Silk materials for biotechnology

Silk materials for biotechnology

10

Ki Hoon Lee Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul, Republic of Korea

10.1

Introduction

Silk has been served as an excellent textile material for more than 5000 years not only because of the luster and touch but also the outstanding mechanical properties. The history of silk has begun in Asia but spread out throughout the world. Still, China is the predominant producer of raw silk, but in the 1960s and 70s, Japan and Korea also shared a large portion of world production. However, as the economic growth of these two countries has grown fast, the increased cost of labor together with the change of national economic fundamentals from agriculture to heavy industry led the corruption of silk industry in the 80s and 90s. During this time of decay, there were tremendous efforts to reinvent the silk industry. One of the efforts was to use silk fiber not in its original form “fiber.” Researchers started to use silk fiber as a resource of protein. This resembles much of the development of cellulose derivatives from wood. Once silk fiber is solubilized, it could be used directly or fabricated into unlimited forms of material ranging from nano to macro in size, from particle to three-dimensional (3D) matrix in shape. As a result, the use of silk has expanded beyond textile including bioengineering and biomedical fields but not limited to those. The nontextile application of silk protein was initiated in cosmetics and nutraceuticals. Sericin has a long history in cosmetics, but fibroin has new ingredients in nutraceuticals. The consumption of fibroin hydrolyzates helped several symptoms of diabetes or obesity. However, I will not discuss these subjects here because of the lack of certified results. In this chapter, various applications of silk proteins in the biotechnological field will be introduced. Starting from how we can obtain the silk proteins as raw material, some principle of fabrication methods will be introduced to produce a specific solid form. In the application of silk proteins, the requirements for each application will be introduced and how silk proteins meet those requirements will be explained. Detailed results will not be described here because there are plenty of good reviews on each application. The aim of this chapter is to provide new insights into the ancient materials (Omenetto and Kaplan, 2010).

10.2

Extraction of fibroin and sericin

Silkworms produce silk proteins in the form of a bicomponent fiber having sheath and core structure, where the core is fibroin and the sheath is sericin. Fibroin is the protein Advances in Textile Biotechnology. https://doi.org/10.1016/B978-0-08-102632-8.00010-4 Copyright © 2019 Elsevier Ltd. All rights reserved.

240

Advances in Textile Biotechnology

that we use as a textile in most cases. Sericin is usually removed to get the luster and touch of silk fabric but sometimes left and fixed to change the touch of silk fabric. Anyhow, we can obtain two different types of protein from the silkworms.

10.2.1

Fibroin

To get fibroin, we first have to remove the sheath layer, sericin. The removal of sericin can be done easily by various alkaline solutions, but sodium carbonate solution at elevated temperature is recommended. One drawback of this sodium carbonate method is the degradation of fibroin which is unavoidable to remove the sericin perfectly. More mild degumming method is reducing the concentration of sodium carbonate by adding soap or using sodium bicarbonate. Once the sericin is removed, the core layer, fibroin, is dissolved using a high concentration of salt solution. Usually, chaotropic salts are used to break the intermolecular hydrogen bonding of fibroin molecules. Calcium chloride and lithium bromide are the most frequently used salts for the dissolution of fibroin (Table 10.1). After the dissolution with a salt solution, the salt in excess is removed by dialysis before further use. It generally requires at least 2 days for the complete removal of salt. Finally, one can get the fibroin solution ready to use. One should take care that the molecular weight of the extracted fibroin depends on the degumming and dissolution method such as chemical reagent, processing time, and temperature (Table 10.1). Storage condition of the as-prepared fibroin solution is important, and it is recommended to store at 4 C to prevent premature crystallization of fibroin. Sometimes, solvents other than water may require, but unfortunately, that the native fibroin fiber cannot be dissolved directly with general solvents. In such case, the fibroin solution from above is lyophilized and then dissolved once again to a proper solvent.

10.2.2

Sericin

If one wishes to obtain sericin, care should be done because sericin is prone to hydrolysis. Currently, there are two extraction methods for sericin, and its feature is summarized in (Table 10.2). Boiling the cocoon in a pressurized vessel would be the easiest Table 10.1 Common dissolution methods of fibroin. Chaotropic salt

Concentration

Temperature

Time

Remarks

CaCl2

1 M CaCl2 8 M H2O 2 M EtOH

60e80 C

5e180 min

Quick dissolution Suitable for commercial scale Time-dependent degradation

LiBr

9.3 M

25e70 C

3e15 h

Minimum degradation Suitable for academic research

Silk materials for biotechnology

241

Table 10.2 Common extraction methods of sericin. Reagent

Concentration

Temperature

Time

Remarks

Distilled water

e

80e120 C

30e60 min

No need for dialysis Suitable for commercial scale Time- and temperaturedependent degradation and extraction yield

Ureamercaptoethanol

Urea 8 M Mercaptoethanol 5%(v/v)

80 C

10 min

Minimum degradation Dialysis at elevated temperature to prevent gelation Suitable for academic research

way to get a sericin solution. No need for dialysis is advantageous for this method, but the degradation of sericin will occur. Use of high concentration of urea minimizes the degradation of sericin during extraction, but time-consuming dialysis at elevated temperature (>40 C) is required. Sericin can also be recovered from the degumming process of silk, but the degradation of molecular weight is unavoidable, and chemicals should be removed by dialysis. Sericin solution higher than 1 wt% tends to form a gel. This can be prevented by keeping the solution at a high temperature of about 50 C. After lyophilization of the sericin solution, it could be dissolved in some different solvents such as fibroin. The lyophilized sericin powder can be best stored in a desiccator to avoid contact with a humid environment. In the humid condition, sericin tends to form a b-sheet structure which reduces its solubility. After the extraction of sericin, fibroin fiber is left over, but we do not use this fibroin because not all sericin is removed during the extraction (Table 10.2).

10.3

Fabrication

The fabrication of silk proteins into useful forms is the first step for a successful application in biomedical fields. The overall process for fibroin and sericin is shown in Fig. 10.1. Whether it is dissolved or extracted, we first obtain both fibroin and sericin

242

Advances in Textile Biotechnology

(a) Cocoon Degumming

Fibroin fiber

Dissolution dialysis

Fibroin solution

Lyophilization

Fibroin powder

Dissolution

Fibroin solution

In organic solvent

Fabric

Fiber, film, sponge, hydrogel, particles, etc.

(b) Cocoon Extraction

Sericin solution

Lyophilization

Sericin powder

Dissolution

Sericin solution

In organic solvent

Fiber, film, sponge, hydrogel, particles,etc.

