Materials Science & Engineering C 102 (2019) 171–185

Contents lists available at ScienceDirect

Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

Review

Graphene and its derivatives: Opportunities and challenges in dentistry T

a,⁎ a b c,d a a
M. Tahriri , M. Del Monico , A. Moghanian , M. Tavakkoli Yaraki , R. Torres , A. Yadegari ,
⁎
L. Tayebia,
a
Marquette University School of Dentistry, Milwaukee, WI 53233, USA
b
Department of Materials Engineering, Imam Khomeini International University, Qazvin 34149-16818, Iran
c
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore
d
Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 2 Fusionopolis Way, 138634, Singapore

A R T I C LE I N FO A B S T R A C T

Keywords: The emerging science of graphene-based engineered nanomaterials as either nanomedicines or dental materials
Graphene in dentistry is growing. Apart from its exceptional mechanical characteristics, electrical conductivity and
Graphene oxide thermal stability, graphene and its derivatives can be functionalized with several bioactive molecules, allowing
Dentistry them to be incorporated into and improve different scaffolds used in regenerative dentistry. This review presents
Biocompatibility
state of the art graphene-based engineered nanomaterial applications to cells in the dental field, with a particular
Stem cells
Tissue engineering
focus on the control of stem cells of dental origin. The interactions between graphene-based nanomaterials and
cells of the immune system, along with the antibacterial activity of graphene nanomaterials are discussed. In the
last section, we offer our perspectives on the various applications of graphene and its derivatives in association
with titanium dental implants, membranes for bone regeneration, resins, cements and adhesives, as well as
tooth-whitening procedures.

1. Introduction
Carbon is an abundant element that has important applications in
the fields of science and technology. Many various carbon allotropes
can be synthesized by altering the combinations of sp, sp2, and sp3
hybridization [1–3], and a variety of carbon structures and nanos-
tructures have been introduced to date. Generally, carbon nanos-
tructures are mainly composed of sp2 hybridized carbon atoms ar-
ranged in a hexagonal lattice, but there are many different potential
structures, morphologies and properties.
There are four main categories of carbon nanostructures: zero, one,
two and three-dimensional. Examples of zero-dimension carbon na-
nostructures are fullerenes, carbon dots and graphene quantum dots.
One-dimensional carbon nanostructures include nanotubes and nano-
fibers. Two-dimensional carbon nanostructures, which have been re-
cently introduced, have interesting characteristics. Graphene is a pro-
minent two-dimensional nanostructure. Lastly, three-dimensional
carbon nanostructures are composed mainly of one and two dimen-
sional-structures, such as three-dimensional graphenic superstructures
and nanotube-graphene hybrids [4–7].
Graphene is the thinnest and strongest material in existence [1]. It is
mainly composed of two-dimensional sheets less than 10 nm thick.
These sheets are made up of sp2 hybridized carbon atoms that are
bonded in a honeycomb-like lattice. Graphene and its derivatives have
many applications in the fields of science and technology due to its
physical and chemical properties such as electrical conductivity,
transparency, biocompatibility, superior mechanical strength and high
surface area [8,9]. Graphene is used to enhance strength and mechan-
ical properties of composite and nanocomposites and has potential
applications in disease diagnosis, cancer therapy and targeting, bioi-
maging and drug and gene delivery [4–7]. In addition, graphene shows
improved biocompatibility compared to other classes of carbon na-
nostructures due to its unique surface characteristics [10]. The toxicity
of graphene is based on physical and chemical characteristics, including
the number of layers, chemical functional groups, surface charge den-
sity and more. There are many reports on biocompatibility and toxicity
of graphene-based nanostructures, but it has yet to be discussed com-
prehensively [11].
2. History of graphene and its derivatives
More than 80 years ago, there was a hot argument about the sta-
bility or instability of two dimensional (2D) materials, when Landau
and Peierls [12,13] claimed that 2D nanocrystal could not exist due to

⁎
Corresponding authors.
E-mail addresses: mohammadreza.tahriri@marquette.edu (M. Tahriri), lobat.tayebi@marquette.edu (L. Tayebi).

https://doi.org/10.1016/j.msec.2019.04.051
Received 16 January 2019; Received in revised form 11 April 2019; Accepted 14 April 2019
Available online 16 April 2019
0928-4931/ © 2019 Elsevier B.V. All rights reserved.
M. Tahriri, et al.
thermodynamic instability. Later, the same claim was supported by
experimental results obtained by Mermin [14], when they claimed that
the melting point of nanocrystals decreases dramatically with decrease
in their thickness; hence, the existence of 2D nanocrystal is under
question. Therefore, the observed 2D atomic materials were considered
as an integral part of a three dimensional (3D) system, which have been
grown epitaxially on top of monocrystals with matching crystal lattices
[15,16]. So, without considering such a 3D base, 2D materials were
presumed not to exist. This theory remained until 2004, when graphene
was discovered experimentally [17].
For easy understanding of what graphene is, it could be considered
as a smaller part of graphite with a few layers. Hence, by this point of
view, the graphene and its properties would not be new and its history
could back to about 500 years ago. Even in the middle ages, we have
used graphite to make the pencil for writing. The extraordinary prop-
erties of graphite, including thermal conductivity (∼3000 W/mK), in-
plane electrical (∼104 Ω−1 cm−1) and mechanical stiffness of the
hexagonal network (1060 GPa) enable graphite to be used in different
industries for different applications, where the annual demand for it
reaches to 1 million tons worldwide [18–20].
The anisotropy of graphite materials has facilitated the scientists in
different field. The sp2 atomic orbitals involved s, px and py, with 120°
angle for strong CeCeC bond make chicken-wire-like layers. The re-
maining pz orbitals could overlap with the three neighbor C atoms and
form two different bands, called valence band (filled π orbitals) and
conduction band (empty π* orbitals). As each carbon has four electrons
involved in the bonds with three neighboring carbons, three of them
take part in sigma bond (σ) and one of them takes part in one-third of
the π bond. Hence, the interaction between the planes is very weak
because no chemical bond exists between them. This property leads to
honeycomb pattern and anisotropic physico-mechanical properties of
graphite, which is function of direction and makes an extreme differ-
ence for in-plane and out-of-plane directions [19].
Graphene has two main derivatives, namely graphene oxide (GO)
and reduced-graphene oxide (rGO). GO could be obtained by oxidation
of graphite and rGO could be synthesized by reduction of GO (Fig. 1).
GO has variety of functional groups (e.g., hydroxyl, carboxyl and epoxy
groups) that allow GO to be attached to different types of molecules,
especially biomolecules for wide range of bio-applications. As such, GO
is of interest to be used as an improved alternative due to its better
mechanical and bioactivity in bio-applications [21–23]. The rGO have
oxygen-containing groups and some defects that result in a very similar
structure to pristine graphene, where the ratio of oxygen-containing
Fig. 1. Key milestones on graphene development [25].
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Materials Science & Engineering C 102 (2019) 171–185
groups and surface defects could be controlled by degree of reduction
reaction [24].
3. Structure of graphene and its derivatives
Graphene and its derivatives have very similar structures that form
2D materials; however, they have some small differences that cause
different physico-chemical properties. In this section, the structure of
graphene and its two main derivatives has been discussed.
3.1. Graphene
As the main critical element in graphene and its derivatives is
carbon, understanding of carbon and its bonding is important. Each
carbon atom has four valence electrons that could be shared through
covalent bonds with other elements. These electrons could be hy-
bridized in different forms, namely sp, sp2 and sp3. The 2D structure of
graphene is a result of sharing sp2 hybridized orbitals of one carbon
with three neighboring carbon atoms (Fig. 2).
In graphene, the resulted in-plane bond is based on sp2 hybridized
orbital, consisting of s, px and py, while pz is free and is perpendicular to
the plane. Hence, the 2D plane has sigma bond with very short in-
teratomic length (about 1.42 Å), which make it stronger than sp3 hy-
bridized orbital in diamond. Such a strong CeCeC in-plane bond is the
reason of dramatic mechanical resistance of graphene monolayer, e.g.
an intrinsic tensile strength and a Young's modulus of 130.5 GPa and
1 TPa, respectively [26]. Additionally, the half-filled π band causes zero
band gap between the valence and conductive bands, resulting free-
moving electron in the monolayer of graphene. Also, the weak π bonds
result a weak Van der Waals interaction between the monolayer, which
enable them to move on each other under very weak shear stress [1,27].
The surface functionalization of graphene sheets is very similar to
the methods available for functionalization of carbon nanotubes
(CNTs). However, the ultrasound treatment before any functionaliza-
tion seems essential in order to separate the graphene sheets from each
other and make them ready for further surface processes. “Hot” re-
agents are radicals that could react mainly with unreactive sp2-carbons.
These hot reagents could be diazonium salts [28–30], fluorine [31,32],
radicals [33] and nitrenes [34], while the control of degree of reaction
is hard. In contrast, controlled reaction rate could be achieved using
cycloaddition reactions based on benzyne [35] or azomethine ylides
[36,37], which have lower reactivity. The functionalization of edges
also could be performed using mild Friedel-Crafts acylation approach
M. Tahriri, et al. Materials Science & Engineering C 102 (2019) 171–185

