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The mystery of the whelk egg capsule protein : electrospinning, mechanical testing, and being outsmarted.. Corbett, Carla M. 2010

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THE MYSTERY OF THE WHELK EGG CAPSULE PROTEIN -ELECTROSPINNING, MECHANICAL TESTING, AND BEING OUTSMARTED BYAN INVERTEBRATEbyCARLA M. CORBETTBSc., The University of British Columbia, 2006A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Zoology)THE UNIVERSITY OF BRITISH COLUMBIA(Vancouver)August 2010 Carla M. Corbett, 2010iiABSTRACT     Whelks are carnivorous marine snails known for their elaborate and durable eggcapsules.  The mechanically complex capsules have been previously studied, and shownto have mechanical behaviour similar to keratin.  The mature protein has an initial stifflinear elastic region at low strain, followed by a rubbery yield region with a fullyrepeatable order of magnitude decrease in stiffness.  The material properties of theprotein mature in distinct stages, with long-range elasticity developing first, followed bythe development of the stiff Hookean region.  As the capsule matures, it is massaged by agland in the foot of the snail, which probably enables cross-linking.     This study sought to mimic the development process using electrospinning to createfibres with charge-based assembly, then adding a cross-linking step to encourage the stiffspring behaviour to form.  An electrospinning protocol was developed and parameterswere optimized.  The technique was applied successfully, and the resulting proteinnanofibres could be cross-linked.  The electrospun protein fibres were shown to havecomposition and secondary structures similar to the native protein.  However, themechanical properties of the cross-linked nanofibres were more similar to a transitionalstage in the egg capsule’s maturation sequence than they were to the mature capsule.  Thefibres did not exhibit the bimodal behaviour seen in the native polymer.iiiTABLE OF CONTENTSAbstract ................................................................. iiTable of Contents ......................................................... iiiList of Tables ............................................................ viList of Figures ........................................................... viiAcknowledgements ........................................................ ixDedication ............................................................... x1     Introduction ........................................................... 11.1 The goal ........................................................11.2 Background..................................................... 11.3 Egg capsule formation ............................................. 31.4 Structure of the capsule ............................................ 41.5 Structure of the protein ............................................ 61.6 Mechanical properties ............................................. 71.7 Maturation model ................................................. 91.8 More mechanical properties ........................................ 121.9 Polarized light microscopy ......................................... 131.10   FTIR ........................................................ 141.11   Biomimetics and electrospinning ................................... 151.12   History of electrospinning ........................................ 161.13   Electrospinning technique .........................................181.14   Electrospinning parameters ........................................191.15   Hypothesis .................................................... 21iv2 Methods ............................................................ 222.1 Preparation of solutions ........................................... 222.2 Selection of TCEP ............................................... 222.3 Nanofibre electrospinning ......................................... 232.4 Preparation of SEM samples ....................................... 252.5 Collection of SEM images ......................................... 252.6 Polarized light microscopy ......................................... 262.7 Effect of PEO .................................................. 272.8 Measurement of fibres ............................................ 272.9 FTIR spectroscopy ............................................... 282.10   Preparation of samples for testing .................................. 292.11   Testing apparatus ............................................... 312.12   Testing procedure .............................................. 333 Results ............................................................. 363.1 Effect of TCEP ................................................. 363.2 Effect of parameters .............................................. 373.3 Polarized light microscopy ......................................... 393.4 Effect of PEO .................................................. 403.5 Measurement of fibres ............................................ 423.6 FTIR spectra ................................................... 453.7 Results of mechanical testing ....................................... 47v4 Discussion ........................................................... 504.1 Electrospinning parameters  ........................................ 504.2 PEO .......................................................... 544.3 Nanofibre diameter .............................................. 554.4 Birefringence of fibres ............................................ 564.5 Matrix Squeeze ................................................. 574.6 Self assembly ................................................... 584.7 FTIR to identify ................................................. 594.8 FTIR to determine structure ........................................ 604.9 Cross-linking ................................................... 624.10   Mechanical testing .............................................. 634.11   Mechanical results .............................................. 644.12   Future directions ............................................... 66References .............................................................. 68viLIST OF TABLESTable 1     Blend solution concentration series ................................... 28Table 2     Characteristic amide I band frequencies................................ 29Table 3     Composition of protein and electrospun fibres ........................... 45Table 4     Young’s modulus and hysteresis...................................... 49Table 5     Secondary structure of common structural proteins ....................... 61viiLIST OF FIGURESFigure 1       Whelk morphology ............................................... 2Figure 2       Production of egg capsules ......................................... 3Figure 3       Stress/strain curve for 15 extension cycles ............................. 7Figure 4       Capsule maturation timeline ........................................ 8Figure 5       Model of WECB mechanics through maturation ........................ 10Figure 6       Reversible α-helix to β-sheet transition during straining .................. 11Figure 7       Stress recovery ................................................. 12Figure 8       Electrospinning technique diagram .................................. 19Figure 9       The Kato Tech NEU-010 ......................................... 24Figure 10     Diagram of modified electrospinning technique ........................ 30Figure 11     Electrospun fibres span the gap ..................................... 31Figure 12     Microscope-based micro-tensile testing apparatus ....................... 32Figure 13     Samples were glued to a glass micro-beam ............................ 34Figure 14     Diagram of SDS-PAGE gel ....................................... 37Figure 15     Effect of electrospinning parameters ................................. 38Figure 16     Birefringence of electrospun fibres .................................. 39Figure 17     Electrospun PEO fibres at different concentrations ...................... 41Figure 18     Results of washing electrospun fibres ................................ 42Figure 19     Mean diameters of electrospun fibres ................................ 42Figure 20     Mean diameters of PEO fibres ..................................... 43Figure 21     Mean diameters of analogous solutions  .............................. 44Figure 22     Mean diameters of fibres before and after washing ...................... 44viiiFigure 23     FTIR spectra ................................................... 46Figure 24     Stress-strain curves  ............................................. 48Figure 25     Hysteresis loops ................................................ 48ixACKNOWLEDGEMENTSIt’s over!I have so many people to thank for so much.First of all, thanks to Bob, the world’s best supervisor, for the incredible patience, theamazing support, and the awesome sense of humour.Thanks to John, my grand-supervisor, for getting me into this in the first place.  Wait,should I be thanking you for that?Thanks to my dad, who took over my tractor payments so I could actually finish mythesis without having to declare bankruptcy.  I promise I’ll pay you back.Thanks to my mom, for the endless baking – it’s all about healthy nutrition.Thanks to all my family and friends for their help on the farm, you guys made it possiblefor me to get to the lab.A humble thank you for the endless love, support, affection, and patience from my sweetHabib, who has been sorely neglected during the course of my graduate work.  That poorhorse sat through more practice runs of my defense presentation than any human wouldever have done.Finally, thanks to Cam for not only putting up with me, but deciding to marry me while Iwent through all the lovely stress of pulling this together.  You must be nuts, honey.CxDEDICATIONFor Gramps11     Introduction1.1     The goal     The aim of this study was to try to solve a mystery.  A very cool self-assemblingbiomaterial goes through a poorly understood maturation process during whichinteresting mechanical properties develop.  We believe that the process involves charge-based assembly and a cross-linking step.  In order to understand the maturation process,we worked to recreate the polymer from its precursor, using the electrospinningtechnique.  Electrospinning a solution of the immature material induces the formation ofnano-scale fibres, which are aligned and assembled by charge.  Adding a cross-linkingagent to the electrospun nanofibres should alter the mechanical properties of the fibresand take them one step further in the maturation process.  Ideally, we will come close tothe properties of the native material, and this will help elucidate the maturation process.If we can mimic the process and produce the material in manageable form, we canharness its mechanical properties for industrial or medical applications.1.2     Some background on whelks and egg capsules     Whelks are a species-rich group of marine Prosobranch snails found in temperatewaters.  Whelks are fascinating not only because they are voracious carnivores with adeceptively benign appearance, or because of their alternative lifestyle (they are serialhermaphrodites who first develop sexually as males, then mature and realize their fullpotential as females), or because an enormous whelk is the first evil boss encountered inthe video game Final Fantasy VI, but also for their creative industry in producing“mermaids’ necklaces”, as their strings of egg cases are sometimes called.     Whelks are known for these strings of pale, disk-shaped egg capsules, commonlyfound on beaches along the east coast of Canada and the United States.  The egg cases are2highly resilient and are composed primarily of an insoluble protein polymer (whelk eggcapsule protein, WECP).  The polymer shares properties with other major structuralproteins: it has hierarchical ordering like collagen, long-range elasticity like elastin, andmechanical behaviour like keratin.Figure 1.  Whelk morphology.  Whelks are carnivorous marine snails found all over the world in a varietyof shapes and sizes.  Females deposit developing embryos in highly resilient proteinaceous egg cases thatshow species-specific morphology.     The egg capsules are physically and chemically stable, and are able to withstand theharsh marine environment for months over winter while the embryos develop.  In fact,when the embryos come full term, looking like scaled-down versions of adult whelks, thejuveniles escape from the egg capsule by means of a mucosal plug, leaving the capsuleitself unharmed.     The capsules have fascinating mechanical properties.  They are reversibly elastic andcan be repeatedly stretched to high extensions without apparent damage.  At smallextensions, the material acts like a stiff spring, with most of the elastic strain energyrecovered by recoil.  However, at larger extensions the material yields and becomesrubbery, with much lower resilience and elastic stiffness.  These dramatic changesarequickly and completely reversible for extensions of 80% in repeated loading cycles.Shockingly, the stiff spring behaviour can even rapidly reform in the rubbery region if theextension is interrupted and the strain held constant.3     Many adaptive functions have been suggested for these energetically expensive eggcapsules, including protection against desiccation, predation, UV irradiation, osmoticstresses, and the impact loading encountered in the intertidal zone (Ojeda and Chaparro,2004; Pechenik, 1979).  However, none of these potential roles has yet been blessed withstrong experimental support, so the protective role of the capsules remains controversial.1.3     Egg capsule formation     A precursor protein of WECB is found in the nidamental gland, where capsuleformation is initiated.  Embryos are wrapped in a soft, pliable sack by nidamental glandmesodermal secretory cells before leaving the genital tract.  This preliminary capsule ispassed out of the body and under a depression in the foot called the ventral pedal gland(VPG).  Here the capsule is massaged and manipulated by the muscle surrounding thegland for about an hour before it is deposited on the substratum.  The capsules may bedeposited individually, or joined to a growing strand that is then attached to thesubstratum as a unit.Figure 2.  Production of egg capsules.  (a) Embryos travel from the ovary down the reproductive tract to thenidamental gland, where they are wrapped in a soft sack – the immature egg capsule.  Egg capsules areformed from protein synthesized and stored in the nidamental gland.  (b) Embryos are packaged intoimmature capsules, which pass out of the body and into a depression in the foot called the ventral pedalgland, where they are processed before deposition on the substratum.ab4     During the manipulation period some unknown physical and/or chemical processrenders the capsule relatively rigid and insoluble.  Capsules removed from a whelk beforetreatment in the VPG are soluble and have no tensile integrity.  This suggests that theVPG processing involves polymer stabilization via covalent crosslinking (Rapoport andShadwick, 2007).  Interestingly, egg capsule production can be induced in gravid femalesby injection with an extract of the circumesophageal ganglion, but the resulting capsulesremain “unhardened”, much like the natural capsules without treatment by the VPG.These observations suggested that the immature capsule, the whelk egg capsule glandprotein (WECGP), would be useful in future studies on the natural polymerizationprocess.1.4     Structure of whelk egg capsules     In an early study, Hunt (1966) reported that the egg capsule of the whelk Buccinumundatum was composed of 77.5% amino acids and 5-6% sugars.  At that time, the sourceof the egg capsule material was unknown.  We now know that the egg capsules areextracellular constructs composed of over 80% protein – the rest is lipids andcarbohydrates (Sullivan and Maugel, 1984).     Early descriptive work on whelk egg capsules found a large degree of species-specificmorphological variation, which has since been attributed to morphological variation inthe VPG itself.  That is, differently shaped glands produce differently shaped capsules.  Ithas also been demonstrated that within a species, snails of different size produce scaledversions of egg capsules of similar morphology.     Unlike a typical amorphous rubbery polymer, whelk egg capsules are highlybirefringent, with a layered structure and a fibrous appearance.  Light microscopy showsthat the capsules are made of numerous distinct layers of material, and transmissionelectron microscopy (TEM) shows that the layers have striated patterns, similar to5collagen, keratin, and some chitins (Rapoport and Shadwick, 2002). Rapoport andShadwick (2002) also found that the capsules are composed of fibres approximately 0.3µm in diameter and that the laminae of the capsule walls have a characteristic bandingpattern of 48 to 52 nm.     Early X-ray diffraction studies showed that the fibrils that form the striated patternhave an α-helical conformation (Flower et al., 1969).  Rawlings (1999) showed that thecapsules have hierarchical layers of ordered fibres.  These observations suggest astructure analogous to an intermediate filament (IF)-based material such as hard α-keratin, but there are important differences in structure and formation (Rapoport andShadwick, 2007).     WECB is composed of discrete plywood-like sheets of fibrous material arranged atdifferent angles and exhibits isotropic mechanical behaviour in the plane formed by thetransverse and longitudinal axes of the capsule (Rapoport and Shadwick, 2007). Hard α-keratin, on the other hand, is a complex cylindrically arranged composite with concentrichierarchical layers around a major axis of fibrillar orientation (Hearle, 2000).  Theprecursor of WECB, WECGP, is an extracellular material stored in glandular vesicles,while hard α-keratin is derived from epithelial cells in a complex biogenic process, andcontains cellular remnants (Van Steensel et al., 2000).     The egg capsules have their fibrous hierarchical arrangement at all stages ofprocessing, even as mechanical integrity is developing (Rapoport and Shadwick, 2007).Scanning electron microscopy (SEM) comparisons show that the mechanical integritydoes not arise from any gross structural changes resulting from the manipulations of theVPG – the capsule structure is completed in the nidamental gland, and VPG treatmentsomehow causes it to become an insoluble protein polymer (Rapoport and Shadwick,2007).  VPG activity likely induces the formation of cross-links and possibly adds matrix(Rapoport and Shadwick, 2007).61.5     Structure of whelk egg capsule protein     Amino acid analysis shows that the WECP is rich in both acidic and basic amino acids(Hunt, 1966; see also Rapoport and Shadwick, 2002).   Flower et al. (1969) proposed thata number of α-helices either aggregate into a large, complex unit, or form a multi-strandcoiled-coil.  Their X-ray diffraction and electron microscopy results suggested that the α-helical units do not have a precise repeat pattern, but have some degree of orientationabout their long axis (Flower et al., 1969).     Flower (1973) stated that two protein structures, “ribbons” and “filaments”, are usedby the whelks to produce egg capsules.  The two protein structures were supposed to beproduced and stored separately (Flower, 1973).   However, Goldsmith et al. (1977) useddigestions, amino acid composition studies, and electron microscopy to conclude that thesoluble precursor protein (which they named “precapsulin”) was likely only one speciesof protein being cross-linked into dimers, trimers, and tetramers.     The egg capsules resist both enzymatic degradation and common protein solvents(such as formic acid, sodium dodecyl sulphate (SDS), and urea), which also suggests thatthe material contains cross-links.  Cross-links likely give the material its mechanicalproperties as well.  Hunt (1966) determined that the insolubility of the material shouldnot be attributed to disulphide cross-links, because the capsule has a low concentration ofsulphur-containing amino acids.  Rapoport and Shadwick (2002) found that cysteine isalso completely absent in Busycon, and is only present in trace amounts in other species.However, a very small number of disulphide cross-links may be sufficient to give thematerial its properties.  Agents that selectively reduce disulphide bonds, such asdithiothreitol (DTT) and Tris [2-carboxyethyl] phosphine (TCEP), were effective inbreaking WECGP into its monomeric subunit.71.6     Mechanical properties of whelk egg capsules     The mechanical properties of the capsules are intriguing.  They are reversibly elasticand can be repeatedly stretched to high extensions (over 80%) without apparent damage.At small extensions (>3-5%), the material acts like a stiff spring, with most of the elasticstrain energy recovered by recoil.  This initial stiff portion of the stress-strain curve iscalled the “Hookean” region, due to its linear elastic response.  The Hookean region ischaracterized by a relatively high modulus of elasticity and low hysteresis.     At larger extensions the capsule yields and becomes rubbery, with much lowerresilience and elastic stiffness.  This portion of the stress-strain curve is plateau-like, hasa relatively low modulus and high hysteresis, and is called the “yield” region.  Thedramatic changes between the two regions are completely reversible, in a second or less,for extensions of 80% in repeated loading cycles.  Remarkably, the stiff spring behaviourof the Hookean region quickly reforms in the yield region if the extension process isinterrupted.Figure 3. 15 extension cycles for a 6mm wide strip of mature egg capsule from B. canaliculatum.  RegionA is called the Hookean region due to its linear elastic behaviour, region B is the yield region.  The dashedline at approximately 3% strain is the yield point, where the material undergoes a reversible, order ofmagnitude decrease of elastic modulus, from 87.9 MPa in A to 3.9 MPa in B.  The arrows indicate thedirection of the strain during the extension cycles.  Reproduced with permission from Journal ofExperimental Biology, Rapoport and Shadwick 2007.8     Rapoport and Shadwick (2002) proposed that two hierarchical levels of stabilizationexist in the material, one giving high stiffness seen at small extensions, the other givingthe elasticity seen at high extensions.  The first, high Young’s modulus level ofstabilization is labile to strain and high temperature, but is fully recoverable upon returnto its native condition (Rapoport and Shadwick, 2002).  The second, low modulus level isstable, and is likely to exist in the immature capsule prior to cross-linking (Rapoport andShadwick, 2007).  Qualitative observations suggest that the bimodal behaviour of WECPdevelops sequentially, with the simpler rubber-like behaviour of the yield regiondeveloping first, and the Hookean region developing later in the capsule maturationprocess (Rapoport and Shadwick, 2007).Figure 4.  Development of mechanical properties in WECP.  Capsule maturation is divided into threedistinct phases: (I) pre-pedal manipulation, (II) pedal manipulation, and (III) post-pedal manipulation.Prior to pedal manipulation, the capsule is immature, uncrosslinked, soluble, and lacks mechanicalintegrity.  During pedal manipulation, the ventral pedal gland (VPG) subjects the capsule to a muscularmassage, and the capsule undergoes a transitional state where it is elastic and has some crosslinking.  Post-pedal manipulation, the mature capsule with fully developed mechanical properties is deposited onto thesubstrate.  Note that force scales are different in II and III.  Multiple curves represent different specimens.Reproduced with permission from the Journal of Experimental Biology, Rapoport and Shadwick 2007.91.7     A model of WECP mechanics through maturation     The awesome model for the structure and mechanics of WECP proposed by Rapoportand Shadwick (2007) starts with charge-based self-assembly of macrofibrils composed ofa staggered head to tail arrangement of IF-type coiled-coil protein structures.  Thisalignment of coiled-coils is responsible for the repeat striation patterns seen in electronmicroscopy (Rapoport and Shadwick, 2007).  The macrofibrils may be interconnected,and are arranged in layers of varying orientations throughout the thickness of the material(Rapoport and Shadwick, 2007).  The layers appear to be discontinuous and interdigitatewith other layers, appearing to be laid down like disordered strokes of a broad paint brush(Rapoport and Shadwick, 2007).  The layers do not necessarily span the entire length ofthe egg capsule, so tensile loading results in shearing and sliding of successive layers,leading to material failure (Rapoport and Shadwick, 2007).     Immediately following formation in the egg capsule gland, the material is soluble,white in colour, and probably held together by weak noncovalent interactions (Rapoportand Shadwick, 2007).  During treatment in the VPG, the muscular massaging actionbrings layers of the protein closer together, at first enabling only a low density of cross-links – during this period, the material behaves like a pliant rubber (Rapoport andShadwick, 2007).     The cross-links that form in the VPG between the coiled-coils are responsible for theheat- and strain-labile stabilization that signals the completion of the capsule maturationprocess (Rapoport and Shadwick, 2007).  After processing in the VPG, cross-link densityis sufficient to transfer mechanical stress down to the coiled-coils, which are the smallesthierarchical level of the material (Rapoport and Shadwick, 2007).  The result is theHookean region of the mechanical response, as a network of stiff coils are strained(Rapoport and Shadwick, 2007).  Unraveling of the coiled-coils begins at the transitionbetween the Hookean and yield regions, and continues throughout the yield region(Rapoport and Shadwick, 2007).  However, this unraveled conformation is not stable10under physiological conditions, and the material quickly returns to its initial state oncethe stress is removed (Rapoport and Shadwick, 2007).Figure 5.  Model of WECP mechanics through maturation.  (A) the general, uncrosslinked structure ofsheets of microfibrils formed by charge-based self-assembly and laid down in varying orientations.  (B)tensile loading (indicated by arrows) of the immature capsule results in shearing and sliding of the layers ofsheets, leading to material failure.  (C) during treatment in the ventral pedal gland (VPG), the muscularaction brings successive sheets closer together, allowing crosslinks to form.  (D) after processing in theVPG, the crosslink density is sufficient to transfer stresses down to the smallest hierarchical level of thematerial.  Reproduced with permission from the Journal of Experimental Biology, Rapoport and Shadwick2007.     Didier (2009) incorporated data from birefringence measurements and X-raydiffraction to add detail to the model at the molecular level.  Birefringence decreases atthe transition to the yield region, indicating a loss of overall order in the structure as α-helices are pulled apart (Didier, 2009).  β-sheet signals were detected in the strained11material but the α-helices spontaneously reform on recoil, indicating that the β-sheetsformed are not stable (Miserez et al., 2009).     Further evidence of the reversible α-helix to β-sheet transformation was gatheredusing wide-angle X-ray scattering analysis of the capsule wall during deformation(Miserez et al., 2009).  Classical features of α-helices were detected at 0% strain, and a β-sheet formation was indicated at 70% strain (Miserez et al., 2009).  After unloading, theX-ray diffraction pattern returns to that of an unstretched specimen, with no β-sheetfeatures remaining (Miserez et al., 2009).Figure 6.  Reversible α-helix to β-sheet transition during straining.  As the material is loaded the α-helicesare pulled apart, first into random coils, then into β-sheets.  Domain 1 is from 0 to 5% strain, domain 2 isfrom 5 to 20% strain, domain 3 is from 20 to 60% strain and domain 4 is over 60% strain.  Reproducedwith permission from Nature Materials, Miserez et al., 2009.     This X-ray data led to the development of a more detailed model (Figure 6) with thereversible α-helix to β-sheet transition divided up into four domains (Miserez et al.,2009).  Domain 1, from unstrained to 5% strain, is dominated by α-helices.  The helicesbegin to unfold into random coils at the yield point of the material, and a decrease instiffness is seen (Miserez et al., 2009).  Domain 2 is from 5 to 20% strain, has a lowermodulus and is dominated by the extension of random coils (Miserez et al., 2009).12Domain 3 is from 20 to 60% strain, where the proportion of random coils decreases asthey are replaced by β-sheets (Miserez et al., 2009).  Domain 4 is β-sheet dominated,with random coils having mostly disappeared and recrystallized into β-sheets (Miserez etal., 2009).  The increased modulus of domain 4 is due to the inherently stiffer nature of β-sheets, as well as their alignment along the loading axis during stretching (Miserez et al.,2009).1.8     More mechanical properties of whelk egg capsules     Interestingly, WECP recovers Hookean behaviour in the yield region if a specimen isstretched, then held at constant strain (Rapoport and Shadwick, 2002).  