Silk Fibroin As Biomaterial for Bone Tissue Engineering

Acta Biomaterialia xxx (2015) xxx–xxx

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Acta Biomaterialia

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Review article Silk ﬁbroin as biomaterial for bone tissue engineering ⇑ Johanna Melke a,b, Swati Midha c, Sourabh Ghosh c, Keita Ito a,b,d, Sandra Hofmann a,b,e, a Orthopaedic Biomechanics, Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands b Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands c Department of Textile Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India d Department of Orthopaedics, UMC Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands e ETH Zurich, Institute for Biomechanics, Vladimir-Prelog-Weg 3, HCI E355.1, CH-8093 Zurich, Switzerland article info abstract

Article history: Silk fibroin (SF) is a fibrous protein which is produced mainly by silkworms and spiders. Its unique Received 13 June 2015 mechanical properties, tunable biodegradation rate and the ability to support the differentiation of Received in revised form 24 August 2015 mesenchymal stem cells along the osteogenic lineage, have made SF a favorable scaffold material for Accepted 6 September 2015 bone tissue engineering. SF can be processed into various scaffold forms, combined synergistically with Available online xxxx other biomaterials to form composites and chemically modified, which provides an impressive toolbox and allows SF scaffolds to be tailored to specific applications. This review discusses and summarizes Keywords: recent advancements in processing SF, focusing on different fabrication and functionalization methods Bone tissue engineering and their application to grow bone tissue in vitro and in vivo. Potential areas for future research, current Silk fibroin Regenerative medicine challenges, uncertainties and gaps in knowledge are highlighted. Drug delivery Scaffold Statement of significance

Silk fibroin is a natural biomaterial with remarkable biomedical and mechanical properties which make it favorable for a broad range of bone tissue engineering applications. It can be processed into different scaffold forms, combined synergistically with other biomaterials to form composites and chemically modified which provides a unique toolbox and allows silk fibroin scaffolds to be tailored to specific applications. This review discusses and summarizes recent advancements in processing silk fibroin, focusing on different fabrication and functionalization methods and their application to grow bone tissue in vitro and in vivo. Potential areas for future research, current challenges, uncertainties and gaps in knowledge are highlighted. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Contents

1. Introduction ...... 00 2. Bone biology ...... 00 3. Silk fibroin as a biomaterial...... 00 3.1. Structure and mechanical properties ...... 00 3.2. Biocompatibility ...... 00 3.3. Degradation ...... 00 4. Silk fibroin processing methods ...... 00 4.1. Gels ...... 00 4.2. Porous sponges ...... 00 4.3. Electrospinning ...... 00 4.4. 3D bioprinting ...... 00

⇑ Corresponding author at: Eindhoven University of Technology, Department of Biomedical Engineering, Orthopaedic Biomechanics, PO Box 513, 5600 MB Eindhoven, The Netherlands. E-mail addresses: [email protected] (J. Melke), [email protected] (S. Midha), [email protected] (S. Ghosh), [email protected] (K. Ito), [email protected] (S. Hofmann). http://dx.doi.org/10.1016/j.actbio.2015.09.005 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

4.5. Silk fibroin composite scaffolds ...... 00 5. Interaction of osteoclasts with silk fibroin ...... 00 6. In vivo bone regeneration with silk fibroin...... 00 7. Drug delivery for osteogenesis ...... 00 8. Conclusion and outlook ...... 00 Acknowledgements ...... 00 References ...... 00

1. Introduction cells [16], which dissolve crystalline HA through release of hydrochloric acid and a mixture of proteases which degrades the Bone tissue engineering (TE) is a promising strategy to regener- organic bone matrix rich in collagen fibers [17]. Their role in bone ate bone and is regarded as a future alternative to current clinical remodeling involves the removal of cracks after fracture or micro treatments. Not only can tissue engineered constructs be trans- damage occurs but they also function as immune cells and can planted as a graft, but also pave the way for three-dimensional secrete cytokines that can affect surrounding cells [18,19]. Their (3D) tissue models which help to investigate tissue abnormalities differentiation and function is controlled by osteoprotegerin, and to analyze them both at the cellular and molecular level receptor activator of nuclear factor (NF)-kappaB (RANK) and RANK [1,2]. In this whole process, the patient’s own cells could be used ligand (RANKL) [20]. Osteoblasts are derived from mesenchymal which would be a key tool for personalized medicine. The goal is stem cells (MSCs) located in the bone marrow and differentiate to create 3D bone tissues by combining cells, scaffolds and to some towards the osteogenic lineage [21]. Once they become encapsu- extent also growth factors or mechanical stimuli. One of the main lated within their own matrix, they acquire a stellate morphology challenges is the choice of an appropriate biomaterial which can and are referred to as osteocytes [22,23]. Osteocytes comprise mimic the natural bone tissue matrix with its mechanical and bio- more than 90% of all bone cells in adult animals and are considered logical characteristics to support tissue development. Various to be the major cell type responsible for sensing mechanical strain materials have been tested for bone TE purposes [3,4]. Silk fibroin and translating it into biochemical responses which affect bone (SF) has been proven to be a promising biomaterial for scaffold fab- remodeling [24]. In vivo, osteocytes communicate to adjacent rication in general and its remarkable mechanical properties pre- osteocytes via a dense and interconnected canalicular network destine it for bone TE applications [5]. There is a steady increase containing cell processes for rapid signal transduction [25]. When in number of publications and citations on the use of SF as scaffold mechanically activated, they produce bone morphogenetic pro- material for bone TE applications over the last 10 years which sup- teins, Wnts, prostaglandin E2 and nitric oxide, which can modulate ports its significance and potential as a biomedical material for the recruitment, differentiation, and activity of osteoblasts and bone TE. This review seeks to highlight its characteristics and the osteoclasts [26–30]. This ability makes osteocytes the essential various modification options of SF as a biomaterial in bone TE. mediator in orchestrating bone remodeling in response to mechanical stimulation [31]. MSCs are a valuable cell source for tissue regeneration because of their ability to self-renew and to differen- 2. Bone biology tiate along the osteogenic lineage [32]. Their recruitment, homing and subsequent differentiation also plays an important role in the Bone TE relies on our knowledge of bone structure, composi- repair of bone fractures [33]. Readily accessible sources of adult tion, mechanics and tissue formation which makes it crucial to MSCs are bone marrow, adipose tissue or peripheral blood, of have a fundamental understanding of bone biology. Approximately which bone marrow mesenchymal stem cells (BMSCs) have been 35% of the bone tissue is made of an organic part while the remain- most studied [34]. BMSCs can be expanded in vitro to 50 popula- ing 65% is inorganic matrix [6]. The organic extracellular matrix tion doublings [35] which allows to increase their number such (ECM) of bone consists of complex self-assembled macromolecules that a sufficient amount can be prepared for terminal differentia- such as collagens, which make up 90–95% of the organic ECM, tion and tissue regeneration. osteocalcin, osteopontin, osteonectin, bone sialoprotein, hyaluro- nan and proteoglycans [7]. The inorganic mineral phase of bone consists of hydroxyapatite (HA) as well as carbonate and inorganic 3. Silk fibroin as a biomaterial salts [8]. Guiding stem cells along the osteogenic lineage is a critical step in bone regeneration and it is well known that the ECM The behavior of cells which adhere to a surface is strongly influ- plays a main role in regulating the stem cell fate [9–11]. Not only enced by the biomaterial characteristics. Molecular structure, does this network serve as a scaffold for cells; it also helps in mechanical properties and surface topography on micrometer immobilizing growth factors and cytokines [12]. The water content and nanometer scale will influence adhesion, migration, prolifera- in cortical bone can vary between 18% and 30% and shows a direct tion, differentiation, and cell signaling [10,36–42]. In addition to relationship to bone porosity, which increases through aging and soluble factors, sensing a biomaterial’s physical properties plays a osteoporosis [13]. Nano-composite structures made of collagen decisive role in cellular function and fate. The following paragraph and HA contribute to the strength and hierarchical architecture will address the biomaterial characteristics of SF. of bone [14]. The overall bone structure is divided into cortical bone, which is more compact, and cancellous bone, which appears 3.1. Structure and mechanical properties more sponge-like and whose pores are filled with bone marrow or fat. Bone is a highly dynamic tissue which is continually renewed Silk is composed of two major proteins: SF (fibrous protein) and and remodeled through formation and resorption processes of sericin (globular protein). SF is a protein isolated from different bone-forming osteoblasts and bone-resorbing osteoclasts as an animals in the form of an aqueous protein solution. The ability to adaption to mechanical loads, regulatory factors such as hormones produce silk has evolved multiple times among insects such as and cytokines, and other environmental parameters [15]. Osteo- Bombyx mori, spiders, mites and beetles with diverse functions clasts are multinucleated cells derived from hematopoietic stem [43]. With such a variety of silk, it is not surprising that its

Please cite this article in press as: J. Melke et al., Silk fibroin as biomaterial for bone tissue engineering, Acta Biomater. (2015), http://dx.doi.org/10.1016/j. actbio.2015.09.005 J. Melke et al. / Acta Biomaterialia xxx (2015) xxx–xxx 3 molecular composition and structure can vary among the different from the cocoon yields degummed silk fibers with a similar species. SF of B. mori, the african wild silk moth Gonometa postica Young’s modulus but an altered tensile strength of 450–700 MPa and G. rufobrunnea were compared amongst each other and at an elongation of 12–24% [62]. These remarkable mechanical revealed a significantly higher amount of polar (serine), acidic characteristics and its high resistance to deformation make SF a (aspartic acid) and basic (arginine) amino acids in Gonometa SF favorable biomaterial for load bearing composites. However, the which can influence cell attachment and also alter chemical reac- material properties of SF fibers cannot be directly compared with tivity for side chain modifications [44]. Indian tropical tasar silk the processed scaffolds as their mechanical properties will vary of Antheraea mylitta or chinese oak tasar silk of Antheraea pernyi, depending on different parameters such as processing techniques, possess Arginine-glycine-aspartic acid (RGD) motifs in their amino composition, matrix stiffness, b-sheet content as well as scaffold acid sequences which is known to mediate cell attachment morphology and topology. (reference: GenBank: AY136274.1, Submitted (on JUL-2002) to the EMBL/GenBank/DDBJ databases). There is a growing interest 3.2. Biocompatibility for silk from genetically engineered organisms. Monster SilkTM of Kraig Biocraft Laboratories, Inc., a composite fiber of spider silk Due to its chemical composition and structure, silkworm SF has proteins and silk from B. mori, is spun by transgenic silkworms. been shown to be highly biocompatible [63]. The role of SF as bio- This chimeric silk has been reported to be stronger and more flex- material was first assessed in 1995, showing the attachment and ible than commercial grade silk [45]. Such diversities should also growth of fibroblast cells on B. mori SF matrices [64]. Years later be taken into account as they can play a role in scaffold fabrication it has been demonstrated that a negligible inflammatory response and the ultimate cell response in TE applications. Most studies so was exhibited with a high biocompatibility with blood [65–67].SF far have focused on SF from B. mori as it has been used for textiles has been recognized by the US Food and Drug Administration over thousands of years and most of our knowledge on how to pro- (FDA) as a biomaterial in 1993 and is widely used as suture mate- cess silk is from this species. It is also easy to obtain in large quan- rial. Different silk-based medical devices have been approved by tities. Therefore only few studies have investigated the biomedical the FDA by now such as the long term bioresorbable surgical mesh application of genetically engineered SF or SF from other species. SeriscaffoldÒ and SeriACLTM, a SF based ligament graft. Since silk can Conventionally, one of the first steps in the fabrication of SF easily be absorbed by the human skin, it has been investigated as a scaffolds is the removal of the sericin component. Allergic reac- biomaterial for wound dressing and products for clinical applications to silk have been reported that were attributed to sericin, tion are being developed (Akeso Biomedical Inc.) [68,69]. which first resulted in its exclusion from biomedical applications [46–49]. However, it seems that these adverse reactions were 3.3. Degradation caused by the combination of sericin and fibroin as mere sericin got established as a biocompatible material in the last years Degradation plays an important role for biomaterials used in [50,51]. The glue-like protein sericin which coats fibroin, is bone TE. Depending on the target tissue, scaffolds should degrade removed by a thermochemical treatment of the cocoons, also at a controlled degradation rate to support new bone formation. known as degumming. In the degumming process, cocoons of Therefore the controlled manipulation and understanding of B. mori are immersed in boiling water, sometimes with salt or degradation rates is one of the main goals in materials science. detergent to increase the efficiency, which leaves the fibroin fibers Being a protein, SF has been shown to be susceptible to proteolytic behind. The SF amino acids sequence consists of two different sub- degradation by various enzymes such as protease XIV, alpha- units, a light and a heavy chain, which are connected through a chymotrypsin and collagenase IA [70,71]. Concerns have been disulfide bond. On both chains, the glycoprotein P25 is noncova- raised about the degradation of the SF b-sheet structure since lently attached to a set of 6 light and heavy chains [52,53]. While amyloid b-fibrils are associated to Alzheimer disease. However, the light chain consist of a non-repetetive, more hydrophilic SF degradation products showed no significant cytotoxicity to sequence, the amino acid sequence of the heavy chain consists of neuronal cells in in vitro assays [72]. As SF is being degraded, the repetitive hydrophobic blocks of Gly-Ala-Gly-Ala-Gly-Ser and resulting amino acids can be absorbed in vitro or in vivo which is repeats of Gly–Ala/Ser/Tyr dipeptides, which form 12 crystalline favorable in biomedical applications. SF degradation can be influ- domains [54]. This high glycine content allows for a tight packing enced by the content of water-insoluble silk II and water soluble into extremely stable b-sheet nanocrystals, which can be induced silk I structures, the former being induced e.g. during methanol e.g. by potassium phosphate or methanol treatment [55,56]. The treatment or stretching. With increasing silk II amount, and there- crystalline domains have a header and terminal sequence and are fore increasing amount of b-sheet structures, the degradation time linked by 11 spacers with 42–43 residues each which are nearly has been shown to increase as well [73]. A faster degradation of SF identical [54]. The main molecular interactions in these crystalline films was achieved by preparing water insoluble SF film with lower b-sheets are hydrogen bonds. Even though they are one of the b-sheet content from a concentrated aqueous solution by either weakest known interactions, hydrogen bonds contribute greatly subsequent water-based annealing or a very slow, controlled dry- to the rigidity and tensile strength of silk. Through silk’s nano- ing process [73,74]. Of these two methods, SF films prepared by a confinement, the crystals are loaded in uniform shear and rupture slow drying process, showed the lowest b-sheet content which is followed by a slip–stick motion as the strand slides and reforms resulted in the fastest degradation [74]. The degradation behavior hydrogen bonds which leads to their most efficient use [57]. is also influenced by the SF source. When comparing SF from mul- Reforming those weak hydrogen bonds gives SF the ability to berry and non-mulberry silkworms, different degradation rates self-assemble and self-heal. SF can self-assemble into larger fibrous have been observed. Non-mulberry SF was less prone to degrada- structures which are characterized by a high degree of hierarchical tion due to the compact crystal structure and high a-helix and b- molecular order and also their enhanced mechanical properties sheet contents [75]. However, so far only a few studies compared [58–60]. Silk of B. mori obtained in the laboratory by forced silking the degradation of SF among different species or genetically mod- of silk worms has a young’s modulus of 12.4–17.9 GPa and an ulti- ified organisms which produce SF. Using transgenic silk fused with mate tensile strength of 360–530 MPa at an elongation of 18–21% a matrix metalloproteinase (MMP-2) cleavage site, a faster [61]. Since the degumming process exposes silk to an unnatural degradation by matrix metalloproteinase-2 was achieved [76].To environment, the SF structure and its mechanical properties slow down degradation, it was shown that protease inhibitors inte- change slightly. Removal of the water-soluble sericin protein coat grated into silk based systems, can decrease the degradation rate

[77]. This enhanced regulation of degradation also opens up new possibilities for controlled drug release applications.