Figure 10.1 Processing flow chart of silk proteins from the cocoon to the end product: (a) fibroin and (b) sericin.

in an aqueous solution state. Unfortunately, the concentration of the initial solution is not high enough to get proper viscosity required in some fabrication methods. In addition, both fibroin and sericin solutions will undergo gelation at room temperature indicating denaturation of the protein. Therefore, we need to use a solvent other than water, which can dissolve enough amount of protein to achieve proper viscosity with minimal degradation. For fibroin, hexafluoroisopropanol (HFIP) is the most frequently used organic solvent because no degradation occurs during the fabrication process. Formic acid is also frequently used as a solvent, but degradation causes inferior properties compared with HFIP dissolved fibroin. In the case of sericin, LiCl/DMSO or tetrafluoroacetic acid (TFA) was used for the same purposes. However, if we consider the safety concerns of organic solvents, the use of these solvents should refrain especially in biomedical applications. In such cases, a synthetic water-soluble polymer such as poly(ethylene oxide) (PEO) is added to increase the viscosity of the silk protein solution.

Silk materials for biotechnology

243

Regardless of the types of solvent used for the fabrication of silk protein, there is an important structural transition of silk protein during the fabrication. In the solution state, silk protein molecules are solvated and have a minimum degree of intermolecular interaction. During the process of fabrication from the solution state into any solid forms, the solvents are removed, and it causes a significant increase of intermolecular interaction between the silk protein molecules. The result of intermolecular interaction depends on the chemical nature of fibroin and sericin. Fibroin consists of a highly repetitive sequence of hydrophobic amino acids, which are interrupted 11 times by a nonrepetitive sequence of hydrophilic amino acids. If the solvents are removed, the highly repetitive sequence has a high tendency to form the antiparallel b-sheet structure through intra- and intermolecular hydrogen bonding. This peptide chain folding into a b-sheet structure is irreversible; thereby, the solid state became insoluble. Furthermore, the b-sheet structure acts as physical cross-linking in the matrix. Therefore, the rate and degree of the b-sheet formation has a tremendous effect not only on the physiochemical properties of the final fibroin product but also on the biological properties such as degradation and cellular responses. Therefore, the control of b-sheet structure is the key to fibroin fabrication. There are many ways on how to induce the b-sheet structure, and it is summarized in Fig. 10.2. Whether being chemical or physical, any external stimuli that are favorable for increasing the possibility of proteineprotein interaction result in b-sheet structure. Meanwhile, sericin has more hydrophilic and less repetitive amino acid sequences. Similar to fibroin, sericin can also form the b-sheet structure, but it cannot be maintained permanently. Therefore, further cross-linking is required to make the final product insoluble.

10.3.1 Film Fibroin film can be made both from aqueous and organic solvents. The removal of the solvent by evaporation results in a transparent film. In general, fibroin film from aqueous solution is still soluble, which requires another step to make it insoluble. Immersing the film in alcohol solution such as methanol induces an irreversible structural transition from random coil to the b-sheet structure (Fig. 10.3). The formation of b-sheet structure acts as a physical cross-link, and the film became insoluble. This is a unique property of fibroin where additional toxic chemical cross-linkers Solution Solvated intramolecular random soluble

Solid Alcohols moisture stress-induced self-assembly

Aggregated intermolecular β-sheet insoluable

Figure 10.2 Structural transition of fibroin during fabrication.

244

Advances in Textile Biotechnology MeOH or crosslinking agent

Evaporation

Silk solution + Active ingredient

Soluble film

Insoluble film

Figure 10.3 Preparation of silk protein films.

are required for most of the other protein materials. On the other hand, a film prepared from formic acid solution has already b-sheet structure without alcohol treatment. Some active component, such as enzyme, antibody, and drug, can be loaded by dissolving it into the fibroin solution unless the active components maintain their activity during the film formation. The use of an organic solvent may induce denaturation of enzymes and antibodies. A prolonged exposure at high temperature to facilitate the evaporation of the solvent can also affect the solubility and activity of the active component. Sometimes, agglomeration between fibroin and active components can also be observed. Sericin can also be cast into a film, but the sole sericin film is too brittle. An addition of a plasticizer such as glycerol can solve this problem, but the film became hygroscopic.

10.3.2

Fiber from wet spinning

Of course, silk is a fiber, and it can be used directly as itself or in the form of woven, knitted, or nonwoven fabrics. However, there are needs for fibers that have different shapes or diameters other than the natural one. This can be accomplished by the artificial spinning of fibroin solution. In general, the fibroin solution should have a proper viscosity to be spun. If HFIP and formic acid are used as a solvent, the concentration of fibroin of less than 10 wt% would be enough for spinning, while higher than 20 wt% is required for spinning from aqueous fibroin solution. The low concentration of fibroin, which means a high amount of solvent in otherwise, limits the spinning process only for the wet spinning where the exchange of solvent and nonsolvent leads to the formation of fiber (Fig. 10.4). Typical nonsolvents (or coagulant) are alcohols such as methanol or ethanol. Once spun into the coagulant, there is no need for further cross-linking because the coagulant induces a structural transition from random coil to b-sheet structure simultaneously. Further drawing process can be performed to enhance tensile properties by the orientation of b-sheet crystals. Unfortunately, the “regenerated” fibroin fibers (like the regenerated cellulose) have lower tensile properties than the native one, probably because of (1) degradation of fibroin during dissolution and spinning, (2) insufficient crystallization and orientation of fibroin at spinning, or (3) low concentration of fibroin in the dope solution. More details about the regenerated silk fibers could be found in another review paper (Koeppel and Holland, 2017). On the other hand, sericin cannot be spun into fiber unless blended with other polymers such as polyvinyl alcohol (PVA). In addition, extensive cross-linking is required to make the fiber insoluble.

Silk materials for biotechnology

245

Silk solution (5–15wt%) in HFIP or formic acid

Coagulant

Figure 10.4 Wet spinning of silk protein. HFIP, hexafluoroisopropanol.

10.3.3 Fiber from electrospinning With the beginning of the 21st century, electrospinning became an innovative spinning technology, which can produce nanofibers (more likely submicrofibers) with a simple apparatus (Fig. 10.5). Fibers from electrospinning have wide application in filtration, composites, and medical applications. Up to now, enormous numbers of organic polymers were subjected to electrospinning, and fibroin was one of the first natural polymers to be electrospun. The same dope solution from wet spinning can be used for electrospinning, but organic solvent such as HFIP and formic acid is favored because of the high volatility of the solvent. However, the use of organic solvent should be limited if possible because most of the electrospun fibroin fibers find its application in the medical field. Electrospinning from aqueous fibroin solution is possible but cannot be spun without the addition of another polymer such as PEO or PVA. The electrospun fibroin fibers are collected in a sheet form like a paper, and further treatment with alcohol is required to make the sheet insoluble. For the application of electrospun fibroin fibers in tissue engineering, 3D scaffolds are desired, but conventional electrospun fibers have two-dimensional shapes. This can be solved Silk solution (5–15wt%) in HFIP or formic acid

High V

Figure 10.5 Electrospinning of silk protein. HFIP, hexafluoroisopropanol.

