Advanced Review

Virus-based scaffolds for tissue engineering applications Xia Zhao,1 Yuan Lin1 and Qian Wang2∗ One of the major research directions of tissue engineering is to develop artificial scaffolds that can mimic extracellular matrix (ECM) and support the growth of functional cells for the repair of damaged tissues and organs. Recently, virus particles have expanded as nanosized building blocks for materials applications. Viruses represent monodispersed supramolecular assemblies with organized three-dimensional architecture, which can be isolated in high yield and purity with batch-to-batch consistency. In addition, virus particles can be re-engineered by chemical and genetic modification to incorporate multivalent functional ligands with high density and ordered arrangement. In this review, we highlight that the self-assembly of the reengineered viruses can form two-dimensional and three-dimensional scaffolds, which can be employed to support cell growth and regulate cellular functions such as adhesion, spreading and proliferation. In particular, the application of virus-based scaffolds for directed differentiation of pluripotent stem cells for bone and neural regeneration is discussed. Finally, the in vivo behaviors of virus nanoparticles will be discussed for the consideration of tissue engineering applications. © 2014 Wiley Periodicals, Inc. How to cite this article:

WIREs Nanomed Nanobiotechnol 2014. doi: 10.1002/wnan.1327

INTRODUCTION

T

he goal of tissue engineering is to exploit substitutes to replace or repair damaged tissues and organs.1 In general, it aims at developing a scaffold which can provide environmental cues to support cells growth and promote tissues regeneration.2 In other words, a scaffold for tissue engineering would provide a cell growth supporting environment and display signal molecules to control cellular behaviors such as adhesion, migration, proliferation, and differentiation3 [Figure 1(b)]. In this review, we highlight the recent development in using virus-based scaffolds for tissue engineering applications. The extracellular matrix (ECM) consists of a complex of myriad biomacromolecules, which ∗ Correspondence

to: [email protected]

1 State

Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China

2 Department

of Chemistry and Biochemistry, University of South Carolina, Columbia, SC, USA Conflict of interest: The authors have declared no conflicts of interest for this article.

provides the structural and biochemical support to the surrounding cells and play an important role in the maintenance of cell and tissue structure and function.2 A cell adhesion motifs arginine-glycine-aspartic acid (RGD), which derives from fibronectin and other ECM proteins, is identified as a typical cell adhesion ligand that can interact with cell surface receptor integrin and mediate cell adhesion.4 Such cell–ECM interaction transduces the information received by the receptor into intracellular event and regulates cell behaviors and functions through the receptor-mediated signaling. However, it was reported that cell behaviors can be controlled by topological cues of the underlying substrates,5 including the ligand-display density and the nanoscale topological features of substrates.6,7 For example, Huang and coworkers revealed that cell adhesion induced by RGD ligands was dependent on the local order of ligand arrangement. They confirmed the existence of a critical local inter-ligand spacing of ∼70 nm, above which the cell adhesion would be strongly reduced. Such cell adhesion was turned off by ordered RGD arrangement and turned on by disordered RGD arrangement.8 Therefore, a nanoscale platform

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FIGURE 1 | (a) Three-dimensional structures of viruses that have been developed as platforms for tissue engineering. CPMV, cowpea mosaic virus; TYMV, turnip yellow mosaic virus; TMV, tobacco mosaic virus; M13 bacteriophage. The structures of CPMV, TYMV, and TMV are generated using PyMol (www.pymol.org) with coordinates obtained from RCSB Protein Data Bank (www.pdb.org). M13 is reproduced with permission from Ref 20; copyright 2007 The National Academy of Sciences of the USA. (b) Schematic illustration of viral nanoparticles-based cell-matrix interaction. ECM, extracellular matrix. (c) Viral nanoparticles-based 2D films and 3D scaffolds with random and aligned structures for cell supporting. The figure is reproduced with permission from Refs 67, 69, 51, 84; copyrights from Elsevier Ltd.; American Chemical Society and the Royal Society of Chemistry. (d) Conventional chemical bioconjugation strategies targeting the endogenous amino acids on viral nanoparticles. (e) Representative examples of genetic engineering strategy to insert exogenous peptide on viral nanoparticles. The figure is reproduced with permission from Ref 84; copyright 2009 American Chemical Society.

with well-controlled architecture will provide a great opportunity to study the cellular responses to nanostructured scaffolds and afford a potential insight to design biomaterials for tissue engineering applications. Recently, viruses have emerged as powerful platforms for various applications, especially in the field of biomedicine and biomaterials.9 In general, viruses consist of protein capsids that encapsulate their genomic materials. Their capsids comprise multiple coat proteins which can self-assemble into well-organized architectures with uniform sizes and

shapes. Among all viruses, much attention has been drawn to plant viruses and bacteriophages due to their availability, stability and biosafety. In addition, two-dimensional (2D) and three-dimensional (3D) scaffolds can be easily achieved by assembly of mono-dispersed virus particles [Figure 1(c)]. Moreover, multiple functionalities can be displayed on viral capsids with an ordered spatial arrangement through chemical conjugation10 and genetic engineering11 [Figure 1(d) and (e)]. Because of these specific properties, viral nanoparticles have been recognized as good candidates for ligand displaying and ECM mimicking.

