Graphene Oxide/Polymer-Based...

1 Graphene Oxide/Polymer-Based Biomaterials

2 Duygu Ege,* Ali Reza Kamali, and Aldo R. Boccaccini

3 Since its discovery in 2004, derivatives of graphene have been developed and4 heavily investigated in the field of tissue engineering. Among the most extensively5 studied forms of graphene, graphene oxide (GO), and GO/polymer-based6 nanocomposites have attracted great attention in various forms such as films, 3D7 porous scaffolds, electrospun mats, hydrogels, and nacre-like structures. In this8 review, the most actively investigated GO/polymer nanocomposites are9 presented and discussed, these nanocomposites are based on chitosan, cellulose,10 starch, alginate, gellan gum, poly(vinyl alcohol) (PVA), poly(acrylamide), poly(e-11 caprolactone) (PCL), poly(lactic acid) (PLLA), poly(lactide-co-glycolide) (PLGA),12 gelatin, collagen, and silk fibroin (SF). The biological and mechanical performance13 of such nanocomposites are comprehensively scrutinized and ongoing research14 questions are addressed. The analysis of the literature reveals overall the great15 potential of GO/polymer nanocomposites in tissue engineering strategies and16 indicates also a series of challenges requiring further research efforts.

17 1. Introduction

18 Graphene, a single layer of sp2-bonded carbon atoms in a19 hexagonal lattice, is one of the most popular nanomaterials of the20 last decade due to its excellent physical, electrical, and thermal21 properties.[1,2] Graphene is the strongest material ever measured.22 It has a high surface area of upto 2630m2 g� 1, Young’smodulus of23 1 TPa, fracture toughness of 130GPa, thermal conductivity of24 5MWK� 1, and relative conductivity of 720 k Sm� 1.[3,4]

25 Graphene oxide (GO) which is a derivative of graphene is the26 oxidized form of graphene with hydroxyls, epoxides, diols, ketones,

1andcarboxyls functionalgroups.Thepresence2of oxygenon the edges andbasal planes ofGO3increases its hydrophilicity and enhances its4water dispersibility in comparison with gra-5phene.[1] It is alsonon-toxic and is biocompati-6ble at low concentrations.[5,6] Studies so far7have shown thatGOgenerallyupregulates the8proliferation and differentiation of cultured9secondary and stem cells.[3,7,8] However, a10dosage above 50μgml� 1 of GO was found to11be cytotoxic.[9,10] Recently, the large-scale12production of graphene-based materials has13been achieved, which has facilitated their14widespread use in many applications includ-15ingbiomedicineand thecommercializationof16these interesting materials.[11–14]

17Due to its excellent physical and biological18properties, GO has found applications in the19field of biomaterials and tissue engineering.20GO improves the physical properties of21polymers even at low amounts. Its carboxyl,22hydroxyl, and epoxy groups enhance the interfacial interaction23between GO and the polymer matrix. This facilitates the stress24transfer from the polymer matrix to GO. A large aspect ratio, high25strength, and highmodulus of GO also contribute to the significant26improvement of GO-reinforced polymers.[3] However, a good27dispersion of GO in the polymer matrix is needed to achieve28desirable physical and biological properties.[1] Natural polymers are29biodegradable, non-toxic with low cost, good processability, and30renewability as opposed to widely used synthetic polymers, which31may release toxic by-products during their degradation.[6] On the32other hand, the mechanical properties of natural polymers33deteriorate upon chemical processes during their extraction.[6] In34general, polymer nanocomposites are of scientific and industrial35interest. Some of the most commonly investigated polymers with36GO for tissue engineering include collagen, gelatin, alginate,37chitosan, cellulose, silk fibroin (SF), polyvinyl alcohol (PVA),38polyacrylamide, poly(e-caprolactone) (PCL), poly(lactic acid) (PLLA),39and poly(lactide-co-glycolide) (PLGA).[15]

40In this review paper, the research on natural, pseudo-natural41and synthetic GO/polymer-based scaffolds including films,42electrospun mats, fibers, 3D porous scaffolds, and hyrogels43that are prepared for biomedical applications are presented and44discussed. Then, themechanical properties of these scaffolds are45given and compared in tables and figures. Finally, the future46potential of these scaffolds and their limitations are provided.

472. GO/Polymer Nanocomposites

48Thus far, the produced structures include films, electrospun49mats, 3D porous scaffolds, fibers hydrogels and nacre-like50structures.[6–8,16–34] A search from 2000 through 2017 was

Dr. D. EgeInstitute of Biomedical Engineering, Bo�gaziSci University, Rasathane St.,Kandilli 34684, Istanbul, TurkeyE-mail: [email protected]

Prof. Dr. A. R. KamaliSchool of Metallurgy, Northeastern University, Shenyang, China

Prof. Dr. A. R. BoccacciniDepartment of Materials Science and Engineering, Institute ofBiomaterials, University of Erlangen-Nuremberg, 91058 Erlangen,Germany

The ORCID identification number(s) for the author(s) of this articlecan be found under https://doi.org/10.1002/adem.201700627.

The copyright line of this paper was changed 29 September 2017 afterinitial publication.

© 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co.KGaA, Weinheim. This is an open access article under the terms of theCreative Commons Attribution-NonCommercial License, which permitsuse, distribution and reproduction in any medium, provided the originalwork is properly cited and is not used for commercial purposes.

DOI: 10.1002/adem.201700627

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1 performed with the search engines Web of Science, Scopus, and2 Google Scholar. The search terms were as follows: chitosan,3 cellulose, starch, alginate, gellan gum, poly(vinyl alcohol), poly-4 (acrylamide), poly(caprolactone), poly(lactic acid), poly(lactide-co-5 glycolide), gelatin, collagenandsilkfibroin, andgrapheneoxide. In6 total, 38 papers on GO/polymer composites were found on7 electrospun mats, 3D scaffolds, hydrogels, and nacre-like8 structures. Figure 1 shows the GO/polymer nanocomposites that9 were produced for tissue engineering applications.10 According to Figure 1, the produced structures are electro-11 spun mats, 3D scaffolds, hydrogels, and nacre-like structures.12 The electrospun mats were studied for potential skin, muscle,13 bone, and neural tissue engineering applications. The 3D14 scaffolds were produced with many different methods including15 freeze drying, supercritical CO2-assisted phase inversion, 3D16 printing, selective laser sintering, and chemical crosslinking.17 The hydrogels were mainly produced for soft tissue engineering18 applications including cartilage, muscle, and peripheral nerve19 tissue regeneration applications.[6–8,16–34]

20 Table 1 shows the production route for different GO/polymer21 scaffolds and the findings of the studies are presented.22 FromTable 1, it is observed that GO incorporation upto 10wt%23 was achieved for GO/polymer scaffolds. However, much lower24 amounts were sufficient for cell adhesion, proliferation, and25 differentiation and improvement of mechanical and thermal26 properties. Further addition of GO in scaffolds led to deterioration27 of physical and biological properties. Overall, addition of GO in28 polymeric scaffolds was effective for stimulating osteogenic,29 myogenic, neural, and chondrogenic differentiation.30 Additionally, there are only several research papers with GO/31 polymer nanocomposites. There is higher number of research on32 electrospunmats. Themostly studied polymers are PCL andPLGA.33 So far, themost extensively studied polymers for production of GO-34 based hydrogels are PVA, polyacrylamide, gelatin, and alginate.

35 2.1. Natural Polymers

36 The widely studied GO/natural polymers are polysaccharides37 and proteins. Polysaccharides are extensively investigated in the

1field of biomedical engineering.[50] To improve their physical and2biological properties, they are blended with each other or other3materials. After recognition of the high potential of GO in tissue4engineering, GO and polysaccharides were studied together for5potential biomedical applications. The most extensively studied6polysaccharides are cellulose, chitosan and alginate followed by7starch and gellan gum. The studied protein-based polymers are8gelatin, collagen, and SF.[15]

92.1.1. Chitosan

10Chitosan is a biocompatible, biodegradable, antibacterial,11pseudo-natural cationic polymer that is derived from approxi-12mately 50% deacetylation of chitin which is one of the most13abundant natural polysaccharides.[51,52] It is considered to be the14most commonly used natural polymer for various tissue15engineering applications.[53] Chitosan has many functional16groups and in an acidic environment, its amino groups can17be protonated to form electrostatic interactions with anion18groups.[52] Chitosan is easily converted to hydrogels and can be19molded into any desired shape. It has the ability to support the20attachment and migration of osteoblast cells and enhances the21formation of mineralized bone matrix.[54–56] GO/chitosan22nanocomposites were prepared in the form of films, nacre-like23structures, 3D porous scaffolds, and hydrogels.

24Chitosan Films25GO is incorporated into chitosan to improve its physical and26biological properties. There is considerable number of studies on27preparation of GO/chitosan films. Han et al.[57] fabricated about280.6-mm-thick GO/chitosan films with incorporations of 6.25,2911.76, and 16.67wt% GO. Rheological studies have shown that30the viscosity of the mixture increased with the addition of GO.31The addition of GO beyond 18wt% did not produce successful32results due to the high viscosity of the mixture. FTIR results33revealed that the interaction between GO and chitosan occurred34via hydrogen bonding between GO and the hexatomic ring of35chitosan. This interaction was evident from the peaks at 168036and 1608 cm� 1. The strong interaction betweenGO and chitosan37was also evident from the improved thermal stability of GO/38chitosan films compared to chitosan films. Moreover, with the39addition of GO, the crystallinity of chitosan decreased which was40observed from the decrease in the intensity of the XRD peak of41chitosan at 14.9�. The mechanical tests in both dry and wet42conditions also supported the results of FTIR and TGA. For pure43chitosan, a drastic reduction of tensile strength was observed in44the wet state in comparison to the dry state. With the addition of45GO, the drop of tensile strength was much less significant in the46wet state than in the dry state. The mechanical properties of GO/47chitosan in the wet state were found to be advantageous for48biomedical applications.[57]

49Similarly, Yang et al.[58] prepared nanocomposites of GO/50chitosan. In this study, the amount of GO in chitosan was kept at510.3, 0.5, and 1wt%. Chitosan was dissolved in 0.5 v/v% aqueous52acetic acid and GO solution was added to the acetic acid solution.53The authors stated that an electrostatic interaction occurred54between the polycations of chitosan and the negative charges on55the surface of GO in addition to hydrogen bonding. This

Duygu Ege is an Assistant Professorat Bo�gaziSci University, Institute ofBiomedical Engineering. She has aPhD in Medical Materials fromUniversity of Cambridge and aMaster of Engineering in MaterialsScience and Engineering fromImperial College, London. Herresearch so far has concentrated onthe production and processing of

novel biomaterials including bioactive and bioinertceramics, carbonaceous materials, and polymers for bonereplacement, implant coatings, nerve regeneration,muscle tissue engineering, and drug delivery devices.

