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Review

Graphene for the development of the

next-generation of biocomposites for dental and
medical applications

Han Xie a , Tong Cao a , Francisco Javier Rodríguez-Lozano b ,

Emma Kim Luong-Van c , Vinicius Rosa a,c,∗
a Faculty of Dentistry, National University of Singapore, Singapore
b School of Dentistry, University of Murcia, Spain
c Centre for Advanced 2D Materials and Graphene Research Centre, National University of Singapore, Singapore

a r t i c l e i n f o a b s t r a c t

Article history: Objective. Graphene and its derivatives, graphene oxide (GO) and reduced graphene oxide
Received 17 October 2016 (rGO), are 2D carbon-based materials with remarkable physical, chemical and biological
Received in revised form properties. Graphene sheets have high specific surface area and mechanical strength. More-
16 March 2017 over, they have been shown to influence the differentiation of stem cells and to improve
Accepted 11 April 2017 properties of biomaterials.
Available online xxx Methods. Here, we present the recent achievements on the use of graphene and its deriva-
tives to improve properties and enhance bioactivity of biomaterials. We also discuss the
Keywords: biosafety constraints to be solved to translate these carbonaceous materials to the clinic.
Carbon Results. Graphene and its derivatives can be functionalized and further modified with several
Graphene oxide bioactive molecules. They can be combined with several biomaterials used in regenerative
Tissue engineering and reconstructive dentistry and medicine. The resultant graphene-modified composites
Bone regeneration often present improved physico-mechanical properties and enhanced bioactivity. Moreover,
Implant graphene-modified composites are promising candidates to deliver growth factors, drugs
Polymers and others bioactive compounds.
Bioceramics Significance. Graphene can improve the physical, chemical and mechanical properties of
Bioglass biomaterials. As it can be functionalized and combined with several biomolecules, graphene
Biomaterials holds enormous potential to be used as drug carriers or substrates and scaffolds for cell-
Biocompatibility based tissue engineering strategies.
© 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

∗
Corresponding author at: Faculty of Dentistry, National University of Singapore, 11 Lower Kent Ridge Road, 119083, Singapore.
Fax: +65 6778 5742.
E-mail address: denvr@nus.edu.sg (V. Rosa).
http://dx.doi.org/10.1016/j.dental.2017.04.008
0109-5641/© 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Xie H, et al. Graphene for the development of the next-generation of biocomposites for dental and medical
applications. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.04.008
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Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00
2. Graphene and bioceramics: opportunities for bone regeneration and tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Improving mechanical properties and bioactivity of polymer-based composites with graphene . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Graphenes as additives or coatings leading to the next generation of metals for biomedical applications . . . . . . . . . . . . . . . . 00
5. Is graphene a cytotoxicity material? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

In this context, emerges graphene emerges (Fig. 1). It is a

1. Introduction
single atomic sheet of conjugated sp2 carbon atoms arranged
in a honeycomb pattern with extremely high mechanical
The combination of two or more materials with different
strength and modulus of elasticity. Moreover, graphene has
compositions, morphologies and properties can result in com-
unparalleled electronic properties and offers a large surface
posites with tailored physical and chemical characteristics,
area that can be chemically functionalized [6–9]. Graphene
and increased mechanical properties or bioactivity. Due to
has two derivatives, namely graphene oxide (GO) and reduced
the enhanced capabilities, composites are widely used in den-
graphene oxide (rGO). GO can be prepared by oxidation of
tistry and other biomedical areas as restorative materials, drug
graphite. It presents several functional groups (e.g., hydroxyl,
carriers, prosthetic parts and others [1,2].
carboxyl and epoxy groups) that can be used to combine GO
Although composite materials can present several advan-
to several biomolecules and materials [10–12]. As such, GO is
tages that single-component materials fail to express, they
an interesting alternative to improve the mechanical prop-
may also present aspects that require further improve-
erties and the bioactivity of biomaterials, or as carriers for
ments. For instance, thermoplastic resin composites can
biomolecules and drugs. The second derivative, rGO, can be
be less toxic than the thermosetting ones but are prone
produced by removing the oxygen-containing groups of GO
to slow crack growth [3]. Also, ionomeric polymer–metal
with the recovery of a conjugated structure [13]. Although this
composites present large displacements when submitted to
process results in a material that resembles pristine graphene,
low applied voltage but are costly as their manufacturing
oxygen-containing groups and defects in different proportions
depends on noble metals such as platinum and silver [4].
are found on the rGO surface [14].