246

Advances in Textile Biotechnology

by electrospinning the fibroin solution directly to the coagulant bath. The final scaffold has a sponge-like structure, but the walls of the sponge are made of fibroin nanofibers (Ki et al., 2007). A tubular form of electrospun fibroin can be prepared for small diameter blood vessel and nerve guide conduit. At first, the fibroin solution will be electrospun against a rotating metallic wire, and removing the wire after spinning result in a tube has the same inner diameter as the wire’s diameter. Various techniques of electrospinning have been developed so far, and fibroin could be applied to most of all those techniques. Meanwhile, the electrospinning of sericin was performed recently using TFA-based solvent system.

10.3.4

Hydrogel

Hydrogel is a polymer network structure that retains a high amount of water inside. The network structure is stabilized through chemical or physical bonding between polymer chains. When we look at the amino acid composition of fibroin, glycine and alanine possess more than 70% of all amino acids. Because both amino acids are relatively hydrophobic, fibroin in water is unstable and tends to interact with each other by hydrophobic interaction. The hydrophobic interaction will locate the fibroin chains in proximity that allows intermolecular hydrogen bonding, and it results in a thermodynamically stable b-sheet structure. Once the b-sheet structure is formed, the fibroin solution is now in a gel form and became opaque. The whole procedure will take several days, but it can be accelerated by incubating at increased temperature, lowering the pH near to the pI of fibroin, and conducting sonication (Fig. 10.6). For example, when the fibroin solution was sonicated for 3 min, the gelation time was less than 3 h, which is far faster than doing nothing. Sericin can also undergo gelation when incubated at room temperature. The gelation mechanism of sericin resembles that of fibroin where a significant increase of b-sheet structure was observed. Unlike the fibroin, sericin gel is partially reversible.

10.3.5

Porous 3D scaffold

In tissue engineering, a 3D scaffold is required to induce proper cell responses. When the fibroin solution is lyophilized, a sponge structure is developed having enormous numbers of pores. The pore was previously occupied by ice, which sublimated during

Time temperature pH sonication Silk solution

Figure 10.6 Preparation of silk hydrogel.

Silk hydrogel

Silk materials for biotechnology

Silk solution

247

Removal of porogen (polymer or salt)

Lyophilization

Solid silk

Silk sponge

Figure 10.7 Preparation of silk sponges.

the heating in a depressurized chamber (Fig. 10.7). Therefore, to control the size or distribution of the pore, it should be controlled during the freezing step where the size of ice crystals is determined. However, this can provide only a limited window to control the properties of the scaffold. Another way to induce a pore structure is mixing an immiscible polymer such as PEO with fibroin. The mixture of PEO and fibroin can be fabricated into film or fiber followed by methanol treatment. The methanol treatment can insolubilize fibroin but not the PEO. Therefore, PEO can be selectively removed by washing with water leaving a pore structure. However, the interconnectivity of pores is low, which may cause some problems in cell migration and substances diffusion. A promising method for the development of 3D scaffold is salt-leaching method. The size, distribution, and interconnectivity of the pores can be controlled by the size of the salt particles (Fig. 10.7).

10.3.6 Particles Various methods can prepare silk protein particles in the range of micro- to nanometer size. Simple dripping the silk solution with a syringe into the coagulant will result in a particle having 1e2 mm in diameter. Particles having micrometer can be prepared by various spray drying techniques on a commercial scale. Electrospraying is another possible method for microparticle preparation (Kim et al., 2015). Silk particles having less than 100 nm can also be prepared by precipitation or bottom-up self-assembly of silk protein molecules. The formation of silk protein particle can be divided into two strategies, top-down and bottom-up (Fig. 10.8). First, particles can be formed by the removal of solvent from the droplet. In this method, droplets of silk solution are generated by dripping, spraying, and emulsion. Once the droplets are formed, the solvent can be evaporated or substituted with nonsolvent. The size of the particle, here, depends on the size of the initial droplet and the concentration of silk protein in the

Bottom-up

Top-down Silk droplet Evaporation coagulation

Silk self-assembly

Silk particle

Figure 10.8 Different approaches for the preparation of silk particles.

Silk molecule

248

Advances in Textile Biotechnology

droplet, which both parameters are proportional to the size of the particle. The rate of evaporation or the solvent to nonsolvent exchange mostly determines the morphology of the particle. Another method is allowing the self-assembly of silk protein molecules, especially in the case of fibroin. As mentioned before, fibroin has a polypeptide chain of alternating highly repetitive sequences of small hydrophobic amino acids with nonrepetitive hydrophobic amino acids. Therefore, a micelle-like structure of fibroin was proposed like the ABA-type surfactant. A controlled manner of inducing the micelle structure can lead to the formation of fibroin nanoparticles. It can be achieved by adding polar solvent or salts, which can interrupt the watereprotein interaction and induce proteineprotein interaction. However, a sudden addition of a large amount of inducing agent will yield agglomeration instead of the separated individual nanoparticle.

10.4 10.4.1

Biotechnological applications Enzyme immobilization

The beginning of the use of silk proteins as a material in the biotechnological field was the support for the immobilization of enzymes. For example, glucose oxidase (GOD) was dissolved in the fibroin solution and cast into a film. The film was further treated with alcohol to make the film insoluble. The immobilized GOD retained its activity quite well and had increased operational stability. Here, the immobilized GOD is entrapped in the polymer cage of fibroin, and such an immobilization technique suffers from loss of enzyme through the loose polymer structure. However, on treatment with alcohols, the fibroin molecules form intermolecular hydrogen bonding which eventually acts as a physical cross-link in the polymer matrix. This physical cross-link prevented the loss of GOD and retained the activity during the repeated uses. This early study is important because it gave us an entirely new concept of how we can use the silk protein in biotechnology. So far, various forms of silk proteins are utilized for the support of the enzyme (Table 10.3). In general, enzymes are immobilized on the surface of the fiber by chemical covalent bonding. As both fibroin and sericin consist of all 20 amino acids, there are functional groups such as amino and carboxyl groups for covalent bonding with enzymes. The easiest way to achieve enzyme immobilization through covalent bonding is activation of amino groupecontaining amino acids with glutaraldehyde. Alternatively, carboxylate groupecontaining amino acids can be another target of enzyme immobilization. In such case, standard EDC/NHS chemistry can be applied in an organic solvent. The immobilized enzyme usually suffers from reduced activity compared with the free enzyme. This drawback is compensated by increasing the amount of enzyme immobilized. For a reason, the supports having high surface areas are recommended, and nanofibrous mats satisfy such requirements. Indeed, enzyme immobilization was one of the first applications of electrospun fibroin nanofibers. In enzyme immobilization, hydrophilic supports have advantages over hydrophobic one because it can provide better microenvironment to the enzyme. In this concept, sericin has great potential than fibroin not only because it is more hydrophilic but

