3D Printing of Regenerated Silk Fibroin and Antibody-Containing

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3D Printing of Regenerated Silk Fibroin and AntibodyContaining Microstructures via Multi-Photon Lithography Matthew B Dickerson, Patrick B. Dennis, Vincent P. Tondiglia, LLoyd J. Nadeau, Kristi M. Singh, Lawrence F. Drummy, Benjamin P. Partlow, Dean P. Brown, Fiorenzo G Omenetto, David L Kaplan, and Rajesh R. Naik ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00338 • Publication Date (Web): 05 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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ACS Biomaterials Science & Engineering

3D Printing of Regenerated Silk Fibroin and Antibody-Containing Microstructures via Multi-Photon Lithography Matthew B. Dickerson1, Patrick B. Dennis1, Vincent P. Tondiglia1, Lloyd J. Nadeau1, Kristi M. Singh1, Lawrence F. Drummy1, Benjamin P. Partlow2, Dean P. Brown1, Fiorenzo G. Omenetto2, David L. Kaplan2, and Rajesh R. Naik3*

1. Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson AFB, OH 2. Biomedical Engineering Department, Tufts University, Medford, MA 3. Human Performance Directorate, Air Force Research Laboratory, Wright-Patterson AFB, OH *Correspondence: [email protected]

Keywords: Silk, Fibroin, Multi-photon lithography, Hydrogel, IgG

Abstract. Regenerated silk fibroin, a biopolymer derived from silkworm cocoons, is a versatile material that has been widely explored for a number of applications (e.g., drug delivery, tissue repair, biocompatible electronics substrates, and optics) due to its attractive biochemical properties and processability. Here, we report on the free-form printing of silk-based, 3D microstructures through multi-photon lithography. Utilizing multi-photon lithography in conjunction with specific photoinitiator chemistry and post-print crosslinking, a number of microarchitectures were achieved including self-supporting fibroin arches. Further, the straightforward production

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of high fidelity and biofunctional protein architectures was enabled through the printing of aqueous fibroin/immunoglobulin solutions.


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Introduction Silk from the cocoon of the domesticated silkworm, Bombyx mori, is a material historically prized for its properties as a textile. However, new and potentially disruptive technological applications for silk have emerged from the sustained efforts of contemporary researchers.1-3 The relative success of silk in these new applications is based, in part, on the availability and affordability of B. mori silk cocoons due to their continued and widespread use in textiles. Processing of the silkworm cocoons yields silk fibers that are primarily composed of fibroin proteins (light and heavy chain).4 These proteins can be solubilized in concentrated lithium bromide solutions and the chaotropes subsequently removed from the proteins by dialysis.5 This procedure yields aqueous solutions of regenerated silk fibroin (RSF).5 RSF has a number of unique attributes that have propelled the use of this material forward for a variety of biomedical and biologically-related applications.6

When B. mori

silkworms are forced to spin fiber at high rates, the properties of their silk approach the high modulus and toughness of spider dragline silk.7 Though RSF materials are not as strong as such high-rate reeled (native) fibroin fibers, RSF materials display robust and controllable mechanical properties.1-4 immunogenicity,

Silk is a FDA-approved biomaterial and RSF materials exhibit low excellent

biocompatibility,

high

bioresorbability,

and

tailorable

biodegradability.3, 4, 6 In addition to biodegradability, many of the material properties of RSF can be modified by selecting processing conditions to yield materials of controlled molecular weight distribution, protein conformation, and degree of crystallinity.4, 5, 8 The benign nature of RSF solutions facilitates the mixing of liquid silk with other biological components, ranging from eukaryotic cells to small molecules, to create materials with additional biofunctionalities.9,

10

Furthermore, RSF has proven versatile in protecting such labile biological additives from



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degradation under austere environmental conditions.9,

10

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As summarized in several recent

review papers, the “cocooning” of sensitive biological components (e.g., vaccines, enzymes, antibodies or antibiotics) in RSF has important implications in improving the expeditionary nature of therapeutics by mitigating the need for cold storage.9, 10 The stabilization of therapeutic proteins and small molecules by solid RSF materials aligns well and augments the capabilities of silk as a platform for controlled release and biosensor applications. 3, 11 In addition to its impressive mechanical and biochemical properties, one of the driving forces for the proliferation of interest in RSF for technological applications is the ease in which silk solutions maybe processed into a variety of material formats.1-5 Examples of architectures produced from RSF solutions include hydrogels, films, foams, microparticles, tubes, scaffolds, bulk matrices and fibers of varying diameter.1-5 Significantly, these newly formed silk structures maybe rendered water insoluble by manipulating the secondary structure of the protein (inducing β-sheet formation) through biocompatible processing schemes (i.e., water vapor annealing).5 Fibroin may also be converted into a water insoluble material through enzymatic or photoinitiated cross-linking reactions that form tyrosine-tyrosine (dityrosine) covalent bonds.12 The ability to control the solubility of RSF under benign conditions is significant as it allows for the biofunctionality of incorporated biological components to be preserved within a stable silk matrix.12 As highlighted above, RSF solutions allow for the fabrication of silk-based materials in a variety of form factors that are more appropriate for technological insertion than the native B. mori fiber. For example, aqueous silk can be spun or solution cast to generate thin films which possess robust mechanical properties, low roughness, and tailorable degradation rate.5 Such films can serve as dielectric materials or substrates for biodegradable, biocompatible, and


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transient electronics that might be used for implantable devices, monitoring food quality, and minimizing electronic waste in the environment.3,

13-19

The high optical transparency of silk

films coupled with the ability to pattern RSF by soft lithography (i.e., PDMS micro-replica mold casting) with features below 100 nm have been exploited to demonstrate an impressive array of optical and photonic devices.3 Such casting techniques, when mated with RSF-solutions doped with biofunctional molecules can be used to produce responsive optical sensors or silk-based materials for controlled release applications (e.g., microneedles).3, 20 Patterning of planar RSF films and substrates may also be conducted by reactive ion etching and nanoimprinting to produce patterns and features at the micro- and nano-scales, respectively.3, 21 In addition to these contact-based techniques, the high-resolution control of 2D silk architectures has recently been accomplished through electron-beam- or photolithography.22-24 In these lithographic approaches, areas of RSF exposed to an e-beam or light source are chemically altered such that the silk becomes water soluble (i.e., a positive tone resist) or insoluble (i.e., a negative tone resist). This change in protein solubility allows for pattern development by rinsing away areas of soluble fibroin with water, revealing the patterned and water insoluble silk. For example, Kim and colleagues reported that RSF could serve as high resolution resist (either positive or negative tone) in e-beam lithography, achieving ~30 nm spatial resolution.22 Furthermore, the eco-friendly nature of this nanofabrication enabled the production of biofunctionalized silk nanostructures through the patterning of RSF resists doped with green fluorescent protein (GFP) or the enzyme horse radish peroxidase (HRP).22 22 Beyond e-beam patterning, silk films may also be patterned with light-based (photo) lithographic methods. For example, fibroin films have been patterned with micron scale spatial resolution utilizing deep-UV lithography.23

Patterning in deep-UV lithography is achieved by the



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photodegradation of the fibroin protein chain, which reduces the crystallinity of the silk and renders it water soluble (i.e., positive tone RSF resist).23 In a similar vein, the ability of fibroin to undergo multiphoton absorption can be exploited for the laser machining of RSF hydrogels into 3D micropatterns.25 RSF may also act as a negative-tone resist for photolithographic patterning.24

