Graphene oxide: A new direction in dentistry

Applied Materials Today 19 (2020) 100576

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Graphene oxide: A new direction in dentistry Mohammed Zahedul Islam Nizami a,b , Shogo Takashiba b , Yuta Nishina a,c,∗ a

Research Core for Interdisciplinary Sciences, OkaYama University, Room #309, 3-1-1 Tsushima-Naka, Kita-ku, 700-8530, Okayama, Japan Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 700-8525, Okayama, Japan c Graduate School of Natural Science and Technology, Okayama University, 700-8530, Okayama, Japan b

a r t i c l e

i n f o

Article history: Received 10 April 2019 Received in revised form 19 January 2020 Accepted 20 January 2020 Keywords: Graphene oxide Dentistry Biocompatibility Drug delivery Oral biofilm infection Oral cancer

a b s t r a c t This inclusive review summarizes the recent advances in the application of graphene oxide (GO) and functionalized GO in oral and dental research. GO possesses several extraordinary physical, chemical, optical, electrical, and mechanical properties. Because of its high surface area and oxygenated functional groups, GO exhibits excellent interaction ability with metals and ions as well as organic species. The current review reveals that GO has been used to produce a variety of functionalized nanocomposites, scaffolds, and advanced nanoparticle carriers. Accordingly, GO shows potential in a variety of research fields, such as tissue engineering, materials engineering, biomaterials, and drug delivery, indicating that the application of GO to biomedicine is particularly promising. More specifically, the recent application of GO in dentistry has provided outstanding results in antimicrobial action, regenerative dentistry, bone tissue engineering, drug delivery, physicomechanical property enhancement of dental biomaterials, and oral cancer treatment. The biocompatibilities of GO and its nanocomposites make them potential units in bone regeneration, osseointegration, and cell proliferation. Furthermore, its antibiofilm and antiadhesion properties have inspired researchers to develop GO for biofilm and caries prevention, as well as implant surface modification and as a quorum sensing inhibitor. This updated review is wide-ranging and provides a useful source for additional information on GO and its composites in dental research and applications. © 2020 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Literature survey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 GO and its nanocomposites in dental applications [23–78] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 3.1. Antimicrobial uses and cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 3.2. Regenerative dentistry and bone tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3.3. Drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.4. Physicomechanical property improvement of dental biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.5. Oral cancer treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4. Preparation of GO materials for dental applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4.1. GO and rGO preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 4.2. GO-metal composites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 4.3. GO-metal nanocomposites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 4.4. Organic functionalization of GO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 4.5. Others. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Abbreviations: GO, Graphene Oxide; rGO, Reduced Graphene Oxide; Gp, Graphene; NPl, Nanoplatelets; NS, Nanosheets; HAp, Hydroxyapatite. ∗ Corresponding author at: Research Core for Interdisciplinary Sciences, Okayama University, Room #309, 3-1-1 Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan. E-mail address: [email protected] (Y. Nishina). https://doi.org/10.1016/j.apmt.2020.100576 2352-9407/© 2020 Elsevier Ltd. All rights reserved.

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M.Z.I. Nizami, S. Takashiba and Y. Nishina / Applied Materials Today 19 (2020) 100576

1. Introduction Among the various nanocarbon materials that have featured extensively in recent academic and industrial researches, two-dimensional nanocarbons such as graphene have attracted increasing attention because of their excellent thermal, mechanical, and electronic properties [1–3]. Graphene oxide (GO) is a graphene analog mainly composed of sp3 -bonded carbon atoms connected with oxygen functional groups, and it possesses several extraordinary physical, chemical, optical, electrical, and mechanical properties [4–8]. GO has shown outstanding potential in many research fields, including biomedical applications. There are several review articles that deal with the application of GO in engineering, biomaterials, biomedical, drug delivery, and tissue engineering, demonstrating its utility in nanoelectronics [9], composite materials [10–14], energy devices (batteries, fuel cells, supercapacitors, and hydrogen storage) [15–17], sensors [18], and catalysts [19]. The demand for research into the bioapplication of GO and its derivatives is due to its many fascinating properties, such as its high specific surface area 2630 m2 /g, mechanical strength Young’s modulus, ≈1100 GPa, intrinsic biocompatibility, low cost, scalable production, and easy biological/chemical functionalization [20,21]. Consequently, the biomedical application of GO, especially in dentistry, is a vibrant area in graphene-based materials research. In recent years, several fascinating studies have explored the use of GO for dental applications, such as drug/gene delivery, antibacterial materials, biocompatible scaffolds for cell culture, and as a means to improve the physicochemical properties of dental biomaterials and cements. In 2017, Xie et al. reviewed the biomedical application of graphene [22], mainly focusing on the mechanical properties of graphene composites for dentistry and bone regeneration. We herein present a complimentary review to that of Xie’s, in which recent research developments concerning the use of GO in different dentistry fields are summarized.

2. Literature survey This review includes studies of GO and GO composites used in dental fields published in the English language. Abstracts, editorials, letters, and literature reviews were excluded. The EMBASE and MEDLINE (National Library of Medicine) databases were searched through PubMed. In the search, the keywords consisting of “graphene oxide” and “dental research”, “dental material”, or “dentistry” were employed. Other keywords “nanocomposite” and “nanoparticles” were also used as search refinement. These keywords would cover as much information about graphene oxide in dentistry as possible without overlooking related researches. After reading all the manuscripts, the following five areas or research were selected as categories.

Antimicrobial applications and cytotoxicity Regenerative dentistry and bone tissue engineering Drug delivery Physicomechanical property improvement of dental biomaterials 5. Oral cancer treatment

1. 2. 3. 4.

The categorized applications of GO are summarized in individual tables in each section. To simplify the text, abbreviations of the material names are also given in the tables.