4. Silk ﬁbroin processing methods

Not only the choice of scaffold material but also scaffold morphology – and with that the choice of the material processing [88] [99,100,103] [101,102,104–106] [121,122,129] [123,125–127] method – is a decisive factor in bone TE. Scaffolds provide biome- [82–87] chanical support for cells until they are organized into a functional tissue through migration, proliferation, differentiation and deposition of extracellular matrix. Therefore they have to meet certain requirements. Three-dimensional scaffolds need to offer appropriate mechanical properties, pore size, pore orientation, porosity, interconnectivity and surface chemistry to promote the development of osteoblastic cells [78–81]. Also the influence of scaffold design parameters on the transport of nutrients and metabolic waste products has to be taken into account. The application of Electrical impulse might be unsuitable for cell encapsulation, small pore size Pore size is limited Difficult to design precise pore orientation and pore interconnectivity Process depends on many variables which hampers reproducibility Low resolution and porosity Small pore sizes SF as sutures and wound dressings has inspired materials scientists to develop a wide range of SF scaffolds. SF can be processed into hydrogels, sponges, fibers, particles, microspheres, tubes and electrospun fiber mats with tunable properties to study cell–cell and cell–biomaterial interactions. The most relevant processing

methods for bone TE (Table 1) will be discussed in the following in vivo sections.

4.1. Gels Temperature reversible, gelation of low concentrated solutions (1%), injectable Good porosity and interconnectivity, also lamellar structures are possible Simple, adaptable, easily controllable pore size and geometry Tunable fiber thickness and porosity, good interconnectivity Controllable micro- and macro- geometry, cells can be encapsulated Injectable for applications, cells can be encapsulated Hydrogels are water-swollen three-dimensional polymer networks which provide remarkable options for the delivery of cells and cytokines in TE. Especially for clinical applications they -helices are favorable as they offer the advantage of being injectable. The a hydrogelation of SF is induced in aqueous SF solutions through high temperatures, low pH, high ionic strength, vortexing, sonication, freeze gelation or electrogelation [82–86]. During the gelation process, structural changes of the SF occur from a disordered state to a b-sheet conformation which physically cross-links and stabilizes the gel [87]. Electrogelation is an exception as it leads to random structures and a-helices rather than b-sheets and is temperature reversible [88]. Also sonication-induced gelation can be reversed to some extent. Sonication imparts local vibrational movement of SF macromolecules, which alters the hydrophobic interaction and facilitates self-assembly of SF macromolecules to -sheet conformation which physically cross-links form b-sheet crystals. After gelation, further sonication can convert b the gel back into a solution due to the cleavage of disulfide bonds, modulation in hydrophobic inter- and intra-chain interactions and b-sheet conformations [89]. However, a gel is formed again shortly after the sonication stops. SF hydrogels with interesting mechanical properties can be formed through freeze gelation techniques, removed through ice sublimation which induces porosity leached out fluid jet and spun into 2D and 3D scaffolds and print them with acrosslinked 3D by printer, enzymes the or printed high constructs temperatures can be and stabilizes the gel which use a frozen regenerated SF solution that is immersed into Structural changes of the SFdisordered in state an to aqueous a solution occur from a nano- or microspheres which leads to random structures and a suitable solvent while being kept below its freezing temperature. After several hours, gelled scaffolds form [86]. SF cryogels were obtained from frozen SF solutions at subzero temperatures by adding ethylene glycol diglycidyl ether which induced b-sheet crystal- lization, inducing gelation. These gels exhibited remarkable mechanical properties such as an elasticity that allowed them to resist complete compression without any crack development (Fig. 1B), while hydrogels formed at 50 °C fractured already under low deformation (Fig. 1A). Cryogel scaffolds produced from a 12.6% SF solution showed a very high compressive modulus of 50 MPa Porogen leaching A SF solution is poured over porogens and solidified; then the porogen is 3D printing, rapid prototyping CAD (computer-aided design) software can be used to create 3D models which makes them a potential scaffold for bone regeneration [90]. ionic strength, vortexing, sonication, freeze gelation Electrogelation An electric fields induces assembly of SF nanoparticles into larger Sonication-induced SF hydrogels have been shown to successfully encapsulate human MSCs while supporting their proliferation, growth and cellular functions [84]. The aqueous SF solution constructs was shortly sonicated, mixed with the hMSCs and then incubated Sponges Freeze drying Ice crystals formed within the aqueous SF solution through freezing are Fibers3D printed Electrospinning SF nanofibers are formed by the creation and elongation of an electrified FormatHydrogels Fabrication technique High temperatures, low pH, high Description Advantages Disadvantages References Table 1 at 37 °C to allow for complete gelation. In a similar approach, Silk fibroin (SF) scaffold processing techniques for bone tissue engineering applications.

Fig. 1. Silk ﬁbroin hydrogels (A) and cryogels (B) formed at 50 and À18 °C, respectively, undergoing a compression test; CSF = 4.2%, EGDE = 20 mmol/g, TEMED = 0.10%. Adapted with permission [90]. Copyright 2013 American Chemical Society.

hMSCs were encapsulated in a blend of SF and SF-poly-L-lysine. The gas foaming or freeze-drying [99,100]. Processing parameters can hydrogel induced osteogenic differentiation of hMSCs even when be varied by using different solvents for regeneration of SF no osteogenic stimulants were added to the cell culture medium including organic solvents such as hexafluoro-2-propanol (HFIP) [91]. Furthermore, injectable SF hydrogels have been demonstrated or aqueous solution. HFIP-SF scaffolds are relatively persistent to accelerate the remodeling processes in rabbit distal femurs [92]. towards degradation and may take up to two years to degrade These osteogenic features were improved by using sonication- in vivo. Whereas scaffolds derived from aqueous solutions can induced SF hydrogels with incorporated vascular endothelial degrade completely in 2–6 months [101]. There are different opin- growth factor (VEGF) and bone morphogenic protein-2 (BMP-2). ions on which fabrication method is most suitable for bone TE. It In vivo studies in rabbits demonstrated that VEGF and BMP-2 in was reported that HFIP-derived SF scaffolds pre-seeded with these hydrogels promote angiogenesis and new bone formation, adipose-derived stem cells (ASCs), demonstrated a better bone tis- respectively, and that the combinations of those two factors had sue formation with increased levels of osteopontin, collagen type I, an additive effect on bone regeneration [93]. Such gels could be bone sialoprotein, increased calcium deposition and mineralized used to deliver various growth factors in a minimally invasive ECM volume. In this study, the amount of alkaline phosphatase approach to regenerate irregular or poorly accessible cavities in (ALP) activity and calcium deposition after 7 weeks were compara- bone. Finally, injectable fibrin network containing activated ble to cells cultured in decellularized trabecular bone [102]. How- platelets have also been combined with SF. The resulting gel ever, different results were obtained using scaffolds with showed increased mechanical properties, slower degradation, and lyophilization-induced bone lamellar-like structure [103]. In this more controllable growth factor release of VEGF, platelet-derived study, methanol treatment was compared to water annealing and b growth factor and transforming growth factor beta 1 (TGF-b1) as steam sterilization for the induction of -sheet structures. Cell cul- compared to control groups without SF [94]. It is known that ture of hMSCs under osteogenic conditions showed higher ALP levels as well as mineralized matrix after 42 days in scaffolds platelet-rich plasma with bone grafts significantly improves the derived from aqueous solutions. Also, Kim et al. reported enhanced quality of bone [95–98]. Therefore, the combination of platelet- osteogenesis of hMSCs on water-based scaffolds as compared to rich plasma with SF could be a beneficial bone TE strategy. HFIP-derived scaffolds after 28 days of culture [104]. It was also reported that aqueous-based SF scaffolds exhibited higher osteo- 4.2. Porous sponges conductivity compared to SF dissolved in HFIP when they were implanted into cortical defects of sheep. After 4 weeks of implanta- Porous sponge-like scaffolds have been widely used in bone TE. tion, new trabecular bone formed inside the pores of the aqueous Their 3D porous structure allows for cell attachment, proliferation, SF scaffold while the HFIP-SF scaffolds mainly contained necrotic and migration, and facilitates nutrient and waste transport either cells and no trabecular bone [105]. It is argued that these observa- by diffusion in a static environment or perfusion e.g. in bioreactor tions might be associated with an increased surface roughness of setups. The pores can be formed in different sizes using porogens, the aqueous SF scaffolds at a micro-scale. This is probably due to

Please cite this article in press as: J. Melke et al., Silk ﬁbroin as biomaterial for bone tissue engineering, Acta Biomater. (2015), http://dx.doi.org/10.1016/j. actbio.2015.09.005 6 J. Melke et al. / Acta Biomaterialia xxx (2015) xxx–xxx the partial dissolution of the NaCl particle surface as well as differ- The progressing mineralization of ECM on RGD decorated SF scaf- ent degradability and structural changes [104,106]. folds with small (106–212 lm), medium (212–300 lm), and large Using porous SF scaffolds, it was shown that the osteogenic dif- pore sizes (300–425 lm) was demonstrated by weekly micro com- ferentiation of hMSCs can be enhanced by a combination of low puted tomography (lCT) imaging [108]. The scaffolds were seeded oxygen levels (5%) and increased lysine and proline concentrations with hMSCs and cultured in bioreactors which allowed to asepti- in the cell culture medium which altered the metabolic rate of the cally scan the constructs repeatedly. The hMSCs were differenti- differentiating cells [107]. Changes in glucose consumption rate, ated along the osteogenic lineage and produced bone-like tissue lactate synthesis rate, and the use of lysine, proline, glutamate, which resembled the initial scaffold geometries and featured and glutamine seemed to be coupled to osteogenesis of hMSCs. trabecular-like structures (Fig. 2). In a similar setup, it was shown

Fig. 2. SEM images (A–C) of silk fibroin scaffolds decorated with RGD sequences with small (106–212 lm), medium (212–300 lm) or large (300–425 lm) pore diameters. Micro-CT images (D–L) taken from the same scaffolds seeded with hMSC after 5 weeks of cultivation under osteogenic conditions in spinner flasks, showing the constructs as seen from the top (D–F), from the side (G–I) and at higher magnification (J–L). Bar length: 500 lm (A–C), 2 mm (D–I), 1 mm (J–L). Reprinted with permission [108]. Copyright 2013 Elsevier.

