Processing Silk Hydrogel and Its Applications in Biomedical Materials Hai-Yan Wang and Yu-Qing Zhang Silk Biotechnology Laboratory, School of Basic Medical and Biological Sciences, Soochow University, Suzhou 215123, P R China DOI 10.1002/btpr.2058 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com)

This review mainly introduces the types of silk hydrogels, their processing methods, and applications. There are various methods for hydrogel preparation, and many new processes are being developed for various applications. Silk hydrogels can be used in cartilage tissue engineering, drug release materials, 3D scaffolds for cells, and artificial skin, among other applications because of their porous structure and high porosity and the large surface area for growth, migration, adhesion and proliferation of cells that the hydrogels provide. All of these advantages have made silk hydrogels increasingly attractive. In addition, silk hydroC 2015 Amerigels have wide prospects for application in the field of biomedical materials. V can Institute of Chemical Engineers Biotechnol. Prog., 000:000–000, 2015 Keywords: silk fibroin, silk hydrogel, tissue engineering, drug release materials, artificial skin

Introduction Hydrogels, also called aqua gels, are networks of polymer chains. The network structure is a reticular crosslinking structure that consists of hydrophobic groups and hydrophilic residues. Hydrogels are water-insoluble, with water being a dispersion medium. Generally speaking, hydrogels, whether natural or synthetic polymers, are highly absorbent. The moisture content of hydrogels can be as high as 70%. Various naturally derived materials and synthetic materials have been used to form hydrogels. The polymers can be classified into natural and synthetic materials according to their sources. Composite hydrophilic polymers include polyethylene, alcohol, acrylic acid and related materials. Natural hydrophilic polymers include polysaccharides (starch, cellulose, alginic acid, chitosan, etc.) and polypeptides (collagen, poly L-lysine, poly L-glutamic acid, silk protein, etc.). Silk is a famous natural fiber produced by the silkworm (Bombyx mori) cocoons (Figure 1a). It is composed of two types of protein (fibroin and sericin). Fibroin forms the thread core, and the glue-like sericin surrounds the fibroin fibers and joins them together (Figure 1b). Fibroin whose diameter is about 5210 lm (Figure 1c) is composed of both light and heavy chains, with an approximately two-third crystalline portion and an amorphous region that represents approximately one-third of fibroin.1 Silk fibroin has two crystalline forms, silk I and silk II, which are the dimorphs of silk fibroin. Silk I is the conformation of solid-state fibroin before spinning.2 Silk II, the structure of the fiber after spinning, is mainly an anti-parallel b-sheet.3 Sericin, a glue-like protein, holds two fibroin fibers together to form the environmentally stable fibroin-sericin composite cocoon structure. The second structure of sericin is mainly an amorphous region, with one part being a b-sheet, and a-helices

Correspondence concerning this article should be addressed to Y. Zhang at e-mail: [email protected] C 2015 American Institute of Chemical Engineers V

barely exist in the structure.4 In recent years, silk fibroin from Bombyx mori silkworms has been the dominant source for silk-based biomaterials. Different forms of silk fibroin have been gradually studied for new biomaterials, especially biomedical applications, because of the slow degradability, biocompatibility and remarkable mechanical properties of the material. Silk hydrogel is a kind of hydrogel which can be processed by sericin or silk fibroin (Figure 2). Sericin can be separated from silk fibroin in vitro. Sericin has to be removed from fibroin for the fiber to acquire softness, whiteness, luster, smoothness, and dyeability. Typically, sericin is the abandoned portion. However, current research indicates that sericin can be formed into a gel.5 Silk fibroin can be dissolved into regenerated fibroin solution, which is transparent, homogeneous, and metastable. The regenerated silk fibroin solution can turn into a gel in the presence of acids, ions and other additives.6–10 In addition, there are many other factors that affect this gelling process, such as temperature and pH. The gelation time decreases significantly as the silk fibroin concentration or temperature increases or as the pH decreases. The pore size of gels can be regulated by the silk fibroin concentration, environmental temperature and pH and Ca21 concentrations.10 In the formation process of gels, the amorphous region of the silk fibroin takes on a b-sheet structure.11,12 During the dehydration reaction of silk fibroin molecules, a stable hydrogel is formed because of the effect of intramolecular and intermolecular hydrogen bonds.13–15 Moreover, regenerated silk fibroin can mix with other biopolymers, such as chitosan and gelatin, to improve the hydrogel properties.16,17 The blended scaffolds have good interconnectivity and high porosity Recently, related articles have mainly focused on silk fibroin hydrogel. Silk fibroin hydrogel is a silk-based biomaterial that has a controllable degradation lifetime. The molecular weights, mechanical properties, secondary structures, and morphological characteristics of silk fibroin hydrogel are 1

