SILK is the sticky material surrounding fibroin and

                                  SILK

Silk
consist of two main proteins sericin and fibroin which is emitted by silkworm.
Silk fibroin is produced by domestic silkworm Bombyx mori and from spiders (Nephila clavipes
and Araneus diadematus) it is
also a natural protein.

Chemical structure:

R=H,
glycine
R=CH3, alanine
R=CH2OH,
serine

 

Both the proteins have 18 similar amino acids such as
glycine, alanine and serine in variable amounts. Sericin is the sticky material surrounding
fibroin and fibroin is the structural centre of silk. Fibroin is largely made
up of amino acids Gly-Ser-Gly-Ala-Gly-Ala and forms beta pleated sheets,
?-keratin1. There are many different silk polymorphs which generally seen in
(silk I ) water soluble state and comes in glandular state before
crystallization ,(silk II) which is often seen in spun  silk state and air/water assembled
interfacial silk usually in helical structure (silk III). Silk I is usually
exposed to heat or physical heat spinning to convert it to silk II, it can be
easily done as silk II structure consist of ?- sheet secondary structure. Silk
I in aqueous condition when exposed to methanol or potassium chloride, the
surface of the ?-sheet structure is asymmetrically divided into hydrogen side
chains and methyl side chains. Hydrogen bonds and van der Waals forces
interacts with the methyl group and hydrogen groups to make the inter-stacking
sheets of crystal to be thermodynamically stable2. Silk II structure at the
later stage deny water and becomes less or completely not soluble in several
solvents very mild acidic and basic conditions.

The
structure represents a tight packing of stacked sheets of hydrogen bonded in an
anti-parallel chain of protein. Hydrogen bonds form between chains, and side
chains form above and below the plane of the hydrogen bond network. Fibroin
contains a high proportion of three ?- amino acids (G;
Gly, 45%, R=H), alanine (A; Ala, 29%, R=CH3), and serine (S; Ser,
12%, R=CH2OH) the approximate molar weight of these amino acids is
3:2:1 while, the remaining 13% consist of Tyrosine, valine, aspartic acid etc.
Glycine has a high proportion (50%) which allows it to tight packing this is
because its R-group has only one hydrogen and, so it is not sterically
constrained. Alanine and serine has many interceded hydrogen bonds and are
strong and resistant to breaking. The less crystalline forming regions are
known as linkers which consists of fibroin heavy chain they are situated in
between 42-44 amino acid residues in length. All linkers do have identical
amino acid residues which are charged amino acid residues found in crystalline
region. Primary sequence of proteins is highly repetitive which provides
homogeneity in the secondary structure. Primary sequence generates hydrophobic
proteins which are in natural co-polymer block design. The interspace is filled
with many hydrophobic and hydrophilic domains, large hydrophobic domains
interspace with smaller hydrophilic domains to bolster the assembly of silk and
improves the strength and resiliency of fibre.

Physical form

Silk
fibre is known for its smooth and soft structure which is not slippery, as compared
to numerous synthetic fibres .They are also known for being lightweight,
breathable, hypoallergenic and good absorbency. The average diameter of depends
upon the type and variety of silk fibroin and the condition of spinning. The diameter
for a bave of b.mori  is 15-25?m and for other
silk like Tasar is about 65?m for a bave.The diameter of the filaments
of silk fibroins are different for each variety as the location of cocoonare
coarser on the outside and are finer at the inside. The cross section of filament
of b.mori  fibroin is a swollen triangle of 10?  across while some other silk fibroin
like Tasar fibroin have wedge shaped cross section. Longitudinally, the
filament of b.mori  appears to be solid rod without any marking,
while Tasar silk has longitudinal weaves.

