© 2011 Nature America, Inc. All rights reserved. S required to process the silk fibroin solution into the time within the arrows indicates the time the silk fibroin extraction process takes 4 d and processingaqueous-based approaches. Overall, from silk fibroin using both organic solvent– and Figure 1 hydrophobic in rich bond ( chain ( chain a light of cocoon by weight. The silk fibroin consists silkworm total the of 25–30% for account that proteins adhesive are Sericins teins. pro sericin with coated protein fibroin applications. potential of range a for biomaterials silk-based of variety a of implementation and design the with help can protocols following The in shown are studied been have that formats the material of Some materials. into different cessed pro further be can that solution aqueous an into dissolved are filament. Once this adhesive protein is removed, the fibroin fibers the surface of fibroin, the silk filament core protein, in the cocoon expressed in the middle silk gland of glycoproteins soluble of is a group Sericin fibers. core the fibroin from removed be must component sericin the cocoons, raw the From required. is protein core the of preparation and extraction this however,polymer; natural for medical applications, adequate for sources cost reasonable and abundant are there industry, tile vent sol or an organic solution aqueous from either formats material of degradation modified be chemically it can which with ease the its biocompatibility, include In erties. properties particular, these gained attention as a biomaterial because of several desirable prop have used silk as a suture material for centuries, and it has recently torically been highly regarded for its strength and luster. Physicians mori Bombyx I in tissue engineering and of applications. In this protocol, we include methods to extract silk from growth factors. controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize S Published online 22 September 2011; doi:10.1038/nprot.2011.379 TuftsBiomedical Engineering, of Department University, Medford, Massachusetts, USA. Correspondence should be addressed to D.L.K. ([email protected]). N Danielle Rockwood, Rucsanda C Preda, Tuna Yücel, Xiaoqin Wang, Michael L Lovett & David L Kaplan Materials fabrication from 1612 the material of choice. composites, composites, fibers, microspheres and thin films. NTRO protocol ilk ilk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has ilk ilk fibroin, derived from Native 6

. Because of large-scale cultivation of silkworms for the tex the for silkworms of cultivation large-scale of . Because | 6 VOL.6 NO.10VOL.6 . Silk fibroin is a block copolymer copolymer block a is fibroin Silk . D M

| UCT Schematic of material forms fabricated w B. mori B. ~390 kDa) linked kDa) by ~390 a disulfide I (silkworm) silk is a unique material, which has his which is silk a material, unique (silkworm) ON in in vivo A M silk is composed of silk silk of composed is silk variety of aqueous or organic solvent-processing methods can be used to generate silk biomaterials for a range | 2011 w ~26 kDa) and a and heavy kDa) ~26 and its to ability be processed into multiple |

β natureprotocols Bombyx mori -sheet-forming -sheet-forming in in vitro Figure Figure disease models, as well as for drug delivery. B. mori cocoons, is a widely used and studied protein polymer for biomaterial applications. 1 - .

7 . These proteins cover T 1– hese hese materials can be used directly as biomaterials for implants, as scaffolding 5 , rate slow its ­ - - - - Bombyx mori these domains lie subdomains that are rich in glycine, alanine, alanine, glycine, in rich are that subdomains lie domains these Within valine. or glycine-X threonine serine, of alanine, is composed X where repeats, primarily are regions crystalline spacers. The or segments linker hydrophilic small by linked blocks but the rate rate the but also a degradable material. Highly crystallized silk degrades slowly, applications. for biomedical polymer MPa 28–50 PLA trast, collagen has an ultimate tensile strength of 0.9–7.4 MPa and of strength tensile ultimate poly( and collagen as such biomaterials degradable polymeric used commonly to superior is silk worm Kevlar including best materials, the synthetic than greater is fibers silk of toughness The toughness. and strength mechanical high with materials protein-based the the assembles to form strong and resilient materials. The dominance of and serine tyrosine In addition to the impressive mechanical properties, silk fibroin is β

-sheet-forming regimes within the fibroin structure imparts imparts the fibroin within structure regimes -sheet-forming

B. mori 5–10 Days 5–10

3 Days in vivo in cocoons to fabricate hydrogels, tubes, sponges, 9 . Therefore, silk fibroin is an excellent candidate candidate excellent an is . fibroin Therefore, silk depends on the implantation site, mechanical implantation the on depends 6

2–5 Days 2–5 1 Day . The result is a hydrophobic protein that self-

B. mori 2 Days 2 1 Da

y silk fibroin silk fibers is 740 MPa.is 740 fibers silk In con 8 . In terms of strength, silk strength, of terms In . l -lactic acid) (PLA). The The (PLA). acid) -lactic

- -

© 2011 Nature America, Inc. All rights reserved. in overall in domains. In general, the degradation rate decreases with an increase of fibroin, as well as post-processing treatments, related to the content degradation rate of silk can be altered byerosion the surface modeby of processingmediated was the degradation enzymatic overall chains; the along locations multiple at protein silk the cleaved enzyme the time incubation increasing with that shown been has weeks, it 12 to and up protease XIV in incubated cells by metabolized generated peptides the with proteases, by mediated is degradation Silk material. silk the prepare to used processing the of features and environment used unlessotherwise noted. Note: The sources for the reagents and equipment described inthese protocols are givenonlyasexamples. Equivalent materials canbe TA the rate of degradation of silk biomaterials directly affected the the affected directly biomaterials silk of degradation of rate the Tissue Tissue engineering Drug Drug delivery Implant devices Disease models A B pplication β LE -sheet crystals and degree of organization of the noncrystalline 1 |

Biomedical applications of silk scaffolds. β -sheet content. However, it has also been shown that that shown been also has However,content. it -sheet Small molecule Growth factor delivery Drug delivery Mandibular defects Femur defects Anterior cruciate ligament kidney disease Autosomal dominant polycystic Breast cancer Skin Cervical tissue Vascular tissues Corneal Soft tissue Cartilage Bone T issue type 9 . B.mori 1 0 . In addition, the the addition, In . yarns have yarns been Electrospun Electrospun fibers Tubes Patterned silk films Hydrogels Aqueous sponges HFIP sponges Electrospun fibers Aqueous sponges HFIP sponges Electrospun fibers Aqueous sponges HFIP sponges Material format Spheres Spheres Spheres Aqueous sponges HFIP sponges Fibers Aqueous sponges Aqueous sponges HFIP sponges Electrospun fibers Aqueous sponges 2 64,6 2 3 7 34,68–7 3 1 5 1 5 0 bated in a humid environment for several hours. For more detailed Water annealing is the process in which the silk materials are incu wants to avoid an alcohol, the use of water can be annealing used. researcher the if but quick and anneal simple is immersion water Alcohol ing. by or ethanol or methanol as such alcohol an in Crystallinity can be induced via two methods, either by immersion formats described here, the final step is often to induce crystallinity. (1,1,1,3,3,3-hexafluoro-2-propanol, HFIP). long-term storage or used to produce materials in organic solvent protocols. the for lyophilized Alternatively, of be can solution the is an aqueous solution of pure silk fibroin that can be used in many are included in the ‘Silk fibroin extraction’ protocol. The end result 5 4 41,43,4 1 6 2 7 7 0 Once the extracted silk fibroin is processed into one of the several 5 48–5 43,45,4 66,6 6 61,6 5 2 3 7 54–5 5 3 58,5 4 1 6 31,5 7 2 0 9 6 6 3 natureprotocols Invivo controls. plastic culture tissue and lagen colto response the to similar was fibroin them implanting and on films MSCs the rat ing evaluated was films silk to response inflammatory the fibroin to assure biocompatibility. The from removed be must sericin Therefore, responses immune the of cause the was subsequent research has shown that sericin thought to cause allergies in some patients, was silk responses. Although immune tial coated in wax to prevent fraying and poten cal material for many applications. a solid basis for utilizing silk as a biomedi activity have been biological established, and provide and rate degradation as such silk fibroin processing, material properties osteogenesis rate of cells (hMSCs) and consequently altered the to those tissue of culture plastic similar response, inflammatory an of tive tumor necrosis factor- of levels have to shown was silk which in assessed been PLA response when compared with collagen and treated to remove the sericin. These steps steps These sericin. the remove to treated the raw silk from the cocoons first must be tocol. For each of the silk material formats, these studies are included this within pro in used formats material the generate to devices and drug release. The methods used implantable models, disease engineering, applications include four categories: tissue ( years recent in growing materials, cell and tissue studies have been bio of range a to silk of applications The the proceduresof Overview cutaneous in vivo several in used been has fibroin silk tion, metabolism of human mesenchymal­metabolism of stem As a suture material, silk fibers are often 1 2 . The inflammatory potential has also in in vivo studies for brain , silk showed a lower inflammatory ,showedlower a silk inflammatory in in vitro 1 6 and bone 1 2 . The in vitro in

| with hMSCs or hMSCs by with seed VOL.6 NO.10VOL.6 in in vitro 1 1 . Thus, links between 1 α with macrophages macrophages with 7 1 applications. , a cytokine indica 4 , soft tissue protocol Table response to silk | 2011 1 3 1 . In addi ). These These ). 1 5 | , sub

1613 9 ------.

© 2011 Nature America, Inc. All rights reserved. phy. For samples that have been d the values presented here will vary izedby avariety of Thisvalue can increase for shorter degum a has kDa.100 centereddistributionnearbroad weight molecular the min, 30 for phoresisand gel permeation chromatogra weighthas been determined by gel electro source of the silk.the and Fortime boiling example,the of thelength the molecularon protocol can be used changes. without bave fibers (raw silk textile yarn) in Step 5, and the be used in the process. If necessary, silk cocoons can appropriately ( be replaced with scaled be can volumes the required, is material more however, if protocol is designed to produce one batch from 5 g of silk cocoons; Our gels. and fibers films, earlier—sponges, listed types material the of any obtain to order in point starting the is protocol This Silk fibroin extraction water annealing. through induction crystalline of analysis systematic a describes information,we referby reader study to Hu the the the final aqueous-based solution takes 4 d. Figure 2 1614 ming times or decrease when degummed for protocol Squeeze outexcesswater and allowtodryovernight The resulting solution can be character be canresultingsolution The Remove silksolutionfrom Bombyx mori

| VOL.6 NO.10VOL.6 cocoons

| cassette Schematic of the silk fibroin extraction procedure. Going from the raw material (cocoons) to silk Fig. |

2011 2 techniquesbut some of ). Only cocoons that look undamaged should Cut cocoonsanddispose silk fibersandincubateat Add 9.3MLiBrontopof | natureprotocols 60 °Cfor4 of worm Centrifuge twice

depending e gummed h - - - - Add silk/LiBrtodialysi Figure 3 concentrated silk Dip mandrelinto Boil cocoonsfor30minin

remainder of the solution Store at4°C cassette 0.02 MNa et al. et

| Schematic for making dipped silk tubes. 1 8 , which 2 CO s 3 Allow silkbeadtorundown the mandrelandthen back tothetip of of the tube walls can also be varied by increasing or decreasing the tubes with various inner diameters can be obtained. The thickness ( tubes create thin-walled Dip method. nol overnight and then them rinse in sterile water before use. are required, incubate the tubes in a solution of 70% (vol/vol) etha There are tubes for tubes.twosilk sterilized preparing methods If Silk tubes (Step 25, options A and B) as applications, of in variety summarized a for used be then can silk Purified Dialyze againstultrapure Rinse fibersfor20min Repeat asnecessary water for48 times The first protocol focuses on a simple dip method to h Figures 3 Figures 3 methanol for5–10 Dip mandrelinto including sterile filtration, filtration, sterile including methods, by of a number be sterilized can solutions silk Aqueous h. 24 additional an within concentrated be also can tions solu Aqueous needed. be will d 3 tional addi an then required, is silk lyophilized T ential scanning calorimeter to determine the be prepared and thermally tested in a differ addition,thin thefilms silk solutionof can times boiling shorter for versa vice and timeslonger forweredegummed that samplesdecreasedvaluefor0.76. This was emissionvaluemin,the 30 for degummed For assessed. was nm 307 at band the and spectrophotometer fluorescence a in nm 280 at excited were copy.Briefly, dilute samples(0.1%, wt/vol) protein degradation via of fluorescenceamount the measure reproducibly and spectr also established a simple method to quickly longer times format are noted within each protocol. protocol. each within noted are format material each for sterilization of preferred methods The use. before the product sterilize final to simpler is it cases, some In system. each for out worked be should timing sterilization steam or dosing tion irradia therefore and fibroin, silk the of weight molecular the degrade to shown been have results), (unpublished claving as proce such dures, rigorous More autoclaving. and g (178 °C) and The extraction process takes 4 d. If If d. 4 takes process extraction The Fig. – 1 1 . 3 ; option A). With method,this silk s Dry for 1 30 3 . Wray and co-workers s γ T -irradiation m (192–203 °C)

Dry tubesovernightan ape ta were that samples soak insoapsolution to remove 20,2 γ -irradiation -irradiation 1 and auto and 1 9 . 1 3 1 have 3 In . d o­s ------

