Feature Article

Recombinant Spider Silk Proteins for

Applications in Biomaterials

Kristina Spiess, Andreas Lammel, Thomas Scheibel*

Due to their extraordinary mechanical and biochemical properties, silks have long been in
focus of research. In vivo, fibers are formed from silk proteins, in vitro, however, a variety of
materials can be produced in addition to fibers including capsules, particles, films, foams, and
gels. The versatility of silk proteins, along with their
biocompatibility, biodegradability, and potential for
processing in aqueous solution under ambient con-
ditions make silk-based materials good candidates
for biomedical applications such as drug delivery sys-
tems and scaffolds for tissue engineering. Here, we
summarize recent progress in research employing
recombinantly produced engineered spider silk
proteins with a focus on the fundamentals of silk
protein processing. We highlight recombinant spider
silk films and particles as morphologies that represent
model systems with adjustable material properties
controlled by process parameters.

Introduction prey, yielding outstanding materials with high tensile

Silk from the silkworm Bombyx mori has been used to
produce textiles for thousands of years due to its lustre, light
weight, and tear resistance. During the past few decades,
spider silks have also aroused increasing interest in
material science due to some extraordinary material
properties. During 400 million years of evolution, spider
silk has been optimized for several tasks such as catching
K. Spiess, T. Scheibel
Lehrstuhl Biomaterialien, Fakultät Angewandte
Naturwissenschaften, Universität Bayreuth Universitätsstr. 30,
95440 Bayreuth, Germany
Fax: (þ49) 921 55 7346;
E-mail: thomas.scheibel@bm.uni-bayreuth.de
A. Lammel
Department Chemie, Technische Universität München, Lehrstuhl
Biotechnologie, Lichtenbergstraße 4, D-85747 Garching, Germany
a
These authors made equivalent contributions to this manuscript
strength combined with high extensibility.[1–3] In contrast
to silkworms, breeding spiders and harvesting their silk has
been limited due to their cannibalistic nature. Through
advances in biotechnology it is now possible to produce
spider silk proteins recombinantly as well as to engineer
silk genes and therefore proteins with properties tailored
for specific requirements. Among the different types of
spider silks, dragline silk, which composes the frame and
radii of the web, is the most extensively studied and the best
characterized.[4–8] In general, dragline silk is mainly
composed of two types of protein.[7] In the case of silk
from the spider Araneus diadematus the main proteins are
ADF3 (A. diadematus fibroin) (which is relatively hydro-
philic) and ADF4 (which is relatively hydrophobic)
(Figure 1A).[7] The outstanding properties of spider silk
fibers can be explained by the hierarchical and sophisti-
cated composition of structural elements of these two
proteins with crystalline regions of different dimensions
being embedded in an amorphous or pre-oriented matrix.[9]