 

Classifications:

Silk
has used in textile industry are different from the those used in biomedical
application some types of silk used in biomedical application is listed in
sequence:

        1.Silk worm silk (Bombyx mori):

Silk obtained from cocoon of Bombyx mori are commonly used
for biomedical and textile production. Sericulture is commonly known for
breeding of silks for commercial scale production of raw silk.   Cocoon of Bombyx mori consist of two major
fibrous proteins. Silk from silkworm is used for decades together for various
biomedical applications and various clinical repair needs like sutures due to
is greater tensile strength and good mechanical properties. Biocompatibility is
a major concern with silkworm silk due to contamination of various residual
protein fibres like sericin. Sericin protein in silkworm have water soluble
glycoprotein and consist of 25-30% of cocoon weight overall, due to 18 amino
acid, polar groups and hydrophilic protein. Various recent studies proved and
suggested that core silk protein fibre (fibroin) exhibits very good mechanical
and biocompatible properties. Silkworm silk are commonly used for designing
scaffolds and culture medium in tissue engineering. It is demonstrated many
antioxidant properties both invitro and in vivo, which proves that sericin has
good immunological properties that safe for many tissue applications which
include vehicle for drug delivery, wound healing, immunological response,
antitumor effect, cryopreservation and various metabolic effects in human
system. Physiochemical properties like functional properties of sericin protein
fibre depends upon the extraction method and process used for sericin isolation
and lineage of the silkworm which can increase the biocompatibility of the
fibre for biomedical applications.

2.Spider
Silk (Nephila clavipes):

Spider silk generally consist of 7 diverse silk glands, each
has a different purpose of production and have different mechanical properties
and biodegradability. Commercial production of spider silk is hampered due to
nature of spidroins due to very less production of silk
and hence it is not extensively used in textile industry neither much in
biomedical applications. Dragline silk from Nephila clavipes which is commonly cloned for natural and
synthetic genes encoding recombinants to limit the use of native organism.
Dragline silk consist of polyalanine
and glycine–glycine-R region where R is often referred to tyrosine, glutamine or leucine. As the Spider Silk are commonly known for
good absorbance energy due extraordinary strength and extendibility. Various
strategies of productions are demonstrated and conducted to increase the
repetitive production of Spider silk. Spider Silk is commonly known in
biomedical applications due to its ability to heal wound as well as to stop
excessive haemorrhage. Several redissolution methods and procedures are carried
to demonstrate the application of spider silk in restoring and repairing the
functions of damaged tissue like tendons.

Characterization:

Silk
is a strong fibre it’s tenacity is between 3.5-5gm/den. The strength is greatly
affected by moisture, the strength of wet silk is 75-85%, which is higher than
the strength of dry silk. The colour of the silk could be brown, yellow, green
or grey as it has good affinity towards
dye with bright lustre. Elastic recovery is not good in silk and the elongation
at break is 20-25%. Specific gravity of silk is 1.24 to 1.34. Standard moisture
regain percentage is 11% but can absorb up to 35%. Silk can withstand higher
temperature, it remains unaffected for prolonged periods at 140?C and it
decomposes at 175?C. Sunlight tends to encourage the decomposition of silk by
atmospheric oxygen. Environmental stability’s silk proteins are due to hydrogen
bonding which enhances biocompatibility and mechanical properties, it can also
be genetically tailored to control the sequencing which make it more beneficial
for any tissue engineering and biomedical applications. It has controlled proteolytic
biodegradability and can be morphologically
flexible. Immobilization of growth factors can be generated by changing the
amino acid.

Biodegradation
is an important characteristic that influences and dominate the use of silk
fibre in various regenerative biomedical applications.

Biodegradation:

Biodegradation is the breakdown of any polymer
material into many smaller fragments or compounds. There are many factors that
influences the biodegradation of silk which includes chemical, physical and
biological factor. Classification of silk fibroin into physio-chemical,
biological and mechanical properties can be decided by the enzymatic
degradation. Enzymes are the vital factors in the degradation behaviour of silk
fibroin. Characteristics of silk biodegradation varies with enzymes. Enzymatic
biodegradation happens in two step processes. The first step is to adsorption
of enzymes, which depends on the enzymes on the surface whether they have the
surface binding domain and second step is hydrolysis of ester bond. At the
second process, the silk biomaterial is completely engulfed by enzymes and the
final product obtained is amino acids in the silk fibroin. This silk
biomaterial can be used in various biomedical application and can be used in
cell culture medium for scaffolds in tissue engineering. Biodegradation gives a
significant change in the molecular weight once the degradation process is
over. Incubation of the enzymes in the silk biomaterial decreases the sample
weight as well as the degree of polymerization. Different enzymes act
differently on the silk biomaterials and hence the sample weight and rate of
polymerization also varies with enzymes.  

