SilkChemical structure R=H, glycine R=CH3, alanine R=CH2OH, serine Silkconsist 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 clavipesand Araneus diadematus) it isalso a natural protein. Both the proteins have 18 similar amino acids such asglycine, alanine and serine in variable amounts. Sericin is thesticky material surrounding fibroin and fibroin is the structural centre ofsilk.
Fibroin is largely made up of amino acids Gly-Ser-Gly-Ala-Gly-Ala andforms beta pleated sheets, ?-keratin1. There are many different silkpolymorphs which generally seen in (silk I ) water soluble state and comes inglandular state before crystallization ,(silk II) which is often seen inspun silk state and air/water assembledinterfacial silk usually in helical structure (silk III). Silk I is usuallyexposed to heat or physical heat spinning to convert it to silk II, it can beeasily done as silk II structure consist of ?- sheet secondary structure.
SilkI in aqueous condition when exposed to methanol or potassium chloride, thesurface of the ?-sheet structure is asymmetrically divided into hydrogen sidechains and methyl side chains. Hydrogen bonds and van der Waals forcesinteracts with the methyl group and hydrogen groups to make the inter-stackingsheets of crystal to be thermodynamically stable2. Silk II structure at thelater stage deny water and becomes less or completely not soluble in severalsolvents very mild acidic and basic conditions. Thestructure represents a tight packing of stacked sheets of hydrogen bonded in ananti-parallel chain of protein. Hydrogen bonds form between chains, and sidechains form above and below the plane of the hydrogen bond network. Fibroincontains 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 is3:2:1 while, the remaining 13% consist of Tyrosine, valine, aspartic acid etc.Glycine has a high proportion (50%) which allows it to tight packing this isbecause its R-group has only one hydrogen and, so it is not stericallyconstrained.
Alanine and serine has many interceded hydrogen bonds and arestrong and resistant to breaking. The less crystalline forming regions areknown as linkers which consists of fibroin heavy chain they are situated inbetween 42-44 amino acid residues in length. All linkers do have identicalamino acid residues which are charged amino acid residues found in crystallineregion. Primary sequence of proteins is highly repetitive which provideshomogeneity in the secondary structure. Primary sequence generates hydrophobicproteins which are in natural co-polymer block design. The interspace is filledwith many hydrophobic and hydrophilic domains, large hydrophobic domainsinterspace with smaller hydrophilic domains to bolster the assembly of silk andimproves the strength and resiliency of fibre. Physical formTheaverage diameter of depends upon the type and variety of silk fibroin and the conditionof spinning.
The diameter for a bave of b.mori is 15-25?m and for othersilk like Tasar is about 65?m for a bave.The diameter of the filamentsof silk fibroins are different for each variety as the location of cocoonarecoarser on the outside and are finer at the inside. The cross section of filamentof b.
mori fibroin is a swollen triangle of 10? across while some other silk fibroinlike Tasar fibroin have wedge shaped cross section. Longitudinally, thefilament of b.mori appears to be solid rod without any marking,while Tasar silk has longitudinal weaves.Classifications Silkhas used in textile industry are different from the those used in biomedicalapplication some types of silk used in biomedical application is listed insequence: 1.Silk worm silk (Bombyx mori):Silk obtained from cocoon of Bombyx mori are commonly usedfor biomedical and textile production. Sericulture is commonly known forbreeding of silks for commercial scale production of raw silk. Cocoon of Bombyx mori consist of two majorfibrous proteins.
Silk from silkworm is used for decades together for variousbiomedical applications and various clinical repair needs like sutures due tois greater tensile strength and good mechanical properties. Biocompatibility isa major concern with silkworm silk due to contamination of various residualprotein fibres like sericin. Sericin protein in silkworm have water solubleglycoprotein and consist of 25-30% of cocoon weight overall, due to 18 aminoacid, polar groups and hydrophilic protein.
