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CHAPTER 2
LITERATURE REVIEW
This chapter includes literature related to tissue engineering,
materials and structure of scaffold, and method of production of scaffold used
for tissue engineering applications. The literature related to the application of
silk for tissue engineering applications, characteristics of different varieties of
silks are included. Different forms of scaffolds, especially the nano fibrous
scaffolds, produced by the electrospinning method are discussed in detail.
2.1 TISSUE ENGINEERING
2.1.1 Current Organ Repairing Therapies
Tissue loss or organ failure, resulting from traumatic or non-
traumatic destruction, gives rise to a major health problem that directly affects
the quality and length of patient’s life. These circumstances often call for
surgical treatments to repair, replace, maintain, or augment the functions of
the affected tissue or organ using some additional functional component that
facilitates an improved life to the patients. They have been traditionally
treated with the help of tissues or organs procured from the donors.
Depending on the location of implantation, the procured tissue or organ (also
called graft) is termed as autograft, allograft, or xenograft as shown in
Figure 2.1 If the graft is implanted in the same patient, it is termed as
autograft. Autografts (tissues removed from the patient) are typically
considered to be the gold standard in treating injuries. Autografts possess the
necessary amount of initial mechanical strength and promote cell proliferation
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and new tissue growth. There is no risk of rejection or disease transmission
associated with autografts, since the tissue comes from the patient. However,
autografts have disadvantages as well (Murugan and Ramakrishna 2007).
Autografts require additional surgery for tissue harvest, which may cause
donor site morbidity (Freeman and Kwansa 2008). In the case of allograft and
xenograft, there have been concerns about infection and second site morbidity
of such tissues, and this type of graft may be rejected by host body due to the
immune response to tissue (Jackson et al 1990).The procurement of living
tissue/organ is complex and expensive, and requires additional surgery. This
clearly indicates the need for a potential solution to overcome the limitations
of traditional therapies and, at the same time, to increase the accessibility and
long-term survivability of the tissue implants (Yarlagadda et al 2005).
Figure 2.1 Conventional medical therapies
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2.1.2 Tissue Engineering Concepts
Tissue Engineering is the application of principles and methods of
engineering and life sciences, towards fundamental understanding of the
structure/function relationships in normal and pathological mammalian tissues
and the development of biological substitutes to restore, maintain or improve
body functions. The key challenges in Tissue Engineering are synthesis of
new cell adhesion-specific materials and development of fabrication methods
to produce three-dimensional synthetic or natural biodegradable polymer
scaffolds with tailored properties. In tissue engineering, the scaffold serves as
a three-dimensional (3D) template for cell adhesion, proliferation and
formation of an extracellular matrix (ECM), as well as a carrier of the growth
factors or other bio- molecular signals. The scaffold fundamentally needs
some properties, such as porosity, pore size distribution, mechanical strength,
and required rate of degradation and bio-compatibility (Chen et al 2004).
2.2 FUNDAMENTAL NEEDS OF SCAFFOLDS
The fundamental needs of scaffolds are:
Biocompatibility: acceptance within the body without causing
bio-fouling, in which the body attacks the implant, or the cells
do not grow on the material.
Biodegradability: ability to degrade in the body into
compatible by-products without causing inflammatory
responses.
Mechanical integrity: ability to maintain the original structure
and mechanical properties upon exposure to the body’s
environment, i.e., 37°C, pH 7.4, phosphate buffer saline
solution
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High porosity: ability to allow the transfer of nutrients/oxygen
and removal of wastes via diffusion.
Bioactivity: ability to transform or conform, depending upon
the influence from the internal milieu that surrounds the
scaffold seeded with cells (McCullen 2006).
2.3 BIOPOLYMERS FOR SCAFFOLD PREPARATION
2.3.1 Bio-degradable Polymer
Biodegradable polymers are solid polymeric materials and devices
which break down due to macromolecular degradation with dispersion in
vivo, however no proof exists for their elimination from the body (this
definition excludes environmental, fungi or bacterial degradation).
Biodegradable polymeric systems or devices can be attacked by biological
elements, so that the integrity of the system and in some cases but not
necessarily, of the macromolecules themselves, is affected and produces
fragments or other degradation by-products. Such fragments can move away
from their site of action but not necessarily from the body (Vert et al 1992,
Hutmacher 2000).
2.3.2 Bio-resorbable Polymer
Bio-resorbable polymers are solid polymeric materials and devices
which show bulk degradation and further resorb in vivo; i.e., polymers which
are eliminated through natural pathways, either because of simple filtration of
degradation by-products or after their metabolization. Bio-resorption is thus a
concept, which reflects the total elimination of the initial foreign material and
of bulk degradation by-products (low molecular weight compounds) with no
residual effects (Vert et al 1992).
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2.3.3 Bio-erodible Polymer
Bio-erodible polymers are solid polymeric materials or devices,
which show surface degradation and further, resorb in vivo. Bio-erosion too is
thus a concept, which reflects total elimination of the initial foreign material
and of surface degradation by-products (low molecular weight compounds)
with no residual side effects (Vert et al 1992).
2.3.4 Bio-absorbable polymer
Bio-absorbable polymers are solid polymeric materials or devices,
which can dissolve in body fluids without any polymer chain cleavage or
molecular mass decrease. For example, it is the case of slow dissolution of
water-soluble implants in body fluids. A bio-absorbable polymer can be bio-
resorbable, if the dispersed macromolecules are excreted (Vert et al 1992).
Both synthetic polymers and biologically derived (or natural) polymers have
been extensively investigated as biodegradable polymeric biomaterials. The
biodegradation of polymeric biomaterials involves the cleavage of
hydrolytically or enzymatically sensitive bonds in the polymer leading to
polymer erosion (Katti et al 2002). Depending on the mode of degradation,
polymeric biomaterials can be further classified into hydrolytically degradable
polymers and enzymatically degradable polymers. Natural polymers can be
considered as the first biodegradable biomaterials used clinically. The rate of
in vivo degradation of enzymatically degradable polymers however, varies
significantly with the site of implantation, depending on the availability and
concentration of the enzymes. The chemical modification of these polymers
can also significantly affect their rate of degradation. Natural polymers
possess several inherent advantages, such as bioactivity, the ability to present
receptor-binding ligands to cells, susceptibility to cell-triggered proteolytic
degradation and natural remodeling. The inherent bioactivity of these natural
polymers has its own downsides. These include a strong immunogenic
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response associated with most of the polymers, the complexities associated
with their purification and the possibility of disease transmission (Nair and
Laurencin 2007).
2.4 HYDROLYTICALLY DEGRADABLE SYNTHETIC
POLYMERS
Hydrolytically degradable polymers are polymers that have
hydrolytically labile chemical bonds in their back-bone. The functional
groups susceptible to hydrolysis include esters, orthoesters, anhydrides,
carbonates, amides, urethanes, ureas etc.
2.4.1 Aliphatic Polyesters
Poly ( -esters) are thermoplastic polymers with hydrolytically
labile aliphatic ester linkages in their backbone. Although all polyesters are
theoretically degradable, as esterification is a chemically reversible process,
only aliphatic polyesters with reasonably short aliphatic chains between ester
bonds, can degrade within the required time for most of the biomedical
applications. Poly ( -esters) comprises the earliest and most extensively
investigated class of biodegradable polymers. The uniqueness of this class of
polymers lies in its immense diversity and synthetic versatility. Poly ( -ester)
can be developed from a variety of monomers via ring opening and
condensation polymerization routes, depending on the monomeric units.
Bacterial bioprocess routes can also be used to develop some poly ( -esters).
Various synthetic routes for developing polyesters have been recently
reviewed by Okada (2007) and Davachi et al (2011).
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2.4.2 Polyglycolide
Polyglycolide is the simplest linear aliphatic polyester, which is one
of the first biodegradable synthetic polymers investigated for biomedical
applications. It is prepared by the ring opening polymerization of a cyclic
lactone, glycolide. Polyglycolide is a highly crystalline polymer, with a
crystallinity of 45–55%, and therefore, exhibits a high tensile modulus with
very low solubility in organic solvents. The glass transition temperature of the
polymer ranges from 35 to 40 C, and the melting point is higher than 200 C.
