Bioinspired Polymeric Nanocomposites for Regenerative Medicine

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continuously evolving. The success of many constructs is often limited by the lack of biological complexity gen-erated, leading to researchers inves-tigating new methods to emulate native tissue environments. Often-times, inspiration for scaffold archi-tecture or utilized biomaterials stems from structures preexisting in nature, considering millions of years have resulted in the emergence of highly sophisticated and effi cient mate-rials. [ 1–3 ] For example, shark skin and lotus leaves have been investigated for inspired surface design due to the anisotropic fl ow 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.

DOI: 10.1002/macp.201400427

Bioinspired Polymeric Nanocomposites for Regenerative Medicine

The design and fabrication of bioinspired nanomaterials for tissue-engineering applications requires a fundamental understanding of the interactions between polymers, nanostruc-tures, and cells. Most biomimetic polymeric nanocomposites consist of two or more types of polymers or of polymers combined with different nanomate-rials to obtain composite structures with desired properties. In this Talents & Trends article, the focus is on bioinspired poly meric nanocomposites surrounding three major strat-egies. Firstly, biomimetic structures composed of a fi brous architecture are discussed. Secondly, the emerging trends in designing complex nanocomposites with multiple functional-ities 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 fi eld of bioin-spired polymeric nanocomposites are highlighted.

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

1. Introduction

The traditional paradigm of tissue engineering includes three inte-gral components: cells, growth fac-tors, 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

Biology offers the best models for strategies to rationally design high-performance biomaterials with properties similar to those of nat-ural materials, such as bone, carti-lage, 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 infl uence long-term cellular activities. Both bottom-up and top-down approaches are consid-ered 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 require-ments for consideration that will

Young Talents in Polymer Science

James K. Carrow , Akhilesh K. Gaharwar*

Macromol. Chem. Phys. 2015, 216, 248−264

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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MacromolecularChemistry and Physics

be implemented cohesively during material design. Akin to nature, bio-material design processes strike a balance between complexity and unifi cation of the individual compo-nents. The design and fabrication of bioinspired nanomaterials for tissue-engineering applications requires a fundamental understanding of the interactions between polymers, nanostructures, and cells. Most bio-mimetic polymeric nanocomposites consist of two or more types of poly-mers, or polymers combined with dif-ferent nanomaterials, to obtain com-posite structures with desired proper-ties (Figure 1 ). A range of structures, including interpenetrating, fi brous scaffolds and nanocomposite bioma-terials, which mimics the structural and physical properties of the ECM is engineered. Biomimetic materials are used for a range of biomedical applications, including regenera-tive 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 proper-ties of the natural tissue, along with the biological infl uence of cells on the synthetic biomaterial proper-ties. For example, bone comprises a hierarchical structure that provides the tissue with its advantageous mechanical and functional proper-ties, yet also signifi cantly hampers replication in an in vitro setting. [ 11 ] Therefore, to recapitulate native complexity, bioactive materials can motivate specifi c cellular activity in a spatially confi ned manner. Among popular fi llers, nanoceramics, such as synthetic silicates, nano-hydroxyapa-tite (nHA), and bioactive glass, instill tissue-engineering scaffolds with a supplementary infl uence over stem-cell behavior. While the underlying mechanisms of their bioactivity are still under evaluation, evidence points to the combination of degra-dation products, surface interactions

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 fi elds, including materials science, chem-istry, biology, and engineering of polymeric biomaterials and nano-composites. 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.

modifi cations can employ bioinspira-tion via mimesis of naturally occur-ring molecular processes or micro-environment induction of cellular behavior (Figure 1 ). Through revers-ible physical interactions (e.g., elec-trostatic, dipole, hydrogen-bonding) or chemical crosslinks (e.g., thiol-based, radical polymerization), supe-rior distribution of stresses imparted onto a polymeric composite scaffold can be achieved. Similarly, degrada-tion profi les of networked polymer composites can be extended to allow suffi cient cell migration and tissue formation in vivo.

