Comprehensive Review on the Use of Graphene-Based Substrates for

Review www.acsami.org

Comprehensive Review on the Use of Graphene-Based Substrates for Regenerative Medicine and Biomedical Devices Sachin Kumar and Kaushik Chatterjee* Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India ABSTRACT: Recent research suggests that graphene holds great potential in the biomedical field because of its extraordinary properties. Whereas initial attempts focused on the use of suspended graphene for drug delivery and bioimaging, more recent work has demonstrated its advantages for preparing substrates for tissue engineering and biomedical devices and products. Cells are known to interact with and respond to nanoparticles differently when presented in the form of a substrate than in the form of a suspension. In tissue engineering, a stable and supportive substrate or scaffold is needed to provide mechanical support, chemical stimuli, and biological signals to cells. This review compiles recent advances of the impact of both graphene and graphene-derived particles to prepare supporting substrates for tissue regeneration and devices as well as the associated cell response to multifunctional graphene substrates. We discuss the interaction of cells with pristine graphene, graphene oxide, functionalized graphene, and hybrid graphene particles in the form of coatings and composites. Such materials show excellent biological outcomes in vitro, in particular, for orthopedic and neural tissue engineering applications. Preliminary evaluation of these graphene-based materials in vivo reinforces their promise for tissue regeneration and implants. Although the reported findings of studies on graphene-based substrates are promising, several questions and concerns associated with their in vivo use persist. Possible strategies to examine these issues are presented. KEYWORDS: graphene, tissue engineering, cytotoxicity, stem cells, medical implants

1. INTRODUCTION

there is little consensus in the reported literature despite years of research.5 Tissue engineering is rapidly emerging as a novel medical strategy for repair and regeneration of damaged tissues and organs of the human body. The science of tissue engineering combines the knowledge of cell biology and biomaterials toward regenerating a functional tissue. Key to the progress in the field of tissue engineering is the development of substrates that mimic the cellular microenvironment and provide physicobiochemical cues to enable cell attachment, proliferation, and differentiation. The ability of stem/progenitor cells to maintain pluripotency and undergo differentiation is shown to be influenced by such cues from the microenvironment.6 As a result, for stem cell-based tissue regeneration, it is important to control the chemical and physical properties of the material to stimulate and guide the fate of the stem cells. For cell therapy, which does not involve an implanted biomaterial, engineered substrates can potentially serve as better alternatives to conventional plastic dishes for expansion and directed differentiation of stem cells ex vivo. Because surface properties of graphene and graphene-derived particles can be easily tuned, they offer exciting opportunities to stimulate cells for

Over the past decade, among various carbonaceous nanoparticles, graphene has emerged as one of the most widely studied nanomaterials becaues of its extraordinary properties. It is believed that graphene and its derivatives will play a critical role in nanotechnology in the future across various technological domains. Graphene is a two-dimensional (2D) nanoparticle containing a single layer of carbon atoms packed in a honeycomb crystal lattice with sp2 hybridization. Since the discovery of graphene, a large number of techniques for the production of graphene and graphene-derived nanoparticles have been reported.1,2 Common methods include mechanical exfoliation, chemical exfoliation, and chemical vapor deposition.3 Some methods, such as chemical exfoliation, yield graphene oxide (GO) rich in hydrophilic oxygenated functional groups on graphene sheets. GO can be reduced thermally or chemically to prepare reduced graphene oxide (RGO), which has fewer oxygen-containing functional groups. Over the years, the fascinating mechanical, electrical, thermal, and chemical properties of graphene-derived nanoparticles have engendered strong interest in developing graphene-based biomaterials including applications such as drug and gene delivery, imaging, and tissue engineering, among others.4 However, the use of graphene and its derivatives in the clinic has been limited due to concerns of potential cytotoxicity, and © 2016 American Chemical Society

Received: August 5, 2016 Accepted: September 23, 2016 Published: September 23, 2016 26431

DOI: 10.1021/acsami.6b09801 ACS Appl. Mater. Interfaces 2016, 8, 26431−26457

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ACS Applied Materials & Interfaces

Figure 1. Representative findings showing the cytocompatibility of different graphene particles in suspension form. (i) (a−d) Graphene oxide causes dose- and size-dependent cytotoxicity. Reproduced with permission from ref 22. Copyright 2010 Elsevier. (ii) (a) Nonfunctionalized graphene agglomerates and accumulates on the cell membrane, leading to toxicity; (b) functionalized hydrophilic graphene disperses well and is internalized by the cells with minimal toxicity. Reproduced with permission from ref 23. Copyright 2011 The Royal Society of Chemistry. (c) GO and RGO with differing oxygen content and surface charge have different impacts on cell viability. (d) RGO particles synthesized by different processing techniques induce different levels of toxicity in cells. Reproduced with permission from ref 24. Copyright 2014 The Royal Society of Chemistry.

interest in the use of graphene for engineering biomaterials for tissue regeneration. A related application wherein graphene-based materials may find use is as engineering biomaterials for implantable medical devices. Millions of devices are implanted globally as substitutes

maximizing the desired biological response. Graphene-based particles with different physicochemical properties are known to influence the interactions of a material surface with biomolecules, cells, and tissue.7,8 Thus, there is rapidly growing 26432


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Table 1. Compilation of Reported Effects of Cell Response to Different Graphene-Based Nanoparticles in Suspended Form parameter studied size

surface chemistry

concentration

yes

no

yes

A549 lung cancer cells

GO and graphene11

yes

yes

yes

human erythrocytes and skin fibroblasts

graphene and COOHfunctionalized graphene23,25 graphene26

no

yes

yes

no

no

yes

monkey kidney cells (Vero) and macrophages (RAW 264.7) macrophages (RAW 264.7 cells)

GO and rGO24

no

yes

yes

mouse spermatozoa

GO and dextran-functionalized GO27

no

yes

no

HeLa cervical cancer cells

hydroxyl-functionalized graphene (G-OH)28,29

no

yes

yes

ARPE-19 cells

CNT, graphene, GO30

yes

yes

yes

mouse embryonic stem cells (mESCs)

material GO

22

cell line

remarks cytotoxicity: small > medium > large GO ROS: small GO ≫ large GO hemolysis: small GO > large GO cytotoxicity: graphene ≫ GO cytotoxicity: graphene≫ COOH-graphene concentration-dependent cell membrane damage and ROS generation cytotoxicity: (N2H4-RGO) > (HT-RGO) > (GTP-RGO) dispersion: dextran-f-GO > GO no cytotoxicity for dextran-f-GO and GO proliferation: dextran-f-GO > GO solubility: G-OH ≫ graphene DNA damage and ROS: graphene ≫ G-OH cell differentiation: GO ≫ graphene ≥ CNT

dependent on surface chemistry, particle size, shape, and concentration.19−21 A number of papers have reviewed the cytotoxicity and biocompatibility of graphene and its derivatives in the form of suspensions and their use in drug delivery and bioimaging applications.19,31 This article focuses on the effect of graphene and graphene-derived particles when used to prepare solid substrates for tissue regeneration and biomedical devices. The associated biological response to these different biomaterials, which is markedly different from that to graphene in suspension, is discussed.

for a wide variety of tissues and organs in the human body. Despite their widespread clinical use, there is considerable scope for improved performance through enhanced mechanical properties, corrosion and wear resistance, biointegration, and resistance to bacterial infections, among others. There is evidence that graphene-based substrates may offer novel approaches to overcome several of these challenges as further described below. With the increasing interest in the use of graphene in biomedical applications, a number of studies have attempted to examine the interaction of graphene particles with different cells and animal models to elucidate the underlying mechanisms of toxicity. Studies have shown that suspended hydrophobic graphene particles, which have poor dispersion in aqueous media, appear to be more toxic than hydrophilic GO or functionalized graphene.9 Graphene particles tend to agglomerate rapidly in cell culture medium with increasing concentration and largely cover the cell surface. This in turn limits nutrient supply and subsequently induces oxidative stress, which triggers apoptotic pathways or programmed cell death. In contrast, GO and chemically functionalized graphene (functionalized with carboxyl, hydroxyl, tween, dextran, chitosan, polyethylene glycol, proteins, etc.) result in stable dispersions. They also tend to adsorb proteins on their surface, limiting direct interaction with cells, thereby minimizing cytotoxicity.10 Also, the cellular uptake of very small sized nano-GO and/or functionalized graphene shows limited cytotoxicity compared to that of nonfunctionalized graphene.11,12 A number of in vivo studies using animal models have revealed that graphene and functionalized graphene nanoparticles show little cytotoxicity.13,14 However, small fractions of graphene particles were found to accumulate in organs such as lungs, liver, spleen, and kidney, which may result in inflammation.15,16 The cytotoxicity of graphene nanoparticles is largely determined by molecular interactions and tend to vary with the cell line.17,18 Figure 1 compiles the collective response of different graphene-based particles in suspension on biocompatibility in terms of particle size, concentration, chemistry, and processing methods. Table 1 summarizes recent studies on the effect of different graphenebased nanoparticles exposed to cells in the form of suspensions. Hence, these reports collectively suggest that the cytotoxic effect of suspended graphene-based materials are highly

2. GRAPHENE OR REDUCED GRAPHENE OXIDE (RGO) FILMS Hydrophobic graphene, when used in the form of a suspension, can induce significant toxicity.11,23,26 On the contrary, graphene or RGO particles, when used to prepare substrates for supporting cell growth, are reported to induce minimal cytotoxicity. Figure 2 shows different parameters affecting cell response to graphene-based particles in suspension and as a substrate. As discussed above, graphene particles in suspension may agglomerate and cover the cell surface, limiting nutrient supply and inducing oxidative stress. Small and well-dispersed

Figure 2. Schematic shows different parameters of graphene in suspension and as a supportive substrate that are believed to influence cellular response. 26433


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ACS Applied Materials & Interfaces Table 2. Multifunctional Nature of Graphene Film Exploited for Tissue Engineering application

material

cytocompatibility cytocompatibility and gene transfer

graphene film41 hybrid RGO and CNT film55

mouse fibroblasts (L-929) NIH3T3 fibroblasts

biocompatibility cell attachment, proliferation, focal adhesion, cell morphology, and gene transfection

neural tissue engineering

graphene film coated with laminins45 graphene film37

human neural stem cells (hNSCs) mouse neural stem cells (mNSCs) mouse hippocampal cells

cell morphology and differentiation

graphene film34,42

bone tissue engineering

antibacterial activity

graphene 3D foam and 2D film40 graphene films coated on PDMS, PET, glass slide, and silicon wafer32 graphene hydrogel52 graphene nanogrid pattern film38 graphene paper57

cell line

cell behavior

cell growth and connections cell viability, growth, and morphology neuronal differentiation

mouse neural stem cells human mesenchymal stem cells (hMSCs)

cell morphology and differentiation

rat bone marrow stromal stem cells hMSCs

cell adhesion, morphology, differentiation proliferation and differentiation

E. coli and adenocarcinomic human alveolar basal epithelial cells (A549)

antibacterial activity and cytocompatibilty of mammalian cells

property influencing biological outcome strong mechanical property nanotopographic rough surface provided by CNTs on RGO hydrophobic hybrid film enhanced gene transfection efficiency electrical conductivity high electrical conductivity, large surface area, and nanotopography surface topography (ripples and wrinkles), crystallinity, and electrical property more charge transport and surface area in 3D than 2D mechanical property, lateral stress transfer from graphene to cytoskeleton corrugated and porous surface of graphene hydrogel exerted tension on cytoskeleton nanotopographic features, adsorption of osteogenic factors, and physical stresses charge transfer and strong and sharp graphene-generated oxidative stress and physical rupture of bacteria

the cells. These observations suggest that the crystallinity of graphene plays an important role in neuronal attachment, outgrowth, and axonal specification.42 In neural tissue regeneration, electrical signals can play an important role in driving cell function and have been shown to facilitate nerve cell regeneration.43 It is reported that electrical stimuli help in calcium-mediated differentiation of neurons.44 Researchers have shown that cells grown on a conductive graphene surface enhance neuronal growth and also facilitate neuronal differentiation.36,45 Several dedicated reviews are available on graphene for neural tissue engineering applications, highlighting the unique properties of graphene to address challenges in the field.46−48 Enhanced neuronal differentiation on a graphene substrate was attributed to the electrical coupling at the cell−material interface, resulting in upregulation of calcium signaling pathways.45 Such conducting substrates can be used to stimulate cells by electrical current, as has been shown for various substrates where electrical stimulation of neurons enhanced cell function.49,50 Currently, in tissue engineering, graphene is considered as an inductive/conductive surface for cellular neurogenesis. The hexagonal arrangement of carbon atoms in a single-layer graphene (SLG) is considered as an important cue for induction of cellular neurogenesis.36,47 Graphene substrates also adsorb biomolecules like growth factors, dexamethasone, ascorbic acid, and β-glycerophosphate through electrostatic, hydrogen, and π−π interactions between the graphene surface and the biomolecules.35 Adsorption of these osteogenic molecules on the graphene surface promotes stem cell differentiation. Furthermore, when human mesenchymal stem cells (hMSCs) were grown on a stiff graphene film, the lateral stress on the cytoskeleton resulted in not only strong anchoring of the cells but also helped in stress-mediated differentiation.32 Recently, Lin et al. showed that surface coating of SLG on different kinds of substrates, such as coverslips, silicone, and polydimethylsiloxane (PDMS), resulted in better cytocompatibility, adhesion, spreading, proliferation, and cytoskeletal remodeling of fibroblasts. Interestingly, the underling substrate stiffness was found to regulate biological outcome. Stiffer substrates had stronger adhesion and resulted

graphene particles can enter cells and may interact with intracellular biomolecules. In contrast, cells on graphene-based substrates are believed to sense the stiffness,32 roughness33 and nanotopographical features34 of graphene. Surface-functionalized graphene film or substrate may provide sites for adsorption of biomolecules 35 and formation of focal adhesions36 for enhanced cell attachment and proliferation. In addition, the conductive nature of a graphene film can also influence cellular behavior.37 Moreover, one can tailor the surface patterning38 and wettability39 and fabricate 3D scaffolds40 of graphene films for controlling the cell response. All of these reports clearly show that graphene in the form of a substrate interacts differently with cells compared to suspended graphene particles. Biocompatibility of graphene-derived materials depends on their physical and chemical properties. However, the choice of the synthesis route results in graphene with different physicochemical properties, which may elicit differences in the cellular response. Chen et al. demonstrated the cytocompatibility of a graphene film using mouse fibroblasts (L-929). Because the graphene film was strong and easy to handle, it provided a stable platform for the adhesion and proliferation of the cells. L-929 cells showed the same doubling time as cells on a commercial polystyrene tissue culture plate, suggesting normal proliferation of the L-929 cells on the graphene film. This study demonstrated the biocompatibility of a graphene film prepared by chemical routes.41 Subsequently, a number of studies have been reported on the multifunctional nature of graphene that may be utilized for tissue engineering (Table 2). Foremost, the electrical conductivity of a graphene substrate was shown to affect proliferation, migration, and differentiation of nerve cells.34 Primary hippocampal neurons grown on a conducting graphene monolayer showed enhanced adhesion and outgrowth of the neurons. In addition, neurons on graphene without poly-L-lysine coating show remarkably well-developed neuritic architecture similar to those cultured on conventional poly-L-lysine-coated glass coverslips.42 Interestingly, neurons cultured on disordered graphene sheets showed limited neuronal attachment and growth; highly ordered and crystalline graphene sheets allowed direct electrical contact with 26434


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(CNT) can offer significant benefits. Thin hybrid films of RGO and CNT supported adhesion and proliferation of NIH-3T3 fibroblasts. The cells showed enhanced focal adhesions on the hybrid film due to the unique nanoscopic roughness presented by the CNTs on the graphene film.55 A significant clinical challenge with biomedical implants is bacterial colonization resulting in infections that can lead to discomfort, rejection, and repeat surgery.56 For tissue engineering applications, graphene sheets offer significant benefits as they show antibacterial activity, preventing bacterial growth with minimal cytotoxicity to mammalian cells.57 Hu et al. demonstrated that GO and RGO papers prevent the formation of E. coli colonies on its surface. E. coli on graphene-based papers showed loss of membrane integrity due to the physical disruption and oxidative stress generated by the graphene films.57 Although a number of studies have shown that graphene and RGO have the ability to support cell attachment and proliferation, a few studies showed that cells behave differently on a graphene surface with a controlled oxidation state. Highly reduced graphene showed significantly decreased cell activity. On the other hand, nonreduced GO or partially oxidized GO surface showed better cellular activity.58 Another limitation with graphene and RGO is hydrophobicity, which affects protein adsorption, thereby limiting cell attachment.

