Silk fibroin biomaterials for tissue regenerations

Advanced Drug Delivery Reviews 65 (2013) 457–470

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Silk fibroin biomaterials for tissue regenerations☆ Banani Kundu a, 1, Rangam Rajkhowa b, 1, Subhas C. Kundu a,⁎, Xungai Wang b, c,⁎⁎ a b c

Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur-721302, India Australian Future Fibres Research and Innovation Centre, Deakin University, Geelong, Victoria3217, Australia School of Textile Science and Engineering, Wuhan Textile University, Wuhan, China

a r t i c l e

i n f o

Article history: Accepted 25 September 2012 Available online 5 November 2012 Keywords: Silk Fibroin Biomaterials Scaffolds Tissue regeneration

a b s t r a c t Regeneration of tissues using cells, scaffolds and appropriate growth factors is a key approach in the treatments of tissue or organ failure. Silk protein fibroin can be effectively used as a scaffolding material in these treatments. Silk fibers are obtained from diverse sources such as spiders, silkworms, scorpions, mites and flies. Among them, silk of silkworms is a good source for the development of biomedical device. It possesses good biocompatibility, suitable mechanical properties and is produced in bulk in the textile sector. The unique combination of elasticity and strength along with mammalian cell compatibility makes silk fibroin an attractive material for tissue engineering. The present article discusses the processing of silk fibroin into different forms of biomaterials followed by their uses in regeneration of different tissues. Applications of silk for engineering of bone, vascular, neural, skin, cartilage, ligaments, tendons, cardiac, ocular, and bladder tissues are discussed. The advantages and limitations of silk systems as scaffolding materials in the context of biocompatibility, biodegradability and tissue specific requirements are also critically reviewed. © 2012 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Silks of silkworms: source, chemistry and structure . . . . . . . . . . Characteristics of silk fibroin as biomaterial . . . . . . . . . . . . . . 3.1. Mechanical properties . . . . . . . . . . . . . . . . . . . . 3.2. Biocompatibility . . . . . . . . . . . . . . . . . . . . . . . 3.3. Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Water based processing . . . . . . . . . . . . . . . . . . . . 3.5. Manipulation of silk properties through structural re-adjustments Morphological diversification of silk biomaterials for tissue regeneration 4.1. Native silk structures . . . . . . . . . . . . . . . . . . . . . 4.2. Regenerated silk morphologies . . . . . . . . . . . . . . . . 4.2.1. Films . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Electro-spun and wet-spun fibers . . . . . . . . . . . 4.2.3. Hydrogels . . . . . . . . . . . . . . . . . . . . . . 4.2.4. 3-D porous scaffolds . . . . . . . . . . . . . . . . . 4.2.5. Particles . . . . . . . . . . . . . . . . . . . . . . . Applications of silk fibroin biomaterials for tissue regeneration . . . . . 5.1. Vascular tissue regeneration . . . . . . . . . . . . . . . . . . 5.2. Neural tissue regeneration . . . . . . . . . . . . . . . . . . 5.3. Skin tissue regeneration . . . . . . . . . . . . . . . . . . . . 5.4. Bone tissue regeneration . . . . . . . . . . . . . . . . . . . 5.5. Cartilage tissue regeneration . . . . . . . . . . . . . . . . .

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☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Bionics — biologically inspired smart materials”. ⁎ Corresponding author. Tel.:+91 3222 283764; fax: +91 3222 278433. ⁎⁎ Correspondence to: Australian Future Fibres Research and Innovation Centre, Deakin University, Geelong, Victoria3217, Australia. Tel.:+61 613 5227 2894; fax: +61 613 5227 2167. E-mail addresses: [email protected] (S.C. Kundu), [email protected] (X. Wang). 1 Authors contributed equally. 0169-409X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.addr.2012.09.043

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5.6. Ligament and tendon tissue regeneration 5.7. Cardiac tissue regeneration . . . . . . 5.8. Ocular tissue regeneration . . . . . . . 5.9. Hepatic tissue regeneration . . . . . . 5.10. Spinal cord tissue regeneration . . . . . 5.11. Inter-vertebral tissue regeneration . . . 5.12. Bladder tissue regeneration . . . . . . 5.13. Tracheal tissue regeneration . . . . . . 5.14. Eardrum tissue regeneration . . . . . . 6. Future prospects . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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1. Introduction The limited supply of donors and increasing morbidity have put new demands on tissue engineering (TE) as a treatment of organ failures [1]. The TE approach involves regenerating tissue within suitable scaffold with the goal of implanting the constructed tissue at the target site. The regeneration of functional tissue requires a suitable microenvironment that closely mimics the host site for desired cellular responses [1]. Such an environment is typically provided by 3-D tissue engineering scaffold that acts as an architectural template [2]. Apart from biocompatibility, which is the essential prerequisite for any biomaterial, matching the degradation time with that of the tissue regeneration is also a critical requirement for a cell scaffolding material. Such a match can maintain the mechanical properties and structural integrity of the engineered tissue in all stages of its regeneration process. In addition, degraded products of the biomaterial should be safely metabolized and cleared from the host body. Materials like polymers, metals and ceramics are widely used as cell scaffolds for tissue engineering. Both synthetic and natural polymers have been trialed, though each has its own limitations. While the former allows easy processing and modifications, the later offers better cyto- and bio-compatibility [3]. There is no universal biomaterial that meets the scaffolding requirements for all the tissues. Different issue constructs require biomaterials with specific physical, mechanical and degradation properties. Hence there is on-going search for universal biomaterial for regeneration therapy. Protein, being a component of natural tissues, is a rational choice for applications in tissue engineering. Structural proteins such as collagen, elastin, elastin-like-peptides, albumin and fibrin are used as sutures, tissue scaffolds, haemostatic and drug delivery agents [4]. Silk fibroin of silkworms is a commonly available natural biopolymer with a long history of applications in the human body as sutures. Currently silk sutures are used in lips, eyes, oral surgeries and in the treatment of skin wounds [5]. Increasingly, silk fibroin is exploited in other areas of biomedical science, as a result of new knowledge of its processing and properties like mechanical strength, elasticity, biocompatibility, and controllable biodegradability [5]. These properties of silk fibroin are particularly useful for tissue engineering. Furthermore, recent studies evaluates silk as a part of flexible electronic devices for real-time physiological and functional recording and optical systems for diagnosis and treatments [6,7]. Silk possesses excellent (ca. 95%) optical transparency throughout the visible range with remarkable surface smoothness and aqueous processing, all of which facilitates its application in optics and photonics biosensor [7,8]. Such silk based systems are implantable and have necessary functionality and sensitivity required for advanced applications. Several reviews are published on the fabrication, structure, and the application of silk based biomaterials [2,5,9,10]. In view of growing applications of silk in new areas of tissue engineering and knowledge on characteristics of silk constructs, a further and more detailed review is now warranted.

