Smart Materials in Medicine 2 (2021) 280–291

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Smart Materials in Medicine

journal homepage: www.keaipublishing.com/en/journals/smart-materials-in-medicine/

Graphene oxide as a promising material in dentistry and tissue regeneration:

A review
Xuanyu Qi a, Fei Jiang a, b, c, Mingliang Zhou a, Wenjie Zhang a, **, Xinquan Jiang a, *
a
Department of Prosthodontics, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, College of Stomatology, Shanghai Jiao Tong
University, National Center for Stomatology, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology, Shanghai Engineering Research
Center of Advanced Dental Technology and Materials, No. 639 Zhizaoju Road, Shanghai, 200011, China
b
Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, No. 140, Han Zhong Road, Nanjing, 210029, China
c
Department of Polyclinic, Affiliated Hospital of Stomatology, Nanjing Medical University, No. 136, Han Zhong Road, Nanjing, 210029, China

A R T I C L E I N F O 1 . A B S T R A C T

Keywords: Current treatments for common dental diseases such as caries, periodontal diseases, tooth defects, missing teeth
Dental materials and bone defects cannot replenish dental tissue with artificial alternatives while maintaining the biological and
Nanomaterials mechanical properties of natural tissue. In recent years, in addition to existing conventional treatments, tissue
Tissue regeneration
engineering and regenerative medicine have also been applied in this field to recover real tissue. Graphene oxide
Graphene oxide
(GO) is considered to be a promising material in dentistry and related tissue regeneration due to its outstanding
properties. In this review, we first describe the physicochemical properties of GO and then explain the biological
properties that derive from the physiochemical properties, including biocompatibility, controlled release of
medicine, antibacterial properties, osteogenic differentiation properties and odontogenic differentiation proper-
ties. We also highlight the potential applications of graphene oxide in dentistry and related tissue regeneration.
For bacterial resistance in dentistry, GO has a wide range of applications. For reconstructing tooth defects,
conventional dental materials such as resin and adhesives have been optimized with GO. To treat missing teeth,
GO surface modification of dental implants is adopted to enhance not only the antibacterial properties but also
osseointegration. To repair maxillofacial bone defects, GO can enhance the osteogenic properties of engineered
tissue scaffolds through both surface and composite modifications. Some obstacles that inhibit the progression of
this technology from the laboratory to the clinic have also been described.

1. Introduction
Dentistry is a field with multiple problems to be solved by optimizing
materials. In modern dentistry, both the early prevention of caries and
the development of new and efficient restorative materials are sought.
Although tremendous efforts have been made to promote oral hygiene
and fluoridation, the prevention of early-stage tooth decay lesions is still
a challenge for dental research and public health [1]. As a result, treating
tooth defects resulting from caries has become one of the most common
treatments in dentistry. The mechanical properties of materials applied in
treating tooth defects, such as resin-based composites, are related to their
clinical application and the longevity of the restoration [2].
More serious than caries and some caused by caries, periodontitis or
trauma, tooth loss is a problem that negatively affects human life quality.
With a survival rate greater than 89 %, dental implants have been a
conventional treatment for patients suffering from missing teeth [3–5].
However, there is still an 11 % failure rate in dental implant treatment
and removal, which brings patients both physical and mental discomfort
along with additional economic loss [6,7]. Many factors result in implant
failures, and a major factor is the harmful influences of anaerobic plaque
bacteria on peri-implant tissues [8]. To address this problem, mechanical
cleaning to remove biofilms from implants, antibiotic therapy to kill in-
fectious bacteria and regenerative surgery to rebuild bone-implant in-
terfaces are routinely adopted [9–13]. Reliable antibacterial surface
modification strategies and efficient cleaning methods should be
improved for future applications. In addition, conventional dental im-
plants made of titanium cannot fully meet clinical needs due to their
limited osseointegration and osteoinductive properties, especially under

* Corresponding author. Shanghai Jiao Tong University, School of Medicine Department of Prosthodontics, 639 Zhizaoju Road, Shanghai, 200011, China.
** Corresponding author. Shanghai Jiao Tong University, School of Medicine Department of Prosthodontics, 639 Zhizaoju Road, Shanghai, 200011, China.
E-mail addresses: zhangwenjie586@126.com (W. Zhang), xinquanjiang@aliyun.com (X. Jiang).