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998 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com DOI: 10.1002/mabi.201000071
Recombinant Spider Silk Proteins for Applications in . . .
The amorphous matrix is likely to be responsible for the
extensibility of dragline silk, while crystalline regions are
responsible for their strength.[10,11] The extent and type of
structural features depend on the amino acid sequence of
the underlying proteins (primary structure). Figure 1B
provides an overview of typical silk motifs of dragline silk
proteins, which have been identified as being responsible
for specific structure formation. Polyalanine motifs align to
form b-crystallites (tightly packed b-sheets) whereas
polyglycine motifs form primarily amorphous regions.
Our approach of recombinant silk protein production
(reviewed in ref.[12]) is based on multimerization of amino
acid consensus motifs (Modules A, Q, and C as illustrated in
Figure 1C) derived from the repetitive part of the natural
proteins ADF3 and ADF4 leading to the engineered proteins
eADF3 and eADF4.
In the following sections we will outline how unraveling
the natural assembly process has given rise to the
development of various new processing techniques yield-
ing novel silk morphologies. We will further describe how
material properties can be adjusted and controlled by
variation of process parameters using exemplarily the
morphologies of silk particles and films.
The Natural Silk Assembly Process
The formation of silk fibers is a sophisticated process which
involves a series of tightly controlled parameters.[1] In
nature, silk proteins are produced and excreted by
specialized cells lining the epithelium of a gland; in case
of dragline silks this is the major ampullate gland (Figure 2).
The proteins are stored as the so-called dope in the glands’
lumen at remarkably high concentrations (up to 50 wt.-%)
without aggregation, and assemble on their way through
the spinning duct into stable and tough fibers which are
then released to the exterior (reviewed in ref.[1]). This
assembly process is characterized by a liquid-solid phase
transition accompanied by structural changes of the
proteins; in the ampulla the core domains of the proteins
are thought to possess no defined secondary structure, but
the proteins are most likely to be arranged in micelle-like
assemblies, resulting from their amphiphilic nature.[13,14]
The final fiber, however, is rich in b-sheet crystallites
aligned parallel to the long axis of the fiber. Several
biochemical and physical factors promote the structural
transition. A slight acidification of the spinning dope
(from pH 7.2 in the gland to pH 6.3) most likely leads to a
neutralization of abundant acidic side chains, allowing
tighter interactions of the proteins.[15,16] Recently, the
aminoterminus of the proteins has been identified as pH-
sensitive regulator of self-assembly.[17] Hydrophobic inter-
molecular interactions are further enhanced by a change in
the ionic composition inside the duct: the more chaotropic
Macromol. Biosci. 2010, 10, 998–1007
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Kristina Spiess studied biology at the University
of Regensburg (Germany), where she received
her Diploma in 2005. After five months working
on an industrial collaboration project in the
group of Thomas Scheibel at the Technische
Universität München (TUM, Germany) she
started her PhD in his research group and moved
with him to the University of Bayreuth
(Germany), where she is currently finishing her
PhD studies. She is an associate member of the
International Graduate School of Science and
Engineering at TUM. Her research focuses on
characterization and functionalization of recom-
binant spider silk proteins and their processing
into different morphologies for medical appli-
cations.
Andreas Lammel studied mechanical engineer-
ing in the field of medical and microtechnology
at Technische Universität München (Germany)
and chemical engineering at Rice University in
Houston (Texas). He is a fellow of the Inter-
national Graduate School of Science and Engin-
eering at TUM as well as of the international
doctorate program Material Science of Complex
Interfaces within the Elite Network of Bavaria.
Since 2007 he has worked on his PhD under the
supervision of Thomas Scheibel. His research
focuses on the development of silk protein based
drug delivery systems.
Thomas Scheibel is full professor and chair of
biomaterials at the Universität Bayreuth in
Germany. He received both his Diploma of bio-
chemistry and a Dr. rer. nat. from the Universität
Regensburg and his habilitation from the Tech-
nische Universität München in Germany. He was
a Kemper Foundation postdoctoral fellow and a
DFG postdoctoral fellow at the University of
Chicago. He gained several awards including
the Biomimetics award of the German Bundes-
ministerium für Bildung and Forschung (BMBF)
in 2006, and the ‘‘Innovation by nature award’’
of the BMBF in 2007. He is one of ten recipients
of the 2006 innovation tribute of the Bavarian
prime minister, received the Heinz-Maier-Leib-
nitz Medal in 2007, and the Karl-Heinz-Beckurts
Award in 2008.
(‘‘salting-in’’) ions sodium and chloride are exchanged by
the more kosmotropic (‘‘salting-out’’) ions phosphate and
potassium. In the next step, water is resorbed from the
solution at the distal part of the duct by endothelial cells,
increasing the protein concentration, which additionally
favors intermolecular interactions. Finally, the viscous
solution is subjected to increasing elongational flow and
shear forces, which are thought to further align the
molecules.[13,18] This finding is supported by the fact that
higher flow rates lead to better aligned fibers.[19] In vitro
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Figure 1. Model of the structure of a spider dragline silk. (A) A. diadematus spider
dragline silk consists mainly of two proteins, ADF3 and ADF4. On the molecular level,
spider silk exhibits crystalline regions of different dimensions embedded in an amor- acid,
phous matrix (figure adapted from ref. [9]). (B) Typical silk motifs and their correspond- liquids,
[11]
ing putative secondary structure (adapted from ref. ). (C) Engineered spider silk
modules adopted from ADF3 and ADF4 for recombinant production (figure adapted etc.). Any point along the side of the
from ref. [7]).
similar results were obtained: after addition of potassium
phosphate (500 mM) to eADF3 spherical aggregates were
formed.[20,21] When accompanied by a pH shift and
simultaneous elongational flow—applied by the geometry
of the employed microfluidic setup—eADF3 formed
fibers.[13,21,22]
Processing into Various Morphologies
As described above, the fundamental principal that is
crucial for the processing of materials from silk proteins in
Figure 2. Natural assembly of spider silk proteins into fibers.
Dragline silk proteins are stored in the spinning gland. Upon
