Bioinspired Polymeric Nanocomposites for Regenerative Medicine

Macromolecular Chemistry and Physics Talents & Trends Young Talents in Polymer Science Bioinspired Polymeric Nanocomposites for Regenerative Medici...
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Macromolecular Chemistry and Physics

Talents & Trends

Young Talents in Polymer Science

Bioinspired Polymeric Nanocomposites for Regenerative Medicine James K. Carrow , Akhilesh K. Gaharwar* The design and fabrication of bioinspired nanomaterials for tissue-engineering applications requires a fundamental understanding of the interactions between polymers, nanostructures, and cells. Most biomimetic polymeric nanocomposites consist of two or more types of polymers or of polymers combined with different nanomaterials to obtain composite structures with desired properties. In this Talents & Trends article, the focus is on bioinspired polymeric nanocomposites surrounding three major strategies. Firstly, biomimetic structures composed of a fibrous architecture are discussed. Secondly, the emerging trends in designing complex nanocomposites with multiple functionalities are assessed. Finally, some of the most critical challenges that come with the design and fabrication are highlighted in bioprinting. Finally, the emerging trends in the field of bioinspired polymeric nanocomposites are highlighted.

1. Introduction The traditional paradigm of tissue engineering includes three integral components: cells, growth factors, and scaffolds, which produce a favorable regenerative response. With the increase in our understanding of the extracellular microenvironment (ECM) and its role in developmental biology, our approaches to material synthesis and scaffold design are J. K. Carrow, Prof. A. K. Gaharwar Department of Biomedical Engineering Texas A&M University, College Station TX 77843, USA E-mail: [email protected] Prof. A. K. Gaharwar Department of Materials Science and Engineering, Texas A&M University College Station, TX 77843, USA

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continuously evolving. The success of many constructs is often limited by the lack of biological complexity generated, leading to researchers investigating new methods to emulate native tissue environments. Oftentimes, inspiration for scaffold architecture or utilized biomaterials stems from structures preexisting in nature, considering millions of years have resulted in the emergence of highly sophisticated and efficient materials.[1–3] For example, shark skin and lotus leaves have been investigated for inspired surface design due to the anisotropic flow characteristics and superhydrophobic properties of each respectively.[4] Both of these naturally occurring “engineered” arrangements illustrate the nano- and microscale components, leading to macroscale function.

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Biology offers the best models for strategies to rationally design high-performance biomaterials with properties similar to those of natural materials, such as bone, cartilage, nacre, or silk.[5–8] To translate our fundamental understanding of nature into products that are useful in a clinical setting, the chemical, physical, and biological properties of newly developed bio-nanomaterials need to be optimized to support, regulate, and influence long-term cellular activities. Both bottom-up and top-down approaches are considered by materials scientists to design biomimetic components for tissue engineering.[2–4,9,10] At each level (i.e., nano, micro, macro), mechanisms underlying cellular interactions will vary, leading to a variety of requirements for consideration that will

DOI: 10.1002/macp.201400427

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be implemented cohesively during material design. Akin to nature, biomaterial design processes strike a balance between complexity and unification of the individual components. The design and fabrication of bioinspired nanomaterials for tissueengineering applications requires a fundamental understanding of the interactions between polymers, nanostructures, and cells. Most biomimetic polymeric nanocomposites consist of two or more types of polymers, or polymers combined with different nanomaterials, to obtain composite structures with desired properties (Figure 1). A range of structures, including interpenetrating, fibrous scaffolds and nanocomposite biomaterials, which mimics the structural and physical properties of the ECM is engineered. Biomimetic materials are used for a range of biomedical applications, including regenerative medicine, wound dressing, drug delivery, gene therapy, and immune engineering. In order to design biomimetic nanomaterials for the repair of native tissues, we need to consider and compare the structures and properties of the natural tissue, along with the biological influence of cells on the synthetic biomaterial properties. For example, bone comprises a hierarchical structure that provides the tissue with its advantageous mechanical and functional properties, yet also significantly hampers replication in an in vitro setting.[11] Therefore, to recapitulate native complexity, bioactive materials can motivate specific cellular activity in a spatially confined manner. Among popular fillers, nanoceramics, such as synthetic silicates, nano-hydroxyapatite (nHA), and bioactive glass, instill tissue-engineering scaffolds with a supplementary influence over stemcell behavior. While the underlying mechanisms of their bioactivity are still under evaluation, evidence points to the combination of degradation products, surface interactions

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Akhilesh K. Gaharwar is an assistant professor in the Department of Biomedical Engineering and Department of Materials Science & Engineering at Texas A&M University, where he directs the Inspired Nanomaterials and Tissue Engineering Laboratory. His research experience spans diverse fields, including materials science, chemistry, biology, and engineering of polymeric biomaterials and nanocomposites. His research group is developing advanced biomimetic nanostructure and integrating nanomaterials and stem cells for the development of functional tissue engineering. He received his Ph.D. in Biomedical Engineering at Purdue University and postdoctoral training at Massachusetts Institute of Technology and Harvard University.