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Until now, many different types of viruses have been employed in tissue engineering applications.12 In this review article, we focus on four different virus particles, i.e., cowpea mosaic virus (CPMV), turnip yellow mosaic virus (TYMV), tobacco mosaic virus (TMV), and M13 bacteriophage [Figure 1(a)]. CPMV is about 27–29 nm in diameter and comprises 60 copies of two-protein subunits that arranged in a icosahedral manner.13,14 Similarly, TYMV is an icosahedral plant virus with an average diameter of 28 nm, consisting of 180 copies of coat proteins.15,16 TMV is a rigid rod-shaped virus that is 300 nm in length and 18 nm in diameter, consisting of 2130 identical protein subunits,17,18 while M13 is a filamentous virus that is 880 nm in length and 6.6 nm in diameter.19,20

REENGINEERING VIRUS PARTICLES FOR CELL ADHESION AND PROLIFERATION As cell adhesion is the first and most important step for cells to sense the microenvironment of supportive scaffold, it is critical to engineer biomaterials to control the cell adhesion behavior.21 It has been reported that wild-type (WT) CPMVs adsorbed onto substrate surface by layer-by-layer technique were able to promote the adhesion and growth of NIH-3T3 fibroblast,22 owing to the interaction of CPMV with surface vimentin on certain mammalian cells.23 However, most viruses derived from plants and bacteria cannot interact with mammalian cells due to the lack of cell binding ligands. It is well known that an RGD motif derived from fibronectin and other ECM proteins can bind to cell surface integrin and mediate cell adhesion.4 Meanwhile, it is easy to anchor RGD ligands on virus surfaces via chemical conjugation or genetic engineering. The multivalency of viruses would allow multiple display of cell signaling motifs to enhance the binding affinity with cell surface receptors. As shown in Figure 1(d), using glutamic/aspartic acids, lysines, and cysteines, the traditional bioconjugation techniques have been well developed to incorporate functional groups on virus surface.10,24 Bruckman et al. have developed a highly efficient CuI -catalyzed azide–alkyne cycloaddition (CuAAC) reaction to introduce the RGD peptides on TMV surface. When a surface was coated with modified TMVs, RGD-displayed TMVs could promote NIH-3T3 cells attachment and induce the cell proliferation more effectively than native TMVs. In comparison, the attachment of polyethylene glycol (PEG) motifs on TMV prohibited the cell attachment effectively.25

Based on the genomic sequences availability, cell binding motifs could also be introduced onto the surface of viruses by genetic modification. For example, the exterior surface of M13 bacteriophage could be displayed with exogenous peptides26 [Figure 1(e)]. Merzlyak et al. have inserted the RGD into the major coat protein pVIII of M13 to promote the attachment of NIH-3T3 cells.27 Similar genetic modification strategy was developed by Lee et al., to construct TMV mutants displaying cell binding motifs such as RGD, P15, DGEA and PHSRN3 with differential adhesion strengths to mammalian cells.28 Moreover, genetically modified TMV-RGD could be co-electrospun with polyvinyl alcohol (PVA)29,30 to form fibrous substrates for supporting cell growth.31 As shown in Figure 2(a), baby hamster kidney (BHK) cells cultured onto the PVA-TMV/RGD fibrous substrate affording much higher cell density. RGD peptides enhanced cell adhesion and promoted cell spreading over the fibers to form prominent F-actin filaments, while cells grown on the fibers without RGD showed round shape morphology and randomized actin structures [Figure 2(b)]. These studies demonstrated that the functionalization of virus particles with cell binding motifs provided a promising strategy to employ viruses for cell-culturing applications.

DIRECTING CELL GROWTH ORIENTATION Cell orientation is very critical for many tissues to achieve their in vivo biological functions, including vascular tissue,32 musculoskeletal system,33 myocardial tissue,34 corneal stroma,35 tendons,36 and nerves.37 Therefore, construction of smart materials that mimic the microenvironment of ECM to guide the cell alignment is crucial for tissue engineering.38,39 One-dimensional (1D) viruses with high aspect ratio, such as M13 and TMV, have become attractive building blocks for developing biomaterials with long-range orientation to guide cell-oriented growth. Zan et al. reported a fluid flow assembly to align TMV in horizontal orientation inside capillary tubes.40 As shown in Figure 3(a), TMV particles were aligned along the long axis of the capillary with high surface coverage, which could be controlled by adjusting the flow rate, the property of substrate surface and the concentration of TMV. The resulting hierarchically ordered TMV films were able to support C2C12 myoblasts attachment and proliferation. It is well known that the myogenesis of C2C12 cells can include the fusion of multinucleated myoblasts and subsequent differentiation to myotubes.41,42 As shown in Figure 3(b), under the

