Silk Protein Lithography as a Route to Fabricate Sericin Microarchitectures

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Silk Protein Lithography as a Route to Fabricate Sericin Microarchitectures

Nicholas E. Kurland , Tuli Dey , Congzhou Wang , Subhas C. Kundu , and Vamsi K. Yadavalli *

Dr. N. E. Kurland, C. Wang, Dr. V. K. Yadavalli Department of Chemical and Life Science Engineering Virginia Commonwealth University 601 W Main Street , Richmond VA , USA 23284 E-mail: [email protected] Dr. Tuli Dey, Dr. S. C. Kundu Department of Biotechnology Indian Institute of Technology Kharagpur , India 721302

DOI: 10.1002/adma.201400777

with sericin), [ 15 ] sericin has faced an uphill climb in its overall acceptance in the biomaterials fi eld.

In contrast to insoluble fi broin, the water soluble nature of sericin presents opportunities as a structurally- and chemically-distinct renewable protein for the “green synthesis” of engi-neered micro and nanostructures. Sericin’s biocompatibility makes it ideal for applications involving cells, enhancing cell attachment, improving cellular growth, and accelerating pro-liferation when added to culture media. [ 16,17 ] Reports on using sericin for bioelectronics applications have further opened up exciting prospects. [ 18 ] However, the limited ability to form sericin into complex patterns presents a challenge for its fab-rication into biologically-relevant architectures. [ 19 ] Strategies such as electrospinning of fi bers, and the formation of 2D fi lm and 3D hydrogel architectures are typically employed, mostly without the ability to accurately control spatial arrangement of the protein. [ 20–23 ] Patterning or micro and nanostructure fabrication via techniques including soft lithography [ 24 ] and imprinting [ 25 ] have not been readily translated to sericin. Fur-ther, these techniques may be constrained in the formation of intricate architectures or require mold fabrication and multiple transfer steps. [ 26 ]

Photolithography provides a route to pattern materials and fabricate circuits of extraordinary complexity with microstruc-tured spatial morphologies. The application of optical lithog-raphy to proteins has however, been relatively uncommon and primarily based on indirect methods – light induced acti-vation, [ 27 ] deposition or passivation to form patterns, followed by covalent protein immobilization, [ 28 ] or attachment to litho-graphically-patterned substrates. [ 29 ] One approach to directly pattern proteins is to alter them chemically or biochemically via site-specifi c and residue-specifi c techniques to render them intrinsically photoreactive. [ 30–32 ] Recently, our group reported on the integration of silk fi broin with photolithog-raphy to create precise protein patterns for use in cell culture and tissue engineering applications. The use of light enables rapid and direct fabrication without use of high temperature or harsh processing. [ 26,33,34 ] Here, for the fi rst time, we show the formation of precise protein microarchitectures using silk sericin as the structural biomaterial. A water-based photolithog-raphy approach is used to form high resolution protein struc-tures that are mechanically stable, biocompatible for cellular growth, and yet completely biodegradable. Without signifi cantly altering protein structure and function, facile biochemical modifi cations result in a “silk sericin protein photoresist” that readily reacts with commercially available photoinitiators to be crosslinked in the presence of light. [ 35 ] Via photolithography, protein features can be patterned at a low micron scale (µm)

Precise spatial patterns and micro and nanostructures of pep-tides and proteins have widespread applications in tissue engi-neering, bioelectronics, photonics, and therapeutics. [ 1–4 ] High resolution fabrication platforms using materials from bio-logical precursors have been proposed as feasible routes not only for sustainable bionanomanufacturing, but also to form organic devices to interface and interact with biological sys-tems. [ 5,6 ] Within this framework, natural silk biopolymers pro-vide green alternatives to synthetic materials with advantages of mechanical strength, optical properties, biocompatibility and controllable degradation. [ 7,8 ] Silk from silkworms occurs in a self-assembled fi brous confi guration, in which the mechani-cally robust fi broin protein core is surrounded by a glue pro-tein-sericin. Conventionally, pure fi broin and sericin or their blends with other materials, have been used to fabricate hydro-gels, fi bers, particles or fi lms. [ 9 ] Recently, exciting opportunities for silks in photonics, implantable bioelectronics and nano-structured scaffolds have been reported, revealing the need for innovative approaches to multi-scale fabrication with precision and manufacturing scalability. [ 10,11 ]