Please cite this article in press as: J. Melke et al., Silk fibroin as biomaterial for bone tissue engineering, Acta Biomater. (2015), http://dx.doi.org/10.1016/j. actbio.2015.09.005 J. Melke et al. / Acta Biomaterialia xxx (2015) xxx–xxx 7 that initial cell pre-cultivation can maximize ECM mineralization sponge-like structures which only offer a 2-D surface in a micro- by hMSCs on SF scaffolds, but only with smaller pore diameters scopic view and usually have lower porosities. in the range of 112–224 lm [109]. In bone TE, micro-CT imaging gives the opportunity of monitor- 4.4. 3D bioprinting ing 3D mineralization without destroying the bone tissue engineered construct. However, when using SF as scaffold material, Current designs and fabrication methods fail to control the the underlying scaffold structure remains invisible as the material material’s resorption properties and lack pre-defined internal is not radio-opaque. SF scaffolds cannot be segmented from the cell geometry. Most scaffolds produced these days are randomly por- culture medium after taking up water. Therefore it is not possible ous with no defined internal architecture which hinders vascular to monitor degradation processes or to investigate the influence of ingrowth, subsequently resulting in poor osseo-integration within single scaffold structures on ECM mineralization over time. the construct. Also in vivo experiments to investigate the influence Several studies have shown that porous SF scaffolds can miner- of this internal geometry on cellular behaviour are therefore lim- alize spontaneously in the presence of culture media, which ited. This is mainly because the scaffold architecture produced by strongly affects the comparability of studies analyzing how cells conventional strategies such as porogen leaching, gas foaming, form bone-like tissue [110,111]. The choice of FBS has been shown freeze drying and melt molding is often process driven, rather than to be a decisive factor in this process for both acellular and cell- design driven. Advanced rapid prototyping has developed a whole seeded SF scaffolds [112]. Many theories concerning the mineraliz- array of approaches such as 3D bioprinting which creates arbitrary ing mechanism of SF have been put forth. Vetsch et al. have attrib- shaped, customised scaffolds with pre-defined internal architec- uted the inherent mineralization of silk to the abundant b-sheet ture using the solid freeform approach that might help to over- crystalline regions present in the structure which act as nucleating come the above-mentioned drawbacks [123]. sites to facilitate HA deposition, similar to the mineralizing mech- Bioprinting is regarded as the future technique for combining anism of collagen in native bone tissues [112]. On the other hand, biomaterials, cells and to some extent also supporting components there is evidence that the amorphous linkages within the b-sheets into 3D biological constructs to reconstruct deficient tissues or to of SF, which resemble the anionic, non-collagenous proteins, facil- model tissues and organs in a healthy and diseased state. The goal itate the deposition of HA on these nucleation sites [113]. The is to plan the precise positions of cells with computer aided design hypothesis was supported by in vitro evidence showing extensive and then print them individually or layer-by-layer. Bioprinting is a apatite formation only in the low molecular weight (2–10 kDa), relatively new method and mostly uses biocompatible hydrogels as electronegative fragments of the fibroin chain. Another supporting they allow cell encapsulation in a gelated, hydrated and mechani- study by Jung et al. has demonstrated enhanced mineralization on cally supportive 3D environment [124]. Only a few studies have such hydrophilic, electronegative fragments of SF by seeded hMSCs been conducted so far using SF as a material for bioprinting pro- [114]. Furthermore, this pro-osteogenic property of the low molec- cesses. Recently, an inkjet printing process was developed and used ular weight fibroin fragments has been attributed to the suppres- to fabricate cell hosting SF nests [125], where patterned SF nests sion of the Notch signaling pathway [114]. measuring 70–100 lm in diameter and modified with anionic and Improved mineralization in vitro and in vivo was observed with cationic side chains acted as anchored nests for the incubation genetically modified SF which had a poly-glutamic acid site and and proliferation of Escherichia coli cells. Using 3D printing, material was produced in transgenic silkworms through systematic trans- stiffness can be modified effectively by altering the b-sheet content formation [115]. In bone, mineralization occurs by self-assembly which consequently affects material degradation. It was shown at the charged acidic domains of non-collagenous proteins recently that direct write silk-gelatin scaffolds (Fig. 3A) processed [116,117]. Upon implantation of these modified, porous scaffolds by sonication, possessed higher b-sheet (25.4%) content over into epicondyle defects in rabbit femurs, mineralization and bone cross-linked structures using tyrosinase enzyme (14.2%) (Fig. 3B) formation occurred earlier as compared to native SF scaffolds. [126]. Subsequently, biological activity of the material was assessed by culturing human nasal inferior turbinate tissue-derived mesenchymal progenitor cells which demonstrated enhanced osteo- 4.3. Electrospinning genic differentiation only on sonicated scaffolds possessing higher b-sheet content (Fig. 3C–E), whereas their tyrosinase cross-linked Electrospinning allows for the production of polymer fibers counterpart demonstrated chondrogenic differentiation as a result with diameters on a micrometer and nanometer scale which are of the lower b-sheet content and consequently lower material stiff- able to mimic nanoscale properties of fibrous ECM components. ness. This study suggested a strong significance of controlling the It is a simple and inexpensive process as its setup consists only structural parameters of SF scaffolds such as their secondary of a syringe pump, a high voltage source, and a collector. With this conformation and consequently matrix stiffness to direct the cell technique, fine mats from B. mori SF with a fiber diameter of less population towards a specific lineage. than 800 nm were formed by electrospinning with polyethylenox- With the recombinant spider silk protein eADF4(C16), which ide [118]. Electrospinning with formic acid as solvent resulted in mimics the repetitive core sequence of dragline SF, 3D printing fibers with an average diameter of 80 nm [119]. Cell culture exper- without crosslinking agents or thickeners was shown by pre- iments showed that after 14 days of incubation, the electrospun SF gelling the solution overnight at 37 °C and 95% relative humidity. mats supported extensive BMSC proliferation and matrix coverage The adhesion of different cell types which were seeded after the [120]. To mimic the ECM for TE applications, a 3D environment is printing process, was tested and revealed that osteoblasts showed desirable. The importance of this was shown by the use of electro- a much better adhesion than fibroblasts, myoblasts, HeLa cells or spun 3D SF scaffold which increased the adhesion and proliferation keratinocytes [127]. Thus, in the future this recombinant spider of preosteoblasts and the ALP activity of osteoblasts [121]. In vivo silk protein could prove useful for 3D bioprinting of cells for bone studies in rats demonstrated an increased bone regeneration in TE. 3D porous electrospun SF scaffolds compared to non-porous controls and commercially available porous three-dimensional poly- 4.5. Silk fibroin composite scaffolds lactic acid (PLA) scaffolds [122]. It is assumed that the 3D fibrous structure provides a more natural surface for cell adhesion and For bone TE applications, SF is often used in combination with allows easier circulation of nutrients and waste compared to other biomaterials which have been shown to be beneficial for

Fig. 3. (A) Schematic showing 3D printed SF-gelatin constructs produced by direct writing. (B) Characterization of SF-gelatin ink demonstrating significantly increased b- sheet content in sonicated constructs over tyrosinase cross-linked constructs. (C) SEM image of custom-made 3D filamentous architecture and osteogenic differentiation of hMSC laden constructs confirmed by alizarin Red S (D) and ALP staining (E). Abbreviations – SF: silk fibroin, hMSC: human mesenchymal stem cells, SEM: scanning electron microscopy, ALP: alkaline phosphatase. Adapted with permission [126]. Copyright 2014 Elsevier. bone regeneration such as calcium-phosphate based inorganic nanoparticles of HA supported growth and osteogenic differentia- components or collagen which are both found in bone in vivo tion of hMSCs when cultured in osteogenic medium whereby the [128–130]. The signaling cascades in response to HA are not yet combination of BMP-2 and HA resulted in the highest calcium completely understood. So far it is known that, upon interaction deposition [140]. Next to HA, different composites containing SF with HA, MSCs trigger signaling cascades such as ERK/Sox9, BMP/ have been investigated and proven to be osteoconductive, includ- Smad, Wnt, TGF-b, MAPK, and Notch signaling pathways [131]. ing chitosan and aloe vera [137,141]. An interesting biomaterial Recently it was suggested that nano-HA induced osteogenic differ- combination for bone regeneration and tissue engineering is SF entiation and inhibited adipogenic differentiation of seeded BMSCs and demineralized bone matrix (DBM) which mainly consist of col- via upregulation of IL-1a; an autocrine/paracrine soluble factor lagen and BMP-2 and has been widely used for bone regeneration that promotes bone formation by regulating collagen and osteo- in clinical applications. DBM powder or particles are difficult to pontin synthesis [132]. HA can be incorporated into porous SF scaf- handle which impedes its clinical application and usually requires folds by direct deposition using an alternate soaking process of the use of a biocompatible, viscous carrier such as putty. Better

CaCl2 and Na2HPO4, by mixing it with a porogen like NaCl or by handling can be achieved through the incorporation of DBM into mixing HA with the regenerated SF solution [133,134]. The osteo- 3D SF scaffolds which also promoted osteogenesis in rat BMSCs conductivity of the resulting composite materials leads to higher and showed increased toughness and strength, probably through formation of tissue engineered bone as compared to unmodified the particle incorporation [142]. SF scaffolds. This can be attributed to the osteoconductivity of To meet the compressive properties of bone e.g. to function as HA as well as the presence of nucleation sites for new mineral. direct load-bearing support, without adding a different biomaterial Nanohydroxyapatite (nanoHA) has also been incorporated into SF to SF, self-reinforced SF composites have been explored. SF micro- hydrogels using ethanol as gelling agent. The resulting SF/nanoHA fibers as well as SF microparticles have been used to increase the hydrogels had a higher compression modulus as well as increased mechanical strength and robustness of SF scaffolds [143–146].SF osteogenic potential and could serve as filling material for small scaffolds containing microparticles (3 lm) had a compressive bone defects [135]. The incorporation of HA, due to its osteocon- modulus of 2.82 ± 0.40 MPa and showed slower enzymatic degra- ductivity and scaffold reinforcing properties, has also found dation as well as increased surface roughness [144]. SF scaffolds application in electrospinning [136–139]. NanoHA was surface- with incorporated large (400–600 lm) and medium (150– modified by c-glycidoxypropyltrimethoxysilane to prevent 200 lm) fibers even achieved compressive modulus values in the aggregation and mixed with aqueous SF solution, resulting in bead- range of approximately 10 MPa [145], which was comparable to less nanofibers with peak strengths at a nanoHA content of 20 wt.% the compressive modulus of cancellous bone (10 MPa) and about [139]. Modification of electrospun mats with BMP-2 and/or 10 times lower than cortical bone (100 MPa), thus forming the

Fig. 4. SEM images of single and co-cultures of osteoblasts (OB) and osteoclasts (OC) on silk fibroin (SF), chitosan and polylactic acid (PLLA) after 10 days of culture. (A) Vapor treated SF film with OB:OC, (B) vapor treated SF film with OC, (C) vapor treated SF film with OB, (D) methanol treated SF film with OB:OC, (E) methanol treated SF film with OC, (F) methanol treated SF film with OB. (G) chitosan film with OB:OC, (H) chitosan film with OC, (I) chitosan film with OB, (J) PLLA film with OB:OC and (K) PLLA film with OC, (L) PLLA film with OB, Scale bar = 75 lm. Reprinted with permission [147]. Copyright 2009 Elsevier. toughest silk-based regenerated material developed so far. It is formed a homogeneous layer with interspersed TRAP positive cells. argued that the scaffold’s strength is a result of the fibers bonding This indicates that SF may be a favorable biomaterial to study the to the matrix and transferring load during compression which cell-cell communication of osteoclasts and osteoblasts. On the con- decreases stress buildup. These incorporated fibers not only trary, it was reported that SF hydrolysate inhibits RANKL-induced increased the rigidity but also the surface roughness of the scaf- formation of TRAP in RAW 264.7 cells, which is a murine monocyte folds and enhanced the differentiation of hMSCs towards the macrophage cell line, in a time and dose dependent manner. It is osteogenic lineage. not clear to which extent SF lysate would be present during a cell culture of osteoclasts on SF scaffolds. However, SF lysate may be 5. Interaction of osteoclasts with silk fibroin used as a natural compound to prevent bone loss by reducing osteoclastogenesis. The use of SF based scaffolds for bone TE is strongly connected to showing that MSCs can differentiate along the osteogenic lin- 6. In vivo bone regeneration with silk fibroin eage on them and mineralize their ECM. However, for a functional tissue-engineered system which is intended to serve as a human The ability of a biomaterial to perform specific functions in in vitro model for drug discovery or testing, not only bone forming patients cannot be evaluated only with in vitro tests as it is difficult cells have to be taken into account but also bone resorbing cells. to extrapolate from the in vitro to the in vivo situation. Therefore, in Only a few publications have dealt with the interaction of osteo- bone TE, both biocompatibility and bone promoting activity of the clasts and SF so far. With SF films it was demonstrated that vapor orthopaedic material should be demonstrated in in vivo studies as stabilized SF and methanol stabilized SF support the growth of well. To assess its bone regeneration capacity, SF has been tested as murine osteoblasts and osteoclasts in both single and co-cultures porous scaffold, electrospun material and hydrogel in different ani- (Fig. 4) [147]. The study also showed that when monocytes were mal models comprising mainly mice, rats, rabbits and sheep with cultured as a single cell type, cells that were positive for the osteo- calvarial, mandibular or femoral defects. clast marker tartrate resistant acid phosphatase (TRAP), formed Studies were conducted to explore differences between colla- aggregates. However, osteoclasts and osteoblasts co-cultures gen and silk proteins in bone regeneration. The presence of GER

