Efficacy Of Proinflammatory Cytokines in The Primary Molar Pulpotomy

One of the main goals of pediatric dentistry is to preserve the functions and structures of primary teeth until the time of exfoliation. Especially carious lesions are the main factor in the early loss of primary teeth. Today, primary teeth can be kept in the mouth by applying different treatments depending on the degree of inflammation caused by carious lesions in the pulp tissue.Coronal pulpotomy application in asymptomatic primary teeth with deep carious lesions near the pulp is one of the most common methods to achieve the goal of retaining the tooth in place. The purpose of the pulpotomy technique is to remove the affected pulp tissue and maintain the normal function of the unaffected root pulp tissue until the tooth is naturally ready to exfoliate.

Studies have shown that the degree of pulp inflammation and the materials used are effective in the success of this treatment. In studies evaluating pulpal inflammation, many biomarkers have been shown to play significant roles at different levels of inflammation. Recently, Presepsin has been studied as a biomarker for detecting bacterial infections. However, there is no study in the literature on the use of Presepsin as a biomarker in endodontic treatments. In our study, it is thought that Presepsin biomarker could be detected in cases of acute or chronic infection in pulp tissue and could be considered as one of the mediators of pulpal inflammation.

Based on this, the aim of our study is to investigate whether the materials used in covering the pulp or the level of inflammation in the remaining pulp tissue is more important for the success of pulpotomy treatment. The inflammation level in the remaining pulp tissue will be measured using IL-6, IL-8, and Presepsin. Then, the one-year success of treatment in different groups where pulp tissue is randomly covered with MTA, NeoMTA, Biodentine, and Zinc oxide eugenol will be demonstrated. Thus, it will be evaluated whether materials previously found to be quite successful in other studies achieve success in pulps with high inflammation levels.

Tooth decay is a commonly encountered chronic infectious disease in children. Coronary pulpotomy treatment is generally preferred for teeth with deep decay lesions that are clinically and radiographically asymptomatic. The success of this treatment method is highly dependent on the inflammatory status of the pulp and the materials used. In predicting the clinical success of pulpotomy treatment, the severity of pulp inflammation must also be evaluated. A molecular approach that aids in the clinical distinction between reversible and irreversible pulpitis can increase the success rate of vital pulp therapy by accurately assessing the inflammation status of the pulp. Although various mediators' increases in advanced pulpal inflammation have been examined in many previous studies, the role of many biomolecules involved in pulpal inflammation has not yet been determined. Due to the complex nature of inflammatory pulp disease, it is unlikely that a small number of mediators would provide sufficient information to diagnose advanced inflammatory disease. Therefore, studies aimed at identifying new mediators remain relevant in dentistry. Presepsin, discovered in 2004, is a biomarker with high sensitivity and specificity that is highly important in the early diagnosis of bacterial diseases in medicine. When dental literature is reviewed, it is observed that presepsin has not been evaluated for the detection of bacterial inflammation in the pulp, and it is believed that this biomarker could be used for the early detection of pulpal inflammation. Thus, with the ability to measure presepsin levels using patient kits, pulpal inflammation can be quantitatively evaluated, aiding in the provision of accurate pulp treatments.The aim of our research is to investigate whether presepsin, a new biomarker that can be rapidly detected in bacterial infections, can also be used as a mediator in the detection of pulpal infections, and to evaluate the levels of the reliable markers of pulpal inflammation, IL-8 and IL-6, comparing the effect of these mediators on the success of treatment with the most successful materials used in pulpotomy treatment, MTA, Neo MTA, Biodentine and Zinc oxide eugenol.

This study was conducted in accordance with the Ataturk University Faculty of Medicine Ethics Committee approval (Date: 05.2021/ Session no:04/ Decision no:66) and guidelines of the Helsinki Declaration. Also the parents of child patients were comprehensively briefed about the procedures before the treatment and their written consents were obtained. Initially, a total of 75 children aged 4-9 years old (36 girls and 39 boys) with no systemic disease and presenting at least one primary second molar indicated for pulpotomy were included in the study and 4 pulpotomy agents (MTA, Biodentine™, NeoMTA™ and ZOE) were randomly divided into four study groups of 30 teeth and pulpotomy was applied to a total of 120 teeth.

In order to randomly distribute the study groups, Research Randomizer (Version 4.0) computer software from http://www.randomizer.org/ was used to generate a list before starting pulpotomy and pulpotomy agents were used accordingly.