https://doi.org/10.1016/j.smaim.2021.08.001
Received 10 June 2021; Received in revised form 27 July 2021; Accepted 3 August 2021
Available online 8 August 2021
2590-1834/© 2021 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
X. Qi et al.
poor or inadequate bone conditions. Materials that positively promote
osseointegration and have osteoinduction properties are urgently needed
to decorate implants.
In addition to existing conventional treatments, tissue engineering
and regenerative medicine have also been applied in dentistry. Tissue-
engineered bone grafts represent an ideal choice for bone augmenta-
tion and create both robust and sufficient bone for dental implantation.
Maxillofacial bone defects resulting from traumatic injuries [14] or
dental and surgical treatments [15,16] require tissue engineered scaf-
folds with optimized properties, including biocompatibility, osteoin-
duction, and controlled drug release, to achieve bone regeneration with
higher efficiency.
Graphene is an allotrope of carbon composed of single-atom-thick
carbon nanosheets with a honeycomb structure and was first success-
fully prepared by Geim and Novoselov in 2004 [17]. When modified by
oxygen-containing groups such as hydroxyl (-OH), epoxy (-O-), carboxyl
(-COOH) and carbonyl (C– – O), the graphene sheet transforms into a
derivative known as graphene oxide (GO). As a result of the modification,
the highly conjugated graphene structure is damaged, but the GO
nanosheets are also rendered with better chemical stability and solubility
in water [18]. GO has many attractive physicochemical and biological
properties. First, it has been reported that GO has great biocompatibility,
can promote cell adhesion and proliferation, and even induce the
directional differentiation of stem cells [19,20] Remarkably, GO alone
can induce the osteogenic differentiation of stem cells [21]. Second, GO
has been indicated to have excellent antibacterial properties through
different mechanisms [22–25]. Third, thanks to a large surface area (on
both sides of the sheet) for the physical adsorption of nucleobases [26,
27] and aromatic compounds via surface adsorption, hydrogen bonding,
and other types of interactions [28], GO also has potential for drug de-
livery [29]. In summary, the properties of GO wonderfully meet the re-
quirements of dentistry and related tissue engineering.
In this review, we first describe the physicochemical properties and
biological properties of GO and then focus on the application of GO in
different aspects of dentistry and related tissue regeneration. We further
summarize the future possible development direction of GO nano-
materials in dentistry and related tissue regeneration.
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2. The physicochemical properties of GO
GO has a high specific surface area (890 m2 g
1) [30], and abundant
functional groups, such as hydroxyl and epoxy groups, as well as slight
amounts of carboxyl, carbonyl, phenol, lactone, and quinone [31], were
clearly observed by FT-IR spectroscopy [32–34]. These functional groups
were created by the oxidation of graphene, which also increased the
hydrophilicity of the surface. The GO properties are enormously changed
due to the presence of these oxygenated groups, which helps the for-
mation of stable dispersion in aqueous media and other polar solvents
[35–37]. Biochemical and bioconjugation reactions were therefore
allowed to occur at both its basal plane and its edges [33], which pro-
moted the functionalization of the GO surface with proteins, antibodies
and DNA fragments [38,39]. Functional moieties (i.e., hydroxyl and
carboxyl groups) on GO can establish chemical bonds with polymers in
scaffold matrices, which can provide scaffolds with enhanced interfacial
strength [40]. GO has also been reported to enhance the mechanical
properties of various materials, including chitosan [41,42], gelatin [43,
44], chitin [45], collagen [46], hydroxyapatite [47,48] and PEEK [49].
A high physical adsorption capacity for proteins was observed on GO.
The mechanism depends on the GO morphology, oxidation degree [50],
hydrophobicity and type of absorbed protein [51–53]. Regarding the
type of protein, polypeptides can be adsorbed on the GO surface via four
mechanisms; the main mechanism is hydrophobic-hydrophobic interac-
tion. 1) Hydrophobic-hydrophobic interactions benefit from the sp2 hy-
bridization of GO and are affected by the electron density of proteins:
Bovine albumin shows reliable adsorption to GO, while low molecular
weight heparin, which has a low electron density, exhibits a decreased
hydrophobic interaction [54]. 2) van der Waals interactions are the
second mechanism by which GO bonds with molecules; these in-
teractions can be weakened by the oxygenated moieties formed during
the oxidation process [55]. 3) Third, electrostatic interactions are also
common when GO serves as a vehicle, and when pH < 6.0, sorption can
be enhanced [56]. 4) The fourth mechanism involves π–π stacking in-
teractions due to the abundant π electrons on the basal plane of the GO
surface, as described for the individual hexagonal cells of the GO basal
planes and the glucose oxidase [57].
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Fig. 1. Bone regeneration of Ti ring implants with or without GO coating and BMP-2/SP loading in a mouse calvarial defect model. Notes: Histological analysis with
Goldners trichrome staining of mouse calvarial defects with implants 8 weeks after treatment. (A) Lower magnification (  12.5) images of the implants. The black
arrowheads and red arrows indicate the implant and newly formed bone, respectively. Scale bars ¼ 1 mm. (B) Higher magnification (  100) images of the implants.
Scale bars ¼ 100 μm. (C) The area of new bone formation was determined using histomorphometric analysis (n ¼ 5). *P < 05 between designated two groups.
Abbreviations: Ti, titanium; GO, graphene oxide; BMP-2, bone morphogenetic protein-2; SP, substance P[68]. Copyright © 2014, Dove Medical Press Limited.

GO can also mechanically form a covalent conjugation on its surface
with substances, which is general in functionalization associated with GO
[58]. The binding strength between GO and other substances is therefore
enhanced, and the stability improves even with varying pH, organic
solvents, and storage conditions [59].
3. The biological properties of GO
3.1. Biocompatibility
Biocompatibility is a fundamental requirement for both implants
and tissue engineering scaffolds and is also the basis for the design and
improvement of novel materials. An increasing number of studies have
shown that GO has some cytotoxicity, but its toxic manifestation is
concentration-dependent and correlates with the shape of the GO [60,
61]. Wang et al. [62] cocultured three different concentration gradients
of GO with human fibroblasts and found that GO had no toxic effect on
human fibroblasts at doses below 20 μg/mL, while doses above
50 μg/mL showed some cytotoxicity. Further in vivo studies in mice
showed no significant toxicity in the low (0.1 mg) and medium
(0.25 mg) dose groups, while the high (0.4 mg) dose group showed
chronic toxicity mainly in the lung, liver and kidney, leading to mor-
tality and lung granuloma formation in mice. In addition, the testing of
the GO cytotoxicity in mammalian cells indicated that microsized GO
led to higher cytotoxicity than nanosized GO, the reason of which may
be the faster sedimentation rate and the formation of compact GO ag-
gregates on top of the adherent cells in the wells, inhibiting the nutrient
availability for the growth of cells [63]. Remarkably, GO has an auto-
degradation pathway in aqueous conditions. After prolonged water
exposure, GO lattice sizes have been shown to decrease over time due to
C–C bond cleavage; the resulting structure is similar to humid acid, a
benign degradation product of all organic matter [64] This property
gives GO moderate biocompatibility, and it can be cleared in vivo on a
timescale of months.
3.2. Controlled release property
The therapeutic efficacy of drugs usually relies on the drug delivery.
Owing to its excellent biocompatibility, low toxicity, and large dosage
loading capacity [65,66], GO group has been considered as a method in
therapeutic medicine for the delivery of therapeutic medicine with high
efficiency. Many kinds of medicines have been successfully loaded on GO
and released in a controlled manner for bone tissue engineering; These
medicines include BMP-2 [67–69], aspirin [70], vancomycin [71], and
siRNA [72].
The mechanism behind the ability of GO to load and sustain protein
release is divided into two parts. First, the ionized groups on the GO
enable protein delivery by binding through electrostatic interactions
[73–75]. Second, the hydrophobic domains on GO can interact with
those of proteins by hydrophobic π–π stacking [76,77]. La et al. coated
titanium substrates with GO through layer-by-layer assembly of posi-
tively (GO-NH3þ) and negatively charged (GO-COO
) GO sheets and
loaded BMP-2 on the outermost coating layer of GO-COO
. Compared to
the hBMMSCs cultured on Ti substrates and directly loaded with BMP-2,
GO enhanced the in vitro osteogenic differentiation when it was used as a
coat, and it led to the higher conformational protein stability, higher
bioactivity, and increased local concentration of BMP-2 on surfaces
modified with GO. When implanted into mouse calvaria, the GO group
showed a sustained release of BMP-2, which resulted in more robust bone
formation [67]. To improve the stem cell recruitment of the material,
substance P was then introduced into the same group, and it was reported
to be effective in promoting recruitment of MSCs to damaged tissues.
Loading of BMP-2 and SP with GO further enhanced bone formation in
vivo [68] (Fig. 1.). Recently, through layer by layer (LBL) self-assembly
techenique, polydopamine(PDA), GO, and type I collagen(Col I) was
loaded on the surface of Ti-based implants. Furthermore, the encapsu-
lated amount and release rate of bioactive substances including silver
(Ag) ions and bovine serum albumin (BSA) could be precisely controlled
by the layer number of the nanofilms [66].

