Composites: Part B 38 (2007) 324337 www.elsevier.

com/locate/compositesb

Spider and mulberry silkworm silks as compatible biomaterials

Osnat Hakimi
a

a,*

, David P. Knight b, Fritz Vollrath b, Pankaj Vadgama

Interdisciplinary Research Centre in Biomedical Materials, Queen Mary University of London, London E1 4NS, United Kingdom b Department of Zoology, University of Oxford South Parks Road, Oxford OX1 3PS, United Kingdom Received 2 June 2006; accepted 28 June 2006 Available online 15 November 2006

Abstract Silks are a diverse family of natural materials with extraordinary mechanical properties such as high tensile strength and extensibility, as well as reported biological compatibility. In recent years, the reported exceptional nature of silk lead to increased interest in silk for biomedical applications. The aim of this review is to assess the potential and compatibility of silk bres and silk-based materials for biomedical purposes. It will do so by reviewing silk properties, structure and formation, with special focus on spider and mulberry silkworm silk bres, as well as the application of silk in the biomedical eld. The review will begin by introducing the general characteristics of silk, and a consideration of properties of particular relevance to the use of silk in biomedical applications: degradation and tensile properties. Subsequently, the formation of silk in vivo will be outlined, as well as the current understanding of silk structure. A comparison of the structural dierences between spider and silkworm silks will follow. Some of the dierent types of silk produced by orb weaving spiders, their main functions and structural features will be described. This will be followed by an introduction to supercontraction, a phenomenon that has only been observed in spider silks, and is considered to be one of the major obstacles to the use of native spider silk for medical applications. Finally, there will be an account of previous biomedical applications of silk. It is the intention of this review to point out the wide range of excellent and valuable properties observed in dierent silk types, and to propose that silks versatility is possibly its strongest advantage. However, in the context of biomedical research and development, there are still major limitations and difculties with native silk bres. It is suggested in this review that an articially produced silk or silk-like material formed to possess specic desired properties will allow the overcoming of present limitations. 2006 Elsevier Ltd. All rights reserved.
Keywords: Silk; A. Fibres; B. Mechanical properties; E. Assembly

1. What is silk? Silks are brous protein polymers that are spun into bres by some arthropods such as silkworms, spiders, scorpions, mites and eas [1,17]. They use it for a variety of functions, including formation of protective shelters, structural support, reproduction and capturing food [17]. Silks dier in composition, structure and properties depending on their specic source and function [1,18,95]. Spiders and insects secret glycine-rich silks characterised by their unique synthesis and processing features, as well as strength and extensibility. However, silks produced by
*

spiders dier in many aspects from those produced by mulberry silkworms. A summary of the main dierences between spider and mulberry silkworm silks is given in Tables 1, demonstrating the mechanical superiority of dragline spider silk, but the better water resistance and availability of silkworm silks. The choice of silk for a particular application must therefore include careful consideration of the desired properties. 2. Physical properties of silk The physical properties of silk distinguish them from all other natural materials known to date. Most importantly, silks are unique for their combined extensibility and high tensile strength [27,86,100]. As can be seen in Table 2, the

Corresponding author. E-mail address: o.hakimi@qmul.ac.uk (O. Hakimi).

1359-8368/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.compositesb.2006.06.012

O. Hakimi et al. / Composites: Part B 38 (2007) 324337 Table 1 A comparison of some properties of spider and silkworm silks Properties Number of silk types per species Functions [95] Spider silk Up to eight silk types Mulberry silk One type of silk Cocoon formation

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Making a web to catch prey, safety line (dragline), support for the web, wrapping prey and egg cocoon formation Mechanical properties [27] Strength rmax (GPa) 1.1 (Araneus MA silk) 0.27 (Araneus MA silk) Extensibility emax Coating of thread Glycoprotein mixture, not fully characterised Major structural Spidroin (sometimes called spider-broins) protein Behaviour in water Supercontraction of dragline (swelling in width and contraction of length, little change in mechanical properties) Mass production Not yet achieved, small quantities extracted by pulling silk from spiders or collecting egg cocoons Table 2 Tensile properties of silks and other materials [27] Silk type Major ampullate Viscid Cocoon Elastin Kevlar 49 bre Species Araenus Araenus Bombyx Extension (%) 27 270 18 15 2.7 Tensile strength (GPa) 1.1 0.5 0.6 0.002 3.6 Toughness (MJ m3) 160 150 70 2 50

0.6 (B. mori) 0.18 (B. mori) Sericins, antigenic proteins (B. mori) Fibroin Little change in silk shape/length B. mori, the domesticated silkworm, has been grown for mass production for millennia

assessing the tensile characteristics of a particular type of silk, but also an issue to consider when using natural silk for biomedical applications. 2.1. Extensibility of silk The extraordinary extensibility of silk (Table 2) has not been fully explained. Some attribute the extensibility of the silk thread to the ability of the amorphous regions to stretch and move from their random coil/other conformation into a more extended helix or b-strand conguration [27]. In particular, there is a suggested model describing b-spiral secondary structures formed by the motif GPGXX in spidroin 2 (spider capture thread), which can act as springs [34]. This theory has been supported by the apparent correspondence between the frequency of the motif and the extensibilities of capture and dragline spider silks [34], as well as by some evidence from Nephila dragline of the production of helical regions during bre stretching which disappear upon relaxation [104]. It has been suggested that the helices are formed reversibly in poly-alanine blocks [104]. Others models propose the nanobrillar morphology of the thread as a major feature contributing to extensibility, by allowing greater bending of the bre without failure [74]. Also, there is evidence that as stress on the thread increases, the bres and nanobrils (sub-brils) contract in width and align more with the threads axis [30]. 2.2. Tensile strength and toughness of silk Toughness is dened as the ability of a material to deform plastically and dissipate energy before fracturing. The toughness of silk has been attributed to its nanobrillar composition, with strong interactions between nanobrils in the thread [73], as well as the ne channels described in the thread which might be helping to disperse energy [100]. Strength has also been credited to the presence of bsheet packed into crystalline regions and hydrophobic interactions between those crystalline regions, which bind