In research reported by Applegate and co-workeers, a photoinitiator (flavin-

mononucleotide (FMN)) was utilized to catalyze the transition of soluble RSF to a cross-linked hydrogel upon exposure to 450 nm light.24 The RSF/FMN system achieved a spatial resolution on par with that reported for the shadow mask (~50 µm).24 Such photoinitiated reactions can also be used to produce silk-based hydrogels in bulk formats.24, 26-28 A variety of printing-based methods have also been utilized to pattern silk architectures. Printing techniques allow for the production of relatively sophisticated RSF patterns while offering the ability to modify and completely switch designs with relative ease through softwarebased changes. Tao and colleagues recently demonstrated the inkjet printing of RSF and RSF doped with enzymes, antibiotics, and polydiacetylene vesicles conjugated with antibodies.29 Inkjet printing could produce 20 µm spots and a variety of functional structures, including colorimetric sensors for the detection of bacterial contamination.29 Direct ink writing (DIW) of concentrated RSF solutions has been utilized to print silk-based optical waveguides that exhibit relatively low losses.3 In this later report, the architectural agility of printing was used to print both straight and curved waveguides.3 Beyond 2D structures, printing also opens possibilities in the additive manufacturing of sophisticated 3D structures.30 Extrusion based additive manufacturing techniques such as DIW rely on sudden changes in the mechanical properties of the printed RSF-based inks. These changes can be achieved by rapidly crystallizing the extruded silk via the printing RSF directly


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into methanol or using self-curing polyol/RSF bionks.31-34 Direct bioprinting of silk hydrogels including those based on recombinant spider silks and RSF/gelatin blends has also been explored to produce scaffolds for tissue engineering.35-37 While such extrusion-based bioprinting methods allow for access to a broad range of geometries (including patient-specific tissue scaffolds), their spatial resolution has been limited (10s-100s of microns) to date. Recently, the 3D micropinting of RSF has been demonstrated utilizing multi-photon lithography, a technique capable of true free-form additive manufacturing with submicron resolution.38 Multi-photon lithography (MPL) is a versatile technique that allows for the direct write, high-resolution fabrication of complex, 3D structures via the spatially controlled polymerization of materials.39-43

In this method, a pulsed laser is focused into a solution containing a

photoinitiator dye and the monomer (i.e., a negative resist). Within the focus of the laser, the illumination is high enough to excite the dye through multiphoton absorption. The excited dye induces the local polymerization of the monomer, producing a small volume of cross-linked material.39-43 Solid, 3D structures are produced by translating the laser focus through the monomer pool in a programmed pathway.39-43 Utilizing the MPL process, self-supporting and complex structures based on 3D image files (including tissue engineering scaffolds and miniature replicas of iconic architecture and art) may be created with spatial resolution below 50 nm. 39-43 A variety of monomer resist chemistries can be utilized in the MPL process, including synthetic acrylates and epoxies which can produce solid polymer structures for optical and metamaterial applications.39,

40

Biocompatible 3D hydrogel structures, including those featuring spatially-

defined chemistries, may be produced by MPL by using monomers and macromers based on poly(ethylene glycol)-based acrylates, organosilicon hybrids, natural polysaccharides, and proteins.39-46 The ability to spatially organize biomolecules through MPL is significant, as it



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offers functionalities beyond that available through the synthetic resist palette. Indeed, the inherent bioactivity of proteins is often retained in MPL printed structures and the benign, aqueous-based solutions utilized are compatible with other labile biologicals, including cells.41 MPL printed protein (e.g., bovine serum albumin (BSA), gelatin, and collagen) structures have been utilized as biocompatible substrates to localize, entrap, and guide the growth of a variety of cell types, including neurons, fibroblasts, and bacteria.47-54 Catalytically-active microstructures have been directly produced through the polymerization of enzymes (i.e., alkaline phosphatase (AP)) within protein microstructures or through secondary AP functionalization of micro 3D printed structures (via biotin-streptavidin coupling).55,

56

Additionally, microarchitectures

produced from cross-linked proteins are rich in functional groups (e.g., carboxylic acids, hydroxyls, and amines), which may be utilized as handles for further functionalization, electrostatically-driven assembly, or to direct biomimetic mineralization processes.38, 57, 58 Silk fibroin is chemically compatible with the MPL process as fibroin is enriched in tyrosine residues (heavy chain fibroin contains 275 tyrosine residues) that undergo localized photo-initiated crosslinking (producing dityrosine linkages) to yield silk hydrogels.38 Silk fibroin is well suited to the fabrication of complex microarchitectures and the unique attributes of this protein (e.g., biocompatibility, robust mechanical strength, and optical transparency) couple well with the traditional applications of MPL-written structures (i.e., optics and structures for cell culture). Indeed, Sun and co-workers have recently demonstrated that MPL can be utilized to fabricate 3D silk microarchitectures, including structures functionalized with noble metals.38 Furthermore, RSF brings additional functionalities (e.g., the stabilization of biofunctional elements) beyond that available from BSA, the protein most commonly explored in MPL to date.


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These combinations of attributes hint at the possibilities that may arise through the MPL-based production of silk-based microstructures, especially those with persistent bioactivity.9-11, 59 In this article, we report on the process parameter windows for the MPL printing of 3D RSF microstructures, including the secondary processing of written structures and the creation of functionalized silk-based materials that retain the biorecognition activity of co-written antibodies.

Specifically, we describe the dependence of silk structure build height on

photoinitiator selection and concentration, laser writing parameters, RSF concentration, and hydration. The relationships established between processing parameters and silk microstructures for simple structures served as a basis for establishing methods to create true 3D RSF microarchitectures featuring spanning elements.

We anticipate that establishing these

processing-structure relationships will facilitate the further development of devices that feature MPL printed and silk-stabilized biological sensing elements.

Materials and Methods Materials- Unless otherwise stated, the chemical reagents utilized in this study were purchased from Sigma Aldrich (St. Louis, MO) and used without further purification. Fibroin solutions were prepared from Bombyx mori cocoons (Tajima Shoji Co. (Yokohama, Japan)) following Na2CO3-based degumming, LiBr-based dissolution, and purification processes previously detailed in the literature.5 Following dialysis against 18.2 MΩ water, insoluble materials were removed from the fibroin solution by repeated centrifugation at 5K rpm and filtration through a 5 µm syringe filter. The silk fibroin solution was concentrated by reverse dialysis at 4°C, against an aqueous, 20 wt% 8 kDa PEG solution utilizing 3.5 kDa MWCO tubing (Fisher Scientific Inc.) prepared according to the manufacturer’s instructions. Fibroin “resist” solutions containing a



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final photo-sensitizer concentration of 10 mM were created by mixing concentrated (50 mM) aqueous solutions of rose bengal or Ru(bpy)32+ with RSF.