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Fig. 2. Live/dead fluorescent staining images. (A, B) S. mutans, (C, D) F. nucleatum, and (E, F) P. gingivalis cells were treated with GO nanosheets and isotonic saline
(control) for 2 h. Representative fluorescence microscopy images of bacterial cells stained with SYTO 9 (green channel) and PI (red channel) for 15 min in the dark. All
treated cases had the same GO dose of 80 μg/mL. Scale bar ¼ 50 μm. (G) TEM images of S. mutans, F. nucleatum, and P. gingivalis cells after incubation with GO
nanosheet dispersion (right side) for 2 h and after incubation with saline solution for 2 h as a control (left side). All treated cases had the same GO dose of 80 μg/mL.
Scale bar ¼ 500 nm. Reprinted with permission from Ref. [90]. Copyright © 2015, American Chemical Society.

Apart from titanium, in recent years, our group has also chosen a GO
delivery system as a modification of silk fibroin electrospun scaffolds in
bone tissue engineering. To avoid the shortcomings of BMP-2, which
includes a complex preparation process, low production rate and side
effects at large doses, we chose a new polypeptide known as P24 with a
simple synthesis procedure and the ability to increase osteogenic dif-
ferentiation as the loaded medicine. The negatively charged BMP-2
polypeptide-grafted GO was assembled on positively charged CS-coated
SF electrospun scaffolds through electrostatic interactions. The scaf-
folds both exhibited satisfying cellular performance as well as excellent
bone regeneration properties in critical-sized bone defects [69].
Chemical medicine is another example of molecules that can be
loaded on substrates with GO via π–π stacking for the enhancement of
bone regeneration properties. Ten et al. conjugated GO to modified ti-
tanium with the aid of 3-APTES functional groups and then loaded
aspirin, which has a benzene ring, onto the Ti-GO surface via π-π stack-
ing. The release of aspirin was sustained for three days, and in vitro cell
studies revealed that aspirin-loaded Ti-GO materials promoted MC3T3-
E1 cell proliferation and osteogenic differentiation [70]. In recent
years, inspired by mussels, Han et al. introduced polydopamine (PDA),
which has a structure similar to that of mussel adhesion proteins, to
GO-based titanium modification. The molecular mechanism is thought to
involve the Michael addition and/or a Schiff base reaction, hydrogen
bonding, or π-π stacking [78]. Because of its universal adhesiveness, PDA
served as an intermediate layer and provided biointeractive and chemi-
cally reactive surfaces for secondary modification of GO [71]. Finally,
chitosan-coated GelMS (CGelMS) that encapsulated BMP-2 and vanco-
mycin (Van) were assembled on the GO/Ti scaffold via electrostatic in-
teractions, which not only facilitated CGelMS immobilization but also
favored cell activity. The scaffold achieved independent release of BMP-2
and Van, which was demonstrated by the superior in vitro and in vivo
osteogenic properties [71].
Finally, GO has also been studied in gene delivery systems with safety
and high transfection activity. Pristine GO has been reported to be able to
absorb single-stranded DNA and RNA through π–π stacking [79]. Poly-
ethylene glycol (PEG) and polyethylenimine (PEI) dual-functionalized
graphene oxide (GO) (nGO-PEG-PEI) are promising siRNA vectors.
Zhang et al. successfully synthesized GO that was dually-functionalized
by PEG and PEI as a novel siRNA nanovector for implant surface bio-
modification through cathodic electrodeposition. When loaded with
osteogenic Ckip-1 siRNA, the coated implant enhanced the osteogenic
differentiation of stem cells in vitro and new bone formation in vivo [72].
To overcome the problem that GO cannot be used as a gene carrier to load
double-stranded DNA (dsDNA) due to its poor loading ability [80–82],
Santo et al. employed microfluidic mixing and synthesized hybrid NPs
consisting of GO nanosheets coated with a double layer of canonic lipids
[83]. We are looking forward to the application of the results of the study
in bone regeneration.
3.3. Antibacterial properties
Graphene-based materials share a similar antibacterial mechanism
with CNTs; this mechanism has been considered to have synergy of both
“physical” and “chemical” effects [84]. The physical mechanisms include
a sharp edge-mediated cutting effect and cell entrapment [85–87].
Smaller-sized GO with a higher edge density has a stronger cutting effect,
while larger-sized GO with wider lateral dimensions has a higher po-
tential to entrap bacterial cells [88,89]. He et al. observed common
dental pathogens, including S. mutans, P. gingivalis and F. nucleatum,
exposed to GO nanosheet dispersion [90] (Fig. 2). The TEM images
indicate a common intracellular density decrease in the GO-treated
groups, which indicates the loss of intracellular substances and is
considered the result of the destruction of the cell wall and membrane.
Oxidative stress is a chemical mechanism that contributes substantially
to the antibacterial activity of GO. Graphene-based materials are able to
oxidize GSH; this process induces superoxide anion-independent oxida-
tive stress in bacterial cells [91]. In contrast to the physical mechanisms,
oxidative stress has no significant relationship with GO size; however, it
can be substantially inhibited by reductive agents. In addition to the
physical mechanism, there is a parabolic relationship between the size
and antibacterial activity of GO [88], and further studies remain to be
conducted to optimize the GO size for the highest degree of antibacterial
activity.
3.4. Osteogenic differentiation properties
The use of GO as a potential material for tissue engineering and
regenerative medicine has increased rapidly in recent years, particularly
in directing the fate of stem cells [92]. Many studies have demonstrated
the advantages of GO in bone tissue engineering. La et al. showed that GO
can reduce the required dose and optimize the use of bone morphoge-
netic protein 2 (BMP-2) to induce osteogenic differentiation in MSCs