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Figure 1. Graphs of silk. (a) Picture of silkworm cocoons; (b) The schematic diagram of silk fiber; c) SEM graph (35K) of degummed silk fibroin.

sericin is dialyzed continuously for 48 h against running water to remove salts and some impurities using a cellulose semi-permeable membrane. Dissolution of fibroin

Figure 2. Schematic illustrating silk-elastin-like protein polymers physically crosslinked with different kinds of drugs. Fluorescent dyes include rhodamine, fluorescein, PAMAM (poly-amidoamine) dendrimer.

different than those of silk fibroin.18 Using further processing, such as freeze drying, this novel material can be applied to cell culture and tissue engineering.19–21 Moreover, silk hydrogel has also been used as a slow-release carrier of bioactivators, such as plasmid DNA, growth factors and viruses.22,23 In recent years, tissue engineering has become a hot area of research. Different forms of silk fibroin, such as films, fibers, hydrogels and 3D scaffolds, are used as biomaterials.24–26 Among these forms, silk hydrogels are gaining a high level of attention and popularity in the tissue engineering field. Hydrogels provide a large surface area for growth, migration, adhesion and proliferation of cells27–31 because of their porous structure and high porosity. It has been reported that blended scaffolds support the growth and adhesion of feline fibroblasts. Chondrocytes can be cultured on silk fibroin hydrogels, and the hydrogels can repair rabbit cranial defects.32,33 In this paper, the main preparation methods of silk hydrogels, along with their classification and applications, are summarized.

Preparation of Regenerated Liquid Silk Protein Degumming methods Sericin consists of 18 types of amino acids, most of which have strong polar side groups, such as carboxyl, hydroxyl, and amino groups. Approximately one-third of the total amino acids are characterized as serine, which gives sericin high hydrophilicity. During the degumming process, sericin is solubilized or hydrolyzed with the help of various agents and media, causing an approximate 20225% weight loss in silk. The different degumming methods are described in Table 1. After these degumming treatments, the resulting

Fibroin is the structural protein of silk fiber. It is rich in glycine (43.7%), alanine (28.8%) and serine (11.9%) and is composed of a heavy chain (325 kDa) and a light chain (25 kDa). The heavy and light chains are linked by a disulfide bond. The light chain has a non-repetitive sequence and only plays a marginal role in the fiber. The heavy chain is highly periodic. The highly repetitive sections are composed of glycine (45%), alanine (30%), and serine (12%) in an approximately 3:2:1 ratio and dominated by [Gly-Ala-Gly-Ala-GlySer]n sequences.36 Silk fibroin is insoluble in water. It can be dissolved in some salt solutions. Here, some of the most commonly used dissolving methods are presented (Table 2). After processing with the salt solutions described above, the silk fibroin is filtered through deionized water or centrifuged at 10,000 rpm for 15 min, and the filtered solution or supernatant are dialyzed continuously for 48 h against running water to remove salts, smaller molecules and some impurities using a cellulose semi-permeable membrane (cutoff 5 kDa).

Silk Hydrogel Silk hydrogel mainly includes pure hydrogel, blended hydrogel with natural polymers or artificial macropolymers. A pure silk hydrogel is made of a single material (sericin or silk fibroin). Pure sericin hydrogel has already been studied, although there have only been a few relevant reports. It can be formed by adding ethanol into sericin solution.45 A single material usually has some defects. Many researchers improve the hygroscopic properties and mechanical properties of silk hydrogel by efficient addition of different biomaterials. Blended hydrogel biomaterials contain at least two different types of materials combined with chemical or physics methods.46 The connected reticular structure can consolidate and enhance the mechanical properties of hydrogel.47 Blended hydrogel is formed by silk fibroin or sericin mixed with other materials. These materials include synthetic and natural hydrophilic polymers. Synthetic hydrophilic polymers Synthetic polymers are widely chosen for hydrogel fabrication as their properties can be precisely controlled while

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Table 1. The Most Commonly Used Degumming Methods for Silk Fiber Types Degumming Methods Bath ratio (v/w)

Degumming agent/media

alkaline sol.

Na2CO3 solution

50

about 0.2% (w/v) Na2CO3

acidic sol.

Alkaline electrolyzed H2O Organic acids

30 20

salt sol.

