Accepted Manuscript Review article Silk protein-based hydrogels: Promising advanced materials for biomedical applications Sonia Kapoor, Subhas C. Kundu PII: DOI: Reference:

S1742-7061(15)30210-5 http://dx.doi.org/10.1016/j.actbio.2015.11.034 ACTBIO 3986

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

10 March 2015 8 November 2015 17 November 2015

Please cite this article as: Kapoor, S., Kundu, S.C., Silk protein-based hydrogels: Promising advanced materials for biomedical applications, Acta Biomaterialia (2015), doi: http://dx.doi.org/10.1016/j.actbio.2015.11.034

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Silk protein-based hydrogels: Promising advanced materials for biomedical applications

Sonia Kapoora and Subhas C. Kundub,* a

University Institute of Engineering and Technology, Panjab University, Chandigarh, 160014, India. b

Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India.

*Corresponding Author:

Professor S. C. Kundu Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India. Tel.: +91-3222-283764 Fax: +91-3222-278433 Email: [email protected]

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Abstract Hydrogels are a class of advanced material forms that closely mimic properties of the soft biological tissues. Several polymers have been explored for preparing hydrogels with structural and functional features resembling that of the extracellular matrix. Favourable material properties, biocompatibility and easy processing of silk protein fibers into several forms make it a suitable material for biomedical applications. Hydrogels made from silk proteins have shown a potential in overcoming limitations of hydrogels prepared from conventional polymers. A great deal of effort has been made to control the properties and to integrate novel topographical and functional characteristics in the hydrogel composed from silk proteins. This review provides overview of the advances in silk protein-based hydrogels with a primary emphasis on hydrogels of fibroin. It describes the approaches used to fabricate fibroin hydrogels. Attempts to improve the existing properties or to incorporate new features in the hydrogels by making composites and by improving fibroin properties by genetic engineering approaches are also described. Applications of the fibroin hydrogels in the realms of tissue engineering and controlled release are reviewed and their future potentials are discussed. Key words: Silk proteins, Fibroin, Hydrogel, Biomaterial, Biomedical applications.

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1. Introduction Biomaterials play a significant role in regenerative medicine as implants, sutures, contact lenses, hip joints, vascular grafts, wound dressing materials, drug releasing stents and several other biomedical devices [1]. Recent developments that have offered the ability to control material properties have led to tremendous success in the field of tissue engineering and therapeutic medicine [2]. Hydrogels have come to the forefronts as an important form of materials for biomedical applications [3-7]. Hydrogels are threedimensional polymeric networks that are capable of absorbing large amounts of water while maintaining their structural integrity. They are usually classified into two categories namely physical gels (where the polymeric network is held together by secondary forces that include hydrogen bonds, ionic as well as hydrophobic interactions) and chemical hydrogels (where covalent linkages form the network). Since the introduction of hydrogels as biomaterials in the 1960s [8]), they have gained considerable attention and have found numerous applications in tissue engineering, drug delivery, implantable devices, biosensors and bio-nanotechnology. The primary reason of the widespread applicability of hydrogels for biomedical applications is the close resemblance of their physical and mechanical properties to that of biological tissues [7]. Several natural and synthetic polymeric materials have been explored to prepare hydrogels. Proteins, the fundamentally important macromolecules of living systems, have evolved to perform very specific biochemical, mechanical and structural roles. The increased understanding about the structural and functional features of a variety of proteins and advancement in techniques to manipulate them has opened avenues to utilize them for unconventional purposes [9-11]. Due to their inherent advantages including biocompatibility, ease of large-scale production via recombinant DNA technology and facile manipulation by chemical or enzymatic means, several proteins have been evaluated for their performance as biomaterials (Table 1) [9-11]. Protein polymers display a variety of properties, which prove especially useful for biomedical applications [9-11]. They undergo hierarchical self-organization that can be mimicked outside cellular environment, thus, offering a bottom-up approach for assembling biomedical devices [48]. Proteins often contain several domains that assist in cell signalling through their interaction with other proteins or ligands [49,50]. The biomaterials prepared from them

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can, therefore, be anticipated to retain this function. Proteins can be integrated with synthetic polymers easily to further enhance their material properties or can be made to adhere on synthetic surfaces, providing a hybrid interface [10,51,52]. Moreover, rational genetic engineering approaches provide us with a possibility to introduce novel features such as self-assembly, sensitivity to specific stimuli, biorecognition, and controlled degradation in proteins [53,54]. Among the naturally occurring fibers, silk occupies a special position because of its properties. Silk refers to protein fibers produced by several species of phylum Arthropoda. Several insects and spiders are able to spin silk fibers; the silks from the domesticated mulberry silkworm, Bombyx mori, and spiders, Nephila clavipes and Araneus diadematus have been extensively characterized [55-60]. Of late, various forms of silk-based materials including films, scaffolds, sponges, tubes, electrospun fibers and hydrogels have been evaluated for several applications [61,62]. This review focuses on the advances achieved in making hydrogels from silk proteins. It describes several methods that have been used for making silk protein hydrogels, structural and functional characteristics and applications of these hydrogels.

2. Structure and properties of silk Silk proteins from different silkworm species exhibit variations in their structure and properties [55,59,63]. Silk proteins can be isolated from the cocoons or the silk glands of silkworms [64,65]. Silk obtained from silkworms is mainly composed of two classes of proteins, fibroin and sericin. B. mori fibroin protein consists of a glycoprotein named P25 and a light (26 kDa) and a heavy chain (325 kDa), linked by a disulphide bond [60]. On the other hand, fibroin protein of Antheraea mylitta silkworm is composed of two similar-sized polypeptides with an estimated molecular weight of 197 kDa each which are linked together by a disulfide bond [66]. Sericin proteins, having a molecular weight ranging between 10 and 300 kDa, act as glue like coating to keep fibroin chains together. Bundles of nanofibrils form fibroin filaments that constitute 70-75% of weight of silk fiber; the remaining weight is sericin. In general, the primary sequence of fibroin consists of highly repetitive motifs [55,59,67]. For B. mori, the primary repetitive sequence is the hexapeptide GAGAGS [55,59,67]. These predominant hydrophobic

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blocks lead to extensive hydrogen and hydrophobic interactions throughout the protein chains resulting in homogeneous secondary structure. These interactions impart crystallinity to the protein which, in turn, enhances environmental stability of fibroin [68]. These highly crystalline regions (containing repetitive alanine or glycine rich sequences) associate with less organized regions of 34-40 amino acids domains which are interspersed between the crystalline regions [67] and result in the remarkable strength and elasticity of silk protein fibers [69]. Not only do fibroin fibers have good mechanical strength, they also possess excellent process ability and have been processed into several forms including films, sponges, mats, gels, scaffolds and tubes [70]. Silk-based biomaterials have been found to be highly biocompatible with various cell types. For example, adhesion and proliferation studies of human and animal cell lines on films formed from native silkworm fibroin and regenerated silk fibroin [71-73] have suggested it to be a suitable matrix for cell growth. Although, the adherence of cells on silk surfaces has been found to vary depending on the source of protein and the processing conditions, nevertheless, silk-based biomaterials provide good support for cell adhesion and proliferation and do not cause any major cell toxicity [73]. It is well emerging that fibroin does not cause activation of immune response. In fact, it evokes a minimum foreign body response [68,75] and has been used as a suture material for many centuries. Silk proteins degrade via enzymatic and non-enzymatic means [76]. The degradation of silk biopolymer has been found to be slower as compared to several natural polymers [68,74,76]. Due to its high crystallinity, it may take several days or up to weeks to degrade in vivo with the rate of absorption depending on factors like implantation site, the mechanical environment, the type of silk (virgin or processed), the diameter of the silk fiber and the secondary structure [68,74]. Due to the slow degradation rate of fibroin, silk-based devices provide a suitable material that can support the neo-forming tissues for long duration. Moreover, the degradation products of silk fibroin materials have been shown to be harmless to the human body [76]. In addition to tailoring the properties of silk proteins by chemical and physical modifications, their properties can be modulated according to the requirements through genetic engineering. Biocompatibility and cell adhesion of silk proteins has been improved by integrating sequences from other proteins like collagen and fibronectin

