Silk sheets of crystal to be thermodynamically stable. Silk

Silk

Chemical structure

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

 

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. 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, ?-keratin. 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 stable. 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

There
are many silk fibres with compatible biomedical properties which are of the
following types of silk fibre:

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. Silk is lightweight, breathable, hypoallergenic and good
absorbency.

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.moori 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 are abled to 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.