To provide additional function-ality, a range of nanoparticles can be incorporated within the poly-meric network to fabricate bioin-spired nanocomposite structures. Most nanomaterials can be divided into four different categories: zero-, one-, two-, and three- dimensional nanomaterials (Figure 2 ). Zero-dimensional (0D) nanomaterials are atomic clusters mostly composed of metallic elements. One-dimensional (1D) nanomaterials include metal nanorods, nanotubes, ceramic crys-tals, polymer nanofi bers, and self-assembled structures. Most two-dimensional (2D) nanomaterials have included layered structures such as graphene, synthetic clays, and double layered hydroxides (LDH), whereas three-dimensional (3D) nanomate-rials include polycrystals and spher-ical particles. Due to the difference in

with cellular membranes, and charge. Novel microscale technolo-gies have emerged in order to fabri-cate polymeric-based structures with additional nanofi llers, thus providing investigators with an architecture or material inspired from nature. [ 9 ]

Integrated composites not only establish additional sources of bio-active factors, they can also fortify the polymer network via physical or chemical interactions. [ 1,12–15 ] While many naturally based polymers dem-onstrate useful cytocompatibility or synthetics, enabling extensive tailor-ability to the polymer chain, often a purely polymeric system lacks the necessary mechanical strength or degradation characteristic in vivo, particularly for load-bearing regen-erative applications. Most exten-sively investigated natural polymers for biomedical applications include collagen, gelatin, starch, cellulose, algin ate, chitosan, and fi brin, 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 seba-cate) (PGS). To overcome the limita-tions of a basic polymeric scaffold, additional nanomaterials are inte-grated into the architecture to form a nanocomposite with the combi-natorial benefi ts of each biomate-rial, including bioactivity, adhesive-ness, environmental-sensitivity, and mechanical improvements. [ 16 ] These



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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 encapsu-lated stem cells; however, these same materials present in the extracellular environment can act as nucleation sites for the deposition of mineral-ized matrix, effectively mimicking those found on collagen fi brils in bone tissue. [ 17,18 ] While multiple

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 nano-materials are introduced to polymeric

surface to volume ratio, these nano-materials interact with polymers via substantially different mechanisms and result in unique property com-binations compared with their micro and nano counterparts. The dimen-sionality of incorporated nanomate-rials will stimulate specifi c 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

Figure 1. To improve scaffold outcomes, bioinspired composite materials can provide additional benefi ts 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 modifi cation 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 specifi c lineages.





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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 fi eld of poly-meric nanocomposites for biomedical applications.

2. Biomimetic Fibrous Scaffolds

The fi brous confi guration of native ECM has been exposed as crucial toward stem-cell behavior, especially in the niche during potency main-tenance. [ 23 ] Notwithstanding topo-graphical characteristics of many polymeric fi brous scaffolds altering cell behavior through mechanotrans-duction pathways, control of cell activity toward specifi c lineages is challenging as oftentimes the syn-thetic polyesters used lack inherent bioactivity. Therefore, the enrich-ment of a fi brous scaffold to form a composite material is desirable in

situations requiring specifi c stem-cell directing.

In an attempt to improve elec-trospun scaffold properties, groups have fabricated composites through direct mixing of polymer and nano-clay (synthetic silicate nanoparticles) solutions prior to electrospin-ning. [ 24–26 ] In our investigations, we noted changes to poly(caprolactone) (PCL) fi ber morphology and size in which the fi bers reduced in diameter and showed increased roughness (Figure 3 ). Moreover, biomineraliza-tion in the presence of SBF and osteo-genic activity were found to signifi -cantly increase with greater amounts of nanoclay, indicating a potential for these inorganic nanomaterials in musculoskeletal tissue engineering. Similar results were likewise pro-duced where silicate nanoparticles were embedded within a PCL elec-trospun scaffold, highlighting the capabilities of these bioinspired nanocomposites for hard-tissue regeneration. [ 27 ]

groups have integrated both polymer and additive prior to scaffold poly-merization, some have investigated the effects of creating nucleation sites after polymer-network forma-tion through incubation with simu-lated body fl uid (SBF) in the presence of carboxyl groups on the polymer chain. [ 19–22 ]