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Fig. 3. (A) Radiographic evaluation of the implants on various samples after treatment for 4 weeks; (B) micro-CT tomography analysis of various samples after
treatment for 4 weeks; (C) Masson staining of longitudinal sections from various samples after treatment for 4 weeks; (D) VG staining of transverse sections from
various samples after treatment for 4 weeks [112]. Copyright © 2019 American Academy of Periodontology.
[93]. GO has been proven to induce BMSC, PDLSC and DPSC osteogenic
differentiation in vitro [94–96]. However, the molecular mechanisms
have not reached a consensus. It has been shown that GO can promote
osteogenic differentiation of MSCs by enhancing the immunomodulatory
properties of MSCs [97]. Some researchers believe that hydroxyl and
epoxy groups on the planes and carboxylic acid groups at the edges
enhance interactions of GO with molecules and proteins in osteogenic
induction medium through hydrophobic and electrostatic interactions
[98–100], potentially enhancing stem cell osteogenic differentiation
[101,102].
3.5. Odontogenic differentiation properties
Inspired by the properties of GO to promote osteogenic differentia-
tion, our group tried to investigate its potential odontogenic differenti-
ation properties. First, we prepared a modified titanium-based material
using the microarc oxidation (MAO) technique and self-assembling GO.
The upregulation of markers in DPSCs cultured on the group with GO,
including dentin sialophosphoprotein (DSPP), dentine matrix protein 1
(DMP-1), ALP, OPN, and OCN, demonstrated a positive correlation with
GO content on coatings [103]. A similar study was reported by Ahn et al.,
who built mesoporous bioactive glass nanoparticle (MBN)/GO compos-
ites. Both the mRNA and protein levels of odontogenic markers showed
upregulation in hDPSCs in response to growth on the MBN/GO com-
posites. Furthermore, they also revealed that the Wnt/β-catenin signaling
pathway is involved in the mechanism behind the odontoblast differen-
tiation property of the material. Radunovic et al. synthetized collagen
membranes coated with GO. Unlike the group that did not have a GO
coating, the GO-coated group exhibited not only biocompatibility and
osteoblast differentiation but also odontoblast differentiation [104].
4. The application of GO in dentistry and related tissue
engineering
4.1. Cooperation of conventional dental materials and GO
In modern dentistry, tremendous efforts have been made to prevent
oral diseases and promote oral hygiene. Restorative materials with
excellent biological properties, improved mechanical properties and
longer service life have always been sought. The mechanical influence of
GO in dental materials should be confirmed before introducing it in
practice and utilizing its multiple biological properties. Conventional
materials in dentistry, such as resin-based composites [47], silane
primers [105], and adhesives [106,107], have been combined with
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Smart Materials in Medicine 2 (2021) 280–291
graphene oxide to confirm the influences on mechanical properties.
Alshahrani et al. used SEM, micro-Raman spectroscopy and microtensile
bond strength analysis to assess the differences between GO-added ad-
hesive and control adhesive. The results indicate that the GO-modified
adhesive shows comparable bond strength and durability of resin
dentine bonds [107]. GO was also added to an adhesive containing 5 wt%
HA at 0.5 wt% (HA-GO-0.5 %) and 2 wt% GO (HA-GO-2%). The
HA-GO-2% group showed uniform diffusion in adhesive and comparable
resin tag development to the controls. The addition of GO has been
proven to provide a higher μTBS and adequate durability [106]. Apart
from adhesives, the primers that are used for surface pretreatment and
therefore require long-term bonding of resin composites to dental pros-
theses remain to be optimized. Khan et al. infused GO sheets into
commercially available silane premiers and influenced the surface
morphology, which was observed to exhibit increased surface roughness
and slightly increased water contact angle measurements. The highest
enclosed mold shear bond strength (EM-SBS) values were found, with
mean (SD) EM-SBS values of 26.4 (3.7) MPa and 21.5 (5.1) MPa after 2
and 4 months of storage, respectively, which indicates improved me-
chanical properties [105]. Remarkably, Nizami et al. assessed the effi-
cacy of functionalized graphene oxide (f-GO) nanocomposites on the
decalcification of dentin, such as GO-silver (Ag), GO-Ag-calcium fluoride
(CaF2), GO-CaF2, GO-zinc, and GO-tricalcium phosphate (Ca3(PO4)2)
[108]. The abilities of GO-Ag, GO-Ag-CaF2, and GO-CaF2 nano-
composites were most preventive for decalcification. In addition, GO-Ag
and GO-Ag-CaF2 almost completely inhibited S. mutans growth.
Furthermore, these f-GO nanocomposites exhibited less or no discolor-
ation of dentin, although commonly used silver diamine fluoride causes
discoloration of dentin to black. The research represents an attractive and
interesting direction of GO clinical transformation in dentistry, which
innovatively play to the strengths of GO and make up for the weaknesses
of traditional dental materials.
4.2. Antibacterial applications of GO
Apart from the GO aqueous dispersion mentioned before, the appli-
cation of GO coatings or mixed components utilizing other mechanisms
in dental biomaterial and tissue engineering scaffolds for the enhance-
ment of antimicrobial adhesives has also been explored. Introducing
either hydrophilic or zwitterionic surfaces generating a hydration layer
on the surface are two usually adopted strategies. A physical or energetic
barrier preventing microbial adhesion can be created by this tightly
bound water layer [107,109]. Oxygen-containing functional groups such
as the hydroxyl (-OH) and carboxyl (-COOH) groups on GO represent the
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Fig. 4. The GO-Cu nanocomposite-coated CPC scaffolds. (a) The appearance of the CPC scaffold under a stereoscopic zoom microscope. (b) The appearance of the GO-
coated CPC scaffold under a stereoscopic zoom microscope. (c) SEM observation of the GO-Cu-coated CPC scaffold, and nanoscale Cu particles can be observed under
higher magnification. (d) Release kinetics of the Cu ions from the GO-Cu-coated CPC scaffolds (n ¼ 6; *represents p < 0.05; **represents p < 0.01) [127].© 2016
WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Implantation of GO scaffold to extraction socket. (e) Photograph of implanted scaffold. (g) Macroscopic view at 2
weeks postsurgery. (f) Radiographic images immediately after operations. (h) Radiographic image at 2 weeks postsurgery. The socket applied with the GO scaffold
showed increased radiopacity. Abbreviation: GO, graphene oxide [129]. Copyright © 2016, Dove Medical Press Limited.