in higher proliferation of fibroblasts.51 Thus, substrate stiffness modulates interactions between live cells and graphene. More recently, three-dimensional (3D) graphene foams have been developed to prepare scaffolds for tissue engineering. It is believed that foams mimic 3D architecture of tissues in vivo. Li et al. reported the use of 3D porous graphene scaffolds for nerve tissue engineering.40 Graphene foam showed enhanced proliferation and upregulation of neuronal markers compared to those of a 2D graphene substrate. This is attributed to the fact that 2D planar graphene offers limited surface area for charge transport in comparison to the larger effective surface area of the 3D graphene foam.40 Interestingly, graphene can form a self-supporting graphene hydrogel (SGH). SGH was shown to be biocompatible, supporting the attachment, spreading, and proliferation of rat bone marrow stromal stem cells in vitro.52 Subcutaneous implantation of SGH in rats elicited minimal fibrous capsule formation with the formation of new blood vessels. In addition, SGH stimulated osteogenic differentiation of stem cells even in the absence of osteogenic factors. It was proposed that SGH provides a corrugated and porous surface for the formation of focal adhesions, which exerts tension on the cytoskeleton, resulting in downstream gene expression. The SGH swelled and cracked in vivo, suggesting its degradation. In another study, Ming et al. demonstrated the use of graphene hydrogel as a substrate for cell growth.53 A porous graphene hydrogel scaffold was found to be compatible with MG63 cells, facilitating cell attachment, spreading, and proliferation on its surface. The cells showed extended filopodia on the graphene hydrogel scaffold. Rough surface features of the graphene hydrogel are believed to have stimulated interactions between the cells and the material. All of these reports demonstrated that the multifunctional properties of graphene can be harnessed to enhance the cellular activity of different cells. Surface features like roughness, curvature, and wrinkled morphology play a critical role in cell adhesion and proliferation. It has been shown that cells grown on graphene with the same chemical properties but with variations in the surface features elicit differences in the biological outcome. Neural cells when grown on the wrinkled surface of a 3D graphene foam led to better interactions with cells, resulting in better cell attachment in comparison to that with the smoother 2D graphene surface.40 In recent times, the use of patterned substrates for guiding stem cell fate is attracting significant interest in tissue engineering. Akhavan et al. showed that a graphene nanogrid pattern enhanced osteogenic differentiation of hMSCs.38 The nanogrid pattern provides nanotopographic network features for enhanced adsorption of biomolecules. The combination of adsorbed biomolecules and physical stress induced from the nanogrid pattern guided the stem cells toward the osteogenic lineage. In a similar study, Balikov et al. showed interplay of electrical stimulation and physical pattern of graphene substrate to regulate hMSC behavior. External electrical stimulation on a patterned graphene surface influences hMSCs to express more neurogenic markers MAP2 and β3-tubulin in comparison to osteogenic transcription factor RUNX2 and osteopontin.54 The combination of external electrical stimuli and physical patterns induces neuron-like morphology. Results of this study thus provided novel understanding of the interplay of electrophysical stimuli on the regulation of stem cell fate on graphene substrates. Leveraging the potential advantages offered by graphene-based materials for biomedical applications. Ryoo et al. reported that a hybrid film of RGO and carbon nanotubes

3. GRAPHENE OXIDE (GO) FILMS Many studies have reported that GO in the form of a suspension is more biocompatible than that of graphene or RGO. As a result, numerous cell studies have been performed on GO or GO-derived substrates or films for tissue engineering. Table 3 lists a few properties of GO films most commonly used for tissue engineering. GO films influence cell spreading and morphology and guide stem cell differentiation due to the presence of hydrophilic oxygenated functional groups on its surface.35,59 The presence of epoxide, hydroxyl, and carboxyl groups on basal planes and edges of GO facilitate greater interaction and adsorption of serum proteins than those of graphene/RGO.35 The interaction of GO with proteins or other biomacromolecules facilitates effective cell attachment, proliferation, and differentiation.35 In another report, Chen et al. demonstrated that GO film supported fast adhesion and proliferation of mouse induced pluripotent stem cells (iPSCs) in comparison to a graphene substrate due to the hydrophilic nature of GO. In a subsequent study, they showed iPSCs spontaneously differentiated on a GO surface, whereas they remained mostly undifferentiated on a graphene surface.39 This study further demonstrated that, although graphene and GO films had similar physical properties like surface thickness, roughness, and surface coverage, the observed difference in iPSC differentiation on GO may be attributed to the polar nature of the GO film.39 Hence, differences in surface functional groups of the graphene substrate can profoundly influence cell behavior. Studies on the use of GO substrates to control stem cell differentiation have led researchers to innovative designs for engineering substrates for stem cell engineering. Kim et al. demonstrated that different surface patterns of GO could be used to control the fate of human adipose-derived mesenchymal stem cells (hADMSCs). The linear line pattern of GO influenced the hADMSCs to differentiate into osteoblasts, whereas a grid pattern of GO induced conversion of hADMSCs to ectodermal neuronal cells.60 Extending biocompatibility tests 26435


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corrugated surface feature proliferation and differentiation in vitro and in vivo

protein/biomolecule adsorb due to π−π stacking, electrostatic, and hydrogen bonds

to different GO substrates, researchers also studied the biocompatibility of 3D GO foam. Serrano et al. showed how a 3D interconnected porous free-standing GO scaffold could be used for neuronal regeneration.61 The GO scaffold provided an artificial 3D substitute for tissue engineering by mimicking the intrinsic 3D architecture in vivo. Because it is now established that GO films can enhance cell proliferation and differentiation, many groups are preparing GO coatings on different biomaterials to improve their bioactivity. Studies have shown that polymeric scaffolds coated with GO exhibit improved cell proliferation and differentiation. Nishida et al. showed in their work that a collagen scaffold coated with 0.01% GO yields corrugated features on the surface. MC3T3E1 preosteoblasts on a GO-coated collagen scaffold showed spread and elongated morphology in comparison to those of a neat collagen scaffold.62 They proposed that the GO coating may provide nanoscale surface features to facilitate cell attachment and proliferation. Surface morphology of an implanted biomaterial greatly influences cell attachment and spreading to facilitate the tissue regeneration process. In particular, biomaterials covered with nanosized particles offer more surface area for adsorption of biomolecules and better cell adhesion.63 Nishida et al. also observed that the GO-coated collagen scaffold in vivo promoted tissue in-growth with blood vessel formation around it.62 Figure 3 compiles the utility of the different properties of graphene-based substrates for various tissue engineering applications. All these studies demonstrate that both graphene and GO substrate as a film, pattern grids, hydrogel, or foam can support cell growth and even induce differentiation. The multifunctional nature of graphene/RGO and GO substrates have been shown to influence the behavior of different cell lines, suggesting that they can be used as an effective substrate for various cell types. Graphene and GO as supportive substrates provide a range of properties like electrical, mechanical, and topographical features, high surface area for biomolecule adsorption, and hydrophilic nature of GO, which are favorable for tissue engineering applications. As suspended particles, graphene may not provide all of the cues mentioned above for cellular functions, thereby underscoring that cellular response to graphene differs between a suspension and a solid substrate. In addition, graphene in suspension has shown toxic effects on cells, whereas no reports were found on the toxic effect of supportive graphene substrates on cells, clearly suggesting that suspended graphene interacts differently than that of a graphene substrate. Thus, the use of graphene-based substrates for tissue regeneration is a promising approach that requires further studies for maximizing clinical outcomes. In tissue engineering, the implanted scaffolds should ideally offer a cell support surface to achieve proper integration of the regenerated tissue with the neighboring tissues at the implant site. Because graphene and GO films/substrates have been shown to be cytocompatible, which improve cell attachment, proliferation, and differentiation, coatings prepared with a thin film of graphene or GO on the implant material provide surfaces suitable for strong cell adhesion and should lead to enhanced integration in vivo. Although some studies in this area have been reported, more in depth studies are required.64,65

GO coating on collagen scaffold


GO and graphene films35

62

human neural stem cells (hNSCs) rat embryonic neural progenitor cells (ENPCs) human bone marrowderived MSCs mouse preosteoblasts (MC3T3-E1) GO film deposited on poly(caprolactone) (PCL) electrospun nanofiber59 porous GO free-standing scaffold61

differentiation into osteogenic lineage

synergetic effect provided by 3D topographic feature of PCL nanofiber and unique chemical surface property of GO for protein adsorption and cell adhesion 3D architecture, roughness, and partial redox state generated by thermal treatment of GO scaffold

hydrophilicity and polar functional groups of GO geometrical patterns

proliferation and differentiation morphology, differentiation, and neuronal network formation cellular morphology, differentiation, and axonal regeneration adhesion, morphology, viability, and neuronal/glial differentiation mouse iPSCs hADMSCs

cell behavior cell line

GO and graphene film GO micropatterned films60

bone and neural tissue engineering neural tissue engineering

39

material application

Table 3. Properties of Graphene Oxide Films Used in Tissue Engineering Applications

property influencing biological outcome


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Figure 3. Utility of multifunctional properties of graphene-based substrates for different biomedical applications. Reproduced in part with permission from ref 34. Copyright 2011 Elsevier. Reproduced in part with permission from ref 38. Copyright 2013 Elsevier. Reproduced in part with permission from ref 39. Copyright 2011 Elsevier. Reproduced in part with permission from ref 40. Copyright 2013 Nature Publishing Group. Reproduced in part with permission from ref 41. Copyright 2008 WILEY-VCH Verlag. Reproduced in part with permission from ref 58. Copyright 2012, WILEYVCH Verlag.

4. FUNCTIONALIZED GRAPHENE-DERIVED PARTICLES The ability to tune the surface chemistry of GO is its significant advantage, and many routes are now available.66,67 Chemical modification routes are rapidly gaining attention for the design of more effective tissue engineering substrates. The potential ability of GO functionalized with chemical moieties to promote cellular attachment, proliferation, and differentiation is highly desired in tissue engineering. A variety of methods to improve biocompatibility and bioactivity of graphene nanoparticles have been reported. These techniques discussed below primarily encompass surface functionalization of small chemical groups, grafting of polymer chains, coating of polymer, and decoration of metallic and ceramic particles on the graphene surface. 4.1. Small Molecule (or Chemical)-Functionalized Graphene-Based Substrates. The chemical composition of a biomaterial surface plays a critical role in material cell

interactions modulating the adsorption of biomolecules, cell adhesion, and ultimately the biological response to the material. Many studies have shown how amine, hydroxyl and carboxyl surface-functionalized substrates show better biological outcomes.68−71 Most approaches for chemical functionalization of graphene make use of GO. Because GO is rich in oxygencontaining functional groups on its surface, it is highly soluble in water and many other polar solvents. The presence of oxygen-containing functional groups can be utilized for interactions with many organic and inorganic chemicals, which help in easy chemical functionalization of GO sheets. Current research is focused more on functionalization of graphene with chemical functional moieties to achieve improved dispersion and enhance its physicochemical properties. Surface charge, energy, and polarity of small chemical functional groups are envisaged to influence biological response at the molecular level. Recent studies show that chemically 26437


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impart functionality for biological applications. Another motivation for functionalization/grafting of GO with biopolymers is to attenuate the toxic effect of graphene when they are used in suspension. Grafting or surface coatings of polymers on graphene-derived particles offer novel routes to address biomedical needs such as specific drug encapsulation and controlled release to influence cell proliferation and differentiation. Surface grafting has emerged as a promising strategy in tissue engineering, which enables cells to contact specific groups on biomaterial surfaces. Surface grafting, functionalization, or coating of polymer on a GO surface usually requires complex chemical processing.76 However, there have been few studies on using polymer-grafted graphene/GO substrate for tissue engineering. A 3D porous thin substrate of GO grafted with poly(styrenesulfonate) (PSS) and poly(acrylamide) (PAM) was tested for cell and blood compatibility. Cells on polymercoated graphene showed firm attachment with well spread morphology. Furthermore, the cells showed spread pseudopodia on the coated graphene surface. Cells at the interface undergo a dynamic change in morphology to attain maximum cell−material interactions. 3D graphene foam coated with laminin was reported to show elongated structure with multinucleated myoblasts (C2C12) on its surface, suggesting their emergent differentiation into myotube cells.77 Myoblasts are known to respond to electrical stimulation. Stimulation with an electrical pulse induces contraction and motion of myoblasts on conductive graphene foams.77 These results may present new routes for developing electrically conductive graphene platforms for regeneration of muscle tissue. Overall, 3Dfunctionalized surfacea were a more effective microenvironment for the cells at the interface with much higher density of surface-exposed functional molecules compared to that of a 2Dfunctionalized surface.78 Also, GO grafted with PSS and PAM showed limited platelet adhesion on the surface and no signs of hemolysis. This study elucidates that surface modification of GO yields a versatile and multifunctional materials surface.79 Biopolymers have attracted great interest in bone tissue engineering due to their excellent biocompatibility and bioactive properties. The presence of charged proteins in the extracellular matrix is believed to facilitate nucleation of hydroxyapatite (HA) on its surface.80 To mimic the in vivo environment and provide a biointerface with a charged proteinaceous bioactive surface for interaction with cells and ions for mineralization, researchers have developed an interesting approach to surface functionalize GO with biopolymers such as gelatin.81 A composite of GO functionalized with gelatin (GO-Gel) showed biomimetic mineralization of HA on its surface. Preosteoblasts (MC3T3-E1) cultured on the GO-Gel showed higher cellular activity and expression of alkaline phosphatase (ALP) activity, indicating osteogenic activity of GO-Gel. GO was shown to provide an effective substrate for cell attachment. In addition, gelatin functionalization of GO enhanced cell attachment, proliferation, and mineralization. The GO-Gel composite having charge and amino acid side chains (aspartates and glutamates) on gelatin helped for better attachment and biomineralization.81 In another study, Liu et al. designed a biomimetic substrate for bone tissue engineering using GO functionalized with the polysaccharide carrageenan (GO-Car). GO-Car substrates showed an effect similar to that of GO-Gel, helping in better cell attachment and HA mineralization.82 The presence of a high amount of sulfate groups on the carrageenan surface

functionalized graphene in the form of suspensions leads to better cytocompatibility in comparison to graphene or even GO.25,28,29 Because this present review is focused on graphenebased substrates, we will discuss the works reporting the use of chemically functionalized graphene substrates for tissue engineering. Only a few studies have been reported on the use of functionalized graphene substrates for tissue engineering, although chemical functional groups on graphene surfaces can improve cell−material interactions, leading to better biological outcomes. Wang et al. reported that fluorinated graphene promotes MSC proliferation and high polarization. Furthermore, it changes the morphology of MSCs toward the neuronal lineage. The presence of fluorine groups on graphene provides electrostatic interactions, increased surface wettability, and nanoscale roughness to the cells.72 Differentiation of MSCs toward the neuronal lineage was attributed to a polarization effect of the carbon−fluorine bond, resulting in a change to the cytoskeletal and nuclear alignment through electrostatic interactions at the cell−material interface.72 Guo et al. have showed that amine group implantation on the graphene surface improved its surface wettability and showed superior cell viability with an excellent anticoagulation property. Furthermore, they showed that an amine-functionalized graphene substrate showed the least platelet adhesion with no demonstrable thrombogenecity. Improved biological response to the amine-functionalized graphene was attributed to nonspecific interactions between amine groups on the graphene and cell surface proteins.73 Our group recently showed that the presence of oxygen-rich functional groups and amine groups on amine-functionalized GO improves surface wettability and helps in binding calcium and phosphate ions, resulting in enhanced mineral deposition by hMSCs.74 In another study by Liu et al., COOH+ ions implanted on a graphene substrate showed a better anticoagulation property; COOH+-functionalized graphene prevented platelet adhesion on its surface, suggesting antiadhesive capacity of COOH+-functionalized graphene. The anticoagulation property of COOH+-functionalized graphene correlated with the surface wettability of the substrate.75 Surface functionalization of graphene substrates provides additional chemical and physical properties for enhanced cell response. The addition of small chemical moieties on graphene or GO altered the cell−material interactions. In contrast, surface-functionalized graphene in suspension remains largely suspended in medium, limiting effective cell−material interactions. The presence of small chemical moieties on the graphene surface imparts bioactivity, resulting in biomineralization on the graphene surface by functionalized chemical moieties. Taken together, small chemical-functionalized graphene shows potential use as a substrate for tissue regeneration. The presence of charged chemical moieties on graphene may improve interactions with proteins and drugs, leading to more attachment of cells in vitro and in vivo. However, more work on the limited studies on in vitro biological response is needed, and detailed in vivo studies must be performed to elucidate the utility of functionalization using small chemical moieties for tissue regeneration. 4.2. Polymeric Group-Grafted Graphene and Graphene Oxide Substrates. Owing to a large surface area and the presence of oxygenated functional groups, 2D GO sheets serve as ideal platforms for grafting or loading different bioactive polymers, drugs, and peptide molecules. The goal of surface grafting of a desired polymer on graphene is typically to 26438


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assembled and reduced to form PVA/RGO composites.89 The PVA/RGO composite film showed a combination of superior mechanical and electrical properties. Furthermore, they studied the biocompatibility of PVA/RGO composite films using human umbilical vein endothelial cells (HUVECs); the PVA/ RGO composite showed more cell adhesion than TCPS plates without any observable cytotoxicity. Interestingly, Guo et al. demonstrated that a hybrid conducting system of poly(3,4ethylenedioxythiophene) (PEDOT)-reduced graphene oxide (RGO) microfiber enhanced cell proliferation and good neural differentiation of MSCs. In addition, the hybrid microfiber when induced with electrical pulse by a triboelectric nanogenerator (TENG) dramatically improved neural differentiation of MSCs. This study underscores the potential of electrical stimulation by a self-powered TENG to a graphene hybrid system for nerve regeneration.90 All these studies demonstrated that functionalization of graphene with a biopolymer or a synthetic polymer can provide surface charge and different chemical moieties such as amine, carboxyl, hydroxyl, and sulfate on the graphene surface. As discussed above, a polymer-functionalized GO/graphene can more effectively induce stem cell differentiation due to the synergetic effect of graphene and the macromolecules. In addition, functionalization of graphene substrate enhances the hemocompatibility, biocompatibility, and antibacterial activity of a material. Such materials hold great promise in tissue engineering applications. Furthermore, functionalized graphene has the ability to interact weakly with protein molecules without affecting the function of proteins. Hence, implantation of functionalized graphene substrates in vivo will likely promote adsorption of proteinaceous growth factors on its surface, resulting in enhanced tissue regeneration and integration. Designing such a biofunctionalized graphene biomedical substrate can overcome critical challenges associated with implants that require the incorporation and release of the administration of various growth factors to improve the integration of the graft. However, the incorporation of labile growth factors limits material processing routes. A functionalized graphene substrate that can interact with and adsorb biomolecules on its surface for cellular bioavailability can be a superior alternative. 4.3. Metallic Nanoparticle-Decorated GrapheneBased Substrate. Studies have demonstrated that one can decorate metallic particles on the GO surface using oxygenated functional groups like epoxy, hydroxyl, and carboxyl on the basal plane of GO.91 Decorating graphene with metallic particles on its surface improves particle stability by preventing restacking or aggregation of graphene sheets and the metallic nanoparticles. Hybrid graphene metallic particles not only showed enhanced dispersion in culture medium but also significantly reduced toxicity.92,93 Surface decoration of metallic nanoparticles on the graphene surface can provide physical cues, such as altered nanotopography, wettability, and stiffness to cells. Graphene hybrids decorated with metallic particles possess excellent physical and chemical properties due to the synergetic effects of graphene and metallic nanoparticles.94 A variety of green and facile synthesis strategies to prepare various graphene and metallic particles have been reported in recent years.95−97 Zhang and co-workers synthesized a strong and stable RGO-decorated silver nanoparticle (RGO/AgNP) hybrid film.98 The hybrid film showed uniformly decorated nanosilver particles on the RGO surface. Decorating both sides of graphene with silver nanoparticles enhances surface

