Computational Assessment of Biocompatibility and Toxicity of Graphene and Its Derivatives for Dental Adhesives

Abstract

Background/Objectives: Graphene and its derivatives have garnered attention for their unique properties that could enhance dental biomaterials. Understanding their interactions with biological systems is crucial for optimizing their application in dentistry. This study aimed to comprehensively evaluate the biocompatibility, molecular interactions, and toxicity profiles of graphene and its derivatives for potential dental applications using in silico approaches. Methods: The study employed molecular-docking simulations, 100 ns molecular dynamics (MD) simulations, pharmacophore modeling, and in silico toxicity assessments. Key bone-related proteins and receptors were selected to assess the potential of graphene-based materials in dental restorative and regenerative therapies. Results: Molecular-docking simulations revealed strong interactions of Graphene Quantum Dots (GQDs) and sulfur-doped graphene with critical bone-related receptors, suggesting their potential for reinforcing dentin and promoting bone regeneration. MD simulations demonstrated stable complex formations, with occasional fluctuations indicating areas for material optimization. In silico toxicity assessments indicated favorable profiles for high-purity graphene and selected doped graphenes (nitrogen-, fluorine-, and sulfur-doped), while graphene oxide (GO) exhibited concerning toxicity levels, highlighting the importance of mitigating strategies. Conclusions: Graphene and its derivatives exhibit promising biocompatibility and molecular interaction profiles relevant to dental applications. Challenges such as GO’s toxicity and occasional instability in simulations suggest the need for further research into surface modifications and material refinement. These findings pave the way for advancing graphene-based dental materials toward clinical implementation, potentially revolutionizing dental prosthetics and treatments.

1. Introduction

Graphene, composed of individual layers of carbon atoms in a two-dimensional honeycomb lattice, is becoming popular in various biomedical fields owing to its unique mechanical, thermal, and electrical characteristics [1,2]. Graphene, along with graphene oxide (GO) and reduced graphene oxide (rGO) derivatives, has been investigated in recent years for use in dental adhesives. These substances possess several advantages, including increased mechanical strength, thermal stability, and antibacterial properties, all of which are essential for maintaining and successfully restoring teeth [3,4]. Dental adhesives are important in restorative dentistry because they help to bond dental substrates and restorations. The performance of these adhesives directly affects the efficacy of dental procedures [5,6]. Similar to most adhesive technologies, marginal leakage, secondary caries, and short-term durability remain major issues. The application of graphene-based materials in dental adhesives can alleviate these issues by enhancing their physical and biological characteristics [7,8]. Graphene’s potential in the field of dental medicine has been explored due to its unique properties. Previous studies have shown that graphene oxide and reduced graphene oxide can improve adhesive bonding to dental substrates, reduce polymerization shrinkage, and provide superior antimicrobial activity against cariogenic bacteria like Streptococcus mutans and Lactobacillus acidophilus [9,10]. These properties stem from the unique nanoscale structure of graphene and its ability to interact effectively with biological molecules. Moreover, the oxygen-containing functional groups on GO and rGO enhance their hydrophilicity, which is beneficial for adhesion to water-based dental adhesives, promoting better interfacial bonding with dental tissues [3,11]. However, despite these advantages, there are limitations to the use of graphene-based materials in dental adhesives. One significant challenge is their potential cytotoxicity, especially when used at high concentrations, which can be problematic for oral tissues in the long term. The size of graphene sheets also poses challenges in terms of their dispersion in adhesive matrices, potentially leading to poor homogeneity and mechanical properties of the adhesive [12,13]. Additionally, the biocompatibility of these materials depends on the specific surface chemistry and the nature of the graphene modifications, which must be carefully controlled to minimize cytotoxic effects while maximizing adhesive performance.

In addition, the biocompatibility and toxicity of adhesives are issues because they directly interact with sensitive tissues of the mouth. Dental adhesives are placed near dental pulp, gingivae, and other mucosal surfaces of the mouth during restorative procedures [14,15]. Assuring that these materials do not cause toxic reactions or harm patients is critical for their safety and efficacy. Biocompatibility is a term used to describe the ability of a material to perform its function without damaging its biological systems. For dental adhesives, this means interacting with oral tissues so as to prevent irritation, allergic reaction, or tissue loss [16,17]. Toxicity testing is equally important because the adhesive component or its degradation products can have toxic consequences at cellular and systemic levels. Because dental adhesives remain in the mouth for a long period of time, any leaching of harmful compounds may negatively impact oral health or general well-being [18,19].

The primary hypothesis of this study was to computationally assess the biocompatibility and toxicity of graphene and its derivatives for use as dental adhesives. We employed molecular docking and dynamics simulations to investigate how these nanomaterials interact with biological molecules. Additionally, we utilized pharmacophore modeling to quantify the active functional groups of graphene-based dental adhesives, further enriching our understanding of their binding potential and cellular activity. In silico toxicity testing was performed to identify and quantify the safety profile of these materials for use in dental treatment. This research aims to establish a clearer understanding of how graphene and its derivatives can enhance the biocompatibility, stability, and safety of dental adhesives, thereby addressing critical challenges in restorative dentistry.

2. Materials and Methods

This study aimed to determine the biocompatibility and toxicity of graphene and its modified derivatives, which are important components of dental adhesive. The method involves finding suitable receptors that suit oral biocompatibility, designing fine 3D structures, and reducing the energy consumption of graphene and its products. Molecular docking and dynamic simulations were used to investigate the molecular interactions of these materials and their dynamic interactions with major biological receptors. Toxicity was also measured using computer algorithms to predict the potentially toxic ingredients or compounds. This strategy was designed to comprehensively describe the biocompatibility and toxicity of graphene and its derivatives in the oral environment.

