Evaluation of Dental Materials and Oral Disease-Related Proteins in Dentistry: Efficacy of Electrophoresis as a Valuable Tool

Abstract

Electrophoresis is a versatile technique that allows for the separation of molecules based on their size and electrical charge. In the field of dentistry, electrophoresis is widely used in various applications, including the analysis of dental materials and proteins associated with diseases of the oral cavity. Through electrophoresis, it is possible to evaluate the size and distribution of filler particles within resin matrices, providing valuable information on the mechanical properties and durability of composite materials used in dental restorations. Furthermore, this technique has significantly contributed to the study of proteins implicated in oral diseases, such as dental caries and periodontitis. By effectively identifying and separating these proteins, researchers gain a deeper understanding of the mechanisms underlying these conditions, facilitating the development of innovative therapeutic strategies. Overall, the application of electrophoresis in dentistry has emerged as an indispensable tool for comprehensive analysis of dental materials and characterization of proteins associated with oral diseases.

1. Introduction

Shortly, electrophoresis is a powerful analytical technique widely used in dental research for the separation and analysis of various biomolecules [1]. In this chapter, we will mention how electrophoresis is employed to study proteins, nucleic acids, glycosylated proteins, phosphorylated proteins, and other relevant molecules in dental materials and oral health research. Understanding the composition, modifications, and interactions of these biomolecules is critical for unraveling the molecular mechanisms underlying dental diseases, identifying potential biomarkers, and improving dental materials’ performance.

1.1 Overview of electrophoresis

Electrophoresis is a widely used technique in dentistry research for the separation and analysis of dental materials and oral disease-related proteins [1, 2]. It is based on the principle of applying an electric field to migrate charged molecules in a medium, such as a gel or a capillary, according to their size, charge, or isoelectric point. Electrophoresis allows the separation, identification, and quantification of various components in complex mixtures, including proteins, nucleic acids, carbohydrates, and ions [3]. Different electrophoretic techniques are employed in dental research, depending on the specific objectives and requirements of the analysis. Gel electrophoresis, such as polyacrylamide gel electrophoresis (PAGE) and agarose gel electrophoresis, is commonly used for the separation of proteins and nucleic acids based on their size and charge [4]. Capillary electrophoresis (CE) offers high resolution and efficiency for the separation of small molecules and ions [5]. Two-dimensional electrophoresis (2DE) combines two separation dimensions, such as isoelectric focusing (IEF) and sodium dodecyl sulfate (SDS)-PAGE, enabling more comprehensive protein profiling [6]. Electrophoresis has revolutionized dental material analysis by allowing the assessment of material components, such as monomers, additives, and degradation products. It aids in the determination of molecular weight distribution, polymerization kinetics, and leaching characteristics of dental materials. Additionally, electrophoresis techniques have been instrumental in protein analysis, allowing the identification, characterization, and quantification of disease-related proteins in oral fluids, tissues, and biofluids [7].

1.2 Significance of dental material analysis

Dental material analysis plays a crucial role in dentistry research and clinical practice [8]. The selection and evaluation of dental materials are essential for the success and longevity of various dental treatments, including restorative procedures, prosthetic devices, and orthodontic appliances [8, 9, 10]. Dental materials must possess desirable physical, chemical, and biological properties to ensure their safety, biocompatibility, and effectiveness in clinical applications. Therefore, thorough analysis and characterization of dental materials are necessary to assess their quality, performance, and potential risks. By employing analytical techniques, researchers and clinicians can examine the composition, structure, and properties of dental materials [11]. This analysis helps in identifying any potential flaws, defects, or limitations in the materials, as well as understanding their behavior under different conditions. It allows for the development of improved dental materials with enhanced properties, such as increased strength, improved esthetics, and reduced toxicity [12]. Moreover, the analysis of dental materials aids in standardization, quality control, and regulatory compliance in the dental industry, as follows:

1.2.1 Analysis of proteins in dental research

Proteins play a pivotal role in dental research, serving as key structural components and orchestrating various biological processes. Electrophoresis techniques are commonly used to analyze proteins in dental materials and biological samples. Several classes of proteins are of particular interest [13, 14, 15]:

• Dental tissue proteins: Dental tissues, such as enamel, dentin, and cementum, have unique protein compositions. Electrophoresis can be used to study the protein profiles of these tissues, providing insights into their structural and functional properties. For example, amelogenins are essential enamel matrix proteins involved in enamel formation and can be analyzed using electrophoresis.

• Salivary proteins: Saliva contains a diverse array of proteins with various functions, including antimicrobial properties and lubrication of oral tissues. Electrophoresis can help identify and quantify specific salivary proteins associated with oral health and disease.

• Inflammatory proteins: Inflammatory responses are critical in periodontal disease and dental caries. Identifying and quantifying inflammatory proteins, such as cytokines and chemokines, in gingival crevicular fluid (GCF) or dental plaque can help elucidate the molecular basis of these diseases. Electrophoresis can be used to analyze the expression levels of these inflammatory proteins.

• Matrix metalloproteinases (MMPs): MMPs are involved in tissue remodeling and degradation of extracellular matrix components. Dysregulation of MMPs is associated with periodontal tissue destruction. Electrophoresis can be used to analyze MMP expression levels in diseased tissues.