Various recent studies proved andsuggested that core silk protein fibre (fibroin) exhibits very good mechanicaland biocompatible properties. Silkworm silk are commonly used for designingscaffolds and culture medium in tissue engineering. It is demonstrated manyantioxidant properties both invitro and in vivo, which proves that sericin hasgood immunological properties that safe for many tissue applications whichinclude vehicle for drug delivery, wound healing, immunological response,antitumor effect, cryopreservation and various metabolic effects in humansystem. Physiochemical properties like functional properties of sericin proteinfibre depends upon the extraction method and process used for sericin isolationand lineage of the silkworm which can increase the biocompatibility of thefibre for biomedical applications.2.SpiderSilk (Nephila clavipes):Spider silk generally consist of 7 diverse silk glands, eachhas a different purpose of production and have different mechanical propertiesand biodegradability.
Commercial production of spider silk is hampered due tonature of spidroins due to very less production of silkand hence it is not extensively used in textile industry neither much inbiomedical applications. Dragline silk from Nephila clavipes which is commonly cloned for natural andsynthetic genes encoding recombinants to limit the use of native organism.Dragline silk consist of polyalanineand glycine–glycine-R region where R is often referred to tyrosine, glutamine or leucine.
As the Spider Silk are commonly known forgood absorbance energy due extraordinary strength and extendibility. Variousstrategies of productions are demonstrated and conducted to increase therepetitive production of Spider silk. Spider Silk is commonly known inbiomedical applications due to its ability to heal wound as well as to stopexcessive haemorrhage. Several redissolution methods and procedures are carriedto demonstrate the application of spider silk in restoring and repairing thefunctions of damaged tissue like tendons.CharacterizationSilkis a strong fibre it’s tenacity is between 3.5-5gm/den. The strength is greatlyaffected by moisture, the strength of wet silk is 75-85%, which is higher thanthe strength of dry silk.
The colour of the silk could be brown, yellow, greenor grey as it has good affinity towardsdye with bright lustre. Elastic recovery is not good in silk and the elongationat break is 20-25%. Specific gravity of silk is 1.24 to 1.34. Standard moistureregain percentage is 11% but can absorb up to 35%. Silk can withstand highertemperature, it remains unaffected for prolonged periods at 140?C and itdecomposes at 175?C. Sunlight tends to encourage the decomposition of silk byatmospheric oxygen.
Silk is lightweight, breathable, hypoallergenic and goodabsorbency. Environmentalstability’s silk proteins are due to hydrogen bonding which enhancesbiocompatibility and mechanical properties, it can also be genetically tailoredto control the sequencing which make it more beneficial for any tissueengineering and biomedical applications. It has controlled proteolyticbiodegradability and can be morphologicallyflexible. Immobilization of growth factors can be generated by changing theamino acid.Biodegradationis an important characteristic that influences and dominate the use of silkfibre in various regenerative biomedical applications.Biodegradation:Biodegradation is the breakdown of any polymermaterial into many smaller fragments or compounds.
There are many factors thatinfluences the biodegradation of silk which includes chemical, physical andbiological factor. Classification of silk fibroin into physio-chemical,biological and mechanical properties can be decided by the enzymaticdegradation. Enzymes are the vital factors in the degradation behaviour of silkfibroin. Characteristics of silk biodegradation varies with enzymes. Enzymaticbiodegradation happens in two step processes. The first step is to adsorptionof enzymes, which depends on the enzymes on the surface whether they have thesurface binding domain and second step is hydrolysis of ester bond. At thesecond process, the silk biomaterial is completely engulfed by enzymes and thefinal product obtained is amino acids in the silk fibroin.
