Analysis Of The Biomarkers Found In Different Pulpal And Periradicular Diseases

Introduction

A vital tooth presents, intact blood supply which in turn maintains nutrition, innervations and immune competency. An assault on pulpo-dentinal component results in an inflammatory reaction, leading to either repair or pulp degeneration. Various vital pulp therapies, like indirect/direct pulp capping, pulpotomy, and partial pulpotomy are carried out in presence of specific indications. The natural progression of these procedures leads to, either healing or degeneration of the pulp, undertaking a lengthy course.

The clinical diagnosis of pulpal and periradicular diseases, is primarily based upon clinical findings, radiographic presentation and findings of the specific test. Definitive diagnosis of these conditions can only be made on histopathologic examination, which is not possible in most of these situations. Identification of biomarkers of the pulpal and the periradicualr diseases could be of immense help in such situations. Based upon the identification of these biomarkers, the progression of vital pulp therapy may be predicted. Similarly, these biomarkers could be a definitive pointer towards the presence of specific disease. Hence the awareness of the specific biomarkers of a disease, would help in identifying and correlating the signs and symptoms with the presence of a particular disease.

During the inflammatory play out, specific molecules are exposed at the site. Various pulpal events can be gauged through measurable levels of protein makers, which when related with the flow of pulpal blood, dentinal fluid, periapical fluid and gingival crevicular fluid could point towards the underlying disease.

This review study was carried out, so as to identify the various biomarkers found in different pulpal and periradicular diseases.

Materials and methodology

Literature regarding biomarkers of various pulp and periradicular diseases was searched on PubMed, Google, EBSCO, Research Gate and Medline databases for articles, using the key words Biomarkers, Inflammatory mediators, Pulpal periradicular inflammations.

Inclusion and Exclusion: Articles that met the following criteria were included in the final selection: articles that concerned human permanent mature teeth, articles with a clear clinical diagnosis of inflammatory pulp disease and studies that included a search for inflammatory mediators, and articles that were published in English. Exclusion criteria were applied to disqualify the following articles: an article reporting a technical development, studies in which the clinical diagnosis was not described and a study with immature teeth. Biomarker and their concepts A biomarker (biologic marker) is a measurable indicator of a specific biological state, particularly one relevant to the risk for, the presence of, or the stage of a disease. The National Institutes of Health (NIH) Biomarkers Definitions Working Group, defined a biomarker as, “a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention".

According to the National Library of Medicines, the medical subject heading term (MeSH term) definition for a biological marker is a measurable and quantifiable biological parameter that serves as an indicator for health- and physiology-related assessments.

Biomarkers are biochemical substances expressed by tumor cells or normal body cells either due to the cause or due to effect of the malignant process. They are substances that can be detected in higher than normal amounts in the blood, urine, or body tissues of patients with certain type of conditions. A biomarker may be produced by tumour/ infection itself or by the body in response to the tumour. They may be present as intracellular substances in tissues or may be released into the circulation and appear in serum.

On the basis of numerous studies, following were found to be widely accepted biomarkers

Matrix metalloproteinase (MMP)-9 (MMP-9), it directly degrades extracellular matrix (ECM) proteins and activates cytokines and chemokines to regulate tissue remodeling.

• Interleukin-2 (IL-2) is a type of cytokine signaling molecule in the immune system. It is a protein that regulates the activities of white blood cells (leukocytes, often lymphocytes) that are responsible for immunity.
• Interleukin 6 (IL-6), promptly and transiently produced in response to infections and tissue injuries, contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions.
• Interleukin-8 (IL-8) is a chemoattractant cytokine produced by a variety of tissue and blood cells. It attracts and activates neutrophils in inflammatory regions.
• Interleukin 10 (IL-10) is a cytokine with potent anti-inflammatory properties that plays a central role in limiting host immune response to pathogens, thereby preventing damage to the host and maintaining normal tissue homeostasis.
• Tumor necrosis factor (TNF, tumor necrosis factor alpha, TNFα, cachexin, or cachectin) is a cell signaling protein (cytokine) involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction.
• Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, innterferons are released by a virus-infected cell, enabling nearby cells to heighten their anti-viral defenses.
• Dentin matrix acidic phosphoprotein 1 is a protein that in humans is encoded by the DMP1 gene. This protein is critical for proper mineralization of bone and dentin, and is present in diverse cells of bone and tooth tissues.
• Extracellular matrix (ECM) is a collection of extracellular molecules secreted by support cells, that provide structural and biochemical support to the surrounding cells.

