|Year : 2016 | Volume
| Issue : 3 | Page : 273-279
Evaluation of chemokines in gingival crevicular fluid in children with dental caries and stainless steel crowns: A clinico-biochemical study
Naveen Kommineni Kumar, Veera Kishore Kasa Reddy, Prathyusha Padakandla, Harshini Togaru, Swathi Kalagatla, Sarath N Chandra
Department of Pedodontics, C.K.S. Teja Institute of Dental Sciences, Tirupati, Andhra Pradesh, India
|Date of Web Publication||25-Jul-2016|
Naveen Kommineni Kumar
Department of Pedodontics, C.K.S. Teja Institute of Dental Sciences, Tirupati - 517 507, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aims and Objectives: The study was conducted to detect the presence of macrophage inflammatory protein-1α (MIP-1α) and MIP-1β and estimate their levels in gingival crevicular fluid (GCF) in children with dental caries and stainless steel crowns. Materials and Methods: A total of 80 children with primary dentition were selected and categorized into four groups with twenty in each group; Group 1 - healthy subjects, Group 2 - dental caries, Group 3 - dental caries involving the pulp, and Group 4 - stainless steel crowns. GCF samples were collected by an extra-crevicular method with microcapillary pipettes. The GCF samples were quantified by ELISA and the levels of MIP-1α and MIP-1β were determined. Results: MIP-1α and MIP-1β were detected in all the samples. Highest mean concentration in GCF was obtained for Group 3 followed by Groups 2 and 4 while the lowest concentration was seen in Group 1. This suggests that MIP-1α and MIP-1β levels in GCF increased proportionately with the inflammation. Conclusions: GCF serves as a noninvasive diagnostic fluid to measure biomarkers released during dental caries initiation and progression. MIP-1α and MIP-1β chemokines can be considered as novel biomarkers, in biological mechanism underlying the pathogenesis and inflammation in children with dental caries and stainless steel crowns.
Keywords: Chemokines, dental caries, gingival crevicular fluid, macrophage inflammatory protein-1α, macrophage inflammatory protein-1β, pulpal inflammation, stainless steel crowns
|How to cite this article:|
Kumar NK, Reddy VK, Padakandla P, Togaru H, Kalagatla S, Chandra SN. Evaluation of chemokines in gingival crevicular fluid in children with dental caries and stainless steel crowns: A clinico-biochemical study. J Indian Soc Pedod Prev Dent 2016;34:273-9
|How to cite this URL:|
Kumar NK, Reddy VK, Padakandla P, Togaru H, Kalagatla S, Chandra SN. Evaluation of chemokines in gingival crevicular fluid in children with dental caries and stainless steel crowns: A clinico-biochemical study. J Indian Soc Pedod Prev Dent [serial online] 2016 [cited 2021 Jan 20];34:273-9. Available from: https://www.jisppd.com/text.asp?2016/34/3/273/186754
| Introduction|| |
Dental caries is an infectious disease with multifactorial etiology. Bacteria causing dental caries will colonize the oral cavity and lead to the process of inflammation. These carious lesions induce both innate and adaptive immune responses in the host. As a response to inflammation, the cytokines are likely to be released into the systemic circulation. Animal models indicated that the proinflammatory cytokine concentrations were higher within the serum in periapical lesions. The proinflammatory cytokines concentrations are elevated in serum and gingival tissues of humans with periodontal inflammation, and may contribute to a systemic hyperinflammatory state, which is a risk factor for several systemic diseases. It is well-known that in oral cavity disorders, the levels of cytokines are increased in saliva. It may be argued that the expression of cytokines in unstimulated whole saliva may be due to the leakage of gingival crevicular fluid (GCF) into the oral cavity. Hence, it is assumed that the level of cytokines is increased in GCF of individuals with dental caries. CCL20 expression in human inflamed pulp is observed mostly in macrophages that have accumulated in the area adjacent to caries lesions.
Steel crowns are indicated in pediatric dentistry to treat teeth surfaces when they have been severely affected by dental caries, development defects, traumatic dental fractures, and following pulp therapies. The association between stainless steel crowns and gingival inflammation has not been fully explained in the literature. It has been reported that gingivitis often occurs around primary teeth restored with steel crowns due to various factors such as poorly adapted, inadequately contoured margins and subgingivally located crowns.
