Acta Biomaterialia 31 (2016) 17–32

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Review article

Silk protein-based hydrogels: Promising advanced materials

for biomedical applications
Sonia Kapoor a,b, Subhas C. Kundu a,⇑
a
Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India
b
University Institute of Engineering and Technology, Panjab University, Chandigarh 160014, India

a r t i c l e i n f o a b s t r a c t

Article history: Hydrogels are a class of advanced material forms that closely mimic properties of the soft biological tis-
Received 10 March 2015 sues. Several polymers have been explored for preparing hydrogels with structural and functional fea-
Received in revised form 8 November 2015 tures resembling that of the extracellular matrix. Favourable material properties, biocompatibility and
Accepted 17 November 2015
easy processing of silk protein fibers into several forms make it a suitable material for biomedical appli-
Available online 18 November 2015
cations. Hydrogels made from silk proteins have shown a potential in overcoming limitations of hydro-
gels prepared from conventional polymers. A great deal of effort has been made to control the properties
Keywords:
and to integrate novel topographical and functional characteristics in the hydrogel composed from silk
Silk proteins
Fibroin
proteins. This review provides overview of the advances in silk protein-based hydrogels with a primary
Hydrogel emphasis on hydrogels of fibroin. It describes the approaches used to fabricate fibroin hydrogels.
Biomaterial Attempts to improve the existing properties or to incorporate new features in the hydrogels by making
Biomedical applications composites and by improving fibroin properties by genetic engineering approaches are also described.
Applications of the fibroin hydrogels in the realms of tissue engineering and controlled release are
reviewed and their future potentials are discussed.

Statement of Significance

This review describes the potentiality of silk fibroin hydrogel. Silk Fibroin has been widely recognized as
an interesting biomaterial. Due to its properties including high mechanical strength and excellent bio-
compatibility, it has gained wide attention. Several groups are exploring silk-based materials including
films, hydrogels, nanofibers and nanoparticles for different biomedical applications. Although there is a
good amount of literature available on general properties and applications of silk based biomaterials,
there is an inadequacy of extensive review articles that specifically focus on silk based hydrogels. Silk-
based hydrogels have a strong potential to be utilized in biomedical applications. Our work is an effort
to highlight the research that has been done in the area of silk-based hydrogels. It aims to provide an
overview of the advances that have been made and the future course available. It will provide an over-
view of the silk-based hydrogels as well as may direct the readers to the specific areas of application.
Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2. Structure and properties of silk. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3. Silk-based hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.1. Fibroin hydrogels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.1.1. Preparation and characterization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.1.2. Sol–gel transition mechanism of fibroin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.2. Silk fibroin composite hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

⇑ Corresponding author.
E-mail address: kundu@hijli.iitkgp.ernet.in (S.C. Kundu).

http://dx.doi.org/10.1016/j.actbio.2015.11.034
1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
18 S. Kapoor, S.C. Kundu / Acta Biomaterialia 31 (2016) 17–32

3.3. Hydrogel fabrication from genetically engineered silk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3.3.1. Silk-elastin like hydrogel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4. Biomedical applications of silk-based hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.1. Tissue engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.1.1. Bone tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.1.2. Cartilage tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4.2. Controlled release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4.2.1. Controlled drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4.2.2. Controlled release of DNA for gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5. Challenges and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

1. Introduction Among the naturally occurring fibers, silk occupies a special

Biomaterials play a significant role in regenerative medicine as
implants, sutures, contact lenses, hip joints, vascular grafts, wound
dressing materials, drug releasing stents and several other biomed-
ical devices [1]. Recent developments that have offered the ability
to control material properties have led to tremendous success in
the field of tissue engineering and therapeutic medicine [2].
Hydrogels have come to the forefronts as an important form of
materials for biomedical applications [3–7]. Hydrogels are three-
dimensional polymeric networks that are capable of absorbing
large amounts of water while maintaining their structural integ-
rity. They are usually classified into two categories namely physical
gels (where the polymeric network is held together by secondary
forces that include hydrogen bonds, ionic as well as hydrophobic
interactions) and chemical hydrogels (where covalent linkages
form the network). Since the introduction of hydrogels as biomate-
rials in the 1960s [8]), they have gained considerable attention and
have found numerous applications in tissue engineering, drug
delivery, implantable devices, biosensors and bio-
nanotechnology. The primary reason of the widespread applicabil-
ity of hydrogels for biomedical applications is the close resem-
blance of their physical and mechanical properties to that of
biological tissues [7].
Several natural and synthetic polymeric materials have been
explored to prepare hydrogels. Proteins, the fundamentally
important macromolecules of living systems, have evolved to per-
form very specific biochemical, mechanical and structural roles.
The increased understanding about the structural and functional
features of a variety of proteins and advancement in techniques
to manipulate them has opened avenues to utilize them for
unconventional purposes [9–11]. Due to their inherent advan-
tages including biocompatibility, ease of large-scale production
via recombinant DNA technology and facile manipulation by
chemical or enzymatic means, several proteins have been evalu-
ated for their performance as biomaterials (Table 1) [9–11]. Pro-
tein polymers display a variety of properties, which prove
especially useful for biomedical applications [9–11]. They
undergo hierarchical self-organization that can be mimicked out-
side cellular environment, thus, offering a bottom-up approach
for assembling biomedical devices [48]. Proteins often contain
several domains that assist in cell signalling through their inter-
action with other proteins or ligands [49,50]. The biomaterials
prepared from them can, therefore, be anticipated to retain this
function. Proteins can be integrated with synthetic polymers
easily to further enhance their material properties or can be
made to adhere on synthetic surfaces, providing a hybrid inter-
face [10,51,52]. Moreover, rational genetic engineering
approaches provide us with a possibility to introduce novel fea-
tures such as self-assembly, sensitivity to specific stimuli,
biorecognition, and controlled degradation in proteins [53,54].
position because of its properties. Silk refers to protein fibers pro-
duced by several species of phylum Arthropoda. Several insects and
spiders are able to spin silk fibers; the silks from the domesticated
mulberry silkworm, Bombyx mori, and spiders, Nephila clavipes and
Araneus diadematus have been extensively characterized [55–60].
Of late, various forms of silk-based materials including films, scaf-
folds, sponges, tubes, electrospun fibers and hydrogels have been
evaluated for several applications [61,62]. This review focuses on
the advances achieved in making hydrogels from silk proteins. It
describes several methods that have been used for making silk pro-
tein hydrogels, structural and functional characteristics and appli-
cations of these hydrogels.
2. Structure and properties of silk
Silk proteins from different silkworm species exhibit variations
in their structure and properties [55,59,63]. Silk proteins can be
isolated from the cocoons or the silk glands of silkworms [64,65].
Silk obtained from silkworms is mainly composed of two classes
of proteins, fibroin and sericin. B. mori fibroin protein consists of
a glycoprotein named P25 and a light (26 kDa) and a heavy chain
(325 kDa), linked by a disulphide bond [60]. On the other hand,
fibroin protein of Antheraea mylitta silkworm is composed of two
similar-sized polypeptides with an estimated molecular weight
of 197 kDa each which are linked together by a disulfide bond
[66]. Sericin proteins, having a molecular weight ranging between
10 and 300 kDa, act as glue like coating to keep fibroin chains
together. Bundles of nanofibrils form fibroin filaments that consti-
tute 70–75% of weight of silk fiber; the remaining weight is sericin.
In general, the primary sequence of fibroin consists of highly repet-
itive motifs [55,59,67]. For B. mori, the primary repetitive sequence
is the hexapeptide GAGAGS [55,59,67]. These predominant
hydrophobic blocks lead to extensive hydrogen and hydrophobic
interactions throughout the protein chains resulting in homoge-
neous secondary structure. These interactions impart crystallinity
to the protein which, in turn, enhances environmental stability of
fibroin [68]. These highly crystalline regions (containing repetitive
alanine or glycine rich sequences) associate with less organized
regions of 34–40 amino acids domains which are interspersed
between the crystalline regions [67] and result in the remarkable
strength and elasticity of silk protein fibers [69]. Not only do
fibroin fibers have good mechanical strength, they also possess
excellent process ability and have been processed into several
forms including films, sponges, mats, gels, scaffolds and tubes
[70]. Silk-based biomaterials have been found to be highly biocom-
patible with various cell types. For example, adhesion and prolifer-
ation studies of human and animal cell lines on films formed from
native silkworm fibroin and regenerated silk fibroin [71–73] have
suggested it to be a suitable matrix for cell growth. Although, the
 S. Kapoor, S.C. Kundu / Acta Biomaterialia 31 (2016) 17–32 19

Table 1
Proteins that have been used to prepare hydrogels.