2.1. Selection of Proteins That Are Biocompatible with Graphene and Its Derivatives

In our study, we aimed to evaluate the biocompatibility of graphene-based dental materials by selecting proteins that are crucial for bone metabolism, tissue morphology, and cellular interactions within the oral environment. The criteria for protein selection were carefully controlled to ensure their relevance to dental applications. We focused on receptors that play a significant role in bone health and cellular interactions due to their proximity to dental materials used in restorative dentistry. These receptors were chosen based on their documented interactions with dental substances, their involvement in bone tissue metabolism, and their influence on local inflammatory responses within the oral cavity. Proteins such as alkaline phosphatase (AP), bone morphogenetic protein-2 (BMP2), type I collagen (COL1A1), fibronectin, osteocalcin, osteonectin, osteopontin, osteoprotegerin, osterix, and RUNX2 were selected because they are known to play critical roles in bone matrix formation, cellular adhesion, and inflammation within the context of dental applications. The 3D structures of the selected receptors were sourced from the Protein Data Bank (PDB) [20] and AlphaFold [21] to ensure accuracy and reliability in subsequent computational analyses. To identify the active sites of these receptors, we used CASTp 3.0 [22] and PDBSum [23], which are advanced computational tools. This deliberate choice of receptors, driven by an intimate sense of their roles in bone metabolism, cellular interactions, and inflammatory activation, provides the basis for exhaustively testing the biocompatibility of graphene and its derivatives within the dense oral microenvironment.

2.2. Optimization of Molecular Structure and Energy of Graphene-Based Dental Adhesives

Three-dimensional molecular maps of graphene and its constituents were carefully drawn in three dimensions to determine their spatial distributions, which are essential for predicting their interactions with biological receptors. Such detailed modeling sought to describe the structural complexity driving these interactions. To further achieve accuracy, the 3D structures of the ligands were remodeled using Chem3D version 20.1.1, a molecular-modeling program created by PerkinElmer Inc., Waltham, MA, USA. This optimization also ensured that the ligand structures accurately reflected their complex three-dimensional shape [24]. Energy minimization was also conducted using the MM2 force field [25,26] in ChemDraw Professional 20.1.1 (PerkinElmer, Inc.). This dual-energy minimization improved the structural and energetic stability of the ligands, providing good starting points for further computational calculations. This rigorous approach fine-tuned the 3D structures of graphene and its alternatives and prepared them for molecular-docking simulations and detailed interactions. With these refinements, this study aimed to prepare precise ligands for a detailed study of their behavior in a complex biological landscape [27].

2.3. Molecular-Docking Simulations

For the molecular-docking simulations, we used the HADDOCK standalone [28] with its powerful interface. This tool allowed them to create detailed protein–ligand simulations that involved understanding the interactions between graphene and its derivatives with target receptors. The selection of the best possible docking solutions for each complex had two criteria: as many meaningful clusters as possible; and the highest HADDOCK score, indicating high binding affinity. The HADDOCK score accurately estimated the level of protein–ligand interactions in the complex. To further refine the calculation, PRODIGY [29] predicted the binding affinity G (free energy change) in kilocalories per mole (kcal/mol). This additional approach provides insights into the thermodynamics of protein–ligand interactions and is useful for understanding complex stability and energetics [30,31]. Molecular docking, which considers the extensibility of ligands and receptors, provides precise information regarding binding strengths, binding positions, and detailed molecular interactions at the atomic scale [32,33]. This is an important step in determining early biocompatibility, providing a comprehensive description of the interactions between graphene and its derivatives with biomolecular substrates. These simulation results serve as a starting point for future studies on molecular dynamics and biocompatibility.

2.4. Pharmacophore Modeling for Evaluating Active Functional Groups in Graphene and Its Derivatives

At this stage, pharmacophore modeling plays a crucial role in the detection of active functional groups in graphene and graphene derivatives in complex receptor networks. The aim of this study was first and foremost to identify the key chemical characteristics that enable ligands (graphene and its derivatives) to interact with their receptors. Using powerful algorithms from LigandScout 4.5 [34], pharmacophore modeling was critical for identifying and characterizing these vital chemical components. By identifying the primary pharmacophoric factors, this modeling method uncovered the molecular factors that determine biocompatibility. It offers specific information about the functional groups involved in the interactions between the dental elements and biological receptors under investigation. Such a refined study was not only about looking at ligand–receptor interactions, but also exploring the subtlety of molecular identification. By pinpointing the essential functional groups, the pharmacophore model helped unravel the molecular complexities that control graphene biocompatibility.

2.5. Molecular Dynamics (MD) Simulations

Based on information from molecular docking, the process advances to molecular dynamics simulations. This computational approach allows for a more detailed description of how ligand–receptor complexes act dynamically over time. These simulations investigate structural stability, malleability, and conformational evolution, and they can provide insight into how graphene and its derivatives work in a dynamic oral environment. Using GROMACS 2023.3, MD simulations were performed for selected ligand–protein complexes [35]. The general AMBER force field (GAFF2) [36] was used to charge the ligands, and partial charges were computed using the Austin Model 1 semi-empirical molecular orbital method in combination with Bond Charge Correlations (AM1-BCCs) [37]. The simulation box was set as a dodecahedron at a distance of 2.0 nm from the protein’s largest principal radius to the edge of the box using periodic boundary conditions. Protein simulations were performed using the AMBER99sb force field [38], with a simulation box consisting of SPC water and counterions. Short-range nonbonded interactions were shorted to 2.0 nm, and long-range electrostatics were calculated using the Particle Mesh Ewald (PME) algorithm [39]. The simulation method had several steps: minimizing the steepest descent until the maximum force of the system was lower than 1000 kJ/mol/nm and 1 ns restrained NVT and NPT simulations. Initially, a Berendsen thermostat at 310 K and a Berendsen barostat at 1 bar were used. The simulation ended with an unbound 100 ns simulation using the Berendsen thermostat and Parrinello–Rahman barostat [40,41]. This long simulation time allowed for a detailed understanding of ligand–receptor interactions [42,43], which provided dynamic descriptions of the behavior of graphene and its derivatives within a simulated oral system over a longer period. This experiment provides the most detailed analysis of the behavior of graphene and its derivatives over the long-term using various environments and simulation steps.

