processing of bombyx mori silk for biomedical...

78

© 2014 Woodhead Publishing Limited

3 Processing of Bombyx mori silk for

biomedical applications

B. D. LAWRENCE , Seryx Biomedical Inc., USA

DOI: 10.1533/9780857097064.1.78

Abstract : This chapter highlights various processing methodologies that may be utilized to create a variety of structural forms from the protein fi broin, which is derived from the Bombyx mori silkworm cocoon. Modulation of fi broin material properties will be considered with respect to induced protein secondary structure formation. Additional considerations will be explored for sericin removal and fi broin extraction. A review of recent scaffold processing techniques will be reported, which include materials such as native fi bers, electrospun fi bers, sponge scaffolds, hydrogels, and microspheres. Finally, a perspective on the future trends relating to fi broin protein based technologies will be discussed.

Key words : silk fi broin, biomaterial, scaffold, tissue engineering, biopolymer.

3.1 Introduction

The use of silk is ubiquitous in society as one of the most useful and oldest

natural fi bers in the world with an evolutionary history of over 380 million

years (Shear et al ., 1989). Similar to how humans use concrete, metals, and

plastics to build the world around us, arthropods have employed nearly 40

000 different silk proteins to produce varying structures such as webbing,

nests, cocoons, and underwater air sacks (Kaplan, 1994). Historically, humans

have harnessed the fi bers produced by the domesticated Bombyx mori silk-

worm for uses in textile applications due to the extraordinary mechani-

cal and visually appealing properties of the material (Shao and Vollrath,

2002). Commercial grade silk fi bers are derived from the mulberry leaf-fed,

domesticated B. mori silkworm cocoon, which is formed during the pupae

phase of its life cycle (Jin et al ., 2002; Valluzzi et al ., 2002). The Bombyx mori silkworm cocoon is composed primarily of three proteins, which consist of

the glue-like glycoprotein sericin and heavy and light chains of the struc-

tural fi brous protein fi broin (Kaplan, 1994).

It has been shown that fi broin may be resolubilized into an aqueous

solution, and then formed into a number of different geometrical forms

Property of Elsevier

Table 3.1 Selected structures previously formed from silk fi broin solution with specifi c material characteristics and associated

references

Formed structure Functional

description

Processing methodology Example applications Selected references

Native fi bers Ropes or fi brous mats

providing uniaxial

strength

Extracted from cocoon

by boiling in sodium

carbonate solution then

drying

Ligament, tendon, and soft tissue

repair

Altman et al., 2002,

2003; Vunjak-

Novakovic et al.,

2004

Electrospun

fi bers

Fibrous mat or

tubular scaffolds

composed of

nano- to micron-

sized fi ber

diameters

Silk solution is ejected

onto a fl at or tubular

surface through the

use of an electrostatic

charge differential

Wound healing, cardiovascular

grafts, soft tissue repair,

intervertebral disc formation,

peripheral nerve regeneration

Jin et al., 2002, 2004;

Kim et al., 2012;

Schneider et al.,

2009; Soffer et al.,

2008; Sukigara

et al., 2003;

Wharram et al.,

2010; Zhang et al.,

2012

Films Thin sheets of

isotropic material

with highly

controlled

length, width,

and thickness

dimensions

Silk solution is cast

upon a molding

substrate and water

is evaporated to grow

a continuous fi lm

structure

Cell culture substrates,

corneal tissue, optics,

sensors, electrical insulator,

drug stabilization, nerve

regeneration, tube formation

Bray et al., 2012;

Horan et al., 2005;

Hwang et al., 2012;

Lawrence et al.,

2008; Minoura

et al. , 1995; Rogers

et al., 2012; Zhang

et al., 2012

(Continued)


Formed structure Functional

description

Processing methodology Example applications Selected references

Sponge scaffolds Molded

scaffolds with

interpenetrating

pores

Silk solution is blended

with leachable solutes

(i.e. salt or beads),

cast using a mold, and

then leached using the

appropriate solvent

system

Bone, cartilage, soft tissue repair,

in vitro 3D culture systems,

adipose

Liu et al., 2012;

Mandal et al., 2012;

Nazarov et al.,

2004; Papenburg

et al., 2009;

Swinerd et al.,

2007; Wang et al.,

2008; Yan et al.,

2012

Hydrogels Filamentous

scaffold

interspersed by

high amounts of

water

Silk solution is subjected

to an agitating

force (i.e. ultrasonic

cavitation or vortex

mixing) which induces

gelation process

Dermal void fi lling, cell

encapsulation, intervertebral

disc

Etienne et al., 2009;

Kim et al., 2004;

