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Protein-based materials in load-bearing tissue-engineering applications

Proteins such as collagen and elastin are robust molecules that constitute nanocomponents in the hierarchically organized ultrastructures of bone and tendon as well as in some of the soft tissues that have load-bearing functions. In the present paper, the macromolecular structure and function of the proteins are reviewed and the potential of mammalian and non-mammalian proteins in the engineering of loadbearing tissue substitutes are discussed. Chimeric proteins have become an important structural biomaterial source and their potential in tissue engineering is highlighted. Processing of proteins challenge investigators and in this review rapid prototyping and microfabrication are proposed as methods for obtaining precisely defined custom-built tissue engineered structures with intrinsic microarchitecture. Keywords:  biomaterial • load bearing • protein • regeneration • scaffold • tissue engineering

The need for organs and tissues is ever increasing due to the aging of the population, diseases, accidents and the limited availability of donors. Tissue engineering and regenerative medicine seek to construct substitutes to revitalize compromised tissues and improve organ functionality. The tissue-engineering idea is based on harnessing in vitro cell methods and bioactive agents with the goal of replacing or supporting the function of defective or injured body parts [1] . Polymeric scaffolds carry great importance in tissue engineering as they protect the cells against dynamic forces exerted by the body and provide appropriate surfaces for cell adhesion, proliferation and guide tissue formation with their inherent biochemical and topographical cues. In the biological tissues, cells reside on and interact with the extracellular matrix (ECM), which is composed of glycosaminoglycans and a fibrillar network of collagen and elastin (Figure 1A & B) . In order for the substitute materials to perform under predominantly load-bearing conditions they should match the compressive and tensile mechanical strength, and the

10.2217/RME.14.52 © 2014 Future Medicine Ltd

Esen Sayin1,2, Erkan Türker Baran*,2 & Vasif Hasirci1,2,3 METU, Department of Biotechnology, Ankara, Turkey 2 BIOMATEN, METU Center of Excellence in Biomaterials & Tissue Engineering, Ankara 06800, Turkey 3 METU, Departments of Biological Sciences, Ankara, Turkey *Author for correspondence: Tel.: + 90 312 2105194 Fax: + 90 312 2101542 erkantur@ metu.edu.tr 1

elasticity of the target tissues. Another important criterion of a tissue-engineered construct must be the resistance to fatigue or to cyclic stresses caused naturally by pulsatile pressure in blood vessels, tensile stresses on tendon and compressive loads on bone tissues. The degradation profile should also comply with the healing timeline of the tissue to maintain mechanical stability of the wound site. Proteolytic enzymes in body fluids and hydrolytic cleavage lead to degradation of protein scaffolds in vivo. For example, scaffolds for bone tissue engineering are expected to disappear typically after 12–18 months without any immunological reactions [2] . For structural integrity and to extend the degradation time, the protein scaffold should be stabilized with physical or chemical crosslinking. The crosslink is expected to prolong the degradation time of protein scaffolds depending on the type of crosslinker and dosage. For example, medical collagen scaffolds implanted into rats were reported to be completely degraded in 8 weeks when chemically crosslinked, while enzymatically crosslinked scaffolds showed only a little degradation after

Regen. Med. (2014) 9(5), 687–701

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A

D

C Nucleus

Collagen triple helix

Anchored cell

Desmosine crosslink

Filopodia

α-helix

Elastin ECM

E α-helix

B Cell membrane Filopodia

F-actin

Fibronectin

Talin

Para cell

Coil rope Cell membrane complex Matrix

Integrin ECM Macrofibril

Keratin assembly

Ortho cell

Figure 1.  Cell-biomaterial surface interaction governing mechanical signaling in a cell. (A) In this signaling process, the integrin receptors on the cell membrane bind to the fibronectin sequences found on ECM proteins. (B) Any mechanical stimuli originating from this anchorage and engagement of signaling proteins and cytoskeleton elements is transferred into the nucleus. (C) Schematic presentation of left-handed tropocollagen molecule (length 87 Å, diameter 10 Å), composed of right-handed helix fibrils. (D) Schematic presentation of elastin molecule composed of tropohelices crosslinked by desmosine links. (E) Schematic presentation of keratin ultrastructure showing self assembly of smaller units into larger fibrillar structures. ECM: Extracellular matrix.

24 weeks [3] . Likewise, when implanted in rats, the 3D scaffolds made of regenerated fibroin, which was stabilized physically, started to disintegrate in a few weeks and was resorbed after 1 year [4] . This review focuses on the high potential structural proteins have for the preparation of load-bearing scaffolds for tissue engineering. The first section describes potent protein classes according to their biological origin by highlighting their unique properties through structure–function relationships. The second section summarizes major structural forms of protein-based scaffolds and tissue-engineering constructs by highlighting their inherent advantages for tissue development and guidance. In this section, we examine the processing strategies for the production of such structures and the latest developments in the field of processing of proteins. Finally, the recent studies on the use of protein-based biomaterials in the construction of load-bearing tissues are discussed. Materials Mammalian proteins

Collagen is a structural protein that is found abundantly in bone, tendon, ligament and skin. It has high tensile strength, adhesiveness and cohesiveness

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properties. Collagen is composed of three left-handed helices twisted to form a right-handed triple helix (Figure 1C) . Gly–X–Y repeats are found in the fibrous structure where one of X and Y is generally proline or hydroxyproline [5] . It carries cell adhesion sequences, such as arginine–glycine–aspartic acid (RGD), which ensures the continuation of cellular activity and maintenance of phenotype for cell types such as fibroblasts and chondrocytes. Collagen isolated from tissues can trigger very low levels of inflammatory and antigenic responses. Recently, recombinant human collagen has come into use in tissue-engineering applications [6] . Elastin can be isolated from animal skin and can also be produced by recombinant DNA technology. It has great importance in terms of bringing elasticity to vascular tissues, skin and cartilage. From the mechanical point of view this adds extension and elastic recoil features to tissues and durability against cyclic loading. In its formation soluble tropoelastin molecules covalently bind to each other and become insoluble elastin (Figure 1D) . Keratin, another widely distributed mammalian protein, is a structural, insoluble protein and its monomers build up the intracellular intermediate filaments. It is found in epithelial cells and in the structures

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Protein-based materials in load-bearing tissue-engineering applications 

connected to the epidermis, such as hair, nails, wool and horns [7] . It mediates cell–matrix interactions due to its RGD and LDV sequences (Figure 1E) [8] .

sericin with hot detergent treatment increases its biocompatibility when used as films, porous matrices and hydrogels [9] .

Non-mammalian proteins

Chimera proteins

Silk has been employed in the production of sutures, membranes, scaffolds, controlled release systems and substrates for cell growth owing to its low inflammatory and antithrombotic nature. Its selfassembled crystalline β-sheets are the reason for its superior material properties (Figure 2A) . Silk is a fibrous protein and has strength, toughness and extensibility (Figure 2B) . It is obtained from insects and spiders. Silk has a high molecular weight but this value significantly changes depending on the source species. The major source of silk is the silkworm Bombyx mori and it is made up of two components, namely sericin and fibroin. Sericin is a glycoprotein and keeps the two fibroin fibers attached to each other in the natural silk structure. Removal of

The use of recombinant DNA technology in tailoring physical and biological attributes of proteins is a significant development in biomaterials science. In this approach gene sequences of different proteins from various organisms can be combined in a single synthetic DNA and, subsequently, it is expressed in high yield when it is transferred into a suitable vector. In designing high strength protein scaffolds, the desired properties of certain proteins, such as the strength, elasticity and active cell adhesion, can be mimicked on a chimera protein. Researchers have produced elastin-like proteins (ELPs) (elastin-like recombinamers) [10] , silk-like proteins [11] , collagen-like proteins [12] , gelatin-like proteins [13] and resilin-like proteins [14]

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B

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Hydrophobic part Hydrophilic part

Micelle formation

5.7 Å

Microfiber formation

C Growth factors, ECM proteins, ELP, SLP, CLP, GLK, RLP Cell adhesion, mineralization, proteolytic and polysaccharide-binding regions