2.6. Computational Assessment of Graphene and Its Derivatives for Toxicity

A large component of this process involves the application of sophisticated computational methods to test the safety profile of graphene and its derivatives. These tools have become integral to toxicity analysis because they use sophisticated algorithms to determine molecular descriptors that encapsulate chemical effects. This makes it possible to take an in-depth look at the crystalline structure of graphene and its variants to anticipate what may be toxic components or compounds within their molecular structures. OSIRIS DataWarrior V6.1.0 [44], a database and predictive platform with considerable resources, helped us to better understand the toxicological effects of graphene and its derivatives. It analyzed various molecular characteristics to determine which structural elements were likely to be dangerous, helping us better understand the safety of these dental materials. The QikProp module in the Schrödinger Software Suites also played a role in the toxicity study, utilizing powerful algorithms to examine molecular compositions [45]. To forecast the toxicity of potentially toxic elements or compounds, QikProp provides information that can help assess the safety of graphene and its derivatives. The toxicity test added a crucial dimension to this study and provided vital information on the risks associated with using graphene and its derivatives in dental work.

3. Results

3.1. Selection of Proteins Relevant to the Biocompatibility of Graphene and Its Derivatives

A careful selection was made for proteins that are crucial for biocompatibility analysis of graphene and its analogs.

Table 1. Results from RCSB PDB for target protein preparation and active site determination via CASTp 3.0 and PDBSum.

Name	PDB/UniProt ID	Resolution (Å)	Chain	Weight (kDa)	Sequence Length	Active Site (Residue Number)
AP	2GLQ [46]	1.60	A	53.57	484	15, 18, 19, 22, 68, 72, 73
BMP2	4UI1 [47]	2.35	A	51.34	114	293, 295, 296, 328, 329, 330, 331, 332, 333, 334, 335, 336, 338, 339, 344, 347, 348, 350, 351, 358, 393, 394, 395
COL1A1	5CTD [48]	1.60	C	21.06	72	50, 53, 54, 57, 70, 71
Fibronectin	1FNH [49]	2.80	A	29.63	271	101, 102, 131, 156, 158, 159, 160, 177, 178, 179, 180, 257, 259, 260
Osteocalcin	1Q8H [50]	2.00	A	5.85	49	16, 19, 38, 39, 42, 43
Osteonectin	1BMO [51]	3.10	A	55.23	233	65, 67, 77, 78, 79, 80, 81, 82, 83, 103, 107, 110, 111, 114, 115, 120
Osteopontin	1MOY [52]	1.55	A	13.80	130	64, 65, 67, 68, 69
Osteoprotegerin	3URF [53]	2.70	B	38.38	171	9, 10, 22, 29, 32, 48, 49, 50, 51, 52, 53, 54, 57, 58, 60, 64, 67, 70, 81, 82, 83, 85, 92, 116
Osterix	6X46 [54]	N/A	A	14.35	121	17, 23, 33, 37, 51, 57, 67, 71, 76
RUNX2	6VGD [55]	4.20	D	59.70	177	117, 164, 165, 200, 210, 212

shows the results of the selection process. These selectively selected receptors are involved in bone metabolism, tissue morphology, and other cellular processes critical for dental and bone health. These were chosen for their relevance in the study of how graphene-based dental materials are related to key biomolecular targets. This choice ensured an all-inclusive approach for assessing the possible effects of these materials on biological systems, with a particular focus on dental applications and bone health.

The mechanism by which graphene and its derivatives react with bone-related receptors is key for dental applications, especially in restorative and regenerative dentistry. Alkaline phosphatase (AP) is a crucial component of tooth mineralization that facilitates the deposition of calcium and phosphate ions to build enamel and dentin [56]. Graphene can affect AP in dental tissues and alter mineralization mechanisms that are critical to tooth strength and integrity. BMP2 plays a critical role in bone repair and regeneration, including osseointegration of dental implants. Dental restorations made from graphene-derived materials can also engage in BMP2, modulating the osteogenic signaling pathways involved in bone healing around implants [57,58]. These interactions can be analyzed to design dental biomaterials that support implants and allow their rapid integration into adjacent bone tissues. Collagen type I alpha 1 (COL1A1) plays an important structural role in dentin and is responsible for its stability and durability [59]. The interaction of graphene with COL1A1 may alter the formation and mineralization of the dentin matrix, affecting the mechanical behavior of dental restorations. Fibronectin, which is involved in cell recruitment and synchronization, regulates periodontal tissue integrity and wound healing [60]. The interactions between graphene-derived proteins and fibronectin may drive periodontal regeneration, which may facilitate periodontal treatment and tissue engineering in dentistry. Osteocalcin, osteonectin, osteopontin, and osteoprotegerin are involved in various processes of bone metabolism and tissue remodeling that are vital for dental and periodontal functions [61,62]. The chemical bonds between graphene and these proteins may affect the function of periodontal ligaments, root surface resurfacing, and general periodontal tissue homeostasis. Furthermore, transcription factors such as osterix and RUNX2 govern odontoblast differentiation and tooth development [63,64]. Understanding the effects of graphene derivatives on these factors would result in dental products with increased dentinogenesis and pulp tissue regeneration.

3.2. Refinement of the Molecular Structures and Energy Optimization of Graphene-Based Dental Adhesives

Graphene dental adhesives were chosen for this experiment because different graphene modifications can have different effects on dental adhesive properties (

Table 2. Chemical structures and molecular weights of graphene and its modifications.