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[77,78]. For instance, Morgan group have prepared scaffolds by blending fibroin and synthetic spidroin, which contains RGD sequences, thus, leading to easy and inexpensive incorporation of sequences to promote cell adhesion and differentiation [79]. Mechanical strength, biocompatibility, stability to heat and humidity, high permeability to oxygen and other molecules along with the opportunity to control the material characteristics through methanol treatment and genetic engineering has made silk protein-based materials much sought after for biomedical purposes [61,80-82]. 3. Silk-based hydrogels In vivo, the cells grow, divide, perform their functions, communicate with each other and migrate. All these functions, in one way or the other, are supported by a matrix – the extracellular matrix (ECM) that provides mechanical support as well as physicochemical cues to cells to perform these functions [83]. Extracellular matrix is composed of fibrous proteins (laminin, collagen and fibronectin) and proteoglycans. The protein chains form a mesh like network and provide the mechanical support while the proteoglycans occupy the interstitial sites of this polymeric network [83]. Dynamic microrestructuring of ECM occurs continuously and essentially maintains the tissue homeostasis [84]. Hydrogels have been widely accepted as near prototypes of the ECM and have been found to be suitable 3-dimensional matrices for cell growth [7]. The hydrogels prepared earlier have been more of a passive 3D platform for cell growth. The significance of properties like mechanical stiffness of hydrogels in mechanotransduction and of biochemical and topological cues in promoting physiological functions of cells has been identified lately [85]. Thus, the emphasis has now shifted to fabricating the hydrogels that reflect the essence of cellular microenvironment and can mimic the key aspects of ECM. As noted above, fibroin-based material promotes cell attachment and proliferation [73]. In particular, the silk from wild silkworm like Antheraea pernyi contains RGD sequences which are known to be the recognition sequences for integrin receptors that mediate the interaction between cells and ECM [86,87]. In addition, the mechanical properties including extensibility and toughness of fibroin fibers, particularly that from wild silkworm fiber are even higher than polymers like elastin and Kevlar [68,75]. The superior mechanical strength of hydrogels prepared from silk fibers may

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thus provide opportunities to overcome the limitations that are often faced while using the hydrogels obtained from other natural polymers [62].

3.1 Fibroin hydrogels 3.1.1 Preparation and characterization In general, hydrogels are obtained by physical or chemical cross-linking of the polymeric chains. Since the performance of hydrogels in the in vivo setting primarily depends on their properties, it is essential to determine the fundamental characteristics of a hydrogel before it can be explored for its biomedical potential. The major properties that need to be characterized for a hydrogel include its porosity, water content and swelling

behaviour,

size

and

shape,

mechanical

and

rheological properties,

biocompatibility and biodegradation. Not only is it essential to determine the initial properties of hydrogels, but equally important is to consider how these properties change with time while the hydrogel is still in use. These properties and their dynamics influence the spatiotemporal behaviour of the hydrogel, thus governing its utility for a specific application. Since the processing conditions greatly influence the characteristics of the hydrogels, it is desirable to control the processing of hydrogels to exert a strict control over the resultant properties. As discussed below, it is becoming increasingly possible to fine tune the properties of silk hydrogels. Fig.1 schematically outlines the general steps to fabricate silk hydrogels. Table 2 lists the methods that have been used to produce silk protein fibroin hydrogels. Predominance of hydrophobic amino acid groups like glycine, serine and alanine [100] in fibroin makes gelation possible without addition of any gelling agent. For example, Ayub et al. prepared fibroin gel of mechanical strength ranging from 1 kg/cm2 to 30 kg/cm2 by keeping fibroin solution at 20 °C, at a relative humidity of 56-64% [101]. Slow rate of gelation of fibroin has been one of the major challenges in preparing silk gels. For example, 2% fibroin solution (pH 6.4-6.8) kept at 37 °C has been reported to form gel after about 30 days [88]. This limitation has been overcome by stimulating gel formation in fibroin by changing temperature, pH or ionic concentration by addition of salts like CaCl2 or KCl [88,89,98,102] and by using several methods like shearing, sonication, removal of bulk water by osmotic stresses, vortexing, heating and exposure to solvents,

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gases and surfactants like sodium dodecyl sulfate and sodium N-lauroyl sarcosinate [90,96,97,103-105]. Silk fibroin solution treated at higher temperature (60 °C) gels faster than the solution kept at 37 °C [84,85,101]. Similarly, an increase in fibroin concentration as well as a decrease in pH towards its isoelectric point (3.8) also results in faster gelation. Not only the gelation rate, but the intrinsic hydrogel properties like mechanical properties and pore-size of silk-gels can be easily manipulated by controlling external conditions. It has been seen that the breaking stress of gels prepared at 55 °C increased to about 19 N/m2 as compared to about 8 N/m2 for the gels prepared at 15 °C suggesting that the fibroin gels prepared at higher temperature have better mechanical properties [102]. Similar impact of temperature on mechanical strength of gels were showed by Kim et al. who found that 16% fibroin solution, which undergoes gelation at 60 °C, has double the compressive strength (3 MPa) than a solution of same concentration that undergoes gelation at 37 °C (1.5 MPa) [88]. This may be due to increased crystallization at higher temperatures. The gel strength has also been shown to vary with pH of the solution; the gels prepared at pH 7.0 have showed highest strength. Non-porous fibroin hydrogels have been prepared by adding glycerol (30% (v/v)) to silk solution [107]. The gels containing glycerol are slow in losing moisture [107], suggesting that certain agents like glycerol can modulate the moisture retention capacity of silk hydrogels. When hydrogels are used as matrices for encapsulating cells or other bioactive molecules, extremely mild methods of preparation are used. Any reagent or intermittent process should not be detrimental to cell viability or to the activity of entrapped bioactive agent. Sonication has been used to fabricate fibroin hydrogels encapsulating cells [91,92,95] or bioactive molecules such as growth factors [94]. The concentration of the protein solution is crucial for such a process. Viscosity of the solution should allow uniform propagations of waves throughout the solution or else it may trigger heterogeneous gelation. Uniform mixing ensures a homogeneous distribution of the cells/bioactive molecules in the matrix. Lately, vortexing and electric current have also been used to fabricate silk hydrogels [93,108]. The treatments including sonication and vortexing promote β-sheet transformation in silk, which ultimately result in gel formation [92-94].