mimicked the natural charged proteins present in the extracellular matrix. Enhanced cell attachment and proliferation was attributed to the hydrophilic nature along with the ability of carrageenan on the GO surface to provide cell−ECM interactions. In another approach, 3D scaffolds of fibrinogen nanofibers (GO-NF) adsorbed on the GO surface were prepared using layer-by-layer (LBL) assembly. GO-NF showed biomimetic mineralization of HA on its surface when immersed in simulated body fluid (SBF) and further showed enhanced fibroblast attachment and proliferation in comparison to control samples of TCPS and GO.83 Self-assembled fibrinogen was reported to help in biomineralization and cell adhesion.84 Hence, the presence of fibrinogen nanofibers on the GO surface provides many amino, hydroxyl, and carboxyl groups, which help in mineralization of HA from SBF. GO as a support helped in the formation of tubular HA particles on the GO-NF surface. Growth of HA on the GO-NF surface improved hydrophilicity of the scaffold, which promoted cell attachment and proliferation.83 The use of GO functionalized with a biopolymer, which provides a biomimetic environment for bone cells, has great potential for preparing osteoinductive scaffolds for orthopedic applications. In a unique study, Mulvaney et al. prepared a GO film by spin coating that was first functionalized with amine moieties and subsequently surface-functionalized with a wide range of biomolecules like peptides, antibodies, globular proteins, and DNA. These GO surface-functionalized biomolecules were found to be bioactive, supporting nucleic acid hybridization and immunoassay on its surface. Furthermore, these surface-functionalized GO films facilitated cell growth and function.85 Literature suggests graphene and graphene-derived particles to have a strong tendency to noncovalently adsorb surfactants on their surface.86,87 Grafting of Tween on GO improves its dispersion in various polar and nonpolar solvents. Good cytocompatibilty of Tween-grafted GO films with HeLa and PC12 cells was also reported.88 Highly stable hybrid Tween/ RGO paper prepared by the Park group showed biocompatibility with three different mammalian cell lines and also inhibited nonspecific binding of bacteria on the hybrid Tween/ RGO substrate.87 The difference in cell attachment between mammalian and bacterial cells on the hybrid Tween/RGO paper was assigned to chemical dissimilarity between the mammalian cell membrane and the bacterial cell wall. Surface coating of Tween on the RGO surface not only helped in biocompatibility but also improved its dispersion in water. In the present scenario, the major scientific challenge is to design multifunctional biomaterials to engineer high performance medical implants. Conventional biomaterials typically suffer from certain disadvantages. They have limited lifetimes, and thus the device may need replacement. Moreover, the biomaterial is typically designed to engender one type of biological response, such as inducing cell differentiation, directing immune cell response, or inducing bacterial cell death, which is a significant limitation for medical devices and tissue scaffolds alike. In contrast, there is now greater emphasis on engineering multifunctional biomaterials to concurrently address multiple clinical challenges. Researchers are increasingly working on novel approaches utilizing surface-decorated and -functionalized graphene particles to make multifunctional composites. Li et al. reported a multifunctional bioinspired nacre-like composite prepared by adsorption of poly(vinyl alcohol) (PVA) on GO sheets that were subsequently self26439


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4.4. Ceramic-Decorated Graphene-Based Substrate. In bone tissue engineering, biomaterials incorporating calcium phosphate including HA and its other forms have long received significant attention due to the unique bioactivity and osteoconductivity imparted by the ceramics.103 Although a few studies have demonstrated osteogenic properties of graphene, it is possible to further enhance the osteogenic activity by decorating or growing HA nanoparticles on the graphene surface. Wang et al. reported biocompatible and bioactive properties of both GO and chitosan-functionalized GO sheets decorated with HA. Both these HA-decorated graphene particles in the form of suspensions showed high cell proliferation. Chitosanfunctionalized GO decorated with HA influenced osteoblasts to express more ALP.104 This study offered new prospects of using GO in combination with ceramic HA particles. Thereafter, researchers have started to work on using GO-decorated ceramic substrates for tissue engineering. Neurons cultured on arrays of silica microbeads with nanotopographical surface features resulted in accelerated bidirectional growth of neurons.105 Similarly, Solanki et al. prepared nanoparticle-based nanotopographical features with GO. Negatively charged GO sheets were coated on to an array of silica nanoparticles to form GO−silica hybrid structures.106 Human neural stem cells (hNSCs) grown on these GO−silica hybrid structures showed enhanced differentiation and aligned growth of axons in comparison to the use of either silica or GO. The presence of GO on the surface of silica nanoparticles with nanotopographical features imparted by GO is believed to have promoted neural stem cell differentiation. Hence, developing such graphene-based hybrid biomaterials could be very effective for the treatment of neural damage. In another study, the use of films of graphene prepared in combination with HA nanoparticles (graphene/HA film) showed good cytocompatibility with osteoblasts. Interestingly, it was reported that the presence of graphene resulted in low expression of ALP; however, it induced an increase in the osteopontin level in the cells. These results suggested cell activation mediated by mechanotransduction.107 Furthermore, a GO−CaCO3 hybrid film was prepared and then transformed to form graphene incorporated CaCO3 (G−CaCO3) by chemical reduction by Kim et al.108 When the films were incubated in SBF, more HA crystals formed on both of the hybrid films compared to the number on bare GO and graphene films. With the help of Raman spectroscopy analysis, they confirmed the coexistence of CaCO3 and the new HA crystals on the hybrid films. Subsequently, the in vitro cell studies revealed that the osteoblasts have different morphologies on GO−CaCO3 and G−CaCO3 compared to those of GO and graphene films. Osteoblasts on GO and graphene films exhibit a large area with circular morphology, whereas hybrid films showed elongated and polynomial cell morphologies. The presence of a CaCO3 microsphere with nanometer-scale thickness on the graphene surface provided additional surface roughness for cell attachment and spreading. Osteoblasts are known to prefer a rough surface for better attachment and elongation.109 Furthermore, with a keen observation, Kim and co-workers showed that osteoblasts on GO and graphene films exhibit an abundant, well-organized, aligned cytoskeleton with stress fibers compared to cells on the hybrid films.108 Stress fibers are highly expressed when cells are grown on a 2D film substrate in contrast to cells on a 3D matrix.110,111 It suggested that the presence of CaCO3 on the graphene substrate not only

wettability of the hydrophobic RGO. Because graphene-based hydrophilic surfaces have significant potential for use in biomedical applications, the cytocompatibility and antibacterial tests were performed on the hybrid film. The RGO/AgNP film showed good cytocompatibilty with mouse osteoblasts, and the cells displayed excellent proliferation over time. Surfacedecorated metallic nanoparticles served as attachment sites for the cells. An improved antibacterial property of the RGO/ AgNP hybrid film was attributed to the synergistic effect of RGO sheets and silver particles. Recently, Tam et al. showed the multifunctional nature of hybrid GO-Ag film in terms of chemo-physical evidence to explain the antibacterial behavior of GO-Ag and for detection of organic dyes in aqueous media. Thus, the GO-Ag hybrid system as a multifunctional material can be used for biomedical as well environmental monitoring applications.99 In another similar study, Hussain et al. demonstrated cytocompatibility and antibacterial property of RGO sheets decorated with nanogold particles. The bactericidal property was attributed to cell membrane damage caused by the hybrid film on contact with the bacterial cells.100 Rizo et al. showed that reduction of hybrid coating of graphene oxide-gold (GO/Au) nanoparticles on a silicon substrate having silver nanowires (Ag NWs) provides a conducting and antibacterial substrate. After chemical reduction, contact between the Ag NW and RGO resulted in enhanced charge transfer between them. As a result, the conductivity of the substrate increased significantly.101 Silicon substrates having graphene and Ag NW on its surface destroy the bacterial cell membrane, thereby inhibiting bacterial cell proliferation on its surface. In another study, surface decoration of GO with silver nanoparticles in the presence of chitosan was proposed for food packaging and biomedical applications.102 A few studies showed that decorating or depositing graphene sheets on a nanometallic surface can also influence cell response. Similar to a graphene hybrid film, graphene deposited on metallic surfaces also enhance the biological performance of a biomaterial. Although limited, reports have highlighted that graphene−metallic hybrid nanoparticles uniquely provide a combination of beneficial properties that are highly desirable for biomedical applications. Hybrid graphene substrates not only provide structural, electronic, and mechanical properties of graphene nanoparticles but also exhibit additional beneficial features and often synergistic properties that significantly augment their utility for tissue engineering. Release of silver ions from graphene−silver hybrid particles have been shown to enhance the antibacterial property of graphene. Slow and steady release of metallic ions from a graphene hybrid substrate/graft are directly available for cellular uptake; as a result, there is no need for highly concentrated drugs with bioactive metallic components that may further cause toxicity at high concentration. Thus, the development of a graphene− metallic hybrid system for local delivery limits the use of high concentrations of metallic ion-based drugs to target tissues. In addition, decorating graphene with some bioactive metallic particles (cobalt, copper, iron, manganese, zinc), which are essential for protein function, can be an interesting strategy for new developments in tissue engineering. Controlled release of such bioactive metallic ions from the graphene surface may enter cells and activate metalloproteins for better cellular response. However, the graphene−metallic hybrid system and its application in tissue engineering is a relatively new area; hence, there is need for more sustained and comprehensive research. 26440


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Figure 4. (i) Small molecule chemical functionalization (amine, fluorine) on graphene sheets influencing (a) surface wettability and nanotopographic roughness. Reproduced with permission from ref 72. Copyright 2012 WILEY-VCH Verlag. (b) Promoted cell proliferation on an aminefunctionalized graphene surface. Reproduced with permission from ref 73. Copyright 2013, Springer. (ii) Polymer-grafted graphene sheets showing an antiplatelet adhesion property and platelets remaining in a quiescent state. Reproduced with permission from ref 79. Copyright 2014 The Royal Society of Chemistry. (iii) Metallic (silver) nanoparticle decoration on a graphene-derived surface demonstrating biocompatibility with enhanced osteoblast proliferation. Reproduced with permission from ref 98. Copyright 2015 The Royal Society of Chemistry. (iv) Surface-decorated ceramic particles on a graphene surface promoting high osteogenic differentiation due to differences in cell morphology and cytoskeleton arrangement of ceramic-coated graphene sheets. Reproduced with permission from ref 108. Copyright 2011 WILEY-VCH Verlag.

graphene/HA hydrogel, suggesting good affinity of the cells for the composite. Figure 4 and Table 4 summarize the beneficial effects of different functionalized graphene films for tissue engineering applications. Overall, the studies showed how chemically modified graphene can act as a perfect substrate for tissue engineering. Chemical modifications add characteristic chemical properties

provided additional roughness but also served like a 3D matrix for the cells. Xie et al. prepared a 3D free-standing graphene/ HA hydrogel.112 The 3D interconnected porous hydrogel showed good mechanical and electrical properties. Bioactivity of the hydrogel was evaluated using mouse multipotent mesenchymal stromal cells (MSCs). MSCs showed elongated morphology with filamentous extensions interacting with the 26441


26442

antibacterial

neural tissue engineering

blood compatible material

musculoskeletal tissue engineering cytocompatibility


application

HeLa, Vero cells, embryonic bovine (EB) cells, Crandell−Rees feline kidney cells, and bacteria mouse fibroblast cells (L929), human umbilical vein endothelial cells (EAHY 926), and platelets human blood platelets blood platelet, human umbilical vein endothelial cells (HUVECs), and human hepatocytes bone marrow-derived mesenchymal stem cells (MSCs)

Tween

PEDOT gold nanoparticle

silver and gold nanoparticle

RGO90 RGO100

GO101

graphene72

COOH+ poly(styrenesulfonate) and poly(acrylamide) fluorine mouse mesenchymal stem cells (MSCs) cervix carcinoma, human (HeLa) and bacteria (Staphylococcus aureus, Bacillus subtili, E. coli, and Pseudomonous aeruginosa) Escherichia coli

mouse myoblasts (C2C12)

laminin

amine

mouse mesenchymal stromal cells (MSCs)

mouse preosteoblasts (MC3T3-E1)

human osteoblasts

HA

CaCO3

graphene75 GO79

GO and RGO87,88 graphene73

GO 3D hydrogel112 graphene foam77

GO/RGO

HA

GO107

108

silver nanoparticle

RGO98 Escherichia coli (E. coli) and mouse preosteoblasts (MC3T3-E1)

fibroblasts (L929)

fibrinogen

mouse preosteoblasts (MC3T3-E1)

GO83

gelatin

cell line


82

chemical functional groups

carrageenan

GO

GO

81

material

antibacterial activity

viability, proliferation, morphology, platelet adhesion, and hemolysis platelet adhesion, hemolysis blood and cell compatibility, proliferation, and differentiation proliferation, differentiation, and cell alignment proliferation and differentiation biocompatibility, antibacterial activity

cytocompatibility

myotube formation, contraction, and motion

cell adhesion, proliferation, ALP activity, and differentiation cell viability, morphology, and cytoskeleton organization cell viability and morphology

adhesion, proliferation, and ALP activity adhesion, morphology, proliferation, and differentiation biocompatibility and biomimetic mineralization antibacterial and cytocompatibility

cell behavior

polarization effect of carbon−fluorine bond, surface wettability, and nanoscale roughness electrical conductivity bacterial membrane damage on contact hybrid film charge transfer between nanoparticles

positive surface charge and nonspecific interactions surface wettability polymer-grafted GO mimicked heparin

amphiphilic nature

electrical conductivity

3D interconnected porosity and cell affinity

surface roughness

charged group from fibrinogen and hydrophilicity of GO-NF-HA synergetic effect of RGO sheets and silver particles mechanotransduction

presence of charge and amino acid side chains on gelatin hydrophilicity and sulfate groups on carrageenan


Table 4. Graphene Surface Modified with Different Chemicals, Polymers, and Metallic and Ceramic Nanoparticles for Biomedical Applications

ACS Applied Materials & Interfaces Review


Review


Similarly, Wang et al. demonstrated composite films of chitosan reinforced with 6 wt % of RGO showed good electrical conductivity of 1.2 S m−1.116 On the other hand, significantly higher content is needed for other fillers, such as ceramic and metallic particles, to enhance the mechanical and electrical properties of these polymers. Furthermore, the use of a low content of graphene in the polymers minimizes potential toxicity as it is slowly released from the degradable polymeric matrix. The addition of GO increases the surface wettability of the composites, resulting in improved cell attachment and proliferation compared to the polymers, many of which tend to be hydrophobic. A hydrophilic surface typically exhibits favorable protein adsorption that can provide potential adhesion sites for cells.31,117 Furthermore, the incorporation of graphene in the polymer provides favorable nanotopographical cues for cell adhesion and has been implicated in topography-mediated cell differentiation.118 Zuo et al. have shown that chitosan polymer films reinforced with GO were cytocompatible with C3H10T1/2 mouse mesenchymal stem cells.113 In another similar study by Depan et al.,119 chitosan chemically linked with the GO composite scaffolds showed significant improvement in the mechanical properties. Chemical interactions between GO and chitosan favored a strong interfacial interaction enabling effective stress transfer and yielding a stronger scaffold. Studying the biological response to the GO−chitosan composite scaffolds, they observed better cell infiltration and the formation of highly dense colonies with strong cell−cell interactions inside the porous scaffold. The use of chitosan with its structural similarity to glycosaminoglycans, a major component of the ECM of bone, in combination with bioactive GO was highly effective in enhancing the activity of bone cells. In another study, the presence of GO in the chitosan matrix enhanced cell proliferation, and importantly, the release of GO during degradation of chitosan did not elicit a toxic effect on the cells.119 For bone tissues, a collagen substrate reinforced with HA is often preferred because they are the major constituents of bone. HA is not very effective for enhancing the modulus and strength of the collagen matrix. The effect of GO reinforced in collagen matrix on the osteogenic differentiation of stem cells was investigated.120 The GO-incorporated collagen matrix supported mesenchymal stem cell attachment and proliferation with no observable toxicity. Cells on the GO−collagen composite surface were well spread. Interestingly, more osteogenic genes were expressed on the GO−collagen surface. It was suggested that the reinforcement of polymeric matrices by GO can impart osteogenic properties to the composite. Mechanically strong GO-based materials are also expected to show better tribological performance for orthopedic applications because graphene materials are derived from selflubricating graphite that exhibits low friction and wear rate. Therefore, GO particles as filler have been used to improve wear resistance of a polymer for load-bearing applications. Reinforcement of RGO or GO in HMWPE has been shown to improve strength, hardness, and tribological performance of UHMWPE.121−123 In a recent study, our group reported the ability of RGO to scavenge free radicals in a gamma irradiated polyethylene matrix, yielding a harder surface through enhanced interfacial interactions for potentially longer lasting liners in the acetubular cups of prosthetic joints.123 However, some studies showed decreased cell viability due to

of functionalized molecules to graphene in addition to intrinsic properties of graphene. Grafting or coating in turn helps graphene by improving dispersion and selectivity. Chemically grafted functional groups help in stimulating cell response, and graphene provides a strong and suitable environment for cell proliferation. Thus, surface functionalization of graphene can translate the chemical, physical, and biological properties of graphene, which can lead to breakthroughs for overcoming challenges in regenerative medicine. Graphene can act as a protective, conductive, and bioactive layer on metallic surfaces, thereby enhancing the performance of biomedical implants. Graphene coated or decorated with bioactive ceramics may be used as a new class of coating materials for biological implants, which promotes osseointegration of the scaffold or implants in vivo.