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Figure 1. a) Produced GO/polymer nanocomposite structures for potential tissue engineering applications, pie charts show the percentage of researchon each polymer for b) 3D porous scaffolds, c) electrospun mats, and d) hydrogels.





Table 1. Structure, method of production, chemical interactions, cell line, and advantages of addition of GO in polymeric scaffolds in electrospunmats and 3D porous structures, hydrogels (RGO, reduced GO; PAM, polyacrylamide; BC, bacterial cellulose; CMC, carboxymethyl chitosan;GelMa, gelatin methacrylate; PU, polyurethane; HADSC, human adipose derived stem cells).

GO content/polymerStructure/processing

route Type of cells Advantages of GO

0.02 wt% GO/PVA[35] ESM by electrospinning N/A Tensile strength increase by 42 times with 0.02 wt% GO

0.1 wt%GO/PCL[25] ESM by electrospinning N/A 53% increase in tensile strength

0.3 wt%GO/PCL[36] ESM by electrospinning N/A Increased tensile strength, modulus and energy at break and bioactivity.

0.1–1 wt% GO/PCL[37] ESM by electrospinning N/A 0.1 wt% GO increased mechanical properties and 1 wt% GO weakened the ESM.

0–2 wt%GO/BC/chitosan[16] ESM by electrospinning N/A GO improved the tensile strength and modulus. Water vapor permeability decreased with the

addition of GO.

0–1 wt%GO/PLLA[34] ESM electospinning N/A Improved mechanical properties

0.1–1 wt% RGO/PVA[38] Hydrogel by crosslinking

with γ-ray irradiation

N/A Irradiation improved the mechanical properties.

0.5–1.5 wt% GO /PVA[39] Hydrogel by freeze-

thawing method

N/A 1 wt% GO improved tensile strength and elongation at break

1 wt%GO/PLGA[32] ESM by electrospinning MSC Improves hydrophilicity, protein adsorption ability, adhesion, and proliferation of MSC and

osteogenic differentiation

10 wt%GO/collagen[8] 3D porous structure by

chemical crosslinking

hMSC GO enhanced osteogenic differentiation.

0.05–1 mg ml� 1 GO/

GelMa[40]

Hydrogel by 3D printing hMSC GO improved cell adhesion, proliferation, and chondrogenic differentiation

0.5–3 wt% GO/chitosan[41] 3D porous structure by

freeze-drying

MC3T3-E1 GO improve mechanical properties, and pore formation and bioactivity

0.1 wt% GO/CMC[42] 3D porous structure by


HADSC Increase of osteoinductivity; osteogenesis related genes were up regulated

0.5–1 wt% GO/chitosan/

HYA[43]

3D porous structure by


MC3T3-E1 Improvement of porosity, density, swelling, degradation, and biomineralization

0.5–3 wt% GO/PVA/

gelatin[20]


freeze-drying

MC3T3-E1 With addition of GO, a smoother porous structure was achieved and the mechanical properties

were also enhanced.

0.1–1 wt% GO/SF[19] 3D porous structure by

freeze-drying

MC3T3-E1 GO decreased pore size, and improved mechanical properties. A higher cell viability was

achieved for scaffolds with 0.1 wt%, 0.2 wt% and 0.5 wt% GO

RGO and GO coating with

0.1 wt% GO/collagen[7]



MC3T3-E1 RGO coated scaffolds showed higher bioactivity than GO coated scaffolds. Compressive

strength of RGO and GO coated scaffolds were higher than pure collagen scaffolds

3 wt% GO/PVA[135] Hydrogels by chemical

crosslinking

chondrocyte GO/PVA hydrogels supported chondrocyte adhesion and growth and found promising for load

bearing applications

0–500 μg ml� 1 GO/RGD/

PLGA[30]

ESM by electrospinning C2C12 GO stimulated myogenic differentiation of C2C12 cells

0–500 μg ml� 1 GO/RGD/

PLGA[31]

ESM by electrospinning C2C12 GO enhanced growth and differentiation of C2C12 cells

10 μg ml� 1–1 mg ml� 1 GO/

alginate[44]

Hydrogel by cell

microencapsulation

C2C12 25–50 μg ml� 1 cell laden hydrogels improved cell viability

100μg ml� 1 GO/gelatin[102] Hydrogel by cell

microencapsulation

C2C12 C2C12 grew and proliferated in GO/gelatin hydrogels. GO stimulated myogenic differentiation

0 and 2 wt% GO/PLGA[29] ESM by electrospinning PC12 2 wt% GO improved Tg and mechanical properties more than 1 wt% GO. 2 wt% GO also

improved hydrophilicity, neuronal cell viability, and proliferation

0–1 wt% GO/PCL[144] ESM by electrospinning mMSCs,

PC12

0.3 and 0.5 wt% GO dramatically enhanced differentiation of MSC and PC12 cells

0.1–1 mg ml� 1 GO/PCL[45] ESM by electrospinning neural stem

cells

GO/PCL promoted differentiation of neural stem cells into mature oligodendrocytes.

0.2–1.2 mg ml� 1 GO/

PAM[46]

Hydrogel by in situ radical

polymerization

Schwann

cells

0.4 mg ml� 1 GO led to highest cell attachment and proliferation

0.5–1 mg ml� 1 GO/ Hydrogel by UV NIH3T3 GO enhanced 3D cell spreading and proliferation. GO also improved electrical and mechanical

(Continued)





1 interaction was evident from XPS results which showed an2 increase of protonated amines from 7.1 to 11.3% with the3 addition of 1wt% GO in chitosan.4 Hydrogen bonding and electrostatic attraction between GO5 and chitosan also constrained the segmental motion of chitosan6 chains. This constraint led to an increase in the Tg of chitosan7 from 175.4 to 180.4 �C. The mechanical properties improved8 with the addition of GO in chitosan in terms of the Young’s9 modulus, tensile strength, and elongation at break. The10 significantly improved mechanical properties were attributed11 to the unidirectional homogeneous dispersion of GO in the12 chitosan matrix and strong interfacial interactions.[58]

13 Different crosslinking methods were used to improve the14 mechanical properties of the nanocomposite films. Shao et al.[59]

15 crosslinked GO and chitosan at 100 �C in order to enhance the16 mechanical properties of the nanocomposites. The authors17 suggested the potential of GO crosslinked chitosan nano-18 composites in the field of biomaterials.[59] Genipin was also used19 as a crosslinker between GO and chitosan by Li et al.[60] This20 study showed that the presence of GO leads to a more compact21 arrangement of chitosan matrix due to hydrogen bonding which22 hindered the water containment and swelling of the nano-23 composites. Therefore, by adding GO, the swelling of24 hydrophilic polymers can be controlled. In addition, the25 incorporation of GO also improves the resistance of nano-26 composite films against enzymatic degradation. For genipin27 crosslinked chitosan/GO films, the highest tensile strength was28 obtained in samples containing 1wt% GO. A further rise in GO29 led to the aggregation of GO sheets and lowering of the tensile30 strength of the nanocomposites.[60]

31 A Simulated Body Fluid study was carried out for GO/32 chitosan films. The presence of GO’s functional groups33 influenced the biomineralization and that of chitosan’s � NH2

34 influenced the apatite deposition. Cell culture studies with35 osteoblasts showed that the GO/chitosan nanocomposite was36 more effective for cellular activity, leading to more extensive37 spreading and more distinct actin stress fibers than GO. A38 higher level of vinculin focal adhesion contacts was also observed39 in this case compared to the case of pristine GO. Fibronectin40 expression was also higher for the GO/chitosan nanocomposite41 than pristine GO, which indicated synthesis, mineralization, and42 organization of the extracellular matrix. In line with these43 results, cell viability was also found to be the highest for GO/44 chitosan among these study groups. Alizarin red staining, which45 is the strongest indicator of osteoblast differentiation and bone46 formation, showed that the highest mineralization was for GO/

1chitosan. Therefore, the GO/chitosan hybrid nanocomposite was2observed to be superior in terms of its cellular activity than3pristine GO.[61]

4Mazaheri et al.[62] also fabricated GO/chitosan films with 1.5,53, and 6wt% GO. The antibacterial performance of the films was6examined against Staphylococcus aureus as a gram-positive7bacterium after a 3 h incubation period. The antibacterial activity8of the films increased with an increase of GO. This result was9explored from the interaction of both the membrane and sharp10edges of the GO sheets with the bacterial cell membrane. In11addition, cell culture studies were carried out with mesenchymal12stem cells and the surface density of the cytoskeleton fibers and13the number of cell nuclei had the maximum value for the 1.5wt14% GO/chitosan films. This result was attributed to the moderate15toxicity caused by higher amounts of GO on the cells.16Artificial nacre was also constructed with 70, 75, 80, 90, and1795wt% GO/chitosan.[63] The tensile strength and toughness of18the artificial nacre was 526.7MPa and 17.7MJm� 3, respec-19tively. These values were four and 10 times higher than natural20nacre, respectively. In addition, a very high electrical conduc-21tivity was achieved. Due to its high mechanical properties and22electrical conductivity, the authors suggested that it has23potential for artificial muscles and tissue engineering applica-24tions. Figure 2 shows the mechanical properties of GO/polymer25nanocomposites.

Table 1. (Continued)

GO content/polymerStructure/processing

route Type of cells Advantages of GO

gelatin[47] crosslinking properties

5 mg ml� 1 GO/CMC[48] Hydrogel by blending in

distilled water

NIH-3T3 GO incorporation improved compressive strength and modulus. Also GO incorporated

hydrogels were biocompatible

0.5–5 wt%GO/PLLA/PU[22] 3D porous structure by

3D printing

NIH3T3 GO enhanced mechanical and thermal properties. Small amount of GO enhanced cell

proliferation

0.02–0.1 g RGO/PVA[49] Hydrogel by chemical

crosslinking

A431 GO incorporated hydrogels had good biocompatibility and improved mechanical performance

Figure 2. Comparison of the mechanical properties of natural nacre andother GO-based nanocomposites, such as GO-PMMA, RGO-PVA, andRGO-SL, GO–Mg2þ, GO–Ca2þ, GO–Fe3þ, RGO-Poly(acrylic acid-co(4-acrylamidophenyl)boronic acid) (PAPB), GO–GA, GO-borate, PGO-PEI,RGO-PCDO, RGO-PDA, and other GO/chitosan nanocomposites withlow GO content. (reproduced with permission from ref.[63] 2015 ACSPublications).





