Silk around fibroin and fibroin is at the centre

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

 

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

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Chemical
structure:

 

Both the proteins sericin
and fibroin have eighteen similar amino acids such as glycine,
alanine and serine in variable amounts. Sericin is a sticky material which covers around fibroin
and fibroin is at the centre of silk. Fibroin contains of mainly amino acids
Gly-Ser-Gly-Ala-Gly-Ala and forms beta-pleated sheets known as ?-keratin 1.
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 disclosed 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 disclosed 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 stable 2. 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 are formed in between of each chains, and the side chains form opposite
sides(above and below) of the plane surface where the hydrogen bond network are
situated . 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.