Advanced Drug Delivery Reviews 65 (2013) 457–470

Contents lists available at SciVerse ScienceDirect

Advanced Drug Delivery Reviews

journal homepage: www.elsevier.com/locate/addr

Silk fibroin biomaterials for tissue regenerations☆

Banani Kundu a, 1, Rangam Rajkhowa b, 1, Subhas C. Kundu a,⁎, Xungai Wang b, c,⁎⁎
a
Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur-721302, India
b
Australian Future Fibres Research and Innovation Centre, Deakin University, Geelong, Victoria3217, Australia
c
School of Textile Science and Engineering, Wuhan Textile University, Wuhan, China

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

Article history: Regeneration of tissues using cells, scaffolds and appropriate growth factors is a key approach in the treat-
Accepted 25 September 2012 ments of tissue or organ failure. Silk protein fibroin can be effectively used as a scaffolding material in
Available online 5 November 2012 these treatments. Silk fibers are obtained from diverse sources such as spiders, silkworms, scorpions, mites
and flies. Among them, silk of silkworms is a good source for the development of biomedical device. It pos-
Keywords:
sesses good biocompatibility, suitable mechanical properties and is produced in bulk in the textile sector.
Silk
Fibroin
The unique combination of elasticity and strength along with mammalian cell compatibility makes silk fibro-
Biomaterials in an attractive material for tissue engineering. The present article discusses the processing of silk fibroin into
Scaffolds different forms of biomaterials followed by their uses in regeneration of different tissues. Applications of silk
Tissue regeneration for engineering of bone, vascular, neural, skin, cartilage, ligaments, tendons, cardiac, ocular, and bladder tis-
sues are discussed. The advantages and limitations of silk systems as scaffolding materials in the context of
biocompatibility, biodegradability and tissue specific requirements are also critically reviewed.
© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
2. Silks of silkworms: source, chemistry and structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
3. Characteristics of silk fibroin as biomaterial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
3.1. Mechanical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
3.2. Biocompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
3.3. Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
3.4. Water based processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
3.5. Manipulation of silk properties through structural re-adjustments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
4. Morphological diversification of silk biomaterials for tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
4.1. Native silk structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
4.2. Regenerated silk morphologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
4.2.1. Films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
4.2.2. Electro-spun and wet-spun fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
4.2.3. Hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
4.2.4. 3-D porous scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
4.2.5. Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
5. Applications of silk fibroin biomaterials for tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
5.1. Vascular tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
5.2. Neural tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
5.3. Skin tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
5.4. Bone tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
5.5. Cartilage tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Bionics — biologically inspired smart materials”.
⁎ Corresponding author. Tel.:+91 3222 283764; fax: +91 3222 278433.
⁎⁎ Correspondence to: Australian Future Fibres Research and Innovation Centre, Deakin University, Geelong, Victoria3217, Australia. Tel.:+61 613 5227 2894; fax: +61 613 5227
2167.
E-mail addresses: kundu@hijli.iitkgp.ernet.in (S.C. Kundu), xwang@deakin.edu.au (X. Wang).
1
Authors contributed equally.

0169-409X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.addr.2012.09.043
458 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470

5.6. Ligament and tendon tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465

5.7. Cardiac tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
5.8. Ocular tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
5.9. Hepatic tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
5.10. Spinal cord tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
5.11. Inter-vertebral tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
5.12. Bladder tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
5.13. Tracheal tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
5.14. Eardrum tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
6. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466

1. Introduction This review article is focused on recent research based on silk fi-

The limited supply of donors and increasing morbidity have put
new demands on tissue engineering (TE) as a treatment of organ fail-
ures [1]. The TE approach involves regenerating tissue within suitable
scaffold with the goal of implanting the constructed tissue at the
target site. The regeneration of functional tissue requires a suitable
microenvironment that closely mimics the host site for desired
cellular responses [1]. Such an environment is typically provided by
3-D tissue engineering scaffold that acts as an architectural template
[2]. Apart from biocompatibility, which is the essential prerequisite
for any biomaterial, matching the degradation time with that of the
tissue regeneration is also a critical requirement for a cell scaffolding
material. Such a match can maintain the mechanical properties and
structural integrity of the engineered tissue in all stages of its regen-
eration process. In addition, degraded products of the biomaterial
should be safely metabolized and cleared from the host body.
Materials like polymers, metals and ceramics are widely used as
cell scaffolds for tissue engineering. Both synthetic and natural poly-
mers have been trialed, though each has its own limitations. While
the former allows easy processing and modifications, the later offers
better cyto- and bio-compatibility [3]. There is no universal biomate-
rial that meets the scaffolding requirements for all the tissues. Differ-
ent issue constructs require biomaterials with specific physical,
mechanical and degradation properties. Hence there is on-going
search for universal biomaterial for regeneration therapy.
Protein, being a component of natural tissues, is a rational choice
for applications in tissue engineering. Structural proteins such as
collagen, elastin, elastin-like-peptides, albumin and fibrin are used
as sutures, tissue scaffolds, haemostatic and drug delivery agents
[4]. Silk fibroin of silkworms is a commonly available natural biopoly-
mer with a long history of applications in the human body as sutures.
Currently silk sutures are used in lips, eyes, oral surgeries and in the
treatment of skin wounds [5]. Increasingly, silk fibroin is exploited
in other areas of biomedical science, as a result of new knowledge
of its processing and properties like mechanical strength, elasticity,
biocompatibility, and controllable biodegradability [5]. These proper-
ties of silk fibroin are particularly useful for tissue engineering.
Furthermore, recent studies evaluates silk as a part of flexible elec-
tronic devices for real-time physiological and functional recording
and optical systems for diagnosis and treatments [6,7]. Silk
possesses excellent (ca. 95%) optical transparency throughout the vis-
ible range with remarkable surface smoothness and aqueous process-
ing, all of which facilitates its application in optics and photonics
biosensor [7,8]. Such silk based systems are implantable and have
necessary functionality and sensitivity required for advanced applica-
tions. Several reviews are published on the fabrication, structure, and
the application of silk based biomaterials [2,5,9,10]. In view of grow-
ing applications of silk in new areas of tissue engineering and knowl-
edge on characteristics of silk constructs, a further and more detailed
review is now warranted.
broin in the field of tissue regeneration and evaluates its prospects
for further development in therapeutic related applications. The re-
view starts with a brief overview of silk protein. The silk protein fibro-
in structure and morphologies are included as these aspects are
highly relevant to the applications of silk biomaterials in tissue engi-
neering. The different silk based platforms are discussed, followed by
an account of their applications in tissue regeneration.
2. Silks of silkworms: source, chemistry and structure
Silk proteins are present in glands of silk producing arthropods
(such as silkworms, spiders, scorpions, mites and bees) and spun into
fibers during their metamorphosis. Silkworm's silk is an established
fiber extensively used in the textile industry. On the other hand, the
cannibalistic nature of spiders restricts the commercial production of
spider silk [5]. Additionally, the yield of fiber from a single silk cocoon
is 600–1500 m, compared to only ~137 m from the ampullate gland of
a spider and ~12 m from the spider web [11]. Spider silks are also het-
erogeneous in nature. Therefore, silk based biomaterials are commonly
prepared from silkworm silk. Of note is the silk produced by Bombyx
mori, a member of the Bombycidae family. B. mori silk is also known
as mulberry silk. Another silk producing family is Saturniidae and the
silk is known as non-mulberry silk.
Silk has several major advantages over other protein based bioma-
terials, which are derived from tissues of allogeneic or xenogeneic or-
igins. As such, the risk of infection is high for those materials.
Processing of such materials is also expensive due to the stringent
protein isolation and purification protocols. In contrast, silk is an
established textile fiber and nearly 1000 metric tons of silk are pro-
duced and processed annually. Silk fiber purification is routinely car-
ried out using a simple alkali or enzyme based degumming
procedure, which yields the starting material for sericin free silk
based biomaterials. It is also economically advantageous to use silk
for biomedical applications, because of available large scale process-
ing infrastructure of traditional silk textile industries.
Silk possesses large molecular weight (200–350 kDa or more) with
bulky repetitive modular hydrophobic domains, which are interrupted
by small hydrophilic groups [12]. The N and C termini of silk fibroin are
highly reserved [5]. Silk fibroin of B. mori is composed of a heavy (H),
and a light (L) chain linked together by a disulfide bond [13]. A
25 kDa glycoprotein, named P25, is also non-covalently linked to
these chains [14]. The hydrophobic domains of H chains contain
Gly-X (X being Ala, Ser, Thr, Val) repeats and can form anti-parallel
β-sheets. The L-chain is hydrophilic in nature and relatively elastic.
P25 protein is believed to play significant role in maintaining the integ-
rity of the complex [15,16]. H-fibroin, L-fibroin, and P25 are assembled
in the ratio of 6:6:1 in mulberry silk [17]. Non-mulberry silks lack light
(L) chain and P25 [2,16]. Instead, they contain heavy (H) chain
homo-dimers with a molecular weight of ~330 kDa formed by individ-
ual proteins (~160 kDa) [18]. Non-mulberry Saturniidae silks exhibit a
 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470 459

Fig. 1. Schematic representation of biomaterials fabricated from silk fibroin: (a) hydrogels; (b) lyophilized powder; (c) 3D porous scaffolds; (d) native silk mat; (e) silk microparticles.