5. POLYMER GRAPHENE-DERIVED COMPOSITES As discussed above, a number of studies have suggested that graphene or GO films or substrates are cytocompatible when tested using a variety of mammalian cells.39−41 This provides a great opportunity for integrating or reinforcing graphenederived particles into a polymer matrix to augment tissue regeneration. In section 4.2, we discussed how the graphene surface can be grafted or coated with polymers to develop novel routes to address needs such as drug encapsulation and controlled release to influence cell proliferation and differentiation. In this section, we discuss the effect of graphenederived particles added or reinforced in a polymer matrix to improve physical, chemical, and biological properties of the polymer composite. Biodegradable polymers that are typically used in tissue engineering have poor mechanical and electrical properties, limiting their use in a broad range of regeneration applications, in particular, for bone, muscle, nerve, and cardiac tissues. Hence, the use of graphene-derived nanoparticles to reinforce the polymer matrix has immense potential to enhance the mechanical and electrical properties of these polymer composites. Improved mechanical and electrical properties of graphene particles can be utilized to provide mechanical support in load bearing tissues and electrical stimuli to cells, respectively. A significant advantage of graphene is that its addition as a filler at low fraction can markedly enhance mechanical, thermal, and electrical properties. Loading 5 wt % of GO in chitosan resulted in 2.5- and 4.6-fold increases in tensile strength and Young’s modulus, respectively. The GO−chitosan composite film showed a shift in glass transition temperature from118 to 158 °C compared to neat chitosan, suggesting that the addition of GO enhances mechanical and thermal properties of chitosan.113 Also, studies have shown that the addition of very low content (0.1 wt %) of graphene in ultrahigh molecular weight polyethylene (UHMWPE) enhances fracture toughness and the tensile strength of UHMWPE by 54 and 71%, respectively.114 Similarly, Upadhyay et al. demonstrated that the addition of 3 wt % of polyethylene-grafted graphene oxide sheets (PE-g-GO) in high density polyethylene (HDPE) matrix improved the yield strength, modulus, and elongation at failure of HDPE by 30, 40, and 26%, respectively. 115 The strengthening mechanism of the polymer-graphene mainly depends on the concentration and state of dispersion of graphene particles in the polymer matrix. Li et al. have shown that a poly(vinyl alcohol)/graphene oxide (PVA/GO) film prepared by the solution-casting method followed by reduction resulted in increased electrical conductivity of the film.89 26443


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ACS Applied Materials & Interfaces incorporated graphene particles; the decrease in cell viability was attributed to the sharp edges of graphene particles exposed at the surface or leaching of graphene from the polymer matrix.114 However, this observed decrease in cell viability was less than 20% different with respect to the untreated controls on a TCPS plate. With 2D flat GO polymer composite surfaces showing good biocompatibility, researchers have also evaluated the biocompatibility of graphene-incorporated polymer composites in 3D formats. Initial cell−scaffold interactions underlying cell attachment and growth on the scaffold can subsequently drive cell differentiation and eventually tissue formation.124 The properties of the scaffold surface play a critical role in controlling initial events. Incorporation of GO in an electrospun polymer matrix is reported to improve the surface properties of the polymer/GO scaffold.125 A 3D electrospun polymer with GO as filler showed better cell proliferation and more cell spreading and differentiation on composite fibers. In our recent study, the biological response to graphene in polymer composites in the form of flat 2D substrates was investigated, and a comparison with macroporous 3D scaffolds126 was carried out. Osteoblasts showed better initial proliferation on the 2D PCL/GO composite in comparison to that in the 3D porous scaffolds. 2D discs showed more spread and randomly distributed osteoblasts, whereas the 3D scaffold induced organization of the osteoblasts into aggregates as a result of the macroporous architecture of the scaffolds. Wellorganized cells in the 3D scaffold with better cell−cell contacts resulted in increased osteogenesis in the 3D porous scaffolds compared to that in the 2D substrates. In the 3D scaffolds, the sharp protruding edges of the scaffold retarded initial cell proliferation, which was not observed on the smoother 2D surfaces. These results highlight the importance of the biomaterial architecture in directing biological response and its role in profoundly influencing the cellular response to graphene in a polymer matrix. Lu et al. demonstrated that chitosan−PVA polymer blend fibers reinforced with graphene particles showed complete and rapid wound healing in mice and rabbits. Furthermore, they proposed that the presence of free electrons in graphene inhibited the multiplication of prokaryotic cells, whereas it did not affect the proliferation of eukaryotic cells, thus preventing microbial growth on the composite fiber and resulting in fast wound healing.127 Although reports have shown 3D porous graphene foams to be biocompatible and promote neural and osteogenic differentiation,128−130 potential clinical practice of implanting a 3D graphene foam is limited by its extreme brittleness and lack of flexibility, leading to difficulty in handling. As an alternative, researchers are developing polymeric graphene foams that offer improved handling with enhanced elasticity with minimal compromise of electrical properties. The bioactivity of a polymer−graphene foam was evaluated by studying biomineralization of the foam immersed in SBF.131 Addition of a polymer having oxygen groups in the graphene foam induces strong Ca−P deposition due to the favorable electrostatic interactions between the oxygen atom and calcium ion. Formation of the Ca−P-rich apatite layer is dependent on the material chemistry of the surface.132 Thus, it appears that incorporation of polymer into graphene foam can further improve the bioactivity of the graphene foam. In a different study, polymer surfaces coated with GO or RGO have been utilized to improve the surface properties of polymers for better cell attachment and differentiation.133

Surface coating using bioactive GO helps to overcome the bioinertness of the polymer surface. The presence of GO on the surface increases surface wettability and also imparts an antibacterial property. Wang et al. showed that a polyethylene terephthalate (PET) surface coated with large surface area graphene enhances bioactivity of PET-based artificial ligaments.133 Furthermore, they observed better bone healing with newly formed bone on the graphene-coated PET artificial ligaments in vivo in a rabbit model. Graphene with excellent electrical and biological surface activity improves adsorption of serum proteins, growth factors, and biomolecules, which regulate cell attachment, ECM synthesis, growth, and differentiation, thereby accelerating bone healing. In another approach, flexible and free-standing graphene and GO hybrid polyaniline papers were synthesized by polymerization of aniline using GO and graphene papers as the substrates. The hybrid paper showed an excellent combination of electrochemical properties and cytocompatibility and thus is a promising candidate material for use in biosensing applications.134 Stem cells on a graphene surface are often observed to exhibit elongated morphology with multiple branching or stretching of its pseudopodia. It is well established that cell morphology regulates cellular function.135 The surface chemical composition of graphene facilitates adsorption of protein and growth factors from cell culture medium and bodily fluids, which subsequently controls cell morphology and adhesion.69 There have been several reports correlating cell morphology as an early marker of stem cell fate.32,136,137 Elongated and highly branched morphology of stem cells has been associated with osteogenic differentiation.137 Thus, elongated morphology with filopodial extensions suggests not only strong adhesion of cells but also indicates differentiation on the graphene surface. We have demonstrated the role of functionalization of graphene in polymer composites on stem cell response.74 Amine-functionalized graphene oxide (AGO) was prepared by modification with methylene dianiline (MDA). In vitro results showed that the PCL/AGO composite significantly enhanced stem cell proliferation with elongated branched morphology leading to osteogenic differentiation. AGO reinforced in PCL having oxygen and amine groups imparted hydrophilicity to PCL for better cell attachment and proliferation. Also, the presence of amine groups that facilitate the nucleation of hydroxyapatite resulted in more cell-mediated mineral deposition on the PCL/AGO composites. Interestingly, the functional groups on AGO, in combination with the mechanical rupture induced by graphene, imparted bactericidal activity to the composites. To further maximize the advantages offered by the amine groups on graphene, we recently prepared polyethylenimine (PEI)-functionalized GO (GO_PEI). Most recently, we showed that PEI-functionalized GO (GO_PEI) having more amine groups than AGO when incorporated in a PCL matrix significantly improved stem cell proliferation and mineralization.138 PCL/GO_PEI showed high adsorption of osteogenic factors due to weak interactions between the amine and oxygen groups on GO_PEI and the biomolecules, resulting in enhanced osteogenic differentiation. In addition, the GO_PEI exposed at the surface of the composite provided bactericidal activity with significant reduction in counts of E. coli colonies due to the synergistic effect of the sharp edges of GO_PEI along with the presence of cationic PEI molecules. Thus, these studies underscore the importance of chemical functionalization of graphene in polymer composites for tissue regeneration. 26444


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Figure 5. (i) Reinforced GO in electrospun PLGA matrix showing enhanced biomolecules (protein and dexamethasone (DEXA)) adsorption on its surface, which in turn influences stem cell proliferation and osteogenic differentiation; (ii) schematic showing amine-functionalized GO in a PCL matrix imparting bioactivity by promoting hMSC differentiation and mineral deposition while also providing antibacterial property to the composites by killing the adhered bacteria; and (iii) porous polymer scaffold reinforced with metallic (strontium) decorated graphene resulting in high proliferation of osteoblasts and mineral deposition on the scaffold. Reproduced with permission from ref 144. Copyright 2015 The Royal Society of Chemistry.

Ma et al. demonstrated that poly(lactic acid) (PLA) nanofibers reinforced with bioactive HA and GO nanoparticles showed better osteoblast attachment and growth. Cells on the PLA/HA/GO composites were reported to exhibit mature and plumper morphology. The enhanced biological activity of PLA/ HA/GO was attributed to the synergetic effect of HA and GO. HA helped to attract the osteoblasts, whereas GO supported cell growth by providing a hydrophilic and rough surface on the scaffold.118 Similarly, Nair et al. showed that addition of GO to a porous gelatin−HA scaffold helped in strengthening the scaffold and also enhanced osteogenesis.140 Also, they have

In an alternative approach, instead of functionalizing GO with polypeptide, the Jeong group reported the use of injectable GO/polypeptide thermogel as a 3D scaffold.139 Thermogel of GO/polypeptide influenced adipogenic differentiation of mesenchymal stem cells by enhancing expression of adipogenic biomarkers (PPAR-γ, CEBP-α, LPL, AP2, ELOVL3, and HSL). They also demonstrated that incorporation of GO in a polypeptide helps in adsorption of insulin and adipogenic differentiation factors.139 Thus, designing such an injectable graphene-based thermogel composite scaffold can be a promising material for 3D tissue engineering matrix. 26445


26446

PLGA125

musculoskeletal tissue engineering biosensor wound healing

polyaniline134 chitosan−PVA127

PCL126,147

PVA145

hMSCs and E. coli


GO and graphene graphene

mouse fibroblast cell line L929 animal model (mouse and rabbit) E. coli, agro-bacterium and yeast

human tonsil-derived mesenchymal stem cells GO and GO decorated with gold mouse fibroblasts L929 nanoparticles (GO_Au) GO and RGO human cord blood-derived mesenchymal stem cells and osteoblasts GO human mesenchymal stem cells

GO

polypeptide139

PCL138

RGO and RGO−strontium hybrid (RGO _Sr) PEI-functionalized GO (GO_PEI) and GO

PCL144

PLA118 gelatin−HA140

cytocompatibility In vivo wound healing and antibacterial activity

cell attachment, proliferation, and differentiaion

morphology and differentiation

cellular toxicity

adipogenic differentiation

morphology, focal adhesion, proliferation, ALP activity, mineralization, and antibacterial

proliferation and mineralization

mouse preosteoblast (MC3T3-E1) and focal adhesion, cell morphology, and differentiation animal study on rabbits morphology, proliferation, differentiation, and hMSCs and E. coli antibacterial activity mouse preosteoblasts (MC3T3-E1) attachment, proliferation, and differentiation human adipose-derived MSCs osteogenic differentiation

graphene MDA-functionalized GO (AGO), GO, and RGO GO, HA GO

no cell studies reported

graphene

cytocompatibilty, cells infiltrated, cell−cell interactions, cell morphology, and mineralization ALP activity and osteogenic differentiation

cell behavior

poly(vinylidene fluoride) (PVDF) and PCL131 polyethylene terephthalate (PET)133 PCL74

mouse mesenchymal stem cells C3H10T1/2 rat bone marrow-derived MSCs no cell studies reported

cell line

GO GO

GO

filler

collagen UHMWPE121

120

chitosan

material

113,119

cardiac and bone tissue engineering

cytocompatibility


application

electrochemical property electronic property

hydrophilicity, biomolecule adsorption

conductivity, dielectric permittivity, surface wettability, and roughness

hydrophilicity, adsorption of osteogenic factors antibacterial activity by mechanical rupture and chemical groups adsorption of insulin and differentiation factors nanofibrous morphology

surface roughness, hydrophilicity adsorption of biomolecules and bone− mimetic matrix surface wettability and ion release

surface wettability, electrical and surface activity hydrophilicity and HA nucleation

structural similarity to glycosaminoglycans bone mimetic collagen and strong GO strong mechanical and lubricating properties conductivity and chemical groups


Table 5. List of Polymer Composites Reinforced with Graphene and Its Derivatives to Enhance Physical, Chemical, and Biological Performances of the Composites

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On the basis of the above reports, it has been observed that graphene in many forms, ranging from simple GO/RGO to chemical/polymer-functionalized graphene and hybrid metallic/ceramic-decorated graphene, has been used as reinforcing filler in polymer matrices to impart multifunctionality to polymeric biomaterials. Studies showing functionalized graphene and hybrid metallic/ceramic graphene-decorated particles in polymer matrix influence cell response due to biomolecule adsorption and cell interaction with functional groups or ceramic particles on the graphene surface. In addition, the release of metallic ions from the hybrid polymer composites showed antibacterial activity and enhanced cell response. The advancement in nanotechnology has opened new avenues to tune the properties of graphene to efficiently utilize its properties for biological applications. Thus, the use of functionalized graphene in polymeric composites not only provides mechanically strong substrates but also imparts additional properties such as enhancement in cell proliferation and differentiation along with antibacterial activity. Whereas functionalized and hybrid graphene-based composites have shown promising responses in vitro, more work is needed to understand the biological responses in vivo toward clinical use.

shown that the release of GO in PBS from the scaffold was below the toxic limit, and that it thus did not compromise cell viability. Intrinsic properties of GO along with the bonemimicking matrix comprising HA and gelatin influences osteogenic differentiation of hADMSCs, even in the absence of osteogenic growth factors. GO helps in the adsorption of ascorbic acid from culture medium, which helps in capturing of calcium ions to promote osteogenic differentiation. 141 Mohandes et al. demonstrated that a polyethylene glycol (PEG) matrix incorporated with GO and HA nanorods showed improved bioactivity by forming a new apatite layer when soaked in SBF.142 Marques et al. observed that the presence of GO led to the uniform distribution of HA particles in the poly(L-lactic acid) (PLLA) matrix in comparison to reinforcement with HA alone, which resulted in substantial improvement in the mechanical properties of the hybrid composite.143 They proposed the potential application of PLLA/HA/GO hybrid nanocomposites for fabricating resorbable bone screws. Hence, these studies suggest that the reinforcement of GO in a polymer matrix containing HA can synergistically enhance the bone-mimetic architecture and properties, thereby inducing osteogenic differentiation of stem cells. A more recent effort in the use of graphene-based biomaterials is the use of a graphene particle surface decorated with metallic nanoparticles for preparing biomedical polymer composites. Such polymer nanocomposites are envisaged to show unique properties as a result of synergism between the polymer, graphene, and metallic particles. In a recent study, we prepared a multifunctional 3D macroporous PCL scaffold reinforced with strontium-decorated graphene hybrid nanoparticles. Bioactivity of the scaffold was evaluated using mouse osteoblasts. A PCL scaffold containing the hybrid particles showed enhanced cell proliferation and mineralization due to improved wettability and release of strontium ions from the hybrid nanoparticles.144 Strontium ions are known to stimulate bone formation and minimize its resorption. In another similar study by Yu et al., electrospun PVA nanofibers incorporating glycine-modified GO decorated with nanogold were prepared. Glycine modification of GO was shown to enhance the biochemical properties of GO. Furthermore, the surface decoration with gold and silver nanoparticles improved the stability of graphene sheets, preventing restacking and agglomeration. Gold-decorated glycine-modified graphene in the PVA fibers resulted in improved electrical, mechanical, and thermal properties of the composite. Biocompatibility of the nanofiber composite mat showed good cytocompatibility with L929 cells.145 On the other hand, reinforcement of hybrid graphene sheets decorated with silver nanoparticles (RGO_Ag) in the PCL matrix improved surface wettability, increased surface roughness and conductivity, and showed low steady release of silver ions and enhanced biocompatibility and antibacterial property of PCL. Overall, the addition of hybrid RGO_Ag particles in PCL was shown to impart multifunctional properties for biomedical applications.146 Figure 5 presents different types of graphene-derived polymer composites used in tissue engineering applications. Table 5 compiles the list of different types of polymer composites reinforced with graphene or its various derivatives to improve the physical, chemical, and biological performances of composites. All of these studies collectively suggest that the use of graphene particles decorated with bioactive metallic particles in polymer composites hold tremendous promise in the biomedical field.