3.5 Å

D

Chitin-binding region Exon 1

5.7 Å

Exon 2

Elastic repeats (N-terminal)

Exon 3 Elastic repeats (C-terminal)

Figure 2. Molecular organization of potential proteins for load-bearing application. (A) Antiparallel β-sheet structure at regions comprised mostly of glycine and alanine (red: oxygen; blue: nitrogen; dark gray: carbon; light gray: hydrogen). Spacing between the β-sheets depends on the side chains. (B) Scheme for fibroin fiber formation. (C) Scheme showing different combinations of proteins and various functional amino acid sequences incorporated within a chimera protein. (D) Scheme of resilin structure. Beta turn formation is observed mostly at N and C terminals, and alpha helix is found mainly at the chitin binding regions. CLP: Collagen-like protein; ECM: Extracellular matrix; ELP: Elastin-like protein; GLK: Gelatin-like protein; RLP: Resilin-like protein; SLP: Silk-like protein. For color images please see online www.futuremedicine.com/doi/full/10.2217/rme.14.52

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Review  Sayin, Baran & Hasirci and they all have sequences repeated in their primary structure, which give them various capabilities. For example, chimeric proteins combine different functions when used in the design of synthetic ECMs and these can help us control cell migration, differentiation and adhesion (Figure 2C) [15] . Additionally, many alternative fusion proteins have been designed such as resilin–elastin–collagen-like polypeptide, which has sequences from each protein [16] , or vitronectin– insulin-like growth factor fusion protein, which promotes cell growth and migration [17] , and similarly other ECM protein fused variants that enhance cell survival, proliferation, spreading and adhesion by using MAP-integrated fibronectin, laminin, type IV collagen-derived peptides [18] , as well as cell adhesion sequence RGD [19] . Silk-like proteins can be designed by using different repeats from silkworm or spider silk to obtain mechanically strong and elastic biomaterials. In a recent study Albertson et al. studied the effect of repeat size (n = 1–3) of the sequence (GAGQQGPG SQGPGSGGQQGPGGQ) nGPYGPSA8 based on the spider species of Argiope aurantia, on the elasticity and strength of synthetic protein structures [20] . Regardless of repeat times, for all protein types specific mechanical properties could be enhanced by post-spin stretch; however, these treatments led to very little improvement in extensibility. In another chimeria application, silk elastin-like proteins were produced from B. mori silk (GAGAGS) and mammalian elastin (GVGVP) domains to combine the strength and elasticity [21] . Elasticity, biodegradation, interactions with cellular receptors, gelation and also responsiveness to temperature, pH and ionic strength can all be incorporated through the use of these domains [22] . Resilin, an insect protein, maintains high-frequency-requiring actions, such as flight, sound production and jumping (Figure 2D) [23] , and has recently attracted the attention of biomaterials scientists. Resilin is a rubber-like protein and possesses high levels of energy storage, low stiffness, high extension and resilience [14] . In light of these it can be noted that the composition and spatial control of synthetic peptides can be useful for the production of biomimetic scaffolds and to obtain functional tissues. Forms & methods of production Fiber structures

At nanoscale the proteins can self assemble into highstrength fibrillar structures by close packing and helical twist of complementary, small and bulky amino acids. At macroscale too, the alignment of biopolymers and proteins into fiber structure by extruding through a narrow orifice and extension of fibers by spinning pro-

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cess or by other physical forces is an efficient method to reinforce and strengthen the mechanical properties, as the molecular chains are aligned anisotropically in one direction and packed tightly. For instance, the electric field orientation, genipin crosslinking and ethanol treatment enhanced the wet ultimate tensile stress of collagen threads up to a value of 109 MPa, which is close to the level of strength provided by natural tendon tissue [24] . Woven or knitted type of scaffolds manufactured from biocompatible fibers present unique mechanical properties and improved mechanical strength by providing effective interconnecting voids for tissue ingrowth. Natural silks are structural proteins that comprise light weight (1.3 g/cm3), high tensile strength (up to 4.8 GPa), elasticity (approximately 35%) and thermal stability (up to 250°C) [25] . Therefore, the use of knitted fibers of silk fibroin has been one of the most preferred biomaterial for constructing scaffolds intended for load-bearing applications. The use of knitted mesh showed that this structure can mediate effective repair of tissues such as ligament/tendon/cartilage and pipe-like organs with their high mechanical properties [26] . Incorporation of a knitted structure of silk fibers and a collagen sponge matrix composite protein scaffold was shown to improve ligament regeneration by affecting ligament matrix gene activation and collagen expression [27] . A knitted silk scaffold composite carrying rat mesenchymal stem cells (MSCs) has been recommended as a tissue-engineered sling for long-term stress urinary incontinence treatment [28] . The Young’s modulus obtained of silk sling group and silk/rat MSC sling constructs were reported to be 3 and 4 MPa, respectively. Silk scaffolds based on microporous silk and knitted silk seeded with mesenchymal cells was used in ligament regeneration. The maximum tensile load of 250 N observed with the knitted scaffolds was comparable to the values of human anterior cruciate ligament experiences during normal physical activity. Collagen fibers are common macromolecular forms in animals, providing the structures that support mechanical loads. For that reason, biomaterials based on collagens are natural candidates for tissue engineering [29] . In a study it was reported that extruded collagen fibers can attain mechanical properties similar to that of human ligaments (tangent modulus of 359.6 MPa, peak stress of 36.0 MPa) [30] . Alternatively, threaded collagen fiber stitches were bonded to poly(glycolic acid) to make a composite scaffold that could provide support for engineering of hollow organs, such as a bladder tissue [31] . The biomechanical studies demonstrated that the composite construct was elastic and maintained the preconfigured struc-

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tures and in an athymic mice model the load at break (35.8 N) was found to be comparable to that of native bladder (18.5 N). Gentleman and his coworkers combined collagen fiber scaffolds with fibroblast-seeded collagen gels, which showed the tensile and viscoelastic behavior closely mimicked the natural ligament [32] . The physical properties of these constructs did not deteriorate during the culture, and for the constructs with seeded fibroblasts the peak strength was greater. Caves et al. fabricated collagen microfibers as a thin lamellae consisting of a continuous mesh form by embedding the fibers with a recombinant ELP [33] . The mechanical properties of the produced sheet exceeded that of a number of native human tissues, including urinary bladder, pulmonary artery and aorta. Nonwoven fiber scaffolds, especially when prepared using random nanofibers, mimic the ECM proteins (50–500 nm diameter fibers) and can create in vivolike conditions by enabling cells to take a 3D orientation [34] . For that reason, wet spinning and electrospinning techniques were used in the production of protein-based micro/nanofibers and designed natural microarchitectures to enable cell ingrowth and effective cellularization of the scaffold. The spinning procedure has been the most effectively used method to

Review

produce fiber forms of biomaterials. In this method, a polymer melt or a viscous solution is ejected through an orifice to form the fiber structures. In wet spinning, natural and synthetic polymers are injected through a syringe needle or narrow orifice into a coagulating bath to precipitate the injected polymer in the form of fibers (Figure 3A) . During or after precipitation these threads can be further reinforced by drawing on a spinneret. The manufacturing of fibrous collagen structures with high yardage was studied by Meyer et al. by wet spinning and melt spinning [35] . The fibers produced by wet spinning were relatively stronger than the ones from melt spinning. Um et al. indicated that the crystallinity of wet spun regenerated filaments was hardly affected by the draw ratio, whereas the crystalline and amorphous orientation of regenerated silk fibroin filament was improved with the increased drawing ratio, hence increased tensile properties [36] . When draw was applied both during takeup and post-spinning, fibroin fibers displayed ductile and stable behavior. Typical values of the mechanical parameters of regenerated silk fibroin fibers were E = 8.7 GPa, σ = 120 MPa and ε = 35%. In an attempt to mimic the biological efficiency of the natural spinneret organs of the silkworm and the

A

B

C

D

E

F

Figure 3. Different forms of tissue-engineering scaffolds produced by various processing methods. (A) SEM image of wet spun poly(3-hydroxybutyrate-co-3-hydroxyvalerate), magnification x100, side view. (B) SEM image of electrospun collagen fibers crosslinked with dehydrothermal treatment, magnification x3000. (C) SEM image of electrospun silk fibroin fibers, magnification x500. (D) SEM image of collagen sponge produced via lyophilization, magnification × 250. (E) SEM images of micropatterned silk film stabilized with methanol showing groove and ridge patterns, magnification x700. (F) SEM image of 3D PCL construct made with rapid prototyping, magnification x50.