1 Chitosan 3D Porous Scaffolds2 In addition to films, 3D porous scaffolds of GO/chitosan were3 also prepared with the incorporation of 0.5 and 3wt% GO.4 Dinescu et al.[41] stated that with the addition of GO, more5 defined and highly interconnected pores were established. MTT6 assay with murine pre-osteoblasts showed that 0.5wt% GO/7 chitosan and 3wt% GO/chitosan scaffolds led to higher8 metabolic activity and proliferation of the cells than pure9 chitosan.[41]

10 Depan et al.[61] produced high-strength 3D scaffolds with11 covalent linkage between GO and chitosan. These scaffolds12 possessed a high elastic modulus of over 6GPa and a hardness of13 approximately 1.2GPa. The results also suggested that the14 presence of GO with high polarity was mainly responsible for15 high cell attachment on the scaffolds due to the presence of16 pendant hydroxyl groups on GO.17 Figure 3 shows the interaction between GO and chitosan,18 degradation studies with chitosan, and GO/chitosan films and19 the SEM images of porous GO/chitosan scaffolds.[57,58,64]

20 Moreover, carboxymethyl chitosan and GO nanocomposites21 were developed to enhance physicochemical properties and22 osteoinductivity. Carboxymethyl chitosan was utilized because of23 its carboxyl and amino groups which led to the formation of24 scaffolds with improved physical properties and cellular activity.25 Carboxymethyl chitosan and GO scaffolds with amide linkages26 provided osteogenic differentiation of seeded cells which was27 attributed to the high density of functional groups on the scaffold28 surface.[42]

29 In another study, Unnithan et al.[43] incorporated hyaluronic30 acid (HYA) with chitosan/GO in 3D scaffolds and subsequently,31 the scaffolds were crosslinked with EDC. HYA is immuno-32 neutral and hydrophilic, and it has negatively charged linear D-33 glucuronic acid. Therefore, it has electrostatic interactions with34 chitosan. HYA is a major constituent of connective, epithelial,35 and neural tissues and the natural extracellular matrix. It is also36 a major constituent in the early fracture callus in lacunae. HYA37 enhances bone regeneration along with other osteogenic38 materials.[43] Studies showed that blending HYA with chitosan39 increased the degradation rate of the scaffolds which was40 associated with higher hydrophilicity on HYA. The tensile41 strength of GO/HYA/chitosan was approximately 10MPa,42 which is stated as an optimal value for tissue engineering43 applications. MC3T3 cells had good attachment and well44 spreading on the scaffolds. CCK assay showed that with a rise in45 the wt% of HYA from 0.5 to 1wt%, the proliferation of cells46 slightly increased.[43]

47 Chitosan Hydrogels48 Fan et al.[48] crosslinked carboxymethyl chitosan with konjac49 glucomannan (KGM) to produce hydrogels via Schiff-base50 reaction between the free amino group of carboxymethyl51 chitosan and the aldehyde groups of KGM. To improve the52 mechanical properties of the hydrogels, GO was also incorpo-53 rated into the hydrogels. The gelation time of the hydrogels54 decreased from 26 to 19 s with the addition of 5mgml� 1 GO. In55 addition, the swelling ability of hydrogels decreased with the56 addition of GO due to the introduction of hydrogen bonds57 between GO and the polymer chains. The mechanical properties58 of hydrogels were also enhanced by the addition of GO.

1Hydrogels were also biocompatible and they were suggested for2wound dressing.3Overall, GO/chitosan scaffolds are extensively studied. 3D4porous scaffolds show potential for bone tissue engineering,5nacre-like structures were found suitable for artificial muscles6and tissue engineering and GO/chitosan hydrogels were7suggested for soft tissue engineering applications.

82.1.2. Cellulose and Starch

9In this section, the research on two very similar polymers namely10cellulose and starch are covered together. Both cellulose and11starch are formed from the same monomeric unit, glucose.12Cellulose is the most abundant polysaccharide, and it is13inexpensive with good processability, renewability, and ease of14physical and chemical modification.[52] It has good mechanical15properties, good hydrolytic stability, low toxicity, and excellent16compatibility to the body environment.[65,66] Bacterial cellulose17(BC), methyl cellulose, and carboxymethyl cellulose are18commonly studied types of cellulose in tissue engineering.19BC differs from plant cellulose in its higher purity, crystallinity,20and tensile strength.[67] To further improve their physical and21biological properties, GO has been incorporated in different22types of cellulose. To date, different types of cellulose and its23derivatives have been blended with GO including BC and24carboxymethyl cellulose. Films, electrospun fibers, and 3D25porous scaffolds were developed for different types of cellulose.

26Cellulose Films27Carboxymethyl cellulose which is an anionic polysaccharide was28blended with GO to prepare films. Carboxymethyl cellulose is29formed from the substitution of the 2, 3, and 6 hydroxyl groups30of cellulose with carboxymethyl groups. It has high viscosity31which has advantages in biomedical engineering.[68,69] Yadav32et al.[70] blended carboxymethyl cellulose, alginate, and GO as33they are compatible because of the formation of hydrogen bonds.34By solution casting different wt% of GO in 25% carboxymethyl35cellulose/75% alginate, 0.04-mm thick films were obtained. Both36the tensile strength and Young’s modulus of the films increased37with the incorporation of 1wt% GO into 25% carboxymethyl38cellulose/75% alginate at the expense of flexibility of the films.

39Cellulose Electrospun Fibers40Azarniya et al.[16] prepared electrospun GO/chitosan/BC nano-41fibrous nanocomposites for potential skin tissue engineering42applications. BC and chitosan have structural similarities, and43this feature leads to good miscibility of the two materials. In the44preparation, 10% poly(ethylene oxide) was added in the BC/45chitosan blends in order to improve the spinnability and46uniformity of the fibers. With an increase of chitosan of the47nanocomposites, the conductivity of the jet increased. Moreover,48with an increase of GO content to 2%, the viscosity of the49solution increased and electrospinning became difficult.[16]

50Cellulose 3D Porous Scaffolds51A few attempts were made at the production of 3D porous52scaffolds with different types of cellulose, which were developed53for tissue engineering applications. Shao et al.[71] prepared





Figure 3. a) Electrostatic interaction between GO and chitosan (reproduced with permission from ref.[58] 2010 ACS Publications). Photographs of purechitosan (Left) and 18 wt% GO/chitosan (right) films immersed in distilled water for b) 10 min and c) 30 Days (reproduced with permission from ref.[57]2011 Elsevier) d) SEM images of d–f) 1 wt% GO/chitosan and g–I) 3 wt% GO/chitosan scaffolds illustrating the porous nature and interconnected porestructure (reproduced with permission from ref.[61] 2014 Elsevier).





1 freeze-dried BC/GO nanocomposites for potential biomedical2 applications. Upto 8wt% GO was incorporated in BC to induce3 anti-bacterial activity to the nanocomposites. SEM images4 revealed a nano-porous 3D network in pure BC samples. In5 the pristine form, BC fibrils were randomly arranged with empty6 spacing. With the addition of GO, BC fibrils became more7 compact. In the nanocomposites, GO exhibited a layered8 structure due to the applied vacuum and its high aspect ratio9 whereas pure BC was randomly arranged without any10 preferential orientation. In addition, MTT assay showed that11 GO did not have a significantly negative impact on the12 proliferation of HEK293 cells. BC/GO nanocomposites were13 found to decrease bacterial attachment more significantly than14 BC. E.coli attachment reduced by 95.61% and S. aureus15 attachment decreased by 65.35% in 1 h. This result was16 attributed to the high electron donor component of BC/GO. A17 high electron donor component led to a lower zeta potential18 since a negative charge is repellent to the negatively charged19 bacteria. This negative charge causes the repulsion of bacteria20 from BC/GO surfaces. Therefore, the authors proposed BC/GO21 nanocomposites as promising biomaterials due to their anti-22 bacterial properties and good biocompatibility.[71]

23 Baldino et al.[72] prepared GO/cellulose acetate nanocompo-24 sites by supercritical CO2-assisted phase inversion. When the wt25 % of GO increased from 2 to 9wt%, the pore size in the26 nanocomposites increased. A wrinkled GO structure was27 observed in TEM images due to surface tension during the28 evaporation process. FESEM showed that the morphologies of29 cellulose acetate and GO/cellulose acetate were similar. To30 conclude, GO/cellulose has been blended together with different31 polymers including alginate and chitosan to prepare scaffolds for32 tissue engineering. The research so far indicate that these33 studies are at an early stage therefore further cell culture studies34 and mechanical testing is required to thoroughly examine their35 potential for different tissue engineering applications.36 Starch is composed of D-glucopyranose polymers linked37 together by α-1,4 and α-1,6 glycosidic bonds.[73] Starch is38 biodegradable, renewable and inexpensive, it is one of the most39 abundant carbohydrates in the nature, and to date, it has been40 heavily investigated for tissue engineering applications.[52,74]

41 However, starch also has some disadvantages such as strong42 hydrophilicity, high brittleness, and low thermal stability.[52]

43 Therefore, starch is often blended together with other polymers.44 GO is also incorporated in starch in order to improve its physical45 and biological properties.[67,75,76] GO/starch nanocomposites46 were produced in the form of films.

47 Starch Films48 Li et al.[52] easily prepared films of upto 2wt% GO in pea starch49 by dissolving them in distilled water, followed by solution50 casting. Strong hydrogen bonding formed between pea starch51 and GO. With an increase in the wt% of GO, both the strength52 and stiffness increased at the expense of elongation at break. A53 lower elongation at break was associated with restraint of54 slippage movement among starch molecules due to GO.[52] In55 another study, films of chitosan and oxidized starch were56 prepared. The mechanical testing showed that the chitosan/57 oxidized starch nanocomposites had a higher Young’s modulus58 and stress at break than chitosan/starch films. However, the

1elongation at break was lower for the chitosan/starch films than2the chitosan/oxidized starch films.[77] Moreover, the addition of3GO improved thermal stability and reduced moisture up-4take.[52,77] Since there is only very few number of papers, GO/5starch nanocomposite research is at an infant stage and more6investigation is needed to understand its true potential in tissue7engineering.