Fig. 2. A) (a) Atomic structure of a carbon atom. (b) Energy levels of outer electrons in carbon atoms. (c) The formation of sp2 hybrids. (d) The crystal lattice of
graphene, where A and B are carbon atoms belonging to different sub-lattices, a1 and a2 are unit-cell vectors. (e) Sigma bond and pi bond formed by sp2 hybridization
[27]; B) different approaches for surface functionalization of graphene sheet: (a) Non-covalent, (b) intercalation and (c) covalent; C) possible routes for the mod-
ifications of biomaterials with graphene [45]; D) TEM image of a graphene nanosheet [48]; E) Raman spectra of graphene with different layers [48].

[38]. In general, all covalent functionalization of the graphene are
based on sharing sp2 orbitals and changing it to sp3 hybridized orbitals,
where it affects the local symmetric and electronic structure, conse-
quently [39].
In addition to covalent functionalization, the non-covalent approach
is of interest in bio-applications, where usually formation of polymer/
graphene nanocomposite is of interest. The interaction between the
graphene and polymeric compounds could be due to Van der Waals
forces, π-π interaction and electrostatic interactions as well as chemical
binding, which is explained previously. The interaction between the
graphene and polymers is always based on the combination of Van der
Waals forces and other interactions because of the structure of gra-
phene. In addition, the polymers with π-bond, e.g. PVA and PMMA
[40], could have π-π interactions with graphene [41]. These non-
covalent interactions offer stability of the graphene, as well as im-
proving the thermal, electrical and mechanical properties [42–44]. Si-
milar to polymers, graphene nanosheets could be added to bioceramics
or metallic structures to improve their bioactivity, mechanical and
thermal properties [45].
The graphene nanosheet consists of only a layer of carbons; hence,
the characterization methods are of importance and it is difficult to
perform adequate characterization. Therefore, scientists usually use a

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series of techniques, mostly based on usual methods that include optical
microscope, SEM, TEM, AFM, Raman spectroscopy, etc. to identify and
characterize the graphene nanosheets. Gao et al. [46] have developed
an easy nondestructive colorimetric method using optical microscope,
based on the change in the contrast of the graphene with different
layers to estimate the number of graphene layers on silica substrate.
Additionally, the SEM has been also used to see the graphene na-
nosheet. Although specific silicon wafer must be used for graphene
characterization, the graphene has more contrast in SEM images com-
pared with optical microscope [47]. Moreover, the atomic force mi-
croscopy (AFM) is not a good choice to estimate the number of layers of
graphene nanosheets, mostly due to the adsorption of a monolayer of
water on the surface of graphene at ambient condition and hence,
causing an error in tip attraction/repulsion. Therefore, it is very hard to
reach the theoretical thickness of graphene (about 0.34 nm) using AFM
[17]. Among the characterization techniques, TEM could provide in-
formation not only about the morphology of the graphene, but also
accurate number of layers using the analysis of edge of samples by
cross-section TEM (Fig. 2D). TEM-assisted with electron diffraction
pattern also shows a hexagonal pattern for graphene nanocrystal
structure [48]. Raman spectroscopy also could help to provide some
information about the number of layers in graphene sample. As seen in
Fig. 2E, the Raman peak at around 1600 cm−1 decreases with a de-
crease in number of layers in graphene nanosheets, but the peak at
2700 cm−1 increases [48].
3.2. Graphene oxide
Graphene oxide is the product of oxidation of graphene; therefore, it
is a complex mixture of materials. Hence, due to lack of analytical
techniques to precisely characterize this amorphous material with
berthollide character (i.e. nonstoichiometric atomic composition), pre-
senting an unambiguous model for GO structure is still subject of con-
siderable debate. Despite these obstacles, different researchers have
proposed different models. Despite the primary developed models (e.g.
Hofmann [49], Ruess [50], Scholz-Boehm [51] and Nakajima-Matsuo
[52,53]) that are usually based on the consideration of lattice-based
model, new models have focused on an amorphous and nonstoichio-
metric alternative. Anton Lerf et al. [54–56] have developed their
model based on the experimental results obtained by solid NMR and
different X-ray analysis techniques, where the GO sheets could have a
hydrogen bond between each other (Fig. 3a and b). In another work,
Szabó et al. [57] have proposed a model with corrugated quinoidal
structure interrupted by trans-linked cyclohexyl regions, which is the
improved model previously suggested in the Scholz-Boehm model.
According to FTIR and DRIFT spectroscopy results, they have reported
that the 1714 cm−1 peak in IR spectra of GO is not indicative of car-
boxylic group, but rather of single ketones and/or quinones [56,57]. In
overall, it could be considered that GO contains different functional
groups [58], mainly hydroxyl, carboxyl or epoxy, which could be stu-
died well through use of different analysis techniques, especially FT-IR
[59–61] and XPS [62–65].
Besides, it should be highlighted that variation in starting materials
for synthesis of GO or the method and degree of oxidation process
might lead to a product with different functional groups and structure,
leading to the possible reason of current doubt in exact model structure
for graphene oxide. Nevertheless, in addition to the NMR-based char-
acterization (Fig. 3b), other techniques, such as thermal gravimetric
analysis (TGA) at very low rate of temperature evolution (1 °C/min),
could be used to characterize the thermal stability of GO. TGA curve of
GO shows a high rate of decomposition around 226 °C, which could be
attributed to the decomposition of the labile oxygen-containing moi-
eties. The TGA curve and its slope might be different for various sam-
ples, which could be due to the difference in chemical structure of
obtained GO from different methods [66,67].
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Materials Science & Engineering C 102 (2019) 171–185
3.3. Reduced graphene oxide
Graphene oxide containing different oxygen groups could be re-
duced and result in a product called reduced-graphene oxide (rGO).
This process could be performed using different approaches, including
chemical reduction, microwave-assisted reduction, photo-reduction,
thermal reduction and solvothermal reduction. The general properties
of rGO are very similar to the graphene nanosheet because the GO has
lost its oxygen-containing groups through the reduction reaction. Shin
et al. [70] have monitored the XRD pattern of the GO sample in dif-
ferent reduction conditions using different concentrations of sodium
borohydride (NaBH4) as the reduction agent. As seen in Fig. 4, the main
peak of XRD pattern of GO sample increases from 10.7° and reaches the
value of 23.9°, with increase in NaBH4 concentration. This shift in XRD
peak and reaching the value very close to the original value of graphene
implies that oxygen-containing groups are being lost and that the final
rGO has very similar crystal structure to the graphene nanosheet. In
addition, a color change occurs after reduction process—the brown
color of GO solution turns to black, indicating formation of rGO [71].
Ma et al. [72] have investigated the FT-IR of the rGO and have observed
that not all functional groups existed in GO could be removed during
reduction process, causing rGO to still have some functional groups
such as eOH, eCOOH, eCeO and eC]O. Similar observation has been
reported by different researchers. Other analyses also indicate that al-
though rGO is the reduced form of graphene oxide and should be ide-
ally very similar properties to graphene nanosheet, the properties of
rGO depends on reduction conditions. The result will be properties
between that of GO and graphene. For example, TGA analysis of rGO
obtained in air and N2 using photothermal reduction method by Guo
et al. [73] shows the enhanced thermal stability of rGO compared with
GO, while it is slightly less thermally stable than graphite. In addition,
the XPS study has confirmed the same results: reduction of oxygen-
containing group in rGO compared with GO [73–76].
4. Synthesis and preparation of graphene
Graphene can be produced by three major techniques: exfoliation,
chemical vapor deposition and chemical-based techniques. One of
graphene's critical features is the number of layers of graphene sheets;
in order to achieve the desired properties of graphene sheets, the sheets
must be separated from each other or else they are prone to aggregate
to form rudimentary graphite structures. Thus, the methods of synthesis
promote the exfoliation of graphene sheets [77].
4.1. Mechanical exfoliation/cleavage
In order to harness the superior properties of graphene, the sheets
must be individually separated [78]. Graphite is composed of graphene
sheets held together by Van der Waals interactions, and the method of
exfoliation takes advantage of this fact by breaking those weak inter-
actions [79–81]. Exfoliation is involved in other various techniques,
including liquid phase exfoliation, exfoliation of graphite oxide, un-
zipping carbon nanotubes, mechanical exfoliation and more.
4.2. Chemical vapor deposition (CVD)
Another method of producing graphene is called chemical vapor
deposition (CVD). In this method, graphene is synthesized by a graphite
target or catalytic decomposition of hydrocarbons on the surface of a
metallic catalyst. An advantage of this method is the absence or low
amount of metallic residuals [82]. CVD is one of the best methods for
producing single-layer graphene or graphene with few layers. In addi-
tion, this method allows for the synthesis of heteroatom-doped gra-
phene nanostructures; graphene may be doped with nitrogen, sulfur,
phosphorous, iodine, brome, fluorine and more, which improves its
performance in catalytic and enzymatic applications, as well as bio-
M. Tahriri, et al. Materials Science & Engineering C 102 (2019) 171–185

