Noyola Sánchez, Paulina¹; Guerrero Velázquez, Celia²; Hernández Troncoso, Rita Stephanie¹; Malespin García, Karla Elizabeth¹; Chávez Maciel, José María¹; Rodríguez Montaño, Ruth²

Language: English/Spanish [Versión en español]
References: 29
Page: 169-177
PDF size: 242.66 Kb.

ABSTRACT

Introduction: In the mixed dentition phase, there is a control between the rash and dental reabsorption, using osteoclastogenesis and osteogenesis. These processes are regulated by the IL-23/IL-17 axis and the RANK/RANKL/OPG system. Il-23 is known to play a crucial role in the production of IL-17 and RANKL among other cytokines. IL-17 activates different immune and non-immune cells such as fibroblasts and these can secrete RANKL. This indicates that IL-23, directly and indirectly, stimulates the production of RANKL which gives way to the activation of osteoclasts. Objective: To evaluate IL-23 saliva levels in children in the mixed dentition phase with molar class I and II. Material and methods: Saliva sample was obtained and stored at -80 ^oC until IL-23 was determined by ELISA. Results: IL-23 concentrations in saliva showed no significant difference between children with molar class I and II dentition. Conclusion: So far, these findings indicate that the regulation of IL-23 in this physiological process does not differ according to the type of molar occlusion.

INTRODUCTION

Tooth eruption is the process by which developing teeth emerge into the oral cavity. This process begins as soon as the formation of the crown and root of the deciduous teeth is completed, followed by the process of tooth replacement, where the deciduous teeth fall out while the permanent teeth emerge. This transitional phase is known as dentition, in which deciduous and permanent teeth are present and is classified into early mixed dentition and late mixed dentition.¹

Once the teeth erupt, dental occlusion (cusp relationship between the upper and lower first molars) is established and is classified into three types: molar class I, molar class II, and molar class III, with molar class I and II being the most prevalent.²

In the mixed dentition phase, there is a control between dental eruption and resorption, through osteoclastogenesis and osteogenesis. These two physiological processes depend on the regulation of various cytokines, mainly the IL-23/IL-17 axis and the RANK/RANKL/OPG system that promote osteoclast differentiation while IFN-α, IFN-β, IL-3, IL-4, IL-10 deregulate osteoclast differentiation.³

Receptor activating receptor nuclear factor kappa B (RANK) is a type I transmembrane protein ubiquitously expressed in skeletal muscle, thymus, liver, colon, small intestine, adrenal gland, osteoclasts, mammary gland epithelial cells, prostate, and pancreas.⁴ Moreover, the ligand gene for RANK (RANKL) gives rise to splice variants encoding two forms of type II transmembrane proteins and one form of the secreted protein.⁵ Although high RANKL expression can be found in lymph nodes, thymus, and lungs, only low levels of RANKL can be detected in the spleen, bone marrow, peripheral blood, leukocytes, heart, placenta, skeletal muscle, stomach, or thyroid. The binding of RANKL to its receptor RANK provides the crucial signal to drive osteoclast development from hematopoietic progenitor cells, as well as to activate mature osteoclasts. OPG negatively regulates RANKL binding to RANK and thereby inhibits bone turnover by osteoclasts. As increased osteoclastic activity is observed in patients with osteoporosis, metastasis, or rheumatoid arthritis, the RANK-RANKL-OPG axis appears to be the therapeutic target for various bone diseases,⁴ conversely, osteoprotegerin (OPG), which is a signaling receptor for RANKL, can regulate osteoclastogenesis by inhibiting RANKL.^6,7

IL-23 is a heterodimeric cytokine composed of two subunits linked by a disulfide bridge: a soluble p40 subunit and a tetra-helical bundle subunit p19.^8,9 The receptor to IL-23 consists of a subunit called IL-23R that forms a complex with the beta 1 subunit of the receptor to IL-12 (IL-12Rβ1). Signaling by IL-23R induces phosphorylation of Janus Kinase 2 (JAK2) and tyrosine kinase 2 (tyk2), which activates STAT3, allowing upregulation of RORγT and subsequently increases the expression of proinflammatory cytokines. IL-23 plays a crucial role in the induction and function of Th17 cells.^10-12 Moreover, the binding of IL-23 to its receptor on Th17 cells activates RORγT+ which induces overexpression of IL-23R, thus providing positive feedback for the maintenance and propagation of these cells.¹²