1.2.2 Analysis of nucleic acids in dental research

Nucleic acids, including DNA and RNA, are essential biomolecules in dental research, providing information about genetic factors, microbial presence, and gene expression in oral health and disease. Electrophoresis is commonly used to study nucleic acids in dental research [16, 17]:

• Analysis of dental microbiome: Electrophoresis-based methods, such as polymerase chain reaction (PCR) and gel electrophoresis, can be used to study the oral microbiome by identifying specific bacterial DNA sequences associated with dental diseases. For instance, 16S rRNA gene sequencing can be performed on gel-separated PCR products to characterize the oral microbial community.

• Genetic analysis of dental conditions: Nucleic acid electrophoresis can aid in the identification of genetic variations associated with dental conditions such as amelogenesis imperfecta and dentinogenesis imperfecta. Genetic testing using gel electrophoresis can reveal specific mutations linked to these dental disorders.

1.2.3 Other biomolecules of interest in dental research

Beyond proteins and nucleic acids, other biomolecules are relevant in dental research. Electrophoresis can be applied to study [18, 19]:

• Glycosylated proteins: Glycosylation is an essential posttranslational modification that influences the functions of dental proteins. Lectin-based electrophoresis techniques can be employed to analyze glycosylated proteins in dental tissues. For example, lectin blotting can detect specific carbohydrate structures on proteins separated by gel electrophoresis.

• Phosphorylated proteins: Phosphorylation is crucial in signal transduction pathways in dental cells. Phospho-specific antibodies or Phos-tag SDS-PAGE can be used to detect and analyze phosphorylated proteins. Electrophoresis combined with immunoblotting allows the detection of phosphorylated protein bands.

1.3 Role of protein analysis in oral disease research

Proteins play a vital role in the pathogenesis, progression, and diagnosis of various oral diseases. Understanding the protein composition and alterations associated with oral diseases is essential for advancing our knowledge of their underlying mechanisms and developing effective diagnostic and therapeutic strategies [20]. Protein analysis provides valuable insights into disease-related biomarkers, protein-protein interactions, and signaling pathways involved in oral diseases. Oral diseases, such as periodontal diseases, dental caries, and oral cancers, involve complex molecular processes that impact the composition and expression of proteins. Biomarker discovery and validation are crucial for early detection, prognosis, and monitoring of oral diseases [21]. Proteomic analysis techniques allow the identification and quantification of disease-specific proteins, providing potential targets for intervention and personalized treatment approaches. Furthermore, protein analysis facilitates the evaluation of treatment outcomes, the assessment of disease progression, and the monitoring of therapeutic responses [22, 23].

1.4 Preparing samples for electrophoresis

Preparing samples for electrophoresis involves specific steps depending on the type of electrophoresis and the type of samples you are working with (e.g., DNA, RNA, proteins) [24, 25]. Here’s a general guide on how to prepare samples for different types of electrophoresis:

1.4.1 DNA electrophoresis (agarose gel electrophoresis)

Agarose gel electrophoresis is commonly used to separate DNA fragments based on their size [24]. Here’s how to prepare samples for this type of electrophoresis:

Materials needed:

• DNA samples

• DNA ladder (molecular weight markers)

• Agarose powder

• Tris acetate EDTA (TAE) and tris borate EDTA (TBE) buffer

• Ethidium bromide or a DNA-specific stain

• Gel loading buffer

Steps:

1. Sample extraction: Extract DNA from your biological material using appropriate DNA extraction methods.

2. Quantification: Measure the concentration of your DNA samples using a spectrophotometer or fluorometer.

3. DNA denaturation (if needed): For double-stranded DNA, denature the samples by heating them at around 95°C for a few minutes and then cooling them on ice. This step converts double-stranded DNA into single-stranded DNA.

4. Prepare the agarose gel: Mix agarose powder with TAE or TBE buffer and heat the mixture to dissolve the agarose. Pour the liquid agarose into a gel tray and insert a comb to create wells.

5. Prepare loading buffer: Mix your DNA samples with a gel loading buffer. The loading buffer will add density to your samples, helping them sink into the wells.

6. Load the samples: Carefully load the DNA samples and the DNA ladder (molecular weight markers) into the wells of the agarose gel using a micropipette.

7. Electrophoresis: Submerge the gel tray in an electrophoresis tank filled with TAE or TBE buffer. Apply an electric field and run the electrophoresis until the DNA bands have separated according to their size.

8. Visualization: Stain the DNA with ethidium bromide or a DNA-specific stain and visualize the separated bands under UV light.

1.4.2 Protein electrophoresis (SDS-PAGE)

SDS-PAGE is used to separate proteins based on their molecular weight [25]. Here’s how to prepare protein samples for SDS-PAGE:

Materials needed:

• Protein samples

• Protein molecular weight markers

• SDS-PAGE running buffer

• SDS-PAGE sample buffer

• Reducing agent (e.g., β-mercaptoethanol or DTT)

Steps:

1. Sample extraction: Extract proteins from your biological material using appropriate protein extraction methods.

2. Protein quantification: Determine the protein concentration using methods, such as the Bradford assay or the BCA assay.

3. Protein denaturation and reducing: Mix your protein samples with an SDS-PAGE sample buffer containing a reducing agent (e.g., β-mercaptoethanol or DTT). The reducing agent helps to denature the proteins and break disulfide bonds.

4. Boiling: Heat the protein samples in the SDS-PAGE sample buffer at around 95°C for a few minutes to ensure complete denaturation.