Protocols for Treatment and Sample Storage:

For a better visibility a rubber-dam is placed over the tooth requiring pulpotomy following local anesthesia. Endodontic cavity was accessed by first removing the carious tissue, unroofing the pulp chamber with diamond round bur (no:18), removing the coronal tissue using a sterile tungsten carbide round bur (no:14) mounted low-speed contra-angled handpiece and clearing the remaining pulpal tissue with an excavator.

Before managing the pulpal hemorrhage, a sterile cotton pellet was placed over the pulp tissue for 30-45 sec. in order to obtain pulpal blood samples and specimens were collected into heparin coated tubes (Vacutainer, BD,USA) with 1mg saline solution and they were stored at -20 oC (-4.0°F) for 6 months until the day of testing.

Then pulpal hemostasis was achieved by placing 0.5 % NaOCl over the canal orifices for 30 sec. Primary molars, in which pulpal hemorrhage was controlled were randomly assigned to 4 different study groups (ProRoot MTA, Biodentine™, NeoMTA™, and ZOE respectively) and they were treated with pulpotomy. Glass ionomer cement was applied over all pulpotomy medications used in the study and pulpotomized teeth were restored with stainless steel crowns (SSCs).

Thawing Process and Assessment:

Samples were thawed at room temperature and centrifuged at 4000 rpm for 15 min. Then cotton pellets were removed from the test tubes and levels of presepsin, IL-6 and IL-8 were measured using enzyme-linked immunoassay (ELISA) \[Human presepsin ELISA kit (Cat.No:E3754Hu, BT LAB,China),Human IL-6 ELISA Kit (Cat.No:E0090Hu,BT LAB,China), Human IL-8 ELISA Kit (Cat.No:E0089Hu, BT LAB,China)\] test in accordance with the manufacturer's instructions. Kit measurement ranges for presepsin, IL-6 and IL-8 were 5-1000ng/L, 2-600ng/L and 5-1000ng/L respectively. The inter- assay CV were \<10% for all biomarkers.

Clinical \& Radiographic Assessment Method:

Primary second molars were assessed clinically and radiographically at the post- treatment 6th and 12th months.

Spontaneous or stimulated pain, swellings in vestibular sulcus, fistula, pathological mobility, sensitivity to percussion and palpation, regional lymphadenopathy adjacent to the pulpotomized teeth were clinically assessed and presence of any of these symptoms were regarded as failed pulpotomy.

Radiolucency in the periapical or furcation areas, internal and external root resorptions, calcified root canals, widened periodontal ligament were radiographically evaluated and presence of any of these symptoms were also regarded as failure.

Additionally, presence of lesions in periapical or furcation areas in radiographs were also accepted as failure and these teeth underwent root canal treatment or extraction. Furthermore, teeth with internal root resorption were regarded as radiographically failed and they were just followed-up until any symptom occurred.

Statistical Analysis Descriptive statistics (count, ratio, mean, standard deviation, minimum and maximum variables) were used to summarize the set of data in the present study. Fisher's exact test was employed to analyze the relationship between pre-treatment clinical findings and dental pathologies developed at the post-treatment 12th month when pathologies became more apparent. Relationship between the mean levels of pulpal inflammatory biomarkers and dental pathologies occurred at the post-treatment 12th month was analyzed using Mann-Whitney U test. Mann-Whitney U test was also used to compare the mean levels of inflammatory biomarkers and clinical findings. Kruskal Wallis test was used to compare the differences between the mean scores of inflammatory biomarkers by the pulpotomy agents used.

Threshold values were calculated with Receiver Operating Characteristics (ROC) analysis and then these obtained values were used to determine the highest biomarker scores in the pulpotomy material groups. All study analysis were performed with IBM® SPSS® Statistics 25 at a significance level of 0.05.

Eligibility Criteria:
Inclusion Criteria:

1. Healthy children between the ages of 6-9
2. Children who do not have any systemic disease
3. Primary second premolars in which physiological root resorption has not started.
4. Cases where tooth decay at the interfaces exceeds ½ of the dentin thickness.
5. Primary second premolars with provoked pain

4. Lack of percussion sensitivity 5. Absence of radiolucent image in the periapical and furcal areas on x-ray 6. Clinical absence of abscess and fistula

Exclusion Criteria:

1. Children with systemic diseases
2. Having percussion sensitivity
3. Radiolucent appearance in the periapical and furcal areas on x-ray
4. Clinical presence of fistula
5. Having night pain