passage of the spinning dope through the spinning duct, the
proteins are subjected to a change of physico-chemical
parameters leading to a liquid-solid phase transition necessary
for fiber formation (figure adapted from ref. [1]).
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K. Spiess, A. Lammel, T. Scheibel
aqueous solution is the induction of
phase separation. The two key para-
meters involved in phase separation are
the protein concentration in solution and
the method with which desolvation of
the protein is triggered. The triggers can
be chemical such as pH and the amount/
type of additives (salts, e.g., potassium
phosphate, non-solvents, e.g., methanol,
polymers, e.g., poly(ethylene glycol), etc.)
or physical conditions like mechanical
shear and temperature.[6,7,21,23–26]
To visualize the influence of chemical
and physical parameters, Figure 3A
shows a qualitative ternary phase
diagram at constant temperature and
pressure. Each corner of the triangle
represents 100% of the respective com-
ponent: silk protein, solvent (e.g., formic
[27]
hexafluoroisopropanol,[28] ionic
[29]
and water[23,30]) and non-
solvent (e.g., methanol,[6] toluene,[31]
triangle represents the composition of
the binary mixtures constituted by the
two components at the corresponding corners. Therefore, a
state inside the phase diagram can be expressed by the silk
concentration and the solvent quality expressed by the
ratio of solvent to non-solvent.
The Spinodal represents the set of conditions denoting
the boundary of absolute instability of a solution where
small fluctuations in composition lead to a spontaneous
phase separation via spinodal decomposition. The region
between the Spinodal and Binodal is the metastable region
where the Binodal denotes the boundary condition at
which two distinct phases may coexist. As can be seen in the
phase diagram, depending on the initial protein concentra-
tion, a different decrease in solvent quality is necessary to
cross the Spinodal to obtain a new stable state. This state
can be in form of a gel or particles. A gel is formed if the
concentration is high enough to build a continuous network
by which the liquid component is immobilized.[32] If the
concentration is below a certain threshold, particles are
formed by nucleation and growth.[18,20] A film can be
obtained by simple evaporation of the solvent. If porogens
are introduced into the silk protein solution and the solvent
is evaporated, porous structures or foams can be produced.
For fiber formation, phase separation has to take place in
combination with mechanical shearing and elongation.[21]
As outlined above, depending on the processing route,
phase transition of spider silk proteins is controlled by
chemical and physical process parameters leading to
different materials morphologies. The characteristics of
the different morphologies along with their material
DOI: 10.1002/mabi.201000071
Recombinant Spider Silk Proteins for Applications in . . .
Figure 3. Phase behavior and morphologies of spider silk assemblies. (A) Qualitative ternary phase diagram of silk protein-solvent-non-
solvent system (figure adapted from ref. [94]). (B) Morphologies useful for potential applications as drug carriers. (C) Morphologies useful for
potential applications as scaffolds in tissue engineering.
properties show potential for different biomedical applica-
tions. For example, silk particles and microcapsules are
envisioned as drug carriers (Figure 3B), whereas silk films,
foams, and non-wovens could be used as scaffolds in tissue
engineering (Figure 3C).
In the following, we will use spider silk particles as an
example of potential drug carriers, outlining their specific
properties. Furthermore, using spider silk films as an
example, we will illustrate how material properties can be
controlled and designed according to the desired needs for a
specific application.
Drug Carriers
In order to achieve constant drug levels in plasma during
therapy, colloidal micro- and nanoparticulate carriers have
been developed on the basis of various synthetic and
natural polymers.[33–40] These systems enable a reduction
of toxic side effects and the number of doses, while
improving cellular uptake and bioavailability.[41–45] Stable
silk protein particles can be obtained in a simple all-
aqueous process important for encapsulation of sensitive
compounds. Detailed studies of the thermodynamic
assembly process of eADF4(C16) (which is composed of
16 repeats of module C, Figure 1C) into microparticles
revealed that solid particles with high b-sheet content and
smooth surfaces are formed upon addition of kosmotropic
salts such as potassium phosphate.[20,21,46] The conversion
of monomeric soluble spider silk proteins to solid silk
particles can be solely triggered by potassium phos-
phate[7,20,21,23,25,46,47]; a fact that is very important in terms
of biocompatibility of the drug-delivery system considering
both the material and its processing.
The particle size (250 nm to 3 mm) and size distribution
can be controlled by mixing intensity and the concentration
Macromol. Biosci. 2010, 10, 998–1007
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of either eADF4(C16) or potassium phosphate
(Figure 4A).[46] A detailed investigation regarding the
applicability of eADF4(C16) particles employing 12 differ-
ent model drugs revealed that spider silk particle loading
and release follows the mechanism depicted in Figure 4D
(unpublished results). In the first step, drug molecules are
attracted to the particle by electrostatic forces (1). After
particle surface saturation, low molecular weight drugs
start to diffuse into the biopolymer matrix (2). Drug
molecules interact with the matrix via attractive hydro-
phobic and electrostatic interactions (3). Figure 4B shows
the obtained loading efficiencies of eADF4(C16) particles
with methyl violet (MV) as a function of molar ratio. Up to a
molar ratio of MV: eADF4(C16)
10 the loading increases
linearly with the amount of MV added. Above a molar ratio
of 10 the loading reaches a plateau, leading to a decrease of
loading efficiency. Upon incubation of loaded particles in
release media, drug molecules are transported to the
particle surface due to concentration gradient driven
transport processes (4). With time, drug molecules are
slowly released from the surface (in an equilibrium
process), leading to constant release rates for > 30 d at
physiologic conditions (37 8C, pH 7.4) (5) (shown for day 0 to
5 in Figure 4C). The release can be triggered by pH enabling
accelerated release behavior at lower pH (Figure 4C)
(unpublished results).
Silk Films—Controlled Processing Allowing
Different Applications
In contrast to particles, which can be used as mobile carriers,
films are more suitable for applications in which a
stationary phase is needed. It has been shown previously
that recombinantly produced engineered spider silk
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 K. Spiess, A. Lammel, T. Scheibel