Stresses dropalmost immediately, indicating a rapid molecular reorganization, probably due to thereformation of coiled-coils (Rapoport and Shadwick, 2007).  These coiled-coils (andlikely H-bonds in and among these structures) will even reform under slow continuousextension (Rapoport and Shadwick, 2007).  However, the idea that coils were reformingwas not supported by birefringence measurements (Didier, 2009).Figure 7.  Stress recovery in the native capsule.  The specimen was held at constant strain for 5 minutes onthe extension cycle during the yield phase (arrow).  Stress drops almost immediately, and the materialrecovers Hookean behaviour when strain is resumed, with an initial slope similar to that in the initial stiffregion.  Reproduced with permission from Biomacrmolecules, Rapoport and Shadwick 2002.13     Microscopic tests on teased-apart fibres of the egg capsule showed that the mechanicalproperties are intrinsic to the macromolecules, and are not due to the laminar structure ofthe capsule walls (Didier, 2009).  Mechanical testing of microscopic fibre samplesproduced stress-strain curves similar to macroscopic samples, and the toe region andtertiary regions were described (Didier, 2009).  The toe region preceeds the Hookeanregion, and represents the taking up of slack in the fibrous protein matrix as the fibres areloaded (Didier, 2009).  The tertiary region produced a higher modulus by pulling moredirectly on the primary molecular chains, beyond the point of mechanical failure in thelaminar structure of macroscopic samples (Didier, 2009).1.9     Polarized light microscopy     Polarized light microscopy is a useful technique for evaluating structure that reducesthe likelihood of methodological artifacts because it does not require any physicalmodification of the sample.  Birefringence is an optical effect generated by orderedstructures on the molecular level, and can therefore be used as an index of order. Thereare two ways in which order produces birefringence: 1.) form birefringence, generated bythe difference in refractive index between a material and the medium it is in, and 2.)intrinsic birefringence, caused by light refracting in two directions when passing throughparallel structures that are closer together than the wavelength of the light.  Totalbirefringence is the sum of the form birefringence and the intrinsic birefringence.  Anisotropic material should have no intrinsic birefringence, while an anisotropic (highlyordered) material should have high intrinsic birefringence (Aaron and Gosline, 1980).     Rapoport and Shadwick (2002) demonstrated that WECGP has an ordered structurewith an α-helical conformation.   Birefringence scans of whelk egg capsules showed thatthe material was highly birefringent, and that the layers of the capsule are randomlyoriented (Rapoport and Shadwick, 2007).   Didier (2009) showed that isolatedmicrofibres from the egg capsule show an increase in birefringence when they are14strained in the Hookean region, then decrease in birefringence once they are strained pastthe yield point, into the rubbery region.1.10     Fourier transform infrared spectroscopy     Fourier transform infrared spectroscopy (FTIR) has been applied in the study of themolecular conformation of many different proteins, including keratin (Aluigi et al., 2008,Kreplak et al., 2004).  FTIR has also been used to examine the structure and compositionof other biological materials such as lipids, biomembranes, carbohydrates, and animalcells and tissues, and is used in blood and skin analysis and clinical screening tests forcervical and colorectal cancers (Stuart and Ando, 1997).  FTIR has also been used toanalyze drugs, wine, and artificial sweeteners (Stuart and Ando, 1997).     The infrared spectra of proteins exhibit nine absorption bands associated with theircharacteristic amide group, called amide A, amide B, and amides I-VII, in order ofdecreasing frequency.  Amide A, I, and II have been used most frequently forconformational studies, and amide I is the most useful for the analysis of the secondarystructure of proteins (Stuart and Ando, 1997).  Amide I represents 80% of the C=Ostretching vibration in the amide group (Stuart and Ando, 1997).  The exact frequency ofthis vibration depends on the nature of hydrogen bonding involving the C=O and N-Hgroups, which is determined by the secondary structure of the protein (Stuart and Ando,1997).  Because proteins often have a variety of domains in different conformations, theamide I band is usually a complex composite made up of a series of overlappingcomponent bands representing α-helices, β-sheets, turns, and random structures (Stuartand Ando, 1997).     Protein secondary structures have been assigned characteristic FTIR frequencies basedon proteins that were well characterized by X-ray crystallography (Stuart and Ando,1997).  Curve fitting the amide I band gives the fractional areas of the component bands,which are directly proportional to the relative amounts of the secondary structure they15represent (Stuart and Ando, 1997).  The percentages of α-helices, β-sheets, and turns areestimated by adding the areas of all the component bands assigned to those structures,and then expressing the sum as a fraction of the total amide I area (Stuart and Ando,1997).     The secondary structure of electrospun WECGP was of interest because the currentmodel has a network of coiled-coils as the smallest hierarchical level of the nativecapsule material (Rapoport and Shadwick, 2007).  Straining of the coiled-coils results inthe Hookean region of the mechanical response, unraveling of the coils results in therubbery yield region, and reformation of the coils enables the material to recover oncestress is removed (Rapoport and Shadwick, 2007).  For the electrospun fibres to havesimilar mechanical properties to the native capsule, it follows that the secondary structureshould also be similar.1.11     An introduction to biomimetics and electrospinning     The development and fabrication of new materials based on biological models(biomimetics) is of broad scientific, medical, and industrial interest.  Becausebiomaterials are synthesized at ambient temperatures and in aqueous conditions usinghighly specialized molecules, they have enormous potential for the design of newmaterials.  With self-assembly, materials can be produced with less energy and lessdependence on fabrication machinery or environmentally damaging processing steps.  Asthe culmination of millions of years of natural mechanical design, biomaterials offer awealth of information and a great opportunity for materials design and biomimeticapplications.     Electrospinning is a novel technique that produces micro- and nano-scale fibres byapplying a high voltage electric field to a solubilized polymeric solution.  Electrospinninghas been successfully applied to many synthetic polymers, as well as naturallybiosynthesized protein polymers such as silk, collagen, elastin, and keratin (eg. Aluigi et16al., 2008; Chen et al., 2007; Li et al., 2005; Matthews et al., 2002; Sukigara et al., 2003,2004, and 2005).  The whelk egg capsule protein seemed an ideal candidate for thistechnique.     Electrospun fibres have great potential for materials design and biomimeticapplications.  Electrospinning has emerged as a leading technique for generatingbiomimetic scaffolds for tissue engineering.  The ultra-fine fibre scaffolds producedclosely mimic the topology of the extracellular matrix, and cells attach and proliferate onthe scaffolds (Li et al., 2006; Min et al., 2004).  Other applications for electrospunnanofibres include wound dressing materials, membrane filters, aerosol filters, textilecomposites, drug delivery, biological membranes, artificial blood vessels for vasculargrafts, formation of carbon nanotubes, and production of electrically conducting orinsulating nanofibrous material.  Ultimately, the goal of this project is to be able tofabricate self-assembling elastomeric biomaterials with useful tensile properties based onthe molecular design of the whelk egg capsule.1.12     History of electrospinning     Creating fibres by electrifying a fluid can be traced back to the late 1800’s, whenelectrostatic machines were available in many laboratories (Reneker and Fong, 2006).  J.Zeleny published a paper in 1917 that described the observation of liquid jets created byelectrical forces (Zeleny, 1917), but the observation of these jets is much older.  G.I.Taylor wrote that W. Gilbert observed in about 1600 that a drop of water on a dry surfaceis drawn up into a cone when a piece of charged amber is held above it (Taylor, 1969).     Electrospinning as a technique to make fibres was first patented by A. Formhals in1934 (US patent 1,975,504).  This followed research in the 1920’s that established thatpolymers are long linear molecules (Reneker and Fong, 2006).  Formhals envisionedelectrospinning as a technique for making textile fibres, but the textile industry came touse other methods (Reneker and Fong, 2006).17     Despite further publications and patents issued from the 1930’s to 1990, interest inelectrospinning only developed in the 1990’s, along with broad interest innanotechnology and nanomaterials in general (Reneker and Fong, 2006).  Electrospinningis now being applied to materials science as an efficient method to manufacture micro-and nano-sized fibres of many polymers.     Several aspects of electrospinning nanofibres make the process appealing.  Theequipment required for electrospinning is simple, not overly expensive, and readilyavailable.  Nanofibres have a very high surface area to volume ratio, which is key tomany of their applications.  The process is reproducible, although obtaining a uniformfibre diameter and collecting the fibres can be problematic.  Electrospinning also offers asimplified manufacturing process, allowing a microporous structure to be sprayeddirectly onto a substrate, such as a fabric, screen, or living tissue (Schreuder-Gibson andGibson, 2006).  The electrospinning process does not involve extreme temperatures,enabling the addition of temperature sensitive components such as drugs and biologicalmaterials (Schreuder-Gibson and Gibson, 2006).     Parameters such as concentration, field strength, and distance between the source andcollector influence the size and consistency of the spun fibres.  These parameters must beoptimized for reliable fibre formation and in order to produce suitable specimens fortensile testing and other applications.  Less than optimal electrospinning parametersproduce fibres of non-uniform diameter, droplets instead of fibres, or “beads on a string”- droplets connected by fibres.181.13     The electrospinning technique     The electrospinning apparatus is composed of three main components: a high voltagegenerator, a syringe feeding the polymer solution to a capillary tip, and a collector..     Electrospinning occurs in three stages: 1.) jet initiation, 2.) jet elongation, and 3.) jetsolidification or fibre formation.  During the jet initiation stage, electrostatic forcesovercome the surface tension of the polymer solution. The charge induced on the polymersolution deforms the spherical drop on the capillary tip to a conical shape, called a Taylorcone.  At a critical voltage, charge imbalance results in the formation of an electricallycharged jet.  The jet is emitted from the capillary tip towards the electrically groundedcollector.     The jet will remain stable over a certain distance, where it will travel in a straight line.Then it becomes unstable, bends, and follows a looping, spiral course.  Jet elongationoccurs during this unstable, looping travel, when electrical forces stretch the jet tothousands to millions of times its original length (Fang et al., 2006).  The charges in thejet are evenly distributed and repel each other, contributing to elongation (Fang et al.,2006).  Solvent evaporation occurs throughout jet initiation and elongation, furtherdecreasing the diameter of the jet.     The final stage of jet solidification occurs when the viscosity of the polymer solutionbecomes so great that there is no further elongation of the jet (Fang et al., 2006).    Theresult is the deposition of a single continuous fibre filament on the collector (Yamashitaet al., 2007).  Both the stretching and the accompanying evaporation of the solvent causea reduction in the diameter of the jet and therefore of the fibres collected.  The driedfibres are deposited either randomly or in an aligned manner on the surface of thecollector.19Figure 8. Whelk egg capsule gland protein is solubilized and placed in a syringe with a flat-tipped needle.When a high voltage is applied, the electrical forces overcome the surface tension of the solution at theneedle tip.  This results in the formation of a jet, which travels from the needle to a grounded target.1.14     Electrospinning parameters     The characteristics of electrospun fibres depend on both the properties of the polymerfluid (concentration, viscosity, conductivity, surface tension) and on the operatingconditions of the electrospinning process (distance between capillary tip and collector,flow rate, strength of electric field, bore diameter of needle, rate of deposition).Electrospinning solvents should be highly volatile to ensure that polymeric nanofibres aredeposited dry on the collector to prevent resolubilization.  Formic acid was chosen for itsvolatility and convenience – it leaves no residual salt when it evaporates and it effectivelysolubilizes WECGP.     The electrospinning process uses an electric field to control the formation anddeposition of polymers on a grounded target.  The distance between the polymer solutionsource and the target can be increased to ensure complete evaporation of the solvent sothat electrospun fibres are deposited dry on the collector and do not resolubilize upondeposition. Yang (2006) found that increasing the distance from the needle to the targetdoes not necessarily enhance solvent evaporation, but does reduce the electric fieldstrength.  These two parameters should therefore be considered together.20     Katti et al. (2004) refer to the two parameters together as the electrospinning voltage,measured in kV/cm.  They found that the diameter of the electrospun fibres decreasedwith an increase in electrospinning voltage (Katti et al., 2007).  Above 1 kV/cm there wasno significant change in fibre diameter (Katti et al., 2007).     Higher gauge (smaller bore diameter) needles were found to produce smaller diameternanofibres (Katti et al., 2007).  