-ray Computed Tomography (micro-ct) by an Xradia micro XCT200 (Carl Zeiss X-ray Microscopy, Inc., USA). An X-ray tube with a voltage of 40 kv and a peak power of 10 W was used. 361 equiangular projections (exposure time: 8 seconds/projection) over 180 degrees were taken for one complete tomographic reconstruction. Phase retrieval tomography with 3D reconstruction algorithm was introduced to obtain clear projections and a final 3D visualization. The size of reconstructed 3D images was voxels with a 4.3 µm voxel size along each side. Fourier transform infrared spectroscopy (FTIR) spectra were recorded in a wavenumber range of cm -1 using a Bruker VERTEX 70 instrument in an attenuated total reflectance (ATR) mode (4 cm -1 resolution, 64 scans). Compressive mechanical properties of silk scaffolds were obtained using an Instron 5967 Computerized Universal Testing Machine (Instron Corp, USA) with a 100 N loading cell. Cylindrical scaffolds with a diameter of 10 mm and height of 4 mm were measured at a crossing-head speed of 5 mm/min (the scaffolds were compressed approximately 85% of their original height) (six samples were measured for each group). Compressive stress and strain were graphed, and the compressive modulus was calculated as the slope of the initial linear section of the stress-strain curve. For cyclic compression, the scaffolds were compressed around 10% of their original height, with 15 cycles at a frequency of 1 Hz. Average peak stress was calculated by the peak stress of first cycles. Cell Capturing and Neurite Outgrowth of Rat Embryonic Dorsal Root Ganglion Neurons in 3D SF Scaffolds. Rat Embryonic Dorsal Root Ganglion Neurons (DRGs; Lonza, USA) were cultured in Primary Neuron Basal Medium (PNBM; Lonza, USA) supplemented with PNGM SingleQuotsTM (Lonza, USA) and 150 ng/ml of Nerve Growth Factor (NGF; Sigma-Aldrich, Australia). Scaffolds (diameter around 10 mm and thickness around 3 mm) were placed in 24-well plates (Greiner Bio-One) after sterilization in an environment of 75% ethanol vapor. DRGs suspended in cell medium were evenly seeded onto scaffolds at a density of /well. DRG-seeded scaffolds were maintained in vitro under standard culture conditions (37, 5% CO2) with medium change every 3-5 day. (1) Cell capturing of scaffolds. At fixed time-points (6, 12 and 24 h) after seeding, the viability of DRGs captured by scaffolds was analyzed using MTS assay (Promega, USA) 4

5 following the manufacturer s instruction with absorbance measured at 490 nm on a microplate reader (SH-1000Lab, Corona Electric Co., Ltd, Japan). (2) Immunostaining for neurite outgrowth of DRGs. After 21 days of culture, the scaffolds were rinsed with PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich, Australia) for 30 min at ambient temperature. Following rinsing with PBS, the scaffolds were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Australia) for 30 min and then rinsed with PBS again. Subsequently, the scaffolds were incubated in 10% Normal Goat Serum blocking solution (Life Technologies, Australia) for 10 min to block non-specific binding, followed by rinsing with PBS. Then the scaffolds were incubated with Anti-Neurofilament-200 antibody from rabbit (1:50; Sigma-Aldrich, Australia) overnight at 4. After rinsing with PBS, the scaffolds were incubated with Goat-anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate (1:200; Life Technologies, Australia) for 1 hour. Finally, the treated samples were analyzed using a confocal fluorescence microscope (Leica TCS SP5 Confocal Microscope, Leica Microsystems, Wetzlar). Cell Capturing, Growth, in vitro Vascularization and Collagen Deposition of Human Umbilical Vein Endothelial Cells in 3D SF Scaffolds. Human Umbilical Vein Endothelial Cells (HUVECs; Life Technologies, Australia) were cultured in Medium 200 with Low Serum Growth Supplement (LSGS; Life Technologies, Australia). Scaffolds (diameter around 10 mm and thickness around 3 mm) were placed in 24-well plates (Greiner Bio-One) after sterilization in an environment of 75% ethanol vapor. HUVECs suspended in cell medium were evenly seeded onto scaffolds at a corresponding density ( /well, /well and /well for in vitro cell adhesion, proliferation and vascularization study, respectively). Cell-seeded scaffolds were maintained in vitro under standard culture conditions (37, 5% CO2) with medium change every 2-3 day. (1) Cell capturing and growth in scaffolds. At fixed time-points (2, 4 and 8 hours for cell capture assay; 2, 4 and 6 days for cell proliferation assay) after seeding, the viability of cells in scaffolds was analyzed using MTS assay (Promega, USA) following the manufacturer s instruction with absorbance measured at 490 nm on a microplate reader (SH-1000Lab, Corona Electric Co., Ltd, Japan). After 3 days of culture, the cell-scaffold composites were rinsed with PBS, and fixed in 4% paraformaldehyde (Sigma-Aldrich, Australia) for 30 min at ambient temperature. After rinsing with PBS, the composites were permeabilized with 0.1% Triton X-100 (Sigma- Aldrich, Australia) for 10 min, followed by rinsing with PBS. The composites were then incubated in Image-iT FX Signal Enhancer Ready Probes reagent (Life Technologies, 5