Figure 4. Spider silk based drug carrier. (A) Influence of protein concentration and mixing intensity on spider silk particle size. Particles are
produced by salting out with potassium phosphate (figure adapted from ref.[46]). (B) Loading and loading efficiency of eADF4(C16) with
methyl violet particles as a function of molar ratio. (C) In vitro release kinetics of methyl violet over a period of 5 d at different pH as
indicated. (D) Drug loading and release mechanism of spider silk particles. Constant release is driven by transport processes influenced by
electrostatic and hydrophobic interactions as well as concentration gradients: (1) attraction, (2) saturation and diffusion, (3) binding, (4)
transport to the surface (5) release (unpublished results).

proteins can be assembled into transparent and stable
films.[48–50]
Biomedical Applications of Silk Films
Possible applications studied so far include the use of silk
films as devices for controlled substance (e.g., drugs) release,
as biochemical sensors and mainly as cell supporting
scaffolds for tissue engineering. Most of these studies have
been carried out with silk fibroins of the silkworm B. mori,
however, similar applications can be envisioned for
recombinant spider silk proteins due to the similarity in
their physico-chemical properties. Controlled release has
been demonstrated for substances which were either
directly integrated into the silk film during layer-by-layer
deposition,[51] or loaded onto microparticles, which were
embedded in or coated by a silk film, resulting in retarded
release kinetics.[52,53] Preparation of transparent films with