Fig. 3. (a) Different proposed structure model for graphene oxide [56], (b) H-NMR and C-NMR spectra of graphene oxide [58], (c) TGA analysis of GO and ozonated
GO [66] and (d) absorbance spectra of graphene and graphene oxide [68,69].

Fig. 4. (a) The XRD pattern of GO and rGO as a product of chemical reduction of GO using different concentration of NaBH4 [70], (b) FT-IR spectra of rGO [72] and
(c) TGA curves for GO, rGO and graphite [73].

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M. Tahriri, et al.
detection and energy conversion [83].
4.3. Hummer's method
Lastly, chemical-based techniques are one of the most productive
methods for the synthesis of graphene and its derivatives. The most
well-known approach is called the Hammer's method, in which graphite
is used as a carbon precursor and oxidized in concentrated sulfuric acid
in the presence of KMnO4, another oxidant. After maintaining the re-
action at a desired temperature, water and oxygen peroxide are added
to separate the graphene oxide sheets, which are subsequently washed
with hydrochloric acid (to remove residual Manganese) and water (to
reach neutral pH) [79]. Chemical-based techniques can be used to
produce large amounts of graphene, though some of these approaches
require special or precious materials. Another chemical-based method
for the synthesis of graphene is called electrochemical modification of
graphene. In this method, graphene sheets are synthesized by applying
constant voltage to a graphite anode and cathode in an electrolytic
solution of deionized water and ionic liquids, such as imidazolium [84].
5. Biological properties of graphene
5.1. Graphene biocompatibility
When using graphene, as with any material being used in a bio-
medical application, the first essential step is considering the material's
biocompatibility [85]. The biocompatibility of the material must be
excellent in order to eliminate the possibility of creating an adverse
effect within the living tissues [86]. Not only is avoiding an adverse
effect within the living tissues a necessary factor in determining a
material successful in regard to biocompatibility, but making sure that
the material can lead to successful tissue engineering techniques is a
key factor as well [87,88]. If the tissue engineering techniques derived
from the use of a certain material can result in the desired tissue lineage
obtaining stem cell differentiation, then one can conclude that the
material has the required excellent biocompatibility [89–108]. What
this infers is that this biocompatibility can be obtained both in vitro and
in vivo, however, in order for the material to have excellent bio-
compatibility, the results obtained in vitro must be reproduced in vivo as
well [109]. Once the results are produced in vivo, they must maintain
the natural oral environment produced by the body in regard to elim-
inating the possibility of bacterial growth on a scaffold composed of the
desired material, consequently avoiding an inflammatory response. The
oral cavity naturally forms bacterial biofilms, making the aforemen-
tioned quality of the material in regard to avoiding the possibility of
bacterial growth on the scaffold an essential part of excellent bio-
compatibility as well. Graphene and graphene-based nanomaterials
themselves are becoming increasingly popular in the dental field,
making the evaluation of these materials a vital part of their continued
use in dentistry in order to ensure that they produce excellent bio-
compatibility [110]. Before stating that graphene has an excellent
function in vivo, it must be evaluated based on its physical and chemical
qualities in vitro, mainly in regard to its toxicological potential, ensuring
that the factors that result in the cytotoxicity of graphene are ac-
knowledged and eliminated in order to avoid the interruption of future
in vivo translational efforts and studies [110–113]. In previous evalua-
tions of graphene-based nanomaterials in vitro, the shape and con-
centration of graphene proved to be the most critical components in
measuring the degree of its cytotoxicity and how the nanomaterials
interact with biological systems and cells [110]. Tests evaluating the
mitochondrial functions of cells such as the 3-(4,5-Dimethylthiazol-2-
yl)-2, 5-Diphynltetrazolium bromide (MTT) assay are used to determine
that the different shapes of graphene sheets and Single Wall Carbon
Nanotubules (SWCNT) make FLG sheets more toxic than SWCNT at low
concentrations and less toxic at higher concentrations of graphene
[112,114]. This proves that a higher concentration of graphene reverses
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Materials Science & Engineering C 102 (2019) 171–185
its cytotoxic effect due to the different shape of graphene compared to
SWCNT, allowing for the continuation of studies on graphene based on
its physical qualities to be carried out [110]. Although in vivo experi-
ments with graphene are few and the results of the in vitro experiments
vary based on the physiochemical properties of different graphene-
based nanomaterials, it can certainly be deduced from these tests that
the hydrophobic forms of graphene-based nanomaterials are more toxic
than the most hydrophilic forms. The hydrophobic forms are also more
likely to be internalized since they are more prone to accumulating on
the surface of cell membranes. However, it can also be determined that
when GO is compared to rGO, rGO is less toxic than GO, proving that by
maintaining the solubility of GO and controlling its reduction, it be-
comes possible to use it in many biomedical applications (including
dentistry) due to the created ability to minimize its toxicity [110].
Therefore, the biocompatibility of graphene and graphene-based na-
nomaterials cannot yet be assumed or generalized based on these
findings, but rather should be continued to be tested in both in vitro and
in vivo settings based on each material's ability to create excellent
biocompatibility resulting from their different physiochemical proper-
ties [110].
5.2. Graphene-stem cells interactions
In relation to the biocompatibility of a material being used in den-
tistry, an ideal material used for creating scaffolds must be able to result
in a scaffold that maintains the natural environment of the oral cavity.
This material must also be able to allow stem cells to form, proliferate
and differentiate into specific tissue lineages [110]. The use of gra-
phene-based nanomaterials in dentistry has been tested and proven to
stimulate cellular biomineralization and osteogenic differentiation
based on these materials' ability to increase osteoconductivity [115].
The main stem cells that are created, proliferated and differentiated
when graphene is introduced to an oral cavity are mesenchymal stem
cells (MSCs). Upon this discovery, the use of graphene to create, pro-
liferate and differentiate other stem cells was tested and resulted in
many beneficial uses and combinations of graphene with other growth
factors and cells to be discovered. When tested in vitro through the
chemical vapor deposition (CVD) method, graphene was found to in-
crease human MSCs when combined with different substrates and was
able to proliferate with no signs of cytotoxicity [116]. Without the use
of additional growth factors, graphene-coated scaffolds allow for the
growth and proliferation of human MSCs, while also resulting in a
quicker differentiation into osteoblasts. The level of the osteogenic
marker, osteocalcin, as well as the calcium mineralization level, are
used to make this growth, proliferation and differentiation of human
MSCs on graphene-coated scaffolds discoverable [110]. Going off of this
fact alone, one can determine that in regard to graphene-stem cell in-
teractions, the previously mentioned concerns of the toxicity of gra-
phene do not apply to this specific use of the material and that graphene
is actually very conducive to the growth of MSCs when used on a
graphene-coated scaffold in the oral cavity. After this discovery, tests
with graphene GO substrates in the creation of MSCs are conducted in
relation to the adipogenic and osteogenic differentiation of human Bone
Marrow MSCs (BM-MSCs). Graphene proves to induce BM-MSCs dif-
ferentiation into osteoblasts, attesting to the osteogenic benefit of using
graphene, however, when used for adipogenic differentiation, the re-
sults show a suppression of graphene but a strong enhancement on GO
substrates [110]. This again speaks to the fact that although graphene is
beneficial in certain biological applications, a generalization about its
overall benefits cannot yet be applied to any setting. Although a gen-