© 2011 Nature America, Inc. All rights reserved. concentrated silk solution as it is extruded through a small di sterile gels, we suggest that the solution be be solution the that gels, suggest we sterile To produce protocols. gel pH and (e-gel) interest, we refer the reader of to is the gel electrogel adhesive an If gels. stiffer obtain orderto in process gelation the after night ommend incubating the gels at 37 °C over within a few minutes. In some cases, we rec tively simple and weak gels can be obtained F) 25, (Step option (Step 25, option E) current electrical direct of application the option C) solutions silk ( aqueous gel to protocols mechanisms of variety a through produced be can hydrogels Silk options C–F) Silk hydrogelation protocols (Step 25, applicable) (if mine tube structure and to determine whether pores were formed conduits and in bioreactor systems create pores to PEO as such polymer sacrificial a byadding or geometries, ing wind different creating by tube, resulting the of diameter inner the change to order in size mandrel the changing by applications tubes resulting the to the nature the of spinning process, gel-spun tubes can add texture of Because formed. is tube the once methanol of addition the by completed is gelation Further gel. to induced is silk the needle, ( mandrel nique in which concentrated silk fibroin is extruded onto a rotating Gel-spun tubes. a fluorescent label and a confocal microscope and the diffusion havecharacteristics been studied with the use of morphology, overall for (SEM) microscopy electron scanning of formed. These tubes have been previously characterized by the use been have tubes the after water in out leached then and silk solution the to added be can kDa) 900 (PEO, oxide polyethylene of of thickness wall approximate 200 an with tubes formed have we method, this With mandrel. the on deposited layers of number Figure 4 sterilized and that the gelation protocol protocol gelation the that and sterilized Fig. Concentrate silkinPEG µ

m 5 ) through vortexing (Step 25, 25, (Step vortexing through ) solution

2 | 2 Schematic for making gel-spun tubes. . In addition, if porous tubes are required, various amounts 2 6 Fig. , sonication (Step 25, option D) 2 3 . These tubes have been evaluated for use as vascular 2 4 3 . ; option B). By applying high shear forces to the the to forces shear high applying By B). option ; The latter protocol a describes gel spinning tech 2 2 8 9 or by lowering the pH . Each method is rela is .method Each 2 3 2 . Silk tubes can be tailored to individual individual to tailored be can tubes Silk . 5 . Here we present present we Here . Extrude thesilkonto mandrel 22,2 4 . toused SEM can be deter 2 7 - - - ,

2 2 Figure 5 .

| Apply methanol Methods of preparing silk hydrogels. Vortexed gels a meter - - -

curve, for e-gels is around 1 mJ) values, adhesion is,that of force-strain areathe under normal the dynamic mechanical analyzer using stainless steel fixtures (the work silk adhesives,of the adhesive wereproperties using a determined the stiffness of a 5% (wt/vol) silk gel is around 100 kPa for the and determining the gel; of rheology stiffness for example, structure, secondary for spectroscopy infrared transform Fourier e-gels), for °C (~206 point melting the verify to calorimetry ning scan differential including techniques, few a with characterized sonication sonication time can be varied from 5 to 30 s at 10–20% amplitude. The silk concentration can be varied from 1% to 20% (wt/vol), and ing, sonication conditions should be optimized first cells.without in the gel. Once again, to ensure a proper time window for cell seed method is also useful when cells or compounds is a lag time between the sonication and the gelation,onset are of this to be encapsulated Sonication. vortexing conditions should be optimized first cells. without prior to gelation. To ensure a proper time window for cell seeding, Cells may be encapsulated into the gel after the vortexing step but 1-ml solution, generally probe around a 0.75 with ml issolution recovered the contact to to form needing the gel. without Vortexing. solution andremove Sonicated gels Soak tubeinsoap from mandrel This protocol produces silk gels from simple equipment This is a simple method to produce silk gels – natureprotocols humid humid environment. Silk hydrogels can be or stored capped in a keeping them tightly care to prevent the gels fromout by drying Take°C). (20–25 °C temperature room at 4 or either at stored be can they pared, pre been have gels the Once cycle. wet a in autoclaved be can solution silk the gels, sonicated of case the In necessary. if ing, filter after (wt/vol) 8% to back it trating the solution and then subsequently concen filter the solution, we recommend diluting 0.2- a through passing by sterilized be can solutions silk Aqueous field. sterile a in out carried be Electrogels + 28,2 9 . µ m filter. If it is difficult to difficult is it filter.If m

| VOL.6 NO.10VOL.6 protocol 2 | 6 2011 . In the case pH gels 2 2 7 6 . As there . From a From . |

1615 - - - - -

© 2011 Nature America, Inc. All rights reserved. PBS or water.orPBS Filmthickness morpholand films or concentrating for thicker or concentrating films films. In tion of the silk solution, diluting for thinner concentra the changing by controlled be siloxane (PDMS) mold. Film thickness can polydimethyl the prepare to used grating the changing by modified be can grooves 40 thick generally are protocol latter the ally it measuring by SEM. manually measuring either it with a micrometer or by by visu assessed be can ogy (vol/vol) ethanol overnight 70% and then rinsed in in sterile incubated be can they bioactive molecules incorporate to flexibility the H) offer techniques these based, option water 25, ( (Step solutions aqueous from nonpatterned films silk and G) option 25, (Step patterned both prepare to protocols present release.Herewe and ing, pattern cells, embed molecules of interest and test degradation Films provide a way to screen for materials Silk films (Step 25, options G and H) tunable adhesion. haveto advantageous is it which in applications in used be can it gel, this of nature reversible the of Because disassemble. to begin will earlier formed had that gel the and electrode positive newly had a negative voltage previously, the gel will begin to form on the switched. Thus, if a positive voltage is applied to the electrode that adhesive gel current. Electric adding cell culture medium CO 5% and °C 37 at incubated then and silk cated ml. A volume of 50 10 × 50 of to concentration up a brought and harvested were cells stem below. mesenchymal Separately,methods human by autoclaving 4% (wt/vol) silk and sonicating 5 ml it of using the gels sonicated into cells gel. haveexample,we encapsulated an As the in encapsulation effective and homogeneous provide to tion gela to the prior step and sonication after the added be can Cells Figure 6 1616 the within induced be can pores addition,

Patterned Nonpatterned protocol The patterned silk films generated from generated films silk patterned The

3 | 1 VOL.6 NO.10VOL.6 . The pattern depth and number of of number and depth pattern The . Add silkto substrate

| Schematic for making patterned and nonpatterned silk films. 2 8 . This gel is reversible if the polarity of the voltage the is of polarity the . reversibleis gel if This The material produced in this protocol is an an is protocol this in produced material The | 2011 µ 3 0 l l of the cell suspension was added to the soni . To sterilize silk films, overnight | natureprotocols Dry 2 7 . Fig. µ m

6 Water anneal - - ­ - ). As the processing is is processing the As ).

for 1day in in vitro Figure 7 Dissolve lipidin chloroform 2 or for 2 h before h 2 for

in in vivo | Schematic of silk microsphere preparation using DOPC.

Dry under nitrogen 6 cells per per cells solution tolipid Add silk/dru test from substrate Remove film film - - -

g the size of the microspheres and SEM to image the image to SEM and microspheres the of size the to determine spheres,nanometer the diffraction size of light laser have been used, including dynamic techniques lightfew scattering a spheres, to these determine To characterize thefilm. the casting to PVA or prior silk the solution tothe bysonicating or solutions the spheresof can be controlled by the changing concentration of 20 and nm 300 between range size a with spheres scaffolds, hydrogels or films. also be mixed with silk solution and then be prepared into porous can microspheres Resuspended up. scaling before process the in molecules.among various We suggest that small batches be tested differ will efficiency loading the therefore and silk the with ently SEM or ( alcohol, and (polyvinyl another polymer PVA; Step 25, option J) silk the between through use microspheres of phase of separation ( required when resuspended be can microspheres the and removed is lipid interest I) of option molecule 25, a (Step with solutions silk aqueous-based late ~2 ~2 is method this with prepared spheres of size particle average The emulsifier an as DOPC using microspheres silk into interest of process. the in solvents organic of avoidance and simplicity Fig. Lipid emulsifie The second protocol, which utilizes PVA, will generate silk silk generate will PVA, utilizes which protocol, second The The first protocol is an example on how to incorporate molecules µ the silk m, which can be determined with either optical microscopy optical either with determined be can which m, 8 ). This second method is particularly useful because of its its of because useful particularly is method second This ). 3 3 . As for drug loading, different drugs will interact differ interact will .drugs different loading, Asdrug for Fig.

s Freeze/thaw cycles then lyophilize 7 silk/drug microspheres Water isremovedbut ). The second procedure describes the formation formation the describes procedure second ).The remain coatedin a lipidlaye 3 3 . Once vesicles have been generated, the the generated, been have vesicles Once . hshcoie DP) o encapsu to DOPC) phosphocholine, acid fatty lipid (1,2-dioleoyl- The first process uses described compounds. an unsaturated therapeutic mol or small ecules factors, growth release and encapsulate to nano used be and can that micro- spheres the silk of describe preparation protocols following The Silk microspheres (Step 25, options I and J) information, please refer to Jin by immersion in water overnight. For more removed be can PEO the annealed, water casting. Once the films have been dried and PEO into the aqueous of silk solution prior to amounts various mixing by films silk r Resuspend in methanol methanol phase Centrifuge and suspended in remove lipid µ

sn m morphology. -glycero-3- et et al. 3 when needed 4 Resuspend . The size size The . 3 2 . 3 3 3 - - - - 4 .

© 2011 Nature America, Inc. All rights reserved. • • Electrogelated gels • • Vortexed gelsandsonicated gels • • • Silk andgel-spuntubes tubes • • • Concentrating silkfibroin • • • • Extraction REAGENTS M (vol/vol) 70% in mats the immersing by sterilized be can mats mandrel rotating interest of fibers are much smaller in diameter compared with conventionally spun produced fibers the but simple, relatively is required equipment large surface areas can be produced ( with fibers small-diameter which by process a is Electrospinning Electrospun silk fibers (Step 25, option K) be determined for each drug. on depending case molecular each weight, charge to and hydrophobicity, specific is and therefore efficiency it must Drug-loading drug. to silk 100:1 of ratio mass a loading with start we general, PVA. the with mixing to Inprior solution silk the to drug the ing drug model ­loading determine to used also was microscopy Confocal Figure 8 • Aqueous silksolution, 8% (wt/vol) pH gels Aqueous silksolution, 8%(wt/vol) Ultrapure water Aqueous silksolution, 8%(wt/vol) soap Dish http://www.fishersci.com/) Methanol (Acros Organics, cat. no. 176840010, Concentrated silkfibroin (PROCEDURE Step 24B) Ultrapure water http://www.sigmaaldrich.com/) PEG (10,000MW, Sigma-Aldrich, cat. no. P6667, Fibroin solution (7–8%, wt/vol); PROCEDURE Step 22) http://www.sigmaaldrich.com/) Lithium bromide (LiBr, Sigma-Aldrich, cat. no. 213225, Ultrapure water http://www.sigmaaldrich.com/) Sodium carbonate (Sigma-Aldrich, cat. no. 451614, Silk cocoons (Tajima or Shoji equivalent) Add aqueoussilk ATER solution toPVA 3 5 , and the process offers the ability to incorporate molecules I

3 | ALS 4 Schematic of silk microsphere preparation using PVA. . Drugs may be encapsulated in these spheres by add by spheres these in encapsulated be may Drugs . 3 6 . In addition, fibers can be aligned either by using a using by either aligned be can fibers addition, In . (sonication optional) Silk andPVAphase 3 7 separate or by inducing the Hall Effect Hall the inducing by or Dry silk/PVAfilm Fig. 9 ; Step 25, option K). The

Figure 9

with PEO Mix silk 3 8 . Electrospun Electrospun . silk/PVA film

Dissolve | Method to prepare electrospun silk fibers. Draw solution into asyringe Centrifuge and remove PVA -

• • Patterned silkfilms • Silk films • ther increase its mechanical properties mechanical its increase ther method, reinforcing agents can be added to tothe silk matrix fur processing this Through strength. mechanical greater have and pores the along surfaces havesmoother HFIP-basedsponges The processing. during matrix silk the into incorporated is molecule requiring an solvent.organic This may interest a be of bioactive if not of benefit the have and pores the between interconnectivity determined via determined liquid via displacement morphology and pore structure of these the materials. Porosity characterize can be to used be can SEM diameter. and height both in mm ~12 are that scaffolds nine least at produce will protocol are that 10 mm in height, and 15 mm in diameter secondthe and sponges silk aqueous-based produce will protocol first The ing. immersing them in 70% (vol/vol) ethanol overnight or by autoclav specifications. Both of these sponge types may be sterilized by either sponges shown that aqueous-based sponges degrade faster than HFIP-based cat. no. 184, SILELAST KIT0.5KG, http://www.ellsworth.com/) Polydimethylsiloxane (PDMS; Sylgard 184, Ellsworth Adhesives, Aqueous silksolution, 8%(wt/vol) Aqueous silksolution, 8%(wt/vol) http://www.sigmaaldrich.com/) Hydrochloric (0.3MHCl, acid Sigma-Aldrich, cat. no. H1758, Apply apositivevoltageto the syringetoinitiateajet when necessary 1 Resuspend 1 , which enables the user to tailor these scaffolds to project + Soak fibersinmethanolfirstand then watertoremovethePEO natureprotocols aqueous-based sponges have excellent excellent have sponges ­aqueous-based r FPbsd scaffolds HFIP-based or protocols to form aqueous-based tions, as applica shown in several for material scaffolding provide a versatile Silk poroussponges 3D Silk sponges (Step 25, options L and M) and orientation. diameter morphology, fiber the examine uniform.fairly appear SEM can tobe used should and nm 800 than less of diameters tropic mat of silk fibers. The fibers will have them rinsing in either sterile water or PBS. subsequently then and overnight ethanol This This protocol produces a nonwoven iso 4 0 . 4