Proteins Functions in nature Typical Advantages Dis- Applications Reference

Mechanical advantages (s)
Properties of
protein fiber
Collagen Major protein of the 1.2 GPaa Good biocompatibility Mechanical Tissue regeneration, Corneal [12–16]
extracellular matrix weakness and replacement, Cartilage repair,
and provides rapid gene therapy, growth factor
structural support degradation delivery
Gelatin Degraded form of NA Extremely biocompatible, lower Mechanical Differentiation of pluripotent [17–22]
collagen immunogenicity as compared to weakness cells; Growth factor delivery,
collagen, forms gels by temperature drug delivery, tissue
change, Pharmaceutically approved as a engineering, wound dressing
coating material
Fibrin/fibronectin Component of the 1–10 MPab Very closely mimics the properties of the Limited Wound healing, Angiogenesis, [23–29]
extracellular matrix soft tissues, promotes cell adherence mechanical Tissue engineering
strength
Elastin Provides elasticity to 1.1 MPac Self assembles under physiological Calcification of Tissue engineering [30–32]
the tissues conditions elastin
implants,
Rapid
degradation
Fibroin Basic material to 7 GPad High mechanical strength Slow gelation Tissue repair, repair of bone [33–38]
form cocoons, nets, rate defects, delivery of therapeutic
traps by insects and molecules
silkworms
Whey proteins Protein components 0.2 kPae Cheap availability, by-product of milk Mechanical Controlled drug release [39–41]
of milk apart from industry, preparation of gel does not weakness
casein and provide need chemical cross-linkers
nutrition
Sericin Acts as a glue to bind 0.9 MPaf Easily available as by-product of silk May result in Wound dressing material [42–44]
fibroin fibrils industry immunogenic
reactions
a
Young’s modulus, E, of collagen from mammalian tendon [45].
b
Young’s modulus, E, of uncross-linked fibrin fibers [26].
c
Young’s modulus, E, of elastin fiber from bovine ligament [46].
d
Bombyx mori cocoon silk [47].
e
Equilibrium modulus of a typical hydrogel made from 12% whey protein solution [40].
f
Maximum stress of a typical hydrogel in wet state at failure [42].

adherence of cells on silk surfaces has been found to vary depend-
ing on the source of protein and the processing conditions, never-
theless, silk-based biomaterials provide good support for cell
adhesion and proliferation and do not cause any major cell toxicity
[73]. It is well emerging that fibroin does not cause activation of
immune response. In fact, it evokes a minimum foreign body
response [68,74,75] and has been used as a suture material for
many centuries.
Silk proteins degrade via enzymatic and non-enzymatic means
[76]. The degradation of silk biopolymer has been found to be
slower as compared to several natural polymers [68,74,76]. Due
to its high crystallinity, it may take several days or up to weeks
to degrade in vivo with the rate of absorption depending on factors
like implantation site, the mechanical environment, the type of silk
(virgin or processed), the diameter of the silk fiber and the sec-
ondary structure [68,74]. Due to the slow degradation rate of
fibroin, silk-based devices provide a suitable material that can sup-
port the neo-forming tissues for long duration. Moreover, the
degradation products of silk fibroin materials have been shown
to be harmless to the human body [76].
In addition to tailoring the properties of silk proteins by chem-
ical and physical modifications, their properties can be modulated
according to the requirements through genetic engineering. Bio-
compatibility and cell adhesion of silk proteins has been improved
by integrating sequences from other proteins like collagen and
fibronectin [77,78]. For instance, Morgan group have prepared
scaffolds by blending fibroin and synthetic spidroin, which con-
tains RGD sequences, thus, leading to easy and inexpensive incor-
poration of sequences to promote cell adhesion and differentiation
[79]. Mechanical strength, biocompatibility, stability to heat and
humidity, high permeability to oxygen and other molecules along
with the opportunity to control the material characteristics
through methanol treatment and genetic engineering has made
silk protein-based materials much sought after for biomedical pur-
poses [61,80–82].
3. Silk-based hydrogels
In vivo, the cells grow, divide, perform their functions, commu-
nicate with each other and migrate. All these functions, in one way
or the other, are supported by a matrix – the extracellular matrix
(ECM) that provides mechanical support as well as physicochemi-
cal cues to cells to perform these functions [83]. Extracellular
matrix is composed of fibrous proteins (laminin, collagen and
fibronectin) and proteoglycans. The protein chains form a mesh
like network and provide the mechanical support while the proteo-
glycans occupy the interstitial sites of this polymeric network [83].
Dynamic microrestructuring of ECM occurs continuously and
essentially maintains the tissue homeostasis [84]. Hydrogels have
been widely accepted as near prototypes of the ECM and have been
found to be suitable 3-dimensional matrices for cell growth [7].
The hydrogels prepared earlier have been more of a passive 3D
platform for cell growth. The significance of properties like
mechanical stiffness of hydrogels in mechanotransduction and of
biochemical and topological cues in promoting physiological func-
tions of cells has been identified lately [85]. Thus, the emphasis has
now shifted to fabricating the hydrogels that reflect the essence of
20 S. Kapoor, S.C. Kundu / Acta Biomaterialia 31 (2016) 17–32

Fig. 1. Schematic representation of hydrogel fabrication from silk fibroin protein: Cocoons and the silk glands are main sources of silk fibroin proteins. Larvae and cocoons of
domesticated mulberry silkworm, Bombyx mori and non-mulberry tropical tasar silkworm, Antheraea mylitta are shown. Silk fibroin protein is isolated from middle silk glands
and cocoons of different silkworms and is regenerated. The regenerated silk fibroin protein is processed through various treatments to get hydrogel. A typical image of fibroin
hydrogel fabricated in our laboratory is shown.

