3D Printing of Hierarchical Silk Fibroin Structures - ACS Applied

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3D Printing of Hierarchical Silk Fibroin Structures Marianne R. Sommer, Manuel Schaffner, Davide Carnelli, and André R. Studart ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11440 • Publication Date (Web): 29 Nov 2016 Downloaded from http://pubs.acs.org on December 1, 2016

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ACS Applied Materials & Interfaces

3D Printing of Hierarchical Silk Fibroin Structures Marianne R. Sommer1, Manuel Schaffner1, Davide Carnelli1, André R. Studart1* 1

Complex Materials, Department of Materials, ETH Zurich, 8093 Zurich, Switzerland

* Corresponding author: Vladimir-Prelog-Weg 5, 8093 Zurich, Switzerland, Phone: +41 44 633 7050, Fax: +41 44 633 1545, E-mail address: [email protected] Keywords: hierarchical structure, silk fibroin, 3D printing, controlled architecture, pore templating particles

Abstract Like many other natural materials, silk is hierarchically structured from the amino acid level up to the cocoon or spider web macroscopic structures. Despite being used industrially in a number of applications, hierarchically structured silk fibroin objects with a similar degree of architectural control as in natural structures have not been produced yet due to limitations in fabrication processes. In a combined top-down and bottom-up approach, we exploit the freedom in macroscopic design offered by 3D printing and the template-guided assembly of ink building blocks at the meso and nanolevel to fabricate hierarchical silk porous materials with unprecedented structural control. Pores with tunable size in the range 40 – 350 µm are generated by adding sacrificial organic microparticles as templates to a silk fibroin-based ink. Commercially available wax particles or monodisperse polycaprolactone made by microfluidics can be used as microparticle templates. Since closed pores are generated after template removal, an ultrasonication treatment can optionally be used to achieve open porosity. Such pore templating particles can be further modified with nanoparticles to create a hierarchical template that results in porous structures with a defined nanotopography on the pore walls. The hierarchically porous silk structures obtained with this processing technique can potentially be 1 ACS Paragon Plus Environment


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utilized in various application fields from structural materials to thermal insulation to tissue engineering scaffolds.

Introduction Fibrous silk proteins are abundantly available in nature and perform various functions1. The silk found in cocoons, for instance, not only protects silkworms from predators but also regulates gas exchange and temperature2. Fulfilling a more structural purpose, spider silk is the main building block used in orb-webs to capture prey1, 3, 4. A common feature of these different types of silk is an underlying hierarchical structure spanning all the way from its macroscopic arrangement down to the single amino acid level. The amino acid sequence dictates the folding and assembly of the proteins over several length scales from the bottom up into fibrils and fibers or filaments, often incorporating more than one protein type. Macroscopically, silk cocoons and spider webs are then organized in specific geometries for optimized functionality5. Because of their unique hierarchical structure and associated properties, silk-based materials have been used to create man-made objects for centuries. Silk fibroin from Bombyx mori is the most studied and industrially used type of silk. Its applications range from biomaterials6 to optical components7 to food preservation8. Due to its unique structure, silk fibroin shows some remarkable properties such as high strength and stiffness reaching up to 690 MPa and 17 GPa, respectively9, slow biodegradation and the ability to control gas transport. Despite being extensively researched, processing silk fibroin into elaborate hierarchical structures has so far been a major challenge. Since most standard material manufacturing techniques rely on subtractive processes, the fabrication of silk-based objects with controlled architecture from the nanometric up to the macroscopic scale is difficult. In two dimensions, silk films have been nano- and microstructured using stamps or masks10-12. In a more 3-dimensional approach, capsules in the micrometer range were fabricated 2 ACS Paragon Plus Environment