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Recombinant Spider Silk Proteins for Applications in . . .
distinct patterned surfaces offers potential applications for
use as biochemical sensors. This could be accomplished by
incorporation of biologically active components[54] or an
additional responsiveness to refractive indices of the
surrounding medium.[55] However, due to its biomedical
relevance, most effort has been put into the development
and investigation of novel scaffolds suitable for tissue
engineering. The culturing of a fibroblast cell line (L929) and
human bone marrow stem cells on regenerated B. mori silk
films revealed good cell adhesion, growth, and differentia-
tion[56] and allowed these films to be classified as ‘‘non-
cytotoxic’’.[57] Silk films have either been employed directly,
e.g., as wound dressings,[58] or as a coating of commercially
available porous membranes, where fibroblast adhesion
and proliferation, scaffold strength, and hydrophilicity
were increased.[59] By demonstrating the cultivation and
differentiation of different cell types (like osteoblast-like
cells,[60,61] mesenchymal stem cells,[62] (corneal) fibro-
blasts,[63–65] or HepG2 cells[66]) on silk substrates, it has
been suggested that silk films are suitable for regeneration
of corneal tissue or skin or bone. Induction of bone tissue
growth could be further enhanced by addition of cell
adhesive peptides like RGD.[60,61,67] Biomineralization of
silk films (with, e.g., hydroxyapaptite) was also exploited to
improve bone regeneration.[68] Other attempts to improve
the biocompatibility or specifity of silk matrices for tissue
engineering include the formation of blends with for
example chitosan,[69] collagen,[70] keratin,[71] PLA,[72]
PEG,[73] or hyaluronan[62] in order to modify mechanical
or degradation behavior or to prevent cell adhesion for
antithrombotic effects.
Processing Parameters
Various material properties are important when preparing
silk-based materials for specific applications, including the
mechanical properties of the material (e.g., different
Table 1. Distribution of secondary structure elements of films of eADF4(C16), eADF3((AQ)12) and blends of both as determined by
deconvolution of the amide I bands after FTIR-spectroscopy.
Protein Post-treatment a-helix
eADF4(C16) – 19.0
MeOH 12.9
KxH3xPO4 13.2
eADF3((AQ)12) – 23.7
MeOH 18.4
KxH3xPO4 9.9
eADF4/eADF3 – 20.5
MeOH 8.2
Films were analysed either as-cast or after treatment with methanol (MeOH) or potassium phosphate (KxH3xPO4) (adapted from ref. [48]).
Macromol. Biosci. 2010, 10, 998–1007
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
requirements for tissue engineering devices for skin
substitutes, soft tissues, bone, or ligaments), degradation
behavior and chemical stability, surface topography and
porosity, and additional functionalities. The versatility of
silk proteins provides the option of varying and controlling
several of these properties in the final assembly, mainly
through the processing conditions.
Silk films are generally produced by casting, dip- or spray-
coating of protein solutions onto a substrate. Evaporation of
the solvent yields a silk film that is stabilized via (non-
covalent) intermolecular interactions, and if required, the
films can be peeled off the surface. Silk films may be further
structurally or chemically modified by subsequent post-
treatments and/or subsequent modifications (mediated for
example through effector molecules). There are two main
components determining the overall properties of the
resulting film: the structure on the molecular level (secon-
dary structure of the underlying proteins and intermolecular
interactions[48,74]) and the macroscopic structure, which
provides the materials’ interface with its environment.
Control of Material Properties through the Molecular
Structure of the Silk Proteins
The secondary structure is determined and influenced by
the primary structure of the employed protein, the solvent,
and post-treatment conditions. We have shown that films
cast from different recombinant spider silk proteins
(mimicking the dragline proteins ADF3 and ADF4 from
the garden spider A. diadematus) show a secondary
structure composition depending on the used silk pro-
teins/blends (Table 1).[48] For a given protein, the solvent
used (most often water, hexafluoro-2-propanol (HFIP) or
formic acid) will determine the predominant secondary
structure in the as-cast films. Silk films prepared from
aqueous solution mainly consist of random coil struc-
ture,[71,75,76] whereas films prepared using HFIP as the
b-sheet b-turn Random coil
35.0 32.2 13.8
45.2 23.8 18.0
52.1 25.5 9.2
27.7 30.1 18.5
34.8 29.2 17.6
36.5 32.0 21.6
20.5 35.7 23.3
46.4 19.9 25.5
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 K. Spiess, A. Lammel, T. Scheibel