Likewise, IL-23 induces the production of IL-17 and RANKL among other cytokines.^5,12 IL-17 is part of a family of 6 isoforms named "A" to "F" of which isoform A and F can form dimers. IL-17 can activate different immune and non-immune cells such as fibroblasts and these, in turn, can secrete RANKL.^5,13 This indicates that IL-23, directly and indirectly, stimulates RANKL production.

On the other hand, it is known that occlusal force varies according to the dental relationship between the maxilla and mandible.¹⁴ In this sense, an increase in RANKL expression has been observed in rats in teeth with occlusal trauma than without occlusal trauma.¹⁵ Likewise, we have previously evaluated RANKL concentrations in permanent and deciduous teeth in children with mixed dentition, and no significant differences were observed between the two groups of dentitions, which is why it is considered that RANKL remains constant during the process of tooth replacement.¹⁶

Since IL-23 and IL-17 stimulate the production of RANKL and this molecule participates in bone remodeling and the dental eruption process, the objective of this study was to initially evaluate the levels of IL-23 in the saliva of children in the mixed dentition phase and to determine if there is variation when molar class I or II is present.

MATERIAL AND METHODS

A cross-sectional study was carried out in the Pediatric Dentistry Clinic of the University of Guadalajara. All the parents or guardians of the children seen at the Pediatric Dentistry Clinic were invited to participate. The objective of the study and the procedure for obtaining the sample was explained to the parents or guardians. All parents or guardians who agreed to have the children participate in the study were given written informed consent following the Helsinki 2013 treaty.

Children with an age range of five to eight years with early mixed dentition, erupted lower and upper first molars were included in the study. The type of occlusion was evaluated according to Angle's classification² and they were divided into two groups: class I molar and class II molar. Patients with caries, dental trauma, periodontal disease, amelogenesis or dentinogenesis imperfecta, systemic diseases, presence of syndromes, oral habits or having received orthopedic or orthodontic treatment, or having taken anti-inflammatory drugs were not included.

SALIVA COLLECTION

The patient was asked to salivate into a sterile 100 mL container for approximately 1 to 2 minutes. Then 20 μL of phosphate buffered saline (PBS) with protease inhibitor (Complete, Roche Diagnostic GmbH) was added. The tubes were then vortexed for 5 seconds. They were centrifuged at 12,000 rpm for 15 minutes at 4 ^oC. 1mL of saliva supernatant was collected in 1.5 mL microtubes and stored at -80 ^oC until ELISA for IL-23.

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

100 μL of saliva was added in triplicate to the wells of 96-well plates of the IL-23R DuoSet^® ELISA Kit (R&D Systems Minneapolis MN, USA) and the ELISA was performed according to the manufacturer's specifications. The optical density of each well was determined using the WHY101 microplate reader (Poweam Medical Systems Co., Nanjing, Jiangsu, China) set at 450 nm with wavelength correction at 540 nm. The IL-23 concentrations of each sample were calculated from the standard curve according to the assay kit and the concentrations were expressed as pg/mL.

STATISTICAL ANALYSIS

Statistical analysis was performed with the SPSS v.25 program. The normality of the data was evaluated with the Shapiro-Wilk test due to the sample size since the data showed non-normal behavior, the Mann-Whitney U test was performed to identify differences between IL-23 concentrations and the variables of age, weight, height, and BMI between molar class I and II. To evaluate differences between female and male sex, a χ² test was performed. Finally, correlations between IL-23 levels and the variables evaluated a Spearman correlation was performed. A p ≤ 0.05 was considered significant.

RESULTS

SOCIODEMOGRAPHIC DATA

Nineteen patients with class I molar and 10 patients with class II molar with early mixed dentition and age range 7 to 8 years were included. There was a tendency to be overweight in patients with class II molar dentition, however, none of the children had systemic diseases or diseases in the oral cavity (Table 1).