toughness of some silk species is higher than that of Kevlar bres. This mechanical superiority is thought to be dependent on the molecular structure, thread substructure (supra-molecular structure) as well the formation process [27,77,101]. Silk proteins have a very large average molecular weight [8]. They are hydrophobic in their solid state, and in some cases their mechanical properties are greatly inuenced by water [71,83]. Silks are thermally stable at very low as well as very high temperatures [84,103,107]. Spider silks are mechanically superior to any insect silk known to date [97]. The native moth cocoon silk produced by mulberry silkworms is sti but also brittle in comparison [100]. However, it has been found that silk articially reeled from immobilised silkworms under controlled conditions is superior to the natural cocoon silk, approaching that of Nephila spider dragline silk [81]. Moreover, silks collected from freely walking spiders dier from silks obtained by forced silking, and one suggested explanation to those dierences is that spiders resist forced silking by applying an internal friction brake, which might be inuencing the mechanical properties of the thread [78]. Silks show variability in their mechanical properties when produced under dierent conditions, [36,99,101]. Factors known to inuence silk properties include climate and ambience, as well as reeling speed and the insect/spiders nutrition [50,63,75,100,101]. However, silks in general present highly variable properties, even for the same thread from the same spider [72,95]. This is a challenge when

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molecules together in the bre [34,61]. Crystalline regions containing poly-Ala b-sheets are known to form more hydrophobic interactions, and therefore have a higher binding energy than poly-Gly-Ala b-sheets [34]. This is in accord with the lower tensile strength of mulberry silkworm (rich in poly-Gly-Ala blocks) compared to spider dragline silk (rich in poly-Ala blocks). It is also possible that higher crystallinity of Bombyx mori compared to spider dragline silk (see Table 4) is responsible for its brittle nature. Thiel et al. [90] describe disrupted b-sheets interspersed with small poly-alanine crystals in major ampullate silk, which could be the origin of their superior toughness. Anyhow, it appears that the overall toughness of silk threads is connected to its structure at all hierarchical levels [100]. 2.3. Degradation of silk A wide range of factors inuence resorption rates of polymers in vivo. These include crystallinity, hydrophobicity, molecular weight, and the existence of proteolytic cleavage sites. Evidence suggests that silk bres are susceptible to both proteolytic degradation and gradual absorption over long periods in vivo [1]. An absorbable biomaterial is dened as one that loses most of its tensile strength within 60 days in vivo [1]. As silk broin (SF, silk from the silkworm B. mori) takes over 60 days to degrade in vivo, it is ocially regarded as a nondegradable material [1]. It was estimated that SF bres lose most of their tensile strength within one year in vivo, and are non-detectable at the site within two years [1]. Dragline spider silk, in comparison, degrades very rapidly when implanted onto the skin [96], which may pose a problem for its use as a scaold. When silk is absorbed, the rate of absorption depends on the implantation site, the mechanical environment, the health/physiological status of the patient, type of silk (virgin or processed), the diameter of the silk bre and the secondary structure. These variables have not been studied in detail yet, so there is no clear understanding of the relationship between structure, processing and degradability [1]. In vivo degradability studies are expensive and often require long bureaucratic procedures. In vitro systems oer a cheaper and simpler alternative settings for assessing the degradability of biomaterials [23]. Horan et al. [39] measured the degradation of B. mori yarns by protease XIV over period of up to 70 days. Loss of mechanical integrity has been measured through mass loss, bre diameter change and failure strength, and results indicated a predictable long term degradation characteristics in vitro. It has also been shown that the enzyme a-chymotrypsin can digest dissolved broin proteins, but not broin sheets [60]. This could possibly be the result of dierent degrees of crystallinity in the native bres versus the sheets or due to reduced accessibility to cleavage sites in the sheets compared to the bres. A comparative study of the degradation of SF in vitro by three dierent enzymes showed that

protease XIV was the most ecient in degrading broin sheets [60]. This was determined by measuring the average molecular weight of the degradation products, as well as the percentage of free amino acids [60]. The exceptional material properties of silk described above make it a highly desirable material for biomedical applications. For example, as a material with slow predictable degradability, silk broin could be valuable for applications that require support and the transfer of load from the scaold to the developing tissue. However, there are still gaps in our understanding of the origin of those properties, as well as their variation across silk species and types. Accumulation of more data about silks from dierent species and types will allow better estimation of the range of properties available in natural silks, while deciphering the origin and nature of each tensile phenomenon could allow the eventual design of tailored silks (or silks produced with specic desired properties according to their application) for varied purposes. 3. Silk production Remarkable mechanical properties of silk lead to much interest in their origin, and hence the formation of silk in vivo. Importantly, the synthesis of this tough material by insects and spiders is achieved under surprisingly mild processing conditions, compared to synthetic man-made materials with comparable properties, which are processed from harsh solvents or at very high temperatures [47]. This is a key issue because of health and safety issues as well as the ecological considerations involved with scaling up the production of a material. Ultimately, a detailed understanding of silk formation in vivo may facilitate biomimetic production of tough silk-like bres. 3.1. Silk synthesis and spinning The solid silk thread is produced from an aqueous solution at room temperature under low pressure and generally mild processing conditions [99]. The changes in molecular interaction and orientation that transform water-soluble silk into an insoluble bre are still not completely understood [5]. It is thought that a combination of ow-induced forces, active dehydration, gradual acidication, potassium and copper ions are involved [12,15,17,21,47,49,98,109]. It has been described as a two-phase process, the rst phase of which is nucleation and the second an aggregation growth process. Nucleation is the formation of a nucleus as a result of random coil conversion to insoluble b-sheet, which propagates rapidly once started. The nucleation phase is thought to be thermodynamically unfavourable, unlike the thermodynamically favourable aggregation phase [59]. An understanding of the principles that govern the assembly of silk proteins is essential not only for developing a biomimetic process but also for understanding how