3. GO and its nanocomposites in dental applications [23–78] The development of biomaterials for dental applications requires multidisciplinary research, including medicinal chemistry, biology, and materials chemistry. Different tissues in the oral and maxillofacial regions and teeth express specific chemical, physical, mechanical, thermal, and electrical characteristics. Therefore, materials design is extremely important. The outstanding biomechanical properties of GO have prompted scientists to use it in dental and restorative dentistry research. However, in general, a material composed of a single component cannot comply with all the properties required for dental applications designated by the American Dental Association (ADA) and FDI World Dental Federation specification. Therefore, composite materials with multiple components are required to fulfill the various requirements of artificial biomaterials. Because of its high surface area and oxygen functional group content, GO can be combined with a variety of metallic nanoparticles and bioactive molecules to endow it with specific functions. 3.1. Antimicrobial uses and cytotoxicity Functionalized GO and GO composites have been widely studied as antimicrobial agents with low cytotoxicity for dental application, as summarized in Table 1. Briefly, Jin et al. [23] reported the outstanding antibacterial and anti-adherence properties of GO-Ag-Ti multiphase nanocomposite, as well as its relatively low cytotoxicity. The cytotoxic effects of GO, T-rGO, and N–GO on human dental follicle stem cells were assessed by Olteanu et al. [24], and they found that GO shows the lowest cytotoxic effect, followed by N-GO, while T-rGO exhibits high cytotoxicity. In an antibiofilm study, Kulshresha et al. [25] observed a significant reduction in Streptococcus mutans biofilm formation in the presence of GO-Zn. GO-Zn was also demonstrated to be harmless to HEK-293 (human embryonic kidney cell line) cells. These promising results indicate that GO-Zn can be used as a coating agent for dental implants. Furthermore, Qian et al. [26] reported that a M-GO-Ti surface exhibited slow release behavior and excellent antibacterial activity under the synergistic effects of contact killing and release killing. M-GO-Ti’s cytocompatibility was shown to be excellent, and coculture test results suggested that, in the presence of Staphylococcus aureus, human gingival fibroblast cells on M-GO-Ti exhibited optimal cell adhesion and the maximum cell surface coverage. These characteristics are highly important for implant restoration in clinical applications. He et al. [27] also suggested that GO would be an effective antibacterial material. They found that a higher GO concentration is more effective against dental pathogens. It was revealed that GO can disrupt the cell walls and membranes and remove plasma, thus killing the bacteria. The cytocompatibility of GO was determined by Rosa et al. [28], who observed that GO-based substrates enhance stem cell proliferation, attachment, and gene expression to upregulate mineral-producing cells. Thus, they suggested further study with the aim of improving the functional properties of existing dental materials as well as introducing new dental biomaterials. GO-L-cys-Ag composite, rGO-Ag, and GO-Ag also show excellent antibiofilm and antimicrobial effects, as reported by Chandraker et al. [29]. The antimicrobial activities against oral pathogens of rGp-NS-Ag composites were compared with those of plain AgNPs and rGp by Peng et al., and rGp-NS-Ag composites were found to exhibit improved antimicrobial properties [30]. The therapeutic potential of GO-Ag for restraining the onset of biofilm formation in bacteria was demonstrated by Kulshrestha et al. [31]. Bai et al. [32] showed that the rGO-FHAp biocomposite could be highly effective as a dental implant material because of its enhanced mechanical properties, dissolution resistance, biocompatibility, and antibacte-

M.Z.I. Nizami, S. Takashiba and Y. Nishina / Applied Materials Today 19 (2020) 100576

3

Table 1 Antimicrobial and cytotoxicity studies of GO and its composites reported in the literature. Study subject

Material(s)

Findings

Ref

Cytotoxicity and antimicrobial activity

GO-Ag-Ti shows outstanding antibacterial properties.

[23]

Antibacterial activity on Staphylococcus aureus, Streptococcus mutans, and Escherichia coli

Minocycline hydrochloride on a GO-modified titanium surface (M-GO-Ti)

Antimicrobial action against dental pathogens

Graphene oxide (GO)

Cytotoxicity and differentiation potential using dental pulp stem cell (DPSC)

Graphene oxide (GO)

Antibacterial activity

Graphene oxide sheets decorated with l-cysteine functionalized silver nanoparticles (GO-L-cys-Ag) Reduced graphene nanosheet silver composite (rGp-NS-Ag) Graphene oxide-silver composite (GO-Ag)

GO shows the lowest cytotoxic effect, followed by N-GO, while T-rGO exhibits high cytotoxicity. Microscopic studies and antibiofilm assays revealed a significant reduction of biofilm in the presence of GO-Zn. M-GO-Ti shows slow release behavior and exhibits excellent antibacterial activity with the synergistic effect of contact killing and release killing. GO shows effective antibacterial action against dental pathogens. GO allows DPSC attachment and proliferation and increases the expression of several genes that are upregulated in mineral-producing cells. GO-L-cys-Ag destroys the cell membrane of Escherichia coli.

[24]

Antibiofilm behavior on Streptococcus mutans

Graphene oxide-sliver-titanium multiphase composite (GO-Ag-Ti) Graphene oxide (GO); thermally treated GO (T-rGO); N-doped GO (N-GO) GO/zinc oxide composite (GO-Zn)

[29]

rGp-NS-Ag exhibits antimicrobial action.

[30]

GO-Ag acts on the Streptococcus mutans biofilm formation cascade. Biofilm-inhibition concentrations of GO-Ag are nontoxic. rGO-FHAp is useful as a dental implant material.

[31]

Cytotoxicity to human dental follicle stem cells

Antimicrobial activities against oral pathogens Antibiofilm activity

Enhanced mechanical properties, dissolution resistance, biocompatibility, and antibacterial activity Antibacterial effect Antimicrobial and antibiofilm properties. Cytotoxicity study Cytotoxicity and hemocompatibility

Reduced graphene oxide/fluoro-hydroxyapatite (rGO-FHAp) Silver decorated magnetic graphene oxide (MGO-Ag) Zinc-oxide-decorated graphene oxide (GO-Zn) Hydroxyapatite/graphene oxide (HAp-GO) Polylactic acid on graphene oxide and graphene nanoplatelets (PLA-GO and PLA-Gp-NPl)

rial activity. Zhang et al. [33] found that MGO-Ag exhibits excellent antibacterial activity against Escherichia coli and S. aureus. ZnOrGO represents a powerful tool for killing both the planktonic and biofilm forms of S. mutans, as reported by Zanni et al. [34], suggesting that ZnO-rGO is highly effective for controlling S. mutans growth and, therefore, caries development. They also suggested utilizing graphene-based materials for the improvement of dental resin composites and the associated dental adhesives. Ramadas et al. [35] reported that HAp-GO nanocomposites exhibit excellent biocompatibility and could be utilized in orthopedic, drug delivery, and dental applications. GO can improve the biocompatibility and mechanical properties of poly (lactic acid) (PLA), as demonstrated by Pinto et al. [36]. They claimed that GO has a positive influence on cell adhesion, proliferation, and tissue regeneration rate. They also stated that Gp-NPl reduces thrombogenicity, which could decrease postoperative complications by blood-clot formation. As a summary of antimicrobial and cytotoxicity of GO, the antimicrobial effects, cytotoxicity with risk-benefit assessment, and mechanical strength of GO provide ample reasons for researchers to consider GO in dental biomaterial research. These characteristic features of GO are practically applicable in dental biomaterials, instruments, equipment, and preventive care materials. Mechanism of antimicrobial activity by GO and its derivative remains controversial. It has been suggested that the physical and chemical factors of GO are involved in antimicrobial action [37–40]. For example, sharp edges of the nanosheets have the potential to break the bacterial cell membrane to leak out the intracellular matrix that leads to bacterial death. Other possible mechanisms are proposed: 1) wrapping of bacteria by GO sheets that prevent its growth; 2) overproduction of reactive oxygen species (ROS) to stimulate a chain reaction and lead to the breakdown of the cell membrane; 3) GO acts as an electron acceptor that pumps the electron away from the bacterial cell membrane creating ROS-