Extending high-resolution microfabrication techniques to natural biopolymers, and silk proteins in particular, provides a powerful avenue for their transformation into complex 2- and 3D devices. [ 1,3,4,12 ] Challenges lie in the development of tech-niques to fashion structural components with desired spatial resolution, complexity or mechanical properties. To operate at these length scales, techniques include “bottom up” or “top down” approaches such as molding via soft-lithography, casting, electrospinning, embossing, inkjet printing and imprinting. [ 5,13 ] To date, fi broin has comprised the bulk of such research owing to its processability, high mechanical strength and biocompat-ibility. In contrast, fabrication methods based on the sericin protein, typically discarded during the fi broin purifi cation process, have been comparatively limited in scope. [ 8 ] Despite reports on its intrinsic biocompatibility, [ 14 ] or that the perceived infl ammatory behavior of silk sericin is often when it is present in conjunction with fi broin (or that of fi broin when it co-exists

Adv. Mater. 2014, DOI: 10.1002/adma.201400777

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resolution at the bench top ( Figure 1 a). Periodic microfabri-cated protein structures can be formed over macroscale areas (cm) to form structurally-induced iridescent protein holograms in complex patterns. This presents an advance in developing novel sensor labels and optical materials using biodegradable biomaterials. We further demonstrate that these high resolu-tion microstructures can function as bio-friendly cellular sub-strates for the spatial guidance of cells without use of cell-adhe-sive ligands. The controllable degradation of this mechanically robust biomaterial provides opportunities in fabricating sus-tainable, nanostructured scaffolds and fl exible microdevices. Using benign solvents further addresses a pressing energy and environmental challenge in the development of chemical pro-cess systems for high resolution fabrication.

Silk sericin was extracted from Bombyx mori silkworm cocoons, [ 36 ] and modifi ed via the reagent 2-isocyanatoethyl methacrylate (IEM) to yield a photocrosslinkable silk sericin protein photoresist (SPP). [ 37 ] Direct conjugation of a photoactive isocyanate to the hydroxyl-rich sericin (∼36 mol% Ser, 9 mol% Thr, 3 mol% Tyr) was used. [ 38 ] Specifi cally, the methacrylate moiety has been extensively characterized in biomaterials. [ 39,40 ] Facile processing also ensures that the novel photoactive sericin

conjugate can be inserted into existing fabrication steps to form stable protein microstructures. [ 41 ] The product was purifi ed and conjugation of the multifunctional acrylate moiety was veri-fi ed. The degree of substitution was estimated from the weight increase of sericin-methacrylate recovered following the IEM coupling reaction, which corresponds to a conversion of ∼11% of amino acids to methacrylate moieties. From this observa-tion, a stoichiometry of 2.3 moles of IEM to moles of primary hydroxyl-containing amino acids is required in order to achieve a desired degree of substitution. This substitution degree was maintained at a low population of amino acids converted, leaving the bulk of serine groups untouched to preserve regions associated with favorable cellular interactions in sericin. [ 17,42 ] Controlling the degree of substitution provides a means of pro-ducing architectures of variable crosslinking densities. Alterna-tively, the presence of unmodifi ed amino acids also enables the production of multifunctional biomaterial architectures via con-current or subsequent modifi cation strategies. [ 43 ]

The successful conversion of sericin to a photoreactive form (SPP) was verifi ed by identifying characteristic peaks from the methacrylate moiety via ATR-FTIR spectroscopy (Supplemental Information, Figure S1). Native sericin displays intrinsic amide


Figure 1. (a) Schematic showing the fabrication of microstructures using silk protein lithography. Patterns are developed in aqueous media resulting in large area patterns with microstructural features (b). Features with line widths down to a few microns can be easily created over large areas. (c-e) SEM images showing different kinds of patterns formed by photolithography. Scale bars = 20 µm. Stars are 50 µm end to end (shown magnifi ed in inset).