1 | . In addition, it has been been has it addition, In . Table VOL.6 NO.10VOL.6 The resultingfiberswillbe under 800nmindiameter 1 . Here we provide 4 protocol 0 ( Fig. | 2011

3

9

1 ( 1 Fig. ). The The ). |

1617

1 0 ) - - - - © 2011 Nature America, Inc. All rights reserved. • • • • • • • • Extraction EQUIPMENT • • • • • HFIP-based sponges • • • Silk sponges • • • • Electrospinning • • • Microspheres (PVA) • • • • • • • Silk microspheres usingDOPC Figure 10 1618 Ultrapure water PVA (MW 30–70 kDa; Sigma, cat. no. P8136, http://www.sigmaaldrich.com/) Aqueous silksolution, 8%(wt/vol) Ultrapure water Drug, or molecule proteininterest small of Nitrogen gas http://www.sigmaaldrich.com/) R4828000-4C, no. cat. (Sigma-Aldrich, Methanol Chloroform (Fisher Scientific, cat. no. C298-1, http://www.fishersci.com/) cat. no. 850375P, http://www.avantilipids.com/) 1,2-Dioleoyl- Aqueous silksolution, 8%(wt/vol) protocol cat. no. 02-591-33, http://www.fishersci.com/) beakerPlastic (2liters; Fisher Scientific, cat. no. 140002000, http://www.fishersci.com/) Glass beaker (2liters; Kimble Chase Kimble, cat. no. 140001000, http://www.fishersci.com/) Glass beaker (1liter; Kimble Chase Kimble, cat. no. 1400050, http://www.fishersci.com/) Glass beaker (50ml; Kimble Chase Kimble, http://www.fishersci.com/) Spatula (Fisher Scientific, cat. no. 14-373-25A, cat. no. 14-513-56, http://www.fishersci.com/) bar(FisherLarge stir Scientific, cat. no. 14-513-62, http://www.fishersci.com/) bar(FisherSmall stir Scientific, http://www.staples.com/) Titanium scissors (Staples, cat. no. 487905, http://www.fishersci.com/) Methanol (Acros Organics, cat. no. 176840010, Ultrapure water http://www.fishersci.com/) NaCl (Fisher Scientific, cat. no. BP358-1, http://www.sigmaaldrich.com/) Sigma-Aldrich, cat. no. 105228-100G, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; Lyophilized silkfibroin Ultrapure water http://www.fishersci.com/) (FisherSalt Scientific, cat. no. BP358-1, Aqueous silksolution, 6–8%(wt/vol) Methanol (Acros Organics, cat. no. 176840010, http://www.fishersci.com/) Ultrapure water http://www.sigmaaldrich.com/) Polyethylene oxide (PEO, 900kDa; Sigma-Aldrich, cat. no. 189456, Aqueous silksolution, 8%(wt/vol) silk solutionto Add aqueous the glassvial

| VOL.6 NO.10VOL.6

| Schematic for making aqueous-based silk sponges. sn -glycero-3-phosphocholine (DOPC; Avanti Polar Lipids, evenly distributesalt the silk.Rotateto | 2011 Pour saltover | natureprotocols

silk togel

Allow

Figure 11 Dissolve silkin HFIP

Remove salt | Method for preparing HFIP-based silk sponges.

solution oversalt Add silk/HFIP • • • • • • • • • • • • • Concentrating silkfibroin • • cat. no. 7751030, http://www.fishersci.com/) Lyophilizer lyophilized (optional: useonly if silkisrequired, Labconco, Fixed-angle rotor (Eppendorf, cat. no. 022637207, http://www.fishersci.com/) Centrifuge (Eppendorf 5804R, cat. no. 022623501, http://www.fishersci.com/) Conical tubes (50 ml; BD Medical, cat. no.Needles (18G; BDMedical, cat.14-432-22, no. 305195, http://www.fishersci.com/) http://www.fishersci.com/) (20ml;Syringe BDMedical, cat. no. 309661, http://www.fishersci.com/) fishersci.com/) Dialysis Scientific, cassette buoy (Thermo cat. no. 66432, http://www. http://www.fishersci.com/) Beaker (1liter; Kimble Chase Kimble, cat. no. 140001000, http://www.fishersci.com/) Beaker (100ml; Kimble Chase Kimble, cat. no. 14000100, http://www.fishersci.com/) Slide-A-Lyzer dialysis cassette (0.5–3.0ml; Scientific, Thermo cat. no. 66330, Needles (18G; BDMedical, cat. no. 305195, http://www.fishersci.com/) (3ml;Syringe BDMedical, cat. no. 309585, http://www.fishersci.com/) (10ml;Syringe BDMedical, cat. no. 309604, http://www.fishersci.com/) Kimwipes (Kimberly-Clark, cat. no. 34256, http://www.fishersci.com/) Scientific, cat. no. ULT2586-9SI-A, http://www.fishersci.com/) Freezer ( scaffold foruse Store orcut

− Remove salt

80 °C; optional: use only if lyophilized80 °C; optional: useonly if silkisrequired, Thermo allow HFIPtoevaporate Once silkpenetratesto the bottomofvial, • • • • • • • • • cat. no. 66110, http://www.fishersci.com/) Scientific, (Thermo 3–12 mlcapacity Slide-A-Lyzer dialysis cassette 3500MWCO, cat. no. 08-557C, http://www.fishersci.com/) Graduated cylinder (50ml; Fisher Scientific, cat. no. 380000000, http://www.fishersci.com/) Hot handprotectors Products, (Bel-Art Orion HR-200, http://www.americanweigh.com/)Analytical balance (A&D Weighing, modelcat. no. 08-732-112, http://www.fishersci.com/) (Fisher Scientific, boat Small weigh 08-732-113, http://www.fishersci.com/) Medium (Fisher Scientific, boat weigh cat. no. cat. no. 08-732-115, http://www.fishersci.com/) (Fisher Scientific, boat Large weigh 100H, http://www.fishersci.com/) Hot plate (Fisher Scientific, cat. no. 11-100- Aluminum foil Scaffold isready Add methanolto induce β -sheets

Remove methanol

© 2011 Nature America, Inc. All rights reserved. • Silk microspheres usingDOPC • • • • • • • • • Patterned silkfilms • • • Silk Films • pH gels • • • Electrogelated gels • • • • Sonicated gels • • • • Vortexed gels • • • • • • • tubes Gel-spun • • • • • Silk tubes • • • • http://www.fishersci.com/) Round-bottomed (Fisher tubes Scientific, glass cat. no. 14-961-29, http://www.fishersci.com/) Needle BDMedical, unimportant; (gauge cat. no. 305195(18G), http://www.fishersci.com/) Ethanol (70%, vol/vol); Ricca Chemical, cat. no. 2546701, Aluminum foil http://www.fishersci.com/) Pipette (5ml; BD Vacutainer Labware Medical, cat. no. 357543, Paper grocery) cup(local Kimwipes (Kimberly-Clark, cat. no. 34256, http://www.fishersci.com/) Tweezers (Fisher Scientific, cat. no. 08-880, http://www.fishersci.com/) Vacuum oven (Fisher Scientific, cat.cat. no.no. NT43-208, http://www.edmundoptics.com/) 13-262-280A, http://www.fishersci.com/)Optics, 600 grooves mm change depending on requirements; anexample here isshown (Edmund size, grating: grooves groovesDiffraction number of and depth of can http://www.fishersci.com/) Vacuum desiccator Products, (Bel-Art cat. no. 420200000, Tweezers (Fisher Scientific, cat. no. 08-880, http://www.fishersci.com/) cat. no. 08-757-12, http://www.fishersci.com/) Petri dish, non–tissue culture treated (100mm; Fisher Scientific, http://www.fishersci.com/) (1.8ml; Fisher Scientific,Glass vials cat. no. 03-339-26A, Conical tubes (50 ml; BD Medical, cat. no. cat.14-432-22, no. 1987, http://www.surepure.com/) http://www.fishersci.com/) Platinum (0.032indiameter, wires 1foot; Surepure Chemetals, PowerDC supply (Agilent E3634A, http://www.agilent.com/) Conical tubes (15 ml; BD Medical, cat. no. 352096, http://www.fishersci.com/) http://www.fishersci.com/) Tapered Microtip (1/8-inch diameter, Branson, cat. no. 101-148-062, http://www.fishersci.com/) Externally Threaded Disruptor Horn (1/2-inch, Branson, cat. no. 101-147-037, Ultrasonicator (Branson 450, cat. no. 101-135-022, http://www.fishersci.com/) (3M, tape Duct cat. no. 3939, http://www.fishersci.com/) Glass (Fisher vials Scientific, cat. no. 03-339-26A, http://www.fishersci.com/) (1ml;Syringe Air Tite Products, cat. no. A1, http://www.fishersci.com/) Vortexer (Fisher Scientific, cat. no. 02-215-365, http://www.fishersci.com/) Kimwipes (Kimberly-Clark, cat. no. 34256, http://www.fishersci.com/) (e.g., 0.5 mm, McMaster-Carr, cat. no. 8908K21, http://www.mcmaster.com/) Stainless steel wire; gauge dependwill on required inner diameter http://www.mcmaster.com/) (27gauge;Blunt-tip McMaster-Carr, needle cat. no. 75165A688, http://www.mcmaster.com/) (30gauge;Blunt-tip McMaster-Carr, needle cat. no. 75165A31, http://www.mcmaster.com/) (14gauge;Blunt-tip McMaster-Carr, needle cat. no. 75165A672, Luer-Lok syringe (1 ml; BD Medical, cat. no. 309628,Mandrel rotation system, custom made tohttp://www.fishersci.com/) rotate andtransverse Styrofoam Tweezers (Fisher Scientific, cat. no. 08-880, http://www.fishersci.com/) Conical tube (15 ml; BD Medical, cat. no. 352096, http://www.fishersci.com/) Feather (EMS, scalpel cat. no. 72042-20, http://www.fishersci.com/) http://www.mcmaster.com/) inner diameter (e.g., 0.5mm, McMaster-Carr, cat. no. 8908K21, Stainless steel cut to wire depend on 3-inch required lengths; will gauge http://www.fishersci.com/) Microcentrifuge (2mlcapacity, tubes Fisher Scientific, cat. no. 05-408-138, Aluminum foil http://www.fishersci.com/) Small dialysis Scientific, cassette buoy (Thermo cat. no. 66430, Large stir bar (Fisher Scientific, cat. no. 14-513-56, http://www.fishersci.com/)