In this article, we focus on work on bioinspired polymeric nanocom-posites surrounding three major strategies. Specifi cally, we discuss the different types of polymeric nano-composites based on different types of structure. First we will discuss biomimetic structures composed of a fi brous architecture. Secondly, inves-tigations designing complex hydrogel nanocomposites with multiple func-tionalities will be examined and dis-cussed 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 appli-cations and challenges in bioprinted

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.





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through coatings or grafts on syn-thetic polymers. [ 29,30 ] Binding of metallic nanoparticles would result in a multifunctional electrospun scaf-fold, while protein adhesion would affect cell spreading and cytoskeletal rearrangement. Another grafting pro-cedure modifi ed a collagen type-II scaffold through a biomimetic recom-binant protein of fi bronectin module III and cadherin-11 (rFN/Cad-11), to promote adhesion and chondro-genesis of MSCs. [ 31 ] Thus, these com-posite materials again fulfi ll the mul-timodal aspect of materials science

regenerative-medicine design via the multiple mechanisms of cellular interaction.

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

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

Figure 3. Electrospinning PCL–nanoclay(synthetic silicate) composite fi bers results in a reduction of fi ber diameter with increased nanoclay content. Additionally, the added nanoclay provides enhanced nucleation sites for hydroxyapatite crystal formation on the fi ber exterior due to enhanced surface roughness, eventually leading to a signifi cant 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 there-fore the potential of these nanocomposites for osteo-regenerative applications. Reproduced with permission. [ 25 ] Copyright 2014, Mary Ann Liebert, Inc.





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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, gener-ating a construct with both physical and chemical inspiration.

3. Bioinspired Polymeric Nanocomposites

Creating a 3D microenvironment capable of supporting cell prolifera-tion and differentiation is a major focus of hydrogel research. The implanted material must fulfi ll a variety of responsibilities, including mechanical stability, cytocompat-ibility, and bioactivity, but many pure polymer systems are incapable of satisfying all requirements, hence the emergence of nanocomposite net-works. Here we will focus on those nanocomposites inspired by nature.

3.1. Polymeric Nanocomposite Hydrogels

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 micro-environment; however, formulating a hydrogel with mechanical stability and bioactivity is a persisting chal-lenge. [ 10,33,34 ] A range of hydrogels has been synthesized to mimic native tissue structure and physical proper-ties using both natural and synthetic polymers. [ 1,16 ] Subsequently, to pro-vide bioactivity, electrical conduc-tivity, and stimuli-responsiveness, a range of nanoparticles has been incorporated within the polymeric network. Carbon-based nanomate-rials such as carbon nanotubes and graphene oxide can provide electrical conductivity; inorganic nanoparticles

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

fi brous scaffolds for hard-tissue applications, in which they mim-icked calcium phosphate nanocrys-tals on collagen fi bers through in situ

Figure 4. Shown is the basic process for electrospun composite and drug reservoir gen-eration. A schematic and the SEM image illustrate localization of dexamethasone within the PEOT/TPBT fi bers. The fl uorescence images were acquired via replacing dexametha-sone with a fl uorescent molecule, Texas Red, as they have comparative molecular weights. Merging of optical and fl uorescence microscopy images display concentration of com-posite materials within the beaded fi ber structure. Reproduced with permission. [ 32 ] Copy-right 2014, Elsevier.





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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 poly-mers, 3D polymeric scaffolds with bioactive properties for regenerative medicine can fabricated.

