Multifaceted Biomedical Applications of Functional Graphene Nanomaterials to Coated Substrates, Patterned Arrays and Hybrid Scaffolds

Because of recent research advances in nanoscience and nanotechnology, there has been a growing interest in functional nanomaterials for biomedical applications, such as tissue engineering scaffolds, biosensors, bioimaging agents and drug delivery carriers. Among a great number of promising candidates, graphene and its derivatives—including graphene oxide and reduced graphene oxide—have particularly attracted plenty of attention from researchers as novel nanobiomaterials. Graphene and its derivatives, two-dimensional nanomaterials, have been found to have outstanding biocompatibility and biofunctionality as well as exceptional mechanical strength, electrical conductivity and thermal stability. Therefore, tremendous studies have been devoted to employ functional graphene nanomaterials in biomedical applications. Herein, we focus on the biological potentials of functional graphene nanomaterials and summarize some of major literature concerning the multifaceted biomedical applications of functional graphene nanomaterials to coated substrates, patterned arrays and hybrid scaffolds that have been reported in recent years.


Introduction
Over the last several decades, the development of nanoscience and nanotechnology has made dramatic progress in research towards the understanding of nanomaterials. In addition, numerous attempts have been devoted at that time to employ nanomaterials in various research and industrial fields. It is commonly acknowledged that nanomaterials are defined as materials having at least one dimension, such as length, thickness, width, or diameter, of smaller than 100 nm in size and the plenty of attentions has been paid to various nanomaterials, including carbon nanotube, nanoparticle, polymeric nanofiber, quantum dot, graphene and nanocomposite [1][2][3][4]. Nanomaterials exhibit unique properties, including extraordinary physicochemical, fluorescent, electrical and thermomechanical Moreover, these stimulating effects of graphene nanomaterial-coated substrates on cell behaviors have also been examined in various cell types, including mesenchymal stem cells (MSCs), neural stem cells (NSCs), induced pluripotent stem cells (iPSCs) and myoblasts. Nayak et al. documented that the graphene coatings on substrates can effectively accelerate the differentiation of human MSCs without hampering cell proliferation. The human MSCs were successfully differentiated into osteogenic lineages only on graphene-coated regions. Moreover, it was shown that the graphene-coated polymeric substrates, including polydimethylsiloxane (PDMS) and polyethylene terephthalate (PET), could also increase osteogenic differentiation. In general, cellular behaviors are strongly dependent on the stiffness of substrates and the osteogenic differentiation is commonly promoted on stiff substrates rather than soft substrates, such as polymeric substrates [53][54][55][56]. However, the differentiation of human MSCs towards osteogenic lineage was increased on Moreover, these stimulating effects of graphene nanomaterial-coated substrates on cell behaviors have also been examined in various cell types, including mesenchymal stem cells (MSCs), neural stem cells (NSCs), induced pluripotent stem cells (iPSCs) and myoblasts. Nayak et al. documented that the graphene coatings on substrates can effectively accelerate the differentiation of human MSCs without hampering cell proliferation. The human MSCs were successfully differentiated into osteogenic lineages only on graphene-coated regions. Moreover, it was shown that the graphene-coated polymeric substrates, including polydimethylsiloxane (PDMS) and polyethylene terephthalate (PET), could also increase osteogenic differentiation. In general, cellular behaviors are strongly dependent on the stiffness of substrates and the osteogenic differentiation is commonly promoted on stiff substrates rather than soft substrates, such as polymeric substrates [53][54][55][56]. However, the differentiation of human MSCs towards osteogenic lineage was increased on graphene-coated polymeric substrates (i.e., soft substrates) regardless of stiffness of underlying substrates, indicating that the graphene coatings can be a driving force of osteogenic differentiation. Nanomaterials 2017, 7, 369 4 of 21 graphene-coated polymeric substrates (i.e. soft substrates) regardless of stiffness of underlying substrates, indicating that the graphene coatings can be a driving force of osteogenic differentiation. Meanwhile, Lee et al. investigated the molecular origin of accelerated differentiation on graphene nanomaterial-coated substrates by comparing the binding abilities of graphene and GO to different growth factors [29]. They showed that the different binding interactions of graphene and GO with growth factor agents play a significant role in determining the stem cell growth and differentiation (Figure 2a,b). The osteogenic differentiation of human bone marrow-derived MSCs was enhanced on the graphene-coated PDMS substrates through π-π stacking interactions between graphene and osteogenic inducer, including dexamethasone and β-glycerophosphate, while GOcoated PDMS substrates could greatly enhance adipogenic differentiation via hydrogen bonding and electrostatic interactions with insulins (Figure 2c,d).