For collection and analyzing the levels of various biomakers, the following analysis can be used

Gingival Cervicular Fluid: can be collected by various techniques.

A) Absorption Method: Either paper strips or paper points are used to collect the sample. They are inserted into gingival sulcus or peridontal pockets. The time to which the paper strips or paper points are left in the sulcus varies between 30 seconds and 1 minute.

B) Microcappillary pipetting: is done by keeping the micro capillary pipettes at the entry of the pocket. The lengthy duration needed is considered as one of the limitations of this technique, which could increase the possibility of saliva and blood contamination.

C) Washing method: due to its technique sensitive difficulties and the high rate of blood contamination, this method is not preferred.

Pulpal Blood: After the isolation of the tooth with a rubber dam, caries is removed, and the pulp is exposed with an excavator and/or a low-speed round bur. Blood from the exposed surface of the pulp is collected with a sterile cotton pellet. The pellet is held at the exposed site for 45–60 seconds to allow absorption of the pulpal blood. The pellets are then placed in 1 mL saline in heparin-coated tubes.

Periapical Fluid: After drying the root canal with sterile paper points, a fine paper point is inserted approximately 2 mm above the apical foramen, to collect the periapical tissue fluid. The paper point is kept in that position for 30 s and then transferred into a sterile micro-centrifugation tube and immediately after frozen at 80 C until further processing.

Methods to isolate biological markers are Immunohistochemistry (IHC), Polymerized Chain reaction (PCR), EnzymeLinked ImmuneSorbent Assay (ELISA) techniques, Reverse transcription polymerase chain reaction (RT-PCR), Multiplex assay, Microarray, Western Blot, Radioimmunoassay (RIA), Zymography, Flow cytometry, Limulus amoebocyte assay (LAL), and specific enzyme assays. The diagnostic efficiency of biomarkers depends on variety of factors relating to its sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV).

1. 1) ELISA (Enzyme-Linked Immunosorbent Assay): It is a technique used to detect the presence of an antibody or antigen in a variety of samples. This assay can be used to analyze a specific analyte or screen for groups of compounds that cross-react with the antigen that is coated on a solid support surface (e. g. , microtiter plate). Even though background reduction and signal enhancement are potential limitations of ELISA analyses, these assays demonstrate a high degree of specificity and sensitivity.
2. 2) Western Blot: It is a core technique in cell and molecular biology. It is used to detect the presence of a specific protein in a complex mixture extracted from cells or tissue. In this method, a gel is used as electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide, followed by an electrophoretic transfer onto a membrane and an immune staining procedure to visualize a certain protein on the blot membrane.
3. 3) Immunohistochemistry (IHC): It is a lab test that uses antibodies to test for certain antigens (markers) in a sample of tissues. The antibodies are usually linked to an enzyme or a fluorescent dye. When the antibodies bind to the antigen in the tissue sample, the enzyme or dye is activated, and the antigen can then be seen under a microscope.

Various Conditions and Biomarkers: Specific Condition Biomarker Sample collection Method Irreversible pulpitis Matrix metalloprotinase 9 Dentinal fluid MMP-9 assay Irreversible pulpitis IL2, IL6, IL8, IL10, TNF-a, IFN-c Pulpal blood Elisa Reversible pulpitis Dentin matrix acidic phosphoprotein 1 Extraction of teeth and pulp extraction Immunohistochemistry Periradicular Cyst Extracellular matrix molecules Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues Microarray analysis Pulpal necrosis Interleukin 18, 1, 6 Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues Microarray analysis CondensiveOsteitis Fox P3 primarily infected root canals with apical periodontitis Enzyme-linked immunosorbent assay.