cellular microenvironment and can mimic the key aspects of ECM.
As noted above, fibroin-based material promotes cell attachment
and proliferation [73]. In particular, the silk from wild silkworm
like Antheraea pernyi contains RGD sequences which are known
to be the recognition sequences for integrin receptors that mediate
the interaction between cells and ECM [86,87]. In addition, the
mechanical properties including extensibility and toughness of
fibroin fibers, particularly that from wild silkworm fiber are even
higher than polymers like elastin and Kevlar [68,75]. The superior
mechanical strength of hydrogels prepared from silk fibers may
thus provide opportunities to overcome the limitations that are
often faced while using the hydrogels obtained from other natural
polymers [62].
3.1. Fibroin hydrogels
3.1.1. Preparation and characterization
In general, hydrogels are obtained by physical or chemical
cross-linking of the polymeric chains. Since the performance of
hydrogels in the in vivo setting primarily depends on their proper-
ties, it is essential to determine the fundamental characteristics of
a hydrogel before it can be explored for its biomedical potential.
The major properties that need to be characterized for a hydrogel
include its porosity, water content and swelling behaviour, size
and shape, mechanical and rheological properties, biocompatibility
and biodegradation. Not only is it essential to determine the initial
properties of hydrogels, but equally important is to consider how
these properties change with time while the hydrogel is still in
use. These properties and their dynamics influence the spatiotem-
poral behaviour of the hydrogel, thus governing its utility for a
specific application. Since the processing conditions greatly influ-
ence the characteristics of the hydrogels, it is desirable to control
the processing of hydrogels to exert a strict control over the resul-
tant properties. As discussed below, it is becoming increasingly
possible to fine tune the properties of silk hydrogels.
Fig.1 schematically outlines the general steps to fabricate silk
hydrogels. Table 2 lists the methods that have been used to pro-
duce silk protein fibroin hydrogels. Predominance of hydrophobic
amino acid groups like glycine, serine and alanine [100] in fibroin
makes gelation possible without addition of any gelling agent. For
example, Ayub et al. prepared fibroin gel of mechanical strength
ranging from 1 kg/cm2 to 30 kg/cm2 by keeping fibroin solution
at 20 °C, at a relative humidity of 56–64% [101]. Slow rate of gela-
tion of fibroin has been one of the major challenges in preparing
 S. Kapoor, S.C. Kundu / Acta Biomaterialia 31 (2016) 17–32
Table 2
Methods for preparation of silk fibroin protein hydrogels.
Method Mechanical propertiesa
Compressive % strain
modulus at
(MPa) failure
Temperature changes 1.2b 50%
Lowering of pH towards isoelectric NA NA
point of fibroin
Shearing forces induced by 1.6c NA
sonication and vortexing of
fibroin solution
CO2 acidification 64.0 kPad
Phase separation induced by NA 16%
freezing and freeze thawing
a
Typical mechanical properties of the hydrogels prepared by different methods as reported in literature.
b
Gel prepared from 8% fibroin solution at 37 °C.
c
Gel prepared from 8% fibroin solution at power output = 30% amplitude.
d
Gel prepared from 4% fibroin solution processed for 8 h.
silk gels. For example, 2% fibroin solution (pH 6.4–6.8) kept at 37 °C
has been reported to form gel after about 30 days [88]. This limita-
tion has been overcome by stimulating gel formation in fibroin by
changing temperature, pH or ionic concentration by addition of
salts like CaCl2 or KCl [88,89,98,102] and by using several methods
like shearing, sonication, removal of bulk water by osmotic stres-
ses, vortexing, heating and exposure to solvents, gases and surfac-
tants like sodium dodecyl sulfate and sodium N-lauroyl sarcosinate
[90,96,97,103–106]. Silk fibroin solution treated at higher temper-
ature (60 °C) gels faster than the solution kept at 37 °C [84,85,101].
Similarly, an increase in fibroin concentration as well as a decrease
in pH towards its isoelectric point (3.8) also results in faster gela-
tion. Not only the gelation rate, but the intrinsic hydrogel proper-
ties like mechanical properties and pore-size of silk-gels can be
easily manipulated by controlling external conditions. It has been
seen that the breaking stress of gels prepared at 55 °C increased
to about 19 N/m2 as compared to about 8 N/m2 for the gels pre-
pared at 15 °C suggesting that the fibroin gels prepared at higher
temperature have better mechanical properties [102]. Similar
impact of temperature on mechanical strength of gels were
showed by Kim et al. who found that 16% fibroin solution, which
undergoes gelation at 60 °C, has double the compressive strength
(3 MPa) than a solution of same concentration that undergoes gela-
tion at 37 °C (1.5 MPa) [88]. This may be due to increased crystal-
lization at higher temperatures. The gel strength has also been
shown to vary with pH of the solution; the gels prepared at pH
7.0 have showed highest strength. Non-porous fibroin hydrogels
have been prepared by adding glycerol (30% (v/v)) to silk solution
[107]. The gels containing glycerol are slow in losing moisture
[107], suggesting that certain agents like glycerol can modulate
the moisture retention capacity of silk hydrogels.
When hydrogels are used as matrices for encapsulating cells or
other bioactive molecules, extremely mild methods of preparation
are used. Any reagent or intermittent process should not be detri-
mental to cell viability or to the activity of entrapped bioactive
agent. Sonication has been used to fabricate fibroin hydrogels
encapsulating cells [91,92,95] or bioactive molecules such as
growth factors [94]. The concentration of the protein solution is
crucial for such a process. Viscosity of the solution should allow
uniform propagations of waves throughout the solution or else it
may trigger heterogeneous gelation. Uniform mixing ensures a
21
Specific conditions Comments Reference
(s)
4, 25, 37, 60 °C have generally Higher temperature improved the mechanical [88–90]
been used strength, Method not suitable for direct
encapsulation of cells and temperature sensitive
peptides and bioactive proteins
Citric acid, Citrate–phosphate Neutralization of acid is needed, method is not [36,89]
buffer and HCl are mainly used suitable for direct encapsulation of pH sensitive
molecules and cells
Power output = 20–50% Method found suitable for making injectable gels [91–95]
amplitude, time = 5–30 s, and for encapsulating cells prior to final setting of
3200 rpm using vortexer gel
2 wt% fibroin solution, pressure Ease of fabrication; hydrogels free of mineral acids [96,97]
CO2 = 60 bar or chemical cross-linkers
Water soluble organic solvent Generally leads to formation of hydrogels with [98,99]
heterogeneous porosity
homogeneous distribution of the cells/bioactive molecules in the
matrix. Lately, vortexing and electric current have also been used
to fabricate silk hydrogels [93,108]. The treatments including son-
ication and vortexing promote b-sheet transformation in silk,
which ultimately result in gel formation [92–94].
3.1.2. Sol–gel transition mechanism of fibroin
Various groups have worked to understand the molecular
mechanisms involved in the gelation of fibroin. Initially, it was pro-
posed that b-sheet transformation is responsible for gelation of silk
solution [109,110] and it has later been shown that there is higher
b-sheet content in silk gels as compared to silk solution and silk
films [101]. Matsumoto et al. have provided a detailed insight into
the gelation mechanism of silk fibroin [103]. FTIR and CD analysis
have shown that regenerated silk solution obtained after dialysis
contains about 10–20% b-sheet fractions (depending on the con-
centration of protein). During initial gelation, only weak interac-
tions take place between the protein chains without any
secondary structure changes. Thus, there is no significant increase
in the b-sheet fraction. Until this stage, silk gelation is a reversible
process [111]. However, as soon as about 15% gelation is complete,
strong interactions follow, resulting in a continuous increase in b-
sheet content [111]. These thermodynamically stable cross-links
ultimately result in gelation of the fibroin solution. At this stage,
b-sheet content reaches as high as 50% [103]. Non-linear micro-
scopic techniques, namely photon excited fluorescence (TPEF)
and second harmonic generation imaging have also showed that
fibroin undergoes structural transformation to b-sheet as hydrogel
formation takes place [112].
Factors like pH, temperature and ionic strength have been used
to control the gelation rate of fibroin. The ultrasensitivity of fibroin
gelation to pH arises because of its structure [103]. Silk is an
amphiphilic protein consisting of hydrophilic N- and C-termini
and large hydrophobic domains interspersed with very short
hydrophilic stretches. The heavy chain N-terminus, rich in acidic
amino acids is expected to have a pI of about 4.59 while C-
terminus is rich in basic amino acids and has a pI of about 10.53.
On the other hand, C-terminus of the light chain is rich in acidic
groups and has a pI of about 5.06. As pH is lowered, more and more
carboxyl groups get protonated, reducing charge-charge repulsion
and thus resulting in faster gelation [103]. Thus, a decrease in neg-
22 S. Kapoor, S.C. Kundu / Acta Biomaterialia 31 (2016) 17–32
Fig. 2. Approaches to improve the properties of silk hydrogels: (a) Properties of fibroin hydrogels can be modulated by varying the processing conditions, (b) Fibroin-
composite hydrogels can be prepared by combining fibroin with synthetic or natural polymers and (c) silk proteins can be genetically engineered to introduce ‘designer’
features into them.
ative charge by protonation of acidic amino acid side chains pro-
motes refolding of protein chains to a more ordered state that is
accompanied by an exclusion of water. A similar increase in
hydrophobic interaction is observed when temperature and ionic
strength of fibroin solution are varied. The profound effect of tem-
perature on gelation rate of fibroin has been attributed to the
reduction of local dielectric properties caused by temperature-
induced dehydration [103]. Two-Dimensional Raman Correlation
Spectroscopy and 13C Solid-State NMR have shown the occurrence
of transition of secondary structure of silk from random coils to b-
sheet in presence of calcium ions [113]. Thus, b-sheet transition
mainly triggers the gelation of fibroin and the conditions that have
been used to prepare fibroin hydrogels promote this transition in
silk, thus, facilitating gel formation. Lately, it has been shown that
the sol–gel transition of fibroin can be reversibly controlled even in
b-sheet-rich silk by regulating its self-assembly process in aqueous
solution [114]. Such reversible gelation may impart an ability to
exert a temporal control in encapsulating molecules in hydrogels
by assembly and subsequently controlling their release by disas-
sembly of hydrogels.
3.2. Silk fibroin composite hydrogels
In addition to modulating the properties of silk hydrogels by
varying external conditions as described above, other approaches
are also used to fine tune their material properties (Fig. 2). These
include producing silk fibroin hybrid hydrogels and fabricating
hydrogels using genetically engineered silk. Table 3 lists the poly-
mers that have been used to fabricate fibroin-composite hydro-
gels and the advantages of the resulting matrices. Regenerated
silk fibroin has been blended or chemically cross-linked with sev-
eral synthetic polymers. For instance, in a continued effort to
improve the mechanical properties of silk hydrogels, our labora-
tory combined polyacrylamide with silk to obtain hydrogels
whose properties can be manipulated by changing the ratios of
the two components [115]. Our data suggested that the gels pos-
sessed high mechanical strength and were cytocompatible. In
other studies, Young’s modulus of polyvinyl alcohol-silk fibroin
50:50 blend gels (146.7 MPa) increased three fold as compared
to that of fibroin only gel (50 MPa) [117]. Additionally, the poros-
ity of the hydrogels increased on increasing the freeze–thaw
cycles, corroborating that in addition to the polymer, method of
blending affects the ultimate properties of the hydrogels and thus
needs to be optimized [117,118]. In addition to improving the
physical properties of the hydrogels, novel features including
stimuli-sensitivity and reversibility of sol–gel transition have
been introduced by fabricating silk composite hydrogels. Kang
et al. first reported that poloxamer 407, a type of pluronic, can
influence gelation of fibroin [119]. Pluronics are a class of non-
ionic tri-block copolymers consisting of a central hydrophobic
chain of polyoxypropylene and two terminal chains of hydrophi-
lic polyoxyethylene. Varying the length of chains can vary their
properties. Gelation of fibroin poloxamer system containing 3%
fibroin was found to be reversible as poloxamers have reversible
sol–gel properties at different temperatures due to variation in
interactions between the hydrophobic and hydrophilic chains
[119]. Thus, incorporation of poloxamer resulted in tempe
rature-sensitive protein hydrogels.
Silk has been combined with natural polymers as well to fabri-
cate composite hydrogels. Mixed protein hydrogels comprising of
fibroin and collagen or gelatin have been prepared [127–131].
Since gelatin hydrogels as such are not stable due to dissolution
of gelatin at 37 °C, mixing of silk fibroin with gelatin proved advan-
tageous, as the presence of silk fibroin b-sheets provided stability
 S. Kapoor, S.C. Kundu / Acta Biomaterialia 31 (2016) 17–32 23