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FIGURE 2 | (a) BHK cell density analyses after 1 and 12 h incubation on electrospinning PVA, PVA-TMV, and PVA-TMV/RGD fibers. (b) Fluorescence microscopy images and Field emission scanning electron microscopy images of BHK cells after 1 h incubation on fibrous substrates. Colour representation: nucleus (blue), F-actin (red). The figure is reproduced with permission from Ref 31; copyright 2011 The Royal Society of Chemistry. PVA, polyvinyl alcohol; TMV, tobacco mosaic virus; RGD, arginine-glycine-aspartic acid.

differentiation condition, C2C12 differentiated into the myotubes in both high and low coverage surfaces. The degrees of the myotubes alignment were quantified by the angles of myotubes with respect to the flow direction, and evaluated by the half height peak width (HPW). Cells cultured on the control surface coated with random TMV showed no alignment with a HPW of 180∘ . As comparison, the alignment of C2C12 myotubes with a HPW of 34∘ was observed on the high-coverage aligned TMV films. But the low coverage of TMV films displayed no significant alignment with (HPW = 126∘ ), which might be attributed to that a low density of TMV was insufficient to guide the C2C12 cells. However, when functionalized with RGD peptide, the low coverage TMV-RGD films could also induce a moderate alignment (HPW = 48∘ ). Furthermore, the differentiation of myoblasts was quantified by the fusion index and maturation index. Both the fusion index and maturation index for cells grown on TMV-RGD (83.7 ± 4.4 and 80.2 ± 2.9%, respectively) are significantly higher than TMV and control groups [Figure 3(c) and (d)]. These results demonstrated that the aligned TMV film could induce the highest level of differentiation for myotubes.43,44 The perpendicular alignment of smooth muscle cells (SMCs) related to the vessel direction is critical for engineering blood vessels.45,46 Lin et al. reported a hierarchical TMV pattern could be achieved to be perpendicular to the long axis of a capillary by controlled evaporation of a TMV solution.47 As shown in Figure 3(e), after drying in an open-ended capillary, TMV formed multiple stripe patterns, which were parallel to the contact line, but perpendicular to the long axis of the capillary. At low TMV

concentrations (0.01 mg/mL < [TMV] < 0.05 mg/mL), these stripes were composed of single layer of TMV [Figure 3(f)]. A multilayer of TMV stripe patterns could be formed at higher concentrations of TMV (0.05 mg/mL < [TMV] < 0.5 mg/mL). SMCs cultured inside such TMV-patterned capillary could align perpendicularly to capillary long axis and tended to be spindle-shaped, which is the typical morphology for contractile phenotype SMCs.48 However, SMCs cultured in the capillary tube without TMV patterns showed the epithelioid or rhomboid morphology, which is the typical morphology of synthetic phenotype.49 The distance between cell stripes could also be controlled [Figure 3(g)]. Therefore, this strategy can potentially be used in development of novel scaffolds for the engineering of particular tissues and organs. Based on their innate ability to organize into liquid crystals structures at high concentration,28,50 the filamentous M13 bacteriophages were also employed to form the aligned structure for directing cell growth. Rong et al. have generated a thin M13 film with the alignment of M13 along their long axis via the convective flow assembly.51 When modified with RGD peptide by chemical conjugation, the M13-RGD film could promote the growth of NIH-3T3 fibroblasts along the alignment direction of M13. Similar work has been carried out by Chung et al. and revealed that genetically engineered M13-RGD matrix could guide and stimulate the growth of the target NIH-3T3 fibroblasts.27 The mechanism for aligned M13 film directing the cell orientation was further explained by Wu et al.52 In their work, both BHK and NIH-3T3 cells cultured on aligned M13 films could spread in

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FIGURE 3 | (a) Schematic illustration of flow assembly for alignment of TMV in capillary tube and AFM image of aligned TMV with horizontal orientation to the tube. (b) Fluorescent images of C2C12 cells with differentiation for 7 days on the tubes with different neural progenitor cells. Colour representation: anti-MHC (red), nucleus (blue). Scale bars: 100 μm. (c) The fusion index and (d) maturation index for C2C12 cells in the tubes with different NPs. **P < 0.01 and NS = not significant. (e) Schematic illustration of TMV self-assembly into aligned stripes in capillary tubes and optical images of these stripe patterns at positions 1 and 2 in the inset. (f) 3D AFM image and an enlarged view of stripe patterns. (g) Florescence images of smooth muscle cell cultured at positions 1 and 2 inside the capillary tube. Colour representation: F-actin (green), nucleus (blue). Panels (a–d) are reproduced with permission from Ref 40; copyright 2013 American Chemical Society. Panels (e–g) are reproduced with permission from Ref 47; copyright 2009 Wiley-VCH. TMV, tobacco mosaic virus; RGD, arginine-glycine-aspartic acid, MHC, myosin heavy chain.