Component	Chemical Structure	Molecular Weight (g/mol)
Graphene-based dental adhesive
High-purity graphene		838.9
Graphene oxide (GO)		1042.9
Reduced graphene oxide (rGO)		946.9
Nitrogen-doped graphene		839.9
Fluorine-doped graphene		817.5
Sulfur-doped graphene		966.3
Graphene Quantum Dot		886.8
NH2-functionalized graphene		929.0
COH-functionalized graphene		934.9
CCOOH-functionalized graphene		1103.0

). Graphene has excellent mechanical, thermal, and electrical conductivities and has already been proven useful for dental composites [53,54]. Chemical doping and functionalization of graphene are believed to optimize its suitability and performance for dental applications. The following explains why particular graphene-based constituents were chosen, as well as their chemical compositions and molecular weights.

Table 3. Comparison of the energetic parameters of high-purity graphene and its modifications following MM2 energy minimization.

Compound	Stretch	Bend	Stretch–Bend	Torsion	Non-1,4 VDW	1,4 VDW	Total Energy (kcal/mol)
High-purity graphene	5.098	3.173	0.123	−165.458	−11.667	110.020	−58.710
Graphene oxide (GO)	566.126	420.514	−20.856	323.780	602.940	317.374	2209.879
Reduced graphene oxide (rGO)	631.345	775.620	−39.638	433.057	509.372	412.552	2722.309
Nitrogen-doped graphene	9.341	21.029	0.236	−74.801	−2.903	106.955	66.953
Fluorine-doped graphene	324.172	983.567	−5.558	280.435	419.107	132.248	2133.973
Sulfur-doped graphene	86.922	1503.161	−37.279	103.188	−6.753	135.782	1787.181
Graphene Quantum Dot	159.045	272.033	−0.403	70.054	82.269	188.230	771.229
NH2-functionalized graphene	8.879	22.195	0.197	−129.026	−2.733	109.129	8.704
COH-functionalized graphene	7.851	14.181	0.054	−154.853	−11.379	103.407	−40.558
CCOOH-functionalized graphene	15.578	33.394	0.579	−51.459	15.810	116.980	164.901

presents a detailed comparison of the energetic parameters of high-purity graphene and its diverse post-MM2 energy-minimization modifications. This painstaking structural and energy minimization step plays an essential role in explaining how these materials are stable and kinetically governed, providing a critical edge in understanding their potential for dental adhesion. Pure graphene has become a good and reliable candidate with relatively low energy on various scales. This stability is also supported by an earlier study that revealed the stability of the molecular geometry of graphene through its special two-dimensional lattice configuration [65]. Significantly, high-purity graphene had positive values for stretch, bend, and 1.4 VDW interactions—all indicators of a clear and energetically preferable molecular structure.

In contrast, GO and rGO had much higher energies than high-purity graphene for all the parameters. This conclusion corroborated the findings of Park et al., who emphasized the greater flexibility and molecular snarl-up brought on by the oxygen-rich functional groups in GO and the reduction in rGO [66]. Nitrogen-doped graphene showed intermediate energy values, indicating that nitrogen doping conferred structural rigidity without disrupting overall stability. This result is consistent with another study that stressed the importance of nitrogen doping for improving the mechanical strength and stability of graphene materials [67]. Fluorine-doped graphene has high energies, especially at stretch and bend values, suggesting greater molecular flexibility and deformation. Sulfur-doped graphene has a slightly more complex character, and different energy values have been reported for different parameters. This is in line with the work of Yang et al., who described the different ways in which sulfur doping alters the structural and electronic properties of graphene and how it can affect the mechanical properties of materials [68]. The quantum dots in graphene were found to have relatively high energies, suggesting a molecular weight distribution that was at least somewhat malleable and stable. This was confirmed in a study that explored the distinctive characteristics and uses of Graphene Quantum Dots for various applications, such as biomedicine and energy storage [69]. NH2-functionalized graphene had relatively low energies compared to pure graphene, suggesting that amine functionalization preserved the lattice stability of graphene but added specific intercalations. COH- and CCOOH-functionalized graphene have very different energy profiles, with COH retaining a lower total energy and desirable stretch and bend parameters. Meanwhile, CCOOH exhibited higher energies across most parameters, suggesting its structural instability. These energy minimization findings, therefore, provide a key clue to the structural stability, malleability, and modification of graphene, and they provide a way for future research on their relationship to dental adhesive monomers and their effect on composites.

3.3. Molecular-Docking Simulations

The results of molecular-docking simulations highlight the interactions between graphene and its derivatives with key bone-related receptors, providing crucial insights into their biocompatibility for dental applications, particularly in interim prosthetic restorations. The HADDOCK score, binding affinity (ΔG), energy contribution, and interaction details are summarized in

Table 4. Results of molecular-docking simulations showing the top complexes of graphene and its derivatives with each target receptor.