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This review article is focused on recent research based on silk fibroin in the field of tissue regeneration and evaluates its prospects for further development in therapeutic related applications. The review starts with a brief overview of silk protein. The silk protein fibroin structure and morphologies are included as these aspects are highly relevant to the applications of silk biomaterials in tissue engineering. The different silk based platforms are discussed, followed by an account of their applications in tissue regeneration. 2. Silks of silkworms: source, chemistry and structure Silk proteins are present in glands of silk producing arthropods (such as silkworms, spiders, scorpions, mites and bees) and spun into fibers during their metamorphosis. Silkworm's silk is an established fiber extensively used in the textile industry. On the other hand, the cannibalistic nature of spiders restricts the commercial production of spider silk [5]. Additionally, the yield of fiber from a single silk cocoon is 600–1500 m, compared to only ~137 m from the ampullate gland of a spider and ~12 m from the spider web [11]. Spider silks are also heterogeneous in nature. Therefore, silk based biomaterials are commonly prepared from silkworm silk. Of note is the silk produced by Bombyx mori, a member of the Bombycidae family. B. mori silk is also known as mulberry silk. Another silk producing family is Saturniidae and the silk is known as non-mulberry silk. Silk has several major advantages over other protein based biomaterials, which are derived from tissues of allogeneic or xenogeneic origins. As such, the risk of infection is high for those materials. Processing of such materials is also expensive due to the stringent protein isolation and purification protocols. In contrast, silk is an established textile fiber and nearly 1000 metric tons of silk are produced and processed annually. Silk fiber purification is routinely carried out using a simple alkali or enzyme based degumming procedure, which yields the starting material for sericin free silk based biomaterials. It is also economically advantageous to use silk for biomedical applications, because of available large scale processing infrastructure of traditional silk textile industries. Silk possesses large molecular weight (200–350 kDa or more) with bulky repetitive modular hydrophobic domains, which are interrupted by small hydrophilic groups [12]. The N and C termini of silk fibroin are highly reserved [5]. Silk fibroin of B. mori is composed of a heavy (H), and a light (L) chain linked together by a disulfide bond [13]. A 25 kDa glycoprotein, named P25, is also non-covalently linked to these chains [14]. The hydrophobic domains of H chains contain Gly-X (X being Ala, Ser, Thr, Val) repeats and can form anti-parallel β-sheets. The L-chain is hydrophilic in nature and relatively elastic. P25 protein is believed to play significant role in maintaining the integrity of the complex [15,16]. H-fibroin, L-fibroin, and P25 are assembled in the ratio of 6:6:1 in mulberry silk [17]. Non-mulberry silks lack light (L) chain and P25 [2,16]. Instead, they contain heavy (H) chain homo-dimers with a molecular weight of ~330 kDa formed by individual proteins (~160 kDa) [18]. Non-mulberry Saturniidae silks exhibit a

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Fig. 1. Schematic representation of biomaterials fabricated from silk fibroin: (a) hydrogels; (b) lyophilized powder; (c) 3D porous scaffolds; (d) native silk mat; (e) silk microparticles.

higher Ala/Gly ratio and poly-alanine blocks, which form β-sheets [19]. They also have higher ratios of basic/acid, polar/non-polar, bulky/ non-bulky and hydrophilic/hydrophobic ratios of amino acids [20,21]. As a result of such variations, there are significant differences in the mechanical properties, bioactivity and the degradation behavior between mulberry and non-mulberry silks [22]. Apart from the primary organization, secondary structure and the hierarchical organization of silk fibroin determine many of its biomaterial properties. The hydrophobic domains of the silk polymeric chains consisting of the repeated amino acid sequence which are assembled into nano-crystals (β-sheet). The hydrophilic links between these hydrophobic domains consist of bulky and polar side chains and form the amorphous part of the secondary structure [23,24]. The chain conformation in amorphous blocks is random coil, which gives elasticity to silk [25,26]. The critical factors that determine the mechanical properties of any particular silk are the precise control of size, number, distribution, orientation and spatial arrangement of crystalline and non-crystalline domains at the nanometer scale [5,27]. Nano-crystals contribute to the outstanding mechanical properties of silk, despite of defects in the microstructure in the form of vacuoles and micro-voids [23,28]. Apart from the secondary structure, a hierarchical supra molecular organization is also evident in silk fibers [24]. Spider and silkworm silks are composed of microfilament bundles (0.5–2 μm), each of which is made of nano-crystals and/or semi crystalline domains [29–31]. Despite some variation in the primary organization and structural features at the nanometer scale between silk types, all silkworm silk fibers follow similar hierarchical structural arrangements. 3. Characteristics of silk fibroin as biomaterial The major advantage of silk compared to other natural biopolymers is its excellent mechanical property. Other important advantages include good biocompatibility, water based processing, biodegradability

and the presence of easy accessible chemical groups for functional modifications. These advantages are elaborated in the following sections. 3.1. Mechanical properties Silk offers an attractive balance of modulus, breaking strength, and elongation, which contributes to its good toughness and ductility (Table 1). Silk fibers are tougher than Kevlar, which is used as a bench mark in high performance fiber technology [25,32]. The strength-to-density ratio of silk is up to ten times higher than that of steel [33]. Spider silk fibers in particular have high extensibility and exhibit marked strain hardening behavior [34]. Strain hardening refers to increase in stress as a fiber is extended in the plastic region beyond the yield point. Such behavior is particularly important for energy absorbing materials. Stress–strain curves of the wild silkworm silk fibers exhibit shape and strain hardening similar to that of spider or dragline silks [22,35]. Considering the good strength and toughness of silk fibers, it is no surprise that silk has been exploited to Table 1 Tensile properties of silk polymeric fibers. Source organisms

Tensile strength (g/den)

Tensile modulus (g/den)

Breaking strain (%)

References

Bombyx mori Antheraea mylitta Philosamia cynthia ricini Coscinocera hercules Hyalophora euryalus Rothschildia hesperis Eupackardia calleta Rothschildia lebeau Antheraea oculea Hyalophora gloveri Copaxa multifenestrata

