Laser-based three-dimensional multiscalemicropatterning of biocompatible hydrogels forcustomized tissue engineering scaffoldsMatthew B. Applegatea, Jeannine Coburna, Benjamin P. Partlowa, Jodie E. Moreaua, Jessica P. Mondiaa,Benedetto Marellia, David L. Kaplana, and Fiorenzo G. Omenettoa,b,1

aDepartment of Biomedical Engineering, Tufts University, Medford, MA 02155; and bDepartment of Physics, Tufts University, Medford, MA 02155

Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved August 14, 2015 (received for review May 13, 2015)

Light-induced material phase transitions enable the formationof shapes and patterns from the nano- to the macroscale. Fromlithographic techniques that enable high-density silicon circuit in-tegration, to laser cutting and welding, light–matter interactions arepervasive in everydaymaterials fabrication and transformation. Thesenoncontact patterning techniques are ideally suited to reshape softmaterials of biological relevance. We present here the use of rela-tively low-energy (< 2 nJ) ultrafast laser pulses to generate 2D and3D multiscale patterns in soft silk protein hydrogels without exoge-nous or chemical cross-linkers. We find that high-resolution featurescan be generated within bulk hydrogels through nearly 1 cm of ma-terial, which is 1.5 orders of magnitude deeper than other biocom-patible materials. Examples illustrating the materials, results, and theperformance of the machined geometries in vitro and in vivo arepresented to demonstrate the versatility of the approach.

ultrafast lasers | biomaterials | silk | micromachining | tissue engineering

The ability to controllably shape biomaterials on the microscalein two, and especially three, dimensions is important given the

utility of these structures in guiding cellular growth, differentiation,gene expression, and regeneration (1–4). The use of soft, bio-compatible materials, however, poses challenges in fabrication dueto their mechanical characteristics. Widely adopted biomaterialmicrofabrication techniques such as soft- and photolithography arelargely limited to two dimensions. The recent advent of 3D printingtechnology has exploited the interaction of light with materials torapidly prototype parts for a variety of industries, and has expandedto significantly impact the biomedical field (5–7). Microscale 3Dprinting has also shown promise for tissue engineering and re-generative medicine applications (8, 9). Here we will present atechnique for generating voids as small as 5 μm in diameter withina biocompatible hydrogel using multiphoton absorption (MPA) oflight that shares many similarities with 3D printing. Furthermore,we demonstrate that this technique functions in the absence ofexogenous photoinitiators or chemical cross-linkers, thereby avoidingpotentially biologic incompatibility that can otherwise limit the utilityof such processes.MPA is a process that occurs under extremely intense illumination

where two or more low-energy photons are absorbed simultaneouslyby a material (10). To achieve photon densities high enough forMPA, very short laser pulses must be tightly focused within a ma-terial. If the material is transparent to the low-energy photons, verylittle of the light is absorbed at the surface, allowing a focal spot to beformed, and MPA to occur, deep within the material. Multiphoton-induced structural modification leading to void formation has beeninvestigated in a variety of biocompatible materials including colla-gen, poly(vinyl-alcohol) (PVA), poly(methyl methacrylate), andgelatin hydrogels (11–13). Poly(ethylene glycol) hydrogels cross-linked with a photolabile bond can be selectively degraded to induce3D structures (14). Collagen, due to its turbidity, is unsuitable for3D patterning with features limited to a few tens of micrometersbelow the surface (15). Transparent materials such as PVA have very

high threshold power requirements necessitating the use of highnumerical aperture objectives, or amplified femtosecond pulses toinitiate MPA for void formation. Extremely high light intensitiesfound in these amplified pulses can locally change a material’s re-fractive index, resulting in self-focusing of the beam. Self-focusinglimits the depth at which a tight focal spot can be formed and haslimited MPA-induced void formation to less than 200 μm below thesurface of the material (12). Some natural proteins including amyloid(16) and silk fibroin (17) are much more efficient multiphoton ab-sorbers than their amino acid composition would suggest. The hy-pothesis here was that the large multiphoton cross-section of thesenatural materials will allow the initiation of MPA at low thresholdpowers, potentially reducing the effects of self-focusing.Silk fibroin collected from the domesticated Bombyx mori silk-

worm has been under steady investigation for decades because ofits suitability as a material for biomaterials and tissue engineering.Silk is cytocompitable, biodegradable, and able to stabilize labilecompounds such as enzymes and drugs (18). Silk fibroin has alsobeen studied as an optical material due to its transparency to visiblelight and low surface roughness, giving it the ability to conform tonanoscale structures such as diffraction gratings (19–21) or togenerate 3D photonic crystals (22). Previous work involving pho-tomodification of silk has thus far only considered surface modi-fication of dried films (23). Extending this work into the thirddimension requires silk to take on a different form. Recently, ahighly transparent elastomeric silk fibroin hydrogel has been de-veloped, which is ideally suited to multiphoton laser micro-machining (Fig. 1A, Inset) (24). These gels are robust enough to beeasily handled, amenable to cell growth, and well tolerated upon