Polyglycolide is not soluble in most organic solvents; the exceptions are
highly fluorinated organic solvents such as hexafluoro-isopropanol
(Morentet al 2011). Common processing techniques such as extrusion,
injection and compression molding can be used to fabricate polyglycolide into
various forms; its high sensitivity to hydrolytic degradation requires careful
control of processing conditions (Sabir et al 2009). Porous scaffolds,
electrospun nanofibrous scaffolds and foams can also be fabricated from
polyglycolide.
An excellent fibre forming ability of polyglycolide was initially
investigated for developing resorbable sutures. The first biodegradable
synthetic suture called DEXON that was approved by the United States Food
and Drug Administration (USFDA) in 1969 was based on polyglycolide.
Non-woven polyglycolide fabrics have been extensively used as scaffolding
matrices for tissue regeneration, due to their excellent degradability, good
initial mechanical properties and cell viability on the matrices, and their
ability to help regenerate biological tissue. The polymer is known to lose its
strength in 1–2 months when hydrolyzed, and loses mass within 6–12 months.
In the body, polyglycolides are broken down into glycine, which can be
excreted in the urine or converted into carbon dioxide and water via the citric
acid cycle (Terasaka et al 2006).
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2.4.3 Polycaprolactone
Polycaprolactone is an aliphatic polyester and hydrophobic
polymer, prepared by the ring opening polymerization of caprolactone. It is
readily degraded by a variety of bacteria and moth. The ring-opening
polymerization of e-caprolactone yields a semi crystalline polymer with a
melting point of 58–63°C and a glass transition temperature of -60°C. The
repeating molecular structure of PCL homopolymer consists of five non polar
methylene groups and a single relatively polar ester group (Vroman and
Tighzer 2009). This structure gives PCL, the unique properties that are similar
to polyolefin, because of its high olefinic content, while the presence of
hydrolytically unstable aliphatic-ester linkage causes the polymer to be
biodegradable. Polycaprolactone has a slower biodegradation in in vivo than
PLA due to its higher crystallinity and hydrophobic property. It is used for
soft and hard tissue engineering and drug delivery. The main drawback of the
PCL polymer is that it has low cell attachment, as it is a hydrophobic
polymer. After blending starch, cellulose and PLA polymers with the PCL,
enhanced cell attachment could be achieved (Yu et al 2006).
2.4.4 Poly (dioxanone)
Poly(dioxanone) is a polyether – ester aliphatic polyester; it is
synthesized by the ring opening polymerization of p-dioxanone, resulting in
the first clinically tested mono filament synthetic suture, that is known as
PDS' marketed by Ethicon. P-Dioxanone or 1,4-dioxan-2-one, abbreviated as
PDO, is a colorless crystal or liquid. Poly (dioxanone) exhibits a crystalline
fraction of 55% and a glass-transition temperature between 10°C and 0°C
(Middleton and Tipton 2000). PDS is used for a suture for a long period, and
provides higher flexibility, higher strength retention, slow absorption rates
and lower inflammatory response rates, when compared to Vicryl (poly
(glycolic-co-lactic acid)) and Dexon (poly (glycolic acid)). It is mostly
15
preferred for application as sutures, because of the flexibility and easier
knotting capacity. In addition, vascular prostheses made of PDO have been
shown to be less thrombogenic than both PGA and Dacron® synthetic grafts
(Pillai and Sharma 2010). PDO is a synthetic bio-resorbable polymer, that
offers several advantages over the more traditional bio-resorbable polymers
like poly(glycolic acid), poly(lactic acid) and poly(lactic-co-glycolic acid),
because it has a slower resorption rate, and induces a lower inflammatory
response (Smith et al 2008).
2.4.5 Poly (trimethylene carbonate)
Poly(trimethylene carbonate) (PTMC)) aliphatic polycarbonates are
employed in many biomedical applications, owing to their high
biocompatibility, facile bio-degradation, low toxicity, and superior
mechanical properties as compared to those of structurally similar polyesters
(Nederberg et al 2007). It is manufactured by the ring opening polymerization
of trimethylene carbonate. The polycarbonate derived from trimethylene
carbonate (TMC or 1,3-dioxan-2-one) has been investigated quite extensively
for its potential utilization as a biodegradable polymer in important
biomedical and pharmaceutical applications, such as sutures, drug delivery
systems and tissue engineering (Gangly 2006). High-molecular-weight PTMC
an amorphous, rubbery polymer at room temperature shows good mechanical
performance, combining high flexibility with high tensile strength (Pego et al
2002). The advantage of PTMC is slow degradation; hence it is suitable for
long term medical applications.
2.4.6 Poly-3-hydroxybutyrate
Poly-3-hydroxybutyrate (PHB) is a bacterial polyester and of a
hydrophobic nature; it is obtained from many types of bacteria, and was
discovered in the year 1920. The bacteria (Bacillus megaterium) have the
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ability to synthesize and polymerize the monomer of hydroxybutyric acid.
Polyhydroxybutyrate is a naturally occurring -hydroxyacid (linear polyester).
Its ability to degrade and resorb in the human body environment makes it a
suitable matrix for bioactive and biodegradable composite implants that will
guide tissue growth and be replaced eventually by newly formed tissue. The
PHB can also be used as surgical sutures, bone implant material, and drug
release system (Chen and Wang 2002). The brittleness of the PHB is largely
due to the presence of large crystallinity in the form of spherulites, which
form upon cooling from the melt. PHB can be injection molded or extruded,
provided care is taken to lower the melting temperature, and minimize the
residence time. Injection molded PHB bars often show high crystallinity and
higher melting temperature, especially, below the glass transition temperature.
It suffers from some disadvantages, including a narrow processability
window, relatively low impact strength, higher brittleness, and a hydrophobic
nature (Park et al 2001).
2.4.7 Polyurethanes
Polyurethane (PUR) elastomers are multi-block copolymers with an
alternating sequence of hard and soft-block locks. Polyurethane (PU)
elastomers have been used extensively in biomedical applications because of
their excellent biocompatibility and mechanical properties (Hung et al 2009).
Segmented polyurethanes are elastomeric block copolymers that generally
exhibit a phase-segregated morphology made up of soft rubbery segments,
and hard glassy or semi crystalline segments (Lligadas et al 2007). The
Melting temperature of polyurethane is in the range of 125-138°C. Segmented
polyurethanes can also be designed to have chemical linkages that are
degradable in a biological environment, and there has been some interest in
developing degradable polyurethanes for medical applications, such as
scaffolds for tissue engineering. Polyurethane semi crystalline polymer
17
possesses many useful properties; but it also exhibits drawbacks such as
hydrophobicity, low resiliency, and swelling and deformation upon
degradation. It can also be blended with amorphous lactic acid to overcome
above drawbacks (Vainio et al 1997).
2.4.8 Poly (ester amide)
Aliphatic segmented poly (ester amide)s, comprising a
crystallizable amide phase and a flexible amorphous ester phase, were
investigated for potential use in biomedical applications. Segmented poly
(ester amide) s are prepared by the melt condensation of preformed bisamide-
diols, 1,4-butanediol, and dimethyl adipate. Poly (ester amide)s are an
emerging group of biodegradable polymers that may cover both commodity
and specialty applications. These polymers have ester and amide groups in
their chemical structure, which are of a degradable character and provide
good thermal and mechanical properties (Galan et al 2011).
2.4.9 Pseudo Poly (amino acid)
As part of the continuing efforts to develop improved biomaterials,
a method for synthesizing a new class of poly (amino acids) was recently
proposed. These polymers, named “pseudopoly (amino acids)”, are different
from conventional poly (amino acids)s in that the polymer backbone is
formed by utilizing the side-chain functional groups on the monomeric -L-
amino acids or dipeptides. Such an approach offers the opportunity to create
polymers from naturally occurring metabolites, but without some of the
potential disadvantages of conventional poly (amino acids)s resulting from
the repeating amide bonds, e.g. poor mechanical strength and enzymatic
degradation. When degradable polymers are used as implant materials in
patients, the potential toxicity of the polymer degradation products and their
subsequent metabolites become a major anxiety. For this reason, poly amino
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acids (PAA) were particularly attractive candidates for biomedical
applications (Pulapura et al 1992). Synthetic PAAs that contain one or at most
two different amino acid residues were virtually non-immunogenic and were
found to degrade in vitro and in vivo to their respective amino acid building
blocks, which are non-toxic and natural metabolites. They provide good
structural stability and biocompatibility for biomedical applications
(Mallakpour et al 2011).
