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Table of Contents
REVIEW ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 1  |  Page : 10-17

Immunocytochemistry and western blot test for the in-situ detection of biomarkers of osteogenesis


Department of Oral Biology, Faculty of Dentistry, Universitas Indonesia, Indonesia

Date of Submission18-Sep-2021
Date of Decision03-Nov-2021
Date of Acceptance13-Dec-2021
Date of Web Publication21-Feb-2022

Correspondence Address:
Priska Natassya
Department of Oral Biology, Faculty of Dentistry, Universitas Indonesia,
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/SDJ.SDJ_101_21

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  Abstract 

There are two ways that bones can form: intramembranous and endochondral ossification. Specific osteogenic markers, such as insulin-like growth factor (IGF-1), osteocalcin (OCN), Runt-related transcription factor 2 (RUNX2), and osterix (OSX), accompany osteoblast differentiation from an undifferentiated state to a functional state. IGF-1 hormones are the main regulators in growth, differentiation, and apoptosis in cells and tissues mediated by IGF-1 receptors (IGF-1R). Biomolecular technology aims to study nucleic acids and their regulation and expression of proteins. Techniques that can be used when analyzing proteins include the Bradford protein assay, immunocytochemistry, immunohistochemistry, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the western blot test. To better understand the biomarkers of osteogenesis, the use of in-situ detection is suggested, such as immunocytochemistry and the western blot test. For this review, the author adhered to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) standards. The research examined 50 articles; only 4 articles were selected for this review. In the immunocytochemical test, IGF-1 expression was found in the nucleus and IGF-1R expression in the cell membrane, and it was found that RUNX2, OSN, and OCN are important for osteogenesis. During the western blot test, it was found that the addition of IGF-1 in dental stem cells could increase the expression of RUNX2, OSX, and OCN proteins. Based on this information, it appears that both immunocytochemistry and western blot tests can detect the main biomarkers of osteogenesis.

Keywords: IGF-1, immunocytochemistry, OCN, OSX, RUNX2, western blot


How to cite this article:
Natassya P, Bachtiar EW. Immunocytochemistry and western blot test for the in-situ detection of biomarkers of osteogenesis. Sci Dent J 2022;6:10-7

How to cite this URL:
Natassya P, Bachtiar EW. Immunocytochemistry and western blot test for the in-situ detection of biomarkers of osteogenesis. Sci Dent J [serial online] 2022 [cited 2022 Oct 2];6:10-7. Available from: https://www.scidentj.com/text.asp?2022/6/1/10/338000




  Background Top


Bone is a calcified connective tissue structure that forms during the proliferation and differentiation of osteoprogenitor cells into mature osteoblasts. The morphology (cuboidal appearance) of osteoblasts (bone-forming cells) and their connection with bone matrices are used to identify them.[1] Proliferation, extracellular matrix (ECM) production, and mineralization are the three primary stages of osteoblast development.[1],[2]

Intramembranous ossification and endochondral ossification are the two methods by which bones develop. When mesenchymal cells migrate and proliferate in the intramembranous bone formation pathway, sheets of mesenchymal connective tissue are generated. This is how bone is formed. Cartilage is produced through a process that involves the condensation of chondrocyte-producing mesenchymal progenitor cells and their differentiation into chondrocytes, which is followed by trabecular bone formation.[3]

In addition to the transcription factors [SRY-box transcription factor 9, Runt-related transcription factor 2 (RUNX2), osterix (OSX), and β-catenin], parathyroid hormone-related protein and Indian hedgehog (IHH) play an important role in the endochondral ossification at the Primary Ossification Center.[3] Changes in cell morphology and the expression of adhesion molecules, ECM proteins (collagen type I; COLI), and specific osteogenic markers—such as osteocalcin (OCN),[1] RUNX2,[4] and OSX[5]—accompany osteoblast differentiation from an undifferentiated to a functional state.