Biodegradation
is an essential factor for biomedical application, but it comes with various
disadvantages with degrading silk fibroin i.e. low molecular weight and
non-compact structure. Biodegradation helps enzymes to bind the surface of the
silk fibroin where they dominate the surface with hydrolysis. Biodegradation
depends on the both methods and structural characteristics like pore size,
processing condition, silk fibroin concentration and host immune system during
the degradation process. Both preparation methods and structural
characteristics are closely related with each other with increased surface
roughness or distribution of crystallinity. Hence rate of degradation can be
regulated by changing the crystallinity, pore size, porosity and molecular
weight. Degradability of silk fibroin can be altered by different processing
conditions; different processing condition may influence the silk material to
variable extent. Of which, chemical modification also affects the
biodegradation apart from concentration of enzymes and availability.

Function:

The
following are the general functions of Silk as a biomaterial listed in
sequence:

Immunological
Response

Immunological response is normally evaluated as inflammatory response as
an expression which releases cytokines. Silk fibre is known for its
hypersensitivity reaction due to sericin has attributed its application in immune
response. Subsequent studies have shown different immunological responses of
sericin. Recent study related to immunological response have examined the potential of silk as a
biomaterial for inflammation and their in vitro extracts. The author found that soluble sericin
are immunologically inert in culture murine macrophage cells while insoluble
fibroin protein induces release of Tumour Necrosis Factor-?. In his
demonstration sericin does activates the immune system but it covers the
fibroin protein fibre. The author confirms the low inflammatory response of the
silk as a biomaterial as dominant macrophage is his examination does not allow
the bacterial lipopolysaccride to respond.

Antioxidant

Investigating the effects of free radicals in the body, can lead to
major consequences the products as it may not be neutralized by a superior
antioxidant system. Study suggests that the antioxidant properties of sericin
of inhibits lipid peroxidation in rodent brain homogenate. The study highlights
the interest of antityrosinase activity in the
biomaterial. Cocoon of B.mori has natural pigment which is
known for antityrosinase activity.
Furthermore, antityrosinase activity of pigments and sericin is responsible for
antioxidant property. The antioxidant properties of sericin protein is due to
high serine and threonine content where the hydroxyl group acts a method to remove
chemical substance from the blood stream. Various study also demonstrated the
presence of polyphenols and flavonoids
in sericin is responsible for sericin antioxidant roles. Herewith making
sericin as a natural and safe ingredient for food and cosmetic industries.

Supplement
in Culture Media and Cryopreservation

Cell line for culture media
should always be viable only then they are considered in tissue engineering and
regenerative medicine. Most commonly used media BSA (Bovine Serum Albumin) are
commonly affected by virus hence cryopreservation is the common method used for
cell lines. Serum used here is of highest cost and hence possible examination
and research is conducted to make the cell culture serum-free. Sericin from
cocoon is tested for with BSA alone in the culture media on various mammalian
cells. The test proved that sericin promotes cell viability and did not change
after autoclaving, proving its use in the culture media emphasis cell
proliferation. Sericin used to substitute BSA, preserve less mature cell lines
and undifferentiated cells but it neglects to act in similar manner in case of
differentiated cells. 

Wound
Healing

Cell proliferation
and migration are studied in the properties of sericin and studies has
eventually proved the properties of sericin in wound healing as it increases
the population of fibroblast and keratinocytes cells in the injured area. It
also increases in the production of collagen essential for healing process. In
clinical study, antibiotic cream with sericin accelerated wound closure and the
average time required to close the wound is comparatively lesser than any other
antibiotic creams (without sericin). Topical usage of sericin in antibiotic
creams promotes skin hydration and less irritation and skin pigmentation.