The difference was significant between 16 and 20 gaugeneedles (internal diameters of 1.19 mm and 0.58 mm, respectively), but there was nosignificant difference between 18 and 20 gauge needles (internal diameter of 18 gauge is0.84 mm) (Katti et al., 2007).     Poly(ethylene oxide) (PEO) is often added to the electrospinning solutions to enhancefibre formation.  PEO is a soluble synthetic polymer known for its ease of processing inelectrospinning applications.  Like most polymers, PEO solutions must reach a certaincritical concentration before electrospinning will produce continuous fibres.  If thesolution is too dilute, droplets result, and if it is too concentrated, the solution becomestoo viscous to undergo the plastic stretching necessary for fibre formation (Gupta, 2004).     The density of electrospun fibres deposited on the collecting target was found toincrease linearly with time, indicating a uniform rate of deposition (Katti et al., 2007).However, as the fibre size becomes very small, the yield of the electrospinning processbecomes very low.  This is the major technical barrier for scale-up processing, such asmanufacturing electrospun fabrics for clothing (Fang et al., 2006).  Another majortechnical problem for mass production is the assembly of spinnerets.  Straightforwardmulti-jet arrangements cannot be used because adjacent electrical fields tend to interferewith each other (Fang et al., 2006).  Issues with increasing the product yield have heldelectrospinning back from many industrial applications in the past, but increased interestin nanotechnology raises expectations for the future.211.15     Hypothesis     Enough was already known about the biology of the whelk egg capsule protein tomake some predictions about what would happen when the electrospinning techniquewas applied to the immature, solubilized protein.  We knew it was unlikely thatelectrospinning alone would yield the mature protein, since there is nothing in thetechnique to act in place of the hour-long muscular massage that the egg capsules receivein the VPG.  We knew from the maturation timeline that a transitional state existsbetween the immature and the mature protein, and that the electrospun fibres would atbest be in a similar state.  If they were, we planned to introduce a cross-linking step totake the fibres closer in their mechanical properties to the mature protein.222     Methods2.1     Preparation and characterization of the blend solutions     Adult female whelks (Busycon canaliculatum) were dissected and their nidamentalglands removed.  Glands not used immediately were stored at –80ºC.  Glands wereplaced in a solution of 25 mM TCEP (Alfa Aesar, Ward Hill, MA, lot # E25R045, MW286.65) in 88% formic acid and homogenized using a glass mortar and pestle.  WECGPwas solubilized and subsequently dialyzed against 0.01 M acetic acid in Spectra/Pormolecularporous membrane tubing (molecular cut off 3,500) for 5 days at 4ºC.  Thedialysis solution was replaced daily and was on a stir plate on low setting.  The solutionwas then freeze-dried for 3 days in a Labconco FreeZone 4.5 to obtain the purifiedprotein.     Blend solutions were prepared by first adding 25 mM TCEP to 88% formic acid, thenadding the freeze-dried protein to make an 8 wt% WECGP solution.  Poly(ethyleneoxide) (PEO, MW 300,000 from Alfa Aesar) was added directly to the 8 wt% WECGPsolution in order to obtain 2 wt% PEO, and 10 wt% total polymeric concentration.  Thesame protocol was used to make blend solutions of other concentrations.2.2     Selection of TCEP as the reducing agent     The egg capsule gland was obtained by dissecting the gland out of an adult female.The gland was homogenized in 6 M guanidine solution using a glass mortar and pestleand centrifuged for 30 minutes at 13500 rpm.  The supernatant was then dialyzed against0.01 M acetic acid for 24 hours in Spectra/Por dialysis tubing (Spectrum Labs, MWCO3500) and freeze-dried.     A stock solution of freeze-dried WECGP in double strength sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (10 mg/ml) was23produced.  Solutions of 0.5 and 20 mM ethylenediaminetetraacetic acid, disodium saltdehydrate (EDTA, BioRad), 0.5 and 20 mM mercaptoethanol (Sigma), 0.5 and 20 mMdithiothreitol (DTT, Sigma), and 0.5 and 20 mM TCEP (Alfa Aeasar) were preparedusing the protein/buffer solution.  The stock solution was used as a control. 5 µl samplesof all solutions were separated on a 14% gel, run at a constant current of 25 mA.Molecular weights were determined with a broad-range protein standard (BenchmarkPre-Stained Protein Ladder).     Another stock solution of freeze-dried WECGP in double strength SDS-PAGE samplebuffer (10 mg/ml) was produced and used as a control.  Different amounts of TCEP orDTT were added to the protein/buffer solution, resulting in solutions with 5, 10, 25, and50 mM TCEP and 0.5 and 20 mM DTT.  5 µl samples of all solutions were separated ona 14% gel, run at a constant current of 25 mA.   Molecular weights were againdetermined with a broad-range protein standard (Benchmark Pre-Stained Protein Ladder).2.3     Nanofibre electrospinning and properties     All fibres were generated using the NEU-010 Nanofibre Electrospinning Unit (KatoTech Co., Ltd.  Kyoto, Japan, Figure 10), and all experiments were carried out at roomtemperature. The syringe cradle of the NEU-010 was built for 20 ml syringes, but 1 mlsyringes were used in this study.  The machine was adapted to accommodate the smallersyringes by adding an extending block to the syringe pump.     About 0.5 ml of blend solution was loaded into a 1 ml syringe linked to a blunt-tippedneedle (18 gauge, inside diameter 0.84 mm).  The anode of the 40 kV power supply wasconnected to the metal needle.  The cathode (collecting screen) was a rotating stainlesssteel cylinder of 8.5 cm diameter.  The target mandrel was not rotating and traversemovement was shut off in order to concentrate the sample in a small target area.24Figure 9.  (a) The Kato Tech NEU-010 Nanofibre Electrospinning Unit.  The apparatus holding the syringecradle and pump has been adjusted to maintain a set distance of 10 cm between the source needle and thecylindrical target mandrel.  (b) Closer view of syringe cradle and extending block attached to the syringepump to accommodate the 1cc syringes used in this study.  The anode of the power supply is also visible,clipped to the blunt-tipped needle.     The target mandrel was covered with an aluminum sheet to obviate the task ofremoving the electrospun samples.  The foil allowed the target surface to remain suitablygrounded.  A voltage of 20 kV was applied and a constant volume flow rate of 0.2ml/min was maintained.  The collecting screen was placed at a distance of 10 cm fromthe syringe tip to ensure the complete evaporation of the solvent.abSyringe cradlePower supplyBlunt-tipped needleTarget mandrelExtending blockSyringe pump252.4     Preparation of SEM samples     Nanofibres were collected on the aluminum sheet as non-woven mats of randomlyoriented fibres.  The fibres adhered to the sheet and were not easily removed.  Weak acid,weak base, distilled water and ethanol solutions had no effect on fibre adhesion.  Thealuminum sheet with the nanofibres deposited on the surface was therefore cut andmounted directly on aluminum SEM specimen stubs with silver adhesive solution.  Latersamples were electrospun directly onto SEM specimen stubs, which were attached to thetarget mandrel through punctures in the aluminum foil.  This method resulted in easierimaging due to a smaller range of fluctuations in the height of the specimen.2.5     Collection of SEM images     Nanofibre morphology was examined by scanning electron microscopy using aHitachi S-4700 Field Emission SEM with an acceleration voltage of 1.0 kV and workingdistance of about 4mm.  At this acceleration voltage the S-4700 is capable of 2.5 nmresolution.     Samples on SEM specimen stubs were viewed uncoated, or sputter coated with 4 ± 1nm of gold using a Cressington Sputter Coater 208HR and a Cressington ThicknessController mtm20 (Cressington Scientific Instruments, Watford, England).  Uncoatedsamples tended to melt when exposed to the electron beam for any length of time, makingthem difficult to image.  Coated samples were more durable and therefore were used forimaging and measurements.     Charging was observed when visualizing many of the samples.  Diameter of the fibres,interconnectedness of the fibres, thickness of the fibre mat, attachment of the mat to thespecimen stub, and concentrations of the blend solutions could all affect charging of thesamples.262.6     Polarized light microscopy     Blend solutions as described above were electrospun and collected on a modifiedtarget.  6 mm by 6 mm windows were cut in the aluminum foil that was wrapped aroundthe target mandrel, and glass microscope slides were attached behind the windows.Electrospun fibres seek a grounded target, so many of the fibres spanned the window inthe foil and were suspended above the slide, but some fibres were collected and observed,and photographed in air using plane polarized light.     Birefringence measurements were taken on smaller samples of fibres using a WildSenarmont 546 compensator with a 546nm interference filter.  These fibres weresuspended freely from the target mandrel.  They were collected by very gently liftingthem onto a drop of immersion oil on a piece of cover slip.     Birefringence is the angle of retardation, or how much the sample rotates the plane ofpolarized light, divided by the path length, or thickness of the sample.  Retardation hadbeen calibrated for the scope as:Γ = θ * 3.03 nm/degwhere Γ is the optical retardation, measured using a Senarmont λ/4 compensator plate,and θ is the degree of rotation.  Birefringence is then calculated as:Birefringence = B = Γ         twhere Γ is the retardation and t is the thickness of the sample.272.7     Determining the effect of PEO addition     PEO was added to 88% formic acid to produce solutions of 2 wt%, 3 wt%, 4 wt%, 5wt%, and 10 wt%.  These solutions were electrospun using the same parameters andprotocol described above for the blend solutions.  The solutions that formed fibres hadthe fibre diameters measured according to the protocol described below for the series ofblend solutions.     PEO is highly soluble in water.  Therefore, rinsing with water was consideredadequate to wash away any PEO that was not bound or incorporated into fibres.  Sampleswere imaged on the SEM, washed, and imaged again.  SEM specimen stubs that weresubjected to washing with water all had samples electrospun directly onto the stubs andwere not sputter-coated.  Stubs were thoroughly rinsed with distilled water and allowed toair dry.  They were then dried further under vacuum before being transferred to the SEMspecimen chamber.  Diameters of the fibres were measured and washed samplescompared to the pre-washed samples.2.8     Measurement of electrospun fibres     Two concentration series of blend solutions were imaged and measured for fibrediameter, one with a constant weight percent of protein (A-D) and one with a varyingamount of protein (D-G).  All blend solutions contained 25mM TCEP and used 88%formic acid as the solvent.     Images were collected at 500 times and 5000 times magnification for overall images,and 10,000 times, 25,000 times, and 50,000 times magnification for measurement.  The25K images were preferred for measurement since that magnification had more fibresavailable for measurement than the 50K images, and created less measurement error thanthe 10K images.  However, good quality 10K and 50K images were used when necessaryin order to collect enough measurements.28Stub A8 wt% WECGP5 wt% PEOStub B8 wt% WECGP4 wt% PEOStub C8 wt% WECGP3 wt% PEOStub D8 wt% WECGP2 wt% PEOStub E7 wt% WECGP3 wt% PEOStub F6 wt% WECGP4 wt% PEOStub G5 wt% WECGP5 wt% PEOTable 1.  Blend solution concentration series used for producing electrospun fibres for diametermeasurement.     Diameters of the nanofibres were acquired from SEM images of random locations onthe specimen stubs, using the image analysis software ImageJ (National Institute ofHealth).  The SEM images selected for measurement were zoomed in to 200% foranalysis.  All images were calibrated using the built-in scale bar from the SEM image asa known distance.  For each sample the diameter distribution was evaluated from 100measurements.  All fibres measured were within the field of focus, and had visible anddistinct edges.  Fibre orientation in the field of view was irrelevant, and fibres that metthe criteria were randomly selected for measurement.2.9     FTIR spectroscopy     To investigate the composition of the electrospun fibres, FTIR spectra were obtainedusing a Nicolet 6700 FTIR using the provided OMNIC software suite (Thermo Scientific,Waltham, MA, USA).  Data was acquired from solid samples of PEO, freeze-driedWECGP, and electrospun fibres from a blend solution of 8% WECGP and 2% PEO(solvent was 88% formic acid with 25mM TCEP).  Samples were clamped directly intothe Nicolet 6700 without treatment or coating, maintaining the integrity of the solids.29Frequency  (cm-1)Assignment1621-1640β-structure1641-1647Random coil1651-1657α-helix1658-1671Turns and bends1671-1679β-structure1681-1696Turns and bendsTable 2.  Characteristic amide I band frequencies of protein secondary structures.  These characteristicfrequencies were used to determine the positions of the peak intervals in the curve-fitting procedure.  FromStuart and Ando, 1997.     The amide I band (1600-1700 cm-1) of each spectrum was subjected to a curve fittingprocedure using six Gaussian distributions centred at the frequencies of well-characterized secondary structures (Stuart and Ando, 1997).  During the fitting the peakheights were not constrained, but the peak positions were kept to a limited interval.Integrating the area of each peak and then normalizing it to the total area of the amide Iband determined the contribution of each peak to the amide I band.  The fitting procedurewas done using Microsoft Excel and the peak fitting module of the OriginPro 8.1software package (OriginLab, Massachusetts, USA).2.10     Preparation of electrospun samples for mechanical testing     Testing the mechanical properties of a single electrospun fibre would be ideal but isunrealistic without an omnipotent technician.  Fibres were therefore measured as thick,semi-aligned non-woven mats.  Since fibres spun onto aluminum foil on the collectingmandrel proved all but impossible to remove, mats spanning the gap between two insectpins that protruded perpendicularly from the target mandrel were used (Figure 11).30Figure 10.  Modified electrospinning apparatus for collection of samples for mechanical testing.  Twoinsect pins were mounted beneath the aluminum foil that covers the target mandrel.  