5. Prepare the SDS-PAGE Gel: Assemble the SDS-PAGE gel according to the manufacturer’s instructions or your lab’s protocol.

6. Load the samples: Load the denatured protein samples and the protein molecular weight markers into the wells of the SDS-PAGE gel.

7. Electrophoresis: Submerge the gel in an electrophoresis tank filled with SDS-PAGE running buffer. Apply an electric field and run the electrophoresis until the protein bands have separated according to their size.

8. Visualization: Stain the proteins with Coomassie Brilliant Blue or other compatible protein stains, or use silver staining for more sensitive applications.

It’s important to note that electrophoresis protocols can vary depending on the specific application and the type of samples being analyzed. Always follow established protocols and optimize the procedure for your particular experiment to ensure accurate and reliable results.

1.4.3 Separation and purification steps

Before performing electrophoresis, separation, and purification steps are often necessary to obtain high-quality samples and accurate results. The steps for separation and purification depend on the type of sample and the target molecules you are working with (e.g., DNA, RNA, proteins) [19, 24, 26]. Here are some common separation and purification methods used before electrophoresis:

DNA electrophoresis (agarose gel electrophoresis): For DNA electrophoresis, the following separation and purification steps are commonly performed:

1. DNA extraction: This is the initial step to isolate DNA from the biological material (e.g., cells, tissues, blood). Various extraction methods, such as phenol-chloroform extraction, column-based purification, or commercial DNA extraction kits, can be employed.

2. PCR or DNA digestion (optional): If you are working with specific DNA fragments, you may need to perform polymerase chain reaction (PCR) or DNA digestion (restriction enzyme digestion) to amplify or cut the DNA into the desired fragments.

3. DNA cleanup: After PCR or DNA digestion, it’s common to perform a DNA cleanup step to remove any leftover primers, enzymes, or other impurities that could interfere with the electrophoresis results. DNA cleanup can be done using purification kits or by precipitation methods.

RNA electrophoresis: For RNA electrophoresis, the following steps are typically performed:

1. RNA extraction: RNA is extracted from the biological material (e.g., cells, tissues, bacteria, or viruses) using methods, such as phenol-chloroform extraction, column-based purification, or commercial RNA extraction kits.

2. RNA integrity check: Before proceeding with electrophoresis, it is essential to assess the integrity of the RNA. This can be done using techniques such as gel electrophoresis or capillary electrophoresis with RNA-specific dyes.

3. RNA cleanup: Similar to DNA cleanup, RNA cleanup is performed to remove impurities that might interfere with the electrophoresis results. Various purification methods can be used, such as column-based cleanup or precipitation methods.

Protein electrophoresis (SDS-PAGE): For protein electrophoresis, the following steps are typically performed:

1. Protein extraction: Proteins are extracted from the biological material using appropriate protein extraction methods, such as cell lysis, tissue homogenization, or subcellular fractionation.

2. Protein quantification: The concentration of proteins in the samples is determined using methods, such as the Bradford assay or the BCA assay.

3. Protein denaturation and reduction: Proteins are denatured and reduced using an SDS-PAGE sample buffer containing a reducing agent (e.g., β-mercaptoethanol or DTT). This step ensures that the proteins are in a linear, denatured state for electrophoresis.

4. Protein cleanup: Sometimes, a protein cleanup step is performed to remove unwanted contaminants or interfering substances that could affect the electrophoresis results. Cleanup methods can include precipitation, dialysis, or column-based purification.

These separation and purification steps are critical for obtaining pure and concentrated samples of DNA, RNA, or proteins, which will ultimately lead to successful and interpretable electrophoresis results. The choice of specific methods and techniques will depend on the experimental requirements and the quality of the starting biological material.

1.5 Preparation of samples for electrophoresis of dental materials

Preparing samples for electrophoresis of dental materials involves specific steps to ensure accurate and reliable results. The preparation process may vary depending on the type of dental material you are working with, such as dental composites, cements, or adhesives [1, 2, 8, 9, 10, 11, 12]. Here’s a general outline of the sample preparation process:

1. Sample collection: Collect the dental material you want to analyze. This could be a cured dental composite, set dental cement, or a specific component of a dental adhesive. Ensure that the sample is representative of the material you want to study.

2. Homogenization or grinding: If the dental material is in a solid form, you may need to homogenize or grind it to create a homogeneous sample. This step is crucial for ensuring even distribution and consistent results during electrophoresis.

3. Solubilization or extraction: Depending on the type of dental material, you might need to extract or solubilize the relevant components. For instance, if you are working with dental composites or adhesives, you might need to extract the monomers or polymer matrix. Some dental cements may need to be dissolved to release the proteins or other components of interest.

4. Protein precipitation (if analyzing proteins): If analysis focuses on proteins present in the dental material, it may need to perform a protein precipitation step to concentrate the proteins and remove interfering substances. This step helps improve protein detection and separation during electrophoresis.

5. Protein denaturation and reduction (For SDS-PAGE): If you are using SDS-PAGE to analyze proteins, the samples need to be denatured and reduced before loading onto the gel. This step involves heating the samples in the presence of a denaturing agent (such as SDS) and a reducing agent (such as β-mercaptoethanol or DTT) to ensure that the proteins are in a linear, denatured state for electrophoresis.