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to assemble silk (and possibly other proteins) into nonspun dope. Below is a short summary of the available literature on the production of silk in silkworms and spiders. Silk production pathways in both spiders and insects are very complex and highly optimised [99]. Metal ions have been suggested to play a role in partial unfolding of Heavy chain broin (H-broin) while it moves through the gland lumen [79]. However, drastic elongation is not a favourable thermodynamically [8]. Copper ions have been shown to interact with B. mori SF to initiate conformation transition [109]. This interaction is thought to occur between the copper ions and some acidic residues conserved in the Nterminal of all H-broins studied to date [79]. Alternatively, it is possible that charge suppression of these residues by pH reduction is what allows aggregation to occur [8]. In spiders, silk is synthesised in the tail region of a specialised gland and stored in the ampulla [12,46]. The stored silk, in the form of a highly concentrated liquid crystalline solution is than passed through a narrow duct, close to the end of which it is thought to assemble into nanobrils [15,52]. Hydrophobic and hydrophilic interactions between the building blocks of spidroin molecules are thought to assist the assembly [52]. During the formation of the bre it is thought to exist in an intermediate form described as a liquid crystalline state [11,52]. Finally, the assembled silk is passed into the anterior of three pairs of spinnerets located near the tip of the spiders abdomen and hence out through a spigot [24]. 3.2. Transgenic production of silk proteins in unicellular systems Since the potential of spider silk has been realised, some eorts have been made to make some spider silk articial protein. In order to succeed, production of both the feedstock dope and spinning process must be carefully imitated [100]. Production of the spinning dope is a challenge already embarked upon by some molecular biologists, via expression of genes and production of recombinant silk proteins [57,100]. Attempts have been made to express silk proteins in E. coli, by inserting silk coding genes into bacterial plasmids [36,58]. However, genes containing repetitive segments are potentially unstable due to recombination, leading to deletion [36,103]. The enormous size of silk proteins and their mRNAs is another obstacle [33]. Finally, the very unusual amino acid composition may make great demand on the supply of glycine and alanine tRNAs, bringing transcription to an early halt in transgenic expression. Lazaris et al. have used transfected mammalian cells to express soluble spider silk (major ampullate) protein analogous, and proceeded to spin them from concentrated aqueous solutions. Expression of the spider silk proteins was only partially successful, as the cells showed preference to smaller monomer (60 kD) over the larger desired pro-

teins (250, 750 kD). In addition, the resulting spun bres were often brittle and presented unstable and inconsistent tensile properties [57]. More recently, Huemmerich et al. [42] have successfully employed the baculovirus expression system to produce two dragline silk components from the spider Araneus diadematus in an insect cell line. The group produced baculoviruses containing partial cDNA of spidroin proteins. These His6 tagged genes were than transferred into the cell line derived from the fall armyworm Sepodoptera frugiperda, and antibodies were than used to detect and purify the expression products. One interesting nding of this study was that one of the spidroin components based on Spidroin 2 (MaSp2) spontaneously assembled inside the cells into laments ranging from 200 to 1000 nm. That this did not occur outside the cell possibly indicates that folding and assembly of spidroin proteins requires intracellular conditions particular to the cytosol of spider silk gland cells. Silk protein synthesis appears to be a key challenge in the eort to produce man-made silk bres. Although recent work described here has increased our general understanding of the process, there are still some major problems to be tackled, such as the spontaneous deletion of repetitive genes and premature aggregation of the proteins in the dope. Production of a silk spinning dope in unicellular systems is therefore more likely to be achieved in the far rather than the near future. 4. Structure of silk It is now clear that there is a strong connection between the structure of silk bres and their physical (and mechanical) properties. Structural organisation from protein sequence, through protein folding to the assembly of the brils appears to play a role in the toughness and elasticity of silk bres [22]. Relating structural features and patterns to physical properties is valuable as it could contribute to the ability to control the properties of future man-made silk analogous (for example, an ability to control mechanical properties by introducing a certain structural features to the thread during spinning could be benecial). However, an understanding the structure of silk threads is still a challenge. Below is a review of the main structural features of silkworm and spider silks, starting at the overall thread organisation and going down to the protein makeup. 4.1. Structure of silk threads Most natural silk threads known to date are thought to consist of an inner silk core of polymer protein, a protein skin, and some type of coating. The core exhibits nanobrils, with some assembled into bundles called microbrils [53,74,76,100]. Generally, the coating functions as glue, but there is some evidence that it may also act as a fungicidal or

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bactericidal agent [95]. It may also have a role during the extrusion process. Micro-morphological studies show that the substructure of spider silks is very similar to that of mulberry silkworm silks [52,74,97]. For example, both are constructed from oriented nanobrils and both contain elongated tubular cavities or vacuoles called canaliculi [24]. The role of these canaliculi may be to facilitate controlled crack formation in the bres when it is stretched or loaded [95]. Although thread diameter varies across silk types and species, the mean width of nanobrils appears to be independent of the bre size, ranging 90170 nm [74].