alignment with the direction of the M13 assembly. Interestingly, BHK cells showed much better directionality than NIH-3T3. When cells were removed from the M13 films, oriented fibronectin and collagen of the denuded ECM were observed for both BHK and NIH-3T3 cells, indicating the topographical feature of patterned M13 film guided ECM protein orientation and influenced the directionality of cells. Additionally, much more ECM proteins were secreted from NIH-3T3 cells compared to that from BHK cells, which had significant impacts on the cell morphology, i.e., that the poor capability in ECM protein secretion of BHK cells resulted in much better directionality along the patterned substrate. These results suggested that ECM protein secreted by cells could accommodate their directionality with the topography of the underneath substrate.

VIRUS ASSEMBLIES AS SCAFFOLDS FOR BONE TISSUE ENGINEERING Developing substitutes to induce bone regeneration is one of the most widely studied topics in tissue

engineering.53 It is well known that bone marrow stromal cells (BMSCs) that exist within bone marrow stroma54 can be cultured ex vivo and induced to differentiate into osteoblast, chondrocytes, adipocytes, tenocytes, and neural cells.55 Therefore, developing active scaffolds for osteoblastic differentiation of BMSCs is a viable approach for bone tissue engineering. As the interactions between BMSCs and underlying substrates play a crucial role in regulating the osteogenic or chondrogenic processes,56,57 some interesting studies have been carried out to modulate the osteoblastic differentiation of BMSCs using engineered viruses as platforms.

WT Viruses Provide Nanotopographic Cues for BMSCs Differentiation Initial study carried out by Wang and coworkers focused on the coating of TYMV to promote the adhesion and differentiation of BMSCs.58 When grown on TYMV-coated surface, the osteocalcin gene, a crucial factor to the process of osteogenic differentiation and mineralization,59 was dramatically upregulated at day 14 in osteogenic media, which was 7–14 days earlier

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FIGURE 4 | (a) Gene expression in the BMSCs seeded on TMV under osteogenic conditions. **P < 0.005 and *P < 0.05. (b) Histochemical staining for osteogenic markers alkaline phosphatase (inset) and alizarin red in BMSCs cultured on TMV for 14 days under osteogenic conditions. (c) Immunostaining of BMP2 in BMSCs cultured on TCP and TMV for 8 h. Colour representation: nucleus (blue), BMP2 (green). (d) Immunostaining of osteogenic markers osteocalcin and osteopontin in BMSCs grown on TMV-phos. Colour representation: nucleus (blue), actin (green), osteocalcin (red), and osteopontin (pink). (e) SEM images of a single cell inside TMV-PAH. (f) Alkaline phosphatase activity assay of BMSCs (P < 0.05). (g) Calcium deposition of BMSCs quantified on day 6 (P < 0.05). Panels (a) and (b) are reproduced with permission from Ref 61; copyright 2009 Elsevier Ltd. Panel (c) is reproduced with permission from Ref 63; copyright 2012 The Royal Society of Chemistry. Panel (d) is reproduced with permission from Ref 67; copyright 2010 Elsevier Ltd. Panels (e–g) are reproduced with permission from Ref 69; copyright 2012 American Chemical Society. BMSC, bone marrow stromal cell; TMV, tobacco mosaic virus; BMP2, bone morphogenetic protein 2; TCP, tissue culture plastic; PAH, porous alginate hydrogels.

than cells grown on standard tissue culture plastic (TCP). In addition, mineralized nodules comprising of osteoblast-like cells were also observed around day 14. These results demonstrated that the nanoscale surface topography generated by TYMV-coating significantly altered the cellular behavior and regulated the differentiation of BMSCs. To better understand the cellular interactions with fibrillar proteins and the subsequent effects on cellular processes, a rod-shaped plant virus TMV that mimic the structure of major ECM components, was explored to study the differentiation process of BMSCs.60 Initially, WT TMVs were coated on a 2D substrate, followed by an assessment of the ability to promote BMSCs differentiation.61 As shown in Figure 4(a), cells grown on TMV coating surface displayed maximum up-regulation of osteo-specific genes (osteocalcin, osteopontin, and osteonectin) at day 14 which was 7 days earlier than that on TCP. Along with the differentiation process, BMSCs were mineralized with highly enriched calcium deposits stained by alizarin red at 14 days, and a common osteogenic differentiation marker alkaline phosphatase (ALP) shown positive [Figure 4(b)]. Further DNA microarray analyses showed that hundreds of genes were affected by TMV substrate. Among

them, bone morphogenetic protein 2 (BMP2), a pivotal growth factor for regulation of osteogenic differentiation of BMSCs,62 was found to be upregulated on TMV-coated surfaces compared to TCP surfaces.61 More importantly, the highest expression of BMP2 in both mRNA and protein were detected in BMSCs cultured on TMV-coated substrates after 8 h of osteo-induction and maintained high levels even after 24 h [Figure 4(c)].63 Thus, it was concluded that the rod-like TMV could promote the osteogenic differentiation process of BMSCs by regulating gene expression profile via the early onset of BMPs expression.