4.3–5.2 2.5–4.5 1.9–3.5 5 ± 1.2 2.7 ± 0.9 3.3 ± 0.8 2.8 ± 0.7 3.1 ± 0.8 3.1 ± 0.8 2.8 ± 0.4 0.9 ± 0.2

84–121 66–70 29–31 87 ± 17 59 ± 18 71 ± 16 58 ± 18 54 ± 14 57 ± 15 48 ± 13 39 ± 6

10.0–23.4 26–39 28.0–24.0 12.1 ± 5.1 11.1 ± 5.8 9.5 ± 4.4 11.8 ± 5.5 15.5 ± 6.7 14.5 ± 6.6 19.3 ± 6.9 4.1 ± 2.7

[22] [22] [22] [202] [202] [202] [202] [202] [202] [202] [202]

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develop scaffolds for load bearing tissue engineering. However, in current design of silk based biomaterials, the wide choice of mechanical properties available from using different silk types is not fully explored. A biomaterial implant fails either due to poor mechanical properties needed for the application, or higher than optimum mechanical properties due to inappropriate stress concentration at the implant tissue interface. Therefore, variations in mechanical properties of different type of silk provide good choice to match material properties. In general, the wide choice of extensibility and elasticity of silks, good strength and strain hardening from different varieties of silk provide outstanding advantage to developing a range of silk based biomaterials. It is important to note that despite excellent mechanical properties of native silk fibers, most of the silk materials developed from silk fibroin solution are weak and brittle. For example, dry tensile strength of silk film is about 0.02 GPa and elongation at break is less than 2% compared to native fibers that have a tensile strength of about 0.5–0.6 GPa and elongation at break 10–40% [22,36]. Such difference can be attributed to the lack of appropriate secondary and hierarchical structure in the regenerated materials as compared to the native fibers [37,38]. Recent studies show that there are scopes to significantly improve the strength of regenerated silk products to the level of native fibers or even higher through manipulation of structure during regeneration [39–41]. Such investigations are still at the proof of concept stage and relevant mostly to regenerated fibers. More attempts are warranted to improve both strength and elongation of regenerated silk materials. The prospects of application of silk in a range of tissue engineering applications will improve if mechanical properties can be tailored to the required level based on application needs.

3.2. Biocompatibility The long history of success of silk sutures has made silk a known biocompatible material [31,32]. But, like any other non-autologous biomaterials causing foreign body response, some adverse immunological events associated with silk proteins cannot be ruled out, particularly due to its non-mammalian origin. Some incidents of delayed hypersensitivity of silk sutures in rare cases are suggested to be due to the presence of silk gum-like protein sericin [33,34]. Further studies employing isolated silk sericin and sericin based biomaterials have provided no clear evidence to suggest sericin as the source of adverse effects [35]. Detailed investigations are needed to specifically identify the source of any cyto-toxic non fibroin elements in silk and develop appropriate diagnostic method. The immunogenicity and antigenicity of silk scaffolds are well tested. Silk fibroin bio-conjugates during the treatment of musculo-skeletal disease have shown well tolerated response [42]. There are no signs of infection during the subcutaneous implantation of electro-spun fiber mats in rats for up to 8 weeks, although some typical accumulation of phagocytes and lymphocytes is observed. Additionally, there is only minimal inflammation according to microscopy of hematoxylin and eosin stained tissues [43]. Silk 3-D scaffolds when subcutaneously implanted in Lewis rats generate very mild immune response at the end of one year; overall expression levels for all the genes assigned to immune response including TNF-α IFN-δ, IL-4, IL-6 and IL-13, become undetectable for most type of silk sponges [44]. A pig model used in ligament tissue engineering shows no evidence of malfunction after 24 weeks of in-vivo culture [45]. Overall, these studies give a wide spread acceptance that properly degummed and sterilized silk products have good biocompatibility and can be compared with other commonly used biomaterials, such as poly (lactic acid) and collagen [42]. As a consequence of extensive studies in recent years, some silk based materials are received regulatory approval for use in expanded biomaterial devices for plastic and reconstructive surgery. For example, ISO 10993 biocompatibility testing

under good laboratory practices (GLP) shows that silk based Seri Fascia surgical mesh meets the biocompatibility requirements [46]. Despite the encouraging results, there still remain some questions about long term safety of silk biomaterials in the human body. First, silk sutures remain in the body only for a limited time until removed depending on the wound healing period. As silk products for tissue engineering are required to be in contact with tissues for a prolonged time period, the long term responses of innate and adaptive immune system based on location of implant site and type of construct using appropriate in-vivo models warrant further investigation. Second, there may be concerns on the immune reaction in response to degraded products of silk biomaterials, depending on their size and morphology [35]. It is recognized that one of the major causes of failure of any biomaterial implant is the generation of particulate debris, which may trigger the immune system. Report shows that fractions of silk fibers are able to induce mild pro-inflammatory cytokine production and increased phagocytosis [37]. Similarly the digestion of C-terminal of A. pernyi silk with α-chimotrypsin results weak cellular attachment and low growth, suggesting degradation reduces cyto-compatibility [47]. The degraded products of silk fibroin may also cause amyloidogenesis as reported by Lundmark et al. [48]. Their observation suggests the potentiality of B. mori solution to facilitate the accumulation of amyloid, resulting in tissue degeneration. Hence, long term investigations on degraded products are necessary in order to fully alleviate any concerns for the use of silk scaffolds in clinical applications. 3.3. Biodegradation The silk biodegradation is studied based on mass loss, change in morphology and analysis of degraded products in-vitro. Similarly, degradation is tested in animal models by testing mechanical properties of silk after implantation for certain time and studying structural integrity by histological examinations, fluorescent staining and various biochemical assays (Table 2). As implanted construct, regenerated silk fibroin biomaterial degrades much faster than fibers. The degradation rate is dependent upon the secondary structure of silk resulting from preparation of regenerated silk materials [49]. The term biodegradability is often used to discuss the disintegration of silk materials. According to the definition of Vert et al., biodegradability is the degradability of an implantable polymer by biological elements giving fragments, which can move away from the site through fluid transfer but not necessarily from the body [50]. On the other hand, bio-sorption is total elimination of the initial foreign material either through filtration or metabolization of the degraded bio-products [50]. Evidence of in-vivo degradability of silk is shown by Wang et al. [44]. Their study reveals that, upon implantation in Lewis rats, water based 3-D scaffolds were disintegrated in a few weeks and completely disappeared after 1 year. Host immune system has significant impact on the degradation of 3-D silk fibroin porous scaffolds and degradation of silk sponge is shown to be mediated by macrophages, suggesting that silk is not only biodegradable but also bio-resorbable [44]. In in-vitro models, protease XIV from Streptomyces griseus is the most widely used model enzyme for silk [51–55], followed by α-chymotrypsin from bovine pancreas [52,53,56]. The rate of enzymatic degradation further can be regulated by gamma-radiation [57]. Degradation of the silk systems is shown to be mediated also by cells in-vitro. The osteoblasts and osteoclast cells could erode silk films via expression of metalloproteinases (MMPs) and integrin [58]. Such results are encouraging because native extracellular matrix is continuously remodeled in-vivo by synchronous proteolytic degradation via MMPs and matrix regeneration [59]. The in-vitro studies are useful to compare the biodegradability among different silk materials and structures. However, the type and the concentration of enzymes at the site of the material implant may vary significantly. This may put limitations on predicting degradation of silk compared to synthetic biomaterials that degrade hydrolytically