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Review  Sayin, Baran & Hasirci spider, capillary spinning equipment was used to spin aqueous solutions of silk fibroin in air [37] . The resultant fiber had a breaking strength of 46 MPa, which could reach 359 MPa after a preliminary post-draw in 80% (v/v) aqueous ethanol solution. In a similar study, natural fiber matrix directly drawn from wild nonmulberry tropical tasar silkworm, Antheraea mylitta, was used for tissue-engineering scaffold production [38] . The mechanical strength of that silk fibroin without sericin was, however, comparatively lower (467 MPa) in comparison to domestic B. mori silk fibroin fibers (740 MPa). A silk-based scaffold was also developed using winding equipment with a fiber network similar to criss-cross structures [39] . The data showed a ninefold increase in compressive modulus (20 MPa) of the silk and silk–chondroitin sulfate scaffolds when the materials were seeded with human nasal cartilage cells cultured for up to 4 weeks. During electrospinning, polymer solution is ejected from a syringe via a high voltage application to a metal capillary tube. As the electric field ejects the solution at a constant rate, the liquid component evaporates leaving dry fibers behind before they reach the collect or surface. The orientation of the fibers has a significant influence on cellular responses and especially on their alignment. Using electrospinning both synthetic and natural polymers can be processed into nano and microfibers (Figure 3B & C) . To date, protein-based matrices, such as those from collagen [40] , silk [41] , gelatin [42,43] , elastin [43] and fibrinogen [44] , have been constructed using electrospinning. Nanofibrous scaffolds prepared using proteins are not only present in a matrix, such as natural ECM, that is suitable for cell ingrowth but they also provide significant mechanical strength, which is important for load-bearing tissue-engineering constructs. McKenna et al. proposed the use of an electrospun recombinant human tropoelastin scaffold for vascular grafts as a material with ideal structural properties and biocompatibility [45] . This scaffold had tensile strength, elastic modulus and burst pressures of 0.36 MPa, 0.91 MPa and 485 mmHg, respectively; values that are close to that of the native tissue. Electrospun gelatin resulted in nanofiber mats that exhibited improved elastic modulus (990 MPa) and tensile strength (21 MPa) when the structure was crosslinked with genipin, although the flexibility of the mat was considerably lower. Interestingly, the electrospinning of a silk elastin-like protein resulted in a ribbon-like morphology by forming a selfstanding, nonwoven fiber mesh that was characterized by tensile strength and initial modulus of 30.8 MPa and 0.88 GPa, respectively [46] . By using a similar strategy, an electrospun synthetic human tropoelastin:type I collagen composite scaffold was suggested for dermal

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tissue engineering [47] . Young’s modulus values of 169, 160 and 841 MPa for respective 80T20C, 60T40C and 50T50C compositions indicated the existence of a critical ratio of 50:50. Several researchers used extrusion to obtain collagen fibers [48,49] . The extrusion technique was also used to fabricate small diameter tubes of anisotropic collagen fibrils [50] . When collagen conduits were crosslinked with glutaraldehyde and with carbodiimide they could withstand high pressures (approximately 200 torrs). In a similar technique, the extruded collagen type-I/PEG fibers were wound on a rotating and translating spool to make a structure of layered-fibers [51] . The resultant collagen fiber structure could withstand a stress of 25–49 MPa, a value that is close to the strength of native tendon tissue. Macroporous structures

Freeze-drying is a widely used technique owing to the resultant high porosity and mechanical strength features that are desirable for load-bearing scaffolds. Solutions of water-soluble polymers can be directly poured to a mold and freeze-dried and this is simpler in preparation than the organic solution required polymers. For the latter type of polymer solutions, aqueous solution should be mixed with them in order to obtain a homogeneous emulsion that can provide even pore distribution. Finally, application of vacuum and sublimation of water results in high porosity structures (Figure 3D) . Silk fibroin foams can present promising scaffold properties with significant compressive strength. In their study, Kim et al. studied the effect of silk fibroin concentration and the particle size of the porogen (NaCl) on the structural properties of the porous 3D scaffolds [52] . It was shown that by using 10% aqueous fibroin solutions, the scaffolds had porosities of 90% compressive strength and modulus of 320 and 3330 kPa, respectively. Instead of using porogens, the incorporation of hydroxyapatite (HA) microparticles (inorganic constituent of natural bone) into silk could lead to sponge matrices with osteogenic and osteconductive properties that are essential for in vitro formation of bone [53] . Furthermore, HA incorporation in silk sponges at 1.6, 3.1 and 4.6% resulted in an increased Young modulus of 594, 865 and 1005 kPa, respectively (340 kPa in the absence of HA). Films

Micropatterning on films can help the cellular orientation needed to achieve complex tissue formation (Figure 3E) . Solvent casting of polymer solution is a widely accepted method, and besides, this pro-

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Protein-based materials in load-bearing tissue-engineering applications 

tein can itself be directly micropatterned by crosslinking through UV exposure over a mask [54] . The micromolding is also useful for scaffold preparation using thin membranes or films that are stacked together to produce thicker structures. A cell/hydrogel micromolding approach was used to fabricate relatively large (0.5–2 cm 2) and thick (127–384 mm) skeletal muscle networks with dense, aligned and highly differentiated muscle fibers [55] . Using this approach a cell/hydrogel mixture was cast inside microfabricated PDMS molds with staggered microposts to create porous tissue networks. Hu et al. investigated the growth of C2C12 myoblasts and human bone marrow stem cells on silk–tropoelastin films with controlled roughness and micro/ nanoscale topological patterns [56] . An increase in tropoelastin fraction decreased the strain compared with pure silk, which had the highest stiffness (27 MPa). The high surface roughness by micro/nanopatterns and increased tropoelastin content favored the proliferation and differentiation of hMSCs, while myoblast cells preferred low surface roughness and high stiffness. ELP membranes were surface patterned with grooves and posts (200 nm in lateral dimensions and up to 10 μm in height) to create self-supporting membranes and produce layered scaffolds [57] . As shown by nanoidentation tests, Young’s modulus, the stiffness, of patterned membranes experienced a significant decrease from the dry state (4.6 GPa) to the hydrated state (5.5 MPa) and the stiffness increased to higher values at higher temperatures. 3D printed forms

Scaffold design is a crucial part of the tissue-engineering process. The microarchitecture of the constructs is important in the tissue formation as the physical and biochemical cues guide cell commitment and correct tissue texture. With the advances in the additive techniques or rapid prototyping (RP) technology, it is possible to make scaffolds with precise geometries and finely controlled physical properties [58] . By using rapid prototyping, horizontal layers are added over each other to build up a scaffold by a computer-controlled mechanical process (Figure 3F) . To date, naturally derived biomaterials were rarely used in RP. This is partly because a protein material does not show thermoplastic behavior for melt extrusion like the synthetic polymers used. Therefore, novel strategies have been adopted for protein processing by RP. In the construction of heart valves carrying human interstitial cells, the scaffolds of collagen type-I was prepared by rapid prototyping [59] . Sacrificial moulds were designed using CAD software and a 3D inkjet printer. In the latter stage, the collagen dispersion was cast into