Silk protein

Form

Fabrication

Enzyme

Immobilization

Reference

Fibroin

Membrane

Casting

Urease

Covalent bonding (GA)

Moon et al. (2017)

Membrane

Freeze-drying

Catalase

Covalent bonding (tyrosinase)

Wang et al. (2015)

Film

Casting

Organophosphorus hydrolase

Entrapment

Dennis et al. (2012)

Film

Casting

Glucose oxidase, lipase, horseradish peroxidase

Entrapment

Lu et al. (2009)

Sphere

Spray-drying

Lipase

Adsorption

Ferreira et al. (2017)

Thin layer

Coating

Acetylcholinesterase

Adsorption

Xue et al. (2012)

Nanoparticle

Precipitation

Neutral protease

Covalent bonding (GA)

Zhu et al. (2011)

Nanoparticles

Precipitation

L-Asparaginase

Entrapment

Zhang et al. (2011)

Nanofibrous mat

Electrospinning

a-Chymotrypsin

Covalent bonding (GA)

Lee et al. (2005a)

Microparticle

Spray-drying

L-Asparaginase

Covalent bonding (GA)

Zhang et al. (2004)

Fiber

Fixation on silk fiber

Trypsin

Covalent bonding (EDC/ NHS)

Lee et al. (2005b)

Sericin

Silk materials for biotechnology

Table 10.3 Silk proteins for the support of enzymes.

249

250

Advances in Textile Biotechnology

also because it possess a higher number of reactive amino acids. However, there are fewer examples of sericin in this field because of inferior mechanical properties and limitation in fabrication methods.

10.4.2

Medical application

The most promising application of silk would be its use in biomedical applications. The use of silk as raw material for a commodity product such as wood cellulose would be impossible because of the available quantity and price of the raw material. However, the quantity and price of the raw material will be a less critical factor when choosing a material if it has the unique features that meet certain criteria for a specific application. The biomedical field is a typical field where such rule of selection is adopted. Silk proteins, especially fibroin, have been studied as biomaterial extensively during the last decades. Fibroin has not only good mechanical properties but also proper biocompatibility. As shown previously, it can be fabricated into any forms as required in the biomedical field. More importantly, both mechanical and biological properties of fibroin can be controlled by various methods.

10.4.2.1 Suture The use of silk as a suture has a long history, and silk sutures are commercially available. Braided silk suture has been used as a nondegradable or nonabsorbable suture. Here, one should be noted that nondegradable suture does not mean that the silk suture can be used for permanent uses. The tensile strength of the suture would continuously decrease even though there is no change in appearance. On the other hand, there were continuous efforts to make regenerated silk sutures to overcome some defects of multifilament sutures such as the possibility of infection. Regenerated silk sutures were prepared by conventional wet spinning using HFIP or formic acid as a solvent and methanol as a coagulant. In the case of suture, the knot strength is an important factor along with the tensile strength. Unfortunately, regenerated fibroin filament is too brittle, and sometimes even the knots cannot be formed. Therefore, mixing with another polymer is necessary (Lee et al., 2007).

10.4.2.2 Bone Although silk is protein, the rate of in vivo degradation is too slow to classify silk as a degradable material. This is because of the unique amino acid composition of fibroin which has only a limited number of amino acids capable of serving hydrolysis sites for proteolytic enzymes. In tissue engineering, one of the requirements of an ideal scaffold is to have similar degradation rate with the ingrown rate of the reconstructed tissue. In this aspect, fibroin is a suitable scaffold material for the tissues where the regeneration is slow. Moreover, the degradation rate of fibroin-based material is controllable by various kinds of method, which allows the adjustment of the degree of b-sheet structure development. In general, the high degree of the b-sheet structure results in prolonged degradation time and enhanced mechanical properties. Another potential

Silk materials for biotechnology

251

of fibroin in bone tissue engineering is that it can control the growth of hydroxyapatite formation. Therefore, the best results of the use of fibroin in the biomedical field come out from the bone regeneration. Films, hydrogels, nonwoven mats, and 3D porous materials of fibroin have been studied for the bone tissue engineering (Table 10.4). Recent studies in this area prefer the use of 3D porous structures, and various other organic and inorganic materials are blended with fibroin. As organic materials, polymers are added to tune the mechanical properties and the degradation time, and therapeutic agents or peptides are incorporated to induce bone regeneration. Inorganic materials, such as hydroxyapatite nanoparticles, not only increase the mechanical properties of scaffold but also enhance the bone regeneration efficiency.

10.4.2.3 Blood vessel The key aspect in the development of artificial blood vessel is maintaining patency in small diameter vessels. Various strategies were suggested to achieve the goal, but probably the best way is to cover the inner surface of the artificial blood vessel with the native endothelial cells. Fibroin has advantages here because of good cytocompatibility, which means the ingrowth of endothelial cells from the healthy blood cell can be promoted. However, this endothelialization takes time to cover the whole length of the new vessel. Therefore, an antithrombogenic agent such as heparin should be incorporated with fibroin to prevent clot formation before a complete endothelialization. In addition, some mechanical properties of fibroin need to be improved especially the elastic properties because blood vessels continuously and repeatedly expand and contract. A knitted mesh of silk fiber, film or electrospun nanofibers of fibroin was studied to develop an artificial vessel (Table 10.5). Recent studies revealed that multilayer scaffold has high potential in this field. Fibroin can be used either as the inner and/or outer layer of the vessel. As an inner layer, fibroin acts as a drug-releasing layer and/or promotes endothelial cell ingrowth. As an outer layer, fibroin can induce the proliferation of fibroblasts and smooth muscle cells.

10.4.2.4 Nerve conduit Although some article reports that fibroin is beneficial in nerve cell proliferation, the actual nerve guide conduits do not require cell activation as a primary factor. Various fabrication methods have been adopted to make a nerve conduit from fibroin (Table 10.6). The main role of nerve guide conduits is to guide the growth of the proximal and distal end of the nerve cell to regenerate properly. The orientation of nanofibers induces the direction of nerve cell growth. Therefore, electrospinning techniques for fiber orientation are adopted for the nerve conduit fabrication. The nerve guide conduits can release some growth factor, but the most important is that it should be degraded without inducing any changes to the microenvironment. In addition, the inflammatory response should also be minimized to prevent the scar formation. Fibroin degrades into small peptides or amino acid, and it has been reported that these products do not cause any problems in vivo. Fibroin is also a well-known material that has a little or moderate level of inflammatory responses (Thurber et al., 2015).