Significance

In this paper we present results on 3D, multiscale laser machiningof soft, transparent biomaterials suited for cellular growth and/orimplantation. We use an ultrafast laser to generate high-resolution,3D structures within the bulk of a transparent soft-biomaterialformulation that can support cell growth and allow cells topenetrate deep within the material. The structure is created bymultiphoton absorption which, thanks to the clarity of the silkgels, is possible nearly 1 cm below the surface of the material.This depth represents an ∼10× improvement over other mate-rials. The ability to create micrometer-scale voids over such alarge volume has promising applications in the biomedical fieldand its efficacy was demonstrated both in vitro and in vivo.

Author contributions: M.B.A., B.M., D.L.K., and F.G.O. designed research; M.B.A. and J.E.M.performed research; J.C., B.P.P., J.E.M., and J.P.M. contributed new reagents/analytictools; M.B.A., J.E.M., B.M., and F.G.O. analyzed data; D.L.K. and F.G.O. supervised theproject; and M.B.A. and F.G.O. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: fiorenzo.omenetto@tufts.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1509405112/-/DCSupplemental.

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implantation. Importantly, these gels are greater than 90% water,which allows material disrupted during MPA to be depositedaround the outside of the machined region without fouling.Here we present the exploration of laser-induced void for-

mation in silk hydrogels (hereafter referred to as multiphotonmicromachining). We find that relatively low-energy (sub-2 nJ perpulse) infrared (λ = 810 nm) pulses at a high repetition rate(80 MHz) can be used to form voids within the hydrogels in threedimensions. The gels have a linear absorption peak at 270 nm,suggesting this to be a three-photon absorption process (Fig. S1).The short time between pulses (12.5 ns) implies that the heatdeposited by the first pulse that arrives does not have time todiffuse away before another pulse hits, leading to thermal ac-cumulation at the focus of the beam which disrupts the silkstructure forming voids. The voids formed survive the rigors ofhandling, cell growth, and subdermal implantation. We furtherfind that it is possible to form voids within the gels nearly 1 cmbelow the gel surface. To our knowledge, this represents thegreatest depth of multiphoton-induced void generation reported,exceeding by 1.5 orders of magnitude the deepest ablation in anymaterial yet tested (12).A custom-built 3D laser writing workstation was constructed

to study multiphoton micromachining (Fig. 1A). Ultrashort (∼ 100 fs)laser pulses at a pulse repetition frequency of 80 MHz were fo-cused into the bulk of a silk hydrogel using a 10× (N.A. = 0.3)

microscope objective (Fig. S2). The sample was mounted on athree-axis micropositioning stage. The sample could then bemoved so the beam was focused in different locations within thematerial. Generation of complex 3D patterns within the materialwas achieved by computer control over the stage translation.The relationship between pulse energy and void size was

characterized by micromachining a series of lines on the topsurface of a gel ∼1 mm thick (Fig. 1D). Each line was made by asingle pass of the laser at a constant speed of 50 μm/s withvarying pulse energies. After machining, the lines were imagedvia atomic force microscopy (AFM). We found the minimumpulse energy necessary to observe structural changes in the silkgel to be ∼0.25 nJ per pulse. At this power, the average trenchdimensions were 1.5-μm full width at half maximum (FWHM) inwidth and 100 nm in depth. These dimensions increased to 2.5-μmFWHM and 600 nm in depth when the pulse energy was raisedto 5 nJ (Fig. 1C). AFM measurements confirmed that the changein appearance of the machined region was due to material re-moval and not local changes in refractive index (Fig. 1B).The depth at which features could be micromachined was

tested by forming a gel inside a plastic fluorescence cuvette.Features were micromachined inside the gel at various depthsand subsequently imaged by rotating the cuvette 90° and exam-ining the features using bright-field microscopy. Visible featureswere found in the gel up to 8 mm below the surface (Fig. 1E and