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3.1.2 Sol-gel transition mechanism of fibroin Various groups have worked to understand the molecular mechanisms involved in the gelation of fibroin. Initially, it was proposed that β-sheet transformation is responsible for gelation of silk solution [109,110] and it has later been shown that there is higher βsheet content in silk gels as compared to silk solution and silk films [101]. Matsumoto et al. have provided a detailed insight into the gelation mechanism of silk fibroin [103]. FTIR and CD analysis have shown that regenerated silk solution obtained after dialysis contains about 10-20% β-sheet fractions (depending on the concentration of protein). During initial gelation, only weak interactions take place between the protein chains without any secondary structure changes. Thus, there is no significant increase in the βsheet fraction. Until this stage, silk gelation is a reversible process [111]. However, as soon as about 15% gelation is complete, strong interactions follow, resulting in a continuous increase in β-sheet content [111]. These thermodynamically stable cross-links ultimately result in gelation of the fibroin solution. At this stage, β-sheet content reaches as high as 50% [103]. Non-linear microscopic techniques, namely photon excited fluorescence (TPEF) and second harmonic generation imaging have also showed that fibroin undergoes structural transformation to β-sheet as hydrogel formation takes place [112]. Factors like pH, temperature and ionic strength have been used to control the gelation rate of fibroin. The ultrasensitivity of fibroin gelation to pH arises because of its structure [103]. Silk is an amphiphilic protein consisting of hydrophilic N- and C-termini and large hydrophobic domains interspersed with very short hydrophilic stretches. The heavy chain N-terminus, rich in acidic amino acids is expected to have a pI of about 4.59 while Cterminus is rich in basic amino acids and has a pI of about 10.53. On the other hand, Cterminus of the light chain is rich in acidic groups and has a pI of about 5.06. As pH is lowered, more and more carboxyl groups get protonated, reducing charge-charge repulsion and thus resulting in faster gelation [103]. Thus, a decrease in negative charge by protonation of acidic amino acid side chains promotes refolding of protein chains to a more ordered state that is accompanied by an exclusion of water. A similar increase in hydrophobic interaction is observed when temperature and ionic strength of fibroin solution are varied. The profound effect of temperature on gelation rate of fibroin has

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been attributed to the reduction of local dielectric properties caused by temperatureinduced dehydration [103]. Two-Dimensional Raman Correlation Spectroscopy and

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C

Solid-State NMR have shown the occurrence of transition of secondary structure of silk from random coils to β-sheet in presence of calcium ions [113]. Thus, β-sheet transition mainly triggers the gelation of fibroin and the conditions that have been used to prepare fibroin hydrogels promote this transition in silk, thus, facilitating gel formation. Lately, it has been shown that the sol-gel transition of fibroin can be reversibly controlled even in β-sheet-rich silk by regulating its self-assembly process in aqueous solution [114]. Such reversible gelation may impart an ability to exert a temporal control in encapsulating molecules in hydrogels by assembly and subsequently controlling their release by disassembly of hydrogels.

3.2 Silk fibroin composite hydrogels In addition to modulating the properties of silk hydrogels by varying external conditions as described above, other approaches are also used to fine tune their material properties (Figure 2). These include producing silk fibroin hybrid hydrogels and fabricating hydrogels using genetically engineered silk. Table 3 lists the polymers that have been used to fabricate fibroin-composite hydrogels and the advantages of the resulting matrices. Regenerated silk fibroin has been blended or chemically cross-linked with several synthetic polymers. For instance, in a continued effort to improve the mechanical properties of silk hydrogels, our laboratory combined polyacrylamide with silk to obtain hydrogels whose properties can be manipulated by changing the ratios of the two components [115]. Our data suggested that the gels possessed high mechanical strength and were cytocompatible. In other studies, Young’s modulus of polyvinyl alcohol-silk fibroin 50:50 blend gels (146.7 MPa) increased three fold as compared to that of fibroin only gel (50 MPa) [117]. Additionally, the porosity of the hydrogels increased on increasing the freeze-thaw cycles, corroborating that in addition to the polymer, method of blending affects the ultimate properties of the hydrogels and thus needs to be optimized [117,118]. In addition to improving the physical properties of the hydrogels, novel features including stimuli-sensitivity and reversibility of sol-gel transition have been introduced by fabricating silk composite hydrogels. Kang et al. first

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reported that poloxamer 407, a type of pluronic, can influence gelation of fibroin [119]. Pluronics are a class of non-ionic tri-block copolymers consisting of a central hydrophobic chain of polyoxypropylene and two terminal chains of hydrophilic polyoxyethylene. Varying the length of chains can vary their properties. Gelation of fibroin poloxamer system containing 3% fibroin was found to be reversible as poloxamers have reversible sol-gel properties at different temperatures due to variation in interactions between the hydrophobic and hydrophilic chains [119]. Thus, incorporation of poloxamer resulted in temperature-sensitive protein hydrogels. Silk has been combined with natural polymers as well to fabricate composite hydrogels. Mixed protein hydrogels comprising of fibroin and collagen or gelatin have been prepared [127-131]. Since gelatin hydrogels as such are not stable due to dissolution of gelatin at 37 °C, mixing of silk fibroin with gelatin proved advantageous, as the presence of silk fibroin β-sheets provided stability to the resulting silk fibroin/gelatin (SF/G) blend hydrogels [128]. Differential scanning calorimetry and dynamic rheological analysis indicated that the presence of silk fibroin β-sheets increased the dynamic elastic modulus of the G/SF hydrogels, which in turn stabilized them at higher temperatures [128]. Interestingly, blended hydrogels have been found to be stimuli-sensitive with the swelling of hydrogel and dissolution of gelatin from hydrogels being higher at 37 °C compared to that at 20 °C [128]. Collagen-fibroin composite hydrogels have been prepared by cross-linking collagen and fibroin by using optimized amounts of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) [131]. In addition to improving the mechanical strength and thermal stability, the gel exhibited gel-to-sol transition in medium of pH 4.0. The reversible gelation characteristic can be attributed to an increase in electrostatic repulsion between protein chains with decrease in pH value [131]. Hyaluronan (HA), a major glycosaminoglycan of the extracellular matrix, has also been used together with fibroin to fabricate a composite hydrogel [134,135]. HA solution has chemically been cross-linked using poly(ethylene glycol)-diacrylate in presence of a prefabricated electrospun mesh of fibroin [134] or has physically been blended with fibroin through ultrasonication [135]. The presence of fibroin acts as mechanical reinforcement and slows degradation of the otherwise mechanically weaker, fast degrading HA hydrogel.

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Different variations of fibroin-based covalent or non covalent interpenetrating networks (IPNs) and semi-IPNs (SIPNs) have also been synthesized. Interpenetrating networks are materials composed of two polymers, each existing as an independent network [140]. The two networks are polymerized or cross-linked such that the resultant networks are interlocking. Combining two networks provides an opportunity to create composite materials that otherwise cannot be obtained by mixing polymers due to phase separation. IPNs result in unique properties, which are not present in the individual polymer networks [141,142]. Semi IPNs of silk fibroin and modified poloxamer 407 having an acrylate-terminated polyethylene oxide (PEO) derivative, modified polyethylene glycol (PEG) macromer and PEG have also been prepared [120,143,144]. The SF (silk fibroin)/PEG SIPN has been prepared using a photopolymerization method. The SIPN hydrogels have higher compression resistance and mechanical strength as compared to SF or polymer (PEG/ poloxamer) hydrogels. The increase in strength makes SIPN hydrogels desirable for biomedical applications, especially skin grafting and wound dressing [143]. Our lab has synthesized photo-crosslinked PVA/SF hydrogels. PVA methacrylate, obtained by reacting PVA with 2-isocyanatoethyl methacrylate, was freeze dried. The resuspended solution was blended with fibroin and blend solutions were photopolymerized giving rise to semi-IPNs [117]. Using a similar approach Xiao et al. fabricated protein IPNs where gelatin methacrylate and SF have been independently crosslinked in the interpenetrating network [130]. Gelatin methacrylate has been polymerized by UV-irradiation in the presence of Irgacure 2959, a photoinitiator and the SIPN has subsequently been exposed to methanol to introduce cross-links in SF. Introducing SF improved the mechanical properties of hydrogels and it has been seen that exposing SF to methanol further enhanced the strength of hydrogels [125]. In yet another study, Gil et al. have made SIPN consisting of poly(N-isopropylacrylamide) (PNIPAAm) and

fibroin [122]. PNIPAAm is one of the most thoroughly investigated

thermoresponsive polymers. It shows a sharp phase transition when the temperature is increased above or decreased below its lower critical solution temperature i.e. 32 ºC [145]. This type of stimuli-sensitive polymer has many applications in devices that require an on-off mechanism. The major drawback of this system is its low de-swelling rate due to formation of a skin layer [146]. Various strategies have been suggested for