Please cite this article in press as: J. Melke et al., Silk fibroin as biomaterial for bone tissue engineering, Acta Biomater. (2015), http://dx.doi.org/10.1016/j. actbio.2015.09.005 10 J. Melke et al. / Acta Biomaterialia xxx (2015) xxx–xxx peptide sequence (GFP*GERGVEGPP*GPA) present in collagen I bone-related outcomes with cell-free naive SF matrices, surface which acts as essential recognition site for the binding of a2b1 properties of SF matrices have been altered. For instance, rather integrin, important in mediating osteoblast differentiation, could than loading with pre-differentiated cells, rapid mineralization of not be identified on B. mori SF [148]. However, when directly com- scaffolds was induced by incorporation of mesoporous bioactive pared in vivo, in terms of their efficacy in promoting guided bone glass [159], known to accelerate apatite deposition in contact with regeneration, it was revealed that the absolute volume of new bone fluids in situ [160]. When implanted in mouse calvarial defects formation did not vary significantly in SF and commercial Bio- in vivo, bioactive glass incorporated in SF scaffolds facilitated the GideÒ collagen membranes (8.75 ± 0.80 and 8.47 ± 0.75 mm3 mineralized tissue formation (7 mm3), compared to unmodified respectively) after 8 weeks of implantation in critical size rat cal- SF scaffold where only peripheral bone deposition occurred varial defects [149]. Moreover, the SF-derived membranes reduce (2.5 mm3). A similar in vivo response of unmodified SF was the risk of transmitting infections, providing a viable alternative observed by Meinel and co-workers when B. mori SF scaffolds were for facilitating bone regeneration. implanted in mice calvarium [161]. The enhanced osteo- Porous SF scaffolds exhibit architectures with hierarchical orga- regenerative ability of the bioactive glass/SF scaffolds is mainly nization which is comparable to cortical and trabecular bones and attributed to the consistent release of silica from bioactive glasses can facilitate osteogenesis within the defect site. For instance, 3D which subsequently up-regulates the bone-related markers in electrospun SF scaffolds resembling ECM-like structure with high osteoblasts [162]. Others included polyaspartic acid in SF scaffolds porosity and controlled pore sizes (200–400 nm) showed signifi- followed by controlled calcium phosphate deposition to develop cantly higher bone coverage over commercial PLA scaffolds pos- pre-mineralized SF scaffolds, which resulted in increased expres- sessing comparable morphology [122]. After 7 weeks of sion of BMP-2 in seeded human bone stem cells in vitro [163]. implantation in a critical sized rat calvaria defect, mean normal However, when implanted in the mandibular defects of canine, bone area of 78.30% was attained in SF scaffolds as opposed to only polyaspartic acid/SF scaffolds formed nominal traces of bone depo- 49.31% in 3D PLA scaffolds, marking the efficiency of electrospun sition along the defect periphery, while substantial bone formation SF scaffolds over commercial PLAs for the treatment of large bone could only be achieved with the addition of pre-seeded autologous defects. Another group prepared nanofibrous membranes of such BMSCs [153]. electrospun SF and demonstrated excellent biocompatibility, with Recent advancements in the bone regeneration field have high- complete defect coverage after 8 weeks of in vivo implantation in lighted the absence of the RGD peptide motif in mulberry B. mori rabbit calvarial defects [150]. Also recently prepared resorbable silk, a cell adhesion ligand typically present in nonmulberry screws of SF targeted for maxillofacial regeneration promoted sat- A. mylitta [GenBank: AY136274.1], as one of the probable reasons isfactory bone remodeling in rat femoral defects 8 weeks post- for the low bone promoting ability of B. mori SF. Cell-free A. mylitta implantation comparable to commercially available poly-lactic- derived 3D SF sponges implanted in premaxillary defects of rat co-glycolic acid fixation systems [151]. model demonstrated considerably higher bone volume over empty It was demonstrated that native B. mori SF scaffolds without any control defects (64.89 ± 7.46 mm2 vs. 27.28% ± 9.2% mm2) after pre-seeded osteogenic cells, showed insufficient bone regenerative 6 weeks of implantation [164]. In agreement with the above argu- potential required for complete in vivo healing of large femoral ment, a recently conducted study compared the repair of critical defects in mice [152]. A similar outcome was observed when these size rat calvarial defect with A. mylitta and B. mori lyophilized scaf- scaffolds were pre-seeded with un-differentiated hMSCs and folds (Fig. 5) [165]. The results provided evidence of progressive implanted. However, with pre-differentiated mesenchymal stem mineralization with bony union in A. mylitta implanted defects cells, a major portion of the defect was healed with newly deposited within 6 months, while the B. mori scaffolds only provided nominal bone tissue. Therefore, a series of subsequent studies investigated traces of mineral deposition within the same time span. However, the osteogenic potential of premineralized or apatite-coated SF scaf- a shortcoming in the study was that B. mori scaffolds were fabri- folds in the mandibular defects of rats [153] and canines [154]. cated by dissolving the cocoons while A. mylitta SF proteins were Increased bone formation was observed in premineralized con- directly isolated from the gland due to the difficult dissolution of structs over uncoated ones in all the studies. Another observation A. mylitta cocoons in common solvents [166]. The translation of was that the addition of BMP-2; a growth factor which plays a signif- A. mylitta scaffolds to clinical settings for bone regenerative proce- icant role in promoting osteogenic mechanisms in skeletal develop- dures still remains a challenge due to limited pre-clinical trials and ment [155], either exogenously incorporated on the scaffold [156] or the debate about the existence of RGD motifs [166]. transduced in the pre-seeded osteogenic cells [153], further acceler- For facilitating a congruent bone ingrowth, vascularization of ated the amount of new bone formation and increased the bone the construct seems to be a pre-requisite. The development of mineral density of the implanted constructs. Karageorgiou and angiogenesis concomitantly with osteogenesis will not only aid co-workers demonstrated the upregulation of the osteogenic in the provision of oxygen and nutrients for osteogenic and marker genes cbfa-1, osteopontin and osteocalcin together with a endothelial cells, but also contribute towards the survival and considerably higher bone formation (0.28 ± 0.15 mm2 vs. differentiation of their counterpart [167]. Sun and co-workers 0.12 ± 0.05 mm2) on SF scaffolds with incorporated BMP-2 and efficiently demonstrated the in vitro co-existence and extensive pre-seeded hMSCs as compared to only pre-seeded hMSCs without matrix formation by hMSCs and human microvascular endothelial BMP-2, when implanted in mice calvarial defects [156]. Besides cells on SF/HA direct write composites with controlled pore BMP-2, epigenetic modification of osteogenic precursors with addi- morphologies [168]. When SF scaffolds with pre-formed tional sequences such as PHF8, a major H4K20/H3K9 demethylase microcapillary-like networks developed in vitro by co-culturing involved in craniofacial and bone development [157], and SATB2 human dermal microvascular endothelial cells (HDMECs) and (special AT-rich sequence-binding protein 2) modified induced human osteoblasts were implanted in the subcutaneous tissue of pluripotent stem cells [158] on SF constructs have been shown to mice, an extensive infiltration of host capillaries, identified with promote healing of critical size defects of mice calvariae. histological evaluation and immunohistochemical staining with Though pre-culturing of osteogenic cells on SF has demon- human-specific antibodies, were distinctively visible in the con- strated excellent osteoinductivity in vivo, the procedure might struct [169]. Significantly reduced capillary invasion from the host not be as suitable for clinical use. The isolation protocols for occurred in SF scaffolds when HDMECs alone were cultured autologous cells are invasive with lengthy culture times, making (8.81 ± 2.11 vessels/mm2) which clearly suggested the synergistic it rather inconvenient for patients. Therefore to further accelerate relationship of osteoblasts with endothelial cells. Determining

Fig. 5. Bone regeneration in Antheraea mylitta (Am) and Bombyx mori (Bm) scaffolds. (A) X-ray radiographs 6 months post-surgery showing defect coverage with neo bone formation in Am constructs (left panel) and no bone formation in Bm constructs (right panel). Centre image shows surgical site drilled with 5 mm bilateral calvarial defects. (B, C) Histological analysis of constructs 3 months post-surgery stained with H&E (B) and Alizarin Red S (C) showing moderate immuno-inflammatory responses with enhanced bone deposition in Am (arrows) (left panel). In Bm constructs, profuse infiltration of immuno-inflammatory cells is conspicuous with minimal traces of bone matrix deposition (right panel). M: Month. Adapted with permission [165]. Copyright Wiley 2015.

the underlying key signaling mechanisms that trigger such cross- mature lamellar bone was deposited within 6 months [165]. This talks between the two cell types can prove to be very crucial for moderate immunogenicity attained in SF scaffolds has been attrib- the development of novel scaffold materials. uted in vitro to the silk II conformation possessing a high b-sheet SF has largely been favored in in vivo bone regenerative studies content in 3D scaffolds when compared to silk I conformation in and its immuno-inflammatory response after implantation has 2D SF films, as well as differences in hydrophilicity–hydrophobi therefore been investigated as well. For instance, when RGD- city, topographical features, matrix stiffness and exposed surface decorated SF was implanted in mouse calvarial defects, new bone area [170]. deposition was evident consisting of both mature lamellar and immature woven bone [108]. A moderate level of typical immuno-inflammatory cells including macrophages, giant cells 7. Drug delivery for osteogenesis and foreign cells (lymphocytes and neutrophil granulocytes) were interspersed with the fibrous tissue and new bone tissue was Due to its slower degradation rate, SF has been shown to pos- observed via histopathology. However, this moderate range of sess outstanding properties as a carrier for bioactive drug delivery immune reaction juxtaposed with the SF material falls within the in several therapeutic applications, especially bone regeneration expected range for such a defect. A comparable level of immuno- [171–175]. In an attempt to mimic the physiological bone hierar- inflammatory response was observed when A. mylitta SF scaffolds chy, various morphologies of SF have been investigated for the with natural RGD sequences were implanted within a similar rat delivery of bone specific growth factors (e.g. BMP-2, platelet- calvarial model. Moderate levels of immune cells, typically macro- derived growth factor), genes and enzymes which include porous phages and giant cells penetrated during the initial period, did not 3D scaffolds, planar films, SF particles and electrospun fibers, with impede infiltration of osteogenic precursors and subsequently a electrospun fibers being considered the most superior as they

Please cite this article in press as: J. Melke et al., Silk fibroin as biomaterial for bone tissue engineering, Acta Biomater. (2015), http://dx.doi.org/10.1016/j. actbio.2015.09.005 12 J. Melke et al. / Acta Biomaterialia xxx (2015) xxx–xxx allow the fabrication of SF into fibers in the range of several differentiation could be identified on unmodified SF films cultured nanometers to micrometers [176–180]. SF has been widely used in osteogenic stimulants. The phenomenal tendency of osteogene- to evaluate its potential for the delivery of bioactive molecules, sis exhibited by hMSCs on BMP-2 loaded SF was primarily a func- especially BMP-2, known to play a crucial role in promoting tion of the immobilized protein and not the soluble BMP-2 in osteogenesis. The delivery of BMP-2 using SF has been extensively media as demonstrated by cell culture inserts. evaluated for the application in bone regeneration [155]. BMP- To promote successful bone regeneration, efficient vasculariza- 2-loaded SF particles have successfully induced the formation of tion is a pre-requisite. Therefore, besides BMP-2, the angiogenic ectopic bone formation in rats, with increase in bone density con- growth factor VEGF and its controlled delivery play a vital part in comitant with the delivery rate of BMP-2. Moreover, BMP-2-loaded bone regeneration. It has been reported that sustained delivery of SF groups showed superior bone formation over BMP-2 alone. The VEGF could aid in bone regeneration by promoting neovasculariza- reasons for the observed phenomenon could be many. Direct tion and osteoblast differentiation [184]. Farokhi and co-workers administration of growth factors without immobilization on a car- fabricated electrospun poly(lactic-co-glycolic acid) nanofibers on rier would result in a shorter half-life of the growth factor, slower the surface of freeze-dried SF/calcium phosphate substrates loaded penetration into the tissue and rapid diffusion of the delivery with VEGF [176]. The controlled release profile of VEGF measured molecule from the site [181]. Therefore, repeated administration in vitro over a 28-day period, classified the setup as a sustained would be needed to consistently maintain the required concentra- delivery system with 83% sustained bioactivity post-release. The tion of the growth factor over long periods of time, which would be delivery system demonstrated good cytocompatibility in vitro, clinically impractical and very expensive [181,182]. Moreover, characterized by improved cell adhesion, proliferation and ALP higher concentrations of the drug than required could have delete- activity of human osteoblast cells. In vivo, the induced neo-bone rious effects on the target tissue as well as ectopic sites [172]. This formation resulted in coverage of 8-mm critical size calvarial underlines the indispensable role of SF as an efficient carrier in defects of rabbits. As VEGF and BMP-2 are important factors increasing the efficacy of BMP-2 as a drug delivery molecule to pro- involved in angiogenesis and osteogenesis, Zhang et al. evaluated mote bone regeneration. A BMP-2 delivery system composed of the potential of sonication-induced SF hydrogels as a carrier to electrospun polycaprolactone nanofiber mesh tubes with the cen- encapsulate dual factors and test their efficacy in the repair of rab- tral hollow chamber injected with a SF hydrogel, was developed bit maxillary sinus floor augmentation [93]. In vivo, while BMP-2 and tested for the treatment of an 8 mm critical size rat femoral alone mediated great bone formation and VEGF triggered neo- segmental defect [183]. The results demonstrated effective bone vascularization, cumulatively they promoted both osteogenesis formation in BMP-2 containing groups with biomechanical proper- and angiogenesis 12 weeks post-surgery. ties (0.030 ± 0.001 Nm/deg torsional stiffness and 0.31 ± 0.02 Nm maximum torque) comparable to native femurs and significant degradation of the SF hydrogels after 12 weeks of implantation. It was further demonstrated that the release of BMP-2 from SF scaf- 8. Conclusion and outlook folds pre-seeded with hMSCs led to higher bone formation than with SF alone [156]. In this study, BMP-2 was adsorbed on the sur- SF is a natural biomaterial with unique biomedical and mechan- face of SF scaffold by continuous immersion for 6 h in 0.05 mg/ml ical properties which make it favorable for a wide range of bone TE of BMP-2 solution. These BMP-2-adsorbed scaffolds were subse- applications. It can be combined with several other biomaterials quently incubated in vitro in media and the release kinetics of such as HA to create composite scaffolds which mimic the natural the drug was quantified. Results revealed that 75% of BMP-2 was bone environment and increase the scaffold’s osteogenic potential. released into the media from the scaffold surface within the 1st Being a protein with tunable degradation and diverse modification week of incubation followed by no release thereafter. Thus it options, SF possesses exceptional properties as a carrier for drugs may not be logical to define it as a sustained release method. and their sustained release in therapeutic applications. SF can be Clearly, sustained release of BMP-2 from the carrier surface is an processed into different scaffolds such as injectable and printable important aspect in bone TE and would benefit from a technique gels, porous sponges and electrospun 2D and 3D constructs. With that can efficiently immobilize growth factors on the scaffold’s sur- novel processing techniques it can be expected that new SF based face in a way that leads to a sustained release of the molecule scaffolds will be developed in the future and open up even more in situ. Another packaging strategy was to prepare SF particles with possibilities for bone TE applications. Clinical practice shows that different sizes and components by a self-assembly procedure, most fractures will heal naturally and do not require TE strategies. resulting in complex calcium carbonate encrustation particles Complex or non-union fractures might be more relevant for bone and functionalized with BMP-2 [178]. This process was used to reg- TE with SF. Creating solid organs and achieve reasonable tissue ulate a sustained release of BMP-2 to the MSCs to trigger osteoge- dimensions is a goal we are still far away from. However, these nesis. The results showed higher ALP activity and expression of the constructs will have to have a structure and SF with its versatile osteogenesis-related genes Runx2 and osteocalcin, which led to scaffold types offers a broad toolbox to choose from. In vivo studies enhanced differentiation of MSCs along the osteogenic lineage. using SF scaffolds for bone TE applications have proven the mate- The combination of BMP-2 with calcium components in the rial’s osteogenic potential. Nevertheless, these studies were done SF/calcium carbonate particles specifically induced higher mostly in small animals that do not sufficiently predict their per- osteogenesis by synchronized activity. This was evident from the formance in humans. More research will have to be done before comparatively reduced osteogenic ability of control groups SF can be used for clinical trials and commercialized for bone TE carrying BMP-2 carriers under similar conditions. applications. It is likely that SF will contribute to creating sophisti- In another study, BMP-2 was immobilized on SF planar films by cated bone tissue models to study ECM mineralization, mineral utilizing carbodiimide chemistry [177]. When hMSCs were resorption and vascularization as well as to investigate bone dis- cultured on these BMP-2 decorated films in the presence of eases and possible therapeutic drugs. Most of the in vitro studies osteogenic stimulants, enhanced differentiation towards the osteo- have focused on MSCs alone, their osteogenic differentiation and genic lineage was evident by the significantly upregulated ALP ability to mineralize their ECM. A better understanding is needed activity, increased calcium deposition, and higher expression of regarding 3D co-culture systems to create dynamic tissues which collagen I, bone sialoprotein, osteopontin, osteocalcin, BMP-2, are able to remodel similar to bone. However, the future of SF in and cbfa1 genes. On the contrary, nominal traces of osteogenic bone TE, where mechanically stable and long-term degradable