MGO-Ag exhibits excellent antibacterial activity against Escherichia coli and Staphylococcus aureus. GO-Zn represents a powerful tool for killing both the planktonic and biofilm forms of Streptococcus mutans. HAp-GO exhibits excellent biocompatibility. Low concentrations of GO and Gp-NPl can be incorporated safely into PLA to improve its mechanical properties for biomedical applications.

[25] [26]

[27] [28]

[32]

[33] [34] [35] [36]

independent oxidative stress (in this case, basal plane of GO kills bacteria while masking the basal plane of GO renders it inactive) [41,42]. 3.2. Regenerative dentistry and bone tissue engineering Table 2 summarizes recent studies on the use of GO and its composites in regenerative dentistry and bone tissue engineering. Lee et al. [43,44] suggested that rGO-HAp can be effectively utilized as a dental and orthopedic bone filler. They demonstrated the potential of rGO-coated hydroxyapatite composites in enhancing the spontaneous differentiation of hMSCs, demonstrating superior bioactivity and osteoinductive potential. rGO-HAp grafts were found to have the ability to enhance new bone formation significantly in full-thickness calvarial defects without inflammatory reactions. Zhang et al. [45] found that dGO-PEG-PEI enhances in vivo osseointegration and suggested utilizing dGO-PEG-PEI as a delivery system for siRNA-based implant biomodification to improve osteogenesis. Kim et al. [46] also reported that rGO-BCP was effective for osteogenesis. Their results suggested that rGO-BCP could be a biocompatible bone graft material. They also found that new bone formation is enhanced in line with the concentration of rGO in the composite and suggested further studies to confirm the optimal concentration of rGO. GO is biocompatible and exhibits high bone-formation capability for the scaffold, as demonstrated by Nishida et al. [47]. In their in vitro and in vivo study, GO was shown to enhance the physical properties of a collagen scaffold and to stimulate biological activity. A GO scaffold resulted in a nearly five-fold increase of bone formation when compared with the collagen scaffold, thus indicating that GO is biocompatible and has a high bone-forming capability for the scaffold. Consequently, the application of GO scaffolds in bone-tissue-engineering therapy was suggested. Zhou et al. [48] evaluated and compared the bioactivities

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M.Z.I. Nizami, S. Takashiba and Y. Nishina / Applied Materials Today 19 (2020) 100576

Table 2 Regenerative dentistry and bone tissue engineering studies of GO and its composites reported in the literature. Study subject

Material(s)

Findings

Ref

Osteogenic differentiation of human mesenchymal stem cells (hMSCs)

Reduced graphene oxide-coated hydroxyapatite composites (rGO-HAp)

[43]

Osteogenesis of MC3T3-E1 preosteoblasts

Reduced graphene oxide/hydroxyapatite nanocomposites (rGO-HAp) Polyethylene glycol and polyethyleneimine dual-functionalized graphene oxide (dGO-PEG-PEI) Biphasic calcium phosphate coated with reduced graphene oxide (rGO-BCP) Graphene oxide (GO)

rGO-HAp stimulates the spontaneous differentiation of hMSCs and shows excellent bioactivity and osteoinductive potential. rGO-HAp enhances new bone formation in full-thickness calvarial defects without inflammatory responses. dGO-PEG-PEI enhances in vivo osseointegration.

[45]

rGO-BCP is found to be effective in osteogenesis.

[46]

GO is biocompatible and exhibits high bone-formation capability for the scaffold. The combination of GO and PDLSCs provides a promising construct for regenerative dentistry. The Dex-rGO-MPCR-TNZ results in significantly enhanced growth and differentiation of MC3T3-E1 cells into osteoblasts. GO-SFs exhibit excellent potential for therapeutic use in regenerative dentistry. Gp exhibits potential for use as a substrate for craniofacial bone tissue-engineering research. GO coating is homogeneously distributed on the PB granule surface and demonstrated to endow PB with increased resistance to fracture load. nHAp-MWCNT-GO nanocomposites are suitable for biomedical applications, with a proven antibacterial effect and no osteoblast cytotoxicity.

[47]

siRNAs for improving osteogenesis

As bone graft materials in bone regeneration Effects of GO application to a 3D collagen scaffold Bioactivity of human periodontal ligament stem cells (PDLSCs) Graphene oxide for dental applications

In the mesenchymal phenotype, the viability, adhesion, and proliferation rate of PDLSCs Inducing odontogenic and osteogenic differentiation in dental pulp stem cells (DPSC) Production of GO-coated porcine bone (PB) granules and characterization Applications of Nanohydroxyapatite/multiwalled carbon nanotube (nHAp-MWCNT-GO) nanocomposites in bone tissue engineering Enhancement of osteoblast alkaline phosphatase (ALP) activity for promoting new bone formation Enhancement of cell proliferation and osteodifferentiation of rat bone mesenchymal stem cells (rBMSCs). Osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in graphene substrates Applications in bone tissue engineering

Synthesis and characterization of poly-ethylenimine (PEI) conjugated graphene oxide (GO-PEI) for biomedical application Design, preparation, and characterization of new hybrid graphene-hydroxyapatite based scaffolds for bone regeneration

Graphene oxide-coated titanium (GO-Ti) Dexamethasone loaded and reduced graphene oxide coated multipass caliber-rolled Ti alloy of Ti13Nb13Zr (Dex-rGO-MPCR-TNZ) Graphene oxide (GO); Silk fibroin and GO (GO-SF) composite Monolayer graphene (Gp) Graphene oxide (GO)

nHAp-MWCNT-GO nanocomposites

[44]

[48] [49]

[50] [51] [[52]]

[53]

Poly(d, l-lactic acid) with graphene/multiwalled carbon nanotube oxides (PDLLA/MWCNTO-GO) GO and rGO-coated titanium (GO-Ti; rGO-Ti) and dexamethasone-loaded, GO and rGO coated titanium (DEX-GO-Ti; DEX-rGO-Ti) Two- and three-dimensional graphene (2D Gp; 3D Gp)

PDLLA/MWCNTO-GO is noncytotoxic and significantly enhances osteoblast ALP activity, thus, promoting new bone formation. GO-Ti and rGO-Ti are both excellent carriers for DEX and promote proliferation and accelerated osteogenic differentiation of rBMSCs. 2D Gp and 3D Gp play a role in enhancing the osteoblastic differentiation of PDLSC.