2.5 NATURAL BIODEGRADABLE POLYMERS
2.5.1 Chitin and Chitosan
Chitin is the most abundant natural amino polysaccharide, and is
estimated to be produced annually almost as much as cellulose. Chitin and its
de-acetylated derivative chitosan, are natural polymers composed of randomly
distributed -(1-4)-linked D-glucosamine (de-acetylated unit) and N-acetyl-D-
glucosamine (acetylated unit) (Ravikumar 2000). Chitin functions naturally as
a structural polysaccharide, like cellulose, but differs from cellulose in its
properties. Chitin is highly hydrophobic and is insoluble in water and most of
the organic solvents. It is soluble in hexafluoro iso-propanol, hexafluoro-
acetone, chloro alcohols in conjugation with aqueous solutions of mineral
acids. Chitin is a white, hard, inelastic, nitrogenous polysaccharide found in
the outer skeleton of insects, crabs, shrimps, and lobsters, and in the internal
structure of other invertebrates. Chitin is obtained basically from prawn/crab
shells; the chemical treatment of chitin produces chitosan. Chitin and chitosan
are recommended as suitable materials for wound dressing, since these natural
polymers have excellent properties such as biodegradability, biocompatibility,
non-toxicity, and adsorption (Dutta et al 2004). The reaction of chitosan is
considerably more versatile than cellulose due to the presence of NH2 groups.
The natural polysaccharide chitosan and its quaternized derivatives possess
high intrinsic activity against bacteria and fungi, low toxicity,
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biodegradability and the ability to affect macrophage functions. Chitosan-
containing materials might contribute to faster wound healing processes,
making them good candidates for wound dressing applications. However,
these scaffolds have a low mechanical strength under physiological
conditions, thus limiting their applicability (Ignatova et al 2009).
2.5.2 Collagen
Collagen is an important biomaterial in medical applications due to
its special characteristics, such as biodegradability and weak antigenecity
(Zhang et al 2005). Collagen is found to make up one quarter of all the total
protein in the body. It possesses a fibrous structure; collagen is able to impart
structure and strength to body tissues, such as tendons, ligaments and skin.
Collagen proteins are comprised of polypeptide chains ( -chains) that form a
unique triple-helical structure that is 300 nanometers long and 1.5 nanometers
in diameter. There are more than twenty disparate collagen types that exist in
animal tissue, five of which are known to form fibres; Types I, II, III, V, and
XI. These types tend to self-assemble into periodic, cross-striated fibres,
which can reach centimeters in length and tens of microns in diameter. Type I
collagen is the predominant fibre-forming collagen type and is found in
bones, skin, teeth, and tendons. Type II collagen, considered the second most
abundant, is found in cartilaginous tissue, developing cornea, and vitreous humor
(Kadler et al 1996). The major disadvantage of collagen nanofibrous matrix may
be the loss of its structural integrity in an aqueous environment such as in the
human body. However, the combination of collagen with biocompatible
polymers that enhance biological interactions with cells and speed up tissue
regeneration, could improve the dimensional stability of scaffolds (Yeo et al
2008).
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2.5.3 Silk
Silk is popularly known in the textile industry for its lustre and
mechanical properties (Kaplan et al 1998). Silk was discovered in China
around 2700 B.C. Silk is traditionally manufactured by sericulture. This
ancient art was practiced in China, Korea and Japan since the fourth century,
and in the sixth century this technique reached Europe via the Silk Route
(Hyde 1984). Silk is now produced across Asia and Europe, although the
main sources are Japan, China and India. Silk has been of interest for over
5000 years not only for its properties of texture, tenacity and dyeing, but also
for its use in cosmetics, creams, lotions, makeup, powders, bath preparations
and pharmaceuticals (Brooks 1989).
Silks are generally defined as protein polymers that are spun into
fibres by some Lepidoptera larvae such as silkworms, spiders, scorpions,
mites and flies (Altman et al 2003). Silk is a natural filament produced by the
silkworm, Bombyx mori, which has been used traditionally in the form of
filaments in textiles for thousands of years. This silk contains a fibrous
protein termed fibroin (both heavy and light chains) that forms the thread core
and glue-like proteins termed sericin that surround the fibroin filament to
reinforce them together. The fibroin is a highly insoluble protein containing
up to 90% of the amino acids glycine, alanine, and serine, leading to anti-
parallel -pleated sheet formation in the filament (Asakura et al 1994). Silk
has been used as a textile material for a long period. After the removal of the
sericin from silk fibroin, it has been considered as the starting raw material for
non-textile applications, especially in the biomedical, cosmetic and
biotechnological fields, such as surgical sutures, wound cover materials,
controlled drug release carriers, tissue engineering scaffolds and repair
materials for skins, bones, ligaments etc. (Altman et al 2003). Silk fibroin has
more mechanical strength than other synthetic bio materials and it has higher
combined strength and toughness due to the presence of an anti-parallel -
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sheet structure. The most extensively used silk is obtained from different types
of silk worm such as mulberry silk (Bombyx mori), and non- mulberry silks such
as eri (Attacus ricinii), muga (Antherae assama) and tassar (Antherae perni). The
mulberry silk belongs to the Bombycidae family, whereas the non –mulberry silk
belongs to Saturniidae.
2.5.3.1 Life cycle of mulberry silk
The life cycle of the mulberry silk worm (Bombyx mori) is shown
in Figure 2.2. In about 50 days, it completes its four-step metamorphosis; egg
or embryo, larva, pupa and adult (moth). The worms consume food (mulberry
leaves) only at the larval stage. Pupation occurs at the end of spinning (cocoon
formation). Silkworm silk is produced basically at one stage in the life cycle,
during the fifth larval instars just before the molt to pupa (Asakura et al 1997).
Figure 2.2 Life cycle of mulberry silk
2.5.3.2 Composition of silk gland
The silk worm extrudes two proteins, namely, fibroin and sericin.
The fibroin forms the core while the sericin is deposited as a coating. The
22
glands of the silk worm in which the proteins are secreted are shown in Figure
2.3.The silk gland consists of three sections, the rear, middle and front. The
rear section is generally 200 to 250mm long and has a diameter of 0.4 to
0.8mm. The fibroin is secreted in this section. The middle section is about 60
to 65 mm long and has a diameter of 1.2 to 2.5mm. Depending on the
products of secretion and other characteristics, the middle section of the silk
gland is in turn divided in to the rear portion, the central portion with a distal
and proximal section, and the front portion. The frontal section is 35 to 40
mm long and has a diameter of 0.05 to 0.3 mm, and has the function of
conducting the silk proteins. The fibroin is secreted in the posterior section of
the silk gland, and transferred to the middle section in which it is stored as a
viscous liquid. The bulk of the sericin is produced in the middle section
together with the pigments, which impart colour to coloured silks. The rear
section of the silk gland, which is narrow and highly convoluted, secretes the
fibroin as a 12- 15% polymer solution. The fibroin then passes in to the broader
middle section, where it is enveloped by sericin, which is synthesized in this
portion of the silk gland. The silk proteins get concentrated in to 30% in this gland.
The water present in the fibroin solution passes in to the sericin layer (Glurajani
1993).
Figure 2.3 Silkworm gland
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2.5.3.3 Composition of the mulberry silk filament
Gulrajani et al (1988) reported that the silk fibre is almost a pure
protein fibre composed of two types of proteins, viz., sericin and fibroin.
Sericin is chemically a non-filamentous protein. Besides sericin, raw silk also
contains other natural impurities, namely, fat and waxes, inorganic salts and
colouring matter as shown in Table 2.1.