Auxiliary component

252

Table 10.4 Silk fibroin for bone tissue engineering. Fabrication

Form

Solvent

Functional ingredient

Insolubilization

Partial dissolution

Nonwoven silk fiber

Formic acid

Fuchs et al. (2009)

pH-induced gelation

Hydrogel

Water

Fini et al. (2005)

Gelation with porogen

Disc

Water

Riccio et al. (2012)

Electrospinning

Mat

Formic acid

MeOH

Kim et al. (2005)

Electrospinning with porogen

Sponge

Formic acid

MeOH

Ki et al. (2008)

Freeze-drying

Sponge

Water

Electrospinning

Mat

Formic acid

Molding with porogen

Porous brick

Water

Electrospinning

Mat

Water

Freeze-drying

Sponge

Water

Chitosan

Reference

Li et al. (2017) MeOH

Wei et al. (2011a)

Poly(aspartic acid)

Hydroxyapatite coating

MeOH

Zhao et al. (2009)

Poly(ethylene oxide)

Modified hydroxyapatite

MeOH and water vapor

Kim et al. (2014)

Bone morphogenetic protein-2eloaded silk microparticle, stromal cellederived factor-1, nanohydroxyapatite

MeOH

Shen et al. (2016)

Advances in Textile Biotechnology

Nanohydroxyapatite coating

Fabrication

Solvent

Auxiliary component

Double-raschel knitted silk graft with fibroin

Water

PGDE

Inner layer: fibroin film reinforced by silk mesh Outer layer: Freeze dried sponge

Water

Functional ingredient

Heparin

Silk fiber assembly fixed with fibroin

Insolubilization

Reference

Chemical coupling

Yagi et al. (2011)

EtOH

Liu et al. (2013)

EtOH

Enomoto et al. (2010)

Coating wire by dipping and crystallization

Water

Poly(ethylene oxide) (PEO)

MeOH

Lovett et al. (2007)

Electrospinning

Water

PEO

MeOH

Zhang et al. (2009)

Electrospinning

Formic acid

MeOH

Marelli et al. (2010)

Multi nozzle electrospinning

Formic acid

Gelatin

EtOH

Wang et al. (2009)

Random or oriented electrospinning

Hexafluoroisopropanol

PCL, PDO

MeOH

McClure et al. (2009)

Aqueous gel spinning

Water

MeOH

Lovett et al. (2010)

Freeze drying

Water

MeOH

Zhu et al. (2014)

Alcohol

Heparin

Silk materials for biotechnology

Table 10.5 Silk fibroin for artificial blood vessel.

253

Table 10.6 Silk fibroin for nerve guide conduits. Functional ingredient

254

Auxiliary component

Fabrication

Form

Solvent

Insolubilization

Reference

Fiber winding and freeze-drying

Inner: silk fiber Outer: silk sponge

Water

MeOH

Yang et al. (2007)

Fiber chopping

Silk fibers in chitosan conduits

Coating wire by dipping and crystallization

Tube

Water

Poly(ethylene oxide) (PEO)

MeOH

Ghaznavi et al. (2011)

Random or oriented electrospinning

Rolled into tube

Hexafluoroisopropanol

P(LLA-CL)

EtOH

Wang et al. (2011)

Film casting, random or oriented electrospinning

Electrospun fibers on membrane rolled into tube

Water

PEO for electrospinning

Nerve growth factor, neurotrophic factor

Water vapor

Madduri et al. (2010)

Electrospinning

Multitubes assembly

Water

PEO

Nerve growth factor, ciliary neurotrophic factor

Water vapor

Dinis et al. (2015)

Freeze-drying, electrospinning

Multilayer tube

Formic acid for electrospinning

Single-wall carbon nanotubes, fibronectin

MeOH

Mottaghitalab et al. (2013)

Freeze-drying

Rolled tube

1.5% acetic acid solution

MeOH

Wei et al. (2011b)

Gu et al. (2014)

Swan cellederived ECM

Advances in Textile Biotechnology

Chitosan

Silk materials for biotechnology

255

10.4.2.5 Ligament reconstruction When we consider the mechanical properties of silk fiber, ligament and tendon replacement with silk would be one of the first potential applications of silk in the biomedical field. Here, we use the native silk fiber instead of the regenerated fiber because of the importance of mechanical properties (Table 10.7). Knitted silk mesh or braided silk fiber tube was used for the replacement, but better results were obtained when secondary material or stem cells are incorporated. The secondary material such as collagen fills the gap between fibers and facilitates the ingrowth of the natural tissue. Preseeding of the stem cells also resulted in better tissue responses.

10.4.2.6 Wound healing One of the first in vivo studies of fibroin in the biomedical field was verifying the effect of fibroin in wound healing because of the relatively simple experiment procedure. The films, sponges, and nanofibrous mats of fibroin were applied as a wound dressing material (Table 10.8). The favorable properties of fibroin in the attachment, proliferation, and differentiation of cells lead to faster wound closure. Swellable properties of fibroin also might help the wound closure without significant scar formation because of maintaining humid conditions. Antimicrobial reagents are typical additives to prevent Table 10.7 Silk fibers in ligament reconstruction. Degumming

Preparation methods

Preseeding

Reference

Sodium carbonate

30 Fibers  6 bundles  3 strands  6 cords  1 ACL matrix

Altman et al. (2002)

Hot water

Silk fibroin knitted sheath with braided core

Fare et al. (2013)

Sodium bicarbonate

Fibroin-coated silk knit mesh wrapping silk cord

Mesenchymal stem cells

Fan et al. (2009)

Sodium carbonate

Fibroin-coated silk knit mesh

Bone marrowederived mesenchymal stem cells and anterior cruciate ligament fibroblast

Liu et al. (2008)

Sodium carbonate

Collagen-coated silk knit mesh

Bone marrow stroma cells

Chen et al. (2008)

Sodium carbonate

Collagen-coated silk knit mesh embedded in collagen hydrogel containing stroma cellederived factor-1 alpha

Shen et al. (2010)

Table 10.8 Silk fibroin for wound healing material. Solvent

Electrospinning

Nanofibrous mat

Formic acid

Electrospinning

Nanofibrous mat

Hexafluoroisopropanol

Electrospinning

Nanofibrous mat

Water

Poly(ethylene oxide) (PEO)

Electrospinning, salt-leaching, cold-plate electrospinning

Nanofibrous mat, nanofibrous sponge

Water

PEO

Casting, electrospinning

Film, porous film, nanofibrous mat

Water

PEO

Casting

Film

Acetic acid

Chitosan, alginate dialdehyde

Freeze-drying

Sponge

Water

Freeze-drying

Sponge

Water

Freeze-drying

Sponge

Water

Genipin

Functional ingredient

Insolubilization

Reference

Antimicrobial peptide

MeOH

Song et al. (2016)

Chitosan

Glutaraldehyde vapor

Cai et al. (2010)

Fibronectin

98% MeOH

Chutipakdeevong et al. (2013)

95% EtOH

Sheikh et al. (2015)

Water vapor

Gil et al. (2013)

Schiff base formation

Gu et al. (2013)

Gentamycinloaded gelatin microsphere

90% MeOH

Lan et al. (2014)

Elastin, gentamicin

90% methanol

Vasconcelos et al. (2012)

Chondroitin sulfate, hyaluronic acid

EDC/NHS

Yan et al. (2013)

Epidermal growth factor, silver sulfadiazine

Advances in Textile Biotechnology

Form

256

Auxiliary component

Fabrication

Silk materials for biotechnology

257

infection during the wound closure. So far, the biomedical application of sericin was not highlighted when compared with fibroin. It was because of the biocompatibility problem of sericin, and the complete removal of sericin was the crucial first step for the success of fibroin in biomedical application. However, during the last decade, the biocompatibility of sericin was reinvestigated, and it was found that sericin did not cause a severe problem when used alone. Nonetheless, care should be taken when using sericin in the biomedical field. Among the limited application of sericin in this field, wound dressing using sericin was the most widely studied area. Sericin can facilitate collagen I production during wound closure.