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Fig. 2. Confocal laser micrographs showing the attachment and morphology of cells on silk fibroin biomaterials. The cells were stained with rhodamine for the actin filaments and Hoechst for nucleus. (a) Hepato-carcinoma cells on the porous B. mori silk fibroin scaffold; (b) human osteosarcoma cell line (MG-63) on the surface of silk fibroin; (c) feline embryo fibroblasts (AH 927) on the surface of silk fibroin hydrogel. All the cells revealed well stretched healthy morphology on silk biomaterial surfaces.

and have minimum site-to-site or patient-to-patient variations [4]. The silk has clear advantages over other biomaterials in several aspects of biodegradation. For synthetic biomaterials such as polyglycolides and polylactides, which are approved by regulatory authorities, degraded products are resorbed through metabolic pathways but release of acidic by-products is an issue of concern. There are no such issues associated with silk. In addition, those synthetic materials may decrease mechanical properties very early during degradation [4,60]. On the other hand, retaining strength over a long time by many silk systems can be an advantage for silk, particularly in tissue engineering, where slow degradation and load bearing capacity are required. Despite such advantages, deep understanding on degradation and clearing mechanisms of silk requires further investigations, which would encourage the development of silk as an important biodegradable and bio-resorbable material. 3.4. Water based processing The silk fibroin is water soluble in its α-helical and random coil forms. Solubility can be maintained over days and even weeks depending on the storage temperature, pH and concentration of silk solution [61]. Hence, silk based systems can be prepared using water

based solution under mild manufacturing conditions such as room temperature, neutral pH and without application of high shear force. Such conditions are favorably exploited for loading sensitive drugs into silk implants [62,63]. Mild processing conditions are also helpful for photonic or electronic devices or biosensors, which may be incorporated within a silk based system or coated with silk for improved bio-integration in-vivo. Conformational transition of α-helix and random coil to highly stable β-sheets is required in silk products to provide good resistance to dissolution, thermal and enzymatic degradation. This can be achieved through water vapor annealing, mechanical stretching and ultrasonic treatments, hence avoiding the use of harmful chemicals. These processing advantages and good structural stability make silk a promising polymeric system for bio-related applications. 3.5. Manipulation of silk properties through structural re-adjustments Silk structure can be adequately tuned during spinning or regeneration to obtain different secondary structures to manipulate material properties. For example, forced extrusion of silk gland protein through silkworm spinnerets attain appropriate altered fiber microstructure with significantly high fiber toughness [64]. Such options, if optimized for other forms of silk based materials, may offer the advantage of matching their load bearing properties with that of the

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Table 2 Investigatios carried out on biodegradation of silk materials. Material forms

In-vitro/In-vivo

Type of study

• Mass loss, microscopy, secondary structure, mechanical properties of substrate • Particle size, amino acids and molecular weight of degraded products • Tissue ingrowth and biomechanical analysis tissues B.mori 3-D sponge • In-vitro using protease XIV • Mass loss • Intramuscular and subcutaneous • Degradation rate vs. cell metabolism and bone implantation in rats regeneration from MSC 3-D sponge with different pore size and • Intramuscular and subcutaneous • Structural integrity of sponge and invasion by giant β-sheet contents implantation in Nude and Lewis rats cell and macrophage based on histology and gene expression of tissue samples by real time RT-PCR Knitted silk mesh and silk sponge composite • Anterior cruciate ligament of pig • Macroscopic view, tensile strength, μCT scan and histology B. mori cast films and thin crystallized films In-vitro using protease XIV, XXI/ trypsin/ • Mass loss, microscopy and secondary structure α-chymotrypsin/ collagenase of substrate • Mass spectroscopy to determine molecular weight of degradation products • Enzyme degradation vs. drug release B. mori water annealed patterned silk film • Bone cells mediated degradation • Microscopy and essays on metalloproteinases production by cells B. mori electro-spun mat and wet spun fibers • In-vitro using protease XIV, K/actinomyces • Mass loss and microscopy • Fluorescein assisted microscopy • Subcutaneous and intra-muscle implantation in rats B. mori silk solution • Actinase • Molecular weight and amino acid analysis B. mori silk particles • Proteases • Particle size, morphological imaging, mass loss, secondary structure, amino acid analysis B. mori/A. pernyi native fiber/surgical mesh

• In-vitro using protease XIV/collagenase IA,/α-chymotrypsin • Implantation in rats

targeted tissues. Water annealing is also used to induce insolubility in silk products [65,66]. Water annealed films are more flexible and degraded faster than methanol treated films [65]. Excellent opportunities are available to use the proticionic liquid system to manipulate the structure and properties of regenerated silk materials [67]. Moreover, process induced variations in structure and surface topography may affect properties such as biodegradation [49], cell interaction [68] and drug release kinetics [69]. For example, a temperaturecontrolled water vapor annealing (TCWVA) technique is used to program the crystallinity in order to tune thermal, mechanical and biodegradation properties of the silk films [49]. Similarly, slowing down the proteolytic degradation by structural adjustments can reduce the release kinetics of drugs from a silk system [57]. Although windows of opportunities are demonstrated in all these cases, more studies are needed to understand the structure-property relationships for adequate control of material properties. Such control will be a key to the success of silk as a natural biopolymer for tissue regeneration. 4. Morphological diversification of silk biomaterials for tissue regeneration Degumming (removal of glue protein sericin) is the first step in silk fiber processing. To produce various other material formats, degummed silk fiber is dissolved to obtain silk solution. In some cases, where it is difficult to dissolve fibers, fibroin can be extracted directly from glands of silkworms using an appropriate buffer solution [70]. Different solid forms are then prepared from silk solution by liquid solid phase transfer. Schematics of different silk based biomaterials are represented in Fig. 1. Both native fibers and materials regenerated from silk solution are used for tissue engineering. 4.1. Native silk structures Degummed silk fibers can be used to form various twisted structures including rope, cable, braided and textured yarns for tissue regeneration [38]. In addition, cocoons are also used to construct non-woven structures by partially dissolving them to use as a cell supporting template, where the arrangement of filaments in the cocoon is preserved to maintain the porous structure of the mat [37,71]. An alternate way of using