Single and multiwalled carbon nanotubes (SWCNTs, MWCNTs); single and multiwalled graphene oxide nanoribbons (SWGONRs, MWGONRs); graphene oxide nanoplatelets (GO-NPls) Poly-ethylenimine and graphene oxide conjugate (GO-PEI)

These 2D-reinforced materials exhibit improved mechanical properties.

[57]

GO-PEI exhibits exceptional bactericidal activity, enhanced cell proliferation, and notable osteoinductive potentials.

[58]

GO and rGO-HAp improve metabolic cellular response and are very promising for bone regenerative-engineering applications.

[59]

GO; rGO-coated hydroxyapatite granules (rGO-HAp)

[54]

[55]

[56]

Fig. 1. Schematic illustration for multipass caliber-rolled Ti alloy surface-modified with dexamethasone-loaded graphene for minimally invasive dental implants with high mechanical strength and a small diameter “reproduced with permission from Jung et al. [49].”.

M.Z.I. Nizami, S. Takashiba and Y. Nishina / Applied Materials Today 19 (2020) 100576

of GO-Ti and sodium titanate substrates on periodontal ligament stem cells (PDLSCs) and reported that PDLSCs on a GO-Ti substrate show significantly higher proliferation rate, ALP activity, and osteogenesis-related gene expression level compared with those for the Na-Ti substrate. Thus, the combination of GO and PDLSCs could provide a promising construct for regenerative dentistry. Jung et al. [49] performed a study on Dex-rGO-MPCR-TNZ (multipass caliber-rolled Ti alloy with dexamethasone-loaded reduced GO) for dental applications and found that Dex-rGO-MPCR-TNZ significantly enhances growth and differentiation of MC3T3-E1 cells into osteoblasts and expression of osteogenic expression markers. A prototype Dex-rGO-MPCR-TNZ dental implant was implanted into an artificial bone block, and the results suggested that mechanically strong Dex-rGO-MPCR-TNZ could be successfully applied to minimally invasive dental implantations. Fig. 1 shows the potential of GO composites in implant dentistry. It shows rGO being used in surface modification of a high-mechanical-strength MPCRTNZ surface decorated with osteogenic Dex via ␲-␲ stacking for effective osseointegration. This indicates the utility of rGO for minimally invasive dental implantation. Further study may discover minimally invasive implants for abridged jaw conditions. Rodríguez-Lozano et al. [50] evaluated the effects of GO and GO-SF films combined with fibroin in the mesenchymal phenotype, as well as the viability, adhesion, and proliferation rate of PDLSCs. They suggested that the combination of human dental stem cells/fibroin/GO based-bioengineered constructs has excellent potential for therapeutic use in regenerative dentistry. Xie et al. [51] investigated the potential of Gp for craniofacial bone-tissueengineering research. Gp stimulated the expression of osteoblastic genes and proteins and has the potential for use in bone tissue engineering and regeneration. Ettorre et al. [52] demonstrated that GO-coated porcine bone (PB) granules homogeneously distributed on PB granule surfaces increased their resistance to fracture load without cytotoxic effects, demonstrating that GO-coated PB granules could be applied to other bone substitute materials to improve their performances. nHAp-MWCNT-GO nanocomposites have been demonstrated by Rodrigues et al. [53] to be bioactive and antibacterial without cytotoxicity to osteoblasts, thus exhibiting potential for biomedical and bone-tissue-engineering applications. Silva et al. [54] reported enhanced osteoblast ALP activity and greater bone formation using MWCNTO-GO. These novel scaffolds were found to be potential substitutes for those currently used in bone tissue regeneration. DEX-GO-Ti and DEX-rGO-Ti substrates were found to highly regulate the proliferation and osteogenic differentiation of rBMSCs by Ren et al. [55]. Thus, these DEX-GO-Ti and DEXrGO-Ti substrates were suggested as regulators for the bioactivities of Ti implants, and, thus, provide an innovative strategy for Tiimplant surface modification in future dental applications. Xie et al. [56] reported that Gp exhibits both physical and chemical interactions with PDLSCs while enhancing their osteogenic differentiation. They suggested exploring the effects Gp on the mechanisms regulating the osteoblastic differentiation of stem cells. This could be advantageous for the further application of these promising materials as substrates for bone tissue regeneration. Lalwani et al. [57] suggested that SWGONRs, MWGONRs, and GO-NPls 2D nanostructures could lead to whole new classes of ultra-strong, light-weight biomaterials for tissue-engineering applications. Their excellent properties may be exploited for the development of implants with improved mechanical properties for bone tissue engineering in dental and orthopedic applications. Kumar et al. [58] reported that GO-PEI significantly promotes the proliferation and formation of focal adhesions in human mesenchymal stem cells (hMSCs) and is highly effective for inducing stem cell osteogenesis and bactericidal activity. Thus, it can be utilized to prepare resorbable bioactive biomaterials for fabricating orthopedic devices for fracture fixation, tissue engineering, and dental applications. GO and rGO-HAp