higher Ala/Gly ratio and poly-alanine blocks, which form β-sheets [19]. and the presence of easy accessible chemical groups for functional
They also have higher ratios of basic/acid, polar/non-polar, bulky/ modifications. These advantages are elaborated in the following
non-bulky and hydrophilic/hydrophobic ratios of amino acids [20,21]. sections.
As a result of such variations, there are significant differences in the
mechanical properties, bioactivity and the degradation behavior 3.1. Mechanical properties
between mulberry and non-mulberry silks [22].
Apart from the primary organization, secondary structure and the Silk offers an attractive balance of modulus, breaking strength, and
hierarchical organization of silk fibroin determine many of its bioma- elongation, which contributes to its good toughness and ductility
terial properties. The hydrophobic domains of the silk polymeric (Table 1). Silk fibers are tougher than Kevlar, which is used as a
chains consisting of the repeated amino acid sequence which are as- bench mark in high performance fiber technology [25,32]. The
sembled into nano-crystals (β-sheet). The hydrophilic links between strength-to-density ratio of silk is up to ten times higher than that
these hydrophobic domains consist of bulky and polar side chains and of steel [33]. Spider silk fibers in particular have high extensibility
form the amorphous part of the secondary structure [23,24]. The and exhibit marked strain hardening behavior [34]. Strain hardening
chain conformation in amorphous blocks is random coil, which refers to increase in stress as a fiber is extended in the plastic region
gives elasticity to silk [25,26]. The critical factors that determine the beyond the yield point. Such behavior is particularly important for
mechanical properties of any particular silk are the precise control energy absorbing materials. Stress–strain curves of the wild silkworm
of size, number, distribution, orientation and spatial arrangement of silk fibers exhibit shape and strain hardening similar to that of spider
crystalline and non-crystalline domains at the nanometer scale or dragline silks [22,35]. Considering the good strength and tough-
[5,27]. Nano-crystals contribute to the outstanding mechanical prop- ness of silk fibers, it is no surprise that silk has been exploited to
erties of silk, despite of defects in the microstructure in the form of
vacuoles and micro-voids [23,28]. Apart from the secondary struc- Table 1
ture, a hierarchical supra molecular organization is also evident in Tensile properties of silk polymeric fibers.
silk fibers [24]. Spider and silkworm silks are composed of microfila- Source organisms Tensile strength Tensile modulus Breaking References
ment bundles (0.5–2 μm), each of which is made of nano-crystals (g/den) (g/den) strain (%)
and/or semi crystalline domains [29–31]. Despite some variation in
Bombyx mori 4.3–5.2 84–121 10.0–23.4 [22]
the primary organization and structural features at the nanometer Antheraea mylitta 2.5–4.5 66–70 26–39 [22]
scale between silk types, all silkworm silk fibers follow similar hierar- Philosamia cynthia ricini 1.9–3.5 29–31 28.0–24.0 [22]
chical structural arrangements. Coscinocera hercules 5 ± 1.2 87 ± 17 12.1 ± 5.1 [202]
Hyalophora euryalus 2.7 ± 0.9 59 ± 18 11.1 ± 5.8 [202]
Rothschildia hesperis 3.3 ± 0.8 71 ± 16 9.5 ± 4.4 [202]
3. Characteristics of silk fibroin as biomaterial Eupackardia calleta 2.8 ± 0.7 58 ± 18 11.8 ± 5.5 [202]
Rothschildia lebeau 3.1 ± 0.8 54 ± 14 15.5 ± 6.7 [202]
The major advantage of silk compared to other natural biopolymers Antheraea oculea 3.1 ± 0.8 57 ± 15 14.5 ± 6.6 [202]
is its excellent mechanical property. Other important advantages in- Hyalophora gloveri 2.8 ± 0.4 48 ± 13 19.3 ± 6.9 [202]
Copaxa multifenestrata 0.9 ± 0.2 39 ± 6 4.1 ± 2.7 [202]
clude good biocompatibility, water based processing, biodegradability
460 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470
develop scaffolds for load bearing tissue engineering. However, in
current design of silk based biomaterials, the wide choice of mechan-
ical properties available from using different silk types is not fully ex-
plored. A biomaterial implant fails either due to poor mechanical
properties needed for the application, or higher than optimum me-
chanical properties due to inappropriate stress concentration at the
implant tissue interface. Therefore, variations in mechanical proper-
ties of different type of silk provide good choice to match material
properties. In general, the wide choice of extensibility and elasticity
of silks, good strength and strain hardening from different varieties
of silk provide outstanding advantage to developing a range of silk
based biomaterials.
It is important to note that despite excellent mechanical proper-
ties of native silk fibers, most of the silk materials developed from
silk fibroin solution are weak and brittle. For example, dry tensile
strength of silk film is about 0.02 GPa and elongation at break is less
than 2% compared to native fibers that have a tensile strength of
about 0.5–0.6 GPa and elongation at break 10–40% [22,36]. Such
difference can be attributed to the lack of appropriate secondary
and hierarchical structure in the regenerated materials as compared
to the native fibers [37,38]. Recent studies show that there are scopes
to significantly improve the strength of regenerated silk products to
the level of native fibers or even higher through manipulation of
structure during regeneration [39–41]. Such investigations are still
at the proof of concept stage and relevant mostly to regenerated fi-
bers. More attempts are warranted to improve both strength and
elongation of regenerated silk materials. The prospects of application
of silk in a range of tissue engineering applications will improve if
mechanical properties can be tailored to the required level based on
application needs.
3.2. Biocompatibility
The long history of success of silk sutures has made silk a known
biocompatible material [31,32]. But, like any other non-autologous bio-
materials causing foreign body response, some adverse immunological
events associated with silk proteins cannot be ruled out, particularly
due to its non-mammalian origin. Some incidents of delayed hypersen-
sitivity of silk sutures in rare cases are suggested to be due to the pres-
ence of silk gum-like protein sericin [33,34]. Further studies employing
isolated silk sericin and sericin based biomaterials have provided no
clear evidence to suggest sericin as the source of adverse effects [35].
Detailed investigations are needed to specifically identify the source
of any cyto-toxic non fibroin elements in silk and develop appropriate
diagnostic method.
The immunogenicity and antigenicity of silk scaffolds are well tested.
Silk fibroin bio-conjugates during the treatment of musculo-skeletal dis-
ease have shown well tolerated response [42]. There are no signs of infec-
tion during the subcutaneous implantation of electro-spun fiber mats in
rats for up to 8 weeks, although some typical accumulation of phagocytes
and lymphocytes is observed. Additionally, there is only minimal inflam-
mation according to microscopy of hematoxylin and eosin stained tissues
[43]. Silk 3-D scaffolds when subcutaneously implanted in Lewis rats gen-
erate very mild immune response at the end of one year; overall expres-
sion levels for all the genes assigned to immune response including
TNF-α IFN-δ, IL-4, IL-6 and IL-13, become undetectable for most type of
silk sponges [44]. A pig model used in ligament tissue engineering
shows no evidence of malfunction after 24 weeks of in-vivo culture [45].
Overall, these studies give a wide spread acceptance that properly
degummed and sterilized silk products have good biocompatibility
and can be compared with other commonly used biomaterials, such
as poly (lactic acid) and collagen [42]. As a consequence of extensive
studies in recent years, some silk based materials are received regulato-
ry approval for use in expanded biomaterial devices for plastic and re-
constructive surgery. For example, ISO 10993 biocompatibility testing
under good laboratory practices (GLP) shows that silk based Seri Fascia
surgical mesh meets the biocompatibility requirements [46].
Despite the encouraging results, there still remain some questions
about long term safety of silk biomaterials in the human body. First,
silk sutures remain in the body only for a limited time until removed
depending on the wound healing period. As silk products for tissue
engineering are required to be in contact with tissues for a prolonged
time period, the long term responses of innate and adaptive immune
system based on location of implant site and type of construct using
appropriate in-vivo models warrant further investigation.
Second, there may be concerns on the immune reaction in response
to degraded products of silk biomaterials, depending on their size and
morphology [35]. It is recognized that one of the major causes of failure
of any biomaterial implant is the generation of particulate debris,
which may trigger the immune system. Report shows that fractions of
silk fibers are able to induce mild pro-inflammatory cytokine production
and increased phagocytosis [37]. Similarly the digestion of C-terminal of
A. pernyi silk with α-chimotrypsin results weak cellular attachment and
low growth, suggesting degradation reduces cyto-compatibility [47].
The degraded products of silk fibroin may also cause amyloidogenesis
as reported by Lundmark et al. [48]. Their observation suggests the po-
tentiality of B. mori solution to facilitate the accumulation of amyloid,
resulting in tissue degeneration. Hence, long term investigations on de-
graded products are necessary in order to fully alleviate any concerns
for the use of silk scaffolds in clinical applications.
3.3. Biodegradation
The silk biodegradation is studied based on mass loss, change in
morphology and analysis of degraded products in-vitro. Similarly,
degradation is tested in animal models by testing mechanical proper-
ties of silk after implantation for certain time and studying structural
integrity by histological examinations, fluorescent staining and
various biochemical assays (Table 2). As implanted construct, regen-
erated silk fibroin biomaterial degrades much faster than fibers. The
degradation rate is dependent upon the secondary structure of silk
resulting from preparation of regenerated silk materials [49].
The term biodegradability is often used to discuss the disintegration
of silk materials. According to the definition of Vert et al., biodegrad-
ability is the degradability of an implantable polymer by biological ele-
ments giving fragments, which can move away from the site through
fluid transfer but not necessarily from the body [50]. On the other
hand, bio-sorption is total elimination of the initial foreign material ei-
ther through filtration or metabolization of the degraded bio-products
[50]. Evidence of in-vivo degradability of silk is shown by Wang et al.
[44]. Their study reveals that, upon implantation in Lewis rats, water
based 3-D scaffolds were disintegrated in a few weeks and completely
disappeared after 1 year. Host immune system has significant impact
on the degradation of 3-D silk fibroin porous scaffolds and degradation
of silk sponge is shown to be mediated by macrophages, suggesting
that silk is not only biodegradable but also bio-resorbable [44].
In in-vitro models, protease XIV from Streptomyces griseus is the
most widely used model enzyme for silk [51–55], followed by
α-chymotrypsin from bovine pancreas [52,53,56]. The rate of enzy-
matic degradation further can be regulated by gamma-radiation
[57]. Degradation of the silk systems is shown to be mediated also
by cells in-vitro. The osteoblasts and osteoclast cells could erode
silk films via expression of metalloproteinases (MMPs) and integrin
[58]. Such results are encouraging because native extracellular ma-
trix is continuously remodeled in-vivo by synchronous proteolytic
degradation via MMPs and matrix regeneration [59]. The in-vitro
studies are useful to compare the biodegradability among different
silk materials and structures. However, the type and the concentra-
tion of enzymes at the site of the material implant may vary signif-
icantly. This may put limitations on predicting degradation of silk
compared to synthetic biomaterials that degrade hydrolytically
 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470 461

Fig. 2. Confocal laser micrographs showing the attachment and morphology of cells on silk fibroin biomaterials. The cells were stained with rhodamine for the actin filaments and
Hoechst for nucleus. (a) Hepato-carcinoma cells on the porous B. mori silk fibroin scaffold; (b) human osteosarcoma cell line (MG-63) on the surface of silk fibroin; (c) feline em-
bryo fibroblasts (AH 927) on the surface of silk fibroin hydrogel. All the cells revealed well stretched healthy morphology on silk biomaterial surfaces.

