Fabrication of Silk Scaffolds with Nanomicroscaled Structures and

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Fabrication of Silk Scaffolds with NanoMicroscaled Structures and Tunable Stiffness Liying Xiao, Shanshan Liu, Danyu Yao, Zhaozhao Ding, Zhihai Fan, Qiang Lu, and David L Kaplan Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 02 Jun 2017 Downloaded from http://pubs.acs.org on June 4, 2017

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Fabrication of Silk Scaffolds with Nano-Microscaled Structures and Tunable Stiffness Liying Xiaoa,#, Shanshan Liub,#, Danyu Yaoa, Zhaozhao Dinga, Zhihai Fanc, Qiang Lua,*, 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

School of Medicine, Shenzhen University, Shenzhen 518060, People’s Republic of

China c

Department of Orthopedics, The Second Affiliated Hospital of Soochow University,

Suzhou 215000, 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] #

The authors have contributed equally to the first author

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Abstract Detailed control of nano- and microstructures in porous biomaterial scaffold systems is important for control of interfacial and biological functions. Self-assembled silk protein nanostructured building blocks were incorporated into salt-leached scaffolds to control these features. Controllable concentration and pH were used to induce the formation of amorphous silk nanofibers in solution and also to reduce beta-sheet transformation during the more traditional salt-leaching process. These new scaffolds showed nanofibrous-microporous structures, reduced beta-sheet content, and tunable mechanical properties. Bone marrow mesenchymal stem cells grew better and showed differentiation behavior on these nanofibrous scaffolds, suggesting cytocompatibility and support for tunable differentiation via the scaffolds. These results suggested a new strategy of designing bioactive silk scaffolds by combining traditional scaffold formation processes with the controllable self-assembly of silk.

Keywords: Salt leaching, Silk, Self-assembly, Scaffolds, Biomimetic

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Introduction As appealing natural biomaterials, silks are being studied in numerous biomedical applications involving tissue engineering and implantable devices.1,

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biomedical applications, one focus is on forming scaffolds with three dimensional (3D) environments that mimic the native extracellular matrix (ECM).3-6 These scaffolds should support cellular adhesion, growth and proliferation, and also actively control cellular migration, and differentiation in the case of progenitor cells. To achieve this goal, silks are a suitable choice due to their tunable degradation and mechanical properties,7, 8 full degradability in vivo over time to support tissue regeneration,9 and a wide range of conformational compositions which related to mechanical and degradability properties.10 This tunability suggests the possibility to design various biomimetic niches to match the specific microenvironment of various tissues. Recently, a growing number of fabrication strategies such as lithography,11, 12 3D printing,13, 14 modified electrospinning15-17 and multiphoton micromachining18 have been developed to form silk matrices with biomimetic niches, by adjusting silk microstructure, orientation and conformational composition.12, 14, 16 These processes are generally complex, time-consuming and need relatively rigorous processing conditions.19-21 A main challenge remains to achieve similar hierarchical, nano-micro-scaled structures and suitable stiffness to match specific tissues. Improvements of novel fabricating methods are possible to facilitate the development of silk biomimetic scaffolds. 3D microenvironments should be more readily achievable by combining different fabrication methods with new options to modulate 3

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silk self-assembly. Recent advances in silk self-assembly have exploited various silk aggregates with specific nanostructures and secondary conformations.22-26 Silk nanofibers with similar morphological features to the ECM were assembled in aqueous solutions and used to construct porous scaffolds through freeze-drying.27 The silk nanofibers were also aligned under an electric field, to form anisotropic hydrogels.28 However, the present silk nanofibers are mainly composed of stable beta-sheet structures, thus limiting the versatility of the materials. Further fabrication is difficult, resulting in limitations to finely tune the stiffness of the materials. Amorphous silk nanofibers can also be generated via silk self-assembly in aqueous solution.29 These amorphous nanofibers can be transformed into beta-sheet structures, making them unsuitable as building blocks to form biomimetic scaffolds in salt-leaching processes, which induces such transitions. Here, considering that silk self-assembly is a kinetic process where the nano-structural and conformational transitions can be regulated by different factors, such as hydrophilic interactions, molecular mobility, charge distribution, and concentration,30 we hypothesized that a modified combination strategy to tune silk self-assembly during the scaffold forming processes would allow us to achieve biomimetic fabrication with metastable silk nanofibers as the units. To assess the feasibility of this strategy, silk self-assembly was tuned through adjusting concentration and charge density within a salt-leaching process. As a typical silk scaffold fabrication process, salt-leaching has limitations in the control nanostructural features or secondary conformation.31, 32 Here, combining tunable silk 4

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self-assembly, scaffolds with hierarchical nanofibrous-microporous structures and tunable stiffness were prepared through traditional salt-leaching. As a result, improved stem cell proliferation and differentiation were achieved on these new scaffolds.