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I/II/III signals, also observed in SPP, while the modifi ed product shows peaks at 1725 cm −1 (ester carbonyl), 1635 cm −1 (terminal C = C stretch), 1635 cm −1 (–CH 2 – stretch), provided by the methacrylate group. Prior research on this modifi cation strategy showed that the secondary structural content of the polypeptide backbone is preserved with this methacrylate bio-conjugation reaction. [ 33 ] This establishes that, while the conju-gation is successful, the biochemical addition of methacrylate does not adversely affect the core structure of sericin. The resulting conjugate retains many other characteristic proper-ties of unmodifi ed sericin, including a high degree of water-solubility. When SPP monomers are exposed to UV light in the presence of the photoinitiator (Irgacure 2959), the conju-gate becomes insoluble in water—a transition which is attrib-uted to chemical crosslinking between large constituent poly-peptide chains of the sericin protein. In contrast, biomaterials fabricated from pure sericin are readily water soluble unless chemically treated to modify secondary structure, which how-ever greatly limits the complexity of architectures that can be formed. [ 21 ] Finally, SPP behaves similarly to sericin in terms of its diffusion limited aggregation behavior on surfaces, resulting in comparable fractal patterns (Supplemental Information, Figure S2). Such nanoscale morphologies of self-assembly in sericin were earlier modeled as a purely physical phenomena depending on parameters such as particle size, dispersity and charge, showing that these properties are conserved.

Micropatterns of silk sericin on silicon and glass substrates were formed via photolithography, using the protein as a “nega-tive” photoresist crosslinked in the presence of UV light. The protein precursor was cast on the substrate, and exposure through a patterned chrome mask was used for crosslinking. Removal of the unexposed and uncrosslinked protein photore-sist (development step) results in clearly defi ned micro-architec-tures (Figure 1 ). Here, we demonstrate a remarkable advantage wherein the water solubility of SPP greatly aids in the process of photolithography, allowing development using water as the only solvent. This is in contrast to the often toxic and expensive developers used in chemical process systems for high resolu-tion fabrication, thereby offering the opportunity to replace key steps with aqueous-based processing, at considerable environ-mental and safety benefi ts. By controlling the thickness of the protein layer deposited on the substrate, structures of varying height in 3D can be created. Patterns ranging from ∼250 nm to several microns in height are easily fabricated. Protein pat-terns were further verifi ed using Coomassie Blue staining (Figure 1 b). Atomic force microscopy (AFM) and scanning elec-tron microscopy (SEM) were used to analyze the surface topog-raphy and to verify the high-resolution features formed. The versatility of the rapid and large-scale fabrication results in a wide range of features with excellent fi delity (Figure 1 c–e).

Currently, we have shown repeatable photolithographic fab-rication down to single micrometers. By varying the protein loading per substrate we can create features of well-defi ned height profi les, producing features ranging from a 100s of nanometers height upwards to tens of micrometers. Using AFM, an expanded area of a ∼2 µm height feature is shown as the number ‘7’, with a corresponding surface roughness of 2.8 ± 1.9 nm over a 5 µm area ( Figure 2 a, b). The rough-ness of these surfaces is therefore comparable to that of a

glass slide. The edges of the structures formed are clear, with a high fi delity (Supplemental Information, Figure S4). The scal-ability in height and feature aspects allow the fabrication of thin microstructures, appropriate for cellular adhesion, up to thick-walled microwells. Furthermore, the ability to vary feature height and spatial resolution of SPP allows fabrication of cel-lular scaffolds over a large range of aspect ratios. Lines, curves or interconnected shapes can be used to produce large scale arrays enabling fabrication of thousands of similar structures. Indeed, the scalability of photolithography over large areas (sev-eral cm) with microstructural topology, can be used to form periodic microstructural arrays in the form of a biodegradable protein hologram. Here, the large scale design is comprised of thousands of microscale features to produce diffraction with light and interesting optical properties. In the designs shown, squares of 10 µm were demonstrated that create “structure-induced iridescence” in the pattern, similar to the diffrac-tion patterns observed in natural structures or compact discs ( Figure 3 ). [ 44 ] In contrast, fi gures without periodic microstruc-tures did not show any specifi c iridescent behavior. Via the for-mation of micro and nanoscale gratings, easily achieved using photolithography, these proteins can be transformed into low cost biofriendly devices for photonic applications.

The photocrosslinked sericin forms mechanically robust and stable microstructures as shown above. While sericin is not particularly known for its mechanical properties in comparison to fi broin (an order of magnitude higher modulus), photo-crosslinking is expected to yield an improvement in mechanical properties in comparison to native sericin, due to the presence of a fl exible crosslink between polypeptide chains. To study the mechanical implications of crosslinking, AFM nanoinden-tation studies were conducted to yield elastic modulus values for SPP, in comparison to native sericin. To date, mechanical properties of sericin fi lms have not been thoroughly investi-gated, and data consists of studies on blends with other mate-rials, indicating a relatively low modulus (kPa -MPa). [ 45 ] In our experiments, crosslinked sericin showed an elastic modulus of 673 ± 106 MPa, which is slightly (∼7%) higher than that of native sericin (630 ± 66 MPa) (Figure 2 c, d). This may be attributed to crosslinks between adjacent protein chains, which enhance network elastic modulus. This improvement in mechanical stability of the crosslinked protein, coupled with its water insolubility in the crosslinked form, therefore enables the patterned protein to satisfy load-bearing requirements in dif-ferent tissue engineering applications.