−

1 , 50×mm, grating, diffraction 1,000nmruled

2

3

• • • • • • • • • Microspheres (PVA) • • • • • • • • • • • • • • • • • • • Silk Sponges • • • • • • • • • • • Electrospinning • • • • • • • • Conical tube (15 ml; BD Medical, cat. no. 352096, http://www.fishersci.com/) (5ml;Syringe BDMedical, cat. no. 309603, http://www.fishersci.com/) http://www.fishersci.com/) filterSyringe unit(pore size 0.45 Hot plate (Fisher Scientific, cat. no. 11-100-100H, http://www.fishersci.com/) Small stir bar (Fisher Scientific, cat. no. 14-513-62, http://www.fishersci.com/) http://www.fishersci.com/) Graduated cylinder (10ml; Fisher Scientific, cat. no. 08-557A, http://www.fishersci.com/) Glass beaker (50ml; Kimble Chase Kimble, cat. no. 1400050, Spatula (Fisher Scientific, cat. no. 14-373-25A, http://www.fishersci.com/) Small weigh boat (Fisher Scientific, cat. no. 08-732-112, http://www.fishersci.com/)http://www.fishersci.com/) 5417R,Microcentrifuge (Eppendorf cat. no. 022621700, Lyophilizer (Labconco, cat. no. 7751030, http://www.fishersci.com/) http://www.fishersci.com/) Freezer ( Small stir bar (Fisher Scientific, cat. no. 14-513-62, http://www.fishersci.com/) http://www.fishersci.com/) Beaker (100ml; Kimble Chase Kimble, cat. no. 14000100, Conical tubes (50 ml; BD Medical, cat. no. Conical14-432-22, tubes (15 ml; BD Medical, http://www.fishersci.com/) cat. no. 352096, http://www.fishersci.com/) http://www.fishersci.com/) Microcentrifuge (2.0ml; tube Fisher Scientific, cat. no. 02-681-258, http://www.fishersci.com/) Microcentrifuge (1.5ml; tube Fisher Scientific, cat. no. 054-08-129, Conical tubes (50 ml; BD Medical, cat. no. Hot14-432-22, plate (Fisher Scientific, cat. no.http://www.fishersci.com/) 11-100-100H, http://www.fishersci.com/) Large stir bar (Fisher Scientific, cat. no. 14-513-56, http://www.fishersci.com/) http://www.fishersci.com/) beakerPlastic (2liters; Fisher Scientific, cat. no. 02-591-33, (5ml;Syringe Air Tite Products, cat. no. A5, http://www.fishersci.com/) http://www.fishersci.com/) Cylindrical molds(5ml; Fisher Scientific, cat. no. 03-338-1E, http://www.americanweigh.com/) balance (A&D Analytical Weighing, HR-200, Orion model Small weigh boat (Fisher Scientific,Sieve cover (Fisher Scientific, cat.cat. no. 04-888B, http://www.fishersci.com/) no. 08-732-112, http://www.fishersci.com/)http://www.fishersci.com/) 850 Sieves (Fisher Scientific,pore various sizes, e.g., 1mm, cat. no. 04-884-1AJ; Alligator clips (McMaster-Carr, cat. no. 7236K252, http://www.mcmaster.com/) wire Insulated electrical http://www.fishersci.com/) Reciprocating shaker (MaxQ, Scientific, Thermo cat. no. 11-675-152, Nonstick aluminum foil (Reynolds Wrap) http://www.glassmanhv.com/) High voltage EH, supply (GlassmanSeries cat. no. EH30P3, Syringe pump (Fisher Scientific, cat. no. 14-831-200,http://www.mcmaster.com/) http://www.fishersci.com/) (16gauge,Blunt-tip needle McMaster-Carr, cat. no. 75165A552, (10ml;Syringe BDMedical, cat. no. 309604, http://www.fishersci.com/) Hot plate (Fisher Scientific, cat. no. 11-100-100H, http://www.fishersci.com/) Small stir bar (Fisher Scientific, cat. no. 14-513-62, http://www.fishersci.com/) http://www.fishersci.com/) (20ml; Fisher Scientific, vials Glass scintillation cat. no. 03-337-15, http://www.fishersci.com/) Reciprocating shaker (MaxQ, Scientific, Thermo cat. no. 11-675-152, http://www.fishersci.com/) Tapered Microtip (1/8-inch diameter, Branson, cat. no. 101-148-062, cat. no. 101-147-037, http://www.fishersci.com/) Externally Threaded Disruptor Horn (1/2-inch, Branson, Ultrasonicator (Branson 450, cat. no. 101-135-022, http://www.fishersci.com/) Tweezers (Fisher Scientific, cat. no. 08-880, http://www.fishersci.com/) Parafilm (Pechiney Plastic Packaging, cat. no.cat. no. 08-757-12, http://www.fishersci.com/)PM996, http://www.fishersci.com/) Petri dish, non–tissue culture treated (100mm; Fisher Scientific, Conical tubes (50 ml; BD Medical, cat. no. 14-432-22, http://www.fishersci.com/) µ m, 04-884-1AK; 600

−

80 °C; Scientific, Thermo cat. no. ULT2586-9SI-A, natureprotocols µ m, 04-884-1AM; 500 µ m; Millipore, cat. no. SLHA033SS,

| VOL.6 NO.10VOL.6 µ m, 04-884-1AN; protocol

|

2011

|

1619 © 2011 Nature America, Inc. All rights reserved. 13| fore, multiply the amount of the dried silkfibroin by4toobtainthe total volume of 9.3MLiBr needed ( fibroin available. As 20% of the solution willbe silk, 80%willbeLiBr. Thatis, a ratio of 1:4(1gto4ml)silkLiBr. There 12| Dissolve silk fibroin in of the boiling step on the label. temperature. For long-term storage, place it in a clean plastic bag or wrap it in aluminum foil. Be sure to indicate the length  11| 10| 9| 8| ! 7| ! silk. Discard the sodiumcarbonate solution inthe sink. 6| indicate thisonthe batchlabel.Increasing the boiling time willdegrade the fibroin.  promote good dispersion of fibroin. 5| ! 4| 3| cocoon pieces into alarge weigh boat. 2| should not beused. ! 1| Fibroin extraction PROCE • • • • • HFIP-based sponge 1620 http://www.fishersci.com/) (20ml; Fisher Scientific, vials Glass scintillation cat. no. 03-337-15, http://www.americanweigh.com/) balance (A&D Analytical Weighing, HR-200, Orion model http://www.fishersci.com/) (Fisher Scientific, boat Small weigh cat. no. 08-732-112, Sieve cover (Fisher Scientific, cat. no. 04-888B, http://www.fishersci.com/) cat. no. 04-884-1AN; http://www.fishersci.com/) 850 Sieves (Fisher Scientific,pore various sizes, e.g., 1mm, cat. no. 04-884-1AJ; protocol

CAUT CAUT CAUT CAUT

PAUSE CR Prepare a9.3MLiBrsolution. Calculatethe amount of 9.3Mlithiumbromide needed toprepare a20%(wt/vol)solution basedonthe amount of dried Allowthe silkfibroin to dry inafume hood overnight. Afterthe third wash,remove the silk,squeezeitwelland then spread itoutonacleanpiece of aluminum foil. µ

Repeat Steps7and 8twice for atotalof three rinses. Rinse the fibroin inwater for 20minwhile gently stirring onastirplate. Place fibroin ina1-literbeaker filledwith1liter ofultrapure water and astirbar. Remove the silkfibroin withaspatulaand coolitbyrinsing inultrapure coldwater. Squeeze excess waterout of the Add the cocoonpieces once the waterstartstoboil and continue boiling for 30min.Occasionally, stirwithaspatulato Add the measured sodium carbonate to the water and let it completely dissolve (to prepare a 0.02 M solution of Na Measure 4.24gof sodiumcarbonate inamedium weigh boat. Meanwhile, cutcocoons withtitaniumscissorsinto dime-sized pieces and disposeof silkworms. Measure out5gof Prepare a2-literglassbeaker filledwith2liters of ultrapure water, coveritwithaluminum foil and heat until boiling. | I m, cat. no. 04-884-1AK; 600 VOL.6 NO.10VOL.6 T D I I I I I ON ON ON ON URE CAL

PO Ifyouare using aplastic beaker, ensure thatthe hot platehascooled. Silkfibroin and solution willbe hot; usehand protectors. Ifwaterisboiling, add sodiumcarbonate slowlytoavoid boiling over. Donot leavethe beaker unattended whileheating and boiling. Becauseof high temperatures, plastic beakers

I STEP NT s Degummed silk fibroin, in which the sericin has been removed, can be stored indefinitely at room | 2011 To increase reproducibility, boilfor exactly 30mineverytime. Ifboiling for longer orshorter times, ●

T | IMI natureprotocols L iBr iBr N µ G m, cat. no. 04-884-1AM; 500 ● 2.5hactive, overnight drying(day1)

T    IMI 86 . . 85 N G mo 4.5 h (day 2) g l      

3 9

mo L µ l m,   

  

1 , 000 1 L ml • • • • • • • •    Conical tubes (50 ml; BD Medical, cat. no. Fume hood 14-432-22, http://www.fishersci.com/) Hot plate (Fisher Scientific, cat. no. 11-100-100H, http://www.fishersci.com/) Large stir bar (Fisher Scientific, cat. no. 14-513-56, http://www.fishersci.com/) http://www.fishersci.com/) beakerPlastic (2liters; Fisher Scientific, cat. no. 02-591-33, (5ml;Syringe Air Tite Products, cat. no. A5, http://www.fishersci.com/) http://www.fishersci.com/) PM996, no. cat. Packaging, Plastic (Pechiney Parafilm http://www.fishersci.com/) (1.8ml; Fisher Scientific,Glass vials cat. no. 03-339-26A, ) ( X = ______g oof Li Br X ).

2 SO

3 ). - © 2011 Nature America, Inc. All rights reserved. the silkwillbe stablefor yearsatroom temperature and canbereconstituted inHFIP. The concentrated solution (20–30%, or concentrated (option B). Forstorage for longer than1month, the silk solution should belyophilized. Inthisform, 24| L therefore another batch will need to be extracted. eventually gel but gelation times will vary. Once the silk has gelled, it cannot be used for protocols that require solution and  culents ordark particulates, itisbesttorecentrifuge toremove them. tinted yellowbutshould berelatively clearand slightly more viscousthanwater. Ifthere are impurities such aswhitefloc  it by0.5ml.Thiswillyield the weight pervolume percentage. of the silksolution tothe boatand allowittodry at60°C.Once the silkisdry, determine the weight of the silk and divide 23| 22| centrifuge tube. Besure toleaveany whiteflocculent orbrown matter behind. 21| 20| ing on the volume, either split it between two19| tubes (if more than 40 ml) or fill one tube and use a counterbalance of water. following day (i.e., six changes within48h). netic stirplate. Change the waterafter1h,4thatevening, the next morning and night, as wellasinthe morning onthe 18| Remove the extra needle once allthe airhasbeenpurged. the cassette. Moreover, haveanadditional needle and insert itinto another topportof the cassettetoallowairescape. tion whenever possibletoavoid the induction of step willbeeasier ifthe solution iskept warmbefore adding tothe cassette. It isimportant toavoid shearing the solu  17| 16| Dialysis and centrifugation ? highly viscousbutshould not contain any intact fibers, as determined byvisual assessment. color and willbetransparent. Blackbitsfrom the silkwormmay bevisiblebutwillremoved later. Thissolution willbe 15| solution. covered and dissolvedbythe LiBr. It isalsohelpful tousethe smallest glasscontainer thatwillstillhold the silkand LiBr  14| small stirbar. suggest adding only60%of the calculatedvolume of waterand then bringing the solution uptothe final volume. Stirwitha  volumes, werecommend carrying thisoutonice. ! wt/vol) canbe useddirectly for preparing silktubes.

yophilization yophilization and concentration

CAUT TROU

PAUSE CR CR CR CR Remove silk from the cassettes with another 20-ml syringe and an 18-gauge needle. Place silk in a 50-ml conical tube. Depend The fibroin solution (25mlatconcentration 7–8% (wt/vol))caneither beused asisoritcanbelyophilized(option A) To determine the concentration of the silkinsolution, measure the weight of asmall weigh boat.Thereafter, add 0.5ml RepeatSteps20and 21again. Carefully remove tubesfrom the centrifuge and either pourortransfer the silksolution witha25mlpipetteinto another Centrifuge toremove impurities. Placeinacentrifuge and spinat9,000r.p.m. (~12,700 Dialyze against 1literof ultrapure waterper12mlcassette. To ensure mixing, usealarge stirbarand placeonamag Witha20-mlsyringe and an18-gauge needle, insert 12mlof the silk-LiBrsolution into a3–12-mldialysis cassette. Hydrate dialysis cassettesinwaterfor afewminutes. Letfibroin dissolveinan oven at60°C for 4h.Once the silkfibroin iscompletelydissolved, itwillappearamberin Pack silkfibroin tightly into a50-mlglassbeaker and add the required amount of LiBrsolution ontop. I I I I T T T T I I I I I B ON CAL CAL CAL CAL LES

PO Adding LiBrtowaterresults inanexothermic reaction; bemindful of the heat generated. When preparing large

H I STEP STEP STEP STEP NT OOT The silk solution can be stored at 4 °C for at least a month. Depending on the purity, stored silk will Abatchof 5gof silkcocoons generally yields 25mlof 7–8%(wt/vol)silksolution. The solution willbe Becareful not topuncture ortouch the dialysis membrane. The solution willbeveryviscous, and this The LiBrmust beadded tothe silkrather thanadding silktothe LiBrsothatthe silkwilleventually be LiBrhasalowdensity and itsvolume should betaken into account whilepreparing the solution. We I N G ●

T IMI N G 49 h (days 2–4) β -sheet withinthe silk.Therefore, onlyusethe needle when injecting into natureprotocols g ) at4°Cfor 20min.

| VOL.6 NO.10VOL.6 protocol | 2011

-

|

1621 - - -

© 2011 Nature America, Inc. All rights reserved.