The pins protrudedperpendicularly from the mandrel, pointing towards the source needle.  Electrospun fibres spanned the gapbetween the pins, avoiding deposition upon and adhesion to the foil.     Electrospun protein blend fibres were collected from the gap between the insect pinsand either immediately fixed to the mechanical testing apparatus, or subjected to a cross-linking step first.  The cross-linking procedure was simple submersion of the sample in a1.25% glutaraldehyde solution for a period of 1 hour.  At the end of the hour the samplewas removed and allowed to air dry for 24 hours before being fixed to the testingapparatus.  Other samples were immediately fixed to the apparatus, tested, and thensubjected to the cross-linking step while still mounted in the testing apparatus.     A sample of 7 wt% PEO in formic acid was electrospun for mechanical testing todetermine whether it could contribute to the mechanical properties of fibres from theprotein blend solutions.  This was spun directly onto aluminum foil on the target mandrel.PEO does not adhere to the foil as strongly as WECGP and can be removed as a thickmat using a razor blade.  A pair of razor blades, fixed together to cut 7.0 mm sections,was used to cut test-sized strips of width 7.0 mm ± 0.1 mm and length 12.0 mm ± 0.1mm.  The strips were measured using Vernier calipers.  Thickness was measured using adigital micrometer, and ranged from 0.300 to 0.450 mm, ± 0.005 mm.31Figure 11.  Electrospun fibres that spanned the gap between two insect pins were collected for mechanicaltesting.2.11     Mechanical testing apparatus     The mechanical properties of the mat samples were tested using a microscope-basedmicro-tensile tester (Figure 12).  The apparatus was originally developed by Gosline et al.(1995) for use on hydrated spider silk fibres, which are usually only microns in diameterand require extremely small forces to test.  The apparatus uses thin glass rods as forcesensing elements.  The rods are mounted on microscope slides and force is determined bymeasuring the deflection in the beam using a video dimension analyzer attached to themicroscope. The micro-tensile tester is capable of measuring nano-Newton forces whenusing very thin glass beams (Savage et al., 2003).     Beam theory is used to convert the deflections of the beam into force values:Force = F = 3xEI         L3Paired insect pinsElectrospun fibres spanningthe gap between the pinsPower supplyElectrospun jet32where x is the stretch of the sample, E is the Young’s modulus of glass (5.72 ± 0.06 x1010 N/m2, Fudge et al., 2003), I is the second moment of area of the beam (bendingmoment), and L is the length of the beam (Fudge et al., 2003).  Force and deflection havea linear relationship for beam deflections up to approximately 10% of the length (Fudgeet al., 2003), so glass micro-beams were chosen to minimize deflection during a test.Figure 13.  The microscope-based micro-tensile testing apparatus used to measure force in electrospunsamples.  A video dimension analyzer (VDA) tracked the movement of the glass micro-beam.  Reproducedwith permission from Biomacromolecules, Savage et al., 2003.     The diameters of the glass beams were slightly tapered instead of perfectly constant.For a uniformly tapered cylinder the second moment of area is:I = π (r1)3(r2) 4where r1 is the radius at the point where the beam is fixed, and r2 is the radius at the pointwhere the sample is attached (Fudge et al., 2003).     Samples electrospun to span the gap between two insect pins were glued with 24 hepoxy (J.B. Weld) to the glass micro-beam at one end and to a moveable cover slip on the33other.  The cover slip was attached to a micrometer mounted under the microscope.  Avideo camera (Panasonic WV-BL600) was mounted on the microscope and connected toa video dimension analyzer (VSA-303, PIM, San Diego, USA), which tracked thedeflection of the micro-beam.  The VDA generates an electronic window that can followthe movement of the contrast boundary created by the edge of the glass rod, and providedvoltage outputs that are proportional to the movement of the rod.  A monitor (PanasonicColour Video Monitor CT-133/YC) was used to follow the deflection of the glass beam.The stretch of the sample was isolated for each point by subtracting the deflection of thebeam from the movement of the micrometer.     The specimen slide had a water chamber surrounding the micro-beam and cover slip,which was filled with distilled water before testing to keep the sample hydrated andcompletely submerged.  Prior to testing, the sample length and width, the beam lengthand diameter at the fixed point and sample attachment point were all measured using afilar micrometer eyepiece.  The microscope system was calibrated with a calibration slidewith 0.01mm increments (Bausch and Lomb, USA).2.12     Mechanical testing procedure     It is not simple to perform accurate measurements of tensile properties of nanofibremats because of the intrinsic weakness and difficulty to manage this kind of material.With patience, stoicism, and stubborn pig-headedness, measurements were repeatedsuccessfully on 6 different samples.  Handling of the samples was kept to a minimum.     Samples ranged from 1.87 to 3.68 ± 0.05 mm in length, and 0.493 to 0.243 ± 0.05 mmin width.  Width and thickness of the samples was not uniform.  Thickness could not bemeasured directly once the sample was mounted and so was estimated by eye as onequarter of the width.34     Once firmly attached to the testing apparatus, samples were extended in 20 µmincrements starting at slack length.  Samples were subjected to one of the followingtreatments: 1.) extension until material failure, 2.) extension to a set point then aFigure 13.  Electrospun samples were glued to a glass micro-beam at one end and a cover slip attached to amicrometer on the other.controlled recoil to get a hysteresis loop, or 3.) hold for a set time during extension beforecontinuing the hysteresis loop.  In materials testing, hysteresis is the proportion of strainenergy that is lost by damping through viscous interactions within the material.     Force was recorded in mN and converted to engineering stress (MPa = 106 N/m2)using the estimated cross sectional area of the sample:Stress = σ = F         Awhere F is force in Newtons and A is the cross sectional area in square meters.Glass rodCover slipElectrospun sample35     Strain was calculated using the formula:Strain = ε = (L – L0)       L0where L is the measured length of the sample and L0 is the original length of the sample.Stress-strain curves were generated from the data using Microsoft Excel and SigmaPlot.The initial Young’s modulus was calculated for each stress-strain curve by finding theinitial slope.  Hysteresis was calculated for each stress-strain curve by taking the ratio ofthe area under the extension curve to the area under the relaxation curve.363     Results3.1     The effect of TCEP addition     Phosphines selectively reduce disulphide bonds and are essentially non-reactivetoward other common protein functional groups. TCEP was chosen as a reducing agentbecause it is odorless, non-volatile, resistant to air oxidation, and able to reducedisulphide bonds at low pH.  SDS-PAGE was used in order to test the efficacy of TCEPin comparison to DTT, which was used in earlier work on WECGP (Rapoport, 2003).The gel showed that at 25 mM TCEP, disulphide bonds in the protein were completelyreduced and the WECGP was broken down into its 50 kDa monomer.  This was similarto the effect of 20 mM DTT, shown by Rapoport (2003) to fully break down the proteininto its monomeric subunit.  The same amount of protein was applied to all gels, butwithout the addition of reducing agents such as TCEP or DTT, WECGP does not travelfar past the 5% stacking gel (Figure 14).  The decreased amount of protein visible in thelanes without reducing agents shows that the large molecular weight protein aggregateswere too large to enter the 14% running gel.     All WECGP electrospinning blend solutions contained 25 mM TCEP in order to breakthe protein down into its monomeric 50 kDa subunit.  Without the TCEP, the WECGPsolution was difficult to homogenize, tended to aggregate, and did not form fibres.37Figure 14.  Diagram of SDS-PAGE gel showing the effect of several concentrations of TCEP and twoconcentrations of DTT on the breakdown of WECGP to its 50 kDa monomer.  Lane 1 is the marker, lane 2is 5 mM TCEP, lane 3 is 10 mM TCEP, lane 4 is 25 mM TCEP, lane 5 is 50 mM TCEP.  Lane 6 was leftempty.  Lane 7 is 0.5 mM DTT, a concentration insufficient for monomer formation, and lane 8 is 20 mMDTT, a concentration sufficient for complete breakdown of the protein into its 50 kDa monomer.  A TCEPconcentration of 25 mM or greater has a comparable effect to 20 mM DTT and is therefore also consideredsufficient for complete breakdown into the monomeric subunit.3.2     The effect of electrospinning parameters     Electrospinning was attempted at distances of 5 – 30 cm, in increments of 5 cm, withapplied electric field strengths of 5 – 40 kV, in increments of 5 kV.   At distances longerthan 15 cm, fibres were not reliably deposited on the target and extensive and tediouscleanup of the equipment was required.  Field strengths greater than 30 kV caused theelectrospun jet to fluctuate wildly at any distance.     At distances shorter than 5 cm a spark was discharged, indicating breakdown of theelectric field between the charged syringe needle and the grounded target.  Field strengthslower than 15 kV deposited wet material on the collecting mandrel, resulting inunattractive blobs of resolubilized protein, and no fibres (Figure 15 b).  The “beads-on-a-string” fibre morphology was produced as the electric field approached the optimum38strength for fibre formation (Figure 15 c).  Voltage polarity was not altered during thecourse of the experiments.     Syringe pump speeds over several orders of magnitude were attempted, from 10 ml/hdown to 0.01 ml/h.  A greater pump speed is desirable to enhance the rate of fibreproduction, but only very slow speeds were successful in producing uniform nanofibres.Higher speeds deposited wet material on the target mandrel, forming films or blobs ofresolubilized protein similar to those produced at inadequate field strengths (Figure 15 a).Figure 15.  SEM images showing the effect of different electrospinning parameters.  (a) polymer filmproduced by overly high syringe pump speed, (b) resolubilized protein blobs produced by insufficientelectric field strength, (c) beads-on-a-string formed by low electric field strength, and (d) uniform fibresformed when electrospinning parameters are optimized.dcba39     Some short, very fine fibres were observed at the ends of broken fibres, andsometimes coming off of beads or fibres.  An example of these fibres can be seen in thetop left corner of Figure 15 d.     The parameters that produced the most uniform fibres (Figure 15 d) were 20 kV ofelectricity and 10 cm between source and collector, or 2 kV/cm.  The syringe pump speedthat produced the most uniform fibres was 0.02 ml/h.  These parameters were used for allsubsequent electrospinning of WECGP.3.3     Polarized light microscopy     Isolated microfibres teased directly from the egg capsules had a resting birefringenceof 5 x 10-4 or greater (Didier, 2009).  As expected, electrospun fibres from solubilizedwhelk egg capsule gland protein were also found to be birefringent (Figure 16), althoughless so than the microfibres.   Electrospun fibres immersed in oil to eliminate formbirefringence were found to have an intrinsic birefringence of 6 x 10-6 or greater.   Figure 16.  Polarized light microscopy images demonstrate that fibres electrospun from whelk egg capsulegland protein are birefringent, like fibres from the native egg capsule.  Image (a) was taken in air and showsthe total birefringence, which includes form birefringence.  Image (b) was used to calculate thebirefringence since the sample was in oil, which penetrates to give only the intrinsic birefringence of theelectrospun fibres.a b403.4     The effect of PEO addition     Poly(ethylene oxide) (PEO) was added to the WECGP solution in order to improve itsprocessability. On its own, PEO formed nanofibres at concentrations of 5 wt% and 10wt% in 88% formic acid (Figure 17, a-b).  At 4 wt% in 88% formic acid, PEO formedfibres with the “beads on a string” morphology, and at 2 wt% and 3 wt%, PEO did notform nanofibres at all, only droplets (Figure 17, c-e).     2 wt% PEO was the proportion chosen for the WECGP electrospinning blend solution.This enhanced processability sufficiently for fibre formation although the PEO would beunable to form fibres on its own, indicating that PEO was not solely responsible for theproduction of fibres from the blend solutions.     Washing of samples to remove the PEO appeared to cause collapse of the fibrescaffolds, but the fibres themselves seemed to remain intact (Figure 18).  Some breakageof fibres was observed, probably due to the scaffold collapse.     Because of its solubility in water, PEO had no effect on the mechanical testing of theelectrospun fibres.  Electrospun PEO test strips dissolved upon contact with the glue usedto attach samples to the force transducer, and what remained of the samples pulled apartimmediately.  They appeared to lack any mechanical integrity and could not be tested.  Itis highly unlikely that they contribute anything to the mechanical properties of the fibresspun from the blend solutions.41Figure 17.  Scanning electron micrographs of Poly(ethylene oxide) (PEO) nanofibres.  PEO was used toenhance fibre formation in the electrospinning WECGP blend solution.  When electrospun on its own, PEOformed fibres at 10 wt% (a) and 5 wt% (b).  At 4 wt% (c), PEO produced the “beads-on-a-string”morphology, and at 3 wt% (d) and 2 wt% (e) PEO failed to form fibres.a bc de42Figure 18.  SEM micrographs of nanofibres electrospun from a blend solution of 8 wt% WECGP, 2 wt%PEO before (a) and after (b) washing with distilled water.  Washing seemed to collapse the scaffold but didnot appear to damage the fibres.3.5     Measurement of electrospun fibres     With the overall polymeric concentration of the protein blend solutions held constant,the mean fibre diameter increased as the wt% PEO increased and the wt% WECGPdecreased (Figure 19 a).  The mean diameter increased from 0.0834 µm to 0.1731 µm asthe wt% PEO increased from 2 to 5 wt%. Figure 19.  Mean diameters of electrospun fibres, ± SE.  Means were taken from a minimum of 100individual fibre measurements.  (a) constant total polymeric concentration, with wt% WECGP decreasingand wt% PEO increasing, (b) constant protein concentration, with wt% PEO increasing.a ba b43     With the concentration of WECGP in the blend solutions held constant at 8 wt%, theaverage diameter of the fibres did not change significantly at 2, 4, or 5 wt% PEO (Figure19 b).     