82.1.3. Alginate and Gellan Gum

9In this section, alginate and gellan gum-based GO nano-10composites will be delivered together as these are complemen-11tary polymers. Alginate which was discovered in 1880s is a12naturally occurring anionic biocompatible polymer with low13toxicity and relatively low cost. It is typically obtained from brown14seaweed, and it has been extensively investigated for many15biomedical applications. Alginate contains blocks of (1,4)-linked16β-D-mannuronate (M) and α-L-guluronate (G) residues. Alginate17gels can be easily prepared by the addition of divalent cations18such as Ca2þ.[78,79] Unfortunately, alginate is non-degradable in19physiological conditions so its degradation requires alterations to20avoid interference with tissue regeneration. Additionally,21alginate has strong hydrophilicity and low thermal stability.22Moreover, alginate gels do not have adequate mechanical23properties for load-bearing in the initial stages of bone24regeneration.[80] GO/alginate nanocomposites were mainly25produced in the form of films and there is also research on26production of nacre-like structures and hydrogels.

27Alginate Films28To overcome drawbacks of pure alginate, alginate/GO nano-29composites were developed.[81,82] Ionita et al.[81] produced GO/30alginate films by casting to improve the mechanical properties31and physical integrity of GO. In addition, by inducing electro-32activity, cell growth could be stimulated. Sodium alginate was33dissolved in double-distilled water and ultrasonicated GO34suspension was slowly added to the sodium alginate solution.35Then, approximately 70-μm thick films were cast onto petri36dishes at the ambient temperature. A homogeneous distribution37of GO sheets could be obtained due to oxygen functional groups38in the basal planes and borders of the GO sheets. Moreover,39interfacial bonding occurred between the hydroxyl groups of40alginate and oxygen functional groups of GO sheets. Figure 441shows the interfacial bonding between GO and alginate. FTIR42confirmed the interfacial bonding between the hydroxyl and43oxygen groups as an alginate peak due to � OH stretching and as44a GO peak due to � H stretching of GO at 3347 and 3401 cm� 1,45respectively, leading to a broadened and shifted peak at463331 cm� 1. Figure 4 shows the chemical interaction between47GO/alginate nanocomposites.[81]

48XRD studies confirmed the solely physical interaction of49alginate and GO with the formation of nanocomposites and d50spacing of GO increased from 8.06 to 17 A when incorporated in51alginate which confirmed the intercalated structure of the52nanocomposite. In addition, the incorporation of GO led to53further broadening of the broad diffraction peak at 13�. This54result indicated hindrance of a relatively ordered arrangement of55alginate with the incorporation of GO, which induced a rough





1 morphology to the films. GO sheets were uni-directionally2 aligned parallel to the surface of the films and agglomeration3 occurred. Finally, a “brick and mortar” structure formed with4 aligned and uniform intercalating GO sheets with alginate5 chains. Tensile strength tests confirmed that even a small6 amount of GO significantly enhanced themechanical properties.7 This significant increase was related to hydrogen bonding8 between GO and alginate and to the “brick andmortar” structure9 of the nanocomposite. This structure led to a uniform stress10 distribution and avoided any stress concentration center.11 To further improve the interaction between sodium alginate12 andGO, Nie et al.[82] used tetraethylenepentamine tomodify GO.13 This modification led to many nitrogen active sites. When14 nitrogen groups were protonated, they formed electrostatic15 interactions with the COO� groups on sodium alginate chains.16 When up to 2wt% modified GO (MGO) was incorporated into17 sodium alginate, the FTIR spectrum indicated a stronger18 interaction of sodium alginate and GO due to the � NH2 and19 � NH groups in the GO structure. MGO was well exfoliated into20 nanosheets, which was similar to GO. SEM images revealed that21 MGO had a wrinkled laminar structure in alginate, which was22 similar to GO/alginate. MGO had much more abundant groups23 between layers than GO which was possibly due to existence of24 nitrogen-containing functional groups. Moreover, the thickness25 of MGO was greater than that of GO in the nanocomposite26 which was also due to presence of nitrogen groups. The authors27 declared that GO aggregated with a loading of 1.5wt% whereas28 MGO aggregated with a loading of 2wt%. A much stronger29 interaction was suggested for MGO/sodium alginate than for30 GO/alginate due to stronger electrostatic interactions between31 MGO and sodium alginate. Moreover, MGO/sodium alginate32 exhibited a much greater tensile strength and Young’s modulus33 than those of GO/sodium alginate due to improved electrostatic34 interactions between sodium alginate and MGO.[82]

35 As mentioned earlier, the hydrophilicity of alginate is very36 high. Moisture absorption studies showed that the moisture

1uptake was much lower for GO/sodium alginate and MGO/2sodium alginate than for sodium alginate. MGO/sodium3alginate’s moisture uptake was relatively lower than that of4GO/sodium alginate which was attributed to greater interfacial5interaction betweenMGO and sodium alginate thanGO/sodium6alginate. The dispersibility of MGO/sodium alginate and GO/7sodium alginate films was assessed by the measurement of UV-8vis percent transmittance values. The transmittance of MGO/9sodium alginate was greater than that of GO/sodium alginate10films which was explained by fewer aggregate domains inMGO/11sodium alginate than in GO/sodium alginate to obstruct the12passage of light.13Hu et al.[83] produced nacre-like nanocomposites with 67%14GO/alginate nanocomposites by a wet spinning method. Ultra-15strong nacre-like fibers could be obtained with wet-spinning.16Application of wet-spinning assembly technique may enable17large-scale production of high strength biomaterials. Finally, cell18laden GO/alginate hydrogels were also prepared. Burgo et al.[44]

19produced GO/alginate hydrogels withmicroencapsulated C2C1220myoblast cells. In vivo studies demonstrated that these21structures were functional for the enhancement of the22hamatocrit levels in mice. Overall, the literature shows that23although GO/alginate scaffolds in the form of films, nacre-like24structures, and hydrogels are promising for biomedical25applications.26Gellan gum was discovered in 1978. It is an anionic high27molecular weight bacterial exopolysaccharide and it is secreted28by Pseudomonas elodea. It ismade of a tetrasaccharide repeating29unit of one l-rhamnose (Rhap), one d-glucuronic acid (GlcpA),30and two d-glucoses (Glcp). Recently, gellan gum has been31investigated as a material for biomedical applications because of32its biocompatibility and low cytotoxicity and its structural33similarity to glycosaminoglycans.[84,85] It has high water holding34capacity and mucoadhesive potential, which makes it useful in35oral, ophthalmic, nasal, and other formulations. Moreover, it has36been successfully employed in biomedical research as an

Figure 4. Schematic representation of GO/alginate nanocomposite film structure (reproduced with permission from ref.[81] 2013 Elsevier).





1 absorbing material for wound healing and for stomatology and2 as a cell carrier for tissue engineering, particularly in cartilage3 regeneration.[85,86] So far, GO/gellan gum nanocomposites were4 mainly produced in the form of films.

5 Gellan Gum Films6 Many attempts have beenmade to mimic nacre and gellan gum/7 GO films were one of the successful efforts.[87–96] Vacuum8 filtration was used to rapidly self-assemble GO/gellan gum into9 aligned nacre-like films. The produced films were strong and10 flexible. Figure 5 shows the structure of prepared gellan gum-GO11 films.[97]

12 Figure 5 demonstrates that hydrogen and coordinate bonds13 were found between gellan gum macromolecules and GO. The14 ionic and coordinate bonds are responsible for the high15 toughness of gellan gum/GO films. In addition, the presence16 of hydrogen bonds between gellan gum and GO led to high17 stiffness and strength. Since these films also had high18 biocompatibility, they show potential in tissue engineering19 and medical devices.[97]

20 Finally, Mun et. al.[98] found that mixing gellan gum with21 sodium alginate significantly improved the tensile strength and22 Young’s modulus. This result was attributed to the strong23 interaction of molecules via hydrogen bonding which also24 improved the miscibility of the polymers. The incorporation of25 0.75wt% GO further improved the mechanical properties. A26 further increase in the concentration of GO, reduced the ductility27 and tensile strength compared to 0.75wt% GO incorporated28 films. FTIR results revealed that GO acted as a crosslinking29 agent between gellan gum and alginate molecules which30 improved the miscibility of the molecules of gellan gum and31 alginate. FESEM also supported these results as strong32 interfacial adhesion was evident from the breaking of GO33 sheets rather than them being pulled out.[98] Overall, limited34 research shows that GO/gellan gum has potential tissue35 engineering applications.

12.1.4. Collagen and Gelatin

2Collagen is one of the main components of bone, cartilage,3tendons, ligaments, and skin.[99] Gelatin is obtained from4collagen by acidic or basic hydrolysis or thermal degradation.[6]

5This sequence leads to rupture of the collagen triple helix into6the random coil structure of gelatin. It is a biodegradable,7biocompatible, hydrophilic with low cost. Gelatin is used in8tissue engineering, drug delivery, and gene therapy and as a9vascular prosthesis and wound dressing.[100] However, it is10moisture sensitive, with poor mechanical properties, including a11low toughness. Therefore, it is used with reinforcement fillers or12non-covalent/covalent crosslinking although residual crosslink-13ing agents could lead to toxic side effects.[1,3] Gelatin loses its14fibrous structure when it is exposed to a high degree of moisture.15Therefore, a crosslinking agent is needed to improve its water16stability.[1] Since the applications of collagen and gelatin are17similar, in this section, the research on GO/gelatin and GO/18collagen scaffolds are both analyzed simultaneously.

19Gelatin Films20Moisture sensitivity and low toughness limits gelatin’s biomedi-21cal engineering applications. To improve gelatin’s physical22properties, up to 2wt% GO was blended in with gelatin. Wan23et al.[3] prepared GO/gelatin nanocomposites via solution24casting. With the addition of GO, the strength, modulus, and25ductility simultaneously increased. Tg rise was also observed26with an increase in the wt% of GO. This effect was attributed to27hydrogen bonding between the GO sheets and the polar groups28of gelatin macromolecular chains. In 1wt% GO in gelatin, GO29nanosheets were fully exfoliated whereas in 2% GO in gelatin,30GO was partially exfoliated. Therefore, in this nanocomposite,312wt% GO in gelatin had a higher reinforcement effect than 2wt32% GO in gelatin. Biomineralization studies proved that anionic33functional groups in GO attract Ca2þ cations deposition onto the34film, which is followed by the formation of minerals with PO4

3� .

Figure 5. Fabrication of the bio-inspired gellan gum (GG)-GO nanocomposite film (reproduced with permission from ref.[97] 2015 Elsevier).