Modified Viruses Provide Biological Cues for BMSCs Differentiation It is well known that multivalent interactions between cell surface receptors and the binding ligands are important for cellular function.64,65 During cell–ECM interaction, ligand density and arrangement not only play a role in initial adhesion, but also control the signaling processes leading to different differentiation pathways.66 TMV was used as a model to display multivalent functional ligands in high density and highly ordered arrangement for BMSCs differentiation. Kaur et al. have introduced functional phosphate for calcium incorporation onto each of 2130 TMV protein subunits via the CuAAC reaction.67 TMV-phosphate

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FIGURE 5 | (a) Schematic illustration of the formation of the phage film by using layer-by-layer method and the MSCs growth on the phage film. (b) Fluorescent images of protein expression in MSCs cultured on M13 thin films. Colour representation: nucleus (blue), actin (green), osteopontin and osteocalcin (red). The figure is reproduced with permission from Ref 73; copyright 2011 Elsevier Ltd. MSC, marrow stromal cell; OPN, osteopontin; OCN, osteocalcin.

substrate displayed significantly higher upregulation of osteocalcin and osteopontin during BMSCs differentiation as compared to TMV substrate. As shown in Figure 4(d), during the differentiation process, BMSCs cultured on TMV-phosphate-coated substrates transformed into a more polygonal-like shape at 14 and 21 days, compared to a well-spread morphology at 7 days. When deposited on Ti substrate, TMV-phosphate showed improved differentiation of BMSCs,67 highlighting the potential application for bone tissue engineering. Other modified TMV viruses were also developed to promote the BMSCs differentiation for bone tissue engineering. As reported by Lee et al., multivalent display of RGD on genetically modified TMV-RGD1 mutant could induce rapid onset of key bone differentiation makers for BMSCs within two days in serum-free osteogenic media, such as osteocalcin, BMP-2 and calcium sequestration.68 The nanotopographic features of TMV, along with its multivalent display of ligands make it an extremely effective platform to develop novel biomaterials for bone tissue engineering with enhanced osteogenic differentiation and matrix mineralization. In order to investigate whether the enhanced osteogenic differentiation potentials can be translated to 3D scaffolds, Luckanagul et al. constructed the porous alginate hydrogels (PAH) functionalized with TMV (TMV-PAH) and TMV-RGD1 mutant (RGD-PAH).69 As shown in Figure 4(e), the BMSCs could adhere to the hydrogels and spread out. RGD-PAH showed significant improvement in cell attachment in 8 h, and highest cell metabolic activity at day 8 as compared to PAH and TMV-PAH due to the RGD sequence. In addition, RGD-PAH accelerated an early osteogenesis of BMSCs as the ALP activity was significantly higher among three samples on day 3 [Figure 4(f)]. The amount of calcium deposited in both TMV-PAH and RGD-PAH were significantly

higher than that in PAH [Figure 4(g)]. Moreover, the osteospecific markers such as osteocalcin accumulated at day 10 for PAH and day 13 for both TMV-PAH and RGD-PAH, indicating that virus incorporation into 3D matrices did not impair the differentiation potential of BMSCs into osteogenic lineage. Engineered M13 bacteriophages were also exploited for osteogenic differentiation. Yoo et al. reported a M13 matrix engineered with collagenderived DGEA-peptides could induce the osteogenic differentiation of pre-osteoblasts with outgrown morphology and osteogenic protein expression.70 Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into osteoblasts, chondrocytes, and adipocytes,71 which have been broadly studied for their potential applications in the repair of bone regeneration.72 In order to investigate the cellular behaviors of MSCs and direct their differentiation into osteoblasts, Mao and coworkers constructed aligned films with genetically engineered M13 to see how MSCs respond to different peptide sequences.73 One peptide was PDPLEPRREVCE that derived from osteocalcin,74 and another was YGFGG, the core domain of osteogenic growth peptide.75 These peptides were fused to the major coat protein of M13 phages respectively, and displayed on the side walls of M13 surface (PD-phage and YG-phage). As shown in Figure 5(a), phage films were prepared through electrostatic interaction between positively charged poly-lysine and negatively charged phage, showing the micro-textured surface morphology with alternating phage bundles and grooves arranged in parallel. As reported, grooved and micro-textured surfaces could direct the morphogenesis of cells with a preferred orientation.76 MSCs cultured on WT phage and YG-phage film displayed a spindle-like cellular morphology with significant elongation and alignment along the phage bundles. While cells seeded