Table 2.1 Composition of mulberry silk
Component Percentage
Fibroin 70-80
Sericin 20-30
Wax matter 0.4-0.8
Carbohydrate 1.2-1.6
In organic matter 0.7
Pigment 0.2
Total 100
2.5.3.4 Structure of mulberry silk filament
Mulberry silk has been shown to be composed of two protein-
monofilaments (named brins) embedded in a glue like sericin coating. A
similar structure has been observed in other silkworms’ silk. The brins are
fibroin filaments made up of bundles of nanofibrils, approximately 5 nm in
diameter, with a bundle diameter of around 100 nm. The nanofibrils are
oriented parallel to the axis and are thought to interact strongly with each
other (Hakimi et al 2007). A schematic representation of the structure of
mulberry thread is shown in Figure 2.4.
24
Figure 2.4 Structure of mulberry silk filament
2.5.3.5 Physical properties of mulberry silk fibroin
Mulberry silk fibres are generally 10-20 m in thickness (size) and
each fibre is actually a duplet of two individual fibres, each with its own silk
coating (sericin) and an inner core (fibroin). The fibroin consists of thousands
of parallel fibrils (100-400 nm), which after ion-etching can be viewed by a
scanning electron microscope. The fibrils give the microfilament its grainy
structure. The fibres also contain small quantities of carbohydrate, wax and
inorganic components, which play significant roles as structural elements
during fibre formation. Mulberry silk fibres are not circular in the cross
section, but appear triangular. The fine structure of the silk fibres gives them
the dynamic qualities of excellent lustre, colour, exquisite texture and
excellent temperature retainability (Ayutsede 2005).
25
2.5.3.6 Chemical properties of mulberry silk fibroin
Mulberry has two silk glands that constitute approximately
one-quarter of the worm’s mass, and produce liquid silk. This polymer is
composed of a 350 kDa (kilo Dalton) fibroin heavy chain (H-fibroin), a
25 kDa fibroin light chain (L-fibroin), and a family of proteins called sericin,
that bind the two threads together as they emerge from the glands, and harden
in contact with the air. The silk thread is pulled from the gland and can attain
a length of more than a kilometer (Wurm 2003). The elements of the
supramolecular structure of silk fibres are macro fibrils with a width of up to
6.5×105 nm, which in turn, consist of helically packed nanofibrils of 90 -170
nm diameter. Nanofibrils play an important role to enhance tensile strength.
Silk fibres produced by cultivated Bombyx mori silkworm consist
mainly of two proteins, sericin and fibroin; they also contain minor amounts
of residues of other amino acids and various impurities: fats, waxes, dyes, and
mineral salts. Depending on the cocoon strain, the fibroin content is
66.5- 73.5, and the sericin content, 26.5- 33.5 wt % (Sashina et al 2006). The
primary structure of mulberry silk consists of 12 repetitive regions called
crystalline regions and 11 non repetitive interspaced regions called
amorphous regions (Zhou et al 2001). The remarkable properties of silk fibres
are attributed to the distribution of microcrystalline and amorphous domains,
which are formed in the process of spinning by protein–protein interactions.
The overall composition of the silk fibroin in mol% consists of glycine
(42.9%), alanine (30.0%), serine (12.2%), tyrosine (4.8%), and valine (2.5%)
(Asakura and Yao 2002). The mole fraction of glycine, alanine, serine, and
tyrosine residues combined is 90%; their sequence is represented by the
general formula: Gly-Ala-Gly-Ala-Gly-Ser-Gly-Ala-Ala-Gly- [-Ser-Gly-(Ala-
Gly)n]8-Tyr- (Shimura et al 1982, Shimura et al 1976). The pendant fragments
26
of the fibroin macromolecules are nonpolar hydrophobic aliphatic
hydrocarbon (alanine, leucine, isoleucine, valine, proline) and aromatic
(phenylalanine) substituents, polar hydrophilic hydroxyl-containing residues
of serine, threonine, and tyrosine, carboxyl groups of aspartic and glutamic
acids, amino groups of lysine, and guanidine groups of arginine. The
secondary structure of fibroin is stabilized by various kinds of interactions.
Hydrogen bonds arise between functional groups of peptide macro chains and
between side fragments of macromolecules (Baker et al 1984). Three main
kinds of secondary structures of natural silk fibroin are distinguished: -
helical and -folded structures (silk I and silk II respectively) in crystalline
areas and as disordered conformation of random globules in amorphous areas.
The fibroin of natural mulberry fibres contains 56±5% macromolecules in the
-folded form and 13±5% macromolecules in the -helical form. Thus, the
fraction of highly ordered (crystalline) areas of the polymer reaches 60 -70%
in the silk fibroin (Trabbic and Yager 1998).
2.5.4 Eri Silk
Samia cynthia ricini (eri silk) (Family:Lepidoptera:Saturniidae),
the Indian eri silkworm, contributes significantly to the production of
commercial silk, and is widely distributed in the Brahmaputra river valley in
North-Eastern India (Vijayan et al 2006). Eri silkworm (Samia ricini) is a
traditional source of food in northeast India, where it is grown primarily for
silk and food uses (Longvah et al 2011). The eri silkworm is polyphagous in
nature and feeds on leaves of several food plants (Rajesh Kumar and Gangwar
2010). The eri silk worm feeds on the leaves of a variety of plants, but ‘Castor
plant’ (Ricinuscommunis) is the most important host plant. The leaves of
plants like kesseroo (Heteropanaxfragrans), ‘Gomari’ (Gmelinaarborea),
‘Gulancha’ (Tinosporacordifolia) etc., are also used (Mishra et al 2003). The
27
study shows that the castor plant feed yields more silk than other plant feeds.
Eri silkworm is multivoltine in character, and can be reared indoors. It is
generally hardy and not susceptible to diseases. It belongs to the family
Saturnidae and species ricini. The most widely domesticated eri silk worm is
Samia cynthia ricini (Kulkarni 2007).
2.5.4.1 Life cycle of eri silk
Eri silk worm has four stages; the egg, larva, pupa and the adult or
moth shown in Figure 2.5. The moth lays white eggs, which turn grey then
black just before hatching. Eggs hatch in seven days in hot weather, but may
take as long as 24 days in cold weather. Female moths lay eggs in clusters that
may contain as many as 100 or more eggs. During the first to the third larva
stage, the head is black and shiny but eventually will turn greenish-yellow or
yellow with a brownish patch on each cheek, when they reach the fourth and
fifth stage. In the fifth stage, the larvae eat enormously and grow very quickly
to their maximum stage of development. The well fed, full grown larvae are
cylindrical and about 90 -100 mm long. The general body colour is white,
which turns yellow before spinning (Capinera 2008).
28
Figure 2.5 Life cycle of eri silk worm
Figure 2.6 Composition of the eri silk gland
29
2.5.4.2 Composition of eri silk gland
Similar to the mulberry silk, the eri silk filament is extruded from
the silkworm; it consists of two portions, the fibroin and the sericin. The
sericin is glue like gum that covers the core filament. Both fibroin and sericin
are produced by very large flattened cells lining a pair of long tubular silk
glands as shown in Figure 2.6. The silk gland is divided into three regions, the
thin and flexuous posterior part, and the wider middle and anterior parts. The
silk fibroins are synthesized in the posterior part of the silk gland, and then
transported down the lumen into the middle part of the silk gland, in which it
is stored in a concentrated state as a weak gel before spinning. It has been
pointed out that the silk press part is important in the process of fibre
formation from the liquid silk fibroin (Asakura et al 2007).
Table 2.2 Chemical composition of eri silk bave (Manuals on sericulture 1987)
Component Percentage %
Fibroin 72.2
Sericin 5-11.9%
Fat 1.3
Moisture 14.6
Eri silk is mainly made up of fibroin, sericin and fat. Sericin is a
non filament which is dissolved in water, whereas the fibroin is a filament
covered by the sericin. The percentage of chemical composition of eri silk is
listed in Table 2.2. Eri silk contains low amount of sericin of 4.96% as
compared to 10% in mulberry silk, 8.62% in tassar silk and 7.88% in muga
silk (Mishra 2000).