and have minimum site-to-site or patient-to-patient variations
[4].
The silk has clear advantages over other biomaterials in several
aspects of biodegradation. For synthetic biomaterials such as
polyglycolides and polylactides, which are approved by regulatory
authorities, degraded products are resorbed through metabolic
pathways but release of acidic by-products is an issue of concern.
There are no such issues associated with silk. In addition, those
synthetic materials may decrease mechanical properties very
early during degradation [4,60]. On the other hand, retaining
strength over a long time by many silk systems can be an advan-
tage for silk, particularly in tissue engineering, where slow degra-
dation and load bearing capacity are required. Despite such
advantages, deep understanding on degradation and clearing
mechanisms of silk requires further investigations, which would
encourage the development of silk as an important biodegradable
and bio-resorbable material.
3.4. Water based processing
The silk fibroin is water soluble in its α-helical and random coil
forms. Solubility can be maintained over days and even weeks
depending on the storage temperature, pH and concentration of silk
solution [61]. Hence, silk based systems can be prepared using water
based solution under mild manufacturing conditions such as room
temperature, neutral pH and without application of high shear force.
Such conditions are favorably exploited for loading sensitive drugs
into silk implants [62,63]. Mild processing conditions are also helpful
for photonic or electronic devices or biosensors, which may be incorpo-
rated within a silk based system or coated with silk for improved
bio-integration in-vivo. Conformational transition of α-helix and ran-
dom coil to highly stable β-sheets is required in silk products to
provide good resistance to dissolution, thermal and enzymatic degra-
dation. This can be achieved through water vapor annealing, mechani-
cal stretching and ultrasonic treatments, hence avoiding the use of
harmful chemicals. These processing advantages and good structural
stability make silk a promising polymeric system for bio-related
applications.
3.5. Manipulation of silk properties through structural re-adjustments
Silk structure can be adequately tuned during spinning or regener-
ation to obtain different secondary structures to manipulate material
properties. For example, forced extrusion of silk gland protein
through silkworm spinnerets attain appropriate altered fiber micro-
structure with significantly high fiber toughness [64]. Such options,
if optimized for other forms of silk based materials, may offer the ad-
vantage of matching their load bearing properties with that of the
462 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470
Table 2
Investigatios carried out on biodegradation of silk materials.
Material forms In-vitro/In-vivo
B. mori/A. pernyi native fiber/surgical mesh • In-vitro using protease XIV/collagenase• Mass loss, microscopy, secondary structure, mechanical
IA,/α-chymotrypsin
• Implantation in rats
B.mori 3-D sponge • In-vitro using protease XIV
• Intramuscular and subcutaneous
implantation in rats
3-D sponge with different pore size and • Intramuscular and subcutaneous
β-sheet contents implantation in Nude and Lewis rats
Knitted silk mesh and silk sponge composite • Anterior cruciate ligament of pig
B. mori cast films and thin crystallized films In-vitro using protease XIV, XXI/ trypsin/ • Mass loss, microscopy and secondary structure
α-chymotrypsin/ collagenase
B. mori water annealed patterned silk film • Bone cells mediated degradation
B. mori electro-spun mat and wet spun fibers • In-vitro using protease XIV, K/actinomyces • Mass loss and microscopy
• Subcutaneous and intra-muscle
implantation in rats
B. mori silk solution • Actinase
B. mori silk particles • Proteases
targeted tissues. Water annealing is also used to induce insolubility in
silk products [65,66]. Water annealed films are more flexible and de-
graded faster than methanol treated films [65]. Excellent opportuni-
ties are available to use the proticionic liquid system to manipulate
the structure and properties of regenerated silk materials [67]. More-
over, process induced variations in structure and surface topography
may affect properties such as biodegradation [49], cell interaction
[68] and drug release kinetics [69]. For example, a temperature-
controlled water vapor annealing (TCWVA) technique is used to
program the crystallinity in order to tune thermal, mechanical and
biodegradation properties of the silk films [49]. Similarly, slowing
down the proteolytic degradation by structural adjustments can reduce
the release kinetics of drugs from a silk system [57]. Although windows
of opportunities are demonstrated in all these cases, more studies are
needed to understand the structure-property relationships for ade-
quate control of material properties. Such control will be a key to the
success of silk as a natural biopolymer for tissue regeneration.
4. Morphological diversification of silk biomaterials for
tissue regeneration
Degumming (removal of glue protein sericin) is the first step in silk
fiber processing. To produce various other material formats, degummed
silk fiber is dissolved to obtain silk solution. In some cases, where it is dif-
ficult to dissolve fibers, fibroin can be extracted directly from glands of
silkworms using an appropriate buffer solution [70]. Different solid
forms are then prepared from silk solution by liquid solid phase transfer.
Schematics of different silk based biomaterials are represented in Fig. 1.
Both native fibers and materials regenerated from silk solution are
used for tissue engineering.
4.1. Native silk structures
Degummed silk fibers can be used to form various twisted structures
including rope, cable, braided and textured yarns for tissue regeneration
[38]. In addition, cocoons are also used to construct non-woven struc-
tures by partially dissolving them to use as a cell supporting template,
where the arrangement of filaments in the cocoon is preserved to main-
tain the porous structure of the mat [37,71]. An alternate way of using
Type of study Ref.
[46,52,203]
properties of substrate
• Particle size, amino acids and molecular weight of
degraded products
• Tissue ingrowth and biomechanical analysis tissues
• Mass loss [203,204]
• Degradation rate vs. cell metabolism and bone
regeneration from MSC
• Structural integrity of sponge and invasion by giant [44]
cell and macrophage based on histology and gene
expression of tissue samples by real time RT-PCR
• Macroscopic view, tensile strength, μCT scan and [45]
histology
[43,53–56,205–209]
of substrate
• Mass spectroscopy to determine molecular weight
of degradation products
• Enzyme degradation vs. drug release
• Microscopy and essays on metalloproteinases [58]
production by cells
[43,210,211]
• Fluorescein assisted microscopy
• Molecular weight and amino acid analysis [212]
• Particle size, morphological imaging, mass loss, [213,214]
secondary structure, amino acid analysis
silk filaments directly in tissue engineering is making a knitted silk struc-
ture to reinforce 3-D porous tissue engineering scaffolds. Such reinforce-
ment improves the mechanical properties of scaffolds for applications in
load bearing tissue engineering, such as ligament [45,72].
4.2. Regenerated silk morphologies
To prepare silk solution for regenerating different silk formats, con-
centrated solutions of chaotropic salts such as LiBr, CaCl2/ethanol/
water, LiSCN [73–75] or ionic liquids [67,76] are commonly used. These
solutions usually fail to dissolve non-mulberry silk fibers [77,78]. At-
tempts using Ca(NO3)2 at 105 °C and LiSCN at 40 °C–55 °C to dissolve
Chinese oak tasar silk fiber are reported [79,80]. However as the total dis-
solution in such cases still remains a challenge, gland proteins are used
mostly for further processing.
4.2.1. Films
Silk fibroin films can be produced by casting the aqueous [81],
acidic [39,81] and ionic [82] silk solution. Fabrication of silk films by
spin coating and Langmuir-Blodgett (LB) process is also reported
[82,83]. Additionally, manual or spin assisted layer by layer deposi-
tion techniques have been used to produce very thin films [84]. As
stability of such cast films is low, techniques like controlled drying
[85], water annealing [49], stretching [86], and alcohol immersion
are used to improve β-sheet crystallinity. It is often necessary to con-
trol surface properties of silk films for guided and enhanced cell
growth or to change the optical properties. Lithography and advanced
printing systems are employed to achieve such features [8,87].
4.2.2. Electro-spun and wet-spun fibers
The large surface area and porous structure of electro-spun silk
nano-fiber mats are useful for cell seeding [86]. 3-D constructs of
nano-fibers are used as blood vessel grafts and nerve guides [88,89].
Wet spinning [39] or micro-fluidic solution spinning [90] are also
employed in making regenerated silk fibers. Wet-spun fibers are typ-
ically in the micrometer scale in fiber diameter, and can be produced
on a much larger scale than nano-fibers. Advantages of such
regenerated fibers over the native silk fibers include the ability to
 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470
tune fiber morphology and properties based on application, and
incorporation of bio-molecules while regenerating from silk solution.
4.2.3. Hydrogels
Silk hydrogels are formed through sol–gel transition of aqueous
silk fibroin solution in the presence of acids, dehydrating agents,
ions, sonication or lyophilization [44,91–93]. Sol–gel transition can
be accelerated by increasing the protein concentration, temperature,
and addition of Ca 2+[94]. Silk hydrogels can be useful for injectable
or non-injectable delivery systems. Mechanical properties of silk
hydrogels are found to be suitable for preparing scaffolds for load
bearing tissue engineering such as cartilage regeneration [95].
4.2.4. 3-D porous scaffolds
Porous 3-D sponges are ideal structures for tissue engineering
scaffolds as they closely mimic the in-vivo physiological micro-
environment. Silk scaffolds are prepared by freeze drying, porogen
leaching and solid free form fabrication techniques [96–98]. The
freeze dried sponges possess pore sizes below 100 μm and the pore
sizes can be controlled by adjusting the freezing temperature, pH of
the solution and amount of organic solvents [97]. Repeated freezing
and thawing processes can increase pore sizes from 60 to 250 μm
[99]. A better control over pore structure can be obtained from
solvent casting/particle leaching or gas-foaming methods [98]. Due
to good control over porosity and pore sizes, porogen leached 3-D
silk scaffolds are commonly used in tissue engineering applications,
predominantly bone and cartilage [100].
To obtain good mechanical and biological outcomes, composite
silk 3-D scaffolds are prepared by incorporating inorganic [101] or or-
ganic [102] fillers. Usually fillers are incorporated during scaffold fab-
rication process to ensure their homogeneous distribution. However,
particle addition after fabrication is also reported [101]. The challenge
in composite design is the compatibility between the components.
Poor compatibility between components results in inhomogeneous
mixtures, phase separation and adverse tissue reactions [103]. To en-
sure good compatibility, silk–silk composite scaffolds are fabricated
by incorporating milled silk particles in porous silk sponge, resulting
in significant improvement in compressive modulus from less than
50 kPa to about 2.2 MPa [104]. Further modifications to scaffolds in-
clude reinforcement with fine silk fibers to gain further improvement
in modulus to about 13 MPa [105]. Such mechanical properties may
be sufficient for regenerating cancellous bone, but still way short in
meeting the practical requirements of load bearing bone tissue engi-
neering. To obtain implantable tissue construct without using metal
support, further development is still required in design of scaffolds
for bone regeneration. Silk composites reinforced with knitted silk
mesh are used as ligament scaffolds. It is revealed that after
Table 3
The limitations of presently available commercial products or approaches.
Targeted organ or General practice
approaches
Autologous grafts
Homograft
Conventional prostheses of polytetrafluoroethylene (ePTFE)
and polyethylene terephthalate
Peripheral nerve grafts Autologous nerve grafts
Neurolac®, CultiGuide®, SaluBridge®, Neurotube®,
Surgisis®,
NeuroMatrix®–Neuroflex® NeuraGen® Revolnerv®
Cornea Synthetic keratoprosthesis
Skin Allograft
Xenografts
Ligament Autograft
Allografts and xenografts
Heart Heart transplantation left ventricular assist devices
463
24 weeks post implantation period, there is uniform distribution of