Complex	HADDOCK Score	Binding Affinity ΔG (kcal/mol)	ΔG Score	Cluster Size	RMSD	Van der Waals Energy	Electrostatic Energy	Desolvation Energy	Restraints Violation Energy	Buried Surface Area	Z-Score
AP/Graphene Quantum Dot	−38.7 +/− 2.0	−9.65	86.41	4	1.0 +/− 0.0	−28.6 +/− 1.7	−61.0 +/− 8.3	−4.2 +/− 0.5	1.7 +/− 0.2	718.2 +/− 13.4	−1.8
BMP2/sulfur-doped graphene	−38.0 +/− 2.9	−11.05	33.78	4	0.1 +/− 0.1	−31.9 +/− 1.3	−111.8 +/− 7.0	−7.2 +/− 0.6	122.5 +/− 20.1	560.5 +/− 11.1	−1.7
COL1A1/Graphene Quantum Dot	−39.2 +/− 0.4	−8.27	124.33	45	1.4 +/− 0.1	−34.7 +/− 0.4	−27.8 +/− 2.0	−1.9 +/− 0.2	1.4 +/− 0.3	758.0 +/− 15.4	−1.4
Fibronectin/Graphene Quantum Dot	−31.0 +/− 2.4	−9.09	110.62	5	1.3 +/− 0.0	−31.3 +/− 2.2	−44.8 +/− 9.3	2.4 +/− 0.2	24.0 +/− 11.6	761.6 +/− 18.4	−1.5
Osteocalcin/sulfur-doped graphene	−28.8 +/− 2.1	−9.82	60.94	5	0.9 +/− 0.0	−17.9 +/− 1.2	−141.6 +/− 11.5	−1.5 +/− 0.1	47.5 +/− 1.1	419.6 +/− 13.4	−1.9
Osteonectin/Graphene Quantum Dot	−32.3 +/− 1.0	−9.88	81.82	8	0.1 +/− 0.1	−23.9 +/− 0.7	−100.8 +/− 7.4	−3.4 +/− 0.4	50.5 +/− 6.1	737.4 +/− 22.6	−1.9
Osteopontin/Graphene Quantum Dot	−36.1 +/− 2.2	−9.25	91.64	5	0.2 +/− 0.1	−25.6 +/− 1.6	−103.7 +/− 5.4	−0.2 +/− 0.2	0.4 +/− 0.3	640.1 +/− 9.6	−1.4
Osteoprotegerin/sulfur-doped graphene	−11.6 +/− 1.0	−9.65	68.85	8	0.4 +/− 0.1	−13.6 +/− 2.4	−89.9 +/− 0.5	−3.8 +/− 0.3	148.5 +/− 16.4	430.4 +/− 5.8	−1.7
Osterix/Graphene Quantum Dot	−43.6 +/− 0.8	−9.38	87.95	7	0.6 +/− 0.1	−29.7 +/− 0.9	−91.5 +/− 8.7	−4.8 +/− 0.3	0.0 +/− 0.0	650.6 +/− 16.8	−1.6
RUNX2/Graphene Quantum Dot	−42.0 +/− 0.1	−8.24	120.10	20	0.1 +/− 0.1	−29.6 +/− 0.2	−41.5 +/− 3.1	−8.2 +/− 0.1	0.1 +/− 0.0	566.0 +/− 3.7	−1.3

. These metrics highlight the strength and nature of the molecular interactions between graphene derivatives and selected receptors, thereby guiding their potential use in dental biomaterial development.

Among the derivatives of graphene explored in this study, Graphene Quantum Dots and sulfur-doped graphene have emerged as promising candidates for interaction with key receptors relevant to dental and bone health. These derivatives consistently exhibited strong binding affinities and favorable interaction energies with critical biomolecular targets in the molecular-docking simulations. Graphene Quantum Dots demonstrated robust interactions with several receptors that are essential in dental applications. For instance, they exhibited high HADDOCK scores and low binding-affinity energies (ΔG) when interacting with COL1A1 (HADDOCK score −39.2 +/− 0.4, ΔG = −8.27 kcal/mol), osteonectin (HADDOCK score −32.3 +/− 1.0, ΔG = −9.88 kcal/mol), osterix (HADDOCK score −43.6 +/− 0.8, ΔG = −9.38 kcal/mol), and RUNX2 (HADDOCK score −42.0 +/− 0.1, ΔG = −8.24 kcal/mol). These findings suggest the potential applications of Graphene Quantum Dots in reinforcing dentin structure, promoting bone remodeling, and facilitating tooth development, highlighting their versatility in dental biomaterials. Similarly, sulfur-doped graphene exhibited notable binding affinities with BMP2 (HADDOCK score −38.0 +/− 2.9, ΔG = −11.05 kcal/mol), osteocalcin (HADDOCK score −28.8 +/− 2.1, ΔG = −9.82 kcal/mol), and osteoprotegerin (HADDOCK score −11.6 +/− 1.0, ΔG = −9.65 kcal/mol). These interactions underscore the potential to enhance osteogenic processes that are critical for dental implant integration and bone mineralization. The strong HADDOCK scores indicate stable interactions between sulfur-doped graphene and these receptors, suggesting its efficacy in supporting bone health and dental material integration. Overall, the molecular-docking simulations highlight Graphene Quantum Dots and sulfur-doped graphene as promising candidates for the further exploration of dental biomaterials. Their strong binding affinities and favorable interaction energies with key receptors provide a foundation for future experimental studies to validate their potential applications in enhancing dental prosthetics and promoting oral health. The results of the complete docking simulation are presented in Supplementary Data S1.

Figure 1 shows the main molecular interactions between Graphene Quantum Dot (GQD) and osteonectin, and between sulfur-doped graphene (SG) and BMP2. These two complexes were shown to be the most stable owing to their free binding energy values, which are crucial for understanding the biocompatibility and effectiveness of graphene-based materials in dentistry. The O/GQD-GQD complex was primarily stabilized by van der Waals interactions, which are important for the binding of the ligand to the receptor [70]. Two standard hydrogen bonds were also found between the residues Thr101 and His107. Hydrogen bonds effectively fix protein–ligand complexes through directional and specific interactions [71]. Four other carbon–hydrogen bonds with other residues were observed, further stabilizing the complex. These hydrogen bonds imply that GQD and osteonectin share a strong, highly targeted interaction that sustains the structure and function of the protein. In addition, the complex exhibited salt-bridge interactions with Lys100 and Lys120. Salt bridges and electrostatic bridges between charged residues at opposite positions dramatically improve the stability of protein–ligand complexes [72,73]. In dentistry, these strong recombinations suggest that GQDs can effectively interact with osteonectin and promote mineralization and repair.