30
2.5.4.3 Physical properties of eri silk
Eri cocoons are usually white in colour; however, brick red colour
cocoons are also available. Eri silk cocoons cannot be reeled as they are made
up of entangled layers, and are therefore spun like cotton into yarn. The
reeling process does not involve the killing of silk moth, as it is an open-
mouthed cocoon (Sarkar 1980). The fineness of eri silk ranges from 14 to
16µ. The average filament length of eri silk is approximately 450 meters. Eri
silk is durable and strong with a typical texture. Eri silk is similar to cotton
and has a unique aesthetic appeal. It appears like wool mixed with cotton and
the softness of silk. Each eri cocoon weighs about 1 to 5 g with a shell weight
of 0.2 to 0.7 g. The denier (d) of the filament is 2.2 to 2.5 d, with a tenacity of
3 to 3.5 g/d. Eri silk has an elongation percentage of up to 20-22. It has
excellent thermal properties, and can be substituted for wool. The moisture
retention capacity is 11 %. Eri silk is more crystalline than any other
non-mulberry silks (Sreenivasa et al 2005); also, eri silk has a higher
elasticity, strong durability, and immunity against disease and insects.
2.5.4.4 Chemical properties of eri silk fibroin
Eri silk fibroin consists mainly of repeated similar sequences (about
100 times) of alternative appearances of the polyalanine (Ala) 12–13
region and
the Gly-rich region (Nakazawa et al 2009). Eri silk is a protein, which is
containing two major amino acid residues alanine and glycine. Samia cynthia
ricini is a wild silkworm and the amino acid composition of the silk fibroin is
different from that of the silk fibroin of the domesticated silkworm Bombyx
mori. The sum of Gly and Ala residues in eri silk is 82% which is similar to
mulberry silk (71%), but the relative composition of Ala and Gly is reversed
(Asakura et al 1999). The primary structure of eri silk fibroin is composed of
alternate blocks of polyalanine regions and glycine-rich regions. The alanine
is dominant in the crystalline region of the silk fibroin, whereas the glycine
31
(Gly) motif provides elasticity to the amorphous region of the silk fibroin.
The eri silk fibroin is in the form of - helix and random coil structure in the
silkworm gland as (silk I structure) shown in Figure 2.7 and the silk II
structure is present in the spun silk filament after spinning, which is attributed
to the - sheet in the silk fibroin (Rousseau et al 2006, Asakura et al 1999).
Three major polypeptides are found in the eri silk with different molecular
weights of 97 KDa, 45KDa and 66 KDa. The molecules of 66 KDa represent
sericin, where as 97KDa and 45KDa indicate the presence of polypeptides,
which are connected by a disulfide bond in the silk fibroin. The eri silk fibroin
aqueous solution contains 70% alanine in a helix structure, and the rest of
alanine in the form of a random coil structure. The silk liquid transition from
- helix to structure by a thermal or mechanical method was observed by
Asakura et al (1988) and Nakazawa et al (1999).
Figure 2.7 – Helix structure of silk fibroin (Nakazawa and Asakura 2003)
32
2.5.4.5 Difference between mulberry silk and eri silk fibroin properties
2.5.4.5.1 Amino acid composition
Mulberry and eri silk fibroins contain three major amino acids viz.,
glycine, alanine and serine as shown in Table 2.3. The total composition of
the three amino acids of the eri silk fibroin (84.26%) is higher than that of the
mulberry fibroins (82.8%). The eri silk fibroin has higher hydrophilic to
hydrophobic ratio than the mulberry silk fibroin, which indicates that the eri
silk fibroin contains less amount of hydrophobic amino acid than the
mulberry silk fibroin. The eri silk possesses higher amount of sulfur content
amino acids (cystine and methionine) than the mulberry silk fibroin, which
helps to connect the heavy and light weight chain in the silk fibroin. The eri
silk fibroin possesses a higher ratio of basic to acidic amino acids, as well as a
substantially greater proportion of positive arginine and negative aspartic acid
residues. The positive charged amino acid supports cell growth and
attachment on the eri silk fibroin scaffold better than on mulberry silk (Sen
and Babu 2004, Mai-ngam et al 2011).
2.5.4.5.2 Moisture regain
The moisture regain percentage of eri silk fibroin is higher than that
of the mulberry silk fibroin due to the higher proportion of amino acids with
bulky side groups, and also the higher hydrophilic to hydrophobic amino acid
ratio (9.06–9.85) for eri silk fibroin, compared to that of the mulberry silk
(5.29–6.22). Moisture regain is crucial for cell attachment and spreading on
the silk fibroin scaffold (Sen and Babu 2004).
33
Table 2.3 Amino acid composition of eri and mulberry silk fibroin
Amino acids Eri silk (mol %) Mulberry silk (mol
%)
Aspartic acid 3.89 1.64
Glutamic acid 1.31 1.77
Serine 8.89 10.38
Glycine 29.35 43.45
Hystidine 0.75 0.13
Arginine 4.12 1.13
Threonine 0.18 0.92
Alanine 36.33 27.56
Proline 2.07 0.79
Tyrosine 5.84 5.58
Valine 1.32 2.37
Methionine 0.34 0.19
Cystine 0.11 0.13
Isoleusine 0.45 0.75
Leucine 0.69 0.73
Phenylalanine 0.23 0.14
Tryptophan 1.68 0.73
Lysine 0.23 0.23
2.5.4.5.3 Thermal behavior
Thermal analysis is a useful tool for monitoring the important
processing parameters and properties of textiles. In addition to other factors,
the response of a fibrous polymer to thermal treatment depends on its
chemical architecture and microstructure. Thermal treatments bring about
34
morphological changes in silks that will have a bearing on the mechanical and
other properties. The thermal stability of silk varies from mulberry to wild
silk. The thermal stability of wild silk fibroin ( tassar, muga and eri) is higher
than that of the mulberry silk fibroin, because of the higher ratio of
bulky/non-bulky amino acids (0.24-0.32) in wild silk fibroins compared to
that of mulberry (0.17-0.18), and also the presence of higher amount of
(Ala)n sequences in the crystalline regions (Babu and Sen 2007). Thermal
stability is essential for sterilizing purposes in biomedical applications.
2.5.4.6 Mulberry and non – mulberry silk fibroin for tissue
engineering applications
The silk protein fibroin, isolated from the cocoon of the
domesticated mulberry silkworm, Bombyx mori, is used extensively in
biomaterial design and tissue culture. The study by Acharya et al (2009) on in
vitro cell culture on Antheraea mylitta (A.mylitta) as a substrate, showed the
higher mechanical strength of A. mylitta , better adherence, growth and
proliferation patterns of feline fibroblast cells on antheraea mylitta fibroin
films compared to that of mulberry fibroin films. The antheraea mylitta silk
fibroin scaffolds are used for tissue engineering applications in different
forms, such as film, foam, salt leach, sponge and nanofibre. The Antheraea
pernyi silk (Oak silkworm) fibroin supported the attachment and growth of
human bone marrow mesenchymal stem cells (hBMSCs). Compared to the
mulberry silk fibroin, the Antheraea pernyi (A. pernyi) silk fibroin contains a
special [Arg - Gly - Asp] (RGD) tripeptide sequence, which favours the cells
to attach. It also contains a certain amount of amino acid with positive
charges, thus the A. pernyi silk fibroin is more beneficial for cells of human
beings and many kinds of mammals to adhere and proliferate than the of
mulberry silk fibroin (Tao et al 2009). The Antheraea pernyi silk film made of
nanofibre was similar to the extracellular matrix (ECM) on the nanoscale,
35
which promoted cell migration and proliferation (Mao et al 2012). Tassar silk
fibroin (TSF) nanofibre is the most favourable silk fibroin material for
supporting the attachment and growth of neurons (Qu et al 2010), and the TSF
is more compatible for the development of neurons than mulberry silk fibroin,
suggesting the potential use of tassar silk fibroin for preparing the tissue-
engineered nerve guides to treat nerve injuries or diseases. Tassar electrospun
nanofibre is very suitable for developing human embryonic stem cells
(hESCs)-derived neural precursors (NPs) (Wang et al 2012). The Antheraea
assama (muga) fibroin based micro / nanofibrous nonwoven scaffold
possessed good bio-compatibility, and blood compatibility and the scaffold
was found to be nontoxic and efficient in supporting cell adhesion and growth
(NareshKasoju et al 2009). Eri silk fibroin films exhibited greater adhesion,
proliferation rate and spreading of cells than mulberry fibroin films. The
better cell supporting properties of eri silk fibroin may be mainly governed by
the initial non-specific binding between the serum proteins and the substrate.
