INTRODUCTION:

Orthodontic biomechanics studies the biological effects of dental supportive tissue due to the mechanical force during the orthodontic treatment.¹ Orthodontic treatment increases the mechanical force to periodontium, in result, the teeth move as desired. Periodontium is a dental supportive tissue functioned as a shock absorber against masticatory pressure. There seems to be a biological process between mechanical strength and tooth movement. The fundamental of orthodontic treatment is basically the remodeling ability of periodontium.²

An orthodontic tooth movement is obtained through alveolar bone remodeling and periodontal tissue in response to the mechanical forces. The application of orthodontic forces on the teeth causes alveolar bone resorption in the compression area and bone formation in the tensile area.³Bone is constantly undergoing a remodeling process which is a complex process including resorption and bone formation. Bone remodeling requires the coordination of three cell types, namely: osteocytes, osteoblasts and osteoclasts. A mechanical force can induce osteocytes acting as mechanoreceptors to detect the changes in blood flow in bone canaliculi and respond through transmission signals to osteoblasts, then osteoblasts stimulate osteoclast differentiation and bone resorption.^4-7

Sclerostin (SOST) is predominantly the discretion by mature osteocytes and expressed in osteoclasts which is found as antagonists of the canonical (βcatenin) WNT pathway.⁸ The binding of WNT co-receptor, Low density lipoprotein Receptor-related Protein 5/6 (LRP5/6) and SOST stimulate phosphorylation and decrease β-catenin and inhibits osteoblast activation.⁹ SOST gene mutations cause Van Buchem's disease or sclerosteosis, with characteristic of hyperostosis.¹⁰ SOST inhibition is assumed can increase and enhance the bone formation and ameliorate the bone resorption. The prevention of sclerostin effects on osteoblasts has been very well illustrated in the relationship between sclerostin and osteoclasts. The previous study states that sclerostin increases Receptor Activator Nuclear Kappa Ligand (RANKL) expression in osteocyte cells.¹¹ SOST expression by osteocytes or osteoclast is regulated by the mechanical responses. SOST expression will tend to increase when the mechanical process increases, while, SOST expression decrease when the mechanical process decreases. SOST mechanosensory characteristics indicate that SOST is a key protein in bone remodeling during the mechanical stimulation. Meanwhile, orthodontic tooth movement is a biological process stimulated by a mechanical process.^12,13

In addition to SOST, Nuclear Factor Activated T Cell-1 (NFATC1) are now considered as a potential bone remodeling regulator. NFATC1 plays an important role as osteoclastogenic cytokines in the inflammatory conditions. Activation of T cell cytokine secretion has the potential to stimulate the differentiation of human bone marrow stromal cell into osteoblasts, as well as the stimulation of IL-6 by osteoblasts.¹⁴ T cell movement is also known to stimulate osteoclast formation. T cells derived from RANKL are confirmed to be able to stimulate osteoclastogenesis in vitro.¹⁵

Green tea is a type of tea enriched with polyphenol flavoid consisting of Epigallocatechin Gallate (EGCG), Epigallocatechin (EGC), Epicatechin gallate (ECG) and Epicatechin (EC).¹⁶ The content of EGCG is mostly found in green tea.¹⁷ EGCG is beneficial for bone regeneration, promising component of bone remodeling, and ameliorating the bone resorption. EGCG can decrease osteoclast differentiation via RANKL signals.^18,19 The hypothesis of this study was EGCG could decrease the NFATC1 and SOST expression during OTM. The aim of this study was to elucidate the influence of EGCG post oral administration in NFATc1 and SOST expression during OTM in Wistar Rat (R. Novergicus).

MATERIAL AND METHODS:

Ethical Clearance and Study Design:

The true experimental with post-test only was carried out. Lameshow’s formula was used to determine the sample size. Meanwhile, the random sampling method was used to pick the sample. This study was approved with number 086/HRECC.FODM/III/2019 by Faculty of Dentistry, Airlangga University, Indonesia

Green Tea Extract Preparation:

We bought the Green Tea leaf (C. Sinensis) from Perkebunan Nusantara XII Company (PTPN), Lawang, Malang, Indonesia. Green tea leaf was dried and boiled in 200 mL distilled water for 30 minutes then filtered. EGCG was extracted from green tea leaf according to previous method.^20,21

Orthodontic Tooth Movement Animal Model:

The sample consisted of 28-healthy male wistar rats (Rattus Novergicus), 16-20 weeks-old with weight 200-250g were randomly selected. The samples were divided into 4 groups (Table 1). The OTM was carried out by means of the fix orthodontic appliance including NiTi close coil spring with 6mm length (American Orthodontics Corporation, USA) placed between the incisor and maxillary molars with 10g/mm² light force was measured by means of tension gauge (American Orthodontics Corporation, USA) (Figure 1). That appliance was fixated by means of 0.07 stainless steel ligature wire (American Orthodontics Corporation, USA).³

The administration of Epigallocatechin Gallate:

The administration of EGCG was done per orally by means of oral gavage, a stomach tube with dose of 300mg/1000g body weight (bw) every day. All samples were sacrificed after 14 days by rodent anaesthesia (60 mg/ bw of ketamine and xylazine 3 mg/bw) (Sigma Aldrich, US). Rat's premaxillae were dissected and placed in 10% formalin (OneMed, Indonesia) for four days. Rat’s premaxillae were decalcified for 1 month by means of 14% Ethylenediaminetetraacetic Acid (EDTA) (OneMed, Indonesia). The tissue processing was performed then clearing and impregnation were carried out.