10.4.2.7 Drug delivery Drugs always have adverse effects, and the development of a drug delivery system (DDS) was one of the efforts to reduce those defects. The aim of DDS is to deliver the drug to the target site at a minimum therapeutic concentration during or at the required time. Silk proteins were studied for drug carriers from the beginning of the nontextile application. At physiological pH, both fibroin and sericin are negatively charged because their pI is below 5. Therefore, the charge of the drug will have a significant effect on its releasing profile. Drugs loaded in the silk protein can be released either by dissolution or diffusion mechanism. In the case of fibroin matrix, the release of the drug is dominated by diffusion mechanism because of the low degradation rate of fibroin. On the other hand, the release of drug from sericin matrix is promoted by the dissolution of sericin. During the oral administration of the drug, there is a dramatic change of pH from acidic to neutral as the drug passes through the stomach and intestine, respectively. Sericin was found to be more stable in stomach condition than in intestine Oh et al. (2011). This is advantageous for the drugs which have a severe adverse effect to the stomach as the release of drug from the sericin matrix is inhibited. Drug loading protocol is important to increase the loading efficiency of the drug. For example, fibroin requires alcohol treatment to make the carrier insoluble. If the drugs are loaded before this step, the drugs are lost during the alcohol treatment. In such a case, a postloading process is recommended.

10.5

Conclusion

Since the beginning of the use of silk other than the textile application, biotechnology field was the most studied and promised field for silk proteins. Notably, in tissue engineering, researchers tried to find an alternative source for scaffold materials that could replace the collagen. Silk proteins have many advantages over other protein materials in purity, availability, and processibility. However, for the commercial application of silk in biotechnology, there are many things to be overcome in the future. The first step would be the stable and reliable supply of the raw material. Unfortunately, there are only a limited number of silk protein suppliers. It means every research groups extract silk protein by themselves, which sometimes results in an inconsistency in the results. The fabrication method should also be well-established

258

Advances in Textile Biotechnology

because various properties, especially the biodegradation behavior, of the final product are affected by the processing history of silk. Silk may be thought of as old material because of its long history. However, silk is indeed a masterpiece of nature. We still do not know the exact mechanism of silk spinning. Novel applications of silk in various engineering fields are reported every year. There are impressive results in electronics and photonics also. Silk is a fascinating subject of the biotechnology, too. Bioengineering of the genetic information of silk protein can lead to producing various kinds of tailor-made proteins for specific applications. It is not easy to predict how the silk will be used in the future in our daily lives, but it is sure that silk is one of the highlighted materials in the biotechnology field.

References Altman, G.H., Horan, R.L., Lu, H.H., Moreau, J., Martin, I., Richmond, J.C., Kaplan, D.L., 2002. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials 23 (20), 4131e4141. Cai, Z., Mo, X., Zhang, K., Fan, L., Yin, A., He, C., Wang, H., 2010. Fabrication of chitosan/silk fibroin composite nanofibers for wound-dressing applications. International Journal of Molecular Sciences 11 (9), 3529e3539. Chen, X., Qi, Y.-Y., Wang, L.-L., Yin, Z., Yin, G.-L., Zou, X.-H., Ouyang, H.-W., 2008. Ligament regeneration using a knitted silk scaffold combined with collagen matrix. Biomaterials 29 (27), 3683e3692. Chutipakdeevong, J., Ruktanonchai, U.R., Supaphol, P., 2013. Process optimization of electrospun silk fibroin fiber mat for accelerated wound healing. Journal of Applied Polymer Science 130 (5), 3634e3644. Dennis, P.B., Walker, A.Y., Dickerson, M.B., Kaplan, D.L., Naik, R.R., 2012. Stabilization of organophosphorus hydrolase by entrapment in silk fibroin: formation of a robust enzymatic material suitable for surface coatings. Biomacromolecules 13, 2037e2045. Dinis, T.M., Elia, R., Vidal, G., Dermigny, Q., Denoeud, C., Kaplan, D.L., Egles, C., Marin, F., 2015. 3D multi-channel bi-functionalized silk electrospun conduits for peripheral nerve regeneration. Journal of the Mechanical Behavior of Biomedical Materials 41, 43e55. Enomoto, S., Sumi, M., Kajimoto, K., Nakazawa, Y., Takahashi, R., Takabayashi, C., Asakura, T., Sata, M., 2010. Long-term patency of small-diameter vascular graft made from fibroin, a silk-based biodegradable material. Journal of Vascular Surgery 51 (1), 155e164. Fan, H., Liu, H., Toh, S.L., Goh, J.C.H., 2009. Anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold in large animal model. Biomaterials 30 (28), 4967e4977. Fare, S., Torricelli, P., Giavaresi, G., Bertoldi, S., Alessandrino, A., Villa, T., Fini, M., Tanzi, M.C., Freddi, G., 2013. In vitro study on silk fibroin textile structure for Anterior Cruciate Ligament regeneration. Materials Science and Engineering: C 33 (7), 3601e3608. Ferreira, I.M., Yoshioka, S.A., Comasseto, J.V., Porto, A.L.M., 2017. Immobilization of Amano lipase from Pseudomonas fluorescens on silk fibroin spheres: an alternative protocol for the enantioselective synthesis of halohydrins. RSC Advances 7, 12650e12658. Fini, M., Motta, A., Torricelli, P., Giavaresi, G., Nicoli Aldini, N., Tschon, M., Giardino, R., Migliaresi, C., 2005. The healing of confined critical size cancellous defects in the presence of silk fibroin hydrogel. Biomaterials 26 (17), 3527e3536.