(B) (B)

by gel spinning, wherein the fibroin is extruded onto a rotating mandrel (option B, (prepared in Step 24B) you can prepare silk tubes25| by either a simple dip method to create thin-walled tubes (option A, ( 1622

(xvii) (viii) (xiii) Placethe cassetteinto 10%(wt/vol)PEGsolution and coveritwithaluminum foil. (xiv) (xvi) protocol A (xii) Attach the dialysis buoy onto the topof the cassette. (vii) (xv) Remove the concentrated silksolution afterthe desired amount of time using a3-mlsyringe and an18-gauge needle. (iii) (iii) (xi) Remove the needle/syringe from the cassette. (vi) (iv) Draw out10mlof 7–8%(wt/vol)silksolution withthe 10-mlsyringe without aneedle. (ix) Afterairisremoved, withdraw the extra needle from the cassette. (iv) (ii) (ii) Hydrate dialysis cassetteina100-mlbeaker filledwithultrapure water for afewminutes. ) (v) (v) Attach the 18-gauge needle tothe syringe filledwithfibroin solution. (x) (i) Divide the aqueoussilksolution into 50-mlconical tubeswithno more than20mlpertube. (i) The silk fibroin can now be used to prepare a number of different materials ( C L

| oncentrating oncentrating silk fibroin (optional) yophilization (optional) VOL.6 NO.10VOL.6 cassette atthe edges and do not touch the membrane. available, silkcanalsobefrozen at allow the airtoescape. dissolve, move toStep24B(ii). magnetic stirplate. The solution should beclearonce the PEGiscompletelydissolved. Whilewaiting for the PEGto  Measure the solution concentration by weighing out a small weigh boat and then adding 0.1 ml of concentrated silk up toaweek,before itgels. Onlyconcentrate silk when itisintended tobeusedrelatively soon.   resulting silk solution should be very viscous and will appear slightly cloudy when compared with thevisually starting determined solution. whether multiple beads form without coalescing. If this occurs, then the solutionit in isthe not intended ready. protocol. The For example, for dipped tubes, a mandrel can be dipped into thegel solution after it andhas been then stored it can for be too long (approximately 1–2 weeks). The solution can be checked by attemptingdays will allowto use it to reach the required concentration. It is suggested that the silk be checkedcassette daily, and allow as thethe dialysissolution tocan proceed for more time or it can be stored at 4 °C. We find that determinestoring whetherthe silk the silkfor is aconcentrated few enough. If the silk is collected too early, it can either be replaced into a silk in the cassette if it is left in the PEG solution for too long. With experience, the researcher will be able to visually may need to be altered for each batch. The concentration time is not linear, so care must be taken to avoid gelling the solution after dialyzing against 10% (wt/vol) PEG for 20–22 h. The amount of time used to concentrate the solution  divided by the volume used (0.1 ml) will yield the weight per volume percent. tips or cutting off the tip of the pipette. Dry the solution at 60 °C and then weigh the dried film. The weight of the silk solution to the boat. Because of the high viscosity of concentrated silk, we suggest using either large-orifice pipette  longer feelcold. Remove from lyophilizer, capand store atroom temperature (20–25°C). Place vertically ina Add 100gof PEGto900mlof ultrapure watertoprepare a10%(wt/vol)PEGsolution. Mixwithalarge stirbarona As the cassettebecomes inflated, purge excess airbyinserting another 18-gauge needle into the other topportto Indicate the time and date thatthe cassettewasadded tosolution. Fordip-method tubes, the silkisgenerally Insert the tipof the needle into atopportof the cassette. Finish filling the cassettewiththe remaining fibroin solution. Although the cassette indicates a maximum volume of Remove the cassettefrom the waterand tapthe bottomof the cassetteonapapertoweltodry. Besure tohold the Remove the needle and transfer the solution to2-ml microcentrifuge tubes. Slowly insert the fibroin solution into the cassette. Place frozen samplesonalyophilizer for 2–3duntil allof the waterisremoved from the solution. The tubewillno Fold aKimwipe overthe topof the tubeand attachwitheither arubberband ortape. Keep capsfor lateruse. concentrated for 20 h. For the gel-spun tubes, the solution must be more viscous and therefore is concentrated for ~22 h. 3.0 ml,10mlcanbeinserted. During the concentration process thisvolume willdecrease. 

CR CR PAUSE CR CR PAUSE I I I I T T T T I I I I CAL CAL CAL CAL

PO PO | 2011

I STEP I STEP STEP STEP NT NT Labeland store at4°Cfor afewdays. Concentrated silkcanonlybestored for afewdays, possibly The lyophilizedmaterial canbestored atroom temperature indefinitely. |

Depending on the initial silk concentration, 10 ml of silk fibroin will yield 2–4 ml of concentrated silk natureprotocols Avoid high shear thatmay allowpremature alignment of the Becareful not tointroduce bubblestothe solution, asthey are verydifficult to remove later. Becareful toinsert justthe tipof the needle toavoid puncturing the membrane.

−

80 °Cfreezer for several hours until the solution iscompletelyfrozen. Ifa

●

T IMI N G

− 3 d

● 20 °Covernight orplacedinliquid nitrogen until the solution isfrozen.

T IMI N G G 21–25 21–25 h

Fig. Fig.

1 ). Using the concentrated solution

4

). Alternatively, silk solution may be used β -sheets inthe silkfibroin.

−

80 °Cfreezer isnot Fig.

3

) or

© 2011 Nature America, Inc. All rights reserved.

( storage conditions). (B) Gel-spuntubes silk fibers (option K, films (options G and H, to prepare hydrogels via vortexing, sonication, electrical current or pH change (options C–F,

(

(viii) (viii) (viii) C A (vii) Allowthe methanol to dry, and then placethe mandrel into asolution of soapywater. (vii) When silkisdry, placethe wires coatedwithsilkinto a15-mlconical tubefilledwithsoapywaterand soak for 1h. (iii) Letexcess silkbeadsdrip off of the wire backinto silksolution. (iii) Remove the needle and replace witha30-gauge needle. (ix) (xi) (vi) (vi) (iv) (iv) Startthe mandrel rotation system. (ix) ) Vortexed gels (ii) (ii) ) Dippedtubes (x) Store the silktubeina plastic Petri dishatroom temperature for upto1year(depending ontubetreatment and (x) (v) (v) (i) Placethe mandrel in the mandrel rotation system. (i) (i) Adjust the concentration of an8% (wt/vol)silksolution to2–5% (wt/vol). including airbubbles. ? wire (trytocoatevenly). When the beadscoalesceatthe bottomof the wire, flipitand allowthe silktorunback down toward the end of the beads of silktoevenlyrundown the wire. ­readily move, continue tosoakitinthe soapsolution. are difficultto remove. Ifbubblesare present, they willcreate holes inthe resulting silktubes.  amount tothe concentrated silksolution. Gently stirwithastainlesssteelwire. 1 htoremove residual soap. induce couple of years. They are brittle, sowesuggest thatthey behydrated inwaterbefore being manipulated. ? scrunching the tube, then placethe tubebackinto soapywaterand waitbefore trying again.  making anexcess number of tubes, assome of the tubesmay crack during the drying process.  Fill aplastic Petri dish(10cm)withultrapure water. Placethe tubeinwaterand incubate in a shaking waterbathfor When the tubehassoftened, grab the tubeuniformly withaKimwipe and gently pullitoff. Ifthe tubedoes not Store silktubesinaplastic Petri dishatroom temperature. These tubescanbestored for several months uptoa Dry tubesbysticking the invertedsilk-coatedwire into styrofoam and placeitinafume hood overnight. We suggest Attach a14-gauge needle ona1-mlsyringe and slowly draw upthe concentrated silksolution. Besure toavoid (Optional) Ifporous tubesare required, prepare asolution of 7–8%(wt/vol)PEOand carefully add the appropriate Once the silkhasbeenlaid onto the mandrel, applymethanol ontopof the silkwithaneedle and syringe inorder to Fill aplastic Petri dish(10cm)withultrapure water. Placethe tubesinthe waterand incubate onashaking water Place the wires inavial of methanol for 5–10s. Squeeze the gel solution outof the needle and onto the mandrel. The fibershould beuniform without any beadsor Dip stainlesssteelwire into concentrated silksolution. Remove the wire from the silkand invertthe wire, allowing the Remove the tubefrom the wire using tweezers. The tubeshould slide off easily. Ifitisdifficultto remove without Repeat dipping process and methanol treatment 2–4times oruntil the wire issufficiently coatedwithsilk.The silk After soaking, remove the tubes from the soapy water and cut the end of the silk tube with a scalpel or a razorsoaking,remove a blade. or After and the fromthe cut scalpel water end the a tubes ofsoapy with thetube silk ? and motor. layers of transverse the number of the on speed the depend will method this with obtained thickness wall The and layer reversed. one rotation after the then stopped be should system rotation mandrel the required, is fiber a If direction same generated. the be in can running design cross-hatched a forth, and back oscillates mandrel the and constant is rotation  replace the needle witha 27-gauge needle. discontinuities. To obtainauniform fiber, a generous amount of pressure is required. Ifsolution does not come out, bath for 1htoremove residual soap. may appearuneven, butitwillshrinkasdries.

TROU

TROU

TROU CR PAUSE CR CR I I I T T T β I I B B B I CAL CAL -sheet formation withinthe silkfibroin. CAL LES LES LES

PO

H H ● H I

● STEP STEP Fig. 9 STEP NT OOT OOT OOT

●

T T Fig. 6 Dried tubescanbestored for several days atroom temperature. IMI IMI

T It isimportant not tointroduce bubblestoeither solution asbothare highly viscousand bubbles Remove the silk-coatedwire from methanol and allowtodry for ~30s. I I I IMI The winding pattern can be altered through the use of the mandrel rotation system. If the the If system. rotation mandrel the of use the through altered be can pattern winding The N N N ); or silk sponges that are either aqueous-based (option L, N N G G G G G ); silk microspheres using the DOPC (option I, N 1d 2minpertubewithovernight dryand1hsoak G 5minpertubeplusovernight dry

Fig. 7 ) or PVA (option J, Fig. 10 natureprotocols

Fig. 5 ) or HFIP-based (option M, ); nonpatterned or patterned silk Fig. 8

) method; electrospun

| VOL.6 NO.10VOL.6 protocol | Fig. 11 2011

|

). 1623 © 2011 Nature America, Inc. All rights reserved.

(F) pHgels ( 1624 (D) (G)

protocol E (vii) (iii) (iii) (iii) Closecapand stirbyinverting the vial. (iii) (iii) (vi) (iv) Turn off the powersupplywhen additional gel no longer collectsonthe positiveelectrode. (vi) (iv) (iv) (iv) ) (ii) (ii) Add 5mlof 4%(wt/vol)silksolution toa15-mlconical tube. (ii) (ii) (ii) (v) Collectthe gel onthe positiveelectrode. (v) Incubate the sonicated solutions at37°Covernight, ifnecessary, toallowhydrogelation. (v) (v) (i) (i) (i) (i) E S S

| lectrogels (e-gels) ilk films volume of 1ml,butthe volumes canbescaledupif the volumetric ratio of silktoacid ismaintained at10:1. onicated gels  Add 0.9mlof 8%(wt/vol) aqueoussilksolution to a glassvial. Thisprotocol hasbeenestablished for atotalsolution VOL.6 NO.10VOL.6 ? emanates outward. visible gel willform atthe positiveelectrode, locking insome oxygen bubblesatthe electrode surface asthe gel ? highly recommended. ! to the instrument. Becareful not totouch the horn onthe sides of the tubeduring sonication. ! If required, the turbid solution can be pipetted into a well plate or a Petri dish immediately after sonication, prior to gelation. increase drying times. increasing acid concentration. Inaddition, wefound thatthe 0.3MHCl gels were the most adhesive. the user. We havestudied gels formed with0.1,0.3and 1.0MHCland havefound thatthe stiffness increased with the topportion of the tube. vortex time and speedmay need tobealtered. It isnot necessary touseglassvials.  silk solution orthe concentration of silk. ally 50 be cutusing ablade (before connecting the needle) toremove the skinlayeratthe tip. inject a5%(wt/vol)gel through a21-gauge needle. Ifinjection requires substantial force, the tipof the syringe can the openend. The the gelation time. Forlong-term storage, store at4°C. ? to beincreased ordecreased.  ­increase the solution turbidity. Gels canbeinjectedthrough 21-gauge (orlarger diameter) needles ifnecessary. Minimal force should besufficient to Immerse apairof platinum wire electrodes into the silksolution. Ifthe electrodes do not reach the solution, cutoff Add 10mlof 7–8%(wt/vol)aqueoussilksolution toa50-mlconical tube. Sonicate the solution at50%amplitude (21W)for 30s. Dilute 8%(wt/vol)aqueoussilksolution to4%(wt/vol). Add 4mlof 8%(wt/vol) aqueous silksolution into a100mmPetri dish.The films produced bythis method are gener Incubate turbid solution overnight in the syringe or in a well plate to allow gelation. Incubation at 37 °C will decrease Collect the turbid solution using a1-mlslip-tipsyringe avoiding any possiblesolid, sticky phase. Transfer 1mlof silksolution into aglassvial and closetightly. (Optional) Ifastiffergel isrequired, collectthe e-gel into asyringe byremoving the plunger and placing the silkin Allow the films towateranneal for 1 d. Place the dry films inthe desiccator and applyvacuum tothe vacuumport. Fill the bottomof the vacuumdesiccator withwater. Apply ~25V Vortex the solution inthe glassvial for ~7minat3,200r.p.m. (maximum speedsetting). Thistreatment should Secure the vial inanupright position onthe vortexer withduct tape. Allow todry overnight without covering the dish.Any modifications toeither the silkconcentration orvolume may Add 0.1mlof 0.3MHClto the vial. The molarity of the HClcanbeincreased ordecreased depending onthe needs of that waterannealing induces 

CAUT CAUT

TROU TROU

TROU

CR CR CR I I I T T ● T I I I µ I B B I B ON ON CAL

CAL CAL ● m thick and canbeeasilyremoved from the polystyrene dish.Forthicker films, either increase the amount of LES LES LES T IMI |

Extended exposure toultrasonication may causedamage toone’s hearing. Use of protective earmuffs is The sonicating horn must beimmersed insolution whilethe powerisapplied inorder toprevent damage 2011 T DC

IMI H H H STEP STEP STEP tothe platinum electrodes overafewminutes. Within seconds of the application of the voltage, a N ● OOT OOT OOT G

N | 10min T

G Thisprotocol isfor 1mlof low-concentration silksolution. Larger volumes may beused, butthe natureprotocols Silksolutions may varybetweenbatches and asasolution ages; therefore, the vortex time may need IMI The water-annealing stepiscritical to prevent the films from dissolving inwater. We have found I I I

β N N 2d N ● -sheet content of the silk isincreased when arapid shear force isapplied tothe gel. G G G

N T G IMI 1d N G

15min β

-sheet similarly toadding methanol but toalesserextent.