In addition to hormones that have an influence on growth hormone (GH), insulin-like growth factor 1 (IGF-1) is a key regulator of somatic growth and organ development.[6] IGF-1, also known as somatomedin C, is a tiny protein with an amino acid sequence identical to insulin.[7]

IGF-1 is made up of 70 amino acids linked together by three intramolecular sulfide bridges in a single chain.[7] IGF-1 plays a role in regulating proliferation, differentiation, apoptosis, and cell migration that affects the development of tissues such as bones, muscles, and nerves.[8] IGF-1 is the primary regulator of growth, differentiation, and death in certain types of cells and organs via IGF-1 receptors (IGF-1R).[9]

Phosphoinositide 3 kinase, protein kinase B (Akt), mechanistic rapamycin (mTOR), mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase are all signaling pathways that IGF-1 mediates.[10]

RUNX2, also known as core-binding factor subunit alpha-1, is an osteogenesis-related transcription factor. During skeletal development, RUNX2 is required for chondrocyte maturation and osteoblast differentiation. RUNX2 is also linked to the osteoblast phenotype and can be found on chromosome 6p21.[4]

In addition to regulating glucose metabolism, testosterone production, and muscle mass, OCN has been found to suppress bone growth. Because osteoblasts are responsible for its production, it is the most abundant non-collagenous protein found in the bone.[11]

Osterix, also known as Sp7, is an osteoblast-specific transcription factor that belongs to the SP/KLF family of transcription factors. OSX is overexpressed in osteoblast-lineage cells, chondrocytes, and cancer tissues.[12]

Biomolecular technology aims to study nucleic acids and their regulation and expression of proteins. The study regarding regulation and replication of DNA is called genomic. The earliest technology in the study of the genome was the detection of chromosomes by lysing cells. With this method, researchers saw that the number of human chromosomes was 46, or 23 pairs. Previously, the extraction and purification of nucleic acids were complicated, time-consuming, labor-intensive, and had a low overall throughput; now there are many specialized methods for extracting pure biomolecules, such as solution-based and column-based protocols.[13]

Proteins are made up of 20 amino acids that can be arranged into different sequences and combinations. The number and sequence of amino acids in the amino acid chains change between proteins. Protein can be found throughout the body. Purification of proteins is required to determine their unique properties, such as size, charge, shape, and function.[13]

Techniques that can be used to analyze proteins include the Bradford protein assay, immunocytochemistry, immunohistochemistry, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and the western blot test. In other studies, looking at protein expression via IGF-1, RUNX2, OCN, and OSX, researchers often use immunocytochemistry techniques with fluorescent staining and western blot techniques.[8],[10] By using in-situ methods, researchers can detect proteins more accurately. The purpose of this review is to explore more about immunocytochemistry and western blot techniques for the in-situ detection of biomarkers concerning osteogenesis.


  Materials and Methods Top


The authors followed the criteria established in the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines for this review [Figure 1].
Figure 1: PRISMA diagram

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Patient Intervention Control Outcome

The question was based on the heterogeneity of the available data in the existing literature regarding immunocytochemistry and western blot techniques for the in-situ detection of biomarkers of osteogenesis. The following primary question was formed: How are immunocytochemistry and western blot techniques used for the in-situ detection of biomarkers of osteogenesis?

Search strategy

A systematic search was performed on PubMed and MEDLINE in June 2021 using the following search terms: “Osteogenesis” AND “Biomarker” AND “Protein” AND “Techniques” AND “Tooth” AND “Immunocytochemistry” AND “western blot” AND “Human.” Based on the findings, an additional manual search considering the references of the included studies was executed.

Inclusion and exclusion criteria

Only English-language full-text papers published between January 2016 and June 2021 were taken into consideration and only those papers examining the expression of osteogenesis in humans that specifically used immunocytochemistry and western blot test methods. The exclusion criteria were studies on animals and review papers. The whole systematic review process, including literature search, abstract/title/full-text screening, and extraction of data, was performed by two individuals.


  Results Top


The systematic research revealed 50 articles, and from those 50 articles, 20 articles were excluded after checking for duplication. From the remaining 30 articles, 10 articles were excluded because the research was not focussed on humans. From the remaining 20 articles, 8 articles were excluded because the full text was not available. After reading the final 12 articles, only 4 were selected for this review [Table 1].
Table 1: Paper result

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  Discussion Top


IGF-1 and IGF-1R

As a broad-spectrum growth promoter, IGF-1 plays a critical role in the formation of healthy teeth.[8] IGF-1 is a hormone that mediates the effects of GH and plays a key role in controlling somatic cell growth and development.[6] There are several ways that IGF-1 assists mesenchymal stem cells in the bone marrow in becoming osteogenic (bone-forming).[14]