Matsumoto et al.,

2006; Park et al.,

2012; Wang et al.,

2008

Microspheres Nano- to micron-sized

sphere structures

Silk solution is dissolved

within a solvent system

and post processed

to produce desired

particulate size

Drug delivery, vascular

applications, chemical

processing

Breslauer 2010;

Lammel et al., 2010;

Wang et al., 2007,

2010; Wenk 2008;

Wenk et al., 2011

Table 3.1 Continued


Processing of Bombyx mori silk for biomedical applications 81

(Jin and Kaplan 2003; Rockwood et al ., 2011) (Table 3.1). This silk solu-

tion is termed ‘regenerated’ silk to indicate the natural origins of the

fi ber, which has subsequently been processed to produce a formable bio-

polymer solution (Altman et al ., 2002). It is well documented that regen-

erated B. mori derived silk is highly biocompatible within the body, and

also demonstrates an impressive range of material properties based on a

variety of processing protocols (Altman et al ., 2003; Minoura et al ., 1995;

Omenetto and Kaplan, 2010; Vepari and Kaplan, 2007). To that end, over

the last two decades numerous investigators have been devoted to further

understanding the potential of regenerated silk fi broin solution for use

in biomedical applications in relation to tissue engineering, regenerative

medicine, and drug delivery applications, which will be the focus of this

chapter.

3.2 Modulation of silk biomaterial properties

Regenerated silk fi broin solution is produced by dissolving silk cocoons into

water through the use of chaotropic agents, such as heavy salts, to disrupt the

high degree of hydrogen bonding that exists between the individual protein

molecules (Jin and Kaplan, 2003; Keten et al ., 2010). The regenerated silk

solution can then be reformed into a variety of three-dimensional geom-

etries (Rockwood et al ., 2011). Silk fi broin has previously been described

as an engineering grade biopolymer due to the degree of control over its

protein secondary structure formation (Lawrence et al ., 2008b). Fibroin

offers great potential for use in medically related applications due to the

high degree of biocompatibility and non-infl ammatory properties when

implanted within the body (Meinel et al ., 2005; Panilaitis et al ., 2003; Wang

et al ., 2008b). This chapter will explore various aspects of the material prop-

erties of silk fi broin and the multitude of applications this protein has found

in biomedical related applications.

Recent work has shown that constructs formed from silk solution are bio-

compatible in vivo , and have proven to be both non-infl ammatory and non-

immunogenic upon implantation within the body (Etienne et al ., 2009; Fini

et al ., 2005; Huang et al ., 2012; Mandal et al ., 2012; Meinel et al ., 2005; Pra et al ., 2005). Current animal trials are underway to assess the use of silk solution in

the creation of scaffolds for bone, ligament, and nervous tissue (Mandal et al ., 2012; Wang et al ., 2006; Yang et al ., 2011). In addition, a number of scaffolds

are being developed as in vitro tissue analogs for corneal, intervertebral disc,

cardiac, breast, skin, and articular cartilage (Altman et al ., 2010; Etienne et al ., 2009; Harkin et al ., 2011; Lawrence et al ., 2009; Park et al ., 2012; Patra et al ., 2012; Yan et al ., 2012). As with traditional tissue engineering approaches, the

silk scaffolds are typically seeded in vitro with a specifi c cell type, and then cul-

tured over time to produce tissue analogs (Kim et al ., 2005). It has been shown


82 Silk Biomaterials for Tissue Engineering and Regenerative Medicine

that the silk fi broin protein can be degraded through a number of naturally

occurring proteolytic enzymes (Horan et al ., 2005; Li et al ., 2003). The hydro-

lyzed silk protein is believed to be cleared from the tissue through cellular

phagocytic pathways (Desjardins, 2005). Silk fi broin protein is primarily com-

posed of glycine and alanine amino acids that can be reused for new protein

synthesis post-degradation (Kaplan, 1994). As a result, silk degradation prod-

ucts do not collect in the local environment to cause an induced infl ammatory

response, which is commonly associated with other synthetic biomaterials like

poly-(lactic-co-glycolic acid) (PLGA) (Onuki and Bhardwaj, 2008).

The degradation rate and by-product formation of silk fi broin is directly

related to the secondary structure content of the protein (Chen et al ., 2001;

Hu et al ., 2006, 2011; Mo et al ., 2006). By increasing or decreasing the presence

of these structures the silk degradation rate can be adjusted from minutes

to years (Kim et al ., 2010; Wang et al ., 2008b). This signifi cant range of mate-

rial processing is a key attribute of fi broin protein and is one of the impor-

tant reasons for the potentially broad application to biomedical applications.