The greater compositions of hydrophilic and positively charged amino acids
of the non-mulberry fibroin molecules may result in appropriate hydrophobic
and electrostatic interactions, allowing the adsorption of protein layer with
proper composition and conformation for cell adhesion and spreading. Eri silk
fibroin provides better biomaterial scaffold design than the more commonly
used mulberry fibroin (Mai-ngam et al 2011). When compared with mulberry
fibroin, wild silk materials are used very limitedly in biomaterial applications,
because most of the wild silk varieties are rare in the world, apart from the
Asian region. Among the wild silks, the tassar silk fibroin is used for tissue
engineering in different forms, but the muga silk and eri silk fibroins are
hardly used for tissue engineering. Mai-ngam et al (2011) carried out research
on eri silk fibroin scaffold for tissue engineering in the form of films.
36
2.6 MINERALS AND ANTIBIOTICS USED AS BIO
MATERIALS
2.6.1 Hydroxyapatite
Hydroxyapatite (Hap) (Ca10 (PO4)6 (OH), is an important inorganic
biomaterial which has attracted the attention of researchers related to the
biomaterial field in recent years. Due to its chemical and structural similarity
with the mineral phase of bones and teeth, hydroxyapatite (Hap) has been
used clinically for many years. It has good biocompatibility in bone contact as
its chemical composition is similar to that of bone material (Porous
hydroxyapatite for artificial bone applications). Hydroxyapatite exhibits
excellent biocompatibility with soft tissues such as skin, muscle and gums
making it an ideal candidate for orthopedic and dental implants or
components of implants (Zhou and Lee 2011). The Hap has higher
crystallinity and higher chemical stability (Agrawal et al 2011). The
advantage of the electro spun biopolymer with Hap is higher strength and
enhanced hydrophilicity of the scaffold (Ito et al 2005). Hydroxyapatite
ceramics have been used as tissue engineering material for cell culture and
tissue repair due to their biocompatibility and osteo-conductivity. However,
brittleness and fatigue failure in the body of Hap ceramics limit their clinical
applications only for repair and substituting purposes. It has been found that
each category of bio-ceramic and polymer cannot fulfill well the demand of
bone repair application by itself. Therefore, hydroxyapatite/polymer bio-
composite offers a possible combination of the advantages of the two
biomaterials (Jie and Yubao 2004).
37
2.6.2 Amoxicillin Drug
Amoxicillin is a broad spectrum antibiotic effective against various
types of microorganisms, but it possesses a short biological half life of about
60 minutes. Hence, repeated administration is needed to maintain the blood
plasma concentration of amoxicillin (Ramesh et al 2010). In order to reduce
the adverse effects due to frequent dosing, there is a need of a controlled
release formulation. Hence, the nanoparticles of amoxicillin were prepared by
using naturally available, nontoxic, low cost polymer obtained from natural
biodegradable polymers. Amoxicillin has been used for various infections,
including septic absorptions, urinary tract infections, upper and lower
respiratory infections, skin and soft tissue, and gastro-intestinal tract
infections (Patel et al 2007). The amoxicillin is a poorly soluble broad-
spectrum antibiotic; hence, this drug is added with bio degradable polymers to
maintain a sustainable drug release.
2.7 DIFFERENT FORMS OF SILK BIOMATERIAL
Silk biomaterials can be prepared directly from silk, or regenerated
from silk fibroin solutions. An alternative way is to convert silk into ultrafine
particles through milling. Figure 2.8 shows a schematic diagram of processing
silk into various forms of diverse morphologies, which are used for tissue
engineering, wound dressing and drug delivery (Rajkhowa et al 2010).
38
Figure 2.8 Different forms of silk biomaterial
2.8 TYPES OF SCAFFOLDS AND PROCESSING TECHNIQUES
2.8.1 Felts or Meshes
Polyglycolic acid (PGA) in the form of tassels and felts were
utilized as scaffolds to demonstrate the feasibility of organ regeneration.
Meshes consist of either woven or knitted three-dimensional patterns of
39
variable pore sizes are used as scaffolds. The advantageous characteristic
features of meshes are large surface area for cell attachment and rapid
diffusion of nutrients in favour of cell survival and growth. However, they
lacked the structural stability necessary for in vivo use, which led to the
development of bonding techniques (Yang et al 2001, Mikos et al 1993).
2.8.2 Bonding
The bonding was prepared by different techniques such as chemical
bonding and composite. PGA fibres were aligned in the shape of the desired
scaffold and then embedded in PLLA- methylene chloride solution. After the
evaporation of the solvent at room temperature, the PLLA-PGA composite
was heated above its melting temperature and then cooled in a room
atmosphere to produce the desired scaffold. This technique is not suitable for
producing fine porosity control (Salgado et al 2004).
2.8.3 Phase Separation
The polymer is dissolved in solvents such as molten phenol,
naphthalene or dioxane at a low temperature (Lo et al 1996). Liquid – liquid
or solid – liquid phase separation is induced by lowering the solution
temperature. The subsequent removal of the solidified solvent rich phase by
sublimation leaves a porous polymer scaffold. One major advantage is to
incorporate the bioactive molecules into the matrix, without decreasing the
activity of the molecules, due to the harsh, chemical and thermal
environments. A slight change in the parameters, such as types of polymer,
polymer concentration, solvent/non solvent ratio, and most importantly, the
thermal quenching strategy, significantly affects the resultant porous scaffold
morphology (Nam and Park 1999).
40
2.8.4 Solvent Casting and Particulate Leaching
This is one of the methods of the porous scaffold preparing
technique. This method involved mixing of water-soluble salt particles into a
biodegradable polymer solvent solution. The mixture was then casted into the
desired shape mould, and the solvent was removed by vacuum drying and
lyophilisation. The water-soluble salt particles were then leached out with
water to leave a porous structure. This method is characterised by its simple
operation and adequate control of the pore size, and the porosity of material is
determined by the amount of salt, and the size of the particle added to the
solution. In this method, the distribution of salt is not uniform within polymer
solution (Mikos et al 1994). The cast solvent and particulate method is used to
produce a two dimensional structure, with a thickness between 500 and
2000µm. In this method, it is very difficult to produce a scaffold of a
thickness of more than 3000µm (Yang et al 2001).
2.8.5 Melt Molding
This process involves physically mixing a polymer with the defined
amount of calibrated leachable particles and loading this powder into a mould.
This is followed by the application of heat and pressures that result in the
melting of the polymer. The compression maximizes the packing of the
mixture. The heating process causes the fusion of the polymer, and promotes
the formation of a continuous polymeric network that gives mechanical
stability to the structure. The last stage consists of immersing the moulded
polymer-porogen composite in a solvent that selectively dissolves the porogen
agent. This methodology has been successfully applied in the production of
natural origin starch-based scaffolds. The drawback of the melt moulding
scaffold processing is the requirement of high temperature (Gomes et al 2002,
Correlo et al 2009).
41
2.8.6 Polymer- Ceramic Composite Foam
In solvent-casting technique the hydroxyapatite short fibres and the
porogen are dispersed in a PLGA/ methylene chloride solution. After the
solvent evaporation, leaching of the porogen leaves the open-cell porous
composite foam of PLGA reinforced with hydroxyapatite short fibres. With a
certain range of fibre content, these scaffolds have superior compressive
strength compared to non -reinforced materials of the same porosity (Yang et
al 2001).
2.8.7 Gas Foaming
Highly open porous biodegradable poly(L-lactic acid) (PLLA)
scaffolds for tissue regeneration were fabricated, by using ammonium
bicarbonate as an efficient gas foaming agent, as well as a particulate porogen
salt. A binary mixture of PLLA-solvent gel containing dispersed ammonium
bicarbonate salt particles, in a paste form, was cast in a mould, and
subsequently immersed in a hot water solution, to permit the evolution of
ammonia and carbon dioxide within the solidifying polymer matrix. This
resulted in the expansion of pores within the polymer matrix to a great extent,
leading to well interconnected macro porous scaffolds, having mean pore
diameters of around 300–400 µm. The major disadvantage of the gas foaming
technique is the failure to obtain uniform porosity in the scaffold (Nam et al
2000, Salerno et al 2009).