- © 2011 Nature America, Inc. All rights reserved.

(xviii) (I) Microspheres (D (H)

(xvii) Allowthe films to dry for 10minand gently peelfilms from the moldusing tweezers. (xiii) Add 100 (viii) (viii) Gently move the Petri dishtoa60°Covenfor 4horleaveatroom temperature for over24h. (xvi) Allowthe films towateranneal for 1 d. (xiv) Allowthe films to dry overnight. (vii) (xii) Placethe PDMS molds inaPetri dish. (vii) Store atroom temperature for upto1year(depending onfilmtreatment and storage conditions). (vii) Once the grating iscovered, remove any bubbleswithaneedle. (xv) (iii) (iii) Mixwithaplastic pipette for 1min. (vi) (xi) (iv) (vi) (ix) (ix) Remove the PDMS from the diffraction grating. Becareful not toripthe PDMS. (vi) Slowlypourthe PDMS overthe diffraction grating; trynot tointroduce bubbles. (iv) PlacePDMS inavacuum ovenfor 30–60mintoremove airbubbles. (ii) (ii) (x) (x) (v) Placethe diffraction grating inaPetri dish. (v) (i) Add 200mg of DOPClipid powder toaglasstest tube. (i) P atterned silkfilms solution withnitrogen gas flow. When the filmis nearly dry, the flow ratecanbe increased. inside achemical fume hood. ! For 1mold (5cm×5cm)use 4.5gof baseand 0.5gof curing agent (9:1ratio). pressurized airfrom the bench top.   diffraction grating). box of Kimwipes, apapercup,needle, aSylgard baseand curing agent, abottleof 70%(vol/vol)ethanol and of the glasstube. proper safetyequipment. ! the lipid filmis hydrated and eventually the lipid filmwilldisappear from the glass, leaving itclear. tion, the waterwillslowlydissolvethe lipid filmfrom the glasstubesurface. The solution willbecome more turbid as  14-mm disksfrom the PDMS. ? Once the layerisrehydrated, dilutewith3mlof ultrapure water, making sure toget the entire lipid layeroff the wall Quickly move the tubetoa37°Cwater bathfor 15min(check that the contents are totallythawedatthe end of Hydrate the lipid filmwiththe silkand drug solution (orcontrol solution, ifapplicable). Byadding the aqueoussolu (Optional) the PDMS mold canbepunched tovarying diameters depending onneed. Forthisprotocol, punch out In ahood, add 1mlof chloroform and waituntil the DOPCiscompletelydissolved;the solution willturnclear. Prepare workarea bylaying down asheet of aluminum foil and collecting the required materials (aplastic pipette, a Leave the tubetodry inthe hood for 30min. Gently remove the filmfrom the dish. Fill the bottomof the vacuumdesiccator withwater. Submerge the tubeinliquid nitrogen for 15minoruntil the liquid nitrogen stopsboiling. Transfer the solution from yourglasstubetoa15-ml conical tubefor freeze-thawing. Roll the tubetoevenlycoatthe interior of the tube withathin,homogenous layer. Whilerolling the tube, dry out Clean the mold withaKimwipe and ethanol. Place papercuponbalance, weigh outanappropriate amount of baseand then curing agent from the Sylgard kit. In a1.5-mlmicrocentrifuge tube, add 200 each 15-minthaw). ultrapure waterfor acontrol sample. lated to1mlof 8%(wt/vol)silksolution. Ifdesired, alsoprepare asolution of 1mlof 8%(wt/vol)silk with 200 damage present onthe casting face.   To indicate the patterned side of the film,placeanedge of the nonpatterned side onto a Post-it note. Store at room temperature until use.

CAUT CAUT

TROU

CR CR PAUSE PAUSE CR I I I T T T I I I I I B ON ON CAL CAL CAL µ LES

PO PO l of 8%(wt/vol)silksolution onto each14-mmdisk. Chloroform isahazardous substance known tobecarcinogenic; wearproper safetyequipment and work Liquid nitrogen is extremely cold and exposure to it may cause burns. Be careful when handling it and wear

H I I STEP STEP STEP NT NT OOT OPC GlasstubescanbecoatedwithDOPCand stored at PDMS molds canbestored atroom temperature and usedindefinitely. Discontinue usewhen there is Ensure thatthe diffraction grating isfree of debris. Spray withethanol, wipeand blow-dry with Ifyouare reusing amold, skiptoStep25H(xii). Placethe dry films inthe desiccator and applyvacuumtothe vacuumport. I ) N ●

● G

T

T IMI IMI N N G G 6hfor generating amold,2dfor casting 4.5d

µ

l of 10mg ml

−

1 solution of the drug, protein orchemical tobeencapsu

−

20 °Cuntil needed. natureprotocols

| VOL.6 NO.10VOL.6 protocol | 2011

|

µ

1625 - l of - © 2011 Nature America, Inc. All rights reserved. (K)

1626 (J) Microspheres ( (xviii)Transfer the solubleportion toanew 2-mlmicrocentrifuge tube.

(xxii) (xvii) (xiii) Pipettethe dilutedsolution into the 100mlof ultrapure waterveryslowly(drip bydrip) withfast stirring. (viii) Peel off the filmfrom the dishand placeone filmina50-mlconical tube. (xiv) (xxi) (xix) Centrifuge the tubesat10,000r.p.m. for 5minat4°Cinamicrocentrifuge. (xvi) protocol (xii) (vii) (xx) Discard the methanol supernatant into aproper wastebottleand dry the pelletovernight inthe hood. Store at4°C. (xv) Lyophilizefor 2–3d. (iii) (iii) (vi) Pour the solution into aPetri dishand allowittocoverthe bottomof the dishevenly. (iv) Dilutesilksolution to5%(wt/vol)withultrapure water. (xi) (xi) (ix) (ii) (ii) (v) (x) (i) (i) E

| lectrospun fibers VOL.6 NO.10VOL.6 that may cause premature crystallization of the silk.  the overall polymer concentration. too low, onlybeadswillform. Hence, wehaveincluded PEOinthisprotocol topromote fiber formation byincreasing the polymer molecules toentangle and form ajet. Ifeither the concentration ormolecular weight of the polymer is  aqueous solution of 6.4%(wt/vol) silk/1%(wt/vol)PEO. ultrapure water. can bestored at4°Cfor 1year.  proper mixing. it atroom temperature.  Prepare a 5% (wt/vol) PVA solution by adding 0.25 g of PVA to 4 ml of ultrapure water. Heat to 60 °C to dissolve the PVA. they canbedispersedbysonicating the solution for about10sat30%amplitude (~20W). or methanol-soluble drug isbeing encapsulated). Optional: usethe suspension (containing bothnano- and microspheres) orlyophilizeand store the dry material. ? distribution of spheres). Transfer the dilutedsolution toa50-mlconical tubeand freeze itat Fill a100-mlbeaker with50 mlof ultrapure water. Add astirbarand move the beaker toastirplate. Dry the solution overnight inafume hood. Add 1mlof 5%(wt/vol)silkand 4mlof 5%(wt/vol)PVA into a15-mlconical tubeand gently mix.Whileslowly Transfer the solution into the 10-mlgraduated cylinder and adjustthe volume of the solution upto5mlwith Prepare asolution of 5%(wt/vol)PEOinwatera20-ml glassscintillation vial. Use asmall stirbartoensure Repeat Steps9–10twice more tocompletethree freeze-thaw cycles(inorder tocreate amore homogenous size Resuspend the pelletin5mlof ultrapure water. Sonicate for 15sat15%amplitude, ifnecessary. Centrifuge at11,000r.p.m. for 20minat4°Cand discard the supernatant. To resuspend the microspheres, firstwashthe microspheres byadding 2ml of watertothe pelletand then centrifuge Slowly stirthe blended solution for ~10 minat4°Ctoobtainahomogenous solution. Avoid high shear during mixing Add 20mlof deionized watertothe tube;shake for 30minatroom temperature todissolvethe film. Filter the PVA solution witha0.45- Remove the waterand resuspend microspheres inthe desired waterorbuffer. Ifthe microspheres haveaggregated, Incubate the samplefor 30minatroom temperature (anincubation time of only10minisrecommended ifaprotein After lyophilization, transfer 30mg of lyophilizedproduct into alarge 2-mlmicrocentrifuge tubeand add 2mlof pure Add 5mlof the 5%(wt/vol)PEO(900,000gmol at 10,000r.p.m. for 5minat4°C. methanol isaflammable solvent; therefore, sources of ignition should be removed before use. ! methanol. solution pertubeasthe solution may expand during freezing. highly recommended. ! to the instrument. Becareful not totouch the horn onthe sides of the tubeduring sonication. ! moving the probe upand down, sonicate the solution for 30sat25%amplitude.

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PO PO PO

| Methanol isahazardous substance; werecommend using the proper safetyequipment. Inaddition, Extended exposure toultrasonication may causedamage toone’s hearing. Use of protective earmuffs is The sonicating horn must beimmersed insolution whilethe powerisapplied inorder toprevent damage 2011

H I I I STEP P NT NT NT OOT

V A Store the PVA solution atroom temperature for no more than3months. The PEOwilltake time to dissolveinto solution, sothisshould bedone ahead of time. Thissolution Afterdrying, the filmcanbestored for afewweeks. Coverand sealthe dishwithParafilm and store |

) Ahigh viscosity, becauseof polymer concentration and molecular weight, must bemet inorder for natureprotocols ● I ● N

T G

IMI T IMI N N G G 1d 2d µ m filtration unit.

−

1 ) solution into 20mlof 8% (wt/vol)silksolution to generate an

−

80 °Cfor atleast5h.Donot exceed 40mlof

© 2011 Nature America, Inc. All rights reserved.

(M) HFI (

(xiii) (viii) (viii) (xiv) Drythe fiber mats inachemical fume hood. L (xii) (vii) (vii) (xii) (iii) (vi) (iv) (vi) Mount the syringe onthe syringe pump. (iv) Draw up10mlof the silk/PEOsolution into a10mlsyringe. (xi) (ix) (ix) (xi) Collectthe silkfibersuntil the desired thickness isachieved. ) (ii) (ii) (x) (v) (v) Attach a16-gauge needle tothe syringe. (x) (i) (i) A queous-based sponges The number of scaffolds caneasilybescaledupbyfilling additional molds. scaffolds may bevaried bychanging the molds. The ratio of salttosilkinsolution should bemaintained at25:1. larger, use8% (wt/vol)silksolution. Forsmaller saltparticles, use6%(wt/vol)silksolution. The sizeand shapeof the from the mats. larger thanthe required pore volume. the saltispartially dissolvedwhilethe silkgels. Therefore, itisimportant tostartwithsaltparticles thatare slightly  mesh onthe bottom.Add saltand shake vigorously. Repeatuntil the desired amount of saltiscollected. ? both topically and ifinhaled. Wear proper safetyequipment and workinside achemical fume hood. ! lyophilized silk. ? dimensions, wesuggest disposablebiopsy punches toobtainuniform disks. per 2litersof water). sources of ignition before use. ! covered withaluminum foil issufficient. electrospun fibersaslong asthey canbeelectrically ground. Asimplesurface thatconsists of apiece of cardboard scaffolds and store them atroom temperature. charged surfaces such asthe needle orpositivevoltage lead. ! Adjust the solution flow rate (0.01–0.03mlmin Before use, cuthydrated scaffolds into the desired dimensions. Autoclave tosterilize. To cutthe scaffolds tosmaller Prepare the saltwiththe particle sizesof interest. Stackthe sieves withthe largest mesh ontopand the smallest Prepare 17%(wt/vol)silk/HFIP solution. Foravolume of 10mlfinal solution, pour9ml of HFIPover1.7g of Once the silkhasgelled, remove the lids and placethe molds ina2-literbeaker withultrapure water (three containers Close the capand allowthe solution tosettleovernight atroom temperature. The silkfibroin should gel in1–2 d. Tap the container gently onthe bench toptoremove airbubbles. Slowly pourthe saltontopof the fibroin solution inthe mold while rotating the container sothatthe saltisuniform. Place acollection surface atadistance of 7–20cmfrom the tipof the needle. Many surfaces canbeusedtocollect Wrap the lid withParafilm and keep itat room temperature overnight oruntil allthe silkisdissolved. With a5-mlsyringe, aliquot fibroin solution into plastic containers, 2mlper mold. Note: Forsaltparticles 750 Weigh 4gof saltinweighing boats, one for eachmold. Store the scaffolds inultrapure waterin50mltubesat4°Cuntil needed for use. Forlong-term storage, dry the Remove the scaffolds from the molds and replace them infresh waterfor anadditional day. Change the water2–3times perday for 2d(4–6washes intotal). Transfer the beaker toastirplateand stir. Incubate the methanol-treated fiber mats inultrapure waterona reciprocating shaker overnight to remove the PEO Set the current toslightly above0A.Turn onthe high voltage and syringe pump. Attach the positivevoltage leadtothe needle onthe syringe and the ground leadtothe collection surface. Immerse the fiber mats ina90%(vol/vol) methanol/water solvent for 20min to obtainwater-insoluble fiber mats. being supplied tothe tipwithout allowing excess solution todrip surface, the fiberdiameter canbe reduced. Finally, the flow rate should beadjustedsothat the solution isconstantly mass transport withthe higher electrical force. Byincreasing the distance betweenthe spinneret and the collecting section. It hasalsobeennoted thatwithanincrease involtage the fiberdiameter increases because of additional evident onthe fibers. At the higher limit,silkfibers transition from a round morphology toaribbon-shaped cross-  capillary tipand the collection screen (7–20cm)toobtainastablejet.