The ability to control silk material properties offers a number of advantages

over other biopolymer systems like collagen, chitosan, and alginate. The for-

mation of silk structures begins with fi broin proteins aggregating into pro-

tein globules in solution (Jin and Kaplan, 2003; Keten et al ., 2010; K ö nig and

Kilbinger, 2007). The fi broin globules then aggregate to form larger bulk mac-

romolecular structures that can then be modifi ed through a variety of process-

ing methods (Jin and Kaplan, 2003). The silk material properties can then be

controlled through inducing protein secondary structure formations, such as

alpha-helices and beta-sheets, through a variety of post-processing techniques

(Chen et al ., 2001; Hu et al ., 2006, 2011; Mo et al ., 2006) ( Fig. 3.1 ).

The formation and organization of these structures modulates the total

hydrogen and hydrostatic bonding within the bulk structure of the material,

which affects the material properties at the macroscale . A variety of silk pro-

cessing methods have been developed to produce a multitude of structures,

and range from the use of physical factors, such as mechanical stress and heat,

to the use of chemicals, from water to organic solvents, in order to induce con-

trolled secondary structure formation (Agarwal et al ., 1997; Gupta et al ., 2007;

Jin et al ., 2005; Lu et al ., 2010; Mandal et al ., 2012). As a result, fi broin material

properties such as degradation rate, hydrophobicity/hydrophilicity, transpar-

ency, mechanical strength, porosity, oxygen permeability, and thermal stabil-

ity may be altered (Agarwal et al ., 1997; Horan et al ., 2005; Jin et al ., 2004b;

Kim et al ., 2005; Motta et al ., 2002; Tretinnikov and Tamada, 2001). In this

regard, silk proteins can be considered as an engineering class of biopolymers

in which the material properties can be defi ned for a given application.

Water plays an important role in processing regenerated silk fi broin mate-

rials. The control of regenerated silk fi broin material properties is primarily

achieved through modulation of hydration state (Agarwal et al ., 1997; Hu



et al ., 2011; Lawrence et al ., 2010; Motta et al ., 2002; Sohn et al ., 2004). Water

acts as a lubricant, which allows for protein chain movement and secondary

structure formation (Hu et al ., 2007, 2008, 2011). As a result, processing of

regenerated silk materials can be achieved through aqueous based processes,

however, organic solvents can be utilized as well (Chen et al ., 2001; Hu et al ., 2011; Mandal et al ., 2012). The use of water vapor to control protein second-

ary structure formation is commonly referred to as water-annealing (Hu et al ., 2011; Jin et al ., 2005). The extent of secondary structure modifi cation is con-

trolled through enhancing water vapor content available for interaction with

the fi broin protein chains (Hu et al ., 2007; Lawrence et al ., 2008b; Mo et al ., 2006). With more extensive secondary structure formation there is an increase

in internal hydrogen bonding which modulates the apparent material proper-

ties, which may be specifi ed for a given application (Hu et al ., 2008).

Previous research indicates that beta-sheet content can be modulated

between 10% and 60% of total protein secondary structure through various

water-annealing processes (Lawrence et al ., 2008b). For example, placing

newly cast silk fi lms in a chamber with water placed in the basin to increase

chamber humidity will cause the formation of up to 24% beta-sheet content,

however if this is used in conjunction with high pressure and temperature

steam, the beta-sheet formation will increase to 60% (Lawrence et al ., 2008b).

Unprocessed silk

Methanol solventtreatment

Water vaportreatment Silk secondary structures:

β-sheet

α-helix

Turn

Random coil

3.1 An illustration that represents how silk fi broin material properties

may be modulated through protein secondary structure formation

using methanol (MeOH) solvent or water vapor (water-annealing)

treatments.



Further work has demonstrated that exposing the silk material to increasing

water vapor content will drop the glass transition temperature ( T g ) from 180

to 40°C (Agarwal et al ., 1997; Hu et al ., 2006, 2011). Practical applications of

this drop in T g have been utilized to produce rapid surface imprinting methods

and also for modifying silk scaffold degradation in vitro and in vivo (Amsden

et al ., 2010; Arai et al ., 2004; Wang et al ., 2008b). These specifi c examples pro-

vide compelling evidence for the large range of material processing windows

attainable for controlling fi broin protein secondary structure formation.

3.3 Silk fibroin materials and their use in biomedical applications

The silk fi broin protein is a structuring molecule that has the ability to be

processed into numerous forms through a variety of techniques. Due to the

material’s inherent biocompatibility, the protein has been recently utilized as a

scaffold for developing a number of biomedical product ideas for use in clinical

applications. A few examples of such clinical applications are explored below.