The specific binding affinity of GO for biomolecules can significantly promote the myoblast growth and myogenic differentiation. Ku et al. studied the myoblast behaviors on GO-and rGOcoated glass substrates and indicated that the GO-and rGO-coated substrates could enhance myogenic differentiation as well as supporting cell adhesion and proliferation ( Figure 3) [32]. They suggested that the enhanced myogenic differentiation was attributed to both the unique physicochemical properties of graphene derivatives, such as ripples and wrinkles and the adsorption ability for serum proteins in culture media. Moreover, it was confirmed that the GO-coated substrates Reproduced with permission from [29]. Copyright American Chemical Society, 2011.
Meanwhile, Lee et al. investigated the molecular origin of accelerated differentiation on graphene nanomaterial-coated substrates by comparing the binding abilities of graphene and GO to different growth factors [29]. They showed that the different binding interactions of graphene and GO with growth factor agents play a significant role in determining the stem cell growth and differentiation (Figure 2a,b). The osteogenic differentiation of human bone marrow-derived MSCs was enhanced on the graphene-coated PDMS substrates through π-π stacking interactions between graphene and osteogenic inducer, including dexamethasone and β-glycerophosphate, while GO-coated PDMS substrates could greatly enhance adipogenic differentiation via hydrogen bonding and electrostatic interactions with insulins (Figure 2c,d).
The specific binding affinity of GO for biomolecules can significantly promote the myoblast growth and myogenic differentiation. Ku et al. studied the myoblast behaviors on GO-and rGO-coated glass substrates and indicated that the GO-and rGO-coated substrates could enhance myogenic differentiation as well as supporting cell adhesion and proliferation ( Figure 3) [32]. They suggested that the enhanced myogenic differentiation was attributed to both the unique physicochemical properties of graphene derivatives, such as ripples and wrinkles and the adsorption ability for serum proteins in culture media. Moreover, it was confirmed that the GO-coated substrates were more favorable for myogenic differentiation because GO has more oxygen-containing functional groups on its surface than rGO, which leads to further increase in serum protein adsorption. were more favorable for myogenic differentiation because GO has more oxygen-containing functional groups on its surface than rGO, which leads to further increase in serum protein adsorption. In other studies, the graphene nanomaterial-coated substrates hold the potentials for neural cells [30,46,48,57]. In particular, it is worth noting that the graphene nanomaterials have superior electrical properties as compared with the standard graphite materials [58,59]. Qiu et al. demonstrated that the conductivity of graphene nanomaterials is quite a bit higher than that of graphite materials (highly oriented pyrolytic graphite crystal, HOPG), while the resistivity of graphene nanomaterials is much lower as compared with that of graphite materials [59]. They described that the conductivities were found to be 92, 407 and 2138 (Ω·cm) −1 for the HOPG sample, the multi-layer graphene sample and the sample with a single-layer to few-layer graphene, respectively. On the other hand, there is also a significant increase in the carrier concentration, especially in the samples containing a mixture of single-layer to several layers of graphene: 3.58 × 10 18 , 14.9 × 10 18 and 46.3 × 10 18 cm −3 for HOPG sample, multi-layer graphene sample and samples containing a mixture of single-layer to several layers of In other studies, the graphene nanomaterial-coated substrates hold the potentials for neural cells [30,46,48,57]. In particular, it is worth noting that the graphene nanomaterials have superior electrical properties as compared with the standard graphite materials [58,59]. Qiu et al. demonstrated that the conductivity of graphene nanomaterials is quite a bit higher than that of graphite materials (highly oriented pyrolytic graphite crystal, HOPG), while the resistivity of graphene nanomaterials is much lower as compared with that of graphite materials [59]. They described that the conductivities were found to be 92, 407 and 2138 (Ω·cm) −1 for the HOPG sample, the multi-layer graphene sample and the sample with a single-layer to few-layer graphene, respectively. On the other hand, there is also a significant increase in the carrier concentration, especially in the samples containing a mixture of single-layer to several layers of graphene: 3.58 × 10 18 , 14.9 × 10 18 and 46.3 × 10 18 cm −3 for HOPG sample, multi-layer graphene sample and samples containing a mixture of single-layer to several layers of graphene, respectively. In addition, it was revealed that the superior electrical properties of graphene nanomaterials result in highly sensitive detection of the micromolar concentration of dopamine-a neurotransmitter-on graphene-coated surfaces by Raman spectroscopy and microscopy.