Growth factors and paracrine signaling molecules such as fibroblast growth factor, transforming growth factor, bone morphogenetic protein, and IGF become mediators in the interaction between epithelial tissue and mesenchyme during tooth formation.[16] Osteo/odontogenic differentiation can be stimulated by many growth stimuli, including apical-papilla stem cells (SCAP).[16]

Insulin receptor (IR), IGF receptor 1 (IGF-1R), and IGF receptor 2 (IGF-2R) are three members of the IR family, which is an activator of tyrosine kinase second classes. The metabolic activity of vertebrates is affected by IR activation. The activation of IGF-1R causes cell proliferation and differentiation. IGF-2R is a monomer that lacks tyrosine kinase activity and is physically and functionally distinct from IR and IGF-1R.[17]

In vitro, IGF-1 acts as a mitogenic factor for MSCs.[18] IGF-1 can help dental pulp stem cells (DPSCs) proliferate and differentiate into osteoblasts. The stimulation of the mTOR signaling pathway, which leads to the overexpression of RUNX2, OSX, and OCN, has a stimulatory impact and can cause cascade of impact toward IGF-1 when the mTOR signaling pathway is blocked.[14]

Runt-related transcription factor 2

RUNX2 belongs to the RUNX family, which also includes RUNX1 and RUNX3. It contains the DNA-binding domain runt.[19] This gene is controlled by two separate transcription factors: the distal promoter (P1) and the proximal promoter (P2). This results in RUNX2 mRNAs with different 5' regions, type I and type II, which are generated by the proximal promoter and distal promoter, respectively. RUNX2 mRNA isoforms have different 5' ends, but their 3' ends are the same.[4]

RUNX2 is expressed in uncommitted mesenchymal cells, and its expression is increased in preosteoblasts, peaks in immature osteoblasts, and then drops in mature osteoblasts.[19] Overexpression of RUNX2 in chondrocytes speeds up chondrocyte development in all types of cartilages, including permanent cartilage, causing joint formation problems.[20]

RUNX2 controls chondrocyte proliferation via modulating IHH expression directly. It also plays a role in the etiology of osteoarthritis by determining whether chondrocytes become those that create transitory or permanent cartilage. RUNX2 is necessary for the development of osteoblasts as well as the proliferation of osteoprogenitors.[20]

Osterix

Osterix has long been known as an important transcription factor in the formation and mineralization of osteoblasts. According to the current research, OSX not only controls intramembranous bone formation, but also influences endochondral ossification by participating in terminal cartilage differentiation.[12]

It is expressed in osteoblasts and chondrocytes (although at lower levels) but not in osteoclasts.[21] Despite the fact that OSX is not expressed in osteoclasts, scientific studies have revealed that it has a number of effects on them. The receptor activator of NF-kappa B ligand (RANKL) signaling is the major determinant of osteoclast growth and activation, whereas osteoprotegerin protects bone from excessive resorption by binding to RANKL.[12]

Osteocalcin

OCN, one of the few osteoblast-specific genes, is the second-most-abundant protein in bone, being behind collagen type I. With significant upregulation in both matrix synthesis and mineralization, it is thought to play a crucial role in osteoblast cell growth.[21]

Non-collagenous protein OCN is the most common non-collagenous protein in bone.[11] OCN expression is regulated by RUNX2, an essential transcription factor for osteoblast development.[19]

In bone, OCN controls the alignment of biological apatite parallel to collagen fibrils, which is required for bone strength in the longitudinal direction of the long bone.[11]

Immunocytochemistry test on IGF-1

The detection of tissue components in situ via particular antigen–antibody interactions in which antibodies have been labelled with visible labels is known as an immunocytochemical test. Cell staining is an effective approach for revealing the presence and location of a specific chemical of interest within the cell.[22]

Immunocytochemistry is a method that uses antibodies to identify proteins or molecules in cells that can be seen with a microscope. By employing particular primary antibodies that bind to proteins or antigens, immunocytochemistry detects the expression of proteins or antigens in cells. Because of its specificity, this primary antibody may be seen under a fluorescent microscope when it interacts with a secondary antibody that has been conjugated with a fluorophore. The advantage of this method is that researchers can observe whether the cells in the sample express the antigen in question.[23] Immunocytochemistry does not require special equipment—for example, it can be performed using a light microscope, which is available in almost every laboratory.[24]