Synthetic silicates strongly interact with polymeric networks resulting in the formation of physi-cally crosslinked networks and sig-nifi cantly increasing the mechanical stiffness. [ 1,42 ] In a recent investiga-tion, we assessed that addition of silicates to neutral polymer such as poly(ethylene oxide) (PEO), aug-menting cell adhesion and spreading, while improving overall material stability through electrostatic inter-actions between the nanoparticle surface and the electronegative oxygen atoms within the polymer backbone. [ 43,44 ] This is mainly attrib-uted to the exfoliated structure of the silicate nanoparticles within the hydrogel network. [ 15,45 ] The exfoli-ated hydrogels, containing silicate and polymer, result in the formation of a highly organized layered struc-ture 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 fi bers made from viscoelastic PEO-silicate nanocom-posite hydrogels. [ 46 ] Then the swollen hydrogel network was pulled to obtain hydrogel fi bers. Although the exact interactions between the sili-cate nanoparticles and the polymer are still not known, it is believed that hydrogen bonding, electro-static interactions, ionic bonds, and physical entanglement of the polymer chains with the nanopar-ticle surfaces play a major role in enhancing the physical and chemical

such as hydroxyapatite, bioglass, and synthetic silicates can provide bio-active properties; metal and metal oxide nanoparticles can provide anti-microbial and magnetic properties; and polymeric nanoparticles can pro-vide controlled release of biological signals to control cellular behavior. These nanoparticles physically and/or covalently interact with polymers and result in unique property combi-nations. Developing nanocomposite hydrogels with tailored functionality has opened up new possibilities in developing advanced biomaterials for various biomedical and biotech-nological applications. These nano-composite hydrogels are currently explored in the area of stem-cell ther-apies, tissue engineering, cellular and molecular treatments, immunomodu-lation, and cancer research. [ 35,36 ]

We have designed nanocomposite hydrogels to mimic some of the phys-ical, chemical and biological charac-teristics of native tissues. Compara-tive to polymeric networks composed of linear polymer chains, hyper-branched polyester (HPE) hydrogels have the potential to fabricate bio-mimetic structures with tunable physical and chemical properties. [ 37 ] These dendritic macromolecules can be functionalized with a variety of peripheral chemical groups for tai-lored 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 bio-mimetic microstructure for cellular adhesion and proliferation. We have previously developed HPE hydrogels capable of sustained release of bio-active molecules, dexamethasone acetate, in which the architecture and substrate stiffness infl uenced cell–matrix interactions. [ 37 ] Internal cavities in the HPE molecule acted as drug reservoirs, while acrylation of hydroxyl-terminated HPE via stoi-chiometric addition of acryloyl chlo-ride generated photo-crosslinkable materials. These are both favorable

facets of the design process as a multitude of chemical cues can be integrated within the dendritic mol-ecules; 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 pro-long therapeutics release for control-ling 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 counter-parts because of their high surface-to-volume ratio. [ 1,38,39 ] The rapid and recent advances in ultra-thin mate-rials have raised tantalizing ques-tions about their interactions with biological moieties. Since 2D nano-materials 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 thera-peutics, imaging, and disease-related diagnostics.

Our recent reports indicate that these synthetic silicates strongly interact with stem cells and induce osteogenesis without using any addi-tional 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 osteo-related genes and proteins, such as RUNX2, osteocalcin, and osteo-pontin, and result in production of mineralized ECM. [ 18 ] Due to strong bioactive characteristics, these nano-particles are proposed for a range of musculoskeletal tissue-engineering approaches. These silicates are





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be tuned. In our recent study, we showed that addition of silicate nanoparticles results in signifi -cant control over cellular functions (Figure 5 ). [ 43,44 ] It is expected that the presence of magnesium on silicate nanoparticles might facili-tate integrin-mediated cell attach-ment. Human mesenchymal stem cells (hMSCs) seeded on PEO-silicate nanocomposites demonstrated good cytocompatibility and enhanced osteogenic differentiation, indicating a potential for regenerative medi-cine. [ 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.