Ceramic–polymeric composites can be bioactive but may
Pristine graphene can be obtained via several routes (e.g.
induce allergic reactions and present low mechanical prop-
micro-mechanical exfoliation of graphite, chemical vapor
erties [5]. Even with current advances, the development of
deposition (CVD), epitaxial growth on SiC and others). The
new materials and methods to create the next generation of
graphene produced by these methods presents an almost
biocomposites with improved capabilities is of high interest.

Fig. 1 – Key milestones on graphene development.

Please cite this article in press as: Xie H, et al. Graphene for the development of the next-generation of biocomposites for dental and medical
applications. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.04.008
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Fig. 2 – Potential improvements provided by graphene in
biomaterials and key areas to be developed to translate the
biocomposites to clinical reality.
perfect hexagonal structure with outstanding physical, chem-
ical and mechanical properties, and can be transferred to
several substrates [15–17]. Alternatively, GO and rGO can be
produced via cost-effective chemical methods using graphite
as raw material [13,14]. They can be dispersed in stable
aqueous solutions to assemble macroscopic structures at
large-scale [10,13]. Nonetheless, their deposition and posi-
tioning in pre-defined geometries are very challenging. This
is particularly important for the fabrication of devices that
require assembling materials with precision in micro- or nano-
sized architectures [7].
The properties of graphene-related materials and their
abilities to be functionalized and combined with biomolecules
and materials offer several opportunities to design bio-
composites with tailored properties. Thus, the objective of
this paper is to present the potential of graphene to pro-
duce polymer-, ceramic- and metal-based composites with
enhanced mechanical properties and enriched bioactivity for
several dental and biomedical applications (Fig. 2).
2. Graphene and bioceramics:
opportunities for bone regeneration and tissue
engineering
Bone tissue regeneration and mineralization are major chal-
lenges in the reconstruction of congenital defects, tumor
resections and fractures. The spontaneous bone regenera-
tion is often hindered by the volume of bone loss, infections,
metabolic disorders or impaired blood supply [18]. One of
the most common strategies to increase bone volume is
based on the use of grafts. Approximately 2.2 million bone
grafting procedures are performed yearly worldwide repre-
senting a market of about $2.5 billion [19]. Autologous bone
grafts are considered the “gold standard” for bone regenera-
tive treatment but their harvesting require additional surgical
procedures that can result in donor-site morbidity, pain and
Please cite this article in press as: Xie H, et al. Graphene for the development of the next-generation of biocomposites for dental and medical
applications. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.04.008
3
increase treatment cost [20,21]. Xenografts can overcome
some of these disadvantages but they lack viable cells and
present high batch-to-batch variability. Moreover, there are
risks of immunogenicity and infection transmission [22].
Alternatively, inert or bioactive ceramic-based synthetic
bone substitutes are used in the clinical practice. Bioceram-
ics can be inert (e.g., alumina and zirconia-based ceramics) or
bioactive (e.g., hydroxyapatite and other calcium phosphates).
The latter category usually present high tissue compatibil-
ity and osteoconductivity that can favor the physiological
processes involved in the formation of calcified matrix. Typ-
ically, bioceramics stimulate the differentiation of stem cells
or osteoprogenitor cells into osteoblastic-like cells, resulting in
calcification [21,23,24]. Despite these advantages, they can be
inherently brittle, present slow resorption rates and are diffi-
cult to shape [21,25]. Hence, the development of strategies that
can overcome some of these limitations is of great interest.
Notably, graphene and its derivatives can be combined with
bioceramics resulting in composites with enhanced mechan-
ical properties and improved osteogenic potential in vitro and
in vivo.
Hydroxyapatite (HAp) particles have a great potential to
induce cell differentiation into the osteolineage [21,25,26].