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the mold, which can be removed with dissolving in a solvent [60] . By this type of sacrificial RP processing macrosized channels in fibroin matrix was created by a 3D inkjet printer [61] . Projection stereolithography based on CAD is another type of additive technology used to engineer 3D scaffolds that mimic the microarchitecture of tissues. The projection stereolithography system has been used successfully in the fabrication of 3D scaffolds from photocrosslinkable gelatin methacrylate (GelMA) [62] . A multilayered tissue-engineering principle was applicable to the construction of human skin by mimicking skin layers. For this purpose, 3D free-form fabrication with direct cell dispensing was used and collagen hydrogel incorporating fibroblasts and keratinocytes were used in this application [63] . Engineered load-bearing tissues The scaffolds made partially or totally of proteins can be produced in various forms for use in loadbearing tissues (Tables 1 & 2) . We discuss below the use of protein scaffolds in the construction of hard and soft tissues requiring specific mechanical properties depending on the tissue type. Hard tissues

Bone is a dynamic tissue that undergoes continuous remodeling and is constituted of organic components (collagen fibers) and inorganic components (HA) (Figure 4A) . Collagen provides flexibility and resistance to cracking and HA introduces stiffness. Similar to the natural tissue, the scaffolds that are designed for bone tissue engineering should have osteoconductivity and evoke osteoinductive responses at the implant site. Generally, additional materials are incorporated into the protein to give the scaffolds the fundamental features of the native bone. Composites were preferred in a sponge form; for example, into the gelatin and chitosan sponges sintered and nonsintered HA were added to increase their mechanical strength and make the composition similar to the bone. In vitro studies with a human osteosarcoma cell line (Saos-2) reported enhanced attachment and proliferation on scaffolds carrying sintered HA [64] . Silk has become increasingly popular in bone tissue engineering owing to its high strength. Recent approaches involved silk modified with different peptides to achieve better mineralization. A combination of bone sialoprotein and spider silk fusion protein induced bone formation by hMSC because sialoprotein component could nucleate calcium phosphate and prompt osteogenic differentiation [65] . Despite the low mechanical strength of collagen, it is widely used as a bone scaffold material due to its biocompatibility. Therefore, several strategies have been

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Table 1. Processing methods and mechanical properties of different protein scaffolds designed for hard tissue engineering. Tissue

Protein

Scaffold morphology

Processing method

Mechanical strength

Ref.

Bone

Silk/silica fusion protein

Film

Solvent casting

–

[9]

 

Spider silk/bone sialoprotein fusion protein

Film

Solvent casting

–

[65]

 

Collagen with chitosan and HA

Sponge

Lyophilization

–

[77]

 

Silk with nanoHA or/and BMP-2

Fiber

Electrospinning

–

[78]

 

Gelatin with chitosan and sintered HA

Sponge

Lyophilization

Compressive strength: 3.17 MPa

[64]

 

 

 

 

Young’s modulus: 0.31 GPa

 

Cartilage

Silk fibroin with chondroitin sulfate

Fiber

Winding

Compressive modulus: ~20 MPa

[39]

 

Silk fibroin

Hydrogel

Sonication

Compressive modulus: ~170 kPa

[73]

 

Collagen with chondroitin sulfate and hyaluronan

Sponge

Lyophilization

–

[79]

Tendon

Silk fibroin and collagen

Knitted

Machine knitting and lyophilization

Young’s modulus: ~35 MPa

[75]

 

Fibrin glue

Gel

–

Young’s modulus: ~130 N/mm2

[80]

 

Silk fibroin with PLGA releasing bFGF

Knitted

Machine knitting (silk fibroin), electrospinning (PLGA)

Failure load: 83 N

[81]

Meniscus

Silk fibroin

Trabecular structure

Salt leaching and lyophilization

Compressive modulus: ~15 MPa

[82,83]

HA: Hydroxyapatite; PLGA: Poly(lactic-acid co-glycolic acid).

tried to remedy this weakness. An ECM-associated protein, SPARC, which is responsible for the mineralization of collagen during bone formation, was shown to enhance HA nanoparticle nucleation on the collagen scaffold [66] . Xia et al. reported a biomimetic approach that utilized precipitation of mineralized collagen fibers and also formation of isotropic equiaxed or unidirectional lamellar architecture via controlled freeze casting [67] . Resilience, flexibility and resistance to compressive loads are characteristics of cartilage tissue. Generally, the cartilage tissue shows alymphatic and aneural properties. Owing to its low metabolic activity and quantity of cells and its avascular nature, which result in scarcity of circulating nutrients and progenitor cells, a slower repair process than bone is observed under normal physiological conditions [68] . Cartilage ECM is mainly composed of type II collagen and proteoglycans. The demand for cartilage tissue substitutes as a result of sports injuries and diseases such as osteoarthritis is high.

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Collagen is the most commonly utilized protein material in cartilage tissue engineering. Type I and II collagen scaffolds were seeded with auricular, articular and meniscal chondrocytes and all were shown to express lubricin, which has antiadhesive properties and maintains joint lubrication [69] . Gelatin, a denatured form of collagen, has been employed in many different forms, such as raw and photoactive forms. Photocurable GelMA, with or without acrylated hyaluronic acid (HA-MA), was used in entrapping human chondrocytes. It was seen that the compressive moduli of control GelMA constructs increased by 26 kPa after 8 weeks culture, constructs with hyaluronic acid methacrylate (HA-MA) and chondroitin sulfate methacrylate (CS-MA) increased by 114 kPa [70] . Similarly, Shin et al. reported that fibroblast cells encapsulated in double-network hydrogels composed of photocrosslinked gelatin and gellan gum molecules presented the high strength needed for load-bearing tissues [71] . Silk fibroin is another potential protein material used in various cartilage constructs. In one such

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Protein-based materials in load-bearing tissue-engineering applications 

study, it was noted that silk matrix designed for articular cartilage improved compressive stiffness, modulus, DNA, glycosaminoglycan (GAG) and collagen content when used with high cell densities [72] . Furthermore, silk hydrogels were shown to possess mechanical strength compatible with that of cartilage tissue and was much better than the biomechanically matching but nonbiodegradable and immunogenic agarose scaffolds [73] . Saha et al. presented an interesting result showing that fibroin obtained from different species can be osteoinductive or chondroinductive for cells that migrate into scaffold from the osteochondral defect [74] . Tendon is a connective tissue and attaches muscles to the bones. Tendon injuries are frequently suffered by athletes and active people. It is viscoelastic and has low healing capacity and high tensile load resistance. Collagen I is the major constituent of tendon (Figure 4B), but it also contains elastin, GAGs, proteoglycans and glycoproteins, such as fibronectin. Fabrication of collagen scaffolds with mechanical properties close to that of natural tendon is a significant challenge. Knitted silk–collagen sponges seeded with human embryonic stem cell-derived mesenchymal stem cells were studied as potential scaffolds for tendon tissue engineering [75] . Mechanical stimuli

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applied to these scaffolds in vitro led to immature, fibroblast-like morphology and expression of tendonassociated genes, while in vivo, mechanical stimulus resulted in high levels of cell alignment and collagen deposition. Meniscus is a fibrocartilaginous tissue and is composed of two semicircular, wedge-shaped parts. Meniscus tissue contains a white-colored avascularized and red-colored vascularized part. In meniscus, collagen type I-rich ECM also involves GAGs, collagen type II, III, V and VI. Meniscus injuries are frequently seen in young patients and removal of meniscus tissue after tear decreases their standard of living. In order to substitute meniscus, three layered, wedge-shaped silk matrices were designed and human fibroblast cells and human articular chondrocytes were seeded onto the outer and inner sides, respectively [76] . Addition of TGF-b3 to the culture medium improved cell proliferation and ECM secretion, preserved chondrocytic phenotype and expressed high amounts of S-GAG, type I and type II collagen. Soft tissues

Skin is a multilayered smooth tissue that comprises keratinocytes, melanocytes and fibroblasts in the epidermis, lower epidermis and lower dermal parts of the

Table 2. Tissue-engineered soft tissues. Tissue

Protein

Scaffold morphology

Processing method

Mechanical strength

Skin

Tropoelastin

Nanofiber

Electrospinning

Tensile strength: 150, 220 kPa

[85]

 

Silk fibroin with chitosan

Hydrogel

Immersion in ethanol

Storage modulus: ~6 × 105 Pa

[86]

 

Keratin

Sponge

Lyophilization

–

[89]

 

Sericin

Membrane

Solvent casting

Tensile strength: ~9 MPa

Bladder

Silk fibroin

Gel

Gel spinning.