Fig. 1. Overview of the micromachining process.(A) Schematic of the multiphoton micromachiningworkstation. (Inset) Photograph of the transparentsilk hydrogel. (B) Three-dimensional AFM image ofone of the lines in D. (C) Graph relating line di-mensions with pulse energy. Error bars represent 1SD (n = 4). (D) Microphotograph of lines machinedinto the upper surface of a silk gel at pulse energiesranging from 0.25 nJ (Bottom) to 5 nJ (Top). (E) End-on view of 30-μm-wide lines machined into a silk gel.Light was incident from the bottom of the image.Ruler on right side measures depth from the surfaceof the sample. Due to the large area involved, thisimage was stitched together from a series of micro-photographs. (Inset) Detail of the cross-section ofone line.

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Fig. S3). Deeper features should be possible using a longerworking distance objective with a similar numerical aperture. Weattribute this large maximum machining depth to the clarity ofthe silk and the large multiphoton cross-section of the protein,which allows low-powered pulses to be used to initiate MPAwithout significant self-focusing (Fig. S3). We estimated thecritical power for self-focusing to be greater than 6 MW, which ismore than 100× more power than is found in the pulses used formicromachining (Fig. S4). This combination of qualities is, to thebest of our knowledge, unique to silk and enables multiphotonmicromachining to occur at such large depths. Deep, high-res-olution features such as these, combined with the ability to dopethe silk with growth factors and other compounds, could be usedfor the generation of complex 3D patterned cell scaffolds to formmicroenvironments for different cell types within the same scaffold.With maximum penetration depth of nearly 1 cm and a lateral

resolution on the order of 5 μm, silk hydrogels are an excellentsubstrate for multiphoton micromachining. Given the limits oftravel of the micropositioning stage, the total addressable vol-ume of our workstation was greater than 100 cm3. Within thisvolume, individual voxels as small as 125 μm3 could be removedat will, with the removed material deposited along the outeredges of the machined regions.To explore the practicality of this technique to generate

complex 3D structures, test patterns were micromachined intothe bulk of the silk gel. The first was a helix consisting of twoturns with an outer diameter of 200 μm (Fig. 2A). The structure

started roughly 500 μm below the surface and extended 400 μmfurther into the gel. The second pattern chosen was a blood-vessel–like branching pattern (Fig. 2E). This structure was situ-ated 300 μm from the surface and had a vertical extent of 100 μm.To image these patterns, the silk was stained with Rhodamine Bafter multiphoton micromachining and tomographic images werecollected using confocal microscopy. The Rhodamine-stainedsilk fluoresced brightly whereas the machined regions were dark,indicating removal of the hydrogel in these regions. In mostcases, the edges of the machined features showed evidence ofgreater material removal than the bulk of the features. Thispattern was due to the control program, which paused lateralmotion of the micropositioning stage at the end of each linebefore closing the shutter so the edges of the features were al-ways exposed to more pulses than the center. Increased fluo-rescence was also visible around the edges of the features, whichwe attribute to the deposition of removed material along theborders. We also observed this phenomenon when imaging usingthe autofluorescence of silk for contrast rather than exogenousstains (Fig. S5).To be useful in biomedical applications, a material must be

nontoxic and support cell growth. To ensure that the machinedregions were not harmful to cells in culture, we prepared sterilegels by filtering the silk through a 0.22-μm pore filter and mixedthe solution in a 35-mm-diameter plastic Petri dish under sterileconditions for gelation. Before removing the dishes from thehood the lids were covered with parafilm to maintain sterility. All

Fig. 2. Overview of two test patterns machined into the gel. (A) Three-dimensional model of a helical pattern input into the control program. (B) Confocalmicroscope image of a cross-section of the helix showing the machined region in black. (Scale bar, 100 μm.) Please see video reconstruction in Movie S1.(C) Reslice of the confocal stack along the dashed line in B. (Scale bar, 100 μm.) (D) Three-dimensional reconstruction of the segmented confocal data showingthe machined feature. (E) Three-dimensional model of a branching pattern input into the machining control program. (F) Three-dimensional reconstructionof resulting machined region made by segmenting the confocal images. (G) Confocal slice showing a cross-section of the micromachined region. (Scale bar,100 μm; same for H and I.) (H and I) Cross-sections of the confocal volumes at the indicated lines.