Urea buffer (pH 7.0)

30

Surfactant

Neutral soap solution

100

pH 11.50 15% and 30% (w/v) citric acid sol. 8 M urea (0.04M Tris-SO4 and 0.5 ME) 0.5% (w/v) soap

Nonionic detergent

20

silk protein surfactant

80

enzyme

Protease degradation

50

physics

Heatingi High pressure water Microwave assistant

30 30 50–100

IR heating

202150

a)

Noigen HC nonionic detergent (0.2%) 0.2% silk protein surfactant 1% alkalase 2.5 l (Kosindo Chemical) water water different solution (Savinase,HCl, soap, etc.) water

Processing conditions

Ref. 34

Twice for 0.5 h at 100 C 100 C for 30 min 98 C for 30 min

35 36

80 C for 2 h

37

35 times for 0.5 h at 100 C 98 C for 30 min

38

3 times for 30 min at 100 C 30 min at 60 C 100 C for 5 to 0 min 120 C for 5 to 30 min power ranging from 30–100% 100120 C for 60 to 120 min

38 39 36 36 36,40 41 42

ME: mercaptoethanol; a) purchased from Dai-ichi Kogyo Seiyaku Co., Ltd. Japan.

Table 2. The Most Commonly Used Dissolving Methods for Silk Fibroin Fiber Solvent system Bath ratio (v/w) Ratio of dissolving agent Ajisawa’s ternary solvent system

10  20

LiSCN/NaSCN solution

30  100

LiBr/LiBr alcohol-H2O sol.

CaCl2/ethanol/water (1:2:8 M) Saturated aqueous LiSCN/ NaSCN sol. (ca. 9 M)

20

Nitrate solution

20

Ionic liquids

25

9.3 M LiBr solution or LiBr alcohol-water (LiBr/ethanol/ water, 45:44:11 weight ratio) (Ca(NO3)2
4H2O/CH3OH, 75:25 weight ratio) [bmim][Ac]

Temperature

Ref.



80 C

36

Room temperature

43

25 C

45, 39

88 C

45

95 C

44

[bmim][Ac]: 1-butyl-3-methylimidazolium acetate

Figure 3. Schematic representation of SF-HPC hydrogel formation and the structural features of the two biopolymers. HPC: Hydroxypropyl cellulose; SF: Silk fibroin; T: Temperature; LCST: Lower critical solution temperature

being readably reproducible.48 The synthetic hydrophilic polymers include polyethylene, polyacrylic acid, its derivatives and carbon nanotubes, etc. Functionalized silk fibroin and multiwall carbon nanotubes hydrogels could be fabricated by the polymerization of acrylamide and N,N’-methylenebisacrylamide in silk fibroin solution containing carboxylated multiwall carbon nanotubes with potassium persulphate/triethanol amine redox system as initiator. Carbon nanotubes significantly improved the

mechanical properties of the blended hydrogel and suggested the fundament for the use of these silk fibroin biomaterials in bone tissue engineering applications.49 Silk-elastin-like protein-based polymers that are genetically engineered and derived from silk and elastin are often used. Aqueous solutions of silk-elastin-like protein copolymers are liquid at room temperature, allowing bioactive agents to be incorporated prior to administration. The ability of silk-elastin-like protein polymers to form physically crosslinked and stable networks in physiological conditions makes them promising candidates for biomedical applications such as drug delivery. Dinerman et al.50 investigated the influence of solute hydrophobicity and charge on partitioning and diffusion in physically crosslinked networks of a genetically engineered silkelastin-like protein (SELP) polymer. A series of fluorescent dyes were used to assess the impact of solute charge and hydrophobicity on release behavior (Figure 2). The results suggested that controlling solute release from SELP hydrogels by modifying the surface charge and hydrophobicity of drugs or drug/polymer conjugates was very important. Gong et al. developed a novel thixotropic injectable hydrogel by blending regenerated silk fibroin and hydroxypropylcellulose (Figure 3). The results demonstrated that the blend hydrogel could protect encapsulated cells against high shear force

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during injection, suggesting that the blend hydrogel is a promising vehicle for cell delivery.51 Hu synthesized a novel superabsorbent hydrogel using crosslinking graft copolymerization of acrylamide and acrylic acid onto the chain of silk sericin. N,N’-methylenebisacrylamide, as crosslinker, and potassium persulfate-sodium sulfite, as a redox initiation system, were used. The swelling capability of the blended hydrogel was measured in solutions with a pH range of 1 to 13, and the hydrogel displayed a pH-dependent character. The water absorbency of the hydrogel in aqueous chloride salt solutions had the Al31>Mg21>Ca21> Na1 order at the investigated concentration.52 Sericin also could form a composite hydrogel by blended with polyacrylamide for dermal reconstruction and with N-isopropylacrylamide for cell culture.53,54