6. CERAMIC GRAPHENE-DERIVED COMPOSITES Bioceramics such as HA and calcium silicates exhibit good osteoconductive and osteoinductive properties, which offer a clinical advantage over polymeric materials. In section 4.4 above, we discussed how decorating or growing ceramic nanoparticles on the graphene surface can improve bioactivity of hybrid graphene−ceramic particles resulting from synergetic effects. In this section, we discuss the effect of graphene-derived particles reinforced in bulk ceramic matrix and the resulting biological outcome. Compared to polymeric materials, the low fracture toughness and intrinsic brittleness of bioceramics limit their use in loadbearing applications.148 There have been many reports on polymer−graphene composites for bone tissue engineering. The addition of GO or RGO not only improved physical, chemical, and mechanical properties but also significantly enhanced the biocompatibility of the polymers. All of these results greatly encouraged researchers to use graphene or graphene-derived particles to improve mechanical and biological properties of ceramic materials. Zhang et al. showed that graphene nanosheets (GNS) incorporated in HA resulted in significantly enhanced osteoblast attachment.149 Interestingly, the osteoblasts showed elongated morphology with uniform coverage of the HA/GNS composite in comparison to that of neat HA. The presence of GNS in HA provided additional nanotopographic surface features for better cell attachment and spreading. It has been demonstrated that the presence of graphene particles on a biomaterial substrate can be detected by cells as additional focal points to adhere and subsequently lead to better cell attachment and spreading. In another study by Meharli et al., reinforcement of RGO in calcium silicate (CS) showed significant improvement in the mechanical properties, osteoblast proliferation rate, and alkaline phosphate activity on the CS/RGO composite surface.150 Furthermore, the composite showed more bone-like apatite layer formation on its surface after incubation in SBF. It has been demonstrated that the exposed graphene particles at the surface in contact with SBF serve as a nucleation site for apatite formation.149 In orthopedics, the formation of bone-like apatite on the biomaterial surface is believed to be critical for forming strong 26447


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ACS Applied Materials & Interfaces Table 6. List of Ceramic−Graphene Composites Used for Biomedical Applications application bone tissue engineering

cytocompatibility

material HA149

filler

calcium silicate150

graphene nanosheets RGO

58S bioactive glass159

graphene

β-tricalcium phosphate bioceramic162

Fe3O4/GO

45S5 Bioglass160 alumina154

graphene graphene

cell line

cell behavior



adhesion and morphology

nanotopography

human osteoblasts (hFOB)

adhesion, proliferation, and ALP activity biocompatibility and bioactivity cytocompatibilty, proliferation, and ALP activity biomineralization in SBF viability test

nucleation of minerals

human bone osteosarcoma (MG-63 cells) human osteosarcoma MG-63 and rat bone marrow stromal cells

mouse preosteoblast (MC3T3-E1)

synergetic effect paramagnetic, synergetic effect, and ion release conductivity surface morphology, microstructure, and grain size

toughness, intrinsic brittleness, and flexural strength) and biological outcomes for ceramic materials. HA is often used for bone tissue engineering due to its similarity to the chemical composition of natural apatite in bone. The reinforced graphene on the surface of bioactive ceramics provides focal adhesion sites for cell attachment. The addition of graphene imparted electrical conductivity to Bioglass, which facilitates bone tissue regeneration due to physioelectrical signal transfer. Interestingly, not much work is done on the use of functionalized graphene in the ceramic matrix to further improve biomineralization, adsorption of protein, and other biomolecules/drugs and cell−material interactions for a significantly improved biological outcome. However, few attempts have been reported on graphene/ceramic composites, possibly due to the difficulties of processing them under high temperature and pressure, as well as the challenge of dispersing graphene in the ceramic/Bioglass matrix. Hence, there is a strong interest in developing innovative approaches for processing to reinforce graphene particles in ceramic/Bioglass matrices without compromising the structure and properties of graphene.157,161

interactions between the material surface and the living tissue.151 Similarly, there have been many reports on using graphene to reinforce a ceramic matrix to overcome low fracture toughness with enhanced bioactivity.152,153 A recent report showed that alumina (Al2O3) ceramic reinforced with graphene platelets resulted in significantly better flexural strength and fracture toughness. These alumina composites also showed better biocompatibility, allowing cell proliferation on the composite surface. However, at a few places on the surface, the sharp edges of the exposed graphene platelets were implicated in preventing cell proliferation. Thus, the cells on the composites attached, proliferated, and covered the sample except where the sharp edges of graphene were exposed on the alumina surface.154 Among ceramic biomaterials, Bioglasss has received special attention for bone tissue regeneration and has shown excellent bioactivity and bone-binding ability.155,156 As an orthopedic implant, Bioglass helps in binding to the bone tissue facilitated by the dissolution of Si, Ca, and P ions from the ceramic to stimulate bone cell proliferation and activated osteogenic gene expression.157 On the other hand, similar to other ceramics, the major limitations of Bioglass include low fracture toughness and intrinsic brittleness. 158 To overcome these limitations, researchers are increasingly using strong and biocompatible graphene-derived particles as a superior reinforced filler to improve the mechanical properties of Bioglass. Gao et al. showed that reinforced graphene in 58S bioactive glass significantly improves compressive strength and fracture toughness by 105 and 38%, respectively; in addition, the 58S bioactive glass composite retains its bioactivity and biocompatibility.159 In another similar study, the addition of graphene nanoparticles in 45S5 Bioglass showed 9 orders of increase in the electrical conductivity that may be attributed to the formation of a percolating network of graphene particles in the bioglass matrix. Conducting 45S5 Bioglass graphene composites showed excellent in vitro biomineralization of HA on its surface when immersed in SBF.160 All of these studies suggest the promising potential of developing a strong and electrically conductive Bioglass graphene composite scaffold for orthopedic applications. Table 6 lists a few ceramic graphene composites used for biomedical applications. Figure 6 demonstrates that the reinforcement of graphene in the ceramic matrix results in improved cytocompatibility and mechanical strength along with bioactivity showing enhanced ALP activity by cells on graphene/ceramic composites. All of these reports showed that ceramic graphene composites can improve mechanical properties (like fracture

7. GRAPHENE-BASED COATINGS ON METALLIC BIOMATERIALS FOR IMPLANTS Related to tissue regeneration, another growing interest in biomaterials research is the development of bioactive coatings for metallic biomedical devices for use in the human body.163,164 Surface coating of advanced graphene-derived particles on metallic implants holds great promise for revolutionizing the performance of metallic bioimplants.165 Metallic implants coated with graphene particles with a large surface area and specific surface activity for adsorbing proteins and other biomolecules can help cell attachment and integration. Furthermore, surfaces of metallic bioimplants are often modified to improve corrosion resistance, surface texture, and bioactivity.166,167 Jia et al. self-assembled different types of graphene-derived particles with different lateral size and surface properties on an orthopedic titanium (Ti) surface. Large (micrometer-sized) GO sheets and small (nano-sized) poly(dopamine)-functionalized RGO (RGO-PDA) were selfassembled on the Ti surface through an evaporation-assisted electrostatic assembly. They showed that self-assembly of GO and RGO-PDA improves wettability and protein adsorption ability of Ti due to the presence of hydrophilic GO and chemical moieties of PDA on the Ti surface. Antibacterial test of GO and RGO-PDA-coated Ti surfaces showed significant reduction of live bacterial cells.168 The antibacterial property of 26448


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Figure 6. (i) Graphene/ceramic (GLP/Al2O3) composites demonstrating cytocompatibility in vitro and (ii) reinforced RGO in calcium silicate improving mechanical properties (fracture toughness) and biological responses in terms of enhanced ALP activity by osteoblasts on the composite surface.

graphene flakes and charge transfer, leading to ROS production. MC3T3-E1 cells cultured on self-assembled GO and RGO-

graphene (GO and RGO-PDA)-coated surfaces was attributed to the presence of nanoknives/nanoneedle-like sharp edges of 26449


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Figure 7. (i) Coating of graphene on germanium imparts (a) cytocompatibility allowing spreading and growth of rBM-MSCs and (b) antibacterial property by killing E. coli resulting in a lower colony count on agar plates. Reproduced with permission from ref 182. Copyright 2015 The Royal Society of Chemistry. (ii) Osteoblast morphology at day 1 on (a) Ti, (b) Ti coated with HA, and (c) Ti coated with gelatin, GO, and HA. Osteoblasts on Ti coated with gelatin, GO, and HA exhibit fusiform structure with extended filopodia, leading to strong binding of osteoblasts on the Ti surface. Reproduced with permission from ref 175. Copyright 2014 Elsevier. (iii) Enhanced bone formation on implanted Ti coated with GO with BMP-2 in vivo. (a) Protocol for implantation of Ti or Ti/GO with and without BMP-2 implant rings in mouse calvarial defects; (b,c) Microcomputed tomography (micro-CT) image and histological analysis showing (arrow) new bone formation at the calvarial defect site after 8 weeks of implantation. Reproduced with permission from ref 173. Copyright 2013 WILEY-VCH Verlag.

PDA Ti surface evoked better focal adhesions, leading to strong cell attachment and proliferation. Among coated surfaces, the RGO-PDA surface mediated strong anchorage and excellent stretching during cell adhesion. The study showed that the presence of PDA helps in adsorption of more serum proteins

and that their favorable confirmation on the RGO-PDA-coated Ti surface assisted in better attachment and spreading.168 Furthermore, self-assembly of GO and RGO-PDA on the Ti surface yielded a conducive microenvironment for osteogenic differentiation to deposit mineral on the surface.168 26450


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Table 7. List of Different Metallic Surfaces Coated with Different Graphene-Based Nanoparticles for Improved Corrosion Resistance and Better Bioactivity application orthopedics

material

cell line

cell behavior

GO and RGOPDA


titanium170,171

graphene-HA

Ti alloys173,174

GO loaded with BMP-2 GO-gelatin-HA

L929 mouse fibroblasts and MG63 human osteosarcoma cells hMSCs and animal study (mouse) mouse preosteoblasts (MC3T3-E1) MG-63 human osteosarcoma cells MSCs and Escherichia coli

Ti175

cytocompatibility

coating material

titanium168

316L stainless steel180 germanium (Ge)182 Ti178

polypyrrole (PPy)/ GO graphene sodiumdecorated GO (NaGO)

human dermal fibroblasts

Coating a thin graphene film on the metal surface using different techniques can enhance corrosion resistance by forming a protective layer on the surface when used in vivo. Reports show that coating the Ti substrate with a ternary coating of GO-hyaluronic acid-HA (GO-HY-HA) improved the corrosion resistance of the Ti substrate.169 Furthermore, electrodeposition of a bioactive HA/graphene composite on the Ti surface resulted in enhanced modulus and surface hardness. Coating the Ti surface with an HA/graphene composite induces the formation of an apatite layer on the surface with enhanced corrosion stability.170,171 Ti-based biomaterials, although cytocompatible, suffer from a lack of bioactivity and have poor tribological properties.172 The Ti substrate coated with GO loaded with bone morphogenic protein-2 (BMP-2) showed enhanced osteogenic differentiation of hMSCs compared to that of the unmodified Ti substrate. Furthermore, in vivo studies revealed that implantation of the Ti surface with BMP resulted in new bone formation in a mouse model.173 Studies also showed that surface-coated GO on a Ti alloy substrate reduced friction and imparted better wear resistance.174 Yan et al. showed that nanotubes of TiO2 on the titanium surface coated with a hybrid composite of GO cross-linked gelatin and HA provided a porous surface morphology. The hybrid coating imparted better corrosion resistance than a coating of HA alone due to the reinforcement effect of GO cross-linked gelatin, making the coating more compact.175 Cytocompatibility tests showed that more osteoblasts proliferated on the Ti surface with nanotubes of TiO2. The nanotube surface of TiO2 promoted cell adhesion, biomineralization, and cell differentiation.176,177 After coating with GO cross-linked gelatin HA, osteoblast proliferation was significantly enhanced. Also, cells showed tight adhesion to the surface with the hybrid film. This enhanced bioactivity was assigned to the combined effect of GO and gelatin in supporting cell attachment and proliferation.175 In another interesting study, sodium decorated on a GO (NaGO) surface was used to cover a Ti implant to enhance its life. NaGO on the Ti surface minimized corrosion by acting as a passive layer, thereby extending its lifetime. Surface-coated NaGO on the Ti surface increased surface roughness and provided wrinkled morphology for better cell response.178 The presence of strong and stiffer GO with a nanoscale wrinkled surface can provide mechanical stimulus leading to a change in intracellular structure and phenotype.179


adhesion, differentiation, mineralization, and antibacterial viability and proliferation

hydrophilicity, surface roughness, and protein adsorption

osteogenic differentiation

bioactivity and osteogenic activity

synergetic effect

adhesion, differentiation, and nanotopography and porous mineralization morphology attachment and spreading hydrophilicity osteogenic differentiation and antibacterial activity viability and morphology

electrical conductivity, biomolecule adsorption, and charge transfer passive layer, surface roughness, wrinkled morphology, and mechanical stimuli

Thus, graphene-based surface coatings on Ti-based alloys offer exciting opportunities to enhance the performance of the next generation of Ti-based biomedical devices. Kumar et al. showed that a hybrid coating of polypyrrole (PPy)/graphene oxide (GO) nanocomposite on 316L stainless steel (SS) surfaces significantly improves surface wettability and corrosion resistance. PPy exhibits corrosion resistance due to its excellent stability in water, which hinders electron transfer. The PPy coating acts as a barrier for the electrolyte to reach the metal surface, and the presence of GO strengthened the polymer network, creating a more compact film over the steel surface for enhanced corrosion resistance. Furthermore, in their study, 316L SS coated with PPy/GO showed better cell adhesion with healthy morphology, exhibiting polygonal shape and filopodial extensions. The better cell attachment and proliferation was attributed to the presence of the hydrophilic GO particles in the PPy coating.180 Electronic biosensors are being extensively used to meet various clinical needs.181 Surface properties of the biosensors are critical for determining their functioning. Recent advancements in graphene research have encouraged scientists to investigate the use of graphene to modify the surface of biosensors to maximize their function. In a work by Li et al., a monolayer graphene film was grown on the surface of electronic biosensor germanium (Ge) by chemical vapor deposition (CVD).182 The graphene overlayer protects Ge from corrosion and improves biocompatibility. MSCs grown on the graphene-coated Ge surface showed upregulation of osteogenic genes. Taking their study further, Li et al. demonstrated that the graphene coating can provide antibacterial property to implantable Ge-based biosensors.182 Figure 7 shows cytocompatibilty, antibacterial, and in vivo bone formation on a metallic surface coated with graphenederived particles. Table 7 lists different metallic surfaces coated with different graphene-based nanoparticles for improved corrosion resistance and better bioactivity. All of these studies have demonstrated that a protective graphene layer on the metallic surface ensures improved functionality as a biomedical implant. The use of a bioactive, metallic-decorated, graphene-coated metal surface for in vivo studies could be an interesting approach to harness the synergetic effect of graphene and bioactive metallic-decorated nanoparticles. Little work has been reported for the in vivo evaluation of graphene-coated implants, underscoring the need 26451


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these materials will have to be subjected to more comprehensive studies. As the substrate degrades, graphene may elute out over time, and the risk, if any, that it may pose needs to be thoroughly characterized in long-term animal experiments. Graphene particles released from the scaffolds as the polymer degrades may be taken up in cells via various endocytosis pathways. Thus, there is a need for better understanding of the uptake of the eluted graphene from the scaffold. Recent reports have indicated that cellular uptake of protein-coated graphene depends on the size of the graphene sheets. Small nanosheets of graphene enter cells mainly through clathrin-mediated endocytosis, whereas large graphene sheets enter by phagocytotic uptake.183 Similar studies need to be extended for various types of graphene-derived particles eluting from scaffolds during degradation to provide fundamental understanding of interactions at the interface of 2D nanostructures and biological systems. In addition to a tissue of interest, the in vivo biodistribution of the released functionalized graphene from the scaffold or substrate must also be assessed with special attention to their accumulation and excretion from the body. Graphene can be tracked by various imaging techniques such as IR, Raman, and so forth, and also by tagging with fluorescent dyes and MRI contrast agents, among others. For the intracellular fate of the particles to be monitored, recent advances in single molecule microscopy can be utilized, where there is considerable scope of tracking their fate as they enter the cell and also eventually excreted, if at all. Zwaag et al. reported an interesting study utilizing super-resolution microscopy to study the uptake of carboxyl- and amine-functionalized polystyrene nanobeads in mammalian cells.184 Furthermore, in their study, they elucidated the location of nanoparticles within the cell and interaction of the nanoparticles with cellular components at nanoscale resolution. Similar strategies could be utilized for functionalized graphene. The emerging high-throughput omicsbased techniques such genomics, proteomics, and metabolomics can also be utilized to reveal the molecular pathways. Through a genomic study on 84 genes of human peripheral immune cells upon incubation of GO having different lateral dimensions, small GO sheets showed significant upregulation of critical genes of immune cells, implicating immune responses compared to that of large GO sheets.185 Furthermore, wholegenome microarray analysis was conducted on the impact of small GO sheets on T-cells and monocytes and showed energydependent pathway modulation. Such findings can pave the foundation for further studies on the biological response of released graphene from composites over time. Nevertheless, the findings by different groups suggest that graphene-based biomaterials hold exciting promise in tissue regeneration, underscoring the need for continued investigations.

for such studies to establish their efficacy for clinical use. Thus, coating of GO and polymeric films on metallic implants may provide synergistic performance from both GO and polymer together to enhance biocompatibility and corrosion resistance.

8. CONCLUDING REMARKS AND FUTURE PERSPECTIVES The rapid increase in reports on graphene-based biomedical products suggests that graphene holds great potential in the biomedical field. However, the use of graphene and its derivatives in the clinic has been limited due to concerns of potential cytotoxicity, and there is little consensus in the reported literature. Many reports have reviewed cytotoxicity and biocompatibility of graphene and its derivatives in the form of suspensions and their use in drug delivery and bioimaging applications. However, there are no reviews detailing the cytotoxicity and biocompatibility of graphene and its derivatives as supporting substrates for tissue regeneration and the associated cell response. This report presents a rigorous survey on the use of different graphene-based substrates for tissue engineering applications. The studies described here demonstrate that there is strong evidence that biological response to graphene-based substrates is markedly different than that to suspended graphene-based particles. Furthermore, multifunctional properties of graphene-based substrates are being widely used to influence cell response. Directing cell response on graphene substrates requires precise control over physical and chemical properties of the substrate, and the unique properties of graphene have been leveraged to explore different routes. These include the use of small molecules and polymers to functionalize graphene and the use of metallic- and ceramicdecorated hybrid graphene substrates to modulate cell adhesion, assembly, proliferation, and differentiation. The unique properties of small chemical moieties, functionalized biopolymers, and surface-decorated ceramic/metallic particles on graphene imparts additional physical and chemical surface features, facilitating better interaction between cells and material. Furthermore, the use of pristine graphene/RGO, GO, functionalized graphene (using polymer and biopolymer), and metallic/ceramic-decorated hybrid graphene nanoparticles as reinforcing material for the preparation of multifunctional polymeric, ceramic, and metallic composites for tissue engineering, and as coatings on implants, offers several advantages. Reinforcement of graphene and graphene-derived nanoparticles in polymer, ceramic, and metal matrices showed improvement in mechanical, electrical, surface wettability, and nanotopography features, resulting in (i) better cell attachment with more focal adhesion points, (ii) enhanced protein, growth factors, drug and other biomolecule adsorption, influencing better cell proliferation, biomineralization, and differentiation, and (iii) reduced corrosion and release of drug or metallic ions for enhanced integration of the implant at the material interface. In vitro biocompatibility reports have demonstrated that these materials have excellent potential for tissue regeneration and device integration. However, the performance of these graphene-based substrates in vivo is not well understood, especially to address potential concerns of toxicity. A few studies as mentioned above have evaluated such graphenebased materials in vivo, although for only a short duration of implantation. These studies have shown favorable outcomes, such as faster wound healing, enhanced osteogenesis, and the formation of new blood vessels. However, prior to clinical use,

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +91-8022933408. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Department of Science and Technology (DST), India for financial support. K.C. acknowledges the Ramanujan fellowship from DST. We acknowledge the work of 26452


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ACS Applied Materials & Interfaces all the researchers in this field that helped us compile this review and apologize to others whose contributions were inadvertently omitted.