LEVELS OF IL-23 IN THE SALIVA OF CHILDREN WITH MIXED MOLAR CLASS I AND II DENTITION

No significant difference was observed in the saliva IL-23 levels in the group with molar class I (59.76 ± 21.66) pg/mL and molar class II (51.03 ± 20.91) pg/mL (Figure 1). However, class I molar patients showed a tendency to have higher levels of IL-23 in saliva than class II molar patients.

CORRELATIONS

A Spearman correlation analysis was performed and no correlation of IL-23 with the variables of age, weight, height, and BMI was found. However, a significantly positive correlation was observed between age and weight, age and height, as well as between weight and height. On the other hand, we observed a significant negative correlation between height and BMI (Table 2).

DISCUSSION

During the mixed dental eruption phase, the type of occlusion that is established once the lower first molar contacts the upper first molar can be known. The stomatognathic system of class I individuals presents characteristics of balanced skeletal bases and its functions are performed normally. However, in class II and III molar individuals, there is a structural imbalance that predisposes some functions to be modified.¹⁷ In addition, a malnutrition relationship has been observed in subjects with malocclusions.¹⁸

On the other hand, it is known that when there are chewing movements a force is exerted that varies according to the type of occlusion and dental relationship.¹⁴ In this sense, an increase in the expression of RANKL has been observed in rats in teeth with occlusal trauma than without occlusal trauma¹⁵ although in permanent and deciduous teeth in children with mixed dentition no significant differences were observed between the two groups of teeth, which is why it is considered that RANKL remains constant during the process of tooth replacement.¹⁶

It is also known that the force exerted by dental occlusion is different from that exerted by orthodontic treatment, in this sense, IL-23 and IL-17 have been evaluated in the gingival crevicular fluid of patients with orthodontic treatment and an increase in these cytokines was observed 24 hours after the orthodontic force was applied, compared to baseline levels.¹⁹

It should be noted that IL-23 is a proinflammatory cytokine that participates in the activation and maturation of Th17 cells. This cytokine has been studied in different pathologies, mainly in those involving bone diseases such as periodontitis, rheumatoid arthritis, Sjogren's syndrome, among others.^20-23 However, IL-23 has not been evaluated in physiological developmental processes, such as the dental eruption process.

Based on this study, it was decided to evaluate IL-23 in saliva, in the mixed dentition phase of children with molar class I or molar class II, because in the mixed dentition phase osteoclastogenesis and osteogenesis are active and these processes are mainly regulated by RANKL which is stimulated by IL-23 and its receptor (IL-23R).²⁴

So the results of this study showed similar concentrations of IL-23 between molar class I and II with a tendency to be elevated in molar class I patients. So far, the findings of this pilot study indicate that the molecular regulation of IL-23 at the systemic level, in this physiological process does not differ from the dental arrangement presented in each phase of tooth eruption. On the other hand, the biological saliva sample provides information only at a systemic level; however, the LCG could provide more information on this behavior at a localized and systemic level. Therefore, we considered further evaluation of IL-23 in liquid samples (LCG), since it is a biological sample that has been used for the evaluation of different cytokines such as IL-23, IL-17, TNF-α, IL-8, IL-6, IL-2, IL-4, IL-10, RANTES, IL-1, IL-5, RANKL among others. In addition, the evaluation of cytokines in LCG provides information on the area where the dental eruption process takes place. It is worth mentioning that IL-23 has been evaluated in LCG of patients with periodontitis, diabetes mellitus, rheumatoid arthritis among other pathologies, as well as in testing dental materials such as implants^25-29 however, this cytokine has not been evaluated during the physiological process of dental eruption. It is worth mentioning that it is considered to increase the sample size to obtain conclusive results of IL-23 in the mixed dentition process.

Regarding correlations, we expected to find some kind of correlation of IL-23 with class I and II molar groups. However, no correlation of any kind was observed, indicating that this behavior may be because no significant differences were observed between the two study groups. Again, we consider that we should first increase the sample size of the study in saliva samples and also perform the evaluation of IL-23 in the LCG and then analyze the correlations of this cytokine for molar class.