HAp has been combined with graphene for scaffolds and coat-
ings with enhanced capabilities. For instance, the addition of
a third component (e.g., chitosan, gelatin or polyethylene gly-
col) to the HAp/GO derives nanocomposites that can release
significantly higher quantities of Ca and P ions compared to
pure HAp particles or nanorods [26,27]. The higher release
of these ions may be related to the lower crystallinity of the
modified material that increases the rate of dissolution of the
nanocomposite [27]. Several improvements in physical and
mechanical properties have been observed from the combi-
nation of graphene-related materials to HAp. For example, the
addition of 2 and 5 wt% of GO to a HAp coating increased the
coating adhesion strength to titanium from 1.5 (control) to 2.7
and 3.3 MPa, respectively. Moreover, the GO-modified coating
presented higher corrosion resistance as compared to the HAp
alone [28]. It was possible to increase HAp’s elastic modulus
by 47% and the fracture toughness by more than 200% when
combined with 1 wt% rGO [29]. Also, the microhardness of the
HAp increases from 322 to 425 HV with the presence of 1 wt%
rGO. Also, the addition of 1.5 wt% increases the elastic mod-
ulus from 87 to 123 GPa and fracture toughness from 0.8 to
1.5 MPa m1/2 [30]. GO nanoflakes incorporated in gelatin/HAp
scaffolds presented higher yield and compressive strengths
comparing to the original gelatin/HAp [31]. The addition of
1 wt% of graphene nanosheets (GNS) to HAp increased the
fracture toughness from 0.5 to 1.0 MPa m1/2 and hardness from
5.5 to 7.2 GPa [32].
Indeed, HAp is not the only bioceramic that can be
improved by graphene-related materials. Bioactive glasses are
reactive glass–ceramic materials that present good biodegrad-
ability and bone-bonding ability. The incorporation of 0.5 wt%
graphene to 58S bioglass via selective laser sintering increase
the compressive strength from 23.6 to 48.7 MPa and the frac-
ture toughness from 1.4 to 1.9 MPa m1/2 [33]. Notably, the
presence of rGO in HAp and bioglass influences their prop-
erties in a concentration-dependent manner. The addition of
0.5 and 1 wt% rGO as a reinforcement particle to 45S5 Bio-
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glass increases the fracture toughness from ∼0.5 (control) to
0.8 MPa m1/2 and 1.2 MPa m1/2 , respectively [34].
The enhancements of mechanical properties of bioceram-
ics by the controlled addition of graphene family materials
are likely to be caused by three-dimensional crack deflec-
tion, bridging and sheet pull-out mechanisms [30,34,35]. The
2D sheet-like structure of graphene allows a large contact
area, and thus possibly greater bond strength, between the
graphene and the ceramic grains, reducing crack propagation
along grain boundaries. Despite the different chemical proper-
ties of graphene, GO and rGO, successful dispersion of each of
these graphene materials in ceramics has been demonstrated.
Importantly, graphene is able to withstand harsh processing
conditions such as high temperature (e.g. up to 1150 ◦ C) and
pressure, which may be used during formation of ceramic
composites [32]. It should be noted that high temperatures
lead to the in situ reduction of GO to rGO [34]. Graphene-based
materials also influence the crystal formation of bioceramics.
The presence of 1 wt% graphene nanosheets (GNS) in HAp-
based composites induced the formation of uniform apatite
layers that are thicker than those observed for HAp alone.
Interestingly, GNS changed the spatial distribution of the crys-
tals, which grew near or inside the pores in the pure Hap,
while crystals permeated the whole surface of the GNS/HAp
composites [32]. rGO sheets were able to act as a nucle-
ation surfaces for HA crystal growth from solution, resulting
in an intimate contact between the graphene and the HA
through van der Waals bonding rather than chemical reaction
[29].
Graphene-modified bioceramics often present enhanced
bioactivity. Mesenchymal stem cells (MSCs) cultured in a col-
loidal dispersion of rGO-coated HAp presented higher alkaline
phosphatase (ALP) activity and calcium nodule deposition
after 21 days comparing to those cultured with HAp or rGO
alone. Moreover, the MSCs treated with the rGO-coated HAp
presented high osteopontin (OPN) and osteocalcin (OCN)
protein expression [36]. As the latter protein is commonly
expressed in late stages of osteoblastic differentiation [37],
it may be feasible that the synergy between rGO and HAp
promotes the osteogenic differentiation of these cells [36].
Moreover, MSCs seeded in gelatin/HAp scaffolds presented
round morphology while those in the GO-modified version
spread and attain a cell sheet-like appearance after 24 h. The
GO-modified scaffold induced higher ALP activity after seven
days and higher OPN protein expression after 21 days com-
pared to the control [31].