Materials and methods Preparation of aqueous silk solution Bombyx mori cocoons were used to prepare silk solutions as described previously.33 Cocoons were boiled in 0.02 M Na2CO3 solution for 20 min and followed by rinsing process with distilled water to extract sericin proteins. The degummed silk was dissolved in a 9.3 M LiBr solution at 60 oC for 4 h, yielding a 20 wt% solution. This solution was dialyzed in deionized water using a dialysis tube (molecular weight cutoff 3,500) for 3 days. Then the solution was centrifuged at 9,000 r/min for 20 min to remove silk aggregates. The final concentration was about 5 wt%, determined by weighing the remaining solid after drying at 60 oC. Preparation of concentrated silk solution Fresh aqueous silk solution was treated by a concentration-dilution process.26 As shown in our previous study,34 two lids with different number of holes were covered over the fresh silk solutions to control the drying rate. Then the fresh solution (5 wt%) was slowly concentrated to about 20 wt% over 6 d at 60 °C or fast concentrated to about 20 wt% within 2 d at 60°C, and diluted to 5 wt% with distilled water. pH adjustment of silk solution 5

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The pH of the fresh aqueous silk solution, slowly concentrated silk solution and rapidly concentrated silk solutions were adjusted to near the isoelectric point of silk, 4.2, to eliminate charge repulsion and induce silk assembly.35 The 5 wt% silk solutions were adjusted to pH 4.2 by the addition of 0.1 M hydrochloric acid (HCl). Preparation of silk porous scaffolds Except the treated silk solution, salt-leached scaffolds were prepared according to same procedures reported elsewhere.33, 36 Here, 4 g of granular NaCl (particle size 350-450 µm) was added into 2 mL of silk solution (5 wt%) in a cylindrically shaped containers (diameter 1 cm) without mixing. The mixture was covered and placed at room temperature for 24 h, and then immersed in water (1 L) for 72 h to extract the salt and hydrochloric acid. Then, the scaffolds were freeze-dried for subsequent characterization. The scaffolds prepared from the fresh silk solution with pH adjustment, fast concentrated silk solution with pH adjustment and slowly concentrated silk solution with pH adjustment were termed F-P-S, C2-P-S and C6-P-S, respectively. As controls, the scaffolds derived from the silk solution without pH adjustment are termed F-S, C2-S and C6-S, respectively. Scanning electron microscopy (SEM) The morphology of the scaffolds was observed using scanning electron microscopy (SEM, Hitachi S-4800, Hitachi, Tokyo, Japan) at 3 kV. Samples were mounted on a copper plate and sputter-coated with gold prior to imaging.27 Porosity 6

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The porosity of the silk scaffolds was measured by liquid displacement.31,

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Ethanol (96 %) was used as the displacement liquid as it permeates through silk scaffolds easily. The silk scaffold was immersed in a known volume (V1) of ethanol for 5min until it became saturated (V2). The ethanol-impregnated scaffold was then removed and the residual ethanol volume was recorded as V3. The porosity of the scaffold was calculated by ℇሺ%ሻ =

V1 − V3 × 100 V2 − V3

Fourier transform infrared spectroscopy (FTIR) FTIR analysis of silk scaffolds was performed with a Nicolet FTIR 5700 spectrometer (Thermo Scientific, FL, USA), equipped with a MIRacle™ attenuated total reflection (Ge crystal).27 For each measurement, 64 scans were co-added with a resolution of 4 cm-1. The wavenumber was ranged from 400 to 4000 cm-1. X-ray diffraction (XRD) The crystallinity of the scaffolds was measured with X-ray diffraction (XRD) (X' Pert-Pro MPD, PANalytical BV, Almelo, Holland) using monochromated Cu Kα radiation (30 mA, 40 kV) with a scanning speed of 6o/min.38 Silk Degradation The scaffolds were incubated in phosphate saline (PBS) at 37 °C to evaluate degradation property.33,

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Samples (40 ± 5 mg) were soaked in PBS solution at

scaffold/solution weight ratios of 1:99. At designated time points (1, 3, 6, 9, and 12 7