Ref. [46,52,203]

[203,204]

[44]

[45] [43,53–56,205–209]

[58] [43,210,211]

[212] [213,214]

silk filaments directly in tissue engineering is making a knitted silk structure to reinforce 3-D porous tissue engineering scaffolds. Such reinforcement improves the mechanical properties of scaffolds for applications in load bearing tissue engineering, such as ligament [45,72]. 4.2. Regenerated silk morphologies To prepare silk solution for regenerating different silk formats, concentrated solutions of chaotropic salts such as LiBr, CaCl2/ethanol/ water, LiSCN [73–75] or ionic liquids [67,76] are commonly used. These solutions usually fail to dissolve non-mulberry silk fibers [77,78]. Attempts using Ca(NO3)2 at 105 °C and LiSCN at 40 °C–55 °C to dissolve Chinese oak tasar silk fiber are reported [79,80]. However as the total dissolution in such cases still remains a challenge, gland proteins are used mostly for further processing. 4.2.1. Films Silk fibroin films can be produced by casting the aqueous [81], acidic [39,81] and ionic [82] silk solution. Fabrication of silk films by spin coating and Langmuir-Blodgett (LB) process is also reported [82,83]. Additionally, manual or spin assisted layer by layer deposition techniques have been used to produce very thin films [84]. As stability of such cast films is low, techniques like controlled drying [85], water annealing [49], stretching [86], and alcohol immersion are used to improve β-sheet crystallinity. It is often necessary to control surface properties of silk films for guided and enhanced cell growth or to change the optical properties. Lithography and advanced printing systems are employed to achieve such features [8,87]. 4.2.2. Electro-spun and wet-spun fibers The large surface area and porous structure of electro-spun silk nano-fiber mats are useful for cell seeding [86]. 3-D constructs of nano-fibers are used as blood vessel grafts and nerve guides [88,89]. Wet spinning [39] or micro-fluidic solution spinning [90] are also employed in making regenerated silk fibers. Wet-spun fibers are typically in the micrometer scale in fiber diameter, and can be produced on a much larger scale than nano-fibers. Advantages of such regenerated fibers over the native silk fibers include the ability to

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tune fiber morphology and properties based on application, and incorporation of bio-molecules while regenerating from silk solution. 4.2.3. Hydrogels Silk hydrogels are formed through sol–gel transition of aqueous silk fibroin solution in the presence of acids, dehydrating agents, ions, sonication or lyophilization [44,91–93]. Sol–gel transition can be accelerated by increasing the protein concentration, temperature, and addition of Ca 2+[94]. Silk hydrogels can be useful for injectable or non-injectable delivery systems. Mechanical properties of silk hydrogels are found to be suitable for preparing scaffolds for load bearing tissue engineering such as cartilage regeneration [95]. 4.2.4. 3-D porous scaffolds Porous 3-D sponges are ideal structures for tissue engineering scaffolds as they closely mimic the in-vivo physiological microenvironment. Silk scaffolds are prepared by freeze drying, porogen leaching and solid free form fabrication techniques [96–98]. The freeze dried sponges possess pore sizes below 100 μm and the pore sizes can be controlled by adjusting the freezing temperature, pH of the solution and amount of organic solvents [97]. Repeated freezing and thawing processes can increase pore sizes from 60 to 250 μm [99]. A better control over pore structure can be obtained from solvent casting/particle leaching or gas-foaming methods [98]. Due to good control over porosity and pore sizes, porogen leached 3-D silk scaffolds are commonly used in tissue engineering applications, predominantly bone and cartilage [100]. To obtain good mechanical and biological outcomes, composite silk 3-D scaffolds are prepared by incorporating inorganic [101] or organic [102] fillers. Usually fillers are incorporated during scaffold fabrication process to ensure their homogeneous distribution. However, particle addition after fabrication is also reported [101]. The challenge in composite design is the compatibility between the components. Poor compatibility between components results in inhomogeneous mixtures, phase separation and adverse tissue reactions [103]. To ensure good compatibility, silk–silk composite scaffolds are fabricated by incorporating milled silk particles in porous silk sponge, resulting in significant improvement in compressive modulus from less than 50 kPa to about 2.2 MPa [104]. Further modifications to scaffolds include reinforcement with fine silk fibers to gain further improvement in modulus to about 13 MPa [105]. Such mechanical properties may be sufficient for regenerating cancellous bone, but still way short in meeting the practical requirements of load bearing bone tissue engineering. To obtain implantable tissue construct without using metal support, further development is still required in design of scaffolds for bone regeneration. Silk composites reinforced with knitted silk mesh are used as ligament scaffolds. It is revealed that after