3.3.1 Sericin extraction and fi broin solubilization

Silk fi bers were traditionally used as medical sutures before the development

of biodegradable synthetic polymers (Altman et al ., 2003). However, silk

sutures would in many cases need to be removed due to their slow biodeg-

radation rate and the potential for infl ammatory reactions to their presence

(Altman et al ., 2003). This infl ammation was largely found to be a reaction

to the presence of the glycoprotein sericin fi ber coating, which could be

removed through cleaning the fi bers with detergents (Barker, 1975; Santin

et al ., 1999). Once the sericin was removed the regenerated structural silk

fi broin protein was well tolerated by the body (Meinel et al ., 2005; Panilaitis

et al ., 2003; Santin et al ., 1999; Wang et al ., 2008b). As a result both native silk

fi bers free of sericin and regenerated fi broin protein solution offer desirable

material properties for designing silk-based biomedical devices.

Silk fi broin solubilization has been documented since the beginning of

the twentieth century (Matthews, 1913), but only recently has the use of

regenerated silk solution for biomedical applications been studied (Altman

et al ., 2002; Minoura et al ., 1995). The most ubiquitous method to produce

regenerated silk fi broin solution is through the use of NaCO 3 and heavy

salts like LiBr (Lawrence et al ., 2012b; Rockwood et al ., 2011) ( Fig. 3.2 ). In

addition, mixtures with urea and ionic liquid solutions have been used to

produce silk solution (Gupta et al ., 2007; Shaw, 1964); these processes will

not be described here, however, for brevity.

Silk fi bers may be cleaned of contaminating sericin proteins by boiling

cocoons or fi bers in 0.02M NaCO 3 in less than 1 h. The extracted fi bers should



be thoroughly rinsed with pure water, and then allowed to dry using clean con-

vective air. The dried fi bers can then be dissolved using a 9.3M LiBr solution

to disrupt hydrogen bonding between the fi broin protein chains (Lawrence

et al ., 2012b). The solution should then be allowed to thoroughly dissolve

for up to 4 h at 60°C to ensure complete dissolution, while making sure the

reaction container is covered to prevent water evaporation. The heavy salts,

in this case LiBr, can then be removed from the silk solution through dialy-

sis against pure water over a period of 48 h. Typically the molecular weight

cut-off for the dialysis membrane is 3500 Da, which is permeable enough to

allow for the LiBr salts and water to travel freely while retaining the 25 and

390 kDa fi broin light and heavy protein chains, respectively (Kaplan, 1994).

Final silk solution concentrations range from 6% to 10% wt./vol. content,

however dialysis against high molecular weight water-soluble polymers, like

polyethylene glycol (PEG), allow for concentrations of the silk solution of

over 20% wt./vol. (Kim et al ., 2005; Mandal et al ., 2012; Nazarov et al ., 2004).

3. Rinse and dryfibroin fiber extract

Structures formed fromsilk fibroin solution:

Films

Tubes

Scaffolds

Hydrogels

Microspheres

Electrospun fibers

Microfluidic devices

4. Dissolve fibersinto LiBr solution

2. Boil cocoons inNaCO3 solution

1. Collect Bombyxmori cocoons

7. Use silk solutionor store at 4°C

6. Centrifuge silksolution at 10 000 g

5. Dialyze silksolution for 48 hrs

3.2 Schematic diagram detailing the key steps for preparing silk fi broin

solution from Bombyx mori silkworm cocoons.



The silk solution can then be stored in a 4°C refrigerated environment, where

the material has shown stability for up to two months post production (Le

et al ., 2008; Matsumoto and Lindsay, 2008).

3.3.2 Native fi bers

Native silk fi bers derived from the B. mori silkworm cocoon provide the raw

material that is used to produce regenerated silk solution. Silk fi bers have

found wide utility in the medical world as sutures over the past millennia

(Altman et al ., 2003). As mentioned earlier these fi bers are composed of

the fi broin protein, which provides robust mechanical strength in the native

silkworm cocoon. Recently, the utility of ‘extracting’ the native fi ber from

the sericin coating proteins has been invaluable for the use of this protein in

medical applications (Altman et al ., 2003). The extracted silk fi bers have rel-

atively high tensile strength recorded between 500 and 740 MPa, a modulus

between 5 and 17 GPa, and an elongation to break ranging from 4% to 20%

(Altman et al ., 2003). This is in the order of 5–10 times higher when com-

pared to other synthetic and natural polymers. Native silk fi bers can then be

further used for the design of medical devices.

Recently, silk fi broin native fi bers have been combined to produce scaf-

folding structures that have found utility in soft tissue repair, such as liga-

ments (Altman et al ., 2002; Vunjak-Novakovic et al ., 2004). Specifi cally a

silk fi ber mesh can be twisted together to produce a rope structure that

mimics the natural mechanics of the anterior cruciate ligament (ACL)

(Altman et al ., 2002). The structure can then be implanted in vivo to serve

as a scaffold to regenerate new ligament tissue (Vunjak-Novakovic et al ., 2004). Extending from this work these fi bers can then be resolubilized into

regenerated silk solution as described above to produce a number of geo-

metrical structures, which are described further below.