–

[93]

 

Fibrinogen

Nanofiber

Electrospinning

–

[102]

Vessel

Collagen, silk fibroin

Hydrogel Rotor milling (collagen), (microfibers) microfiber (silk fibroin)

Ultimate tensile stress: ~40 kPa

 

Collagen

Gel

–

Ultimate tension: ~90 N/m

[103]

Cornea

Recombinant human collagen type III

Flat sheet

Solvent casting

Tensile strength: 1.7 MPa

[100]

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Ref.

[101]

[95]

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Collagen fibril

Artey and capillaries

Nerve fiber

Collagen fiber Primary fiber bundle

Vein

Tertiary fiber bundle

Collagen fibrils 300 nm in length Hydroxyapatite crystals (Ca10(PO4)6(OH)2)

Osteon

Cross-section of bone

Secondary fiber bundle

Adventitia Intima

Elastin layer

Media

Epitenon

Figure 4. Supramolecular protein organization and hierarchial structure of various tissues. (A) Collagen and HA in the bone structure are highly ordered to form the osteons. Opposite alignment of collagen fibrils in the consecutive layers of the osteon is the source of resistance against twisting forces. (B) The ultrastructure of tendon. (C) Endothelium and thin layer of connective tissue (tunica intima), muscle cells and elastic tissue (tunica media) and fibrous connective tissue (tunica adventitia) constitute the blood vessel (artery). HA: Hydroxyapatite.

skin, respectively. Materials for skin tissue engineering should favor regeneration of natural tissue and their mechanical and physical characteristics, such as elasticity and tensile strength, should match that of natural skin. Mechanical tensile strength values of stratum corneum, epidermis and dermis are reported as 1.9 MPa, 102 MPa and 10.2 MPa, respectively [84] . Besides, materials should be wettable and they should cover and adhere to the injury site, prevent fluid loss and bacterial infections. Many other proteins were employed in skin tissueengineering scaffolds, such as recombinant human tropoelastin [85] , silk fibroin [86] , gelatin [87,88] and human hair keratin [89] . More recently, scaffolds made of chimera proteins were prepared for skin substitution. For example, the use of ELP and keratinocyte growth factor (a member of the FGF family, which has a role in epithelization) fusion protein, which can selfassemble into nanoparticles, induced the healing of the wounds of diabetic mice; they achieved higher reepithelialization and granulation [90] . Bladder is a highly elastic tissue that can adapt to hourly swelling and contraction and accommodate large volumes of urine. Atala and colleagues carried out an important clinical study with a composite of collagen and polyglycolic acid and it was understood

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that implanted composite scaffold performed better than only collagen scaffold in vivo in terms of bladder compliance and capacity [91] . In another in vivo study, addition of collagen to poly(lactic acid-co-3-caprolactone) scaffolds decreased the inflammatory reactions [92] . Alternatively, silk fibroin was used in bladder tissue engineering and the expression of contractile proteins that indicate bladder wall regeneration was recorded [93] . Silk fibroin also presented lower levels of fibrosis and inflammatory response in comparison to small intestinal submucosa (SIS) and polyglycolic acid matrices. In vessel tissue engineering, tubular structures with hemocompatible surfaces were designed to allow pulsatile blood flow. To perform suitably, fabricated vascular substitutes should have similar mechanical properties to the blood vessels (Figure 4C) . Collagen type I can be employed in the form of a hydrogel carrying cells inside the molded tubular blood vessel structure. For example, Miwa et al. designed a graft that spatially mimicked the native vessel by creating medial and adventitial layers that carried encapsulated smooth muscle cells and fibroblasts, respectively [94] . However, inadequate mechanical strength of this structure required incorporation of stronger and hydrophobic polyethylene terephthalate.

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Protein-based materials in load-bearing tissue-engineering applications 

Alternatively, fibroin microfibers were used to reinforce the collagen hydrogel [95] , but it was shown that the processing parameters of the silk influenced its hemocompatibility [96] . Smooth muscle and endothelial cells encapsulated in fibrin gel yielded approximately the same mechanical strength as the natural small diameter vessels after remodeling [97] . Cornea is a highly innervated tissue that is composed of epithelium, stroma and endothelial cell layers from the outside to the inside. Keratocytes, collagen fibers and proteoglycans are found in the stromal layer, which constitutes the main bulk of the cornea. Fabricated matrices need to be transparent, have adequate mechanical strength, biocompatibility, and allow diffusion of gases and glucose, innervation, epithelium formation and inhibition of retrocorneal fibrous membrane development [98] . Orthogonally aligned collagen lamellae stacks are frequently seen in load-bearing tissues, such as bone and cornea. By using the biomimetic approach Tanaka et al. created collagen lamellae stacks by repeated flow casting of layers in parallel/orthogonal directionalities (2–5 μm layer thickness). The films demonstrated

Review

good optical character and the measured mechanical properties were higher than the values recorded from natural cornea (3.81 N/mm 2 tensile strength at break and 3–13 N/mm 2 in elastic modulus) [99] . Conclusion & future perspective The structural biomacromolecules involved in load bearing by natural hard and soft tissues, the mammalian proteins, collagen, elastin and keratin constitute a significant biomaterial group that are preferred for use in the scaffolds owing to their high biocompatibility and relatively good mechanical properties. Nonmammalian proteins, such as arthropod silk fibroins, on the other hand, have been an alternative to the mammalian proteins in the construction of scaffolds as they had similar biocompatibility and superior mechanical strengths, especially in fiber form. Although proteins can be processed into various physical forms, such as foams, membranes and other bulky structures, the spun protein nanofiber and microfiber meshes provided relatively better mechanical properties required by load-bearing tissues. Silk fibroin materials have increasingly become more used materials in bone

Executive summary Proteins as scaffolds • Functional constructs are possible with intrinsic sequences of proteins that can serve for missions such as cell adhesion and differentiation. • Proteins contribute to mechanical strength of scaffolds as they make possible more elastic, resilient, flexible or tough structures.

Materials • Collagen and elastin are found at load-bearing tissues and for this reason they are utilized for tissue engineering of these body parts. • Silk has toughness, strength and extensibility. Owing to its availability and superior mechanical properties it is a widely accepted biomaterial. • Chimeric proteins have tailor-made mechanical properties and they can also show responsiveness to temperature, pH and ionic strength. • Cell migration, growth, differentiation and spreading can be added to the functions of chimeric proteins.

Forms & methods of production • The spinning process is an efficient method to reinforce and strengthen the mechanical properties of collagen and silk fibroin. • While nonwoven fibrous structures obtained by electrospinning and wet spinning provide an effective nutrient transfer and cell infiltration, respectively, weaving and knitting provide high tensile strength constructs. • Application of freeze-drying and the use of leachable porogens results in high porosity protein foams resistant to compressive stresses. • Although the recent advances in rapid prototyping technology enables scaffolds with precise geometries and finely controlled physical properties, naturally derived biomaterials, including proteins, were rarely used in rapid prototyping.

Engineered load-bearing tissues • Hydroxyapatite is incorporated into the protein materials to give scaffolds fundamental features of the native bone. • Collagen and fibroin protein materials are successfully utilized for cartilage and tendon constructs where high tensile strength is required. • Both elasticity and tensile strength become important for soft tissues, therefore, the composite scaffolds that include elastin-like proteins or synthetic polymers together with collagen and silk are preferred.

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Review  Sayin, Baran & Hasirci tissue engineering owing to their high strength in spun and knitted fiber forms, especially when combined with inorganic HA. Chimera protein technology is likely to enable the creation of a new group of proteins that can combine the strength and biocompatibility of selected proteins. In respect to material processing, the so-called additive technologies, which are currently in state-of-art form thus preventing their widespread application, will play important roles in the precise building of protein structures that mimic tissue-like microstructures.