Fibres electrospun from PEO alone increased in average diameter as the wt% PEOincreased from 4 to 10 wt% (Figure 20).  No fibres were produced at 2 or 3 wt% PEO.At 4 wt% the fibres had the undesirable beads-on-a-string morphology, which restrictedthe number of fibres available for diameter measurements.  Therefore, the mean fibrediameter for the 4 wt% PEO solution is based on 40 individual fibre measurements,rather than the 100 measurements that comprised all other means.Figure 20. Mean diameters (± SE) of electrospun PEO fibres.  Means were taken from a minimum of 40individual measurements.     There were no similarities found in average fibre diameters of analogous solutions(Figure 21).  At a total polymeric concentration of 10 wt%, the average diameter of PEOfibres was 0.171 µm, more than double the average diameter of the 10 wt% protein blendfibres with the least PEO.  Fibres produced from a 5 wt% PEO solution had an averagediameter of 0.065 µm, while fibres produced from a blend solution of 5 wt% protein and5 wt% PEO had an average diameter of 0.173 µm.44       Figure 21.  Mean diameters (± SE) of analogous solutions.  (a) electrospinning solutions with a totalpolymeric concentration of 10 wt%, (b) electrospinning solutions that contained 5 wt% PEO.     The mean diameter of the washed fibres was 0.095 µm compared to a mean of 0.083µm for the pre-washed fibres (Figure 22).  This indicates that no material was lost fromthe fibres in washing with distilled water.  Washing caused a collapse of the electrospunfibre scaffold, so the slight increase in average diameter may be due to a flattening of thefibres during the scaffold collapse.Figure 22.  Mean diameters of fibres (±SE) electrospun from an 8wt% WECGP, 2wt% PEO solution,before and after washing with distilled water.  Means were each calculated from 100 individual fibrediameter measurements.  Unwashed samples were measured dry, and washed samples were dried undervacuum before measurement.a b453.6     FTIR spectra     The FTIR spectra for PEO was overlaid on the spectra for electrospun fibres from theblend solution containing 8 wt% WECGP and 2 wt% PEO.  The spectra for the freeze-dried WECGP was overlaid on the same electrospun blend fibre spectra.  The spectra forthe electrospun fibres was similar to that of the protein, and not similar to that of the PEO(refer to Figure 23).     The component curves of the amide I band for the freeze-dried WECGP andelectrospun fibres were determined using a curve fitting procedure.  In this way thecontributions from peaks centred at the frequencies of well-characterized secondarystructures were determined.  Fortunately the amide I region of WECGP falls in a regionthat is not disturbed by PEO absorptions, so it can be used for structural study of theprotein (Aluigi et al., 2008).  Solvent interactions, which can affect the accuracy of theassigned frequencies for the component bands, do not need to be considered becausesolid samples were used.Freeze-dried WECGPElectrospun fibresBand position(cm-1)Bandassignments% ContentBand position(cm-1)Bandassignments% Content1621-1640β-Sheet19 1621-1640β-Sheet181641-1647Random coil23 1641-1647Random coil221651-1657α-Helix21 1651-1657α-Helix211658-1671Turns/bends16 1658-1671Turns/bends171671-1679β-Sheet12 1671-1679β-Sheet131681-1696Turns/bends8 1681-1696Turns/bends9Table 3.  Composition of freeze-dried whelk egg capsule gland protein (WECGP) and electrospun fibres.Percents that do not add up to 100 are due to rounding.46Figure 23.  FTIR spectra (a) comparison of electrospun fibres from protein blend solution (green) to PEO(red), and (b) comparison of electrospun fibres from blend solution (red) to freeze-dried WECGP (purple).Differences in intensity between electrospun and protein spectra in (b) are within error.abAmide AAmide IIAmide IAmide AAmide IIAmide ICO2CO2Green = Electrospun    blend fibresRed = PEORed = Electrospunblend fibresPurple = WECGP473.7     Results of mechanical testing on electrospun fibres     Electrospun protein blend fibres were mounted on the testing apparatus andmechanically tested in one of three ways: 1.) as they were collected on the insect pins, 2.)after being cross-linked with glutaraldehyde, or 3.) draw-processed, cross-linked on thetesting apparatus, and re-tested.     The electrospun fibres were white in colour, and became brownish-yellow if they werecross-linked.  Samples that were not cross-linked would shear and slip during testing anddid not return to their original length after testing.  Draw-processed and then cross-linkedsamples were brittle and failed before 40% extension.  Hysteresis data could not becollected for draw-processed samples as none of the samples survived the load cycle.Cross-linked samples did not slip and shear, or did so to a much lesser extent thanuncross-linked samples (Figure 24).  Neither the cross-linked nor the uncross-linkedsamples had stress-strain curves resembling those of the mature native protein, but thecross-linked samples appeared to be similar to the elastic transitional state in thematuration process of the capsule. The stiffly elastic Hookean region of the native proteinwas notably absent in the electrospun fibres.     The Young’s modulus of the cross-linked electrospun fibres was 2.43, similar to thatof the native capsule’s yield region, where the modulus is 3.91 (Rapoport and Shadwick,2002).  Without being cross-linked, the electrospun fibres had a modulus of 0.15, anorder of magnitude below the yield region of the capsule.  The initial, stiffly elasticHookean region of the capsule has a modulus of 87.9, 30X greater than the cross-linkedelectrospun fibres.     Hysteresis was calculated to be 0.51 and 0.69 for the cross-linked and uncross-linkedelectrospun fibres, respectively (Figure 25).  Both of these hysteresis values are greaterthan the native capsule, indicating that more energy was lost and dissipated as heat duringthe loading and unloading cycle.  Hysteresis loops did not change when a timed hold wasinserted during extension.480.01.02.03.04.00.000.200.400.600.801.00StrainStress (MPa)0.000.100.2000.050.10.150.20.250.30.350.40.450.5StrainStress (MPa)Not cross-linkedCross-linked    00.511.522.533.540.000.100.200.300.400.500.600.700.800.90StrainStress (MPa)Cross-linkedTested, then cross-linkedNot cross-linkedFigure 24.  Stress-strain curves for electrospun WECGP samples.  (a) cross-linked fibres, (b) fibres testedand then cross-linked after draw-processing.  Before being cross-linked, the sample slipped atapproximately 35% strain.  (c) curves for cross-linked, draw-processed and then cross-linked, and uncross-linked fibres.  Note different scales.-0.0050.0000.0050.0100.0150.0200.0250.0300.000.100.200.300.400.500.60StrainStress (MPa)0.0000.1000.2000.3000.4000.000.100.200.300.400.500.600.70StrainStress (MPa)Figure 25.  Hysteresis loops for (a) uncross-linked and (b) cross-linked electrospun WECGP protein blendfibres.  Note difference in scales.aba bcE = 0.0501, R2 = 0.9891Hysteresis = 0.505E = 0.3586, R2 = 0.9886Hysteresis = 0.69049MaterialModulus (MPa)HysteresisNot cross-linked0.15 0.69Draw-processed, then cross-linked0.56 -Cross-linked2.43 0.51Native capsule, Hookean region87.9 0.20Native capsule, yield region3.91 0.37Table 4.  Young’s modulus and hysteresis values for electrospun whelk egg capsule gland protein fibres,and for the native capsule.  Capsule data from Rapoport and Shadwick, 2002.504     Discussion4.1     Electrospinning parameters and the electrospinning of WECGP     Samples of nanofibres have been produced in different conditions, but the optimalparameters for electrospinning the whelk egg capsule gland protein seem to be 1.) adistance of 10cm from needle tip to collecting target, 2.) an applied electric field of 20kV, and 3.) a flow rate of 0.02 ml/hour, controlled by the syringe pump.     Increasing the distance between the needle tip and the target mandrel may allow forgreater evaporation of the solvent but also decreases the strength of the electric field.  Ifthe distance is too short, the solvent will not fully evaporate and the jet will land on thetarget still wet, forming globular droplets rather than fibres.  This can be ameliorated bylowering the ambient humidity or by choosing a solvent with a larger difference betweenits vapour pressure and that of air (Yang et al., 2006).  If ambient humidity is low,increasing the distance between source and target will result in lower fibre diameters asmore solvent evaporates, but if ambient humidity is high, there will be no increase insolvent evaporation (Yang et al., 2006).   No attempts were made to adjust the ambienthumidity during the course of this study since formic acid proved volatile enough toevaporate appropriately.     On the other hand, if the vapour rate of the solvent is too fast, the fibre diameter willincrease (Yamashita et al., 2007).  There are also other techniques to remove unwantedsolvent residue.  Spasova et al. (2007) placed electrospun samples under vacuum toremove solvent residue in order to improve conditions for cell growth on theirelectrospun fibre scaffolds.     In this study, short distances between needle tip and target produced either a proteinfilm or globular protein deposits on the collecting foil, but no fibres.  The film was due toresolubilization of the protein on the target, caused by incomplete evaporation of the51solvent.  Globular protein deposits were likely due to clumping at the needle tip, whichwas caused by inadequate electric field strength.  Increasing the electric field strength atshort distances produced a spark between the charged needle and grounded target.  Atdistances of less than 5 cm between needle and target, a spark was discharged when theelectric field was applied, even at low electric field strength.  This indicates breakdown ofthe electric field.     At distances between source and target of greater than 15 cm, fibres were no longerreliably deposited on the collector.  The amplitude of the whipping motion of theelectrified polymer jet increases with distance from the needle, making it less likely thatnanofibres will be collected in a small area.  Working with PEO, Yang et al. (2006) foundthat short distances between source and target prevented fibre formation, and longdistances resulted in a larger distribution of fibre diameters.  Electric field strengthsgreater than 30 kV also increased the amplitude of the whipping motion, and caused thepolymer jet to fluctuate wildly.  This also led to poor deposition of nanofibres on thecollector, and an impressive mess within the electrospinning chamber.     In their topical review of electrospinning design, Teo and Ramakrishna (2006)summarized the advantages and disadvantages of the rotating mandrel as a means ofcollecting electrospun fibres.  The advantages were the simplicity of the set-up and theability to collect large areas of fibres, but the drawback was that highly aligned fibres aredifficult to fabricate, as observed in this study (Teo and Ramakrishna, 2006).  Themandrel of the Kato Tech NEU-010 was not able to rotate quickly enough to producealigned nanofibres of WECGP.  The mandrel was also so large (8 cm diameter) that whenit was rotating, very low fibre density was produced even when electrospinning for manyhours.  This was not helped by the slow flow rate (0.02 ml/h) used to produce WECGPnanofibres.     Teo and Ramakrishna’s (2006) review of another electrospinning set-up involving apair of parallel electrodes sparked the idea for the paired insect pins used in this study tocollect samples for mechanical testing.  They found that using parallel electrodes as a52collecting device made highly aligned fibres easy to obtain, and that the aligned fibreswere easily transferable to another substrate (Teo and Ramakrishna, 2006).  The pairedinsect pins were mounted parallel to each other and perpendicular to the target mandrel,pointed at the source needle.  The pins were mounted beneath the aluminum foil thatcovered the target mandrel and emerged by piercing the foil, effectively increasing theavailable surface area of the grounded target.  Electrospun fibres that spanned the gapbetween the pins could be transferred for use in mechanical testing with minimalhandling.  Upon visual inspection, these fibres showed a marked increase in alignmentcompared to fibres deposited directly onto foil, probably because fibres of differentorientations failed to span the gap between the pins and thus were deposited elsewhere.     Several unsuccessful attempts were made over the course of this study to electrospinWECGP nanofibres directly onto glass.  Li et al. (2005) electrospun protein fibres ontocover glasses that were attached to a brass grounded target.  In this study, however, fibreswere not convinced to land on a glass target, whether it was attached to a grounded target,covered in foil with only a small window of glass exposed, or even if the glass wassputter-coated with gold before being attached to the grounded target.     In an attempt to eliminate handling of samples prior to mechanical testing, a smallforce transducer was made using an 80 nm glass rod and a cover slip attached to amicrometer, which slid on a drop of vacuum grease.  These were mounted onto amicroscope slide and sputter-coated, first with 8 nm of gold, then with a further 8 nm.The entire apparatus was grounded, but the fibres stubbornly refused to span the gapbetween the rod and cover slip.     Two distinct size groupings of nanofibres were often observed on the SEM images.The main group was composed of uniform fibres, which were measured to determine theaverage diameters of the blend solutions.  The second group was at least an order ofmagnitude smaller in diameter, was generally short in length, and tended to emerge frommain group fibres and beads.  These fibres were less stable when viewed under the SEMand would quickly melt and curl under the electron beam.  Yang et al. (2006) proposed53that short, very fine fibres observed at the ends of broken fibres, or seen coming off ofbeads and other fibres may be caused by strong electrical forces on fibres with littleremaining solvent.     The egg capsule gland has been used in this study as a source of protein forelectrospinning because it is composed of the precursor protein.  The precursor proteinhas not been mechanically manipulated or cross-linked by the whelk, so it is more solublethan the mature capsule.  However, “soluble” here is a relative term.  The gland proteinwas solubilized using a mortar and pestle, brute force, and blood, sweat, and tears in 88%formic acid.  