Table 1: Exprimental group design in this study.

No	Group	Description
No	Group	OTM	EGCG
1	Control Negative (K-)	-	-
2	Control Positive (K+)	Yes, 14 days	-
3	Treatment 1 (T1)	Yes, 14 days	EGCG start at Day 7 to Day 14
4	Treatment 2 (T2)	Yes, 14 days	Yes, 14 days

Immunohistochemical examination and analysis:

Samples were then examined by Immunohistochemical (IHC) staining by means of a 3.3'-diaminobenzidine stain kit (DAB) (Sigma Aldrich, US) with counter staining Hematoxylin Eosin (Sigma Aldrich, US). Monoclonal antibodies anti-SOST (sc-130258) and anti-NFATC1 (sc-7294) were used in this study (Santa Cruz Biotechnology™, US). The observation and examination of expression number of NFATC1 and SOST in osteoclast and osteocye of alveolar bone was carried out by 3 observers in 5 different visual fields by means of Nikon H600L light microscope (Japan) at 400x magnification with a 300 megapixels Fi2 DS digital camera and image processing software Nikon Image System (Nikon, Japan).

Statistical Analysis.

Descriptive statistics are given as means ± Standard Deviation (SD). One-way Analysis of Variance (ANOVA), and Tukey Honest Significant Different (HSD) (p<0.05) was done based on Shapiro-Wilk and Levene's test (p>0.05) to compare the SOST and NFATC1 expression between groups. The data were analysed by Statistical Package for the Social Sciences 20.0 software (SPSS) (IBM corporation, Illinois, USA).

The Shapiro-Wilk and Levene’s test showed data were normal and homogen (p>0.05). The positive expression of NFATc1 in both tensile and compression side can be seen in figure 2A and 2B. The highest number of NFATc1 was found in the K+ compression area and tensile area. The lowest expression number of NFATC1 expression was found in T2 groups compression area and tensile area (Figure 2C and 2D). The highest number of SOST was found in the K+ compression area and tensile area. The positive expression of NFATC1 in both tensile and compression side can be seen in figure 3A and 3B. The lowest expression number of SOST expression was found in T2 groups compression area and tensile area (Figure 3C and 3D). There were significant different of NFATC1 and SOST expression in compression and tensile area between groups (p<0.05).

In this present study, we found that EGCG could decrease the expression of NFATC1 and SOST. The expression of NFATC1 and SOST was lower in T2 group in both compression and tensile side compared to K+ group. The mechanical force, the bones homeostasis and the regulation of SOST and NFATC1 expression are the pivotal keys in biological transitions of tooth movement.^12,22 OTM is obtained from the periodontal ligament remodeling and alveolar bone response in the balance of mechanical processes by increasing bone resorption in the compression area and bone formation in tensile area.^1-3 There are a significant difference of NFATc1 and SOST expression in compression and tensile areas between groups. EGCG as a bioactive component which can decrease bone resorption by inhibiting osteoclast differentiation and function via regulation of RANK/RANKL/OPG pathway.^23,24Furthermore, it is discovered that there is an enhancement expression of NFATc1 in compression area which indicates osteoclastogenesis. This reveals that NFATc1 is a crucial transcription factor for osteoclast differentiation. During the osteoclastogenesis, NFATc1 is induced by RANKL and this induction corresponds to the Tumor Necrosis Factor Receptor-Associated Factor Regulation of Nuclear Factor κB (TRAF6-NF-kB) and C-Fos pathways.²² Moreover, SOST expression is decreasing during post EGCG administration in the tensile area which indicates the bone formation occurs in this area. The increased of SOST expression related with certain bone disease such as osteoporosis. The higher expression of SOS indicated the high rate of bone resorption and osteoclastogenesis.²⁵EGCG component is an action to decrease osteoclasts through in vivo and reduce osteoclastogenesis²⁴. EGCG reduce the mitogen-activated protein kinase (MAP kinase) activation and inhibit the production of interleukin-6 (IL-6) so the bone resorption rate decreased.^26,27 NFATC1 expression decreased post EGCG administration implicate that osteoclastic differentiation was inhibited and bone resorption was reduced.^28,29The effects of EGCG is to ameliorate bone resorption through stabilization of collagen, decrease Interleukin (IL)-6 production, inhibit osteoclast differentiation through RANKL signaling, and stimulate osteoclast apoptosis.³⁰ Based on this study result, it can be concluded that daily oral administration of EGCG for 14 days can reduce the SOST and NFATc1 expression during OTM in compression area and tensile area during OTM. Further study and analysis is needed with better setting to confirm this study.

ACKNOWLEDGEMENT:

This study was funded by Penelitian Dasar Unggulan Perguruan Tinggi Negeri supported by Ministry of Research, Technology and Higher Education Republic of Indonesia with appointment number 554/UN3.14/LT/2019. The authors are grateful to the authorities of Faculty of Dental Medicine Airlangga University and Faculty of Pharmaceutical Science, Widya Mandala Catholic University, Surabaya, Indonesia for the support.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 11.08.2019 Modified on 17.10.2019

Research J. Pharm. and Tech. 2020; 13(4):1730-1734.

DOI: 10.5958/0974-360X.2020.00312.1