Moreover, these superior electrical properties also allow graphene nanomaterials to be used in stimulating neural cells. Park et al. found that the differentiation of human NSCs into neurons was increased on graphene-coated substrates ( Figure 4). Meanwhile, Tang et al. cultured NSCs on graphene-coated substrates and investigated the neural excitation by monitoring spontaneous Ca 2+ oscillations, which represents neural signal transmission [57]. The results indicated that the NSCs were able to form functionally active neural networks on graphene-coated substrates and the neural network activities, such as the intracellular spontaneous and synchronous Ca 2+ oscillations and spontaneous synaptic currents, were significantly improved on the graphene-coated substrates. Hence, it is indicated that graphene nanomaterial-coated substrates are a typical strategy for biomedical applications of graphene nanomaterials. graphene, respectively. In addition, it was revealed that the superior electrical properties of graphene nanomaterials result in highly sensitive detection of the micromolar concentration of dopamine-a neurotransmitter-on graphene-coated surfaces by Raman spectroscopy and microscopy. Moreover, these superior electrical properties also allow graphene nanomaterials to be used in stimulating neural cells. Park et al. found that the differentiation of human NSCs into neurons was increased on graphene-coated substrates ( Figure 4). Meanwhile, Tang et al. cultured NSCs on graphene-coated substrates and investigated the neural excitation by monitoring spontaneous Ca 2+ oscillations, which represents neural signal transmission [57]. The results indicated that the NSCs were able to form functionally active neural networks on graphene-coated substrates and the neural network activities, such as the intracellular spontaneous and synchronous Ca 2+ oscillations and spontaneous synaptic currents, were significantly improved on the graphene-coated substrates. Hence, it is indicated that graphene nanomaterial-coated substrates are a typical strategy for biomedical applications of graphene nanomaterials. In addition to these, graphene-coated substrates or materials can be also applied for other biomedical applications, such as biosensor, implantable electrode, antibacterial system and composite graft material [50][51][52][60][61][62][63][64][65]. Several studies related to the other biomedical applications of graphene nanomaterial-coated substrates are summarized in Table 1. In addition to these, graphene-coated substrates or materials can be also applied for other biomedical applications, such as biosensor, implantable electrode, antibacterial system and composite graft material [50][51][52][60][61][62][63][64][65]. Several studies related to the other biomedical applications of graphene nanomaterial-coated substrates are summarized in Table 1.

Graphene Nanomaterial-Patterned Arrays
Up to now, much research has indicated that the beneficial effects of graphene nanomaterials in biomedical applications, such as promoting effects on cellular behaviors, including cell adhesion, proliferation, development, spreading and differentiation. Along with those findings previously reported, there have been considerable efforts to use the unique physicochemical and topographical properties of graphene nanomaterials in biomedical applications [64,66,67]. In particular, distinctive rippled or wrinkled features of graphene nanomaterials can provide specific topographical guidance cues for directing cell behaviors. The precisely controlled cell migration or orientation plays a crucial role in determining cell responses and fates [68][69][70]. Therefore, the studies concerning the regulation of cellular behaviors by graphene nanomaterials have been recently proposed and investigated for biomedical applications.
The graphene nanomaterial-patterned arrays have been especially spotlighted as a novel strategy for guiding and stimulating cellular behaviors, because the graphene nanomaterials can provide desirable topographical guidance cues as well as biochemical cues [71][72][73][74][75][76][77][78][79]. Bajaj et al. fabricated rectangular island-shaped graphene patterns on SiO 2 /Si substrate using photolithography techniques and examined the myogenic differentiation of C2C12 skeletal muscle myoblasts ( Figure 5) [71]. It was shown that most myotubes were formed on graphene patterns, while few cells were differentiated into myotubes on the SiO 2 /Si substrate without graphene patterns (Figure 5a). In addition, the island-shaped graphene patterns were able to induce the spontaneous alignment of myotubes, which leads to a maximized the contractile power for muscle contractions (Figure 5b). Moreover, they evaluate the functionality of myotubes on graphene patterns and revealed that the myotubes on graphene patterns were mature and highly functional.