capability of introducing hydrophilicity [110]. Qian et al. processed pure
titanium with NaOH and APS to endow it with a positively charged
surface, which caused GO, with its oxygen-containing functional groups
and negatively charged surfaces, to adhere to the titanium surfaces
through nucleophilic substitution reactions and electrostatic interactions
[111]. Minocycline hydrochloride was then loaded on the GO-modified
titanium surface, which showed slow release behavior; the observed
superior antibacterial activity against S. aureus, S. mutans and E. coli is
considered to be a synergistic effect of contact killing by GO and release
killing by minocycline hydrochloride [111]. Furthermore, they estab-
lished a peri-implantitis model in beagles and treated them with implants
that were equipped with abutments coated with graphene oxide film and
loaded with minocycline hydrochloride [112]. Compared to other
groups, the MH/GO/Ti group demonstrated the least marginal bone loss
in radiographic analysis and nearly no neutrophils in the histological
analysis [112] (Fig. 3).
The oral cavity, including teeth, root canals, mucosa, periodontal
tissues, saliva and dentures, is a complex ecosystem. The oral bacteria
themselves and substances produced or gathered by them can form oral
bacterial biofilms [113]. Pure GO nanosheets have been found to be toxic
to the development of mature biofilms from planktonic cells [114].
Bacterial biofilms have high pathogenicity because they are less sus-
ceptible to antibiotics and even more resistant to physical treatments
[115–117]. Qin et al. substantially removed polymicrobial biofilms that
remained on previously contaminated titanium surfaces by utilizing
256 μg/mL GO dispersion with brushing. The bone marrow–derived
mesenchymal stem cell osteogenic potential was regained or even
enhanced on the titanium surfaces treated in vitro, which represents
another application of GO nanosheets in addition to killing planktonic
pathogens and inhibiting the formation of bacterial biofilms, and this
application might provide a new strategy for treating periimplantitis
[118]. Di Giulio et al. reported that during the biofilm formation, GO
reduces the S. aureus and P. aeruginosa growth of 55.05 %  4.73 and
44.18 %  3.91 compared to the control. In mature biofilm, GO affects S.
aureus and P. aeruginosa by reducing their growth of 70.24 %  4.47 and
63.68 %  17.56, respectively [22].
Graphene consists of a single layer of tightly packed carbon atoms and
possesses unique optical properties, which gives it the capability to
absorb light irradiation and release it as heat [119–123]. Therefore, the
photothermal effect has become another mechanism of GO activity in
bacterial therapy. Wu et al. applied magnetic nanoparticle-functionalized
graphene as a photothermal agent for effective bacterial capture and
killing [123]. Ran et al. demonstrated a hyaluronidase-triggered photo-
thermal platform based on AgNPs and graphene oxide (GO), which
resulted in high mortality of S. aureus as well as low toxicity to
mammalian cells under locally raised temperatures upon illumination
with NIR light [124].
4.3. Osteogenic GO coating on dental implants and tissue-engineered
scaffolds
Successful dental implant treatment is highly dependent on stable
osseointegration and reliable infection inhibition. In the process of dental
implant treatment, failure may occur under the negative effects of oral
bacteria such as E. coli, C. albicans, S. aureus, and S. mutans or marginal
bone loss due to the poor osteoinduction property of conventional tita-
nium material. Among the nanocarbons that share antibacterial proper-
ties, graphenic materials also hold exceptional promise in biomaterials
due to their unique physicochemical properties, which include hydro-
philic functional groups, considerable mechanical strength, large surface
area and excellent biocompatibility [119,120]. In recent years, many
publications have illustrated the enhanced osteogenic properties of
GO-containing composites. Biomimetic modifications could also offer
mechanical and chemical environments encouraging osteogenesis.
GO-based materials have become a promising regeneration scaffold
competitor for bone tissue engineering.
Due to the attractive properties of GO, some studies have introduced
it as a coating on dental and orthopedic implants as well as on bone tissue
engineering scaffolds. A pioneering study demonstrated that graphene
and GO can accelerate MSC osteogenic differentiation to different de-
grees due to π–π stacking, hydrogen bonding, and electrostatic in-
teractions with proteins [121], which may be the underlying mechanism
supporting the coating application.
As a widely accepted material applied in dental and orthopedic im-
plants, Ti has been modified in many strategies to enhance its biocom-
patibility and osteointegration. GO has been proven to be an attractive
surface decoration on titanium, which is usually deposited electropho-
retically [122] or physically [123]. Recently, Carlo et al. activated tita-
nium surfaces with piranha solution or using a UV/ozone lamp and
coated them with GO via covalent bond formation. In the presence of a
GO coating, the titanium discs showed outstanding properties in pro-
moting the osteogenic differentiation of DPSCs, which was indicated by