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(20) Chowdhury, S. M.; Lalwani, G.; Zhang, K.; Yang, J. Y.; Neville, K.; Sitharaman, B. Cell Specific Cytotoxicity and Uptake of Graphene Nanoribbons. Biomaterials 2013, 34, 283−293. (21) Nezakati, T.; Cousins, B. G.; Seifalian, A. M. Toxicology of Chemically Modified Graphene-Based Materials for Medical Application. Arch. Toxicol. 2014, 88, 1987−2012. (22) Chang, Y.; Yang, S.-T.; Liu, J.-H.; Dong, E.; Wang, Y.; Cao, A.; Liu, Y.; Wang, H. In Vitro Toxicity Evaluation of Graphene Oxide on A549 Cells. Toxicol. Lett. 2011, 200, 201−210. (23) Sasidharan, A.; Panchakarla, L.; Chandran, P.; Menon, D.; Nair, S.; Rao, C.; Koyakutty, M. Differential Nano-Bio Interactions and Toxicity Effects of Pristine Versus Functionalized Graphene. Nanoscale 2011, 3, 2461−2464. (24) Hashemi, E.; Akhavan, O.; Shamsara, M.; Rahighi, R.; Esfandiar, A.; Tayefeh, A. R. Cyto and Genotoxicities of Graphene Oxide and Reduced Graphene Oxide Sheets on Spermatozoa. RSC Adv. 2014, 4, 27213−27223. (25) Sasidharan, A.; Panchakarla, L. S.; Sadanandan, A. R.; Ashokan, A.; Chandran, P.; Girish, C. M.; Menon, D.; Nair, S. V.; Rao, C.; Koyakutty, M. Hemocompatibility and Macrophage Response of Pristine and Functionalized Graphene. Small 2012, 8, 1251−1263. (26) Li, Y.; Liu, Y.; Fu, Y.; Wei, T.; Le Guyader, L.; Gao, G.; Liu, R.S.; Chang, Y.-Z.; Chen, C. The Triggering of Apoptosis in Macrophages by Pristine Graphene Through the MAPK and TGFBeta Signaling Pathways. Biomaterials 2012, 33, 402−411. (27) Zhang, S.; Yang, K.; Feng, L.; Liu, Z. In Vitro and In Vivo Behaviors of Dextran Functionalized Graphene. Carbon 2011, 49, 4040−4049. (28) Yan, L.; Lin, M.; Zeng, C.; Chen, Z.; Zhang, S.; Zhao, X.; Wu, A.; Wang, Y.; Dai, L.; Qu, J. Electroactive and Biocompatible Hydroxyl-Functionalized Graphene by Ball Milling. J. Mater. Chem. 2012, 22, 8367−8371. (29) Lin, M.; Zou, R.; Shi, H.; Yu, S.; Li, X.; Guo, R.; Yan, L.; Li, G.; Liu, Y.; Dai, L. Ocular Biocompatibility Evaluation of HydroxylFunctionalized Graphene. Mater. Sci. Eng., C 2015, 50, 300−308. (30) Yang, D.; Li, T.; Xu, M.; Gao, F.; Yang, J.; Yang, Z.; Le, W. Graphene Oxide Promotes the Differentiation of Mouse Embryonic Stem Cells to Dopamine Neurons. Nanomedicine 2014, 9, 2445−2455. (31) Pinto, A. M.; Gonçalves, I. C.; Magalhães, F. D. Graphene-Based Materials Biocompatibility: A Review. Colloids Surf., B 2013, 111, 188−202. (32) Nayak, T. R.; Andersen, H.; Makam, V. S.; Khaw, C.; Bae, S.; Xu, X.; Ee, P.-L. R.; Ahn, J.-H.; Hong, B. H.; Pastorin, G. Graphene for Controlled and Accelerated Osteogenic Differentiation of Human Mesenchymal Stem Cells. ACS Nano 2011, 5, 4670−4678. (33) Aryaei, A.; Jayatissa, A. H.; Jayasuriya, A. C. The Effect of Graphene Substrate on Osteoblast Cell Adhesion and Proliferation. J. Biomed. Mater. Res., Part A 2014, 102, 3282−3290. (34) Li, N.; Zhang, X.; Song, Q.; Su, R.; Zhang, Q.; Kong, T.; Liu, L.; Jin, G.; Tang, M.; Cheng, G. The Promotion of Neurite Sprouting and Outgrowth of Mouse Hippocampal Cells in Culture by Graphene Substrates. Biomaterials 2011, 32, 9374−9382. (35) Lee, W. C.; Lim, C. H. Y.; Shi, H.; Tang, L. A.; Wang, Y.; Lim, C. T.; Loh, K. P. Origin of Enhanced Stem Cell Growth and Differentiation on Graphene and Graphene Oxide. ACS Nano 2011, 5, 7334−7341. (36) Lee, J. S.; Lipatov, A.; Ha, L.; Shekhirev, M.; Andalib, M. N.; Sinitskii, A.; Lim, J. Y. Graphene Substrate for Inducing Neurite Outgrowth. Biochem. Biophys. Res. Commun. 2015, 460, 267−273. (37) Tang, M.; Song, Q.; Li, N.; Jiang, Z.; Huang, R.; Cheng, G. Enhancement of Electrical Signaling in Neural Networks on Graphene Films. Biomaterials 2013, 34, 6402−6411. (38) Akhavan, O.; Ghaderi, E.; Shahsavar, M. Graphene Nanogrids for Selective and Fast Osteogenic Differentiation of Human Mesenchymal Stem Cells. Carbon 2013, 59, 200−211. (39) Chen, G.-Y.; Pang, D.-P.; Hwang, S.-M.; Tuan, H.-Y.; Hu, Y.-C. A Graphene-Based Platform for Induced Pluripotent Stem Cells Culture and Differentiation. Biomaterials 2012, 33, 418−427.

REFERENCES

(1) Georgakilas, V. Functionalization of Graphene, 1st ed; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2014. (2) Kuila, T.; Bose, S.; Mishra, A. K.; Khanra, P.; Kim, N. H.; Lee, J. H. Chemical Functionalization of Graphene and its Applications. Prog. Mater. Sci. 2012, 57, 1061−1105. (3) Zhu, Y.; Murali, S.; Cai, W.; Li, X.; Suk, J. W.; Potts, J. R.; Ruoff, R. S. Graphene and Graphene Oxide: Synthesis, Properties, and Applications. Adv. Mater. 2010, 22, 3906−3924. (4) Ahadian, S.; Ramalingam, M.; Khademhosseini, A. In Biomimetics: Advancing Nanobiomaterials and Tissue Engineering; Ramalingam, M., Wang, X., Chen, G., Ma, P., Cui, F. Z., Eds.; John Wiely & Sons, Inc. Hoboken: NJ, 2013; Chapter 12, pp 279−299. (5) Wu, S.-Y.; An, S. S. A.; Hulme, J. Current Applications of Graphene Oxide in Nanomedicine. Int. J. Nanomed. 2015, 10, 9−24. (6) Higuchi, A.; Ling, Q.-D.; Ko, Y.-A.; Chang, Y.; Umezawa, A. Biomaterials for the Feeder-Free Culture of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells. Chem. Rev. 2011, 111, 3021−3035. (7) Goenka, S.; Sant, V.; Sant, S. Graphene-Based Nanomaterials For Drug Delivery and Tissue Engineering. J. Controlled Release 2014, 173, 75−88. (8) Dubey, N.; Bentini, R.; Islam, I.; Cao, T.; Neto, A. H. C.; Rosa, V. Graphene: A Versatile Carbon-Based Material for Bone Tissue Engineering. Stem Cells Int. 2015, 2015, 1−12. (9) Feng, L.; Liu, Z. Graphene in Biomedicine: Opportunities and Challenges. Nanomedicine 2011, 6, 317−324. (10) Wojtoniszak, M.; Chen, X.; Kalenczuk, R. J.; Wajda, A.; Łapczuk, J.; Kurzewski, M.; Drozdzik, M.; Chu, P. K.; Borowiak-Palen, E. Synthesis, Dispersion, and Cytocompatibility of Graphene Oxide and Reduced Graphene Oxide. Colloids Surf., B 2012, 89, 79−85. (11) Liao, K.-H.; Lin, Y.-S.; Macosko, C. W.; Haynes, C. L. Cytotoxicity of Graphene Oxide and Graphene in Human Erythrocytes and Skin Fibroblasts. ACS Appl. Mater. Interfaces 2011, 3, 2607−2615. (12) Zhang, H.; Peng, C.; Yang, J.; Lv, M.; Liu, R.; He, D.; Fan, C.; Huang, Q. Uniform Ultrasmall Graphene Oxide Nanosheets with Low Cytotoxicity and High Cellular Uptake. ACS Appl. Mater. Interfaces 2013, 5, 1761−1767. (13) Jaworski, S.; Sawosz, E.; Kutwin, M.; Wierzbicki, M.; Hinzmann, M.; Grodzik, M.; Winnicka, A.; Lipińska, L.; Włodyga, K.; Chwalibog, A. In Vitro and In Vivo Effects of Graphene Oxide and Reduced Graphene Oxide on Glioblastoma. Int. J. Nanomed. 2015, 10, 1585. (14) Yang, K.; Gong, H.; Shi, X.; Wan, J.; Zhang, Y.; Liu, Z. In Vivo Biodistribution and Toxicology of Functionalized Nano-Graphene Oxide in Mice after Oral and Intraperitoneal Administration. Biomaterials 2013, 34, 2787−2795. (15) Zhang, X.; Yin, J.; Peng, C.; Hu, W.; Zhu, Z.; Li, W.; Fan, C.; Huang, Q. Distribution and Biocompatibility Studies of Graphene Oxide in Mice after Intravenous Administration. Carbon 2011, 49, 986−995. (16) Rao, C. N. R.; Sood, A. K. Graphene: Synthesis, Properties, and Phenomena, 2nd ed; Wiley-VCH, Verlag GmbH: Weinheim, Germany, 2013. (17) Sanchez, V. C.; Jachak, A.; Hurt, R. H.; Kane, A. B. Biological Interactions of Graphene-Family Nanomaterials: an Interdisciplinary Review. Chem. Res. Toxicol. 2012, 25, 15−34. (18) Caffo, M.; Merlo, L.; Marino, D.; Caruso, G. Graphene in Neurosurgery: The Beginning of a New Era. Nanomedicine 2015, 10, 615−625. (19) Guo, X.; Mei, N. Assessment of the Toxic Potential of Graphene Family Nanomaterials. J. Food Drug Anal. 2014, 22, 105−115. 26453


Review


Adipose-Derived Stem Cells Using Combinatorial Graphene HybridPattern Arrays. ACS Nano 2015, 9, 3780−3790. (61) Serrano, M. C.; Patiño, J.; García-Rama, C.; Ferrer, M. L.; Fierro, J.; Tamayo, A.; Collazos-Castro, J. E.; del Monte, F.; Gutierrez, M. C. 3D Free-Standing Porous Scaffolds Made of Graphene Oxide as Substrates for Neural Cell Growth. J. Mater. Chem. B 2014, 2, 5698− 5706. (62) Nishida, E.; Miyaji, H.; Takita, H.; Kanayama, I.; Tsuji, M.; Akasaka, T.; Sugaya, T.; Sakagami, R.; Kawanami, M. Graphene Oxide Coating Facilitates the Bioactivity Of Scaffold Material for Tissue Engineering. Jpn. J. Appl. Phys. 2014, 53, 06JD04. (63) Saptarshi, S. R.; Duschl, A.; Lopata, A. L. Interaction of Nanoparticles with Proteins: Relation to Bio-Reactivity of the Nanoparticle. J. Nanobiotechnol. 2013, 11, 26. (64) Lee, H.; Lee, N.; Seo, Y.; Eom, J.; Lee, S. Comparison of Frictional Forces on Graphene and Graphite. Nanotechnology 2009, 20, 325701. (65) Hauert, R. An Overview on the Tribological Behavior of Diamond-Like Carbon in Technical and Medical Applications. Tribol. Int. 2004, 37, 991−1003. (66) Georgakilas, V.; Otyepka, M.; Bourlinos, A. B.; Chandra, V.; Kim, N.; Kemp, K. C.; Hobza, P.; Zboril, R.; Kim, K. S. Functionalization of Graphene: Covalent and Non-Covalent Approaches, Derivatives and Applications. Chem. Rev. 2012, 112, 6156− 6214. (67) Wang, Y.; Li, Z.; Wang, J.; Li, J.; Lin, Y. Graphene and Graphene Oxide: Biofunctionalization and Applications in Biotechnology. Trends Biotechnol. 2011, 29, 205−212. (68) Phillips, J. E.; Petrie, T. A.; Creighton, F. P.; García, A. J. Human Mesenchymal Stem Cell Differentiation on Self-Assembled Monolayers Presenting Different Surface Chemistries. Acta Biomater. 2010, 6, 12−20. (69) Webb, K.; Hlady, V.; Tresco, P. A. Relationships Among Cell Attachment, Spreading, Cytoskeletal Organization, and Migration Rate for Anchorage-Dependent Cells on Model Surfaces. J. Biomed. Mater. Res. 2000, 49, 362. (70) Barrias, C. C.; Martins, M. C. L.; Almeida-Porada, G.; Barbosa, M. A.; Granja, P. L. The Correlation Between the Adsorption of Adhesive Proteins and Cell Behaviour on Hydroxyl-Methyl Mixed Self-Assembled Monolayers. Biomaterials 2009, 30, 307−316. (71) Maia, F. R.; Bidarra, S. J.; Granja, P. L.; Barrias, C. C. Functionalization of Biomaterials with Small Osteoinductive Moieties. Acta Biomater. 2013, 9, 8773−8789. (72) Wang, Y.; Lee, W. C.; Manga, K. K.; Ang, P. K.; Lu, J.; Liu, Y. P.; Lim, C. T.; Loh, K. P. Fluorinated Graphene for Promoting NeuroInduction of Stem Cells. Adv. Mater. 2012, 24, 4285−4290. (73) Guo, M.; Li, M.; Liu, X.; Zhao, M.; Li, D.; Geng, D.; Sun, X.; Gu, H. N-Containing Functional Groups Induced Superior Cytocompatible and Hemocompatible Graphene by NH2 Ion Implantation. J. Mater. Sci.: Mater. Med. 2013, 24, 2741−2748. (74) Kumar, S.; Raj, S.; Kolanthai, E.; Sood, A. K.; Sampath, S.; Chatterjee, K. Chemical Functionalization of Graphene to Augment Stem Cell Osteogenesis and Inhibit Biofilm Formation on Polymer Composites for Orthopedic Applications. ACS Appl. Mater. Interfaces 2015, 7, 3237−3252. (75) Liu, X.; Cao, Y.; Zhao, M.; Deng, J.; Li, X.; Li, D. The Enhanced Anticoagulation for Graphene Induced by COOH+ Ion Implantation. Nanoscale Res. Lett. 2015, 10, 1−6. (76) Yan, L.; Zheng, Y. B.; Zhao, F.; Li, S.; Gao, X.; Xu, B.; Weiss, P. S.; Zhao, Y. Chemistry and Physics of a Single Atomic Layer: Strategies and Challenges for Functionalization of Graphene and Graphene-Based Materials. Chem. Soc. Rev. 2012, 41, 97−114. (77) Krueger, E.; Chang, A. N.; Brown, D.; Eixenberger, J.; Brown, R.; Rastegar, S.; Yocham, K.; Cantley, K. D.; Estrada, D. Graphene Foam as a 3-dimensional Platform for Myotube Growth. ACS Biomater. Sci. Eng. 2016, 2, 1234−1241. (78) Tang, Z.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Biomedical Applications of Layer-by-Layer Assembly: from Biomimetics to Tissue Engineering. Adv. Mater. 2006, 18, 3203.