Multi-Photon Lithography- Protein structures were fabricated using a Nanoscribe Photonic Professional 3D printing system (Nanoscribe, Gmbh; Stutensee, Germany), equipped with a 100 mW femtosecond solid state laser operating at wavelength of 780 nm. A high-power objective (Zeiss, Plan-Aprochromat, 100x, oil-immersion, numerical aperture 1.4) was utilized to focus the laser. Unless otherwise noted, typical writing parameters set through the Nanoscribe software were as follows: laser output power 50%, acceleration/deceleration times 0.01 s (to mitigate over development at pattern edges), and a scan speed 50 µm/s. Writing files (.gwl, Nanoscribe proprietary files) were produced using Nanosribe-provided software (DeScribe) from STL files produced in Solid Works 3D CAD program, using 0.2 µm slicing and hatching and files were written using alternating X and Y laser scanning directions (cross-hatched).

Protein

microstructures were built on microscope cover glass substrates that had been cleaned by ultrasonication in isopropanol. To mitigate evaporation, silk fibroin/ Ru(bpy)32+ solutions (~50 µl) were pipetted into well structures created by affixing flat a PDMS ring to the glass substrate (with clear nail polish), which were then capped by a second smaller piece of coverslip glass. Following the writing procedure, the fibroin structures were developed by removing excess silk solution and rinsing with 18.2 MΩ water by gentle pipetting. The coverslip substrate was removed from the Nanoscribe and soaked in 50 mL of 18.2 MΩ water (or buffer) for 30 minutes.

Characterization- For atomic force microscopy (AFM) characterization of dry samples, 3D laser scanning confocal microscopy and FTIR analysis, the silk fibroin microstructures were


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removed from the rinse water and dried by gently blowing off excess water with a dry N2 gas stream. The topography of the dried materials was characterized by atomic force microscopy (AFM) utilizing a Veeco Dimension 3100 in tapping mode.

Characterization of wet and

rehydrated hydrogel structures was conducted with a Dimension Icon utilizing a NanoScope V Controller (Bruker Nano) imaged in aqueous solution using ScanAsyst Fluid+ AFM probes by Bruker.

AFM characterization of the corresponding RSF and RSF/IgG dried samples was

accomplished by rinsing the structures with water (3x), drying with a flowing stream of N2 gas, followed by incubation (30 min) on the benchtop before imaging. These samples (i.e., dried RSF and RSF/IgG structures) were imaged in air using ScanAsyst Air probes by Bruker. FTIR analysis was achieved using a FTIR-ATR attachment (Bruker Alpha –P) with RSF structures patterned over large areas (1 mm2). Fourier self-deconvolution (FSD) of the infrared spectra in the amide I region was performed according to previously published procedures.60 3D laser scanning confocal microscopy was conducted using a VK-X250/VK-X260K system (Keyence).

Secondary Cross-Linking- The production of self-supporting / spanning structures for SEM characterization required secondary cross-linking and preparation steps. Following direct laser write and development, the substrate-supported silk structures were transferred to 25 mm petri dish containing an aqueous 5 mL of 1 mM Ru(bpy)32+, 2 mM ammonium persulfate solution, and exposed to a 150 W halogen light source (Ace I light source, SCHOTT North-America) for 10 min. After secondary cross-linking, the silk structures were rinsed in 18.2 MΩ water. Confocal scanning microscopy was conducted utilizing a LSM 700 confocal laser scanning microscope (Zeiss, Jena, Germany) capable of excitation at 555, 405 and 488 nm. For SEM characterization, a standard biological fixation, staining, and dehydration (using increasing concentrations of



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ethanol) procedure was applied to the silk microstructures.61 Printed fibroin structures were observed using a FEI Quanta scanning electron microscope (SEM).

RSF/Antibody Composite Microstructures- Fibroin/IgG structures were prepared from stock solutions of rabbit serum IgG stocks prepared in TBS at 20 mg/mL, mixed with silk (135 mg/mL), and 50 mM Ru(bpy)32+ to yield a solution of 14.5 mg/mL IgG, 14.5 mg/mL fibroin, and 10 mM Ru(bpy)32+. Labeling of the polymerized IgG was conducted with goat anti-rabbit IgG (H+L) antibody- Oregon Green® 488 conjugate (ThermoFisher Scientific). The fibroin/IgG, IgG (only), or fibroin only (negative control) samples were blocked with 10mg/mL BSA in TBSTween-20, then incubated with goat anti-rabbit Oregon green, diluted 1:2000 in TBS-Tween-20 containing 10 mg/mL BSA for 2 h. The samples were subsequently washed with TBS-Tween20.

Results and Discussion Photoinitiator Screening for the MPL of RSF In our study, silk microstructures were fabricated with a commercial MPL system (Nanoscribe Photonic Professional) that has a manufacturer stated resolution of 600 nm in the Z direction and 200 nm in the X,Y directions, when using high fidelity resist formulations.

For

our work, MPL was conducted with protein and photosensitizer solutions contained within closed reservoirs formed by sandwiching a polydimethylsiloxane (PDMS) ring between 2 glass cover slip substrates (to mitigate evaporation during extended writing campaigns). Following 3D patterning, the polymerized structures were developed by rinsing away unpolymerized RSF with water, followed by drying with a stream of dry N2. Though multiphoton absorption has


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been utilized in the micro laser machining of silk gels, direct write MPL experiments conducted using neat RSF solutions (i.e., without a photoinitiator) did not produce silk structures (data not shown).25 Prior research conducted in the photo-polymerization of proteins have utilized a variety of initiators, including methylene blue, rose bengal, flavin adenine dinucleotide, FMN, and tris(bipyridine)ruthenium(II)2+ (Ru(bpy)32+).24,

27, 38, 62, 63

Here, we initially screened

methylene blue, rose bengal, and Ru(bpy)32+ for use in the MPL of RSF. In addition to their pedigree in the photoinitiated cross-linking of proteins, these dyes are attractive as they are readily available and inexpensive. The appealing aspects of rose bengal and Ru(bpy)32+ are slightly offset by their two-photon absorption (2PA) cross-sections (rose bengal; 10 x 10-50 cm4 s/photon (GM) at 800 nm and Ru(bpy)32+; 4.3 GM at 880 nm).64,

65

Though the 2PA

performance of these dyes are comparable to many organic fluorophores, they are relatively low compared to 2PA cross-sections of photoinitiators specifically designed for MPL with synthetic resin systems (i.e., 1,250 GM at 775 nm).64-66 However, this deficit in 2PA cross-section may be mitigated by increasing the concentration of rose bengal or Ru(bpy)32+ above that which would be used for high 2PA cross-section dyes in MPL resist formulations. The 2PA cross-section of methylene blue is not available in the literature.41

Of the photoinitiators screened here, rose

bengal and Ru(bpy)32+ yielded robust RSF microstructures. Though methylene blue has been successfully utilized to produce 3D protein microstructures in several publications, including silk fibroin, it did not seem to couple well with our system, producing only gossamers of the intended patterns and was excluded from further study (Figure S1).38, 41 To assess the efficacy of RSF as a biomacromolecular monomer for MPL, test microstructures of RSF were prepared and benchmarked to similar structures prepared with BSA (the most commonly pursued protein-based resist for MPL to date) and a proprietary acrylate-