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CR CR -based sponges I I T T I I I I I B B ON ON ON CAL CAL LES LES HFIPislistedbythe Hazardous Materials Information Systemasahealth hazard level3;itcancauseburns Methanol isahazardous and flammable substance; useproper personal safetyequipment and remove all Electrospinning isperformed atahigh voltage. Becareful whilethe high voltage isonand do not touch

H H STEP STEP OOT OOT

It isimportant tonote thatthe pore sizeswillbeslightly smaller thanthe saltparticles used, as The electric fieldstrength is required toinitiate the jetand atthe lowerlimitbead defects are I I N N ● G G

T ● IMI

T IMI N G 8d N G 5d

−

1 ), electric potential (8–15KV)and the distance betweenthe 4 2 . natureprotocols

| VOL.6 NO.10VOL.6

protocol

| 2011

µ m and

|

1627

© 2011 Nature America, Inc. All rights reserved.

Troubleshooting advice canbefound in ? 1628

T (xiii) Change the water2–3times aday for 2–3d. (viii) a (xiv) (xvi) 15 15 (silk processing) 25A(ii) 25A(ii) (tubes) S protocol (vii) (xii) Placethe beaker onastirplateand stir.

(xv) (iii) (vi) (xi) (iv) (ix) TROU tep b (v) (x) le

| VOL.6 NO.10VOL.6 2 is collected. on topand the smallest mesh atthe bottom.Add saltand shake vigorously. Repeatuntil the desired amount of salt each container toreduce solvent evaporation. ? ultrapure water(~6containers per2litersof water).  ! evaporate for 1d. dialysis times may need to be scaled up to ensure penetration of the solvent molecules into theHFIP. center of HFIP the is scaffolds.highly corrosive, so metal should be avoided. In addition, for larger scaffoldsbe scaled withthe a methanol20:1 ratio treatment with the silk and mass in solution. Be careful to choose a mold material that will not interact with (Optional) Ifyouare adding areinforcing agent such ashydroxyapatite, weigh itand add ittothe salthere. Mixwell, Remove extra methanol and disposeof itinaproper wastecontainer. Transfer the containers toa2-literbeaker of In afume hood, add 1mlof silksolution toeachcontainer of saltveryquickly using a5-mlsyringe. Immediately cap Tap the container gently onthe bench topsurface tolevelthe salt inthe container. Weigh 3.4gof NaCl and placeitinaglassshell vial. Repeatfor eight more vials. Remove the scaffolds from the molds and replace them infresh waterfor anadditional day. Scaffolds without While the silkisdissolving, prepare the saltwiththe particle sizesof interest. Stackthe sieves withthe largest mesh When needed for use, cutthem inwateratdesired dimensions and autoclavetosterilize. We suggest slicing off the Once the saltappears‘wet’and the silksolution hasreached the bottomof the vial, uncap the vials toallowHFIP Wrap eachcapwithParafilm and allowsilksolution topenetrate the salt(1–2d). Store scaffolds inultrapure waterin50-mltubesat4°Cuntil needed for use(canbestored for 2–6months) ordry When the silkappearsdry and detached from the container walls, add 1mlof methanol toeachcontainer and recap. ? top and bottomlayersand discarding them, asthe surface hasaskinthatisless porous. reinforcement willbegin tofloatwhen the saltis removed. Allow the methanol toseepthrough the scaffold for 1d. both inthe weigh dishand inthe mold. the scaffolds and store them dry atroom temperature.

B

CAUT |

TROU TROU LES

CR PAUSE Troubleshooting table. I T H I I B B OOT ON CAL LES LES

PO | Keep the scaffolds inthe hood. 2011 I

H H N STEP I NT OOT OOT G present along the mandrel wire and several beads are Silk will not evenly coat the not amber colored LiBr and silk fibroin solution is in lithium bromide (LiBr) Silk does not dissolve after 4 h P The dried scaffolds canbestored atroom temperature for several days. | roblem

If larger scaffolds are required, the mold can be exchanged with a larger one and the salt weight can natureprotocols

I I N N G G T able 2 .

periodically as the silk will gel if it is stored for too long Silk is not concentrated enough, store at 4 °C for a few days, check it Change or clean glassware Check that the oven is set to 60 °C cover the beaker to reduce evaporation. solution to ensure contact between the silk and the LiBr solution. Be sure to Did you pour the LiBr over the silk fibers? If not, you will need to mix the note of the boiling time as batches may vary if the boiling times are different. shorter time, you may need to allow the silk to dissolve in LiBr for longer. Make Did you boil the cocoons in sodium carbonate for 30 min? If you boiled for a If not, add the appropriate amount of LiBr. Did you account for the volume of LiBr when preparing the 9.3 M solution? S olution

(continued)

© 2011 Nature America, Inc. All rights reserved. Step Step 25J, Preparation of microspheres: 2 d Step 25I, Preparation of microspheres (DOPC): 4.5 d Step 25H, Generating molds and casting of patterned silk films: 6 h for generating molds, 2 d for casting Step 25G, Preparation of silk films: 2 d Step 25F, Preparation of pH gels: 10 min Step 25E, Preparation of electrogels: 15 min Step 25D, Preparation of sonicated gels: 1 d Step 25C, Preparation of vortexed gels: 1 d Step 25B, Preparation of gel-spun tubes: 5 min per tube, plus overnight dry Step 25A, Preparation of dipped tubes: 2 min per tube, plus overnight dry and 1 h soak Step 24B, Concentration: 21–25 h Step 24A, Lyophilization: 3 d Steps 16–23, Dialysis and centrifugation: 49 h (days 2–4) Steps 12–15, Dissolve silk fibroin: 4.5 h (day 2) Steps 1–11, Fibroin extraction: 2.5 h to overnight (day 1) ● T Step Step 25K, Electrospinning: 1 d a 25C(iv) 25C(iv) (vortex gels) spinning) 25B(v) (gel 25A(ix) (tubes) based based sponges) 25M(xvi) (HFIP- sponges) 25M(xi) (HFIP-based (sponges) 25L(i),25M(i) microspheres) 25J(ix) (PVA 25G(vi) (thin films) (electrogelated gels) 25E(iii) gels) 25D(iii) (sonicated S

tep b T le IMI 2 N |

G

Troubleshooting table (continued).

in in them HFIP-based sponges have voids while in water HFIP-based sponges dissolved size range of interest are very few crystals within the When shifting the salt, there dissolve after 30 min The film will not completely plate Films will not lift off of the Gel will not form Gel will not form Gel will not form has beads along it wrapped around the mandrel The silk gel fiber being tubes Large gaps are present on the Tubes crack after drying P roblem Try pushing the scaffolds down or centrifuge them before drying treatment time Methanol was not able to penetrate properly. Repeat but extend the methanol within the mesh The sieve may be clogged. Invert the sieve and try to dislodge particles trapped Sonicate the solution for 30 s at 15% amplitude Use non–tissue culture–treated dishes approximately 1 week old the solution and volume. Electrogelation works best on silk solutions that are Gelation time may need to be adjusted depending on the batch of silk, age of of silk, age of the solution and volume Sonication time and amplitude may need to be adjusted depending on the batch of silk, age of the solution and volume Vortexing time and amplitude may need to be adjusted depending on the batch check it periodically as the silk will gel if it is stored for too long is not concentrated enough. Store the solution at 4 °C for a couple of days but result in an uneven tube. Beads along the gel fiber indicate that the silk solution If the fiber is not uniform as it is being wrapped around the mandrel, it will before use pipetting or mixing in PEO (if applicable). Degas the solution in a vacuum oven Bubbles are present in the silk solution. Be careful not to induce bubbles when the tube to dry between layers up and down the mandrel. Also, allow 10 s for methanol treatment and 30 s for If the problem continues, follow the protocol, carefully allowing the bead to go Try a solution of 70% (vol/vol) methanol in water or add more layers next time. S olution natureprotocols

| VOL.6 NO.10VOL.6 protocol | 2011 |

1629 © 2011 Nature America, Inc. All rights reserved. 16. 19. 15. 8. 21. 20. 11. 7. 6. 4. 13. 10. 1. com/reprints/index.html. http://www.nature. at online available is information permissions and Reprints Published online at http://www.natureprotocols.com/. interests. financial CO manuscript and D.L.K. supervised and edited the project. AUT National Science Foundation and the Air Force Office of Scientific Research. Institutes of Health (P41 EngineeringEB002520—Tissue Resource Center), the X. Wang, J.A. Kluge and F. Omenetto. This work was supported by the National contributions to these protocols: E.S. Gil, C. Wittmer, H. J. Kim, E.A. Pritchard, Expected outcomes for the various options described inthe PROCEDUREare summarized inthe INTRODUCTION. ANT Step 25M, Preparation of HFIP-based sponges: 8 d Step 25L, Preparation of sponges:aqueous-based 5 d 1630 5. A 17. 3. 12. 18. 2. 14. 9. protocol c

k M

nowle H PET Wang, Y.Wang, Hu, X., Lu, Q., Kaplan, D.L. & Cebe, P. Microphase separation controlled separation P.Microphase Cebe, & D.L. Kaplan, Q., Lu, X., Hu, Etienne, O.Etienne, material.ancient an for opportunities New D.L. Kaplan, & F.G.Omenetto, beta-sheet crystallization kinetics in fibrous proteins. fibrous in kinetics crystallization beta-sheet annealing.vapor water defects. scaffolds. vivo in fibroin. silk modified Bombyx mori Bombyx A. Kojthung, (2009). fibroin. silk of properties physiological N.-Y. Sung, (2009). 2079–2087 electronics.bio-integrated Biomater.Appl. biomaterials:reproducibility biocompatibility.and vivo in osteogenesis. S.H. Park, (2005). 3385–3393 Science Biol. Mol. Biochem. B spiders. and insects in genes encoding their and proteins fibrous silks, of architecture Comparative C. Riekel, & C. Craig, (2007). 991–1007 biomaterial. a as Silk D.L. Kaplan, & C. Vepari, (2001). formation. bone for biomaterials based applications.antithrombotic and antiadhesion for glycol) poly(ethylene with fibroin silk of modification Surface D.L. Kaplan, & R. Naik, Drummy,L., D., Matheson, C., Vepari, (2010). 1403–1413 regeneration.tissue in FGF-2 of potency and delivery differentiation. and proliferation hMSC on effects the and chemistry coupling diazonium Wray, L.S., Hu, X. & Kaplan,processingEffectofD.L.Wray,silk-based& on X. Hu,L.S., R.L. Horan, Murphy, A.R. & Kaplan, D.L. Biomedical applications of chemically- of applicationsBiomedical D.L. Kaplan, & Murphy,A.R. Sofia, S., McCarthy, M.B., Gronowicz, G. & Kaplan, D.L. Functionalized silk- Functionalized D.L. Kaplan, & G. Gronowicz, McCarthy, M.B., S., Sofia, Meinel, L. Meinel, Wenk,E. Meinel, L. Meinel, Hu, X. Hu, Murphy, A.R., John, P.S. & Kaplan, D.L. Modification of silk fibroin using fibroin silk of Modification D.L. Kaplan, P.S.& John, Murphy,A.R., Kim, D.-H. Kim, Altman,G.H. I OR

C | VOL.6 NO.10VOL.6 I I

N CONTR PATE G G FI study. . dgm

et al. et 329 Biomaterials Bone et al.et et al. et NANC Biomaterials et al. et et al. et al. et D IB ents et al. et et al. et , 528–531 (2010). 528–531 , et al. et et al. et Regulation of silk material structure by temperature-controlledby structurematerial silk of Regulation silk fibroin. silk et al. et UT

et al. et Theuseofsulfonated silkfibroin derivatives controlto binding, RESULTS Biomaterials J. Periodont. J. 39 publishedonline, doi:10.1002/jbm.b.31875 2011).June(21

I In vivo In Biomaterials I Relationships between degradability of silk scaffolds and scaffolds silk of degradability between Relationships Silk based biomaterials to heal critical sized femur sized critical heal to biomaterials based Silk films silk to responses inflammatory The

AL Dissolvable films of silk fibroin for ultrathin conformal ultrathin for fibroin silk of films Dissolvable The authors acknowledge the following researchers for their ONS , 922–931 (2006). 922–931 , Soft tissue augmentation using silk gels: an gels: silk using augmentation tissue Soft Effect of gamma irradiation on the structural and structural the on irradiation gamma of Effect

| Silk-based biomaterials. In vitro In 2011 Effect of gamma radiation on biodegradationof on radiation gamma of Effect I

NTERESTS 26

J. Mat. Chem. Mat. J.

D.N.R., D.N.R., R.C.P., T.Y., X.W. and M.L.L. wrote the 133 degradation of three-dimensional silk fibroin three-dimensionalsilk ofdegradation 29 Biomacromolecules , 147–155 (2005). 147–155 , | degradation of silk fibroin. silk ofdegradation , 3415–3428 (2008). 3415–3428 , Int. Biodeterior. Biodegrad. Biodeterior. Int.