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on the PD-phage films showed several filopodia and were more spread out. It was reported that the elongation and stretching of hMSCs favored the preferential differentiation into osteogenic lineage.77 Further studies were designed to test if the engineered phage films could specifically promote the differentiation of MSCs into osteoblasts.73 As shown in Figure 5(b), when cultured in osteogenic media for 2 weeks, higher expression levels of osteocalcin and osteopontin were observed in MSCs seeded on the WT,YG-, and PD-phage films compared to poly-lysine film. MSCs grown on the PD-phage film aggregated to form calcified nodule-like structures because of strong negative charge (−35 mV) on PD-phage surface, which would attract the calcium ions to induce mineralization. Gene expression analysis confirmed that MSCs on both WT and engineered-phage films showed significantly higher up-regulation of osteoblast gene expression than poly-lysine film. Specially, the YG-phage could significantly up-regulate expression of Runx2, a master transcription factor in MSCs differentiation,78 as compared to WT or PD-phage film, which indicated that the YG peptide regulated the MSCs growth and differentiation into osteoblast cells via Runx2 pathway.79 Further mineralization study showed MSCs on phage films have 1.5 times more calcium-containing matrix cells than films without phage. They proved that the phage films can stimulate the MSCs to undergo differentiation and mineralization in osteogenic media. In their follow-up research, other peptides including RGD and PHSRN were displayed on M13 to study the effect of biological cues on the osteoblastic differentiation of MSCs.80 The results showed that displayed functional peptides could induce the osteoblastic differentiation of MSCs in the primary media without any osteogenic supplements, which could be further enhanced in osteogenic media.

VIRUS SCAFFOLDS FOR NEURAL TISSUE ENGINEERING Nowadays, neural tissue engineering for neural regeneration is the most promising treatment of spinal cord injuries.81 Neural progenitor cells (NPCs) were chosen for in vitro study of neural tissue engineering because of their advantages of generating various kinds of central nervous system cells.82 Scaffolds used in most reports for neural tissue engineering were synthetic polymers81 as well as biomolecules such as fibrous assembly of peptide amphiphiles.83 These scaffolds with longitudinal orientation and functional cell signaling sequences were shown to promote better neural cell adhesion and direct the differentiation of NPCs into neurons, suggesting the importance of

topographic and biological signals of biomaterials for in vitro cell culture and in vivo neural tissue engineering applications. Recently, the filamentous viruses such as M13 have been brought into this field because of their abilities to self-assemble into long-range ordered filamentous structures,50 and display of certain foreign functional peptides on the virus surfaces.26 Pioneering works have been carried out by Lee Group, using genetically engineered M13 as platforms to evaluate the cell response84 [Figure 6(a)]. RGD and IKVAV peptides were first incorporated into the N-terminus of major protein VIII (p VIII) to yield a peptide array spaced at 2.7 nm apart and available for cell receptor interaction.85 NPCs grew in proliferative media supplemented with RGD-, IKVAV-, and WT-phage showed significant proliferation over 5 days but without any phenotypical or morphological changes as compared to positive control (cells grown in the same culture media without phage), indicating non-cytotoxicity of M13 to the NPCs. However, even the RGD-, IKVAV-phages existing in proliferation or differentiation media could not induce any expression of progenitor or neural specific proteins in NPCs. When the M13 mutant was coated on surface of substrates, it could induce the neuronal differentiation with expression of neurofillament makers like b-tubulin III. Moreover, engineered-phage substrates showed much higher fluorescence intensity of phage antibody at cells locations compared to WT-phage substrate, indicating that RGD and IKVAV peptides displayed on phages in multivalent manner could enhance the specificity of interaction with cell receptor. As reported, NPCs behaved differently on various substrates and formed the cell aggregates (neurospheres) when grew on the poorly adhesive surfaces.86 Further studies were carried out to show the cell growth patterns on the various phage substrates by nearest neighbor analysis,87 which can quantify the differences in the spatial distribution of cells. As shown in Figure 6(b), cells on WT-phage substrate exhibited the most clustered pattern, while RGD- and IKVAV-phage substrates showed an independent distribution that similar to positive control (cells on laminin substrates). However, the morphology of cells on M13-RGD surfaces was similar to the laminin control groups, while cells on M13-IKVAV surfaces retained the neurosphere-like aggregate morphology due to the less adhesive. These results revealed that the type of adhesive ligands displayed on the surface have great influence on the spreading and spatial distribution of NPCs. In another study, the effect of alignment of M13 on growth and differentiation of NPCs was