3.3.3 Electrospun fi bers

Electrospinning silk solution is a favored processing methodology for pro-

ducing nanometer- to micron-scale fi bers that result in a high degree of

available surface area for use in creating scaffolds for tissue engineering

and regenerative medicine purposes (Jin et al ., 2002). In brief, electrospun

materials are produced by applying a strong electric fi eld between a poly-

mer solution and a collection device (Soffer et al ., 2008). In the case of silk

fi broin this has been readily accomplished using both organic solvents and

aqueous based processes (Jin et al ., 2002; Sukigara et al ., 2003). The elec-

trospun silk fi bers have found utility in producing scaffolds for a variety of

biological applications such as growing cardiac, bone, nerve, and skin tissue



(Jin et al ., 2004a; Schneider et al ., 2009; Soffer et al ., 2008; Wharram et al ., 2010; Zhang et al ., 2012b).

One area of work has focused on the use of a silk fi broin and poly-

(ethylene oxide) (PEO) solution blend that reduces material brittleness

and enables ready processing and handling of the fi nal material (Jin et al ., 2002). Fiber diameters range between 700 and 900 nm for varying silk/PEO

blends. Further work using silk/PEO blends was undertaken to form elec-

trospun mats for use in culturing human bone marrow stromal cells (hBM-

SCs), which demonstrated excellent cell attachment, spreading, and culture

growth over a 14-day period (Jin et al ., 2004a). These materials compared

favorably with native silk fi ber controls. Building from this work silk/PEO

electrospun fi bers were used to produce tubular scaffolds for use as small-

diameter vascular grafts (Soffer et al ., 2008). Human endothelial and smooth

muscle cells were successfully grown on these scaffolds, and mechanical

testing demonstrated the ability of the constructs to withstand arterial pres-

sures and tensile properties comparable to native vessels (Soffer, 2006).

Similar blended silk/PEO electrospun constructs were developed for

use in wound healing applications (Schneider et al ., 2009; Wharram et al ., 2010). Initial material characterization proved that these scaffolds exhib-

ited aqueous absorption, water vapor transmission, oxygen permeation, and

enzymatic biodegradation profi les applicable for full thickness wound site

regeneration (Wharram et al ., 2010). Further studies used a human skin-

equivalent wound healing model to demonstrate physiological feasibility

(Schneider et al ., 2009). The study showed that the silk/PEO electrospun

fi bers could be loaded with epidermal growth factor (EGF) and release

25% of the molecule content over a one-week period. It was shown that the

electrospun mats increased wound closure by a remarkable rate, increased

by 90% when compared to controls. Additional work has shown successful

implantation of electrospun silk fi broin constructs in vivo using a subdermal

rat model (Kim et al ., 2012). Results indicated that electrospun constructs

demonstrated varying biodegradation profi les based on post-processing with

varying blends of ethanol and propanol concentrations, where higher etha-

nol concentrations showed longer degradation times. In addition, implanted

constructs proved highly biocompatible, and supported cell infi ltration for

materials treated with lower ethanol concentrations. Together these fi ndings

suggest promise for such materials in the use of chronic wound healing.

3.3.4 Silk fi broin fi lms

Silk fi lms offer an elegant and straightforward biomaterial of choice for med-

ical device design. Due to the inherently less complex nature of fi lms these

materials offer rapid characterization and design with regards to scaffold

development. The dynamic protein secondary structure of the fi broin beta-



sheet and alpha-helical structures has been largely determined from studies

utilizing silk fi lm samples (Hu et al ., 2008; Motta et al ., 2002; Tretinnikov and

Tamada, 2001), and they possess the longest history of scientifi c experimen-

tation and use (Matthews, 1913; Minoura et al ., 1990). As a result, silk fi lms

offer the most immediate potential utility for a variety of biomedical appli-

cations (Kaplan and Omenetto, 2012b; Omenetto and Kaplan, 2008). As a

biomaterial of choice they offer a number of advantages for in vitro char-

acterization due their ease of production and consistent material properties

(Lawrence et al ., 2012b; Rockwood et al ., 2011). Silk fi lm material properties

like biodegradation, mechanical properties, chemistry, and optical proper-

ties may be readily modulated for a desired application (Arai et al ., 2004;

Horan et al ., 2005; Karageorgiou and Meinel, 2004; Lawrence et al ., 2008a;

Li et al ., 2003; Motta et al ., 2002). In addition, fi broin fi lms have been shown

to support a multitude of cell types including a variety of cell lines from epi-

thelium, endothelium, and fi broblasts, which allows for the adaptation to a

variety of tissue systems (Arai et al ., 2004; Meinel et al ., 2005; Panilaitis et al ., 2003). In vitro results can be quickly translated to in vivo models due to the

fi broin fi lm possessing a high level of biocompatibility and material consis-

tency (Arai et al ., 2004; Meinel et al ., 2005; Panilaitis et al ., 2003).

