ORIGINAL RESEARCH |
https://doi.org/10.5005/jp-journals-10015-2237 |
Qualitative and Quantitative Evaluation of Cytokines in Gingival Crevicular Fluid of Children with Stainless Steel Crowns: A Clinico-biochemical Study
1,2Department of Pediatric and Preventive Dentistry, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS) (Deemed to be University), Chennai, Tamil Nadu, India
3Department of Pharmacology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai Tamil Nadu, India
Corresponding Author: Mahesh Ramakrishnan, Department of Pediatric and Preventive Dentistry, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS) (Deemed to be University), Chennai, Tamil Nadu, India, e-mail: maheshpedo@gmail.com
Received on: 06 May 2023; Accepted on: 07 June 2023; Published on: 22 August 2023
ABSTRACT
Introduction: Chemokines are chemotactic cytokines that draw particular leukocyte subsets into inflammatory tissues. The gingival crevicular fluid (GCF) serves as a diagnostic fluid to measure these biomarkers released during inflammation. The aim of this study was to evaluate the presence of macrophage inflammatory protein (MIP) 1α and 1β in children with stainless steel crowns (SSC).
Materials and methods: A total of 20 molars in children aged 6–10 years requiring SSC were included in this study (primary and permanent).
A total of 20 teeth (control)—same tooth with clinically healthy gingiva before tooth preparation for stainless steel crown.
A total of 20 teeth (control)—One month after placing the stainless-steel crown.
Gingival crevicular fluid (GCF) samples were collected using microcapillary pipettes from standardized sites of each molar, and quantitative measurement was done. Using the enzyme-linked immunosorbent assay (ELISA) kit, MIP-1α and 1β levels were determined.
Results: Results showed statistically significant differences between both groups (p-value of <0.005). The highest mean concentration of the cytokines was found in group II. It was present at a much lesser concentration in group I, confirming the association of these cytokines with inflammation.
Conclusion: Improperly placed or ill-fitting stainless-steel crowns often predispose to gingival inflammation, recruiting leukocytes into the gingival crevice. In children with SSC, GCF is a noninvasive diagnostic fluid for measuring chemokines like MIP-1α and 1β, which are biomarkers for identifying underlying inflammation.
How to cite this article: Gandhi JM, Ramakrishnan M, Thangavelu L. Qualitative and Quantitative Evaluation of Cytokines in Gingival Crevicular Fluid of Children with Stainless Steel Crowns: A Clinico-biochemical Study. World J Dent 2023;14(6):526-529.
Source of support: Nil
Conflict of interest: None
Keywords: Biomarkers, Gingival crevicular fluid, Inflammation, Macrophage inflammatory protein, Stainless steel crowns.
INTRODUCTION
At inflammatory sites, a variety of inflammatory cell types, including neutrophils, monocytes, and lymphocytes, as well as noninflammatory cell types, release chemokines and cytokines.1 Higher amounts of proinflammatory cytokines have been seen in the serum and gingival tissues of people with periodontal inflammation, which may play a role in the emergence of a more widespread hyperinflammatory condition.2,3 Due to their direct connection to the pathophysiology of inflammation in soft tissues, cytokines have been linked to the initiation and progression of dental caries. Numerous disorders are associated with elevated cytokine levels, which also mediate local and systemic inflammatory responses.2 Salivary cytokines could result from gingival crevicular fluid (GCF) leaking into the oral cavity during periodontal probing, certain subgingival restoration, and crown preparation. Stainless steel crowns (SSC) are pivotal and widely accepted in pediatric dentistry and are indicated after pulpectomy or in class 2 cavities as full-coverage restorations.4 In teeth with steel crowns that have been restored, it has been discovered that there is a significant correlation between gingivitis and the dental biofilm around these crowns.5 Hence, gingival health is affected by the presence of biofilm around SSC. In order to maintain good periodontal health, there should be adequate marginal adaptation of the SSC. These stainless-steel crowns are supposed to be evaluated clinically and radiographically before the cementation. Poorly fitting crowns or improper marginal adaptation can lead to the accumulation of plaque, which would then contribute to gingival inflammation.6 Macrophage inflammatory protein (MIP) 1α and 1β are significant immune system cytokines that are essential to the body’s antibacterial defense system. Considered novel biomarkers in biological defense systems, MIP1α and MIP1β chemokines, both of which have been shown to be potent chemoattractants for inflammatory mediator cells.7 Both cluster of differentiation (CD) 4 and 8 lymphocytes are attracted to inflammatory proteins in a specific manner. MIP1α towards CD8 lymphocytes and MIP1β towards CD4 cells. The amount of GCF was shown to rise as the degree of inflammation increased. Maintenance of oral hygiene also plays a pivotal role in this aspect. While children with good oral hygiene retained healthy gingiva surrounding SSC, children with poor oral hygiene showed a greater incidence of gingivitis. Hence, the need of this study was to evaluate whether the stainless-steel crowns, used so widely in pediatric dentistry, contributed to measuring chemokines like MIP-1α and 1β, which are biomarkers for identifying underlying inflammation.