5

were reported to be promising for regenerative-engineering applications by Perrotti et al. [59]. They suggested performing in vitro and in vivo studies to analyze the biocompatibility, bioactivity, and the microstructure of the regenerated bone. Positive results in these analyses might be beneficial for bone tissue engineering and further orthopedic and dental application. The action of organic-functionalized GO is represented schematically in Fig. 2. The spin-coated poly(␧-caprolactone) (PCL)/graphene composite films promote cell proliferation and regeneration of stem cells and also exhibit antibacterial activity in vitro. This dual-action has potential in orthopedics. GO and its derivatives have promising properties in regenerative dentistry and bone tissue engineering, while molecular pathways, signal transduction, active interaction between GO and the cellular component have not fully understood. Various types of GO analogues are proposed in this field, therefore, mechanistic studies based on the molecular level investigation are needed to optimize the functionalization mode of GO. These reports indicate that GO and its composites were mainly used as a coating agent for biomaterials (e.g., hydroxyapatite and biphasic calcium phosphate) for regenerative dentistry and bone tissue engineering 3.3. Drug delivery (Table 3) Nanosheet structure with high surface area and good water dispersibility is a promising candidate for drug carriers toward specific organs. However, there are a few studies directly related to drug delivery using GO in dental therapy, despite the known potential of GO for drug delivery in other biomedical and pharmaceutical applications. La et al. [60,61] reported that GO is an efficient carrier for the delivery of therapeutic proteins. They applied GO-Ti implants as carriers to deliver BMP-2 for bone regeneration and successfully demonstrated that ionized GO can deliver proteins by binding through electrostatic interactions. Furthermore, they used a GO-Ti substrate for BMP-2 delivery as an osteoinductive and SP as a stem cell recruitment agent for in situ bone regeneration and reported that GO has the potential to sustain the release of BMP-2. They also reported enhanced bone formation on a Ti-implanted site and proposed the possibility of the dual delivery of BMP-2 and SP to improve osteointegration of dental or orthopedic implants. 3.4. Physicomechanical property improvement of dental biomaterials Table 4 summarizes the application of GO and its composites to the improvement of the physicomechanical properties of dental biomaterials. Using Gp-NSs to improve the physicomechanical properties and bioactivity of dental cement was suggested by Dubey et al. [62]. They mixed Gp-NSs with Biodentine (BIO) and Endocem Zr (ECZ) and found that it enhanced their hardnesses and decreased their setting times without interfering with any of their basic properties. Rajesh et al. [63] in their study found that the synergistic interactions between rGO-HAp nanocomposite components enhanced its tensile strain and elasticity. Nahorny et al. [64] showed that MWCNTO-GO combined with nHAp has potential as a protective coating against dentin erosion. PLA-HAp-GO composite may be a promising material for load-bearing orthopedic implants, as reported by Gong et al. [65]. They found that GO and HAp improve the thermal stabilities and hydrophobic properties of PLA-based nanocomposites, and that tensile strength and hardness of PLAHAp-GO increases with increasing GO content. Mehdi et al. [66] evaluated the effect of electrophoretic deposition of CS-rGO composites on a titanium substrate and found that the CS-rGO coating

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Fig. 2. Schematic representation of biological studies showing stem cell and the bacterial response to PCL/graphene composites for potential orthopedic use “reproduced with permission from Kumar et al. [58].”. Table 3 Drug delivery studies of GO and its composites reported in the literature. Study Subject

Material(s)

Findings

Ref

GO as a carrier for therapeutic protein delivery

Graphene oxide (GO)

[60]

GO for the delivery of bone morphogenetic protein-2 (BMP-2) and substance P (SP)

Graphene oxide (GO); GO-Ti

GO is an effective carrier for the controlled delivery of therapeutic proteins, such as BMP-2. The dual delivery of BMP-2 and SP using GO-Ti shows excellent new bone formation on a Ti-implanted site.

[61]

Table 4 Physicomechanical property development of dental biomaterials. Study subject

Material(s)

Findings

Ref

Potential of graphene nanosheets (Gp-NSs) on the bioactivity, physicomechanical, and chemical properties of bioactive cement Physicochemical properties of rGO-HAp nanocomposite

Multilayer graphene nanosheets (Gp-NSs)

[62]

Protective coating for dentin erosion

Multiwalled carbon nanotube/graphene oxide hybrid carbon-based material combined with nanohydroxyapatite (nHAp-MWCNTO-GO) PLA-HAp-GO

Gp-NSs can improve the physicomechanical properties of bioactive cement, but it is not recommended for all materials when effective bonding is a concern. Intercalation of HAp nanorods imparts tensile strain into the rGO layers. The elasticity of the rGO-HAp nanocomposite is improved tenfold compared with that of HAp. nHAp-MWCNTO-GO appears to form a protective layer for dentin against erosive processes.

Morphology, crystallinity, thermal stability, hydrophobic property, and mechanical properties of a polylactic acid/hydroxyapatite/graphene oxide composite (PLA-HAp-GO) Electrophoretic deposition of calcium silicate-reduced graphene oxide composites on titanium substrate Osteogenic differentiation of MC3T3-E1 preosteoblasts

Adherence of HAp on Gp nanosheets Noncytotoxic hybrid ceramic–polymer coating

Enhancing the mechanical strength and fracture toughness of brittle hydroxyapatite (HAp) Increasing the mechanical properties of hydroxyapatite (HAp) Improving the anticorrosion and biocompatibility of NiTi alloy Implant surface modification

rGO-HAp

[63]

[64]

PLA-HAp-GO composites are promising materials for load-bearing orthopedic implants.

[65]

[66]

Graphene oxide (GO); GO-hydroxyapatite (HAp)

CS-rGO coating improves adhesion, hardness, and elastic modulus. The CS-rGO composite coatings exhibit good apatite-forming ability in simulated body fluid (SBF). rGO-HAp hybrid composites are found to be biocompatible, transferable, and implantable scaffolds for bone regeneration. These composites can be utilized as coating and bone fillers for dental implants. The strong grafting of HAP on Gp nanosheets is achieved by keeping the native structure of Gp nanosheets intact. HAp-CS-Gp composites significantly improve morphology, thermal stability, and bioactivity. These are noncytotoxic to peripheral blood mononuclear cells (PBMCs). These composites improve the mechanical properties of HAp with minimal cytotoxicity.

Graphene oxide-reinforced hydroxyapatite (GO-HAp) Graphene-film-wrapped NiTi alloy (Gp-NiTi)

GO-HAp sheets significantly improve the corrosion protection of titanium. Gp-NiTi enhances anticorrosion and biocompatibility.

[71]

GO on Ti6Al7Nb alloy; medical-grade titanium alloys such as Ti6Al7Nb implants (Ti67IMP)

A hybrid composite of nanotubular arrays and GO provides a better connection between the implant and the bone, as well as better cell adhesion.

