Leptin Levels in Gingival Crevicular Fluid during Orthodontic Tooth Movement.

AIM: To evaluate the levels of leptin in gingival crevicular fluid (GCF) during orthodontic tooth movement and to find the role of leptin as a mediator for tooth movement.
MATERIALS AND METHODS: GCF was collected from 25 subjects and the levels of leptin in it were measured. Sample (A) pretreatment, (B) 6 h after applying retractive orthodontic force from right side canine, (C) without retractive force from left side canine, and after 21 days from right canine (D), and left canine (E).
RESULTS: GCF Leptin concentration in pretreatment, 6 h control and 21st day control were all similar. GCF leptin concentration at 6 h test tooth site was increased which was statistically significant. GCF leptin concentration in 21st day test tooth was decreased only a little than the control tooth which shows statistically no difference.
CONCLUSION: Leptin is one of the mediators of orthodontic tooth movement. Copyright:

INTRODUCTION

Orthodontic tooth movement is based on force-induced periodontal ligament (PDL) and alveolar bone remodeling.[1] Cytokines are one among the local biochemical mediators of tooth movement and are secreted mainly by adipocytes.[2] Cytokines have multiple biologic activities including bone remodeling, and thus, they play an important role in tooth movement.[2] Leptin is a 16 KDa nonglycosylated polypeptide hormone and has been classified as pro-inflammatory cytokine.[3] Leptin has been reported to influence various biological mechanisms including the immune and inflammatory response, hematopoiesis, angiogenesis, bone formation, and wound healing.[4] Acute infection, sepsis, and wide range of inflammatory mediators increase leptin synthesis. However chronic stimulation induces suppression of leptin synthesis.[5] Recently it has been suggested that leptin plays a significant role in bone formation by virtue of its direct effect on osteoblast proliferation, differentiation and in prolonging the life span of human primary osteoblasts by inhibiting apoptosis. Thus, leptin at high concentration protects the host from inflammation and infections and maintains the bone level that is very crucial for orthodontic tooth movement. Biochemical analysis of gingival crevicular fluid (GCF) provides a noninvasive model for investigating the cellular response of underlying PDL during orthodontic tooth movement. The purpose of this study was to test the levels of leptin in GCF around a moving tooth and to find if any changes in leptin level occur during orthodontic tooth movement after applying constant continuous force.

MATERIALS AND METHODS

Twenty-five orthodontic subjects including 13 boys and 12 girls in the age group of 16–20 years attending the outpatient Department constitute the sample. Ethical clearance was obtained from the Institutional Ethical Committee.

Inclusion criteria

Orthodontic patients requiring maxillary 1st PM extraction and distal movement of canines

Normal body mass index

No use of anti-inflammatory or antimicrobial drugs within the month preceding the study

Healthy periodontal tissues with generalized probing depth of ≤2 mm with minimal bleeding and no radiographic evidence of periodontal bone loss

No history of chronic medication that may have an effect on leptin levels (oral contraceptives and antipsychotics.

Experimental design

Before the placement of orthodontic appliance, GCF samples were collected from all subjects from left maxillary canines. Then maxillary left and right 1st premolar extractions were done. After leveling the maxillary arch, the left side canine was retracted with 9 mm size (0.012” ×0.030”) NiTi coil spring along 17 × 25 SS wire. The spring delivers a light constant continuous force of 150–200 g. The contralateral canine, i.e. the right maxillary canine did not receive any distal retractive force. GCF was collected using periopaper strips [Figure 2] and measured using Periotron [Figure 1] and stored properly.

Figure 2

GCF collection from test tooth using Perio Strips

Figure 1

GCF volume measurement in Periotron

Timing of the sample

Totally 5 GCF samples were collected from each subject. Pretreatment – from right side maxillary canine (Sample A). After the maxillary arch is aligned up to 17 × 25 SS wire stage, retractive force is applied with 9 size NiTi coil spring to the maxillary left side canine and not to the right side canine. 6 h after applying this distal retractive force GCF is collected from both maxillary right (Sample B) and maxillary left canine (Sample C). After 21 days GCF is again collected from the maxillary right (Sample D) and maxillary left canines (Sample E).

Previous studies did not compare the leptin levels around an absolutely stable tooth and tooth under orthodontic movement. Hence, in this study, pretreatment leptin levels, i.e. leptin levels around the tooth when it is completely stable are also measured. 4–6 h is the critical period during tooth movement when second messengers are released that are very important for cellular functions including differentiation. 21st day is the one in which the appliance is usually reactivated after giving the periodontal tissues a period for repair and regeneration.[6]

Storage

After the measurement of GCF volume, the strips were transferred to Eppendorf tubes (Micro centrifugetubes) and isolated with Parafilm to avoid evaporation. The Eppendorf tubes were subjected to physical agitation in cyclotron to make the solid particles settle down. Then each sample was labeled and stored at − 80°C (Deep freezer) until the assay was performed.

Leptin analysis

Leptin concentration was measured by commercially available enzyme-linked immunosorbent assay [Figure 4]. The total leptin was determined in picograms (pg) [Figure 3]. The calculation of concentration in each sample was performed by dividing the amount of leptin by the volume of the sample (pg/microlitre).