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improving the deswelling kinetics of PNIPAAm [147]. Introduction of fibroin in PNIPAAm has been shown to alter its microstructure, leading to an increase in its deswelling rate that results from reduced phenomena of skin layer formation in the gels, while maintaining the swelling rate [122]. To further improve the swelling/deswelling rates of these composite hydrogels, a novel strategy has been used [123]. The composite hydrogels have been lyophilized and the lyophilized matrix has been exposed to methanol. Freeze-drying introduces temporary macropores in the hydrogels, while exposing the matrix to methanol introduces β-sheets in SF that assists in maintaining the macroporous structure of hydrogels even after swelling [123]. These examples clearly suggest that several of the limitations associated with parent polymers can be overcome and novel properties, which do not exist in the individual parent polymers, can be introduced by preparing fibroin hybrid hydrogels. It should be noted, however, that when combining silk with other polymers and varying the relative amount of the two polymers in the combination, method of blending and cross-linking density may simultaneously affect many properties including strength, porosity, swelling, biocompatibility and morphological properties [120,122,139]. Therefore, while fabricating a hybrid hydrogel, it is essential to ensure that an attempt to improve a particular aspect of hydrogel does not affect other physical or biological properties adversely.

3.3 Hydrogel fabrication from genetically engineered silk Advancement in biochemistry and molecular biology of silk at genetic and protein levels have provided a strong platform for genetic engineering of silk proteins (Figure 2). One of the unparalleled strengths of genetic engineering of proteins is the ability to precisely customize the structure and function according to specific needs. Thus, we can have biomaterials with desirable properties by utilizing tremendous chemical diversity available in the amino acid building blocks [148]. Another reason for cloning silk genes is to obtain large quantities of protein in a cost effective way. Although protein can be obtained in larger amounts from silkworms like B. mori and spiders, the total availability of protein is not sufficient considering the amounts required for various applications. Silk-like proteins have been obtained by creating variants of the repeat sequences of N.

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clavipes and B. mori. Continuous efforts in this direction have resulted in successful production of recombinant silk proteins [149-156]. Various strategies and host systems including insect cells [157], E. coli [158], microbes [159,160], mammalian cells [161], goats [162] and potato and tobacco plants [163,164] have been tried to optimize the yield of protein, to improve the solubility of protein and to allow proper folding and post translational modification of protein. Not only the proteins have been obtained in larger quantities but also, new features, which are not found in native proteins, have been incorporated [149]. The main functional modalities that have been incorporated in these genetically-engineered proteins are oriented towards making the proteins self assembling, stimuli-sensitive, mechanically stable and information rich so that they contain specific epitopes and domains that assist in cell adhesion and signalling. Incorporation of these functionalities ultimately aims to make the genetically engineered protein-based hydrogels more suitable for tissue engineering and drug delivery purposes [165,166]. 3.3.1 Silk-elastin like hydrogel Elastin-like proteins have gained considerable attention due to their striking selfassembling properties, attributed mainly to the transitions that take place in the polymer backbone in the presence of water. Thorough details about elastin-like proteins are provided elsewhere [167-169]. Silk-elastin like polymers (SELPs), developed by Cappello and colleagues, constitute an important class of genetically engineered silk proteins [170]. These synthetic proteins consist of (a) repetitive silk peptide sequence of B. mori (Gly-Ala-Gly-Ala-Gly-Ser) that provides mechanical strength to block copolymer protein and (b) elastin peptide sequence (Gly-Val-Gly-Val-Pro) that provides flexibility to the protein, improves its solubility and determines the cross-link density within the hydrogels [170,171]. Varying the composition of the two constituent peptide blocks provides a means to regulate the properties including strength, immunogenicity, solubility and degradation rate of the resulting biomaterial [172]. The silk-elastin like polymers have been well characterized to determine their properties and their utility for various applications. The general techniques for making and characterizing these polymers have been reviewed in detail [170-174]. Certain members of this family of proteins, called Prolastins, exhibit sol-to-gel transition and hydrogel forming capabilities

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[175]. They mainly consist of two or more silk-like sequences per monomer. Hydrogel formation is primarily regulated by temperature (due to dependence of assembly of elastin-like blocks and hydrogen bonding of silk-like blocks on temperature). Under physiological conditions, the hydrogen bond formation between silk-like units causes protein solution to undergo crystallization, which results in gelation [175]. The major advantage is that no chemical cross-linking or solvent treatment is needed to prepare SELP hydrogels. Thus, SELP hydrogels present novel opportunities to encapsulate bioactive molecules or cells under ambient conditions and form in situ gelling hydrogels for tissue engineering and therapeutic purposes. SELP-47K and SELP-415 K, consisting of four silk-like and seven and fifteen elastin-like blocks, respectively, are main examples of gel-forming proteins. SELP-47K, which undergoes irreversible sol-to-gel transition at body temperature, is the most extensively

investigated

gel-forming

Prolastin

polymer

[173-178].

Swelling

characteristics and equilibrium water content are important parameters of the matrix and primarily govern the release properties of the molecules from hydrogels. Hydrogel swelling characteristics are, in general, controlled by factors like polymer concentration and cross-link density as well as environmental stimuli such as pH, temperature, and ionic strength of solvent. However, in case of SELP-47K, the swelling of the gels is relatively independent of temperature, pH and ionic strength, suggesting that this polymer can be utilized to make hydrogels whose release properties remain constant in spite of external variations [35]. Although hydrogels which maintain similar swelling properties under different conditions are robust, there may be conditions where it is desirable to have hydrogels with stimuli-sensitive swelling and release properties. Swelling properties of SELP-415K have been found to be sensitive to temperature and ionic strength [179]. Physiologically relevant sensitivity to pH- and temperature has been introduced in these polymer gels by strategic replacement of valine in elastin chain with glutamic acid (a charged amino acid) through genetic engineering techniques [180]. Dandu et al. have prepared hydrogels from another silk-elastin like protein (SELP-815K) and have shown that the silk sequences provide mechanical stiffness to the hydrogels whereas the elastin sequences determine the cross-link density within the hydrogels [171]. Recently Silk-

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Elastin like proteins with matrix metalloproteinase (MMP) degradable sequences inserted in between the polymer backbone have been expressed. The degradation behavior of the hydrogels fabricated from this recombinant protein can evidently be finely controlled by environmental cues like the expression of MMPs, which have been found to be overexpressed in certain tumors [150,181]. Recently, in situ gelling injectable SELP-47K and SELP-815K based-hydrogels have been investigated for formulating transarterial embolics to selectively prevent tumoral blood supply as a novel strategy for chemotherapeutics [178]. A 12% w/w solution of SELP-815K fabricated into hydrogel by shearing has been found to exhibit acceptable rheological properties and embolic capability [178]. Although, the research about SELP hydrogels is still in a budding stage, it is clear that hydrogels from silk-copolymers can be made with varied mechanical properties, degradation signals, assembly characteristics and bioactive signals, making them useful for future biomedical applications. 4. Biomedical applications of silk-based hydrogels 4.1. Tissue engineering Engineering the polymeric materials to replace/repair a malfunctioning or damaged tissue or organ is one of the most important advances in the recent decades [182]. The success of hydrogels as tissue engineering matrices lie in the fact that their biochemical composition and properties such as water content, viscoelasticity and mechanical strength can be made to mimic different types of natural tissues. Controlling the factors like monomer concentration, cross-linker concentration and nature of functional groups during processing can optimise the properties of hydrogels. Similarity of hydrogels to natural tissues and their ability to support functions like transport of bioactive molecules such as hormones, growth factors and peptide sequences while maintaining structural integrity make them attractive for tissue engineering applications [183]. Another advantage of using hydrogels for tissue engineering applications is the ability to entrap cells within hydrogels during fabrication, provided that mild conditions are used for preparation so that the cell viability is not affected [184]. Cell entrapment during gelation results in a more uniform distribution of cells, as compared to populating the matrix after gelation, which results in higher cell populations near the surface of the