eralization cannot yet be made, other benefits of the use of graphene in
dentistry, specifically in regard to graphene-stem cell interactions can
be confidently concluded.
One of these benefits is in regard to the human MSC, Dental Pulp
Stem Cells (DPSCs). DPSCs and BM-MSCs have been proven to be
analogous cell populations due to their sharing of the expression profile
M. Tahriri, et al.
for thousands of genes, particularly those in relation to the initiation of
mineralization and bone homeostasis [117]. Due to this similarity be-
tween DPSCs and BM-MSCs, tests with GO-based scaffolds on DSPCs are
performed. These tests compare GO-based scaffolds to glass substrates
and show that although DPSCs are able to adhere to and grow on both
GO-based scaffolds and glass substrates without any significant differ-
ence, mRNA levels prove to be significantly higher in all genes tested
onto GO-based scaffolds as opposed to those tested onto glass substrates
after 14 days of seeding [118–120]. In addition to this, it is proven that
even without any inducers for differentiation, the GO-substrates pro-
vide an up-regulation in regard to the entirety of the measured gene
expression. The GO-substrate is also able to increase the expression of
two genes very closely related to the odontogenic differentiation of
stem cells from dental pulp, DMP-1 and DSPP, showing that the use of
GO-substrates alone may enhance the expression of odontogenic genes.
This would consequently allow for new opportunities for the use of GO
alone or with other dental materials enhance bioactivity and other
biomedical benefits [110].
Another stem cell that is closely related to BM-MSCs in regard to its
function is the Periodontal Ligament Stem Cell (PDLSC). Since they can
differentiate into many different types of specialized cells, PDLSCs can
be used in place of BM-MSCs so that the viability onto different sub-
strates and osteogenic potential can be determined [121]. So far, the
main and most beneficial substrate used with graphene to produce
PDLSCs has been Silk Fibroin (SF) [122]. PDLSCs are able to proliferate
the best in combination with a low amount of graphene and a higher
amount of SF. In fact, the best ratio for PDLSC proliferation is rGO:rSF
at a 1:3 ratio, and GO alone. Further, the information used to arrive at
this most beneficial ratio also concludes that SF is used to confer 3D
characteristics to rGO or GO and to improve its handling. This is per-
formed with the previously obtained information that 3D graphene
scaffold preparations result in higher PDLSC proliferation than 2D
graphene scaffold preparations when bioengineering PDLSCs [123].
When further tested in gene expression analyses, it is shown that gra-
phene-fibroin composites, in the low graphene high fibroin ratio and in
the absence of growth factors, can induce cementoblast differentiation
of human PDLSCs [124].
Continuing with this use of graphene with GO and other substrates,
Dental Follicle Progenitor Cells (DFPCs) are multipotent stem cells that
have the ability to differentiate into odontoblasts, osteoblasts, ce-
mentoblasts and other cells implicated in the teeth, while also having a
high proliferation rate and immunomodulatory properties [125]. In the
only experiment produced in regard to the behavior of human DFSCs on
graphene-based nanomaterials, DFSCs are taken from healthy extracted
teeth and seeded onto GO, Thermally Reduced Graphene Oxide (TRGO)
and Nitrogen-doped Graphene (N-Gr) substrates and analyzed based on
cytotoxicity, cellular and mitochondrial membrane alterations, and
oxidative stress induction for all developed substrates [126]. GO and N-
Gr are concluded to be the most promising fillers for dental nano-
composites, making them promising candidates as fillers that can lead
to a new periodontal ligament after in vivo implantation, since this is
another function of DFPCs [110]. This further attests to the importance
of continued in vitro experimentation before in vivo assumptions can be
made.
In vivo, the interaction of graphene and its composites with the
immune system is very important to consider since once they are ad-
ministered, they immediately come into contact with the immune
system, making the interaction between graphene and immune cells
very important to consider [110]. In lymphocytes, low doses of GO
have no cytotoxic effects on T lymphocytes, while doses over 100 μg/
mL result in apoptosis stemming from oxidative stress [127]. Although
no significant reduction in the immune response of T lymphocytes is
found, at high concentrations GO is absorbed into the cell membrane
without disrupting it, while also causing an increase in the apoptosis of
lymphocytes. Immune ability is suppressed and T cells are damaged in
GO-polyethylenimine, and high doses of GO-polyvinylpyrrolidone (GO-
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PVP) result in much less apoptosis than GO alone and also increase the
biocompatibility of GO. In macrophages, GO-PVP has a better im-
munological biocompatibility than GO alone, since it reduces the ability
of macrophages to phagocyte the compound, internalizing the GO
[128].
Graphene and GO also promote the production of several cytokines.
GO stimulates the production of myeloid differentiation primary re-
sponse gene 88, and 100 μg/mL GO generates the same effects of LPS
stimulation, activating macrophages and producing pro-inflammatory
cytokine [129–131]. With dendritic cells (DCs), GO with DCs alone
down-regulates intracellular levels of LMP7, which is part of an im-
munoproteasome required for processing protein antigens. GO-PVP on
DCs show GO promoting the maturation of DCs, pointing to the fact that
PVP mitigates the effects of GO and causes lower immunogenicity
[128,131]. Similar to the other tests consisting of graphene and gra-
phene-based nanomaterials with different cells, one can see that gra-
phene has different effects on different cells, both harmful and bene-
ficial to its use in dentistry, depending on the different physiochemical
factors, combinations with other materials or lack of combinations at
play.
5.3. Antibacterial activity of graphene
Although excellent biocompatibility is important in deeming a
biomaterial and its consequent scaffold successful, the scaffold's ability
to not harm the natural environment of the oral cavity is also essen-
tial—primarily achieved through inhibition of surface-level bacterial
growth. This material must also be able to promote cell adhesion and
proliferation [132]. As with the other characteristics of graphene, the
antibacterial properties of graphene and graphene-based nanomaterials
are based on the materials' time of exposure, concentration, physical-
chemical properties and the characteristics of the bacteria used in the
tests [133,134]. Graphene nanocomposites have a great antibacterial
ability against Gram-positive and Gram-negative bacteria, while the
general antibacterial effect is the result of graphene's ability to physi-
cally damage microorganisms by penetrating and cutting the cell
membrane, wrapping cells inducing mechanical stress and extracting
phospholipids from lipid membranes. It then produces oxidative stress
through ROS generation and charge transfer phenomena. Graphene
nanosheets can also insert into bacterial membranes, creating a break
through which Van Der Walls forces and the hydrophobic properties of
graphene extract phospholipids from the lipid layers of the bacterial
membranes. This causes irreversible damages, making the effects of
graphene on bacteria very effective [135–139].
Graphene sheets that are aggregated in suspension can isolate the
bacteria from the nutrient-rich surrounding environment [136], while
nanosheets of GO can be functionalized easily and disperse water ex-
cellently. Although these are beneficial qualities of GO, the anti-
bacterial activity of GO nanosheets is highly dependent on their size.
Larger GO nanosheets express stronger antibacterial activity against E.
coli, and GO itself decreases the viability of dental pathogens depending
on the concentration of GO present [140,141]. Graphene and some of
its composites can also act against bacterial biofilms in addition to
single bacteria [142]. Relating to the previously mentioned anti-
bacterial ability of graphene against Gram-positive and Gram-negative
bacteria, this is dose-dependent as well. While high GO concentrations
inhibit the formation of Gram-positive and Gram-negative bacteria
biofilms, low GO concentrations can actually enhance their formation,
creating a completely adverse reaction to the intended [143]. Although
it may seem that graphene itself has an antibacterial effect on bacteria
itself and bacterial biofilms, it has been proven that less than 50 μg/mL