However, the study allowed us to observe positive correlations between age and weight, age and height, as well as weight and height, indicating that these variables increase in a directly proportional manner. Since it is indispensable to measure height, weight, and age mainly to obtain BMI, it was expected to obtain positive correlations between height and weight with BMI. However, a negative correlation was observed between height and BMI, which indicates that when one variable increases, the other decreases. It is possible that the lack of positive correlations with BMI is due to the overweight tendency observed mainly in the group of children with molar class II. It is interesting to study whether the normal weight, overweight, or obesity of the children influences dental malocclusion.

We also intend to evaluate the concentrations of IL-23, IL-17, and RANKL in the deciduous, early mixed, late mixed, and permanent dentition. In this way, we will be able to know how these molecules fluctuate during the whole process of dental eruption.

CONCLUSION

A trend of elevated IL-23 levels was observed in patients with class I molar teeth with no significant difference. So far, this pilot study indicates that the molecular regulation of IL-23 in this physiological process does not differ from the dental disposition presented in each phase of tooth eruption.

REFERENCES

1. Wise GE, Frazier-Bowers S, D'Souza RN. Cellular, molecular, and genetic determinants of tooth eruption. Crit Rev Oral Biol Med. 2002; 13 (4): 323-334.

2. Angle E. Classification of the malocclusion. Dental Cosmos. 1899; 41 (3): 248-264.

3. Amarasekara DS, Yun H, Kim S, Lee N, Kim H, Rho J. Regulation of osteoclast differentiation by cytokine networks. Immune Netw. 2018; 18 (1): e8.

4. Franchi A, Taverna C, Simoni A, Pepi M, Mannelli G, Fasolati M et al. RANK and RANK ligand expression in parotid gland carcinomas. Appl Immunohistochem Mol Morphol. 2018; 26 (7): 478-482.

5. Bettelli E, Korn T, Kuchroo VK. Th17: the third member of the effector T cell trilogy. Curr Opin Immunol. 2007; 19 (6): 652-657.

6. Harokopakis-Hajishengallis E. Physiologic root resorption in primary teeth: molecular and histological events. J Oral Sci. 2007; 49 (1): 1-12.

7. Cordeiro MMR, Santos BZ, Reyes-Carmona JF, Figueiredo CP. Primary teeth show less protecting factors against root resorption. Int J Paediatr Dent. 2011; 21 (5): 361-368.

8. Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol. 2005; 6 (11): 1123-1132.

9. Abdi K, Singh NJ, Spooner E, Kessler BM, Radaev S, Lantz L et al. Free IL-12p40 monomer is a polyfunctional adaptor for generating novel IL-12-like heterodimers extracellularly. J Immunol. 2014; 192 (12): 6028-6036.

10. McGeachy MJ, Chen Y, Tato CM, Laurence A, Joyce-Shaikh B, Blumenschein WM et al. The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-producing effector T helper cells in vivo. Nat Immunol. 2009; 10 (3): 314-324.

11. Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science. 1993; 260 (5107): 547-549.

12. Ghoreschi K, Laurence A, Yang X-P, Tato CM, McGeachi MJ, Konkel JE et al. Generation of pathogenic T(H)17 cells in the absence of TGF-beta signalling. Nature. 2010; 467 (7318): 967-971.

13. Romagnani S. Human Th17 cells. Arthritis Res Ther. 2008; 10 (2): 206.

14. Yoon H-R, Choi Y-J, Kim K-H, Chung C. Comparisons of occlusal force according to occlusal relationship, skeletal pattern, age and gender in Koreans. Korean J Orthod. 2010; 40 (5): 304-313.

15. Yoshinaga Y, Ukai T, Abe Y, Hara Y. Expression of receptor activator of nuclear factor kappa B ligand relates to inflammatory bone resorption, with or without occlusal trauma, in rats. J Periodontal Res. 2007; 42 (5): 402-409.

16. Vizcaíno-Martínez LM, Guerrero VC. Niveles de RANKL solubles en el líquido crevicular gingival de dientes anteroinferiores y primeros molares inferiores de niños con dentición mixta temprana. Impacto Odontologíco. 2018; 6 (3): 17-21.