Please cite this article in press as: J. Melke et al., Silk fibroin as biomaterial for bone tissue engineering, Acta Biomater. (2015), http://dx.doi.org/10.1016/j. actbio.2015.09.005 J. Melke et al. / Acta Biomaterialia xxx (2015) xxx–xxx 13 biomaterials are needed, is promising and has great potential to [27] L. Bonewald, M. Johnson, Osteocytes, mechanosensing and Wnt signaling, bring viable strategies and innovations. Bone 42 (2008) 606–615. [28] J. Klein-Nulend, C.M. Semeins, N.E. Ajubi, P.J. Nijweide, E.H. Burger, Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not Acknowledgements periosteal fibroblasts–correlation with prostaglandin upregulation, Biochem. Biophys. Res. Commun. 217 (1995) 640–648. [29] A. Santos, A.D. Bakker, B. Zandieh-Doulabi, C.M. Semeins, J. Klein-Nulend, The research leading to these results has received funding Pulsating fluid flow modulates gene expression of proteins involved in Wnt from the European Union’s Seventh Framework Programme signaling pathways in osteocytes, J. Orthop. Res. 27 (2009) 1280–1287. 10.1002/jor.20888. (FP/2007-2013) under Grant Agreement No. 336043 and funding [30] A.G. Robling, P.J. Niziolek, L.a. Baldridge, K.W. Condon, M.R. Allen, I. Alam, from the Department of Biotechnology, India (BT/PR8038/MED/ et al., Mechanical stimulation of bone in vivo reduces osteocyte expression of 32/303/2013). Sost/sclerostin, J. Biol. Chem. 283 (2008) 5866–5875, http://dx.doi.org/ 10.1074/jbc.M705092200. [31] J. Klein-Nulend, R.G. Bacabac, A.D. Bakker, Mechanical loading and how it References affects bone cells: the role of the osteocyte cytoskeleton in maintaining our skeleton, Eur. Cell Mater. 24 (2012) 278–291. [32] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, [1] L.G. Griffith, M.A. Swartz, Capturing complex 3D tissue physiology in vitro, Multilineage potential of adult human mesenchymal stem cells, Science 284 Nat. Rev. Mol. Cell Biol. 7 (2006) 211–224. (1999) 143–147, http://dx.doi.org/10.1126/science.284.5411.143 (80-). [2] N.T. Elliott, F. Yuan, A review of three-dimensional in vitro tissue models for [33] X. Wang, Y. Wang, W. Gou, Q. Lu, J. Peng, S. Lu, Role of mesenchymal stem drug discovery and transport studies, J. Pharm. Sci. 100 (2011) 59–74. cells in bone regeneration and fracture repair: a review, Int. Orthop. 37 (2013) 10.1002/jps.22257. 2491–2498. 10.1007/s00264-013-2059-2. [3] S. Bose, M. Roy, A. Bandyopadhyay, Recent advances in bone tissue [34] R. Hass, C. Kasper, S. Böhm, R. Jacobs, Different populations and sources of engineering scaffolds, Trends Biotechnol. 30 (2012) 546–554, http://dx.doi. human mesenchymal stem cells (MSC): a comparison of adult and neonatal org/10.1016/j.tibtech.2012.07.005. tissue-derived MSC, Cell Commun. Signal. 9 (2011) 12. 10.1186/1478-811X- [4] S. Wu, X. Liu, K.W.K. Yeung, C. Liu, X. Yang, Biomimetic porous scaffolds for 9-12. bone tissue engineering, Mater. Sci. Eng. R-Rep. 80 (2014) 1–36, http://dx.doi. [35] W. Wagner, P. Horn, M. Castoldi, A. Diehlmann, S. Bork, R. Saffrich, et al., org/10.1016/j.mser.2014.04.001. Replicative senescence of mesenchymal stem cells: a continuous and [5] N. Kasoju, U. Bora, Silk fibroin in tissue engineering, Adv. Healthc. Mater. 1 organized process, PLoS ONE 3 (2008) e2213. 10.1371/journal. (2012) 393–412, http://dx.doi.org/10.1002/adhm.201200097. pone.0002213. [6] J.A. Buckwalter, M.J. Glimcher, R.R. Cooper, R. Recker, Bone biology. I: [36] M. Ghibaudo, A. Saez, L. Trichet, A. Xayaphoummine, J. Browaeys, P. Silberzan, Structure, blood supply, cells, matrix, and mineralization, Instr. Course Lect. et al., Traction forces and rigidity sensing regulate cell functions, Soft Matter 4 45 (1996) 371–386. (2008) 1836–1843. 10.1039/B804103B. [7] H.I. Roach, Why does bone matrix contain non-collagenous proteins? The [37] R.J. McMurray, A.K.T. Wann, C.L. Thompson, J.T. Connelly, M.M. Knight, possible roles of osteocalcin, osteonectin, osteopontin and bone sialoprotein Surface topography regulates wnt signaling through control of primary cilia in bone mineralisation and resorption, Cell Biol. Int. 18 (1994) 617–628, structure in mesenchymal stem cells, Sci. Rep. (2013) 3. http://dx.doi.org/10.1006/cbir.1994.1088. [38] M.J. Dalby, N. Gadegaard, R. Tare, A. Andar, M.O. Riehle, P. Herzyk, et al., The [8] M.J. Olszta, X. Cheng, S.S. Jee, R. Kumar, Y.-Y. Kim, M.J. Kaufman, et al., Bone control of human mesenchymal cell differentiation using nanoscale structure and formation: a new perspective, Mater. Sci. Eng. R-Rep. 58 (2007) symmetry and disorder, Nat. Mater. 6 (2007) 997–1003. 77–116. 10.1016/j.mser.2007.05.001. [39] L. Liu, B.D. Ratner, E.H. Sage, S. Jiang, Endothelial cell migration on surface- [9] F.M. Watt, B.L.M. Hogan, Out of Eden: stem cells and their niches, Science 287 density gradients of fibronectin, VEGF, or both proteins, Langmuir 23 (2007) (2000) 1427–1430, http://dx.doi.org/10.1126/science.287.5457.1427 (80-). 11168–11173. 10.1021/la701435x. [10] A.J. Engler, S. Sen, H.L. Sweeney, D.E. Discher, Matrix elasticity directs stem [40] Y.-W. Won, A.N. Patel, D.A. Bull, Cell surface engineering to enhance cell lineage specification, Cell 126 (2006) 677–689, http://dx.doi.org/10.1016/ mesenchymal stem cell migration toward an SDF-1 gradient, Biomaterials j.cell.2006.06.044. 35 (2014) 5627–5635. doi: 10.1016/j.biomaterials.2014.03.070. [11] F.M. Watt, W.T.S. Huck, Role of the extracellular matrix in regulating stem cell [41] S.A. Yavari, J. van der Stok, Y.C. Chai, R. Wauthle, Z.T. Birgani, P. Habibovic, fate, Nat. Rev. Mol. Cell Biol. 14 (2013) 467–473. 10.1038/nrm3620. et al., Bone regeneration performance of surface-treated porous titanium, [12] R.S. Taichman, Blood and bone: two tissues whose fates are intertwined to Biomaterials 35 (2014) 6172–6181, http://dx.doi.org/10.1016/j. create the hematopoietic stem-cell niche, Blood 105 (2004) 2631–2639. biomaterials.2014.04.054. [13] A. Techawiboonwong, H.K. Song, M.B. Leonard, F.W. Wehrli, Cortical [42] H.V. Unadkat, M. Hulsman, K. Cornelissen, B.J. Papenburg, R.K. Truckenmüller, bone water: in vivo quantification with ultrashort echo-time MR A.E. Carpenter, et al., An algorithm-based topographical biomaterials library imaging, Radiology 248 (2008) 824–833, http://dx.doi.org/10.1148/ to instruct cell fate, Proc. Natl. Acad. Sci. U.S.A. 108 (2011) 16565–16570. radiol.2482071995. 248705. 10.1073/pnas.1109861108. [14] C. Frantz, K.M. Stewart, V.M. Weaver, The extracellular matrix at a glance, J. [43] T.D. Sutherland, J.H. Young, S. Weisman, C.Y. Hayashi, D.J. Merritt, Insect silk: Cell Sci. 123 (2010) 4195–4200. 10.1242/jcs.023820. one name, many materials, Annu. Rev. Entomol. 55 (2010) 171–188. 10.1146/ [15] P. Ehrlich, L. Lanyon, Mechanical strain and bone cell function: a review, annurev-ento-112408-085401. Osteoporos. Int. (2002) 688–700. [44] V. Mhuka, S. Dube, M.M. Nindi, Chemical, structural and thermal properties of [16] T. Suda, N. Takahashi, N. Udagawa, E. Jimi, M.T. Gillespie, T.J. Martin, Gonometa postica silk fibroin, a potential biomaterial, Int. J. Biol. Macromol. Modulation of osteoclast differentiation, Endocr. Rev. 13 (1992) 66–80. 52 (2013) 305–311. 10.1016/j.ijbiomac.2012.09.010. 10.1210/er.13.1.66. [45] F. Teulé, Y.-G. Miao, B.-H. Sohn, Y.-S. Kim, J.J. Hull, M.J. Fraser, et al., Silkworms [17] H.K. Väänänen, H. Zhao, M. Mulari, J.M. Halleen, The cell biology of osteoclast transformed with chimeric silkworm/spider silk genes spin composite silk function, J. Cell Sci. 113 (Pt 3) (2000) 377–381. fibers with improved mechanical properties, Proc. Natl. Acad. Sci. 109 (2012) [18] Y.-F. Hsieh, M.J. Silva, In vivo fatigue loading of the rat ulna induces both bone 923–928. 10.1073/pnas.1109420109. formation and resorption and leads to time-related changes in bone [46] W. Zaoming, R. Codina, E. Fernandez-Caldas, R.F. Lockey, S.C. Bukanz, Partial mechanical properties and density, J. Orthop. Res. 20 (2002) 764–771. characterization of the silk allergens in mulberry silk extract, J. Allergy Clin. 10.1016/S0736-0266(01)00161-9. Immunol. 97 (2015) 210. 10.1016/S0091-6749(96)80327-7. [19] B.F. Boyce, Z. Yao, L. Xing, Osteoclasts have multiple roles in bone in addition [47] H.K. Soong, K.R. Kenyon, Adverse reactions to virgin silk sutures in cataract to bone resorption, Crit. Rev. Eukaryot. Gene Expr. 19 (2009) 171–180. surgery, Ophthalmology 91 (1984) 479–483. [20] Khosla S. Minireview, The OPG/RANKL/RANK system, Endocrinology 142 [48] C.M. Wen, S.T. Ye, L.X. Zhou, Y. Yu, Silk-induced asthma in children: a report (2001) 5050–5055. 10.1210/endo.142.12.8536. of 64 cases, Ann. Allergy 65 (1990) 375–378. [21] A.G. Robling, A.B. Castillo, C.H. Turner, Biomechanical and molecular [49] D.H. Hollander, Interstitial cystitis and silk allergy, Med. Hypotheses 43 regulation of bone remodeling, Annu. Rev. Biomed. Eng. 8 (2006) 455–498. (1994) 155–156, http://dx.doi.org/10.1016/0306-9877(94)90142-2. 10.1146/annurev.bioeng.8.061505.095721. [50] T.V. Chirila, S. Suzuki, L.J. Bray, N.L. Barnett, D.G. Harkin, T.V. Chirila, et al., [22] T.A. Franz-Odendaal, B.K. Hall, P.E. Witten, Buried alive: how osteoblasts Evaluation of silk sericin as a biomaterial: in vitro growth of human corneal become osteocytes, Dev. Dyn. 235 (2006) 176–190. 10.1002/dvdy.20603. limbal epithelial cells on Bombyx mori sericin membranes, Prog. Biomater. 2 [23] S.L. Dallas, L.F. Bonewald, Dynamics of the transition from osteoblast to (2013) 10. 10.1186/2194-0517-2-14. osteocyte, Ann. N. Y. Acad. Sci. 1192 (2010) 437–443. 10.1111/j.1749- [51] P. Aramwit, S. Kanokpanont, W. De-Eknamkul, T. Srichana, Monitoring of 6632.2009.05246.x. inflammatory mediators induced by silk sericin, J. Biosci. Bioeng. 107 (2009) [24] L.E. Lanyon, Osteocytes, strain detection, bone modeling and remodeling, 556–561, http://dx.doi.org/10.1016/j.jbiosc.2008.12.012. Calcif. Tissue Int. 53 (Suppl. 1) (1993) S102–S106. discussion S106-7. [52] S. Inoue, K. Tanaka, F. Arisaka, S. Kimura, K. Ohtomo, S. Mizuno, Silk fibroin of [25] E.H. Burger, J. Klein-Nulend, Mechanotransduction in bone – role of the Bombyx mori is secreted, assembling a high molecular mass elementary unit lacuno-canalicular network, FASEB J. 13 (Suppl.) (1999) S101–S112. consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio, J. Biol. Chem. [26] A. Santos, A.D. Bakker, H.M.E. Willems, N. Bravenboer, A.L.J.J. Bronckers, J. 275 (2000) 40517–40528. 10.1074/jbc.M006897200. Klein-Nulend, Mechanical loading stimulates BMP7, but not BMP2, [53] K. Tanaka, S. Inoue, S. Mizuno, Hydrophobic interaction of P25, containing production by osteocytes, Calcif. Tissue Int. 89 (2011) 318–326. 10.1007/ Asn-linked oligosaccharide chains, with the H-L complex of silk fibroin s00223-011-9521-1.