1 Gelatin Nanofibers2 A number of studies were conducted on the production of3 electrospun GO/gelatin mats.[1,6,100] Jalaja et al.[1] and Nagarajan4 et al.[6] crosslinked gelatin with dextran aldehyde and gluter-5 aldehyde, respectively. More than 0.5wt% of GO in gelatin led to6 the agglomeration of GO. Crosslinking of gelatin with dextran7 aldehyde improved the water stability of the electrospun mats.8 The addition of GO and crosslinkingwith dextran aldehyde led to9 increased mechanical properties and thermal stability. Interest-10 ingly although GO suspension induced anti-microbial proper-11 ties, GO/gelatin mats supported bacterial growth. No anti-12 microbial activity of GO/gelatin was attributed to the unavail-13 ability of GO sheets to damage bacterial cell walls. Finally, GO/14 gelatin mats showed very good viability and proliferation for L-15 929 fibroblast cells. The biocompatibility of glutaraldehyde16 crosslinked gelatin/GO was assessed with HOS cells. The study17 showed that the addition of GO did not affect the biocompati-18 bility of electrospun mats. The expression of osteoblast genes19 significantly decreased with increase in GO concentration,20 except for SP7. This result was related to the oxidation stress21 exhibited by GO on the cells. MTT assay results for 8 days22 showed that glutaraldehyde slightly decreased the biocompati-23 bility of electrospun mats. Overall, GO addition did not24 significantly reduce the biocompatibility of gelatin.

25 Collagen 3D Porous Scaffolds26 Three-dimensional porous scaffolds were prepared with GO/27 collagen nanocomposites. Kanayama et al.[7] coated 3D scaffolds28 of collagen with GO or RGO. RGO was reduced either with29 ascorbic acid or sodium hydrosulfite solution. Coating collagen30 with GO or RGO significantly enhanced its compressive31 strength. Animal studies proved the high tissue compatibility32 of GO or RGO-coated collagen scaffolds. Moreover, RGO-coated33 collagen scaffolds had higher bioactivity than GO-coated34 collagen scaffolds. Kang et al.[8] also prepared GO/collagen35 scaffolds which were crosslinked with EDC. Conjugation36 increased stiffness and it was found biocompatible. GO/collagen37 scaffolds also increased the osteogenic differentiation of38 mesenchymal stem cells. Therefore, these scaffolds were39 suggested as strong candidates to carry out stem cell research40 and orthopedic regeneration medicine.[8]

41 Gelatin Hydrogels42 Hydrogels were prepared with GO/gelatin-based nanocompo-43 sites. Zhou et al.[40] produced GO-incorporated gelatin-based44 hydrogels with a 3D bioprinting method using a stereo-45 lithography-based printer. The addition of GO led to higher46 protein adsorption; therefore, higher cell proliferation and47 differentiation was observed for GO-incorporated hydrogels.48 This platform was suggested as a suitable candidate for49 promoting chondrogenic differentiation of mesenchymal stem50 cells for cartilage regeneration.51 Gelatin methacrylate (GelMa) which is a photocrosslinkable52 hydrogel is effective for production of 3D cell laden hydro-53 gels.[101,47] Shin et al.[47] produced NIH3T3 fibroblast-laden GO/54 GelMa hydrogels. The methacrylate groups of GelMa were55 crosslinked with UV irradiation. With an increase in UV56 exposure time, the mechanical properties of the hydrogel57 increased. A strong adhesion between GO and acrylic groups

1also led to an improvement of its mechanical properties.2Fibroblasts had similar spreading patterns and interconnected3actin network in GO/GelMa as pure GelMa hydrogels. The4authors suggested that more complex structures can be5produced for soft tissue engineering applications. Finally, Lee6et al.[102] prepared C2C12 cell-laden gelatin/GO hydrogels. The7porous structure of the hydrogels enabled water permeability8and retention with rapid diffusion of essential nutrients of the9cells in the matrix. Therefore, C2C12 myoblasts grew,10proliferated and differentiated in the hydrogels. The study11concluded potential of these hydrogels for skeletal tissue12engineering. All in all, research indicates that electrospun mats13and 3D scaffolds show potential for bone tissue engineering14while GO/gelatin hydrogels are promising for soft tissue15engineering.

162.1.5. SF

17Silk is a naturally occurring fibrous protein and it is commonly18produced by spiders or insects such as silkworms.[103,104] Silk is19degradable and lightweight with excellent thermal and mechan-20ical properties.[105,106] SF is obtained after the extraction of21sericin proteins from silk. SF is non-immunogenic, biocompati-22ble, and capable of supporting cell attachment, spreading,23growth, and differentiation.[104,107] It is a protein with a24secondary molecular structure in the form of a beta-sheet that25combines well with graphene.[17,108] SF has so far been produced26in the form of films, fibers, electrospun mats, and 3D porous27scaffolds.

28SF Films29Hu et al.[109] prepared nanomembranes comprised of alternating30layers of SFand GO by the spin-assisted layer-by-layer technique.315-nm thick SF layers were covered with 0.95-nm thick GO sheets.32The volume fraction of GO on the nanomembranes increased33from 3 to 23.5 by increasing the number of GO layers. Moreover,34the samples were intensely treated with methanol to induce β-35sheet formation in SF. The tensile strength and ultimate stress of36these nanocomposites were 145GPa and 300MPa, respectively.37These values were many folds higher than the reported values in38the literature and they were much greater than the predicted39values by the Halpin–Tsai model. The authors attributed this40difference to an interphase reinforcement mechanism. In41addition, the compressive modulus was lower than the tensile42modulus. This difference has been attributed to delamination of43the layers due to the local wrinkling of GO. The authors derived44an expression for the nanocomposite elastic modulus (E�) at the45interphase region.

E� tð Þ ¼ΔE

1þ exp η tτ� 1

� �� þ ESF ð1Þ

46E� is difference of elastic moduli of the two components; η is47shape factor; τ is effective thickness; and t is distance from GO–48SF.49In a few studies, GO/SF films were also produced by simple50casting. Wang et al.[110] prepared GO/SF nanocomposites by51solution casting. This study indicated that silk II structures





1 emerged with an increase in the wt% of GO film from 0.5 to 1wt2 %. The increase in silk II structures was associated with the3 intermolecular forces between the functional groups of GO and4 the polar groups on the SF molecular chains. Moreover, a very5 high toughness was achieved for SF/GO nanocomposites.6 However, with an increase in the wt% of GO, the elongation7 at break gradually decreased. This result was firstly attributed to8 the hindrance of slippage of SF chains due to the strong9 interaction between GO and SF. Second, the rise of silk II10 component in the nanocomposites was also related to the11 decrease in ductility. Cell culture studies with L-929 cells showed12 that the nanocomposite films were biocompatible and that the13 films could support attachment, proliferation, and differentia-14 tion of the cells.[110]

15 Abdulkhani et al.[111] also incorporated carboxymethyl cellu-16 lose in GO/silk films. They successfully produced GO/SF/17 carboxymethyl cellulose and RGO/SF/carboxymethyl cellulose18 films. TGA and DSC results revealed that RGO was more19 effective than GO in the hindrance of chain mobility of20 carboxymethyl cellulose/SF. Therefore, authors suggested high21 potential of RGO for manufacturing nanocomposites for22 biomedical applications.23 Cell culture studies were also carried out for different GO/SF24 scaffolds. Rodriguez-Lozano et al.[112] studied the effect of GO/25 SFnanocomposite films on periodontal ligament stem cells. The26 morphology of periodontal ligament stem cells was examined for27 10 days after staining with DAPI and phalloidin. The cells had a28 lower growth rate on SF in comparison with GO/SF films. MTT29 cell proliferation assay validated the visual observations. After30 240 h, the cell viability was even higher for GO/SF films than for31 plastic. Moreover, the mesenchymal phenotype of periodontal32 ligament stem cells was evaluated by analysis of the expression33 of mesenchymal surface markers. After a period of 7 days of cell34 culture, 95% of the cells were positive for the mesenchymal35 markers CD74, CD90, and CD105. This result showed that the36 cells could maintain the mesenchymal phenotype of periodontal37 ligament stem cells on GO/SF films.38 Moreover, Vera-Sanchez et al.[113] also demonstrated the39 differentiation of periodontal ligament stem cells into cemento-40 blasts. Both reduced and oxidized forms of GOwere prepared for41 1:1 and 1:3 ratios of GO/SF films. In this study, the authors42 reported that SF had a negative impact on cell spreading and43 adhesion. This result was related to a defective β1 integrin-44 dependent adhesion on fibroin nanocomposites. The prolifera-45 tion rate on RGO /SF films was much higher than that on46 oxidized GO/SF films. The differentiation of periodontal47 ligament cells into cementoblasts was revealed by the over-48 expression of CEMP1, a cementoblast phenotype marker, and49 the down regulation of osteochondroblast-specific genes.[113]

50 SF Spun Fibers51 Aznar-Cervantes et al.[17] prepared electrospunGO/SFand RGO/52 SF samples. With the reduction of GO, the authors induced53 electrical and ionic interaction to support cell proliferation for54 tissue restoration while electrospun SF provided tri-dimension-55 ality and mechanical strength. GO was reduced with the aid of56 ascorbic acid and fibers coated with RGO were electronic57 conductors and were electroactive. Moreover, RGO-coated SF58 was found to be biocompatible.[17]

1Dry spun fibers of GO/SF were also prepared. Zhang et al.[18]

2prepared dry spun GO/SF and studied the physical properties of3the fibers. As shown in Figure 6, SF molecules and GO4interaction occured via intermolecular hydrogen bonding, polar–5polar, and hydrophobic–hydrophobic interactions. As an6increase in GO increases the entanglement density, GO addition7increases the viscosity. FTIR results showed that as the wt% of8GO increased, less β-sheet conformations were found in the9hybrid fibers. This result has been explained with the hindrance10of rearrangement of RSF chains in the presence of GO. The % of11mesophase zones in the hybrid fibers was calculated with12WAXD, and the thickness of themesophase zones was estimated13with SAXS. The % of mesophase zone was optimized for 1%14GO/RSF hybrid fibers. The addition of GO increased the15thickness of interface zones remarkably. Incorporation of GO16significantly improved the mechanical properties of the fibers.17The high strength, breaking energy, and modulus were18attributed to the presence of the mesophase zone and the19homogeneous dispersion of GO. The mesophase zone improved

Figure 6. a) Schematic description of interaction between SF and GOsheets in the solution (reproduced with permission from ref.[18] 2016ACS Publications). b) Tensile stress–strain curves of the GO/SFnanocomposite films with different GO contents. (reproduced withpermission from ref.[110] 2014 RSC Publications).