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by colloidal templating13,

14

. To produce silk fibroin scaffolds, salt crystals, gas bubbles15 or

polycaprolactone particles16 have all been used as pore templating building blocks. Other methods to achieve nanostructured materials are electrospinning17 or gyration18, 19. However, a combination of such interesting nano- and mesostructures with controlled macroscopic architecture has not yet been achieved. 3D printing techniques have recently been shown to be an effective platform to build up biologically-inspired structures with precise control spanning multiple length scales in a layer-bylayer fashion that resembles the way tissues are formed in living systems20, 21. In particular, silk fibroin has been 3D printed for different applications using several techniques. Tissue engineering scaffolds22 and optical waveguides23 were prepared by direct writing of a silk fibroin solution into a methanol bath that causes immediate consolidation of deposited filaments. Ink jet printing of an aqueous silk fibroin solution has been developed as a more biocompatible alternative that also allows for the addition of bioactive molecules to the ink24. Print line widths down to 1 µm were achieved by electrohydrodynamic printing of silk fibroin25. To be able to incorporate cells into the ink, a silk fibroin/gelatin mixture was also developed into a printable hydrogel system, which was either enzymatically crosslinked or sonicated to induce silk fibroin crystallization prior to printing26. In spite of these recent developments, 3D printing has not yet been exploited to create truly hierarchical structures made of silk materials. This is partially due to the fact that 3D printing using conventional inks does not allow for the manufacturing of hierarchical structures of large size within reasonable production timescales. While techniques achieving resolutions down to the nanometer range have been developed27, printing macroscopic materials with such precision is extremely time-consuming and tedious. Also, the maximal printable object size achieved with those techniques has been typically limited to the micrometer range27. To overcome these limitations, smartly designed inks can be used with techniques such as direct ink writing to 3 ACS Paragon Plus Environment


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enable the fabrication of centimeter-sized objects with architectural control down to the nanometer range28. In this approach, the top-down manufacturing capabilities of 3D printing is combined with the bottom-up assembly of building blocks present in the ink to build materials covering several length scales within timescales compatible with engineering applications. In a recent example, hierarchically porous ceramics were produced by 3D printing inks containing emulsions droplets or gas bubbles as templates within the printed filaments29. Such architectured systems appear often in nature, particularly when the unusual combination of high strength and low weight offers a competitive advantage to the living organism20. Exploiting this combined top-down and bottom-up manufacturing approach using other chemical compositions may offer additional functionalities beyond lightweight load-bearing capabilities. For instance, materials made from silk fibroin exhibiting porosity on several length scales are of interest as tissue engineering scaffolds, where larger pores should ensure sufficient nutrient supply to smaller pores that serve as host for new growing tissue30. While conventional 3D printed scaffolds suffer from poor cell seeding efficiencies due to their convex structures31, mesopores in the filaments not only potentially improve cell seeding but also accelerate tissue growth30. Nanostructural features on the walls of such smaller pores can be useful to further enhance cell response and tissue growth32. Here, we use direct ink writing combined with a templating technique to 3D print hierarchical silk fibroin structures with features spanning from the nanometric to the macroscopic scale, as schematically shown in Figure 1. Control at the macroscale is achieved by developing an ink with viscoelastic properties optimized for 3D printing of complex geometries, whereas meso and nanostructural control are obtained by adding to the ink sacrificial template particles covering several orders of magnitude in size. Removal of the template leads to silk-based materials with a hierarchical porous architecture defined by the printed filament spacing and by the size and features of the sacrificial particles. Wax particles sieved to a defined size 4 ACS Paragon Plus Environment

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distribution are first used as a mesostructural template within the printed filament. In a second example, monodisperse polycaprolactone (PCL) particles made by microfluidics can be used to create pores of the same size at the mesoscale. Finally, the PCL particles can be electrostatically coated with latex nanoparticles to generate silk structures exhibiting structural features at three hierarchical levels. The resulting porous structures show close porosity that resemble the foam-like proteinaceous architectures found in feathers, beaks and quills33, making them suitable for structural and thermal applications. Given the controlled biodegradability of silk and the possibility to also create open porosity, the hierarchical structures obtained may also find use in tissue engineering, environmental and biotechnological applications. While we remain far from replicating the exquisite hierarchical organization found in the natural world, the proposed fabrication technology significantly enhances our ability to control the structure of silkbased materials over multiple length scales.

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Figure 1: Process to fabricate 3D printed silk fibroin structures with architectural control over multiple levels of hierarchy, namely the macro-, meso- and nanostructure. PCL particles are produced by microfluidics (aI, actual sample: Figure 3) and decorated with latex nanoparticles through electrostatic adsorption (b, actual sample: Figure 5b). Alternatively, commercially available wax particles can be sieved to a certain size distribution (aII, actual sample: Figure 3) and used as sacrificial template. The latex-modified PCL or wax particles are added to a 3D printing ink made of silk fibroin and a Konjac gum hydrogel (c, actual sample: Figure 5c) and printed (d). After drying and silk fibroin crystallization, the templating particles are dissolved (e, actual sample: Figure 4) and the material is subjected to ultrasonication to open the pore walls and obtain open porous structures (f, actual sample: Figure 6).