GO in a nutrient medium solution has no antimicrobial activity and
actually enhances bacterial growth by acting as a biofilm itself. How-
ever, GO-polyoxyalkyleneamine, which is two antibacterial molecules
in the same concentration, exhibits antibacterial activity where bacteria
are grown in phosphate buffered saline solution [110,144]. What this
M. Tahriri, et al.
shows is that similar to the other applications of graphene in dentistry,
its antibacterial benefits are relative to the conditions of the tests,
particularly the type of material and size of particles, concentration and
state, as well as the type of bacteria and medium used [110]. In addi-
tion to all of this, the result on the antibacterial effects of graphene in
the oral cavity are also dependent on the host susceptibility, diets and
habits that could lead to a break in the balance of the oral micro-
environment [145]. Although the antibacterial activity of graphene
seems to be beneficial, it is all relative and cannot yet be generalized.
5.4. Graphene biodegradability
Biodegradability of graphene and its derivates is one of the crucial
parameters determining the fate of these materials in vivo. To evaluate
how graphene acts inside the body, Prof. Bianco and his group con-
ducted a lot of tests looking to discover if and how graphene was broken
down with the incorporation of a typical human enzyme [146,147].
The enzyme here, myeloperoxidase (MPO), is a peroxide enzyme dis-
charged by neutrophils, cells that are in control of the elimination of
any bacteria or foreign bodies that invade the body, found in the lungs.
In the event that bacteria or a foreign body is identified inside the body,
neutrophils surround it and secrete MPO, in this manner demolishing
the threat. Earlier studies by Rajendra Kurapati et al. [146] and Sourav
P. Mukherjee et al. [148] found that MPO to successfully biodegrade
graphene oxide. But the structure of non-functionalized graphene
(virgin graphene) was viewed as more degradation resistant. In order to
investigate this phenomenon, Prof. Bianco and his team tested the in-
fluences of MPO, ex vivo, on two graphene forms; single- and few-layer
[146]. They used two forms of graphene, single- and few-layer, fabri-
cated by two different techniques in water. They were then taken and
put in contact with myeloperoxidase within the sight of hydrogen
peroxide. This peroxidase was capable to degrade and oxidise them.
This was not so much expected in light of the fact that non-functiona-
lized graphene was more resistant than graphene oxide. The obtained
results concluded that highly dispersible graphene could be degraded in
the body by the action of neutrophils. This would open the new road for
creating graphene and its derivates.
6. Applications of graphene and its derivatives in dentistry
6.1. Implants
Titanium implants are currently considered to be the best replace-
ment for natural teeth due to the fact that they have favorable bio-
compatibility, are reliable and predictable. However, titanium's in-
herently inert quality leaves it susceptible to inducing the development
of fibrous tissue. This can cause the implant to fail, making titanium
implants the best option, but open to improvement [149,150].
The structure of an endosseous implant is made out of a fixture that
is put on the bone, alongside an abutment screwed to the top of the
fixture and the upper prosthesis, as displaced in Fig. 5.
Based on the aforementioned benefit of GO-coating in regard to
DPSCs, GO-Ti implants were created. Each way that the implants were
created, they showed that GO-coating of Ti implants creates an overall
improved benefit over Ti implants alone. These improvements are
mostly in regard to cell osteogenic differentiation, but also with the
biocompatibility and cell proliferation of the implants as well
[110,150,152]. GO-coating of Ti implants has also proven to create
antibacterial properties on these implants, most effectively when the
GO coating is functionalized with antibacterial substances, such as
silver nanoparticles and antibiotics. This further emphasizes how the
antibacterial activity of graphene is highly relative to the substances
that it is combined with. When combined with the antibiotic minocy-
cline hydrochloride, the GO-coating improves the antibacterial activity
against facultative anaerobic or aerobic bacteria due to the synergic
effect of minocycline release-killing and GO contact-killing. GO-silver
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coating on Titanium has been proven to be very helpful against P.
gingivalis and S. mutans. It can then be inferred that the GO-coating with
antibiotics or silver on Ti implants improves the antibacterial qualities
of the Ti implants, preventing infections from arising as a result of the
implant being used in the patient [110,153,154].
Although GO-coating with these different nanocomposites create
antibacterial effects against similar and different types of bacteria de-
pending on which is used, it can be determined that some type of GO-
coating of Ti implants with one of these nanocomposites would be an
improvement to the popular Titanium implant. These improvements
may be able to expand when one considers the ability of PDLSCs to
serve as an alternative to BM-MSCs that was mentioned earlier. PDLSCs
facilitate the use GO as a helpful canvas on which to form the structure
and function of these PDLSCs. PDLSCs seeded onto GO-coated scaffolds
show a higher proliferation rate than on GO alone, and a Na-Ti sub-
strate is beneficial as well. Concluded from this is the fact that a com-
bination of PDLSCs, GO and Na-Ti can create many added improve-
ments and benefits in the field of regenerative dentistry [155]. As with
the rest of graphene and graphene-based nanomaterials' applications in
dentistry, the carrying over of these benefits from scaffolds to implants
in a regenerative manner must be further studied and tested for tangible
benefits to result [110]. Graphene has many potential benefits to im-
plants in dentistry, however, the nanoparticles that it is paired with and
the environmental factors of each patient's oral cavity must be con-
sidered in each case before any further measure is taken.
6.2. Membranes
In order to improve the overall function of Guided Bone
Regeneration (GBR) membranes in oral surgery, the addition of gra-
phene must improve the ability of the membrane to prevent soft tissue
cells from infiltrating into the growing bone [156]. The addition of
graphene must do this while also adhering to the five criteria underline
by Scantlebury: biocompatibility, space-making, cell-occlusiveness,
tissue integration and clinical manageability [157]. When enriching
collagen membranes with GO and testing its effect on Human Gingival
Fibroblasts (HGFs), the presence of GO on collagen membranes resulted
in lower deformability, reduced hydration, higher stiffness and in-
creased roughness in comparison to non-coated membranes. After
3 days of culture and the facilitated adhesion of proteins to the mem-
brane, it was discovered that these changes created by the GO on col-
lagen avoided any type of inflammatory response and overall favored
the proliferation of HGFs (Fig. 6). The experiments performed with GO
on collagen membranes were done using two different amounts of GO:
one at 2 μg/mL and one at 10 μg/mL, both creating the same afore-
mentioned beneficial results to HGFs [158]. When applied to DPSCs,
GO-coating on collagen membranes is discovered to render cells unable
to penetrate into the membrane, while the more concentrated GO
coating results in the formation of a thicker cell layer. These discoveries
are made through Hematoxylin-Eosin staining. GO coating of collagen
membranes is also determined to promote the process of osteoblastic
differentiation, to be compatible with cell viability in a dose-dependent
manner, and to decrease inflammation [158,159]. Again, although it
seems very possible that these GO coated membranes can be used in
order to improve or replace the current GBR membranes used in den-
tistry, different environments, host and environmental factors should be
considered before reaching a definitive conclusion on the application of
graphene in membranes.
6.3. Resins and cements
When considering graphene's role in dental restoration, it is im-
portant to consider the fact that resins, cements and adhesives are the
most commonly used materials. The materials originally used were
polymeric materials, which are prone to the formation of biofilms that
result in dental restoration failure, as well as bacterial adhesion and
M. Tahriri, et al. Materials Science & Engineering C 102 (2019) 171–185