3.3. Bioinspired Hydroxyapatite Nanocomposites

Nano-hydroxyapatite (nHAp), a min-eral found naturally in hard tissue, is another candidate for integration as a bioinspired nanofi ller. [ 51–54 ] nHAp within the matrix could act as nuclea-tion sites for the mineralization of encapsulated cells and provide useful minerals for bone formation. How-ever, 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 frac-ture toughness. By incorporation of nHAp within a polymeric hydrogel network, bioactive nanocomposites are fabricated for non-load-bearing

characteristics of nanocomposite networks. [ 48–50 ] These hydrogel fi bers were subjected to solvent evapora-tion to obtain nanocomposite fi bers with a high aspect ratio. [ 46 ] Cross-polarized images of a semi-dried nanocomposite fi ber suggest an an isotropic orientation of the silicate nanoparticles and polymer chains along the fi ber axis. Scanning elec-tron microscopy (SEM) images show that these fi bers have a smooth and uniform surface morphology. The addition of silicate to PEO induces cell-adhesion properties, and fi bro-blasts aligned themselves along the fi bers, as visualized through Phal-loidin-actin staining. [ 46 ]

After incorporating silicate nano-particles within a neutral polymer, cell adhesion and function can

Figure 5. Increasing silicate nanoparticles content enhances hierarchical microstructure of nanocomposite fi lms and also improves cell adhesion and spreading, potentially through magnesium-mediated integrin binding. The supplementary complexity of the fi lm architec-ture 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.





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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 syn-thetic materials. Again, biological and physical interactions at various scales

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 medi-cine nanocomposites is rosette nano-tubes, from assembling synthetic DNA bases, more specifi cally the

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

Figure 6. The nanocomposite hydrogels composed of nanohydroxyapatite (nHAPs) and PEG can be fabricated via photo-crosslinking. Thin nanocomposite fi bers 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 signifi cantly enhanced cell adhesion of the PEG-nHAp hydrogel. The presence of nHAp provided bioactive attachment sites to the cells, which led to elongated lamellipodia and pseudo-podia. Reproduced with permission. [ 51 ] Copyright 2011, American Chemical Society.





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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 (pDNA VEGF ) was loaded for delivery to damaged cardiac tis-sues. Although nonviral nanopar-ticles have poor transfection effi -ciency when compared with viral systems, GO nanosheets can effi -ciently deliver the genes (pDNA VEGF ) 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 pro-angiogenic activities of the hydrogel, which showed enhanced cellular proliferation. In vivo studies indi-cate that fGO/GelMA hydrogel did not invoke any signifi cant toxicity or infl ammatory response. This inject-able nanocomposite hydrogel (GG’), loaded with fGOVEGF (pDNA VEGF bound to fGO), was shown to facili-tate local myocardial neovasculari-zation at the injected sites, reduce fi brosis, and improve cardiac func-tion 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 pri-marily composed of natural struc-tures, the utilization of metal-based components for research in regener-ative medicine typically falls outside the realm of biomimesis. However examples of successful inclusion

of these inorganic elements within polymeric systems exist and provide alternative avenues for functional-tissue regeneration. For example, interactions between peptides and solid inorganic surfaces can engineer reorganized molecular structures for improved bioactivity or further func-tionalization with synthetic mate-rials, improving implanted-device regenerative performance. [ 63 ] Alter-natively, the conductivity of metallic nanoparticles embedded within a polymeric matrix or nanofi ber (e.g., gold) make them attractive for regenerative processes employing electrical stimulation to mimic neu-ronal 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 Interpen-etrating Networks (IPNs)

Most tissue structures, such as car-tilage 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 net-works (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 sub-nanometer length scale and result in unique property combinations due to extensive polymer–polymer inter-actions. Likewise, there is a funda-mental mimesis of nature through the complexity of the fi ber mor-phology within the hydrogel, as well as structural integrity effectively matching that of native tissue. Due to their impressive mechanical quali-ties, there is great potential for use in