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gel [185], though considerable success has been achieved with the latter strategy as well [186]. Thus, the aqueous environment, ease of transportation of nutrients and entrapped molecules, ease of modification and utility as in situ forming matrices represent important advantages of hydrogels as tissue engineering matrices. The disadvantages may include difficulty in handling, sterilization and lack of mechanical strength [187]. Three main design approaches to use hydrogels as functional matrices include preparing acellular hydrogels, cell-laden hydrogels and tissue-engineered hydrogels, which, in addition to the polymeric scaffolds and cells, contain several other factors like chemical moieties and/or bioactive molecules such as growth factors and peptide sequences to modulate degradation/remodelling of matrix, cell adhesion, cell motility, cell growth and differentiation [187]. Silk hydrogels based on all these approaches have been produced. The real challenge yet lies in replicating their structural and functional properties according to the desired tissue type where the hydrogel is to be implanted and in creating the dynamic chemo-mechanical environment that exists in vivo.

4.1.1 Bone tissue engineering Bone, which mainly consists of collagen protein and hydroxyapatite, provides support and strength for the movements. Critical bone defects or severe bone injuries, which cannot be regenerated naturally, may need external interventions in the form of implants. Implants provide mechanical strength to bear stresses at the site of injury and milieu that accelerate natural tissue growth. Bone tissue engineering is an approach, which differs from permanent bone implants in a sense that the tissue engineered bone is designed to integrate and absorb in the environment where it is implanted. For this, the implant should be biodegradable and bioresorbable [188,189]. The major challenge for successful bone tissue engineering lies in understanding the spatial and temporal distribution of cells and growth factors and their interaction with extra cellular matrix for osteogenesis in diseased conditions. In addition, the implant material should provide mechanical, chemical and structural signals for osteoconduction and osteoinduction, essential for in vivo tissue growth. Several materials have been investigated for bone tissue engineering [188,189]. Metals have been the traditional choice as implant material due to their load bearing capability. However, they suffer from several disadvantages like

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the stress shielding effects, poor biodegradability, and lack of integration with the surrounding tissue [190]. Ceramics, though resemble to the mineral phase of bone, are severely limited in application by their brittle nature and poor biodegradation properties [191]. Amongst natural polymers, collagen-based materials offer opportunity to influence cellular responses but often exhibit poor mechanical strength and undesirable immune responses [192]. Biomaterials fabricated from alginate have adequate mechanical properties, however, they have limitation for different cell-based applications due to lack of cell-specific bioactivities [193]. To address some of these challenges, silk implant material has been evaluated for bone regeneration. Silk proteins can be tailored to make a bioactive material for bone implants to induce direct bone formation and ultimately result in osteointegration, although much work still needs to be done in this direction. Silk fibroin hydrogels have shown superior ability to promote cell metabolism as well as bone remodelling as compared to poly(lactide-co-glycolic acid) (PLGA), an FDA approved polymer, which is a popular choice for comparing the characteristics of a material for tissue engineering applications because

of

its

long

clinical

standing,

favorable

biodegradation

properties,

biocompatibility and a minimal systemic toxicity [193]. The bone-healing rate, proliferation and differentiation of the osteoblasts in presence of silk hydrogels have also been better than the control [37]. In another study, porous silk matrix (prepared by using hexafluoroisopropanol (HFIP) as solvent) was seeded with stem cells derived from human bone marrow and the cells have been allowed to grow under osteogenic conditions with medium being supplemented with ascorbic acid-2-phosphate, dexamethasone, β-glycerolphosphate, and BMP-2 [195]. Various approaches including (1) tissue engineered bone (cells seeded on matrix grown in bioreactor for 5 weeks), (2) scaffolds seeded with cells at the day of surgery, (3) scaffold alone, or (4) no implant (unfilled) were evaluated by implanting in mouse. Clearly visible trabecular structures could be observed through micro-computed tomography after about 5 weeks of implantation in case of the defects treated with the engineered bone, suggesting that silk based implants can be utilised for effective bone regeneration. It is worth noting that the scaffolds prepared from protein obtained from the same source but differing in the preparation method exhibit different osteogenic responses [196]. Differences in bone

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related outcomes such as mineralization and expression of collagen type I, alkaline phosphate, osteopontin and matrix metalloproteinase 13, which regulate the remodelling of ECM have also been observed [196]. The differences in the processing of protein lead to subtle changes in structure and properties of resulting matrix, which ultimately lead to different cell response [197]. Recently, fibroin/sodium alginate hydrogels have been used as template for promoting controlled biomineralization of hydroxyapatite crystals for bone repair [139]. The preliminary success of hydrogels derived from silk fibroin suggests that further studies are warranted. Incorporation of additional biological signals as well as utilization of polymer blends, interpenetrating networks and silk copolymers may provide ideal scaffolds for bone repair. 4.1.2 Cartilage tissue engineering Cartilage, a connective tissue, is mainly composed of type II collagen and is rich in proteoglycans, particularly chondroitin sulphate while the primary cell type is chondrocytes [198]. This tissue contains almost 65-70% water. The clinical impact of defects in cartilage tissue due to congenital abnormalities or trauma is of particular concern due to the low regenerative capability of this tissue, resulting from an absence of blood vessels, nerve tissue, and lymphocytes and the low cell density, slow cell proliferation and slow matrix turnover rate. Therefore, cell-based therapies, in which 3D scaffolds carrying stem cells or differentiated chondrocytes can be implanted at the site of injury, are of significant interest. Due to their close resemblance to cartilage tissue, hydrogels have been considered as a suitable matrix for encapsulating cells and the growth factors (e.g. TGF-β superfamily, IGF, FGF, BMP, PDGF, and EGF). Natural materials like collagen and synthetic material like poly(lactic acid) (PLA) and PLGA have been evaluated for this purpose. Though agarose hydrogels have particularly shown utility as scaffolds for cartilage tissue engineering because of their high mechanical strength, their success is limited by poor biocompatibility, lack of biodegradability and poor host tissue integration [91]. The mechanical strength, frictional properties and the uncertainty of interaction between the matrix and the cells have remained the major issues with most of the scaffolding materials [91]. Aoki and colleagues have prepared fibroin hydrogel and compared its cartilage regeneration performance with collagen gels [33]. They inoculated the chondrocytes isolated from the proximal humerus, distal femur