The chemokines are considered to be one of the inflammatory factors which play a crucial role in mediating the extravasation and accumulation of selective leukocyte subsets in the process of inflammation. Chemokines are chemotactic cytokines that direct the recruitment and subsequent activation of specific types of leukocytes into inflamed periodontal tissues. Chemokines play a vital role in the process of inflammation, physiological and pathological activities, such as lymphoid trafficking, Th1/Th2 cell development, and wound healing. They are secreted by a range of inflammatory cells, such as neutrophils, monocytes, and lymphocytes as well as noninflammatory cell types at sites of inflammation. Cytokines cause selective migration of human monocytes and lymphocytes. Macrophage inflammatory protein-1α (MIP-1α) is preferentially chemotactic for CD8 + T-cell subset.
Expression of MIP-1α in gingival tissue with chronic periodontal diseases has been investigated in the previous studies. Ryu et al. have found that MIP-1α expression in gingival epithelial cells is induced by lipopolysaccharide (LPS), and it is important in initiating inflammation. Garlet et al. have detected higher expression of MIP-1α in inflamed gingival tissue of subjects with chronic periodontitis and aggressive periodontitis. It makes clear that as the severity of periodontal disease increases, MIP-1α levels also increases. MIP-1β belongs to the CC chemokine subfamily. The chemokine MIP-1β (also called CCL4) is considered to be the most abundantly expressed chemokine in periodontal diseases in correspondence to MIP-1α. Both MIP-1α and MIP-1β have shown to be potent chemoattractants for macrophages, lymphocytes, eosinophils, natural killer cells, and dendritic cells.,, Both of these chemokines exert similar effects on monocytes but their effects on lymphocytes differ: MIP-1α selectively attracts CD8 lymphocytes and MIP-1β selectively attracts CD4 cells. However, to date, no study has been done to evaluate the GCF levels of MIP-1α and MIP-1β in subjects with dental caries and stainless steel crowns. The present study is thus the first of its kind to investigate the presence of MIP-1α and MIP-1β levels in such subjects.
| Materials and Methods|| |
Children were selected from outpatient department, Department of Pedodontics, C.K.S. Teja Institute of Dental Sciences, Tirupati, Andhra Pradesh. Healthy male and female children in the age group of 3–5 years with healthy gingiva were included in the study. Children with dental caries and stainless steel crowns were also included in the study. Subjects with intraoral and systemic infections, having received periodontal or antibiotic therapies 6 months before testing, using mouth rinses containing antimicrobials in the preceding 2 months, with diabetes or with other orthodontic appliances, were excluded from this study. All eligible subjects are thoroughly informed as to the nature, methods, risks, and benefits of the study. Their participation was obtained by a clear written consent. The study was carried out after approval of Institute's Ethical Committee's.
Criteria for subject grouping
The selected children were categorized into four groups (twenty subjects each):
- Group 1: (Control group) 3–5 years of age with clinically healthy gingiva (gingival index (GI) <1, plaque index (PI) <1, and probing pocket depth <3) and with deft score <3
- Group 2: 3–5 years of age with healthy gingiva (GI <1, PI <1, and probing pocket depth <3) and deft >3
- Group 3: 3–5 years of age with healthy gingiva (GI <1, PI, and <1 probing pocket depth <3) and with deft >3 with pulp involvement
- Group 4: 3–5 years of age with stainless steel crowns and with deft <3.
GI, PI, probing pocket depth and deft were assessed. Without touching the marginal gingiva, supragingival plaque was removed to avoid contamination and blocking of the microcapillary pipette. A standard volume of 3 ml GCF was collected from each site by placing 1–3 ml calibrated volumetric microcapillary pipette (Sigma-Aldrich Chemical Company, USA: Catalog number p0549) extracrevicularly (unstimulated) for 5–20 min. GCF was collected from the distal sites of primary first molar and around the gingival sulci of teeth with stainless steel crown [Figure 1] and [Figure 2]. The test sites, which did not express standard volume (3 ml) of GCF and micropipettes contaminated with blood or saliva were excluded from this study. The collected GCF was immediately aliquoted and stored at −70°C until the time of the assay. An ELISA was performed to determine the chemokines present in the GCF samples. ELISA employs the quantitative sandwich enzyme immunoassay technique (R and D Systems: Catalog numbers DMP300 and DTM100) [Figure 3] and [Figure 4].