Immunocytochemistry differs from immunohistochemistry, which are based on the type of sample and the testing technique used. Cellular immunocytochemistry can be used to identify proteins, but its success depends on the specificity of antibodies that bind to the protein epitopes employed as immunogens in cells and tissues. His/her specificity is determined by the antibody and the technique utilized. Immunoblotting and immunoprecipitation are the best methods for determining antibody specificity. The steps performed during immunocytochemical testing include fixation, antigen retrieval, permeabilization, immunostaining, counterstaining, and mounting.[23]

In most cases, immunocytochemistry is conducted in four stages. This begins with a firm support, such as a glass slide or a glass bottom plate, where the cells are located. Prior to immunostaining, depending on the cell type and immunization procedure, an incubation period may be necessary. Adherent cells will attach to a firm support surface during incubation, which can take anywhere from half an hour to 24 h depending on the kind of cell. Antibody incubation and fixation are the next steps in immunostaining, followed by permeabilization and antibody incubation. In addition to preserving the protein’s location in the cell, fixation also preserves the protein’s chemical and structural state. Alternatively, the protein can be precipitated using organic solvents or by cross-linking the protein. An acidic solution or detergent is employed to puncture the cell membrane, allowing massive antibodies to flow through. Due to the fact that permeabilization is dependent on immobilization, the process is limited to dead cells. An antibody is allowed to attach to its target antigen in the cell during antibody incubation, and then the plate is washed to eliminate any unbound antibodies. In the third stage, a microscope is used to determine the position of the cells and antibodies attached to the target antigen. An image is taken with a camera or other detector, and then the cell structure is analyzed and annotated in the final phase.[25]

Immunocytochemistry streptavidin biotin peroxidase complex is a method that uses a biotin-labeled secondary antibody capable of recognizing primary antibodies (monoclonal or polyclonal antibodies), a streptavidin conjugate labeled with the enzyme horseradish peroxidase, and a mixture of chromogen substrates to detect antigens in cells or tissues with high sensitivity, so that even low levels of antigen can be detected.[24] This technique is used to examine IGF-1 and IGF-1R expression in cells.[10]

In the immunocytochemical test of IGF-1 using staining, it was found that there was IGF-1 expression in the cell nucleus. In addition to IGF-1, IGF-1R receptors were also found, although on the cell membrane rather than in the nucleus.[10] Staining of IGF-1R was expressed on ameloblast cells, odontoblasts, and dental papilla cells.[8]

Lee et al.[26] found that, on a polyamide film, there was a differentiation between osteogenic lines shown by OCN, OSX, and RUNX2 and the uncoated surface [Figure 2].
Figure 2: Sample of immunocytochemistry staining on IGF-1 (A), OCN (B), OSX(C), and RUNX2 (D)[26],[27]

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Western blot test on IGF-1

Through the use of gel electrophoresis, the western blot test is used to identify particular antibodies in proteins that have been sorted by size. It is made of nitrocellulose or polyvinylidene fluoride depending on the application. Proteins in the gel are induced to migrate across the membrane by an electric current when the gel is put on the membrane. After staining it using antibodies, it will produce a copy of the protein pattern.

The gel is loaded with material that has been prepared. Electrophoresis is a process in which a negatively charged protein travels toward a positively charged anode in a solution. Using a technique called blotting, the proteins are transferred to a membrane to be further analyzed. Membrane blocking is performed after the transfer to prevent undesired membrane–protein interactions in the subsequent phases. As a general rule, a primary protein-specific antibody is employed to probe the membrane, followed by a secondary antibody labeled with a fluorescent dye. A picture is taken and the outcome analyzed.[28]

The western blot test is a popular technique for identifying and quantifying particular proteins in complicated mixtures. This method enables the detection of protein samples immobilized on nitrocellulose membranes in an indirect way. The protein samples are first run with SDS–PAGE and then electrophoretically transferred to the membrane. After the blocking step, the membrane is probed with primary antibodies, both monoclonal and polyclonal, which increase in number compared to the antigen. After sequential washing, the membrane is then incubated with a secondary antibody conjugated with an enzyme that is reactive to the antibody. Finally, the membrane is re-washed with the substrate of the appropriate enzyme to produce a recordable signal.[28]

There are three parameters that influence the outcome of an immunoblotting experiment: the antibody’s affinity for the target protein, the intensity of its interaction, and its concentration. Also, other crucial parameters are selectivity and minimal cross-reactivity. As a result of post-translational changes (such as glycosylation) or interactions with other proteins, the results of a western blot test are not always straightforward to interpret.[28]

In a study conducted by Feng et al.[14] using the western blot test to examine the expression of RUNX2, OSX, and OCN proteins in the DPSC group treated with IGF-1 and left for 21 days, the expression of these three proteins increased, which indicates that the addition of IGF-1 can increase osteogenic differentiation in DPSC.