bial degradation, animal digestion, and other damaging processes. Recent studies show some surprising properties of sericin; dietary sericin has been suggested to suppress the development of colon tumours by reducing cell proliferation, and creating oxidative stress and nitric acid production [110]. It has also been found, under certain conditions, to induce bone-like apatite deposition [89]. However, sericin is still considered an obstacle for B. mori silk biocompatibility, despite the coating being soluble in hot alkaline water and therefore removable [79]. As discussed later in this review, insucient sericin removal is still an issue in many biomedical applications. 4.1.2. Structure of spider silk thread A number of structural models have been suggested for spider silks [5]. Generally, it is thought that the thread has a well-ordered hierarchical structure made of bres and nanobrils [30,51,73]. All spider silks described to date contain only one protein monolament [73], and no covalent cross-links of any type [104]. In some spider silks, a coreskin structure has been described, with a thin outer layer surrounding a core of a possibly homogenous material [24,51,73]. The elongated canaliculi described in other silks may contain uid and act as shock absorbers in dragline silk [80]. A suggested model specically for dragline silk describes well dened single molecules stacked together to form a nanobrillar sub-structure [69]. This model is supported by X-ray diraction studies that showed the protein molecules to be oriented with the main chain parallel to the bre axis [24]. However, non-parallel bril-like regions have also been observed in MA spider silk [30]. 4.2. Types of silk proteins Two major families of silk proteins have been described to date: silk broin (SF) and silk spidroin (also named spider broin). Spidroin is the chief component of spider dragline silk, while broin is the analogue in silkworm silk. Generally, dierent silk glands express broins or spidroins with dierent proportions of crystal forming and amorphous sequence elements, leading to dierent tendencies for crystal formation, and hence dierent mechanical properties [27]. Below is a description of these two protein families, with a summary of their main features in Table 4. 4.2.1. Silk broin Fibroin from the silkworm B. mori is composed of two chains (H-broin [heavy] and L-broin [light]) linked by a disulde bond [79]. Another component of the protein is P25, which has been suggested to act as a chaperone to assist in the transport and secretion of H-broin [79]. Hbroin, L-broin and P25 are thought to be assembled in the ratio 6:6:1 [79]. This assembly appears to be standard in silk secreted by members of the lepidoptera order, with

4.1.1. The structure of B. mori silk thread B. mori silk bre has been shown to be composed of two protein-monolaments (named brins) embedded in a gluelike sericin coating [1,73]. A similar structure has been observed in other silkworms silk [22]. The brins are broin laments made up of bundles of nanobrils, approx 5 nm in diameter, with a bundle diameter of around 100 nm [65,73]. The nanobrils are oriented parallel to the axis of the bre, and are thought to interact strongly with each other [73]. A schematic representation of the structure of B. mori thread is shown in Fig. 1. The sericin coating constitutes 2530% of the weight of B. moris silk bre, and helps in the formation of silk cocoon by gluing the bres together [79,108]. It is made of sericins, gum-like proteins that vary in their molecular weight between 10 and 300 kDa, and are rich in the amino acid serine [108]. It is also thought to contain carotenoids, which are responsible for cocoon pigmentation [88]. Sericins are known to have several extraordinary properties: they resist oxidation, are antibacterial, UV resistant, and can absorb and release moisture easily [108]. These properties are valuable in the protection of silk from micro-

Fig. 1. The structure of B. mori silk thread.

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the exception of the saturniidae family, where L-broin and P25 are absent [79]. The main structural dierences between broin from B. mori and Antheraea pernyi are summarised in Table 3. Silk broin can exist in a number of dierent conformational states: Silk I (a-silk), and silk II. Silk II is the solid broin that can be found in the spun silk thread, while silk I is the dissolved, meta-stable form stored in the silk gland [35]. The conformation of Silk II is fairly well known, and is described in Sections 4.3.2 and 4.3.3. The conformation of Silk I is not fully understood, and is often described as a range of conformations, from lacking secondary structure, through partially ordered to well formed crystals [35,40]. It has been suggested that the more ordered conforTable 3 Structural dierences between broin in B. mori and A. pernyi, based on [79] Properties Main components of broin C-terminus size (Non-repetitive) Central part of H-broin size (regular repeats) Crystalline regions Nature of the repetitive region in H-broin Structure of the repetitive region in H-broin B. mori H-broin, L-broin and P25 58 Residues 5054 Residues

mations are transitional stages before Silk II formation [35]. 4.2.2. Silk Spidroin Spidroins are thought to be the chief components of both the skin and core of spider dragline silk [51]. Spidroins show a predominance of Gly, Ala, and Glu, contain relatively many Leu and Tyr residues, and share a limited set of amino acid motifs [34,84,86]. The crystalline regions of spidroin are primarily alanine-rich and form very tight b-sheets [44]. X-ray diraction studies show crystallite size of around 2 5 6 nm in Nephila clavipes [31]. Glu residues in the so called amorphous regions of the polymer are thought to be responsible for its hydrophilic nature [8,55].

A. pernyi H-broin 34 Residues 2400 Residues

Poly-Gly-Ala blocks Mostly hydrophobic

Poly-Ala blocks Alternations of hydrophobic and hydrophilic motifs

First order repeat: GAGAGS

Tri-peptide motif RGD

Second order repeat: strings of GAGAGS (varied number) followed by a sequence (often GAGS) and periodically disturbed by a block of 1028 residues Third order repeat: 26 2nd order repeat followed by an amorphous sequence of 43 residues (spacers) 12 Units of third order repeat make up H-broin No

First order repeat: relatively long sequences consisting of a hydrophilic string (1122 residues), an hydrophobic string (1214 residues), and alanine Second order repeat: random combination of 36 copies of the rst order repeats followed by two specic copies 12 Units of second order repeats make up H-broin

Yes

Table 4 Structural dierences between broin and spidroin Character b-Sheet crystals content % Major amino acid components Fibroin (B. mori) B. mori: 4050% [27] Gly (46%) Ala (29%) Ser (12%) Spidroin In MA silk: 35% [30] Gly (37.1%) Ala (21.1%) Glu (9.2%) Arg (7.6%) Ser (4.5%) GPGGX GPGQQ (A)n (GA)n GGX [34] 275320 kDa 410 Alanine residues linked together, sometimes with a serine residue [52] Variable in length and composition, rich in glycine [52]