In contrast, BMP2-SG interaction entails a different series of molecular interactions. The π-cation and attractive charge interactions (especially with the sulfur groups, as found on residues Asp335 and His336) attested to the SG’s distinct binding nature. Pi cations stabilize the protein–ligand complex through electrostatic interactions between the aromatic ring of the ligand and positively charged amino acid residues of the protein. The van der Waals and alkyl bonds found in the SG-BMP2 complex further increased the stability and specificity of the interaction. The precise molecular interactions reflected in Figure 1, such as the quantity and type of bonds present, revealed a comprehensive picture of SG’s atomic-scale interaction with BMP2. This interaction profile indicates that SG could effectively alter the activity of BMP2, a protein important for bone formation and repair. Such interactions were necessary for the biocompatibility and use of graphene in dental materials, particularly for bone regeneration and dental implant insertion. Figure 1 helped us to grasp the molecular basis of the graphene derivatives’ interaction with their receptors. Realistic simulations of these interactions provided an ideal basis for further research using molecular dynamics simulations. All the graphene complexes and their derivatives are listed in Supplementary Data S2.

3.4. Pharmacophore Modeling for Evaluating Active Functional Groups in Graphene and Its Derivatives

The results of pharmacophore modeling, as detailed in

Table 5. Construction of pharmacophore models based on optimal docking conformations using 2D and 3D structural data. Hydrophobic interactions are indicated with yellow spheres, and hydrogen bond acceptors are indicated with red arrows.

Compound	2D Pharmacophore	3D Pharmacophore
High-purity graphene
Graphene oxide (GO)
Reduced graphene oxide (rGO)
Nitrogen-doped graphene
Fluorine-doped graphene
Sulfur-doped graphene
Graphene Quantum Dot
NH2-functionalized graphene
COH-functionalized graphene
CCOOH-functionalized graphene

, offer a comprehensive retrospective analysis of the active functional groups participating in the interactions between graphene and its derivatives with the chosen receptors. Two- and three-dimensional structure-based pharmacophore models provide valuable insights into the molecular interactions at receptor-binding sites. These models identify critical hydrophobic interactions, hydrogen bond donors, and hydrogen bond acceptors, highlighting the essential features that drive the binding affinity and stability of the complexes. These pharmacophore models not only depict the spatial arrangement of functional groups but also emphasize their roles in enhancing the interaction strength with specific receptor residues [24].

Because benzene rings form the fundamental structural building blocks of graphene and its derivatives, pharmacophore simulations demonstrate hydrophobic interactions with target receptors. This is important because hydrophobic interactions are essential to prevent graphene-based compounds from locking onto biomolecular targets. Graphene oxides, such as GO, COH-functionalized graphene, and CCOOH-functionalized graphene, have hydrogen bond acceptors owing to the presence of hydroxyl groups. These hydroxyls are critical because they form hydrogen bonds, which serve as acceptors for the active residues of the receptors, and their hydrogen bonding ability boosts the stability and specificity of the interactions of the graphene derivatives with their receptors. These findings provide deeper insight into the functional groups of graphene and graphene derivatives that activate specific receptors. Understanding how the hydroxyl groups and benzene rings contribute to hydrogen bonding and hydrophobic interactions makes it possible to identify the molecular factors that shape the biocompatibility of dental surfaces. These pharmacophore models have clarified the exact molecular characteristics governing the interfaces between graphene derivatives and specific biomolecular partners. This information is essential for the design and optimization of dental materials to improve their biocompatibility and clinical function. By knowing the functional groups and how they play a role in molecular interactions, researchers can modify the properties of graphene dental materials to enhance their suitability and performance for various dental applications. In addition, the pharmacophore models provided opportunities for future work because they highlighted the role of certain molecular aspects in the biocompatibility of graphene derivatives. Such knowledge may provide a roadmap for the further development of new derivatives with better properties and help develop dental materials that are safe and effective for clinical application. By incorporating these complex molecular details into the design, it was ensured that dental materials would interact as optimally as possible with living systems, thus improving their overall performance and dental acceptability.

3.5. Molecular Dynamics (MD) Simulations

Molecular dynamics (MD) simulations provide a deeper understanding of the stability and interactions of graphene and its derivatives with selected receptors. Figure 2 visually represents the root-mean-square deviation (RMSD) values of the simulated complexes over time, offering insights into their dynamic behavior. Most of the generated complexes exhibited RMSD values within the 1.5–2.5 Å range. This range indicates a relatively stable interaction between the graphene derivatives and receptors, suggesting that the complexes maintained their structural integrity throughout the simulation. Such stability is crucial for potential biomedical applications, including dental materials, for which consistent performance and minimal structural changes are desired.

However, the simulations also revealed occasional spikes in RMSD values, reaching 3–4 Å. These spikes showed brief shifts in the intricate structures for a variety of reasons. A possible explanation for these peaks is the intrinsic malleability of the receptor proteins, which may have changed their conformation during their interactions with graphene derivatives. These conformations may temporarily disrupt the stable interactions, resulting in higher RMSD values. A second reason for these spikes might be the dynamic nature of graphene derivatives. For example, functional groups on the surface of doped graphene may vary in their interactions with different receptor sites and temporarily distort it. This pattern was particularly apparent in the GO complexes, where reactive oxygen species might have enhanced their interactions with protein residues, leading to RMSD spikes. The MD simulations also demonstrated the significance of certain interactions in maintaining the stability of the complexes. Graphene Quantum Dot (GQD) complexes with receptors such as osteonectin and RUNX2, for instance, had fewer and smaller RMSD spikes, suggesting stable binding interactions. This stability may have been a result of the strong van der Waals forces and hydrogen bonds that had been discovered during the docking experiments, which were confirmed by dynamic simulations. In contrast, sulfur-coated graphene complexes with receptors such as BMP2 showed higher RMSD changes. Such behavior could be related to specific binding interactions (pi-cation and attractive charge interactions, for example), which might have been easier to break when moving dynamically. These small interactions allowed us to compare the molecular docking and MD simulation outcomes in a way that gave us a complete picture of the biocompatibility and stability of these materials. In general, MD simulations have confirmed the potential of graphene derivatives for dental applications by verifying their stable interactions with important receptors. The RMSD spikes occasionally resulted in opportunities to optimize these points, including the functional groups on graphene, to improve stability and minimize variation. These are important insights for the development of graphene-based dental materials that are biocompatible but dynamically stable, thereby ensuring both long-term clinical performance and safety.