This silkbiomaterial can be used in various biomedical application and can be used incell culture medium for scaffolds in tissue engineering. Biodegradation gives asignificant change in the molecular weight once the degradation process isover. Incubation of the enzymes in the silk biomaterial decreases the sampleweight as well as the degree of polymerization. Different enzymes actdifferently on the silk biomaterials and hence the sample weight and rate ofpolymerization also varies with enzymes. Biodegradationis an essential factor for biomedical application, but it comes with variousdisadvantages with degrading silk fibroin i.e.
low molecular weight andnon-compact structure. Biodegradation helps enzymes to bind the surface of thesilk fibroin where they dominate the surface with hydrolysis. Biodegradationdepends on the both methods and structural characteristics like pore size,processing condition, silk fibroin concentration and host immune system duringthe degradation process. Both preparation methods and structuralcharacteristics are closely related with each other with increased surfaceroughness or distribution of crystallinity. Hence rate of degradation can beregulated by changing the crystallinity, pore size, porosity and molecularweight.
Degradability of silk fibroin can be altered by different processingconditions; different processing condition may influence the silk material tovariable extent. Of which, chemical modification also affects thebiodegradation apart from concentration of enzymes and availability.FunctionThefollowing are the general functions of Silk as a biomaterial listed insequence:ImmunologicalResponseImmunological response is normally evaluated as inflammatory response asan expression which releases cytokines. Silk fibre is known for itshypersensitivity reaction due to sericin has attributed its application in immuneresponse. Subsequent studies have shown different immunological responses ofsericin.
Recent study related to immunological response have examined the potential of silk as abiomaterial for inflammation and their in vitro extracts. The author found that soluble sericinare immunologically inert in culture murine macrophage cells while insolublefibroin protein induces release of Tumour Necrosis Factor-?. In hisdemonstration sericin does activates the immune system but it covers thefibroin protein fibre. The author confirms the low inflammatory response of thesilk as a biomaterial as dominant macrophage is his examination does not allowthe bacterial lipopolysaccride to respond.AntioxidantInvestigating the effects of free radicals in the body, can lead tomajor consequences the products as it may not be neutralized by a superiorantioxidant system. Study suggests that the antioxidant properties of sericinof inhibits lipid peroxidation in rodent brain homogenate.
The study highlightsthe interest of antityrosinase activity in the biomaterial. Cocoon of B.mori has natural pigment which is known for antityrosinaseactivity. Furthermore, antityrosinase activity of pigments and sericin isresponsible for antioxidant property. The antioxidant properties of sericinprotein is due to high serine and threonine content where the hydroxyl group acts a method to removechemical substance from the blood stream. Various study also demonstrated thepresence of polyphenols and flavonoids in sericin is responsible for sericinantioxidant roles. Herewith making sericin as a natural and safe ingredient forfood and cosmetic industries. Supplementin Culture Media and CryopreservationCell line for culture mediashould always be viable only then they are considered in tissue engineering andregenerative medicine.
Most commonly used media BSA (Bovine Serum Albumin) arecommonly affected by virus hence cryopreservation is the common method used forcell lines. Serum used here is of highest cost and hence possible examinationand research is conducted to make the cell culture serum-free. Sericin fromcocoon is tested for with BSA alone in the culture media on various mammaliancells. The test proved that sericin promotes cell viability and did not changeafter autoclaving, proving its use in the culture media emphasis cellproliferation. Sericin used to substitute BSA, preserve less mature cell linesand undifferentiated cells but it neglects to act in similar manner in case ofdifferentiated cells. WoundHealingCell proliferationand migration are studied in the properties of sericin and studies haseventually proved the properties of sericin in wound healing as it increasesthe population of fibroblast and keratinocytes cells in the injured area. Italso increases in the production of collagen essential for healing process.
Inclinical study, antibiotic cream with sericin accelerated wound closure and theaverage time required to close the wound is comparatively lesser than any otherantibiotic creams (without sericin). Topical usage of sericin in antibioticcreams promotes skin hydration and less irritation and skin pigmentation. Antitumor EffectChemotherapyis the most common clinical practice used for cancer treatment due to highcytotoxicity which affects both cancerous and non-cancerous cells. The majorconcern of chemotherapy is the resistance of chemotherapeutic agents. Sericinis therefore used for its low toxicity and biocompatible properties making itan antitumor agent.