Fig. 5. Graphene as new strategy for designing and developing dental implants. (A) Endosseous dental implant structure composition, (B) process osseointegration:
left after 4 weeks, mineralized new bone be contacted implant fixture surface, right after 12 weeks, processing bone remodeling, bone be surrounded by blood,
adipose cell, collagen fiber, monocyte, and so on, (C) numerous titanium implant surface treatment for osseointegration, biocompatibility, and antibacterial effect
[151].

growth. In order to combat the susceptibility to bacterial growth in the
oral cavity that is a direct result of their porous nature, graphene na-
noplates (GNPs) are used as a nanofiller in a commercial dental ad-
hesive [160]. The graphene nanocomposite ultimately results in very
effective inhibition of growth of S. mutans. Although this works well
without changing the normal adhesion properties of the dental ad-
hesive, GNP is grey in color, which is not ideal for its use in dentistry.
An attempt was made to improve the color of the GNPs so that they
could look more natural in the oral cavity by growing the hybrid metal
Zinc Oxide Nanorods (ZnO-NRs) on GNPs so that the antimicrobial
quality of the GNPs would still be present while the ZNO-NRs lightened
the color and contributed biocidal properties to the compound. The
resulting Zinc Oxide Nanorods-Decorated Graphene Nanoplatelets
(ZNGs) are found to combat S. mutans, even when used in very low
amounts. In addition to this, ZNGs prove to be an obstacle to biofilm
growth when their biomass and exopolysaccharide production are
evaluated. Therefore, it can be concluded that since ZNGs decrease S.
mutans growth, they are a viable option when considering how to
control diseases that lead to dental caries.
Going along with this, graphene sheets embedded with gold nano-
particles (Gr-Au-x) through the radiofrequency catalytic chemical vapor
deposition technique over an AUx/MgO catalyst were used as a nano-
filler for other dental nanocomposites on a BisGMA/triethyleneglycol
dimethacrylate matrix. The graphene-gold nanocomposites prove to be
another possible filler for dental nanocomposites, since the large
amount of nanoparticles provides reinforcement that could improve the
physicochemical properties of the adhesives. Glass ionomer cements
with fluorinated graphene also prove to be useful in the inhibition of
bacterial growth, while also improving the mechanical properties of
cements. This also proves to have additional benefits, since it conse-
quently provides compressive strength through increasing microhard-
ness, while also decreasing the friction coefficient [161–164]. In regard
to graphene's possible role in dentistry pertaining to resins, cements and
adhesives, it seems promising when paired with the proper materials.

Fig. 6. Different GO coating concentration on collagen membrane from porcine dermis. (a) SEM images of uncoated, 2 mg/mL and 10 mg/mL GO-coated membranes
(b) Hematoxylin-Eosin staining of uncoated, 2 mg/mL and 10 mg/mL GO-coated membranes with DPSCs after 28 days of culture [158,159].