cells throughout the construct [72]. The findings suggest the suitabil-
ity of silk composites for applications where mechanical properties
are important.
4.2.5. Particles
Silk micro and nano-particles are produced from silk solution by
freeze drying and grinding [106], spray drying [107], jet breaking
[108], self-assembly [109,110] and freeze-thawing [111]. Milling silk
fiber is an alternate approach to make silk particles directly from
fibers without using any chemicals [104]. While milled particles are
used for reinforcing scaffolds to improve mechanical properties and
cellular outcomes, regenerated silk particles are mostly used in drug
carrier applications [104,112–115]. Thus it will be of interest to see
if the particles can play the dual role of improving mechanical proper-
ties of scaffolds and at the same time act as a carrier of growth factors
for rapid tissue regeneration. The challenge here is to make very fine
and uniform silk particles, which is not easy with the milling
approach.
5. Applications of silk fibroin biomaterials for tissue regeneration
Substitution of a human body part with a biomaterial requires good
communication between the host and implanted system for a success-
ful outcome. The limitations of existing implants are summarized in
Table 3. To overcome such limitations, silk based tissue engineering
systems are trialed, which are included in the following sections (cellu-
lar responses on silk biomaterials are represent in Fig. 2.
5.1. Vascular tissue regeneration
Silk based regenerated vascular tissues are clinically used as flow
diverting devices and stents [116,117]. In the case of a study related
to flow-devices, two out of the three patients show promising
outcome, suggesting silk as an attractive option to treat fragile
blood-blister like aneurysms. Silk stents are also employed in the re-
construction of an intra-cranial aneurysm artery. There has been a
successful attempt to fabricate a tubular ~ 3 mm blood vessel from
silk with a thickness of 0.15 mm having an average tensile strength
of 2.42 MPa [118]. The burst strength of silk tubular vessels is found
to be 811 mm Hg compared to 1800 mm Hg of the gold standard sa-
phenous veins [119,120]. The implantation of vascular graft of silk fi-
broin composites of B. mori and transgenic silkworm into rat
abdominal aorta results excellent patency (ca. 85%) after a year
[121]. Composites of silk fibroin and human-like-collagen or
double-raschel knitted silk-poly(ethylene glycol diglycoldiglycidyl
ether) are successfully used to develop vascular constructs [122].
Limitations References
Low quality, vasospasam (spasm of blood vessels) , restricted [215–217]
length
Fast degeneration
Grafts of >5 mm in diameter suffer from thrombosis
Limited graft availability [218–221]
Secondary deformities Dissimilarity in tissue organization and size
restricted to short defects (b3 cm)repair of the small caliber nerves
Limited short-term clinical studies and patient follow-up available
till date
High host rejection rate [222,223]
Inadequate availability [224]
Limited permanent revascularization
Compromises normal healthy tissue and prolonged surgical time [225,226]
Risk of disease transmission and immune rejection
Scarcity of donors [227]
High cost
464 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470
While the former composition is able to provide good tensile strength
to the construct, the latter is able to avoid early thrombosis. An
aqueous gel spinning process is also employed to design a blood
vessel like tubular structure [123].
Critical requirements for designing blood vessels include survival
under the changes in blood pressure, ability to sustain cyclic loading,
compatibility with the adjacent host vessels, and anti-thrombotic lin-
ing [124]. Silk fibroin possesses an anti-thrombotic surface with good
resistance to high shear stress and blood flow pressure [86,125].
Moreover, silk fibers also support the intercellular contact in endo-
thelial cells [126]. The challenge to include a selection of suitable
cell sources still remains. Primary human endothelial cells and endo-
thelial cell lines (HPMEC-ST1.6R and ISO-HAS-1) seeded in silk fiber
are unable to fill the gaps between two adjacent silk fibers [127].
The optimization of co-culture of endothelial cells and smooth muscle
cells to get a well-organized endothelium [89], sulfation of silk for
better anti-coagulant activity [128] and coating of matrigel to
enhance endothelium coverage [89] are suggestive positive develop-
ment towards native vascular tissue construction under current
situation.
5.2. Neural tissue regeneration
The human nervous system is broadly classified into (a) the cen-
tral nervous system (CNS) and (b) the peripheral nervous system
(PNS). The PNS is capable of self-healing minor injuries, while large
damage needs to be treated surgically with nerve grafts harvested
elsewhere in the body. Therefore, in such treatments, tissue engineer-
ing is highly relevant and the compatibility of scaffolding material
with neuro-progenitor cells assumes significance. Silk fibroin
supports the viability of dorsal root ganglia and schwann cells
without affecting their normal phenotype or functionality [129].
Composites of silk fibroin with chitosan or poly(l-lactic acid-co-
caprolactone) are able to bridge a gap of a 10 mm long sciatic nerve
defects in rats [130–132]. Furthermore, an assembly of B. mori silk
fibroin and Spider X® fiber (a spider silk like fiber) is effectively
able to bridge up to 13 mm nerve gap within 12 weeks [133]. The
micro-architecture of scaffolds play important role in the regenera-
tion of nerves. Improved length and axonal outgrowth of chicken em-
bryo dorsal root ganglion sensory neurons and spiral cord motor
neurons are observed on aligned silk nano-fibers, compared to slow
and random orientation of axonal growth on non-aligend fibers
[134]. The orientation of pores and addition of neurotrophic factors
for enhanced neuronal growth are being presently investigated to en-
hance the outcome of silk based nerve grafts.
5.3. Skin tissue regeneration
The skin is the biggest organ in human and acts as barrier to the
infectious organisms. It has limited self-healing capability. Large dam-
age to the skin causes loss of skin integrity, which may lead to death.
The adult human skin consists of two major layers: the epidermis (a
keratinized layer) and the dermis (collagen-rich layer). Appendages
like hair and hormonal glands are raised from the epidermis but root-
ed deeply within the dermis. This complex structure makes the engi-
neering of skin a challenging task. Silk fibroin supports well the
human keratinocytes and fibroblasts [86]. However the complex
structure of native tissue requires a composite scaffolding material.
Bio-mimicking approaches with other natural extra-cellular materials
are proposed. Layering silk fibroin with collagen-I enhances the at-
tachment and dispersion of keratinocytes, while fibronectin coating
supports both keratinocytes and fibroblasts cells adhesion and disper-
sion within the matrix [135]. Structural integrity of silk matrices in-
duced by different treatments also influences greatly the cell
adherence. Both human keratinocytes and fibroblasts prefer water
vapor-treated silk nano-fibrous mats over methanol-treated ones,
suggesting that selecting appropriate processing route is also impor-
tant for silk based biomaterial designing [135]. Other silk composites
successfully used are nano-fibrous silk-chitin [136], silk-collagen
[137] and intermolecular cross-linked recombinant human-like colla-
gen (RHLC) with fibroin [138]. Additionally, silk fibroin-alginate
blended scaffolds sufficiently favor re-epithelization in a full-
thickness rat wound model [139]. These findings suggest that blend-
ed silk fibroin may have better prospect than pure silk fibroin for skin
tissue regeneration.
5.4. Bone tissue regeneration
Bone is a specialized connective tissue composed of calcified ex-
tracellular matrix, which has collagen type I and hydroxyapatite as
the major components [140]. Thus the scaffolding material for bone
tissues must ensure matrix toughness and matrix deposition. In this
context, silk fibroin is a rational choice for its high toughness and me-
chanical strength along with good bio-compatibility. Silk fibroin
based bone tissue engineering is one of the most extensively studied
tissue engineering approaches to date [10]. Porous fibroin scaffold
based bone constructs are able to stimulate advanced development
of bone tissues within 5 weeks, when engineered within a bioreactor
[100]. Silk fibroin scaffolds also promote human mesenchymal stem
cell based healing of femoral defects in nude mice [141]. Furthermore,
the loading of human mesenchymal stem cells within silk fibroin-
polyethylene oxide nano-fibrous composite scaffold with bone
morphogenic protein-2 led to regeneration of bone like tissue [142].
Incorporation of hydroxyapatite nano-particles into silk matrix im-
proves bone regeneration in animals [143,144]. The incorporation of
n-Hap within the fibroin sheet and subsequent culture of rat bone
marrow mesenchymal stem cell (BM-MSCs) demonstrates the cell
supportive nature of embedded Hap crystals and successful osteogen-
ic differentiation of BMMSCs [145]. These composite scaffolds
completely heal the segmental bone defect in Sprague–Dawley rats
after 12 weeks post implantation [143]. Silk fibroin also guides
full-thickness growth of calvarial defect (8 mm-diameters) in rats
within 12 week [100]. Similar results are also obtained when silk hy-
drogel [92,146] or non-woven mats [147] are used. The strategies to
improve bone regeneration using silk-silk composites are already
discussed in the Section 4.2.4.
The vascularization of in-vitro tissue models is always a problem.
The pre-incubation of silk matrices with cells before implantation in
animals increases the vascularization in neo-implant [148]. Under
such condition, the rate of vascularization is directly proportional to
in-vitro incubation time. Co-culture of osteoblasts with endothelial
cells on silk scaffolds results in the formation of micro-capillary
[149] and pre-vascular structures [150]. Furthermore, upon implanta-
tion, these pre-mature micro-capillaries survive the host defense
system and become functional micro-capillaries [151]. Further inves-
tigations on scaffold architectures and pore distribution are needed to
obtain the completely vascularized 3-D bone tissue.
5.5. Cartilage tissue regeneration
Cartilage is a non-vascular, non-innervated connective tissue. The
3-D porous fibroin scaffolds [152–155], electro-spun silk fibers treat-
ed with micro-wave induced argon plasma [156], silk fibroin blended
with chitosan [157] or genipin cross-linked chitosan-fibroin porous
sponges [158] provide adequate support to chondrocytes. Non-
mulberry gland silk fibroin of A. mylitta [159] also acts as promising
material for cartilage development. Insulin-like growth factor 1
(IGF-I) is a regulatory molecule in chondrogenesis [160], thus can
be incorporated within scaffolds for better chondrogenic outcomes
[160]. Bioreactors provide mechanical stimulation and maturation of
cartilaginous constructs [161]. Therefore, the hydrodynamic factors
are important in chondrogenic outcome. Other factors to be taken
 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470
into serious consideration for regenerating the cartilaginous tissues
are cell sources [162], scaffold architectures, pore sizes and pore
distribution.
5.6. Ligament and tendon tissue regeneration
The engineering of ligaments and tendons requires scaffolding
material with excellent combination of mechanical strength, elastici-
ty, toughness and structural integrity. The first successful silk based
engineering of anterior cruciate ligament (ACL) employs twisted scaf-
fold, the mechanical property of which is comparable to human ACL
[38]. Synergistic incorporation of silk fibers within the collagen
matrix [163], coating of silk fibroin on electro-spun poly-lactic-
co-glycolic acid (PLGA) nano-fibers [164], loading basic fibroblast
growth factor (bFGF) and transforming growth factor-β (TGF-β) in
optimal regimen within the scaffold stimulates the biochemical and
mechanical pathways for ligament tissue regeneration [165].
Further approach to design scaffold-free ligament analogs in-
volves incorporation of porcine bone marrow MSCs on laminin coated
silk sutures as anchor points [166]. As a result, the cells within the
construct are self-organized into cohesive rod-like tissues, which
mechanically and histologically resemble the native ligament. Silk fi-
broin scaffold of A. pernyi is also able to repair defects in Achilles ten-
don of an adult New Zealand white rabbit [167]. The recovery of
Achilles tendon in the rabbit shows the feasibility of using