|Figure 2: Collection of gingival crevicular fluid from tooth with stainless steel CROWN|
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|Figure 3: Addition of streptavidin-horseradish peroxidase conjugate (Cat. no. S100180)|
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Data analysis was carried out using the Statistical Package for Social Sciences (SPSS version 20, USA). Multiple comparisons for GI and PI were analyzed by analysis of variance. Further post hoc comparisons were analyzed using Tukey honest significant difference test. For other variables such as probing pocket depth, deft, MIP-1α, and MIP-1β, multiple comparisons were analyzed by Kruskal–Wallis test. Further pairwise comparisons were analyzed using Mann–Whitney U-test.
| Results|| |
As shown in [Table 1], the mean PIPI for Group 1 was 0.39 ± 0.21, for Group 2 was 0.41 ± 0.16, for Group 3 was 0.42 ± 0.14, and for Group 4 was 1.61 ± 0.16. The mean GI was 0.42 ± 0.19 for Group 1, for Group 2 was 0.42 ± 0.15, for Group 3 was 0.41 ± 0.14, and for Group 4 was 1.63 ± 0.14 [Table 1]. The mean probing pocket depth for Group 1 was 1.12 ± 0.27, for Group 2 was 1.13 ± 0.26, for Group 3 was 1.12 ± 0.23, and for Group 4 was 1.19 ± 0.24 [Table 2]. The mean deft for Group 1 was 1.50 ± 0.51, for Group 2 was 5.25 ± 0.96, for Group 3 was 5.75 ± 0.91, and for Group 4 was 1.40 ± 0.50 [Table 2]. The differences in [Table 1] and [Table 2] were highly significant (P< 0.001). All the samples, in each group, tested for the presence of MIP-1α and MIP-1β. The mean total concentration of MIP-1α in the GCF for group 1 was 197.60 pg/µl, for Group 2 was 900.40 pg/µl, for Group 3 was 1286.55 pg/µl, and for Group 4 was 682.55 pg/µl [Table 3] and [Graph 1 [Additional file 1]]. The mean total concentration of MIP-1β in the GCF for Group 1 was 287.85 pg/µl, for Group 2 was 1048.85 pg/µl, Group 3 was 1208.85 pg/µl, and Group 4 was 884.35 pg/µl [Table 3] and [Graph 2 [Additional file 2]]. Statistically significant difference existed between these groups (P = 0.001).
|Table 1: Mean plaque index and mean gingival index for Groups 1, 2, 3, and 4|
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|Table 3: Mean macrophage inflammatory protein-1a and macrophage inflammatory protein-1ß concentrations in for Groups 1, 2, 3 and 4|
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| Discussion|| |
Cytokines are closely associated with the pathogenesis of inflammation in soft tissues,, and evidence indicates that they contribute to the initiation and progression of dental caries., MIP-1α and MIP-1β are the most important cytokines in the human immune system which play a vital role in the antibacterial defense system. Carious lesions have previously been shown to be dominated by a variety of infiltrating immune cells., Cytokines and chemokines are well-studied in the host response to bacterial infection and are expressed by a variety of immune cells. They are characterized by their pleiotropism and pluripotentiality. They play a key role in mediating the immune response by exerting their biological effects on a range of target cells. Certain cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukins (ILs), and epithelial cell-derived neutrophil attractant 78 are predominantly proinflammatory. These cytokines mediate both local and systemic inflammatory responses, with increased levels which are associated with a variety of diseases.Streptococcus mutans level positively correlated with saliva IL-1β concentration and inversely correlated with saliva IL-1 receptor antagonist concentration. So far, only limited studies have been performed on the molecular nature of the dental tissue immune response, and no study has been done to evaluate the presence of MIP-1α and MIP-1β in dental caries and stainless steel crowns. Therefore, the present study is thus the first of its kind to better characterize the molecules involved by analyzing the expression of MIP-1α and MIP-1β.
In this study, GI and PI were found to be high in Group 4 when compared with other groups. When mean concentrations of MIP-1α and MIP-1β in GCF were compared between Groups 1 and 4, high concentrations were found in Group 4. The possible reasons for increase in GCF levels of MIP-1α and MIP-1β in Group 4 could be because of recruitment and retention of leukocyte subsets into gingival crevice in response to plaque accumulation, periodontal pathogens, and their bacterial components like LPS. Furthermore, factors like tissue injury contributed to the release of chemokines. The variability of MIP-1α and MIP-1β concentrations within subjects of each group could be due to their role in different stages of disease process at the time of GCF collection.