2.8.8 Membrane Lamination
Membrane lamination is another solid freeform fabrication
technique (SFF) used for constructing three-dimensional biodegradable
polymeric foam scaffolds with precise anatomical shapes. Membrane
lamination is prepared by solvent casting and particle leaching, and
42
introducing peptides and proteins layer by layer during the fabrication
process. The membranes with appropriate shapes are soaked in the solvent,
and then stacked up in three-dimensional assemblies with a continuous pore
structure and morphology (Maquet and Jerome 1997). The bulk properties of the
final 3D scaffolds are identical to those of the individual membranes. This method
generates the porous 3D polymer foams with defined anatomical shapes, since it is
possible to use computer assisted modeling to design a template with the desired
implant shape. The disadvantages of this technique are the layering of porous
sheets, resulting in lesser pore interconnectivity (Hutmacher et al 2000, Hutmacher
et al 2001), and the fact that it is a time consuming process since, only thin
membrane can be used in this process (Subia et al 2010).
2.8.9 Freeze Drying Method
The freeze drying technique is also used for the fabrication of
porous scaffolds (Whang et al 1995, Schoof et al 2001). This technique is
based upon the principle of sublimation. Polymer is first dissolved in a
solvent to form a solution of the desired concentration. The solution is frozen
and the solvent is removed by lyophilisation under the high vacuum, that
fabricates the scaffold with high porosity and inter connectivity (Mandal and
Kundu 2009a, 2009b). This technique is applied to a number of different
polymers including silk proteins (Vepari and Kaplan 2007, Altman et al 2003),
PGA, PLLA, PLGA and PLGA/PPF blends. The pore size can be controlled by
the freezing rate and pH; a fast freezing rate produces smaller pores. Controlled
solidification in a single direction has been used to create a homogenous 3D-pore
structure (Schoof et al 2001). The main advantage of this technique is that, it
neither requires high temperature nor a separate leaching step. The drawback of
this technique is the smaller pore size, long processing time and poor homogeneity
of the pore structure (Boland et al 2004, Hou et al 2003). The freeze drying
technique is used to make silk protein porous scaffolds for tissue engineering
applications (Mandal and Kundu 2008a, 2008b, 2009, Kundu et al 2008).
43
2.8.10 Knitted Scaffold
A knitted scaffold is a structure made from the interlacing of yarn.
The knitted scaffold was made from silk filament, PLA, PCL and silk etc.
(Chen et al 2010) for tissue engineering applications. The knitted scaffolds
provide a suitable architecture and mechanical properties to withstand the
stresses faced by tissue. The knitted micro fibrous scaffold possesses an
interconnected porous structure allowing better tissue growth and nutrient
supply. The knitted structure is most adequate scaffolding for ligament tissue
engineering (Sahoo et al 2007). The major drawback of the knitted structure is
that controlling and homogeneous cell seeding could be very difficult to
achieve, due to high porosity and larger hole size in the knitted scaffold
(Vaquette et al 2009).
2.8.11 Braided Scaffold
Three-dimensional braiding is defined as a system, where three or
more braiding yarns are used to form an integral braided structure, with a
network of continuous filaments and yarn bundles with fibrous architecture,
oriented in various directions. Three dimensional braiding systems can
produce thin and thick structures in a wide variety of shapes through the
selection of the yarn bundle size. The braided scaffolds are mostly fabricated
for ligament growth due to their tensile strength. The advantage of the braided
scaffold is the interconnected network of porous structure, which supports the
transportation of oxygen and nutrients to the implant site (Cooper et al 2005).
Drawback of the braided structure is poor cell seeding due to the limited
internal space (Ouyang et al 2003).
44
2.8.12 Electrospun Fibrous Scaffold
Electrospinning is a unique process to produce polymeric fibres in
the diameter range of 100 nm -5 m. Fibres produced by this approach are at
least one or two orders of magnitude smaller in diameter, than those produced
by conventional production methods like melt or solution spinning
(Srinivasan and Reneker 1995, Subbiah et al 2005). Based on earlier research
results, it is evident that the average diameter of the electrospun fibres ranges
from 100 nm–500 nm. In textile and fibre science related literature, fibres
having diameters in the range of 100 nm–500 nm are generally referred to as
nanofibres. Electrospun fibres have a small pore size and high surface area to
volume ratio. There is also evidence of sizable static charges in electrospun
materials that could be effectively handled to produce three dimensional
structures.
The stable electrospinning jet was described in detail by Reneker
and Chun (1996) as being composed of four regions: the base, the jet, the
splay and the collection. Electrospinning is aided by the application of the
high electric potential of a few kV magnitudes to a pendant droplet of
polymer solution/melt from a syringe or capillary tube as shown in Figure 2.9
(Kumbar et al 2008). A polymer jet is ejected from the surface of a charged
polymer solution when the applied electric potential overcomes the surface
tension. The ejected jet under the influence of applied electrical field, travels
rapidly to the collector and collects in the form of a non-woven web as the jet
dries (Reneker et al 2000). Before reaching the collector, the jet undergoes a
series of electrically driven bending instabilities in the base region and
emerges from the needle to form a cone known as the Taylor cone (Hsu and
Shivkumar 2004). The shape of the base depends upon the surface tension of
the liquid and the force of the electric field; jets can be ejected from surfaces
that are essentially flat if the electric field is strong enough. The charging of
45
the jet occurs at the base with solutions of higher conductivity being more
conducive to jet formation (Pham et al 2006). The diameter of the electrically
charged jet decreases under electro-hydrodynamic forces, and under certain
operating conditions this jet undergoes a series of electrically induced bending
instabilities during passage to the collection plate, which results in extensive
stretching. The stretching process is accompanied by a rapid evaporation of
the solvent, which leads to a reduction in the diameter of the jet (Sukigara et
al 2003).
Doshi and Reneker (1995) classified the parameters (shown in
Table 2.4) that control the process in terms of solution properties, controlled
variables and ambient parameters. The solution properties include viscosity,
conductivity, surface tension, polymer molecular weight, dipole moment, and
dielectric constant. The fibre diameter can be controlled by varying the
processing parameters, such as polymer solution concentration, viscosity,
applied charge and electric field, type of solvent employed, distance from the
tip of the capillary to the collection plate, flow rate, diameter and angle of
spin of the spinneret (Pham et al 2006). In 1969, Taylor derived the condition
for the critical electric potential needed to transform the droplet of liquid into
a cone (commonly referred to as the Taylor cone), and to exist in equilibrium
under the presence of both electric and surface tension forces as given in
Equation (2.1).
2 2 2
c
2L 3v 4H L 0 117 R
R 2/ . (2.1)
where Vc is the critical voltage, H is the distance between the capillary tip and
the ground, L is the capillary length, R is the capillary radius and is the
surface tension of the liquid (Lyons 2004).
46
Table 2.4 Process parameters of electrospinning (Pham et al 2006)
Process parameters Effect on morphology of fibre diameter
Viscosity/concentration Low concentrations/viscosities yielded defects in the
form of beads and junctions; increasing
concentration/viscosity reduced the defects
Conductivity/solution charge
density
Fibre diameters increased with increasing
concentration/viscosity
Increasing the conductivity aided in the production of
uniform bead-free fibres
Higher conductivities yielded smaller fibres in general
Surface tension No conclusive link established between surface tension
and fibre morphology
Polymer molecular weight Increasing molecular weight reduced the number of
beads and droplets
Dipole moment and
dielectric
Successful spinning occurred in solvents with a high
dielectric constant
Flow rate Lower flow rates yielded fibres with smaller diameters
High flow rates produced fibres that were not dry upon
reaching the collector
Field strength/voltage At too high voltage, beading was observed
Correlation between voltage and fibre diameter was
ambiguous
Distance between tip and
collector
A minimum distance was required to obtain dried fibres
At distances either too close or too far, beading was
observed
Needle tip design Using a coaxial, 2-capillary spinneret, hollow fibres
were produced
Multiple needle tips were employed to increase the
throughput
Collector composition and
geometry
Smoother fibres resulted from metal collectors; more
porous structure was obtained using porous collectors
Aligned fibres were obtained using a conductive frame,
rotating drum, or a wheel-like bobbin collector
Yarns and braided fibres were also obtained
Ambient parameters Increased temperature caused a decrease in the solution
viscosity, resulting in smaller fibres
Increasing humidity resulted in the appearance of
circular pores on the fibres
47
Figure 2.9 Electrospinning set up
2.8.12.1 Nanofibre scaffolds for tissue engineering
The nanofibres are extremely thin fibres with diameters ranging
from microns down to a few nanometers. Such small-size fibres could
physically mimic the structural dimension of the extracellular matrix of a
great variety of native tissues and organs, which are characterized by well
organized hierarchical fibrous structures realigning from nanometer to
millimeter scale. The scaffolds produced provide a highly porous
microstructure with interconnected pores and extremely large surface area to
volume ratios, which are conducive to tissue growth. They are very versatile
and allow the use of a variety of polymers, blends of different polymers, and
inorganic materials as well as the integration of additives, biomolecules and
living cells for tailoring different application requirements. The
electrospinning process is a simple, straightforward and cost-effective method
to make various types of scaffolds (Zhang et al 2005).