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6.4. Teeth whitening
Involving graphene in the discussion surrounding improvements to
teeth whitening requires one to first acknowledge the fact that any
improvements to the process must increase the rate and ease of
whitening and time of treatment, retain the bleach qualities of hy-
drogen peroxide (H2O2) and mitigate the side effects, such as gingival
irritation and dental sensitivity [165,166]. With this in mind, a nano-
composite made of rGO and Cobalt Tetraphenylporphyrin (CoTPP) was
created with the intent of being a catalyst for teeth bleaching [167].
Teeth bleaching with this compound is performed with the standard
photoactivation currently used with hydrogen peroxide and proves that
hydrogen peroxide used with CoTPP/rGO in this manner increases the
whitening effect of H2O2 and lowers the time of treatment, most likely
because hydrogen peroxide's resulting active radicals have a short
lifespan that consists of penetrating tooth structure and initiating a
radical generation mechanism. Additionally, photoactivating hydrogen
peroxide and CoTPP/rGO together creates more reactions between
stains molecules and hydrogen peroxide, while photoirradiation allows
for the radical generation to initiate deeper in the structure [110]. As it
has been noted in previous applications of graphene in dentistry, gra-
phene seems to produce improvements in the dental derivative of teeth
whitening so long as it is paired with the proper materials.
6.5. Dental bacteria detection
Sensors and electronic devices with biomaterials are picking up
enthusiasm for quite a long time. However, such device development
includes mechanical embedding of components by clamps or other
implantation of electrodes into the surface of the tissue. But such large
components over some stretch of time make inconvenience [168]. So
devices which limit inconsistencies between biotic/abiotic surface are
needed. Electronic sensors developed of nanomaterials such as carbon
nanotubes, nanowires and graphene. The single atom thick graphene is
of specifically compelling a direct result of its optical characteristics. It
could be grown on metallic films (Cu/Ni) by chemical vapor deposition
(CVD), trailed by post etching of the underlying metal and employed for
flexible electronic applications and biosensing [168–170]. Due to its
high elastic modulus (~l TPa) [26], intrinsic strength of 42 N m−1 and
high interfacial adhesion, it could be a perfect material for adhesion
onto the tough surfaces. Silk could be utilized as a medium for trans-
ferring these passive electrodes to the tissue's surface because of its high
tensile strength, elastic modulus and ductility. Also, silk can dissolve on
intimate contact. These developed electrodes could act as transistors.
The instruments create to detect the particular type of organism. In fact,
these fabricated graphene nanosensors could biotransfer on to the
surface of a human tooth for recognition of the pathogenic bacteria
(Fig. 7) [168,169].
Fig. 7. (a) Graphene is printed onto bioresorbable silk and contacts are formed containing a wireless coil. (b) Biotransfer of the nanosensing architecture onto the
surface of a tooth. (c) Magnified schematic of the sensing element, explaining wireless readout. (d) Binding of pathogenic bacteria by peptides self-assembled on the
graphene nanotransducer [169].
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6.6. Inhibition of bacterial biofilm formation
Bacterial biofilms play a crucial role in dental pathogenic mechan-
isms because of their capacity to endure numerous conventional anti-
bacterial agents. Therefore, it is vital to find successful techniques to
inhibit the creation of biofilms. Recently, Zisheng Tan and his collea-
gues ascertained that graphene oxide nanosheets could productively
inhibit Streptococcus mutans (S. mutans) biofilm formation over a wide
range of concentrations amid the beginning times of the bacterial bio-
film formation (0–4 h). Interestingly, in the case of mature biofilms
(6 h), GO had only a minimal impact (Fig. 8) [171].
They found that incorporating GO to a developing film could inhibit
the adhesion and activity of bacterial cells during the early stage of
biofilm formation and GO was powerful in killing the S. mutans bac-
teria. Additionally, the sheet-like structure of GO could conjugate with
cells to form an encapsulating inorganic functional layer, which influ-
enced the capability of the bacteria to secrete extracellular polymeric
substance (EPS). This is noteworthy as EPS plays a crucial protective
role in the attachment procedure and resistance against antimicrobial
agents. Lower EPS will prompt simpler destruction of the forming films.
Tragically, inferable from the plentiful EPS secreted, bacteria in mature
biofilms are less sensitive to the presence of GO. Finally, the obtained
results of their work demonstrated that GO nanosheets are powerful in
preventing and treating oral diseases by meddling with the formation of
bacterial biofilms [171].
6.7. Tissue engineering
Graphene's use in tissue engineering is largely analyzed throughout
Sections 5.1 and 5.2 of this paper. Although the approaches to dis-
cussing the uses of graphene throughout these sections are multi-
faceted, they all point to the nanomaterial's ability, or lack thereof, to
maintain the host's environment and promote a certain form of anti-
bacterial, tissue or cellular growth. Although not all of the conclusions
reached as a result of these different types of research arrive at a con-
crete answer to how graphene can be used in tissue engineering, they
all point to the fact that graphene can provide many benefits to tissue
engineering if used in combination with the proper materials, in ideal
conditions and in certain amounts and shapes. The physiochemistry of
these combinations of nanomaterials and how they work with certain
growth-promoting aspects of the oral cavity vary, but these results can
all be used to determine which scaffold, coated with a certain nano-
material structure, should be used. Tissue engineering can be greatly
improved when partnered with graphene, and it is as clear as testing the
scaffolds with different nanomaterials in certain amounts [172].
A more definite example affirming graphene as a very useful na-
nomaterial in tissue engineering is the fact that it has shown cells ad-
hering to graphene films and proliferating on them better than when
M. Tahriri, et al.
Fig. 8. The inhibition effect of GO on biofilm formation [171].
the cells were cultured on a SiO2 substrate [173]. A more general ap-
plication of graphene in tissue engineering can be seen through the fact
that graphene sheets can greatly improve the physical properties of a
given host polymer in small concentrations when it is incorporated
correctly, further demonstrating the usefulness graphene being depen-
dent on how much is used in a given circumstance [59]. Graphene's
aromatic scaffold nature allows it to promote cell attachment, growth,
proliferation, and differentiation. GO is also an aromatic scaffold in
nature, allowing GO nanoflakes—when incorporated into a gelatin-
hydroxyapatite (GHA) matrix—to improve overall mechanical strength
and osteogenic differentiation [174–176]. In this test, the GHA matrix
with GO also show less brittleness than the untreated GHA scaffolds. As
far as the toxicity of graphene is concerned, within this test, the gra-
phene scaffolds immersed in phosphate buffered saline in order to
mimic physiologic conditions release an amount of graphene that fell
within the toxicity limit. The level of osteogenic differentiation of stem
cells cultured on the GO treated GHA scaffolds is comparable to those
cultured on GHA scaffolds with osteogenic supplements, further at-
testing to the likelihood of biocompatible, biodegradable and porous
GO being considered a satisfactory candidate for tissue engineering and
its consequent bone growth. In addition to this, it has been proven that
GO coating of collagen scaffolds improves the surface structure, com-
pressive strength and cell ingrowth of the scaffold. This is also a suc-
cessful application of graphene, since the low concentration of gra-
phene being used is not inhibiting cell proliferation or differentiation in
vitro. This low concentration also allows the biocompatibility and bio-
degradability to be improved as well, further attesting to the im-
portance of determining which concentrations of graphene are most
successful in different situations [172,176].
Graphene in tissue engineering is also very effective due to its
ability to be readily synthesized when paired with other materials. Free-
standing G/HA hydrogels are synthesized using colloidal chemistry,
demonstrating unprecedented homogeneity in their 3D structure in the
process. These G/HA hydrogels are strong, highly porous, electrically
conductive and biocompatible, making them very good options for
scaffold materials in bone tissue engineering [177]. Another GO-Gel
composite is used for the biomimetic mineralization of hydroxyapatite,
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Materials Science & Engineering C 102 (2019) 171–185