Formic acid was chosen for its ability to solubilize the protein, its volatility,and because it vapourizes without leaving a residue.  This protein does not solubilizewithout strong coercion, and even so would not solubilize in formic acid to the criticalconcentration required for electrospinning.     In dilute solutions, polymers form separate hydrated coils that do not interact witheach other.  As the polymeric concentration of the solution increases, the coils go frommoving independently to overlapping and becoming an entangled network.  Thesepolymeric interactions are necessary for the electrospinning of continuous fibres,therefore the electrospinning solution must have a critical concentration of polymer.     Tris 2-carboxyethyl phosphine (TCEP) was added to the formic acid to enable the eggcapsule gland protein to reach the critical concentration for electrospinning.  TCEP actsas a reducing agent to break disulfide bonds in the protein, thus decreasing the viscosityof the solution.  TCEP was selected because it is able to reduce these bonds at lower pHthan dithiothreitol (DTT), and it smells much better than mercaptoethanol.  25 mM wasfound to be sufficient to break the protein into its 50 kDa monomeric subunit.544.2     PEO     Poly(ethylene oxide) (PEO) is added to the electrospinning solution to increase theviscosity, improve processability, and encourage fibre formation.  Nanofibre-producingsolutions of the whelk egg capsule gland protein used for this study consisted of 8 wt%protein, 2 wt% PEO, and 25 mM TCEP solubilized in 88% formic acid.     The change in morphology from beads-on-a-string to continuous nanofibres, whichwas observed with increasing amounts of PEO, is likely the result of the increase insolution viscosity (Aluigi et al., 2008).  On its own, PEO formed nanofibres atconcentrations of 5 wt% and 10 wt%, formed fibres with the “beads on a string”morphology at 4 wt% and did not form nanofibres at all at 2 wt% and 3 wt%.  The lowerviscosities of the latter solutions do not allow sufficient molecular chain entanglements toprevent breakage of the electrically driven jet formed by the electrospinning process(Aluigi et al., 2008).  The fact that PEO did not form fibres at 2 wt% unless WECGP wasalso present indicates that WECGP is the primary component of the fibres electrospunfrom the blend solution, and that PEO was playing a supporting role.     Aluigi et al. (2008) found that nanofibres rich in protein were more homogenous thannanofibres rich in PEO.  This indicates that the blend composition plays an important rolein determining the diameter distribution of the fibres and was one of the reasons theamount of PEO in the blend solution was minimized.  Since the PEO solution series oftendid not form fibres, or formed only very few, in a “beads on a string” conformation, it isdifficult to draw conclusions about whether these fibres were more or less homogenousthan the fibres spun from the protein blend solution.  In addition, only 40 fibre diametersper PEO solution were measured, compared to 100 for each of the protein blendsolutions, which makes the standard error of the means less comparable.554.3     Nanofibre diameter     The diameter of electrospun fibres is influenced by many factors, such as proteinconcentration, viscosity, field strength, distance between polymer source and target, andflow rate.  In this study a distance of 10 cm between the needle tip and the target mandrelwas used, with an applied electric field of 20 kV.  The electrospinning voltage was 2kV/cm, past the upper limit determined by Katti et al. (2007) that produces the smallestdiameter fibres.     As expected, the average diameter of WECGP blend nanofibres decreased as theprotein concentration increased.  Aluigi et al. (2008), looking at the effect of proteinconcentration, found that as the keratin content of their electrospinning solutionincreased, the average nanofibre diameter decreased.  They also found that the diameterdistribution was narrower in keratin-rich nanofibres (Aluigi et al., 2008).  This may bedue to several factors, including changes in the viscosity and conductivity of the solution.When a higher charge density is carried by the polymer jet, it forms smoother, finernanofibres because it produces stronger whipping instability, which enhances filamentstretching (Aluigi et al., 2008).  Lower viscosity has also been shown to promote theformation of smaller diameter nanofibres (Aluigi et al., 2008).     In this study, when the protein concentration was held constant, the amount of PEO inthe polymer blend solution did not significantly change the average diameter of thefibres.  This suggests that once the protein component of the blend solution reaches somecritical concentration, the protein controls the fibre diameter.     Li et al. (2005), found that increasing the syringe pump flow rate from 1 to 3 ml/hcaused a significant increase in fibre diameter for both collagen and gelatin, although afurther increase from 3 to 8 ml/h did not significantly affect the mean diameter.  In thisstudy, the syringe pump flow rate that produced the smallest, most uniform fibres was0.02 ml/h.  Flow rates of this order of magnitude have successfully produced electrospunfibres from many different polymer solutions, but increasing flow rates to scale up56production has been key for many applications, so a slow flow rate has not beenconsidered desirable.  However, WECGP blend solutions did not produce fibres until theflow rates from the literature had been decreased a hundredfold.     When determining the average diameter of the electrospun fibres from the SEMmicrographs, it was not possible to distinguish if one fibre was measured more than once.Fibres are laid down on the collector in a looping deposition pattern, so a single fibre maycross the image field of view repeatedly and thus be measured repeatedly.  However,considering the vast number of fibres deposited in a spinning session, this was assumedto be a rare occurrence that would not have greatly skewed an average of 100measurements.4.4     Birefringence of electrospun fibres     Fibres electrospun from the protein blend solution (8 wt% protein, 2 wt% PEO, and 25mM TCEP solubilized in 88% formic acid) were found to be birefringent, which indicatesthat the fibres have an ordered structure.  Since the process of electrospinning alignsmolecules on the basis of charge, and induces the formation of fibres based on thisalignment, it is not surprising that they have ordered structures.  Birefringence scans ofwhelk egg capsules showed that the native material is highly birefringent (Rapoport andShadwick, 2007), and further work by Didier (2009) demonstrated that individual proteinfibres teased out of the native capsule are also birefringent.     Birefringence tends to increase as a material is strained, and Didier (2009) found thatthis is true for individual fibres of the whelk egg capsule, but only in the Hookean region.After the transition to the yield region the birefringence decreases, indicating a loss ofoverall order in the structure as α-helices are pulled apart (Didier, 2009).  The FTIR dataon the electrospun fibres showed that the fibres had 21% α-helical character, same as thepurified WECGP, so it is possible that the α-helices were also being pulled apart whenthe cross-linked electrospun fibres were strained.57     The samples of electrospun fibres used to measure birefringence contained as fewfibres as possible given the collection method.  Even so, the diameters of the sampleswere between 1.0 and 1.5 µm.  Since we know that the individual electrospun fibres areonly about 100 nm in diameter, each sample measured must have been composed ofmany smaller fibres.  It is difficult to resolve detail on a fibre that is less wide than awavelength of light.4.5     The Matrix Squeeze hypothesis     Applying part of Fudge and Gosline’s “matrix squeeze” hypothesis for intermediatefilaments (IFs) (2004), which argues that the matrix restricts the hydration of IFs, thusregulating water’s role as a plasticizer, we would expect WECP to exhibit lower stiffnessand strength when hydrated.  WECP acts as predicted by this hypothesis – it is hydratedin its native state, and becomes stiffer and stronger when dehydrated, with both its yieldstress and initial modulus increasing by an order of magnitude (Rapoport and Shadwick,2007).  Hydration did not seem to have an effect on yield strain (Rapoport and Shadwick,2007).     Cross-links are thought to restrict the hydration of IFs, allowing H-bonding todominate, increasing modulus and yield stress (Fudge and Gosline, 2004).  Matrix-freeIFs such as hagfish slime threads are very hydration sensitive and swell greatly as theyhydrate, but matrix acts to restrict hydration of IFs by resisting deformation (Fudge andGosline, 2004).  An elastomeric matrix that resists circumferential expansion such ashydration swelling will also resist longitudinal deformation from applied tensile forcesand improve IF recovery (Fudge and Gosline, 2004).     Drying WECP seems to make it more similar to hydrated α-keratin, but because cross-link (or matrix) density in WECP is lower than keratin, WECP does not attain a modulusas great as keratin (Rapoport and Shadwick, 2007).  This difference in cross-link densityis probably due to both the environment in which the materials are expected to operate58and their adaptive functions (Rapoport and Shadwick, 2007).  Stiffer keratins such ashoof, hair, and horn do not need to be diffusive barriers, while whelk embryonicdevelopment may be compromised by a more cross-linked and therefore less diffusivecapsule (Rapoport and Shadwick, 2007).     The cross-linking agent used in this study was glutaraldehyde, which tends to quicklyform many, fairly indiscriminate cross-links.  The density of cross-links was notcontrolled, although it is likely that the native protein has a much more specific cross-linking strategy.  However, even with this uncontrolled cross-linking, the electrospunfibres did not attain the stiffness or strength of the mature egg capsule.4.6     Self-assembly of elastic polymers     Research has so far not been able to find one obvious protein sequence that giveselastomers their elastic recoil properties (Keeley et al., 2002).  The way monomers areorganized into polymeric structures must be crucial for elastomeric properties (Keeley etal., 2002).  Elastin, for example, is synthesized as a monomer, assembled in theextracellular matrix, and stabilized by covalent cross-links derived from side chain lysineresidues (Keeley et al., 2002).  The monomer has a strong tendency for self-aggregation,and this intrinsic ability to self-assemble is seen even in relatively small recombinantpeptides based on human elastin sequences (Bellingham et al., 2003).     Keeley et al. found (2002) that the process of self-assembly aligned the elastinmonomers such that cross-linking of lysine residues could take place.  They showed thatsmall numbers of hydrophobic and cross-linking domains could provide this ability forself-alignment and impart the properties of extensibility and elastic recoil (Keeley et al.,2002).  Bellingham et al. (2003) demonstrated that as few as three hydrophobic domainsflanking two cross-linking domains in recombinant peptides are sufficient to support aself-assembly process that aligns lysines and forms the cross-links of native elastin.  Theyalso showed that these cross-linked polymers have physical and mechanical properties59similar to native elastin (Bellingham et al., 2003).  It is likely that the self-assemblyprocess of WECP similarly aligns monomers such that cross-linking can take place, albeitwith the assistance of a muscular massage in the ventral pedal gland.   It is possible that achemical change takes place at the VPG, but this has been neither confirmed nor ruledout.4.7     Using FTIR to positively identify WECGP in electrospun fibres     The FTIR spectra for freeze-dried WECGP and a sample of fibres electrospun from ablend of 8 wt% WECGP, 2 wt% PEO and 25 mM TCEP in 88% formic acid were nearlyidentical, indicating that WECGP was in fact the dominant component in the fibres.  Theslight shift in intensity was within experimental error.  Three absorbance peakscharacteristic of proteins were strongly mirrored in the two spectra, at amide A, amide I,and amide II.  The largest difference between the spectra was the absorbance peak at2350 cm-1, which is an artifact of atmospheric CO2.     PEO is a synthetic polymeric material that is well known for its “spin-ability”.  SincePEO is so keen to form fibres by electrospinning, it was a useful addition to the proteinblend solution, but simply electrospinning PEO was not the purpose of this study.Qualitative tests of electrospun fibres formed from the blend solution (8 wt% WECGP, 2wt% PEO, 25 mM TCEP in 88% formic acid) had indicated that the fibres were indeedprotein.  For example, the fibres were not water soluble, and PEO dissolves instantly inwater.  However, stronger evidence was desired.  Therefore, the FTIR spectra for PEOwas compared to the spectra for the sample electrospun from the blend solution.  The twospectra showed a similar absorbance peak at 2350 cm-1, the artifact peak for CO2, but hadvery little similarity otherwise.  Not surprisingly, PEO lacked the amide absorbancepeaks characteristic of proteins, which were the dominant peaks in the electrospun fibres’spectrum.  The first large absorbance peak on the PEO spectrum was at 3150 cm-1, whichis not a frequency of interest for proteins.604.8     Determining secondary structural changes using FTIR     The FTIR spectra of any protein will consist of three types of amide bands, atcharacteristic frequencies.  The bands represent the bonds between the amide, alphacarbon, and carbonyl groups that make up the basic structure of all amino acids.  Amide Iis the most intense absorption band in proteins.  It is found between 1600 cm-1 and 1700cm-1, and is primarily due to stretching of the C=O bond in the carbonyl group.  Amide IIis found between 1520 cm-1 and 1540 cm-1, and represents deformation of the N-H bondin the amide group.  Amide III is found between 1230cm-1 and 1270 cm-1, and resultsfrom bending of the C-N bond between the amide and the alpha carbon.  The frequenciesof these amide bands can be used to determine the secondary structure of proteins.  Theamide I band of WECPG is not disturbed by the PEO absorption spectra.  Therefore, itcan be used cleanly to study the structure of WECGP in the nanofibres.     Different protein conformations show different FTIR absorption bands.  α-helicesshow strong absorbance in the amide I band between 1651 and 1657 cm-1.  β-sheets showstrong absorbance in the amide I band between 1621 and 1640 cm-1, and between 1671and 1679 cm-1.  