Repeating motifs

GAGAGS GAGAGY

Size Content of crystalline (hydrophobic) regions Content of non-crystalline (less hydrophobic) regions

[3] $350 kDa [23] Mostly the repeating motifs [48] Negatively charged, polar, bulky hydrophobic and aromatic residues [3]

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4.3. Molecular structure of silk proteins A wide range of studies has been carried out in attempts to unveil the microstructure of silk proteins. Techniques used include various types of NMR, X-ray diraction and transmission electron microscopy (TEM) [5]. Silk proteins were traditionally described as a matrix of rubbery polypeptide with small, rigid, inextensible b-sheets crystallites embedded in it [86,95]. The crystalline regions have been suggested to be associated with the strength of silk, and the surrounding inter-phase with elasticity [56]. However, other suggested models challenge the concept of the amorphous matrix, or suggest a model (for dragline spider silk) of cylindrical sub-units containing a crystalline poly(alanine) core capped by non-crystalline, glycine-rich regions [76,97]. Below is a review of the literature of the general structure of silk proteins, from sequence to folding and assembly. 4.3.1. Primary structure Silks have a highly repetitive primary structure, which gives rise to very regular conformations at the primary level [102]. The repeating units are mostly poly glycine-alanine blocks in broin and poly-alanine with some glycine residues blocks in spidroin [8,26]. High glycine content is thought to allow greater conformational variability [102]. The central region of the protein is mostly hydrophobic and repetitive. The N-terminal and the C-terminal domains are non repetitive and more hydrophilic. In broin, the hydrophilic spacer units are more complex and smaller (around 10%) than the hydrophobic repeating units [8]. They also contain a much higher proportion of charge-carrying amino acids [8,79]. In broin, which is composed of L-broin and P25, Lbroin is mostly hydrophobic, but has two disulphide bridges (one internal and one linking it to the heavy chain) which might be responsible for exposing the hydrophilic N-terminal through a specic conformation [79]. P25 consists of alternating hydrophobic and hydrophilic regions and many charged residues [79]. 4.3.2. Secondary structure Traditionally, silk proteins are thought to be twophased semi-crystalline polymers containing both crystalline and non-crystalline regions [19,31]. Alternatively, silks

are described as repetitive AB block copolymers, with alteration of hydrophobic (crystalline) and less hydrophobic (non-crystalline) blocks [52]. The crystalline regions have been identied as rigid, tightly packed anti-parallel b-pleated sheets [27]. The b-sheets are either poly-Ala, or poly-Gly-Ala, interlocking with adjacent chain via hydrogen bonds [34], (Fig. 2). Wide-angle X-ray diraction measurements show crystal size at around 2 5 7 nm [38,76]. The large gap between measurements has been explained as an eect caused by the non-periodic lattice structure (NPL), where well-ordered small crystallites form less ordered large crystalline regions [5,38]. The non crystalline domains are often described as poorly orientated, randomly coiled sections of the peptide, or amorphous [93]. However, new data indicate that those domains have a preferred secondary structure of glycinerich 31-helix, and a specic orientation, with chains mostly parallel to the bre [93]. The ratio of crystalline to noncrystalline domains is known to vary between spider and silkworm silk, with spider silk containing less b-sheet and more helical regions. This is thought to be important for assembly and mechanical features [82]. An alternative hypothesis proposed for the secondary structure spider silk proteins suggests the existence of three phases: an amorphous phase, highly oriented crystals, and a third, oriented non-crystalline phase [31,76]. 4.3.3. Tertiary structure The combination of extensive hydrogen bonding, high levels of crystallinity, and general hydrophobic nature are all thought to stabilise tertiary structure [2]. The hydrophobic nature of silk is essential to exclude water and produce the high packing density and b-crystallinity necessary for its mechanical function [8]. Silk proteins are therefore insoluble in most solvents, including water and dilute acid and alkali [2]. The crystalline regions of silk proteins are known to be well orientated along the silk bre [32,69,76], and hydrophobic interactions are thought to be responsible for the association between the chains, at least in lepidoptera [79]. Despite reviewing the features of spider and silkworm silks together (for convenience sake), it is apparent that their amino acids and therefore secondary structure is very dierent [82]. This clearly manifests itself in the dierent mechanical properties observed for these silks, as described earlier. However, the highly consistent design details, which are observed in both silk types, are likely to be important for the assembly of silk, and are of interest to evaluate and compare. A full, comparative and well-conrmed model of dierent silk types becomes a real requirement for understanding and in the future possibly controlling its properties. 5. Spider silk

Fig. 2. Diagram showing the interactions of the two dierent b-sheet regions in silk proteins [34].

Spider silks are materials with a long history of evolution and are highly tailored for the functions they full

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[17,63]. However, the study of silk from web spiders is a relatively new eld [97]. Unlike silkworms, spiders produce several kinds of silks diering in material properties [24]. This is paralleled by a huge variation in molecular weight; from 70 to 700 kDa [1]. Araneid spiders (true orb weavers) provide the best studied model for functional studies they have seven dierent types of silk gland, each producing protein with unique amino acid composition [27]. As there is a larger variability among silk sequences of non-orb weavers than among orb weavers [8], it is hard to estimate how many more silk sequences are available in nature. 5.1. Types of spider silk Spider silk proteins dier in the type and proportion of structural modules (motifs), and these dierences can be correlated with their mechanical properties [37,34,68]. Major ampullate (MA) silks are used for dragline silk and Web frame silk [27]. Minor ampullate silks provide additional structural support of the web [37]. Capture silk, the viscid silk that forms the catching spiral, is produced by the agelliform (FL/Flag) gland [27,37]. Egg cocoon silk is produced by the cylindriform (Tubuliform) glands [5]. A summary of silks from dierent glands and their function in the spider species N. clavipes appears in Table 5. Genes coding for various types of spider silk protein from the spider A. diadematus have been studied, and it has been shown that they bear some similarity to the genes coding spidroin I and II from N. calvipes [36]. Another common feature of the studied spidroins is the C-terminal, containing a highly conserved cysteine residue [36]. This indicates crossspecies similarities for silks with similar function, and suggest that the purpose of the silk, rather than it origin, is the biggest factor dening its primary structure. 5.1.1. Major ampullate silks Silk from the major ampullate glands of orb-weaving spiders is designed to act as a shock absorber [32,95].