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24 weeks post implantation period, there is uniform distribution of cells throughout the construct [72]. The findings suggest the suitability of silk composites for applications where mechanical properties are important. 4.2.5. Particles Silk micro and nano-particles are produced from silk solution by freeze drying and grinding [106], spray drying [107], jet breaking [108], self-assembly [109,110] and freeze-thawing [111]. Milling silk fiber is an alternate approach to make silk particles directly from fibers without using any chemicals [104]. While milled particles are used for reinforcing scaffolds to improve mechanical properties and cellular outcomes, regenerated silk particles are mostly used in drug carrier applications [104,112–115]. Thus it will be of interest to see if the particles can play the dual role of improving mechanical properties of scaffolds and at the same time act as a carrier of growth factors for rapid tissue regeneration. The challenge here is to make very fine and uniform silk particles, which is not easy with the milling approach. 5. Applications of silk fibroin biomaterials for tissue regeneration Substitution of a human body part with a biomaterial requires good communication between the host and implanted system for a successful outcome. The limitations of existing implants are summarized in Table 3. To overcome such limitations, silk based tissue engineering systems are trialed, which are included in the following sections (cellular responses on silk biomaterials are represent in Fig. 2. 5.1. Vascular tissue regeneration Silk based regenerated vascular tissues are clinically used as flow diverting devices and stents [116,117]. In the case of a study related to flow-devices, two out of the three patients show promising outcome, suggesting silk as an attractive option to treat fragile blood-blister like aneurysms. Silk stents are also employed in the reconstruction of an intra-cranial aneurysm artery. There has been a successful attempt to fabricate a tubular ~ 3 mm blood vessel from silk with a thickness of 0.15 mm having an average tensile strength of 2.42 MPa [118]. The burst strength of silk tubular vessels is found to be 811 mm Hg compared to 1800 mm Hg of the gold standard saphenous veins [119,120]. The implantation of vascular graft of silk fibroin composites of B. mori and transgenic silkworm into rat abdominal aorta results excellent patency (ca. 85%) after a year [121]. Composites of silk fibroin and human-like-collagen or double-raschel knitted silk-poly(ethylene glycol diglycoldiglycidyl ether) are successfully used to develop vascular constructs [122].

Table 3 The limitations of presently available commercial products or approaches. Targeted organ or approaches

Peripheral nerve grafts

Cornea Skin Ligament Heart

General practice

Limitations

References

Autologous grafts Homograft Conventional prostheses of polytetrafluoroethylene (ePTFE) and polyethylene terephthalate Autologous nerve grafts Neurolac®, CultiGuide®, SaluBridge®, Neurotube®, Surgisis®, NeuroMatrix®–Neuroflex® NeuraGen® Revolnerv®

Low quality, vasospasam (spasm of blood vessels) , restricted length Fast degeneration Grafts of >5 mm in diameter suffer from thrombosis Limited graft availability Secondary deformities Dissimilarity in tissue organization and size restricted to short defects (b3 cm)repair of the small caliber nerves Limited short-term clinical studies and patient follow-up available till date High host rejection rate Inadequate availability Limited permanent revascularization Compromises normal healthy tissue and prolonged surgical time Risk of disease transmission and immune rejection Scarcity of donors High cost

[215–217]

Synthetic keratoprosthesis Allograft Xenografts Autograft Allografts and xenografts Heart transplantation left ventricular assist devices

[218–221]

[222,223] [224] [225,226] [227]

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While the former composition is able to provide good tensile strength to the construct, the latter is able to avoid early thrombosis. An aqueous gel spinning process is also employed to design a blood vessel like tubular structure [123]. Critical requirements for designing blood vessels include survival under the changes in blood pressure, ability to sustain cyclic loading, compatibility with the adjacent host vessels, and anti-thrombotic lining [124]. Silk fibroin possesses an anti-thrombotic surface with good resistance to high shear stress and blood flow pressure [86,125]. Moreover, silk fibers also support the intercellular contact in endothelial cells [126]. The challenge to include a selection of suitable cell sources still remains. Primary human endothelial cells and endothelial cell lines (HPMEC-ST1.6R and ISO-HAS-1) seeded in silk fiber are unable to fill the gaps between two adjacent silk fibers [127]. The optimization of co-culture of endothelial cells and smooth muscle cells to get a well-organized endothelium [89], sulfation of silk for better anti-coagulant activity [128] and coating of matrigel to enhance endothelium coverage [89] are suggestive positive development towards native vascular tissue construction under current situation. 5.2. Neural tissue regeneration The human nervous system is broadly classified into (a) the central nervous system (CNS) and (b) the peripheral nervous system (PNS). The PNS is capable of self-healing minor injuries, while large damage needs to be treated surgically with nerve grafts harvested elsewhere in the body. Therefore, in such treatments, tissue engineering is highly relevant and the compatibility of scaffolding material with neuro-progenitor cells assumes significance. Silk fibroin supports the viability of dorsal root ganglia and schwann cells without affecting their normal phenotype or functionality [129]. Composites of silk fibroin with chitosan or poly(l-lactic acid-cocaprolactone) are able to bridge a gap of a 10 mm long sciatic nerve defects in rats [130–132]. Furthermore, an assembly of B. mori silk fibroin and Spider X® fiber (a spider silk like fiber) is effectively able to bridge up to 13 mm nerve gap within 12 weeks [133]. The micro-architecture of scaffolds play important role in the regeneration of nerves. Improved length and axonal outgrowth of chicken embryo dorsal root ganglion sensory neurons and spiral cord motor neurons are observed on aligned silk nano-fibers, compared to slow and random orientation of axonal growth on non-aligend fibers [134]. The orientation of pores and addition of neurotrophic factors for enhanced neuronal growth are being presently investigated to enhance the outcome of silk based nerve grafts. 5.3. Skin tissue regeneration The skin is the biggest organ in human and acts as barrier to the infectious organisms. It has limited self-healing capability. Large damage to the skin causes loss of skin integrity, which may lead to death. The adult human skin consists of two major layers: the epidermis (a keratinized layer) and the dermis (collagen-rich layer). Appendages like hair and hormonal glands are raised from the epidermis but rooted deeply within the dermis. This complex structure makes the engineering of skin a challenging task. Silk fibroin supports well the human keratinocytes and fibroblasts [86]. However the complex structure of native tissue requires a composite scaffolding material. Bio-mimicking approaches with other natural extra-cellular materials are proposed. Layering silk fibroin with collagen-I enhances the attachment and dispersion of keratinocytes, while fibronectin coating supports both keratinocytes and fibroblasts cells adhesion and dispersion within the matrix [135]. Structural integrity of silk matrices induced by different treatments also influences greatly the cell adherence. Both human keratinocytes and fibroblasts prefer water vapor-treated silk nano-fibrous mats over methanol-treated ones,