Experimental Materials Silk cocoons from Bombyx mori were kindly supplied by Trudel Inc. (Switzerland). Sodium carbonate (anhydrous, puriss.), lithium bromide (≥ 99 %), dichloromethane (≥ 99.5 %), polycaprolactone (Mn 10000 g/mol), polyvinyl alcohol (Mw 13000-23000 g/mol, 87-89% hydrolyzed), ethanol (absolute) and polystyrene latex (carboxylate-modified, fluorescent red) were purchased from Sigma (Switzerland). Methanol (for analysis) and sodium hydroxide (for analysis) were obtained from Merck (Germany). Poly(allylamine hydrochloride) was acquired from Alfa Aesar (Germany) and Konjac gum (Ticagel ®) from Ticgums (USA). Wax beads (Kahlbeads 7625P) were supplied by Kahl GmbH (Germany).

Ink Preparation The Bombyx mori silk cocoons were degummed by boiling twice in 0.02 M sodium carbonate solution for 1 hr and rinsing with water. The resulting silk fibroin was then dissolved in a 9 M LiBr solution at a concentration of 10 wt% and dialyzed against water for 36 hrs. After 6 ACS Paragon Plus Environment

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filtering through a 5 µm syringe filter, the obtained solution was frozen and freeze-dried (Freezone 2.5 Plus, Labconco, USA). Konjac glucomannan was dissolved in water at 80 °C under vigorous mechanical mixing until a concentration of 6 wt% was reached. The solution was then slowly cooled down to 4 °C while still under vigorous mixing. A 16 wt% aqueous solution of silk fibroin was prepared at the same time from the freeze-dried powder prepared as described above. 1 g of the Konjac solution was then mixed with 1 g of the silk fibroin solution. Next, 2 g of pore templating particles were added to 2 g of the Konjac-silk solution and mixed well (Figure 1c). The ink was finally degassed in a planetary mixer (ARE-250, Thinky, USA) before the printing experiments.

Fabrication of Pore Templating Particles As-received wax beads (Figure 1aII) were sieved through a 200 µm mesh and only particles passing the sieve were used for printing. PCL particles were produced by emulsifying a dichloromethane solution containing 10 wt% PCL in water containing 2 wt% polyvinyl alcohol (PVA), followed by slow evaporation of the dichloromethane. Alternatively, PCL particles were also made by microfluidics (Figure 1aII), as reported before

16

. In brief, a flow-focusing glass

capillary microfluidic device was fabricated by pulling (Flaming/Brown micropipette puller P-97, Sutter Instruments, USA) two round borosilicate glass capillaries (outer diameter 1 mm, World Precision Instruments, Germany) and subsequently adjusting the diameter of their openings using a microforge (MF-830, Narishige, Japan). The two capillaries were then aligned within a square glass capillary (inner diameter 1.05 mm, Harvard, USA). Needles (H. Sigrist & Partner AG, Switzerland) were used as inlets and connected to syringes (Hamilton Gastight, Switzerland) using polyethylene tubing (Scientific Commodities Inc, USA). Using lab-scale syringe pumps (PHD2000, Harvard Apparatus, USA), the flow rates were varied from 1 to 5 ml/hr and 5 to 30 ml/hr for the inner and outer phases, respectively. The exact device 7 ACS Paragon Plus Environment


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geometries and flow rates used are displayed in Table 1. Droplets were collected in excess 2 wt% PVA aqueous solution and resulted in solid PCL particles after complete evaporation of the solvent. The obtained PCL particles were then washed 5 times with water (electrical resistivity > 18 MΩ.cm). Table 1: Parameters used to obtain droplets of different sizes (D1 to D3) using a constant collector capillary size of 580 µm Emitter capillary size (µm)

Inner flow rate (ml/h)

Outer flow rate (ml/h)

D1

60

1

30

D2

60

1

10

D3

300

5

5

To modify the templating particles with latex nanoparticles (Figure 1b), PCL particles were first etched in a 1:1 1 M NaOH/methanol solution for 30 minutes and washed with water to create a negative surface charge34. Next, the etched particles were placed in a 1 wt% poly(allylamine hydrochloride) solution for 1 hr, which was followed by washing with water to reverse the surface charge to positive. Finally, a suspension of 0.5 wt% latex nanoparticles in water was added to the PCL suspension and stirred at 30 rpm for 18 hours to promote electrostatic adsorption of the small nanoparticles on the surface of the template particles. Again, the particles were washed with water and dried in air.