Natural hydrophilic polymers Hydrogels processed from naturally derived sources generally lack versatility in terms of tunable chemistry and reproducibility compared with synthetic polymers. However, despite these limitations, natural polymers usually present improved biocompatibility and cellular interactivity, which enhance their utility in the fabrication of biomaterials.55 Hydrogels synthesized from natural macromers generally induce fewer immunogenic reactions, as they are produced from basic molecules already used by the body. Natural hydrophilic polymers include polypeptides, such as collagen, poly L-lysine, poly L-glutamic acid and polysaccharides, including starch, cellulose, alginic acid, and chitosan. Many researchers have studied hydrogel blends of regenerated silk fibroin and natural hydrophilic polymers, for example, collagen, chitosan, and gelatin.17,46 Hydrogel mixing with natural collagen can obviously improve its thermal stability, leading to stronger mechanical strength than that of pure hydrogels. Although they are crosslinked, the hydrogels still maintain the mobility of fibroin molecules. Gelatin derived from porcine skin is a natural gel. It can be blended with a silk fibroin solution and silk fibroin microfibers. The blended hydrogels display a lower swelling ratio and degradation rate and higher compressive modulus than those of gelatin methacrylate.56 Silk fibroin and collagen fibrils have a suitable interfacial adhesion, and the blended scaffolds exhibit improved mechanical properties.57 Combinations of proteins and polysaccharides may mimic the naturally occurring environment of certain tissues. Chitosan is a natural widespread chitin obtained by acetyl stripping chitin. Some authors have observed that composites based on chitosan are mechanically flexible and easily moldable into desirable shapes. The association of chitosan and silk fibroin as a binary organic matrix for hydroxyapatite to prepare scaffolds is attractive for bone tissue engineering.58 Silva et al. first proposed the role of ionic liquids as solvents for the production of hydrogels from blends of silk fibroin and chitosan. The ionic liquids offer the advantage of being homogeneous and presenting short and easy dissolution times for both silk fibroin and chitosan. In vitro assays demonstrated that the composite hydrogels could support the adhesion and growth of primary human dermal fibroblasts.42 In addition, silk fibroin is a natural polymer. It can be added into silk hydrogels. The incorporation of fibroin fiber can improve its mechanical properties. The equilibrium swelling ratio of composite hydrogels is dependent on both the

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temperature and the content of fibroin fiber. Moreover, there are gaps between the fibroin fibers and hydrogel on the hydrogel surface, and they act as the transmission channels that are favorable for the outflow and inflow of water from the interior hydrogel in the course of oscillating swellingdeswelling.

Processing Method A large number of new techniques are being studied to fabricate silk fibroin hydrogels. Gels form by intermolecular and intramolecular cross-linking between protein chains, including hydrogen bonds and hydrophobic and electrostatic interactions.59 Gelation is initially induced by weak interactions that do not involve important secondary structural changes. These weak changes are followed by a virtually irreversible, strong b-sheet formation. The overall processing parameters include temperature, concentration, and pH. These findings suggest that sequence-specific control of fibroins relates both to the hydrophobic regions and the chain end hydrophilic sequence blocks.12 Silk gels can be formed by treatments such as heating, shearing, water evaporation, or solvent exposure. These treatments can be divided into three categories: the manipulation of the physiochemical environment, physical methods, and chemical methods. The manipulation of the physiochemical environment 1. The increase of temperature or protein concentration The increase of temperature or protein concentration is conducive to the cross-linking of silk fibroin chains. The cross-linking effect is enhanced along with an increase of temperature or protein concentration. Thus, silk fibroin macromolecules are more likely to cross-link with each other. 2. Low pH Reports have demonstrated a significant reduction in gelation time, from days to a matter of hours, at a reduced solution pH near to the isoelectric point of silk fibroin (pH 5 3.824.0).6 3. Carbon dioxide method Floren et al. first presented a novel method to fabricate silk fibroin hydrogels by carbon dioxide (CO2). They used high pressure CO2 as a volatile acid. The easy and efficient recovery of CO2 during post-processing led to a remarkably clean production method offering huge benefits for materials processing for biomedical applications. Furthermore, with this novel technique, they revealed that silk protein gelation could occur under high-pressure CO2 lasting for less than 2 hours. The attractive features of the method include reducing processing complexity, no required chemical crosslinkers or residual mineral acids, acceleration of stable silk hydrogel formation, and avoidance of adverse biological responses. Moreover, the method provides a direct manipulation of hydrogel physical properties, which facilitates tailoring for particular biomedical applications.60 Physical methods Physical methods are simple and controllable without the addition of chemical reagents. These include methods such as vortexing and ultrasound.61–63