non-mulberry silks along with mulberry silk as suitable tendon
scaffolds.
5.7. Cardiac tissue regeneration
The loss of cardiomyocytes after injury reduces cardiac function,
which further leads to morbidity and mortality. A possible treatment
is engineering an artificial heart or cardiac patch in-vitro followed by
implantation. Chitosan or hyaluronic acid (HA) loaded silk fibroin
seeded with rat mesenchymal stem cells is able to generate cardiac
patches [168]. Silk protein fibroin 3-D scaffolds of A. mylitta also
show good outcome without the employment of other extra cellular
matrix materials, producing beating patches of rat cardio-myocytes
in-vitro [169]. Critical issues still to be resolved are suitable silk or
silk based composite biomaterials supporting the mechanical
strength of the heart valves, formation of elastin by employing
co-culture system with fibroblasts cells, employment of human se-
rums or platelet lysates during in-vitro culture to minimize the tissue
engineered graft rejection.
5.8. Ocular tissue regeneration
Allograft approach of corneal tissue regeneration suffers from
bio-burden [170], while the use of silk fibroin results in a much better
outcome. Optical transparency of silk fibroin films and stability of
aqueous protein solution at neutral pH are the key characteristics
that favor silk for functional bio-photonic applications over other
synthetic or inorganic materials [6]. Silk films are stacked into a 3-D
porous structure mimicking closely the heli-coidal organization of
cornea in-vivo. When these 3-D structures are cultured with human
and rabbit corneal fibroblasts, cells reveal natural corneal keratocyte
morphology [171]. Silk fibroin implants with embedded rabbit cornea
epithelial cells become translucent within 4 weeks and results in the
formation of new limbus and blood vessels within 8 weeks
post-implantation. Complete regeneration of rabbit cornea occurs
within 16 weeks leaving behind a few opaque pieces of degraded
scaffolds [172]. In addition, the affinity of corneal cells towards a pat-
terned surface [173] and ease in surface patterning in silk films
(discussed in detail in another review elsewhere [174]), open up fur-
ther scopes for silk-biomaterials in ocular regenerative medicine.
Coating of fibroin with collagen IV, fibronectin, and chondroitin
465
sulfate–laminin mixtures enhances the performance of fibroin [175].
Successful pre-clinical reconstruction of human limbal epithelium
on silk membranes is also reported [176].
5.9. Hepatic tissue regeneration
Liver plays crucial role in the metabolism of carbohydrates, pro-
teins, lipids and vitamins. The chief cellular component of hepatic tis-
sue is hepatocytes, which is employed in-vitro to reconstruct the 3-D
tissue or liver patches. The silk composites for hepatic tissue engi-
neering includes functionalized silk with lactose and cyanuric acid
[177], silk fibroin blended films with collagen [178], recombinant
human-like collagen [179], silk fibroin-collagen-heparin scaffolds
[180], and silk micro-particle embedded PLA scaffolds [181]. Blending
of silk fibroin with collagen causes mild inflammation in the omen-
tum of rats [182]. Furthermore, non-mulberry A. assama fibroin
based multi-scale scaffolds provide adequate mechanical support
and help cellular response [183]. Functionalization of A. assama fibro-
in with sulfone results enhanced blood-compatibility [184]. However,
as hepatocytes carry a degree of structural organization, it forms large
cellular aggregates in long term in-vitro culture. These complex cellu-
lar aggregates make it difficult for nutrient diffusion and thus require
further investigation in designing silk scaffold for complete tissue
regeneration.
5.10. Spinal cord tissue regeneration
The grafting of olfactory ensheathing cells (OECs) is one of the
most commonly employed approaches to treat spinal cord injuries.
Silk based spinal cord regeneration is presently at its neo-natal
stage. The culture of OECs on nano-fibrous silk fibroin reveals pros-
pects of silk biomaterials in this area [185]. The diameter of the
nano-fibers possesses regulatory effects on growth of OECs [186];
smaller diameters result in better cellular responses than the larger
fibers.
5.11. Inter-vertebral tissue regeneration
The treatment of degenerative disk disease involves repair of an-
nulus fibrosus, which is one of the major components of inter-
vertebral disk. Porous scaffolds of silk fibroin allow good growth of
bovine annulus fibrosus cells up to 8 weeks in-vitro [187]. The growth
of bovine annulus fibrosus cells on silk fibroin is greatly influenced by
culture condition and average pore sizes of the scaffolding material
(≥600 μm) [188]. However, further investigations are required to
completely mimic the high strength, elastic and tough morphology
of naive inter-vertebral tissues.
5.12. Bladder tissue regeneration
In the treatment of stress urinary incontinence, bladder shaped
scaffolds with autologous urothelial and smooth muscle cells are re-
quired for successful reconstruction of the bladder [189]. Films of
silk fibroin provide good support to transitional epithelial cells of uri-
nary bladders of New Zealand rabbits [190]. Silk films in rabbits result
in the successful repair of short-length defects of 1.5 cm [191]. Fur-
ther animal studies include mice by Cannon et al., [192] and murine
by Mauney et al. [193]. The silk based sling engineered with bone
marrow mesenchymal stem cells exhibits good control on the
leak-point pressure comparable to negative control [194]. This
suggests a hopeful treatment for stress urinary incontinence.
5.13. Tracheal tissue regeneration
The incidence of pre-mature newborns developing tracheal steno-
sis is on the rise as a result of prolonged incubation. In silk based
466 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470
reconstruction of 12 tracheal defects in rabbits, successful generation
of full-thickness fibroblasts layer of 240–302 μm is observed, without
foreign-body granuloma and macro-phagocyte infiltration [195].
Blending of silk fibroin with chitosan enhances perichondrium
wrapping in nude mice, suggesting the suitability of silk based de-
vices in generation of epithelial lining for tracheal transplants [196].
5.14. Eardrum tissue regeneration
Eardrum (tympanic membrane) perforation results pain, infection
and even loss of hearing. Surgical treatment to restore chronic
perforations is myringoplasty, where autologous grafts, allografts,
and synthetic graft materials are commonly used [197]. Recent at-
tempts with silk fibroin membrane have resulted good adhesion
and growth kinetics of human tympanic membrane keratinocytes
[198–200]. Silk membranes provide better healing compared to
conventional paper patch [201]. These findings suggest suitability to
engineer silk based eardrum patches.
6. Future prospects
Tissue regeneration for therapeutics is very critical and target
specific. To complement the functionality of living systems, the
constructed tissue must successfully interact with the body's immune
system. Silk based designs allow easy control on matrix morphology,
degradation rate and conformal adhesion to underlying tissues with
low immune-toxicity and good biocompatibility. Recent advance-
ments in understanding silk structure and processing open up new
opportunities in the use of various forms of silk in tissue regeneration.
Silk systems will be particularly useful for applications where slow
biodegradation and good mechanical properties are critically re-
quired, such as for bone, ligament and muco-skeletal tissues. Success-
ful applications of silk based materials in tissue engineering depends
on further understanding of the long term biocompatibility, biode-
gradability and its degraded products, along with the ability to tune
silk morphologies for tissue specific requirements. Hybrid materials,
incorporating 100% silk in different matrix morphologies show
promising results in this regard.
Acknowledgments
The work is financially supported by the Department of Biotech-
nology and its Bioinformatics Facility, Department of Science and
Technology (SCK), and Council of Scientific Industrial Research (for
awarding Senior Research Fellowship to B. Kundu), Government of
India, New Delhi. R. Rajkhowa and X. Wang acknowledge the support
from the Australian Research Council for their silk related work. The
authors are thankful to Dr. Tuli Dey, Sunita Nayak and Deboki Naskar
for providing the photograph of bone cells on silk.
References
[1] R. Langer, J. Vacanti, Tissue engineering, Science 260 (1993) 920–926.
[2] B. Kundu, S.C. Kundu, Osteogenesis of human stem cells in silk biomaterial for
regenerative therapy, Prog. Polym. Sci. 35 (2010) 1116–1127.
[3] B.L. Seal, T.C. Otero, A. Panitch, Polymeric biomaterials for tissue and organ
regeneration, Mater. Sci. Eng. R 34 (2001) 147–230.
[4] L.S. Nair, C.T. Laurencin, Biodegradable polymers as biomaterials, Prog. Polym.
Sci. 32 (2007) 762–798.
[5] F.G. Omenetto, D.L. Kaplan, New opportunities for an ancient material, Science
329 (2010) 528–531.
[6] F.G. Omenetto, D.L. Kaplan, A new route for silk, Nat. Photonics 2 (2008)
641–643.
[7] M.K. Hota, M.K. Bera, B. Kundu, S.C. Kundu, C.K. Maiti, A natural silk fibroin
protein-based transparent bio-memristor, Adv. Funct. Mater. 22 (2012) 4493–4499.
[8] H. Tao, D.L. Kaplan, F.G. Omenetto, Silk Materials — a road to sustainable high
technology, Adv. Mater. 24 (2012) 2824–2837.
[9] G.H. Altman, F. Diaz, C. Jakuba, T. Calabro, R.L. Horan, J. Chen, H. Lu, J. Richmond,
D.L. Kaplan, Silk-based biomaterials, Biomaterials 24 (2003) 401–416.
[10] N. Kasoju, U. Bora, Silk fibroin in tissue engineering, Adv. Healthc. Mater. 1
(2012) 393–412.
[11] R. Lewis, Unraveling the weave of spider silk, Bioscience 46 (1996) 636–638.
[12] N.A. Ayoub, J.E. Garb, R.M. Tinghitella, M.A. Collin, C.Y. Hayashi, Blueprint for a
high-performance biomaterial: Full-length spider dragline silk genes, PLoS One
2 (2007) e514.
[13] K. Shimura, A. Kikuchi, K. Ohtomo, Y. Katagata, A. Hyodo, Studies on silk fibroin
of bombyx mori. I. Fractionation of fibroin prepared from the posterior silk
gland, J. Biochem. 80 (1976) 693–702.
[14] K. Tanaka, S. Inoue, S. Mizuno, Hydrophobic interaction of P25, containing
Asn-linked oligosaccharide chains, with the H-L complex of silk fibroin
produced by Bombyx mori, Insect Biochem. Mol. Biol. 29 (1999) 269–276.
[15] C.-Z. Zhou, F. Confalonieri, M. Jacquet, R. Perasso, Z.-G. Li, J. Janin, Silk fibroin:
structural implications of a remarkable amino acid sequence, Proteins Struct.
Funct. Bioinf. 44 (2001) 119–122.
[16] F. Sehnal, M. Žurovec, Construction of silk fiber core in lepidoptera,
Biomacromolecules 5 (2004) 666–674.
[17] S. Inoue, K. Tanaka, F. Arisaka, S. Kimura, K. Ohtomo, S. Mizuno, Silk fibroin of
Bombyx mori is secreted, assembling a high molecular mass elementary unit
consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio, J. Biol. Chem.
275 (2000) 40517–40528.
[18] S.-i. Inoue, H. Tsuda, T. Tanaka, M. Kobayashi, Y. Magoshi, J. Magoshi, Nanostruc-
ture of natural fibrous protein: In vitro nanofabric formation of Samia cynthia
ricini wild silk fibroin by self-assembling, Nano Lett. 3 (2003) 1329–1332.
[19] F. Lucas, J.T.B. Shaw, S.G. Smith, Comparative studies of fibroins: I. The amino
acid composition of various fibroins and its significance in relation to their
crystal structure and taxonomy, J. Mol. Biol. 2 (1960) 339–349.
[20] G. Freddi, Y. Gotoh, T. Mori, I. Tsutsui, M. Tsukada, Chemical structure and
physical properties of Antheraea assama silk, J. Appl. Polym. Sci. 52 (1994)
775–781.
[21] K. Sen, M.K. Babu, Studies on Indian silk. I. Macrocharacterization and analysis of
amino acid composition, J. Appl. Polym. Sci. 92 (2004) 1080–1097.
[22] R. Rajkhowa, V.B. Gupta, V.K. Kothari, Tensile stress–strain and recovery behav-
ior of Indian silk fibers and their structural dependence, J. Appl. Polym. Sci. 77
(2000) 2418–2429.
[23] F. Vollrath, D. Porter, Spider silk as a model biomaterial, Appl. Phys. A: Mater. Sci.
Process. 82 (2006) 205–212.
[24] T. Lefèvre, M.-E. Rousseau, M. Pézolet, Protein secondary structure and orienta-
tion in silk as revealed by raman spectromicroscopy, Biophys. J. 92 (2007)
2885–2895.
[25] F. Vollrath, D.P. Knight, Liquid crystalline spinning of spider silk, Nature 410
(2001) 541–548.
[26] F. Vollrath, Strength and structure of spiders' silks, J. Biotechnol. 74 (2000)
67–83.
[27] S. Keten, Z. Xu, B. Ihle, M.J. Buehler, Nanoconfinement controls stiffness, strength
and mechanical toughness of [beta]-sheet crystals in silk, Nat. Mater. 9 (2010)
359–367.
[28] Frische, Maunsbach, Vollrath, Elongate cavities and skin–core structure in
Nephila spider silk observed by electron microscopy, J. Microsc. 189 (1998)
64–70.
[29] S. Putthanarat, N. Stribeck, S.A. Fossey, R.K. Eby, W.W. Adams, Investigation of