Calcium silicate-reduced graphene oxide (CS–rGO) rGO-HAp composites

HAp functionalized graphene nanosheets (Gp-HAp) Graphene and chitosan introduced in hydroxyapatite (HAp-CS-Gp)

[67]

[68] [69]

[70]

[72] [73]

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Fig. 3. Schematic of the potential applications of GO-modified mixed oxide nanotubes on Ti67IMP in orthopedic and dental applications “reproduced with permission from Rafieerad et al. [73].”.

improves adhesion, hardness, and elastic modulus and that CS-rGO coatings exhibit good apatite-forming ability in simulated body fluid (SBF). They also suggested that their results might have the potential for implants and other biomaterial applications. Shin et al. [67] suggested that rGO-HAp hybrid composites can serve as biocompatible, transferable, and implantable scaffolds for bone regeneration, and they established that rGO-HAp composite materials have the potential to promote spontaneous osteogenesis in the absence of any osteogenic factors. Neelgund et al. [68] evaluated the adherence of HAp to GO nanosheets, and strong seeding of HAp on Gp nanosheets was obtained while preserving the native structures of the Gp nanosheets. Marija et al. [69] found that Gp in HApCS composites enhances their morphologies, thermal stabilities, and bioactivities. HAp-CS and HAp-CS-Gp composite coatings both show cytocompatibility with healthy peripheral blood mononuclear cells (PBMCs), although no antibacterial activity was observed. Pulyala et al. [70] enhanced the mechanical strength and fracture toughness of brittle HAp by GO incorporation (GO-HAp). They also demonstrated its cytocompatibility, suggesting that this composite has the potential to be used as bone graft substitutes. Fathyunes et al. [71] found that GO sheets significantly improve the corrosion protection of titanium, and Zhang et al. [72] also showed that Gp enhances its anti-corrosion and biocompatibility. This property is useful for surface modification and surface coating of NiTi alloys, which are widely used in biomedical materials. Dental biomaterials require constant improvement, and GO might be a potential candidate for further study. Fig. 3 illustrates the use of GO-modified mixed oxide nanotubes on Ti67IMP using a hybrid approach, as well as it’s potential in orthopedic and dental applications [73]. The unique properties of GO such as large surface area, high mechanical properties, and functionality pose a promising effect for dental materials. In the presence of GO materials, properties can be improved by anchoring with other dental biomaterials such as polymers, ceramics, and alloys. Functional groups on GO improve the interaction with biomolecules and biomaterials to enhance the physicochemical and mechanical properties of existing materials.

inhibited WNT- and Notch-driven signaling, as well as STAT1/3 signaling and the NRF2-dependent antioxidant response, while little effect was observed on TGF-beta/SMAD-signaling [74]. However, a few studies in this field have been reported. Application of GO in oral cancer treatment has been reported by Kumar et al. [75], who investigated the fabrication of a noninvasive, label-free, and efficient biosensing platform for detection of the oral cancer biomarker CYFRA-21-1. They suggested using a ZrO2 -rGO nanocomposite for investigating the effect of ZrO2 nanoparticles and the role of antigen-antibody interactions in the performance of this immunosensor. Thus, more focus on these types of applications could provide positive outcomes in oral cancer treatment. Lee et. al. [76] applied nGO in anticancer drug delivery and performed both in vivo and in vitro experiments. In their investigation, the effects of ligand density on active tumor targeting ability of nGO were evaluated using folate as a model ligand. KB cells showed that increasing ligand density increased the cellular uptake of nGO almost linearly, but in vivo data of tumor accumulation of nGO showed a low critical ligand density. The higher tumor accumulation of nGO by ligand conjugation above the critical concentration also results in better photothermal tumor ablation in vivo. Excluding in vitro results, they claim the successful applications of nGO as a drug delivery tool for cancer therapy. Zheng et. al. [77] developed GPt nanocomposite and applied for anti-cancer drug delivery system. GPt enhanced cell cycle arrest in the S phase and lead to cell apoptosis. They also suggested that the incorporated Pt on GO quantum dot can increase Pt accumulation inside cells in both normoxia and hypoxia. In vivo and in vitro experiments suggested GPt can be a candidate for anticancer therapy, while further studies are required to establish the pharmacokinetics and application systems. Hou et. al. used GO–N=N–GO/PVA hydrogels encapsulated Curcumin (CUR) and found to be safe in the stomach and enhance the colon-targeting ability and residence time in the colon site. Thus, these composite hydrogels are expected for the treatment of colon cancer with high efficiency and low toxicity (Table 5). 4. Preparation of GO materials for dental applications

3.5. Oral cancer treatment There is considerable potential for the use of GO in oral cancer treatment because it was recently reported that GO treatment

Many researchers have employed numerous methods, such as coating, hydrogel blending, wet/dry-spinning procedures, and 3D printing, to make 2D or 3D graphene-based constructs. GO

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Table 5 GO application in Oral cancer. Study Subject

Material(s)

Findings

Ref

Development of an immunosensor.

Nanostructured zirconia on reduced graphene oxide (ZrO2 –RGO)

[75]

Functionalization of nanographene oxide (nGO) sheets for targeting tumor Development of a tumor-targeting graphene nanocomposite Graphene oxide-based anti-cancer drug for targeted colon cancer treatment

Nanographene oxide (nGO)

ZrO2 -rGO regulates the role of antigen-antibody interactions and exhibits its performance as immunosensor nGO has potential in targeting cancer cells and applied as a drug delivery tool for cancer therapy GPt has the potential to improve the chemotherapeutic efficacy Curcumin-loaded GO-N=N-GO/PVA composite hydrogels exhibit a potential for the treatment of colon cancer.

polyethylene glycol-graphene quantum dots-Pt composite (GPt) GO containing azoaromatic and poly vinyl alcohol (GO-N=N-GO/PVA) composite hydrogels.

and its derivatives can also be tethered to other biomaterials to enhance their mechanical, physical, and electrical properties as dental and biomedical materials and in tissue-engineering applications. Accordingly, we have attempted to summarize the numerous synthesis techniques for GO composites reported by researchers. The following should provide useful guidelines for prospective researchers in this field. 4.1. GO and rGO preparation Most of the carbon atoms in GO are covalently connected to oxygen atoms, and thus the physical properties of GO are different from those of pristine graphene. The preparation of GO is usually performed by one of three methods: Brodie’s method [79], Staudenmaier’s method [80], and Hummers’ method [81]. The number and type of oxygen functional groups in the product (and thus, its physical properties) largely depend on its method of production. Most of the studies reported in recent years have adopted the Hummers’ method, an example of which is shown below: 1. First, 100 g of graphite and 50 g of sodium nitrate are added to 2.3 L of sulfuric acid, which is then stirred with cooling. 2. Then, 300 g of potassium permanganate is gradually added. During this time, the temperature of the reaction solution should not exceed 20 ◦ C. 3. The mixture is left to stand for 30 min in a 35 ◦ C water bath. 4. 4.6 L of water is added, and is heated in a 98 ◦ C water bath for 15 min. 5. Then, the reaction is diluted with water to 14 L. 6. Hydrogen peroxide solution is added to the resulting mixture until bubbling stops. 7. Impurities are removed by filtration and washing. Modifications of Hummers’ method have been reported [82–84] and recently employed for dental applications. Thermally reduced GO (T-rGO) is obtained by transferring freeze-dried GO to a quartz boat that is heated inside a temperature-programmed furnace [24]. In Fig. 4, GO and rGO synthesis is illustrated. 4.2. GO-metal composites The large surface area of GO supports nanomaterials of various chemical structures. Different studies have demonstrated that GO functionalization occurs by encapsulation, wrapping, layering, anchoring, sandwiching ions/particles, and combinations thereof. Fig. 5 shows a general scheme of GO functionalization and types of GO composites. Jin et al. [23] electroplated commercial pure titanium plates in an aqueous electrolyte solution containing GO at room temperature. Qian et al. [26] used pure titanium plates (Ti), and hydroxylated titanium (Ti−OH) was obtained by immersing Ti in aqueous NaOH solution. Positively charged titanium