Table 3
The polymers that have been used in combination with silk fibroin protein to obtain composite hydrogels with improved properties.

Nature of Polymer Method of preparation Characterization Properties/Advantages of the resulting Reference

polymer hybrid hydrogel (s)
Synthetic Polyacrylamide Chemical cross-linking of silk and Swelling, morphological, rheological, Improved mechanical strength [115,116]
acrylamide solution mechanical properties and
biocompatibility
Polyethylene Mixing powdered PEO in silk solution Gelation kinetics, morphological and Enhanced gelation rate of silk solution [88]
oxide followed by heat treatment mechanical properties
Polyvinyl Freezing and Freeze thawing Mechanical properties and porosity Improved mechanical properties [117,118]
alcohol
Poloxamer Photocross-linking of silk and Gelation kinetics, thermal stability, Reversible sol–gel transition of silk [119,120]
poloxamer solution, Physical mechanical and swelling properties solution
blending of silk and poloxamer
solutions
Polyurethane Chemical crosslinking Mechanical, rheological properties and Injectable; improved mechanical [121]
biocompatibility properties, suitability for application in
nucleus pulposus replacement
NIPAAma Physical blending of silk and NIPAAm Swelling and rheological properties Increased deswelling kinetics of [122,123]
solution composite hydrogels as compared to
NIPAAm hydrogels
Polyethylene Physical blending of silk and PEG 300 Gelation kinetics, rheological Injectable, In situ gelling, low initial cell [124]
glycol or PEG 400 solution properties, mechanical properties, attachment, thus suitable as, anti-
degradation kinetics and cell fouling and anti-adhesion surface
attachment
Natural Gelatin Physical blending of silk and gelatin Thermal stability, viscoelastic, swelling Temperature sensitive conformational [125–130]
protein solutions, Crosslinking using and morphological properties transition of gelatin from helix to coil,
Genipin formation of stable gels
Collagen Chemical cross-linking of Thermal, viscoelastic, swelling, Improved mechanical strength, thermal [131]
fibroin/collagen solution morphological properties and stability and substrate stiffness
biocompatibility
Chitosan Chemical/physical cross-linking of Swelling properties and ion and pH pH and ion sensitive hydrogel, [132,133]
silk and chitosan solution sensitivity of gels improvement in release kinetics of
drugs
Hyaluronan Cross-linking of HA in presence of an Gelation kinetics, thermal stability, Improved mechanical properties and [134,135]
electrospinned silk mat, Physical morphology, mechanical and swelling controlled degradation
blending using ultrasonication properties, enzymatic degradation
Methylcellulose Physical cross-linking of silk and MC Morphology,drug release kinetics Control of sol–gel transition of fibroin [136]
solution at 50 °C and drug release properties
Pectin Dialysis against methanol Mechanical Properties, swelling Increased stiffness of hydrogel [137]
properties biodegradability,
biocompatibility
Alginate Calcium ion-induced gelation Porosity, intravital imaging, Mechanical properties of the hydrogel [138,139]
Rheological properties, Immune facilitated stem cell differentiation
response Supports biomimetic crystallization of
hydroxyapatite
a
NIPAAm: N-isopropylacrylamide.