produced by Bombyx mori, Insect Biochem. Mol. Biol. 29 (1999) 269–276. mesenchymal stem cells expressing BMP7, Acta Biomater. 6 (2010) 3021– 10.1016/S0965-1748(98)00135-0. 3028, http://dx.doi.org/10.1016/j.actbio.2010.02.030. [54] C.-Z. Zhou, F. Confalonieri, M. Jacquet, R. Perasso, Z.-G. Li, J. Janin, Silk fibroin: [80] C.M. Murphy, M.G. Haugh, F.J. O’Brien, The effect of mean pore size on cell structural implications of a remarkable amino acid sequence, Proteins Struct. attachment, proliferation and migration in collagen-glycosaminoglycan Funct. Bioinform. 44 (2001) 119–122. 10.1002/prot.1078. scaffolds for bone tissue engineering, Biomaterials 31 (2010) 461–466, [55] C.Y. Hayashi, N.H. Shipley, R.V. Lewis, Hypotheses that correlate the sequence, http://dx.doi.org/10.1016/j.biomaterials.2009.09.063. structure, and mechanical properties of spider silk proteins, Int. J. Biol. [81] P. Joly, G.N. Duda, M. Schöne, P.B. Welzel, U. Freudenberg, C. Werner, et al., Macromol. 24 (1999) 271–275, http://dx.doi.org/10.1016/S0141-8130(98) Geometry-driven cell organization determines tissue growths in scaffold 00089-0. pores: consequences for fibronectin organization, PLoS ONE 8 (2013) e73545, [56] D. Huemmerich, U. Slotta, T. Scheibel, Processing and modification of films http://dx.doi.org/10.1371/journal.pone.0073545. made from recombinant spider silk proteins, Appl. Phys. A Mater. Sci. Process. [82] U.-J. Kim, J. Park, C. Li, H.-J. Jin, R. Valluzzi, D.L. Kaplan, Structure and 82 (2006) 219–222. 10.1007/s00339-005-3428-5. properties of silk hydrogels, Biomacromolecules 5 (2004) 786–792, http://dx. [57] S. Keten, Z. Xu, B. Ihle, M.J. Buehler, Nanoconfinement controls stiffness, doi.org/10.1021/bm0345460. strength and mechanical toughness of b-sheet crystals in silk, Nat. Mater. 9 [83] T. Yucel, P. Cebe, D.L. Kaplan, Vortex-induced injectable silk fibroin (2010) 359–367. hydrogels, Biophys. J. 97 (2009) 2044–2050, http://dx.doi.org/10.1016/j. [58] M. Hedhammar, A. Rising, S. Grip, A.S. Martinez, K. Nordling, C. Casals, et al., bpj.2009.07.028. Structural properties of recombinant nonrepetitive and repetitive parts of [84] X. Wang, J.A. Kluge, G.G. Leisk, D.L. Kaplan, Sonication-induced gelation of silk major ampullate spidroin 1 from Euprosthenops australis: implications for fibroin for cell encapsulation, Biomaterials 29 (2008) 1054–1064, http://dx. fiber formation, Biochemistry 47 (2008) 3407–3417. 10.1021/bi702432y. doi.org/10.1016/j.biomaterials.2007.11.003. [59] A. Sponner, W. Vater, S. Monajembashi, E. Unger, F. Grosse, K. Weisshart, [85] T. Yucel, N. Kojic, G.G. Leisk, T.J. Lo, D.L. Kaplan, Non-equilibrium silk fibroin Composition and hierarchical organisation of a spider silk, PLoS ONE 2 (2007) adhesives, J. Struct. Biol. 170 (2010) 406–412, http://dx.doi.org/10.1016/j. e998. 10.1371/journal.pone.0000998. jsb.2009.12.012. [60] J.M. Gosline, P.A. Guerette, C.S. Ortlepp, K.N. Savage, The mechanical design of [86] N. Bhardwaj, S. Chakraborty, S.C. Kundu, Freeze-gelled silk fibroin protein spider silks: from fibroin sequence to mechanical function, J. Exp. Biol. 202 scaffolds for potential applications in soft tissue engineering, Int. J. Biol. (1999) 3295–3303. Macromol. 49 (2011) 260–267, http://dx.doi.org/10.1016/j. [61] J. Pérez-Rigueiro, M. Elices, J. Llorca, C. Viney, Tensile properties of silkworm ijbiomac.2011.04.013. silk obtained by forced silking, J. Appl. Polym. Sci. 82 (2001) 1928–1935. [87] A. Matsumoto, J. Chen, A.L. Collette, U.-J. Kim, G.H. Altman, P. Cebe, et al., 10.1002/app.2038. Mechanisms of silk fibroin solÀgel transitions, J. Phys. Chem. B 110 (2006) [62] J. Pérez-Rigueiro, M. Elices, J. Llorca, C. Viney, Effect of degumming on the 21630–21638, http://dx.doi.org/10.1021/jp056350v. tensile properties of silkworm (Bombyx mori) silk fiber, J. Appl. Polym. Sci. 84 [88] Q. Lu, Y. Huang, M. Li, B. Zuo, S. Lu, J. Wang, et al., Silk fibroin electrogelation (2002) 1431–1437. 10.1002/app.10366. mechanisms, Acta Biomater. 7 (2011) 2394–2400, http://dx.doi.org/10.1016/ [63] Y. Cao, B. Wang, Biodegradation of silk biomaterials, Int. J. Mol. Sci. 10 (2009) j.actbio.2011.02.032. 1514–1524. 10.3390/ijms10041514. [89] S.K. Samal, D.L. Kaplan, E. Chiellini, Ultrasound sonication effects on silk [64] N. Minoura, S.I. Aiba, M. Higuchi, Y. Gotoh, M. Tsukada, Y. Imai, Attachment fibroin protein, Macromol. Mater. Eng. 298 (2013) 1201–1208, http://dx.doi. and growth of fibroblast cells on silk fibroin, Biochem. Biophys. Res. Commun. org/10.1002/mame.201200377. 208 (1995) 511–516. 10.1006/bbrc.1995.1368. [90] F. Ak, Z. Oztoprak, I. Karakutuk, O. Okay, Macroporous silk fibroin cryogels, [65] M. Santin, A. Motta, G. Freddi, M. Cannas, In vitro evaluation of the Biomacromolecules 14 (2013) 719–727, http://dx.doi.org/10.1021/ inflammatory potential of the silk fibroin, J. Biomed. Mater. Res. 46 (1999) bm3018033. 382–389. 10.1002/(SICI)1097-4636(19990905)46:3<382::AID-JBM11>3.0. [91] R. Calabrese, D.L. Kaplan, Silk ionomers for encapsulation and differentiation CO;2-R. of human MSCs, Biomaterials 33 (2012) 7375–7385, http://dx.doi.org/ [66] H. Sakabe, H. Ito, T. Miyamoto, Y. Noishiki, W.S. Ha, In vivo blood 10.1016/j.biomaterials.2012.06.043. compatibility of regenerated silk fibroin, Sen’i Gakkaishi 45 (1989) 487– [92] M. Fini, A. Motta, P. Torricelli, G. Glavaresi, N.N. Aldini, M. Tschon, et al., The 490. 10.2115/fiber.45.11_487. healing of confined critical size cancellous defects in the presence of silk [67] L. Meinel, S. Hofmann, V. Karageorgiou, C. Kirker-Head, J. McCool, G. fibroin hydrogel, Biomaterials 26 (2005) 3527–3536, http://dx.doi.org/ Gronowicz, et al., The inflammatory responses to silk films in vitro and 10.1016/j.biomaterials.2004.09.040. in vivo, Biomaterials 26 (2005) 147–155. 10.1016/j.biomaterials.2004.02.047. [93] W. Zhang, X. Wang, S. Wang, J. Zhao, L. Xu, C. Zhu, et al., The use of injectable [68] A.R. Padol, K. Jayakumar, N.B. Shridhar, H.D. Narayana Swamy, M. Narayana sonication-induced silk hydrogel for VEGF165 and BMP-2 delivery for Swamy, K. Mohan, Safety evaluation of silk protein film (a novel wound elevation of the maxillary sinus floor, Biomaterials 32 (2011) 9415–9424, healing agent) in terms of acute dermal toxicity, acute dermal irritation and http://dx.doi.org/10.1016/j.biomaterials.2011.08.047. skin sensitization, Toxicol Int 18 (2011) 17–21, http://dx.doi.org/10.4103/ [94] I. Pallotta, J.A. Kluge, J. Moreau, R. Calabrese, D.L. Kaplan, A. Balduini, 0971-6580.75847. Characteristics of platelet gels combined with silk, Biomaterials 35 (2014) [69] S.E. Wharram, X. Zhang, D.L. Kaplan, S.P. McCarthy, Electrospun silk material 3678–3687, http://dx.doi.org/10.1016/j.biomaterials.2013.12.065. systems for wound healing, Macromol. Biosci. 10 (2010) 246–257. 10.1002/ [95] T.L. Aghaloo, P.K. Moy, E.G. Freymiller, Evaluation of platelet-rich plasma in mabi.200900274. combination with anorganic bovine bone in the rabbit cranium: a pilot study, [70] M. Li, M. Ogiso, N. Minoura, Enzymatic degradation behavior of porous silk Int. J. Oral Maxillofac. Implant 19 (2004) 59–65. fibroin sheets, Biomaterials 24 (2003) 357–365, http://dx.doi.org/10.1016/ [96] D. Dallari, L. Savarino, C. Stagni, E. Cenni, A. Cenacchi, P.M. Fornasari, et al., S0142-9612(02)00326-5. Enhanced tibial osteotomy healing with use of bone grafts supplemented [71] R.L. Horan, K. Antle, A.L. Collette, Y. Wang, J. Huang, J.E. Moreau, et al., In vitro with platelet gel or platelet gel and bone marrow stromal cells, J. Bone Joint degradation of silk fibroin, Biomaterials 26 (2005) 3385–3393, http://dx.doi. Surg. 89 (2007) 2413–2420. org/10.1016/j.biomaterials.2004.09.020. [97] S.R. Kanthan, G. Kavitha, S. Addi, D.S.K. Choon, T. Kamarul, Platelet-rich [72] K. Numata, P. Cebe, D.L. Kaplan, Mechanism of enzymatic degradation of plasma (PRP) enhances bone healing in non-united critical-sized defects: a beta-sheet crystals, Biomaterials 31 (2010) 2926, http://dx.doi.org/10.1016/j. preliminary study involving rabbit models, Injury 42 (2011) 782–789, http:// biomaterials.2009.12.026. dx.doi.org/10.1016/j.injury.2011.01.015. [73] H.-J. Jin, J. Park, V. Karageorgiou, U.-J. Kim, R. Valluzzi, P. Cebe, et al., Water- [98] P. Kasten, J. Vogel, F. Geiger, P. Niemeyer, R. Luginbühl, K. Szalay, The effect of stable silk films with reduced b-sheet content, Adv. Funct. Mater. 15 (2005) platelet-rich plasma on healing in critical-size long-bone defects, 1241–1247, http://dx.doi.org/10.1002/adfm.200400405. Biomaterials 29 (2008) 3983–3992, http://dx.doi.org/10.1016/j. [74] Q. Lu, X. Hu, X. Wang, J.A. Kluge, S. Lu, P. Cebe, et al., Water-insoluble silk biomaterials.2008.06.014. films with silk I structure, Acta Biomater. 6 (2010) 1380–1387, http://dx.doi. [99] Y. Tamada, New process to form a silk fibroin porous 3-D structure, org/10.1016/j.actbio.2009.10.041. Biomacromolecules 6 (2005) 3100–3106, http://dx.doi.org/10.1021/ [75] R. You, Y. Xu, Y. Liu, X. Li, M. Li, Comparison of the in vitro and in vivo bm050431f. degradations of silk fibroin scaffolds from mulberry and nonmulberry [100] R. Nazarov, H.-J. Jin, D.L. Kaplan, Porous 3-D scaffolds from regenerated silk silkworms, Biomed. Mater. 10 (2015) 15003. fibroin, Biomacromolecules 5 (2004) 718–726, http://dx.doi.org/10.1021/ [76] G. Huang, D. Yang, C. Sun, J. Huang, K. Chen, C. Zhang, et al., A quicker bm034327e. degradation rate is yielded by a novel kind of transgenic silk fibroin [101] Y. Wang, D.D. Rudym, A. Walsh, L. Abrahamsen, H.-J. Kim, H.S. Kim, et al., In consisting of shortened silk fibroin heavy chains fused with matrix vivo degradation of three-dimensional silk fibroin scaffolds, Biomaterials 29 metalloproteinase cleavage sites, J. Mater. Sci. Med. 25 (2014) 1833–1842, (2008) 3415–3428, http://dx.doi.org/10.1016/j.biomaterials.2008.05.002. http://dx.doi.org/10.1007/s10856-014-5220-6. [102] C. Correia, S. Bhumiratana, L.-P. Yan, A.L. Oliveira, J.M. Gimble, D. Rockwood, [77] E.M. Pritchard, T. Valentin, D. Boison, D.L. Kaplan, Incorporation of proteinase et al., Development of silk-based scaffolds for tissue engineering of bone from inhibitors into silk-based delivery devices for enhanced control of human adipose-derived stem cells, Acta Biomater. 8 (2012) 2483–2492, degradation and drug release, Biomaterials 32 (2011) 909–918, http://dx. http://dx.doi.org/10.1016/j.actbio.2012.03.019. doi.org/10.1016/j.biomaterials.2010.09.055. [103] A.L. Oliveira, L. Sun, H.J. Kim, X. Hu, W. Rice, J. Kluge, et al., Aligned silk-based [78] D.W. Hutmacher, Scaffolds in tissue engineering bone and cartilage, 3-D architectures for contact guidance in tissue engineering, Acta Biomater. 8 Biomaterials 21 (2000) 2529–2543. (2012) 1530–1542, http://dx.doi.org/10.1016/j.actbio.2011.12.015. [79] Y. Zhang, W. Fan, Z. Ma, C. Wu, W. Fang, G. Liu, et al., The effects of pore [104] H.J. Kim, U.-J. Kim, G. Vunjak-Novakovic, B.-H. Min, D.L. Kaplan, Influence of architecture in silk fibroin scaffolds on the growth and differentiation of macroporous protein scaffolds on bone tissue engineering from bone marrow