6. Size separation (electrophoresis): Load the prepared samples onto the appropriate electrophoresis gel. The choice of gel (agarose or polyacrylamide) and the electrophoresis conditions (e.g., voltage, buffer system) will depend on the specific objectives of your study.

7. Staining or detection: After electrophoresis, you may need to stain the gel to visualize the separated components. Coomassie Brilliant Blue, silver staining, or specific protein stains are commonly used for protein detection. For other types of dental materials, alternative staining methods or detection techniques may be employed.

8. Data analysis and interpretation: Analyze the gel images to identify and quantify the components of interest. Compare the results with appropriate controls or standards to validate your findings.

It’s essential to optimize the sample preparation process to obtain reliable and reproducible results. The specific steps and conditions will depend on the dental material you are studying and the objectives of your analysis. Additionally, some dental materials might require additional purification steps, depending on their complexity and the presence of interfering substances. Always follow established protocols and adapt them as needed for your specific research.

2. Electrophoresis principles and techniques

2.1 Gel electrophoresis

Gel electrophoresis is one of the most commonly used techniques in electrophoresis. It involves the migration of charged molecules through a gel matrix under the influence of an electric field. The gel matrix provides a medium for size-based separation of molecules, with smaller molecules migrating faster than larger ones [26].

2.1.1 Polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is widely used for protein separation. It utilizes polyacrylamide gels with different concentrations, which create sieving properties suitable for separating proteins based on their size. Two commonly used variants of PAGE are native PAGE and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Native PAGE preserves the protein’s native structure, while SDS-PAGE denatures proteins and allows for separation based on molecular weight [4, 6]. In dentistry, PAGE is utilized to analyze dental materials, such as composite resins and adhesive systems, to assess their polymerization efficiency, molecular weight distribution, and the presence of impurities or residual monomers. Additionally, PAGE is employed in protein analysis for the identification and characterization of disease-related proteins in oral tissues and fluids [27].

2.1.2 Agarose gel electrophoresis

Agarose gel electrophoresis is commonly used for the separation of nucleic acids, such as DNA and RNA. Agarose gels are formed by the polymerization of agarose, a polysaccharide derived from seaweed. The porosity of the gel matrix can be adjusted by varying the agarose concentration, allowing for the separation of nucleic acid fragments of different sizes [28]. While agarose gel electrophoresis is primarily employed for nucleic acid analysis, it can also be utilized in dental research for the examination of genetic factors related to oral diseases or the evaluation of antimicrobial agents targeting oral pathogens [29].

2.2 Capillary electrophoresis

Capillary electrophoresis (CE) is a high-resolution technique that utilizes a narrow capillary tube as the separation medium. The capillary is typically coated with a polymer or has a charged inner surface to facilitate the separation of analytes based on the charge-to-size ratio. CE offers advantages such as high separation efficiency, short analysis time, and small sample requirements [30]. In dentistry, CE is employed for the analysis of ions, small molecules, and biomarkers in oral fluids and dental materials. It allows for the quantification of metal ion release from dental alloys, the evaluation of antimicrobial agents, and the assessment of salivary biomarkers for oral diseases [31, 32].

2.3 Two-dimensional electrophoresis

Two-dimensional electrophoresis (2DE) is a powerful technique that combines two separation dimensions to achieve increased resolution and separation capacity [33]. The first dimension typically involves isoelectric focusing (IEF), which separates proteins based on their isoelectric points (IP). In the second dimension, proteins are separated based on their molecular weights using SDS-PAGE [34]. The 2DE technique enables the separation and visualization of complex protein mixtures, providing a comprehensive protein profile. In dentistry, 2DE is utilized to analyze oral disease-related proteins, identify biomarkers, and compare protein expression patterns in healthy and diseased oral tissues [35].

2.4 Isoelectric focusing

IEF is a technique that separates proteins based on their IP, which is the pH at which a protein has no net charge. IEF utilizes a pH gradient gel or a capillary with a pH gradient, and proteins migrate until they reach their isoelectric point and become focused in a narrow band [36]. IEF is often used as the first dimension in 2DE to achieve high-resolution separation based on IP. In dentistry research, IEF is employed to study protein charge heterogeneity, identify protein isoforms, and analyze protein modifications [37].

3. Electrophoresis analysis of dental materials

In dentistry, the degradation products can vary depending on the specific dental materials used. Dental materials such as dental composites, dental cements, and dental adhesives may undergo degradation over time due to factors such as oral pH, mechanical stresses, and exposure to saliva and other oral fluids [38]. Here’s an overview of some common dental materials and their potential degradation products:

3.1 Dental composites

Dental composites are tooth-colored restorative materials used to fill cavities and repair teeth. They typically consist of a resin matrix (monomers) and filler particles (such as glass or ceramic) [39]. Electrophoresis analysis of composite materials provides valuable insights into their composition, polymerization characteristics, and degradation products [2, 6, 7]. Electrophoresis techniques, such as PAGE or CE, are utilized to analyze the monomers present in composite materials. The analysis helps in assessing the efficiency of the polymerization process, the presence of unreacted monomers, and the identification of potential leachable components [40]. Additionally, electrophoresis can be used to investigate the degradation products of composite materials over time, providing information on their long-term stability and biocompatibility [41]. The charges and sizes of degradation products in dentistry are highly specific to the materials used. Electrophoresis can be used to analyze the degradation products, but it is not a common method in dental research or clinical practice. Dental materials are typically analyzed through other methods, such as spectroscopy, chromatography, scanning electron microscopy, and mechanical testing. While electrophoresis is a powerful tool for analyzing the degradation products of biomolecules such as DNA, RNA, and proteins, it is not commonly used for analyzing degradation products in dental materials. Dental materials have different chemical compositions, and their degradation products are typically assessed using other specialized analytical techniques [42]. Degradation of dental composites may result in:

• Leaching of monomers: Some monomers used in dental composites, such as bisphenol A glycidyl methacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA), and urethane dimethacrylate (UDMA), may undergo leaching into the oral environment over time.