to the resulting silk fibroin/gelatin (SF/G) blend hydrogels [128].
Differential scanning calorimetry and dynamic rheological analysis
indicated that the presence of silk fibroin b-sheets increased the
dynamic elastic modulus of the G/SF hydrogels, which in turn sta-
bilized them at higher temperatures [128]. Interestingly, blended
hydrogels have been found to be stimuli-sensitive with the swel-
ling of hydrogel and dissolution of gelatin from hydrogels being
higher at 37 °C compared to that at 20 °C [128]. Collagen-fibroin
composite hydrogels have been prepared by cross-linking collagen
and fibroin by using optimized amounts of 1-ethyl-3-(3-dimethyla
minopropyl)carbodiimide hydrochloride (EDC) [131]. In addition
to improving the mechanical strength and thermal stability, the
gel exhibited gel-to-sol transition in medium of pH 4.0. The rever-
sible gelation characteristic can be attributed to an increase in
electrostatic repulsion between protein chains with decrease in
pH value [131]. Hyaluronan (HA), a major glycosaminoglycan of
the extracellular matrix, has also been used together with fibroin
to fabricate a composite hydrogel [134,135]. HA solution has
chemically been cross-linked using poly(ethylene glycol)-
diacrylate in presence of a prefabricated electrospun mesh of
fibroin [134] or has physically been blended with fibroin through
ultrasonication [135]. The presence of fibroin acts as mechanical
reinforcement and slows degradation of the otherwise mechani-
cally weaker, fast degrading HA hydrogel.
Different variations of fibroin-based covalent or non covalent
interpenetrating networks (IPNs) and semi-IPNs (SIPNs) have also
been synthesized. Interpenetrating networks are materials com-
posed of two polymers, each existing as an independent network
[140]. The two networks are polymerized or cross-linked such that
the resultant networks are interlocking. Combining two networks
provides an opportunity to create composite materials that other-
wise cannot be obtained by mixing polymers due to phase separa-
tion. IPNs result in unique properties, which are not present in the
individual polymer networks [141,142]. Semi IPNs of silk fibroin
and modified poloxamer 407 having an acrylate-terminated poly-
ethylene oxide (PEO) derivative, modified polyethylene glycol
(PEG) macromer and PEG have also been prepared [120,143,144].
The SF (silk fibroin)/PEG SIPN has been prepared using a pho-
topolymerization method. The SIPN hydrogels have higher com-
pression resistance and mechanical strength as compared to SF
or polymer (PEG/poloxamer) hydrogels. The increase in strength
makes SIPN hydrogels desirable for biomedical applications, espe-
cially skin grafting and wound dressing [143]. Our lab has synthe-
sized photo-crosslinked PVA/SF hydrogels. PVA methacrylate,
obtained by reacting PVA with 2-isocyanatoethyl methacrylate,
was freeze dried. The resuspended solution was blended with
fibroin and blend solutions were photopolymerized giving rise to
semi-IPNs [117]. Using a similar approach, Xiao et al. fabricated
24 S. Kapoor, S.C. Kundu / Acta Biomaterialia 31 (2016) 17–32
protein IPNs where gelatin methacrylate and SF have been inde-
pendently crosslinked in the interpenetrating network [130]. Gela-
tin methacrylate has been polymerized by UV-irradiation in the
presence of Irgacure 2959, a photoinitiator and the SIPN has subse-
quently been exposed to methanol to introduce cross-links in SF.
Introducing SF improved the mechanical properties of hydrogels
and it has been seen that exposing SF to methanol further
enhanced the strength of hydrogels [125]. In yet another study,
Gil et al. have made SIPN consisting of poly(N-isop
ropylacrylamide) (PNIPAAm) and fibroin [122]. PNIPAAm is one
of the most thoroughly investigated thermoresponsive polymers.
It shows a sharp phase transition when the temperature is
increased above or decreased below its lower critical solution tem-
perature i.e. 32 °C [145]. This type of stimuli-sensitive polymer has
many applications in devices that require an on–off mechanism.
The major drawback of this system is its low de-swelling rate
due to formation of a skin layer [146]. Various strategies have been
suggested for improving the deswelling kinetics of PNIPAAm [147].
Introduction of fibroin in PNIPAAm has been shown to alter its
microstructure, leading to an increase in its de-swelling rate that
results from reduced phenomena of skin layer formation in the
gels, while maintaining the swelling rate [122]. To further improve
the swelling/deswelling rates of these composite hydrogels, a
novel strategy has been used [123]. The composite hydrogels have
been lyophilized and the lyophilized matrix has been exposed to
methanol. Freeze-drying introduces temporary macropores in the
hydrogels, while exposing the matrix to methanol introduces b-
sheets in SF that assists in maintaining the macroporous structure
of hydrogels even after swelling [123]. These examples clearly sug-
gest that several of the limitations associated with parent polymers
can be overcome and novel properties, which do not exist in the
individual parent polymers, can be introduced by preparing fibroin
hybrid hydrogels. It should be noted, however, that when combin-
ing silk with other polymers and varying the relative amount of the
two polymers in the combination, method of blending and cross-
linking density may simultaneously affect many properties includ-
ing strength, porosity, swelling, biocompatibility and morphologi-
cal properties [120,122,139]. Therefore, while fabricating a hybrid
hydrogel, it is essential to ensure that an attempt to improve a par-
ticular aspect of hydrogel does not affect other physical or biolog-
ical properties adversely.
3.3. Hydrogel fabrication from genetically engineered silk
Advancement in biochemistry and molecular biology of silk at
genetic and protein levels have provided a strong platform for
genetic engineering of silk proteins (Fig. 2). One of the unparalleled
strengths of genetic engineering of proteins is the ability to pre-
cisely customize the structure and function according to specific
needs. Thus, we can have biomaterials with desirable properties
by utilizing tremendous chemical diversity available in the amino
acid building blocks [148]. Another reason for cloning silk genes is
to obtain larger quantities of protein in a cost effective way.
Although protein can be obtained in larg amounts from silkworms
like B. mori and spiders, the total availability of protein is not suf-
ficient considering the amounts required for various applications.
Silk-like proteins have been obtained by creating variants of the
repeat sequences of N. clavipes and B. mori. Continuous efforts in
this direction have resulted in successful production of recombi-
nant silk proteins [149–156]. Various strategies and host systems
including insect cells [157], E. coli [158], microbes [159,160], mam-
malian cells [161], goats [162] and potato and tobacco plants
[163,164] have been tried to optimize the yield of protein, to
improve the solubility of protein and to allow proper folding and
post translational modification of protein.
Not only the proteins have been obtained in larger quantities
but also, new features, which are not found in native proteins, have
been incorporated [149]. The main functional modalities that have
been incorporated in these genetically-engineered proteins are ori-
ented towards making the proteins self assembling, stimuli-
sensitive, mechanically stable and information rich so that they
contain specific epitopes and domains that assist in cell adhesion
and signalling. Incorporation of these functionalities ultimately
aims to make the genetically engineered protein-based hydrogels
more suitable for tissue engineering and drug delivery purposes
[165,166].
3.3.1. Silk-elastin like hydrogel
Elastin-like proteins have gained considerable attention due to
their striking self-assembling properties, attributed mainly to the
transitions that take place in the polymer backbone in the presence
of water. Thorough details about elastin-like proteins are provided
elsewhere [167–169]. Silk-elastin like polymers (SELPs), developed
by Cappello and colleagues, constitute an important class of genet-
ically engineered silk proteins [170]. These synthetic proteins con-
sist of (a) repetitive silk peptide sequence of B. mori (Gly-Ala-Gly-
Ala-Gly-Ser) that provides mechanical strength to block copolymer
protein and (b) elastin peptide sequence (Gly-Val-Gly-Val-Pro) that
provides flexibility to the protein, improves its solubility and
determines the cross-link density within the hydrogels [170,171].
Varying the composition of the two constituent peptide blocks pro-
vides a means to regulate the properties including strength,
immunogenicity, solubility and degradation rate of the resulting
biomaterial [172]. The silk-elastin like polymers have been well
characterized to determine their properties and their utility for
various applications. The general techniques for making and char-
acterizing these polymers have been reviewed in detail [170–174].
Certain members of this family of proteins, called Prolastins, exhi-
bit sol-to-gel transition and hydrogel forming capabilities [175].
They mainly consist of two or more silk-like sequences per mono-
mer. Hydrogel formation is primarily regulated by temperature
(due to dependence of assembly of elastin-like blocks and hydro-
gen bonding of silk-like blocks on temperature). Under physiolog-
ical conditions, the hydrogen bond formation between silk-like
units causes protein solution to undergo crystallization, which
results in gelation [175]. The major advantage is that no chemical
cross-linking or solvent treatment is needed to prepare SELP
hydrogels. Thus, SELP hydrogels present novel opportunities to
encapsulate bioactive molecules or cells under ambient conditions
and form in situ gelling hydrogels for tissue engineering and ther-
apeutic purposes.
SELP-47K and SELP-415K, consisting of four silk-like and seven
and fifteen elastin-like blocks, respectively, are main examples of
gel-forming proteins. SELP-47K, which undergoes irreversible sol-
to-gel transition at body temperature, is the most extensively
investigated gel-forming Prolastin polymer [173–178]. Swelling
characteristics and equilibrium water content are important
parameters of the matrix and primarily govern the release proper-
ties of the molecules from hydrogels. Hydrogel swelling character-
istics are, in general, controlled by factors like polymer
concentration and cross-link density as well as environmental
stimuli such as pH, temperature, and ionic strength of solvent.
However, in case of SELP-47K, the swelling of the gels is relatively
independent of temperature, pH and ionic strength, suggesting
that this polymer can be utilized to make hydrogels whose release
properties remain constant in spite of external variations [35].
Although hydrogels which maintain similar swelling properties
under different conditions are robust, there may be conditions
where it is desirable to have hydrogels with stimuli-sensitive swel-
ling and release properties. Swelling properties of SELP-415K have
been found to be sensitive to temperature and ionic strength [179].
 S. Kapoor, S.C. Kundu / Acta Biomaterialia 31 (2016) 17–32
Physiologically relevant sensitivity to pH- and temperature has
been introduced in these polymer gels by strategic replacement
of valine in elastin chain with glutamic acid (a charged amino acid)
through genetic engineering techniques [180]. Dandu et al. have
prepared hydrogels from another silk-elastin like protein (SELP-
815K) and have shown that the silk sequences provide mechanical
stiffness to the hydrogels whereas the elastin sequences determine
the cross-link density within the hydrogels [171]. Recently Silk-
Elastin like proteins with matrix metalloproteinase (MMP) degrad-