provide desirable topographical guidance cues as well as biochemical cues [71][72][73][74][75][76][77][78][79]. Bajaj et al. fabricated rectangular island-shaped graphene patterns on SiO2/Si substrate using photolithography techniques and examined the myogenic differentiation of C2C12 skeletal muscle myoblasts ( Figure  5) [71]. It was shown that most myotubes were formed on graphene patterns, while few cells were differentiated into myotubes on the SiO2/Si substrate without graphene patterns (Figure 5a). In addition, the island-shaped graphene patterns were able to induce the spontaneous alignment of myotubes, which leads to a maximized the contractile power for muscle contractions (Figure 5b). Moreover, they evaluate the functionality of myotubes on graphene patterns and revealed that the myotubes on graphene patterns were mature and highly functional.   (Figure 6a). Their results indicated that both graphene nanogrids (GO and rGO nanoribbon grid) could enhance the actin cytoskeleton proliferations. Meanwhile, in the presence of chemical inducers, including dexamethasone, β-glycerophosphate and ascorbic acid, rGO nanoribbon grid especially accelerated the osteogenic differentiation of human MSCs (Figure 6b). They explained these findings by the fact that the rGO nanoribbon grids can highly adsorb chemical induces in culture media and can also provide physical stress induced by the surface topographical features of rGO nanogrids [29,[80][81][82]. They also proved that those stimulating effects of rGO nanogrids are equally effective on human NSCs [73]. These results indicate that the graphene nanomaterial patterns can be readily applied in biomedical fields. the osteogenic differentiation of human MSCs (Figure 6b). They explained these findings by the fact that the rGO nanoribbon grids can highly adsorb chemical induces in culture media and can also provide physical stress induced by the surface topographical features of rGO nanogrids [29,[80][81][82]. They also proved that those stimulating effects of rGO nanogrids are equally effective on human NSCs [73]. These results indicate that the graphene nanomaterial patterns can be readily applied in biomedical fields. In several studies described above, the excellent biocompatibility and the applicability of graphene nanomaterial patterns in biomedical applications have been demonstrated. However, such patterning of graphene nanomaterials requires elaborate techniques, such as photolithography, dippen lithography and microcontact printing. These techniques, of course, are sufficiently efficient but simpler and more scalable methods have been reported by Wang et al. (Figure 7a) [77]. They simply In several studies described above, the excellent biocompatibility and the applicability of graphene nanomaterial patterns in biomedical applications have been demonstrated. However, such patterning of graphene nanomaterials requires elaborate techniques, such as photolithography, dip-pen lithography and microcontact printing. These techniques, of course, are sufficiently efficient but simpler and more scalable methods have been reported by Wang et al. (Figure 7a) [77]. They simply fabricated wrinkled GO multilayer films by relaxation of GO sheets on pre-stretched elastomers. The wrinkled GO patterns can be easily removed by re-stretching the elastomer substrates and the fabrication process is reversible. In addition, the wavelength and height of GO wrinkled can be controlled by film thickness and pre-stretch. The cell alignment and morphology on the wrinkled GO patterns were also evaluated. It was observed that the fabricated GO wrinkles can effectively induce cell alignment and elongation by contact guidance provided from wrinkled GO patterns (Figure 7b). Hence, it was suggested that the wrinkled GO patterns are promising new approach for functional biomedical applications due to advantages, such as the simplicity and scalability of fabrication. fabrication process is reversible. In addition, the wavelength and height of GO wrinkled can be controlled by film thickness and pre-stretch. The cell alignment and morphology on the wrinkled GO patterns were also evaluated. It was observed that the fabricated GO wrinkles can effectively induce cell alignment and elongation by contact guidance provided from wrinkled GO patterns (Figure 7b). Hence, it was suggested that the wrinkled GO patterns are promising new approach for functional biomedical applications due to advantages, such as the simplicity and scalability of fabrication. On the other hand, intriguing results have been obtained by Kim et al. [76]. Kim et al. ascertained that the stem cell fate can be controlled by manipulating the sizes and geometries of patterned arrays (Figure 8a). They prepared GO-patterned arrays with different sizes and geometries on various types of substrates and examined the differentiation of human adipose-derived mesenchymal stem cells (ADMSCs) on those GO-patterned arrays. Interestingly, the differentiation of human ADMSCs was observed to strongly depend on the geometries of GO-patterned arrays. The GO line patterns promote the elongation and spreading of human ADMSCs following the geometry of GO line patterns, which results in the enhanced osteogenesis of human ADMSCs (Figure 8b). On the contrary, the