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and proximal tibia of 4-week-old Japanese white rabbits into the fibroin-hydrogel sponge, formed by phase separation of freezed fibroin solution and collagen gels [33]. The cells were then cultured for 4 weeks and were found growing in the pores and the outer surface of the fibroin hydrogel and the cell density increased with incubation times. On the other hand, the cell density in the collagen gels was seen to be higher initially, but it did not increase with time. Similarly, the rate of increase of chondroitin sulfate in fibroin hydrogel was higher than that in collagen gels [33]. Positive histological staining for key cartilage extracellular matrix components (chondroitin sulfate and type II collagen) indicated formation of hyaline cartilage-like tissue in the pores of the fibroin hydrogel. Chondrocytes maintain round morphology and retain their differentiated phenotype within the fibroin hydrogel [33]. Cell microaggregates seeded in fibroin hydrogel closely mimic the initial stages of tissue formation and have been found to be very efficient in forming extracellular matrix for cartilage tissue regeneration [199]. To improve the mechanical properties of fibroin hydrogels, recently a novel approach has been used, wherein silk hydrogel has been reinforced with silk microfibers. The composite hydrogel has exhibited mechanical properties comparable to that of the agarose hydrogels with proven mechanical robustness [200]. Bioreactors can be used to stimulate functional regeneration of tissues and to continuously supply the nutrients and growth factors to the cultured cells. This represents an advanced approach holding immense potential for improving cartilage tissue engineering. This approach has been used with fibroin hydrogel [201]. The cells grown in matrix homed within a stirring chamber have been found to exhibit higher DNA and glycosaminoglycan content as compared to the cells not grown in the reactor. Positive histological staining of proteoglycans and collagen in tissue grown in this system vis-àvis the one in a system lacking stirring chamber suggested that stirrer facilitated the maturity of cartilage matrix by providing mechanical stimulation to the cellular microenvironment. This system could support chondrogenesis in vivo and resulted in cartilage regeneration in the rabbits corroborating its clinical potential [201]. Primary cell lines and chondrocytes are mainly used as cell source for cartilage tissue regeneration. Since they are available in limited supply, scientists are now focusing on the use of stem cells that show unlimited potential to differentiate in several tissue types

20

including cartilage [202,203]. Human mesenchymal stem cells (hMSC) encapsulated in SELP-47 K hydrogel and cultured in chondrogenic medium in the presence of TGF-β3 exhibited differentiation and chondrogenesis [204]. The cells remained metabolically active even after 28 days of culture and histological analyses have revealed the formation of extracellular matrix along with the expression of SOX9, and matrix proteins aggrecan and collagen. The studies indicate that fibroin hydrogels have potential for mimicking the extracellular matrix and promoting cartilage regeneration.

4.2 Controlled release 4.2.1 Controlled drug delivery Controlled release implies that the delivery of the active agent should follow a predetermined course of release. It may consist of both sustained (for prolonged duration) and targeted (localized) release. Supplying essential amounts of active molecule at a particular area for required duration is a major challenge in the field of drug release. This is more so in the case of bioactive agents like hormones and peptides, which are highly selective in their action and are sensitive to processing treatments. Silk based hydrogels have been found to be suitable for controlled release. One of the first studies investigating silk fibroin for controlled release has not used a hydrogel but a membrane. In this early study, Chen et al. examined the permeation of pharmaceuticals like 5-fluorouracil, L-(+)ascorbic acid, resorcinol, sodium phenolsulfonate and benzyltrimethylammonium chloride through the fibroin membrane [205]. They proposed that since fibroin membrane consists of weak basic and acidic groups, it can act as an amphoteric ion-exchanger and controlling the pH may control the passage of molecules through it. Since then fibroin has been considered as pH responsive drug delivery material. Hanawa et al. have showed that the release behaviour of benfotiamine, a synthetic derivative of thiamine (vitamin B1), in the glycerol containing fibroin hydrogels is inversely related to the fibroin content in the matrix [206]. This is likely due to the increased number of interchain interactions at higher protein concentration, which resulted in smaller pore diameters and hence slower release. Not only the protein concentration but molecular weight of silk protein affects the release rate as well [36]. High molecular weight proteins (76 kDa) have been shown to effectively slow down the release of buprenorphine in comparison to low molecular

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weight silk protein (18 kDa) when used as matrix [36]. Our laboratory has evaluated fibroin/polyacrylamide SIPN hydrogels for drug release [115]. Two model compounds namely trypan-blue and FITC-inulin have been used to determine the effect of physicochemical properties of hydrogels on release of molecules. Our results have indicated that these composite hydrogels were suitable for sustained release of low and high molecular weight molecules alike. Recently, silk hydrogels have shown potential for sustained release of bevacizumab, a clinically used anti-VEGF therapeutic for certain metastatic cancers [207]. The controlled release of a chemotherapeutic over a long period may eventually reduce the dosing frequency, which increases patient compliance. As noted above, specific Prolastins are important particularly as in situ gel forming delivery systems [169,175,178]. They spontaneously form a gel over a period of time after being injected via fine gauge hypodermic needles [175]. Cappello et al. have characterized the release of many fluorescently labelled model compounds of different molecular weights and a model pharmaceutical protein pantarin from Prolastins of different peptide sequence compositions [175]. The rate of release of entrapped molecules has been observed to depend on the composition as well as the concentration of the recombinant Prolastin. Moreover, the entrapped therapeutic protein retained the bioactivity after being released from gels [175]. The interactions between the entrapped molecule and protein matrix might affect the solute partitioning and hence the release profiles of molecules. This has been shown in the case of cytochrome c, Vitamin B12 and theophylline release from SELP hydrogels [35].

4.2.2 Controlled release of DNA for gene therapy There has been a tremendous increase in understanding the implications of the diseases caused due to genetic mutations. Hence, a lot of effort is being placed on replacing abnormal genes with the correct ones. Successful gene delivery remains the key for gene therapy. Targeting the cells, controlled activation and expression of the genes and getting rid of the side effects remain major challenges for success in this endeavour. Safe and effective delivery systems are thus the main clinical concerns. SELP polymers have recently been evaluated for DNA delivery for tumor therapy [179,181,208-211]. The ease of sol-to-gel transition of these block copolymers enables the loading of DNA

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or viral particles into an aqueous solution, which may be injected by minimal invasive procedures to form solid matrix in and around the tumour at physiological temperature. Megeed et al. have studied the release of plasmid DNA from SELP-47K hydrogels [208]. Lysine (thirteen) and arginine (five) residues present in SELP-47K are protonated at pH 7.4, and can thus interact with the negatively charged phosphate backbone of DNA and affect its release profile. Ionic strength, concentration of the SELP polymer and time for curing the gels have also been found to influence the release rates [208]. In addition, the conformation (supercoiled, open circular or linear) and molecular weight of the DNA significantly influence the release profiles while the concentration of DNA (within a range of 50 or 250 mg/ml) has no impact [209]. Finally, gene expression analysis showed that the released DNA retains its bioactivity even after 28 days of release [209]. Further, SELP matrices have been found to be suitable for localizing and prolonging the release of adenovirus and viral transduction in murine models of breast and head and neck tumour xenografts [210]. Haider et al. have created a new matrix, SELP-415 K, having more elastin units for gene delivery purpose. The release rate of DNA from this polymer matrix increased with an increase in the number of elastin units [179]. Recently, the release behaviour of incorporated plasmid has been studied as a function of biodegradation of the SELP-47 and SELP-415K hydrogels in presence of elastase enzyme [211]. In case of both the hydrogels, the release of DNA is much faster when the release medium contains elastase as compared to controls. Moreover, higher number of elastin blocks in protein resulted in a faster elastase-induced degradation of the matrix, and loss of integrity of hydrogel, thus, resulting in faster DNA release [211]. The release of viral vectors from MMP responsive-SELPs has been evaluated in a mice model of head and neck squamous cell carcinoma for tumor therapy [181]. The rate of release of viral vectors has been found to be dependent on the expression of MMPs, frequently found to be overexpressed in several tumors [181]. Such a system may enable localized delivery of bioactive therapeutics to tumors specifically, thus limiting systemic host toxicity.