Other osteoconductive bioceramics such as !-tri-calcium-
phosphate (!-TCP) and calcium phosphate (CaP) can also
be blended with graphene to further improve their bioac-
tivity in vitro. The combination of GO flakes with !-TCP
scaffolds increased the ALP activity and the expression of
osteogenic-related genes of bone marrow MSCs. Moreover, the
GO-modified scaffold increased the expression of several pro-
teins involved in the canonical Wnt signaling pathway (e.g.
WNT3A, LPP5, AXIN2 and CTNNB) suggesting that this path-
way may be activated during the enhanced differentiation
[38]. GO/CaP nanocomposites increased the protein expres-
sion of ALP and OCN and calcification of MSCs compared to
cells treated with GO or CaP alone [39].
Please cite this article in press as: Xie H, et al. Graphene for the development of the next-generation of biocomposites for dental and medical
applications. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.04.008
Fig. 3 – Possible routes for the modifications of biomaterials
with graphenes.
Most importantly, the enhancements in osteoblastic
differentiation provided by graphene-related materials to bio-
ceramics are not only observed in vitro but also in vivo. It
has been shown that critical-sized calvarial defects created in
rabbits that were treated with a GO-modified !-TCP scaffold
presented higher new bone formation compared to the defects
filled with the unmodified !-TCP scaffold. In fact, after eight
weeks of implantation, the bone volume/total volume ratios
were equivalent to 30% for the !-TCP scaffold and 44% for the
GO-modified one [38]. Similar improvements were observed
with a rGO/HAp graft that presented bone density consider-
ably higher (52%) compared to HAp alone (26%) and untreated
control (17%) in the same type of defect after four weeks [40].
Numerous types of bioceramics are constantly devel-
oped aiming for launch onto the market with the promise
of promoting bone tissue regeneration and mineralization.
Graphene family materials offer new opportunities to improve
mechanical properties and enhance the bioactivity of bioce-
ramics, making these composites interesting alternatives for
bony applications.
3. Improving mechanical properties and
bioactivity of polymer-based composites with
graphene
Polymer-based materials are widely used for dental and
biomedical applications since they can be processed in high
scale, easily shaped, and chemically tuned to attain spe-
cific biological properties [2,41]. Nonetheless, some of the
polymeric materials used in reconstructive and regenera-
tive dentistry and medicine are not suitable for load bearing
areas, suffer lack of remodeling and may induce inflamma-
tory reactions. Moreover, their degradation may induce an
autocatalytic ester breakdown which lowers the pH in the
microenvironment, posing difficulties for cell survival and
differentiation [1,42]. To overcome some of the inherent lim-
itations, graphene and its derivatives can be blended with
polymers via several routes (Fig. 3) to produce composites with
improved capabilities.
Graphene-related materials and polymers can be blended
to produce composites with enhanced mechanical proper-
ties. Different graphene derivatives can be selected to achieve
good dispersion within different polymers, for example GO
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can be easily processed with water-soluble polymers. As
with ceramic composites, the large surface area provided
by the 2D sheet-like structure of graphene likely provides
enhanced interfacial adhesion between the phases, and facil-
itates crack deflection and improved fracture toughness.
Notably, the enhancements can be observed even with a low
filler loading in the polymer matrix. An unfilled epoxy rein-
forced with 0.125 wt% functionalized GNS presented increases
of ∼65% in the fracture toughness and ∼115% in the frac-
ture energy [43]. Similarly, the incorporation of 2 wt% of GO
nanosheets to polyvinylidene difluoride (PVDF) increased the
tensile strength by 92% and the Young’s modulus by 192% [44].
The improvements in properties promoted by graphene
are also observed in biopolymers. The addition of 5 wt% GO
to polycaprolactone (PCL) increased the modulus of elastic-
ity from 344 to 626 MPa due to higher polymer crystallinity
[45]. Films of chitosan blended with 6 wt% rGO sheets with
nacre-like layered structure presented significant increases in
the Young’s modulus (from 2.4 to 6.3 GPa) and tensile strength
(from 88 to 206 MPa) compared to pure chitosan [46]. The
addition of 3 wt% GO to chitosan scaffolds increased the hard-
ness from 0.3 to 1.1 GPa and the modulus of elasticity from
2.6 to 6.7 GPa [47]. Likewise, the combination of 0.2w/v% GO
to carboxymethyl-chitosan increased the hardness from 0.05
to 0.18 GPa and the elastic modulus from 1.0 to 2.8 GPa [48].