the nanofibrils of silk fibers, Polymer 41 (2000) 7735–7747.
[30] P. Poza, J. Pérez-Rigueiro, M. Elices, J. Llorca, Fractographic analysis of silkworm
and spider silk, Eng. Fract. Mech. 69 (2002) 1035–1048.
[31] H. Akai, T. Nagashima, S. Aoyagi, Ultrastructure of posterior silk gland cells and
liquid silk in Indian tasar silkworm, Antheraea mylitta drury (Lepidoptera:
Saturniidae), Int. J. Insect Morphol. Embryol. 22 (1993) 497–506.
[32] F. Vollrath, Spiders' webs, Curr. Biol. (2005) R364–R365.
[33] T. Giesa, M. Arslan, N.M. Pugno, M.J. Buehler, Nanoconfinement of spider silk
fibrils begets superior strength, extensibility, and toughness, Nano Lett. 11
(2011) 5038–5046.
[34] N. Du, Z. Yang, X.Y. Liu, Y. Li, H.Y. Xu, Structural origin of strain-hardening of
spider silk, Adv. Funct. Mater. 21 (2011) 772–778.
[35] Y. Zhang, H. Yang, H. Shao, X. Hu, Antheraea pernyi silk fiber: a potential resource
for artificially biospinning spider dragline silk, J. Biomed. Biotechnol. (2010)
(2010).
[36] R. Rajkhowa, B. Levin, S.L. Redmond, L. Wang, J.R. kanwar, M.D. Atlas, X. Wang,
Structure and properties of biomedical films prepared from aqueous and acidic
silk fibroin solutions, J. Biomed. Mater. Res. Part A 97A (2011) 37–45.
[37] K. Gellynck, P. Verdonk, E. Van Nimmen, K. Almqvist, T. Gheysens, G. Schoukens,
L. Van Langenhove, P. Kiekens, J. Mertens, G. Verbruggen, Silkworm and spider
silk scaffolds for chondrocyte support, J. Mater. Sci. Mater. Med. 19 (2008)
3399–3409.
[38] G.H. Altman, R.L. Horan, H.H. Lu, J. Moreau, I. Martin, J.C. Richmond, D.L. Kaplan,
Silk matrix for tissue engineered anterior cruciate ligaments, Biomaterials 23
(2002) 4131–4141.
[39] S.W. Ha, A.E. Tonelli, S.M. Hudson, Structural studies of Bombyx mori silk fibroin
during regeneration from solutions and wet fiber spinning, Biomacromolecules
6 (2005) 1722–1731.
[40] B. Zuo, L. Dai, Z. Wu, Analysis of structure and properties of biodegradable
regenerated silk fibroin fibers, J. Mater. Sci. 41 (2006) 3357–3361.
[41] C. Jiang, X. Wang, R. Gunawidjaja, Y.-H. Lin, M.K. Gupta, D.L. Kaplan, R.R. Naik,
V.V. Tsukruk, Mechanical properties of robust ultrathin silk fibroin films, Adv.
Funct. Mater. 17 (2007) 2229–2237.
[42] L. Meinel, D.L. Kaplan, Silk constructs for delivery of musculoskeletal therapeu-
tics, Adv. Drug Deliv. Rev. 64 (2012) 1111–1122.
 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470
[43] J.A. Zhou, C.B. Cao, X.L. Ma, L. Hu, L.A. Chen, C.R. Wang, In vitro and in vivo deg-
radation behavior of aqueous-derived electrospun silk fibroin scaffolds, Polym.
Degrad. Stab. 95 (2010) 1679–1685.
[44] Y. Wang, D.D. Rudym, A. Walsh, L. Abrahamsen, H.-J. Kim, H.S. Kim, C.
Kirker-Head, D.L. Kaplan, In vivo degradation of three-dimensional silk fibroin
scaffolds, Biomaterials 29 (2008) 3415–3428.
[45] H. Fan, H. Liu, S.L. Toh, J.C.H. Goh, Anterior cruciate ligament regeneration using
mesenchymal stem cells and silk scaffold in large animal model, Biomaterials 30
(2009) 4967–4977.
[46] R. Horan, D. Bramono, J. Stanley, Q. Simmons, J. Chen, H. Boepple, G. Altman,
Biological and biomechanical assessment of a long-term bioresorbable
silk-derived surgical mesh in an abdominal body wall defect model, Hernia 13
(2009) 189–199.
[47] N. Minoura, S.I. Aiba, M. Higuchi, Y. Gotoh, M. Tsukada, Y. Imai, Attachment and
growth of fibroblast cells on silk fibroin, Biochem. Biophys. Res. Commun. 208
(1995) 511–516.
[48] K. Lundmark, G.T. Westermark, A. Olsén, P. Westermark, Protein fibrils in nature
can enhance amyloid protein A amyloidosis in mice: cross-seeding as a disease
mechanism, Proc. Natl. Acad. Sci. 102 (2005) 6098–6102.
[49] Y. Hu, Q. Zhang, R. You, L. Wang, M. Li, The relationship between secondary
structure and biodegradation behavior of silk fibroin scaffolds, Adv. Mater. Sci.
Eng. (2012), http://dx.doi.org/10.1155/2012/185905.
[50] M. Vert, S.M. Li, G. Spenlehauer, P. Guerin, Bioresorbability and biocompatibility
of aliphatic polyesters, J. Mater. Sci. Mater. Med. 3 (1992) 432–446.
[51] E.M. Pritchard, T. Valentin, D. Boison, D.L. Kaplan, Incorporation of proteinase in-
hibitors into silk-based delivery devices for enhanced control of degradation
and drug release, Biomaterials 32 (2011) 909–918.
[52] R.L. Horan, K. Antle, A.L. Collette, Y. Wang, J. Huang, J.E. Moreau, V. Volloch, D.L.
Kaplan, G.H. Altman, In vitro degradation of silk fibroin, Biomaterials 26 (2005)
3385–3393.
[53] M. Li, M. Ogiso, N. Minoura, Enzymatic degradation behavior of porous silk
fibroin sheets, Biomaterials 24 (2003) 357–365.
[54] Q. Lu, B. Zhang, M. Li, B. Zuo, D.L. Kaplan, Y. Huang, H. Zhu, Degradation mechanism
and control of silk fibroin, Biomacromolecules 12 (2011) 1080–1086.
[55] K. Numata, P. Cebe, D.L. Kaplan, Mechanism of enzymatic degradation of
beta-sheet crystals, Biomaterials 31 (2010) 2926–2933.
[56] T. Arai, G. Freddi, R. Innocenti, M. Tsukada, Biodegradation of Bombyx mori silk
fibroin fibers and films, J. Appl. Polym. Sci. 91 (2004) 2383–2390.
[57] J.N. Wang, Z.-W. Liu, Y.-X. Yang, H.-Y. Huang, Enzymatic degradation behavior of
silk fibroin fiber treated by y-ray irradiation, Textile Res. J. 82 (2012) 1799–1805.
[58] S. Sengupta, S.-H. Park, G.E. Seok, A. Patel, K. Numata, C.-L. Lu, D.L. Kaplan, Quan-
tifying osteogenic cell degradation of silk biomaterials, Biomacromolecules 11
(2010) 3592–3599.
[59] W.P. Daley, S.B. Peters, M. Larsen, Extracellular matrix dynamics in development
and regenerative medicine, J. Cell Sci. 121 (2008) 255–264.
[60] P.A. Gunatillake, R. Adhikari, Biodegradable synthetic polymer for tissue
engineering, Eur. Cells Mater. 5 (2003) 1–16.
[61] T.T. Le Zainuddin, Y. Park, T.V. Chirila, P.J. Halley, A.K. Whittaker, The behavior of
aged regenerated Bombyx mori silk fibroin solutions studied by 1H NMR and
rheology, Biomaterials 29 (2008) 4268–4274.
[62] V. Karageorgiou, L. Meinel, S. Hofmann, A. Malhotra, V. Volloch, D. Kaplan, Bone
morphogenetic protein-2 decorated silk fibroin films induce osteogenic differ-
entiation of human bone marrow stromal cells, J. Biomed. Mater. Res. Part A
71A (2004) 528–537.
[63] V. Karageorgiou, M. Tomkins, R. Fajardo, L. Meinel, B. Snyder, K. Wade, J. Chen, G.
Vunjak-Novakovic, D.L. Kaplan, Porous silk fibroin 3-D scaffolds for delivery of
bone morphogenetic protein-2 in vitro and in vivo, J. Biomed. Mater. Res. Part
A 78A (2006) 324–334.
[64] Z. Shao, F. Vollrath, Materials: Surprising strength of silkworm silk, Nature 418
(2002) 741.
[65] H.J. Jin, J. Park, V. Karageorgiou, U.J. Kim, R. Valluzzi, P. Cebe, D.L. Kaplan,
Water-atable silk films with reduced β-sheet content, Adv. Funct. Mater. 15
(2005) 1241–1247.
[66] Y. Kawahara, K. Furukawa, T. Yamamoto, Self-expansion behaviour of silk fibroin
film, Macromol. Mater. Eng. 291 (2006) 458–462.
[67] N. Goujon, X. Wang, R. Rajkhowa, N. Byrne, Regenerated silk fibroin using protic
ionic liquids solvents : towards an all-ionic-liquid process for producing silk
with tunable properties, Chem. Commun. 48 (2012) 1278–1280.
[68] E. Servoli, D. Maniglio, A. Motta, R. Predazzer, C. Migliaresi, Surface properties of
silk fibroin films and their interaction with fibroblasts, Macromol. Biosci. 5
(2005) 1175–1183.
[69] S. Lu, X. Wang, Q. Lu, X. Hu, N. Uppal, F.G. Omenetto, D.L. Kaplan, Stabilization of
enzymes in silk films, Biomacromolecules 10 (2009) 1032–1042.
[70] B.B. Mandal, S.C. Kundu, A novel method for dissolution and stabilization of
non-mulberry silk gland protein fibroin using anionic surfactant sodium dodecyl
sulfate, Biotechnol. Bioeng. 99 (2008) 1482–1489.
[71] S. Rathbone, P. Furrer, J. Lübben, M. Zinn, S. Cartmell, Biocompatibility of
polyhydroxyalkanoate as a potential material for ligament and tendon scaffold
material, J. Biomed. Mater. Res. Part A 93A (2010) 1391–1403.
[72] H. Fan, H. Liu, E.J.W. Wong, S.L. Toh, J.C.H. Goh, In vivo study of anterior cruciate
ligament regeneration using mesenchymal stem cells and silk scaffold, Biomate-
rials 29 (2008) 3324–3337.
[73] R. Ahmad, A. Kamra, S.E. Hasnain, Fibroin silk proteins from the nonmulberry
silkworm Philosamia ricini are biochemically and immunologically distinct
from those of the mulberry silkworm Bombyx mori, DNA Cell Biol. 23 (2004)
149–154.
467
[74] C. Acharya, B. Hinz, S.C. Kundu, The effect of lactose-conjugated silk biomate-
rials on the development of fibrogenic fibroblast, Biomaterials 29 (2008)
4665–4675.
[75] N.V. Bhat, S.M. Ahirrao, Investigation of the structure of silk film regenerated
with lithium thiocyanate solution, J. Polym. Sci. Part A: Polym. Chem. 21
(1983) 1273–1280.
[76] D.M. Phillips, L.F. Drummy, R.R. Naik, H.C.D. Long, D.M. Fox, P.C. Trulove, R.A.
Mantz, Regenerated silk fiber wet spinning from an ionic liquid solution,
J. Mater. Chem. 15 (2005) 4206–4208.
[77] W. Tao, M. Li, C. Zhao, Structure and properties of regenerated Antheraea pernyi
silk fibroin in aqueous solution, Int. J. Biol. Macromol. 40 (2007) 472–478.
[78] H. Kweon, Y.H. Park, Dissolution and characterization of regenerated Antheraea
pernyi silk fibroin, J. Appl. Polym. Sci. 82 (2001) 750–758.
[79] H.Y. Kweon, Y.H. Park, Structural and conformational changes of regenerated
Antheraea pernyi silk fibroin films treated with methanol solution, J. Appl.
Polym. Sci. 73 (1999) 2887–2894.
[80] M. Li, W. Tao, S. Lu, C. Zhao, Porous 3-D scaffolds from regenerated Antheraea
pernyi silk fibroin, Polym. Adv. Technol. 19 (2008) 207–212.
[81] I.C. Um, H. Kweon, Y.H. Park, S. Hudson, Structural characteristics and properties
of the regenerated silk fibroin prepared from formic acid, Int. J. Biol. Macromol.
29 (2001) 91–97.
[82] M.K. Gupta, S.K. Khokhar, D.M. Phillips, L.A. Sowards, L.F. Drummy, M.P. Kadakia,
R.R. Naik, Patterned silk films cast from ionic liquid solubilized fibroin as
scaffolds for cell growth, Langmuir 23 (2006) 1315–1319.
[83] A. Higuchi, M. Yoshida, T. Ohno, T. Asakura, M. Hara, Production of interferon-β
in a culture of fibroblast cells on some polymeric films, Cytotechnology 34
(2000) 165–173.
[84] X. Wang, H.J. Kim, P. Xu, A. Matsumoto, D.L. Kaplan, Biomaterial coatings by
stepwise deposition of silk fibroin, Langmuir 21 (2005) 11335–11341.
[85] Q. Lu, X. Hu, X. Wang, J.A. Kluge, S. Lu, P. Cebe, D.L. Kaplan, Water-insoluble silk
films with silk I structure, Acta Biomater. 6 (2010) 1380–1387.
[86] X. Zhang, M.R. Reagan, D.L. Kaplan, Electrospun silk biomaterial scaffolds for
regenerative medicine, Adv. Drug Deliv. Rev. 61 (2009) 988–1006.
[87] B.D. Lawrence, Z. Pan, M.D. Weber, D.L. Kaplan, M.I. Rosenblatt, Silk film
culture system for in vitro analysis and biomaterial Design, J. Vis. Exp. (2012)
3646.
[88] M.L. Lovett, C.M. Cannizzaro, G. Vunjak-Novakovic, D.L. Kaplan, Gel spinning of
silk tubes for tissue engineering, Biomaterials 29 (2008) 4650–4657.
[89] X. Zhang, X. Wang, V. Keshav, X. Wang, J.T. Johanas, G.G. Leisk, D.L. Kaplan,
Dynamic culture conditions to generate silk-based tissue-engineered vascular
grafts, Biomaterials 30 (2009) 3213–3223.
[90] M.E. Kinahan, E. Filippidi, S. Koster, X. Hu, H.M. Evans, T. Pfohl, D.L. Kaplan, J.
Wong, Tunable silk: Using microfluidics to fabricate silk fibers with controllable
properties, Biomacromolecules 12 (2011) 1504–1511.
[91] X. Chen, W. Li, W. Zhong, Y. Lu, T. Yu, pH sensitivity and ion sensitivity of
hydrogels based on complex-forming chitosan/silk fibroin interpenetrating
polymer network, J. Appl. Polym. Sci. 65 (1997) 2257–2262.
[92] A. Motta, C. Migliaresi, F. Faccioni, P. Torricelli, M. Fini, R. Giardino, Fibroin
hydrogels for biomedical applications: preparation, characterization and in
vitro cell culture studies, J. Biomater. Sci. Polym. Ed. 15 (2004) 851–864.
[93] N. Guziewicz, A. Best, B. Perez-Ramirez, D.L. Kaplan, Lyophilized silk fibroin
hydrogels for the sustained local delivery of therapeutic monoclonal antibodies,
Biomaterials 32 (2011) 2642–2650.
[94] U.J. Kim, J. Park, C. Li, H.-J. Jin, R. Valluzzi, D.L. Kaplan, Structure and properties of
silk hydrogels, Biomacromolecules 5 (2004) 786–792.
[95] P.H. Chao, S. Yodmuang, X. Wang, L. Sun, D.L. Kaplan, Silk hydrogel for cartilage
tissue engineering, J. Biomed. Mater. Res. B Appl. Biomater. (2010) 84–90.
[96] M. Li, Z. Wu, C. Zhang, S. Lu, H. Yan, D. Huang, H. Ye, Study on porous silk fibroin
materials. II. Preparation and characteristics of spongy porous silk fibroin
materials, J. Appl. Polym. Sci. 79 (2001) 2192–2199.
[97] M. Tsukada, G. Freddi, N. Minoura, G. Allara, Preparation and application of
porous silk fibroin materials, J. Appl. Polym. Sci. 54 (1994) 507–514.
[98] L.D. Harris, B.S. Kim, D.J. Mooney, Open pore biodegradable matrices formed
with gas foaming, J. Biomed. Mater. Res. 42 (1998) 396–402.
[99] M. Li, Z. Wu, C. Zhang, S. Lu, H. Yan, D. Huang, H. Ye, Study on porous silk fibroin
materials. II. Preparation and characteristics of spongy porous silk fibroin
materials, J. Appl. Polym. Sci. 79 (2001) 2129–2199.