A recent interest has been the use of patterned silk fi lms to develop

a multitude of silk scaffolds and devices (Gil et al ., 2010; Kaplan and

Omenetto, 2012a; Lawrence et al ., 2008a, 2009; Omenetto and Kaplan,

2008, 2010; Rogers et al ., 2012; Tsioris et al ., 2011). A nanopatterned fi broin

fi lm surface may be produced by casting silk solution upon a molding sur-

face, then allowing the water to evaporate to form a fi lm, following which

the dried silk fi lm is air-lifted from the molding surface (Lawrence et al ., 2008a; Omenetto and Kaplan, 2008; Perry et al ., 2008). In addition, the

water-annealing technique may be employed post-casting to produce water

insoluble fi lms through the induction of alpha-helical and beta-sheet sec-

ondary structure formations for use in biological applications (Lawrence

et al ., 2008a; Rockwood et al ., 2011).

This technique has been recently used to produce culture surfaces for

investigating corneal cell response to silk fi lm surface topography (Gil et al ., 2010; Lawrence et al ., 2009, 2012a). Results indicated that silk fi lm surface

topography could be used to direct corneal epithelial and fi broblast align-

ment (Gil et al ., 2010; Lawrence et al ., 2009), and that the edge surface of the

patterned topography signifi cantly infl uenced localization of focal adhesion

formations (Lawrence et al ., 2012a). One immediate biomedical application

for silk fi lms is for use in repairing the cornea after injury due to their trans-

parent nature and high degree of biocompatibility (Bray et al ., 2011; Chirila

et al ., 2008; Liu et al ., 2012a).

In addition to the surface patterning capabilities of silk fi broin, the

material can also be employed for immobilizing bioactive compounds for



later use without loss of activity or function (Demura and Asakura, 1989;

Lawrence et al ., 2008a; Liu et al ., 1996; Numata and Kaplan, 2010; Tsioris

et al ., 2011; Zhang et al ., 2012a). Recent work has demonstrated that the

antibiotic tetracycline and the measles, mumps, and rubella vaccine can be

successfully stored within a dried silk fi broin fi lm matrix for six months at

60°C (Zhang et al ., 2012a). Such capability eliminates the need for cold stor-

age and may revolutionize the way many labile molecules are stored and

utilized throughout the world (Zhang et al ., 2012a). In addition, the mole-

cule immobilization work has been combined with the unique surface pat-

terning properties of silk to produce a microneedle delivery system: high

aspect ratio topographic features were produced on a silk fi lm surface that

were strong and sharp enough to pierce the skin (Tsioris et al ., 2011). The

fi broin protein secondary structure is then modifi ed to release a therapeutic

molecule in a continual release profi le after the microneedles are embedded

within the hydrated dermis.

Silk fi lms have also found utility as carrier surfaces for use in biomed-

ical applications. Recently it has been shown that electronic components

may be fabricated upon fi broin fi lm surfaces that can later fully integrate or

degrade naturally with the surrounding environment (Hwang et al ., 2012).

Silk fi broin has been shown to be an optimal insulating material of choice in

which semiconductor materials, like silicon, can be readily integrated to pro-

duce biodegradable and highly functional electronic components (Hwang

et al ., 2012; Kim et al ., 2010; Tao et al ., 2012). The fi broin fi lms can be pro-

cessed to allow for a conformational adhesion to a given surface such as

on brain, bone, and food surfaces (Hwang et al ., 2012; Kim et al ., 2010; Tao

et al ., 2012). Recent work has demonstrated the feasibility of mapping the

feline neural brain surface using sensors that utilize the insulator and carrier

abilities of silk fi broin (Kim et al ., 2010). Similar efforts have utilized such

conformable electronics to adhere to food products to determine spoilage

or bacterial contamination (Tao et al ., 2012). These studies demonstrate the

utility of silk fi broin as a manufacturing material in which continued devel-

opment will provide a sustainable, biodegradable material platform that can

be used for a number of high-tech, medicinal, and environmental applica-

tions (Kaplan and Omenetto, 2012b).

3.3.5 Sponge scaffolds

Regenerated silk fi broin solution may also be processed to produce three-

dimensional sponge scaffolds for use in tissue engineering (Mandal et al ., 2012; Nazarov et al ., 2004). Sponge scaffolds provide a framework of inter-

connected pores with a high amount of surface area within a defi ned three-

dimensional volume, which allows for cell attachment and tissue ingrowth.