stem cells, Biomaterials 26 (2005) 4442–4452, http://dx.doi.org/10.1016/j. silk-based tissue-engineered ligament scaffold, J. Biomed. Mater. Res. A 101 biomaterials.2004.11.013. (2013) 555–566. 10.1002/jbm.a.34333. [105] L. Uebersax, T. Apfel, K.M.R. Nuss, R. Vogt, H.Y. Kim, L. Meinel, et al., [129] N. Panda, A. Biswas, K. Pramanik, S. Jonnalagadda, Enhanced osteogenic Biocompatibility and osteoconduction of macroporous silk fibroin implants potential of human mesenchymal stem cells on electrospun nanofibrous in cortical defects in sheep, Eur. J. Pharm. Biopharm. 85 (2013) 107–118, scaffolds prepared from eri-tasar silk fibroin, J. Biomed. Mater. Res. B Appl. http://dx.doi.org/10.1016/j.ejpb.2013.05.008. Biomater. 00B (2014) 1–12. 10.1002/jbm.b.33272. [106] U.-J. Kim, J. Park, H.J. Kim, M. Wada, D.L. Kaplan, Three-dimensional aqueous- [130] Y. Zhang, C. Wu, T. Friis, Y. Xiao, The osteogenic properties of CaP/silk derived biomaterial scaffolds from silk fibroin, Biomaterials 26 (2005) 2775– composite scaffolds, Biomaterials 31 (2010) 2848–2856. 10.1016/j. 2785, http://dx.doi.org/10.1016/j.biomaterials.2004.07.044. biomaterials.2009.12.049. [107] S. Sengupta, S.-H. Park, A. Patel, J. Carn, K. Lee, D.L. Kaplan, Hypoxia and [131] J.S.J. Kim, S. Park, W. Kang, H.K.H.K.J. Jang, Signaling responses of osteoblast amino acid supplementation synergistically promote the osteogenesis of cells to hydroxyapatite: the activation of ERK and SOX9 2008, 10:138–42, human mesenchymal stem cells on silk protein scaffolds, Tissue Eng. Part A doi: 10.1007/s00774-007-0804-6. 16 (2010) 3623–3634, http://dx.doi.org/10.1089/ten.tea.2010.0302. [132] H. Liu, G. Wei, Y. Fei, H. Shi, S. Xiong, Y. Wu, et al., Composite scaffolds of [108] S. Hofmann, M. Hilbe, R.J. Fajardo, H. Hagenmüller, K. Nuss, M. Arras, et al., nano-hydroxyapatite and silk fibroin enhance mesenchymal stem cell- Remodeling of tissue-engineered bone structures in vivo, Eur. J. Pharm. based bone regeneration via the interleukin 1 alpha autocrine/paracrine Biopharm. 85 (2013) 119–129, http://dx.doi.org/10.1016/j.ejpb.2013.02.011. signaling loop, Biomaterials 49 (2015) 103–112. 10.1016/j. [109] B.W. Thimm, S. Wüst, S. Hofmann, H. Hagenmüller, R. Müller, Initial cell pre- biomaterials.2015.01.017. cultivation can maximize ECM mineralization by human mesenchymal stem [133] J. Jiang, W. Hao, Y. Li, J. Yao, Z. Shao, H. Li, et al., Hydroxyapatite/regenerated cells on silk fibroin scaffolds, Acta Biomater. 7 (2011) 2218–2228, http://dx. silk fibroin scaffold-enhanced osteoinductivity and osteoconductivity of bone doi.org/10.1016/j.actbio.2011.02.004. marrow-derived mesenchymal stromal cells, Biotechnol. Lett. 35 (2013) [110] A. Takeuchi, C. Ohtsuki, T. Miyazaki, H. Tanaka, M. Yamazaki, M. Tanihara, 657–661. 10.1007/s10529-012-1121-2. Deposition of bone-like apatite on silk fiber in a solution that mimics [134] S. Bhumiratana, W.L. Grayson, A. Castaneda, D.N. Rockwood, E.S. Gil, D.L. extracellular fluid, J. Biomed. Mater. Res. A 65 (2003) 283–289, http://dx.doi. Kaplan, et al., Nucleation and growth of mineralized bone matrix on silk- org/10.1002/jbm.a.10456. hydroxyapatite composite scaffolds, Biomaterials 32 (2011) 2812–2820. [111] F. Lin, Y. Li, J. Jin, Y. Cai, K. Wei, J. Yao, Deposition behavior and properties of 10.1016/j.biomaterials.2010.12.058. silk fibroin scaffolds soaked in simulated body fluid, Mater. Chem. Phys. 111 [135] M. Ribeiro, M.A. de Moraes, M.M. Beppu, M.P. Garcia, M.H. Fernandes, F.J. (2008) 92–97, http://dx.doi.org/10.1016/j.matchemphys.2008.03.019. Monteiro, et al., Development of silk fibroin/nanohydroxyapatite composite [112] J.R. Vetsch, S.J. Paulsen, R. Müller, S. Hofmann, Effect of fetal bovine serum on hydrogels for bone tissue engineering, Eur. Polym. J. 67 (2015) 66–77, http:// mineralization in silk fibroin scaffolds, Acta Biomater. 13 (2015) 277–285, dx.doi.org/10.1016/j.eurpolymj.2015.03.056. http://dx.doi.org/10.1016/j.actbio.2014.11.025. [136] N. Panda, A. Bissoyi, K. Pramanik, A. Biswas, Directing osteogenesis of stem [113] B. Marelli, C.E. Ghezzi, A. Alessandrino, J.E. Barralet, G. Freddi, S.N. Nazhat, cells with hydroxyapatite precipitated electrospun eri-tasar silk fibroin Silk fibroin derived polypeptide-induced biomineralization of collagen, nanofibrous scaffold, J. Biomater. Sci. Ed. 25 (2014) 1440–1457. 10.1080/ Biomaterials 33 (2012) 102–108, http://dx.doi.org/10.1016/j. 09205063.2014.943548. biomaterials.2011.09.039. [137] S. Suganya, I.J. Venugopa, S. Ramakrishna, B.S. Lakshmi, V.R.G. Dev, Aloe vera/ [114] S.-R. Jung, N.-J. Song, D.K. Yang, Y.-J. Cho, B.-J. Kim, J.-W. Hong, et al., Silk silk fibroin/hydroxyapatite incorporated electrospun nanofibrous scaffold for proteins stimulate osteoblast differentiation by suppressing the Notch enhanced osteogenesis, J. Biomater. Tissue Eng. 4 (2014) 9–19. 10.1166/ signaling pathway in mesenchymal stem cells, Nutr. Res. 33 (2015) 162– jbt.2014.1139. 170, http://dx.doi.org/10.1016/j.nutres.2012.11.006. [138] E.I. Pascu, J. Stokes, G.B. McGuinness, Electrospun composites of PHBV, silk [115] A. Nagano, Y. Tanioka, N. Sakurai, H. Sezutsu, N. Kuboyama, H. Kiba, et al., fibroin and nano-hydroxyapatite for bone tissue engineering, Mater. Sci. Eng. Regeneration of the femoral epicondyle on calcium-binding silk scaffolds C Mater. Biol. Appl. 33 (2013) 4905–4916. 10.1016/j.msec.2013.08.012. developed using transgenic silk fibroin produced by transgenic silkworm, [139] H. Kim, L. Che, Y. Ha, W. Ryu, Mechanically-reinforced electrospun composite Acta Biomater. 7 (2011) 1192–1201, http://dx.doi.org/10.1016/j. silk fibroin nanofibers containing hydroxyapatite nanoparticles, Mater. actbio.2010.10.032. Sci. Eng. C Mater. Biol. Appl. 40 (2014) 324–335. 10.1016/j.msec.2014.04.012. [116] G.K. Hunter, H.A. Goldberg, Modulation of crystal formation by bone [140] C. Li, C. Vepari, H.-J. Jin, H.J. Kim, D.L. Kaplan, Electrospun silk-BMP-2 phosphoproteins: role of glutamic acid-rich sequences in the nucleation of scaffolds for bone tissue engineering, Biomaterials 27 (2006) 3115– hydroxyapatite by bone sialoprotein, Biochem. J. 302 (1994) 175–179. 3124. 10.1016/j.biomaterials.2006.01.022. [117] N.L. Harris, K.R. Rattray, C.E. Tye, T.M. Underhill, M.J. Somerman, J.A. D’Errico, [141] J.-P. Chen, S.-H. Chen, G.-J. Lai, Preparation and characterization of et al., Functional analysis of bone sialoprotein: identification of the biomimetic silk fibroin/chitosan composite nanofibers by electrospinning hydroxyapatite-nucleating and cell-binding domains by recombinant for osteoblasts culture, Nanoscale Res. Lett. 7 (2012) 1–11. 10.1186/1556- peptide expression and site-directed mutagenesis, Bone 27 (2000) 795– 276X-7-170. 802, http://dx.doi.org/10.1016/S8756-3282(00)00392-6. [142] X. Ding, X. Wei, Y. Huang, C. Guan, T. Zou, S. Wang, et al., Delivery of [118] H.-J. Jin, S.V. Fridrikh, G.C. Rutledge, D.L. Kaplan, Electrospinning Bombyx mori demineralized bone matrix powder using a salt-leached silk fibroin carrier silk with poly(ethylene oxide), Biomacromolecules 3 (2002) 1233–1239, for bone regeneration, J. Mater. Chem. B 3 (2015) 3177–3188. 10.1039/ http://dx.doi.org/10.1021/bm025581u. C5TB00046G. [119] B.-M. Min, G. Lee, S.H. Kim, Y.S. Nam, T.S. Lee, W.H. Park, Electrospinning of [143] R. Rajkhowa, E.S. Gil, J. Kluge, K. Numata, L. Wang, X. Wang, et al., Reinforcing silk fibroin nanofibers and its effect on the adhesion and spreading of normal silk scaffolds with silk particles, Macromol. Biosci. 10 (2010) 599–611. human keratinocytes and fibroblasts in vitro, Biomaterials 25 (2004) 1289– 10.1002/mabi.200900358. 1297, http://dx.doi.org/10.1016/j.biomaterials.2003.08.045. [144] E.S. Gil, J.A. Kluge, D.N. Rockwood, R. Rajkhowa, L. Wang, X. Wang, et al., [120] H.-J. Jin, J. Chen, V. Karageorgiou, G.H. Altman, D.L. Kaplan, Human bone Mechanical improvements to reinforced porous silk scaffolds, J. Biomed. marrow stromal cell responses on electrospun silk fibroin mats, Biomaterials Mater. Res. A 99 (2011) 16–28. 10.1002/jbm.a.33158. 25 (2004) 1039–1047, http://dx.doi.org/10.1016/S0142-9612(03)00609-4. [145] B.B. Mandal, A. Grinberg, E.S. Gil, B. Panilaitis, D.L. Kaplan, High-strength silk [121] C.S. Ki, S.Y. Park, H.J. Kim, H.-M. Jung, K.M. Woo, J.W. Lee, et al., Development protein scaffolds for bone repair, Proc. Natl. Acad. Sci. U.S.A. 109 (2012) of 3-D nanofibrous fibroin scaffold with high porosity by electrospinning: 7699–7704. 10.1073/pnas.1119474109. implications for bone regeneration, Biotechnol. Lett. 30 (2008) 405–410. [146] D.N. Rockwood, E.S. Gil, S.-H. Park, J.A. Kluge, W. Grayson, S. Bhumiratana, 10.1007/s10529-007-9581-5. et al., Ingrowth of human mesenchymal stem cells into porous silk particle [122] S.Y. Park, C.S. Ki, Y.H. Park, H.M. Jung, K.M. Woo, H.J. Kim, Electrospun silk reinforced silk composite scaffolds: an in vitro study, Acta Biomater. 7 (2011) fibroin scaffolds with macropores for bone regeneration: an in vitro and 144–151. 10.1016/j.actbio.2010.07.020. in vivo study, Tissue Eng. Part A 16 (2010) 1271–1279. 10.1089/ten. [147] G.L. Jones, A. Motta, M.J. Marshall, Haj A.J. El, S.H. Cartmell, Osteoblast: tea.2009.0328. osteoclast co-cultures on silk fibroin, chitosan and PLLA films, Biomaterials [123] S. Ghosh, S.T. Parker, X. Wang, D.L. Kaplan, J.A. Lewis, Direct-write assembly 30 (2009) 5376–5384, http://dx.doi.org/10.1016/j.biomaterials.2009.07. of microperiodic silk fibroin scaffolds for tissue engineering applications, 028. Adv. Funct. Mater. 18 (2008) 1883–1889. 10.1002/adfm.200800040. [148] M. Mizuno, R. Fujisawa, Y. Kuboki, Type I collagen-induced osteoblastic [124] A.B. Dababneh, I.T. Ozbolat, Bioprinting technology: a current state-of-the-art differentiation of bone-marrow cells mediated by collagen-a2b1 integrin review, J. Manuf. Sci. Eng. 136 (2014) 61016. interaction, J. Cell. Physiol. 184 (2000) 207–213. 10.1002/1097-4652(200008) [125] R. Suntivich, I. Drachuk, R. Calabrese, D.L. Kaplan, V.V. Tsukruk, Inkjet printing 184:2<207::AID-JCP8>3.0.CO;2-U. of silk nest arrays for cell hosting, Biomacromolecules 15 (2014) 1428–1435. [149] J.-Y. Kim, B.-E. Yang, J.-H. Ahn, S.O. Park, H.-W. Shim, Comparable efficacy of 10.1021/bm500027c. silk fibroin with the collagen membranes for guided bone regeneration in rat [126] S. Das, F. Pati, Y. Choi, G. Rijal, J.-H. Shim, S. Won, et al., Bioprintable, cell-laden calvarial defects, J. Adv. Prosthodont. 6 (2014) 539–546. silk fibroin–gelatin hydrogel supporting multilineage differentiation of stem 10.4047/jap.2014.6.6.539. cells for fabrication of three-dimensional tissue constructs, Acta Biomater. 11 [150] K.-H. Kim, L. Jeong, H.-N. Park, S.-Y. Shin, W.-H. Park, S.-C. Lee, et al., Biological (2015) 233–246, http://dx.doi.org/10.1016/j.actbio.2014.09.023. efficacy of silk fibroin nanofiber membranes for guided bone regeneration, J. [127] K. Schacht, T. Jüngst, M. Schweinlin, A. Ewald, J. Groll, T. Scheibel, Biotechnol. 120 (2005) 327–339, http://dx.doi.org/10.1016/j.jbiotec.2005. Biofabrication of cell-loaded 3D spider silk constructs, Angew. Chem. Int. 06.033. Ed. 54 (2015) 2816–2820. 10.1002/anie.201409846. [151] G.S. Perrone, G.G. Leisk, T.J. Lo, J.E. Moreau, D.S. Haas, B.J. Papenburg, et al., [128] P. He, S. Sahoo, K.S. Ng, K. Chen, S.L. Toh, J.C.H. Goh, Enhanced The use of silk-based devices for fracture fixation, Nat. Commun. 5 (2014) osteoinductivity and osteoconductivity through hydroxyapatite coating of 3385. 10.1038/ncomms4385.