3.6. Computational Assessment of Graphene and Its Derivatives for Toxicity

Computer simulations of the toxicity of graphene and its analogs yielded valuable information about their biological effects (

Table 6. In silico toxicity assessment of graphene and its derivatives.

Compound	Mutagenic	Tumorigenic	Reproductive Effective	Irritant	Total Surface Area	Solvent Accessible Surface Area (SASA)	Hydrophobic Component of SASA (FOSA)	Hydrophilic Component of SASA (FISA)
High-purity graphene	None	None	None	None	284.64	372.83	372.83	0
Graphene oxide (GO)	High	High	None	High	306.37	427.47	301.06	126.41
Reduced graphene oxide (rGO)	None	None	None	None	267.87	385.25	339.79	45.46
Nitrogen-doped graphene	None	None	None	None	277.94	396.83	389.20	7.62
Fluorine-doped graphene	None	None	None	None	298.68	411.12	382.17	8.11
Sulfur-doped graphene	None	None	None	None	276.87	439.51	394.42	0
Graphene Quantum Dot	None	None	None	None	445.87	618.91	290.09	328.82
NH2-functionalized graphene	None	None	None	None	299.16	372.40	288.45	83.95
COH-functionalized graphene	None	None	None	None	294.82	365.13	287.01	78.11
CCOOH-functionalized Graphene	None	None	None	None	332.52	442.53	290.86	151.66

). In silico methods were used to model different toxicity parameters, such as mutagenicity, tumorigenicity, reproductive effects, and irritancy. These results reflect the significant differences in the toxicity of different graphene derivatives, which are important for dental applications. Pure graphene has a neutral toxic profile with no predicted mutagenic, tumorigenic, reproductive, or irritant effects. The combined total surface area and solvent-accessible surface area (SASA) were comparatively mild, indicating a moderate interaction profile with biological systems. This low-toxicity profile emphasized the attractiveness of high-purity graphene as a safe material for use in dental treatment where little or no biological interference was needed. GO had a poor toxicity profile, with high mutagenic, tumorigenic, and irritant effects. Such negative impacts probably arise from oxidative functional groups on the surface, which can react negatively with living molecules. The greater total surface area and large hydrophilic component (FISA) indicate that GO has a higher affinity for biological fluids, potentially making it more toxic. These findings suggest that, although GO could be useful, its biocompatibility needs to be carefully monitored and perhaps lowered by surface alteration or moderation.

In contrast to GO, reduced graphene oxide (rGO) showed no mutagenic, tumorigenic, reproductive, or irritant effects, aligning it more closely with high-purity graphene. The reduction process likely eliminated or transformed the reactive oxygen species, thereby reducing overall toxicity. The surface area and hydrophobic components were balanced, making rGO a promising candidate for biocompatible dental materials, offering the mechanical benefits of graphene with reduced toxicity risks. Other derivatives, such as nitrogen-doped graphene, fluorine-doped graphene, and sulfur-doped graphene, also displayed no adverse toxicity predictions. Their modifications led to slight variations in the surface area and hydrophilic/hydrophobic balance, which can be tailored for specific applications. For instance, the increased solvent-accessible surface area (SASA) of sulfur-doped graphene suggests an enhanced interaction potential, which is beneficial for applications requiring strong adhesion to biological tissues. Graphene Quantum Dots (GQDs) had the highest total surface area and SASA, indicating a high degree of interaction with biological environments. Despite this, the GQDs showed no toxicity across the evaluated parameters, making them particularly suitable for applications that require extensive biological interfacing, such as drug delivery systems or imaging agents in dental diagnostics. Functionalized graphene, including NH₂-functionalized graphene, COH-functionalized graphene, and CCOOH-functionalized graphene, exhibited favorable toxicity profiles with no predicted adverse effects. These functional groups are likely to enhance the biocompatibility by promoting benign interactions with biological molecules. Their varied hydrophilic components (FISA) suggest diverse interaction potentials, allowing for tailored applications in dental materials, where specific binding or interaction profiles are required.

4. Discussion

This was a comprehensive evaluation of the biocompatibility, stability, and toxicity of graphene and its derivatives for dental use. The selection of bone proteins (AP, BMP2, COL1A1, and others important for bone metabolism and tissue formation) made graphene-based dental surfaces a very useful platform for studying the interfaces between these proteins and the rest of the body. Molecular-docking simulations have shown that GQDs and sulfur-doped graphene interact with critical receptors and can be used to reinforce dentin structures and aid in bone remodeling. This is in line with other studies that have documented the potential of graphene to improve bone growth and regeneration. For instance, Guo et al. (2023) showed that graphene oxide can induce osteogenic differentiation of stem cells, making it a candidate for bone tissue engineering [64]. Additionally, Shadjou et al. (2018) reported that graphene-based scaffolds significantly improved bone repair in animal models [65]. MD simulations confirmed that these interactions remained stable and that most of the complexes retained their structural integrity for clinical use.