1 the load transfer efficiency and ultimately improved the2 mechanical performance of the hybrid fibers.3 Figure 6 shows the interaction between GO and SF chains and4 the tensile stress–strain curve of GO/SF films.[18,110]

5 Zhang et al.[114] also studied the microstructural evolution of6 dry-spun SF/GO fibers under tensile loading with the aid of7 Synchrotron radiationWAXD. In the elastic zone, no change was8 observed in the fractions of the crystal, mesophase, amorphous9 phases, and the degree of orientation of the fibers was sustained.10 In the plastic deformation zone, the fraction of crystals increased11 and the fraction of mesophase decreased. This result was12 associated to the conversion of mesophase into crystals. A strain13 higher than 18% led to breakage of the β-sheet crystallites and14 their conversion to mesophase. In addition, the chains in the15 amorphous regions oriented themselves and formed meso-16 phase. This led to a final rise of wt% of mesophase and17 improvement of mechanical properties.

18 SF 3D Porous Scaffolds19 Wang et al.[19] used freeze-drying to fabricate 3D scaffolds of SF/20 GO to study the nanocomposite’s potential in tissue engineer-21 ing. Thus far, the GO/SF scaffolds have only been studied with22 osteoblasts. The authors prepared 0.1, 0.2, 0.5, and 1.0wt% of23 GO/SF scaffolds and added 30wt% glycerol to obtain water24 stable scaffolds. With the incorporation of GO, the pores in the25 scaffolds became more elliptical. GO incorporation in SF26 decreased the pore diameter of the scaffolds and led to27 uniformity of the pore size of the scaffolds. Cell culture studies28 were carried out with MC3T3-E1 cells for 10 days. On day 5, cell29 adherence and proliferation were observed for all the scaffolds.30 On day 10, the highest cell proliferation was found with the 0.1,31 0.2, and 0.5% GO/SF scaffolds.32 Overall, GO/SF was so far mostly used to prepare fibrous33 scaffolds for biomedical applications. The cell culture studies34 show high potential of GO/SF scaffolds for tissue engineering35 applications such as for periodontal ligament and bone36 regeneration.

37 2.2. Synthetic Polymers

38 Synthetic biodegradable polymers are widely investigated for39 biomedical applications due to their generally good mechanical40 properties and adjustable degradation rate.[115] The most widely41 studied synthetic polymers are polyvinyl alcohol, polyacryl-42 amide, and poly(α-hydroxy acids). Poly(α-hydroxy acids) are PCL,43 PLLA, and PLGA are commonly studied especially in non-load-44 bearing applications. The favorable properties of these polymers45 include good biocompatibility, biodegradability, and ease of46 handling.[116–118] Some other very rarely studied synthetic47 polymers for potential biomedical sciences are poly(carbonate48 urethane),[119] poly(acrylic acid),[92] poly(l-lysine),[120] and elas-49 tomers.[121–123]

50 2.2.1. PVA

51 PVA is a biocompatible, highly hydrophilic, water-soluble, and52 biodegradable synthetic polymer that is widely studied in the

1biomedical field.[124] It is recognized as one of the very few2soluble vinyl polymers in water.[125] Thus, far GO/PVA nano-3composites were prepared in the form of films, electrospun4mats, 3D porous scaffolds, and hydrogels.

5PVA Films6In many studies, GO was incorporated in PVA, and most cases,7they were prepared by solution casting.[28,126,127,151] Vacuum8filtration[27,128] and layer by layer self-assembly[129] was also used9to prepare GO/PVA films. Xu et al.[128] prepared micron-thick10films by 3wt% GO in PVA by vacuum filtration, which led to the11alignment of GO sheets in the PVAmatrix. Bao et al.[28] prepared12nacre-like GO/PVA films by vacuum-assisted self-assembly. In13their nanocomposites, approximately 55wt% GO was the14inorganic building block and PVA was used as a soft organic15binder. Liu et al.[130] also covalently bound the two phases with16borate treatment, which increased the Young’s modulus from1725.3 to 42.2GPa.[130] A layer-by-layer self-assembly technique18was also utilized by Zhao et al.[129] to prepare 3-nm thick films of19GO and PVA.20Feng et al.[131] produced nanocomposite films from 0.8wt%21RGO, 27wt% chitosan, and PVA. Chitosan was added in the22matrix to prevent the aggregation of GO in the PVA matrix. GO23led to the enhancement of tensile strength at the expense of24elongation at break. The decrease in elongation at break was25attributed to the restriction of the movement of molecules due to26strong hydrogen bonding between PVA and the chitosan27molecules.[131]

28The oxidation degree of GO is critical to influencing GO’s29structure and properties. Ultimately, the oxidation degree of GO30also effects the properties of GO nanocomposites. Liu et al.[35]

31added 1wt% GO in PVA with different oxidation degrees by32varying the KMnO4 oxidant content. Both the tensile strength33and elongation at break improved with an increase in the34oxidation degree of GO.[35] Kashyap et al.[132] used hydrazine in35order to RGO. It was stated that the decrease in the elastic36modulus of the 0.3wt% GO/PVA nanocomposites was due to37effect of hydrazine on the polymermatrix, which was offset by an38equivalent increase in the modulus due to RGO fillers. Despite39that effect, 0.3wt% GO/PVA had better mechanical properties40than 0.3wt% RGO/PVA nanocomposites.[132]

41Covalently crosslinked GO/PVA nanocomposite films were42prepared by Linares et al.,[133] and 1.5wt% GO incorporated PVA43increased human osteoblast proliferation. Moreover, the nano-44composites reduced apoptosis in human osteoblasts and45intracellular ROS content. Therefore, Linares et al.[133] suggested46its potential applications in tissue engineering and bone implants.

47PVA Nanofibers48Wang et al.[134] and Qi et al.[22] also produced electrospun GO/49PVA nanofibers. Upto 1wt% GO led to an increase of50mechanical properties; however, higher loading of GO resulted51in decreased mechanical performance. The authors claimed the52potential use of GO/PVA nanofibers for non-load bearing53applications in tissue engineering and drug delivery systems. Liu54et al.[24] also incorporated chitosan into GO/PVA nanofibers.55These nanofiber mats had good mechanical, antibacterial, and56physical properties. Therefore, GO/PVA nanocomposites were57suggested for antibacterial biomedical applications.





1 PVA 3D Porous Scaffolds2 Three-dimensional porous GO/PVA scaffolds were also pre-3 pared for potential biomedical applications. Shuai et al.[19]

4 utilized selective laser sintering to produce GO/PVA nano-5 composites with interconnected porous structures. GO loading6 up to 2.5wt% led to good mechanical performance. Osteoblast-7 like cells grew well and proliferated on this 3D porous structure.8 Therefore, this nanocomposite was suggested to show potential9 in bone tissue engineering. Ionita et al.[20] also added gelatin to10 the GO/PVA porous nanocomposites. GO was observed to11 influence pore adjustment, which is important in obtaining 3D12 porous scaffolds with good physical properties. Cell culture tests13 with MC3T3-E1 preosteoblast murine cells indicated the14 cytocompatibility of the scaffolds and suggested future in vivo15 studies for these nanocomposites.

16 PVA Hydrogels17 Recently, GO/PVA hydrogels were also studied for tissue18 engineering applications. Pourjavadi et al.[49] produced salep19 reduced GO-based hydrogels. In addition to reducing GO, salep20 prevented the aggregation of GO nanosheets in the polymer21 matrix. However, the addition of salep also caused a reduction in22 tensile strength. To overcome any deterioration of mechanical23 properties, citric acid, and glycerol were introduced into the24 hydrogels. The incorporation of citric acid also increased the25 degree of swelling due to its carboxylic acid groups. The26 mechanical properties of the hydrogels were found to be27 comparable with soft tissues. Du et al.[135] prepared GO/PVA28 hydrogels with 2,2-(ethylene dioxy)-diethanethiol (EDDET)29 crosslinked GO for potential load-bearing applications. The30 mechanical properties of PVA significantly improved with the31 incorporation of EDDET crosslinked GO. This result was32 attributed to the interpenetrating structure and hydrogen33 bonding. The fracture toughness of the hydrogels was34 1328 Jm� 2, which was higher than that of articular cartilage,35 which is approximately 1000 Jm� 2. Finally, cell culture studies36 with chondrocytes confirmed more favorable cell adhesion and37 proliferation in the presence of GO and EDDET than in PVA38 hydrogels. In another study, Shi et al.[38] gamma irradiated39 freeze-thawed GO/PVA hydrogels. Irradiation led to a reduction40 of GO and crosslinking of the PVA polymer chains. The water41 content decreased as the irradiation dose increased. The42 compressive strength of the nanocomposites increased from43 0.8 to 2.8 kPa with the application of gamma irradiation due to44 crosslinking of the PVA chains. The friction coefficient of the45 hydrogels increased with irradiation dosage, which was46 attributed to the decrease in water content with gamma47 irradiation. Figure 7 shows the interaction between GO and48 PVA chains.[38]

49 Finally, Rui-Hong et al.[39] fabricated GO/regenerated cellu-50 lose/PVA hydrogels. With the incorporation of regenerated51 cellulose, much better mechanical properties than those of pure52 PVA hydrogels can be obtained.With the addition of 1wt%GO, a53 tensile strength of 0.73MPa was obtained with an elongation at54 break of 240%.55 GO/PVA scaffolds have been extensively utilized to prepare56 scaffolds for tissue engineering. Among these nanocomposites,57 porous 3D scaffolds were investigated for bone tissue engineer-58 ing. GO/PVA hydrogels were mainly studied for cartilage tissue

1engineering applications. The hydrogels were strengthened with2different methods including chemical crosslinking, UV irradia-3tion, and addition of fillers. Overall, all of these nanocomposites4were found promising for tissue engineering applications.

52.2.2. Polyacrylamide

6Polyacrylamide is mostly used in the form of hydrogels.7Polyacrylamide hydrogels are biocompatible, soft, and can easily8be processed, therefore they are extensively studied in the field of9tissue engineering.[46]

10Polyacrylamide Hydrogels11Zhang et al.[136] prepared GO/sodium alginate/polyacrylamide12hydrogels. Alginate was incorporated to induce pH sensitivity in13the hydrogels and to promote its mechanical properties. A14compressive strength of 656MPa was obtained for the GO-15incorporated hydrogels. Moreover, Zhu et al.[122] incorporated16agar into the GO/polyacrylamide hydrogels. Agar hydrogels have17a high elastic modulus; however, they are brittle. On the other18hand, polyacrylamide can withstand high elongation at break.19With the addition of agar into the GO/polyacrylamide hydrogels,20excellent mechanical properties were achieved with high fatigue21resistance, and self-healing properties. A fracture strength of22332 kPa and a fracture strain of 4600% were obtained with a23fracture dissipated energy of 11.5MJm� 3. Figure 8 shows the24GO/sodium alginate/polyacrylamide hydrogels without and with25pressure and the GO/polyacrylamide hydrogels under different26modes of stress.[136,122]

27Figure 8 demonstrates that polyacrylamide hydrogels may28have very different mechanical properties. Blending polyacryl-29amide with alginate, a very high strength was achieved and with30addition of agar, a very high elongation at break could be31maintained which can withstand different stress modes.32GO/polyacrylamide hydrogels were also developed by Li33et al.[46] to study its effect on Schwann cell attachment and34proliferation. The addition of 0.4mgml� 1 GO led to optimum35Schwann cell attachment and proliferation. Therefore, the36authors suggested the potential of these hydrogels in repairing37peripheral nerve injury.38All in all, GO/polyacrylamide hydrogels were prepared for39potential tissue engineering applications. GO/polyacrylamide40was blended with different polymers including alginate and agar41and these hydrogels may show potential particularly for various42soft tissue regeneration applications.