Discussion

Diagnosis of the various pulp conditions is largely dependent upon the clinical findings and the findings of the test carried out for the confirmation. Role of biomarkers, as a confirmatory agent and predictor of the prognosis has been accepted, since long. Irreversible pulpitis tends to display TNF-α as an important biomarker. This agent assumes importance since its expressions leads to inhibiting pulp repair and induces apoptosis. It promotes odontoblast survival in chronic diseases, at the same time inhibitting differentiation of progenitor II and odontoblasts. Yang et al found, TNF-α to promote apical papillae cell mineralization in acute conditions but inhibitting mineralization in chronic conditions. Boyle et al found that short term exposure of cells to TNF, induces apoptosis with Vascular Endothelial Growth Factor up-regulation and nuclear factor kappa B signalling.

On the contrary, long-term exposure increases proliferation and shortens the telomere. TNF-α triggers neutrophils to release large amounts of reactive oxygen intermediates, promoting neutrophil degranulation. Although the underlying mechanism is still not clear, TNF- α plays a crucial role in neutrophil survival, through induction of apoptosis. IL especially IL-2, IL-6, IL- 8 and IL-10 can be used as a biomarker in irreversible pulpitis. IL-8 is produced by numerous cells like macrophages, lymphocytes, fibroblast and endothelial cells. Odontoblast exhibit low level of interleukin which increases with pathogens associated molecular pattern stimulation. Thus the increased IL-8 level is the diagnostic of irreversible pulpitis. The elevated level of IL-8 is correlated with enhanced production of PNM within the pulp. IL-8 induces neutrophil hemostasis and release of degradation enzymes as a result of degranulation. IL-8 is the primary regulatory molecule in acute inflammatory response, thus it is correlated as a biomarker in irreversible pulpitis. Its elevated levels, precipitate and exacerbate the acute inflammation response hence justify its enhanced level within the pulp during irreversible pulpitis.

IL-6 is a pro inflammatory cytokine produced mainly by lymphocytes, plasma cells, monocytes and macrophages that plays a role in the immune response and is associated with inflammatory diseases such as rheumatoid arthritis, periodontal diseases and endodontic lesions. An up-regulation of adhesion molecules is caused by IL 6 and induces angiogenesis, leading to increase in vascular permeability and inflammatory oedema. In addition, it induces osteoclast differentiation and bone resorption. The higher levels of IL-6 have been detected in pulpal tissue from irreversible pulpitis and from periapical lesions. A high level of mRNA is detected in irreversible pulpitis when the lesion is larger or symptomatic, recommending that IL-6 might augment tissue injury at the inflammatory site, and perhaps signaling an active state of inflammatory periradicular disease. A cytokine network is activated in response to bacterial irritation and plays a major role in the progress of periapical pathogenesis. As pulpal symptoms are generally explained by increase in intra-pulpal pressure due to oedema, levels of IL-6 can be correlated to the extent of inflammation and oedema in the pulp, in addition to its role as a mediator of host response following tissue injury and infection. Interleukin-10 (IL-10) is an anti-inflammatory cytokine, produced by various immune cells including macrophages and Th2 cells. It is the main inhibitor of cytokine synthesis and macrophage activity. It also inhibits the production of the pro inflammatory cytokines like TNF-a, IL-1b and IL-6 in various cell types. IL-10 production increases during inflammatory processes exerting an immune modulatory role. It has been reported to be an important endogenous modulator of infection-stimulated periapical bone resorption. It can contributes to the maintenance of bone mass through inhibition of osteoclastic bone resorption and regulation of osteoblastic bone formation. Higher levels may indicate that the pulp is attempting to suppress inflammation before it reaches an irreversible stage, as in other inflammatory diseases.