[69] [77] [78]

surfaces bearing amino groups (Ti-NH2 ) were obtained by 3aminopropyltrimethoxysilane (3-APTES) treatment. Later, GO-Ti was obtained by adding GO to the titanium surfaces by nucleophilic substitution and electrostatic interactions with -NH2 on 3-APTES. Finally, GO-Ti was immersed in aqueous minocycline hydrochloride solution, and the samples were named “M-GO-Ti”. Zhou et al. [48] synthesized GO-Ti. Titanium foils were immersed in an aqueous NaOH solution followed by hydrothermal treatment to obtain Ti–OH substrates. The obtained Ti–OH films were immersed in an ethanol solution of 3-APTES and then immersed in GO for 24 h. Fig. 6 shows a schematic preparation method of GO-Ti and rGO-Ti. Layered GO coating of metal substrates has been demonstrated to have a great impact on surface properties. Fig. 7 illustrates the concept of GO multilayer nanocomposites. Coating of a Ti implant surface was performed by a layer-by-layer alignment of positively and negatively charged GO. This alignment exemplifies the potential of depositing thin films with preferred functionality and nanoscale regulation of structure, width, thickness, and electrostatics onto prospective substrates through corresponding exchanges arbitrated either by hydrogen bonding, covalent bonding, or electrostatic interactions. 4.3. GO-metal nanocomposites Kulshrestha et al. [25] used GO-Zn in their study. To synthesize GO-Zn, zinc acetate and GO were dispersed in absolute ethanol followed by sonication and the product was isolated by centrifugation. Then, the product was vacuum dried and mixed with ethylene glycol. The resulting mixture was heated, and the synthesized GO-Zn suspension was centrifuged and dried. Chandraker et al. [29] synthesized GO-L-Cys-Ag. In this process, GO sheets were decorated with amino acid l-cysteine functionalized silver nanoparticles by AgNO3 , trisodium citrate, and NaBH4 . Peng et al. [30] synthesized Ag-NP-decorated reduced graphene nanocomposites (rGp-Ag) in an efficient and convenient chemical reduction method for their research. GO-Ag was synthesized by a nontoxic and eco-friendly route using a floral extract of Legistromia speciosa by Kulshrestha et al. [31]. Furthermore, Ag-NP-decorated magnetic GO was synthesized by doping silver and Fe3 O4 nanoparticles onto the surface of GO by Zhang et al. The product was used as an antibacterial agent [33]. 4.4. Organic functionalization of GO Lee et al. [44] prepared rGO-HAp for their research. A GO suspension was sonicated and then hydrazine hydrate (N2 H4 ·H2 O) was added. Later, the suspension was filtered and washed with ethanol/water solution several times. Finally, rGO nanosheets were prepared by a vacuum drying technique. To prepare rGO-Hap, colloidal dispersion of rGO nanosheets and HAp microparticles was vigorously mixed and slowly air-dried at room temperature. The rGO-coated BCP graft material was obtained by vigorously mixing

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Fig. 4. Schematic of the preparation of GO and rGO.

Fig. 5. (a) Preparation methods of GO composites with nanoparticles. (b) Types of GO composites.

colloidal dispersions of rGO nanoplatelets and BCP microparticles and slowly air-drying at room temperature [46]. Bai et al. [32] synthesized an rGO-FHAp bio-composite. rGO was incorporated into HAp to form a rGO-FHAp composite by in situ chemical synthesis method and spark plasma sintering technology. Gong et al. [65] prepared HAp-GO by a wet-chemical precipitation method. Then, PLA-HAp-GO nanocomposite films were prepared via solution blending and casting method using N, Ndimethyl-formamide and CH2 Cl2 as mutual solvents. CS-rGO was synthesized using an in situ hydrothermal method. CS nanowires were uniformly decorated on the rGO with proper interfacial bonding. CS-rGO was reported to behave like a hybrid composite when deposited on a titanium substrate by cathodic electrophoretic deposition (EPD) by Mehrali et al. [66]. As reported by Shin et al.

[67], in order to prepare rGO-HAp, HAp microparticles were suspended in deionized water and mixed with a colloidal dispersion of rGO nanosheets at a 1:1 wt ratio. The mixed colloidal dispersion was vortexed vigorously, and the solvent was air-dried slowly at room temperature to afford rGO-Hap. Neelgund et al. [68] functionalized GO nanosheets on hydroxyapatite. A GO suspension in ethanol was refluxed to obtain a black suspension of Gp nanosheets. This suspension was then centrifuged and dried. Later, the Gp nanosheets were dispersed in DI water and added to an aqueous solution of Ca (OH)2 . The pH of the was adjusted to ≈9. Finally, the resulting HAp-functionalized Gp nanosheets (fHAp-Gp) were separated by centrifugation and drying. In a study by Pulyala et al. [70], hydroxyapatite was reinforced by adding GO and CNTs, which also significantly altered its wet-

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Fig. 6. Schematic representation of the preparation of GO-APTES-Ti and rGO-APTES-Ti samples “reproduced with permission from Ren et al. [55].”.

tability, hardness, and roughness. Fathyunes et al. [71] attempted to improve the properties of titanium by synthesizing GO sheets according to the modified Hummers’ method and converting a titanium sheet to amorphous TiO2 . To apply a GO-HAp composite coating, a GO suspension was added to an electrolyte. Then, HAp and GO-HAp coatings were electrodeposited onto the TiO2 . Furthermore, HAp nanorods were grown on GO to form HAp-GO by Ramadas et al. through a hydrothermal process [35]. Kumar et al. [75] decorated rGO with nanostructured ZrO2 and then functionalized the product for the fabrication of an APTES/ZrO2 rGO nanocomposite. The EPD technique was then used to deposit a thin film of APTES/ZrO2 -rGO onto pre-hydrolyzed ITO glass. This APTES/ZrO2 -rGO/ITO composite was then dried at room temperature and used as a transducer in the development of an immunosensor. Simple graphene and reduced graphene coatings on HAp plates were also explored by Perrotti et al. [59]. As discussed earlier, GO enhances the adherence and growth of MSCs and osteoblasts, and rGO-HAp nanocomposites enhance osteogenesis, although a smaller particle size of HAp was used. Thus, GO combined with alloplastic bone grafting is also thought to enhance bone regeneration. These GO-organic functionalities represent the state-of-the-art in biomedical applications. Field emission scanning electron microscopy (FE-SEM) evaluation revealed that the surface morphologies of BCP microparticles (BioC) and rGO-BCP bone graft material combine well. Fig. 8 shows that rGO-BCP microparticles have irregular granule-like shapes with diameters of several microns and are partially covered by an interrelated network of rGO nanoplatelets. Such an analysis can provide vital information for the use of organic GO functionalization in the biomedical field.