[152] L. Meinel, O. Betz, R. Fajardo, S. Hofmann, A. Nazarian, E. Cory, et al., Silk endothelial and osteoblast cells and the host vasculature, Biomaterials 31 based biomaterials to heal critical sized femur defects, Bone 39 (2006) 922– (2010) 6959–6967, http://dx.doi.org/10.1016/j.biomaterials.2010.05.057. 931, http://dx.doi.org/10.1016/j.bone.2006.04.019. [170] M. Bhattacharjee, E. Schultz-Thater, E. Trella, S. Miot, S. Das, M. Loparic, et al., [153] X. Jiang, J. Zhao, S. Wang, X. Sun, X. Zhang, J. Chen, et al., Mandibular repair in The role of 3D structure and protein conformation on the innate and adaptive rats with premineralized silk scaffolds and BMP-2-modified bMSCs, immune responses to silk-based biomaterials, Biomaterials 34 (2013) 8161– Biomaterials 30 (2009) 4522–4532, http://dx.doi.org/10.1016/j. 8171, http://dx.doi.org/10.1016/j.biomaterials.2013.07.018. biomaterials.2009.05.021. [171] L. Uebersax, H.P. Merkle, L. Meinel, Insulin-like growth factor I releasing silk [154] J. Zhao, Z. Zhang, S. Wang, X. Sun, X. Zhang, J. Chen, et al., Apatite-coated silk fibroin scaffolds induce chondrogenic differentiation of human mesenchymal fibroin scaffolds to healing mandibular border defects in canines, Bone 45 stem cells, J. Control. Release 127 (2008) 12–21, http://dx.doi.org/10.1016/j. (2009) 517–527, http://dx.doi.org/10.1016/j.bone.2009.05.026. jconrel.2007.11.006. [155] R.N. Wang, J. Green, Z. Wang, Y. Deng, M. Qiao, M. Peabody, et al., Bone [172] X. Wang, E. Wenk, X. Zhang, L. Meinel, G. Vunjak-Novakovic, D.L. Kaplan, Morphogenetic Protein (BMP) signaling in development and human diseases, Growth factor gradients via microsphere delivery in biopolymer scaffolds for Genes Dis. 1 (2014) 87–105, http://dx.doi.org/10.1016/j.gendis.2014.07.005. osteochondral tissue engineering, J. Control. Release 134 (2009) 81–90, [156] V. Karageorgiou, M. Tomkins, R. Fajardo, L. Meinel, B. Snyder, K. Wade, et al., http://dx.doi.org/10.1016/j.jconrel.2008.10.021. Porous silk fibroin 3-D scaffolds for delivery of bone morphogenetic protein- [173] X. Wang, X. Zhang, J. Castellot, I. Herman, M. Iafrati, D.L. Kaplan, Controlled 2 in vitro and in vivo, J. Biomed. Mater. Res., Part A 78A (2006) 324–334. release from multilayer silk biomaterial coatings to modulate vascular cell 10.1002/jbm.a.30728. responses, Biomaterials 29 (2008) 894–903, http://dx.doi.org/10.1016/j. [157] Q. Han, P. Yang, Y. Wu, S. Meng, L. Sui, L. Zhang, et al., Epigenetically modified biomaterials.2007.10.055. bone marrow stromal cells (BMSCs) in silk scaffolds promote craniofacial [174] L. Uebersax, M. Mattotti, M. Papaloïzos, H.P. Merkle, B. Gander, L. Meinel, Silk bone repair and wound healing, Tissue Eng. Part A (2015). 10.1089/ten. fibroin matrices for the controlled release of nerve growth factor (NGF), TEA.2014.0484. Biomaterials 28 (2007) 4449–4460, http://dx.doi.org/10.1016/j. [158] J.-H. Ye, Y.-J. Xu, J. Gao, S.-G. Yan, J. Zhao, Q. Tu, et al., Critical-size calvarial biomaterials.2007.06.034. bone defects healing in a mouse model with silk scaffolds and SATB2- [175] A. Schneider, X.Y. Wang, D.L. Kaplan, J.A. Garlick, C. Egles, Biofunctionalized modified iPSCs, Biomaterials 32 (2011) 5065–5076, http://dx.doi.org/ electrospun silk mats as a topical bioactive dressing for accelerated wound 10.1016/j.biomaterials.2011.03.053. healing, Acta Biomater. 5 (2009) 2570–2578. 10.1016/j.actbio.2008.12.013. [159] C. Wu, Y. Zhang, Y. Zhou, W. Fan, Y. Xiao, A comparative study of mesoporous [176] M. Farokhi, F. Mottaghitalab, M.A. Shokrgozar, J. Ai, J. Hadjati, M. Azami, Bio- glass/silk and non-mesoporous glass/silk scaffolds: physiochemistry and hybrid silk fibroin/calcium phosphate/PLGA nanocomposite scaffold to in vivo osteogenesis, Acta Biomater. 7 (2011) 2229–2236, http://dx.doi.org/ control the delivery of vascular endothelial growth factor, Mater. Sci. Eng., 10.1016/j.actbio.2010.12.019. C 35 (2014) 401–410, http://dx.doi.org/10.1016/j.msec.2013.11.023. [160] S. Midha, T.B. Kim, W. van den Bergh, P.D. Lee, J.R. Jones, C.A. Mitchell, [177] V. Karageorgiou, L. Meinel, S. Hofmann, A. Malhotra, V. Volloch, D. Kaplan, Preconditioned 70S30C bioactive glass foams promote osteogenesis in vivo, Bone morphogenetic protein-2 decorated silk fibroin films induce osteogenic Acta Biomater. 9 (2013) 9169–9182, http://dx.doi.org/10.1016/j. differentiation of human bone marrow stromal cells, J. Biomed. Mater. Res. A actbio.2013.07.014. 71 (2004) 528–537. 10.1002/jbm.a.30186. [161] L. Meinel, R. Fajardo, S. Hofmann, R. Langer, J. Chen, B. Snyder, et al., Silk [178] P. Shi, S.a. Abbah, K. Saran, Y. Zhang, J. Li, H.-K. Wong, Silk fibroin-based implants for the healing of critical size bone defects, Bone 37 (2005) 688– complex particles with bioactive encrustation for bone morphogenetic 698. 10.1016/j.bone.2005.06.010. protein 2 delivery, Biomacromolecules 14 (2013) 4465–4474, http://dx.doi. [162] I.D. Xynos, A.J. Edgar, L.D.K. Buttery, L.L. Hench, J.M. Polak, Gene-expression org/10.1021/bm401381s. profiling of human osteoblasts following treatment with the ionic products of [179] Y.-Q. Zhang, W.-L. Zhou, W.-D. Shen, Y.-H. Chen, X.-M. Zha, K. Shirai, et al., BioglassÒ 45S5 dissolution, J. Biomed. Mater. Res. 55 (2001) 151–157. Synthesis, characterization and immunogenicity of silk fibroin-l- 10.1002/1097-4636(200105)55:2<151::AID-JBM1001>3.0.CO;2-D. asparaginase bioconjugates, J. Biotechnol. 120 (2005) 315–326, http://dx. [163] H.J. Kim, U.-J. Kim, H.S. Kim, C. Li, M. Wada, G.G. Leisk, et al., Bone tissue doi.org/10.1016/j.jbiotec.2005.06.027. engineering with premineralized silk scaffolds, Bone 42 (2008) 1226–1234. [180] M. Haider, V. Leung, F. Ferrari, J. Crissman, J. Powell, J. Cappello, et al., 10.1016/j.bone.2008.02.007. Molecular engineering of silk-elastin like polymers for matrix-mediated gene [164] J.-H. Kim, H.-J. Moon, T.-H. Kim, J.-M. Jo, S.H. Yang, D. Naskar, A novel in vivo delivery: biosynthesis and characterization, Mol. Pharm. 2 (2005) 139–150. platform for studying alveolar bone regeneration in rat, J. Tissue Eng. (2013) 10.1021/mp049906s. 4, http://dx.doi.org/10.1177/2041731413517705. [181] B. Chen, H. Lin, J. Wang, Y. Zhao, B. Wang, W. Zhao, et al., Homogeneous [165] N. Sahu, P. Baligar, S. Midha, B. Kundu, M. Bhattacharjee, S. Mukherjee et al., osteogenesis and bone regeneration by demineralized bone matrix loading Nonmulberry silk fibroin scaffold shows superior osteoconductivity than with collagen-targeting bone morphogenetic protein-2, Biomaterials 28 mulberry silk fibroin in calvarial bone regeneration. Adv. Healthc. Mater. n.d., (2007) 1027–1035, http://dx.doi.org/10.1016/j.biomaterials.2006.10.013. accepted. [182] Y.M. Kolambkar, K.M. Dupont, J.D. Boerckel, N. Huebsch, D.J. Mooney, D.W. [166] S.S. Silva, E.G. Popa, M.E. Gomes, M.B. Oliveira, S. Nayak, B. Subia, et al., Silk Hutmacher, et al., An alginate-based hybrid system for growth factor delivery hydrogels from non-mulberry and mulberry silkworm cocoons processed in the functional repair of large bone defects, Biomaterials 32 (2011) 65–74. with ionic liquids, Acta Biomater. 9 (2013) 8972–8982, http://dx.doi.org/ 10.1016/j.biomaterials.2010.08.074. 10.1016/j.actbio.2013.06.044. [183] T. Diab, E.M. Pritchard, B.A. Uhrig, J.D. Boerckel, D.L. Kaplan, R.E. Guldberg, A [167] J.M. Kanczler, R.O.C. Oreffo, Osteogenesis and angiogenesis: the potential for silk hydrogel-based delivery system of bone morphogenetic protein for the engineering bone, Eur. Cell Mater. 15 (2008) 100–114. treatment of large bone defects, J. Mech. Behav. Biomed. Mater. 11 (2012) [168] L. Sun, S.T. Parker, D. Syoji, X. Wang, J.A. Lewis, D.L. Kaplan, Direct-write 123–131, http://dx.doi.org/10.1016/j.jmbbm.2011.11.007. assembly of 3D silk/hydroxyapatite scaffolds for bone co-cultures, Adv. [184] J.M. Kanczler, P.J. Ginty, L. White, N.M.P. Clarke, S.M. Howdle, K.M. Healthc. Mater. 1 (2012) 729–735. 10.1002/adhm.201200057. Shakesheff, et al., The effect of the delivery of vascular endothelial growth [169] R.E. Unger, S. Ghanaati, C. Orth, A. Sartoris, M. Barbeck, S. Halstenberg, et al., factor and bone morphogenic protein-2 to osteoprogenitor cell populations The rapid anastomosis between prevascularized networks on silk fibroin on bone formation, Biomaterials 31 (2010) 1242–1250, http://dx.doi.org/ scaffolds generated in vitro with cocultures of human microvascular 10.1016/j.biomaterials.2009.10.059.

Please cite this article in press as: J. Melke et al., Silk ﬁbroin as biomaterial for bone tissue engineering, Acta Biomater. (2015), http://dx.doi.org/10.1016/j. actbio.2015.09.005