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based resist (IPL) supplied by Nanoscribe. Rose bengal was used as the photosensitizer for the MPL production of the protein structures in these experiments. Following development, the RSF, BSA, and IPL test structures (step pyramids and simple blocks) were characterized by AFM (Figures 1 and 2). As seen in Figures 1 and 2, the morphologies of the 3D printed microarchitectures are clearly distinguishable as step pyramids and block structures. The step pyramid design featured a 10 x 10 µm base and 5 steps 0.5 µm in height to produce a structure with a maximum height of 2.5 µm. The overall heights of the IPL, BSA, and RSF pyramidal structures after writing, development, and drying were 3.41, 3.08, and 1.24 µm, respectively (see Figure 1). The simple block test structure was designed to feature 10 x 10 µm base and a height of 1 µm. The overall heights of the IPL, BSA, and RSF cubic structures after writing, development, and drying were 2.02, 1.61, and 0.54 µm, respectively (see Figure 2). Clearly, in the dry state in which they were imaged, none of the MPL written structures exhibited perfect accuracy to the original CAD file, indicating that further optimization of writing parameters is required. That the IPL and BSA structures exceeded the intended height of the pyramid and cubic designs indicates that the size of voxel in which there is sufficient energy for polymerization was larger than anticipated (i.e., excessive laser power was used). Though the morphology of the RSF structures reflects an overall resemblance to the test structure designs, the Z heights of the structures are ~50% lower than expected. This reduction in build height is partially due to the shrinkage of the RSF hydrogel structure during drying (see further discussion below). That microstructures produced from RSF at identical concentrations (150 mg/mL) and writing conditions as BSA were not built to similar heights or collapsed to a greater extent than those from BSA would seem to indicate that RSF is not being cross-linked as efficiently as BSA. This is surprising as silk fibroin contains a greater percentage of cross-linkable tyrosine residues


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(5.2%, 275 Y residues, 389.2 kDa MW (fibroin heavy chain)) than BSA (3.5%, 21 Y residues, 66.5 kDa MW). Though this distinction in tyrosine contents between RSF and BSA is somewhat complicated by the fragmentation of the fibroin protein during the silk degumming process. Such processed fibroin (i.e., RSF) will possess a range of molecular weights and tyrosine contents.67 The differences in build height between the synthetic and biomacromolecular resists explored here can be accommodated by adjusting writing parameters to produce (dry) structures of desired height and feature sizes.

Establishing Process Parameter Windows for the MPL of RSF/ Ru(bpy)32+ Though rose bengal was effective as an photoinitiator for the production of step pyramid and block test structures, the dye poses challenges in the generation of structures requiring long write times (>8 h). Specifically, rose bengal induced the bulk gelation of RSF solutions over time (in the absence of light), making the development of MPL-written structures impossible. Prior research also indicates that relatively low concentrations of methylene blue must be used with RSF in order to avoid rapid gelation of the solution.38 Conversely to these systems, RSF solutions containing relatively high concentrations (10 mM) of Ru(bpy)32+ were observed to be stable (no bulk gelation) for >16 hours in the absence of light. In single photon activated protein cross-linking reactions, ammonium persulfate (APS) is typically used to enhance the activity of Ru(bpy)32+.27, 68 While APS can be used in MPL (Figure S2), the intensity of illumination within the focus of the femtosecond laser is sufficiently high to allow for sufficient RSF cross-linking without APS and eliminates complications associated with handling APS-containing solutions in the dark.68



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Given the greater operational flexibility of the RSF/Ru(bpy)32+ formulation, we decided to explore MPL printing parameters of this system in order to identify the critical variables allowing for the rapid and accurate production of silk microarchitectures. To investigate the robustness and spatiotemporal control of RSF/ Ru(bpy)32+ in MPL, arrays of cubic structures (20 x 20 x 2 µm blocks) were written at varying scan speeds and laser powers (Figure 3 and Figure S3). As seen in Figure 3, the build height of the cubic structures is dependent on photon dosage, which is a function of both write speed (laser dwell time) and laser power. Within a given column of block-like test structures (corresponding to write speeds of ≤100 µm/s), sample height is seen to increase with increasing laser power. Similarly, sample height is observed to increase with increasing photon dosage realized by holding laser power constant and decreasing write speed (Figure 3 and Figure S3). As shown in Figure 3, write speeds up to 100 µm/s can be utilized to produce block structures with good spatiotemporal control at laser powers up to 80% of maximum. Write speeds of up to 300 µm/s can be utilized at intermediate laser powers (5060%) to produce structures with expected x-y cubic shapes, though the build heights are substantially lower than the build file (Figure S3). The most rapid write speeds used were observed to produce cubic structures of poor design fidelity at high laser powers (>70%). Conditions featuring rapid write speeds (>100 µm/s) and low laser powers (≤ 30%) combined to yield low photon densities, which in turn produced RSF structures that possessed low Z heights. Under the most extreme of these conditions, 20% laser power and write speeds (≥200 µm/s), the Z height of the RSF cuboids is so low that the structures are difficult to resolve. Considering the results presented in Figure 3, we selected conservative printing parameters (i.e., 50% laser power, 50 µm/s writing speed) as our standard writing condition in order to ensure high spatial control and limit instrument wear.


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In addition to writing speed and laser power, we also explored the influence of Ru(bpy)32+ and RSF concentrations on MPL printed protein microarchitecture (Figures 4 and 5). The printed test structures were arrays of blocks (20 x 20 x 2 µm).

Initial 3D printing

experiments utilized 10 mM Ru(bpy)32+ as a photoinitiator to produce RSF structures with good spatiotemporal control (Figures 3 and S1), making the use of higher concentrations of Ru(bpy)32+ unnecessary. As seen in Figure 4, reducing the concentration of Ru(bpy)32+ to 5 mM had a minimal effect on the resulting build height of the printed cubic test structures. The heights of the silk blocks written from RSF solutions containing 10 mM and 5 mM Ru(bpy)32+ were 617 ± 29 and 560 ± 60 nm, respectively. Further decreasing the concentration of the photoinitiator to 1 mM had a dramatic effect on the printed RSF structures (Figure 4). As seen in Figure 4, the resultant Z-height of the cubic silk structures written at 50% laser power was only 93 ± 1 nm compared to the 600 nm build height of similar samples produced with 10 mM Ru(bpy)32+ . Furthermore, silk structures were difficult to visualize or completely absent (Figure 4) when MPL writing was attempted utilizing RSF/1 mM Ru(bpy)32+ and low laser powers (≤50%). To minimize effects due to small variations in photoinitiator concentration that occurs when making sequential batches of RSF/Ru(bpy)32+ solutions, we standardized our silk-based resist formulations utilizing 10 mM Ru(bpy)32+. Prior work reported for the 3D printing of protein-based structures with MPL have a utilized a range of protein concentrations in biomacromolecule resist formulations, from highly concentrated solutions of BSA (up to 400 mg/mL) to RSF at low concentrations (25 mg/mL).50 38 To discern the effects of RSF concentration on the structure of the MPL written silk microstructures we screened fibroin resist formulations of varying concentrations. In our work, aqueous RSF stock solutions were prepared at concentrations of 200 mg/mL and subsequently



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mixed with photoinitiator solution, setting an upper limit of 150 mg/mL for our working MPL resist formulation. As seen in Figure 5, decreasing the concentration of RSF from 150 mg/mL to 100 mg/mL had no discernable effect on the build height of the test structures (design blocks were 20 x 20 x 2 µm).

The cross-sectional heights (as measured by confocal scanning

microscopy) of the cubic structures produced from 150 and 100 mg/mL MPL solutions were 617 ± 29 and 547 ± 46 nm, respectively.