MA silks contain at least two dierent types of proteins, often named Major ampullate spider silk 1 and 2 (MaSp1/MaSp2) [19,37]. The molecular weights of those proteins are up to several hundred kDa [104], with MaSp2, but not MaSp1, containing proline-rich motifs [42,90]. Dragline silk is the best-studied spider silk, due to the interest in its impressive mechanical properties. It has strength and elongation at break giving it a toughness superior to the best synthetic man-made bres [44,54], and per unit weight it is stronger than high tensile steel [27,36]. Dierent spider species produce dragline with dierent mechanical properties [63]. In fact there is a rather high variability in mechanical properties even in silk produced by the same individual spider under carefully controlled conditions [63]. Dragline thread is thought to consists of three layers [51]: (i) Core and skin form the bulk and thickness of the thread. The core contains several ne, elongated structures called canaliculi. The chief component of the core and skin are spidroin 1 and 2; (ii) a thin coat: thought to be a glycoprotein; (iii) a thin laminar or lamentous layer that covers the surface of the thread. Frische et al. describe elongate cavities parallel to the silk bre axis [24]. 5.1.2. Minor ampullate silks Silks produced in the minor ampullate are characterised by great tensile strength but little extensibility [37]. Similarly to major ampullate silk, it is thought to consist of two proteins, MiSp1 and MiSp2. Dramatic dierence in tensile properties between minor and major ampullate silk has been suggested to origin from dierences in protein conformation [61]. 5.1.3. Cylindriform silks Little work has been done on cocoon silks (egg case silks), which are the produced by the Cylinfriform (Tubuliform) glands. In terms of amino acid composition, silks produced in the cylindrical glands of Araneid orb weavers are reported to be rich in the amino acid Serine [100]. Hu et al. has investigated morphology of egg case silk from the black widow spider Latrodectus hesperus [41]. They have demonstrated that the egg case is composed of bres with two dierent diameters, the larger bres reported at approximately 45 lm and thinner bres at around 500 nm. The thinner threads were thought to originate from the Aciniform gland. However, no evidence has been produced to conrm this. Hue et al have also identied a Cylindriform synthesised protein dominant in the egg case, which they called egg case protein 1 (ECP1). The primary structure of ECP1 appears to contain motifs also present in silkworm broin, and an N-terminal rich in serine [41].

Table 5 Dierent silks produced by the golden orb weaver Nephila clavipes, their gland of origin, spinneret and function [103] Type of silk Dragline Viscid Glue-like Minor ampullate Cocoon Wrapping Attachment Gland Major ampullate Flagelliform Aggregate Minor ampullate Tubuliform Aciniform Pyriform Spinneret Anterior Posterior Anterior and posterior Medial Posterior Anterior Anterior Function of the silk Orb web frame, radii, safety line Prey capture, core bres of adhesive spiral Prey capture, adhesive silk of spiral Orb web frame reinforcement Reproduction Wrapping captured pray, inner egg sac Attachment disk and joining bres

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Interestingly, cocoon silk from the garden spider A. diadematus was found to contain similar motifs to N. clavipes MA silks [5]. TEM studies of cocoon silk of A. diadematus suggest the existence of non-periodic lattice crystals (NPL), also described as orientated amorphous material [5]. 5.1.4. Flagelliform silks Orb weaving spiders produce capture silk (or viscid silk) for the sticky spiral in their webs [6]. Capture silk has a low stiness and only breaks after a 200% extension [36]. It is coated by hydroscopic glue, a viscous liquid containing long-chain glycoproteins, keeping it wet and thus maintaining the bres exceptional elasticity [6,36]. This aqueous coating spontaneously forms evenly spread droplets [36]. The major component of the bre core is agelliform protein, which is a variety of broin [6]. The agelliform protein is rich in the motif Gly-Pro-Gly-Gly-(Xaa)n (with Xaa being Ala, Ser, Tyr or Val), thought to form type II b-turns [33]. The chain of b-turns formed by these repeating motifs has been described as a spiral, and may partially account for the reversible extensibility of agelliform silk [33]. Unlike other spider silks, agelliform silk does not contain the alanine-rich regions responsible for forming the tightly packed b-sheets in MaSp1 and 2 [33]. 5.2. Supercontraction As mentioned earlier, the mechanical properties of silks are greatly inuenced by water [83]. Some spider silk (notably major ampullate silks), when contacted with water, shrinks to around half its original length and doubles in diameter, a phenomenon called super-contraction [36,83]. Super-contraction involves a reversible uptake of solvent by silk [94]. This results in increased bre extensibility and reduced stiness [36,83]. This is thought to be the consequence of hydrogen bonds destruction by water molecules, leading to signicant molecular chain motion and disorientation [38,83,95]. The more hydrogen bonds are destroyed the larger the shrinkage, as the molecule retracts to a random coil [31,83]. However, despite the change in secondary structure, there is no major loss of molecular order, possibly due to the sub-organization of spider silk, and the thread can be re-stretched to recover its mechanical properties [94]. It has been shown that water molecules do not penetrate the crystalline regions [38]. As can be seen in Table 6, non-MA silks appear to show a smaller contraction upon exposure to water [45]. This is thought to be the result of dierences in primary structure [45]. Short MA silk threads were also observed to superTable 6 Contraction of dierent silks upon exposure to water [45] Silk type Major ampullate (N. calvipes) Minor ampullate (N. calvipes) Degummed cocoon (B. mori) Amount of contraction (%) 44 2 52 01