Rheology Rheology measurements were performed on a strain and stress controlled rheometer (MCR501 and MCR702, Anton Paar, Austria). Steady state flow curves were obtained from strain rate-controlled measurements performed at shear rates ranging from 0.1 to 100 s-1 in timecontrolled measurements. For the 8 wt% silk fibroin solution, a double-gap geometry (DG26.7, 8 ACS Paragon Plus Environment

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Anton Paar, Austria) was used. Inks containing 8 wt% silk fibroin and 3 wt% Konjac gum were measured using a cone-plate geometry (CP25/2). To study the recovery behavior of the ink after printing, the shear forces present during extrusion were simulated by shearing the ink for 100 s at a shear rate of 100 s-1, followed by the measurement of the viscosity over time at a shear rate of 0.1 s-1. Strain-controlled oscillatory amplitude sweeps at a frequency of 1 Hz with strains ranging from 10-4 to 10 were obtained on the same respective devices as the flow curves. The ink was further characterized by a stress-controlled measurement of the yield stress (0.01 – 500 Pa) at an integration time of 20 s.

3D Printing The ink was 3D printed using a 3D Discovery equipment (regenHU Ltd, Switzerland) and STL files generated in BioCAD (Figure 1d). Typically, conical needles with an inner diameter of 1.2 mm (H. Sigrist & Partner AG, Switzerland) and a print speed of 10 mm/s were chosen. For experiments performed with particles made by microfluidics, conical needles with an opening of 2.2 mm were used. Extrusion pressures in the range of 1-4 kPa were applied and individually adjusted for each new ink. After printing, the structures were dried at air and treated with 90 vol% methanol for 30 minutes to induce β-sheet formation in the silk fibroin. Finally, the latexdecorated PCL particles or wax beads were dissolved in dichloromethane or chloroform at 60 °C, respectively, to generate the hierarchical porous structures (Figure 1e). To dissolve away the Konjac gum and obtain pure silk fibroin structures, printed structures were autoclaved immersed in water at 121 °C and 1 bar for 20 minutes.



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Sonication Open porous structures were produced by placing samples in an ethanol bath and sonicating them at a distance of 3 mm from the probe (Vibra-cell VCX 130, Sonics & Materials Inc, USA) for 1 minute using 3 s on and off pulses at different amplitudes (Figure 1f).

Infrared Spectroscopy Infrared spectra were recorded on an ATR-FTIR (Cary 670 FTIR Spectrometer, Agilent Technologies, US). 32 scans at a resolution of 2 cm-1 were performed on both samples before and after Konjac gum dissolution.

Imaging The samples were sputter-coated with 6 nm of platinum before imaging in a scanning electron microscope (LEO Gemini, Zeiss, Germany).

Results & Discussion Fine control over the rheology of the ink is necessary for it to retain its shape after extrusion and thus enable printing of distortion-free structures. Overall, facile and homogeneous extrusion combined with shape retention requires inks exhibiting shear-thinning behavior35, fast viscosity recovery after extrusion36, high storage modulus and a well-defined yield stress29, 37-39. The key to prepare silk fibroin solutions displaying these rheological properties was to use Konjac gum as a rheology modifier, a carbohydrate widely used as thickener in food industry. Indeed, when low amounts of Konjac gum are added, the otherwise liquid-like silk fibroin solution becomes shear-thinning, is able to recover its viscosity after experiencing high shear forces and displays a clear initial elastic behavior and yield stress, making it printable (Figure 2).