stability of the respective film. Those rich

in random coil or helical conformation
are water soluble, while b-sheet rich films
are insoluble in water. As for most
applications stability in aqueous solution
is necessary, a subsequent treatment of
water soluble films is applied in order to
render them stable, which is typically
achieved using dehydrating agents like
alcohols[49,50] (usually methanol) or kos-
motropic ions[48] (like potassium phos-
phate, which is known to influence silk
protein structure during the natural
spinning process) and water vapor[80] or
high temperature.[81] All treatments lead
to a structural transition from random
coil/a-helical to a higher content of b-
sheet structure (Table 1). The portion of
induced b-sheet structure can be regu-
lated by the incubation time or by
treatment of the film with solutions of
Figure 5. Processing parameters to control material properties of silk films. (A) Schematic methanol/water mixed at various ratios
presentation of individual steps necessary for film formation and influencing factors. (Figure 5C) (unpublished results).
The material properties can be controlled by variation and combination of distinct The content of b-sheet structure and
parameters at each step. (B) and (C) FTIR-spectra illustrating the influence of different
crystallinity not only determines the
solvents (B) or post-treatments (C) on the secondary structure of eADF4(C16) films
(unpublished results). (D) Functionalization of eADF4(C16) films by covalent coupling of stability of silk films, but also influences
b-galactosidase (þ). Blue precipitate of X-gal (seen as dark color in black and white their mechanical characteristics. Gener-
reprint) indicates enzyme activity. Non-coupled b-galactosidase does not adhere to the ally, an increase in b-sheet structure
silk surface ().[49] (E) Control of porosity of eADF4(C16) films generated by blending increases the elastic modulus and
with different amounts of PEO. Upper panel: 20% (wt.-%) PEO, lower panel: 333% (wt.-%)
strength of the film, but reduces its
PEO. Scale bar: 5 mm.
elasticity[82] (Table 2, films as-cast and
treated with 50% methanol). Not only the
solvent typically yield a-helix-rich structures[48,49,75,77] amount of b-sheets, but also their molecular arrangement is
(Figure 5B). In contrast, films prepared from formic crucial to the final properties.[74] A high amount of b-sheet
acid solutions are rich in b-sheet structure polyalanine regions and long-range order crystals have
(Figure 5B).[71,76,78,79] Interestingly, these different struc- been correlated with stiffness and brittleness in silk
tural conformations of the proteins determine the chemical films.[50,76] Relative humidity of the environment as well

Table 2. Mechanical properties of eADF4(C16) films cast from HFIP and of eADF4(C16) films made in presence of 0, 10, 20, and 40% glycerol
with and without 50% methanol (MeOH) post-treatment (PT).

Material Glycerol smax (Pa) e at sma (%)

% no PT 50% MeOH no PT 50% MeOH

eADF4(C16) – 1.5  107 3.9  107 11 12

7
eADF4(C16) 10 2.3  10 2.6  107 47 21
6 7
eADF4(C16) 20 8.3  10 2.4  10 89 56
eADF4(C16) 40 9.9  106 1.2  107 129 100

The mechanical characteristics (tensile strength and elongation) were assessed in a custom built vertical tensile test setup. Briefly, the top
end of the samples was fixed to a force transducer (FORT25, WPI, Sarosota, USA) and the bottom end to a linear motor (Linmot PS01-23  80
motor, Linmot AG, Switzerland). The pulling velocity for each measurement was 1 mm  s1 and the data was recorded at a rate of 500
datapoints s1.

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Recombinant Spider Silk Proteins for Applications in . . .
as water content of the film also influence the mechanical
properties, with higher water content rendering the film
more elastic, as demonstrated on B. mori fibroin films.[83–85]
Accordingly, addition of hygroscopic plasticizers like
glycerol, which alter intermolecular interactions, renders
spider silk films more elastic (Table 2) and can therefore be
used to further adjust the mechanical properties.[84] As an
example, a content of 40% (v/w) glycerol increases the
elasticity of a eADF4(C16) film about tenfold, accompanied
by a tenfold decrease in elastic modulus and a slight
decrease in strength (Table 2).
Another important topic for the suitability of silk films in
biomedical applications is the degradation of the respective
material. For example, in functional tissue design, the
replacement of an implant by the body’s own tissue is
desired.[86] Silk proteins are degraded by distinct proteases
in vivo, mediated by the foreign body response[87] in case of
implantation, or by enzymes of the intestinal tract in case of
oral administration. In vitro analysis, for instance, revealed
the degradation of eADF4(C16) particles by artificial
intestinal fluid, but not by artificial gastric fluid.[88] Studies
on B. mori fibroin revealed that besides the morphology of
the material (including porositiy) and the presence of
recognition sites for proteolytic enzymes, the secondary
structure and degree of crystallinity also influence the rate
of degradation,[86,87,89] with the crystalline regions being
the most stable structures.
Material Function Controlled by its Macroscopic
Structure
Besides the silk properties determined by the molecular
structure of the underlying proteins, the materials’ macro-
scopic structure has a direct impact on biomedical
applications. Silk fibroin films, for instance, displaying a
distinct surface morphology can be prepared by casting the
films on grooved or patterned substrates,[54,65] with the
structure of the substrate being reflected in the films’
topography. Such patterned film surfaces can be used to
induce aligned cell growth.[65] Another option of generating
altered surface topography on silk films is to apply different
post-treatment conditions. Applying diverse methanol
treatments led to varying roughness of the individual
films and resulted in different growth behavior of
fibroblasts.[90] In general, the topography of a cell support-
ing scaffold has an impact on cell morphology, adhesion,
alignment, proliferation, and differentiation.[90,91] In order
to obtain interfaces for applications ranging from tissue
engineering to controlling diffusion of molecules, porous
films have been prepared by salt leaching, gas foaming, or
freeze-drying.[92] In salt leaching, salt crystals are
embedded during film formation and washed out after
stabilizing the film. Similar to the salt leaching process,
addition of PEG to the protein solution and its subsequent
Macromol. Biosci. 2010, 10, 998–1007
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
removal by a washing step after film formation yields pores
(Figure 5E). The size of the pores as well as their
interconnectivity can be varied by concentration and size
of the employed salts or the concentration of PEG, as shown
for porous films made of eADF4(C16) (Figure 5E).
Taken together, the combination of processing para-
meters allows the generation of tailored silk films.
However, for specific applications, additional features
might be necessary; a task which can be met by combining
silk films with additional functional groups such as
components of the extracellular matrix for better cell
adhesion or differentiation. Modification of surface hydro-
philicity or coupling of (poly-)peptides is also feasible via
chemical coupling through reactive side chains of amino
acids (Figure 5D).[67,93] Our established recombinant
production technology of spider silk proteins offers various
possibilities to introduce new/additional amino acid side
chains by genetic engineering, which can be utilized for
further functionalization.
Summary
Recombinantly produced spider silk proteins possess a high
potential for biomedical applications. Here, we highlighted
the production of silk particles and films showing that the
molecular and macroscopic material properties can be
easily controlled in vitro.
In case of eADF4(C16) particles, one major advantage of
the system is the production and loading within an all-
aqueous process under ambient conditions, which is
extremely important considering encapsulation of labile
compounds and the biocompatibility of the product. In case
of silk films, we showed that molecular structure can be
influenced by solvents and postcasting treatments and that
macroscopic properties like porosity and mechanical
stability can be influenced by additives like PEO and
glycerol. This level of control allows the adaptation of such
films for a large variety of biomedical applications. The
diversity of spider silk proteins and the processing control
along with their biocompatibility and biodegradability
renders them valuable tools for future applications in novel
biomaterials.
Acknowledgements: This work was supported by the Interna-
tional Graduate School of Science and Engineering (IGSSE to AL)
within the Elite Network of Bavaria and funding by the Army
Research Office (grant W911NF-0810284). The authors wish to
thank Eileen Lintz and Dr. John Hardy for critical reading the
manuscript.
Received: February 15, 2010; Revised: April 1, 2010; Published
online: July 5, 2010; DOI: 10.1002/mabi.201000071
Keywords: biopolymers; drug delivery systems; process para-
meters; recombinant spider silks; tissue engineering
www.mbs-journal.de 1005