able sequences inserted in between the polymer backbone have
been expressed. The degradation behavior of the hydrogels fabri-
cated from this recombinant protein can evidently be finely con-
trolled by environmental cues like the expression of MMPs,
which have been found to be overexpressed in certain tumors
[150,181]. Recently, in situ gelling injectable SELP-47K and SELP-
815K based-hydrogels have been investigated for formulating
transarterial embolics to selectively prevent tumoral blood supply
as a novel strategy for chemotherapeutics [178]. A 12% w/w solu-
tion of SELP-815K fabricated into hydrogel by shearing has been
found to exhibit acceptable rheological properties and embolic
capability [178]. Although, the research about SELP hydrogels is
still in a budding stage, it is clear that hydrogels from silk-
copolymers can be made with varied mechanical properties, degra-
dation signals, assembly characteristics and bioactive signals, mak-
ing them useful for future biomedical applications.
4. Biomedical applications of silk-based hydrogels
4.1. Tissue engineering
Engineering the polymeric materials to replace/repair a mal-
functioning or damaged tissue or organ is one of the most impor-
tant advances in the recent decades [182]. The success of
hydrogels as tissue engineering matrices lie in the fact that their
biochemical composition and properties such as water content,
viscoelasticity and mechanical strength can be made to mimic dif-
ferent types of natural tissues. Controlling the factors like mono-
mer concentration, cross-linker concentration and nature of
functional groups during processing can optimise the properties
of hydrogels. Similarity of hydrogels to natural tissues and their
ability to support functions like transport of bioactive molecules
such as hormones, growth factors and peptide sequences while
maintaining structural integrity make them attractive for tissue
engineering applications [183]. Another advantage of using hydro-
gels for tissue engineering applications is the ability to entrap cells
within hydrogels during fabrication, provided that mild conditions
are used for preparation so that the cell viability is not affected
[184]. Cell entrapment during gelation results in a more uniform
distribution of cells, as compared to populating the matrix after
gelation, which results in higher cell populations near the surface
of the gel [185], though considerable success has been achieved
with the latter strategy as well [186]. Thus, the aqueous environ-
ment, ease of transportation of nutrients and entrapped molecules,
ease of modification and utility as in situ forming matrices repre-
sent important advantages of hydrogels as tissue engineering
matrices. The disadvantages may include difficulty in handling,
sterilization and lack of mechanical strength [187]. Three main
design approaches to use hydrogels as functional matrices include
preparing acellular hydrogels, cell-laden hydrogels and tissue-
engineered hydrogels, which, in addition to the polymeric scaffolds
and cells, contain several other factors like chemical moieties and/
or bioactive molecules such as growth factors and peptide
sequences to modulate degradation/remodelling of matrix, cell
adhesion, cell motility, cell growth and differentiation [187]. Silk
hydrogels based on all these approaches have been produced.
25
The real challenge yet lies in replicating their structural and func-
tional properties according to the desired tissue type where the
hydrogel is to be implanted and in creating the dynamic chemo-
mechanical environment that exists in vivo.
4.1.1. Bone tissue engineering
Bone, which mainly consists of collagen protein and hydroxya-
patite, provides support and strength for the movements. Critical
bone defects or severe bone injuries, which cannot be regenerated
naturally, may need external interventions in the form of implants.
Implants provide mechanical strength to bear stresses at the site of
injury and milieu that accelerate natural tissue growth. Bone tissue
engineering is an approach, which differs from permanent bone
implants in a sense that the tissue engineered bone is designed
to integrate and absorb in the environment where it is implanted.
For this, the implant should be biodegradable and bioresorbable
[188,189]. The major challenge for successful bone tissue engineer-
ing lies in understanding the spatial and temporal distribution of
cells and growth factors and their interaction with extra cellular
matrix for osteogenesis in diseased conditions. In addition, the
implant material should provide mechanical, chemical and struc-
tural signals for osteoconduction and osteoinduction, essential
for in vivo tissue growth. Several materials have been investigated
for bone tissue engineering [188,189]. Metals have been the tradi-
tional choice as implant material due to their load bearing capabil-
ity. However, they suffer from several disadvantages like the stress
shielding effects, poor biodegradability, and lack of integration
with the surrounding tissue [190]. Ceramics, though resemble to
the mineral phase of bone, are severely limited in application by
their brittle nature and poor biodegradation properties [191].
Amongst natural polymers, collagen-based materials offer oppor-
tunity to influence cellular responses but often exhibit poor
mechanical strength and undesirable immune responses [192].
Biomaterials fabricated from alginate have adequate mechanical
properties, however, they have limitation for different cell-based
applications due to lack of cell-specific bioactivities [193]. To
address some of these challenges, silk implant material has been
evaluated for bone regeneration.
Silk proteins can be tailored to make a bioactive material for
bone implants to induce direct bone formation and ultimately
result in osteointegration, although much work still needs to be
done in this direction. Silk fibroin hydrogels have shown superior
ability to promote cell metabolism as well as bone remodelling
as compared to poly(lactide-co-glycolic acid) (PLGA), an FDA
approved polymer, which is a popular choice for comparing the
characteristics of a material for tissue engineering applications
because of its long clinical standing, favorable biodegradation
properties, biocompatibility and a minimal systemic toxicity
[193,194]. The bone-healing rate, proliferation and differentiation
of the osteoblasts in presence of silk hydrogels have also been bet-
ter than the control [37]. In another study, porous silk matrix (pre-
pared by using hexafluoroisopropanol (HFIP) as solvent) was
seeded with stem cells derived from human bone marrow and
the cells have been allowed to grow under osteogenic conditions
with medium being supplemented with ascorbic acid-2-
phosphate, dexamethasone, b-glycerolphosphate, and BMP-2
[195]. Various approaches including (1) tissue engineered bone
(cells seeded on matrix grown in bioreactor for 5 weeks), (2) scaf-
folds seeded with cells at the day of surgery, (3) scaffold alone, or
(4) no implant (unfilled) were evaluated by implanting in mouse.
Clearly visible trabecular structures could be observed through
micro-computed tomography after about 5 weeks of implantation
in case of the defects treated with the engineered bone, suggesting
that silk based implants can be utilised for effective bone regener-
ation. It is worth noting that the scaffolds prepared from protein
obtained from the same source but differing in the preparation
26 S. Kapoor, S.C. Kundu / Acta Biomaterialia 31 (2016) 17–32
method exhibit different osteogenic responses [196]. Differences in
bone related outcomes such as mineralization and expression of
collagen type I, alkaline phosphate, osteopontin and matrix metal-
loproteinase 13, which regulate the remodelling of ECM have also
been observed [196]. The differences in the processing of protein
lead to subtle changes in structure and properties of resulting
matrix, which ultimately lead to different cell response [197].
Recently, fibroin/sodium alginate hydrogels have been used as
template for promoting controlled biomineralization of hydroxya-
patite crystals for bone repair [139]. The preliminary success of
hydrogels derived from silk fibroin suggests that further studies
are warranted. Incorporation of additional biological signals as
well as utilization of polymer blends, interpenetrating networks
and silk copolymers may provide ideal scaffolds for bone repair.
4.1.2. Cartilage tissue engineering
Cartilage, a connective tissue, is mainly composed of type II col-
lagen and is rich in proteoglycans, particularly chondroitin sul-
phate while the primary cell type is chondrocytes [198]. This
tissue contains almost 65–70% water. The clinical impact of defects
in cartilage tissue due to congenital abnormalities or trauma is of
particular concern due to the low regenerative capability of this
tissue, resulting from an absence of blood vessels, nerve tissue,
and lymphocytes and the low cell density, slow cell proliferation
and slow matrix turnover rate. Therefore, cell-based therapies, in
which 3D scaffolds carrying stem cells or differentiated chondro-
cytes can be implanted at the site of injury, are of significant inter-
est. Due to their close resemblance to cartilage tissue, hydrogels
have been considered as a suitable matrix for encapsulating cells
and the growth factors (e.g. TGF-b superfamily, IGF, FGF, BMP,
PDGF, and EGF). Natural materials like collagen and synthetic
material like poly(lactic acid) (PLA) and PLGA have been evaluated
for this purpose. Though agarose hydrogels have particularly
shown utility as scaffolds for cartilage tissue engineering because
of their high mechanical strength, their success is limited by poor
biocompatibility, lack of biodegradability and poor host tissue inte-
gration [91]. The mechanical strength, frictional properties and the
uncertainty of interaction between the matrix and the cells have
remained the major issues with most of the scaffolding materials
[91]. Aoki and colleagues have prepared fibroin hydrogel and com-
pared its cartilage regeneration performance with collagen gels
[33]. They inoculated the chondrocytes isolated from the proximal
humerus, distal femur and proximal tibia of 4-week-old Japanese
white rabbits into the fibroin-hydrogel sponge, formed by phase
separation of freezed fibroin solution and collagen gels [33]. The
cells were then cultured for 4 weeks and were found growing in
the pores and the outer surface of the fibroin hydrogel and the cell
density increased with incubation times. On the other hand, the
cell density in the collagen gels was seen to be higher initially,
but it did not increase with time. Similarly, the rate of increase
of chondroitin sulfate in fibroin hydrogel was higher than that in
collagen gels [33]. Positive histological staining for key cartilage
extracellular matrix components (chondroitin sulfate and type II
collagen) indicated formation of hyaline cartilage-like tissue in
the pores of the fibroin hydrogel. Chondrocytes maintain round
morphology and retain their differentiated phenotype within the
fibroin hydrogel [33]. Cell microaggregates seeded in fibroin
hydrogel closely mimic the initial stages of tissue formation and
have been found to be very efficient in forming extracellular matrix
for cartilage tissue regeneration [199]. To improve the mechanical
properties of fibroin hydrogels, recently a novel approach has been
used, wherein silk hydrogel has been reinforced with silk microfi-
bers. The composite hydrogel has exhibited mechanical properties
comparable to that of the agarose hydrogels with proven mechan-
ical robustness [200].
Bioreactors can be used to stimulate functional regeneration of
tissues and to continuously supply the nutrients and growth fac-
tors to the cultured cells. This represents an advanced approach
holding immense potential for improving cartilage tissue engineer-