• Filler particle breakdown: The filler particles in dental composites may undergo wear or degradation, leading to the release of fine particles into the oral cavity.

These monomers are polymerized to form dental materials, which play a crucial role in their mechanical and adhesive properties [42]. It’s important to note that dental materials and their degradation mechanisms are continuously being studied and improved to enhance their performance and longevity in dental restorations and treatments. Dental research aims to develop materials that withstand the oral environment and minimize potential adverse effects from degradation products.

3.2 Dental cements

Dental cements are materials used to bond various dental restorations to teeth, essential in various dental applications, including cementation of crowns, bridges, and orthodontic appliances [43]. They can be classified into different types, such as resin-based cements, glass ionomer cements, and zinc phosphate cements. Electrophoresis analysis aids in the evaluation of the composition, setting reaction, and mechanical properties of dental cements. Electrophoretic techniques, such as PAGE or CE, are employed to assess the composition of dental cements and identify the presence of various components, such as resin monomers, initiators, fillers, and additives. This analysis helps in understanding the role of each component in the cement’s properties and performance. Furthermore, electrophoresis can be utilized to study the setting reaction of dental cements, allowing for the identification and quantification of reaction by-products and the assessment of the cement’s final properties [44]. In addition, the degradation products may include [45]:

• Ion release: Glass ionomer cements, for example, can release fluoride and other ions over time, which can have beneficial effects on the adjacent tooth structure.

• Chemical reactions: Some cements may undergo chemical reactions leading to changes in their properties over time.

3.3 Impression materials

Impression materials are used to capture the precise dental structures for fabricating prosthetic devices and dental restorations [46]. Electrophoresis analysis of impression materials enables the evaluation of their composition, polymerization characteristics, and potential leakage [47]. Electrophoretic techniques, such as PAGE or CE, are employed to analyze the composition of impression materials, including the identification and quantification of various components, such as polymers, initiators, and accelerators. The analysis helps in assessing the material’s consistency, stability, and performance. Additionally, electrophoresis can be used to study the polymerization process of impression materials, evaluating the efficiency and completeness of the polymerization reaction [48].

4. Electrophoresis analysis of oral disease-related proteins

4.1 Periodontal disease biomarkers

Periodontal diseases, including gingivitis and periodontitis, are characterized by inflammation and destruction of the periodontal tissues [49]. Biomarkers are specific molecules, proteins, or genes that can indicate the presence or severity of a disease. In the context of periodontal disease, biomarkers can help in diagnosis, prognosis, and monitoring of the progression of the disease. Protein patterns depict the collective arrangement of proteins within a biological sample, such as GCF or saliva [50]. In the study of periodontal diseases, techniques such as 2D gel electrophoresis and mass spectrometry are commonly employed to examine the protein patterns within GCF or saliva samples [51]. By comparing protein profiles from healthy individuals and those with periodontal disease, researchers can spot potential biomarkers linked to the condition. Electrophoresis analysis aids in the identification and characterization of biomarkers associated with periodontal diseases. This analysis allows for the identification of disease-specific proteins or protein patterns that can serve as potential biomarkers for periodontal diseases [51, 52]. Here are some example proteins and genes that have been studied as potential biomarkers for periodontal disease:

4.1.1 Example proteins as biomarkers

• Matrix metalloproteinases (MMPs): MMPs are enzymes that play a role in tissue remodeling and degradation of extracellular matrix components. Elevated levels of certain MMPs, such as MMP-8 and MMP-9, have been associated with periodontal tissue destruction [53].

• C-reactive protein (CRP): CRP is an acute-phase protein produced by the liver in response to inflammation. Increased levels of CRP have been linked to periodontal disease and may indicate systemic inflammation [54].

• Interleukins (ILs): Various interleukins, such as IL-1β, IL-6, IL-8, and IL-17, are involved in the immune response and have been implicated in periodontal inflammation [55].

• Tumor necrosis factor-alpha (TNF-α): TNF-α is a pro-inflammatory cytokine that plays a role in the inflammatory process of periodontal disease [56].

4.1.2 Example genes as biomarkers

• Interleukin-1 gene cluster (IL1): Genetic variations in the IL1 gene cluster have been associated with increased susceptibility to severe periodontitis [57].

• Toll-like receptor genes (TLRs): TLRs are involved in recognizing microbial components and initiating the immune response. Genetic variations in TLR genes have been linked to periodontal disease susceptibility [58].

• Human leukocyte antigen (HLA) Genes: Certain HLA genotypes have been associated with increased susceptibility to periodontal disease [59].