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Vortexing changes the structure of silk fibroin from random coil to a b-sheet. The novel vortexing technique is simple and versatile.60,64 The sonication method is based on the input of energy to the system. The principle is similar to vortexing (Figure 4). Some materials cannot form physical crosslinks during sonication, and they do not undergo gelation under sonication, including hydroxyapatite macromolecular chains. However, for such materials, gels can be formed via silk physical cross-linking. The structure of silk fibroin also can be changed under electric field. In the presence of an electric current, silk electrogelation involves the transition of silk fibroin structure from random-coil to the helical state, where reversibility is feasible. However further transition to the b-sheet is unidirectional (Figure 4). So far, there are three methods which are applying this principle, such as electrogelation, electrophoretic deposition and electropolymerization. First of all is electrogelation. An aqueous reconstituted silk fibroin solution undergoes a sol2gel transition at low voltage. This process, which

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is termed electrogelation, produces a soft gel with good adhesive properties. Leisk et al. found that the formed E-gel can be returned to the solution state through a reverse electrical process. When the electrode polarity was reversed and 25 VDC was reapplied, the gel disappeared, whereas fresh gel was formed on the new positive electrode (Figure 5). Electrogelation and the reversal to a silk solution could be cycled many times.65 This process is based on local pH changes caused by water electrolysis, which generates H1 and OH2 ions at the anode and cathode electrodes, respectively. Silk fibroin has a pI 5 4.2. E-gels form when the local pH < pI. Kojic et al. were the first to construct an experimental system that allowed for a pH distribution for an applied current and the measurement of E-gel growth. In addition, they developed a finite-element ion electrodiffusion model to explain the observed rectangular pH profile of pH gel  4 surrounded by a pH silk-solution  10. This model relies on the electrodiffusion of the generated OH2 and H1 ions.66 Electrophoretic deposition is another method which has received increasing interest because of short processing times and simple producing apparatus.67,68 Silk fibroin can modify metal substrates by coating on the surface of them through electrophoretic deposition. Zhang et al. used the electrophoretic deposition technique to deposit chitosan/silk fibroin composite coatings onto titanium substrates. The polymer of chitosan/silk fibroin was positively charged. Chitosan experienced a higher pH near the cathode, where chitosan’s amino groups were deprotonated and became insoluble, after voltage was applied. So, chitosan and silk fibroin were codeposited onto the surface of titanium substrates which acted as cathode (Figure 6).69 More recently, the authors reported a novel method for the preparation of electropolymerized silk fibroin hydrogel membranes, which are formed on a nanoporous film as a barrier, based on the principle of electropolymerization, using our lab-made device. When a regenerated silk fibroin solution in Tris buffer was added into the reservoir with negative charge and the silk molecules migrated toward the positive charge at a higher DC voltage, the silk fibroin hydrogel was formed on the barrier film (Figure 7).70 Chemical methods

Figure 4. The schematic diagram of the structure change of silk fibroin by vortexing, ultrasound and electric field.

Gels can be formed by chemical methods. Under the effects of chemical crosslinking agents or other chemical

Figure 5. Silk e-gel process applied to an 8 wt% silk aqueous solution with 25 VDC using mechanical pencil-lead electrodes: Over 3 min the gel forms around the positive electrode with gas evolution at both electrodes and gelation is reversed with the application of reversed polarity DC voltage.

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Figure 6. Schematic representation of the mechanism of chitosan/silk fibroin coatings deposited on titanium substrates (a) chitosan was protonated in hydrochloric acid solution; (b) some silk fibroin formed polyelectrolyte complex with chitosan and the other parts were negatively charged; and (c) chitosan/silk fibroin composite coating was deposited onto the titanium substrate.

Figure 7.

The schematic diagram of the electropolymerized silk fibroin hydrogel membrane formation.70

reagents, the initial hydrogen bonds or covalent bonds of silk fibroin can be broken. 1. Ultraviolet radiation Photo-crosslinked hydrogels are becoming more and more popular because mixed aqueous macromer solutions with cells or other bioactive factors can be delivered in a minimally invasive manner and crosslinked under physiological conditions upon exposure to ultraviolet light.71,72 The operation process is simple and the cells and bioactive molecules can maintain their activity by controlling the intensity of the UV exposure and with the appropriate selection of the photoinitiator.73 Kundu et al. demonstrated the fabrication of a newer type of drug that releases a photo-crosslinked hydrogel based on a semiinterpenetrating network of poly(vinyl alcohol) methacrylate and silk fibroin and possesses release properties and a tailorable structure.74 2. Chemical crosslinking agents The characters of gels formed by chemical crosslinking agents are influenced by the crosslinking monomer, crosslinking agents, and reaction conditions. The usual chemical reagents include glutaraldehyde,75,76 N,N’-methylene bisacrylamide, carbodiimide,77,78 genipin,79,80 epichloro-