with cellular membranes, and charge. Novel microscale technologies have emerged in order to fabricate polymeric-based structures with additional nanofillers, thus providing investigators with an architecture or material inspired from nature.[9] Integrated composites not only establish additional sources of bioactive factors, they can also fortify the polymer network via physical or chemical interactions.[1,12–15] While many naturally based polymers demonstrate useful cytocompatibility or synthetics, enabling extensive tailorability to the polymer chain, often a purely polymeric system lacks the necessary mechanical strength or degradation characteristic in vivo, particularly for load-bearing regenerative applications. Most extensively investigated natural polymers for biomedical applications include collagen, gelatin, starch, cellulose, alginate, chitosan, and fibrin, whereas synthetic polymers include use of poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA), and poly(glycerol sebacate) (PGS). To overcome the limitations of a basic polymeric scaffold, additional nanomaterials are integrated into the architecture to form a nanocomposite with the combinatorial benefits of each biomaterial, including bioactivity, adhesiveness, environmental-sensitivity, and mechanical improvements.[16] These Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

modifications can employ bioinspiration via mimesis of naturally occurring molecular processes or microenvironment induction of cellular behavior (Figure 1). Through reversible physical interactions (e.g., electrostatic, dipole, hydrogen-bonding) or chemical crosslinks (e.g., thiolbased, radical polymerization), superior distribution of stresses imparted onto a polymeric composite scaffold can be achieved. Similarly, degradation profiles of networked polymer composites can be extended to allow sufficient cell migration and tissue formation in vivo. To provide additional functionality, a range of nanoparticles can be incorporated within the polymeric network to fabricate bioinspired nanocomposite structures. Most nanomaterials can be divided into four different categories: zero-, one-, two-, and three- dimensional nanomaterials (Figure 2). Zerodimensional (0D) nanomaterials are atomic clusters mostly composed of metallic elements. One-dimensional (1D) nanomaterials include metal nanorods, nanotubes, ceramic crystals, polymer nanofibers, and selfassembled structures. Most twodimensional (2D) nanomaterials have included layered structures such as graphene, synthetic clays, and double layered hydroxides (LDH), whereas three-dimensional (3D) nanomaterials include polycrystals and spherical particles. Due to the difference in

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Figure 1. To improve scaffold outcomes, bioinspired composite materials can provide additional benefits toward cellular proliferation and induction through a variety of materials and can take multiple forms upon fabrication. Basic materials include multipolymer systems or the introduction of nano-/microparticles into a polymeric network. Two main components from this inspiration are the physical and chemical modification of the base polymers present within the scaffold. The integration of various materials can result in enhanced mechanical stability through additional crosslinking sites, ECM mimesis, or interactions between the cell membrane and material surface. Additionally, these same materials enable spatially controlled protein binding for cellular adhesion, nucleation of mineralized matrix, or provide vital factors for the motivation of stem cells toward specific lineages.

surface to volume ratio, these nanomaterials interact with polymers via substantially different mechanisms and result in unique property combinations compared with their micro and nano counterparts. The dimensionality of incorporated nanomaterials will stimulate specific cellular pathways via multiple channels, providing investigators with a host of tools for controlling cell behavior. Thus, the type of biomaterial used to make a composite structure plays

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a major role in determining the end application of these structures. Polymeric nanocomposites take a variety of forms for applications in regenerative medicine, each with associated tradeoffs resulting from the varying fabrication methods and materials. Persistent efforts to improve cellular and subsequently tissue outcomes have encouraged creative approaches toward material design. The methods by which nanomaterials are introduced to polymeric Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

matrices are not only dependent on these fabrication strategies but also the desired biological response. For example, internalization of many inorganic nanomaterials can alter the differentiation status of encapsulated stem cells; however, these same materials present in the extracellular environment can act as nucleation sites for the deposition of mineralized matrix, effectively mimicking those found on collagen fibrils in bone tissue.[17,18] While multiple

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Figure 2. Illustrated are the various dimensions (0D, 1D, 2D, 3D) and corresponding materials available for implementation within polymeric nanocomposite designs. Each dimension targets explicit cellular pathways through mimicking physical or chemical cellular environments and consequently a multitude of tools is offered for investigators to better control material–cell interactions.

groups have integrated both polymer and additive prior to scaffold polymerization, some have investigated the effects of creating nucleation sites after polymer-network formation through incubation with simulated body fluid (SBF) in the presence of carboxyl groups on the polymer chain.[19–22] In this article, we focus on work on bioinspired polymeric nanocomposites surrounding three major strategies. Specifically, we discuss the different types of polymeric nanocomposites based on different types of structure. First we will discuss biomimetic structures composed of a fibrous architecture. Secondly, investigations designing complex hydrogel nanocomposites with multiple functionalities will be examined and discussed in detail. Lastly, some of the most critical challenges that come with the design and fabrication will be highlighted in bioprinting. We will also express some of the applications and challenges in bioprinted

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constructs from composite bioinks and customized systems. Lastly, we will emphasize emerging trends of technological advancements and their relation to the field of polymeric nanocomposites for biomedical applications.

2. Biomimetic Fibrous Scaffolds The fibrous configuration of native ECM has been exposed as crucial toward stem-cell behavior, especially in the niche during potency maintenance.[23] Notwithstanding topographical characteristics of many polymeric fibrous scaffolds altering cell behavior through mechanotransduction pathways, control of cell activity toward specific lineages is challenging as oftentimes the synthetic polyesters used lack inherent bioactivity. Therefore, the enrichment of a fibrous scaffold to form a composite material is desirable in Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

situations requiring specific stem-cell directing. In an attempt to improve electrospun scaffold properties, groups have fabricated composites through direct mixing of polymer and nanoclay (synthetic silicate nanoparticles) solutions prior to electrospinning.[24–26] In our investigations, we noted changes to poly(caprolactone) (PCL) fiber morphology and size in which the fibers reduced in diameter and showed increased roughness (Figure 3). Moreover, biomineralization in the presence of SBF and osteogenic activity were found to significantly increase with greater amounts of nanoclay, indicating a potential for these inorganic nanomaterials in musculoskeletal tissue engineering. Similar results were likewise produced where silicate nanoparticles were embedded within a PCL electrospun scaffold, highlighting the capabilities of these bioinspired nanocomposites for hard-tissue regeneration.[27]

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Figure 3. Electrospinning PCL–nanoclay(synthetic silicate) composite fibers results in a reduction of fiber diameter with increased nanoclay content. Additionally, the added nanoclay provides enhanced nucleation sites for hydroxyapatite crystal formation on the fiber exterior due to enhanced surface roughness, eventually leading to a significant increase in the local formation of mineralized matrix, as demonstrated by Alizarin Red S staining after 21 days. These results indicated successful motivation of stem cells down osteogenic pathways and therefore the potential of these nanocomposites for osteo-regenerative applications. Reproduced with permission.[25] Copyright 2014, Mary Ann Liebert, Inc.