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days), five samples for each group were rinsed with distilled water and prepared for mass balance assessment. Mechanical properties A bigger cylindrically shaped container (diameter 11 cm, height 30 mm) was used to prepare samples through same salt-leaching process and cut into suitable size (11 mm in diameter and 22 mm in height) for mechanical measurement. To measure the compressive properties of the scaffolds in wet conditions, the scaffolds were first hydrated in water for 4 h and then measured with a cross head speed of 2 mm/min at 25 oC using an Instron 3365 testing frame (Instron, Norwood, MA) with a 500 N loading cell.33 The load was applied until the cylinder was compressed by more than 30 % of its original length. The modulus was obtained by measuring the slope of the stress-strain curve in the elastic region. Five samples were carried out for each test group. In vitro cytocompatibility of scaffolds All animals were supplied by Laboratory Animal Research Center of Soochow University (Suzhou, China). All in vitro studies followed the ethical guidelines of the experimental animals approved by Institutional Animal Care and Use Committee, Soochow University. Bone marrow mesenchymal stem cells (BMSCs) derived from male Sprague-Dawley (SD) rats (age 2 weeks) were used to evaluate the in vitro cytocompatibility of the scaffolds. The scaffolds were punched into small disks (diameter of 8 mm and height of 2 mm) for 96-well plates, and sterilized with 8

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γ-irradiation at the dose of 50 kGy. BMSCs were cultured in Dulbecco’s modified Eagle medium (DMEM, low glucose) supplemented with 10 % fetal bovine serum (FBS), and 1 % IU ml-1 streptomycin-penicillin (Invitrogen, Carlsbad, CA). After reaching 90 % confluence, cells were detached from Petri dish and seeded into the scaffolds at a density of 1.0×105 cell per well. The cell morphology on the scaffolds was examined by confocal microscopy. After 1 and 12 days, the cell-seeded scaffolds were washed three times with PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich, St Louis, MO) for 30 min, followed by further washing. The cells were permeabilized with 0.1% Triton X-100 for 5 min and incubated with Alexa Fluor® 488-phalloidin (invitrogen, Carlsbad, CA) for 20 min at room temperature, followed by washing with PBS and, finally, staining with DAPI (Sigma-Aldrich, St. Louis, MO) for 1 min. Representative fluorescence images of the stained samples were obtained using a confocal microscope (Olympus FV10 inverted microscope, Nagano, Japan) with excitation/emission at 358/462 nm and 494/518 nm. The images of the scaffolds were captured from the surface to a depth of 100 µm in increments of 10 µm. To study cell proliferation on the scaffolds, samples harvested at the indicated time points (from day 1 to 12) were digested with proteinase K buffer solution for 16 h at 56 oC, as described previously.35,

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The DNA content was determined using the

PicoGreenTM DNA assay, following the protocol of the manufacturer (Invitrogen, Carlsbad, CA). Samples (n=5) were measured at an excitation wavelength of 480 nm and emission wavelength of 530 nm, using a BioTeK Synergy 4 spectrofluorometer 9

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(BioTeK, Winooski, VT). The amount of DNA was calculated by interpolation from a standard curve prepared with λ-phage DNA in 10×10-3 M Tris-HCl (pH 7.4), 5×10-3 M NaCl, 0.1×10-3 M EDTA over a range of concentrations. Cell differentiation on the scaffolds The BMSCs derived from SD rats were used to evaluate the in vitro cell differentiation

on

the

scaffolds.

Cell

differentiation

was

studied

via

immunofluorescence staining and Western Blot.40-44 Immunofluorescence staining of cells with desmin (muscle-specific marker) was used to characterize myogenicdifferentiation of BMSCs.45,

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Briefly, the samples were

fixed in PBS containing 4 % parafmaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 30 min, permeabilized with 1 % Triton X-100 in PBS for 10 min, washed three times with PBS, and blocked in PBS containing 3 % BSA for 1 h. Cells were incubated with rabbit polyclonal anti-desmin (Abcam, Cambridge, MA, USA) in blocking buffer for 1 h. Samples were then rinsed three times with PBS containing 0.1 % Tween-20 and incubated with secondary antibodies. DNA and silk scaffolds were identified by staining with DAPI (Sigma-Aldrich, St. Louis, MO, USA). FITC-phalloidin (Invitrogen, Grand Island, NY, USA) was used to stain F-actin. Representative fluorescence images of stained samples were obtained by confocal laser scanning microscopy (CLSM, Olympus FV10 inverted microscope, Nagano, Japan).

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For Western blot analysis, samples were lysed in RIPA lysis buffer (50 mMTris-HCl, pH 7.4, 150 mM NaCl, 0.1 % SDS, 1 % NP-40, and 0.5 % sodium deoxycholate) containing 10 µg mL-1 leupeptin,10 µg mL-1 aprotinin, and 1 mM PMSF. Equal amounts of lysates were electrophoresed in 12 % SDS-polyacrylamide gels. Proteins were transferred to a nitrocellulose membrane. Membranes were blocked with 5 % defatted milk and probed with rabbitanti-desmin (Abcam, Cambridge, MA, USA), then incubated with a horseradish-peroxidase conjugated secondary antibody. The ECL western blotting analysis system was used to detect the substrates. Statistical methods Comparison of the mean values of the data sets was performed using one-way ANOVA. Measures are presented as means ± standard deviation, unless otherwise specified. *P< 0.05 was considered significant, **P