References

Financial & competing interests disclosure The authors acknowledge the support by METU through project BAP-07.02.2013.101. ET Baran gratefully acknowledges the Scientific and Technological Research Council of Turkey (TUBITAK) for the Post Doctoral 2232 BIDEP-TUBITAK Fellowship. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. No writing assistance was utilized in the production of this manuscript.

12

Yoshizumi A, Yu Z, Silva T et al. Self-association of Streptococcus pyogenes collagen-like constructs into higher order structures. Protein Sci. 18(6), 1241–1251 (2009).

13

Werten MWT, Teles H, Moers APHA et al. Precision gels from collagen-inspired triblock copolymers. Biomacromolecules 10(5), 1106–1113 (2009).

Papers of special interest have been highlighted as: • of interest; •• of considerable interest 1

Atala A. Regenerative medicine strategies. J. Pediatr. Surg. 47(1), 17–28 (2012).

2

Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 21(24), 2529–2543 (2000).

14

3

Rothamel D, Schwarz F, Sager M, Herten M, Sculean A, Becker J. Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat. Clin. Oral Implants Res. 16(3), 369–378 (2005).

Li L, Teller S, Clifton RJ, Jia X, Kiick KL. Tunable mechanical stability and deformation response of a resilinbased elastomer. Biomacromolecules 12(6), 2302–2310 (2011).

•

Reports incorporation of both mechanically important and biologically active sequences.

4

Wang Y, Rudym DD, Walsh A et al. In vivo degradation of three-dimensional silk fibroin scaffolds. Biomaterials 29(24–25), 3415–3428 (2008).

15

Gomes S, Leonor IB, Mano JF, Reis RL, Kaplan DL. Natural and genetically engineered proteins for tissue engineering. Prog. Polym. Sci. 37(1), 1–17 (2012).

5

Nair LS, Laurencin CT. Biodegradable polymers as biomaterials. Prog. Polym. Sci. 32(8–9), 762–798 (2007).

16

6

Merrett K, Liu W, Mitra D et al. Synthetic neoglycopolymer-recombinant human collagen hybrids as biomimetic crosslinking agents in corneal tissue engineering. Biomaterials 30(29), 5403–5408 (2009).

Bracalello A, Santopietro V, Vassalli M et al. Design and production of a chimeric resilin-, elastin-, and collagen-like engineered polypeptide. Biomacromolecules 12(8), 2957–2965 (2011).

17

Van Lonkhuyzen DR, Hollier BG, Shooter GK, Leavesley DI, Upton Z. Chimeric vitronectin:insulin-like growth factor proteins enhance cell growth and migration through coactivation of receptors. Growth Factors 25(5), 295–308 (2007).

18

Choi BH, Choi YS, Kang DG, Kim BJ, Song YH, Cha HJ. Cell behavior on extracellular matrix mimic materials based on mussel adhesive protein fused with functional peptides. Biomaterials 31(34), 8980–8988 (2010).

19

Bini E, Foo CWP, Huang J, Karageorgiou V, Kitchel B, Kaplan DL. RGD-functionalized bioengineered spider dragline silk biomaterial. Biomacromolecules 7(11), 3139–3145 (2006).

20

Albertson AE, Teulé F, Weber W, Yarger JL, Lewis RV. Effects of different post-spin stretching conditions on the mechanical properties of synthetic spider silk fibers. J. Mech. Behav. Biomed. Mater. 29, 225–234 (2014).

21

Haider M, Cappello J, Ghandehari H, Leong KW. In vitro chondrogenesis of mesenchymal stem cells in recombinant silk–elastinlike hydrogels. Pharm. Res. 25(3), 692–699 (2008).

22

Hwang W, Kim BH, Dandu R, Cappello J, Ghandehari H, Seog J. Surface induced nanofiber growth by self-assembly of a silk-elastinlike protein polymer. Langmuir 25(21), 12682–12686 (2009).

7

8

Balaji S, Kumar R, Sripriya R et al. Characterization of keratin-collagen 3D scaffold for biomedical applications. Polym. Adv. Technol. 23(3), 500–507 (2012).

9

Mieszawska AJ, Nadkarni LD, Perry CC, Kaplan DL. Nanoscale control of silica particle formation via silk– silica fusion proteins for bone regeneration. Chem. Mater. 22(20), 5780–5785 (2010).

•

Reports mineralization via recombinant incorporation of a naturally found marine diatom peptide.

10

Kilic C, Girotti A, Rodriguez-Cabello JC, Hasirci V. A collagen-based corneal stroma substitute with microdesigned architecture. Biomater. Sci. 2, 318–329 (2014).

11

••

698

Rouse JG, Van Dyke ME. A review of keratin-based biomaterials for biomedical applications. Materials 3(2), 999–1014 (2010).

Teulé F, Cooper AR, Furin WA et al. A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nat. Protoc. 4(3), 341–355 (2009). Introduces a method for recombinant production of silklike protein to be used in protein including scaffolds.

Regen. Med. (2014) 9(5)

future science group

Protein-based materials in load-bearing tissue-engineering applications 

23

Werkmeister JA, Ramshaw JAM. Recombinant protein scaffolds for tissue engineering. Biomed. Mater. 7(1), 012002 (2012).

24

Alfredo Uquillas J, Kishore V, Akkus O. Genipin crosslinking elevates the strength of electrochemically aligned collagen to the level of tendons. J. Mech. Behav. Biomed. Mater. 15C, 176–189 (2012).

39

Bhattacharjee M, Miot S, Gorecka A et al. Oriented lamellar silk fibrous scaffolds to drive cartilage matrix orientation: towards annulus fibrosus tissue engineering. Acta Biomater. 8(9), 3313–3325 (2012).

40

Bürck J, Heissler S, Geckle U et al. Effects of different postspin stretching conditions on the mechanical properties of synthetic spider silk fibers. Langmuir 29, 1562–1572 (2013).

41

Yu Q, Xu S, Zhang H, Gu L, Xu Y, Ko F. Structure– property relationship of regenerated spider silk protein nano/microfibrous scaffold fabricated by electrospinning. J. Biomed. Mater. Res. A. doi:10.1002/jbm.a.35051 (2013) (Epub ahead of print).

42

Dhandayuthapani B, Krishnan UM, Sethuraman S. Fabrication and characterization of chitosan–gelatin blend nanofibers for skin tissue engineering. J. Biomed. Mater. Res. B. Appl. Biomater. 94(1), 264–272 (2010).

43

Han J, Lazarovici P, Pomerantz C, Chen X, Wei Y, Lelkes PI. Co-electrospun blends of PLGA, gelatin, and elastin as potential nonthrombogenic scaffolds for vascular tissue engineering. Biomacromolecules 12(2), 399–408 (2011).

44

Baker S, Sigley J, Carlisle CR et al. The mechanical properties of dry, electrospun fibrinogen fibers. Mater. Sci. Eng. C. Mater. Biol. Appl. 32(2), 215–221 (2012).

45

McKenna KA, Hinds MT, Sarao RC et al. Mechanical property characterization of electrospun recombinant human tropoelastin for vascular graft biomaterials. Acta Biomater. 8(1), 225–233 (2012).

46

Ner Y, Stuart JA, Whited G, Sotzing GA. Electrospinning nanoribbons of a bioengineered silk-elastin-like protein (SELP) from water. Polymer 50(24), 5828–5836 (2009).

47

Rnjak-Kovacina J, Wise SG, Li Z et al. Electrospun synthetic human elastin:collagen composite scaffolds for dermal tissue engineering. Acta Biomater. 8(10), 3714–3722 (2012).

25

Altman GH, Diaz F, Jakuba C et al. Silk-based biomaterials. Biomaterials 24(3), 401–416 (2003).