While bioinspiration for many fibrous scaffolds stems from the replication of the ECM, some have incorporated chemical moieties straight from nature. For example, the catechol functional group present in polydopamine (PDA) and its derivatives allows mussels to adhere to many surfaces and has inspired a fair amount of work in the biomedical field.[28] Specific to fibrous constructs in regenerative medicine, some have introduced this binding moiety for immobilization of other nanomaterials or biological agents

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through coatings or grafts on synthetic polymers.[29,30] Binding of metallic nanoparticles would result in a multifunctional electrospun scaffold, while protein adhesion would affect cell spreading and cytoskeletal rearrangement. Another grafting procedure modified a collagen type-II scaffold through a biomimetic recombinant protein of fibronectin module III and cadherin-11 (rFN/Cad-11), to promote adhesion and chondrogenesis of MSCs.[31] Thus, these composite materials again fulfill the multimodal aspect of materials science Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

regenerative-medicine design via the multiple mechanisms of cellular interaction. We have also explored alternative methods to modify these fibrillar scaffolds to direct cellular processes from incorporated bioactive factors. Through a polyether-ester multiblock copolymer of poly(ethylene oxide terephthalate)-poly(butylenes terephthalate) (PEOT/PBT), amphiphilic beads entrapped dexamethasone for sustained release to direct hMSC differentiation (Figure 4).[32] In this design, PEOT afforded elastomeric

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synthesis of dicalcium phosphate anhydrate and polyester poly(lactic acid) (PLA).[17] The suspension was then electrospun for architecture comparable to natural ECM, generating a construct with both physical and chemical inspiration.

3. Bioinspired Polymeric Nanocomposites Creating a 3D microenvironment capable of supporting cell proliferation and differentiation is a major focus of hydrogel research. The implanted material must fulfill a variety of responsibilities, including mechanical stability, cytocompatibility, and bioactivity, but many pure polymer systems are incapable of satisfying all requirements, hence the emergence of nanocomposite networks. Here we will focus on those nanocomposites inspired by nature. 3.1. Polymeric Nanocomposite Hydrogels

Figure 4. Shown is the basic process for electrospun composite and drug reservoir generation. A schematic and the SEM image illustrate localization of dexamethasone within the PEOT/TPBT fibers. The fluorescence images were acquired via replacing dexamethasone with a fluorescent molecule, Texas Red, as they have comparative molecular weights. Merging of optical and fluorescence microscopy images display concentration of composite materials within the beaded fiber structure. Reproduced with permission.[32] Copyright 2014, Elsevier.

qualities, while PBT imparted stiffness to the network as thermoplastic, crystalline polymer. Another group has investigated nanocomposite

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fibrous scaffolds for hard-tissue applications, in which they mimicked calcium phosphate nanocrystals on collagen fibers through in situ Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Nanocomposite hydrogels are highly hydrated 3D polymeric networks loaded with nanoparticles. These water-swollen polymeric networks attempt to augment cell viability in vivo through hydration, facilitating diffusion of nutrients and waste to an encapsulated cell population, in conjunction with a biomimetic microenvironment; however, formulating a hydrogel with mechanical stability and bioactivity is a persisting challenge.[10,33,34] A range of hydrogels has been synthesized to mimic native tissue structure and physical properties using both natural and synthetic polymers.[1,16] Subsequently, to provide bioactivity, electrical conductivity, and stimuli-responsiveness, a range of nanoparticles has been incorporated within the polymeric network. Carbon-based nanomaterials such as carbon nanotubes and graphene oxide can provide electrical conductivity; inorganic nanoparticles

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such as hydroxyapatite, bioglass, and synthetic silicates can provide bioactive properties; metal and metal oxide nanoparticles can provide antimicrobial and magnetic properties; and polymeric nanoparticles can provide controlled release of biological signals to control cellular behavior. These nanoparticles physically and/ or covalently interact with polymers and result in unique property combinations. Developing nanocomposite hydrogels with tailored functionality has opened up new possibilities in developing advanced biomaterials for various biomedical and biotechnological applications. These nanocomposite hydrogels are currently explored in the area of stem-cell therapies, tissue engineering, cellular and molecular treatments, immunomodulation, and cancer research.[35,36] We have designed nanocomposite hydrogels to mimic some of the physical, chemical and biological characteristics of native tissues. Comparative to polymeric networks composed of linear polymer chains, hyperbranched polyester (HPE) hydrogels have the potential to fabricate biomimetic structures with tunable physical and chemical properties.[37] These dendritic macromolecules can be functionalized with a variety of peripheral chemical groups for tailored drug-delivery applications or for engineering a polymeric matrix with high reactivity or crosslink density. These globular HPE macromolecules can be introduced to provide a biomimetic microstructure for cellular adhesion and proliferation. We have previously developed HPE hydrogels capable of sustained release of bioactive molecules, dexamethasone acetate, in which the architecture and substrate stiffness influenced cell–matrix interactions.[37] Internal cavities in the HPE molecule acted as drug reservoirs, while acrylation of hydroxyl-terminated HPE via stoichiometric addition of acryloyl chloride generated photo-crosslinkable materials. These are both favorable