While the protein can be lithographically patterned and crosslinked using UV light to form water insoluble features, we show that it is also biodegradable over time, exhibiting proteolytic degradation. The stability of the network was ana-lyzed through incubation studies in PBS buffer containing the enzyme protease XIV. As-cast SPP fi lms are water-soluble and dissolve over short time periods (< 4 hours). On the other hand, photocrosslinked SPP fi lms in PBS buffer (control) demon-strate an initial rapid decrease in mass, corresponding to the loss of residual photoinitiator from the network (Figure 2 b). When protease XIV is added to the buffer, the fi lms show a signifi cant reduction in mass, and loss of the fi lm’s structural integrity over a 3-day period. Films initially swell in the aqueous environment, and slowly fragment during incubation. The


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relatively hydrophilic nature of sericin-methacrylate network is implicated in this rapid loss of structural integrity—swelling of the sericin-methacrylate ‘hydrogel’ allows infl ux of the protease enzyme, which then digests the polypeptide backbone, eroding

the bulk of the fi lm. A gradual in vitro degradation establishes the ability of crosslinked sericin-methacrylate architectures to undergo controlled biodegradation and bioresorption. Both of these properties are important considerations for cellular cul-ture and tissue engineering applications, in which the rate of scaffold degradation must ideally match that of tissue regenera-tion. [ 46 ] By controlling the crosslinking degree of the resulting hydrophilic network, it may be possible to create sericin-meth-acrylate architectures of tailored physical (biodegradation and bioresorption) and mechanical (Young’s modulus) properties.

To demonstrate the compatibility of the sericin micropatterns for cell culture applications, microstructured scaffolds were fab-ricated. Scaffolds varied from simple parallel lines (10 µm), to square grids ( Figure 4 ). Feature heights were varied from thin (0.5 µm) to thick features (2 µm), to observe cellular attach-ment and proliferation. In our experiments, human osteoblasts were seeded onto patterned glass substrates and incubated over 96 hours in the presence of osteoblast mineralization media. The process of seeding yields nonspecifi c adhesion and subse-quent spreading, as evident in initial optical images-cells are located at the site of initial seeding, in regions on the scaffold and in between features. Subsequent analysis was conducted over a 96 hour time period, at which point osteoblast minerali-zation occurs. The underlying hydrophobic methacrylate mon-olayer that remains in unexposed regions after development ensures compatibility of the substrate with adhesion in cellular culture. [ 47 ] Osteoblast cells displayed preferential migration and


Figure 3. Structure induced iridescent behavior in periodic microstruc-tures of sericin. Large areas of precisely patterned features can be used to form designs that have the ability to diffract light based on viewing angle. (a) In this example, 10 µm squares were patterned to form the “VCU logo”. (b) Optical microscope and (c) SEM images showing the underlying architecture.

Figure 2. (a) AFM image of a patterned “7” feature on a silicon surface. The feature is around 2.5 µm in height. 5 µm zoomed in surface scan of the marked area of the photopolymerized product. (b) Proteolytic degradation characteristics for crosslinked SPP fi lms indicate a rapid initial degradation in the presence of Protease XIV. In the fi rst 3 days of analysis, fi lms swell in PBS buffer and gradually lose fragments up to day 21. In comparison, control fi lms indicate an initial loss of mass, without signifi cant mass subsequently lost. (c-d) AFM nanoindentation of sericin-methacrylate. Sample nanoindentation curves overlaid. A slight improvement of elasticity (Young’s modulus) with the photocrosslinked sericin having a value of 673 ± 106 MPa (∼7%) higher than that of native sericin (630 ± 66 MPa).