Quantitative analysis by electrophoresis is valuable for comparing protein expression levels between different groups of samples, such as healthy and diseased individuals, and for identifying potential biomarkers associated with specific conditions such as periodontal disease [60]. However, it’s essential to ensure that the quantification process is accurate and reliable by using appropriate controls and validation methods. There are different methods for quantitative analysis in electrophoresis [1, 2, 60]:

1. Densitometry: In traditional gel electrophoresis, such as SDS-PAGE or 2DE, densitometry can be used to measure the intensity of protein bands on the gel. The densitometry data can be quantified using imaging software, providing relative information about protein abundance.

2. Western blotting (Immunoblotting): In Western blotting, after electrophoresis, the separated proteins are transferred to a membrane, and specific antibodies are used to detect and quantify the target proteins. The signal intensity of the protein bands on the membrane corresponds to their abundance in the sample.

3. Difference gel electrophoresis (DIGE): DIGE is a technique that allows for the simultaneous comparison of multiple samples in a single gel run. Different samples are labeled with different fluorescent dyes, and their protein patterns can be quantified and compared.

4. Capillary electrophoresis: In CE, samples are separated in a capillary tube, and the detector measures the migration times and peak heights of the analytes. This information can be used for quantitative analysis.

4.2 Dental caries biomarkers

Dental caries, commonly known as tooth decay, is a prevalent oral disease caused by the demineralization of tooth structures [61]. Electrophoresis analysis facilitates the identification and characterization of biomarkers associated with dental caries [62]. Electrophoretic techniques, such as PAGE or CE, are employed to analyze proteins present in dental plaque, saliva, or biofilms. This analysis helps in identifying specific proteins or protein profiles associated with cariogenic bacteria or host response to dental caries. By comparing the protein expression patterns between caries-free individuals and those with active caries, potential biomarkers for dental caries can be identified. Electrophoresis analysis of dental caries biomarkers contributes to the development of diagnostic tools for early caries detection, risk assessment, and personalized preventive strategies [63].

4.3 Oral cancer biomarkers

Oral cancer encompasses a range of malignancies affecting the oral cavity, including the lips, tongue, gingiva, and oral mucosa [64]. Electrophoresis analysis plays a crucial role in the identification and characterization of oral cancer biomarkers [65]. Using techniques such as PAGE or 2DE, researchers can separate and analyze proteins present in oral cancer tissues, saliva, or serum. This analysis aids in the identification of differentially expressed proteins that are associated with oral cancer development, progression, or metastasis. By comparing protein expression patterns between healthy individuals and those with oral cancer, potential biomarkers can be discovered. Electrophoresis analysis of oral cancer biomarkers contributes to the development of early detection methods, prognostic indicators, and targeted therapies for oral cancer management [66].

5. Advantages and limitations of electrophoresis in dentistry research

5.1 Advantages

5.1.1 High separation efficiency

Electrophoresis offers high separation efficiency, allowing for the resolution of complex mixtures of molecules based on their charge, size, or IP. This enables researchers to analyze and identify individual components within dental materials or disease-related proteins with precision [1].

5.1.2 Versatility

Electrophoresis techniques, such as PAGE, agarose gel electrophoresis, CE, and 2DE, provide a range of options for different types of analyses. This versatility allows researchers to adapt the technique to specific research objectives and sample requirements [1].

5.1.3 Quantification and comparison

Electrophoresis can be used for the quantification of proteins or other analytes present in dental materials or biological samples. By comparing the expression levels of specific proteins or the presence of certain components, researchers can assess differences between healthy and diseased states, evaluate the effectiveness of dental treatments, and monitor disease progression [2]. Quantifying dental materials and biological samples using electrophoresis typically involves measuring the intensity of bands or peaks on the electrophoresis gel or capillary. The quantification process can vary depending on the type of electrophoresis and the specific materials or biomolecules being analyzed [60]. Here are general steps for quantifying dental materials and biological samples using two common types of electrophoresis:

1. Quantification using SDS-PAGE for protein analysis [2, 4]:

   1. Gel electrophoresis: Perform SDS-PAGE to separate the proteins in the sample. Use appropriate molecular weight markers to estimate the size of protein bands.

   2. Gel staining: After electrophoresis, stain the gel with a protein-specific stain, such as Coomassie Brilliant Blue or a fluorescent dye. The stain will bind to the proteins, allowing them to be visualized.

   3. Gel imaging: Capture an image of the stained gel using a gel documentation system or a high-resolution scanner.

   4. Densitometry: Use image analysis software to quantify the intensity of protein bands. Select the region of interest (band) on the gel, and the software will calculate the integrated optical density or intensity of the band.

   5. Standard curve: For more accurate quantification, create a standard curve using known concentrations of purified protein standards. The standard curve can be used to convert the measured intensity of protein bands into protein concentrations.

2. Quantification using agarose gel electrophoresis for nucleic acid analysis [1, 2]:

   1. Agarose gel electrophoresis: Run the DNA or RNA samples on an agarose gel to separate the nucleic acids based on size.

   2. Gel staining: After electrophoresis, stain the gel with a DNA-specific dye (e.g., ethidium bromide or SYBR Green) to visualize the DNA bands.

   3. Gel imaging: Capture an image of the stained gel using a gel documentation system or a high-resolution scanner.

   4. Densitometry (optional for DNA): For more accurate quantification of DNA bands, you can use densitometry software to measure the intensity of DNA bands. However, densitometry is not commonly used for DNA quantification; instead, DNA quantification is typically performed using spectrophotometry or fluorometry before electrophoresis.