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facets of the design process as a multitude of chemical cues can be integrated within the dendritic molecules; likewise, microfabrication through photolithography results in a regulated matrix shape for guiding cell behavior. These hybrid hydrogel networks have huge potential in regenerative medicine as HPE-based nanocomposites can be used for prolong therapeutics release for controlling cellular behaviors. 3.2. Bioactive Silicate-Based Nanocomposites Synthetic silicates are a novel class of ultrathin (or 2D) nanomaterials, with a high degree of anisotropy and functionality, which interact with biological entities in a substantially different manner to their respective 3D nano, micro, and macro counterparts because of their high surfaceto-volume ratio.[1,38,39] The rapid and recent advances in ultra-thin materials have raised tantalizing questions about their interactions with biological moieties. Since 2D nanomaterials for the life-sciences are still in their infancy, deciphering their biological interactions will open our eyes to wide range of biomedical and biological applications such as therapeutics, imaging, and disease-related diagnostics. Our recent reports indicate that these synthetic silicates strongly interact with stem cells and induce osteogenesis without using any additional growth factors.[18,40] Due to the disc shape and charged surface of silicate nanoparticles, cells easily internalize it via cadherin-mediated endocytosis. Upon internalization, these nanoparticles upregulate osteorelated genes and proteins, such as RUNX2, osteocalcin, and osteopontin, and result in production of mineralized ECM.[18] Due to strong bioactive characteristics, these nanoparticles are proposed for a range of musculoskeletal tissue-engineering approaches. These silicates are Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

complex polyions, and one of the components – magnesium – is shown to promote cell adhesion, spreading, and migration.[41] By controlling the interactions of silicates with polymers, 3D polymeric scaffolds with bioactive properties for regenerative medicine can fabricated. Synthetic silicates strongly interact with polymeric networks resulting in the formation of physically crosslinked networks and significantly increasing the mechanical stiffness.[1,42] In a recent investigation, we assessed that addition of silicates to neutral polymer such as poly(ethylene oxide) (PEO), augmenting cell adhesion and spreading, while improving overall material stability through electrostatic interactions between the nanoparticle surface and the electronegative oxygen atoms within the polymer backbone.[43,44] This is mainly attributed to the exfoliated structure of the silicate nanoparticles within the hydrogel network.[15,45] The exfoliated hydrogels, containing silicate and polymer, result in the formation of a highly organized layered structure similar to nacre, when subjected to solvent evaporation in a controlled manner.[46] This self-assembled structure, composed of hierarchical network, mimics brick and mortal structures.[47] The ratio between the silicate and the polymer dictates the formation of such highly organized biomimetic structures. We fabricated and characterized nanocomposite fibers made from viscoelastic PEO-silicate nanocomposite hydrogels.[46] Then the swollen hydrogel network was pulled to obtain hydrogel fibers. Although the exact interactions between the silicate nanoparticles and the polymer are still not known, it is believed that hydrogen bonding, electrostatic interactions, ionic bonds, and physical entanglement of the polymer chains with the nanoparticle surfaces play a major role in enhancing the physical and chemical

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Figure 5. Increasing silicate nanoparticles content enhances hierarchical microstructure of nanocomposite films and also improves cell adhesion and spreading, potentially through magnesium-mediated integrin binding. The supplementary complexity of the film architecture better mimics native-tissue microscale features, improves mechanical stability, and allows sequestration of bioactive molecules for sustained delivery applications. Upper Images: reproduced with permission.[44] Copyright 2012, John Wiley & Sons, Inc. Lower schematic: reproduced with permission.[45] Copyright 2010, John Wiley & Sons, Inc.

characteristics of nanocomposite networks.[48–50] These hydrogel fibers were subjected to solvent evaporation to obtain nanocomposite fibers with a high aspect ratio.[46] Crosspolarized images of a semi-dried nanocomposite fiber suggest an anisotropic orientation of the silicate nanoparticles and polymer chains along the fiber axis. Scanning electron microscopy (SEM) images show that these fibers have a smooth and uniform surface morphology. The addition of silicate to PEO induces cell-adhesion properties, and fibroblasts aligned themselves along the fibers, as visualized through Phalloidin-actin staining.[46] After incorporating silicate nanoparticles within a neutral polymer, cell adhesion and function can

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be tuned. In our recent study, we showed that addition of silicate nanoparticles results in significant control over cellular functions (Figure 5).[43,44] It is expected that the presence of magnesium on silicate nanoparticles might facilitate integrin-mediated cell attachment. Human mesenchymal stem cells (hMSCs) seeded on PEO-silicate nanocomposites demonstrated good cytocompatibility and enhanced osteogenic differentiation, indicating a potential for regenerative medicine.[18] Additionally, the control over selective cell adhesion can be used, from tissue-engineering scaffolds where cell adhesion might be desired to biosensor and cardiovascular stent applications where protein and cell adhesion is undesirable. Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3.3. Bioinspired Hydroxyapatite Nanocomposites Nano-hydroxyapatite (nHAp), a mineral found naturally in hard tissue, is another candidate for integration as a bioinspired nanofiller.[51–54] nHAp within the matrix could act as nucleation sites for the mineralization of encapsulated cells and provide useful minerals for bone formation. However, the applicability of nHAp as a biomaterial in clinical orthopedic and dental applications is limited to only non-stressed loaded regions owing to the brittle nature and low fracture strength, accompanied by low fracture toughness. By incorporation of nHAp within a polymeric hydrogel network, bioactive nanocomposites are fabricated for non-load-bearing

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Figure 6. The nanocomposite hydrogels composed of nanohydroxyapatite (nHAPs) and PEG can be fabricated via photo-crosslinking. Thin nanocomposite fibers can be stretched to extreme elongations even after having been knotted. SEM images from all the nanocomposite samples show highly porous structures with interconnected pores. The addition of nHAp significantly enhanced cell adhesion of the PEGnHAp hydrogel. The presence of nHAp provided bioactive attachment sites to the cells, which led to elongated lamellipodia and pseudopodia. Reproduced with permission.[51] Copyright 2011, American Chemical Society.