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To better quantify the rheology of the printable inks, we evaluate the effect of Konjac gum on the rheological response of silk fibroin solutions, as indicated in Figure 2. An aqueous solution containing 8 wt% of silk fibroin alone behaves purely Newtonian and therefore cannot be printed unless a methanol solution is used as coagulation bath (Figure 2a). The addition of 3 wt% of Konjac gum to an 8 wt% silk fibroin solution not only increases by about 3 orders of magnitude its viscosity at a low shear rate of 1 s-1, but also changes the flow behavior from Newtonian to shear-thinning. The shear-thinning response allows for a reduction of the ink viscosity to a level comparable to that of the silk fibroin solution when the high shear stresses at rates on the order of 102 s-1 developed during extrusion are applied.

Figure 2: (a) Steady state flow curves obtained for an 8 wt% silk fibroin aqueous solution and the developed printing ink made of 8 wt% silk fibroin and 3 wt% Konjac gum. (b) Recovery behavior 11 ACS Paragon Plus Environment


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of the ink after applying cycles of high shear simulating the forces present during printing. (c) Oscillatory measurements showing a liquid-like behavior for the 8 wt% silk fibroin solution (G’’ > G’) and the viscoelastic nature of the ink (crossover between G’ and G’’). (d) Stress-strain curve obtained for the ink, revealing a clear yield stress. Remarkably, the ink containing 3 wt% Konjac and 8 wt% silk fibroin immediately recovers 51 % of its original viscosity after being sheared at 100 s-1 simulating extrusion (Figure 2b). Oscillatory amplitude sweeps of the ink and of the 8 wt% silk fibroin solution reveal that the first behaves viscoelastically, whereas the latter shows a purely viscous response (Figure 2c). The storage modulus of the ink in the linear region lies at 21.5 kPa, which allows printing of bridging structures spanning as much as 1 cm, according to simple beam theory calculations40. In addition to such high storage modulus, the developed ink displays a high yield stress of 283 Pa, which prevents capillary-induced distortion of the deposited filaments (Figure 2d). A yield stress on the order of 100 Pa was shown to be required to avoid such shape distortion effects in print lines with diameters of 200 µm41. From these rheological measurements it can be concluded that the developed ink system exhibits the viscoelastic properties required for extrusion-based 3D printing and thus can be used as matrix for the sacrificial template particles. Beeswax beads ranging from 40 to 200 µm in size with a median size of 88 µm (Figure 3a) or polycaprolactone particles, whose size can be precisely tuned between 164 and 335 µm when made by microfluidics (Figure 3b-c), are used to template pores within the printed filaments. The reason for choosing these relatively large particles is that they create porosity in a size range that would be interesting for biomedical applications. Although both the wax beads and polycaprolactone particles are fairly hydrophobic and thus prone to agglomeration in water, we expect possible agglomerates to be easily destroyed under the shear stresses applied during mixing and printing. In case pores in a much smaller size range are required, smaller particles could be used. Nanometric particles might necessitate the use of a dispersant, however, to 12 ACS Paragon Plus Environment

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reach equally high volume fraction without agglomeration. Assuming a wax density of 0.9 g/cm3 and an ink density of 1.03 g/cm3, the concentration of 50 wt% of beeswax beads used here correspond to 57 vol%. Supposedly, this concentration can be further increased up to approximately 64 vol%, which is the maximum theoretical content expected for random close packing of hard spheres. Possible changes in the solids content should be accompanied by adjustments in concentration of the rheology modifier Konjac gum in order to tune the viscoelastic properties of the ink to the levels required for direct ink writing.



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Figure 3: Pore templating particles used for printing. (a) Representative image of the wax particles sieved through a 200 µm mesh and the corresponding size distribution. (b) Correlation between the sizes of PCL solution droplets directly after production in a microfluidic device and the resulting PCL particle size after evaporation of the solvent. (c) Droplet (D) and resulting PCL