4.5. Others In an osteogenic differentiation study by Lee et al. [43], GO was synthesized by the modified Hummers’ method. Then, a water-soluble calcium phosphate HAp powder was used for synthesizing HAp microparticles. The HAp microparticles were suspended in deionized water and mixed with a colloidal dispersion of rGO-NPs. The mixed colloidal dispersion was vortexed vigorously, and the solvent was removed gradually overnight at room temperature, resulting in the formation of rGO-HAp composite. Furthermore, during their research into periodontal ligament stem cells, Rodríguez-Lozano et al. [50] synthesized GO-SF composite films. A dispersion of GO in water was mixed with an aqueous solution of SF, and the mixture was evaporated at room temperature from the wells of a tissue culture plate. Later, these composites were water annealed, and an opaque, light-brown film was produced. Nahorny et al. [64] prepared a novel coating for preventing dentin erosion. MWCNTs were prepared using chemical vapor deposition (CVD). MWCNT oxidation was then performed in a pulsed-direct current plasma reactor, and the product was named “MWCNTO-GO”. For the synthesis of nHAp-MWCNTO-GO, MWCNTO-GO powder was mixed in an aqueous solution of Ca (NO3 )2 ·4H2 O and (NH4 )H2 PO4 ·NH4 OH (0.1 M). Thereafter, the precipitations were subjected to ultrasound while maintaining a pH above 10. The resulting suspensions were left to age for 120 h and then filtered and extensively washed and dried. MWCNT-GO and ultrasound-assisted preparation of nHApMWCNT-GO nanocomposites have been used in biological applications by Rodrigues et al. [53]. Furthermore, Silva et al. developed honeycomblike scaffolds by combining poly (D, l-lactic

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Fig. 7. Coating of a GO multilayer onto a Ti substrate: Schematic representations of chemically modified GO−COO− and GO-NH3 + and an illustration of a GO-NH3 + /GO-COO− multilayer coating on a Ti substrate “reproduced with permission from La et al. [61].”.

Fig. 8. FE-SEM images of (A and B) BCP microparticles and (C and D) rGO-coated BCP bone graft materials. Arrows indicate rGO nanoplatelets “reproduced with the permission under an open access Creative Commons CC BY 4.0 license [46].”.

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M.Z.I. Nizami, S. Takashiba and Y. Nishina / Applied Materials Today 19 (2020) 100576

acid) (PDLLA) with a large amount of graphene/multiwalled carbon nanotube oxides (MWCNTO-GO, 50 %, w/w) [54]. Xie et al. [56] used 2D Gp and 3D Gp for periodontal ligament cell research. In their study, 2D Gp was obtained by CVD, and CVD was also used to synthesize 3D Gp using Ni foam as a template. After the CVD process, the Ni scaffold coated with graphene was placed in a FeCl3 solution for 72 h at room temperature and rinsed with deionized water for 72 h to remove the etching agents. Lalwani et al. [57] used 2D-nanostructure-reinforced biodegradable polymeric nanocomposites for bone tissue engineering. Single-walled and multiwalled graphene oxide nanoribbons were synthesized from SWCNTs and MWCNTs via a longitudinal unzipping method. Furthermore, Kumar et al. [58] used different GO composites in their bone regeneration research. They synthesized GO by Hummers’ method and PEI conjugated GO via a two-step process, including the grafting of polyacrylic acid (PAA) with GO through free-radical polymerization followed by conjugation of low-molecular-weight PEI (2 kDa) with the carboxylic acid groups of PAA. In research by Pinto et al. [36], after obtaining GO and Gp-NPl, PLA/GO, and PLA/Gp-NPl films were synthesized by doctor blade casting of solvent dispersions. Then, GO and Gp-NPl were dispersed in acetone and chloroform, respectively, using an ultrasonic bath. Later, a PLA/chloroform solution was added and sonicated. Finally, the obtained PLA/GO and PLA/Gp-NPl films were obtained by spreading the PLA/GO dispersion on a polytetrafluoroethylenecoated plate using a blade applicator and drying in a vacuum oven for use in a biocompatibility response study. 5. Conclusions GO and its composites are applied in almost all fields of biomedicine. However, dentistry appears to be a step behind in the use of these unique materials. Graphene is the thinnest, strongest, and stiffest imaginable material. Its large surface area, excellent mechanical properties, and compatibility with a range of different substrates make it a unique material for application in dental biomaterials, regenerative dentistry, tissue engineering, and stem cell research. The biocompatibility of GO and its nanocomposites show potential in bone regeneration and osseointegration, and this biocompatible GO can initiate and facilitate cell adhesion, proliferation, and differentiation of PDL, osteogenic, and oral epithelial cells. However, there are concerns about the biosafety of GO, and there are a lot of discussions underway. The effect of lateral dimension, thickness, and functionalization was clearly observed [85]. For example, few-layered graphene functionalized with polyethylene glycol was gradually exited from organs; in contrast, non-functionalized one remained even after 90 days. In addition, purity, post-production processing steps, oxidative state, dispersion state, synthesis methods, route and dose of administration, and exposure times also affects to the biosafety [86]. Further advance studies are required to develop safer graphene-based nanocomposites and technologies applicable for biomedicines to lessen the risks. Furthermore, the antibiofilm and anti-adhesion properties of GO have inspired researchers to research its application in the prevention of dental biofilm infections, dental caries, and dental erosion as well as for implant surface modification and as an anti-quorum sensing agent. GO has been shown to bond well with HAp, titanium, and potential biomaterials. Therefore, applications of GO in the dental field are expected to be further expanded to new disciplines in addition to the performance improvement of existing dental materials.

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