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FIGURE 6 | (a) Schematic diagram of NPCs response to phage materials. (b) Plot of cell spatial distribution on phage substrates by nearest neighbor analysis. (c) Immunostaining of NPCs grown on the aligned RGD-phage film in proliferation media and differentiation media at day 1. Colour representation: nucleus (blue), actin (green). Bright-field optical micrograph of NPCs on the aligned RGD-phage film at 4 h. (d) Neurite length on the phage matrices and SEM of neural cells on the RGD-phage matrices. Panels (a), (b), and (d) are reproduced with permission from Ref 84; copyright 2009 American Chemical Society. Panle (c) is reproduced with permission from Ref 88; copyright 2010 American Chemical Society. NPC, neural progenitor cell; RGD, arginine-glycine-aspartic acid.

investigated. A centimeter-scale liquid-crystalline phage film was constructed by shearing method.88 Phage particles assembled into fiber bundles and formed a film, and the film showed long-range orientation with periodic anisotropic grooves and ridges aligned parallel to the direction of shear. Moreover, the diameters of the fiber bundles as well as the amplitude and roughness of the topography could be tuned by controlling the assembly conditions. Compared to the control-phage films (RGE- and WT phage), both attachment and proliferation of NPCs were increased on the RGD-phage films. As shown in Figure 6(c), NPCs on the aligned RGD-phage film were observed to grow along the direction of phage fiber bundle alignment in proliferation media. Under the differentiation condition, the aligned RGD phage film could direct NPCs to differentiate into a neuronal lineage and an outgrowth of neurites parallel to the phage alignment was observed.88 These results showed that the biological and topographical cues of the aligned phage films could guide directional growth of NPCs. Based on the above results, aligned three-dimensional phage nanofiber matrices were fabricated to control macroscopic behavior of neural cells.84 Liquid crystalline suspensions (∼15 mg/mL) of phage were mixed with NPCs in media and manually injected into low melting temperature liquid agarose to form long-range ordered structures. NPCs cultured in the aligned 3D phage fibers with proliferation media were able to proliferate over a 7-day period and form multiple colonies. When grew in differentiation media, NPCs were observed to differentiate and

extend neurites in a direction parallel to the long axis of the fibers [Figure 6(d)]. These cell processes could maintain orientation along the long-axis of the fiber while being dispersed throughout its thickness. After 6 days in the differentiation media, both RGD- and IKVAV-phage matrices contained cells with neurites 33 and 23% longer than those in the WT-phage scaffold, respectively. These results proved that oriented liquid crystalline phage structures provided a chemically friendly and a directionally instructive environment for neurite extension.

SAFETY CONSIDERATIONS IN VIRUS-BASED TISSUE ENGINEERING As viruses have shown great potentials in tissue engineering applications, the biosafety must be taken into consideration for future in vivo applications. Many works have been done to investigate the fate of these viral nanoparticles in vivo and evaluate their toxicity, including the blood clearance, bio-distribution, biocompatibility and pathology. Using radiolabeled TMV, Wu et al. reported that the blood clearance and distribution of TMV in mouse.89 When injected into the body, about 97% of TMVs would be cleared quickly from blood in 40 min by the reticulo-endothelial system (RES),90 after that 90% of the particles accumulated in the liver and spleen. The amount of particles in liver and spleen increased within 4 h, and the amount decreased progressively 24 h later because of the clearance from liver and spleen. In addition, the filamentous structure

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M13 bacteriophage with surface modification by single-walled carbon nanotubes were reported to be eliminated from blood circulation by RES in appropriately 60 min, after which most of these particles were observed in liver, spleen and bone.91 Similar works have been carried out by Bruckman et al.92 They compared the in vivo pharmacokinetics of TMV rods and TMV spheres generated from TMV by thermal transition. The blood clearance and biodistribution were consistent with the results from Wu et al. Moreover, both the TMV-rods and re-assembled spheres did not induce any hemolysis or blood clotting at 1 h after injection, showing the blood biocompatibility of TMV. However, rod-shaped TMV was still detectable in liver and spleen 24 h after administration, while spheres were cleared from tissues within 1 day. The different clearance rates of TMV-rod and -sphere showed the shape-dependent in vivo behaviors. In another report, the spherical CPMV particles were still detectable in the liver with significant quantities 48 h after injection93 because of the interaction between viruses and tissue cells.94 These studies suggested that the in vivo behaviors of viruses depended a lot on their properties such as shape, size, and surface chemistries. Further histological examination of the tissues of both TMV89,92 and CPMV93 inoculated animals showed no pathological changes and no signs of overt toxicity such as tissue degeneration, cell necrosis, and apoptosis, which supported the biocompatibility of virus platforms. Except these aforementioned biobehaviors, the metabolism of viral particles in tissue such as the degradation and the effects of the degraded components may also influence the tissue engineering application of virus. Nevertheless, there is no reported studies on this issue in literature. Moreover, the aggregation of viruses in liver and spleen may also effect immunogenic processing and induce immune attack against viruses-based implants. Luckanagul et al. have studied the immunogenicity of TMV when incorporated into 3D porous hydrogel for transplantation in mice. The immune response against TMV was observed to decrease dramatically due to its immobilization in hydrogel, suggesting the feasibility of using viral nanoparticles for in vivo applications.95 Thanks to the facile modification of viruses, it will be possible to avoid potential toxicity

and immunogenicity problem for tissue engineering application by either controlling their surface properties or being embedded into scaffold materials.