GO grid patterns guide human ADMSCs to grow in a bipolar orientation and encourage the On the other hand, intriguing results have been obtained by Kim et al. [76]. Kim et al. ascertained that the stem cell fate can be controlled by manipulating the sizes and geometries of patterned arrays (Figure 8a). They prepared GO-patterned arrays with different sizes and geometries on various types of substrates and examined the differentiation of human adipose-derived mesenchymal stem cells (ADMSCs) on those GO-patterned arrays. Interestingly, the differentiation of human ADMSCs was observed to strongly depend on the geometries of GO-patterned arrays. The GO line patterns promote the elongation and spreading of human ADMSCs following the geometry of GO line patterns, which results in the enhanced osteogenesis of human ADMSCs (Figure 8b). On the contrary, the GO grid patterns guide human ADMSCs to grow in a bipolar orientation and encourage the conversion of mesodermal stem cells to ectodermal neuronal cells (Figure 8c). These different cellular behaviors of human ADMSCs could be attributed to the physicochemical and geometric properties of GO-patterned arrays, indicating that the graphene nanomaterial-patterned arrays are particularly attractive for biomedical applications.
conversion of mesodermal stem cells to ectodermal neuronal cells (Figure 8c). These different cellular behaviors of human ADMSCs could be attributed to the physicochemical and geometric properties of GO-patterned arrays, indicating that the graphene nanomaterial-patterned arrays are particularly attractive for biomedical applications. These guidance effects of graphene nanomaterial-patterned arrays are closely related to their size and shape. Zhang et al. confirmed that the width of GO-patterned arrays can directly affect the cell migration, alignment, morphology and cell adhesion [78]. They found that the cytoskeleton contractility, intracellular traction and actin filament elongation are significantly enhanced when the width of the GO-patterned arrays is similar to the cell dimension, which in turn cell migration is greatly increased. Kim et al. also revealed that the shape of GO-patterned arrays can determine cell morphology, migration distance, speed and directionality [79]. Therefore, graphene nanomaterialpatterned arrays fabricated with sophisticated control of structures and properties can provide unique opportunities for biomedical applications. These guidance effects of graphene nanomaterial-patterned arrays are closely related to their size and shape. Zhang et al. confirmed that the width of GO-patterned arrays can directly affect the cell migration, alignment, morphology and cell adhesion [78]. They found that the cytoskeleton contractility, intracellular traction and actin filament elongation are significantly enhanced when the width of the GO-patterned arrays is similar to the cell dimension, which in turn cell migration is greatly increased. Kim et al. also revealed that the shape of GO-patterned arrays can determine cell morphology, migration distance, speed and directionality [79]. Therefore, graphene nanomaterial-patterned arrays fabricated with sophisticated control of structures and properties can provide unique opportunities for biomedical applications.
Bai et al. reported that the GO and poly(vinyl alcohol) (PVA) composite hydrogels can be easily prepared and the GO/PVA composite hydrogels showed pH-sensitive gel-sol-gel transition behaviors (Figure 9) [83]. The GO/PVA composite hydrogels were decomposed with increasing pH value and gel-sol transition occurred. Meanwhile, when the pH value dropped again, the GO/PVA composite hydrogels underwent sol-gel transition. This could be attributed to the surface negative charge of GO originated from the carboxyl groups. The different pH values led to changes in surface charge densities of GO, which in turn, electrostatic repulsion forces between GO sheets were altered. These pH-sensitive properties make the GO/PVA composite hydrogels exceptionally useful as drug delivery carriers. On the other hand, graphene nanomaterial-hybridized hydrogels can be also utilized as cell scaffolds. Cha et al. showed that the mechanical properties of methacrylated gelatin (GelMA) hydrogels can be controlled by incorporation of methacrylate group-introduced GO (MeGO) and the GO-incorporated GelMA hydrogels showed good biocompatibility with fibroblasts [86]. The fracture strength of GelMA hydrogels could be enhanced by incorporating MeGO, while minimizing the changes in their rigidity. These enhanced mechanical properties were due to the interfacial bonding between GO and polymeric network [102,103]. In addition, the incorporated GO or MeGO did not detrimental effects on the viability and proliferation of encapsulated fibroblasts. Qiu et al. also proved the improving effects of graphene incorporation on the mechanical performance of polymeric hydrogels. They introduced the graphene aerogel into the poly(N-isopropylacrylamide) (PNIPAM) hydrogels. The results demonstrated that the good mechanical strength and electrical conductivity of graphene can significantly increase the mechanical performance of polymer hydrogels, suggesting that graphene nanomaterials can be used as reinforcing nanofillers for polymeric composites.