applications. We have also investigated nanocomposites incorporating this nanomaterial for osteoconductive applications (Figure 6).[51] The compressive strength of poly(ethylene glycol) (PEG) hydrogels was increased upon addition of nHAp nanoparticles. Interestingly, the nanocomposite hydrogels produced were much more extensible due to the combination of covalent crosslinking between the PEG macromolecules and the

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physical interactions between the PEG and nHAp, generating a viscoelastic quality. nHAp has also been fabricated in various other polymer scaffolds for bone regeneration, where the primary concern of the investigators was the imitation of natural structures.[55–57] These groups have assembled polymer scaffolds into collagen-mimetic matrices through natural and/or synthetic materials. Again, biological and physical interactions at various scales Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

contribute to the determination of the overall composition through control over the individual constituents. 3.4. Bioinspired Rosette Nanotube Composites A bioinspired structure also gaining popularity for regenerative medicine nanocomposites is rosette nanotubes, from assembling synthetic DNA bases, more specifically the

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hydrogen-bonding arrays between guanine and cytosine.[58,59] These bases comprise a structure similar to collagen fibers of the ECM, and also have the advantage of tailorability with additional amino acids and peptide sequences for improved bioactivity or cell adhesion within the scaffold. Inspired from the fibrillar arrangement produced by these sequences, the groups encouraged chondrogenesis from human mesenchymal stem cells, leading to a significant increase in glycosaminoglycans (GAG) production, indicating successful chondrocyte differentiation. The capability of transforming 2D materials into 3D implantable structures through polymeric interactions provides new perspectives for influencing cellular behavior. 3.5. Graphene-Enhanced Polymeric Nanocomposites In a combinatorial design strategy, 2D nanomaterials like graphene can be implemented with 3D polymeric hydrogels or scaffolds for tissue engineering, as their unique shape and surface properties can benefit both cell behavior and construct stability. For example, graphene oxide sheets can self-assemble with singlestrand DNA via non-covalent interactions resulting in the formation of 3D structures.[60] The resulting biomimetic hierarchical composition would also maintain graphene's mechanical strength and electrical conductivity, presenting a possible avenue for future tissue-engineering applications. Graphene oxide (GO) nanosheets can be used to deliver genes due to efficient biomolecule loading and cell-internalization properties of graphene. A shear-thinning methacrylated gelatin (GelMA) hydrogel is developed by impregnating PEIfunctionalized GO nanosheets (fGO) (Figure 7).[61] The preliminary investigation using a 22-gauge needle indicates that all the nanocomposite

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hydrogels composition can be easily injected. Due to the high surface area of fGO, pro-angiogenic human vascular endothelial growth factor plasmid DNA (pDNAVEGF) was loaded for delivery to damaged cardiac tissues. Although nonviral nanoparticles have poor transfection efficiency when compared with viral systems, GO nanosheets can efficiently deliver the genes (pDNAVEGF) when ionically bound to PEI which in turn strongly binds to DNA and helps in releasing the nucleic acids after cellular internalization. In vitro studies have been carried out using HUVECs to evaluate the proangiogenic activities of the hydrogel, which showed enhanced cellular proliferation. In vivo studies indicate that fGO/GelMA hydrogel did not invoke any significant toxicity or inflammatory response. This injectable nanocomposite hydrogel (GG’), loaded with fGOVEGF (pDNAVEGF bound to fGO), was shown to facilitate local myocardial neovascularization at the injected sites, reduce fibrosis, and improve cardiac function in an in vivo model of acute myocardial infarction (AMI). This study demonstrated a promising approach in nonviral gene-delivery system, however long-term effects of this treatment on heart function need to be monitored. Other types of carbon based nanoparticles, such as carbon nanotubes, have also been used to fabricate biomimetic nanocomposites.[62] Controlling the nanoparticles–polymer interactions, native tissue-like properties can be achieved. 3.6. Polymeric Nanocomposites Loaded with Metallic Nanoparticles Considering organic materials primarily composed of natural structures, the utilization of metal-based components for research in regenerative medicine typically falls outside the realm of biomimesis. However examples of successful inclusion Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

of these inorganic elements within polymeric systems exist and provide alternative avenues for functionaltissue regeneration. For example, interactions between peptides and solid inorganic surfaces can engineer reorganized molecular structures for improved bioactivity or further functionalization with synthetic materials, improving implanted-device regenerative performance.[63] Alternatively, the conductivity of metallic nanoparticles embedded within a polymeric matrix or nanofiber (e.g., gold) make them attractive for regenerative processes employing electrical stimulation to mimic neuronal pathways.[64,65] Future work is needed to better characterize interactions between stem cells and organometallic composites, as well as to develop stimuli-responsive materials to mimic physiological conditions. 3.7. Mechanically Stiff Interpenetrating Networks (IPNs) Most tissue structures, such as cartilage and muscle, are composed of multiple types of polymers, resulting in the formation of interpenetrating networks. Synthetic mimicry can be obtained by combining two or more polymers together. For example, interpenetrating polymer networks (IPNs), semi-IPNs, and double networks (DNs) are able to mimic the higher mechanical stiffness of highly resilient tissues such as cartilage.[66] These composite structures consist of two or more types of polymeric chains intermingled at the subnanometer length scale and result in unique property combinations due to extensive polymer–polymer interactions. Likewise, there is a fundamental mimesis of nature through the complexity of the fiber morphology within the hydrogel, as well as structural integrity effectively matching that of native tissue. Due to their impressive mechanical qualities, there is great potential for use in