Although chitosan is an insulator, the addition of 1 wt% rGO
amplified the conductivity of the composite film to 0.33 S/m
while the composite film with 6 wt% rGO presented a maxi-
mum conductivity of 1.28 S/m [46].
Apart from the enhancements observed in physical and
mechanical properties, the combination of graphene and poly-
mers can improve the bioactivity and promote stem cell
differentiation.
For instance, the addition of GO to electrospun polylactic-
co-glycolic acid (PLGA) nanofibrous mats increased the
adsorption of dexamethasone, an osteogenic inducer, com-
pared to the unmodified mat. This composite increased the
gene expression of collagen I, ALP and OCN in MSCs in
the presence of dexamethasone. Notably, irrespective of the
presence of dexamethasone, cells in the GO/PLGA scaffolds
presented significantly higher amount of OCN protein after
28 days than those in the control scaffold [49]. Similarly, PCL
scaffolds modified with GO increased the proliferation of MSCs
and induced greater mineral deposition compared to the scaf-
folds modified with rGO and pure PCL [45]. The combination of
rGO to poly-dopamine (PDA) results in composites with a ten-
dency to induce nucleation of hydroxyapatite when soaked in
simulated body fluid. The rGO/PDA-based substrates also pro-
moted higher adhesion and proliferation of osteoblastic cells
as compared to glass. Remarkably, cells on rGO/PDA substrate
spread to an area of 30,000 "m2 while those on glass reached
only 8600 "m2 . Moreover, the pre-osteoblasts cultured on the
rGO/PDA substrate presented higher ALP activity suggesting
an enhanced osteogenic differentiation compared to the con-
trols [50].
Among the natural polymers, chitosan is one of the most
explored for tissue engineering and other applications (e.g.,
sutures, implants and wound dressings). This polysaccha-
ride can present antifungal and antibacterial activity besides
having analgesic and hemostatic potential. Nonetheless, chi-
Please cite this article in press as: Xie H, et al. Graphene for the development of the next-generation of biocomposites for dental and medical
applications. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.04.008
5
tosan per se is not osteoconductive [51]. The addition of
rGO to chitosan can improve specific stem cells functions
(e.g. attachment, calcium nodule deposition and OCN protein
expression) via nanotopographic features [52]. Notably, the
negative charge and polarity of GO increase the adsorption of
serum albumin protein by the GO-modified chitosan scaffold
compared to chitosan alone [53]. Together, these characteris-
tics may play an important role in the improved attachment
and proliferation of pre-osteoblasts in the GO-modified chi-
tosan scaffolds [47,53]. Similarly, bone marrow MSCs seeded
in 0.2w/v% GO/carboxymethyl-chitosan scaffolds presented
higher gene expression of OCN, OPN, and ALP after seven days.
The improved osteogenic potential of the GO-modified com-
posite was confirmed in vivo where it increased the new bone
volume to tissue volume ratio (BV/TV) from 11 to 28% compar-
ing to chitosan alone [48].
Biocomposites made of silk-fibroin combined with both
GO or rGO are promising alternatives for the differen-
tiation of periodontal ligament stem cells (PDLSCs) into
osteo/cementoblast-like cells. Silk-fibroin films loaded with
GO increased the initial adhesion and proliferation of PDLSCs
after seven days as compared to fibroin alone [54]. GO
can also be used to improve the handling and confer
three-dimensional characteristics for scaffolds made of this
biopolymer. Although PDLSCs failed to express definitive
genetic markers of chondroblastic (SOX9) and osteoblas-
tic (SP7/OSX and BGLAP) differentiation, an increase in
the expression of early osteoblast/cementoblasts markers
(COL1A1, RUNX2, BMP2 and ALP) after ten days of culture
was observed. Notably, cells in the scaffold loaded with rGO
presented positive protein expression of CEMP-1 which is
expressed by cementoblasts and their progenitors, even with-
out the use of chemical inducers for the same [55].
Polymers are widely used for drug delivery systems. In
this sense, graphene can be combined with these materials
to design enhanced carriers for biomolecules, peptides and
anticancer agents [56,57]. The sheet-like structure of graphene
provides a large surface area for drug loading. The carbon
lattice structure of graphene allows non-covalent pi–pi inter-
actions between the graphene and hydrophobic molecules,
enabling, for example, the loading of small molecule can-
cer drugs or proteins/peptides with hydrophobic residues.