contract in urea solutions, violently coiling into a right handed loose spiral before swelling [97], probably as the result of the disruption of hydrogen bonds by this chemotropic agent. Spiders are thought to take advantage of supercontraction to restore the shape of the web after deformation by rain, wind or contact with prey. However, this material property is not desirable in most biomedical and technological application and synthetic bres presenting a similar phenomenon are heat treated to prevent it [32]. Supercontraction could possibly be removed by incorporating bres into a water resistant matrix or genetic manipulation to eliminate specic sequences from the protein thought to be responsible for the water absorption [7]. In the meantime, non-super-contracting silks with a smaller degree of water sensitivity, such as spider egg sac and silkworm silks could be more practical for wet applications. 6. Properties of silk as a potential biomaterial 6.1. Biomaterials denition A biomaterial is a non-living material used in a medical device, intended to interact with a biological system [111,9]. The chief function of biomaterials is to repair, replace or augment a tissue [85]. The ability of a biomaterial to perform with the appropriate host response in a specic situation is known as biocompatibility [9]. The advantages and disadvantages of silk as a potential biomaterial is reviewed below. 6.2. Advantages of silk as a biomedical material Good mechanical strength when wet, resistance against enzymatic cleavage, and high oxygen and drug permeability all make silk a desirable biomaterial [67]. Silk is also considered reliable, able to perform under a wide humidity and temperature range [84]. Silk can also be processed to form a variety of new materials with an array of properties. Silk broin, when processed from an aqueous solution has been shown to form a hydrogel, with porosity dependent upon protein concentration and gelation temperature [48]. It can also be used to coat other materials, such as poly-(carbonate)-urethane membranes [16]. In terms of cell compatibility, degummed silks are arguably well suited for cell culture purposes. SF coated membranes (prepared using aqueous broin solutions) permitted greater adherence and consequently greater proliferation of broblasts in comparison to non-coated membranes [16]. Silk broin lms from regenerated B. mori broin has also been shown to be biocompatible to human mesenchymal stem cells in vitro, and even enhanced cell proliferation compared to collagen scaolds. In vivo, inammatory reactions to the silk lms were either similar to or less than to collagen [64].

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6.3. Issues with silk as a biomedical material There are a number of concerns regarding the compatibility of silk for biomedical use. One issue is the slow degradation of silk, which could be an advantage or a disadvantage, as discussed above. Activation of an immune response is a useful measure of biocompatibility [70]. Biomaterials derived from a nonautologous source often elicit a foreign body response (FBR) in vivo [1]. Factors such as implantation site, size, geometry and surface topography inuence the level of the response [1]. The sericin protein coating of silkworm bres are reported to stimulate an immune response [70]. Conventionally, broin is degummed in alkaline baths containing soap. Alkaline or neutral proteases have also been shown to eectively remove the sericin envelope [23]. However, it is generally agreed that a further optimisation of the degumming process is needed, in order to control and corroborate the adequate removal of sericin from B. mori silk while maintaining the integrity of the bre [1,23]. Evidence suggests that fully clean silk bres are largely inert and possibly inhibitory with regard to macrophage activation [70]. An additional grave issue is recent reports that broin may be a risk factor for amyloidogenesis. Dissolved B. mori broin has been shown to accelerate amyloid accumulation in vivo, a process that can lead to tissue degeneration [62]. Another topic to be considered is the tensile variability of natural silk. As indicated earlier, silk produced by a single spider can show variability from segment to segment along the same thread [95]. This might complicate the use of natural silk for load-bearing applications. However, this diculty could be addressed by using a multi-bre thread rather than a single lament, and should not be an issue once silk is manufactured in alternative systems. Silk therefore appears to be highly suitable for numerous biomedical purposes. From load and impact resistant applications to cell adhesion-enhancing coatings, dierent silks oer a great range of desirable assets. The choice of silk type for a specic biomedical application should therefore include a careful consideration of the particular advantages of each silk species, as well as their limitations. 7. Biomedical applications and modications of silks Silk, and especially Bombyx mori silk, has a very long history in biomedical applications. Some of those applications are reviewed here. Silk bres from silkworm have been successfully used as sutures for years, but inammatory reactions caused by macrophages have since been reported [25]. It is now clear that the sericin proteins are the major cause of such biological responses, and bres treated to remove sericin appear to be comparable to other commonly used biomaterials [1]. In commercial sutures, once sericin is removed, it is replaced with a wax or silicone coating [1].