5. Challenges and future prospects Silk proteins represent interesting polymeric biomaterial because of their mechanical properties, thermal stability, biocompatibility, and possibility of control via

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genetic engineering. Several examples mentioned in this work elucidate the potential of using fibroin for producing hydrogels. A wide range of applications including controlled release of active molecules and tissue engineering have also been described (Fig. 3). Advances in fabrication methods, ability to process and sterilize, control of properties like degradation and strength, and possibility of encapsulating cells have made silk hydrogels suitable constructs for complex tissue regeneration. Nevertheless, there are limitations that need to be overcome to explore complete potential of silk hydrogels. A greater understanding of the fundamentals underlying structure-function relationship of these proteins is needed to control the properties according to the specific requirements of design criteria. As more specialized hydrogels are made for advanced applications, rigorous and accurate mathematical modelling will be required to describe the systems and the mechanisms associated with them. Fig. 4 outlays the novel possibilities with silk hydrogels. In situ gelling hydrogels can assist in overcoming the invasive implantation of hydrogels for tissue reconstruction, thus, increasing the patient compliance. Self-assembling silk-like and silk-elastin like proteins provide huge opportunities to design in situ forming gels under mild conditions but we need to incorporate simultaneously “smartness (stimuli-sensitive response character)” in these proteins. The ‘decision making’ silk hydrogels that can be programmed to respond to physical, biochemical and mechanical cues will enable temporal control over several functions including the release of the encapsulated bioactive agents. Consistent with several other natural and synthetic polymers and as described herein, silk hydrogels support encapsulation of cells and therapeutic molecules. They are porous to allow diffusion of nutrients and wastes as well as to allow intercellular communication with secreted molecules. However, they lack many of the signals of the native extracellular matrix (ECM). Included in these signals are insoluble bioactive cues stemming from the proteins, glycoproteins, and proteoglycans andsoluble signals such as cytokines and growth factors that are secreted by the ECM. Future studies will need to carefully consider incorporation of biological signals to improve tissue regeneration. We should gain guidance from the increasing knowledge regarding the importance of glycosaminoglycans and proteoglycans in tissue homeostasis and disease. Studies investigating blends of silk fibroins and other polymers in the form of interpenetrating

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networks and semi-interpenetrating networks and even as block-co-polymers begin to move toward materials that better mimic the native ECM [95,105,139,212]. These blends show improved mechanical characteristics and in some cases include cell adhesion domains. Through incorporation of key features of proteoglycans, the ‘information rich’ silk hydrogels can better mimic the native ECM and will also contain natural binding sites for cytokines and growth factors that are secreted by the regenerating engineered tissue. An interesting feature that has not yet been thoroughly explored in case of the silk hydrogels is the multilayered hydrogels. Instead of hydrogels with bulk properties, novel hydrogels that contain multiple layers can be prepared from silk proteins in near future. Layer by layer assembling techniques can be utilized for this purpose. The properties of each layer can be tailored to provide a hierarchical organization suitable for distinct functions. Such hydrogels will help realizing true compartmentalization in biomaterials that is needed to achieve biologically relevant spatio-temporal complexity. Different cell types or different molecules can be encapsulated in distinct layers to enable complex release patterns. In addition, gradients of the release molecules can be achieved. Several multilayered hydrogel systems have been described recently with natural and synthetic polymers [213-215]. Recently, hydroxyapatite nanoparticle containing silk hydrogels with superior bone regenerating potential have been fabricated [216]. The challenge in getting multilayered silk hydrogels will be to find novel preparation methods that allow the generation of multiple layers. It is needed to make sure that the distinct layers remain separate. Another challenge will be to better understand the properties of boundaries and the interface between each layer.

The utilization of silk proteins for making hydrogels for novel applications in diagnostics including synthesis of microdevices, microchannels and micropatterning for guided cell proliferation and differentiation as well as for targeted release of molecule remains to be explored. In addition to fabricating hydrogels from alternative sources of silk proteins from non-mulberry silkworms including A. mylitta [217], A. assamensies, A. pernyi, Samia recini and spider silks. The efforts are being made to improve the quality of the silk proteins by genetic engineering. As we progress towards finding new

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techniques to observe as well as control microscopic morphological features, it can be anticipated that silk-based hydrogels will enable the development of new therapeutic approaches and help us to meet the demands in regenerative medicine.

Acknowledgements: This work is supported by start-up grant from University Grant Commission, Government of India to SK. We appreciate Ms Sarani Ghoshal for taking interest during the initial stages

of this write up. We are greatly indebted to Dr. Alyssa Panitch, Weldon School of Biomedical Engineering of Purdue University for critical reading, excellent scientific inputs and suggestions for the improvement of the manuscript. SCK’s laboratory has financially been supported by Indo Australia Biotechnology Fund, Department of Biotechnology and its Bioinformatics facility, Indo-Russia Biotechnology Programme, Department of Science and Technology, Indian Council of Medical Research, Govt of India, and Indo US Science and Technology Forum, New Delhi. References [1]. Huebsch N, Mooney DJ. Inspiration and application in the evolution of biomaterials. Nature 2009;462:426-432. [2]. Helary C, Desimone MF. Recent advances in biomaterials for tissue engineering and controlled drug delivery. Curr Pharm Biotechnol 2015;16:635-645. [3]. Khademhosseini A, Langer R. Microengineered hydrogels for tissue engineering. Biomaterials 2007;28:5087-5092. [4]. Lowman AM, Peppas NA. Hydrogels. In: Mathiowitz E, editor. Encyclopedia of controlled drug delivery, New York. Wiley, 1999, 397-418. [5]. Peppas NA, Hilt JZ, Khademhosseini A, Langer R. Hydrogels in biology and medicine: from molecular principles to bionanotechnology. Adv Mater 2006;18:1345– 1360. [6]. Slaughter BV, Khurshid SS, Fisher OZ, Khademhosseini A, Peppas NA. Hydrogels in regenerative medicine. Adv Mater 2009;21:3307-3329. [7]. Tibbitt MW, Anseth KS. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol Bioeng 2009;103:655-663. [8]. Wichterle O, Lim D. Hydrophilic gels for biological use. Nature 1960;185:117118. [9]. Gagner JE, Kim W, Chaikof EL. Designing protein-based biomaterials for medical applications. Acta Biomater. 2014;10:1542-1557. [10]. Krishna OD, Kiick KL. Protein- and peptide-modified synthetic polymeric biomaterials. Biopolymers 2010;94:32-48.

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Figure Legends: Figure 1: Schematic representation of hydrogel fabrication from silk fibroin protein: Cocoons and the silk glands are main sources of silk fibroin proteins. Larvae and cocoons of domesticated mulberry silkworm, Bombyx mori and non-mulberry tropical tasar silkworm, Antheraea mylitta are shown. Silk fibroin protein is isolated from middle silk glands and cocoons of different silkworms and is regenerated. The regenerated silk fibroin protein is processed through various treatments to get hydrogel. A typical image of fibroin hydrogel fabricated in our laboratory is shown. Figure 2: Approaches to improve the properties of silk hydrogels: (a) Properties of fibroin hydrogels can be modulated by varying the processing conditions, (b) Fibroincomposite hydrogels can be prepared by combining fibroin with synthetic or natural polymers and (c) silk proteins can be genetically engineered to introduce ‘designer’ features into them. Figure 3: Applications of silk hydrogels: Silk fibroin based hydrogels are currently being explored for tissue engineering and controlled release of therapeutic molecules. Figure 4: Some future directions for silk hydrogels: (a) Silk hydrogels that undergo spontaneous gelation in physiological conditions at the site of injection will pave way for their non-invasive clinical applications in tissue engineering and drug release, (b) Multilayered hydrogels consisting of separate silk gel layers with distinct properties will enable ‘true’ compartmentalization of the matrix. Such compartmentalized multilayered hydrogel can be used for carrying different ‘payloads’ to achieve spatiotemporally complex release profiles, (c) Integration of biomimetic signals on silk hydrogel surface will enable the ‘information rich’ hydrogels to be targeted to specified regions where they can replicate the cellular microenvironment and facilitate signal dependent cell functions, (d) Multifunctional hydrogels whose properties are optimized for performing different functions simultaneously (for instance, one that supports cell growth and release of growth factors) can play better role in regenerative medicine, (e) Stimuli-sensitive silk hydrogels that respond to physiologically relevant environmental signals will be used as ‘Smart’ decision making biomaterials.