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the increased gene expression of TGFβ and BMP2 at the early stage and
the positive regulation of the RUNX2 transcription factor. Furthermore,
following RUNX2 upregulation, the subsequent SP7 increased only when
DPSCs were cultured on titanium surfaces modified with GO [125]In
addition to the three strategies mentioned above, Li et al. also introduced
large particle sandblasting and acid etching (SLA) combined with the
ultrasonic atomization spraying technique, which is commonly used in
clinical practice, in GO coatings. The GO coating endowed the SLA tita-
nium surface with enhanced proliferation, adhesion and osteogenic dif-
ferentiation of BMSCs in vitro, which has been proven to be involved in
the FAK/P38 pathways. In vivo, the SLA/GO implants also showed
excellent osteointegration performance [126].
GO coating has also been applied in modifying scaffolds for bone
tissue engineering; such scaffolds include CaP [127,128], collagen [129]
and poly(3-hydroxybutyrate-Co-4-hydroxybutyrate) [130]. Our group
fabricated aqueous soluble graphene oxide-copper nanocomposites
(GO-Cu) and used them to coat porous calcium phosphate (CaP) scaf-
folds. The composite scaffolds therefore obtained the ability to release Cu
ions in the long term and were reported to enhance the adhesion and
osteogenic differentiation of rat BMSCs. Remarkably, by activating the
Erk1/2 signaling pathway, the GO-Cu nanocomposites upregulated the
expression of Hif-1α in BMSCs, which resulted in the secretion of VEGF
and BMP-2 proteins. Finally, when transplanted into rats with
critical-sized calvarial defects, the composite scaffolds (CPC/GO-Cu)
promoted angiogenesis and osteogenesis [127](Fig. 4 a-d). Nishida et al.
fabricated a GO-coated collagen sponge scaffold and then implanted it
into a dog tooth extraction socket. Dog bone formation tests showed that
1 μg/mL GO scaffold implantation enhanced bone formation. New bone
formation following GO scaffold implantation was enhanced fivefold
compared to that in control subjects [129] (Fig. 4e-h).
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Smart Materials in Medicine 2 (2021) 280–291
Fig. 5. SEM images of the newly formed
bone growth into the (a), (b), (c) scaffold
HA/rGO-6/0.3 and (d), (e), (f) scaffold HA-6
(M means material, NB means new bone).
Rendered 3D images and representative
tomographic images of micro-CT of the
implanted scaffold (g) HA/rGO-6/0.3 and
(h) HA-6 after 8 weeks. (i) Quantification of
newly formed bone in the implant. BV/TV
refers to bone volume/total volume, Tb. Th
refers to the trabecular thickness and Tb. Sp
refers to the trabecular separation. (j) Dia-
gram of the HA/rGO composite scaffold
substitution and new bone formation mech-
anism. (k) Mechanical test performance of
the surgical femur integrated with scaffold
HA/rGO-6/0.3 after 6 months. Reprinted
with permission from Ref. [148]. Copyright
© 2019, American Chemical Society.
4.4. Tissue-engineered scaffolds consisting of GO composites
Some studies also reported that GO rich in functional groups such as
carbonyls (
COOH) and hydroxyls (
OH) can provide biomimetic
mineralization using simulated body fluid (SBF), which has many active
sites [131]. Remarkably, graphene particles have the ability to induce
stem cell osteogenesis similar to that of bone morphogenic protein
(BMP-2) and other bioactive inorganic materials, such as hydroxyapatite
and nanosilicates [132,133]. In addition, the graphene family can exert
distinct molecular effects on immune cells [134–136], which is advan-
tageous for new immune-based strategies in osteogenesis and bone
regeneration.
GO has been used to form composites with biopolymers such as
collagen [131,137], chitosan [138], gelatin [139] and alginate [140].
The introduction of GO can help enhance the mechanical properties of
biopolymer materials so they can better bear the force and provide
possibilities for different designed constructures when serving as scaf-
folds for bone tissue engineering. Recently, our group prepared
gelatin-reduced graphene oxide (GOG) and constructed composites that
mimicked the procallus by combining GOG with a photocrosslinked
gelatin hydrogel [139]. The bidirectional differentiation of BMSCs,
including osteogenesis and angiogenesis, was proven to be activated
through the Erk1/2 and AKT pathways. Furthermore, due to the large
specific surface area and the strong π-π conjugation with the drug mol-
ecules [141,142] as well as the tendency of GO to be taken up by cells
through endocytosis [136], we also loaded the GOG with methyl vanil-
late (MV), which contributed to the bioactive signals of the biomimetic
procallus by priming the osteogenesis of BMSCs [139]. Another study
demonstrated that the synthesis of injectable hydrogels containing
poly(N-isopropylacrylamide) (PNIPAAm)-based copolymers, graphene
X. Qi et al. Smart Materials in Medicine 2 (2021) 280–291