hydrogen-bonding arrays between guanine and cytosine. [ 58,59 ] These bases comprise a structure similar to collagen fi bers of the ECM, and also have the advantage of tailorability with additional amino acids and peptide sequences for improved bio-activity or cell adhesion within the scaffold. Inspired from the fi brillar arrangement produced by these sequences, the groups encouraged chondrogenesis from human mesen-chymal stem cells, leading to a signifi -cant increase in glycosaminoglycans (GAG) production, indicating suc-cessful chondrocyte differentiation. The capability of transforming 2D materials into 3D implantable struc-tures through polymeric interactions provides new perspectives for infl u-encing 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 benefi t both cell behavior and construct sta-bility. For example, graphene oxide sheets can self-assemble with single-strand DNA via non-covalent inter-actions resulting in the formation of 3D structures. [ 60 ] The resulting biomimetic hierarchical composi-tion 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 effi cient biomolecule loading and cell-internalization properties of graphene. A shear-thinning meth-acrylated gelatin (GelMA) hydrogel is developed by impregnating PEI-functionalized GO nanosheets (fGO) (Figure 7 ). [ 61 ] The preliminary inves-tigation using a 22-gauge needle indicates that all the nanocomposite





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to encapsulated stem cells, consid-ering many biological tissues do not display a uniform structure. There-fore, a mechanically stiff and bio-mimetic IPN can be fabricated with hierarchical design for cartilage regeneration. [ 68 ] The high water con-tent of the hydrogel provides vital lubricity, the mechanical strength provides construct stability in a dynamic region of the body, and the

combination of synthetic and natu-rally based polymers motivates gly-cosaminoglycan (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 bioin-spired interwoven network. [ 69 ] Again, the blending of natural and syn-thetic polymers provides cell-based

biomedical engineering and specifi -cally 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 charac-teristics similar to native tissue. A hydrogel with asymmetric mechan-ical integrity can provide more-effective microenvironment cues

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 signifi cantly 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 fi brosis 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.





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to model the tissue complexity by supporting survival, adhesion, and proliferation of encapsulated cells. We have designed a micro-array of bioadhesive nanocomposite microgels with tunable physical and chemical properties, modular sizes, and tailored adhesive biomol-ecule 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 fabrica-tion of these bio adhesive gels does not require harsh chemical and UV-crosslinking conditions and thus will be benefi cial in providing an effi -cient platform for designing tissue mimics, delivery systems, and high-throughput screening devices.

Another microfabrication tech-nique, microfl uidics, can be used to design a spatially controlled gradient of materials for mimicking tissue gradients. [ 73,74 ] For example, nano-composite gradient scaffolds enable region-specifi c variation of scaffold stiffness, bioactivity, pore intercon-nectivity, and cell adhesiveness. One simple, but effective, method by Hancock et al. employs hydrophobic interactions to generate a surface-tension-driven composite gradient down a pre-wet polymer solution. [ 75 ] For more-viscous polymers, diffu-sion 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 repre-sents 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 benefi t of bioinspired nanofi ller bioactivity. [ 76 ] Functionally graded scaffolds not only mimic architec-ture, but also mechanical distribu-tions, important in bone transitions between cortical and cancellous regions. [ 77 ]

4. Bioengineering Tissue Complexity via Bioprinting

Engineering artifi cial tissues offer great promise for treating patients with organ failures that are associated with disease, injury, and degenera-tion. 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 for-mation 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 tissue-engineering techniques have been applied to generate a range of tissues including cartilage and skin, as these tissues can survive without the pres-ence 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 nutri-ents and oxygen, and the inability to mimic the complex cell-microenviron-mental interactions that regulate the formation of functional tissue.

Recently, the bioprinting tech-nique has shown promising in mimicking tissue complexity by controlling cell–matrix and cell–cell interactions. [ 9,34,80–83 ] This bottom-up approach uses layer-by-layer printing of cell-laden polymeric bioinks. The very essence of bioprinting emanates from bioin-spiration, as materials are meticu-lously printed to mimic the cellular arrangement in the body. The merging of synthetic and natural polymer bioprinting systems can more aptly control material prop-erties, and these hybrid polymer designs strive to incorporate the ben-efi ts of both types of polymers, for

