Processing of Bombyx mori silk for biomedical...
Transcript of 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
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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)
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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
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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
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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
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Processing of Bombyx mori silk for biomedical applications 83
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.
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84 Silk Biomaterials for Tissue Engineering and Regenerative Medicine
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
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Processing of Bombyx mori silk for biomedical applications 85
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.
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86 Silk Biomaterials for Tissue Engineering and Regenerative Medicine
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
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Processing of Bombyx mori silk for biomedical applications 87
(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-
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88 Silk Biomaterials for Tissue Engineering and Regenerative Medicine
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
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Processing of Bombyx mori silk for biomedical applications 89
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
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90 Silk Biomaterials for Tissue Engineering and Regenerative Medicine
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
Property of Elsevier
Processing of Bombyx mori silk for biomedical applications 91
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
Property of Elsevier
92 Silk Biomaterials for Tissue Engineering and Regenerative Medicine
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
Property of Elsevier
Processing of Bombyx mori silk for biomedical applications 93
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.
Altman, AM, Gupta, V, R í os, CN, Alt, EU and Mathur, AB (2010), ‘Adhesion, migra-
tion and mechanics of human adipose-tissue-derived stem cells on silk fi broin–
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F (2010), ‘Rapid nanoimprinting of silk fi broin fi lms for biophotonic applica-
tions’, Advanced Materials , vol. 22 , no. 15, pp. 1746–1749.
Arai, T, Freddi, G, Innocenti, R and Tsukada, M (2004), ‘Biodegradation of Bombyx mori silk fi broin fi bers and fi lms’, Journal of Applied Polymer Science , vol. 91 ,
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(2011), ‘Human corneal epithelial equivalents constructed on Bombyx mori silk fi broin membranes’, Biomaterials , vol. 32 , no. 22, pp. 5086–5091.
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Demura, M and Asakura, T (1989), ‘Immobilization of glucose oxidase with Bombyx mori silk fi broin by only stretching treatment and its application to glucose sen-
sor’, Biotechnology and Bioengineering , vol. 33 , no. 5, pp. 598–603.
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Etienne, O, Schneider, A, Kluge, JA, Bellemin-Laponnaz, C, Polidori, C, Leisk, GG,
Kaplan, DL, Garlick, JA and Egles, C (2009), ‘Soft tissue augmentation using
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Hu, X, Kaplan, D and Cebe, P (2006), ‘Determining beta-sheet crystallinity in fi brous
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Hu, X, Kaplan, D and Cebe, P (2007), ‘Effect of water on the thermal properties of
silk fi broin’, Thermochimica Acta , vol. 461 , no. 1–2, pp. 137–144.
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DL, Zakin, MR, Slepian, MJ, Huang, Y, Omenetto, FG and Rogers, JA (2012),
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Jin, H, Park, J, Karageorgiou, V, Kim, U, Valluzzi, R, Cebe, P and Kaplan, D (2005),
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strength and mechanical toughness of β -sheet crystals in silk’, Nature Materials ,
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(2008a), ‘Bioactive silk protein biomaterial systems for optical devices’,
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