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adhesion to the micropatterned sericin architectures, effectively demonstrating the ability of sericin to selectively promote adhe-sion in a desired pattern. Via fl uorescence microscopy, osteo-blasts tended to specifi cally follow the ‘protein grid’, eventually producing a repeating architecture on top of the sericin scaf-fold. Here, the preservation of serine-rich repeats, implicated as regions for cellular interactions, enabled the sericin patterns to actively promote adhesion and proliferation. The absence of osteoblasts from growth and spreading on methacrylate-func-tionalized Si was interesting, given the high degree of hydro-phobicity of this substrate in comparison to the relatively hydro-philic protein pattern. For this reason, specifi c adhesion to the scaffold could be directly attributed to bioactive properties of sericin. The low surface roughness (∼3 nm) of the sericin fea-tures is further implicated in this preferential adhesion of oste-oblasts, which are known to display a marked improvement in integrin adhesion on low-roughness surfaces. [ 48 ] Similar experi-ments were conducted using fi broblasts (3T3 and L-929 murine fi broblasts) as a system on sericin micropatterns. The results of these are shown in the supplementary information.

In conclusion, we have demonstrated the application of a bio-chemical modifi cation strategy of the water soluble silk protein, sericin to produce a photoactive conjugate, capable of being

rapidly photopatterned into micro and nano-structured components. The high-resolution microfabrication technique provides a pow-erful route for the transformation of complex, mechanically robust 2- and 3D architectures for tissue engineering and optoelectronic applications. Sericin microstructures formed via photocrosslinking act as ideal 2D sub-strates for the spatial guidance of cellular adhesion and proliferation. Sericin scaf-folds simultaneously exhibit a high degree of cytocompatibility to both cultured human osteoblasts and fi broblasts, with specifi c serine-rich cell-binding sequences within the sericin polypeptide chain and favorable nano-topography for cellular attachment. Despite the impartation of photoactivity, the sericin is mechanically stable during the fabrication process, and can be completely enzymatically degraded in vitro. In comparison to fi broin demonstrated earlier, [ 33 ] the water-based pro-cessing of sericin provides unique opportuni-ties in terms of green microfabrication using a traditionally discarded biomaterial. While chemically diverse, both silk proteins may be biochemically adapted for spatial patterning using silk protein lithography. A range of mechanical properties of these proteins from sericin (∼0.6 GPa) to fi broin (∼15 GPa), offers exciting possibilities for a host of applica-tions. The ease of fabrication using modi-fi ed sericin, combined with a stable yet bio-degradable, cytocompatible confi guration, provides a versatile platform as a multifunc-tional biological building block.

Experimental Section Materials : Pure sericin protein (Wako Chemicals, Richmond, VA) and

2-isocyanatoethyl methacrylate (IEM, Sigma-Aldrich, St. Louis, MO) reagents were utilized for chemical conjugation. Sericin hydrolysate with an average molecular mass of 30 kDa is commercially available and prepared by boiling silkworm (Bombyx mori) cocoons under alkaline conditions and then drying the extract to a powder. Lithium chloride salt and anhydrous DMSO were employed for dissolving sericin initially. Subsequent solubilization of product was achieved via 2,2,2-trifl uoroethanol (TFE) using Irgacure 2959 as a photoinitiator (Ciba Specialty Chemicals, Basel, Switzerland). All chemical species were obtained from Acros Organics (Geel, Belgium) unless otherwise specifi ed.

Synthesis of Photoreactive Sericin : SPP was synthesized via chemical conjugation between sericin and IEM in an anhydrous solvent system of 1M LiCl in DMSO. [ 49 ] Lithium chloride and all glassware were thoroughly dried at 150°C for 24 hours prior to use, while silk sericin was dried at 70°C. Sericin (100.0 mg) was suspended at 1% (w/v) in a solution of 1M LiCl/DMSO in a round bottom fl ask and stirred at 60 ºC in a dry N 2 atmosphere for 45 minutes. Immediately after, the IEM was added at a stoichiometric equivalent to reactive hydroxyl-containing amino acids. The reaction was allowed to proceed to completion at 60 ºC for 5 hours. The product was precipitated out into cold ethanol, centrifuged and washed with a mixture of cold ethanol/acetone. Lyophilization yielded a pure white powder.


Figure 4. Fluorescence micrographs of osteoblast proliferation on sericin scaffolds demon-strate selective patterning on microstructures. Rhodamine-conjugated phalloidin staining of osteoblasts on a dense grid (a) displays cellular adhesion over a widespread area, with (b) clear defi ciency of osteoblasts in the grid void space. (c, d) Optical and fl uorescence image of osteoblasts. FITC-phalloidin and DAPI stained osteoblasts are displayed with the ability to selectively follow underlying patterns. Scale bar on all images = 100 µm.