ing. This approach has been used with fibroin hydrogel [201]. The
cells grown in matrix homed within a stirring chamber have been
found to exhibit higher DNA and glycosaminoglycan content as
compared to the cells not grown in the reactor. Positive histological
staining of proteoglycans and collagen in tissue grown in this sys-
tem vis-à-vis the one in a system lacking stirring chamber sug-
gested that stirrer facilitated the maturity of cartilage matrix by
providing mechanical stimulation to the cellular microenviron-
ment. This system could support chondrogenesis in vivo and
resulted in cartilage regeneration in the rabbits corroborating its
clinical potential [201].
Primary cell lines and chondrocytes are mainly used as cell
source for cartilage tissue regeneration. Since they are available
in limited supply, scientists are now focusing on the use of stem
cells that show unlimited potential to differentiate in several tissue
types including cartilage [202,203]. Human mesenchymal stem
cells (hMSC) encapsulated in SELP-47K hydrogel and cultured in
chondrogenic medium in the presence of TGF-b3 exhibited differ-
entiation and chondrogenesis [204]. The cells remained metaboli-
cally active even after 28 days of culture and histological
analyses have revealed the formation of extracellular matrix along
with the expression of SOX9, and matrix proteins aggrecan and col-
lagen. The studies indicate that fibroin hydrogels have potential for
mimicking the extracellular matrix and promoting cartilage
regeneration.
4.2. Controlled release
4.2.1. Controlled drug delivery
Controlled release implies that the delivery of the active agent
should follow a predetermined course of release. It may consist
of both sustained (for prolonged duration) and targeted (localized)
release. Supplying essential amounts of active molecule at a partic-
ular area for required duration is a major challenge in the field of
drug release. This is more so in the case of bioactive agents like
hormones and peptides, which are highly selective in their action
and are sensitive to processing treatments. Silk based hydrogels
have been found to be suitable for controlled release. One of the
first studies investigating silk fibroin for controlled release has
not used a hydrogel but a membrane. In this early study, Chen
et al. examined the permeation of pharmaceuticals like 5-
fluorouracil, L-(+)-ascorbic acid, resorcinol, sodium phenolsul-
fonate and benzyltrimethylammonium chloride through the
fibroin membrane [205]. They proposed that since fibroin mem-
brane consists of weak basic and acidic groups, it can act as an
amphoteric ion-exchanger and controlling the pH may control
the passage of molecules through it. Since then fibroin has been
considered as pH responsive drug delivery material. Hanawa
et al. have showed that the release behaviour of benfotiamine, a
synthetic derivative of thiamine (vitamin B-1), in the glycerol con-
taining fibroin hydrogels is inversely related to the fibroin content
in the matrix [206]. This is likely due to the increased number of
interchain interactions at higher protein concentration, which
resulted in smaller pore diameters and hence slower release. Not
only the protein concentration but molecular weight of silk protein
affects the release rate as well [36]. High molecular weight pro-
teins (76 kDa) have been shown to effectively slow down the
release of buprenorphine in comparison to low molecular weight
silk protein (18 kDa) when used as matrix [36]. Our laboratory
has evaluated fibroin/polyacrylamide SIPN hydrogels for drug
release [115]. Two model compounds namely trypan-blue and
FITC-inulin have been used to determine the effect of physico-
 S. Kapoor, S.C. Kundu / Acta Biomaterialia 31 (2016) 17–32
chemical properties of hydrogels on release of molecules. Our
results have indicated that these composite hydrogels were suit-
able for sustained release of low and high molecular weight mole-
cules alike. Recently, silk hydrogels have shown potential for
sustained release of bevacizumab, a clinically used anti-VEGF ther-
apeutic for certain metastatic cancers [207]. The controlled release
of a chemotherapeutic over a long period may eventually reduce
the dosing frequency, which increases patient compliance.
As noted above, specific Prolastins are important particularly as
in situ gel forming delivery systems [169,175,178]. They sponta-
neously form a gel over a period of time after being injected via fine
gauge hypodermic needles [175]. Cappello et al. have characterized
the release of many fluorescently labelled model compounds of dif-
ferent molecular weights and a model pharmaceutical protein pan-
tarin from Prolastins of different peptide sequence compositions
[175]. The rate of release of entrapped molecules has been
observed to depend on the composition as well as the concentra-
tion of the recombinant Prolastin. Moreover, the entrapped thera-
peutic protein retained the bioactivity after being released from
gels [175]. The interactions between the entrapped molecule and
protein matrix might affect the solute partitioning and hence the
release profiles of molecules. This has been shown in the case of
cytochrome c, Vitamin B12 and theophylline release from SELP
hydrogels [35].
4.2.2. Controlled release of DNA for gene therapy
There has been a tremendous increase in understanding the
implications of the diseases caused due to genetic mutations.
Hence, a lot of effort is being placed on replacing abnormal genes
with the correct ones. Successful gene delivery remains the key
for gene therapy. Targeting the cells, controlled activation and
expression of the genes and getting rid of the side effects remain
major challenges for success in this endeavour. Safe and effective
delivery systems are thus the main clinical concerns. SELP poly-
mers have recently been evaluated for DNA delivery for tumor
therapy [179,181,208–211]. The ease of sol-to-gel transition of
these block copolymers enables the loading of DNA or viral parti-
cles into an aqueous solution, which may be injected by minimal
invasive procedures to form solid matrix in and around the tumour
at physiological temperature. Megeed et al. have studied the
release of plasmid DNA from SELP-47K hydrogels [208]. Lysine
(thirteen) and arginine (five) residues present in SELP-47K are pro-
tonated at pH 7.4, and can thus interact with the negatively
charged phosphate backbone of DNA and affect its release profile.
Ionic strength, concentration of the SELP polymer and time for cur-
ing the gels have also been found to influence the release rates
[208]. In addition, the conformation (supercoiled, open circular or
linear) and molecular weight of the DNA significantly influence
the release profiles while the concentration of DNA (within a range
of 50–250 mg/ml) has no impact [209]. Finally, gene expression
analysis showed that the released DNA retains its bioactivity even
after 28 days of release [209]. Further, SELP matrices have been
found to be suitable for localizing and prolonging the release of
adenovirus and viral transduction in murine models of breast
and head and neck tumour xenografts [210]. Haider et al. have cre-
ated a new matrix, SELP-415K, having more elastin units for gene
delivery purpose. The release rate of DNA from this polymer matrix
increased with an increase in the number of elastin units [179].
Recently, the release behaviour of incorporated plasmid has been
studied as a function of biodegradation of the SELP-47 and SELP-
415K hydrogels in presence of elastase enzyme [211]. In case of
both the hydrogels, the release of DNA is much faster when the
release medium contains elastase as compared to controls. More-
over, higher number of elastin blocks in protein resulted in a faster
elastase-induced degradation of the matrix, and loss of integrity of
hydrogel, thus, resulting in faster DNA release [211]. The release of
27
viral vectors from MMP responsive-SELPs has been evaluated in a
mice model of head and neck squamous cell carcinoma for tumor
therapy [181]. The rate of release of viral vectors has been found
to be dependent on the expression of MMPs, frequently found to
be overexpressed in several tumors [181]. Such a system may
enable localized delivery of bioactive therapeutics to tumors
specifically, thus limiting systemic host toxicity.
5. Challenges and future prospects
Silk proteins represent interesting polymeric biomaterial
because of their mechanical properties, thermal stability, biocom-
patibility, and possibility of control via genetic engineering. Several
examples mentioned in this work elucidate the potential of using
fibroin for producing hydrogels. A wide range of applications
including controlled release of active molecules and tissue engi-
neering have also been described (Fig. 3). Advances in fabrication
methods, ability to process and sterilize, control of properties like
degradation and strength, and possibility of encapsulating cells
have made silk hydrogels suitable constructs for complex tissue
regeneration.
Nevertheless, there are limitations that need to be overcome to
explore complete potential of silk hydrogels. A greater understand-
ing of the fundamentals underlying structure–function relation-
ship of these proteins is needed to control the properties
according to the specific requirements of design criteria. As more
specialized hydrogels are made for advanced applications, rigorous
and accurate mathematical modelling will be required to describe
the systems and the mechanisms associated with them. Fig. 4 out-
lays the novel possibilities with silk hydrogels. In situ gelling
hydrogels can assist in overcoming the invasive implantation of
hydrogels for tissue reconstruction, thus, increasing the patient
compliance. Self-assembling silk-like and silk-elastin like proteins
provide huge opportunities to design in situ forming gels under
mild conditions but we need to incorporate simultaneously
‘‘smartness (stimuli-sensitive response character)” in these pro-
teins. The ‘decision making’ silk hydrogels that can be programmed
to respond to physical, biochemical and mechanical cues will
enable temporal control over several functions including the
release of the encapsulated bioactive agents.
Consistent with several other natural and synthetic polymers
and as described herein, silk hydrogels support encapsulation of
cells and therapeutic molecules. They are porous to allow diffusion
of nutrients and wastes as well as to allow intercellular communi-
cation with secreted molecules. However, they lack many of the
signals of the native extracellular matrix (ECM). Included in these
signals are insoluble bioactive cues stemming from the proteins,
glycoproteins, and proteoglycans and soluble signals such as
cytokines and growth factors that are secreted by the ECM. Future
studies will need to carefully consider incorporation of biological
signals to improve tissue regeneration. We should gain guidance
from the increasing knowledge regarding the importance of gly-
cosaminoglycans and proteoglycans in tissue homeostasis and dis-
ease. Studies investigating blends of silk fibroins and other
polymers in the form of interpenetrating networks and semi-
interpenetrating networks and even as block-co-polymers begin
to move toward materials that better mimic the native ECM
[95,105,139,212]. These blends show improved mechanical charac-
teristics and in some cases include cell adhesion domains. Through
incorporation of key features of proteoglycans, the ‘information
rich’ silk hydrogels can better mimic the native ECM and will also
contain natural binding sites for cytokines and growth factors that
are secreted by the regenerating engineered tissue.
An interesting feature that has not yet been thoroughly
explored in case of the silk hydrogels is the multilayered hydrogels.
28 S. Kapoor, S.C. Kundu / Acta Biomaterialia 31 (2016) 17–32