432.2.3. PCL

44PCL is a hydrophobic, semi-crystalline, resorbable, aliphatic45polyester. Its crystallinity decreases with increasing molecular46weight. The good solubility of PCL and its low melting point47(59–64 �C) and exceptional blend-compatibility have stimulated48extensive research into its potential applications in the49biomedical field.[137] It is regarded as a soft and hard-tissue50compatible material like resorbable sutures, drug delivery51systems, and recently bone graft substitutes. However, its52degradation and resorption kinetics are slow due to its





1 hydrophobicity and high crystallinity which limits its application2 range.[138] PCL can be blended or co-polymerized with other3 polymers, such as PLA or PLGA in order to improve its physical4 properties.[116] GO/PCL nanocomposites were prepared in the5 form of rods and electrospun mats.

6 PCL Films7 In many studies, PCL was grafted onto the GO sheets by in situ8 ring opening polymerization of e-caprolactone. The grafted GO/9 PCL dissolved well in dichloromethane, chloroform, DMF, THF,10 toluene, and ethylene acetate. The homogeneous dispersion of11 GO in the polymer matrix improved the mechanical properties12 of PCL.[139] The aggregation and stacking of GO nanosheets were13 also supported by tethering GO sheets on the PCL chains.[140]

14 GO was also incorporated in PCL and poly((R)-3-hydroxybu-15 tyric acid) (PHB) nanocomposites. Bymixing PHBwith PCL, the16 degradability of PCL was enhanced. Honeycomb-patterned GO/17 PCL/PHB thin films were prepared with breath-figure method.18 5wt% GO and different wt% (5, 10, and 20) of PHB were added19 in PCL. The authors stated that without the presence of PCL in20 the nanocomposites, honey-comb patterned films could not be21 obtained. With the addition of PHB, the biodegradation rate of22 the PCL films increased which was attributed to the loss of PCL23 matrix integrity.[141]

24 One of the important factors that affects the mechanical25 properties of semi-crystalline polymers is their crystallization26 behavior.[142] To improve the mechanical properties of 1wt%27 RGO/PCL nanocomposites, epitaxial crystallization of PCL was28 induced. To induce epitaxial crystallization, nanocomposites29 were heated upto 90 �C and then cooled down to 48 �C at a30 cooling rate of 10 �Cmin� 1 with a dwell time of 30min.31 Subsequently, samples were cooled down to room temperature.32 Both the yield strength and Young’s modulus increased after an33 annealing treatment of the nanocomposites. Wang et al.[142,143]

34 studied the effect of RGO on crystallization and the crystal

1orientation of injection-molded PCL. Up to 1wt%, RGO was2incorporated in the injection-molded nanocomposites. RGOwas3observed to increase crystallization and the orientation of crystals4in the flow direction.[142]

5PCL Nanofibers6There is extensive work on the production of electrospun GO/7PCL nanofibers. For example, Wang et al.[36] added 0.3wt% GO/8PCL in DMF solution. With the addition of GO, the fiber9diameter increased. The mechanical properties of the fibers10improved with the addition of GO as did the biomineralization.11The authors therefore suggested the great potential of GO/PCL12fibrous membranes in biomedical engineering applications.[36]

13In terms of effect of GO on the physical properties of the14nanofibers, many contradictory results were found in the15literature. Ramazani et al.[25] studied the effect of the oxidation16level of GO on the physical properties of GO/PCL electrospun17fibers. With an increase in the GO concentration to 3wt% GO,18the diameter of the fibers decreased[25,37] Ramazani et al.[37] also19prepared aligned GO/PCL nanofibers and with the addition of20upto 1wt% GO nanosheets, a very significant rise in the tensile21strength and elastic modulus was observed. This result was22attributed to hydrogen bonding, CH2-π, and van derWaals forces23between PCL chains and nanosheets.24Song et al.[144] carried out cell culture studies for GO/PCL25fibrous mats. Mesenchymal stem cells and PC12-L cells were26cultured on the nanocomposite nanofibers. Both cells adhered,27spread, and grew well on the fibrous scaffolds. Moreover, the28differentiation of mesenchymal stem cells and PC12 cells into29osteo- and neuro-like cells was enhanced with the incorporation30of 0.3 and 0.5wt% GO in PCL nanofibers. Shah et al.[45] also31observed that GO coated PCL nanofibers provided the physical32cues for selective differentiation of neural stem cells into mature33oligodendrocytes. Finally, Chaudhuri et al.[145,146] stated excellent34differentiation of myoblasts on GO/PCL scaffold meshes.

Figure 7. GO/PVA hydrogels (reproduced with permission from ref.[38] 2016 ACS Publications).





1 Therefore, GO/PCL electrospun mats were found promising for2 bone, muscle, and nerve regeneration applications.

3 2.2.4. PLLA

4 PLLA is a biocompatible and biodegradable synthetic polymer5 being used as a biomaterial in various biomedical applica-6 tions.[147] GO/PLLA and RGO/PLLA nanocomposites were7 compression molded into plates.[148,149] RGO was prepared8 either by adding 1.6 g of glucose in 100ml of DMF with 100mg9 of GO or by adding polyvinyl pyrrolidone (PVP) and GO at a ratio10 of 5:1. Before injection molding, GO and PLA were blended in11 DMFsolution. The electrical conductivities of GO and RGOwere12 measured and the electrical conductivity of glucose-reduced GO13 was found to be much higher. This implied that glucose was a14 much more effective reducing agent than PVP. Moreover, GO/15 PVA molds showed high conductivity. The highest conductivity16 was also measured for glucose-reduced GO/PLLA molds.[148] Li17 et al.[149] prepared GO-grafted PLLA by solution blending and

1compression molding. Mechanical tests were carried out, and2the results suggested a significant increase in flexural strength3and elongation at break for GO-grafted PLLA nanocomposites.4GO/PLLA nanocomposites were also prepared in the form of5electrospun fibers and 3D porous scaffolds.[150]

6PLLA Nanofibers7GO/PCL/PLLA nanofibers were also prepared.[25,147] Although8PCL and PLLA are immiscible, with the incorporation of GO,9their miscibility could be improved. In the solution form, PLLA10and PCL had phase separation. In the blend form of PCL and11PLLA, interestingly, GO was found to be distributed in the PCL12phase. Wang et al.[25] and An et al.[147] examined the strong13antibacterial activity of GO/PLLA/PCL nanocomposites. There-14fore, they suggested their potential in tissue engineering.

15PLLA 3D Porous Scaffolds16In a few studies, GO/PLLA was also blended together with other17materials. Cellulose nanocrystal(CNC)/RGO/PLLA porous scaf-18fold was one of the ternary composite scaffolds. A good

Figure 8. Photographs of GO/sodium alginate/polyacrylamide hydrogels a) without pressure and b) with pressure (reproduced with permission fromref.[136] 2015 RSC Publishing). GO/agar/polyacrylamide hydrogels which can withstand c) winding, d) knotting e) knot stretching, f) twisting, and g andh) free shapeable properties (reproduced with permission from ref.[122] 2016 Wiley).





1 dispersion of 1wt% CNC and 0.5wt% RGO could be achieved in2 PLLA, which led to high tensile strength, thermal stability,3 antibacterial response, and biocompatibility.[150] Chen et al.[22]

4 prepared 3D printed biocompatible GO/polyurethane/PLLA5 nanocomposites were easily prepared. The nanocomposites had6 excellent mechanical properties, thermal stability, and NIH3T37 cell viability. Therefore, research so far shows that fibrous and8 porous 3D scaffolds of GO/PLLA show potential for tissue9 engineering applications.

10 2.2.5. PLGA

11 PLGA is one of the most successfully studied biodegradable12 polymers. In vivo, it is hydrolyzed into the easily metabolized,13 non-toxic monomers, lactic acid, and glycolic acid.[152] PLGA is14 commercially available in different molecular weights and15 copolymer compositions. Depending on the copolymer ratio and16 its molecular weight, the degradation rate varies from several17 months to several years. PLA has also been used to a lesser extent18 than PLGA due to its lower degradation rate.[153] GO/PLGA19 nanocomposites were mainly prepared in the form of electro-20 spun mats.

21 PLGA Nanofibers22 A number of studies were carried out to study the physical and23 biological properties of electrospun GO/PLGA for biomedical24 applications.[29–33] Yoon et al.[29] incorporated upto 2wt% GO in25 PLGA to produce electrospun fibers. The storage moduli of26 PLGA and 1wt% GO-incorporated PLGA were 4.1 and27 217.8MPa, respectively. Moreover, 2wt% GO incorporated28 PLGA nanofibers were also stiff at 30–35 �C, whereas for pure

1PLGA group, the modulus dropped rapidly with increase of2temperature. Cell culture studies were carried out on nanofibers3with PC12 neuronal cells. Neuronal cells on 2wt% GO/PLGA4nanofibers showed more significant neurite outgrowth and5branching than those on pure PLGA and 1wt% GO/PLGA6nanofiber.7Shin et al.[30] extensively studied the myogenic differentiation8of C2C12 cells on GO/PLGA nanofibers.[31,30,33] Shin et al.[30]

9prepared RGD-decorated PLGA fibers and incubated the10samples in growth media containing 10 μgml� 1 GO for 5 days.11The immunogenic differentiation of C212 myoblasts was then12evaluated by immunofluorescence stains for myosin heavy13chains. Cells on pure PLGA matrices had very poor F-actin14development and did not differentiate. C2C12 cells on RGD/15PLGA matrices were well-grown, but they did not differentiate16into mature myotubes. Finally, RGD/PLGA matrices with GO17fully proliferated and differentiated. Therefore, a combination of18GO with RGD/PLGA nanofiber matrices was suggested for19skeletal tissue regeneration and the treatment of muscle20dysfunctions. In another study, Shin et al.[33] incorporated2110mgml� 1 GO in collagen/PLGA nanofiber matrix. Both22collagen and GO supported proliferation of the cells. Moreover,23collagen/GO/PLGA matrices had greater C2C12 differentiation24than GO/PLGA nanofibers.[33]

25Moreover, the effect of electrospun GO/PLGA on mesenchy-26mal stem cell differentiation to osteoblasts has been evaluated.27The adsorption of cell culture proteins onto the surface of28substrates is required for cell adhesion. Therefore, the29incorporation of GO in PLGA increases the protein spreading30capacity. Therefore, incorporation of GO in the matrices31significantly increased cell proliferation. The amount of ALP32and collagen Type 1 extracts from the mesenchymal stem cells

Figure 9. a) Tensile strength and b) modulus of GO/polymer nanocomposite films.