hydrin,81 and 1-(3-dimethylaminopropyl)23-ethylcarbodiimide. Because of its high efficiency in collagenous material stabilization, glutaraldehyde is the most widely used by far among the chemical crosslinking agents. Crosslinking of collagenous samples with glutaraldehyde involves the reaction of the aldehyde groups of glutaraldehyde with free amino groups of hydroxylysine amino acid residues or lysine of the polypeptide chains. Glutaraldehyde is inexpensive, easily available and can effectively crosslink collagenous tissues in a relatively short period of time. However, when released into the host due to biodegradation, glutaraldehyde is toxic. Thus, several agents, including epoxy compounds, genipin and carbodiimides, have recently been tested to crosslink collagen-based materials. To reduce the risk of cytotoxicity, it is necessary to reduce the addition of chemical crosslinking.

Application of Silk Hydrogels Cell culture matrix and extension in tissue engineering (3D scaffold) Hydrogels are a type of three-dimensional reticulated polymer. They can display persistent swelling and

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insolubility in an aqueous solution. Thus, they can play an important role in the transfer of cells and cytokines. The deficit of donor corneal grafts is a major and persistent problem in cornea disease. Tissue engineering provides the potential of producing transplants that can be used to replace native damaged corneas. Guan et al. cultured keratocytes on silk fibroin/chitosan hydrogels. The cells exhibited stellate morphology and a significant increase in the expression of keratocan compared with identical cultures on tissue culture plastics. The acellular membrane was transplanted into the rabbit corneal stromal pockets, and there was no inflammatory complication detected at the macroscopic level.82 Silk hydrogel has been used for repairing of peripheral nerve injury. Tang et al. successfully fabricated the gradient of nerve growth factor-immobilized membranes (G-Ms) and nanofibrous nerve conduits (G-nNCs), which were made of silk hydrogel. Significantly, the neurite turning ratio was 0.4820.11 for the G-M group. Moreover, G-nNCs achieved nerve regeneration results associated with functional and morphological improvements 12 weeks after implantation in rats with a 14-mm gap of sciatic nerve injury, which was superior to that of the uniform NGF-immobilized nNCs (UnNCs).83 Cartilaginous tissues have an inherent lack of regenerative potential. A majority of the engineering concepts are aimed at the regeneration of hyaline cartilage. Current treatment protocols for osteoarthritis, such as abrasion arthroplasty, chondral shaving, chondrogenic cell, chondrocyte transplantation and autologous tissue grafting, or subchondral drilling, have limitations because of the limitations on autologous donor material and suboptimal integration with the defect zone. A biocompatible and mechanically robust biomaterial is needed for successful tissue engineering of cartilage. In particular, polyesters, such as poly (lactic acid) or poly (lactic-co-glycolic acid), have been used.84,85 However, these materials have potential problems. They can cause inflammation due to elevated acidity caused by polymer hydrolysis. The ability to modify the composition, structure, and mechanical properties of these materials are fairly limited. Processing difficulties may lead to tissue response profiles and inconsistent biodegradation rates.86,87 Recently, silk has been shown to provide a new option to fill the niche. Silk scaffolds can foster the deposition of cartilage in a robust and homogenous fashion because of the slower biodegradation compared to collagen scaffolds, and they can be produced using a water-based method without other organic solvents.88 Chao et al. observed that one particular preparation of silk hydrogel yielded cartilaginous constructs with mechanical properties and biochemical content matching constructs based on agarose. The resulting data indicated that silk hydrogels could be used as a tool for the studies of the mechanisms and factors involved in cartilage formation as well as a fully degradable and changeable scaffold for cartilage tissue engineering.89 Hofmann et al. seeded human bone marrow-derived mesenchymal stem cells on different scaffolds and cultured them for 21 days in serum-free chondrogenic medium, which was expected to increase control of serum composition and minimize the risk of disease transmission. The cells proliferated more quickly on the silk fibroin scaffolds than the collagen matrices. Compared with collagen scaffolds, the total content of glycosaminoglycan deposition was three times much higher on the silk scaffolds. Cartilage-like tissue was homogeneously distributed over the