[100] L. Meinel, R. Fajardo, S. Hofmann, R. Langer, J. Chen, B. Snyder, G.
Vunjak-Novakovic, D. Kaplan, Silk implants for the healing of critical size bone
defects, Bone 37 (2005) 688–698.
[101] V.V. Seregin, J.L. Coffer, Biomineralization of calcium disilicide in porous
polycaprolactone scaffolds, Biomaterials 27 (2006) 4745–4754.
[102] A. Hokugo, T. Takamoto, Y. Tabata, Preparation of hybrid scaffold from fibrin and
biodegradable polymer fiber, Biomaterials 27 (2006) 61–67.
[103] M. Wang, Developing bioactive composite materials for tissue replacement,
Biomaterials 24 (2003) 2133–2151.
[104] R. Rajkhowa, E.S. Gil, J.A. Kludge, K. Numata, L. Wang, X. Wang, D.L. Kaplan,
Reinforcing silk scaffolds with silk particles, Macromol. Biosci. 10 (2010)
599–611.
[105] B.B. Mandal, A. Grinberg, E. Seok Gil, B. Panilaitis, D.L. Kaplan, High-strength silk
protein scaffolds for bone repair, Proc. Natl. Acad. Sci. 109 (2012) 7699–7704.
[106] H. Yoshimizu, T. Asakura, Preparation and characterisation of silk fibroin powder
and its application to enzyme immobilization, J. Appl. Polym. Sci. 40 (1990)
127–134.
[107] J.H. Yeo, K.G. Lee, Y.W. Lee, S.Y. Kim, Simple preparation and characteristics of
silk fibroin microsphere, Eur. Polym. J. 39 (2003) 1195–1199.
468 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470
[108] E. Wenk, A.J. Wandrey, H.P. Merkle, L. Meinel, Silk fibroin spheres as a platform
for controlled drug delivery, J. Control. Release 132 (2008) 26–34.
[109] A.S. Lammel, X. Hu, S.H. Park, D.L. Kaplan, T.R. Scheibel, Controlling silk fibroin
particle features for drug delivery, Biomaterials 31 (2010) 4583–4591.
[110] Y.Q. Zhang, S. Wei-De, X. Ru-Li, L.J. Zhuge, W.J. Gao, W.B. Wang, Formation of silk
nanoparticles in water-miscible organic solvent and their characterization,
J. Nanopart. Res. 9 (2007) 885–900.
[111] Z. Cao, X. Chen, J. Yao, L. Huang, Z. Shao, The preparation of regenerated silk fi-
broin microspheres, Soft Matter 3 (2007) 910–915.
[112] D.N. Rockwood, E.S. Gil, S.H. Park, J.A. Kluge, W. Grayson, S. Bhumiratana, R.
Rajkhowa, X. Wang, S.J. Kim, G. Vunjak-Novakovic, D.L. Kaplan, Ingrowth of
human mesenchymal stem cells into porous silk particle reinforced silk compos-
ite scaffolds: an in vitro study, Acta Biomater. 7 (2011) 144–151.
[113] A.B. Mathur, V. Gupta, Silk fibroin-derived nanoparticles for biomedical applica-
tions, Nanomedicine 5 (2010) 807–820.
[114] J. Kundu, Y.I. Chung, Y.H. Kim, G. Tae, S.C. Kundu, Silk fibroin nanoparticles for
cellular uptake and control release, Int. J. Pharm. 388 (2010) 242–250.
[115] X. Wang, T. Yucel, Q. Lu, X. Hu, D.L. Kaplan, Silk nanospheres and microspheres
from silk/PVA blend films for drug delivery, Biomaterials 31 (2010) 1025–1035.
[116] F. Causin, R. Pascarella, G. Pavesi, R. Marasco, G. Zambon, R. Battaglia, M. Munari,
Acute endovascular treatment (b48 hours) of uncoilable ruptured aneurysms at
non-branching sites using silk flow-diverting devices, Interv. Neuroradiol. 17
(3) (2011) 357–364.
[117] M. Leonardi, L. Cirillo, F. Toni, M. Dall'Olio, C. Princiotta, A. Stafa, L. Simonetti, R. Agati,
Treatment of intracranial aneurysms using flow-diverting silk stents (BALT): a single
centre experience, Interv. Neuroradiol. 17 (3) (2011) 306–315.
[118] L. Soffer, X. Wang, X. Zhang, J. Kluge, L. Dorfmann, D.L. Kaplan, G. Leisk,
Silk-based electrospun tubular scaffolds for tissue-engineered vascular grafts,
J. Biomater. Sci. Polym. Ed. 19 (2008) 653–664.
[119] J.M. Orban, L.B. Wilson, J.A. Kofroth, M.S. El-Kurdi, T.M. Maul, D.A. Vorp,
Crosslinking of collagen gels by transglutaminase, J. Biomed. Mater. Res. Part A
68A (2004) 756–762.
[120] T. Nishibe, Y. Kondo, A. Muto, A. Dardik, Optimal prosthetic graft design for small
diameter vascular grafts, Vascular 15 (2007) 356–360.
[121] Y. Nakazawa, M. Sato, R. Takahashi, D. Aytemiz, C. Takabayashi, T. Tamura, S.
Enomoto, M. Sata, T. Asakura, Development of small-diameter vascular grafts
based on silk fibroin fibers from bombyx mori for vascular regeneration,
J. Biomater. Sci. Polym. Ed. 22 (2011) 195–206.
[122] T. Yagi, M. Sato, Y. Nakazawa, K. Tanaka, M. Sata, K. Itoh, Y. Takagi, T. Asakura,
Preparation of double-raschel knitted silk vascular grafts and evaluation
of short-term function in a rat abdominal aorta, J. Artif. Organs 14 (2011)
89–99.
[123] M. Lovett, G. Eng, J. Kluge, C. Cannizzaro, G. Vunjak-Novakovic, D.L. Kaplan,
Tubular silk scaffolds for small diameter vascular grafts, Organogenesis 6
(2010) 217–224.
[124] A. Ratcliffe, Tissue engineering of vascular grafts, Matrix Biol. 19 (2000)
353–357.
[125] S. Enomoto, M. Sumi, K. Kajimoto, Y. Nakazawa, R. Takahashi, C. Takabayashi, T.
Asakura, M. Sata, Long-term patency of small-diameter vascular graft made
from fibroin, a silk-based biodegradable material, J. Vasc. Surg. 51 (2010)
155–164.
[126] B. Bondar, S. Fuchs, A. Motta, C. Migliaresi, C.J. Kirkpatrick, Functionality of endo-
thelial cells on silk fibroin nets: comparative study of micro- and nanometric
fibre size, Biomaterials 29 (2008) 561–572.
[127] R.E. Unger, K. Peters, M. Wolf, A. Motta, C. Migliaresi, C.J. Kirkpatrick,
Endothelialization of a non-woven silk fibroin net for use in tissue engineering:
growth and gene regulation of human endothelial cells, Biomaterials 25 (2004)
5137–5146.
[128] H. Liu, X. Li, G. Zhou, H. Fan, Y. Fan, Electrospun sulfated silk fibroin nanofibrous
scaffolds for vascular tissue engineering, Biomaterials 32 (2011) 3784–3793.
[129] Y. Yang, X. Chen, F. Ding, P. Zhang, J. Liu, X. Gu, Biocompatibility evaluation of
silk fibroin with peripheral nerve tissues and cells in vitro, Biomaterials 28
(2007) 1643–1652.
[130] Y. Yang, F. Ding, J. Wu, W. Hu, W. Liu, J. Liu, X. Gu, Development and evaluation
of silk fibroin-based nerve grafts used for peripheral nerve regeneration,
Biomaterials 28 (2007) 5526–5535.
[131] Y. Wei, K. Gong, Z. Zheng, A. Wang, Q. Ao, Y. Gong, X. Zhang, Chitosan/silk
fibroin-based tissue-engineered graft seeded with adipose-derived stem cells
enhances nerve regeneration in a rat model, J. Mater. Sci. Mater. Med. 22
(2011) 1947–1964.
[132] C.Y. Wang, K.-H. Zhang, C.Y. Fan, X.M. Mo, H.J. Ruan, F.F. Li, Aligned natural–
synthetic polyblend nanofibers for peripheral nerve regeneration, Acta
Biomater. 7 (2011) 634–643.
[133] W. Huang, R. Begum, T. Barber, V. Ibba, N.C.H. Tee, M. Hussain, M. Arastoo, Q.
Yang, L.G. Robson, S. Lesage, T. Gheysens, N.J.V. Skaer, D.P. Knight, J.V.
Priestley, Regenerative potential of silk conduits in repair of peripheral nerve
injury in adult rats, Biomaterials 33 (2012) 59–71.
[134] S. Madduri, M. Papaloïzos, B. Gander, Trophically and topographically function-
alized silk fibroin nerve conduits for guided peripheral nerve regeneration,
Biomaterials 31 (2010) 2323–2334.
[135] B.M. Min, L. Jeong, K.Y. Lee, W.H. Park, Regenerated silk fibroin nanofibers:
water vapor-induced structural changes and their effects on the behavior of
normal human cells, Macromol. Biosci. 6 (2006) 285–292.
[136] C.R. Yoo, I.S. Yeo, K.E. Park, J.H. Park, S.J. Lee, W.H. Park, B.M. Min, Effect of
chitin/silk fibroin nanofibrous bicomponent structures on interaction with
human epidermal keratinocytes, Int. J. Biol. Macromol. 42 (2008) 324–334.
[137] I.S. Yeo, J.E. Oh, L. Jeong, T.S. Lee, S.J. Lee, W.H. Park, B.M. Min, Collagen-based
biomimetic nanofibrous scaffolds: preparation and characterization of
collagen/silk fibroin bicomponent nanofibrous structures, Biomacromolecules
9 (2008) 1106–1116.
[138] H.L. Khor, K.W. Ng, A.S. Htay, J.T. Schantz, S.H. Teoh, D.W. Hutmacher, Prelimi-
nary study of a polycaprolactone membrane utilized as epidermal substrate,
J. Mater. Sci. Mater. Med. 14 (2003) 113–120.
[139] D.H. Roh, S.Y. Kang, J.Y. Kim, Y.B. Kwon, H. Young Kweon, K.G. Lee, Y.H. Park, R.M.
Baek, C.Y. Heo, J. Choe, J.H. Lee, Wound healing effect of silk fibroin/
alginate-blended sponge in full thickness skin defect of rat, J. Mater. Sci.
Mater. Med. 17 (2006) 547–552.
[140] L.G. Griffith, G. Naughton, Tissue engineering-current challenges and expanding
opportunities, Science 295 (2002) 1009–1014.
[141] L. Meinel, O. Betz, R. Fajardo, S. Hofmann, A. Nazarian, E. Cory, M. Hilbe, J.
McCool, R. Langer, G. Vunjak-Novakovic, H.P. Merkle, B. Rechenberg, D.L.
Kaplan, C. Kirker-Head, Silk based biomaterials to heal critical sized femur
defects, Bone 39 (2006) 922–931.
[142] C. Li, C. Vepari, H.J. Jin, H.J. Kim, D.L. Kaplan, Electrospun silk-BMP-2 scaffolds for
bone tissue engineering, Biomaterials 27 (2006) 3115–3124.
[143] G. Wang, H. Yang, M. Li, S. Lu, X. Chen, X. Cai, The use of silk fibroin/
hydroxyapatite composite co-cultured with rabbit bone-marrow stromal cells
in the healing of a segmental bone defect, in: JSBS, British Volume, 92-B, 2010,
pp. 320–325.
[144] H. Kweon, K.-G. Lee, C.-H. Chae, C. Balázsi, S.-K. Min, J.Y. Kim, J.Y. Choi, S.G. Kim,
Development of nano-hydroxyapatite graft with silk fibroin scaffold as a new
bone substitute, J. Oral Maxillofac. Surg. 69 (2011) 1578–1586.
[145] K. Tanaka, N. Kajiyama, K. Ishikura, S. Waga, A. Kikuchi, K. Ohtomo, T. Takagi, S.
Mizuno, Determination of the site of disulfide linkage between heavy and light
chains of silk fibroin produced by Bombyx mori, Biochim. Biophys. Acta, Protein
Struct. Mol. Enzymol. 1432 (1999) 92–103.
[146] M. Fini, A. Motta, P. Torricelli, G. Giavaresi, N. Nicoli Aldini, M. Tschon, R.
Giardino, C. Migliaresi, The healing of confined critical size cancellous de-
fects in the presence of silk fibroin hydrogel, Biomaterials 26 (2005)
3527–3536.
[147] H.J. Kim, U.J. Kim, H.S. Kim, C. Li, M. Wada, G.G. Leisk, D.L. Kaplan, Bone tissue
engineering with premineralized silk scaffolds, Bone 42 (2008) 1226–1234.
[148] S. Ghanaati, R.E. Unger, M.J. Webber, M. Barbeck, C. Orth, J.A. Kirkpatrick, P.
Booms, A. Motta, C. Migliaresi, R.A. Sader, C.J. Kirkpatrick, Scaffold vasculariza-
tion in vivo driven by primary human osteoblasts in concert with host inflam-
matory cells, Biomaterials 32 (2011) 8150–8160.
[149] R.E. Unger, A. Sartoris, K. Peters, A. Motta, C. Migliaresi, M. Kunkel, U. Bulnheim, J.
Rychly, C. James Kirkpatrick, Tissue-like self-assembly in cocultures of endothelial
cells and osteoblasts and the formation of microcapillary-like structures on
three-dimensional porous biomaterials, Biomaterials 28 (2007) 3965–3976.
[150] S. Fuchsa, X. Jianga, H. Schmidtb, E. Dohlea, S. Ghanaatia, C. Ortha, A. Hofmannc,
A. Mottad, C. Migliaresid, C.J. Kirkpatricka, Dynamic processes involved in the
pre-vascularization of silk fibroin constructs for bone regeneration using out-
growth endothelial cells, Biomaterials 30 (2009) 1329–1338.
[151] R.E. Unger, S. Ghanaati, C. Orth, A. Sartoris, M. Barbeck, S. Halstenberg, A. Motta,
C. Migliaresi, C.J. Kirkpatrick, The rapid anastomosis between prevascularized
networks on silk fibroin scaffolds generated in vitro with cocultures of human
microvascular endothelial and osteoblast cells and the host vasculature, Bioma-
terials 31 (2010) 6959–6967.
[152] Y. Wang, D.J. Blasioli, H.J. Kim, H.S. Kim, D.L. Kaplan, Cartilage tissue engineering
with silk scaffolds and human articular chondrocytes, Biomaterials 27 (2006)
4434–4442.
[153] Y. Morita, N. Tomita, H. Aoki, M. Sonobe, S. Wakitani, Y. Tamada, T. Suguro, K.
Ikeuchi, Frictional properties of regenerated cartilage in vitro, J. Biomech. 39
(2006) 103–109.
[154] Y. Wang, U.J. Kim, D.J. Blasioli, H.J. Kim, D.L. Kaplan, In vitro cartilage tissue
engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal
stem cells, Biomaterials 26 (2005) 7082–7094.
[155] K. Gellynck, P.C.M. Verdonk, E.V. Nimmen, K.F. Almqvist, T. Gheysens, G.
Schokens, L.V. Langenhove, P. Kiekens, J. Mertens, G. Verbruggen, Silkworm
and spider silk scaffolds for chondrocyte support, J. Mater. Sci. Mater. Med. 19
(2008) 3399–3409.
[156] H.S. Baek, Y.H. Park, C.S. Ki, J.C. Park, D.K. Rah, Enhanced chondrogenic responses
of articular chondrocytes onto porous silk fibroin scaffolds treated with
microwave-induced argon plasma, Surf. Coat. Technol. 202 (2008) 5794–5797.
[157] N. Bhardwaj, Q.T. Nguyen, A.C. Chen, D.L. Kaplan, R.L. Sah, S.C. Kundu, Potential of
3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan
for in vitro cartilage tissue engineering, Biomaterials 32 (2011) 5773–5781.
[158] S.S. Silva, A. Motta, M.R.T. Rodrigues, A.F.M. Pinheiro, M.E. Gomes, J.O.F. Mano,
R.L. Reis, C. Migliaresi, Novel Genipin-cross-linked chitosan/silk fibroin
sponges for cartilage engineering strategies, Biomacromolecules 9 (2008)
2764–2774.
[159] S. Talukdar, Q.T. Nguyen, A.C. Chen, R.L. Sah, S.C. Kundu, Effect of initial cell
seeding density on 3D-engineered silk fibroin scaffolds for articular cartilage
tissue engineering, Biomaterials 32 (2011) 8927–8937.
[160] L. Uebersax, H.P. Merkle, L. Meinel, Insulin-like growth factor I releasing silk fi-
broin scaffolds induce chondrogenic differentiation of human mesenchymal