(40) Li, N.; Zhang, Q.; Gao, S.; Song, Q.; Huang, R.; Wang, L.; Liu, L.; Dai, J.; Tang, M.; Cheng, G. Three-Dimensional Graphene Foam as a Biocompatible and Conductive Scaffold for Neural Stem Cells. Sci. Rep. 2013, 3, 1−6. (41) Chen, H.; Müller, M. B.; Gilmore, K. J.; Wallace, G. G.; Li, D. Mechanically Strong, Electrically Conductive, and Biocompatible Graphene Paper. Adv. Mater. 2008, 20, 3557−3561. (42) Veliev, F.; Briançon-Marjollet, A.; Bouchiat, V.; Delacour, C. Impact of Crystalline Quality on Neuronal Affinity of Pristine Graphene. Biomaterials 2016, 86, 33−41. (43) Haastert-Talini, K.; Schmitte, R.; Korte, N.; Klode, D.; Ratzka, A.; Grothe, C. Electrical Stimulation Accelerates Axonal and Functional Peripheral Nerve Regeneration Across Long Gaps. J. Neurotrauma. 2011, 28, 661−674. (44) Liu, T.; Shang, S. J.; Liu, B.; Wang, C. H.; Wang, Y. S.; Xiong, W.; Zheng, L. H.; Zhang, C. X.; Zhou, Z. Two Distinct Vesicle Pools for Depolarization-Induced Exocytosis in Somata Of Dorsal Root Ganglion Neurons. J. Physiol. 2011, 589, 3507−3515. (45) Park, S. Y.; Park, J.; Sim, S. H.; Sung, M. G.; Kim, K. S.; Hong, B. H.; Hong, S. Enhanced Differentiation of Human Neural Stem Cells into Neurons on Graphene. Adv. Mater. 2011, 23, H263−H267. (46) Akhavan, O. Graphene Scaffolds in Progressive Nanotechnology/Stem Cell-Based Tissue Engineering of the Nervous System. J. Mater. Chem. B 2016, 4, 3169−3190. (47) Bouzid, T.; Sinitskii, A.; Lim, J. Y. Graphene Platform for Neural Regenerative Medicine. Neural Regener. Res. 2016, 11, 894. (48) Ryu, S.; Kim, B.-S. Culture of Neural Cells and Stem Cells on Graphene. Tissue Eng. Regener. Med. 2013, 10, 39−46. (49) Wang, K.; Fishman, H. A.; Dai, H.; Harris, J. S. Neural Stimulation with a Carbon Nanotube Microelectrode Array. Nano Lett. 2006, 6, 2043−2048. (50) Malarkey, E. B.; Parpura, V. Carbon Nanotubes in Neuroscience. Acta Neurochir Suppl. 2010, 106, 337−341. (51) Lin, F.; Du, F.; Huang, J.; Chau, A.; Zhou, Y.; Duan, H.; Wang, J.; Xiong, C. Substrate Effect Modulates Adhesion and Proliferation of Fibroblast on Graphene Layer. Colloids Surf., B 2016, 146, 785−793. (52) Lu, J.; He, Y. S.; Cheng, C.; Wang, Y.; Qiu, L.; Li, D.; Zou, D. Self-Supporting Graphene Hydrogel Film as an Experimental Platform to Evaluate the Potential of Graphene for Bone Regeneration. Adv. Funct. Mater. 2013, 23, 3494−3502. (53) Lim, H. N. Fabrication and Characterization of Graphene Hydrogel via Hydrothermal Approach as a Scaffold for Preliminary Study of Cell Growth. Int. J. Nanomed. 2011, 6, 1817−1823. (54) Balikov, D. A.; Fang, B.; Chun, Y. W.; Crowder, S. W.; Prasai, D.; Lee, J. B.; Bolotin, K. I.; Sung, H.-J. Directing Lineage Specification of Human Mesenchymal Stem Cells by Decoupling Electrical Stimulation and Physical Patterning on Unmodified Graphene. Nanoscale 2016, 8, 13730−13739. (55) Ryoo, S.-R.; Kim, Y.-K.; Kim, M.-H.; Min, D.-H. Behaviors of NIH-3T3 Fibroblasts on Graphene/Carbon Nanotubes: Proliferation, Focal Adhesion, and Gene Transfection Studies. ACS Nano 2010, 4, 6587−6598. (56) Ribeiro, M.; Monteiro, F. J.; Ferraz, M. P. Infection of Orthopedic Implants with Emphasis on Bacterial Adhesion Process and Techniques Used in Studying Bacterial-Material Interactions. Biomatter 2012, 2, 176−194. (57) Hu, W.; Peng, C.; Luo, W.; Lv, M.; Li, X.; Li, D.; Huang, Q.; Fan, C. Graphene-Based Antibacterial Paper. ACS Nano 2010, 4, 4317−4323. (58) Shi, X.; Chang, H.; Chen, S.; Lai, C.; Khademhosseini, A.; Wu, H. Regulating Cellular Behavior on Few-Layer Reduced Graphene Oxide Films with Well-Controlled Reduction States. Adv. Funct. Mater. 2012, 22, 751−759. (59) Shah, S.; Yin, P. T.; Uehara, T. M.; Chueng, S. T. D.; Yang, L.; Lee, K. B. Guiding Stem Cell Differentiation into Oligodendrocytes Using Graphene-Nanofiber Hybrid Scaffolds. Adv. Mater. 2014, 26, 3673−3680. (60) Kim, T.-H.; Shah, S.; Yang, L.; Yin, P. T.; Hossain, M. K.; Conley, B.; Choi, J.-W.; Lee, K.-B. Controlling Differentiation of 26454


Review

ACS Applied Materials & Interfaces (79) Zhou, H.; Cheng, C.; Qin, H.; Ma, L.; He, C.; Nie, S.; Zhang, X.; Fu, Q.; Zhao, C. Self-Assembled 3D Biocompatible and Bioactive Layer at the Macro-Interface via Graphene-Based Supermolecules. Polym. Chem. 2014, 5, 3563−3575. (80) Chen, Y.; Bal, B.; Gorski, J. Calcium and Collagen Binding Properties of Osteopontin, Bone Sialoprotein, and Bone Acidic Glycoprotein-75 From Bone. J. Biol. Chem. 1992, 267, 24871−24878. (81) Liu, H.; Cheng, J.; Chen, F.; Bai, D.; Shao, C.; Wang, J.; Xi, P.; Zeng, Z. Gelatin Functionalized Graphene Oxide for Mineralization of Hydroxyapatite: Biomimetic and In Vitro Evaluation. Nanoscale 2014, 6, 5315−5322. (82) Liu, H.; Cheng, J.; Chen, F.; Hou, F.; Bai, D.; Xi, P.; Zeng, Z. Biomimetic and Cell-Mediated Mineralization of Hydroxyapatite by Carrageenan Functionalized Graphene Oxide. ACS Appl. Mater. Interfaces 2014, 6, 3132−3140. (83) Wang, J.; Wang, H.; Wang, Y.; Li, J.; Su, Z.; Wei, G. Alternate Layer-by-Layer Assembly of Graphene Oxide Nanosheets and Fibrinogen Nanofibers on a Silicon Substrate For a Biomimetic Three-Dimensional Hydroxyapatite Scaffold. J. Mater. Chem. B 2014, 2, 7360−7368. (84) Areva, S.; Peltola, T.; Säilynoja, E.; Laajalehto, K.; Linden, M.; Rosenholm, J. Effect of Albumin and Fibrinogen on Calcium Phosphate Formation on Sol-Gel-Derived Titania Coatings In Vitro. Chem. Mater. 2002, 14, 1614−1621. (85) Mulvaney, S. P.; Stine, R.; Long, N. C.; Tamanaha, C. R.; Sheehan, P. E. Graphene Veils: a Versatile Surface Chemistry for Sensors. BioTechniques 2014, 57, 21−30. (86) Xu, Y.; Bai, H.; Lu, G.; Li, C.; Shi, G. Flexible Graphene Films via the Filtration of Water-Soluble Noncovalent Functionalized Graphene Sheets. J. Am. Chem. Soc. 2008, 130, 5856−5857. (87) Park, S.; Mohanty, N.; Suk, J. W.; Nagaraja, A.; An, J.; Piner, R. D.; Cai, W.; Dreyer, D. R.; Berry, V.; Ruoff, R. S. Biocompatible, Robust Free-Standing Paper Composed of a TWEEN/Graphene Composite. Adv. Mater. 2010, 22, 1736−1740. (88) Li, N.; Zhang, Q.; Zhang, Q.; Song, Q.; Qi, L.; Wang, L.; Tang, M.; Jin, G.; Cheng, G. Synthesis and Biocompatibility of Amphiphilic Graphene Oxide. Chem. J. Chinese U. 2013, 34, 50−54. (89) Li, Y. Q.; Yu, T.; Yang, T. Y.; Zheng, L. X.; Liao, K. Bio-Inspired Nacre-Like Composite Films Based on Graphene with Superior Mechanical, Electrical, and Biocompatible Properties. Adv. Mater. 2012, 24, 3426−3431. (90) Guo, W.; Zhang, X.; Yu, X.; Wang, S.; Qiu, J.; Tang, W.; Li, L.; Liu, H.; Wang, Z. L. Self-Powered Electrical Stimulation for Enhancing Neural Differentiation of Mesenchymal Stem Cells on Graphene-Poly (3, 4-ethylenedioxythiophene) Hybrid Microfibers. ACS Nano 2016, 10, 5086−5095. (91) Park, S.; Lee, K.-S.; Bozoklu, G.; Cai, W.; Nguyen, S. T.; Ruoff, R. S. Graphene Oxide Papers Modified by Divalent IonsEnhancing Mechanical Properties via Chemical Cross-Linking. ACS Nano 2008, 2, 572−578. (92) Urbas, K.; Aleksandrzak, M.; Jedrzejczak, M.; Jedrzejczak, M.; Rakoczy, R.; Chen, X.; Mijowska, E. Chemical and Magnetic Functionalization of Graphene Oxide as a Route to Enhance its Biocompatibility. Nanoscale Res. Lett. 2014, 9, 1−12. (93) Ma, X.; Tao, H.; Yang, K.; Feng, L.; Cheng, L.; Shi, X.; Li, Y.; Guo, L.; Liu, Z. A Functionalized Graphene Oxide-Iron Oxide Nanocomposite for Magnetically Targeted Drug Delivery, Photothermal Therapy, and Magnetic Resonance Imaging. Nano Res. 2012, 5, 199−212. (94) Ma, J.; Zhang, J.; Xiong, Z.; Yong, Y.; Zhao, X. Preparation, Characterization and Antibacterial Properties of Silver-Modified Graphene Oxide. J. Mater. Chem. 2011, 21, 3350−3352. (95) Shen, J.; Shi, M.; Yan, B.; Ma, H.; Li, N.; Ye, M. One-Pot Hydrothermal Synthesis of Ag-Reduced Graphene Oxide Composite With Ionic Liquid. J. Mater. Chem. 2011, 21, 7795−7801. (96) Turcheniuk, K.; Boukherroub, R.; Szunerits, S. Gold−Graphene Nanocomposites for Sensing and Biomedical Applications. J. Mater. Chem. B 2015, 3, 4301−4324.

(97) Teo, P.; Lim, H.; Huang, N.; Chia, C.; Harrison, I. Room Temperature in Situ Chemical Synthesis of Fe3O4/Graphene. Ceram. Int. 2012, 38, 6411−6416. (98) Zhang, P.; Wang, H.; Zhang, X.; Xu, W.; Li, Y.; Li, Q.; Wei, G.; Su, Z. Graphene Film Doped with Silver Nanoparticles: Self-Assembly Formation, Structural Characterizations, Antibacterial Ability, and Biocompatibility. Biomater. Sci. 2015, 3, 852−860. (99) Dinh, N. X.; Van Cuong, N.; Van Quy, N.; Huy, T. Q.; Mølhave, K.; Le, A.-T. Graphene Oxide/Silver Nanohybrid as Multifunctional Material for Highly Efficient Bacterial Disinfection and Detection of Organic Dye. J. Electron. Mater. 2016, 45, 5321−5333. (100) Hussain, N.; Gogoi, A.; Sarma, R. K.; Sharma, P.; Barras, A.; Boukherroub, R.; Saikia, R.; Sengupta, P.; Das, M. R. Reduced Graphene Oxide Nanosheets Decorated with Au Nanoparticles as an Effective Bactericide: Investigation Of Biocompatibility and Leakage of Sugars and Proteins. ChemPlusChem 2014, 79, 1774−1784. (101) Varela-Rizo, H.; Martín-Gullón, I.; Terrones, M. Hybrid Films with Graphene Oxide and Metal Nanoparticles Could now Replace Indium Tin Oxide. ACS Nano 2012, 6, 4565−4572. (102) Chook, S. W.; Chia, C. H.; Sarani, Z.; Ayob, M. K.; Chee, K. L.; Neoh, H. M.; Huang, N. M. Silver Nanoparticles-Graphene Oxide Nanocomposite for Antibacterial Purpose. Adv. Mater. Res. 2012, 364, 439−443. (103) Hench, L. L. Bioceramics: from Concept to Clinic. J. Am. Ceram. Soc. 1991, 74, 1487−1510. (104) Li, M.; Wang, Y.; Liu, Q.; Li, Q.; Cheng, Y.; Zheng, Y.; Xi, T.; Wei, S. In Situ Synthesis and Biocompatibility of Nano Hydroxyapatite on Pristine and Chitosan Functionalized Graphene Oxide. J. Mater. Chem. B 2013, 1, 475−484. (105) Kang, K.; Choi, S. E.; Jang, H. S.; Cho, W. K.; Nam, Y.; Choi, I. S.; Lee, J. S. In Vitro Developmental Acceleration of Hippocampal Neurons on Nanostructures of Self-Assembled Silica Beads in Filopodium-Size Ranges. Angew. Chem., Int. Ed. 2012, 51, 2855−2858. (106) Solanki, A.; Chueng, S. T. D.; Yin, P. T.; Kappera, R.; Chhowalla, M.; Lee, K. B. Axonal Alignment and Enhanced Neuronal Differentiation of Neural Stem Cells on Graphene-Nanoparticle Hybrid Structures. Adv. Mater. 2013, 25, 5477−5482. (107) Crisan, L.; Crisan, B.; Soritau, O.; Baciut, M.; Biris, A. R.; Baciut, G.; Lucaciu, O. In Vitro Study of Biocompatibility of a Graphene Composite With Gold Nanoparticles and Hydroxyapatite on Human Osteoblasts. J. Appl. Toxicol. 2015, 35, 1200−1210. (108) Kim, S.; Ku, S. H.; Lim, S. Y.; Kim, J. H.; Park, C. B. Graphene−Biomineral Hybrid Materials. Adv. Mater. 2011, 23, 2009− 2014. (109) Kunzler, T. P.; Drobek, T.; Schuler, M.; Spencer, N. D. Systematic Study of Osteoblast and Fibroblast Response to Roughness by Means of Surface-Morphology Gradients. Biomaterials 2007, 28, 2175−2182. (110) Cukierman, E.; Pankov, R.; Stevens, D. R.; Yamada, K. M. Taking Cell-Matrix Adhesions to the Third Dimension. Science 2001, 294, 1708−1712. (111) Hu, J.; Liu, X.; Ma, P. X. Induction of Osteoblast Differentiation Phenotype on Poly (L-Lactic Acid) Nanofibrous Matrix. Biomaterials 2008, 29, 3815−3821. (112) Xie, X.; Hu, K.; Fang, D.; Shang, L.; Tran, S. D.; Cerruti, M. Graphene and Hydroxyapatite Self-Assemble into Homogeneous, Free Standing Nanocomposite Hydrogels for Bone Tissue Engineering. Nanoscale 2015, 7, 7992−8002. (113) Zuo, P.-P.; Feng, H.-F.; Xu, Z.-Z.; Zhang, L.-F.; Zhang, Y.-L.; Xia, W.; Zhang, W.-Q. Fabrication of Biocompatible and Mechanically Reinforced Graphene Oxide-Chitosan Nanocomposite Films. Chem. Cent. J. 2013, 7, 39−39. (114) Lahiri, D.; Dua, R.; Zhang, C.; de Socarraz-Novoa, I.; Bhat, A.; Ramaswamy, S.; Agarwal, A. Graphene Nanoplatelet-Induced Strengthening of Ultrahigh Molecular Weight Polyethylene and Biocompatibility. ACS Appl. Mater. Interfaces 2012, 4, 2234−2241. (115) Upadhyay, R.; Naskar, S.; Bhaskar, N.; Bose, S.; Basu, B. Modulation of Protein Adsorption and Cell Proliferation on Polyethylene Immobilized Graphene Oxide Reinforced HDPE 26455


Review

ACS Applied Materials & Interfaces Bionanocomposites. ACS Appl. Mater. Interfaces 2016, 8, 11954− 11968. (116) Wang, X.; Bai, H.; Yao, Z.; Liu, A.; Shi, G. Electrically Conductive and Mechanically Strong Biomimetic Chitosan/Reduced Graphene Oxide Composite Films. J. Mater. Chem. 2010, 20, 9032− 9036. (117) Pinto, A. M.; Moreira, S.; Gonçalves, I. C.; Gama, F. M.; Mendes, A. M.; Magalhães, F. D. Biocompatibility of Poly (Lactic Acid) with Incorporated Graphene-Based Materials. Colloids Surf., B 2013, 104, 229−238. (118) Ma, H.; Su, W.; Tai, Z.; Sun, D.; Yan, X.; Liu, B.; Xue, Q. Preparation and Cytocompatibility of Polylactic Acid/Hydroxyapatite/ Graphene Oxide Nanocomposite Fibrous Membrane. Chin. Sci. Bull. 2012, 57, 3051−3058. (119) Depan, D.; Girase, B.; Shah, J.; Misra, R. Structure−Process− Property Relationship of the Polar Graphene Oxide-Mediated Cellular Response and Stimulated Growth of Osteoblasts on Hybrid Chitosan Network Structure Nanocomposite Scaffolds. Acta Biomater. 2011, 7, 3432−3445. (120) Zhang, W.; Sun, H.; Mou, Y.; Jiang, X.-X.; Wei, L.; Wang, Q.; Wang, C.; Zhou, J.; Cao, J. Graphene Oxide-Collagen Matrix Accelerated Osteogenic Differentiation of Mesenchymal Stem Cells. J. Biomater. Tissue Eng. 2015, 5, 257−266. (121) Puértolas, J.; Kurtz, S. Evaluation of Carbon Nanotubes and Graphene as Reinforcements for UHMWPE-Based Composites in Arthroplastic Applications: A Review. J. Mech. Behav. Biomed. Mater. 2014, 39, 129−145. (122) Suñer Moreno, S. Carbon Reinforced UHMWPE Composites for Orthopaedic Applications Characterization and Biological Response to Wear Particles. Licentiate thesis; Luleå University of Technology, SwePub, 2013; p 103. (123) Kolanthai, E.; Bose, S.; Bhagyashree, K.; Bhat, S.; Asokan, K.; Kanjilal, D.; Chatterjee, K. Graphene Scavenges Free Radicals to Synergistically Enhance Structural Properties in a Gamma-Irradiated Polyethylene Composite Through Enhanced Interfacial Interactions. Phys. Chem. Chem. Phys. 2015, 17, 22900−22910. (124) Hutmacher, D. W. Scaffolds in Tissue Engineering Bone and Cartilage. Biomaterials 2000, 21, 2529−2543. (125) Luo, Y.; Shen, H.; Fang, Y.; Cao, Y.; Huang, J.; Zhang, M.; Dai, J.; Shi, X.; Zhang, Z. Enhanced Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells on Graphene OxideIncorporated Electrospun Poly (Lactic-Co-Glycolic Acid) Nanofibrous Mats. ACS Appl. Mater. Interfaces 2015, 7, 6331−6339. (126) Kumar, S.; Azam, M.; Raj, S.; Kolanthai, E.; Vasu, K.; Sood, A.; Chatterjee, K. 3D Scaffold Alters Cellular Response to Graphene in a Polymer Composite for Orthopedic Applications. J. Biomed. Mater. Res., Part B 2016, 104, 732−749. (127) Lu, B.; Li, T.; Zhao, H.; Li, X.; Gao, C.; Zhang, S.; Xie, E. Graphene-Based Composite Materials Beneficial to Wound Healing. Nanoscale 2012, 4, 2978−2982. (128) Bressan, E.; Ferroni, L.; Gardin, C.; Sbricoli, L.; Gobbato, L.; Ludovichetti, F. S.; Tocco, I.; Carraro, A.; Piattelli, A.; Zavan, B. Graphene Based Scaffolds Effects on Stem Cells Commitment. J. Transl. Med. 2014, 12, 296. (129) Crowder, S. W.; Prasai, D.; Rath, R.; Balikov, D. A.; Bae, H.; Bolotin, K. I.; Sung, H.-J. Three-Dimensional Graphene Foams Promote Osteogenic Differentiation of Human Mesenchymal Stem Cells. Nanoscale 2013, 5, 4171−4176. (130) Xie, H.; Cao, T.; Gomes, J. V.; Neto, A. H. C.; Rosa, V. Two and Three-Dimensional Graphene Substrates to Magnify Osteogenic Differentiation of Periodontal Ligament Stem Cells. Carbon 2015, 93, 266−275. (131) Wang, J. K.; Xiong, G. M.; Zhu, M.; Ö zyilmaz, B.; Castro Neto, A. H.; Tan, N. S.; Choong, C. Polymer-Enriched 3D Graphene Foams for Biomedical Applications. ACS Appl. Mater. Interfaces 2015, 7, 8275−8283. (132) Li, X.; Lan, J.; Ai, M.; Guo, Y.; Cai, Q.; Yang, X. Biomineralization on Polymer-Coated Multi-Walled Carbon Nano-