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particle (P) diameters reaching up to 350 µm obtained when varying device geometry and flow rates in a microfluidic device. Scale bars 500 µm. Our ability to tune the pore sizes while keeping them monodisperse is illustrated here by preparing solid PCL particles from monodisperse droplets made in a glass capillary microfluidic device. The size of such monodisperse template particles can be adjusted within a broad range from a few tens of micrometers to millimeters by changing the microfluidic device geometry as well as the applied flow rates42-45. Droplets of PCL solution of a defined size lead to solid spherical particles upon evaporation of the solvent (Figure 3b). The initial droplet size shows a strong linear correlation with the dry particle size (R2 = 0.994). From this correlation, we find that the average shrinkage upon solvent evaporation lies at 46 %. This leads to average particle sizes ranging from 165 to 335 µm. Remarkably, shrinkage does not compromise the desired narrow size distribution of the PCL particles. Examples of droplet (D) and corresponding particle (P) size distributions and optical images thereof obtained under 3 different experimental conditions are shown in Figure 3c. For the droplets, polydispersities below 3 % are achieved. After solvent evaporation, the polydispersities are only marginally higher and vary between 2 and 4 %. This shows that we can effectively produce monodisperse PCL particles by microfluidic emulsification of a PCL solution and subsequent slow solvent evaporation through the continuous aqueous phase. Hierarchically porous silk fibroin structures are successfully obtained after 3D printing of inks loaded with sacrificial microparticles followed by removal of such pore templates from printed filaments and grids (Figure 4). Before the dissolution of the sacrificial particles, the silk fibroin is methanol-treated to induce the formation of β-sheets, which is the morphology silk fibroin attains after spinning in nature. This phase transformation increases both the chemical and mechanical stability of the silk fibroin. Figure 4a shows a grid structure obtained after printing of an ink with 50 wt% of beeswax beads ranging from 40 to 200 µm with a median size 15 ACS Paragon Plus Environment


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of 88 µm. The hierarchical object produced after printing and dissolution of the template exhibits a macroscopic shape with feature sizes in the mm-scale defined by the print pattern combined with mesoscale porosity within the filaments templated by the sacrificial particles. Because of their large size in the range of tens to hundreds of micrometers, the achievable resolution and the minimal needle size are dictated by the sacrificial particles rather than the positioning accuracy of the printer.

Figure 4: Structures obtained upon removal of the sacrificial material after printing the developed ink with different kinds of pore-templating particles. (a) Beeswax particles that passed through a 200 µm mesh sieve; (b) uncoated PCL particles (left) and latex-modified PCL particles (middle and right) with an average size of 120 µm; (c) monodisperse 310 µm PCL particles fabricated by microfluidics. Left: macroscopic 3-dimensional print pattern; middle: mesostructure of the single 16 ACS Paragon Plus Environment

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print lines and filaments; right: cross-sections of the print lines and filaments. The inset in the middle-right panel depicts the nanopores created on the walls of the pores for structures made from latex-modified PCL templates. Scale bars: left and middle 5 mm; right 25 µm; inset 1 µm. Another hierarchical level in the form of nano-dimples on the pore walls can be incorporated in the silk porous structures by coating the template microparticles with a layer of latex nanoparticles (Figures 4 and 5). To illustrate this possibility, we use PCL microparticles as template at the mesoscale (Figure 4b). The PCL particles (Figure 5a) are first etched to create a negative surface charge that allows for the electrostatic adsorption of latex nanoparticles. To reverse the charge, a layer of positively charged polyelectrolyte is then adsorbed onto their surface followed by the adsorption of carboxylate-modified latex nanoparticles displaying a negative charge (Figure 5b). For this last step, stirring at low speed (30 rpm) is crucial to reach complete coverage of the templating particles. While higher stirring speeds lead to less homogeneous latex multilayers, no stirring at all leads to incomplete coverage and patchy topographies. The suspension containing the latex and PCL particles was kept under stirring until a clear supernatant was obtained, which typically occurs after about 18 hours. When the thus-modified particles are added to the ink and dried, the latex detaches from the PCL particles and sticks to the ink (Figure 5c). Removal of such hierarchical template creates nanostructured pore walls with dimple sizes that correlate well with the size of the initial latex nanoparticles (Figure 5d). The resulting structures are thus truly hierarchical in nature (Figure S2).