The overall height of silk structures (556 ± 45 nm)

produced with 50 mg/mL silk solutions did not significantly differ from analogous structures printed with more concentrated RSF solutions. However, slight defects (depressions) were prevalent on the top surfaces of the block structures (Figure 5). Further reductions in the concentration of RSF in the MPL resist formulation lead to dramatic changes in the build height of the silk blocks (Figure 5). Indeed, block structures 3D printed with 25 and 10 mg/mL RSF solutions were only 340 ± 21 and 91 ± 2 nm tall, respectively. This reduction in structure height with decreasing fibroin solution concentration is consistent with the results of prior studies that have demonstrated positive correlations between fibroin concentration and film thicknesses produced by casting techniques.5

Conformation of MPL Printed Silk The secondary structure content of the MPL-written RSF microstructures (fabricated with Ru(bpy)32+) was characterized by Fourier transform infrared-attenuated total reflection (FTIRATR). Deconvolution of the amide I region (1575−1725 cm−1) of FTIR spectrums collected for MPL-patterned RSF indicated that these materials contained 33% β-sheet content. This level of β-sheet lies between that of amorphous silk and RSF annealed under humid conditions (at room temperature).14, 69 Electron diffraction patterns collected in conjunction with TEM analysis of


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MPL-patterned RSF indicated the presence of the silk II structure (β-sheet structure) in the silk structures (Figure S4). These results align well with the β-sheet dominated structure reported for fibroin samples 3D printed with MPL utilizing methylene blue as a photoinitiator.38

Direct-Write Printing of RSF/IgG Structures Having developed the methodologies required to successfully produce RSF structures by the MPL direct-write process, we proceeded to explore the introduction of additional biofunctionality into these materials.

Considering the prior success of RSF in stabilizing

therapeutic agents from harsh environments, we chose to evaluate the ability of the MPL process to produce antibody-containing silk structures.70 Antibodies are versatile biomolecules that can be utilized as biorecognition elements for sensor development as well as point-of-care medical diagnostics, and have emerged as important elements for the treatment of pathogens.70-73 Here, solutions containing equal concentrations of RSF and rabbit IgG, as well as RSF (only) and rabbit IgG (only) controls were used in the direct write process.

Following printing and

development, the proteinaceous structures were blocked with BSA (to inhibit non-specific interactions) and incubated with immunoglobulins specific for rabbit IgG (i.e., Oregon Green® 488 dye-conjugated goat anti-rabbit polyclonal IgG). The Oregon Green-conjugated anti-rabbit IgG recognized the structures written with RSF/rabbit IgG and rabbit IgG (only), but did not stain the negative control (RSF only) sample (Figure 6). Though the structures written with only rabbit IgG were recognized by the fluorescent anti-rabbit probe, the morphology of these structures is ragged and departs significantly from the expected box structure (Figures 6C, 6D). In contrast, the RSF only structures (Figures 6E, 6F) are seen to possess the expected box morphology but are not recognized by the anti-rabbit probe. Only with the RSF/rabbit IgG



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printed structure (Figures 6A, 6B) do we see both the expected structural morphology and recognition functionality. Thus, by co-writing RSF with the IgG, the structural integrity of the printed, functionalized hydrogel is significantly increased without increasing non-specific binding to the labeled antibody probe. Inspection of Figure 6B reveals that the anti-rabbit IgG is concentrated on the outside of the structure but is also present, at lower concentrations, within the interior of the RSF-based hydrogel. Prior work with MPL-patterned BSA structures has established that the diffusion of biomacromolecules into hydrogels is substantially slower than the movement of these species in free solution.74 In considering our results (Figure 6B) in the context of this prior work, it would seem reasonable that the rabbit IgG has been incorporated throughout the silk-based structure and that the high concentration of the probe on the outside of the block is a consequence of diffusion kinetics and accessibility to the probe. The efficacy of RSF in stabilizing co-written IgGs and other functional biomolecules is currently under study.

Swelling of RSF and RSF/IgG Microstructures The protein microstructures produced by MPL in our work are written as hydrogels, which is a material class that possesses high water contents, an interconnected network structure, and exhibits characteristic swelling behaviors.75 The small size of the silk-based hydrogels precluded the investigation of hydrogel swelling ratios by mass, as is typically conducted for bulk samples. Instead, we explored the dimensional changes in RSF structures that occur when moving the structures from wet to dry to rehydrated states (Figure 7). For these experiments, MPL was conducted using a write file specifying cubic sample geometry (20 x 20 x 8 µm). RSF-based hydrogels were written utilizing RSF and RSF/IgG concentrations reflective of the resist solutions explored for the production of antibody-functionalized silk structures. Following


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3D printing the fibroin-based microarchitectures were developed and stored in water (RSF sample) or buffer (RSF/IgG sample) solutions that matched the pH and ionic strength of the resist solution. Though it would be informative to image the MPL-written silk microstructures in an “as printed” state while still submerged in aqueous silk solution, this approach did not prove to be feasible. This is due to the behavior of fibroin, which gels in response to the movement of the AFM probe during imaging, a phenomenon previously exploited to pattern RSF on planar substrates.76 Characteristic images of RSF and RSF/IgG hydrogels in the developed (wet), dried, and rehydrated states are presented in Figure 7. The wake-like structures surrounding the RSF and RSF/IgG samples when imaged in water (wet and rehydrated states) (Figure 7) are imaging artifacts caused by the side of the tip hitting the relatively tall hydrogel structures (see Figure S4). The average heights of the RSF hydrogels, as measured by AFM, were 11.53 ± 0.44 µm (wet), 2.41 ± 0.27 µm (dry), and 10.64 ± 1.21 µm (rehydrated). The average heights of the RSF/IgG hydrogels were 9.17 ± 0.73 µm (wet), 2.81 ± 0.09 µm (dry), and 9.92 ± 0.59 µm (rehydrated). The dimensional changes associated with the drying and rehydration of these RSFbased hydrogels is striking in light of prior research investigating photopolymerized silk hydrogels.26 In research reported by Whittaker and colleagues, RSF hydrogels produced with Ru(bpy)32+ exhibited 50% reduction in thickness on drying and recovered to only 58% of the original sample thickness.26 This suggests that the network structure within the MPL printed RSF materials differs from the bulk hydrogels produced via the Ru(bpy)32+/APS cross-linking route. Such differences are not unexpected however, as though these materials are composed of B. mori-derived fibroin, there are a myriad number of experimental and processing variations