Figure 4

Leptin kit

Figure 3

Leptin detection by formation of Coloured products

Leptin values

GCF Leptin concentration in pretreatment, 6 h control, and 21st day control were all similar.

GCF leptin concentration at 6 h test tooth site was increased which was statistically significant. (one-way ANOVA test: P <0.01) (Tukey honestly significant difference [HSD] test: P <0.05)

GCF leptin concentration in 21st day test tooth was decreased only a little than the control tooth which shows statistically no difference (P > 0.05).

Moreover, as an additional finding, the leptin concentration of all the girls was higher than the boys even after correcting for body mass.

Statistical analysis

Descriptive statistics including means and standard deviations were calculated for GCF volume and GCF leptin levels of the test tooth and control tooth [Table 1]. One-way ANOVA followed by Tukey HSD test were used [Table 2]. The data thus collected were assessed using SPSS statistical software.

Table 1

Mean and standard deviations of levels of gingival crevicular fluid leptin (pg/µl) in the test tooth and control tooth throughout the study period

	Pretreatment	6 h	21st day	P
Controltooth	0.75+0.11	0.75+0.07	0.74+0.09	<0.001
Test tooth		0.86+0.08	0.70+0.09

Table 2

One way ANOVA descriptive: Leptin

	n	Mean	SD	P
Group A	25	0.7512	0.11745	<0.001**
Group B	25	0.8604	0.08810
Group C	25	0.7500	0.07890
Group D	25	0.7012	0.09705
Group E	25	0.7424	0.09252

**Denotes significance of 1% level. SD: Standard deviation

DISCUSSION

To our knowledge, this is the first study to determine leptin levels in GCF during orthodontic tooth movement by applying optimal force. In the earlier study[7] tooth movement has been achieved by applying heavy magnitude of force. An increase in leptin secretion during infection and inflammation strongly suggests that it is involved in the cytokine network that governs host defense mechanisms. Leptin may have not only an indirect effect on bone metabolism via its regulation of the HPG axis and subsequent estrogen production but it may also have a direct effect on bone metabolism.

Fetal serum leptin levels are negatively correlated with serum markers of bone resorption, suggesting a possible effect of leptin on overall increase of bone mass by decreasing bone loss.[8] In the peripubertal period,[9] leptin was significantly correlated with bone area but not bone mineral content, suggesting an effect of leptin on cortical bone and periosteum during puberty. All these findings indicate the significant role of leptin in alveolar bone remodeling that is very important for orthodontic tooth movement.

The levels of leptin in GCF were demonstrated to be significantly lower in smokers than in non –smokers. Karthikeyan and Pradeep[10] reported that leptin concentrations in GCF were found to be higher in healthy gingiva than in tissues with periodontitis. Although there are no adipocytes in the gingiva, in a recent study by Johnson and Seri they proposed that this might be caused by entrapment of leptin within the gingiva by diffusion from the microvasculature.[11] As leptin has a role in the inflammatory response, an increased leptin level in healthy gingiva may be a host defense mechanism similar to that which occurs during sepsis. However, during gingival inflammation, the concentration of leptin is decreased as a result of the expansion of the vascular network caused by vascular endothelial growth factor, which may increase the net rate of leptin removal from the gingival tissues.

In the earlier study by Dilslz retraction of canine was done with E -chain. However, studies by Sueri et al.[12] indicate that E- chain exerts a heavy force of 380 g. This may lead to undermining resorption of alveolar bone, thereby delaying tooth movement. Moreover, E- chain gets imbibed in oral fluids and loses its force magnitude quickly before the next activation. All these lead to interrupted force values.

In the present study, retraction of canine was done with NiTi coil springs that deliver light constant continuous force (150–200 g). This leads to direct resorption and optimal rate of continuous tooth movement. In this study, the control tooth shows no changes in leptin levels between pretreatment, 6 h or 21 days. This study shows an increase in GCF leptin levels at 4–6 h at the test tooth site that is statistically significant. This corresponds to the fact that GCF leptin levels increase in acute sepsis. When a retractive force is applied to the tooth with NiTi coil springs, acute inflammatory changes occur in the gingival and periodontal tissues. After 21 days, the test tooth with NiTi coil springs still in place shows a little decrease in GCF leptin concentration than the control side and pretreatment (Baseline) values. But this shows statistically no difference. This indicates that as the forces exerted by the NiTi coil springs are optimal (150–200 g)[13] unlike E-chain that exerts heavy force (380 g),[14] the investing tissues of the teeth recover easily and quickly with minimal hyalinization, aseptic nectrotic area and undermining resorption. In other words, gingival conditions do not worsen but recovers which is indicated by returning of GCF leptin levels to normal or baseline values on the 21st day.

CONCLUSION

From the findings observed in this study, it can be concluded that

When constant, continuous, and optimal orthodontic forces are applied concentration of leptin in GCF is increased in early acute stages.

When the orthodontic forces are maintained within the optimal range for longer period, the gingival tissues recover quickly restoring the normal or baseline leptin values in GCF.

Leptin is one of the mediators of orthodontic tooth movement.

Future studies are required to evaluate the levels of leptin in GCF under various force magnitudes over a long period and to clarify the protective role of leptin in periodontal disease progression. Future interventional studies involving leptin administration are expected to further clarify the pharmacotherapeutic role of leptin in orthodontic tooth movement and periodontal disease progression.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.