Tissue engineering has emerged as a promising alternative
approach to treat the loss or malfunction of a tissue or organ without the
limitations of current therapies. Tissue engineering involves the expansion of
48
cells from a small biopsy, followed by the culturing of the cells in temporary
three-dimensional scaffolds to form the new organ or tissue by using the
patient's own cells as shown in Figure 2.10.This approach has the advantages
of autografts, but without their associated problems of inadequate supply. The
porous three-dimensional temporary scaffolds play an important role in
manipulating cell function and guidance of new organ formation (Chen et al
2002). Tissue engineering holds great promise as an alternative strategy to
current treatment modalities of diseased or otherwise failed tissues. Most
strategies of tissue engineering rely on three-dimensional porous scaffolds to
mimic the natural extracellular matrix (ECM) as templates, onto which cells
attach, multiply, migrate and function. When cells are harvested from a donor
and seeded, scaffolds facilitate the organization of these cells into a three-
dimensional architecture, control cell behavior and subsequently direct the
formation of organ-specific tissue (Johnson et al 2010).
For tissue engineering, various forms of scaffolds such as sponge,
foam, film, woven, knitted and non-woven materials are being used. The
nanofibrous matrix has a much higher surface-to-volume ratio than those of
fibrous non-woven fabrics fabricated with the textile technology, or foam
fabricated with other techniques (Smith et al 2009). As biomaterials for tissue
engineering, electrospun meshes exhibit important advantages when
compared with other scaffolds. First, the interconnectivity of the voids
available for tissue ingrowths is beneficial compared to foams and sponges.
Second, ultra thin fibres produced by electrospinning offer an unsurpassed
surface- to- volume ratio among established tissue scaffolds. The latter is
expected to have important advantages on the availability and activity of
immobilized molecules (e.g. peptides, lectines, enzymes etc.). Thus,
electrospun fibres have been explored as better extra cellular matrix for tissue
engineering, novel carriers for bioactive drugs and filtrations for bimolecules.
49
Besides the three-dimensional structure, biocompatibility and biological
activity is required, and this remains a major challenge for tissue engineering
applications (Burger et al 2006). Several methods have been developed to
fabricate highly porous biodegradable scaffolds including fibre bonding,
braiding, solvent casting, particle leaching, phase separation, emulsion freeze
drying, gas foaming and 3D- printing techniques. However, the simplicity of
the electrospinning process to generate nanofibres makes it an ideal process
for scaffold fabrication (Gandhi 2006).
Figure 2.10 Tissue culture on electrospun nanofibrous scaffolds
2.8.12.2 Other application of electrospun fibres
Electrospun nanofibrous mats are used for various applications as
shown in Figure 2.11 (Huang et al 2003)
50
Figure 2.11 Electrospun nanofibre applications
2.8.12.2.1 Filtration
The nanofibrous filter had been widely used in both households and
industries for removing substances from air or liquids. Filters for environment
protection are used to remove pollutants from air or water. In the armed force,
they are used in uniform garments and isolating bags to decontaminate
aerosol dust, bacteria and even virus. The respirator is another example that
requires an efficient filtration function. A similar function is also needed for
some fabrics used in the medical area (Fang et al 2008). Conventional
mechanical fibrous filters made of microfibres exhibit minimum fractional
collection efficiency for the aerosol particle size ranging between 100 and 500
nm, which is called the most penetrating particle size (MPPS). Simple
theoretical calculations predict that this efficiency may be significantly
increased using nanofibrous media (Podgorsk et al 2006). Electrospun
nanofibres have the diameters that are 5-10 times smaller than the smallest
51
melt blown filter. The nanofibrous filter possesses some special properties
such as smaller fibre size and higher pressure drop, which cause interception
and inertial impaction efficiencies higher than that of conventional melt
blown nonwoven filters (Grafe and Graham 2003).
2.8.12.2.2 Wound dressing
Electrospun nanofibre based wound dressing material potentially
offers many advantages than conventional wound dressing. Nanofibrous mat
provides intrinsic properties such as high surface area and micro porous
structure, which affords quick start signaling pathway and attracts fibroblasts
to the derma layer that can excrete important extracellular matrix components,
such as collagen and several cytokines (e.g., growth factors and antigenic
factors). The electrospun membrane enhances the cell attachment and
proliferation in wound healing (Chen et al 2008). The electrospun nanofibre
mats usually have pores which are small enough to prevent bacterial
penetration. The high surface area is of importance for fluid absorption and
dermal drug delivery. Although plenty of polymers have been successfully
electrospun into nanofibres, reports on electrospun nanofibrous mats suitable
for wound dressings are still scarce (Kanani and Bahrami 2010). Electrospun
nanofibrous membrane wound dressings can also meet the requirement of
high gas permeation, apart from providing effective protection of the wound
against infection and dehydration. Electrospun nanofibre can be easily
incorporated with pharmaceutical compounds, such as antiseptics, antifungal,
vasodilators and cell growth factor etc. But conventional wound dressing is
not provided with the feasibility for drug incorporation; and another major
advantage over conventional wound dressing is that the nano fibres hold a
promise of healing wounds without leaving scars (Zahedi et al 2010).
52
2.8.12.2.3 Drug delivery
Many therapeutic compounds can be conveniently incorporated into
the electrospun fibres through the electrospinning process, which is unlike
common encapsulation methods involving some complicated preparation
processes. At present, both degradable and non-degradable polymers are
under investigation to be developed as drug carriers for local delivery of
antibiotics and anticancer drugs, and electrospun fibrous materials with
different structures are the preferred selection. The advantages of the
electrospinning technique are, maintaining of molecular structure and
bioactivity of the incorporated drugs or bioactive molecules due to the mild
process conditions, and reducing the burst release of drugs in vitro. Ignatious
et al (2010) defined that the release of pharmaceutical dosage from nanofibres
can be designed as rapid, immediate, delayed or modified dissolution
depending on the polymer carrier used for drug delivery. The biocompatible
and biodegradable polymer fibre mats produced by electrospinning methods,
are able to serve both as drug encapsulation vehicles and tissue engineering
scaffolds (Venugopal and Ramakrishna 2005). The release profile can be
finely controlled by the modulation of nanofibre morphology, porosity and
composition. Nanofibres for drug release systems are obtained mainly from
biodegradable polymers and hydrophilic polymers.
2.8.12.2.4 Other applications of nanofibres
Nanofibres fabricated through electrospinning have a specific
surface, approximately one to two orders of magnitude larger than flat films,
making them excellent candidates for potential applications in sensors. The
sensitivity of chemical gas sensors is strongly affected by the specific surface
of the sensing materials. A higher specific surface of sensing material leads to
53
a higher sensor sensitivity; therefore, many techniques have been adopted to
increase the specific surface of sensing films with fine structures, especially to
form nanostructures, taking advantage of the large specific surface of nano
structured materials. Electrospun nanofibrous materials have attracted
considerable interest in the food industry for their utilization as highly
functional ingredients, and high-performance packaging materials. The
nanofibres are used as filtration membranes for environmental remediation,
which minimize the pressure drop and provide better efficiency than
conventional fibre mats. The large surface area-to-volume ratio of the
nanofibre membrane allows greater surface adsorption of contaminants from
air and water, and increases the life-time of the filtration media. It is also used
in solar and fuel cell applications (Ding et al 2009).
Recommended