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Verifi cation of Product via FTIR Spectroscopy : To confi rm the methacrylate conjugation, Fourier transform infrared spectroscopy (FTIR) was conducted on unmodifi ed sericin and SPP using a Nicolet iS10 FTIR spectrometer. Cast fi lms (5.0 mg) were analyzed in attenuated total refl ectance (ATR) mode using a Ge ATR crystal, and data was collected between 4000 – 1000 cm −1 , for 32 scans at a resolution of 1 cm −1 .

Surface Functionalization : Glass slides and silicon surfaces for microfabrication were initially treated with Piranha solution (3:1 98% H 2 SO 4 :30% H 2 O 2 ) to hydroxylate the surface and remove organic contaminants ( Caution: Piranha solution reacts violently with organic materials and must be handled with extreme care ). Following treatment, surfaces were washed with copious deionized water and ethanol and dried at 150 °C. Chemical vapor deposition of 3-(trichlorosilyl) propyl methacrylate (TPM) was conducted in a desiccator (0.4 bar for 12 hours), to form surface functionalized substrates.

Photolithography : Microscale patterns of sericin were fabricated using photolithography. A solution of 2% (w/v) SPP in 2,2,2-trifl uoroethanol (TFE, Sigma Aldrich, St. Louis, MO) was prepared, with 0.5% (w/v) of Irgacure 2959 photoinitiator, and cast at 0.5mg per substrate. TFE is associated with a greater evaporation rate in comparison to water, allowing rapid casting of thick homogenous fi lms. Contact photolithography was conducted using a photomask, with a 3.0 second UV exposure (Lumen Dynamics OmniCure 1000, 320–500 nm fi lter). Development of uncrosslinked and unexposed protein photoresist was performed using deionized water (18.2 MΩ·cm) for 2 hours followed by copious rinsing with deionized water and ethanol. Substrates with the developed protein patterns were then dried in a gentle stream of dry N 2 .

Topographic and Mechanical Analysis of Scaffold Microstructure : Scanning electron microscopy (SEM) was conducted using a Hitachi SU-70 high-resolution fi eld emission microscope, at a 5.0 kV accelerating voltage. Patterned substrates were sputter coated with 15Å platinum (Denton Vacuum Desk V cold sputtering system, Denton Vacuum, Moorestown, NJ). Atomic Force Microscopy (AFM) was performed on an MFP-3D AFM (Asylum Research, Santa Barbara, CA), operating in non-contact mode. The AFM was further used to determine Young’s modulus, through AFM-based nanoindentation using an AC240TS tip (nominal k = 1.5 nN nm −1 , Olympus, Japan). Films (>1 µm thickness) were indented with 10 indents at 3 discrete locations under a constant load of 150 nN. Force-distance curves were analyzed via fi tting tip extension data to a Hertzian model to determine values for Young’s moduli. [ 50 ]

In Vitro Proteolytic Degradation : SPP fi lms (∼5 mg) were incubated at 37 °C in a solution of (1 unit/mg protein) Protease XIV ( Streptomyces griseus , ≥3.5 units/mg, Sigma Aldrich). Films incubated in PBS buffer without protease were used as negative controls. At specifi c intervals, samples were removed from buffer, rinsed with deionized water, and dried for subsequent analysis.

Osteoblast Culture on 2D Sericin Scaffolds : Osteoblast like cells (MG 63) and mouse fi broblast (L929) were obtained from NCCS, Pune India and maintained in DMEM complemented with 10% FBS and 1% penicillin/streptomycin solution. Cells were propagated at 37 °C, supplemented with 5% CO 2 . Osteoblasts were seeded at 5 × 10 4 cells onto sericin patterns (n = 3 substrates), photocrosslinked fi lms, and unpatterned (TPM-treated) glass substrates as controls. Images were acquired via inverted light microscope after 12 hours, and subsequently every 24 hours post-seeding. After 4 days of cell culture, scaffolds were removed from media and fi xed in 4% paraformaldehyde to prepare for staining and fl uorescence imaging. Cell staining was achieved using phalloidin for staining F-actin fi laments, and DAPI to stain the nucleus.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements SEM images were obtained in the Nanomaterials Core Characterization facility at VCU. This work was partly supported by the School of Engineering at VCU and the Department of Biotechnology of the Government of India. Complete experimental details, characterization of the sericin protein photoresist and photolithography are included in the Supporting information.

Received: February 17, 2014 Revised: March 21, 2014

Published online:

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