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machining of the gels was done within the sealed Petri dishes inambient conditions.Parallel lines ∼3 μm in width separated by about 20 μm were

micromachined onto the top surface of a gel through the bulk.Human foreskin fibroblasts were seeded on the surface andobserved using phase contrast microscopy as they attached andspread over the dish. We observed that the cells tended to alignwith the grooves machined into the gel and grew parallel withthese surface features (Fig. 3 A–D). This contact guidance phe-nomenon is well-known and has previously been used to inducealignment of various cell types (25, 26). Because features can bemachined onto the gel through a sealed dish, we hypothesize thatthis could be a convenient method to reorient or disrupt alreadyestablished cell cultures.In tissue engineering, access to oxygen and nutrients within an

artificial tissue is a major challenge that limits cell density withintissue engineered constructs (27). To address this issue, re-searchers have generated scaffolds with interconnected porousnetworks (28). However, such pores are randomly distributed,limiting the amount of control of cell growth and infiltrationthat is possible. Multiphoton micromachining allows fullypredetermined micrometer-scale features to be generated withina construct, allowing spatial control over cell infiltration. To testwhether micromachined features within the silk hydrogels couldbe used to direct cell growth in three dimensions, Y-shapedbranching patterns were machined into the gels such that themain branch intersected the surface, allowing cells and media topenetrate the bulk of the gel (Fig. 4A). Cells were stained with afluorescent dye and confocal images were taken of each featureat days 5, 9, and 14 postseeding. Cell density was assessed atthree locations within each feature: the main branch, the tran-sition region, and the lower branch. By day 9 and continuing today 14, cells were observed in all three regions in 100% of thesmall features. The larger features were less well populated withcells found in 100% of the main branches, 86% of the transition

regions, and only 14% of the lower branches by day 9. On day14, 71% of the large features had cells in the lower branches.One of the large features did not intersect the surface of the geland was omitted from this analysis. No subsurface cells wereobserved in areas that were not laser machined (Fig. 4A).Rather than providing a means for cells to infiltrate a material

from the surface, it is often easier to encapsulate cells within thematerial itself. It has been shown that human mesenchymal stemcells (hMSCs) can be encapsulated within this type of silk hydrogel(24). When cells are encapsulated in this way, the concentrations ofoxygen, nutrients, and growth factors are governed by diffusion,limiting the size of such constructs. Three-dimensional patternedcell-laden hydrogels would have more surface area for the diffusionof oxygen and well-defined patterns could provide an artificial mi-crovasculature, greatly increasing the maximum size at which cellgrowth could be supported. To investigate the ability of multipho-ton micromachining to pattern cell-laden hydrogels, we embeddedhMSCs in the bulk of a thin gel. The word “Tufts” was micro-machined into the gel (Fig. 3E) and, less than 4 h after machining,cells were stained with a live/dead fluorescence assay. Followingstaining the dishes were examined using confocal microscopy. Wefound dead cells in the plane of micromachining with living cellspresent both directly above and below the machined volume (Fig. 3F–J). This was expected as cells are largely transparent to 810-nmlight so they should be unaffected by the beam far from the focus.The high temperatures at the focus of the beam are likely re-sponsible for the dead cells found in the micromachined regions.Finally, we conducted a pilot in vivo study in which three mice

were implanted with two machined gels each. One gel containeda branching pattern with a main branch diameter of 200 μm; thesecond gel contained a branching pattern with a main branchdiameter of 400 μm. One mouse was killed at 2, 3, and 4 wk.Upon subsequent imaging we were able to identify the machinedfeatures in four of the six samples with at least one featureidentified at each timepoint. Cells were found to penetrate the

Fig. 3. Micromachined features in vitro. (A–D) Machined lines on the surface of a gel at day 1, 3, 5, and 8, respectively. Arrows indicate cells growing alongthe machined lines. D shows a fluorescently labeled cells growing in the lines. (Scale bar, 100 μm long.) D shows a slightly different region of the gel as highcell density obscured the features at the location of the other images. (E) Cartoon showing micromachining of a gel laden with hMSCs. (Inset) Bright-fieldimage of the machined region. (Scale bar, 250 μm.) (F–H) Confocal images of the cell-laden gel following live/dead staining 76 μm below, 62 μm above, and inthe plane of machining, respectively. Dashed lines outline the micromachined region. (Scale bar, 250 μm.) (I and J) Close-up of living cells irradiated by thebeam above (I) and below (J) the focal plane.