Table 1
Combination of different native materials and GO.
Native materials Incorporated Cell/bacteria Biological effects GO preparing method Ref.
materials

GO dispersion N/A S. mutans; Antimicrobial activity Modified Hummer's method [90]

P. gingivalis;
F. nucleatum
GO suspensions N/A S. mutans Antimicrobial activity Bath sonication [88]
GO coating N/A fibroblast cells; Biocompatible; High-density atmospheric plasma [162]
S. mutans Antibacterial deposition
PMMA GO E. coli; Sustained antimicrobial-adhesive Purchased [163]
C. albicans;
S. aureus;
S. mutans;
Hyaluronidase Ag NP/GO S. aureus Antibacterial Purchased [124]
composites
GO dispersion N/A S. mutans; Antibacterial; Purchased [118]
P. gingivalis; Reosteogenesis
F. nucleatum;
BMSCs
Titanium plates GO S. aureus; Antibacterial Purchased [111]
S. mutans;
E. coli;
Human gingival
fibroblasts
Type I collagen GO rBMSCs Biomineralization; Modified Hummer's method [46]
Biocompatibility;
Osteogenic ability
Chitosan GO/HAP/Au C3H10T1/2; Biocompatibility; Hummer's method [138]
E.coli; Osteogenic Differentiation;
S.mutans; Antibacterial
S.aureus;
P.aeruginosa
Alginate GO hMSCs Proliferation and survival in oxidative Purchased [140]
environment;
Osteogenic differentiation
Hydrogel Chitosan/GO hDPSCs Proliferation; Differentiation to the Hummer's method [143]
osteoblasts
Fibrin hydrogel GO, IONPs, nHAP NIH-3T3; Biocompatible; Modified Hummer's method [144]
MG-63 cells Osteogenic property;
Calcium deposits
HA rGO MC3T3-E1 cells Increasing ALP; mineralization; osteopontin; Modified Hummer's and Offeman's [147]
osteocalcin methods
β-TCP GO hBMSCs Proliferation; Purchased [150]
Osteogenic property
HA rGO BMSCs Improved adhesion; proliferation; Two-step oxidation of NG [148]
spontaneous osteogenic differentiation flakes
CaP GO hMSCs; monocytes Activation of monocytes; Purchased [151]
Osteogenesis
Poly(L-lactic-co-glycolic Tussah silk fibroin; Mouse MSCs Enhanced adhesion; proliferation; Modified Hummer's method [157]
acid) GO ALP;
Mineral deposition
Titanium GO DPSCs Biocompatibility; Purchased [125]
Osteogenic differentiation
Titanium GO rBMSCs Proliferation; N/A [126]
Adhesion;
Osteogenic differentiation;
Osteointegration
Titanium GO PDLSCs Proliferation; Modified Hummer's method [96]
Osteogenic differentiation;
CaP Cu-GO BMSCs Adhesion; Modified Hummer's method [127]
Osteogenic differentiation
CaP rGO MC3T3-E1 cells Accelerated new bone formation Modified Hummer's and Offeman's [128]
methods
P34HB GO BMSCs Improved cellular performance; Purchased [130]
Osteogenic differentiation
Titanium GO/BMP-2 hBMMSCs Osteogenic differentiation; Modified Hummer's method [67]
Osteointegration
Titanium plates GO/BMP-2/SP hBMMSCs Osteogenic differentiation; Modified Hummer's method [68]
Osteointegration
Silk fibroin GO/P24 BMSCs Biocompatibility; Modified Hummer's method [69]
Adhesion;
Proliferation;
Osteogenic differentiation
Titanium GO/aspirine MC3T3-E1 cells Proliferation; osteogenic differentiation Modified Hummer's method [70]
Titanium GO/BMP-2/ BMSCs Osteogenic property; Modified Hummer's method [71]
vancomycin Antibacterial
Titanium nGO-PEG-PEI/siRNA MC3T3-E1 cells Osteogenic differentiation; Modified Hummer's method [72]
Osteointegration