Lv et al.[9] found that when they administered IGF-1 to DPSCs in an attempt to determine the MAPK signaling pathway’s role in IGF-1-mediated differentiation, the IGF-1-added group showed an increase in RUNX2, OSX, and OCN expressions.

Advantages and disadvantages

The immunostaining detection method used in immunocytochemistry can be either direct or indirect. A primary antibody linked to the reporter directly targets the molecule of interest in the direct method, resulting in a rapid and precise procedure,[25] and the antibodies may be utilized for many studies. The downsides of direct methods include increased time consumption, lack of flexibility, and poor label detection.[23]

Unlabeled primary antibodies are used in the indirect approach to target the desired molecular structure. A secondary antibody that detects the primary antibody is used in the indirect technique.[25] It is less expensive to employ secondary antibodies than direct immunocytochemistry, and there is more labeling than with direct immunocytochemistry as many secondary antibodies can attach to a single primary antibody. The limitations to indirect techniques include the need for additional checks to ensure that secondary antibodies are binding to the correct primary antibodies, as well as the requirement for multiple primary antibodies to be generated from different species of animals for use in one experiment.[23]

Immunocytochemistry procedures are similar to immunohistochemistry and immunofluorescence in that they all employ antibodies to identify specific targets. Immunocytochemistry is often used to stain intact cells that have been isolated from the ECM, whereas immunohistochemistry is used to stain slices of biological tissue and immunofluorescence is used to stain microbiological cells.[29]

Using the western blot test to identify protein weights also has advantages and disadvantages. The main advantage is its specificity, as electrophoresis gel can sort sample proteins by size, charge, and formation. As well as electrophoresis gel, the specificity also depends on the antibody–antigen interaction, as specific antibodies show an affinity for specific proteins. The western blot test also displays great sensitivity, as it can identify even the tiniest protein in a sample and may be used as an early diagnostic tool in recognizing the slightest immunogenic response from a virus or bacterium. However, the western blot test can produce a false positive or false negative result. Other disadvantages are the high cost and required precision in every step for proper identification. The western blot test is a non-quotative test.[30]

The western blot test is sometimes compared with the ELISA test, as both are popular. Both are indirect tests that measure the immune system by detecting proteins. The ELISA test uses absorbance detection for protein and nucleic acid quantification, whereas the western blot test uses gel electrophoresis.[31]

In summary, both immunocytochemistry and the western blot test can detect biomarkers of osteogenesis. Immunocytochemistry uses antibodies from proteins and the cell that can be seen under a microscope, whereas the western blot test identifies the antibodies of the protein and sorts them by size using gel electrophoresis. Researchers can choose techniques to detect the biomarkers of osteogenesis according to their own needs, as both techniques are reliable.

The analysis was limited by the fact that numerous researchers utilized diverse approaches to detect osteogenesis biomarkers, including many new techniques, and that many of the research publications available focussed on animal cells.


  Conclusion Top


In conclusion, bone is a calcified connective tissue structure that forms during the proliferation and differentiation of osteoprogenitor cells into mature osteoblasts. IGF-1, RUNX2, OSX, and OCN play important roles in ossification. To study the expression of IGF-1, RUNX2, OSX, and OCN, researchers need to use different types of laboratory techniques, including immunocytochemistry and the western blot test. Both techniques can reliably detect the biomarkers of osteogenesis. In the immunocytochemical test, IGF-1 expression was found in the nucleus and IGF-1R expression in the cell membrane, and RUNX2, OSN, and OCN were shown to be important for osteogenesis. As for the western blot test, it was found that the addition of IGF-1 in dental stem cells could increase the expression of RUNX2, OSX, and OCN proteins. Thus, both immunocytochemistry and the western blot test can adequately detect the biomarkers of osteogenesis.

Financial support and sponsorship

Nil.

Conflicts of interest

There is no conflict of interest.



 
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