CONCLUSION Mimicking of ECM environment and displaying of biological and topographic cues for cell signaling are critical for the success of tissue engineering. Interest in applying viral nanoparticle to tissue engineering is growing due to their specific capability for modulating cell behaviors. There are several advantages of choosing viruses as platforms to construct materials/platforms for tissue engineering. First, viruses can be modified to present biological cues for cellular behaviors via chemical conjugation and genetic engineering.24,96 Secondly, the multivalent coat proteins can display functional molecules at high density to enhance the cell–material interaction.31,68 Furthermore, a wide variety of strategies have been developed and implemented to assemble viruses into well-defined structures with nanoscale topography in two dimension and three dimension.24 Based on these features, virus-based 2D and 3D biomaterials have shown to be able to enhance cell adhesion and proliferation, direct cell orientation and promote cell differentiation. The examples discussed herein demonstrate the ability of virus-based biomaterial to regulate differentiation of bone marrow stromal cells and neural progenitor cells, suggesting the potential application in bone and neural tissue engineering. Despite these advantages, there are some problems for virus scaffolds in tissue engineering application. For example, virus-based biomaterials do not possess the desirable mechanical property which is necessary for bone tissue engineering and other applications.97 Considering the future clinical application, potential toxicity of the nanoparticles in human body must be addressed. Some preliminary studies in the in vivo behavior of viruses showed a quick clearance from blood and filtered by liver and spleen without overt toxicity such as tissue degeneration or necrosis. It is necessary to further investigate the metabolic pathway of different viral particles. Finally, additional efforts are needed to evaluate the immune responses and the inflammatory reactions.

ACKNOWLEDGEMENT This work was supported by National Natural Science Foundation of China (21374119 and 21104080). XZ is grateful for the support of China Scholarship Council. QW would like to acknowledge the South Carolina EPSCoR-GEAR grant. © 2014 Wiley Periodicals, Inc.

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FURTHER READING Wang F, Cao B, Mao C. Bacteriophage bundles with pre-aligned Ca initiate the oriented nucleation and growth of hydroxylapatite. Chem Mater 2010, 22:3630–3636. Li K, Nguyen HG, Lu X, Wang Q. Viruses and their potential in bioimaging and biosensing applications. Analyst 2010, 135:21–27. Lee LA, Nguyen HG, Wang Q. Altering the landscape of viruses and bionanoparticles. Org Biomol Chem 2011, 9:6189–6195. Chen L, Zhao X, Lin Y, Huang Y, Wang Q. A supramolecular strategy to assemble multifunctional viral nanoparticles. Chem Commun (Camb) 2013, 49:9678–9680. YoungáYoo S, HyunáKim T. Facile patterning of genetically engineered M13 bacteriophage for directional growth of human fibroblast cells. Soft Matter 2011, 7:363–368. Sitasuwan P, Lee LA, Li K, Nguyen HG, Wang Q. RGD-conjugated rod-like viral nanoparticles on 2D scaffold improve bone differentiation of mesenchymal stem cells. Front Chem 2014, 2:31. Wang J, Yang M, Zhu Y, Wang L, Tomsia AP, Mao C. Phage nanofibers induce vascularized osteogenesis in 3D printed bone scaffolds. Adv Mater 2014, 26:4961-4966. Serban MA, Scott A, Prestwich GD. Use of hyaluronan-derived hydrogels for three-dimensional cell culture and tumor xenografts. Curr Protoc Cell Biol 2008, Chapter, Unit-10.14. doi:10.1002/0471143030.cb1014s40. Li WJ, Tuan RS. Fabrication and application of nanofibrous scaffolds in tissue engineering. Curr Protoc Cell Biol 2009, Chapter, Unit-25.2. doi:10.1002/0471143030.cb2502s42. Carlson MW, Alt-Holland A, Egles C, Garlick JA. Three-dimensional tissue models of normal and diseased skin. Curr Protoc Cell Biol 2008, Chapter, Unit-19.9. doi:10.1002/0471143030.cb1909s41. Kharkar PM, Kiick KL, Kloxin AM. Designing degradable hydrogels for orthogonal control of cell microenvironments. Chem Soc Rev 2013, 42:7335–7372. McGann CL, Levenson EA, Kiick KL. Resilin-based hybrid hydrogels for cardiovascular tissue engineering. Macromol Chem Phys 2013, 214:203–213.

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Virus-based scaffolds for tissue engineering applications.

One of the major research directions of tissue engineering is to develop artificial scaffolds that can mimic extracellular matrix (ECM) and support th...
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