Initial studies indicate that the porosity and mechanical properties of these



scaffolds can be readily controlled through salt leaching and gas foaming

processes (Nazarov et al ., 2004). Pores averaged 100 μ m for both process-

ing conditions, and reached porosity void volume ranges between 84%

and 98%. However, compressive strength measured up to 175 kPa for salt

leached scaffolds, and up to 280 kPa for gas foamed structures. Further

work identifi ed that silk fi broin concentration was a critical determinant

of fi nal porosity formation and mechanical properties (Yan et al ., 2012).

Specifi cally, porosity could be dialed in from 80% to 90% by ranging the

fi broin concentration between 8% and 16%, respectively. Such scaffolds

could fi nd utility in the production of cartilage, bone, or connective tissue

engineering applications.

Additional methods to produce silk scaffolds utilize sacrifi cial template

techniques (Swinerd et al ., 2007). It has also been shown that silk fi broin solu-

tion can be mixed with 500 nm diameter polystyrene beads and then dried

to form a solid structure. The dried construct is then treated with metha-

nol to produce the highly stable silk beta-sheet protein secondary structure.

The scaffold is then treated with toluene to leach out the polystyrene beads.

What is left is a spongy scaffold with 400 nm pores interspersed throughout

the scaffold, and could elastically recover from 112 MPa compressive loads

while possessing super hydrophobicity properties that may not be desirable

for in vivo use (Swinerd et al ., 2007).

Follow up in vivo work to assess silk fi broin sponge scaffold biocompati-

bility was undertaken in rat muscle pockets and subcutaneous animal mod-

els (Wang et al ., 2008b). Results indicated that the implanted scaffolds were

highly biocompatible and encouraged tissue ingrowth to varying extents

depending on porosity and solvent processing conditions (i.e., aqueous or

organic solvent) (Wang et al ., 2008b). It was shown that all aqueous pro-

cessed scaffolds degraded within a 2–6-month time frame, while scaffolds

prepared using the organic solvent hexafl uoroisopropanol (HFIP) persisted

beyond one year post-implantation (Wang et al ., 2008b).

Following on from this work, scaffolds were produced that had high com-

pressive strength characteristics of up to 13 MPa within a hydrated envi-

ronment, which could allow for functional bone formation (Mandal et al ., 2012). These scaffolds utilized a novel processing method in which silk

fi bers were combined with NaOH pellets in water to produce a sponge-

like scaffold construct. Subcutaneous in vivo studies in rats demonstrated

a high degree of biocompatibility and neovascularization throughout the

scaffold. In addition, the standard sponge scaffolding processing techniques

can also be combined with other innovative methods. Silk fi broin was com-

bined with gelatin, dried, and then freeze dried to produce a sponge scaffold

matrix upon a molding template surface (Liu et al ., 2012b). The template

was designed to mimic the liver by producing a scaffold with grooves and

substructures patterned on the surface. The scaffolds were then seeded



with primary hepatocyte cultures and, after seeding, the sponge layer was

rolled upon itself to form a three-dimensional structure (Liu et al ., 2012b;

Papenburg et al ., 2009). Analysis of the structures revealed the scaffolds

were highly biocompatible and encouraged tissue growth. Future process-

ing techniques will continue to evolve for producing silk fi broin sponge scaf-

folds for a variety of applied tissue engineering applications.

3.3.6 Hydrogels

Hydrogels offer a tissue culture system where interconnected fi laments

aggregate and stabilize water within a confi ned volume to produce a gel.

Gelation of the silk fi broin solution can be controlled by temperature,

calcium ion concentration, pH, and polymer blending with materials like

PEO to produce a hydrogel (Kim et al ., 2004). Results indicated that gel-

ation time decreased with an increase in protein concentration, decrease

in pH, increase in temperature, addition of calcium, and addition of PEO.

Gelation was linked to beta-sheet secondary structure formation through-

out the hydrogel structure (Kim et al ., 2004; Matsumoto et al ., 2006). It was

shown that above 15% beta-sheet content gelation time increased linearly

with fi broin protein concentration in solution (Matsumoto et al ., 2006). Such

results indicate that silk fi broin hydrogels offer a number of control points

for defi ning material properties.

Building upon this work, the silk fi broin gelation rate was highly con-

trolled through the use of sonication (Wang et al ., 2008a). Gelation can be

induced from minutes to hours depending on sonication parameters, such

as power output and time, and silk fi broin concentration. Interestingly,

the sonicated silk solution had human mesenchymal stem cells (hMSCs)

added while in the solution state, which then encapsulated the cells within

the hydrogel matrix. The cells were found to grow and proliferate within

the gel over a 21-day time period, where cell viability was highest for lower

silk fi broin concentration gels. A subsequent in vivo study implanted silk

fi broin hydrogels subcutaneously within nude mice (Etienne et al ., 2009).