Using the amide I band for calculations, the ratio of the area under theseintervals to the total area under the amide I curve will give the percentage of α-helix or β-sheet in the sample, respectively.     The calculated % α-helix in the electrospun sample was 21% and the calculated % β-sheet was 32%.  The calculated % α-helix in the freeze-dried WECGP sample was also21% and the calculated % β-sheet was 31%.  From these results it appears that βstructures are more prevalent in the protein than helical structures, which is contrary tothe model proposed by Rapoport and Shadwick (2007) and supported by Didier (2009)and Miserez et al. (2009).  However, these results are for the immature protein, and themodel is for the mature capsule.61ProteinCollagenElastinKeratinWECGP% α-helix19 24 42 21% β-sheet28 41 19 31Table 5.  Secondary structure composition in common structural proteins and WECGP.  All data is fromFTIR studies.  Collagen was type I, from bovine skeletal muscle tissue – note that in addition to 19 % α-helix there was an additional 18 % triple helix (Petibois et al., 2006).  Elastin was from human aortic tissue(Bonnier et al., 2008), and keratin was from horsehair (Kreplak et al., 2004).     Although we have evidence that the α-helices of the native protein are pulled apartwhen the egg capsule is strained, it seems that the β-sheets that form as a result of thestrain are not stable (Didier, 2009).  The protein does not irreversibly pull apart; rather, itsα-helices rapidly reform once strain is removed, even generating force while doing so(Didier, 2009).  This is interesting since the α-helix to β-sheet transformation is oftenirreversible since the β-sheet structure forms a new stable state.  It would be interesting touse FTIR to obtain spectra of both the mature protein and electrospun fibres, held atdifferent extensions to look more closely at how the secondary structures are affected bystrain.     Looking at FTIR results analyzing the secondary structure of other structural proteins,we see that keratin has the most α-helix (42%), followed by elastin at 24%, and collagenat 19%.  Keratin, which is very stiff, would be expected to have a high proportion of α-helix, but the collagen data is mysterious since collagen is almost entirely triple helix.The FTIR data from Petibois et al. (2006) indicates that only 18% of the secondarystructure of collagen is triple helix, and that it is 19% α-helix and 28% β-sheet.  Theremust be an interesting explanation.  From the FTIR data, WECGP is unexpectedly closerin α-helical character to elastin than keratin.  This may be related to the fact that theelectrospun fibres, even when cross-linked, did not reproduce the stiff Hookean region ofthe mature protein, but only the rubbery yield region.  The Hookean region is attributedto α-helices, and develops last in the native protein’s maturation sequence.624.9     Cross-linking of electrospun fibres     Electrospun fibre scaffolds from the blend solutions lack the strength of the maturenative protein and were therefore treated to improve their mechanical integrity.  Cross-linking can be used to tailor biomechanical characteristics to match that of the originalmaterial, in this case WECGP.  It does not appear that the native cross-links form to anysignificant degree, if at all, in the electrospun fibres.     Glutaraldehyde, as a cross-linking agent, has many advantages.  It is relativelyinexpensive, cross-links over varying distances, reacts quickly, and reacts with manyamino groups present in proteins (Barnes et al., 2007).  Glutaraldehyde reacts mainlywith primary amines in proteins (Barnes et al., 2007).  Although glutaraldehyde is themost popular agent for cross-linking biological materials, it is known to be cytotoxic andto compromise biocompatibility (Barnes et al., 2007).     Interestingly, Buttafoco et al. (2006) found that the cross-linking process resulted inthe leaching of PEO from fibres originally electrospun from 1:1 by weight type I collagenand PEO.   They also observed that the fibre morphology was not modified after cross-linking (Buttafoco et al., 2006).     Both glutaraldehyde and carbodiimide have been used for cross-linking electrospunmats produced from type II collagen.  Unlike fibres produced by electrospinningWECGP, type II collagen fibres disintegrate upon contact with aqueous solutions,making the cross-linking process more difficult if the nanofibrous morphology was to beconserved (Barnes et al., 2007).  Aluigi et al. (2008) had similar difficulties withelectrospun keratin nanofibres – they were also water soluble and had poor mechanicalproperties since they were not cross-linked.  Mechanical properties of cross-linkedcollagen mats were measured in the hydrated state, as were the WECGP mats in thisstudy, to show behaviour under approximate physiological conditions.63     Barnes et al. (2007) found that the stress-strain curves of cross-linked collagen fibresbehaved more like native collagen than untreated samples. In collagenous structures thereis a non-linear toe region in which the collagen fibres align, then the aligned collagenfibres are collectively stretched.  The non-woven electrospun mats appeared to follow thesame procedure during extension, with the electrospun fibres aligning in a toe region,then being collectively stretched (Barnes et al., 2007).  Barnes et al. (2007) reported thatthe stiffness of the electrospun mat depends upon fibre diameter, orientation, and numberof fibres, as well as the strength of the covalent cross-links and the type and amount ofnon-covalent bonds.4.10     Mechanical testing of electrospun fibres     The simplest method, and the model proposed by Altman et al. (2002) assumes that abundle of multiple fibres acts as a single fibre with an effective radius determined by thenumber and radii of the individual fibres.  This model ignores friction between theindividual fibres, and assumes parallel alignment of the fibres (Altman et al., 2002).However, the silk fibres produced in the very cool study by Altman et al. (2002) wereorders of magnitude larger than those produced in this study, and the silk fibres werehighly controlled for alignment using a clever hierarchical approach.     Due to the variable orientation of electrospun fibre deposition in this study, even withthe greater alignment inherent in the insect pin spanning structure, the samples used formechanical testing were not uniform.  However, the estimation of sample thickness usedto calculate the cross-sectional area should not have contributed too much error to thestress values.  Samples were assumed to be rectangular, and the width was measurable.Assuming a circular cross-section would have created more error, since the estimatedvalue for the radius would have been squared, compounding the effect of the estimate.However, the samples were measured hydrated, immediately before they were tested.Hydration is likely to have caused the sample to swell, especially given the very lowdensity of the fibres in each sample.  Therefore, the cross-sectional area measurements64will all have been overestimated, which results in all the stress values beingunderestimated.  It is not unreasonable to think the actual values could be double whatwas measured in this study.     The addition of synthetic polymers to electrospinning solutions is common and hasbeen shown to affect the mechanical properties of the resulting fibres.  Jeong et al. (2007)electrospun a synthetic polymer onto the surface of tissue-engineered collagen scaffoldsto improve their mechanical strength.  The synthetic polymer, PLGA, was found toincrease the scaffold strength in both the wet and dry states (Jeong et al., 2007).  PEO, onthe other hand, could not have affected the mechanical strength of the electrospun fibresin this study since PEO is water soluble, and all samples were mechanically tested inaqueous conditions.  When an attempt was made to test the mechanical strength of a matof PEO fibres, the mat disintegrated upon contact with the glue, and so could not betested.  Therefore any remnant of PEO that did not immediately dissolve when placed inthe water chamber would dissolve upon contact with the glue, and without being glued tothe strain gauge the PEO would be unable to contribute to the force measurements.4.11     Mechanical results     The native protein of the whelk egg capsule undergoes a maturation process, withdifferent characteristic stress-strain curves for different steps of the process.  If theprotein is mechanically tested early in the process, before the capsule is manipulated bythe ventral pedal gland, the protein lacks mechanical integrity (Rapoport and Shadwick,2007).  At this point the protein is thought to have self-assembled but not yet cross-linked.  The native protein then undergoes a muscular massage in the VPG, where it isthought that layers of the protein are brought close together, enabling crosslinking, orwhere some chemical change may occur.  This produces an elastic, cross-linked,transitional state before the capsule is fully mature.  The electrospun protein fibres appearto be similar to the native protein in this transitional stage.65     The cross-linked electrospun fibres did not yield stress-strain curves similar to themature native protein.  The electrospinning process aligns and assembles the solubilizedprotein monomers according to charge, and the current maturation model starts withcharge-based self-assembly of microfibrils (Rapoport and Shadwick, 2007).  However,there may be a much more specific process during self-assembly that produces the correctlocations and number of locations for cross-linking later in the maturation process.     The Young’s modulus of the cross-linked electrospun fibres was the same order ofmagnitude as the yield region of the mature capsule, but still an order of magnitude belowthe modulus of the egg capsule’s stiffly elastic Hookean region.  Without being cross-linked, the electrospun fibres had a modulus an order of magnitude below even the yieldregion of the capsule.     The electrospun fibres were cross-linked with glutaraldehyde, rather than by amysterious muscular massage.  Glutaraldehyde is likely not as specific, seeing as it reactswith many different amino groups, and it reacts over various distances, which is unlikelyto be the case for the native protein, or the massaging should not be necessary.  Thecross-linked electrospun samples lacked the Hookean region of the stress-strain curves ofthe mature protein.  This region is the last to form in the maturation timeline of theprotein, and it signals that the material properties are fully developed.     All of the cross-linked electrospun collagen structures produced by Barnes et al.(2007) had values of modulus and peak stress at least one order of magnitude less thanthose reported for the native cartilage tissue that the collagen was derived from, and thebreaking strains of the electrospun mats were greater than that of the native cartilage.The breaking stress of cross-linked electrospun samples in this study was an order ofmagnitude less than the native protein.  The breaking strains were the same order ofmagnitude, although the electrospun fibres did not reach the largest strains of the nativecapsule.66     Hysteresis values for the electrospun fibres were greater than those of the native eggcapsule, indicating that the nanofibres lost more energy as heat during their loading andunloading cycle.     There is an important source of error in the mechanical data that should be considered.It is very likely that the cross-sectional areas of the samples were over-estimated sincemeasurements were taken once the samples were hydrated.  Each electrospun sampletested was made of a loose network of semi-aligned nano-fibres.  The samples were verylow mass and density, and it would be generous to assume that they were 10% fibre.Therefore, 90% of the volume of the sample would be the medium, which duringmechanical testing was water.  The result of this is that the stress values determined inthis study are all too low, probably by 1 or 2 orders of magnitude.  The actual cross-sectional area of the fibres would need to be measured to give more accurate data.  If thisassessment of the underestimated stress values is correct, the cross-linked electrospunfibres are closer in their mechanical behaviour to the native protein than it originallyappeared.4.12     Future directions     The mechanical integrity of the native whelk egg capsule protein does not arise fromany gross structural changes in the ventral pedal gland (Rapoport and Shadwick, 2007).Cross-linking during VPG treatment is the most likely source of the stability that givesthe mature capsule its interesting mechanical properties.  The fact that the mechanicalbehaviour develops sequentially gives hope that the mysterious cross-linking step will beidentified and effectively mimicked in the future.     Different cross-linking agents could be tested on the electrospun fibres, and cross-linking agents with more specific actions could shed more light on what is occurring inthe maturation process.  Although the nanofibres are delicate, it may be possible to67mechanically manipulate them in a way that mimics the muscular massage of the VPG tosee if that has an effect on their mechanical properties.     Mechanical testing could be attempted on dry samples of electrospun fibres andcompared to data from dry capsule testing.  Samples could also be subjected to repeatedhysteresis loops in order to determine the conditioned behaviour.  Pre-conditionedsamples could be treated with cross-linking agents, or cross-linking treatments could becarried out while the samples are going through a series of loading and unloading cycles.     In order to more accurately model both the protein and the electrospun fibres, it wouldbe interesting to analyze FTIR spectra collected with the material held at different strains,and with the sample hydrated.  This could determine changes in secondary structure asstrain is increased or water is introduced.  Fibres held at constant strain could also haveFTIR spectra collected over time to gather information on the mechanism for stressrelaxation.     It would also be interesting to monitor the birefringence of the electrospun proteinblend fibres as they are strained, to determine if their behaviour is the same as that ofmicroscopic fibres teased out of the egg capsule.  Even the electrospinning solution couldbe tested for birefringence, and compared to the spun fibres.  Since the charge-alignednanofibres are presumably a more ordered structure than the homogenized solution, wewould expect the fibre birefringence to be greater than that of the solution.     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