Charged molecules can also be loaded onto GO via electrostatic
interactions. To improve biocompatibility and cellular uptake
of the graphene materials, the functional groups present on
graphene oxide can be used to attach stabilizing polymers
such as polyethylene glycol. For instance, GO functionalized
with polyethylene glycol (PEG) can be an efficient vector for the
intracellular delivery of proteins. The GO/PEG can effectively
protect bovine serum albumin from enzymatic hydrolysis
without compromising the serum functions [58]. GO function-
alized with poly N-vinyl caprolactam (GO-PVCL) can be an
effective carrier for the anti-cancer drug camptothecin (CPT).
The GO-PVCL loaded with CPT induced significant cancer cell
death as compared to the controls. In fact, after three days,
the KB cell viability decreased to less than 20% for the loaded
GO-PVCL whereas approximately 50% of the cells remained
viable when incubated with the unloaded version [59]. SN-38
is an active metabolite of irinotecan (CPT-11) that contributes
significantly to its activity. When attached to a GO-PEG com-
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Fig. 4 – PDLSCs can attach, proliferate and secrete extracellular matrix on the surface of a graphene-based scaffold after
5 days (scale bar = 20 !m).
plex, SN-38 presented stronger cancer cell killing potential
compared to CPT-11 when used to treat human colon cancer
HCT-116 cells in vitro [57].
As shown by these and other reports, graphene family
materials can improve significantly the physicomechanical
properties of polymeric composites. Indeed, there are sev-
eral opportunities for further development and optimization
of graphene-modified polymer composites such as the opti-
mizations of reinforcement phase size and concentration,
understanding of the interfacial adhesion between graphene
and the polymeric matrices, processing methods and oth-
ers. Nonetheless, one major drawback of these combinations
is the dramatic color change of the polymers promoted by
the addition of these carbonaceous materials. GO and rGO
solutions are dark even at very low concentrations [60] and
pristine graphene absorbs a significant fraction of white light
(pa = 2.3%) [61]. This may pose a new set of challenges for the
use of these materials in restorative and prosthetic materials
where optical properties are a concern.
4. Graphenes as additives or coatings
leading to the next generation of metals for
biomedical applications
Metals are used in several biomedical applications in pure
form or as alloys due to their high toughness, strength, and
durability. The graphene family materials can improve the
properties of metals even further and confer a bioactive char-
acter to the metal-based composites.
A remarkable study showed that composites made of
alternated nanolayers of metal and graphene achieved
ultra-high strengths of 1.5 (copper/graphene) and 4.0 GPa
(nickel/graphene) tested under nanopillar compression test.
The improvements observed were related to the ability
of graphene to block dislocation propagation across the
graphene–metal interface [62]. Copper–graphene nanocom-
posite foils obtained via electrodeposition presented signifi-
cantly higher hardness and modulus of elasticity as compared
pure copper [63]. Notably, the addition of 0.18 wt% graphene
nanoplatelets to Mg–1%Al–1%Sn via semi powder metallurgy
method increased the ultimate tensile strength (from 236 to
268 MPa) and yield strength (161 to 208 MPa) comparing to the
control [64]. These enhancements are likely to be a result
of the uniform distribution of the second phase within the
Please cite this article in press as: Xie H, et al. Graphene for the development of the next-generation of biocomposites for dental and medical
applications. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.04.008
metal matrix that absorbs the load transferred from the matrix
upon an effective interfacial connection between them [65].
GO sheets deposited on titanate nanowire scaffolds increase
by forty times the Young’s modulus of the structure [66].
These reports show that graphene family materials have the
potential to improve physical properties and mechanical per-
formance of metals and alloys.