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between the bulk silk hydrogels created by Whittaker et al. and the 3D printed materials studied here.26 Beyond differences in overall height, there are a number of morphological changes that occur in the RSF microarchitectures during dehydration and rehydration that merit discussion here. The fibroin-based structures studied in this work are printed on glass coverslip substrates that constrain the motion of the hydrogel near the structure/substrate interface. Due to this local substrate pinning effect, the tops of the RSF-hydrogels contract to a greater extent than do the bottoms of the structures when the samples are dried (Figure 7B & E). For example, the dried RSF structure depicted in Figure 7B is 13.3 and 23.1µm wide at the top and bottom, respectively, giving the sample a truncated pyramidal appearance. The greater prominence of differential shrinkage and substrate pinning in the MPL printed silk structures seen in Figure 7 over those shown in Figure 2 are likely due to the taller height of the Figure 7 samples. The morphology of the top surface of the RSF sample (Figue 7A) has a subdued network-like appearance that is reflective of the porous nature of these hydrogels. This surface topography flattens out when the sample is dried (Figure 7B) and only partially recovers when rehydrated (Figure 7C), though this later loss of surface features maybe due to degradation of imaging fidelity caused by tip contamination during wet AFM rather than pore closure. The top surface of the RSF/IgG hydrogel samples (Figure 7D) possesses a more sponge-like appearance than the purely RSF materials and features micron-sized porosity. This network-like morphology is largely flattened out upon the drying of the RSF/IgG samples but becomes prevalent again upon rehydration of the sample (Figure 7F). Further research is required to determine how the different chemistries and network structures of the RSF and RSF/IgG hydrogels effect the ingress and diffusion of antigens into printed silk microstructures.74


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Printing of Optical and Self-Supporting RSF Structures These initial successes in creating simple RSF and RSF/IgG architectures inspired us to explore the creation of proof-of-concept microarchitectures that derive function from their structure. Considering the prior success of silk as a material for waveguides, photonic crystals, and corneal prosthesis, we gravitated towards the MPL fabrication of silk-based optical elements. 1-3, 24

Indeed, a number of prior studies demonstrated the MPL-based fabrication of microlenses

composed of synthetic resins or BSA, which could be integrated into microfluidic devices or exploited as pH-sensitive dynamic lenses, respectively.77,

78

AFM and optical microscope

images of the RSF-, BSA-, and IPL-based microlenses are presented in Figure 8. The surface topology of the RSF-based lenses (Figure 8C) is observably smoother than those composed of BSA or IPL and may be advantageous in limiting light loss due to wide-angle scatter. All MPLfabricated microlenses focused light, though the focal length of the lenses were different as a consequence of the differential build heights of the IPL-, BSA-, and RSF-based structures (Figure 8). While effective as test structures and simple optics, the blocks, step pyramids, and microlenses presented in Figures 1-8 are best characterized as 2.5D structures that could also be fabricated by soft lithography approaches.3, 20 To demonstrate the true 3D free-form fabrication potential of MPL, we investigated the writing of self-supporting arch structures composed of RSF (Figure 9). Characterization of microarches under conditions reflective of those present during MPL writing was accomplished through confocal microscopy imaging of RSF structures developed and wetted with aqueous silk solutions. As seen in Figure 9A, arches produced from RSF/rose bengal solutions were self-supporting within RSF aqueous solutions, but lacked



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sufficient strength to maintain integrity subsequent to development, drying, and inspection in high-vacuum SEM (Figure 9B). Arches written from RSF/Ru(bpy)32+ solutions maintained structural stability under SEM conditions to a greater extent than those produced with rose bengal, yet still exhibited discernable subsidence of their free-spanning segments (Figure S5). The more robust nature of RSF arches created with Ru(bpy)32+ suggested that the ruthenium-based photoinitiator might be more effective than rose bengal in generating dityrosine cross-links in RSF when used under similar conditions. Based on this observation, we strove to further increase the cross-link density in MPL-written RSF through secondary cross-linking processes to yield self-supporting spans able to survive the rigors of drying and vacuum treatments (i.e., associated with SEM observation). Secondary crosslinking steps, such as heat treatments or UV flood light illumination, are commonly employed post-print processes in additive manufacturing (e.g., stereo lithography) that increase the covalent network density in printed parts.79 Here, we pursued secondary crosslinking through post-print processing based on single-photon initiated polymerization.27,

68

Unreacted tyrosine residues were targeted for

crosslinking by subjecting the developed RSF arches to one-photon (white light) initiated reaction conditions. Specifically, the MPL written and developed RSF arches were submerged in an aqueous solution containing Ru(bpy)32+ and APS (i.e., no extra proteins were present) and exposed to an intense white light source.27, 68 For the sake of clarity, we will refer to MPL written samples that have undergone such secondary Ru(bpy)32+/APS-based cross-linking as MPL-3P (post-print processed). Confocal microscopy of MPL-3P RSF arches in aqueous solution were promising as the structure was seen possess a morphology consistent of the spanning and selfsupporting arch design from the write file (Figure 9C). Prior to SEM characterization, the MPL3P RSF arches were subjected to a standard sample fixation and staining procedure similar to


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that previously utilized to visualize BSA-based microprinted structures.50, 61 The combination of photosensitizer selection, MPL writing conditions, and post-print processing yielded selfsupporting RSF arches that exhibited excellent resemblance to the overall part design morphology and displayed little subsidence (Figure 9D and Figure S7). Much like the RSF hydrogel parts depicted in Figure 7C, the non-surface constrained top of the microarch undergoes a greater degree of drying-related shrinkage than does the base of the structure, yielding a structure with slightly sloped sides. The surface morphology of the RSF microarch is not as smooth the surfaces of the simple fibroin structures (i.e., microlenses) characterized in this study, but are consistent with the SEM-observable topology of MPL-printed silk microbowls previously reported by Sun and coworkers.38

Summary In this manuscript, we have demonstrated the MPL-based printing of 3D silk microstructures, including step pyramids and functional microlenses. By combining the freeform fabrication capability of MPL with post-print crosslinking (MPL-3P), we produced spanning and self-supporting RSF microstructures (arches), which are inaccessible by soft lithography techniques (e.g., nano embossing and PDMS molding) previously utilized to create silk structures at these scales. The straightforward production of protein microstructures with good pattern fidelity and biofunctionality was enabled through the use of aqueous solutions containing RSF and immunoglobulin. The coupling of the attractive attributes of MPL printing and the properties of RSF represents potential advances for both the silk and 3D microprinting fields and the results previously reported and presented here suggest a number of opportunities for future innovation.38 For example, MPL could be utilized to fabricate RSF microarchitectures



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containing stabilized biorecognition elements and microlenses as components of microfluidicbased, optical read-out biosensors.77,

80

Furthermore, the ability to precisely position and

fabricate protein microstructures of different composition within the same architecture by MPL could be leveraged to produce microreactors containing enzyme cascades for use in fuel cells or diagnostic devices.44,

46, 81

Other applications for the RSF/MPL system may include the

fabrication of strongly anti-microbial surfaces, where the chemical functionality of silk (e.g., halogenation

of

nitrogen

moieties)

can

be

combined

with

bactericidal

material

microarchitectures.82, 83 Finally, MPL printing requires only small volumes (typically < 50 µl here) of proteins for structure production, making it a powerful research tool for fabricating polymerized samples of rare or expensive recombinant proteins within protective RSF “cocoon” structures for study with microanalytical techniques.

Supporting Information: Additional AFM and optical microscope characterization of RSF pyramids and blocks, TEM and electron diffraction of MPL RSF structures, plots of cuboid height versus MPL write speed and laser power, and SEM of RSF/Ru(bpy)32+ microarches. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements: This research was supported by the Air Force Office of Scientific Research under program officer Dr. Hugh DeLong. The assistance of Pam Lloyd with microtome sample preparation and Caitlin L. Bojanowski with confocal microscopy imaging is gratefully acknowledged.

We also gratefully acknowledge Maneesh Gupta for helpful discussions.

Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the United States Air Force.