Bai et al. reported that the GO and poly(vinyl alcohol) (PVA) composite hydrogels can be easily prepared and the GO/PVA composite hydrogels showed pH-sensitive gel-sol-gel transition behaviors (Figure 9) [83]. The GO/PVA composite hydrogels were decomposed with increasing pH value and gel-sol transition occurred. Meanwhile, when the pH value dropped again, the GO/PVA composite hydrogels underwent sol-gel transition. This could be attributed to the surface negative charge of GO originated from the carboxyl groups. The different pH values led to changes in surface charge densities of GO, which in turn, electrostatic repulsion forces between GO sheets were altered. These pH-sensitive properties make the GO/PVA composite hydrogels exceptionally useful as drug delivery carriers. On the other hand, graphene nanomaterial-hybridized hydrogels can be also utilized as cell scaffolds. Cha et al. showed that the mechanical properties of methacrylated gelatin (GelMA) hydrogels can be controlled by incorporation of methacrylate group-introduced GO (MeGO) and the GO-incorporated GelMA hydrogels showed good biocompatibility with fibroblasts [86]. The fracture strength of GelMA hydrogels could be enhanced by incorporating MeGO, while minimizing the changes in their rigidity. These enhanced mechanical properties were due to the interfacial bonding between GO and polymeric network [102,103]. In addition, the incorporated GO or MeGO did not detrimental effects on the viability and proliferation of encapsulated fibroblasts. Qiu et al. also proved the improving effects of graphene incorporation on the mechanical performance of polymeric hydrogels. They introduced the graphene aerogel into the poly(Nisopropylacrylamide) (PNIPAM) hydrogels. The results demonstrated that the good mechanical strength and electrical conductivity of graphene can significantly increase the mechanical performance of polymer hydrogels, suggesting that graphene nanomaterials can be used as reinforcing nanofillers for polymeric composites. On the other hand, recently, the natural and synthetic polymers have been extensively used to fabricate biological scaffolds for tissue engineering applications. Polymeric biomaterials have superior biocompatibility and biodegradability but their intrinsic poor thermal and mechanical properties are often quoted as disadvantages. Therefore, much research has been suggested to compensate the poor thermal and mechanical properties of polymeric biomaterials by functionalization of graphene possessing exceptional thermomechanical properties. Shin et al. On the other hand, recently, the natural and synthetic polymers have been extensively used to fabricate biological scaffolds for tissue engineering applications. Polymeric biomaterials have superior biocompatibility and biodegradability but their intrinsic poor thermal and mechanical properties are often quoted as disadvantages. Therefore, much research has been suggested to compensate the poor thermal and mechanical properties of polymeric biomaterials by functionalization of graphene possessing exceptional thermomechanical properties. Shin et al. demonstrated that the impregnation of GO can not only reinforced the poor mechanical properties of polymer nanofiber scaffolds but can also promote myoblast growth and differentiation [93]. Despite excellent biocompatibility of collagen-based scaffold, it suffers from poor mechanical and rapidly degrading properties of collagen. However, the poor mechanical properties, including tensile strength and elastic modulus, of scaffolds could be remarkably improved by the incorporation of GO. These improved mechanical properties of scaffolds could be rationalized by the fact that the oxygen-containing functional groups on GO surface can strongly interact with hydroxyl or amine groups of polymeric substrates, which allows interfacial bonding between GO and polymeric substrates [100,[102][103][104][105]. Moreover, the cellular behaviors of myoblasts, including proliferation and myogenic differentiation, were significantly promoted on the GO-hybridized scaffolds [94,96]. As mentioned above, graphene nanomaterials have great capability in adsorption of serum proteins from culture media, which leads to accelerated myogenic differentiation [29,32]. Furthermore, the promoting effects of graphene nanomaterial-based hybrid scaffolds were confirmed in various types of cells. Shah  demonstrated that the impregnation of GO can not only reinforced the poor mechanical properties of polymer nanofiber scaffolds but can also promote myoblast growth and differentiation [93]. Despite excellent biocompatibility of collagen-based scaffold, it suffers from poor mechanical and rapidly degrading properties of collagen. However, the poor mechanical properties, including tensile strength and elastic modulus, of scaffolds could be remarkably improved by the incorporation