tubes with Different Surface Functional Groups. Colloids Surf., B 2014, 123, 753−761. (133) Wang, C.-H.; Guo, Z.-S.; Pang, F.; Zhang, L.-Y.; Yan, M.; Yan, J.-H.; Li, K.-W.; Li, X.-J.; Li, Y.; Bi, L. Effects of Graphene Modification on the Bioactivation of Polyethylene-Terephthalate-Based Artificial Ligaments. ACS Appl. Mater. Interfaces 2015, 7, 15263−15276. (134) Yan, X.; Chen, J.; Yang, J.; Xue, Q.; Miele, P. Fabrication of Free-Standing, Electrochemically Active, and Biocompatible Graphene Oxide− Polyaniline and Graphene− Polyaniline Hybrid Papers. ACS Appl. Mater. Interfaces 2010, 2, 2521−2529. (135) Folkman, J.; Moscona, A. Role of Cell Shape in Growth Control. Nature 1978, 273, 345−349. (136) Jang, J.-Y.; Lee, S. W.; Park, S. H.; Shin, J. W.; Mun, C.; Kim, S.-H.; Kim, D. H.; Shin, J.-W. Combined Effects of Surface Morphology and Mechanical Straining Magnitudes on the Differentiation of Mesenchymal Stem Cells without using Biochemical Reagents. J. Biomed. Biotechnol. 2011, 2011, 860652. (137) Kumar, G.; Tison, C. K.; Chatterjee, K.; Pine, P. S.; McDaniel, J. H.; Salit, M. L.; Young, M. F.; Simon, C. G. The Determination of Stem Cell Fate by 3D Scaffold Structures Through the Control of Cell Shape. Biomaterials 2011, 32, 9188−9196. (138) Kumar, S.; Raj, S.; Sarkar, K.; Chatterjee, K. Engineering a Multi-Biofunctional Composite using Poly (Ethylenimine) Decorated Graphene Oxide for Bone Tissue Regeneration. Nanoscale 2016, 8, 6820−6836. (139) Patel, M.; Moon, H. J.; Ko, D. Y.; Jeong, B. Composite System of Graphene Oxide and Polypeptide Thermogel As an Injectable 3D Scaffold for Adipogenic Differentiation of Tonsil-derived Mesenchymal Stem Cells. ACS Appl. Mater. Interfaces 2016, 8, 5160−5169. (140) Nair, M.; Nancy, D.; Krishnan, A. G.; Anjusree, G.; Vadukumpully, S.; Nair, S. V. Graphene Oxide Nanoflakes Incorporated Gelatin−Hydroxyapatite Scaffolds Enhance Osteogenic Differentiation of Human Mesenchymal Stem Cells. Nanotechnology 2015, 26, 161001. (141) Kanayama, I.; Miyaji, H.; Takita, H.; Nishida, E.; Tsuji, M.; Fugetsu, B.; Sun, L.; Inoue, K.; Ibara, A.; Akasaka, T. Comparative Study of Bioactivity of Collagen Scaffolds Coated with Graphene Oxide and Reduced Graphene Oxide. Int. J. Nanomed. 2014, 9, 3363. (142) Mohandes, F.; Salavati-Niasari, M. In Vitro Comparative Study of Pure Hydroxyapatite Nanorods and Novel Polyethylene Glycol/ Graphene Oxide/Hydroxyapatite Nanocomposite. J. Nanopart. Res. 2014, 16, 1−12. (143) Marques, P. A.; Gonçalves, G.; Singh, M. K.; Grácio, J. Graphene Oxide and Hydroxyapatite as Fillers of Polylactic Acid Nanocomposites: Preparation and Characterization. J. Nanosci. Nanotechnol. 2012, 12, 6686−6692. (144) Kumar, S.; Chatterjee, K. Strontium Eluting Graphene Hybrid Nanoparticles Augment Osteogenesis in a 3D Tissue Scaffold. Nanoscale 2015, 7, 2023−2033. (145) Yu, Y.-H.; Chan, C.-C.; Lai, Y.-C.; Lin, Y.-Y.; Huang, Y.-C.; Chi, W.-F.; Kuo, C.-W.; Lin, H.-M.; Chen, P.-C. Biocompatible Electrospinning Poly (Vinyl Alcohol) Nanofibres Embedded with Graphene-Based Derivatives with Enhanced Conductivity, Mechanical Strength and Thermal Stability. RSC Adv. 2014, 4, 56373−56384. (146) Kumar, S.; Raj, S.; Jain, S.; Chatterjee, K. Multifunctional Biodegradable Polymer Nanocomposite Incorporating GrapheneSilver Hybrid for Biomedical Applications. Mater. Des. 2016, 108, 319−332. (147) Chaudhuri, B.; Bhadra, D.; Moroni, L.; Pramanik, K. Myoblast Differentiation of Human Mesenchymal Stem Cells on Graphene Oxide and Electrospun Graphene Oxide−Polymer Composite Fibrous Meshes: Importance of Graphene Oxide Conductivity and Dielectric Constant on Their Biocompatibility. Biofabrication 2015, 7, 015009. (148) Rezwan, K.; Chen, Q.; Blaker, J.; Boccaccini, A. R. Biodegradable and Bioactive Porous Polymer/Inorganic Composite Scaffolds for Bone Tissue Engineering. Biomaterials 2006, 27, 3413− 3431. 26456


Review

ACS Applied Materials & Interfaces (149) Zhang, L.; Liu, W.; Yue, C.; Zhang, T.; Li, P.; Xing, Z.; Chen, Y. A Tough Graphene Nanosheet/Hydroxyapatite Composite with Improved In Vitro Biocompatibility. Carbon 2013, 61, 105−115. (150) Mehrali, M.; Moghaddam, E.; Shirazi, S. F. S.; Baradaran, S.; Mehrali, M.; Latibari, S. T.; Metselaar, H. S. C.; Kadri, N. A.; Zandi, K.; Osman, N. A. A. Synthesis, Mechanical Properties, and In Vitro Biocompatibility with Osteoblasts of Calcium Silicate−Reduced Graphene Oxide Composites. ACS Appl. Mater. Interfaces 2014, 6, 3947−3962. (151) Pan, H.; Zhao, X.; Darvell, B. W.; Lu, W. W. Apatite-Formation Ability−Predictor of “Bioactivity. Acta Biomater. 2010, 6, 4181−4188. (152) Liu, Y.; Huang, J.; Li, H. Synthesis of Hydroxyapatite-Reduced Graphite Oxide Nanocomposites for Biomedical Applications: Oriented Nucleation and Epitaxial Growth of Hydroxyapatite. J. Mater. Chem. B 2013, 1, 1826−1834. (153) Baradaran, S.; Moghaddam, E.; Basirun, W.; Mehrali, M.; Sookhakian, M.; Hamdi, M.; Moghaddam, M. N.; Alias, Y. Mechanical Properties and Biomedical Applications of a Nanotube Hydroxyapatite-Reduced Graphene Oxide Composite. Carbon 2014, 69, 32−45. (154) Liu, J.; Yang, Y.; Hassanin, H.; Jumbu, N.; Deng, S.; Zuo, Q.; Jiang, K. Graphene-Alumina Nanocomposites with Improved Mechanical Properties for Biomedical Applications. ACS Appl. Mater. Interfaces 2016, 8, 2607−2616. (155) Labbaf, S.; Tsigkou, O.; Müller, K. H.; Stevens, M. M.; Porter, A. E.; Jones, J. R. Spherical Bioactive Glass Particles and Their Interaction with Human Mesenchymal Stem Cells. Biomaterials 2011, 32, 1010−1018. (156) Roohani-Esfahani, S.; Nouri-Khorasani, S.; Lu, Z.; Appleyard, R.; Zreiqat, H. Effects of Bioactive Glass Nanoparticles on the Mechanical and Biological Behavior of Composite Coated Scaffolds. Acta Biomater. 2011, 7, 1307−1318. (157) Hoppe, A.; Güldal, N. S.; Boccaccini, A. R. A Review of the Biological Response to Ionic Dissolution Products from Bioactive Glasses and Glass-Ceramics. Biomaterials 2011, 32, 2757−2774. (158) Ma, J.; Chen, C.; Wang, D.; Meng, X.; Shi, J. Influence of the Sintering Temperature on the Structural Feature and Bioactivity of Sol−Gel Derived SiO2−CaO−P2O5 Bioglass. Ceram. Int. 2010, 36, 1911−1916. (159) Gao, C.; Liu, T.; Shuai, C.; Peng, S. Enhancement Mechanisms of Graphene in Nano-58S Bioactive Glass Scaffold: Mechanical and Biological Performance. Sci. Rep. 2014, 4, 4712. (160) Porwal, H.; Grasso, S.; Cordero-Arias, L.; Li, C.; Boccaccini, A. R.; Reece, M. J. Processing and Bioactivity of 45S5 Bioglass®Graphene Nanoplatelets Composites. J. Mater. Sci.: Mater. Med. 2014, 25, 1403−1413. (161) Sun, S.; Gao, L.; Liu, Y. Enhanced Dye-Sensitized Solar Cell Using Graphene-TiO2 Photoanode Prepared by Heterogeneous Coagulation. Appl. Phys. Lett. 2010, 96, 3113. (162) Zhang, Y.; Zhai, D.; Xu, M.; Yao, Q.; Chang, J.; Wu, C. 3DPrinted Bioceramic Scaffolds with A Fe3O4/Graphene Oxide Nanocomposite Interface for Hyperthermia Therapy of Bone Tumor Cells. J. Mater. Chem. B 2016, 4, 2874−2886. (163) Zhang, B. G.; Myers, D. E.; Wallace, G. G.; Brandt, M.; Choong, P. F. Bioactive Coatings for Orthopaedic ImplantsRecent Trends in Development of Implant Coatings. Int. J. Mol. Sci. 2014, 15, 11878−11921. (164) Lopez-Esteban, S.; Saiz, E.; Fujino, S.; Oku, T.; Suganuma, K.; Tomsia, A. P. Bioactive Glass Coatings for Orthopedic Metallic Implants. J. Eur. Ceram. Soc. 2003, 23, 2921−2930. (165) Seidi, A.; Ramalingam, M.; Elloumi-Hannachi, I.; Ostrovidov, S.; Khademhosseini, A. Gradient Biomaterials for Soft-to-Hard Interface Tissue Engineering. Acta Biomater. 2011, 7, 1441−1451. (166) Kappelt, G.; Kurze, P.; Banerjee, D. Method for Producing a Corrosion-Inhibiting Coating on an Implant Made of a Bio-Corrodible Magnesium Alloy and Implant Produced According to the Method. Google Patents, 2007. (167) Tang, L. Nanocoating for Improving Biocompatibility of Medical Implants. Google Patents, 2004.

(168) Jia, Z.; Shi, Y.; Xiong, P.; Zhou, W.; Cheng, Y.; Zheng, Y.; Xi, T.; Wei, S. From Solution to Biointerface: Graphene Self-Assemblies of Varying Lateral Sizes and Surface Properties for Biofilm Control and Osteo-Differentiation. ACS Appl. Mater. Interfaces 2016, 8, 17151− 17165. (169) Li, M.; Liu, Q.; Jia, Z.; Xu, X.; Shi, Y.; Cheng, Y.; Zheng, Y.; Xi, T.; Wei, S. Electrophoretic Deposition and Electrochemical Behavior of Novel Graphene Oxide-Hyaluronic Acid-Hydroxyapatite Nanocomposite Coatings. Appl. Surf. Sci. 2013, 284, 804−810. (170) Janković, A.; Eraković, S.; Mitrić, M.; Matić, I. Z.; Juranić, Z. D.; Tsui, G. C.; Tang, C.-y.; Mišković-Stanković, V.; Rhee, K. Y.; Park, S. J. Bioactive Hydroxyapatite/Graphene Composite Coating and its Corrosion Stability in Simulated Body Fluid. J. Alloys Compd. 2015, 624, 148−157. (171) Li, M.; Liu, Q.; Jia, Z.; Xu, X.; Cheng, Y.; Zheng, Y.; Xi, T.; Wei, S. Graphene Oxide/Hydroxyapatite Composite Coatings Fabricated by Electrophoretic Nanotechnology for Biological Applications. Carbon 2014, 67, 185−197. (172) Geetha, M.; Singh, A.; Asokamani, R.; Gogia, A. Ti Based Biomaterials, the Ultimate Choice for Orthopaedic Implants−A Review. Prog. Mater. Sci. 2009, 54, 397−425. (173) La, W. G.; Park, S.; Yoon, H. H.; Jeong, G. J.; Lee, T. J.; Bhang, S. H.; Han, J. Y.; Char, K.; Kim, B. S. Delivery of a Therapeutic Protein for Bone Regeneration from a Substrate Coated with Graphene Oxide. Small 2013, 9, 4051−4060. (174) Li, P. F.; Zhou, H.; Cheng, X.-H. Nano/Micro Tribological Behaviors of a Self-Assembled Graphene Oxide Nanolayer on Ti/ Titanium Alloy Substrates. Appl. Surf. Sci. 2013, 285, 937−944. (175) Yan, Y.; Zhang, X.; Mao, H.; Huang, Y.; Ding, Q.; Pang, X. Hydroxyapatite/Gelatin Functionalized Graphene Oxide Composite Coatings Deposited on TiO2 Nanotube by Electrochemical Deposition for Biomedical Applications. Appl. Surf. Sci. 2015, 329, 76−82. (176) Brammer, K. S.; Frandsen, C. J.; Jin, S. TiO2 Nanotubes for Bone Regeneration. Trends Biotechnol. 2012, 30, 315−322. (177) Ghicov, A.; Schmuki, P. Self-Ordering Electrochemistry: A Review On Growth and Functionality of TiO2 Nanotubes and Other Self-Aligned MOX Structures. Chem. Commun. 2009, 2791−2808. (178) Marimuthu, M.; Veerapandian, M.; Ramasundaram, S.; Hong, S. W.; Sudhagar, P.; Nagarajan, S.; Raman, V.; Ito, E.; Kim, S.; Yun, K. Sodium Functionalized Graphene Oxide Coated Titanium Plates for Improved Corrosion Resistance and Cell Viability. Appl. Surf. Sci. 2014, 293, 124−131. (179) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 2006, 126, 677−689. (180) Kumar, A. M.; Suresh, B.; Ramakrishna, S.; Kim, K.-S. Biocompatible Responsive Polypyrrole/GO Nanocomposite Coatings for Biomedical Applications. RSC Adv. 2015, 5, 99866−99874. (181) Song, S.; Xu, H.; Fan, C. Potential Diagnostic Applications of Biosensors: Current and Future Directions. Int. J. Nanomed. 2006, 1, 433−440. (182) Li, J.; Wang, G.; Zhang, W.; Jin, G.; Zhang, M.; Jiang, X.; Di, Z.; Liu, X.; Wang, X. Graphene Film-Functionalized Germanium as a Chemically Stable, Electrically Conductive, and Biologically Active Substrate. J. Mater. Chem. B 2015, 3, 1544−1555. (183) Mu, Q.; Su, G.; Li, L.; Gilbertson, B. O.; Yu, L. H.; Zhang, Q.; Sun, Y.-P.; Yan, B. Size-Dependent Cell Uptake of Protein-Coated Graphene Oxide Nanosheets. ACS Appl. Mater. Interfaces 2012, 4, 2259−2266. (184) van der Zwaag, D.; Vanparijs, N.; Wijnands, S. P.; De Rycke, R.; De Geest, B. G.; Albertazzi, L. Super Resolution Imaging of Nanoparticles Cellular Uptake and Trafficking. ACS Appl. Mater. Interfaces 2016, 8, 6391−6399. (185) Orecchioni, M.; Jasim, D. A.; Pescatori, M.; Manetti, R.; Fozza, C.; Sgarrella, F.; Bedognetti, D.; Bianco, A.; Kostarelos, K.; Delogu, L. G. Molecular and Genomic Impact of Large and Small Lateral Dimension Graphene Oxide Sheets on Human Immune Cells from Healthy Donors. Adv. Healthcare Mater. 2016, 5, 276−287.

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Comprehensive Review on the Use of Graphene-Based Substrates for

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