, 493–507 (2002). 493–507 , natureprotocols 31 Nat. Mater. Nat. 80

29 J. Biomed. Mat. Res. A Res. Mat. Biomed. J. , 6162–6172 (2010). 6162–6172 , , 1852–1858 (2009). 1852–1858 , , 2829–2838 (2008). 2829–2838 , The authors declare no The authors competing

19

9 , 6443–6450 (2009). 6443–6450 , Food Sci. Biotechnol. Sci. Food J. Biomed. Mat. Res. Mat. Biomed. J. , 511–517 (2010). 511–517 ,

12 Biomaterials , 1686–1696 (2011). 1686–1696 , Prog. Poly. Sci. Poly. Prog. J. Biomed.Mater.J. B Res.

Comp. Chem. Physiol. Chem. Comp.

93 62

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54 18

in vitro in 32 , 139–148 , , 228–233 228–233 ,

and

, 26 31

, , and 42

,

41. 31. 29. 28. 24. 33. 32. 43. 39. 38. 37. 36. 35. 40. 34. 22. 27. 44. 42. 25. 30. 26. 23. 45.

Rockwood, D.N. Rockwood, Lawrence,B.D.,Marchant, Pindrus, J.K.,Omenetto,M.A., Kaplan,F.G.D.L. & Yucel, T., Kojic, N., Leisk, G.G., Lo, T.J. & Kaplan, D.L. Non-equilibrium D.L. Kaplan, & T.J. Lo, G.G., Leisk, N., Kojic,T., Yucel, adhesives. protein for Electrogelation D.L. Kaplan, & Q. Lu, T.,Yucel,T.J., Lo, G.G., Leisk, (2008). encapsulation. cell for fibroin silk of gelation hydrogels. Biomacromolecules prevascularization. anddifferentiation, cell stem mesenchymalhumangradients, oxygen of characterization for system a engineering: tissue for bioreactors modular Simple D.L. Kaplan, & A. Baryshyan, D.N., Rockwood, M.L., Lovett, (2008). 4650–4657 engineering. tissue for tubes silk of spinning Gel Biomaterials microspheres from silk/pva blend films for drug delivery. drug for films blend silk/pva frommicrospheres Release Control J. X. Wang, (2004). 711–717 of films Biomaterial D.L. Kaplan, P.& Cebe, R., Valluzzi, J., Park, H.J., Jin, (2009). 1299–1308 engineering. tissue cornea for biomaterials film Silk delivery. adhesives. fibroin silk Bone Biomaterials scaffolds. silk porous and cells stem mesenchymalhuman using structures cells. stem marrow bone from engineering tissue bone on scaffolds protein macroporous of Influence D.L. Kaplan, & B.H. Min, G., Vunjak-Novakovic, U.J., Kim, H.J., Kim, (2009). medicine.regenerativefor scaffolds Biomater.Acta an scaffolds: composite silk reinforced particle silk porous fibroin. silk from scaffolds biomaterial aqueous-derived Three-dimensionalD.L. Kaplan, & M. Wada, H.J., Kim, J., Park, U.J., Kim, (2007). 2777–2782 macroscopicallyaligned electrospun polymer nanofibers. Kakade,M.V. (2008). cardiomyocytes by expressionpeptide natriuretic atrial decreases scaffolds polyurethane electrospun on Culture J.F. Rabolt, & Woodhouse,K.A. I.C., Parrag, R.E., Akins, D.N., Rockwood, (2006). engineering. tissue bone for scaffolds BMP-2 silk- Electrospun D.L. Kaplan, & H.J. Kim, H.-J., Jin, C., Vepari, C., Li, (2002). 1233–1239 mori Bombyx Electrospinning D.L. Kaplan, & G.C. S.V.,Rutledge,Fridrikh, H.J., Jin, (2010). 1025–1035 regenerated silk fibroin. silk regenerated from scaffolds 3-D Porous D.L. Kaplan, & H.J. Jin, Nazarov,R., (2005). 2775–2785 Wang, X.Q., Yucel, T., Lu, Q., Hu, X. & Kaplan, D.L. Silk nanospheres andnanospheres Silk D.L. Kaplan, & X. Hu, Q., Lu, T.,Yucel, X.Q., Wang, Lovett, M. Lovett, Wang, X.Q., Kluge, J.A., Leisk, G.G. & Kaplan, D.L. Sonication-inducedD.L. Kaplan, & G.G. Leisk, J.A., Kluge, X.Q., Wang, Hofmann, S. Hofmann, biomaterial silk Electrospun D.L. Kaplan, & M.R. Reagan, X.H., Zhang, Kim, U.J. Kim, Hofmann, S. Hofmann, Yucel, T., Cebe, P. & Kaplan, D.L. Vortex-induced injectable silk fibroin silk Vortex-inducedinjectable D.L. Kaplan, P.& Cebe,T., Yucel, Lovett, M.L., Cannizzaro, C.M., Vunjak-Novakovic, G. & Kaplan, D.L. Kaplan, & G. Vunjak-Novakovic, C.M., Cannizzaro, M.L., Lovett, Kim, H.J. Kim, Bombyx mori Bombyx

42 , 1226–1234 (2008). 1226–1234 , J. Control Release Control J. et al. et et al. et et al. et Biophys. J. Biophys. et al. et

silk with poly(ethylene oxide).poly(ethylene with silk 28 28 et al. et et al. et et al. et

Silk microspheres for encapsulation and controlled release.controlled and encapsulation formicrospheres Silk 7 silk fibroin with poly(ethylene oxide). with fibroin silk Structure and properties of silk hydrogels.silk of properties and Structure , 5271–5279 (2007). 5271–5279 , , 1152–1162 (2007). 1152–1162 , Bone tissue engineering with premineralized silk scaffolds. silk premineralized with engineering tissue Bone Silk fibroin microtubes for blood vessel engineering. vessel blood for microtubes fibroin Silk et al. et , 144–151 (2011). 144–151 ,

117 Silk fibroin as an organic polymer for controlled drug controlled for polymer organic an as fibroin Silk Control of Control Electric field induced orientation of polymer chains in chains polymer of orientation induced field Electric Adv.Mater. 5 Tissue Eng. Part C Methods. C Part Eng. Tissue , 786–792 (2004). 786–792 ,

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tissue-engineered bone-liketissue-engineered 170 in vitro in , 406–412 (2010). 406–412 ,

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61 Biomaterials Biomaterials

, 988–1006 , 27

29 in vitro in 29 , 3115–3124 , , 1054–1064 ,

, 4783–4791 , 29

3

, 30 , study.

,

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31 129 ,

, 5 , ,

© 2011 Nature America, Inc. All rights reserved. 57. 47. 55. 58. 56. 46. 53. 49. 48. 50. 52. 51. 54.

House,Sanchez,M., C.C.,Rice, W.L., Socrate,Kaplan, & D.L.CervicalS. tissue Hofmann, S. Hofmann, Zhang, X.H., Baughman, C.B. & Kaplan, D.L. Kaplan, & C.B. Baughman, X.H., Zhang, cells. stem mesenchymaland scaffolds silk aqueous-derived porous 3D with engineering tissue cartilage Biomaterials chondrocytes.articular human and scaffolds silk with engineering tissue scaffolds. cells. stem mesenchymaland scaffolds. 3-D accelerated wound healing. wound accelerated for dressing bioactive topical a as mats silk Biofunctionalizedelectrospun C. Egles, & J.A. Garlick, D.L., Kaplan,X.Y., Wang, Schneider, A., (2010). 2101–2112 engineeringusing silk scaffolds and human cervical cells. grafts. vascular tissue-engineered X.H. Zhang, (2008). 2217–2227 growth. cell vascular for scaffolds fibroin silk electrospun grafts. vascular engineered Biosci. patterns. surface film silk on fibroblasts corneal human of scaffolds. 3D fibroin silk with cells stem mesenchymaladipose-derived and marrow bone human utilizing Biomaterials chondrogenicdevelopment.duringcondensation mesenchymal of model Kim, H.J. Kim, Gil, E.S., Park, S.H., Marchant, J., Omenetto, F. & Kaplan, D.L. Response D.L. Kaplan, & F.Omenetto, J., Marchant, S.H., Park, E.S., Gil, Wang, Y.Z., Blasioli, D.J., Kim, H.J., Kim, H.S. & Kaplan, D.L. Cartilage D.L. Kaplan, & H.S. Kim, H.J., Kim, D.J., Blasioli,Y.Z., Wang, Tigli, R.S. Tigli, Wang, Y.Z., Kim, U.J., Blasioli, D.J., Kim, H.J. & Kaplan, D.L. Kaplan, & H.J. Kim, D.J., Blasioli, U.J., Kim, Y.Z.,Wang, Mauney,J.R. D.L. Kaplan, & S. Sengupta, S., Mondal, M., Laha, S., Ghosh, Soffer,L.

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et al. et 27 30 et al. et et al. et Macromol. Biosci. Macromol. Silk-based electrospun tubular scaffolds for tissue- for scaffolds tubular electrospun Silk-based , 4434–4442 (2006). 4434–4442 , , 6530–6540 (2009). 6530–6540 , Bone regeneration on macroporous aqueous-derived silk aqueous-derived macroporous regenerationon Bone Comparative chondrogenesis of human cell sources in 3D in sources cell human chondrogenesisof Comparative Dynamic culture conditions to generate silk-based generate to conditions culture Dynamic Cartilage-like tissue engineering using silk scaffolds silk using engineering tissue Cartilage-like Engineering adipose-like tissue adipose-like Engineering J. Biomat. Sci.- Poly. Ed. Poly. Sci.- Biomat. J. Acta Biomater.Acta Biomaterials Eng. Tissue Biomaterials

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Subramanian, B.Subramanian, Pritchard, E.M., Szybala, C., Boison, D. & Kaplan, D.L. Silk fibroin Silk D.L. Kaplan, & D. Boison, C., Szybala, E.M., Pritchard, Altman, G.H. Altman, Wang, X.L. Wang, J. Control Release Control J. adenosine.of release sustained for reservoirs powder encapsulated canines. in defects border J. Zhao, (2009). bMSCs. BMP-2-modified and scaffolds ligaments.tissue-engineered of development the in stimulation mechanical and biochemicalSequential ligaments. kidney.diseased and normal for scaffolds. silk 3D on cells epithelial mammary human ofdifferentiation functional function. andmorphogenesis breast modeling for scaffolds silk and cells stromal mammary X.L. Wang, (2007). 10304–10308 model. mouse a in cancer breast human of metastasis J.E. Moreau, (2010). healing. wound for systems material Release engineering. tissue osteochondralfor scaffolds biopolymer epilepsy.for rats. kindled in release Wharram, S.E., Zhang, X.H., Kaplan, D.L. & McCarthy, S.P. Electrospun silk McCarthy,S.P.Electrospun & D.L. Kaplan, X.H., Zhang, S.E., Wharram, Wang, X.Q. Wang, Szybala, C. Szybala, Moreau, J.E., Bramono, D.S., Horan, R.L., Kaplan, D.L. & Altman, G.H. Altman, & D.L. Kaplan, R.L., Horan, D.S., Bramono, J.E., Moreau, Jiang, X.Q. Jiang, Wilz, A. Wilz,

134 et al. et et al. et Tissue Eng. Part A Part Eng. Tissue Biomaterials et al. et al. et et al. et et al. et et al. et , 81–90 (2009). 81–90 , Biomaterials et al. et et al. et Silk polymer-based adenosine release: therapeutic potentialtherapeutic release: adenosine polymer-based Silk Apatite-coated silk fibroin scaffolds to healing mandibular healing to scaffolds fibroin silk Apatite-coated Preadipocytes stimulate ductal morphogenesis andmorphogenesis ductal stimulate Preadipocytes on based system culture tissue human 3D complex A Antiepileptic effects of silk-polymer based adenosine based silk-polymer of effects Antiepileptic Growth factor gradients via microsphere delivery in deliverymicrosphere via gradients factor Growth et al. et Mandibular repair in rats with premineralized silk premineralized with rats in repair Mandibular

144 Tissue-engineered bone serves as a target for target a as serves bone Tissue-engineered Silk matrix for tissue engineered anterior cruciate anteriorengineered tissue for matrix Silk Tissue-engineered three-dimensionalTissue-engineered , 159–167 (2010). 159–167 , natureprotocols

23 Exp. Neurol. Exp.

29 , 4131–4141 (2002). 4131–4141 , Bone

, 3609–3616 (2008). 3609–3616 , 15 Tissue Eng. Part A Part Eng. Tissue Biomaterials

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1631 ,