The results of this study are in accordance with Sharaf and Farsi, who stated that the gingival health is affected due to marginal adaptation of stainless steel crowns and the level of oral hygiene. This is supported by studies carried out by Henderson  and Myers, who reported a high incidence of gingivitis around incorrectly contoured stainless steel crowns. These results disagree with those of Checchio et al. study  in which improper adaptation showed no relationship with periodontal problems. Myers  showed a significant association between crown defects (34%) and clinical evidence of gingivitis (P < 0.001), with extension being the most common error. Many studies supported that there is a correlation between gingivitis and oral hygiene around stainless steel crowns.,, Children with inadequate oral hygiene showed higher frequency of gingivitis, whereas children with proper oral hygiene showed a healthy gingiva around steel crowns. The study by Ramazani et al. showed that gingival health is affected by the presence of biofilm around stainless steel crowns. This is supported by Durr et al., who reported that there is a correlation between accumulation of dental biofilm and gingivitis in teeth restored with steel crowns.
In this study when all groups were compared for GCF concentration of MIP-1α and MIP-1β, the differences were statistically significant with P < 0.001. The mean concentration of MIP-1α and MIP-1β in GCF was found to be higher in Groups 2 and 3 when compared to controls. The possible reasons for increase in GCF levels of MIP-1α and MIP-1β in Groups 2 and 3 could be because of systemic inflammatory response to progressive dental caries. Gornowicz et al. reported that elevated levels of salivary cytokines such as ILs and TNF-α were seen in children with dental caries. Słotwińska and Zaleska  reported that a significant correlation exists between salivary IL-1β concentration and S. mutans levels in the oral cavity, denoting the modulation of cytokine concentration by bacterial antigens in caries pathogenesis. Ruhl et al. measured the levels of IL-1α, IL-6, IL-8, epidermal growth factor, nerve growth factor, and albumin in salivary glands and whole saliva. They stated that IL-1α, IL-6, and IL-8 were present in whole saliva at concentrations significantly higher than in major salivary gland secretions and concluded that the inflammatory cytokines detected in whole saliva did not come only from the secretions of major salivary glands but also from GCF, which was the likely source of these cytokines. This was supported by a study done by Wozniak et al., who stated that whole saliva contains GCF from all periodontal sites providing an assessment of periodontal disease status. Miller et al. reported that there is a positive relationship between clinical parameters of periodontal disease and the levels of IL-1β, matrix metalloproteinase-8 and osteoprotegerin in whole saliva. Zehnder et al. analyzed the transcript levels of cytokines in carious and healthy pulps and their findings indicated that statistically significant levels of ILs were present in pulps of teeth with caries.
The results of this study are contrary to Emingil et al. and Fokkema et al. reported that there is no significant difference found in MIP-1α and MIP-1β levels in GCF samples collected from subjects with periodontitis, gingivitis, and sound periodontal health. Hence, they explained that the low MIP-1α levels in periodontitis group could be because of the lack of macrophages as well as subsets lymphocytes with specific receptors for MIP-1α. Gemmell et al., who demonstrated that significant correlation existed between the levels of MIP-1α and degree of inflammation. Antigens within the root canal are also capable of stimulating a systemic antibody response. This was first demonstrated by Barnes and Langeland  who showed that there is marked systemic antibodies against both antigens with the introduction of bovine serum albumin and sheep erythrocytes into the root canals of monkeys. Dahlén et al., confirmed systemic antibody responses to LPS and other bacterial antigens. Taken together, these observations suggest that both locally and systemically produced antibodies may help to protect the periapex against bacterial invasion, through opsonization or complement-mediated lysis.
| Conclusions|| |
A better understanding of the molecular mediation of dental tissue inflammation will ultimately facilitate improved future diagnosis and treatment. GCF could be used as a noninvasive diagnostic fluid to measure biomarkers released during disease initiation and progression. Molecular diagnosis helps understand the molecular mechanisms underlying the disease and plays a pivotal role in the early detection, delivery of safe and effective therapy for many diseases in the future. Thus, by determining the disease risk at an early stage in subjects with dental caries and stainless steel crowns, preventive measures can be advised and so the progression and spread of disease are controlled.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]