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Figure 7. Injectable nanocomposite hydrogel prepared from graphene oxide (GO) and gelatin (GG’). First, GO is functionalized with branched polyelectrolyte, polyethylenimine (PEI) to form fGO, which is further functionalized with anionic plasmids (DNAVEGF) to form fGO/DNAVEGF (Scale bar: 1 μm). These composite structures are incorporated within a prepolymer of gelatin hydrogel and then subjected to UV to obtain a crosslinked network with a porous network. The therapeutic hydrogel can be easily injected into rat hearts with acute intramyocardial infarction for local gene delivery. The results indicate significantly reduced scar areaof infarcted hearts treated with therapeutic hydrogels. The representative images of the left ventricle myocardial sections stained with Sirius red show the cardiac fibrosis regions (in red). Sham operated and untreated infarcted group were used as controls. The red area represents ECM deposition in the scar tissue and the gray area represents the myocardium. Reproduced with permission.[61] Copyright 2014, American Chemical Society.

biomedical engineering and specifically regenerative medicine. While some therapies rely purely on multi-polymer compositions to mimic ECM,[67] others have designed the IPN architecture to combine chemical as well as physical characteristics similar to native tissue. A hydrogel with asymmetric mechanical integrity can provide moreeffective microenvironment cues

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to encapsulated stem cells, considering many biological tissues do not display a uniform structure. Therefore, a mechanically stiff and biomimetic IPN can be fabricated with hierarchical design for cartilage regeneration.[68] The high water content of the hydrogel provides vital lubricity, the mechanical strength provides construct stability in a dynamic region of the body, and the Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

combination of synthetic and naturally based polymers motivates glycosaminoglycan (GAG) synthesis from encapsulated chondrocytes. To improve clinical relevance, others have taken this hierarchical structure and designed an injectable system for enhanced delivery of the bioinspired interwoven network.[69] Again, the blending of natural and synthetic polymers provides cell-based

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degradation for more-rapid clearance of the scaffold for cellular migration (natural), while including a secondary network for a long-term presence. IPN hydrogels can also include environment-responsive polymeric materials coupled with integrated nanomaterials for transduction of external stimuli. For example, a poly(vinyl alcohol)/polyacrylamide IPN was formed with copper nanoparticles dispersed throughout.[70] This hydrogel in particular could provide a swelling–deswelling capability to an otherwise mechanically robust and hydrated scaffold. Future studies to control the spatial distribution of these nanoparticles within the network could generate a robust hydrogel capable of region-specific growth-factor release for cellular induction, while preserving the scaffold architecture in those regions lacking the inorganic nanoparticles. Translation of IPN constructs from applications other than tissue engineering (e.g., DNA or small-molecule sensing), will similarly offer novel designs. One such composite would be that of a functionalized quasi-IPN with multi-walled carbon nanotubes.[71] Aside from capillary-electrophoresis DNA sequencing, carbon nanotubes within a DN or IPN would provide similar signals to stem cells as would filaments comprising the ECM, and therefore improve differentiation outcomes. 3.8. Spatially Controlled Hydrogel Nanocomposites Microfabrication techniques such as micromolding can also be used to control cellular microenvironments to better mimic tissue complexity. Recently, we have reported bioengineered polymeric nanocomposites for cell- and tissue-engineering applications to modulate cellular function.[72] A rationale to design biomimetic hydrogel scaffolds is

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to model the tissue complexity by supporting survival, adhesion, and proliferation of encapsulated cells. We have designed a microarray of bioadhesive nanocomposite microgels with tunable physical and chemical properties, modular sizes, and tailored adhesive biomolecule composition. An IPN of gelatin ionically crosslinked with silicate nanoparticles was incorporated within maleimide-functionalized poly(ethylene glycol) (PEG-MAL) hydrogels (Figure 8).[72] The fabrication of these bio adhesive gels does not require harsh chemical and UVcrosslinking conditions and thus will be beneficial in providing an efficient platform for designing tissue mimics, delivery systems, and highthroughput screening devices. Another microfabrication technique, microfluidics, can be used to design a spatially controlled gradient of materials for mimicking tissue gradients.[73,74] For example, nanocomposite gradient scaffolds enable region-specific variation of scaffold stiffness, bioactivity, pore interconnectivity, and cell adhesiveness. One simple, but effective, method by Hancock et al. employs hydrophobic interactions to generate a surfacetension-driven composite gradient down a pre-wet polymer solution.[75] For more-viscous polymers, diffusion may be limited on a relevant timescale, opening the door for new microscale technologies to produce similar outcomes. The exploitation of multiple polymer regions within a single gradient construct represents another future development of multi-polymer systems. One could also imagine the use of composite semi-IPNs for enhanced mechanical stability and hydration, with the benefit of bioinspired nanofiller bioactivity.[76] Functionally graded scaffolds not only mimic architecture, but also mechanical distributions, important in bone transitions between cortical and cancellous regions.[77] Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

4. Bioengineering Tissue Complexity via Bioprinting Engineering artificial tissues offer great promise for treating patients with organ failures that are associated with disease, injury, and degeneration. Current approaches to engineer 3D tissue structures are based on encapsulating cells within a porous scaffold and providing structural and molecular clues to facilitate the formation of tissue structures.[78] These scaffolds serve as a synthetic ECM that assists in cellular organization into a 3D architecture by providing appropriate chemical and physical stimuli for facilitating their growth and maturation.[79] These tissueengineering techniques have been applied to generate a range of tissues including cartilage and skin, as these tissues can survive without the presence of extensive vascularization. However, engineering tissues with a complex structure, such as heart and liver tissues, is not possible until numerous challenges regarding their development have been addressed. These challenges include our inability to generate a functional vasculature that can supply the tissue with nutrients and oxygen, and the inability to mimic the complex cell-microenvironmental interactions that regulate the formation of functional tissue. Recently, the bioprinting technique has shown promising in mimicking tissue complexity by controlling cell–matrix and cell–cell interactions.[9,34,80–83] This bottomup approach uses layer-by-layer printing of cell-laden polymeric bioinks. The very essence of bioprinting emanates from bioinspiration, as materials are meticulously printed to mimic the cellular arrangement in the body. The merging of synthetic and natural polymer bioprinting systems can more aptly control material properties, and these hybrid polymer designs strive to incorporate the benefits of both types of polymers, for