Use of sericin as an antitumoral effect proved to have avery less cell proliferation rate, decreasing the oncogenes expression andreducing the oxidative stress. Antioxidant properties of sericin make it remainundigested in the colon which induce lower oxidative stress. Sericin can reducethe cell viability by inducing the apoptosis of tumorous cell byincreasing reducing the activity expression of antiapoptotic protein.Sericin do not induce apoptosis to control cells. MetabolicEffectsConsideringthe antioxidant and hydrophilic properties of sericin, it is considered forvarious metabolic abnormalities. The use of sericin is investigated in variousanimal model for gastrointestinal tracts abnormalities.
Required consumption ofsericin do not cause any harm in the microflora and secondary bile acids, eventhough it reduces the primary bile acid content. Furthermore, sericin can beconsidered as for its modulating immune response and intestinal barrierfunctions.Sericinpromotes vascular modulation. Oligopeptides in sericin have an antagonisticaction on chemical channels by blocking them and promoting muscle relaxation.
Oligopeptides mechanism is also known for agonist interaction with nitic oxideand prostacyclin, which promotes smooth muscle relaxation. Sulphated sericinare investigated for coagulation cascade mechanism to clarify itsanticoagulation mechanism.Variousstudy has proven the promising effect of sericin in lipid metabolism andobesity. Careful examination is being conducted on the effect of sericin onlipid and carbohydrate metabolism in rodent which is fed by high fat diet withan addition of small amount of sericin .
For 5 weeks it did not alter any changes in the body weight and fat weightof the rodent, but showed considerable changes in the serum concentration ofcholesterol, free fatty acids, phospholipids, Very Low Density of Lipoproteins(VLDL) and Low -density lipoprotein (LDL),Hence quality amount as a supplementof sericin is beneficial for metabolic syndrome resulting in high-fat dietconsumption.TissueEngineeringTissueengineering uses biomaterials which can possess strong mechanical and bindingproperties to the scaffold and can provide efficient replacement of the organwithout affecting the surrounding tissues or organ. Sericin fibres are fragileand are difficult to use as scaffolds in tissue engineering they are oftencrosslinked to increase the physical properties.
Sericin/gelatin combinationprovide uniform pore distribution, improved mechanical properties and highswellibility. Sericin membrane of A.mylittacocoon when crosslinked with glutaraldehyde, shows increased physicalproperties, which include non-rapid enzymatic degradation and increasedfibroblast cell viability and attachment. Crosslinking of silk fibre withcrosslinking agents has made silk as a biomaterial in various tissueengineering applications.Vehiclefor Drug DeliveryDelivery system should becompatible 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 pHresponse which is essential for fabrication of small materials. Fabrication ofcrosslinked covalently crosslinked 3D sericin gel are proved to be injectablematerial which promote cell adhesion and provide both physical and chemicalproperties to provide sustained release of drug with long term survival.BiocompatibilityBiocompatibilityis the ability of any biomaterials to adjust with the surrounding tissuewithout causing effecting the immune response of on the adjacent tissue.
Silkfibroin are generally used for clinical and biomedical application for decadesas a suture material. Sutures are generally a wide application of silk as theyhave very good mechanical properties. Biocompatibility of silk was questionedwhen wax coating or silicone coating was done on the surface of silk basedsuture. Sericin glue-like fibre are known for opposite effects whenbiocompatibility and hypersensitivity of silk is concerned.
There are studyconducted in vivo and proved that silk fibre is susceptible to proteolyticdegradation and can also degrade overtime. References1 Zhou CZ, Confalonieri F, Jacquet M, Perasso R, Li ZG, Janin J. Proteins. 2001;44:119–122.