and when MC3T3-E1 cells re cultured on the GO-Gel surfaces, HA mi-
neralization takes place, while improvements in cell adhesion, cell
proliferation and alkaline phosphate activity occur as well. These im-
provements are compared to MC3T3-E1 cells cultured on GO or a glass
surface. Taking this into consideration, the possibility of using GO-Gel
hybrids as osteogenesis promoting scaffolds in bone tissue regeneration
and surgery continues to be reaffirmed [178].
When creating nanofibrous biocomposite scaffolds of polyvinyl al-
cohol (PVA) and GO are assessed based on tensile testing, more facts
about how to use graphene in manner that is beneficial to tissue en-
gineering arise. Mouse MC3T3-E1 cells are used to determine that the
average diameter of the composite fibers decreases with an increasing
amount of GO, and that tensile strength and elasticity modulus are
greater when the content of GO is lower than 1 wt%, but decrease when
GO rises to 3 and 5 wt%. The cells attach to and grow on these scaffolds
without affecting their viability, and are more likely to spread on the
PVA/GO composite scaffolds. This also attests to the useful nature of
graphene in tissue engineering not only because of the benefits it cre-
ates, but also due to the fact that this was done using a simple water
solution processing method that can be repeated [179]. Further testing
using polydopamine-functionalized reduced graphene oxide (RGO-
PDA) creates similar improvements to cell proliferation, adhesion and
osteogenic differentiation [172]. In addition to this, homogeneously
coating highly porous 45S5 Bioglass®–derived ceramic scaffolds with
graphene sheets did not alter the shape and dimension of pores, at-
testing to the benefits of these hybrid sol-gels as well [172,180].
When coated with electrically conductive organic-inorganic hybrid
layers consisting of graphene by a solution method, the graphene na-
noplatelets in the hybrid coatings and presence of graphene in general
do not hinder the bioactivity of the scaffolds in simulated body fluid.
Although both the uncoated scaffolds and scaffolds coated with organic-
inorganic hybrids containing graphene provide an adequate space for
the cell attachment of cultured cells, the fact that the scaffolds coated
with graphene are able to provide this beneficial environment exposes
the fact that these scaffolds are biocompatible and can support cellular
activity. Building off of this foundational discovery, the electrical con-
ductivity created through this coating may be able to increase tissue
M. Tahriri, et al.
growth when an applied electric field is utilized in the carrying out of
cell culture. From here, expression and spreading of the extracellular
matrix can continue as well [172,180]. Here, the presence of graphene
proves to be acceptable to the human body's natural environment,
giving the nanomaterial the opportunity to increase tissue growth in the
treated areas, using its acceptance to eventually benefit and expedite
the tissue engineering process. Bioglass® with graphene nanoplatelets
(BG-GNP) demonstrates the electrical conductivity that graphene con-
tributes to tissue engineering as well. With an increasing concentration
of GNP, the electroconductivity of BG-GNP composites increases as
well. This increase in electrical conductivity occurs without affecting
the bioactivity of the composites, allowing the possibility of fabricating
electrically conductive and bioactive scaffolds for bone tissue en-
gineering to be considered [181].
7. Challenges on the graphene and its derivates
Although there are abundant studies on graphene and its derivates
for bioapplications, this field is still in the early stage and some key
challenges should be addressed before this area can be widely com-
mercialized. Mechanical failure after implantation caused by defects is
one issue in dental implants fabrication that should be prevented [182].
The size and shape of made defects in graphene implants are directly
dependant on their production method [183]. Hence, formed defects
during production of graphene implants for application on large scale
need to be investigated as a first challenge on the graphene and its
derivates [110].
Another challenge on the graphene and its derivates in their clinical
approaches is long-term toxicity and in vivo toxicity mechanisms of
graphene and its derivatives [184]. In addition, the potential toxicity of
graphene and its derivatives in biological systems has been studied
recently [185,186]. It was reported that the purity of graphene and its
derivatives during biofunctionalization process should be considered
properly because graphene-based biomaterials sometimes generate
oxidative debris, which may induce cytotoxicity [187]. In addition,
some investigations report that graphene and graphene oxide (GO) tend
to be toxic to mice in a dose-dependently [188,189], while functiona-
lized graphene oxide (e.g., by biocompatible polymer coating) shows
less in vitro and in vivo toxicity [190]. For example, Bao et al., Singh
et al. and Yang et al. report that functionalizing carbon nanotubes
(CNTs) with biocompatible polymers such as PEG led to decrease in
their toxicity [191–193]. Several reports exhibited that in vitro/in vivo
behavior of graphene and its derivates as well as its toxicity to biolo-
gical systems are influenced by graphene and its derivate's physico-
chemical properties such as the surface functional groups [194,195],
charges, coatings [184], sizes [196] and structural defects [197].
Furthermore, suitable drug loading capability for practical uses of
graphene and its derivatives needs to be further investigated. In other
words, in vitro and in vivo toxicity profiles, biocompatibility and bio-
degradability of graphene and its derivatives should be considered in
order to development of proper in vivo release profiles and drug dis-
tribution and follow by finding exact chemical modification processes
of graphene and its derivatives for cell membrane barrier penetration
and intracellular release for drug delivery [187]. Taken together, two
main challenges on application of graphene and its derivatives are the
production parameters and their in vivo toxicity, which need to be
evaluated precisely.
The enzymatic degradation of graphene is a very vital topic, since
on a basic level, graphene could be some harm. Instead, if there are
microorganisms capable to degrade graphene and its derivates, the
persistence of these materials in the body will be remarkably reduced.
These types of works are needed to examine the nature of degradation
products of the graphene particles. Once graphene is broken down by
enzymes, it could produce harmful subsidiaries. We need to find out the
structure of these subsidiaries and research their influence on health.
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8. Conclusions and future perspectives
The development of graphene and its derivatives as biomedical
materials has become highly interesting research field in the last few
years. Meanwhile, this field of research is still in its infancy stage and
requires proper future research directions to change it into a market-
oriented research area. The functionalization potential of graphene
with various biomaterials and biomolecules make it a promising can-
didate despite its other properties, such as mechanical strength, elec-
trical conductivity and thermal stability. One of the most essential fu-
ture goals for the biomedical therapeutic application of graphene and
its derivatives, such as antibiotic and/or anti-cancer agents, is related to
conceptual understanding of their toxicity profile. Moreover, design of
surface chemistry of graphene and its derivatives for future treatment of
genetic disorders or in vivo gene delivery by using of graphene-based
biomaterials should be explored in more detail.
Despite the usage of bulk graphene-based nanobiomaterials for
novel bio-applications, few attempts have been done to realize cost-
effective, scalable and reproducible syntheses methods of stable and
reliable graphene-based nanomaterials. In addition, new bio-functio-
nalization methods have to be developed to prevent the graphene na-
nomaterials from agglomeration during biomedical applications.
Additionally, graphene and its derivates have been introduced as a
promising method in order to improve characteristics of dental mate-
rials. For example, the biocomposites with tunable physicochemical/
biological properties that can be synthesized by functionalization and
combination of graphene and its derivatives with other biomolecules
and biomaterials in order to obtained specific characteristics, such as
high mechanical properties, large surface area as well as enhanced
bioactivity. Meanwhile, the final properties of biomaterials are directly
dependant on graphene and its derivatives' physicochemical properties,
such as their size, surface functionalization, conditions and parameters
of coating.
Eventually, we believe that the utilization of graphene-based en-
gineered nanomaterials in the dentistry deserves to be profoundly ex-
amined as it can prompt much progressively dependable dental treat-
ments in the near future.
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