26

Wang X, Han C, Hu X et al. Applications of knitted mesh fabrication techniques to scaffolds for tissue engineering and regenerative medicine. J. Mech. Behav. Biomed. Mater. 4(7), 922–932 (2011).

27

Chen X, Qi YY, Wang LL et al. Ligament regeneration using a knitted silk scaffold combined with collagen matrix. Biomaterials 29(27), 3683–3692 (2008).

28

Zou XH, Zhi YL, Chen X et al. Mesenchymal stem cell seeded knitted silk sling for the treatment of stress urinary incontinence. Biomaterials 31(18), 4872–4879 (2010).

29

Kew SJ, Gwynne JH, Enea D et al. Regeneration and repair of tendon and ligament tissue using collagen fibre biomaterials. Acta Biomater. 7(9), 3237–3247 (2011).

30

Gentleman E, Lay AN, Dickerson DA, Nauman EA, Livesay GA, Dee KC. Mechanical characterization of collagen fibers and scaffolds for tissue engineering. Biomaterials 24(21), 3805–3813 (2003).

31

Eberli D, Freitas Filho L, Atala A, Yoo JJ. Composite scaffolds for the engineering of hollow organs and tissues. Methods 47, 109–115 (2009).

32

Gentleman E, Livesay GA, Dee KC, Nauman EA. Development of ligament-like structural organization and properties in cell-seeded collagen scaffolds in vitro. Ann. Biomed. Eng. 34(5), 726–736 (2006).

33

Caves JM, Cui W, Wen J, Kumar V, Haller C, Chaikof EL. Elastin-like protein matrix reinforced with collagen microfibers for soft tissue repair. Biomaterials 32(23), 5371–5379 (2011).

48

Enea D, Henson F, Kew S et al. Extruded collagen fibres for tissue engineering applications: effect of crosslinking method on mechanical and biological properties. J. Mater. Sci. Mater. Med. 22(6), 1569–1578 (2011).

34

Barnes CP, Sell SA, Boland ED, Simpson DG, Bowlin GL. Nanofiber technology: designing the next generation of tissue engineering scaffolds. Adv. Drug Deliv. Rev. 59(14), 1413–1433 (2007).

49

Zeugolis DI, Paul RG, Attenburrow G. Extruded collagen– polyethylene glycol fibers for tissue engineering applications. J. Biomed. Mater. Res. B. Appl. Biomater. 85(2), 343–352 (2008).

35

Meyer M, Baltzer H, Schwikal K. Collagen fibres by thermoplastic and wet spinning. Mater. Sci. Eng. C 30(8), 1266–1271 (2010).

50

Lai ES, Anderson CM, Fuller GG. Designing a tubular matrix of oriented collagen fibrils for tissue engineering. Acta Biomater. 7, 2448–2456 (2011).

36

Um IC, Ki CS, Kweon H, Lee KG, Ihm DW, Park YH. Wet spinning of silk polymer. II. Effect of drawing on the structural characteristics and properties of filament. Int. J. Biol. Macromol. 34(1–2), 107–119 (2004).

51

Kew SJ, Gwynne JH, Enea D et al. Synthetic collagen fascicles for the regeneration of tendon tissue. Acta Biomater. 8(10), 3723–3731 (2012).

52

37

Wei W, Zhang Y, Zhao Y, Luo J, Shao H, Hu X. Bio-inspired capillary dry spinning of regenerated silk fibroin aqueous solution. Mater. Sci. Eng. C 31(7), 1602–1608 (2011).

Kim UJ, Park J, Kim HJ, Wada M, Kaplan DL. Threedimensional aqueous-derived biomaterial scaffolds from silk fibroin. Biomaterials 26(15), 2775–2785 (2005).

53

38

Mandal BB, Kundu SC. Biospinning by silkworms: silk fiber matrices for tissue engineering applications. Acta Biomater. 6(2), 360–371 (2010).

Bhumiratana S, Grayson WL, Castaneda A et al. Nucleation and growth of mineralized bone matrix on silk-hydroxyapatite composite scaffolds. Biomaterials 32(11), 2812–2820 (2011).

•

Presents a method that renders polymer processing unnecessary by using a natural polymer, silk fibroin.

•

Documentation of improved osteoconductivity and mineralization of silk constructs by addition of hydroxyapatite.

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699

Review  Sayin, Baran & Hasirci 54

Kurland NE, Dey T, Wang C, Kundu SC, Yadavalli VK. Silk protein lithography as a route to fabricate sericin microarchitectures. Adv. Mater. 26(26), 4431–4437 (2014).

69

Zhang L, Spector M. Comparison of three types of chondrocytes in collagen scaffolds for cartilage tissue engineering. Biomed. Mater. 4(4), 045012 (2009).

••

Presents an approach for direct production of protein micropatterns by photolithography of photoreactive sericin.

70

55

Bian W, Bursac N. Engineered skeletal muscle tissue networks with controllable architecture. Biomaterials 30(7), 1401–1412 (2009).

Levett PA, Melchels FPW, Schrobback K, Hutmacher DW, Malda J, Klein TJ. A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin, hyaluronic acid and chondroitin sulfate. Acta Biomater. 10(1), 214–223 (2014).

56

Hu X, Park SH, Gil ES, Xia XX, Weiss AS, Kaplan DL. The influence of elasticity and surface roughness on myogenic and osteogenic-differentiation of cells on silk–elastin biomaterials. Biomaterials 32(34), 8979–8989 (2011).

71

57

Tejeda-Montes E, Smith KH, Poch M et al. Engineering membrane scaffolds with both physical and biomolecular signaling. Acta Biomater. 8(3), 998–1009 (2012).

Shin H, Olsen BD, Khademhosseini A. The mechanical properties and cytotoxicity of cell-laden double-network hydrogels based on photocrosslinkable gelatin and gellan gum biomacromolecules. Biomaterials 33(11), 3143–3152 (2012).

72

58

Melchels FPW, Domingos MAN, Klein TJ, Malda J, Bartolo PJ, Hutmacher DW. Additive manufacturing of tissues and organs. Prog. Polym. Sci. 37(8), 1079–1104 (2012).

Talukdar S, Nguyen QT, Chen AC, Sah RL, Kundu SC. Effect of initial cell seeding density on 3D-engineered silk fibroin scaffolds for articular cartilage tissue engineering. Biomaterials 32(34), 8927–8937 (2011).

73

Chao PHG, Yodmuang S, Wang X, Sun L, Kaplan DL, Vunjak-Novakovic G. Silk hydrogel for cartilage tissue engineering. J. Biomed. Mater. Res. B. Appl. Biomater. 95(1), 84–90 (2010).

74

Saha S, Kundu B, Kirkham J, Wood D, Kundu SC, Yang XB. Osteochondral tissue engineering in vivo: a comparative study using layered silk fibroin scaffolds from mulberry and nonmulberry silkworms. PLoS ONE 8(11), e80004 (2013).

••

Showed the significance of mulberry (Bombyx mori) and non-mulberry (Antheraea mylitta) silk fibroin biomaterials on osteochondral tissue regeneration in vivo.

75

Chen JL, Yin Z, Shen WL et al. Efficacy of hESC-MSCs in knitted silk-collagen scaffold for tendon tissue engineering and their roles. Biomaterials 31(36), 9438–9451 (2010).

76

Mandal BB, Park SH, Gil ES, Kaplan DL. Multilayered silk scaffolds for meniscus tissue engineering. Biomaterials 32(2), 639–651 (2011).

77

Pallela R, Venkatesan J, Janapala VR, Kim SK. Biophysicochemical evaluation of chitosan–hydroxyapatite– marine sponge collagen composite for bone tissue engineering. J. Biomed. Mater. Res. A. 486–495 (2011).

78

Li C, Vepari C, Jin H, Joo H, Kaplan DL. Electrospun silkBMP-2 scaffolds for bone tissue engineering. Biomaterials 27, 3115–3124 (2006).

79

Ko CS, Huang JP, Huang CW, Chu IM. Type II collagen– chondroitin sulfate–hyaluronan scaffold cross-linked by genipin for cartilage tissue engineering. J. Biosci. Bioeng. 107(2), 177–182 (2009).