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References [1] Omenetto, F. G., and Kaplan, D. L. New Opportunities for an Ancient Material. Science 2010, 329, 528-531. [2] Omenetto, F. G., and KapLan, D. L. A new route for silk. Nature Photonics 2008, 2, 641643. [3] Tao, H., Kaplan, D. L., and Omenetto, F. G. Silk Materials - A Road to Sustainable High Technology. Advanced Materials 2012, 24, 2824-2837. [4] Pereira, R. F. P., Silva, M. M., and de Zea Bermudez, V. Bombyx mori Silk Fibers: An Outstanding Family of Materials. Macromolecular Materials and Engineering 2015, 300, 1171-1198. [5] Rockwood, D. N., Preda, R. C., Yucel, T., Wang, X., Lovett, M. L., and Kaplan, D. L. Materials fabrication from Bombyx mori silk fibroin. Nat. Protocols 2011, 6, 1612-1631. [6] Kasoju, N., and Bora, U. Silk Fibroin in Tissue Engineering. Advanced Healthcare Materials 2012, 1, 393-412. [7] Shao, Z., and Vollrath, F. Surprising strength of silkworm silk. Nature 2002, 418, 741. [8] Wray, L. S., Hu, X., Gallego, J., Georgakoudi, I., Omenetto, F. G., Schmidt, D., and Kaplan, D. L. Effect of Processing on Silk-Based Biomaterials: Reproducibility and Biocompatibility. Journal of biomedical materials research. Part B, Applied biomaterials 2011, 99, 89-101. [9] Li, A. B., Kluge, J. A., Guziewicz, N. A., Omenetto, F. G., and Kaplan, D. L. Silk-based stabilization of biomacromolecules. Journal of Controlled Release 2015, 219, 416-430. [10] Pritchard, E. M., Dennis, P. B., Omenetto, F., Naik, R. R., and Kaplan, D. L. Review physical and chemical aspects of stabilization of compounds in silk. Biopolymers 2012, 97, 479-498. [11] Wenk, E., Merkle, H. P., and Meinel, L. Silk fibroin as a vehicle for drug delivery applications. Journal of Controlled Release 2011, 150, 128-141. [12] Partlow, B. P., Applegate, M. B., Omenetto, F. G., and Kaplan, D. L. Dityrosine CrossLinking in Designing Biomaterials. Acs Biomaterials Science & Engineering 2016, 2, 2108-2121. [13] Hwang, S.-W., Park, G., Cheng, H., Song, J.-K., Kang, S.-K., Yin, L., Kim, J.-H., Omenetto, F. G., Huang, Y., Lee, K.-M., and Rogers, J. A. 25th Anniversary Article: Materials for High-Performance Biodegradable Semiconductor Devices. Advanced Materials 2014, 26, 1992-2000.



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[14] Dickerson, M. B., Fillery, S. P., Koerner, H., Singh, K. M., Martinick, K., Drummy, L. F., Durstock, M. F., Vaia, R. A., Omenetto, F. G., Kaplan, D. L., and Naik, R. R. Dielectric Breakdown Strength of Regenerated Silk Fibroin Films as a Function of Protein Conformation. Biomacromolecules 2013, 14, 3509-3514. [15] Yin, L., Bozler, C., Harburg, D. V., Omenetto, F., and Rogers, J. A. Materials and fabrication sequences for water soluble silicon integrated circuits at the 90 nm node. Applied Physics Letters 2015, 106, 014105. [16] Hwang, S.-W., Kang, S.-K., Huang, X., Brenckle, M. A., Omenetto, F. G., and Rogers, J. A. Materials for Programmed, Functional Transformation in Transient Electronic Systems. Advanced Materials 2015, 27, 47-52. [17] Brenckle, M. A., Cheng, H., Hwang, S., Tao, H., Paquette, M., Kaplan, D. L., Rogers, J. A., Huang, Y., and Omenetto, F. G. Modulated Degradation of Transient Electronic Devices through Multilayer Silk Fibroin Pockets. ACS Applied Materials & Interfaces 2015, 7, 19870-19875. [18] Tao, H., Hwang, S. W., Marelli, B., An, B., Moreau, J. E., Yang, M. M., Brenckle, M. A., Kim, S., Kaplan, D. L., Rogers, J. A., and Omenetto, F. G. Silk-based resorbable electronic devices for remotely controlled therapy and in vivo infection abatement. Proceedings of the National Academy of Sciences of the United States of America 2014, 111, 17385-17389. [19] Tao, H., Brenckle, M. A., Yang, M., Zhang, J., Liu, M., Siebert, S. M., Averitt, R. D., Mannoor, M. S., McAlpine, M. C., Rogers, J. A., Kaplan, D. L., and Omenetto, F. G. Silk-Based Conformal, Adhesive, Edible Food Sensors. Advanced Materials 2012, 24, 1067-1072. [20] Tsioris, K., Raja, W. K., Pritchard, E. M., Panilaitis, B., Kaplan, D. L., and Omenetto, F. G. Fabrication of Silk Microneedles for Controlled-Release Drug Delivery. Advanced Functional Materials 2012, 22, 330-335. [21] Tsioris, K., Tao, H., Liu, M. K., Hopwood, J. A., Kaplan, D. L., Averitt, R. D., and Omenetto, F. G. Rapid Transfer-Based Micropatterning and Dry Etching of Silk Microstructures. Advanced Materials 2011, 23, 2015-2019. [22] Kim, S., Marelli, B., Brenckle, M. A., Mitropoulos, A. N., Gil, E. S., Tsioris, K., Tao, H., Kaplan, D. L., and Omenetto, F. G. All-water-based electron-beam lithography using silk as a resist. Nature Nanotechnology 2014, 9, 306-310. [23] Park, J., Lee, S. G., Marelli, B., Lee, M., Kim, T., Oh, H. K., Jeon, H., Omenetto, F. G., and Kim, S. Eco-friendly photolithography using water-developable pure silk fibroin. RSC Advances 2016, 6, 39330-39334. [24] Applegate, M. B., Partlow, B. P., Coburn, J., Marelli, B., Pirie, C., Pineda, R., Kaplan, D. L., and Omenetto, F. G. Photocrosslinking of Silk Fibroin Using Riboflavin for Ocular Prostheses. Advanced Materials 2016, 28, 2417-2420. 28 ACS Paragon Plus Environment