Furuzono et al. demonstrated that higher levels of platelet adhesion occurred in untreated silk compared to silk that has been treated (poly 2-Methacryloyloxyethyl phosphorylcholine (MPC)-grafted silk fabric) for improving blood compatibility [25]. U et al. recorded relatively high phagocytic response from macrophages elicited by soluble silk factors from sutures [91]. However, Altman et al. point out that the factors producing such response were unidentied, and could include residual sericin, waxes or silicones used during manufacturing process, or simply the size of the broin particles [1]. The braided nature of silk suture has also been considered as a source of inammation, as it allows the retention of surface debris and bacterial accumulation [105]. Silk has been suggested as an alternative to collagen as a substratum for the cultivation of animal cells [43]. It was found to be almost as ecient as the widely used collagen in enhancing the growth of anchorage-dependent mammalian cells [43]. Recently, use of collagen has been avoided because of the potential prion protein contamination, associated with neuro-degenerative diseases [43]. Minoura et al. [67] studied the attachment and growth of broblast cells on SF from B. mori and A. pernyi. They compared the performance of SF to collagen and showed that growth rates appeared to be at least as good or better on silk. The highest attachment and growth rate were recorded on A. pernyi silk, and were attributed to the tri-peptide cell adhesion sequence Arg-Gly-Asp. They also investigated the performance of A. pernyi silk whose C-terminal region was digested by a-chymotrypsin. Interestingly, it exhibited very low cell attachment and growth ratios [67]. Yamada et al. [106] isolated and identied two sequences from the N-termainal region of B. mori SF that promote growth: [VITTDSDGNE] and [NINDFDED]. They showed that the sequences are part of an amorphous region, are water-soluble, and act in synergy, enhancing each others activity Studies on SF coated scaolds (Polyurethane membranes and foams) have shown that the SF coating enhanced human broblasts cell adhesion and growth compared to non-coated scaolds [16,20]. In other studies, the surface of lms from the synthetic polymer poly (D,L-lactic acid) has been modied by incorporating various molecular weights of SF, and the eect on rat osteoblasts attachment and proliferation was measured [10]. Results indicated that the SF modied lms were more hydrophilic and cell attachment and growth rates were consequently higher than on the control [10]. Galactose residue has been immobilised into SF to form a new glycoconjugate [28]. Hepatocytes (liver cells) were shown to attach to polystyrene dishes coated with the glycoconjugate [28]. Arginine residues on SF have been modied by binding to 1,2-cyclohexanedione, reducing the basic nature on the bre surface [29]. The attachment, but not the growth of broblast cells appeared to improve on the modied, less basic SF [29].

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SF from B. mori has been used as a net to support the growth of various human cells of dierent tissue and type (endothelial, epithelial, broblast, glial, keratinocytes, osteoblast), allowing all types of cells to adhere and spread over the bres [92]. Most of the cells were able to grow and survive on the nets for at least seven weeks, forming tissuelike structure, without changing the structure of the bres [92]. Chen et al. used RGD-modied broin matrices and lms to grow human bone marrow stromal cells, and found that the RGD-coupled matrices signicantly enhanced cell attachment [13]. Chen et al. studied the transport of pharmaceuticals through SF membrane as a function of pH [14]. They concluded that the permeability of SF membranes can be regulated by changing the external pH, and suggested its use as the matrix of a drug delivery system [14]. It has been shown that wounds dressed with transparent broin lm healed quicker than wounds dressed with conventional hydro-colloid dressing. Sugihara et al. [87] found that wounds dressed with silk lm were kept suitably moist, and attributed quicker healing to silks capacity to drain exudates from wounds. Another advantage recorded was the transparency of silk lm allowing direct observation of wound healing. They examined the eect of silk powder on skin healing, and found it more ecient than conventional dressings but slower than the silk lm. These authors also suggested that silk lm has the ability to retain water, proteins and electrolytes, although no direct evidence was produced. In another study, SF nano-bres produced by electrospinning from regenerated SF solution were used as a scaffold for growing primary normal human oral keratinocytes (NHOK). The results of this study indicated that the electrospun SF nano-bres promoted cell adhesion [66]. Shao et al. reported that highly polar solvents such as water and methanol reduced bre modulus and strength because of plasticisation, but that other solvents had no measurable eect [80,82,83] designed a silk-like hybrid from poly-alanine regions of S. cricini SF and the cell adhesion sequence [TGRGDSPA] from bronectin [4]. Containing the RGD cell binding motif, this sequence has been reported to keep the high activity in human lysozyme [4]. The group recorded higher adhesion and growth rates on the new hybrid than on the original SF [4]. It is clear from the vast number of applications, that silk is a versatile material with excellent tensile properties and good biocompatibility. Its versatility also allows diverse modications, a tool not yet fully explored. Better understanding of silk structure in all levels is likely to enhance capabilities in that area. 8. Conclusions Silks are materials with excellent mechanical properties, and particularly superior tensile strength and toughness. As a group of materials they are also characterised by an

exceptionally wide range of properties. Dierent degrees of extensibility, water resistance, thermal stability, and degradation rates are reported for dierent species and types of silks. It is now recognized that many of these properties could be of great value for biomedical applications, specifically those that involve bearing a load, such as orthopaedics. In light of the great variety of silks described to date, it appears that an ultimate compatible material would probably contain various properties selected from the available range according to the specic application, or in another words, a designer silk. Recent work links the physical and mechanical properties of native silk to the molecular make up, assembly and formation process in spiders and insects. A Closer look into the morphological and biochemical variation between dierent silks reveals that they add to the versatility of silk as a biomaterial. They do so chiey by inuencing the nature of its interaction with cells and tissues and eliciting diverse biological responses. They also inuence the possible types of silk modications, including surface alteration or complete regeneration. There are still some major obstacles to the development of silk-based biomedical devices and engineered tissue. Those include the poor availability of most types of spider silk and unresolved biocompatibility issues with mulberry silkworm silk. To overcome those major obstacles, eort has been invested in attempts to produce silk and silk-like bres in vitro. These eorts have mostly focused on synthesising major ampullate Spidroin 1 and 2 proteins in bacterial systems and encouraging the assembly of these proteins to bres. However, there are still some critical problems with the feasibility of forming silk bres in vitro, namely the spontaneous deletion of repetitive genes and the premature aggregation of proteins in the dope. The use of silk for biomedical applications in the near future will therefore require some other approaches. These could include using conventional polymer chemistry to synthesis polymers units that mimic silk, according to desired properties. Alternatively, selecting a native bre with the desired properties for a specic application could also be explored, but that will require breeding an insect or a spider and collecting the spinning dope or formed silk bres. Finally, the regeneration of more readily available silks to form sheets, foams or newly spun bres is an option already being explored. It is clear, however, that further understanding of silk structure in both molecular and supra-molecular levels, as well as its formation process will be essential in order to allow modication, manipulation and ultimately controlling the mechanical and biological properties of man-made silk and silk-like bres. References
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