Results indicated that human fi broblasts embedded within the material had

a 68% survival rate after 12 weeks post-implantation. Revascularization had

occurred and no infl ammatory response was observed after the 3-month

implantation period. These hydrogel materials are anticipated to have uses

in periodontal, maxillofacial, and dermal fi lling applications.

3.3.7 Microspheres

Microspheres describe a general class of particulates that have diameters in

the high nanometer to micron range and assume a spherical shape, which can



be used for a variety of biomedical purposes such as controlled drug release

applications. Silk microspheres can be readily produced by mixing regener-

ated fi broin solution with lipid vesicles that act as templates to effi ciently

load biological molecules in an active form for sustained release (Wang

et al ., 2007b). The lipid could then be subsequently removed by methanol

or sodium chloride treatments that produced silk fi broin microspheres with

beta-sheet structure, and measured 2 μ m in diameter on average. Studies

were then undertaken using horseradish peroxidase (HRP) as a model ther-

apeutic molecule that was encapsulated within the silk microspheres using

freeze–thaw cycles that showed enzymatic activity post encapsulation and

continually released over a two-week time frame (Wang et al ., 2007a, 2007b).

Additionally, silk microspheres could be produced by using a fi broin and

polyvinyl alcohol blend methodology to avoid the complications from the

freeze–thaw cycle and use of organic solvents (Wang et al ., 2010). Fibroin/

PVA blended fi lms were fi rst produced by drying the solution into fi lm form,

the dried fi lm was then rehydrated in water, and residual PVA was removed

by centrifugation. Controlled microsphere diameters ranging from 300 nm

up to 20 μ m were achieved.

An additional processing method was developed in which drug-loaded

silk fi broin microspheres were produced using a laminar jet break-up of

aqueous solution (Breslauer et al ., 2010; Wen et al ., 2011; Wenk et al ., 2008).

Sphere diameters ranging between 100 and 440 μ m were produced depend-

ing on the diameter of the nozzle and treatment to induce water insolubility

using either methanol or water annealing methods (Jin et al ., 2005; Wenk

et al ., 2008). Insulin-like growth factor (IGF) was used as a model drug

for delivery characterization, and showed a seven-week delivery period.

In vitro results indicate that silk microspheres are highly biocompatible, and

released IGF increased MG-63 cell line growth over time (Wenk et al ., 2008).

Additionally, smaller sphere sizes ranging in the hundreds of nanometers

to the lower micron size range can be similarly controlled through the use

of salting-out techniques of potassium phosphate solutions (Lammel et al ., 2010). Silk fi broin microspheres may offer much promise for drug delivery

applications for a variety of applications, and future in vivo studies will fur-

ther aid in promoting their translation to clinical use.

3.4 Conclusion and future trends

The functionality of silk fi broin in the production of biomedical structures

is only limited to the variety of ways to controllably process the varying

properties of the material. As with most silk proteins, the highly modifi able

secondary structure of fi broin in solution can be manipulated to form any

variety of three-dimensional structures (Rockwood et al ., 2011). In addition,

use of fi broin as a biomaterial post-sericin extraction is well documented and



therefore will continue to fi nd itself as a material of choice for biomedical

applications (Altman et al ., 2003; Vepari and Kaplan, 2007). Future develop-

ments of silk fi broin based scaffolds will benefi t from further studies focused

on improving material properties and assessing these materials in vivo. In

addition, promising work utilizing composite methodologies is being under-

taken that has shown much progress in producing fi broin devices for use in

peripheral nerve and intervertebral disc regeneration (Ghaznavi et al ., 2011;

Nectow et al ., 2012; Park et al ., 2012). Progress will continue in utilizing silk

for in situ sensors and electronics for monitoring a variety of medical condi-

tions (Hwang et al ., 2012; Kim et al ., 2010). Although 40 000 other varieties

of silk proteins exist, no other is as highly produced or ubiquitously utilized

as silk fi broin derived from the domesticated B. mori silkworm (Shear et al ., 1989). As the world continues to look for alternatives to synthetic based

polymers, fi broin may be a hopeful candidate for the future development of

biologically based building materials for medical applications and beyond.

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the thermal properties of Bombyx mori silk fi broin fi lms’, Journal of Applied Polymer Science , vol. 63 , no. 3, pp. 401–410.

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Zhang, Q, Zhao, Y, Yan, S, Yang, Y, Zhao, H, Li, M, Lu, S and Kaplan, DL (2012b),

‘Preparation of uniaxial multichannel silk fi broin scaffolds for guiding primary

neurons’, Acta Biomaterialia , vol. 8 , no. 7, pp. 2628–2638.


processing of bombyx mori silk for biomedical...

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