stem cells, J. Control. Release 127 (2008) 12–21.
[161] C. Shangkai, T. Naohide, Y. Koji, H. Yasuji, N. Masaaki, T. Tomohiro, T. Yasushi,
Transplantation of allogeneic chondrocytes cultured in fibroin sponge and
stirring chamber to promote cartilage Regeneration, Tissue Eng. C Methods 13
(2007) 483–492.
 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470
[162] R. Seda Tigli, S. Ghosh, M.M. Laha, N.K. Shevde, L. Daheron, J. Gimble, M.
Gümüşderelioglu, D.L. Kaplan, Comparative chondrogenesis of human cell
sources in 3D scaffolds, J. Tissue Eng. Regen. Med. 3 (2009) 348–360.
[163] X. Chen, Y.Y. Qi, L.L. Wang, Z. Yin, G.L. Yin, X.H. Zou, H.W. Ouyang, Ligament re-
generation using a knitted silk scaffold combined with collagen matrix, Bioma-
terials 29 (2008) 3683–3692.
[164] S. Sahoo, S. Lok Toh, J.C. Hong Goh, PLGA nanofiber-coated silk microfibrous
scaffold for connective tissue engineering, J. Biomed. Mater. Res. B Appl.
Biomater. 95B (2010) 19–28.
[165] J.E. Moreau, J. Chen, R.L. Horan, D.L. Kaplan, G.H. Altman, Sequential growth fac-
tor application in bone marrow stromal cell ligament engineering, Tissue Eng.
11 (2005) 1887–1897.
[166] M. Hairfield-Stein, C. England, H.J. Paek, K.B. Gilbraith, R. Dennis, E. Boland, P.
Kosnik, Development of self-assembled, tissue-engineered ligament from bone
marrow stromal cells, Tissue Eng. 13 (2007) 703–710.
[167] Q. Fang, D. Chen, Z. Yang, M. Li, In vitro and in vivo research on using antheraea
pernyi silk fibroin as tissue engineering tendon scaffolds, Mater. Sci. Eng., C 29
(2009) 1527–1534.
[168] M.C. Yang, S.S. Wang, N.K. Chou, N.H. Chi, Y.Y. Huang, Y.L. Chang, M.J. Shieh, T.W.
Chung, The cardiomyogenic differentiation of rat mesenchymal stem cells on silk fi-
broin–polysaccharide cardiac patches in vitro, Biomaterials 30 (2009) 3757–3765.
[169] C. Patra, S. Talukdar, T. Novoyatleva, S.R. Velagala, C. Mühlfeld, B. Kundu, S.C.
Kundu, F.B. Engel, Silk protein fibroin from Antheraea mylitta for cardiac tissue
engineering, Biomaterials 33 (2012) 2673–2680.
[170] W. Liu, K. Merrett, M. Griffith, P. Fagerholm, S. Dravida, B. Heyne, J.C. Scaiano,
M.A. Watsky, N. Shinozaki, N. Lagali, R. Munger, F. Li, Recombinant human
collagen for tissue engineered corneal substitutes, Biomaterials 29 (2008)
1147–1158.
[171] B.D. Lawrence, M. Cronin-Golomb, I. Georgakoudi, D.L. Kaplan, F.G. Omenetto,
Bioactive silk protein biomaterial systems for optical devices, Biomacromolecules
9 (2008) 1214–1220.
[172] T. Yang, M. Zhang, Biocompatibility of silk fibroin membrane as tissue engineer-
ing corneal scaffold, Int. J. Ophthalmol. 8 (2008) 1557–1559.
[173] B.D. Lawrence, J.K. Marchant, M.A. Pindrus, F.G. Omenetto, D.L. Kaplan, Silk film
biomaterials for cornea tissue engineering, Biomaterials 30 (2009) 1299–1308.
[174] S.C. Kundu, B. Kundu, S. Talukdar, S. Bano, S. Nayak, J. Kundu, B.B. Mandal, N.
Bhardwaj, M. Botlagunta, B.C. Dash, C. Acharya, A.K. Ghosh, Nonmulberry silk
biopolymers, Biopolymers 97 (2012) 455–467.
[175] P.W. Madden, J.N.X. Lai, K.A. George, T. Giovenco, D.G. Harkin, T.V. Chirila,
Human corneal endothelial cell growth on a silk fibroin membrane, Biomaterials
32 (2011) 4076–4084.
[176] L.J. Bray, K.A. George, S.L. Ainscough, D.W. Hutmacher, T.V. Chirila, D.G. Harkin,
Human corneal epithelial equivalents constructed on Bombyx mori silk fibroin
membranes, Biomaterials 32 (2011) 5086–5091.
[177] Y. Gotoh, S. Niimi, T. Hayakawa, T. Miyashita, Preparation of lactose–silk fibroin
conjugates and their application as a scaffold for hepatocyte attachment, Bioma-
terials 25 (2004) 1131–1140.
[178] B. Cirillo, M. Morra, G. Catapano, Adhesion and function of rat liver cells adher-
ent to silk fibroin/collagen blend films, Int. J. Artif. Organs 27 (2004) 60–68.
[179] K. Hu, Q. Lv, F.Z. Cui, Q.L. Feng, X.D. Kong, H.L. Wang, L.Y. Huang, T. Li, Biocom-
patible fibroin blended films with recombinant human-like collagen for hepatic
tissue engineering, J. Bioact. Compat. Polym. 21 (2006) 23–37.
[180] Q. Lu, S. Zhang, K. Hu, Q. Feng, C. Cao, F. Cui, Cytocompatibility and blood com-
patibility of multifunctional fibroin/collagen/heparin scaffolds, Biomaterials 28
(2007) 2306–2313.
[181] Q. Lv, K. Hu, Q. Feng, F. Cui, C. Cao, Preparation and characterization of
PLA/fibroin composite and culture of HepG2 (human hepatocellular liver
carcinoma cell line) cells, Compos. Sci. Technol. 67 (2007) 3023–3030.
[182] Z. She, W. Liu, Q. Feng, Self-assembly model, hepatocytes attachment and
inflammatory response for silk fibroin/chitosan scaffolds, Biomed. Mater. 4
(2009) 045014.
[183] N. Kasoju, R.R. Bhonde, U. Bora, Fabrication of a Biomed. Mater., novel micro–
nano fibrous nonwoven scaffold with Antheraea assama silk fibroin for use in tis-
sue engineering, Mater. Lett. 63 (2009) 2466–2469.
[184] N. Kasoju, U. Bora, Antheraea assama silk fibroin-based functional scaffold with
enhanced blood compatibility for tissue engineering applications, Adv. Eng.
Mater. 12 (2010) B139–B147.
[185] Y. Qian, Y. Shen, Z. Lu, Z. Fan, T. Liu, J. Zhang, F. Zhang, Biocompatibility of silk
fibroin nanofibers scaffold with olfactory ensheathing cells, Zhongguo Xiu Fu
Chong Jian Wai Ke Za Zhi 23 (2009) 1365–1370.
[186] Y. Shen, Y. Qian, H. Zhang, B. Zuo, Z. Lu, Z. Fan, P. Zhang, F. Zhang, C. Zhou, Guid-
ance of olfactory ensheathing cell growth and migration on electrospun silk
fibroin scaffolds, Cell Transplant. 19 (2010) 147–157.
[187] G. Chang, H.J. Kim, D. Kaplan, G. Vunjak-Novakovic, R. Kandel, Porous silk scaf-
folds can be used for tissue engineering annulus fibrosus, Eur. Spine J. 16
(2007) 1848–1857.
[188] G. Chang, H.J. Kim, G. Vunjak-Novakovic, D.L. Kaplan, R. Kandel, Enhancing an-
nulus fibrosus tissue formation in porous silk scaffolds, J. Biomed. Mater. Res.
A 92A (2010) 43–51.
[189] A. Atala, Tissue engineering of human bladder, Br. Med. Bull. 97 (2011) 81–104.
[190] C.X. Liu, Y.F. Liao, H.L. Li, S.B. Zheng, Cytocompatibility of silk fibroin film with
rabbit urinary bladder transitional epithelial cells in vitro, Nan Fang Yi Ke Da
Xue Xue Bao 28 (2008) 216–218.
[191] C.X. Liu, Y.Y. Lin, H.L. Li, S.B. Zheng, Application of silk fibroin film for
repairing rabbit urethral defect, Nan Fang Yi Ke Da Xue Xue Bao 27 (2007)
184–187.
469
[192] G.M. Cannon Jr., J.R. Mauney, E.M. Gong, R.N. Yu, R.M. Adam, C.R. Estrada, Silk as
a novel biomaterial in bladder tissue engineering, J. Pediatr. Urol. 6 (Supplement
1) (2010) S82.
[193] J.R. Mauney, G.M. Cannon, M.L. Lovett, E.M. Gong, D. Di Vizio, P. Gomez Iii, D.L.
Kaplan, R.M. Adam, C.R. Estrada Jr., Evaluation of gel spun silk-based biomate-
rials in a murine model of bladder augmentation, Biomaterials 32 (2011)
808–818.
[194] X.H. Zou, Y.L. Zhi, X. Chen, H.M. Jin, L.L. Wang, Y.Z. Jiang, Z. Yin, H.W. Ouyang,
Mesenchymal stem cell seeded knitted silk sling for the treatment of stress
urinary incontinence, Biomaterials 31 (2010) 4872–4879.
[195] Y. Ni, X. Zhao, L. Zhou, Z. Shao, W. Yan, X. Chen, Z. Cao, Z. Xue, J.J. Jiang, Radiolog-
ic and histologic characterization of silk fibroin as scaffold coating for rabbit
tracheal defect repair, Otolaryngol. Head Neck Surg. 139 (2008) 256–261.
[196] M. Zang, Q. Zhang, G. Davis, G. Huang, M. Jaffari, C.N. Ríos, V. Gupta, P. Yu, A.B.
Mathur, Perichondrium directed cartilage formation in silk fibroin and
chitosan blend scaffolds for tracheal transplantation, Acta Biomater. 7 (2011)
3422–3431.
[197] B. Levin, R. Rajkhowa, S.L. Redmond, M.D. Atlas, Grafts in myringplasty :utilizing
a silk fibroin scaffold as a novel decice, Expert Rev. Med. Devices 6 (2009)
653–664.
[198] B. Levin, S.L. Redmond, R. Rajkhowa, R.H. Eikelboom, R.J. Marano, M.D. Atlas,
Preliminary results of the application of a silk fibroin scaffold to otology,
Otolaryngol. Head Neck Surg. 142 (2010) S33–S35.
[199] R. Ghassemifar, S. Redmond, T.V. Chirila, Zainuddin, Advancing towards a
tissue-engineered tympanic membrane: Silk fibroin as a substratum for growing
human eardrum keratinocytes, J. Biomater. Appl. 24 (2010) 591–606.
[200] B. Levin, S.L. Redmond, R. Rajkhowa, R.H. Eikelboom, M.D. Atlas, Utilising silk
fibroin membranes as scaffolds for the growth of tympanic membrane
keratinocytes, and application to myringoplasty surgery, J. Laryngol. Otol.
(2012), http://dx.doi.org/10.1017/S0022215112001661.
[201] J. Kim, C.H. Kim, C.H. Park, J.N. Seo, H. Kweon, S.W. Kang, K.G. Lee, Comparison of
methods for the repair of acute tympanic membrane perforations: Silk patch vs.
paper patch, Wound Repair Regen. 18 (2010) 132–138.
[202] N. Reddy, Y. Yang, Morphology and tensile properties of silk fibers produced by
uncommon saturniidae, Int. J. Biol. Macromol. 46 (2010) 419–424.
[203] C. Zhao, X. Wu, Q. Zhang, S. Yan, M. Li, Enzymatic degradation of Antheraea
pernyi silk fibroin 3-D scaffolds and fibers, Int. J. Biol. Macromol. 48 (2011)
249–255.
[204] S.H. Park, E.S. Gil, H. Shi, H.J. Kim, K. Lee, D.L. Kaplan, Relationships between
degradability of silk scaffolds and osteogenesis, Biomaterials 31 (2010)
6162–6172.
[205] X. Hu, Q. Lu, D.L. Kaplan, P. Cebe, Microphase separation controlled beta-sheet
crystallization kinetics in fibrous proteins, Macromolecules 42 (2009) 2079–
2087.
[206] X. Hu, K. Shmelev, L. Sun, E.S. Gil, S.H. Park, P. Cebe, D.L. Kaplan, Regulation of
silk material structure by temperature-controlled water vapor annealing,
Biomacromolecules 12 (2011) 1686–1696.
[207] A. Vasconcelos, G. Freddi, A. Cavaco-Paulo, Biodegradable materials based on silk
fibroin and keratin, Biomacromolecules 9 (2008) 1299–1305.
[208] R. Kino, T. Ikoma, S. Yunoki, A. Monkawa, A. Matsuda, G. Kagata, T. Asakura, M.
Munekata, T. Junzo, Biodegradation of multilayer silk fibroin and hydroxyapatite
composite material, Key Eng. Mater. 309–311 (2006) 1169–1172.
[209] E.M. Pritchard, T. Valentin, D. Boison, D.L. Kaplan, Incorporation of proteinase in-
hibitors into silk-based delivery devices for enhanced control of degradation
and drug release, Biomaterials 32 (2011) 909–918.
[210] B. Zuo, L. Liu, Z. Wu, Effect on properties of regenerated silk fibroin fiber
coagulated with aqueous methanol/ethanol, J. Appl. Polym. Sci. 106 (2007)
53–59.
[211] J.H. Kim, C.H. Park, O.J. Lee, J.M. Lee, J.W. Kim, Y.H. Park, C.S. Ki, Preparation and
in vivo degradation of controlled biodegradability of electrospun silk fibroin
nanofiber mats, J. Biomed. Mater. Res. A 100 (2012) 3287–3295.
[212] K. Chen, Y. Umeda, K. Hirabayashi, Enzymatic hydrolysis of silk fibroin, J. Seric.
Sci. Jpn. 65 (1995) 131–133.
[213] W. Xu, G. Ke, X. Peng, Studies on the effects of the enzymatic treatment on silk
fine powder, J. Appl. Polym. Sci. 101 (2006) 2967–2971.
[214] R. Rajkhowa, X. Hu, T. Tsuzuki, D.L. Kaplan, X.G. Wang, Structure and biodegra-
dation mechanism of milled B. mori silk particles, Biomacromolecules 13 (2012)
2503–2512.
[215] Y. Tomizawa, Vascular prostheses for aortocoronary bypass grafting: a review,
Artif. Organs 19 (1995) 39–45.
[216] A. Callow, Arterial homografts, Eur. J. Vasc. Endovasc. Surg. 12 (1996) 272–281.
[217] K.J. Pawlowski, S.E. Rittgers, S.P. Schmidt, G.L. Bowlin, Endothelial cell seeding of
polymeric vascular grafts, Front. Biosci. 9 (2004) 1412–1421.
[218] G.R.D. Evans, Challenges to nerve regeneration, Semin. Surg. Oncol. 19 (2000)
312–318.
[219] A. Beris, M. Lykissas, A. Korompilias, G. Mitsionis, End-to-side nerve repair in
peripheral nerve injury, J. Neurotrauma 24 (2007) 909–916.
[220] M.F. Meek, J.H. Coert, Clinical use of nerve conduits in peripheral-nerve repair:
review of the literature, J. Reconstr. Microsurg. 18 (2002) 097–110.
[221] L. Thomsen, P. Bellemere, T. Loubersac, E. Gaisne, P. Poirier, F. Chaise, Treatment
by collagen conduit of painful post-traumatic neuromas of the sensitive digital
nerve: a retrospective study of 10 cases, Chir. Main 29 (2010) 255–262.
[222] O. Ilhan-Sarac, E.K. Akpek, Current concepts and techniques in keratoprosthesis,
Curr. Opin. Ophthalmol. 16 (2005) 246–250.
[223] D. Myung, W. Koh, A. Bakri, F. Zhang, A. Marshall, J. Ko, J. Noolandi, M. Carrasco, J.
Cochran, C. Frank, C. Ta, Design and fabrication of an artificial cornea based on a
470 B. Kundu et al. / Advanced Drug Delivery Reviews 65 (2013) 457–470

photolithographically patterned hydrogel construct, Biomed. Microdevices 9
(2007) 911–922.
[224] S. Geetha Priya, H. Jungvid, A. Kumar, Skin tissue engineering for tissue repair
and regeneration, Tissue Eng. B Rev. 14 (2008) 105–118.
[225] G. Vunjak-Novakovic, G. Altman, R. Horan, D.L. Kaplan, Tissue engineering of
ligaments, Annu. Rev. Biomed. Eng. 6 (2004) 131–156.
[226] S. Calve, R.G. Dennis, P.E. Kosnik II, K. Baar, K. Grosh, E.M. Arruda, Engineering of
functional tendon, Tissue Eng. 10 (2004) 755–761.
[227] M.P. Prabhakaran, J. Venugopal, D. Kai, S. Ramakrishna, Biomimetic material
strategies for cardiac tissue engineering, Mater. Sci. Eng., C 31 (2011) 503–513.