The aim of this study was to evaluate the presence of MIP-1α and 1β in children with SSC.
MATERIALS AND METHODS
Study Design
The study is a randomized study design clinical trial that followed the standards published by Consolidated Standards of Reporting Trials.
Sample Size Calculation
The sample size for the present trial was based on the Using G*Power 3.1.9.2 software for power analysis indicated that we required a total of 20 participants requiring SSC. With a power of 95%, the sample size was calculated.
Recruitment of Participants
The children were chosen for this study based on the predetermined inclusion and exclusion criteria. Prior to their enrollment, when the patient was found suitable for the study, written informed consent was obtained from the parents or the accompanying guardians. The parents were also explained in detail regarding the nature of the study, treatments given and follow-up period, advantages and risks that may present during the treatment. The parents or guardians were also made aware that they were free to withdraw themselves from the study without any undue effect on the course of the treatment required. Each step of the randomized trial was done and reported based on the “CONSORT” guidelines framed for randomized clinical trials.
This clinical study was conducted in the Department of Pediatric and Preventive Dentistry of Saveetha Dental College and Hospitals, Chennai, from May to December 2022; after receiving ethical approval from the Institutional Review Board. Children aged 6–10 years requiring SSC were included in this study (mixed dentition period). A total of 20 teeth were assessed, which included 12 primary and eight permanent teeth.
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A total of 20 teeth (healthy control group): Tooth with clinically healthy gingiva indicated for SSC.
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A total of 20 teeth (test group): Same tooth 1 month after placing the SSC.
Clinical Procedure
Appropriate tooth preparation was done with occlusal reduction of 1–1.5 mm, followed by proximal reduction. Buccal and lingual surfaces were reduced only if required, and the “snap-fit” of crown placement was checked using the trial-and-error method to select the ideal crown. After placing a 1–3 mL calibrated volumetric microcapillary pipette (Sigma-Aldrich Chemical Company, United States of America) allowed for the collection of a volume of 1.5 mL GCF from each site. GCF was collected from the proximal sites around the gingival sulcus of teeth before placing the stainless-steel crown (group I) (Fig. 1) and with a stainless-steel crown (group II) (Fig. 2). Enzyme-linked immunosorbent assay (ELISA) test was performed to determine the chemokines, MIP-1α and 1β present in the GCF samples. Three investigators were included in this study, two who were entirely conducting the study and 1 investigator from the Department of Microbiology who carried out the ELISA test in the lab.
Fig. 1: Control group (group I)—a collection of GCF in healthy gingiva before placement of SSC
Fig. 2: Test group (group II)—a collection of GCF 1 month after placement of SSC
Dental plaque was measured using a sterile periodontal probe according to Silness and Loe plaque index. Scoring was from 0 to 3 and was based on the Silness and Loe plaque index, where score 0 indicated the absence of microbial plaque and score 3 indicated a large amount of plaque in the sulcus or pocket along the free gingival margin. Gingival indices were also checked for both groups to assess the presence/absence and severity of gingivitis. The scoring was from 0 to 3, where score 0 indicated normal gingiva; natural coral pink gingiva with no evidence of inflammation and score 3 indicated severe inflammation; marked redness, edema or ulceration, and tendency to bleed spontaneously.
Data analysis was done using Statistical Package for the Social Sciences software (version 20). Multiple comparisons were analyzed by the Kruskal–Wallis test.
RESULTS
All the participants of this study included children aged 6–10 years (mixed dentition period) with equal participation of boys and girls requiring SSC. A total of 20 teeth were assessed, which included 12 primary and eight permanent teeth.
Macrophage inflammatory protein (MIP) 1α and 1β inflammatory cytokines were assessed in both groups. The average concentration of MIP1α in the GCF for group I was 287.85 ± 42.20 and 682.55 ± 61.84 in group II. MIP-1β was 417.85 ± 40.83 in group I and 814.55 ± 59.97 in group II. Results showed a statistically significant difference between both groups (p-value of <0.005) (Table 1). The highest mean concentration of the cytokines was found in group II.
| Parameters | Frequency | Mean ± standard deviation (pg/μL) | p-value |
|---|---|---|---|
| MIP1α | 0.001* | ||
| Group I | 20 | 287.85 ± 42.20 | |
| Group II | 20 | 682.55 ± 61.84 | |
| MIP1β | 0.001* | ||
| Group I | 20 | 417.85 ± 40.83 | |
| Group II | 20 | 814.55 ± 59.97 |
*p-value of 0.001, statistically significant data
Plaque index and gingival index were also measured immediately after crown placement; T1 and 1 month postoperatively; T2. Grades 1 and 2 of plaque index were found in all teeth 1 month after crown placement, and gingival index showed grade 1 changes only in T1; immediately after crown placement (Table 2).