Perhaps the most striking advantage that graphene offers
to metallurgy in biology is their capability to interfere
positively in several processes related to osteogenic differenti-
ation and cellular maturation toward osteoblastic phenotype
[12,37,67–69]. These have been observed in studies where
MSCs seeded on various inert substrates coated with CVD-
grown graphene presented higher calcium nodule deposition
comparing to uncoated ones [67,68,70]. We have shown that
periodontal ligament stem cells presented higher calcium
deposition, RUNX2 and OCN gene and protein expression in
graphene-coated glass and graphene scaffold (Fig. 4) com-
paring to the controls even without the use of chemical
inducers for the same [37]. Similar behavior was observed
for dental pulp stem cells that increased spontaneously the
protein expression of both OCN and OPN when cultured on
single-layer graphene without the use of osteogenic medium
[70]. GO and rGO also receive a great share of interest
for promoting the osteogenic differentiation of stem cells
[10,67,71,72]. For instance, bone marrow MSCs on GO-coated
polydimethyl siloxane substrates presented a significant
increase in mineralized deposition compared to those on
uncoated polydimethyl siloxane substrates with the use of
osteogenic medium [67]. Likewise, dental pulp stem cells
cultured on glass coated with GO for 15 days presented signif-
icant increases in the expression of several genes expressed
by mineral secreting cells (e.g., RUNX2 and DMP-1) [10]. It
is hypothesized that the ability of graphene to concentrate
osteogenic enhancing molecules/proteins on its surface via
pi–pi interactions with the carbon lattice, or through electro-
static interactions with functional groups on GO and rGO, may
contribute to its ostogenic properties. Hence, graphene and its
derivatives may be interesting alternatives to improve osteo-
genesis in metals used for implants.
CVD-grown graphene may be directly synthesized or trans-
ferred to metal substrates. The direct synthesis demands
optimal conditions to allow the growth of graphene domains
on the target substrate. Although this is challenging, CVD-
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grown graphene directly synthesized on nitinol shape
memory alloy can increase the ALP activity and expression
of osteogenic-related genes such as OCN, OPN, BMP-2 and
RUNX2 [73]. Alternatively, the transfer of graphene films onto
target substrates can be obtained via several techniques. For
instance, the polymer-assisted dry transfer technique have
successfully transferred CVD-grown graphene to the sur-
face of two-dimensional austenitic stainless steel substrates.
The graphene attached to the stainless steel surface pre-
sented good stability and allowed MSCs proliferation [16].
Despite of these interesting results, the transfer of atomic
thin films onto three-dimensional objects with high shape
complexity remains as one of the most challenging issues in
graphene-related research. Recently our group have developed
a vacuum-assisted dry transfer method to deposit CVD-grown
graphene on metal prosthetic parts of different sizes and
high shape complexities (e.g., dental implants, locking plates
and compression plates). The prosthetic parts were three-
dimensionally coated with the graphene film and allowed the
attachment and proliferation of MSCs for 7 days [17].
Several reports confirm the osteogenic potential of GO on
metal substrates. The presence of 0.1 mg/mL GO coating on
sodium titanate significantly increased the gene expression
of collagen I, RUNX2, OCN and ALP in periodontal ligament
stem cells compared to sodium titanate alone [74]. Titanate
nanowire scaffolds modified with GO grafted with OH, NH2
and COOH accelerate MG-63 cell proliferation as compared to
the unmodified scaffold. Interestingly, only the scaffolds with
decorated GO were capable to increase the ALP activity and
calcium nodule deposition [66].
Another promising area is the use of graphene as plat-
forms for the release of therapeutic molecules at surgical
sites to improve bone formation and integration of implants.
Graphene, GO and rGO are known to bind to dexametha-
sone, serum, insulin, albumin, BMP-2, and others [72,75–77].
The application of rGO-based coating loaded dexametha-
sone onto Ti13Nb13Zr alloy enhanced the differentiation of
MC3T3-E1 cells into osteoblasts. Notably, cells seeded on the
rGO/dexamethasone-coated alloy presented higher expres-
sion of collagen I, OCN RUNX2 and OPN genes after seven days.
Moreover, it was observed higher ALP activity and increased
calcium nodule formation as compared to the uncoated alloy
or unloaded rGO coating [78].
The binding properties and release profile vary according
to the electrical character of the chemical moieties present on
the material. For instance, GO negatively charged with COO
releases only 40% of the BMP-2 attached to it within the first
24 h. However, GO positively charged with NH3 + releases
more than 65% for the same period of time [77]. Moreover, the
characteristics of the compound also affect the profile release
of proteins. La et al., found that nearly 80% of BMP-2 attached
to GO was released within two days, whereas it took ten days
to release similar percentage of their proprietary substance P
[76]. Nevertheless, the cumulative release of both substance
P and BMP-2 reaches more than 80% after 14 days in the dif-
ferent forms of GO [76,77]. In fact, the potential of GO as a
carrier for BMP-2 on titanium implants has been confirmed in
vivo. GO-modified Ti ring implants loaded with BMP-2 placed
on occipital bone of mice increased the new bone area by
three times and new bone volume by approximately four times
Please cite this article in press as: Xie H, et al. Graphene for the development of the next-generation of biocomposites for dental and medical
applications. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.04.008
7
when comparing to bare Ti or Ti/BMP-2 implants used as con-
trols [76].