6 Australia) for 30 min and rinsed with PBS. Subsequently, the composites were incubated with Alexa Fluor 568 Phalloidin (1:100; Life Technologies, Australia) for 1 hour. After rinsing in PBS, the composites were incubated in DAPI (Life Technologies, Australia) in dark for 10 min. As-treated samples were assessed using a confocal fluorescence microscope (Leica TCS SP5 Confocal Microscope, Leica Microsystems, Wetzlar). (2) In vitro vascularization in scaffolds. After 21 days of culture, cell-scaffold composites were rinsed with PBS, and fixed in 4% paraformaldehyde (Sigma-Aldrich, Australia) for 30 min at ambient temperature. Following rinsing with PBS, the composites were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Australia) for 10 min, followed by rinsing with PBS. The composites were then incubated in Image-iT FX Signal Enhancer Ready Probes reagent (Life Technologies, Australia) for 30 min. After rinsing with PBS, the scaffolds were incubated for 10 min with 10% Normal Goat Serum blocking solution (Life Technologies, Australia) to block non-specific binding and then rinsed with PBS. Subsequently, the composites were incubated with CD31 Monoclonal Antibody (1:50; Life Technologies, Australia) overnight at 4. Following rinsing with PBS, the composites were incubated with Goat-anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate (1:200; Life Technologies, Australia) for 1 hour. The scaffolds were rinsed again with PBS and incubated in DAPI (Life Technologies, Australia) in dark for 10 min. As-treated samples were assessed using the confocal fluorescent microscope. (3) Collagen deposition in scaffolds. After 28 days of culture, cell-scaffold composites were rinsed with PBS, and fixed in 4% paraformaldehyde (Sigma-Aldrich, Australia) for 30 min at ambient temperature. Following rinsing with PBS, the composites were stained with Direct Red 80 (Sirius Red; Sigma-Aldrich, Australia) solution (0.1 g/100 ml saturated aqueous picric acid) (Sigma-Aldrich, Australia) for 1 hour at room temperature. Then the composites were rinsed with acidified distilled water (0.5% v/v acetic acid; Sigma-Aldrich, Australia) and PBS, respectively, 3 times. Subsequently, the composites were imaged using a DP71 Microscope (Olympus Corporation, Japan). In vitro Degradation of 3D SF Scaffolds. Samples (21 ± 2 mg) were incubated at 37 in a 5 ml of PBS solution with or without 4.8 U/mL protease XIV (Sigma-Aldrich, Australia), respectively. All solutions were replaced every 24 h. At fixed time-points (1, 5, 10, 15, 21 and 28 d), the solutions were removed and the samples were rinsed with distilled water 3 times. As-treated samples were lyophilized using the freeze dryer. The percentages of weight loss of the samples were calculated with the equation: Pl = (Wi Wt)/Wi 100%, where Pl represents the percentage of weight loss, Wi represents the initial weight of a sample and Wt 6

7 represents the final weight of a sample at a fix time-point. Morphologies of the scaffolds after degradation were observed using SEM. Statistical Analysis. All experiments were carried out in triplicate and data were expressed as mean ± standard deviation (s.d.). Statistical differences were analyzed by one-way ANOVA using statistical software in the Origin 9 software package (OriginLab, USA). Difference with p<0.05 or p<0.01 was considered as statistical significance. 7

8 RESULTS Figure S1. SEM images of 3D silk fibroin (SF) scaffolds with short channels/pores/nanofibers (W&F) and porous wall-like 3D SF scaffolds (W) before treatment with ethanol. (a) SEM images of the W&F scaffold obtained by freezing aqueous silk fibroin at -80. Scale bars: from left to right 200, 30 and 100 µm, respectively. (b) SEM images of the W scaffold obtained by freezing aqueous silk fibroin at -20. Scale bars: from left to right 200, 20 and 100 µm, respectively. 8

9 Figure S2. SEM profiles of macrochannels in the 3D silk fibroin (SF) scaffolds (A(F&C)) with co-aligned nanofibers and macrochannels. (a) A side view of macrochannels in the scaffold. The macrochannels were indicated by yellow arrows where the profiles of macrochannels are clearly seen. (b) A top view of macrochannels (indicated by red arrows) in the scaffold. Scale bars: 200 µm in (a) and 100 µm in (b). 9