Interleukin-18, is primarily produced by allophycocyanins (APCs) and also by osteoblasts, adrenal cortex cells and oral epithelial cells. It is found to be up regulated in various chronic inflammatory diseases. It could play a significant role in progression of periapical disease because of its chemotactic, pro inflammatory, and angiogenic properties. It can also increase the rate of neutrophil activation. MMP-9 has been identified as another biomarker for irreversible pulpitis. MMPs are responsible for extracellular matrix degranulation and are essential in tissue chromostatin. They play an important role during pulpal inflammation, altering the cellular adhesive properties, thereby aid in cellular migration. Destruction of pulp tissue is, in part, regulated by MMPs and tissue inhibitors of these MMPs (ATIMDS). Combination of these, stimulates the protective MMP-8 adaptive MMPs response, as well as precipitates tissue destruction. MMP-9 is considered as biomarker of the pulp tissue breakdown. Its high levels are found in gingival cervicular fluid and serve as a marker of connective tissue breakdown: as seen in periodontal destruction. MMP-9 is secreted primarily by PMN and since pulpitis is a PMN derived inflammatory condition, the former serves as an established biomarker. It was found by Pugin et al that TNF-α and IL-8 can induce rapid MMP-9 zymogens release in whole blood.

Neutrophils which are a primary pillar against pulpal infection, during this process can induce irreversible tissue damage. MMP-s released by neutrophils recruit immune cells to the pulp and release oxygen inter-mediators like superoxol, hydrogen peroxide, hydroxyl radials and other highly potent enzymes. Regular tissue necrosis is thus initiated and is evidenced by micro-abscess formation, leading to pulp necrosis. In CondensiveOsteitis, POX P3 has been implicated as a biomaker. The POX P3 protein binds in specific sites in DNA and regulates the genes involved in governing the immune system. The pox p3 protein thus alters the immune response towards bacteria and virus present in the periapical tissue, leading to response elevated bone production, thus producing radio graphically visible opacity.

IL-1 family members are known to alter the host response to an inflammatory, infectious, or immunological challenge. The four best known members of this family are IL-1a/b, IL-1Ra, and IL-18. IL-1a/b and IL-18 have high inflammatory potential, and dysregulation of their expression can lead to severe patho-biological effects. Accordingly, the expression of these cytokines is highly regulated via both soluble receptors (e. g. , type 2 IL-1 receptor) and natural antagonist proteins (e. g. , IL-1Ra and IL-18 binding protein) as well as alternatively spliced forms of both ligands and receptors. Increased levels of MMP-2 and -9 have been reported in teeth with apical periodontitis and in wall extracts from jaw cysts. Both gelatinases, gelatinase A (MMP-2) and B (MMP-9), are synthesized by both fibroblasts and pulp cells, and their presence favours the degradation of type IV collagens/gelatins (main components of the basement membrane) as well as denatured gelatins, lamins, elastins, fibronectins, and basement membrane zone–associated components.

IL-1 has been established as a biomarker for pulp necrosis. It has precise high inflammatory potential and disregulation of its expression can lead to serve pathobiological effects. IL-6 and IL-18 are the other biomarker for pulp necrosis, owning to the high potentional as a inflammatory mediator.

In apical periodontitis high levels of mmp-2 and mmp-9 have been observed. Their presence favour the degredation of type-IV collagens oblique gelatins (main component the basement memberance). Hence mmp-2 and mmp-9 are considered as biomarkers for apical periodontitis. The mini MIRNS are found to have a higher level of expression in diseased periapical tissues, as compared to healthy pulp. The high sequence conservative across the spceies and tissue specificity make them an idea biomarker. They are seen as a biomarker for endodontics periapical conditions.

In periapical granuloma IL-4 and INTERFERON –ALPHA in radicular cyst are found to have a high level of expression, leading to their status as biomarkers in the respective conditions.

Conclusion

Biomarker is a measurable indicator of the severity or presence of a disease where, clinical evaluation fails to diagnose accurately. In application, Biomarkers consist of various tools and technologies that aid in understanding the prediction, cause, diagnosis, progression, regression or outcome of treatment of diseases. Early diagnosis and management reduce the severity, possible complications of the disease process and plays a cranial role in successful therapy.