Toxicity analysis revealed positive profiles for high-purity graphene and some doped graphene (nitrogen-, fluorine-, and sulfur-doped) with no expected toxicity, which is in accordance with research demonstrating the biocompatibility of these materials for biomedical applications. For instance, Huang et al. (2023) showed that N-doped graphene is highly biocompatible and can promote better cell-to-cell interactions, demonstrating its biomedical use [66]. However, GO is highly toxic, which is worth careful testing and potentially changing to reduce its adverse effects. These results also match those of studies that have focused on the functional groups that mediate the biocompatibility of graphene derivatives. Liao et al. (2018) pointed out that GO’s cytotoxic activity might be quenched via surface functionalization, suggesting an avenue for improved biomedical safety [67]. Functionalized graphene also had good toxicity profiles, suggesting that functional groups might enhance contact with biological molecules for use in dental applications. These findings, together with the results of earlier studies, illustrate that graphene materials can revolutionize dental prosthetics and dental health, and that further experimental validation and clinical trials are needed to develop them for dental applications.

These results for graphene and its derivatives have major clinical implications in dentistry, leading to future dental prosthetics and treatment. The biocompatibility and stability of some graphene materials (especially high-purity graphene and doped variants (nitrogen-, fluorine-, and sulfur-doped)) show that they are suitable for dental applications. These could be used to add mechanical strength to dental composites, enhance the adhesive strength of restorative dentistry, and facilitate a faster healing period around dental implants. For example, the relationship between GQDs and receptors such as the bone receptors osteonectin and BMP2 suggests that they can reinforce dentin and activate bone repair, which is key to the effectiveness and longevity of dental procedures. However, this research also pointed out the flaws and hurdles that need to be addressed. Even so, GO was toxic in some respects and needed to be thoughtfully considered and potentially altered to reduce adverse effects prior to translation into clinical use. These rare RMSD spikes in molecular dynamics simulations are yet another indication that the design of materials is constantly evolving to be stable and robust over the long term in biological environments. In addition, while functionalized graphene has promising biocompatibility data, we need to learn more about the effects of different functional groups on tissue responses over time and incorporation into dental tissues. These issues will have to be addressed in future research, as well as new graphene-based dental applications. These include surface modifications to optimize biocompatibility; preclinical studies to ensure safety and efficacy; and novel applications, including drug delivery systems and tissue engineering scaffolds.

Material scientists, biologists, and clinicians will need to collaborate to bring these innovations out of the laboratory and into the clinic and ensure that graphene dental products are safe, effective, and durable for use in patients. Additionally, a related study by Spinola et al. (2021) demonstrated that the incorporation of multi-walled carbon nanotubes (MWCNTs) into glass ionomer cements can influence the mechanical properties of these materials. For instance, their research showed that the presence of MWCNTs decreased the mean compressive strength values while significantly increasing diametral tensile strength [74]. This finding aligns with our observations for graphene and its derivatives, where surface modifications and inclusion of certain additives enhance specific material properties without adversely affecting overall biocompatibility and stability. Further research should explore surface functionalization with biocompatible and bioactive molecules, optimizing functional groups such as carboxyl and amine groups to enhance stability and reduce toxicity. Additionally, material perfection strategies such as reducing defect densities during synthesis and employing advanced methods like chemical vapor deposition (CVD) should be pursued to improve structural integrity. The development of hybrid materials, combining GO with biopolymers or nanohydroxyapatite, should also be investigated to balance mechanical stability and biocompatibility. Furthermore, in vitro cytotoxicity and in vivo biocompatibility tests will be essential to validate computational predictions and ensure practical applicability. These approaches aim to translate findings into safer, more effective graphene-based dental adhesives, thereby addressing critical challenges in this field.

While the computational and experimental findings offer exciting prospects, the translational aspects of moving graphene-based dental materials from in silico models to clinical applications involve significant challenges. A critical step will be scaling the synthesis and functionalization methods to ensure consistency in material properties without compromising quality. For example, while CVD and hybrid material synthesis have been shown to improve structural and biocompatibility characteristics, their scalability and cost-effectiveness in industrial settings remain unresolved issues. Another major hurdle is ensuring regulatory compliance. Graphene-based materials must meet stringent biocompatibility standards set by regulatory agencies, such as the FDA or EMA, before they can be approved for dental applications. Comprehensive toxicity testing, extending beyond cellular-level assays to long-term animal studies, is required to confirm their safety and potential for human use. Additionally, validation steps must include robust in vitro and in vivo studies that replicate the complex environment of the oral cavity. This includes exposure to saliva, bacterial biofilms, and mechanical stresses that could degrade material properties over time. Computational models, although insightful, cannot fully account for the dynamic biological and chemical interactions in these environments. In vivo testing in animal models and subsequent clinical trials will be necessary to bridge this gap. Material longevity and patient outcomes are further aspects requiring attention. While functionalized graphene and hybrid materials demonstrate promising adhesive strength and biocompatibility, studies evaluating their long-term performance under real-world conditions are scarce. Issues such as material fatigue, resistance to enzymatic degradation, and interactions with adjacent tissues must be thoroughly addressed.

5. Conclusions

Overall, the testing of graphene and its analogs in dentistry demonstrates their potential as versatile materials that can be used in many aspects of dental care. Researchers have found in silico biocompatibility and stability of pure graphene and doped variations (nitrogen-, fluorine-, and sulfur-doped), which can be incorporated into dental materials to increase their mechanical strength and adhesiveness. Such materials, particularly Graphene Quantum Dots (GQDs), demonstrate positive interactions with key bone receptors, such as osteonectin and BMP2, suggesting that they can be used to support the reinforcement of dentin and regeneration of bone, both of which are important for dental treatment success. Moreover, further investigation of how functionalized graphene affects tissue responses and incorporation into teeth is required. Future research should focus on taming surface modifications; rigorous preclinical experiments; and novel applications, such as drug delivery and tissue engineering. Cross-disciplinary collaboration is required to move these innovations from bench to bedside, with graphene dental composites meeting the strict safety, efficacy, and durability criteria for clinical use. Thus, graphene can become a revolutionary force in dental prosthetics and treatment, leading to better oral health for patients worldwide.