suggesting that selecting appropriate processing route is also important for silk based biomaterial designing [135]. Other silk composites successfully used are nano-fibrous silk-chitin [136], silk-collagen [137] and intermolecular cross-linked recombinant human-like collagen (RHLC) with fibroin [138]. Additionally, silk fibroin-alginate blended scaffolds sufficiently favor re-epithelization in a fullthickness rat wound model [139]. These findings suggest that blended silk fibroin may have better prospect than pure silk fibroin for skin tissue regeneration. 5.4. Bone tissue regeneration Bone is a specialized connective tissue composed of calcified extracellular matrix, which has collagen type I and hydroxyapatite as the major components [140]. Thus the scaffolding material for bone tissues must ensure matrix toughness and matrix deposition. In this context, silk fibroin is a rational choice for its high toughness and mechanical strength along with good bio-compatibility. Silk fibroin based bone tissue engineering is one of the most extensively studied tissue engineering approaches to date [10]. Porous fibroin scaffold based bone constructs are able to stimulate advanced development of bone tissues within 5 weeks, when engineered within a bioreactor [100]. Silk fibroin scaffolds also promote human mesenchymal stem cell based healing of femoral defects in nude mice [141]. Furthermore, the loading of human mesenchymal stem cells within silk fibroinpolyethylene oxide nano-fibrous composite scaffold with bone morphogenic protein-2 led to regeneration of bone like tissue [142]. Incorporation of hydroxyapatite nano-particles into silk matrix improves bone regeneration in animals [143,144]. The incorporation of n-Hap within the fibroin sheet and subsequent culture of rat bone marrow mesenchymal stem cell (BM-MSCs) demonstrates the cell supportive nature of embedded Hap crystals and successful osteogenic differentiation of BMMSCs [145]. These composite scaffolds completely heal the segmental bone defect in Sprague–Dawley rats after 12 weeks post implantation [143]. Silk fibroin also guides full-thickness growth of calvarial defect (8 mm-diameters) in rats within 12 week [100]. Similar results are also obtained when silk hydrogel [92,146] or non-woven mats [147] are used. The strategies to improve bone regeneration using silk-silk composites are already discussed in the Section 4.2.4. The vascularization of in-vitro tissue models is always a problem. The pre-incubation of silk matrices with cells before implantation in animals increases the vascularization in neo-implant [148]. Under such condition, the rate of vascularization is directly proportional to in-vitro incubation time. Co-culture of osteoblasts with endothelial cells on silk scaffolds results in the formation of micro-capillary [149] and pre-vascular structures [150]. Furthermore, upon implantation, these pre-mature micro-capillaries survive the host defense system and become functional micro-capillaries [151]. Further investigations on scaffold architectures and pore distribution are needed to obtain the completely vascularized 3-D bone tissue. 5.5. Cartilage tissue regeneration Cartilage is a non-vascular, non-innervated connective tissue. The 3-D porous fibroin scaffolds [152–155], electro-spun silk fibers treated with micro-wave induced argon plasma [156], silk fibroin blended with chitosan [157] or genipin cross-linked chitosan-fibroin porous sponges [158] provide adequate support to chondrocytes. Nonmulberry gland silk fibroin of A. mylitta [159] also acts as promising material for cartilage development. Insulin-like growth factor 1 (IGF-I) is a regulatory molecule in chondrogenesis [160], thus can be incorporated within scaffolds for better chondrogenic outcomes [160]. Bioreactors provide mechanical stimulation and maturation of cartilaginous constructs [161]. Therefore, the hydrodynamic factors are important in chondrogenic outcome. Other factors to be taken

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into serious consideration for regenerating the cartilaginous tissues are cell sources [162], scaffold architectures, pore sizes and pore distribution.

sulfate–laminin mixtures enhances the performance of fibroin [175]. Successful pre-clinical reconstruction of human limbal epithelium on silk membranes is also reported [176].

5.6. Ligament and tendon tissue regeneration

5.9. Hepatic tissue regeneration

The engineering of ligaments and tendons requires scaffolding material with excellent combination of mechanical strength, elasticity, toughness and structural integrity. The first successful silk based engineering of anterior cruciate ligament (ACL) employs twisted scaffold, the mechanical property of which is comparable to human ACL [38]. Synergistic incorporation of silk fibers within the collagen matrix [163], coating of silk fibroin on electro-spun poly-lacticco-glycolic acid (PLGA) nano-fibers [164], loading basic fibroblast growth factor (bFGF) and transforming growth factor-β (TGF-β) in optimal regimen within the scaffold stimulates the biochemical and mechanical pathways for ligament tissue regeneration [165]. Further approach to design scaffold-free ligament analogs involves incorporation of porcine bone marrow MSCs on laminin coated silk sutures as anchor points [166]. As a result, the cells within the construct are self-organized into cohesive rod-like tissues, which mechanically and histologically resemble the native ligament. Silk fibroin scaffold of A. pernyi is also able to repair defects in Achilles tendon of an adult New Zealand white rabbit [167]. The recovery of Achilles tendon in the rabbit shows the feasibility of using non-mulberry silks along with mulberry silk as suitable tendon scaffolds.

Liver plays crucial role in the metabolism of carbohydrates, proteins, lipids and vitamins. The chief cellular component of hepatic tissue is hepatocytes, which is employed in-vitro to reconstruct the 3-D tissue or liver patches. The silk composites for hepatic tissue engineering includes functionalized silk with lactose and cyanuric acid [177], silk fibroin blended films with collagen [178], recombinant human-like collagen [179], silk fibroin-collagen-heparin scaffolds [180], and silk micro-particle embedded PLA scaffolds [181]. Blending of silk fibroin with collagen causes mild inflammation in the omentum of rats [182]. Furthermore, non-mulberry A. assama fibroin based multi-scale scaffolds provide adequate mechanical support and help cellular response [183]. Functionalization of A. assama fibroin with sulfone results enhanced blood-compatibility [184]. However, as hepatocytes carry a degree of structural organization, it forms large cellular aggregates in long term in-vitro culture. These complex cellular aggregates make it difficult for nutrient diffusion and thus require further investigation in designing silk scaffold for complete tissue regeneration.

5.7. Cardiac tissue regeneration The loss of cardiomyocytes after injury reduces cardiac function, which further leads to morbidity and mortality. A possible treatment is engineering an artificial heart or cardiac patch in-vitro followed by implantation. Chitosan or hyaluronic acid (HA) loaded silk fibroin seeded with rat mesenchymal stem cells is able to generate cardiac patches [168]. Silk protein fibroin 3-D scaffolds of A. mylitta also show good outcome without the employment of other extra cellular matrix materials, producing beating patches of rat cardio-myocytes in-vitro [169]. Critical issues still to be resolved are suitable silk or silk based composite biomaterials supporting the mechanical strength of the heart valves, formation of elastin by employing co-culture system with fibroblasts cells, employment of human serums or platelet lysates during in-vitro culture to minimize the tissue engineered graft rejection. 5.8. Ocular tissue regeneration Allograft approach of corneal tissue regeneration suffers from bio-burden [170], while the use of silk fibroin results in a much better outcome. Optical transparency of silk fibroin films and stability of aqueous protein solution at neutral pH are the key characteristics that favor silk for functional bio-photonic applications over other synthetic or inorganic materials [6]. Silk films are stacked into a 3-D porous structure mimicking closely the heli-coidal organization of cornea in-vivo. When these 3-D structures are cultured with human and rabbit corneal fibroblasts, cells reveal natural corneal keratocyte morphology [171]. Silk fibroin implants with embedded rabbit cornea epithelial cells become translucent within 4 weeks and results in the formation of new limbus and blood vessels within 8 weeks post-implantation. Complete regeneration of rabbit cornea occurs within 16 weeks leaving behind a few opaque pieces of degraded scaffolds [172]. In addition, the affinity of corneal cells towards a patterned surface [173] and ease in surface patterning in silk films (discussed in detail in another review elsewhere [174]), open up further scopes for silk-biomaterials in ocular regenerative medicine. Coating of fibroin with collagen IV, fibronectin, and chondroitin