3. Quantification of other biomolecules (e.g., RNA, proteins, or DNA in capillary electrophoresis) [1, 24, 25].

For some advanced techniques such as capillary electrophoresis, quantification can be performed using specialized software that analyzes the migration times and peak heights of the analytes. The software can compare the migration times and peak areas of the sample peaks with those of standard solutions of known concentrations. It’s important to note that the accuracy of quantification using electrophoresis depends on several factors, such as the quality of the gel or capillary run, the staining and detection methods, and the selection of appropriate standards for calibration. Always validate the obtained quantification results and consider using multiple complementary methods for quantification to ensure accuracy and reliability.

5.1.4 Protein characterization

Electrophoresis facilitates the characterization of proteins by providing information on their molecular weight, IP, and charge heterogeneity. This information aids in the identification of disease-related proteins, protein modifications, and protein-protein interactions, contributing to a deeper understanding of the molecular mechanisms underlying oral diseases [20]. Identification of disease-related proteins, protein modifications, and protein-protein interactions using electrophoresis often involves a combination of electrophoretic techniques with other analytical methods [67, 68]. Here’s an overview of how each of these aspects can be achieved.

5.1.4.1 Identification of disease-related proteins

Electrophoresis alone does not provide direct identification of disease-related proteins. Instead, it is often used as a first step to separate complex protein mixtures, followed by additional methods for protein identification [1, 67]. Two commonly used techniques for protein identification after electrophoresis are mass spectrometry and Western blotting.

• Mass spectrometry (MS): After gel electrophoresis, the proteins of interest are typically extracted from the gel, digested into peptides, and analyzed by MS. The latter can provide information about the masses and sequences of the peptides, allowing researchers to identify the corresponding proteins through database searches.

• Western blotting (Immunoblotting): Western blotting is used to detect and quantify specific proteins in a complex mixture. After gel electrophoresis, proteins are transferred to a membrane and probed with specific antibodies against the proteins of interest. The antibodies bind to their targets, and the presence of the protein is detected through chemiluminescence or fluorescence.

5.1.4.2 Identification of protein modifications

Electrophoresis can be combined with specific staining or immunodetection techniques to identify protein modifications, such as posttranslational modifications (PTMs) [60]. Some examples of PTM identification are:

• Phosphorylation: Phosphorylated proteins can be detected using phospho-specific antibodies in Western blotting or specific stains, such as Pro-Q Diamond, in 2D gel electrophoresis.

• Glycosylation: Glycosylated proteins can be identified using lectin staining in gel electrophoresis or through glycoprotein-specific antibodies.

5.1.4.3 Identification of protein-protein interactions

Electrophoresis itself is not a direct method for identifying protein-protein interactions. However, it can be used as a preliminary step in techniques that investigate protein-protein interactions [1, 25]. Two commonly used methods for this purpose are co-immunoprecipitation (Co-ip) and pull-down assays.

• Co-immunoprecipitation (Co-ip): In Co-ip, proteins are first cross-linked or stabilized to preserve protein-protein interactions. The target protein is immunoprecipitated using specific antibodies, and the co-immunoprecipitated proteins are then separated by gel electrophoresis and identified using MS or Western blotting.

• Pull-down assays: Pull-down assays use affinity chromatography to isolate one protein from a complex mixture, along with its interacting partners. After electrophoresis, the interacting proteins can be identified using mass spectrometry or Western blotting.

5.1.5 Biomarker discovery

Electrophoresis plays a crucial role in the discovery of biomarkers associated with dental diseases. By analyzing protein expression patterns or identifying disease-specific proteins, researchers can identify potential biomarkers for early detection, diagnosis, prognosis, and monitoring of oral diseases. This information has the potential to improve patient outcomes and guide personalized treatment approaches [21].

5.2 Limitations

5.2.1 Sample complexity

One of the main limitations of electrophoresis is the complexity of samples, particularly in biological fluids or tissues. The presence of a wide range of proteins, nucleic acids, and other biomolecules can hinder the separation and identification of specific components. Additional sample preparation steps, such as protein enrichment or fractionation, may be required to overcome this limitation [69].

5.2.2 Sensitivity

The sensitivity of electrophoresis techniques can vary depending on the type of analysis and detection method employed. Some low-abundance proteins or analytes may be challenging to detect, especially in complex samples. Sensitivity limitations can affect the ability to identify specific biomarkers or quantify analytes accurately [70].

5.2.3 Interpretation of results

Electrophoresis provides a visual representation of separated components, but the interpretation of results requires expertise and careful analysis. The identification of specific proteins or analytes often requires additional techniques, such as mass spectrometry or immunoassays, to confirm their identity. Additionally, the presence of posttranslational modifications or protein isoforms can complicate the interpretation of electrophoresis results [71].

5.2.4 Standardization and reproducibility

Achieving consistent and reproducible results in electrophoresis can be challenging due to variations in gel preparation, running conditions, staining methods, and data analysis. Standardization of protocols and rigorous quality control measures are necessary to ensure reliable and comparable results across different studies and laboratories [72].

5.2.5 Technical expertise and equipment requirements

Electrophoresis techniques require specialized equipment, such as gel electrophoresis apparatus, power supplies, and imaging systems. Additionally, the interpretation of results and data analysis often necessitate expertise in molecular biology, biochemistry, and biostatistics. These requirements may limit the accessibility of electrophoresis to certain research settings or require collaboration with experienced researchers or core facilities [73].