80

Pataquiva-Mateus AY, Wu HC, Lucchesi C, Ferraz MP, Monteiro FJ, Spector M. Supplementation of collagen scaffolds with SPARC to facilitate mineralization. J. Biomed. Mater. Res. B. Appl. Biomater. 100(3), 862–870 (2012).

Ni M, Lui PPY, Rui YF et al. Tendon-derived stem cells (TDSCs) promote tendon repair in a rat patellar tendon window defect model. J. Orthop. Res. 30(4), 613–619 (2012).

81

Xia Z, Yu X, Jiang X, Brody HD, Rowe DW, Wei M. Fabrication and characterization of biomimetic collagenapatite scaffolds with tunable structures for bone tissue engineering. Acta Biomater. 9(7), 7308–7319 (2013).

Sahoo S, Toh SL, Goh JCH. A bFGF-releasing silk/ PLGA-based biohybrid scaffold for ligament/tendon tissue engineering using mesenchymal progenitor cells. Biomaterials 31(11), 2990–2998 (2010).

82

Yan LP, Oliveira JM, Oliveira AL, Caridade SG, Mano JF, Reis RL. Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications. Acta Biomater. 8(1), 289–301 (2012).

59

60

Sachlos E, Reis N, Ainsley C, Derby B, Czernuszka JT. Novel collagen scaffolds with predefined internal morphology made by solid freeform fabrication. Biomaterials 24(8), 1487–1497 (2003).

•

Describes first production of collagen scaffolds with CAD.

61

Chen CH, Liu J, Chua CK, Chou SM, Shyu V, Chen JP. Cartilage tissue engineering with silk fibroin scaffolds fabricated by indirect additive manufacturing technology. Materials 7(3), 2104–2119 (2014).

62

Gauvin R, Chen YC, Lee JW et al. Microfabrication of complex porous tissue engineering scaffolds using 3D projection stereolithography. Biomaterials 33(15), 3824–3834 (2012).

63

64

65

66

67

68

700

Taylor PM, Sachlos E, Dreger SA, Chester AH, Czernuszka JT, Yacoub MH. Interaction of human valve interstitial cells with collagen matrices manufactured using rapid prototyping. Biomaterials 27(13), 2733–2737 (2006).

Lee W, Debasitis JC, Lee VK et al. Multi-layered culture of human skin fibroblasts and keratinocytes through threedimensional freeform fabrication. Biomaterials 30(8), 1587–1595 (2009). Isikli C, Hasirci V, Hasirci N. Development of porous chitosan – gelatin/ ydroxyapatite composite scaffolds for hard tissue-engineering applications. J. Tissue Eng. Regen. Med. 6, 135–143 (2012). Gomes S, Leonor IB, Mano JF, Reis RL, Kaplan DL. Spider silk–bone sialoprotein fusion proteins for bone tissue engineering. Soft Matter 7(10), 4964 (2011).

Huey DJ, Hu JC, Athanasiou KA. Unlike bone, cartilage regeneration remains elusive. Science 338(6109), 917–921 (2012).

Regen. Med. (2014) 9(5)

future science group

Protein-based materials in load-bearing tissue-engineering applications 

83

Balint E, Gatt CJ, Dunn MG. Design and mechanical evaluation of a novel fiber-reinforced scaffold for meniscus replacement. J. Biomed. Mater. Res. A. 100(1), 195–202 (2012).

84

Hendriks FM, Brokken D, Oomens CWJ, Bader DL, Baaijens FPT. The relative contributions of different skin layers to the mechanical behavior of human skin in vivo using suction experiments. Med. Eng. Phys. 28(3), 259–266 (2006).

85

86

87

88

Rnjak-Kovacina J, Wise SG, Li Z et al. Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering. Biomaterials 32(28), 6729–6736 (2011). Silva SS, Santos TC, Cerqueira MT et al. The use of ionic liquids in the processing of chitosan/silk hydrogels for biomedical applications. Green Chem. 14(5), 1463 (2012). Rahman MM, Pervez S, Nesa B, Khan MA. Preparation and characterization of porous scaffold composite films by blending chitosan and gelatin solutions for skin tissue engineering. Polym. Int. 62, 79–86 (2012). Renò F, Rizzi M, Cannas M. Gelatin-based anionic hydrogel as biocompatible substrate for human keratinocyte growth. J. Mater. Sci. Mater. Med. 23(2), 565–571 (2012).

89

Verma V, Verma P, Ray P, Ray AR. Preparation of scaffolds from human hair proteins for tissue-engineering applications. Biomed. Mater. 3(2), 025007 (2008).

90

Koria P, Yagi H, Kitagawa Y et al. Self-assembling elastinlike peptides growth factor chimeric nanoparticles for the treatment of chronic wounds. Proc. Natl Acad. Sci. USA 108(3), 1034–1039 (2011).

•

Documentation of an example for the therapeutic action of designed chimeric proteins.

91

Atala A, Bauer SB, Soker S, Yoo JJ, Retik AB. Tissueengineered autologous bladders for patients needing cystoplasty. Lancet 367(9518), 1241–1246 (2006).

•

Reports the in vivo success of collagen–polyglycolic acid for reconstruction of bladder for end stage patients.

92

Engelhardt EM, Micol LA, Houis S et al. A collagenpoly(lactic acid-co-ε-caprolactone) hybrid scaffold for bladder tissue regeneration. Biomaterials 32(16), 3969–3976 (2011).

future science group

93

Mauney JR, Cannon GM, Lovett ML et al. Evaluation of gel spun silk-based biomaterials in a murine model of bladder augmentation. Biomaterials 32(3), 808–818 (2011).

94

Miwa H, Matsuda T, Iida F et al. Development of a Hierarchically Structured Hybrid Vascular Graft Biomimicking Natural Arteries. ASAIO J. 39(3), 273–277 (1993).

95

De Moraes MA, Paternotte E, Mantovani D, Beppu MM. Mechanical and biological performances of new scaffolds made of collagen hydrogels and fibroin microfibers for vascular tissue engineering. Macromol. Biosci. 12(9), 1253–1264 (2012).

96

Seib FP, Maitz MF, Hu X, Werner C, Kaplan DL. Impact of processing parameters on the haemocompatibility of Bombyx mori silk films. Biomaterials 33(4), 1017–1023 (2012).

97

Swartz DD, Russell JA, Andreadis ST. Engineering of fibrinbased functional and implantable small-diameter blood vessels. Am. J. Physiol. Heart Circ. Physiol. 288(3), 1451–1460 (2005).

98

Shah A, Brugnano J, Sun S, Vase A, Orwin E. The development of a tissue-engineered cornea: biomaterials and culture methods. Pediatr. Res. 63(5), 535–544 (2008).

99

Tanaka Y, Baba K, Duncan TJ et al. Transparent, tough collagen laminates prepared by oriented flow casting, multicyclic vitrification and chemical cross-linking. Biomaterials 32(13), 3358–3366 (2011).

Review

100 Dravida S, Gaddipati S, Griffith M et al. A biomimetic

scaffold for culturing limbal stem cells: a promising alternative for clinical transplantation. J. Tissue Eng. Regen. Med. 2(5), 263–271 (2008). 101 Nayak S, Talukdar S, Kundu SC. Potential of 2D crosslinked

sericin membranes with improved biostability for skin tissue engineering. Cell Tissue Res. 347(3), 783–794 (2012). •

Provides evidence for biocompatibility of sericin discarded during silk fibroin isolation.

102 McManus M, Boland E, Sell S et al. Electrospun nanofibre

fibrinogen for urinary tract tissue reconstruction. Biomed. Mater. 2(4), 257–262 (2007). 103 Achilli M, Meghezi S, Mantovani D. On the viscoelastic

properties of collagen-gel-based lattices under cyclic loading: applications for vascular tissue engineering. Macromol. Mater. Eng. 297(7), 724–734 (2012).

www.futuremedicine.com

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