degradation for more-rapid clearance of the scaffold for cellular migra-tion (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 nano-particles dispersed throughout. [ 70 ] This hydrogel in particular could provide a swelling–deswelling capa-bility to an otherwise mechani-cally robust and hydrated scaffold. Future studies to control the spatial distribution of these nanoparticles within the network could gen-erate a robust hydrogel capable of region-specifi c growth-factor release for cellular induction, while preserving the scaffold architecture in those regions lacking the inor-ganic 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 pro-vide similar signals to stem cells as would fi laments comprising the ECM, and therefore improve differ-entiation 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 bioen-gineered polymeric nanocompos-ites for cell- and tissue-engineering applications to modulate cellular function. [ 72 ] A rationale to design biomimetic hydrogel scaffolds is





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for functional regeneration. Tissue engineers utilize copolymeric sys-tems to avoid the shortcomings of single-polymer-type systems. While synthetics demonstrate acceptable

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

example, the tunability of synthetic materials with the biomimetic char-acteristics of natural polymers. Due to the inherent complexity of native tissues, both types may be justifi ed

Figure 8. Bioadhesive nanocomposite hydrogels composed of PEG-MAL, gelatin, and silicate nanoparticles (NP). Schematic showing syn-thesis 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 suspen-sion cells and the green cells are anchorage-dependent. The second schematic represents microfabrication of bioadhesive microgels. The bottom fl uorescence images show microfabricated hydrogels coencapsulating suspension cells and anchorage-dependent cells. Scale bar = 200 μm. Reproduced with permission. [ 72 ] Copyright 2014, Springer.





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nanocomposite 3D scaffold could pro-vide 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 biomed-ical engineering. These discoveries have been instrumental in estab-lishing unconventional methods to attack persistent tissue-engineering challenges, yet the formulations of complex tissues, especially those that are highly vascularized, remain unre-alized. New biomimetic approaches aim to extinguish these obstacles (see Figure 9 ). [ 90 ]

Technological advancements have also impacted bioinspired polymeric systems through the generation of microfl uidic devices and 3D printing to signifi cantly enhance control over microarchitecture. To immo-bilize declining protein amounts along the length of the hydrophobic silanized glass-slide surface, one group fabricated a PDA graded sur-face. [ 91 ] As a model protein, fl uores-cein isothiocyanate–bovine serum albumin (FITC-BSA) provided a visu-alization 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 mecha-nism enabled both covalent and non-covalent interactions between the surface and the composite material for bioactivity maintenance as well as long-term stability. Rapid proto-typing and computer-aided design (CAD) improve upon spatial control through mathematical models. [ 92 ] Polymer solutions loaded with nano-fi llers can be extruded into designs mimicking natural microstructures like cuttlefi sh bone [ 93 ] or osteochon-dral defects. [ 94 ] In another study, a biphasic mold for osteochondral replacement was designed using

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, functionaliza-tion of base polymers to improve physical or chemical interactions is possible. One group chemically func-tionalized 3D-printed PLA with multi-walled carbon nanotubes (MWCNTs), inducing stem-cell differentiation into both osteogenic and chondro-genic lineages. [ 89 ] Polymer-nanocom-posite interfaces boosted mechanical strength of the modifi ed 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 H 2 treat-ment, the hydrophilicity of MWCNTs increased as did, consequently, the biocompatibility of the construct. From successful biomimesis, the scaf-fold 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 functional-ized PLA-MWCNT scaffold, tissue for-mation 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 modi-fi cations to PLGA fi bers, as well as physical crosslinking between the polymer and the nanofi ller. Lastly, these nanoparticles shield the scaf-fold from the acidic degradation products of PLGA during hydro-lysis, reducing autocatalysis effects and thus extending the mechan-ical stability for longer periods of time. Again, printing a polymer-hydrogel bioink concurrently with a