[1] M. Heim, D. Keerl, T. Scheibel, Angew. Chem. Int. Ed. 2009, 48,
3584.
[2] F. Vollrath, Int. J. Biol. Macromol. 1999, 24, 81.
[3] F. Vollrath, Rev. Mol. Biotechnol. 2000, 74, 67.
[4] S. Winkler, D. L. Kaplan, Rev. Mol. Biotechnol. 2000, 74, 85.
[5] J. D. van Beek, S. Hess, F. Vollrath, B. H. Meier, Proc. Natl. Acad.
Sci. USA 2002, 99, 10266.
[6] D. Huemmerich, T. Scheibel, F. Vollrath, S. Cohen, U. Gat,
S. Ittah, Curr. Biol. 2004, 14, 2070.
[7] D. Huemmerich, C. W. Helsen, S. Quedzuweit, J. Oschmann,
R. Rudolph, T. Scheibel, Biochemistry 2004, 43, 13604.
[8] B. O. Swanson, T. A. Blackledge, J. Beltrán, C. Y. Hayashi, Appl.
Phys. A: Mater. Sci. Process. 2006, 82, 213.
[9] A. M. Smith, T. Scheibel, Macromol. Chem. Phys. 2010, 211,
127.
[10] J. M. Gosline, P. A. Guerette, C. S. Ortlepp, K. N. Savage, J. Exp.
Biol. 1999, 202. 3295.
[11] C. Y. Hayashi, N. H. Shipley, R. V. Lewis, Int. J. Biol. Macromol.
1999, 24, 271.
[12] C. Vendrely, T. Scheibel, Macromol. Biosci. 2007, 7, 401.
[13] F. Hagn, L. Eisoldt, J. G. Hardy, C. Vendrely, C. Murray,
T. Scheibel, H. Kessler, Nature 2010, 465, 239.
[14] H. J. Jin, D. L. Kaplan, Nature 2003, 424, 1057.
[15] F. Vollrath, D. P. Knight, X. W. Hu, Proc. R Soc. London Ser. B-
Biol. Sci. 1998, 265, 817.
[16] C. Dicko, J. M. Kenney, D. Knight, F. Vollrath, Biochemistry
2004, 43, 14080.
[17] G. Askarieh, M. Hedhammar, K. Nordling, A. Saenz, C. Casals,
A. Rising, J. Johansson, S. D. Knight, Nature 2010, 465, 236.
[18] J. G. Hardy, L. M. Roemer, T. R. Scheibel, Polymer 2008, 49,
4309.
[19] J. Perez-Riguero, M. Elices, J. Llorca, C. Viney, J. Appl. Polym. Sci.
2001, 82, 2245.
[20] U. K. Slotta, S. Rammensee, S. Gorb, T. Scheibel, Angew. Chem.
Int. Ed. Engl. 2008, 47, 4592.
[21] S. Rammensee, U. Slotta, T. Scheibel, A. R. Bausch, Proc. Natl.
Acad. Sci. USA 2008, 105, 6590.
[22] L. Eisoldt, J. G. Hardy, M. Heim, T. R. Scheibel, J. Struct. Biol.
2010, 170, 413.
[23] T. Scheibel, Microb. Cell Fact. 2004, 3, 14.
[24] C. W. P. Foo, E. Bini, J. Hensman, D. P. Knight, R. V. Lewis, D. L.
Kaplan, Appl. Phys. A 2006, 82, 223.
[25] U. Slotta, S. Hess, K. Spiess, T. Stromer, L. Serpell, T. Scheibel,
Macromol. Biosci. 2007, 7, 183.
[26] A. Matsumoto, A. Lindsay, B. Abedian, D. L. Kaplan, Macromol.
Biosci. 2008, 8, 1006.
[27] I. C. Um, H. Kweon, Y. H. Park, S. Hudson, Int. J. Biol. Macromol.
2001, 29, 91.
[28] Z. Chenhua, Y. Juming, M. Hiromi, K. Raghuvansh, A. Tetsuo,
Biopolymers 2003, 69, 253.
[29] D. M. Phillips, L. F. Drummy, D. G. Conrady, D. M. Fox, R. R.
Naik, M. O. Stone, P. C. Trulove, H. C. De Long, R. A. Mantz,
J. Am. Chem. Soc. 2004, 126, 14350.
[30] B. Lawrence, F. Omenetto, K. Chui, D. Kaplan, J. Mater. Sci.
2008, 43, 6967.
[31] K. D. Hermanson, D. Huemmerich, T. Scheibel, A. R. Bausch,
Adv. Mater. 2007, 19, 1810.
[32] S. Rammensee, D. Huemmerich, K. D. Hermanson, T. Scheibel,
A. R. Bausch, Appl. Phys. A 2006, 82, 261.
[33] S. Freiberg, X. X. Zhu, Int. J. Pharm. 2004, 282, 1.
[34] L. Robert, A. P. Nicholas, AIChE J. 2003, 49, 2990.
[35] M. George, T. E. Abraham, J. Controlled Release 2006, 114, 1.
[36] X. Liu, Q. Sun, H. Wang, L. Zhang, J.-Y. Wang, Biomaterials
2005, 26, 109.
Macromol. Biosci. 2010, 10, 998–1007
1006 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
K. Spiess, A. Lammel, T. Scheibel
[37] Y.-W. Won, Y.-H. Kim, J. Controlled Release 2008, 127, 154.
[38] T. M. Allen, P. R. Cullis, Science 2004, 303, 1818.
[39] U. Edlund, A. Albertsson, in: Degradable Aliphatic Polyesters,
A. C. Albertsson, Ed., Springer, Berlin 2002, pp. 67.
[40] R. Arshady, J. Bioact. Compatible Polym. 1990, 5, 315.
[41] N. Yasutomo, N. Yoshiharu, W. Masahisa, K. Shigenori, M. Jun,
J. Appl. Polym. Sci. 2002, 86, 3425.
[42] J. K. Vasir, K. Tambwekar, S. Garg, Int. J. Pharm. 2003, 255, 13.
[43] M. Roser, D. Fischer, T. Kissel, Eur. J. Pharm. Biopharm. 1998,
46, 255.
[44] K. E. Uhrich, S. M. Cannizzaro, R. S. Langer, K. M. Shakesheff,
Chem. Rev. 1999, 99, 3181.
[45] S. K. Daniel, Biotechnol. Bioeng. 2007, 96, 203.
[46] A. Lammel, M. Schwab, U. Slotta, G. Winter, T. Scheibel, Chem.
Sus. Chem. 2008, 1, 413.
[47] J. H. Exler, D. Hummerich, T. Scheibel, Angew. Chem. Int. Ed.
Engl. 2007, 46, 3559.
[48] U. Slotta, M. Tammer, F. Kremer, P. Koelsch, T. Scheibel,
Supramol. Chem. 2006, 18, 465.
[49] D. Huemmerich, U. Slotta, T. Scheibel, Appl. Phys. A 2006, 82,
219.
[50] K. Spiess, S. Wohlrab, T. Scheibel, 2010, In revision.
[51] X. Y. Wang, X. Hu, A. Daley, O. Rabotyagova, P. Cebe, D. L.
Kaplan, J. Controlled Release 2007, 121, 190.
[52] X. Wang, E. Wenk, X. Hu, G. R. Castro, L. Meinel, X. Wang, C. Li,