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gels via the machined features in the 2- and 3-wk sample (Fig. 4and Figs. S6 and S7). In the 4-wk sample, cells were found to haveovergrown the machined feature and not penetrate into the gel. It islikely that the overgrowth in the 4-wk case was not due to the extratime of implantation as no cells were seen to penetrate the gel, butrather occurred relatively soon after implantation.These results are significant as they show that multiphoton

micromachining in silk fibroin hydrogels was capable of directingcell growth and speeding infiltration into an artificial construct.Patterned biocompatible constructs are of great interest in the fieldof tissue engineering, which seeks to artificially recapitulate naturalstructures in the body. One promising avenue to do so is the use ofdecellularization as a means to replace damaged organs (29). Thistechnique involves the harvest of a healthy organ and the removalof all cellular material, leaving behind a structured extracellularmatrix. The resulting decellularized scaffold acts as a template fornew cell growth. However, this technique requires access to ahealthy organ as well as time for cell culture. Whereas this methodcould be used to reduce rejection of donated organs, it does little tohelp those who are still waiting for an organ transplant. Whereasthe micropatterning described here is too small-scale to be used toreplicate an entire organ, it provides a unique combination of high-resolution (micrometer-scale) structuring with the possibility ofgenerating large (nearly millimeter-scale) features. We believe thiscombination of high resolution with large volume of modificationcould prove useful to link large-scale 3D patterning of biologicalmaterials using techniques like 3D bioprinting (8), with techniquesto produce random voids in a material on the 0.1-m scale (28).

In conclusion, silk hydrogels were found to be an attractive sub-strate for photoinitiator-free multiphoton micromachining. Usingonly moderate laser power it was possible to generate voids withinthe bulk material at depths of nearly 1 cm. This approach enabledrapid formation of high-resolution structures over multiple lengthscales in three dimensions and could be carried out in cell-ladenhydrogels without damage to living cells in the volume immediatelyadjacent to the micromachined region. The features are formed in asoft, biocompatible matrix which enables the guidance of cells inthree dimensions and appears to promote infiltration of cells in vivowithout loss of the pattern’s structural integrity. All-aqueous pro-cessing of the material and machining at ambient temperatureswithout harsh solvents or toxic photoinitiators should make it pos-sible to further promote cell infiltration and differentiation usinggrowth factors or other chemical signals. Whereas there are manyoptions to improve the resolution and utility of the micromachiningworkstation, the technique described here allows for rapid proto-typing of mesoscale features in a robust, simple to use, bio-compatible substrate. Three-dimensional patterns that are suitablefor guiding cell growth can be produced over large volumes withhigh resolution. Such patterned gels allow control over cell growthand implantation on the 10-μm scale, allowing the recapitulation ofnative micrometer-scale structures in tissue engineering scaffolds.This approach for the generation of programmable structures usingmultiphoton micromachining in biocompatible silk hydrogels is vir-tually impossible to produce using any other method, opening nu-merous new avenues of investigation into the 2D and 3D patterningof soft materials.

Fig. 4. Cell infiltration into machined features. (A, Top) Three-dimensional model of the pattern machined into hydrogels that were subsequently seededwith cells in vitro. (A, Bottom) Series of confocal images of fibroblasts growing within a Y-shaped machined feature on day 9 after seeding. Each image isseparated by 10 μm in the Z direction. (Scale bar, 100 μm.) (B, Top) Three-dimensional model of the pattern machined into a hydrogel that was subsequentlyimplanted s.c. in mice. Lines marked “(i–iii)” indicate the confocal cross-sections shown in the panels below. The white circles in (B, i) and (B, ii) correspond tothe main branch diameter and approximate location in the construct. The smaller circles in (B, iii) correspond to the secondary branch diameters. Cells hadinfiltrated to the bottom of the main branch (B, ii) and had begun extending down one of the secondary branches (B, iii). (All scale bars, 100 μm.)

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Materials and MethodsMultiphoton Micromachining Workstation. Approximately 100-fs pulses of 810-nmlight from a titanium sapphire oscillator (Tsunami, Spectra Physics) at a repetitionrate of 80 MHz were passed through a computer-controlled shutter and directedinto the rear accessory port of an inverted microscope. The light was focused toan ∼ 5 μm spot through a 0.3-N.A. microscope objective with a working distanceof 1.03 cm onto the sample, which was placed on a computer-controlled XYZtranslation stage (Ludl Electronics). Using a custom LabView application,complex patterns could be micromachined by inputting stacks of binaryimages into the program. Pulse energies were manually adjusted via a half-wave plate and polarizer giving continuous control of pulse energy from0.1 to ∼10 nJ per pulse.