Silk materials for biotechnology

259

Fuchs, S., Jiang, X., Schmidt, H., Dohle, E., Ghanaati, S., Orth, C., Hofmann, A., Motta, A., Migliaresi, C., Kirkpatrick, C.J., 2009. Dynamic processes involved in the prevascularization of silk fibroin constructs for bone regeneration using outgrowth endothelial cells. Biomaterials 30 (7), 1329e1338. Ghaznavi, A.M., Kokai, L.E., Lovett, M.L., Kaplan, D.L., Marra, K.G., 2011. Silk fibroin conduits: a cellular and functional assessment of peripheral nerve repair. Annals of Plastic Surgery 66 (3), 273e279. Gil, E.S., Panilaitis, B., Bellas, E., Kaplan, D.L., 2013. Functionalized silk biomaterials for wound healing. Advanced Healthcare Materials 2 (1), 206e217. Gu, Z., Xie, H., Huang, C., Li, L., Yu, X., 2013. Preparation of chitosan/silk fibroin blending membrane fixed with alginate dialdehyde for wound dressing. International Journal of Biological Macromolecules 58, 121e126. Gu, Y., Zhu, J., Xue, C., Li, Z., Ding, F., Yang, Y., Gu, X., 2014. Chitosan/silk fibroin-based, Schwann cell-derived extracellular matrix-modified scaffolds for bridging rat sciatic nerve gaps. Biomaterials 35 (7), 2253e2263. Ki, C.S., Kim, J.W., Hyun, J.H., Lee, K.H., Hattori, M., Rah, D.K., Park, Y.H., 2007. Electrospun three-dimensional silk fibroin nanofibrous scaffold. Journal of Applied Polymer Science 106 (6), 3922e3928. Ki, C.S., Park, S.Y., Kim, H.J., Jung, H.-M., Woo, K.M., Lee, J.W., Park, Y.H., 2008. Development of 3-D nanofibrous fibroin scaffold with high porosity by electrospinning: implications for bone regeneration. Biotechnology Letters 30 (3), 405e410. Kim, K.H., Jeong, L., Park, H.N., Shin, S.Y., Park, W.H., Lee, S.C., Kim, T.I., Park, Y.J., Seol, Y.J., Lee, Y.M., Ku, Y., Rhyu, I.C., Han, S.B., Chung, C.P., 2005. Biological efficacy of silk fibroin nanofiber membranes for guided bone regeneration. Journal of Biotechnology 120 (3), 327e339. Kim, H., Che, L., Ha, Y., Ryu, W., 2014. Mechanically-reinforced electrospun composite silk fibroin nanofibers containing hydroxyapatite nanoparticles. Materials Science and Engineering: C 40, 324e335. Kim, M.K., Lee, J.Y., Oh, H.J., Song, D.W., Kwak, H.W., Yun, H., Um, I.C., Park, Y.H., Lee, K.H., 2015. Effect of shear viscosity on the preparation of sphere-like silk fibroin microparticles by electrospraying. International Journal of Biological Macromolecules 79, 988e995. Koeppel, A., Holland, C., 2017. Progress and trends in artificial silk spinning: a systematic review. ACS Biomaterials Science and Engineering 3 (3), 226e237. Lan, Y., Li, W., Jiao, Y., Guo, R., Zhang, Y., Xue, W., Zhang, Y., 2014. Therapeutic efficacy of antibiotic-loaded gelatin microsphere/silk fibroin scaffolds in infected full-thickness burns. Acta Biomaterialia 10 (7), 3167e3176. Lee, K.H., Ki, C.S., Baek, D.H., Kang, G.D., Ihm, D.-W., Park, Y.H., 2005. Application of electrospun silk fibroin nanofibers as an immobilization support of enzyme. Fibers and Polymers 6 (3), 181e185. Lee, K.H., Kang, G.D., Shin, B.S., Park, Y.H., 2005. Sericin-fixed silk fiber as an immobilization support of enzyme. Fibers and Polymers 6 (1), 1e5. Lee, K.H., Baek, D.H., Ki, C.S., Park, Y.H., 2007. Preparation and characterization of wet spun silk fibroin/poly(vinyl alcohol) blend filaments. International Journal of Biological Macromolecules 41 (2), 168e172. Li, D.W., Lei, X., He, F.L., He, J., Liu, Y.L., Ye, Y.J., Deng, X., Duan, E., Yin, D.C., 2017. Silk fibroin/chitosan scaffold with tunable properties and low inflammatory response assists the differentiation of bone marrow mesenchymal stem cells. International Journal of Biological Macromolecules 103, 584e597.

260

Advances in Textile Biotechnology

Liu, H., Fan, H., Toh, S.L., Goh, J.C.H., 2008. A comparison of rabbit mesenchymal stem cells and anterior cruciate ligament fibroblasts responses on combined silk scaffolds. Biomaterials 29 (10), 1443e1453. Liu, S., Dong, C., Lu, G., Lu, Q., Li, Z., Kaplan, D.L., Zhu, H., 2013. Bilayered vascular grafts based on silk proteins. Acta Biomaterialia 9 (11), 8991e9003. Lovett, M., Cannizzaro, C., Daheron, L., Messmer, B., Vunjak-Novakovic, G., Kaplan, D.L., 2007. Silk fibroin microtubes for blood vessel engineering. Biomaterials 28 (35), 5271e5279. Lovett, M., Eng, G., Kluge, J.A., Cannizzaro, C., Vunjak-Novakovic, G., Kaplan, D.L., 2010. Tubular silk scaffolds for small diameter vascular grafts. Organogenesis 6 (4), 217e224. Lu, S., Wang, X., Lu, Q., Hu, X., Uppal, N., Omenetto, F.G., Kaplan, D.L., 2009. Stabilization of enzymes in silk films. Biomacromolecules 10, 1032e1042. Madduri, S., Papaloïzos, M., Gander, B., 2010. Trophically and topographically functionalized silk fibroin nerve conduits for guided peripheral nerve regeneration. Biomaterials 31 (8), 2323e2334. Marelli, B., Alessandrino, A., Fare, S., Freddi, G., Mantovani, D., Tanzi, M.C., 2010. Compliant electrospun silk fibroin tubes for small vessel bypass grafting. Acta Biomaterialia 6 (10), 4019e4026. McClure, M.J., Sell, S.A., Ayres, C.E., Simpson, D.G., L Bowlin, G., 2009. Electrospinningaligned and random polydioxanoneepolycaprolactoneesilk fibroin-blended scaffolds: geometry for a vascular matrix. Biomedical Materials 4 (5), 055010. Moon, B.M., Choi, M.-J., Sultan, M.T., Yang, J.W., Ju, H.W., Lee, J.M., Park, H.J., Park, Y.R., Kim, S.H., Kim, D.W., Lee, M.C., Jeong, J.Y., Lee, O.J., Sung, G.Y., Park, C.H., 2017. Novel fabrication method of the peritoneal dialysis filter using silk fibroin with urease fixation system. Journal of Biomedical Materials Research Part B 105, 2136e2144. Mottaghitalab, F., Farokhi, M., Zaminy, A., Kokabi, M., Soleimani, M., Mirahmadi, F., Shokrgozar, M.A., Sadeghizadeh, M., 2013. A biosynthetic nerve guide conduit based on silk/SWNT/fibronectin nanocomposite for peripheral nerve regeneration. PLoS One 8 (9), e74417. Oh, H., Kim, M.K., Lee, K.H., 2011. Preparation of sericin microparticles by electrohydrodynamic spraying and their application in drug delivery. Macromolecular Research 19 (3), 266e272. Omenetto, F.G., Kaplan, D.L., 2010. New opportunities for an ancient material. Science 329 (5991), 528e531. Riccio, M., Maraldi, T., Pisciotta, A., La Sala, G.B., Ferrari, A., Bruzzesi, G., Motta, A., Migliaresi, C., De Pol, A., 2012. Fibroin scaffold repairs critical-size bone defects in vivo supported by human amniotic fluid and dental pulp stem cells. Tissue Engineering Part A 18 (9e10), 1006e1013. Sheikh, F.A., Ju, H.W., Lee, J.M., Moon, B.M., Park, H.J., Lee, O.J., Kim, J.-H., Kim, D.-K., Park, C.H., 2015. 3D electrospun silk fibroin nanofibers for fabrication of artificial skin. Nanomedicine: Nanotechnology, Biology and Medicine 11 (3), 681e691. Shen, W., Chen, X., Chen, J., Yin, Z., Heng, B.C., Chen, W., Ouyang, H.-W., 2010. The effect of incorporation of exogenous stromal cell-derived factor-1 alpha within a knitted silkcollagen sponge scaffold on tendon regeneration. Biomaterials 31 (28), 7239e7249. Shen, X., Zhang, Y., Gu, Y., Xu, Y., Liu, Y., Li, B., Chen, L., 2016. Sequential and sustained release of SDF-1 and BMP-2 from silk fibroin-nanohydroxyapatite scaffold for the enhancement of bone regeneration. Biomaterials 106, 205e216.