H. Merkle, D. L. Kaplan, Biomaterials 2007, 28, 4161.
[53] E. Wenk, A. J. Meinel, S. Wildy, H. P. Merkle, L. Meinel,
Biomaterials 2009, 30, 2571.
[54] B. D. Lawrence, M. Cronin-Golomb, I. Georgakoudi, D. L.
Kaplan, F. G. Omenetto, Biomacromolecules 2008, 9, 1214.
[55] J. J. Amsden, H. Perry, S. V. Boriskina, A. Gopinath, D. L. Kaplan,
L. Dal Negro, F. G. Omenetto, Opt. Express 2009, 17, 21271.
[56] X. Y. Wang, H. J. Kim, P. Xu, A. Matsumoto, D. L. Kaplan,
Langmuir 2005, 21, 11335.
[57] T. L. Liu, J. C. Miao, W. H. Sheng, Y. F. Xie, Q. Huang, Y. B. Shan,
J. C. Yang, J. Zhejiang Univ. Sci. B 2010, 11, 10.
[58] A. Sugihara, K. Sugiura, H. Morita, T. Ninagawa, K. Tubouchi,
R. Tobe, M. Izumiya, T. Horio, N. G. Abraham, S. Ikehara, Proc.
Soc. Exp. Biol. Med. 2000, 225, 58.
[59] C. Cassinelli, G. Cascardo, M. Morra, L. Draghi, A. Motta,
G. Catapano, Int. J. Artif. Organs 2006, 29, 881.
[60] E. Bini, C. W. P. Foo, J. Huang, V. Karageorgiou, B. Kitchel, D. L.
Kaplan, Biomacromolecules 2006, 7, 3139.
[61] A. W. Morgan, K. E. Roskov, S. Lin-Gibson, D. L. Kaplan, M. L.
Becker, C. G. Simon, Biomaterials 2008, 29, 2556.
[62] M. Garcia-Fuentes, A. J. Meinel, M. Hilbe, L. Meinel, H. P.
Merkle, Biomaterials 2009, 30, 5068.
[63] J. S. Chen, G. H. Altman, V. Karageorgiou, R. Horan, A. Collette,
V. Volloch, T. Colabro, D. L. Kaplan, J. Biomed. Mater. Res.
Part A 2003, 67A, 559.
[64] A. Chiarini, P. Petrini, S. Bozzini, I. Dal Pra, U. Armato, Bio-
materials 2003, 24, 789.
[65] B. D. Lawrence, J. K. Marchant, M. A. Pindrus, F. G. Omenetto,
D. L. Kaplan, Biomaterials 2009, 30, 1299.
[66] Q. Lv, K. Hu, Q. L. Feng, F. Z. Cui, J. Appl. Polym. Sci. 2008, 109,
1577.
[67] S. Sofia, M. B. McCarthy, G. Gronowicz, D. L. Kaplan, J. Biomed.
Mater. Res. 2001, 54, 139.
[68] Y. Tamada, T. Furuzono, T. Taguchi, A. Kishida, M. Akashi,
J. Biomater. Sci. Polym. Ed. 1999, 10, 787.
[69] K. Cai, Y. Hu, K. D. Jandt, J. Biomed. Mater. Res. Part A 2007,
82A, 927.
[70] T. Asakura, J. Yao, T. Yamane, K. Umemura, A. S. Ulrich, J. Am.
Chem. Soc. 2002, 124, 8794.
DOI: 10.1002/mabi.201000071
Recombinant Spider Silk Proteins for Applications in . . .
[71] A. Vasconcelos, G. Freddi, A. Cavaco-Paulo, Biomacromole-
cules 2008, 9, 1299.
[72] H. L. Zhu, X. X. Feng, H. P. Zhang, Y. H. Guo, J. Z. Zhang, J. Y.
Chen, J. Biomater. Sci. Polym. Ed. 2009, 20, 1259.
[73] C. Vepari, D. Matheson, L. Drummy, R. Naik, D. L. Kaplan,
J. Biomed. Mater. Res. Part A 2009, 93A, 595.
[74] E. Metwalli, U. Slotta, C. Darko, S. V. Roth, T. Scheibel, C. M.
Papadakis, Appl. Phys. A 2007, 89, 655.
[75] C. Zhao, J. Yao, H. Masuda, R. Kishore, T. Asakura, Biopolymers
2003, 69, 253.
[76] I. C. Um, H. Y. Kweon, Y. H. Park, S. Hudson, Int. J. Biol.
Macromol. 2001, 29, 91.
[77] J. S. Stephens, S. R. Fahnestock, R. S. Farmer, K. L. Kiick, D. B.
Chase, J. F. Rabolt, Biomacromolecules 2005, 6, 1405.
[78] S. W. Ha, A. E. Tonelli, S. M. Hudson, Biomacromolecules 2005,
6, 1722.
[79] B. M. Min, L. Jeong, Y. S. Nam, J. M. Kim, J. Y. Kim, W. H. Park,
Int. J. Biol. Macromol. 2004, 34, 281.
[80] H. J. Jin, S. V. Fridrikh, G. C. Rutledge, D. L. Kaplan, Biomacro-
molecules 2002, 3, 1233.
[81] J. Magoshi, S. Nakamura, J. Appl. Polym. Sci. 1975, 19, 1013.
[82] N. Minoura, M. Tsukada, M. Nagura, Biomaterials 1990, 11, 430.
[83] Q. Lu, X. Hu, X. Wang, J. A. Kluge, S. Lu, P. Cebe, D. L. Kaplan,
Acta Biomater. 2009, 6, 1380.
Macromol. Biosci. 2010, 10, 998–1007
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[84] S. Lu, X. Wang, Q. Lu, X. Zhang, J. A. Kluge, N. Uppal,
F. Omenetto, D. L. Kaplan, Biomacromolecules 2010, 11,
143.
[85] B. D. Lawrence, S. Wharram, J. A. Kluge, G. G. Leisk, F. G.
Omenetto, M. I. Rosenblatt, D. L. Kaplan, Macromol. Biosci.
2010, 10, 393.
[86] C. Vepari, D. L. Kaplan, Prog. Polym. Sci. 2007, 32, 991.
[87] G. H. Altman, F. Diaz, C. Jakuba, T. Calabro, R. L. Horan, J. S.
Chen, H. Lu, J. Richmond, D. L. Kaplan, Biomaterials 2003, 24,
401.
[88] B. Liebmann, D. Hümmerich, T. Scheibel, M. Fehr, Colloids
Surf. A 2008, 331, 126.
[89] A. Leal-Egana, T. Scheibel, Biotechnol. Appl. Biochem. 2010, 55,
155.
[90] E. Servoli, D. Maniglio, A. Motta, R. Predazzer, C. Migliaresi,
Macromol. Biosci. 2005, 5, 1175.
[91] C. J. Bettinger, R. Langer, J. T. Borenstein, Angew. Chem. Int. Ed.
2009, 48, 5406.
[92] Y. Z. Wang, H. J. Kim, G. Vunjak-Novakovic, D. L. Kaplan,
Biomaterials 2006, 27, 6064.
[93] A. R. Murphy, P. S. John, D. L. Kaplan, Biomaterials 2008, 29,
4260.
[94] J. P. Anderson, Protein Based Materials, K. McGrath, D. L.
Kaplan, Eds., Birkhäuser, Boston 1996, p. 371.
www.mbs-journal.de 1007