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X. Qi et al.
oxide (GO) and chitosan through free-radical copolymerization resulted
in materials with porosity, swelling and biocompatibility. Increased
expression of osteogenic genes such as Runx 2 and OCN as well as ALP
activity and calcium deposition in hDPSCs demonstrated osteogenic po-
tential [143]. A similar conclusion was also drawn by Pathmanapan
et al., who incorporated GO with a fibrin hydrogel by the interaction
between the –COOH groups of the GO and the -NH2 groups of fibrin
[144]. Chitosan is an attractive biopolymer with properties such as
nontoxicity, biodegradability, biocompatibility, and flexibility, but its
poor mechanical properties limit its application in tissue engineering.
Some strategies, including constructing nanocomposite polymeric films
and nanoparticles, have been used to improve the mechanical properties
[145,146]. Prakash et al. adopted a hydrothermal method to uniformly
disperse nanorod-like HAP and Au NPs on the surfaces of GO and pre-
pared GO/HAP/Au ternary nanocomposites. Furthermore, they incor-
porated the obtained GO/HAP/Au ternary nanocomposites with
polymeric film by a gel casting technique, the product of which exhibited
good chemical stability, improved mechanical properties, good antibac-
terial properties against infection-causing pathogens and improved
osteoblast cell viability [138].
Other strategies have included combining GO with ceramic materials
to enhance the osteogenesis of composites. The combination of GO with
different kinds of ceramic materials has been reported, including HA
[147–149] and CaP [150,151]. Owing to its similarities to the inorganic
components of bone tissues and its high biocompatibility, degradability
and osteoconductivity [152,153], hydroxyapatite (HA) was chosen
explicitly by many researchers as a partner for GO to create and enhance
the mechanical and osteogenic properties. For instance, Lee et al. soni-
cated prepared reduced graphene oxide (rGO) and mixed it with hy-
droxyapatite (HA). The in vitro behaviors of the composites have been
proven, including promoting the differentiation of MC3T3-E1 preosteo-
blasts and increasing alkaline phosphatase, mineralization, osteopontin
and osteocalcin. Enhanced in vivo formation of new bone in
full-thickness calvarial defects without inflammatory responses after the
implantation of rGO/HA grafts was also observed [147]. In the study
conducted by Zhou et al., when incorporated with rGO, the hierarchically
porous hydroxyapatite hybrid scaffold improved the adhesion, prolifer-
ation and spontaneous osteogenic differentiation of BMSCs. After im-
plantation in vivo, the scaffold was crushed, degraded and wrapped by
the newly formed bone, the breaking strength of which with the healing
of scaffold HA/rGO-6/0.3 was satisfactory (20.0 MPa) and much higher
than that of HA-6 (9.8 MPa)[148]. (Fig. 5).
In addition to HA, calcium phosphate (CaP) has also been introduced
in combination with GO. Wu et al. successfully prepared β-TCP-GO disks
and 3D-printed scaffolds through a simple GO/water suspension soaking
method and heat treatment. Significantly enhanced proliferation, alka-
line phosphatase activity and osteogenic gene expression of human
marrow stromal cells (hBMSCs) was observed in the β-TCP-GO disk group
compared to the control group with only β-TCP and without GO modi-
fication, which was attributed to the activation of the Wnt/β-catenin
signaling pathway. When implanted in the cranial bone defects of rab-
bits, β-TCP-GRA scaffolds led to an increased rate of new bone formation
compared to β-TCP controls in vivo [150]. In addition to MSCs, mono-
cytes have also been demonstrated to be involved in the osteogenic
properties of GO owing to their immunological properties. Bordoni et al.
found that in the presence of monocytes, the ALP activity and the
expression of osteogenic markers increased on the GO-CaP material along
with the overproduction of Oncostatin M, a pro-osteogenic factor pro-
duced by monocytes [151].
Polymeric materials are another matrix that has been studied for
incorporation into GO. GO-doped poly(lactic-co-glycolic acid) nanofiber
scaffolds formed through electrospinning were reported to increase the
adhesion and proliferation of human MSCs [154].
Polydopamine-functionalized rGO increased the osteogenesis of MC3T3-E1
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Smart Materials in Medicine 2 (2021) 280–291
preosteoblasts [155]. The GO incorporated into poly(vinyl alcohol) and the
increased porosity formed by selective laser sintering improved the me-
chanical properties of the composite as well as the attachment and growth of
MG-67 human bone fibroblasts [156]. In recent years, Shao et al. fabricated
an ultrafine nanofiber scaffold by electrospinning a blend of
poly(L-lactic-co-glycolic acid), tussah silk fibroin, and graphene oxide (GO).
The data indicated that the modifications boosted the Young's modulus and
tensile strength by nearly 4-fold and 3-fold, respectively, and enhanced
adhesion and proliferation in mouse mesenchymal stem cells along with
promoted biomineralization-relevant ALP and mineral deposition [157].
In addition to being used in the decoration of single ceramic or
polymeric materials, in recent years, GO has also been incorporated into
composites to enhance their properties by utilizing the different advan-
tages of different materials. The same materials, such as the combination
of gelatin and alginate [158], chitosan and hydrogel [159], and different
kinds of materials, including the combination of hydroxyapatite and silk
fibroin [160], hydroxyapatite and chitosan [161], have been reported to
successfully serve as scaffold materials used in bone tissue engineering.
5. Conclusions and future perspectives
We have highlighted the recent progress in GO as strategies to
improve the properties of dental materials and bone tissue engineered
scaffolds. First, we described its physicochemical properties, which are
the basis of its diverse applications. Then, the biological properties were
discussed, including biocompatibility, controlled release of medicine,
antibacterial properties, osteogenic differentiation properties and odon-
togenic differentiation properties. After a preliminary description of its
applications, we further described the applications of GO in dentistry and
related tissue engineering. Antibacterial properties were discussed due to
their importance in dentistry applications, as dentistry covers areas with
large amounts of bacteria. Researchers then investigated the cooperation
of GO and conventional dental materials mainly in terms of the effects on
the mechanical properties. With the increasing number of attempts at GO
application, GO coatings for dental implants and tissue engineering
scaffolds were proven to promote bone formation. Scaffolds consisting of
GO were then summarized according to the matrices, which included
ceramic, polymeric, biopolymeric materials and composite materials
(Table 1).
Although the integration of GO within scaffolds can reduce problems
related to physical properties, including cytotoxicity, biodistribution,
biotransformation and immune response, there are still no distinct
chemical structures for the prepared GO, and the variation resulting from
methods or laboratory conditions requires a deep exploration to ensure
safety in further clinical use. In addition, recently, the delivery of genes
loaded on GO showed great potential, and this method may have great
potential in dental tissue engineering when used to deliver osteogenic-
related or odontogenic-related genes on implants or scaffolds. With
respect to the novel odontogenic properties of GO, although several kinds
of materials have been studied as substrates in vitro, there is still no in
vivo experiment to verify the properties and investigate the possibility of
their clinical application. In summary, the development of GO is an
exciting and promising material with the potential to revolutionize
clinical practice, which remains to be further explored.
Declaration of competing interest
We declare that we have no financial and personal relationships with
other people or organizations that can inappropriately influence our
work, there is no professional or other personal interest of any nature or
kind in any product, service and/or company that could be construed as
influencing the position presented in, or the review of, the manuscript
entitled, “Graphene Oxide: A Promising Material in Dentistry and Related
Tissue Regeneration.
X. Qi et al.
Acknowledgements
This work was supported by the National Key Research and Devel-
opment Program of China (2016YFC1102900), the National Natural
Science Foundation of China (81921002, 82022015,81900960) and the
Natural Science Foundation of Jiangsu Province (BK20180671).
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