can better mimic the ECM, leading to superior cellular outcomes. [ 84 ] The development of hybrid systems that apply synthetic polymers as scaf-folding for structure and shape with cell-laden naturally based bioink as a fi ller closely follows the work of those groups integrating electro-spun mats into hydrogel networks. This method benefi ts from including synthetic thermoplastic polymers due to the improved mechanical strength that typical hydrogel mate-rials 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 thermo-plastic materials, typically PCL or PLA, enabling researchers to print with a greater amount of bioink materials. Another benefi t of XYZ-controlled nozzles printing thermo-plastic polymers is a similar spatial resolutionas the applied hydrogels. This enables additional control over the fi nal structure, or through electrospinning layers of randomly aligned PCL fi bers to separate sec-tions of hydrogels, allowing varia-tion of printed cell type and hydrogel material at different layers.

While bioprinting has only recently demonstrated its true impact as an exciting fi eld of regenerative-medicine research, recent trends attempting to propel these technolo-gies into areas of even greater clinical relevance have surfaced. To over-come the shortcomings of a purely polymeric system (e.g., insuffi cient mechanical strength, ineffi cient cel-lular stimulation, etc.), nanocompos-ites 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 varia-tion of chemical groups among mul-tiple materials, multi-nozzle printing systems can also enhance both





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As is the case with any implanted material, immunological interactions between the body and nanomate-rials are constantly evaluated. While bioinspired nanomaterials or archi-tectures typically display an inherent biocompatibility, as one would expect coming from nature, confl icts might arise from the fabrication con-ditions, the physical introduction into the body, or from accompanying materials comprising the nanocom-posite. Therefore, confi rming interac-tive pathways between the local cell population and the introduced nano-material as well as possible immu-nostimulation is a necessary facet to any tissue-engineering design. Exten-sive research has focused on these properties, [ 101 ] and while we will not discuss the exhaustive list of immu-nologic factors, there are important variables to consider when designing a bioinspired or any regenerative-medicine system. For example, cell types may prefer specifi c shapes or dimensionality (2D versus 3D) for internalization, leading to activa-tion, which can induce a positive or negative cellular response depending on the cells and material in ques-tion. [ 102 ] In some cases, this capability of immunomodulation can be benefi -cial for tissue regeneration. [ 103 ] Par-ticularly in instances where immune responses limit functional tissue repair, like in osteoarthritis patholo-gies, bioinspired systems may be introduced to diminish this activation and improve tissue-regeneration out-comes. The design of novel inspired biomaterials capable of balancing the regenerative and immunological pro-cesses 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 biomate-rials and creating new technological

for cytocompatibility and develop-mental biology investigations. [ 80 ] While some have employed bioin-spired superhydrophobic surfaces for this combinatorial approach, [ 98 ] others have fabricated composite col-loid/polymer nanoshell systems for encapsulating cells within a specifi c 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-styrene-sulfonate)) in order to uncover signal-transduction pathways, for example. [ 100 ] The ability to confi ne these bioinspired matrices in multi-plexed platforms demonstrates the fl exibility and potential applications for composite biomaterials.

stereolithography of beta-tricalcium phosphate slurry interfaced with a gel-cast collagen solution. [ 95 ] The multi-material/multi-structure included an engineered transitional phase to mimic the bone–cartilage interface and deliver a more-func-tional end product. Minimal surface geometries, like those found in beetle shells, weevils, or butterfl y wings, are likewise feasible architectures for scaffold design. [ 96 ] Future research employing these design mechanisms could introduce multicomponent sys-tems for composite constructs princi-pally to imitate natural ECM. [ 97 ]

Another direction of bioinspired composite research focuses on multiplexing techniques for high-throughput analysis of biomate-rial–cell interactions, improving the effi ciency of experimental processes

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 nanocom-posite arrangement within the construct-like developed gradient scaffolds, rapid proto-typing processes, and bioinspired hydrophobic surfaces. Unique polymer chemistry used in conjunction with nanofi llers establishes a hydrated biomimetic microenvironment suit-able 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 inter-face, but also for high-throughput screening between cells and biomaterials.





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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

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Bioinspired Polymeric Nanocomposites for Regenerative Medicine

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