5.10. Spinal cord tissue regeneration The grafting of olfactory ensheathing cells (OECs) is one of the most commonly employed approaches to treat spinal cord injuries. Silk based spinal cord regeneration is presently at its neo-natal stage. The culture of OECs on nano-fibrous silk fibroin reveals prospects of silk biomaterials in this area [185]. The diameter of the nano-fibers possesses regulatory effects on growth of OECs [186]; smaller diameters result in better cellular responses than the larger fibers. 5.11. Inter-vertebral tissue regeneration The treatment of degenerative disk disease involves repair of annulus fibrosus, which is one of the major components of intervertebral disk. Porous scaffolds of silk fibroin allow good growth of bovine annulus fibrosus cells up to 8 weeks in-vitro [187]. The growth of bovine annulus fibrosus cells on silk fibroin is greatly influenced by culture condition and average pore sizes of the scaffolding material (≥600 μm) [188]. However, further investigations are required to completely mimic the high strength, elastic and tough morphology of naive inter-vertebral tissues. 5.12. Bladder tissue regeneration In the treatment of stress urinary incontinence, bladder shaped scaffolds with autologous urothelial and smooth muscle cells are required for successful reconstruction of the bladder [189]. Films of silk fibroin provide good support to transitional epithelial cells of urinary bladders of New Zealand rabbits [190]. Silk films in rabbits result in the successful repair of short-length defects of 1.5 cm [191]. Further animal studies include mice by Cannon et al., [192] and murine by Mauney et al. [193]. The silk based sling engineered with bone marrow mesenchymal stem cells exhibits good control on the leak-point pressure comparable to negative control [194]. This suggests a hopeful treatment for stress urinary incontinence. 5.13. Tracheal tissue regeneration The incidence of pre-mature newborns developing tracheal stenosis is on the rise as a result of prolonged incubation. In silk based

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reconstruction of 12 tracheal defects in rabbits, successful generation of full-thickness fibroblasts layer of 240–302 μm is observed, without foreign-body granuloma and macro-phagocyte infiltration [195]. Blending of silk fibroin with chitosan enhances perichondrium wrapping in nude mice, suggesting the suitability of silk based devices in generation of epithelial lining for tracheal transplants [196]. 5.14. Eardrum tissue regeneration Eardrum (tympanic membrane) perforation results pain, infection and even loss of hearing. Surgical treatment to restore chronic perforations is myringoplasty, where autologous grafts, allografts, and synthetic graft materials are commonly used [197]. Recent attempts with silk fibroin membrane have resulted good adhesion and growth kinetics of human tympanic membrane keratinocytes [198–200]. Silk membranes provide better healing compared to conventional paper patch [201]. These findings suggest suitability to engineer silk based eardrum patches. 6. Future prospects Tissue regeneration for therapeutics is very critical and target specific. To complement the functionality of living systems, the constructed tissue must successfully interact with the body's immune system. Silk based designs allow easy control on matrix morphology, degradation rate and conformal adhesion to underlying tissues with low immune-toxicity and good biocompatibility. Recent advancements in understanding silk structure and processing open up new opportunities in the use of various forms of silk in tissue regeneration. Silk systems will be particularly useful for applications where slow biodegradation and good mechanical properties are critically required, such as for bone, ligament and muco-skeletal tissues. Successful applications of silk based materials in tissue engineering depends on further understanding of the long term biocompatibility, biodegradability and its degraded products, along with the ability to tune silk morphologies for tissue specific requirements. Hybrid materials, incorporating 100% silk in different matrix morphologies show promising results in this regard. Acknowledgments The work is financially supported by the Department of Biotechnology and its Bioinformatics Facility, Department of Science and Technology (SCK), and Council of Scientific Industrial Research (for awarding Senior Research Fellowship to B. Kundu), Government of India, New Delhi. R. Rajkhowa and X. Wang acknowledge the support from the Australian Research Council for their silk related work. The authors are thankful to Dr. Tuli Dey, Sunita Nayak and Deboki Naskar for providing the photograph of bone cells on silk. References [1] R. Langer, J. Vacanti, Tissue engineering, Science 260 (1993) 920–926. [2] B. Kundu, S.C. Kundu, Osteogenesis of human stem cells in silk biomaterial for regenerative therapy, Prog. Polym. Sci. 35 (2010) 1116–1127. [3] B.L. Seal, T.C. Otero, A. Panitch, Polymeric biomaterials for tissue and organ regeneration, Mater. Sci. Eng. R 34 (2001) 147–230. [4] L.S. Nair, C.T. Laurencin, Biodegradable polymers as biomaterials, Prog. Polym. Sci. 32 (2007) 762–798. [5] F.G. Omenetto, D.L. Kaplan, New opportunities for an ancient material, Science 329 (2010) 528–531. [6] F.G. Omenetto, D.L. Kaplan, A new route for silk, Nat. Photonics 2 (2008) 641–643. [7] M.K. Hota, M.K. Bera, B. Kundu, S.C. Kundu, C.K. Maiti, A natural silk fibroin protein-based transparent bio-memristor, Adv. Funct. Mater. 22 (2012) 4493–4499. [8] H. Tao, D.L. Kaplan, F.G. Omenetto, Silk Materials — a road to sustainable high technology, Adv. Mater. 24 (2012) 2824–2837. [9] G.H. Altman, F. Diaz, C. Jakuba, T. Calabro, R.L. Horan, J. Chen, H. Lu, J. Richmond, D.L. Kaplan, Silk-based biomaterials, Biomaterials 24 (2003) 401–416.

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