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Figure 5: Morphology of the sacrificial templating particles and of the resulting hierarchical silk fibroin structures. PCL particle and its surface before (a) and after (b) latex adsorption. (c) Structures obtained after mixing the latex-decorated particles (white asterisk) with the ink (black asterisk) followed by drying. (d) Upon dissolution of the latex-decorated particles, the resulting pore walls are patterned with latex imprints. Scale bars: 100 µm for (a-b), 50 µm for (c-d) and 2 µm for all the close-ups. The flexibility of the proposed approach makes the choice of the chemical composition of the pore templating particle be only limited by the precondition that it must not dissolve in water or methanol, but be soluble in any other solvent. To avoid clogging of the needle and prevent phase separation during printing at high volume fractions, the particle diameter should also be more than 5 times smaller than the needle opening46. The use for example of relatively large needle sizes of 1.2 mm allows us to print lines with particles up to 200 µm in size. If desired, this range might be extended by increasing the needle opening even further. Since the ink contains 3 wt% Konjac gum, it is also important to note that the material obtained after drying is not pure silk fibroin. Should this be required, the Konjac gum can easily be dissolved away in water. To 18 ACS Paragon Plus Environment

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demonstrate this, structures obtained after removal of the sacrificial template were autoclaved in water and characterized by FTIR before and after autoclaving (Figure S1). Indeed, Konjac gum’s acetyl peak at 1730 cm-1 disappears completely and the methyl peaks between 3000 and 2800 cm-1 are greatly reduced in intensity after autoclaving. This confirms our ability to print hierarchical structures with pure silk fibroin.

Figure 6: The creation of structures with open porosity using ultrasonication. (a) Photographs of a hierarchically porous silk fibroin structure when added to ethanol. Before sonication the structure floats. Sonication at 70 % amplitude leads to immediate sinking of the structure, indicating the creation of open pores. (b-c) SEM micrographs depicting examples of closed and open pores after sonication at 40 and 70 % amplitude, respectively. Scale bars are 50 µm. Besides hierarchical features, the proposed processing route also enables the preparation of structures with interconnected open pores. As the particles are always covered with a layer of ink, only closed porous structures are obtained right after particle removal, even if a high particle loading is used in the ink. For applications calling for open pores, an ultrasonication step can be applied to generate open porosity in the silk fibroin structures47. The incorporation of open porosity through the cavitation effect caused by ultrasonication was probed 19 ACS Paragon Plus Environment


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by placing samples subjected to different sonication intensities in ethanol (Figure 6). Asproduced structures submerged in ethanol float due to the air entrapped within their pores. Sonication of such submerged samples at 40 % amplitude at 20 kHz for 1 minute is not enough to make them sink. But if the sonication amplitude is increased to 70 % the sample quickly sinks, a clear indication of opening of the pores (Figure 6a). Representative SEM images of pores after sonication show that no windows between pores are visible in samples exposed to 40% amplitude, whereas open pores are clearly formed in structures sonicated at 70 % amplitude (Figure 6b-c). A similar trend was previously reported when sonication was proposed to create open porosity in other polymeric porous materials47. Opening of the pores presumably happens by collapsing cavitation bubbles, which create very strong local jets that are able to break open the pore walls47. While the use of ultrasonication to create open porosity is qualitatively demonstrated here, further follow-up work should be carried out to quantify the effect of sonication power and frequency on the final porous structure.

Conclusion In this work, hierarchical silk fibroin structures were created by 3D printing of a silk-based ink containing thoroughly designed sacrificial particles to template porosity. The choice of the sacrificial particle geometry determines the resulting porosity. At the mesoscale, pores can be tailored from a defined polydisperse size range to a very specific monodisperse diameter by using commercially available wax microparticles or polycaprolactone particles obtained from microfluidics, respectively. These microparticles can also be coated with nanoparticles to generate a hierarchical template that leads to architectural control at an even smaller length scale. The closed pores obtained after printing of inks containing such microparticle templates can be optionally opened by an ultrasonication treatment. By combining this bottom-up templatedirected assembly process with the spatially-controlled deposition enabled by top-down direct


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ink writing, we demonstrate that this approach offers a versatile platform for the creation of silk fibroin structures with an unprecedented degree of hierarchical structural control.

Acknowledgements Parts of this research project were funded by the Swiss Competence Center for Energy Research (SCCER - Capacity Area A3: Minimization of energy demand). The authors would also like to thank Dr. Patrick Rühs for performing the rheological measurements and Dr. Rok Simic for helping with the FTIR measurements.

Supporting Information Additional images illustrating the structural hierarchy as well as FTIR measurements are shown in the supporting information.

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3D Printing of Hierarchical Silk Fibroin Structures - ACS Applied

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