1 cultured on GO-incorporated PLGA was higher than that for2 pure PLGA matrices in the presence of dexamethasone.3 Osteocalcin secretions of PLGA and GO/PLGA were higher4 than tissue culture plates, which was attributed to the 3D5 structure of the scaffolds that forced the cells into an osteogenic6 morphology. Finally, substrates with a stiffness greater than7 100 kPa promote osteogenic differentiation. PLGA nanofibers8 have a stiffness of 70–180MPa. In this study, the stiffness of the9 fibers decreased with the incorporation of GO. Therefore, an10 enhancement of cell differentiation into osteoblasts was11 promoted due to the presence of dexamethasone and the greater12 protein loading capacity of GO/PLGA than PLGA, which13 promoted adhesion, proliferation, and ultimately differentiation14 of mesenchymal stem cells into osteoblasts.[32] Overall, so far15 GO/PLGA shows potential for neural, bone, and skeletal tissue16 regeneration.

17 2.3. Mechanical Properties

18 The mechanical properties of nanocomposites are among the19 most important characteristics in determining their suitability20 for biomedical applications. Figure 9a shows the tensile strength21 of the fabricated GO/polymer nanocomposite films in reference22 to the mechanical properties of cortical bone[154] and23 Figure 9b shows the modulus of GO/polymer nanocomposite24 films.25 As shown in Figure 9a, the tensile strength of GO/chitosan,26 GO/gelatin, GO/PLLA, GO/PVA, GO/alginate, and GO/hydrox-27 yethyl cellulose nanocomposite films have similar tensile28 strength to that of cortical bone. Many mechanical properties

1of GO/PVA films can be observed in different studies. The2significant differences put forward in these studies can be3attributed to the effect of wt% of GO in the film. A quick4deterioration in mechanical properties can be observed when wt5% of GO is above an optimal value. Similarly, different GO/6chitosan films have very different tensile strength values. In7addition to the wt% of GO in PVA films, these differencesmay be8due tomany other factors. First, the selected chitosan in the films

Figure 10. a) Tensile strength versus % elongation at break of GO/polymer nanocomposites presented with their modulus (E) and the mechanicalproperties of tendon and articular cartilage[156] b) modulus versus tensile strength of GO/polymer nanocomposites.

Figure 11. Tensile strength versus elongation at break of electrospunGO/polymer mats.





1 may have different degrees of acetylation andmolecular weights.2 It is well-known that, both the modulus and tensile strength of3 chitosan increase with a rise in the degree of acetylation.[155]

4 Second, crosslinking may influence mechanical performance. Li5 et al.[60] crosslinked chitosan with genipin and the tensile6 strength of their nanocomposite wasmuch higher than the other7 GO/chitosan nanocomposite films that were prepared without8 crosslinking.9 Figure 9b shows that in a few studies, films with a modulus10 over 21GPa were achieved. In these studies rather than solution11 casting, other techniques were employed for the production of12 the films. Ultra-high strength nacre-like films were achieved by13 Hu et al.[74] with wet spinning of alginate/GO films, by Kang14 et al.[97] with vacuum filtration of gellan gum/GO films, by Liu15 et al.[130] with vacuum assisted self-assembly of GO/PVA films16 and by Hu et al.[109] with spin-assisted layer-by-layer technique17 for the formation of GO/SF membranes.18 The mechanical properties of the GO/polymer nanocompo-19 site films are analyzed in more detail to better understand the20 mechanical performance of the nanocomposite films.21 Figure 10a shows the tensile strength versus the percentage22 elongation at break graph along with a presentation of moduli of23 nanocomposite films from the available literature.24 Figure 10a shows that GO/polymer nanocomposite films have25 high variation in terms of their mechanical properties. Among26 the nanocomposites, 2wt% GO/pea starch nanocomposites had27 the lowest mechanical properties. Their tensile strength and28 modulus were both much lower than those of cortical bone.29 Notably, 2wt%GO/PCL had very high ductility, which wasmuch30 higher compared to that of cortical bone. There are a few studies31 on the mechanical properties of GO/PVA nanocomposite films.32 GO/PVA nanocomposite films had a high modulus and tensile33 strength with a moderately high elongation at break.34 Figure 10b shows the modulus versus tensile strength of the35 nanocomposite films.[156]

36 Among the prepared nanocomposites, the highest mechani-37 cal properties were for GO/PVA nanocomposites, followed by38 GO/alginate, GO/CMC, and GO/chitosan films. The lowest39 mechanical properties were for GO/pea starch, followed by GO/

1PCL and starch/chitosan films. GO/PVA, GO/chitosan, and GO/2alginate had a tensile strength close to that of cortical bone3however, none of the films had a modulus close with cortical4bone. Figure 11 draws attention to the tensile strength versus5elongation at break of electrospun mats of GO/polymer6nanocomposites.7The % elongation at break of the nanocomposites had a very8high variation. The elongation at break values of the GO/gelatin,9GO/chitosan, and GO/SF scaffolds were similar with cortical10bone which is about 2%. GO/BC/chitosan had the highest11modulus and the second highest tensile strength.12Table 2 shows the mechanical properties of the hydrogels13prepared for tissue engineering applications. If the potential14application of the hydrogels are given in the paper, they are also15specified in the materials and methods column.16According to Table 2, GO/polyacrylamide/alginate hydrogels17have exhibited the highest strength among the studied GO/18polymer-based hydrogels. GO/PVA also had very high strength19values. Therefore, GO/PVA hydrogels were also suggested for20load-bearing applications. Additionally, most studies lack21information about the modulus and elongation at break values.22With measurement of modulus and elongation break, these23hydrogels can be characterized more extensively.24To produce cell-laden hydrogels, the gel stiffness should be25adjusted according to the cell type and incubation time. When26cells are cultured in a hydrogel with high stiffness, there may be27some limitation for cell growth and if the gel stiffness is too low,28the hydrogels will degrade rapidly before achieving the desired29effect.[102] A stiffness of approximately 12 kPa may support30muscle tissue growth.[71,102] Therefore, GO/gelatin hydrogels31were only suggested for soft tissue engineering applications32such as cardiac patches and muscle tissue engineering.[47,93,157]

33Hydrogels with a higher stiffness are suggested for load-bearing34applications such as EDDETcrosslinked GO/PVA hydrogels.[135]

353. Conclusions and Future Directions

36In conclusion, many different polymers are being utilized to37prepare GO/polymer nanocomposites for potential biomedical

Table 2. Mechanical properties of GO/polymer hydrogels and their potential applications according to literature reports.

Materials and mechanical test method Strength Modulus Elongation at break/%

1.8 wt% GO/polyacrylamide/alginate (compression)[136] 65.6 MPa N/A 80

6 wt% RGO/PVA (tension)[49] 40.86 MPa 3.38 MPa 176

Articular cartilage[152] 9–40 MPa N/A 60–120

1.2% GO/polyacrylamide hydrogel for peripheral nerve regeneration (tension)[46] 38 MPa N/A N/A

Skin[152] 1–30 MPa 15–150 MPa 30–115

1 wt% GO/PVA for load-bearing tissue substitutes (compression)[135] 17 MPa 60 kPa N/A

0.1 wt% GO/PVA (compression)[38] 2.8 MPa N/A N/A

1 wt% GO/cellulose/PVA (tension)[39] 0.73 MPa N/A 240

1 mg ml� 1 GO/carboxymethyl chitosan/KGM for wound dressing (compression)[48] 208 kPa 1.11 MPa N/A

GO/polyacrylamide/agar (compression)[122] 332 kPa N/A 4600

2 mg ml� 1 GO/GelMa hydrogel for cardiac patches (tension)[47] 977 kPa 24 kPa 55

1 mg ml� 1 GO/gelatin hydrogel for muscle tissue engineering (rheometry measurement)[102] N/A 3.42 kPa N/A





1 engineering applications. The incorporation of GO in polymer2 was found to improve the physical and biological properties of3 the composites. With the addition of only a small concentration4 of GO, the Young’s modulus and tensile strength was5 significantly improved. In most of the studies, this was6 accompanied with a lower elongation at break.7 Under physiological conditions the mechanical properties of8 polymers are much lower. With the addition of GO, the9 mechanical performance of GO composites were extensively10 improved under physiological conditions. Moreover, by vacuum11 filtration and wet spinning methods more advanced mechanical12 properties were obtained than solvent casting.13 GO/polymer-based scaffolds can be categorized into four14 main categories, namely, 3D porous scaffolds, electrospun mats,15 hydrogels, and nacre-like structures. So far, 3D porous scaffolds16 have been mainly prepared for bone tissue engineering17 applications. Electrospun mats were produced for different18 types of applications including bone, neural, muscle, and skin19 tissue engineering. Poly(α-hydroxy acids) such as PLGA, PLLA20 were so far the most successful materials considered to prepare21 ESMs. GO/polymer hydrogels have been mainly prepared for22 soft tissue engineering such as wound dressing, peripheral23 nerve regeneration, and muscle tissue engineering. Hydrogels24 with higher mechanical strength such as crosslinked GO/PVA25 also show potential for load-bearing applications such as26 articular cartilage regeneration.27 In future studies, to determine the applicability of GO/28 polymer nanocomposites for biomedical applications, mechani-29 cal characterization in wet conditions at 37 �C, further30 degradation studies, and ultimately in vivo studies are needed31 to determine the applicability of GO/polymer nanocomposites.32 In addition, with the incorporation of bioceramics, the nano-33 composite properties may be further developed for bone tissue34 engineering. Overall, GO/polymer nanocomposites are very35 promising for tissue engineering applications and represent a36 successful example of the broad applicability of GO in the37 biomedical field.

38 Conflict of Interest39 The authors declare no conflict of interest.

Keywordsbone, cellulose, chitosan, gelatin, graphene oxide

4041 Received: July 25, 201742 Revised: August 21, 2017

Published online:

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