17. Talley MM, Katagiri KM, Pérez THE. Casuística de maloclusiones clase I, clase II y clase III según Angle en el Departamento de Ortodoncia de la UNAM. Rev Odont Mex. 2007; 11 (4): 175-180.

18. Kikutani T, Yoshida M, Enoki H, Yamashita Y, Akifusa S, Shimazaki Y et al. Relationship between nutrition status and dental occlusion in community-dwelling frail elderly people. Geriatr Gerontol Int. 2013; 13 (1): 50-54.

19. Allgayer S, Macedo de Menezes L, Batista EL Jr. Interleukin 17 (IL-17) and interleukin 23 (IL-23) levels are modulated by compressive orthodontic forces in humans. J World Fed Orthod. 2019; 8 (4): 148-152.

20. Sadeghi R, Sattari M, Dehghan F, Akbari S. Interleukin-17 and interleukin-23 levels in gingival crevicular fluid of patients with chronic and aggressive periodontitis. Cent Eur J Immunol. 2018; 43 (1): 76-80.

21. Liukkonen J, Gürsoy UK, Pussinen PJ, Suominen AL, Konoen E. Salivary concentrations of interleukin (IL)-1beta, IL-17A, and IL-23 vary in relation to periodontal status. J Periodontol. 2016; 87 (12): 1484-1491.

22. Przepiera-Bedzak H, Fischer K, Brzosko M. Serum IL-6 and IL-23 levels and their correlation with angiogenic cytokines and disease activity in ankylosing spondylitis, psoriatic arthritis, and SAPHO syndrome. Mediators Inflamm. 2015; 2015: 785705.

23. Deveci H, Turk AC, Ozmen ZC, Deveci K. Serum interleukin-23/17 levels in ankylosing spondylitis patients treated with nonsteroidal anti-inflammatory drugs: a prospective cohort study. J Interferon Cytokine Res. 2019; 39 (9): 572-576.

24. Fukushima H, Kajiya H, Takada K, Okamoto F, Okabe K. Expression and role of RANKL in periodontal ligament cells during physiological root-resorption in human deciduous teeth. Eur J Oral Sci. 2003; 111 (4): 346-52.

25. Szeremeske MT, De Freitas FN, Figueiredo LC, Pereira da Silva HD, Godoy RF, Mendes DP. Cytokine profiles of healthy and diseased sites in individuals with periodontitis. Arch Oral Biol. 2020; 120: 104957.

26. Javed F, Al-Askar M, Al-Hezaimi K. Cytokine profile in the gingival crevicular fluid of periodontitis patients with and without type 2 diabetes: a literature review. J Periodontol. 2012; 83 (2): 156-161.

27. Arvikar SL, Hasturk H, Strle K, Stephens D, Bolster MB, Collier DS et al. Periodontal inflammation and distinct inflammatory profiles in saliva and gingival crevicular fluid (GCF) compared with serum and joints in rheumatoid arthritis patients. J Periodontol. 2021; 92(10): 1379-1391.

28. Hu Z, Wu D, Zhao Y, Chen S, Li Y. Inflammatory cytokine profiles in the crevicular fluid around clinically healthy dental implants compared to the healthy contralateral side during the early stages of implant function. Arch Oral Biol. 2019; 108: 104509.

29. Kapoor P, Kharbanda OP, Monga N, Miglani R, Kapila S. Effect of orthodontic forces on cytokine and receptor levels in gingival crevicular fluid: a systematic review. Prog Orthod. 2014; 15 (1): 65.

AFFILIATIONS

¹ Especialidad en Odontopediatría. Departamento de Clínicas Odontológicas Integrales. Centro Universitario de Ciencias de la Salud. Universidad de Guadalajara. Guadalajara, Jal. México.

² Instituto de Investigación en Odontología. Departamento de Clínicas Odontológicas Integrales. Centro Universitario de Ciencias de la Salud. Universidad de Guadalajara. Guadalajara, Jal. México.

CORRESPONDENCE

Ruth Rodríguez Montaño. E-mail: ruth_rodriguez21@outlook.com

Received: Diciembre 2020. Accepted: Abril 2021.