Antitumor Effect

Chemotherapy
is the most common clinical practice used for cancer treatment due to high
cytotoxicity which affects both cancerous and non-cancerous cells. The major
concern of chemotherapy is the resistance of chemotherapeutic agents. Sericin
is therefore used for its low toxicity and biocompatible properties making it
an antitumor agent. Use of sericin as an antitumoral effect proved to have a
very less cell proliferation rate, decreasing the oncogenes expression and
reducing the oxidative stress. Antioxidant properties of sericin make it remain
undigested in the colon which induce lower oxidative stress. Sericin can reduce
the cell viability by inducing the apoptosis
of tumorous cell by increasing reducing the activity expression of
antiapoptotic protein. Sericin do not induce apoptosis to control cells.  

Metabolic
Effects

Considering
the antioxidant and hydrophilic properties of sericin, it is considered for
various metabolic abnormalities. The use of sericin is investigated in various
animal model for gastrointestinal tracts abnormalities. Required consumption of
sericin do not cause any harm in the microflora and secondary bile acids, even
though it reduces the primary bile acid content. Furthermore, sericin can be
considered as for its modulating immune response and intestinal barrier
functions.

Sericin
promotes vascular modulation. Oligopeptides in sericin have an antagonistic
action on chemical channels by blocking them and promoting muscle relaxation.
Oligopeptides mechanism is also known for agonist interaction with nitic oxide
and prostacyclin, which promotes smooth muscle relaxation. Sulphated sericin
are investigated for coagulation cascade mechanism to clarify its
anticoagulation mechanism.

Various
study has proven the promising effect of sericin in lipid metabolism and
obesity. Careful examination is being conducted on the effect of sericin on
lipid and carbohydrate metabolism in rodent which is fed by high fat diet with
an addition of small amount of sericin .For 
5 weeks it did not alter any changes in the body weight and fat weight
of the rodent, but showed considerable changes in the serum concentration of
cholesterol, free fatty acids, phospholipids, Very Low Density of Lipoproteins
(VLDL) and Low -density lipoprotein (LDL),Hence quality amount as a supplement
of sericin is beneficial for metabolic syndrome resulting in high-fat diet
consumption.

Tissue
Engineering

Tissue
engineering uses biomaterials which can possess strong mechanical and binding
properties to the scaffold and can provide efficient replacement of the organ
without affecting the surrounding tissues or organ. Sericin fibres are fragile
and are difficult to use as scaffolds in tissue engineering they are often
crosslinked to increase the physical properties. Sericin/gelatin combination
provide uniform pore distribution, improved mechanical properties and high
swellibility. Sericin membrane of A.mylitta
cocoon when crosslinked with glutaraldehyde, shows increased physical
properties, which include non-rapid enzymatic degradation and increased
fibroblast cell viability and attachment. Crosslinking of silk fibre with
crosslinking agents has made silk as a biomaterial in various tissue
engineering applications.

Vehicle
for Drug Delivery

Delivery system should be
compatible and adjustable to the morphology to gain optimal effect of the drug.
Sericin can bind with other molecule due to its chemical reactivity and good pH
response which is essential for fabrication of small materials. Fabrication of
crosslinked covalently crosslinked 3D sericin gel are proved to be injectable
material which promote cell adhesion and provide both physical and chemical
properties to provide sustained release of drug with long term survival.

Biocompatibility

Biocompatibility
is the ability of any biomaterials to adjust with the surrounding tissue
without causing effecting the immune response of on the adjacent tissue. Silk
fibroin are generally used for clinical and biomedical application for decades
as a suture material. Sutures are generally a wide application of silk as they
have very good mechanical properties. Biocompatibility of silk was questioned
when wax coating or silicone coating was done on the surface of silk based
suture. Sericin glue-like fibre are known for opposite effects when
biocompatibility and hypersensitivity of silk is concerned. There are study
conducted in vivo and proved that silk fibre is susceptible to proteolytic
degradation and can also degrade overtime.

 

 

 

References:

1  
Zhou CZ, Confalonieri F, Jacquet M, Perasso R, Li ZG, Janin J. Proteins. 2001;44:119–122.