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entirety of the silk scaffolds. However, on the collagen and crosslinked collagen systems, tissue formation was restricted to the outer rim. This article indicated that silk fibroin scaffolds enable mechanical improvements and compositional features suitable for durable implants to generate or regenerate cartilage, and the scaffolds were suitable biomaterial substrates for autologous cartilage tissue engineering in serumfree medium.90 The use of bone grafts for augmenting or accelerating bone regeneration suffers from significant drawbacks, including the risk of disease transmission and donor site morbidity. These limitations have prompted researchers to study the effects of combining biomaterial scaffolds with biochemical cues to augment bone repair. In recent years silk fibroin has been proposed as implant material and as a scaffold material able to promote tissue regeneration processes.91 Diab et al. investigated the efficacy of a delivery system consisting of an electrospun polycaprolactone nanofiber mesh tube with a silk fibroin hydrogel containing bone morphogenetic protein 2 (BMP-2) by using an acritically-sized rat femoral segmental defect model. The results demonstrated that silk was an effective carrier for BMP-2. The delivery system that contained BMP-2 resulted in more bone formation when compared to the delivery system without BMP-2. Biomechanical properties were also remarkably enhanced in the presence of BMP-2. The results conclude that a BMP-2 delivery system consisting of an electrospun PCL nanofiber mesh tube with a silk hydrogel presents an effective strategy for functional repair of large bone defects and were equal to age-matched intact femurs.92 Fini et al. studied the in vitro and in vivo behavior of an injectable silk fibroin hydrogel through osteoblast cultures and implantation in critical-size defects of rabbit distal femurs. At 12 weeks the re-grown bone of the silk fibroin hydrogel-treated defects appeared more similar to normal bone than that of the control synthetic poly (D,L lactide-glycolide) material -treated defects. Mineral apposition rate and bone formation rate presented significantly higher values for silk fibroin hydrogel-treated defects in comparison with the control group, confirming that silk fibroin hydrogel could accelerate remodeling processes.34 Finding a suitable 3D shape with soft tissue is an obstacle to surgical reconstruction and aesthetic improvement. Classically, a free gingival graft or connective tissue graft used for such indications may lead to different clinical complications.93,94 Etienne et al. embedded human fibroblasts in silk hydrogel material and found that the survival rate of the cells was up to 68.4% and the cells were able to proliferate and synthesize proteins. Histologic analysis revealed revascularization of the area through the augmentation after subcutaneous implantation for 30 days. In addition, the 3D soft tissue was stable. Only a mild inflammatory reaction occurred and disappeared after 12 weeks. The results indicated that silk hydrogel could create lasting 3D soft-tissue augmentation, and the material was a promising candidate for maxillofacial and periodontal therapies.95 An injectable hydrogel can fill a degenerate area in degenerative disc disease. It can minimize surgical defects and decrease the risk of implant migration and subsequent loss of height of the intervertebral disc. Hu et al. proposed a method of processing an injectable silk fibroin/polyurethane composite hydrogel. The cell culture results showed that the bone marrow stromal cells which cultured in the hydrogel had positive cell viability and increased proliferation. Besides, the hydrogel showed good visibility based on X-ray

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assays. In particular, the hydrogel provided the advantage of visibility in CT and T2-weighted magnetic resonance imaging. The blend hydrogel may provide some advantages for future application in nucleus pulpous replacement on the basis of the current study.96 Drug delivery systems In cases where drug penetration into avascular, ischemic and necrotic tissues is limited, controlled-release polymers have the potential to reduce side effects and increase efficacy by releasing drugs locally at a target site of interest.97 Toward this goal, various biodegradable biomaterials have been explored for drug delivery, including natural polymers, such as collagen and chitosan, and synthetic polymers, such as poly (lactide-co-glycolide) and polycaprolactone. However, these polymers suffer from a number of drawbacks, including poor biocompatibility or harsh processing conditions for the synthetic polymers and rapid degradation and poor tenability in the case of the natural polymers. Silk hydrogels have demonstrated excellent properties for drug delivery, including robust mechanical strength, biocompatibility, and controllable rates of biodegradation. The release behavior of these compounds from silk carriers is tunable according to the process of preparing the silk polymer, including molecular weight, carrier morphology, and crystallinity/b-sheet content. Pritchard et al. investigated the release of ampicillin and penicillin from bulk-loaded silk films, bulk-loaded silk hydrogels and drug-loaded silk microspheres suspended in silk hydrogels. The in vivo efficacy of the ampicillinreleasing silk hydrogels was shown in a murine infectedwound model. Silk hydrogels loaded with doxorubicin can be used for reducing primary tumor growth.75 Philipp et al. injected silk hydrogels locally in mice, and the results indicated an excellent antitumor response. In addition, the doxorubicin-loaded silk hydrogels could decrease metastatic spread and were well tolerated.61 The exploitation of sustained delivery systems compatible with protein therapeutics continues to be a substantial unmet need. Lyophilized silk fibroin hydrogels can serve as the matrix for the sustained release of pharmaceutically relevant monoclonal drugs. Guziewicz et al. found that sonication of silk fibroin protein prior to drugs incorporation can avoid exposing the drugs to the sol2gel transition inducing shear stress. The drug release was chiefly altered by hydrophobic/ hydrophilic silk-antibody interactions and then governed by the hydration resistance of the lyogel. Hydration resistance was controlled by altering crystalline density of the matrix. The antibody released from lyogels maintained its biological activity.98 Gene delivery system Although adenoviruses have superior transfection efficiency, their application as gene delivery vectors has been plagued with problems of toxicity, which have delayed the advancement of adenoviruses to clinical use. The family of silk-elastin-like protein polymers (SELPs), one class of hydrogels, has been used for such applications.28,29,33 SELP hydrogels have been investigated in vivo as controlled release materials for viral gene delivery and have distinct advantages compared with direct intratumoral injection in terms of localization of gene expression, prolonging intratu-