Silk materials for biotechnology

261

Song, D.W., Kim, S.H., Kim, H.H., Lee, K.H., Ki, C.S., Park, Y.H., 2016. Multi-biofunction of antimicrobial peptide-immobilized silk fibroin nanofiber membrane: implications for wound healing. Acta Biomaterialia 39, 146e155. Thurber, A.E., Omenetto, F.G., Kaplan, D.L., 2015. In vivo bioresponses to silk proteins. Biomaterials 71, 145e157. Vasconcelos, A., Gomes, A.C., Cavaco-Paulo, A., 2012. Novel silk fibroin/elastin wound dressings 8 (8), 3049e3060. Wang, S., Zhang, Y., Wang, H., Yin, G., Dong, Z., 2009. Fabrication and properties of the electrospun polylactide/silk fibroin-gelatin composite tubular scaffold. Biomacromolecules 10 (8), 2240e2244. Wang, C.Y., Zhang, K.H., Fan, C.Y., Mo, X.M., Ruan, H.J., Li, F.F., 2011. Aligned naturalsynthetic polyblend nanofibers for peripheral nerve regeneration. Acta Biomaterialia 7 (2), 634e643. Wang, P., Qi, C., Yu, Y., Yuan, J., Cui, L., Tang, G., Wang, Q., Fan, X., 2015. Covalent immobilization of catalase onto regenerated silk fibroins via tyrosinase-catalyzed crosslinking. Applied Biochemistry and Biotechnology 177, 472e485. Wei, K., Li, Y., Kim, K.-O., Nakagawa, Y., Kim, B.-S., Abe, K., Chen, G.-Q., Kim, I.-S., 2011. Fabrication of nano-hydroxyapatite on electrospun silk fibroin nanofiber and their effects in osteoblastic behavior. Journal of Biomedical Materials Research Part A 97 (3), 272e280. Wei, Y., Gong, K., Zheng, Z., Wang, A., Ao, Q., Gong, Y., Zhang, X., 2011. Chitosan/silk fibroin-based tissue-engineered graft seeded with adipose-derived stem cells enhances nerve regeneration in a rat model. Journal of Materials Science: Materials in Medicine 22 (8), 1947e1964. Xue, R., Kang, T.-F., Lu, L.-P., Cheng, S.-Y., 2012. Immobilization of acetylcholinesterase via biocompatible interface of silk fibroin for detection of organophosphate and carbamate pesticides. Applied Surface Science 258, 6040e6045. Yagi, T., Sato, M., Nakazawa, Y., Tanaka, K., Sata, M., Itoh, K., Takagi, Y., Asakura, T., 2011. Preparation of double-raschel knitted silk vascular grafts and evaluation of short-term function in a rat abdominal aorta. Journal of Artificial Organs 14 (2), 89e99. Yan, S., Zhang, Q., Wang, J., Liu, Y., Lu, S., Li, M., Kaplan, D.L., 2013. Silk fibroin/ chondroitin sulfate/hyaluronic acid ternary scaffolds for dermal tissue reconstruction. Acta Biomaterialia 9 (6), 6771e6782. Yang, Y., Ding, F., Wu, J., Hu, W., Liu, W., Liu, J., Gu, X., 2007. Development and evaluation of silk fibroin-based nerve grafts used for peripheral nerve regeneration. Biomaterials 28 (36), 5526e5535. Zhang, Y.-Q., Tao, M.-L., Shen, W.-D., Zhou, Y.-Z., Ding, Y., Ma, Y., Zhou, W.-L., 2004. Immobilization of l-asparaginase on the microparticles of the natural silk sericin protein and its characters. Biomaterials 25, 3751e3759. Zhang, X., Wang, X., Keshav, V., Wang, X., Johanas, J.T., Leisk, G.G., Kaplan, D.L., 2009. Dynamic culture conditions to generate silk-based tissue-engineered vascular grafts. Biomaterials 30 (19), 3213e3223. Zhang, Y.-Q., Wang, Y.-J., Wang, H.-Y., Zhu, L., Zhou, Z.-Z., 2011. Highly efficient processing of silk fibroin nanoparticle-L-asparaginase bioconjugates and their characterization as a drug delivery system. Soft Matter 7, 9728e9736. Zhao, J., Zhang, Z., Wang, S., Sun, X., Zhang, X., Chen, J., Kaplan, D.L., Jiang, X., 2009. Apatite-coated silk fibroin scaffolds to healing mandibular border defects in canines. Bone 45 (3), 517e527.

262

Advances in Textile Biotechnology

Zhu, L., Hu, R.-P., Wang, H.-Y., Wang, Y.-J., Zhang, Y.-Q., 2011. Bioconjugation of neutral protease on silk fibroin nanoparticles and application in the controllable hydrolysis of sericin. Journal of Agricultural and Food Chemistry 59, 10298e10302. Zhu, M., Wang, K., Mei, J., Li, C., Zhang, J., Zheng, W., An, D., Xiao, N., Zhao, Q., Kong, D., Wang, L., 2014. Fabrication of highly interconnected porous silk fibroin scaffolds for potential use as vascular grafts. Acta Biomaterialia 10 (5), 2014e2023.