6. Discussion

The use of electrophoresis in dental research brings notable advantages and potential applications for analyzing dental materials and proteins linked to oral diseases. The prior sections of this chapter have presented how electrophoresis enhances our comprehension of dental materials, oral diseases, and personalized patient care in dentistry. Electrophoresis emerges as an effective technique for dental materials analysis. Employing diverse methods such as PAGE, agarose gel electrophoresis, and 2DE, researchers have effectively separated and characterized dental material components based on their charge, size, or IP [1, 2]. This insight has proven invaluable in understanding the composition, purity, and structural attributes of dental materials, offering an assessment of their quality, performance, and compatibility.

Furthermore, electrophoresis techniques have facilitated the identification of specific components within dental materials, such as resin composites, cements, and impression materials, contributing to the development of improved dental materials with enhanced properties and clinical outcomes [63]. Apart from dental materials, electrophoresis has played a significant role in investigating proteins connected to oral diseases [65]. By examining protein expression patterns and identifying disease-specific proteins, researchers have been able to discover and validate potential biomarkers related to oral issues such as periodontal disease, dental caries, and oral cancer. Techniques such as 2DE and IEF have enabled the dissection and characterization of intricate protein mixtures, furnishing crucial insights into protein modifications, interactions, and deviations during disease states. This knowledge has the potential to transform clinical practice by aiding early detection, diagnosis, monitoring of oral problems, and guiding tailored treatment plans for individual patients [69].

While electrophoresis has demonstrated great efficacy in dentistry research, it’s essential to recognize its limitations. The intricacy of samples, especially in biological fluids or tissues, poses challenges in segregating and identifying specific components using electrophoresis techniques [69]. Overcoming this challenge might require preparatory steps such as protein enrichment or fractionation. Moreover, the sensitivity of electrophoresis techniques can fluctuate, making the detection of low-abundance proteins or analytes challenging, particularly in complex samples. Therefore, supplementary techniques such as MS or immunoassays might be necessary to validate and confirm the identity of specific proteins or biomarkers [62, 71].

To propel the field forward, upcoming research should address these limitations and explore novel applications of electrophoresis in dentistry [74]. Integrating multi-omics approaches, encompassing genomics, transcriptomics, proteomics, and metabolomics, can furnish a more comprehensive grasp of the molecular mechanisms behind dental issues and the influence of dental materials [75]. Validating identified biomarkers across larger patient groups and diverse populations is pivotal for their practical use and integration [76]. Moreover, incorporating artificial intelligence (AI) and machine learning algorithms can enhance data analysis, pattern recognition, and predictive modeling, leading to more precise and individualized diagnostic and treatment strategies in the realm of dentistry [77, 78].

7. Future perspectives and conclusions

7.1 Future perspectives

Electrophoresis has been a valuable tool in dentistry research, enabling the analysis of dental materials and oral disease-related proteins. Looking ahead, several future perspectives can be envisioned for the application of electrophoresis in dentistry.

7.1.1 Advancements in electrophoresis techniques

Electrophoresis techniques are continually evolving, driven by technological advancements. Future improvements may include the development of novel gel matrices, better detection methods, and enhanced automation. These advancements would lead to increased sensitivity, resolution, and throughput, enabling more comprehensive and efficient analysis of dental materials and oral disease-related proteins [74].

7.1.2 Integration of multi-omics approaches

The integration of different omics approaches, such as genomics, transcriptomics, proteomics, and metabolomics, holds great potential for advancing dentistry research. By combining electrophoresis analysis with other high-throughput technologies, researchers can obtain a more comprehensive understanding of the molecular mechanisms underlying dental diseases and the effects of dental materials [75].

7.1.3 Biomarker validation and clinical translation

The discovery of potential biomarkers through electrophoresis analysis requires further validation and translation into clinical practice. Future studies should focus on validating the identified biomarkers in larger patient cohorts and diverse populations. Moreover, efforts should be made to develop reliable and user-friendly diagnostic assays that can be readily implemented in dental clinics [76].

7.1.4 Integration of artificial intelligence and machine learning

The integration of AI and machine learning algorithms can enhance data analysis, pattern recognition, and predictive modeling in dentistry research. AI-driven approaches can aid in the identification of complex protein patterns, the prediction of disease progression, and the optimization of dental material properties [77, 78].

8. Conclusions

Electrophoresis has emerged as a valuable asset in dentistry research, shedding light on dental materials and proteins linked to oral diseases. Its benefits encompass efficient separation, versatility, quantification capabilities, protein understanding, and biomarker discovery. Nonetheless, it’s essential to acknowledge limitations such as complex samples, sensitivity, result interpretation, standardization, and technical expertise. Looking ahead, progress in electrophoresis techniques, incorporating multi-omics methods, validating biomarkers, clinical adaptation, and utilizing AI and machine learning offer exciting avenues to amplify electrophoresis’ role in dentistry research. By embracing these forthcoming possibilities and tackling current challenges, researchers can further leverage electrophoresis as a potent tool for studying dental materials and oral disease-related proteins. This, in turn, will advance diagnostics, treatments, and patient care in the field of dentistry.

Conflict of interest

The authors declare no conflict of interest.