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Tables:

Table 1: Proteins that have been used to prepare hydrogels Proteins

Functions in nature

Collagen

Major protein of the extracellular matrix and provides structural support

Gelatin

Degraded form of collagen

NA

Fibrin/ Fibronectin

Component of the extracellular matrix

1-10 MPab

Elastin

Provides elasticity to the tissues

1.1 MPac

Basic material to form cocoons, nets, traps by insects and silkworms

7 GPad

Fibroin

Typical Mechanical Properties of protein fiber 1.2 GPaa

Advantages

Disadvantages

Applications

Reference (s)

Good biocompatibility

Mechanical weakness and rapid degradation

[12-16]

Extremely biocompatible, lower immunogenicity as compared to collagen, forms gels by temperature change, Pharmaceutically approved as a coating material

Mechanical weakness

Tissue regeneration, Corneal replacement, Cartilage repair, gene therapy, growth factor delivery Differentiation of pluripotent cells; Growth factor delivery, drug delivery, tissue engineering, wound dressing

Very closely mimics the properties of the soft tissues, promotes cell adherence

Limited mechanical strength

Self assembles under physiological conditions

Calcification of elastin implants, Rapid degradation

High mechanical strength

Slow gelation rate

Wound healing, Angiogenesis, Tissue engineering

[17-22]

[23-29]

[30-32] Tissue engineering

Tissue repair, repair of bone defects, delivery of therapeutic molecules

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[33-38]

Whey proteins

Protein components of milk apart from casein and provide nutrition

0.2 KPae

Cheap availability, by- product of milk industry, preparation of gel does not need chemical crosslinkers

Mechanical weakness

Controlled drug release

[39-41]

Sericin

Acts as a glue to bind fibroin fibrils

0.9 MPaf

Easily available as by- product of silk industry

May result in immunogenic reactions

Wound dressing material

[42-44]

a

Young’s Modulus, E, of collagen from mammalian tendon [45] Young’s Modulus, E, of uncross-linked fibrin fibers [26] c Young’s Modulus, E, of elastin fiber from bovine ligament [46] d Bombyx mori cocoon silk [47] e Equilibrium modulus of a typical hydrogel made from 12% whey protein solution [40] f Maximum stress of a typical hydrogel in wet state at failure [42] b

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Table 2: Methods for preparation of silk fibroin protein hydrogels Method

Mechanical propertiesa

Specific conditions

Comments

Reference (s)

Compressive modulus (MPa)

% strain at failure

Temperature changes

1.2b

50%

4, 25, 37, 60 °C have generally been used

Higher temperature improved the mechanical strength, Method not suitable for direct encapsulation of cells and temperature sensitive peptides and bioactive proteins

[88-90]

Lowering of pH towards isoelectric point of fibroin

NA

NA

Citric acid, Citratephosphate buffer and HCl are mainly used

Neutralization of acid is needed, method is not suitable for direct encapsulation of pH sensitive molecules and cells

[36,89]

Shearing forces induced by sonication and vortexing of fibroin solution

1.6c

NA

Power output = 20-50% amplitude, time =5-30s, 3200 rpm using vortexer

Method found suitable for making injectable gels and for encapsulating cells prior to final setting of gel

[91-95]

CO2 acidification

64.0kPad

2 wt % fibroin solution, pressure CO2= 60 bar

Ease of fabrication; hydrogels free of mineral acids or chemical crosslinkers

[96,97]

Phase NA 16% Water soluble Generally leads to formation [98,99] separation organic of hydrogels with induced by solvent heterogeneous porosity freezing and freeze thawing a Typical mechanical properties of the hydrogels prepared by different methods as reported in literature. b Gel prepared from 8% fibroin solution at 37 °C. c Gel prepared from 8% fibroin solution at power output = 30% amplitude. d Gel prepared from 4% fibroin solution processed for 8 h.

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Table 3: The polymers that have been used in combination with silk fibroin protein to obtain composite hydrogels with improved properties Nature of polymer

Polymer

Method of preparation

Characterization

Properties/Advantages of the resulting hybrid hydrogel

Reference(s)

Synthetic

Polyacrylamide

Chemical crosslinking of silk and acrylamide solution

Swelling, morphological, rheological, mechanical properties and biocompatibility

Improved mechanical strength

[115,116]

Polyethylene oxide

Mixing powdered PEO in silk solution followed by heat treatment Freezing and Freeze thawing

Gelation kinetics, morphological and mechanical properties

Enhanced gelation rate of silk solution

[88]

Mechanical properties and porosity

Improved mechanical properties

[117,118]

Poloxamer

Photocross-linking of silk and poloxamer solution, Physical blending of silk and poloxamer solutions

Gelation kinetics, thermal stability, mechanical and swelling properties

Reversible sol-gel transition of silk solution

[119,120]

Polyurethane

Chemical crosslinking

Mechanical, rheological properties and biocompatibility

Injectable ; improved mechanical properties, suitability for application in nucleus pulposus replacement

[121]

NIPAAma

Physical blending of silk and NIPAAm solution

Swelling and rheological properties

Physical blending of silk and PEG 300 orPEG400 solution

Gelation kinetics, rheological properties, mechanical properties, degradation kinetics and cell attachment

Increased deswelling kinetics of composite hydrogels as compared to NIPAAm hydrogels Injectable, In situ gelling, low initial cell attachment, thus suitable as, anti-fouling and anti-adhesion surface

Physical blending of silk and gelatin protein solutions, Crosslinking using Genipin Chemical crosslinking of

Thermal stability, viscoelastic, swelling and morphological properties

Temperature sensitive conformational transition of gelatin from helix to coil, formation of stable gels

Thermal, viscoelastic, swelling,

Improved mechanical strength, thermal stability

Polyvinyl alcohol

Polyethylene glycol

Natural

Gelatin

Collagen

[122,123]

[124]

[125-130]

[131]

46

a

fibroin/collagen solution

morphological properties and biocompatibility

and substrate stiffness

Chitosan

Chemical/physical cross-linking of silk and chitosan solution

Swelling properties and ion and pH sensitivity of gels

pH and ion sensitive hydrogel, improvement in release kinetics of drugs

[132,133]

Hyaluronan

Cross-linking of HA in presence of an electrospinned silk mat, Physical blending using ultrasonication

Gelation kinetics, thermal stability, morphology, mechanical and swelling properties, enzymatic degradation

Improved mechanical properties and controlled degradation

[134,135]

Methylcellulose

Physical crosslinking of silk and MC solution at 50 °C

Morphology, drug release kinetics

Control of sol-gel transition of fibroin and drug release properties

[136]

Pectin

Dialysis against methanol

Mechanical Properties, swelling properties biodegradability, biocompatibility

Increased stiffness of hydrogel

[137]

Alginate

Calcium ioninduced gelation

Porosity, intravital imaging, Rheological properties, Immune response

Mechanical properties of the hydrogel facilitated stem cell differentiation Supports biomimetic crystallization of hydroxyapatite

[138,139]

NIPAAm: N-isopropylacrylamide

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