10 Figure S3. 3D scaffolds with co-aligned nanofibers and macrochannels (A(F&C)) from other natural polymers by the facile two-step freezing technology developed in this work. Representative images of A(F&C) scaffolds from SF/gelatin mixture (a) as well as sodium alginate (b) showing a good co-alignment of nanofibers and macrochannels. Red arrows indicate the channels in the scaffolds and yellow arrows indicate the alignment direction of nanofibers on the wall of channels. Scale bars: 20 µm in (a) and 2 µm in inset 1, (b) and inset 2. 10

11 Figure S4. Micro-CT images of 3D silk fibroin (SF) scaffolds with short channels/pores/nanofibers (W&F) and porous wall-like 3D SF scaffolds (W) after treatment with ethanol. The details in structure can be seen clearly in Supporting Information, Figure S5. Both scale bars are 1000 µm. 11

12 Figure S5. SEM images of 3D silk fibroin (SF) scaffolds with short channels/pores/nanofibers (W&F) and porous wall-like 3D SF scaffolds (W) after treatment with ethanol. (a) SEM images of the W&F scaffolds after treatment with ethanol. The scaffolds retained the hybrid structure of short channels/pores/fibers after ethanol treatment. The treatment did not increase interconnectivity of the W&F scaffolds compared with the scaffolds before treatment (Figure S1a). Scale bars: from left to right 100, 20 and 100 µm, respectively. (b) SEM images of the W scaffolds after treatment with ethanol. The scaffolds retained the wall-like porous structure after ethanol treatment. The treatment also did not increase interconnectivity of W scaffolds compared with the scaffolds before treatment (Figure S1b). Scale bars: from left to right 100, 20 and 100 µm, respectively. 12

13 Figure S6. ATR-FTIR spectra of 3D silk fibroin scaffolds. (a) ATR-FTIR spectra of silk fibroin (SF) scaffolds from different freezing-temperatures: -20 (porous wall-like 3D SF scaffolds, W), -80 (3D SF scaffolds with short channels/pores/nanofibers, W&F) and in liquid nitrogen (3D SF scaffolds with radially aligned nanofibers, but without macrochannels, AF). (b) ATR-FTIR spectra of SF scaffolds after post treatment in ethanol. 13

14 Figure S7. Growth of Embryonic Dorsal Root Ganglion Neurons (DRGs) in the porous walllike 3D silk fibroin (SF) scaffold (W) and the 3D SF scaffold with short channels/pores/nanofibers (W&F) after 21 days of culture. The extension and outgrowth of DRG neurites in the W and W&F scaffolds were blocked by surrounding materials, suggesting the scaffolds did not provide DRGs with a suitable 3D environment. Scale bars: 100 and 25 µm in W and W&F, respectively. 14

15 Figure S8. Co-aligned nanofibers and macrochannels in 3D scaffolds facilitate the formation of CD31-positive vessel-like structures by directing the growth, migration and interaction of adherent HUVECs (Figure 5c illustrates how to read the images presented in this Figure). Growth, interaction and vessel-like structures of HUVECs in 3D silk fibroin (SF) scaffolds with radially co-aligned nanofibers and macrochannels (A(F&C)), radially aligned 3D SF nanofibrous scaffolds without macrochannels (AF), 3D SF scaffolds with short channels/pores/nanofibers (W&F) and porous wall-like 3D SF scaffolds (W). Scale bars: 50 µm in A(F&C), W&F and W; 25 µm in AF. 15

16 Figure S9. In vitro degradation of 3D SF scaffolds. (a) The percentage of weight loss of 3D SF scaffolds after in vitro degradation in protease/pbs solution (i.e., Protease degradation) or pure PBS solution (i.e., PBS degradation) for 1, 5, 10, 15, 21 and 28 days, respectively. (b) Morphologies of the A(F&C) scaffold after in vitro degradation in pure PBS solution (i.e., the A(F&C)-PBS scaffold) and protease/pbs solution (i.e., the A(F&C)-protease scaffold), respectively, for 10 days. Blue arrows indicate the cracks on the relatively thin nanofibers and red arrows indicate the pores on nanofibers. Scale bars: 2 µm in A(F&C)-PBS and A(F&C)- protease; 1 µm in the insets 1 and 2. 16

17 Figure S10. Morphologies of the W and W&F scaffolds after in vitro degradation in pure PBS solution (i.e., W-PBS and W&F-PBS scaffolds) and protease/pbs solution (i.e., W- protease and W&F-protease scaffolds), respectively, for 10 days. Red arrows indicate the pores on the scaffolds. Scale bars: 20 µm in W-PBS; 50 µm in W-protease, W&F-PBS and W&F-protease; 10 µm in the insets 1, 2 and 3; 1 µm in the inset 4. 17