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[25] Applegate, M. B., Coburn, J., Partlow, B. P., Moreau, J. E., Mondia, J. P., Marelli, B., Kaplan, D. L., and Omenetto, F. G. Laser-based three-dimensional multiscale micropatterning of biocompatible hydrogels for customized tissue engineering scaffolds. Proceedings of the National Academy of Sciences of the United States of America 2015, 112, 12052-12057. [26] Whittaker, J. L., Choudhury, N. R., Dutta, N. K., and Zannettino, A. Facile and rapid ruthenium mediated photo-crosslinking of Bombyx mori silk fibroin. Journal of Materials Chemistry B 2014, 2, 6259-6270. [27] Whittaker, J. L., Dutta, N. K., Elvin, C. M., and Choudhury, N. R. Fabrication of highly elastic resilin/silk fibroin based hydrogel by rapid photo-crosslinking reaction. Journal of Materials Chemistry B 2015, 3, 6576-6579. [28] Schacht, K., and Scheibel, T. Controlled Hydrogel Formation of a Recombinant Spider Silk Protein. Biomacromolecules 2011, 12, 2488-2495. [29] Tao, H., Marelli, B., Yang, M. M., An, B., Onses, M. S., Rogers, J. A., Kaplan, D. L., and Omenetto, F. G. Inkjet Printing of Regenerated Silk Fibroin: From Printable Forms to Printable Functions. Advanced Materials 2015, 27, 4273-4279. [30] Jose, R. R., Rodriguez, M. J., Dixon, T. A., Omenetto, F., and Kaplan, D. L. Evolution of Bioinks and Additive Manufacturing Technologies for 3D Bioprinting. ACS Biomaterials Science & Engineering 2016, 2, 1662-1678. [31] Ghosh, S., Parker, S. T., Wang, X., Kaplan, D. L., and Lewis, J. A. Direct-Write Assembly of Microperiodic Silk Fibroin Scaffolds for Tissue Engineering Applications. Advanced Functional Materials 2008, 18, 1883-1889. [32] Sun, L., Parker, S. T., Syoji, D., Wang, X., Lewis, J. A., and Kaplan, D. L. Direct-Write Assembly of 3D Silk/Hydroxyapatite Scaffolds for Bone Co-Cultures. Advanced Healthcare Materials 2012, 1, 729-735. [33] Jose, R. R., Brown, J. E., Polido, K. E., Omenetto, F. G., and Kaplan, D. L. Polyol-Silk Bioink Formulations as Two-Part Room-Temperature Curable Materials for 3D Printing. ACS Biomaterials Science & Engineering 2015, 1, 780-788. [34] Rodriguez, M. J., Brown, J., Giordano, J., Lin, S. J., Omenetto, F. G., and Kaplan, D. L. Silk based bioinks for soft tissue reconstruction using 3-dimensional (3D) printing with in vitro and in vivo assessments. Biomaterials 2017, 117, 105-115. [35] Das, S., Pati, F., Choi, Y.-J., Rijal, G., Shim, J.-H., Kim, S. W., Ray, A. R., Cho, D.-W., and Ghosh, S. Bioprintable, cell-laden silk fibroin–gelatin hydrogel supporting multilineage differentiation of stem cells for fabrication of three-dimensional tissue constructs. Acta Biomaterialia 2015, 11, 233-246.



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Figure 1: AFM images and height plots of step pyramid microstructures created by multiphoton lithography. The structures were produced from IPL synthetic resist (A and B), aqueous BSA solutions (150 mg/ml) (C and D), and aqueous RSF solutions (150 mg/mL) (E and F). Rose bengal was used as a photosensitizer in the proteinaceous solutions. Cross-sectional profiles of step pyramids are presented in B, D, and F as black traces. The red traces in B, D, and F represent the cross-section of the intended structure as specified by the writing file. All scale bars represent 5 µm. The Z-axis is A) 7 µm, C) 7 µm, and E) 3 µm.


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Figure 2: AFM images of block microstructures created by multiphoton lithography. The structures were produced from IPL synthetic resist (A and B), aqueous BSA solutions (150 mg/ml) (C and D), and aqueous RSF solutions (150 mg/mL) (E and F). Rose bengal was used as a photosensitizer in the proteinaceous solutions.

Cross-sectional profiles of block

microstructures are presented in B, D, and F as black traces. The red traces in B, D, and F represent the cross-section of the intended structure as specified by the writing file. All scale bars represent 5 µm. The Z-axis is A) 5 µm, C) 5 µm, and E) 1.5 µm.



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Figure 3: Dependence of RSF microstructure height and morphology on the writing speed and laser power used during the MPL process. Structures were produced utilizing aqueous solutions of RSF (150 mg/mL) containing 10 mM Ru(bpy)32+ as a photoinitiator. False color laser scanning microscopy images depict sample height with a Z axis of 2 µm. The scale bar represents 20 µm.


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Figure 4: Dependence of RSF microstructure height and morphology as a function of changes in the photoinitiator (Ru(bpy)32+) concentration and laser power used during the MPL process. Structures were produced utilizing aqueous solutions of RSF (150 mg/mL).

False color laser

scanning microscopy images depict sample height with a Z axis of 2 µm. The scale bar represents 20 µm.



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Figure 5: Changes in RSF microstructure height and morphology as a function of silk fibroin concentration used during the MPL process.

Structures were produced utilizing aqueous

solutions of silk containing 10 mM Ru(bpy)32+ as a photoinitiator. False color laser scanning microscopy images depict sample height with a Z axis of 2 µm. The scale bar represents 20 µm.


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Figure 6: Characterization of functional RSF/IgG microstructures and controls. Fluorescent signals arise from Ru(bpy)32+ dye entrapped within the protein structures during printing (left column) and Oregon Green dye associated with an anti-body probe (right column). Images captured using identical exposure times for each fluorescent signal. Simple blocks (4) 10 x 10 x 1 µm in size are depicted in each image. The microstructures were written with RSF/rabbit IgG (A and B), rabbit IgG (only, no RSF) (C and D), and RSF only (no rabbit IgGs)(E and F). All scale bars represent 20 µm.



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Figure 7: AFM images of RSF and RSF/IgG block microstructures created by multiphoton lithography depicting changes in height as a function of hydration state. A single sample composed of RSF and RSF/IgG are presented in the top and bottom rows, respectively. AFM height maps of a RSF block microstructure imaged A) in water after MPL writing and development, B) in the dry state following dehydration, and C) in the water subsequent to rehydration.

AFM height maps of a RSF/IgG block microstructure imaged D) in water after

MPL writing and development, E) in the dry state following dehydration, and F) in the water subsequent to rehydration . Scale bars are 10 µm. The Z heights of the wet AFM images (A, C, D, F) are 20 µm and those of dry AFM images (B and E) are 7 µm.


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Figure 8: AFM and optical characterization of microlenses created by MPL. The microlenses were produced from monomer solutions of A) IPL (Nanoscribe, Gmbh) resist, B) BSA (150 mg/mL), and C) RSF (150 mg/mL).

Insets are optical microscope images of microlenses

(identical to those imaged by AFM) focusing light. The focal length of the lenses was A) 30 µm, B) 40 µm, and C) 50 µm. Scale bars represent 5 µm. The Z-axis in A) 12 µm, B) 8 µm, and C) is 5 µm.



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Figure 9: Characterization of RSF microarches printed by MPL. A) Confocal microscope fluorescence images of a RSF/rose bengal microstructure submerged in an aqueous solution, showing an intact arch architecture. The fluorescent signal originates from rose bengal dye entrapped within the RSF structure during printing. B) SEM image of a RSF/rose bengal arch that has collapsed during SEM preparation (i.e., drying) and imaging. C) Confocal microscope fluorescence images of a RSF/ Ru(bpy)32+ microstructure submerged in an aqueous solution. The fluorescent signal originates from Ru(bpy)32+ dye entrapped within the RSF structure during printing. D) SEM image of a RSF/ Ru(bpy)32+ arch printed by MPL, subjected to additional crosslinking conditions (MPL-3P) to increase the rigidity of the structure, and dried. Scale bars represent A) 10 µm, B) 5 µm, C) 10 µm, and D) 5 µm.


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TOC graphic:


3D Printing of Regenerated Silk Fibroin and Antibody-Containing

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