| Plaque index | T1 | T2 |
|---|---|---|
| Grade 0: No plaque | 18 | – |
| Grade 1: A not visible thin coating of plaque that is only visible after using the probe | 2 | 14 |
| Grade 2: Moderate accumulation of plaque, visible with the naked eye but not filling interdental space | – | 6 |
| Grade 3: Abundance of plaque, filling interdental space | – | – |
Based on these results, it can be inferred that inflammatory cytokines are increased immediately and 1 month after crown placement, gingival index changes were seen immediately after crown placement, and plaque accumulation was most after 1 month of placement (Table 3).
| Gingival index | T1 | T2 |
|---|---|---|
| Grade 0: No inflammation | 4 | 18 |
| Grade 1: Mild inflammation, a slight change in color, slight edema, no bleeding on probing | 16 | 2 |
| Grade 2: Moderate inflammation, moderate glazing, redness, bleeding on probing | – | – |
| Grade 3: Marked redness and edema, ulceration with a tendency to spontaneous bleeding | – | – |
DISCUSSION
Stainless steel crowns (SSC) have been the most widely used restorative option for primary teeth due to their durability and longevity. The ideal placement of SSCs on primary teeth requires a “snap-fit,” with the margins of the crown accurately adapted to the surrounding tissues with a supra or equigingival preparation.4 Smoothening and polishing the margins of the crowns after crimping and contouring prevents corrosion and plaque accumulation.4,5
In this study, GCF was collected and evaluated in teeth with SSC. It was found that group II had proportionally higher mean MIP-1α and MIP-1β GCF concentrations, and these values exhibited favorable relationships with clinical parameters. The influx of particular leukocyte subsets into the gingival crevice in response to plaque deposition around the stainless-steel crowns is one of the potential causes of the elevated GCF levels of MIP-1β.
The present study correlates with Kumar et al.,7 which evaluated chemokines in GCF of children with dental caries and stainless-steel crowns. The findings were in correlation with this study as chemokines were found to be increased in patients with SSC. Another study with similar findings found an increased presence of chemokines in patients with band and loop space maintainers.8 Studies with findings contrary to this study were by Checchio et al.,9 in which ill-fitting crowns or bands showed no relationship with increased plaque accumulation leading to increased GCF levels and similarly in another study by Emingil et al.,10 where MIP-1α and MIP-1β levels in GCF in samples with gingivitis did not differ significantly.
As mentioned in the results, the highest mean concentration of MIP-1α and 1β were found after crown placement which is due to the inflammatory response caused by the crown placement leading to elevated GCF levels. It may be possible to use measures to stop tissue deterioration caused by ill-fitting crowns in children by minimal or no subgingival preparation and accurate adaptation of crowns. Appropriate tooth preparation should be carried out, which should include sufficient occlusal reduction of 1–1.5 mm to avoid significant occlusal prematurity and proximal reduction to allow the crown to be seated beyond the maximum bulbosity of the crown. The occlusal reduction should follow the contours of the tooth. The preparation should finish with a smooth feather-edge cervically with no step or shoulder. The preparation should be rounded off with no sharp line angles. Spedding described two principles pertaining to length and gingival margin. Appropriate occlusal-gingival crown length should be followed, and crown margins should be contoured circumferentially to follow the natural contours of the marginal gingiva of the tooth. The goal is to extend the crown 1 mm beneath the free margin of the gingival sulcus and to approximate the gingival margins of the crown to the gingival crest around the tooth.11 Choosing the correct size of crown is assisted by measuring the mesiodistal dimension of the tooth with dividers or a graduated periodontal probe.4,12 The selected crown should have a snap-fit. This would then inhibit the accumulation of leucocyte subsets in the GCF.
The novelty of this study is that it is the only clinical study that evaluates the presence of cytokines in a GCF of both primary and permanent teeth with SSC. To avoid drawbacks from the previous studies similar to this study, the teeth examined served as self-control; GCF collection was done in the same tooth with clinically healthy gingiva before placement of stainless-steel crown. There are certain limitations also in this study, such as the sample size being lesser in number, and the other limitation was that presence of only a few cytokines was evaluated; further studies can be done with a larger sample size and with the evaluation of the presence of various other cytokines.
CONCLUSION
Stainless steel crowns (SSC) that are ill-fitting or improperly positioned frequently contribute to gingival inflammation by attracting leukocytes to the gingival crevice. In children with SSC, GCF is a reliable noninvasive diagnostic fluid for measuring chemokines like MIP-1α and 1β, which are biomarkers for identifying underlying inflammation.
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