Graphene and its derivatives have remarkable abilities to
improve the properties of metals, enabling the binding of
biomolecules, and to induce and enhance osteoblastic differ-
entiation. These characteristics place them in the spotlight for
the improvement and development of enhanced metals and
alloys for augmented bone formation.
5. Is graphene a cytotoxicity material?
One area of research that will keep growing aims to estab-
lish the safety levels and adverse effects of different types of
graphene in vitro and in living organisms. In fact, the ques-
tion “is graphene a cytotoxic material?” will remain open,
as the high versatility of the graphene family of materials
does not allow a single and conclusive answer to suits them
all. Although the literature offers valuable data, the variety
of shapes, sizes, chemical characteristics and combinations
obtained with materials and graphene, GO and rGO allow a
wide range of biological outcomes and interpretations [12,69].
For example, CVD-grown graphene film is widely recognized
to allow human stem cells cell attachment and prolifera-
tion without signs of apparent cytotoxicity [17,37,67,68,79].
Nonetheless, graphene sheets in solution can threaten cell
viability, either by accumulation on the membrane induc-
ing high level of oxidative stress, by physical damage (e.g.,
membrane penetration), or by the interaction between the
hydrophobic surface of graphene with cell lipids [80–82]. The
same is true for GO that when in solution can increase the
generation of mitochondrial reactive oxygen species and cell
death in mouse alveolar macrophages, but does not decrease
human cellular proliferation either as a film or when com-
bined with other biomaterials [10,49,54,67,71].
The lack of consensus is also reflected in in vivo studies
where the concentrations and variations of materials tested
can dramatically change the outcomes observed. For instance,
mice intratracheally treated with GO presented a significant
higher number of apoptotic cells and increase in plasma
thrombin antithrombin complexes after 24 h compared to
those treated with graphene dispersed in 2% block copoly-
mer Pluronic [83]. The outcomes also vary according to the
outcome evaluated. For example, the intravenous injections
of GO at a concentration of 20 mg/kg in mice can induce the
formation of micronucleated polychromic erythrocytes [84],
but may not present reproductive side effects in a concentra-
tion as high 25 mg/kg [85]. Hence, when drawing conclusions
regarding the nature of graphene safety and biocompatibility,
it is important to distinguish between the different mate-
rial variations and experimental conditions, rather than make
generalized observations. Future research shall also focus on
determining the safety aspects of specific graphene-based and
modified materials in conditions relevant to their intended
clinical use.
In addition to the biological concerns, other aspects must
be better elucidated such as the long-term stability of these
materials. This is especially concerning for GO-based mate-
rials and coatings, which may detach from substrates or
leach from materials due to the hydrophilic nature. Hence,
DENTAL-2945; No. of Pages 10
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8 d e n t a l m a t e r ia l s x x x ( 2 0 1 7 ) xxx–xxx
it is important to understand how biomaterials that con-
tain graphene and its derivatives behave in humid corrosive
microenvironments, as the particles may harm tissues and
organs if they reach the bloodstream [69,82,83,86].
6. Conclusion
Graphene and its derivatives are versatile materials that
present unique characteristics such as large surface area, high
mechanical properties and can be transferred or deposited
onto different substrates. As they can be functionalized and
combined with several biomolecules and biomaterials, these
carbonaceous materials hold great potential to design bio-
composites with fine-tuned physicochemical and mechanical
properties and to confer to existing biomaterials enhanced
bioactivity and new capabilities.
Acknowledgments
V.R. was supported by the grants from the National
University of Singapore, Singapore (Academic Research
Fund Tier 1, R-221-000-091-112) and National Univer-
sity Health System, Singapore (NUHS Bench-to-Bedside
NUHSRO/2014/017/B2B/02 and NUHS Open Collaborative
Research Grant NUHS O-CRG 2016 Oct-25) and National
Research Foundation, Singapore (NRF-CRP Medium Sized Cen-
tre Programme).
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