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Figure 8. Bioadhesive nanocomposite hydrogels composed of PEG-MAL, gelatin, and silicate nanoparticles (NP). Schematic showing synthesis and fabrication of nanocomposite microgels consisting of 4-arm PEG-MAL loaded with gelatin-silicate NP and then crosslinked with a Michael-type addition reaction. The PEG network provides structural stability and mechanical integrity to the scaffold structure, while the gelatin–silicate NP provides cell-adhesion sites to support cell survival, proliferation, and cell signaling. The red spheres represent suspension cells and the green cells are anchorage-dependent. The second schematic represents microfabrication of bioadhesive microgels. The bottom fluorescence images show microfabricated hydrogels coencapsulating suspension cells and anchorage-dependent cells. Scale bar = 200 μm. Reproduced with permission.[72] Copyright 2014, Springer.

example, the tunability of synthetic materials with the biomimetic characteristics of natural polymers. Due to the inherent complexity of native tissues, both types may be justified

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for functional regeneration. Tissue engineers utilize copolymeric systems to avoid the shortcomings of single-polymer-type systems. While synthetics demonstrate acceptable Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

mechanical strength and fair biocompatibility, they lack cell-recognizable binding sites to improve adhesion or migration; however, hybridization with natural polymers

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can better mimic the ECM, leading to superior cellular outcomes.[84] The development of hybrid systems that apply synthetic polymers as scaffolding for structure and shape with cell-laden naturally based bioink as a filler closely follows the work of those groups integrating electrospun mats into hydrogel networks. This method benefits from including synthetic thermoplastic polymers due to the improved mechanical strength that typical hydrogel materials cannot provide,[85,86] and this system permits numerous bioinks laden with multiple cell types, either differentiated or potent cells, for printing in a single construct.[85] It was possible to print lower-viscosity hydrogels due to the mechanical strength provided by the thermoplastic materials, typically PCL or PLA, enabling researchers to print with a greater amount of bioink materials. Another benefit of XYZcontrolled nozzles printing thermoplastic polymers is a similar spatial resolutionas the applied hydrogels. This enables additional control over the final structure, or through electrospinning layers of randomly aligned PCL fibers to separate sections of hydrogels, allowing variation of printed cell type and hydrogel material at different layers. While bioprinting has only recently demonstrated its true impact as an exciting field of regenerativemedicine research, recent trends attempting to propel these technologies into areas of even greater clinical relevance have surfaced. To overcome the shortcomings of a purely polymeric system (e.g., insufficient mechanical strength, inefficient cellular stimulation, etc.), nanocomposites have been introduced to improve upon these lacking characteristics for those same factors expressed for 2D and 3D scaffolds.[1,87,88] Through force distribution and greater variation of chemical groups among multiple materials, multi-nozzle printing systems can also enhance both

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mechanical integrity and inductivity. These two aspects are crucial for bioprinting design to more suitably mimic native ECM. In order to better replicate this microenvironment, functionalization of base polymers to improve physical or chemical interactions is possible. One group chemically functionalized 3D-printed PLA with multiwalled carbon nanotubes (MWCNTs), inducing stem-cell differentiation into both osteogenic and chondrogenic lineages.[89] Polymer-nanocomposite interfaces boosted mechanical strength of the modified scaffold, with a Young’s modulus similar to that of subchondral bone (30–50 MPa), therefore providing stem cells with a desirable substrate, as well as limiting possible stress-shielding effects.[89] By subjecting MWCNTs with poly(L-lysine) after H2 treatment, the hydrophilicity of MWCNTs increased as did, consequently, the biocompatibility of the construct. From successful biomimesis, the scaffold increased stem-cell proliferation. One could imagine in future work if cells were encapsulated in a hydrogel bioink and printed layer-by-layer simultaneously with the functionalized PLA-MWCNT scaffold, tissue formation could be further improved. Similarly, dispersal of nano-titania in a printed poly(lactic-co-glycolic acid) (PLGA) scaffold established a surface roughness comparable with that of native bone, comparable with nHAp electrospun scaffolds.[88] Osteoblast adhesion and the tensile modulus increased in well-dispersed scaffolds resulting from topographical modifications to PLGA fibers, as well as physical crosslinking between the polymer and the nanofiller. Lastly, these nanoparticles shield the scaffold from the acidic degradation products of PLGA during hydrolysis, reducing autocatalysis effects and thus extending the mechanical stability for longer periods of time. Again, printing a polymerhydrogel bioink concurrently with a Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

nanocomposite 3D scaffold could provide the correct microenvironment for tissue formation in vivo.