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moral gene expression, and efficacy of anticancer treatment.99 SELP hydrogels must also display an improvement in safety compared with a noncontrolled delivery treatment to be a viable treatment option. Gustafson et al. first presented evidence that the safety of subcutaneously injected adenovirus-mediated gene-directed enzyme prodrug therapy is significantly improved by delivery in SELP-815K compared with injection of virus in saline.100 Artificial skin Artificial skin is a human skin substitute that is developed in vitro to repair or replace defective skin tissue. Scientists have already developed a variety of artificial leather materials. The medical profession has successfully used composite skin for large area burns. However, artificial skin is very expensive. Thus, it is necessary to look for alternative materials. In the clinical treatment of skin defects, silk fibroin is known to promote re-epithelialization and collagen synthesis. Furthermore, silk fibroin is considered to be suitable for the generation of biomedical materials because of its minimal adverse effects on the immune system.101 Sawitree et al. studied the effects of silk hydrogels on wound healing compared with a Cutinova hydro dressing. The results indicated that the healing time of wounds dressed with a silk hydrogel was 3 days shorter than those dressed with a Cutinova hydro dressing. The histological findings revealed that there was less inflammation and more advanced granulation in the wounds dressed with a silk hydrogel than with a Cutinova hydro dressing at all stages. The results clearly indicated that the regeneration of the dermis and epidermis of the wound beds covered with the silk hydrogel was faster than with a Cutinova hydro dressing.102 Contact lenses Contact lenses need to direct contact with eyes. Good biocompatibility of the contact lenses materials is very important. Silk fibroin is becoming more and more popular for its slow degradability, biocompatibility and remarkable mechanical properties. The silk-based contact lenses are a nontoxic alternative to glass and plastics. It has been reported that the Japanese have already developed silk-based contact lenses as commercial products.103,104 Islet encapsulation platform Pancreatic islet encapsulation within biosynthetic materials has had limited clinical success due to loss of cell death and islet function. Davis et al. developed a silk-based scaffold to reestablish the islet microenvironment lost during cell isolation. The result indicated that islets remained viable and maintained insulin secretion in response to glucose stimulation, after a 7 day in vitro encapsulation. This work demonstrates that encapsulation in silk with both mesenchymal stromal cells and ECM (laminin and collagen IV) proteins enhances islet function and may have potential as a suitable platform for islet delivery in vivo with further development.62

Perspectives Silk fibroin hydrogel is a novel biomaterial that has been used extensively because of its good biocompatibility and

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controllable biodegradability. Many reports indicate that it has wide application prospects in the field of tissue engineering, such as the reconstruction of skin and bone. Despite this success, it is obvious that much work in this field is still needed. First, systematic research on the effect of processing methods on the hydrogel properties is very important. Although various processing methods have been reported, systematic research is still lacking. On the other hand, we need a thorough understanding of the interaction between silk fibroin hydrogel and cells or tissues from animals. In addition, the mechanical properties, degradability and functionality in vivo of cell-hydrogel composite scaffolds require further exploration. This knowledge will provide a good basis for theoretical and experimental guidance on the construction of tissue-engineered materials and for animals, which will finally make it possible to use such materials clinically.

9

14. 15. 16.

17. 18. 19. 20.

Acknowledgments The authors gratefully acknowledge the earmarked fund (CARS-22-ZJ0504) for China Agriculture Research System (CARS) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, P. R. China.

21. 22. 23.

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