of GO. These improved mechanical properties of scaffolds could be rationalized by the fact that the oxygencontaining functional groups on GO surface can strongly interact with hydroxyl or amine groups of polymeric substrates, which allows interfacial bonding between GO and polymeric substrates [100,[102][103][104][105]. Moreover, the cellular behaviors of myoblasts, including proliferation and myogenic differentiation, were significantly promoted on the GO-hybridized scaffolds [94,96]. As mentioned above, graphene nanomaterials have great capability in adsorption of serum proteins from culture media, which leads to accelerated myogenic differentiation [29,32]. Furthermore, the promoting effects of graphene nanomaterial-based hybrid scaffolds were confirmed in various types of cells.  More recently, studies concerning the development of three-dimensional (3D) scaffolds using graphene nanomaterials have been increasingly reported [31,85,95,97,99,101]. It has been well known that the cellular behaviors, including migration, growth, morphology, differentiation and protein expression, are definitely different in 2D and 3D environments [106][107][108]. Thus, developing 3D scaffolds that mimic the in vivo microenvironment of the natural extracellular matrix is critical to biomedical applications. Jakus et al. suggested the feasibility of 3D printing for the fabrication of graphene-based 3D scaffolds ( Figure 11) [95,99]. They developed 3D printable graphene ink composed of poly(lactic-co-glycolic acid, PLGA) and graphene flakes and fabricated 3D-printed graphene scaffolds. It was verified that the mechanical integrity and electrical conductivity of graphene were maintained in the 3D-printed graphene scaffolds and the viability and proliferation of human MSCs were significantly increased on the 3D-printed graphene scaffolds as compared to the PLGA scaffolds. In addition, the expression of neurogenic relevant genes, such as glial fibrillary acidic protein, neuron-specific class III β-tubulin (Tuj1), nestin and microtubule-associated protein 2, was upregulated in human MSCs on 3D-printed graphene scaffolds. Further in vivo studies using a female BALB/c mouse model validated that the 3D-printed graphene scaffolds did not induce a severe immune response or fibrous capsule formation, indicating that the 3D-printed graphene scaffolds were highly biocompatible. These findings expand the versatility and applicability of graphene nanomaterials for emerging biomedical applications. Collectively, the graphene nanomaterial-based scaffolds can provide a great value for the potentials of graphene nanomaterials in biomedical applications.

Conclusions
Splendid progress in nanoscience and nanotechnology has invigorated interest in application of nanomaterials to biomedical fields. Thanks to this interest, there has also been tremendous advances in research on graphene nanomaterials and their biocompatibility and biofunctionality having been gradually established. In this review, some of recent literature concerning the multifaceted

Conclusions
Splendid progress in nanoscience and nanotechnology has invigorated interest in application of nanomaterials to biomedical fields. Thanks to this interest, there has also been tremendous advances in research on graphene nanomaterials and their biocompatibility and biofunctionality having been gradually established. In this review, some of recent literature concerning the multifaceted biomedical applications of functional graphene nanomaterials was summarized and discussed. According to the recent studies, it is obvious that the functional graphene nanomaterials can be employed in a variety of ways to biomedical applications. In addition, the conventional approaches can be further developed by unique properties of graphene nanomaterials themselves, such as exceptional thermomechanical, excellent physicochemical, outstanding electrical and specific biological properties. In particular, many studies support the fact that graphene nanomaterial-coated substrates, -patterned arrays and hybrid scaffolds are typical approaches for biomedical applications of graphene nanomaterials, which allows us to more easily and intuitively understand the potential of graphene nanomaterials.
Although we have focused on three ways to apply graphene nanomaterials in biomedical applications, there are many other ways to employ them in biomedical fields and the potentials for application of graphene nanomaterials to biomedical fields will continue to evolve. Even if more research remains a significant challenge that should be addressed through comprehensive and systematic studies to fundamentally understand the functional graphene nanomaterials, we envision that the functional graphene nanomaterials will become promising novel candidates, which can open the way to handle unsolved problems in the current biomedical field.