5. Emerging Trends in Polymeric Nanocomposites Over the last decade, innovative research has uncovered biologically relevant nanomaterials for biomedical engineering. These discoveries have been instrumental in establishing unconventional methods to attack persistent tissue-engineering challenges, yet the formulations of complex tissues, especially those that are highly vascularized, remain unrealized. New biomimetic approaches aim to extinguish these obstacles (see Figure 9).[90] Technological advancements have also impacted bioinspired polymeric systems through the generation of microfluidic devices and 3D printing to significantly enhance control over microarchitecture. To immobilize declining protein amounts along the length of the hydrophobic silanized glass-slide surface, one group fabricated a PDA graded surface.[91] As a model protein, fluorescein isothiocyanate–bovine serum albumin (FITC-BSA) provided a visualization of adhered protein in those regions containing PDA, translating to regions capable of cell adhesion or bioactivity depending on the agents conjugated to the PDA. This mechanism enabled both covalent and noncovalent interactions between the surface and the composite material for bioactivity maintenance as well as long-term stability. Rapid prototyping and computer-aided design (CAD) improve upon spatial control through mathematical models.[92] Polymer solutions loaded with nanofillers can be extruded into designs mimicking natural microstructures like cuttlefish bone[93] or osteochondral defects.[94] In another study, a biphasic mold for osteochondral replacement was designed using

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Figure 9. Bioinspiration meshed with technological advancements has advanced scaffold design while also inviting fresh perspectives toward regenerative medicine. Microscale fabrication techniques enable highly spatially controlled architecture as well as nanocomposite arrangement within the construct-like developed gradient scaffolds, rapid prototyping processes, and bioinspired hydrophobic surfaces. Unique polymer chemistry used in conjunction with nanofillers establishes a hydrated biomimetic microenvironment suitable for cell growth. These emerging techniques have not only been employed for tissue regeneration of complex tissues, like the hierarchical structure of the cartilage–bone interface, but also for high-throughput screening between cells and biomaterials.

stereolithography of beta-tricalcium phosphate slurry interfaced with a gel-cast collagen solution.[95] The mu lt i - mat e r i a l / mu lt i - s t r u c t u r e included an engineered transitional phase to mimic the bone–cartilage interface and deliver a more-functional end product. Minimal surface geometries, like those found in beetle shells, weevils, or butterfly wings, are likewise feasible architectures for scaffold design.[96] Future research employing these design mechanisms could introduce multicomponent systems for composite constructs principally to imitate natural ECM.[97] Another direction of bioinspired composite research focuses on multiplexing techniques for highthroughput analysis of biomaterial–cell interactions, improving the efficiency of experimental processes

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for cytocompatibility and developmental biology investigations.[80] While some have employed bioinspired superhydrophobic surfaces for this combinatorial approach,[98] others have fabricated composite colloid/polymer nanoshell systems for encapsulating cells within a specific biomimetic local environment.[99] Variations of polymeric core–shell particles can easily be obtained via addition of external coatings or membranes (e.g., polyelectrolytes, poly(diallyldimethylammonium chloride), or poly(sodium 4-styrenesulfonate)) in order to uncover signal-transduction pathways, for example.[100] The ability to confine these bioinspired matrices in multiplexed platforms demonstrates the flexibility and potential applications for composite biomaterials. Macromol. Chem. Phys. 2015, 216, 248−264 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

As is the case with any implanted material, immunological interactions between the body and nanomaterials are constantly evaluated. While bioinspired nanomaterials or architectures typically display an inherent biocompatibility, as one would expect coming from nature, conflicts might arise from the fabrication conditions, the physical introduction into the body, or from accompanying materials comprising the nanocomposite. Therefore, confirming interactive pathways between the local cell population and the introduced nanomaterial as well as possible immunostimulation is a necessary facet to any tissue-engineering design. Extensive research has focused on these properties,[101] and while we will not discuss the exhaustive list of immunologic factors, there are important variables to consider when designing a bioinspired or any regenerativemedicine system. For example, cell types may prefer specific shapes or dimensionality (2D versus 3D) for internalization, leading to activation, which can induce a positive or negative cellular response depending on the cells and material in question.[102] In some cases, this capability of immunomodulation can be beneficial for tissue regeneration.[103] Particularly in instances where immune responses limit functional tissue repair, like in osteoarthritis pathologies, bioinspired systems may be introduced to diminish this activation and improve tissue-regeneration outcomes. The design of novel inspired biomaterials capable of balancing the regenerative and immunological processes of the body, improving scaffold outcomes, will be a great challenge for future investigations.

6. Outlook The vitality of bioinspired research will continue to rely on designing and developing new functional biomaterials and creating new technological

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tools to control and manipulate cell– matrix interaction at macro, micro, and nano length scales. The nanomaterials currently used in commercial and industrial applications provide a unique opportunity for translation into the biomedical field. Synthetic silicates recently entered the medical field from employment as food additives, adsorbents, and anticaking agents, and have already impacted regenerative-medicine research. Bioinspiration may arise from natural structures found within the cell environment or possibly from unrelated biological anomalies like shark skin or adhesive proteins from marine mussels. Multifunctional approaches integrating physical, mechanical, and chemical factors stimulate several pathways simultaneously, potentially amplifying cellular responses. A major future step for bioinspired regenerative medicine research is concurrent spatial and temporal control over stem-cell development through polymeric and nanomaterial manipulation. Acknowledgements: Note: Reference 72 was corrected on February 2, 2015, after initial publication online. Received: August 13, 2014; Revised: September 16, 2014; Published online: November 18, 2014; DOI: 10.1002/macp.201400427 Keywords: bioinspired structures; hydrogels; nanomaterials; polymeric nanocomposites; tissue engineering [1] A. K. Gaharwar, N. A. Peppas, A. Khademhosseini, Biotechnol. Bioeng. 2014, 111, 441. [2] C. Sanchez, H. Arribart, M. M. G. Guille, Nat. Mater. 2005, 4, 277. [3] P. Fratzl, R. Weinkamer, Prog. Mater. Sci. 2007, 52, 1263. [4] G. D. Bixler, B. Bhushan, Soft Matter 2012, 8, 12139. [5] A. J. Parsons, I. Ahmed, N. Han, R. Felfel, C. D. Rudd, J. Bionics Eng. 2010, 7, S1. [6] Y. J. Zhu, H. Wu, S. F. Sun, T. Zhou, J. J. Wu, Y. Wan, J. Mech. Behav. Biomed. Mater. 2014, 36, 32.

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