Fig. 3. Applications of silk hydrogels: Silk fibroin based hydrogels are currently being explored for tissue engineering and controlled release of therapeutic molecules.

Fig. 4. Some future directions for silk hydrogels: (a) Silk hydrogels that undergo spontaneous gelation in physiological conditions at the site of injection will pave way for
their non-invasive clinical applications in tissue engineering and drug release, (b) Multilayered hydrogels consisting of separate silk gel layers with distinct properties will
enable ‘true’ compartmentalization of the matrix. Such compartmentalized multilayered hydrogel can be used for carrying different ‘payloads’ to achieve spatiotemporally
complex release profiles, (c) Integration of biomimetic signals on silk hydrogel surface will enable the ‘information rich’ hydrogels to be targeted to specified regions where
they can replicate the cellular microenvironment and facilitate signal dependent cell functions, (d) Multifunctional hydrogels whose properties are optimized for performing
different functions simultaneously (for instance, one that supports cell growth and release of growth factors) can play better role in regenerative medicine, (e) Stimuli-
sensitive silk hydrogels that respond to physiologically relevant environmental signals will be used as ‘Smart’ decision making biomaterials.
 S. Kapoor, S.C. Kundu / Acta Biomaterialia 31 (2016) 17–32
Instead of hydrogels with bulk properties, novel hydrogels that
contain multiple layers can be prepared from silk proteins in near
future. Layer by layer assembling techniques can be utilized for
this purpose. The properties of each layer can be tailored to provide
a hierarchical organization suitable for distinct functions. Such
hydrogels will help realizing true compartmentalization in bioma-
terials that is needed to achieve biologically relevant spatio-
temporal complexity. Different cell types or different molecules
can be encapsulated in distinct layers to enable complex release
patterns. In addition, gradients of the release molecules can be
achieved. Several multilayered hydrogel systems have been
described recently with natural and synthetic polymers [213–
215]. Recently, hydroxyapatite nanoparticle containing silk hydro-
gels with superior bone regenerating potential have been fabri-
cated [216]. The challenge in getting multilayered silk hydrogels
will be to find novel preparation methods that allow the genera-
tion of multiple layers. It is needed to make sure that the distinct
layers remain separate. Another challenge will be to better under-
stand the properties of boundaries and the interface between each
layer.
The utilization of silk proteins for making hydrogels for novel
applications in diagnostics including synthesis of microdevices,
microchannels and micropatterning for guided cell proliferation
and differentiation as well as for targeted release of molecule
remains to be explored. In addition to fabricating hydrogels from
alternative sources of silk proteins from non-mulberry silkworms
including A. mylitta [217], A. assamensies, A. pernyi, Samia recini
and spider silks. The efforts are being made to improve the quality
of the silk proteins by genetic engineering. As we progress towards
finding new techniques to observe as well as control microscopic
morphological features, it can be anticipated that silk-based hydro-
gels will enable the development of new therapeutic approaches
and help us to meet the demands in regenerative medicine.
Acknowledgements
This work is supported by start-up grant from University Grant
Commission, Government of India to SK. We appreciate Ms Sarani
Ghoshal for taking interest during the initial stages of this write up.
We are greatly indebted to Dr. Alyssa Panitch, Weldon School of
Biomedical Engineering of Purdue University for critical reading,
excellent scientific inputs and suggestions for the improvement
of the manuscript. SCK’s laboratory has financially been supported
by Indo Australia Biotechnology Fund, Department of Biotechnol-
ogy and its Bioinformatics facility, Indo-Russia Biotechnology Pro-
gramme, Department of Science and Technology, Indian Council of
Medical Research, Govt of India, and Indo US Science and Technol-
ogy Forum, New Delhi.
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