Hydrogel Preparation. Silk fibroin was extracted as described in ref. 30 with adegumming time of 60 min. Gels were prepared by adding 10 units/mL typeVI horseradish peroxidase and 10 μL/mL 1% hydrogen peroxide (24). To fa-cilitate fluorescence imaging, Rhodamine B-stained gels were prepared afterthe desired pattern had been micromachined. These gels were soaked in asolution of 0.1 mM Rhodamine B for 4 h and then were rinsed in 10 changesof deionized water over the following 24 h to remove any Rhodamine notbound to the silk.

Gel Micromachining. Lines were micromachined on the top surface of a thingel at pulse energies ranging from 0.25 to 5 nJ per pulse and were imaged onan MFP-3D-Bio AFM (Asylum Research). The samples were scanned in contactmode under PBS solution using TR800PSA cantilevers with a calibrated springconstant of 0.4 N/m.

Maximum depth of machining was determined by forming a silk gel insidea plastic fluorescence cuvette. Thirty-μm-thick lines were micromachined inthe silk at regular depth intervals. Translation speed varied between 100 and25 μm/s depending on the depth. A side view of the lines was obtained byrotating the cuvette 90° and imaging via bright-field microscopy.

Two-Dimensional Contact Guidance. Silk solutions were filtered through a0.22-μm filter and gelled in a 35-mm Petri dish. Before removal from thehood, dishes were sealed with parafilm to maintain sterility. Lines were thenmicromachined onto the top surface of the gel. Human foreskin fibroblastswere seeded onto the gel and cultured in DMEM with 10% FBS at 37 °C, 5%CO2. Gels were imaged via phase contrast microscopy at day 1, 3, and 5

postseeding. On day 8 the cells were stained with a Live/DeadViability/Cytotoxicity kit (Molecular Probes, Inc.) fluorescence assay and imaged viafluorescence microscopy.

Cell-Laden Hydrogels. Human mesenchymal stem cells (hMSCs) were isolatedfrom fresh bone marrow aspirate (Lonza) as previously described (31). hMSCswere gently mixed into a partially gelled silk hydrogel at a rate of 1,000 cellsper mm3. One hundred μL of the silk/cell mixture was added to each glass-bottomed Petri dish (24). After gelation, micromachining was performed onthe cell-laden hydrogels. Within 4 h of machining the cells were stained witha Live/DeadViability/Cytotoxicity kit (Molecular Probes, Inc.) and examinedvia confocal fluorescence microscopy.

Three-Dimensional Cell Guidance. Sterile gels were prepared as described in 2DContact Guidance above. Three-dimensional Y-shaped branching patterns weremachined into the gel, with the main branch of the Y intersecting the topsurface of the gel. Main branch diameters were 200 μm and 400 μm in the smalland large features, respectively. Human foreskin fibroblasts were seeded ontothe surface of the gel after micromachining. Gels were examined via confocalmicroscopy on day 5, 9, and 14 postseeding. The day before each imagingsession dishes were stained with CytoTraker Green (Molecular Probes).

Implantation. Sterile gels were prepared in 35-mm Petri dishes as describedabove and a 4-mm biopsy punch was used to remove cylinders of gel.Branching patterns (Fig. 4B) were machined into each cylinder. All pro-cedures involving mice were approved by the Tufts University InstitutionalAnimal Care and Use Committee. Animals were anesthetized by isofluraneinhalation during the procedure. Machined gels were implanted s.c. into thelumbar region of three mice. Silk implants and adjacent tissues wereextracted following euthanasia (carbon dioxide asphyxiation) at 2-, 3-, and4-wk postimplantation. Gels were recovered from the mice and fixed in 10%formalin and stained with Phalloidin and DAPI.

ACKNOWLEDGMENTS. The authors acknowledge Dr. Elise Spedden for helpwith the AFM. The authors acknowledge funding from the Office of NavalResearch (N00014-13-1-0596). M.B.A. acknowledges support from the SternFellowship at Tufts University. M.B.A. and B.P.P. received support from theNational Defense Science and Engineering Graduate Fellowship.

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