INTRODUCTION:

Orthodontic tooth movement is a process of adapting the alveolar bone to physiological strains due to a reversible mild mechanical trauma to the periodontal ligament (PDL)¹. Different chemical mediators influence the cellular activity on the compression side and the tension side, resulting in bone resorption on the compression side and bone apposition on the tension side. The constant balance between apposition and resorption will facilitate the remodeling process².

Osteoprotegerin (OPG) is a glycoprotein which becomes a key factor in inhibiting osteoclast differentiation and activation³. OPG acts as a decoy receptor for the receptor activator of nuclear factor kappa-β ligand (RANKL) which competes with RANK, so it does not bind to RANKL, resulting in inhibition of osteoclastogenesis⁴.

OPG expression by osteoblasts is found on the tension side, and its expression depends on various cytokines, hormones, growth factors, and Wingless Int-1 (Wnt)/β-catenin pathway⁵.

Orthodontic tooth movement consists of three phases, namely initial phase, lag phase, and post lag phase. The initial phase starts 24 hours to 48 hours after the first application of orthodontic force, the lag phase starts after 4 days of activation, which usually occurs in 2-3 weeks and can last up to 20 days, while the post lag phase starts from 14 days after orthodontic force implementation⁶. The apposition of alveolar bone on the tension side by osteoblasts begins after 40-48 hours of orthodontic force application, in which the OPG level increases, then the final stage of bone apposition starts on day 7 to 14^7.8. Suparwitri et al. (2019) stated that the OPG level on the tension side peaks on day 14 after orthodontic appliance activation⁹.

The process of bone apposition and remodeling is regulated not only by genetic traits but also by nutritional, mechanical, and hormonal factors^10,11,12. High consumption of fruit, vegetables, and olive oil for dietary intake has been found to reduce the incidence of osteoporosis and fractures in Mediterranean countries ^13,14. Antioxidants include a group of vitamins, minerals, herbs and enzymes that help to protect the body from the formation of free radicals^15,16,17,18. Extra-virgin olive oil (EVOO) is a key component of this diet, containing more than 30 phenolic compounds with antioxidant content^19,20,21,22. Olive oil contains phytoestrogens and natural antioxidants that act as a substitute for estrogen in bone loss prevention ^23,24. An EVOO diet study in pigs for 8 weeks showed a significant increase in the daily bone density, reaching 6.28mg/cm² of bone mineral density²⁵.

At the period of young age, bone resorption and apposition cycle are balanced. Along with age, the balance reduces. At the age of 35, humans reach the peak of their bone production which then gradually declines ²⁶. Aging in men is known to cause a decrease in the production of estradiol (the hormone estrogen) and testosterone ²⁷. The most influential agent that maintains the balance of bone remodeling is the hormone estrogen ²⁸. The estrogen suppresses osteoclast activity by stimulating OPG production by osteoblasts and suppressing IL-6 production^10,29. This study aims to determine the effect of olive oil administration on changes in the OPG expression in the gingival sulcus fluid during an orthodontic tooth movement at young and old ages.

Methods:

The ethical clearance was approved by the Research Ethics Committee of the Faculty of Dentistry, University Gadjah Mada (UGM), Yogyakarta, Indonesia (00144/KKEP/FKG-UGM/EC/2019). The “Animal Research: Reporting of in vivo Experiment” (ARRIVE) guideluneswas used for reporting this study. The experimental animal research used guinea pigs as the research subjects³⁰. 12 healthy male guinea pigs were usedwhich were devidedinto four groups (n = 3). A young guinea pig group (± 4 months, weighing 300-350 g) was not given olive oil (YC), a young guinea pig group was given 0.7ml olive oil (YO), an old group (± 2.5years, weighing 750-800g) was not given olive oil (OC), and an old guinea pig group was given 1.86ml olive oil (OO). Gingival crevicular fluid (GCF) was observed on days 0, 7, and 14. The experimental animals were previously anesthetized using ketamine (35mg/kg BW) and xylazine (5mg/kg BW) intramuscularly. A separator was installed between two lower mandibular incisors followed by etching the labial surface of the teeth and bracket bonding. A 0.018" stainless steel wire and a NiTi open coil spring were installed at a strength of 0.35 N.

Figure 1. Installation of orthodontic device on guinea pigs with SS 0.018 wire and open coil spring.

Sample collection was carried out on day 0 (before orthodontic device installation), then days 7 and 14 in each group of guinea pigs from the tension side. Samples were obtained by paper point insertion into the lower incisors of the crevicular on the mesial surface with 1 mm depth for 60 seconds. Three paper point samples were obtained with 60 seconds time interval to increase the volume of the gingival crevicular fluid. The paper points were enclosed into 1.5ml Eppendorf tube containing 350µL of saline fluid and centrifuged for 5 minutes (2000rpm) to completely elute liquid component of the gingival crevicular fluid. The paper points were obtained and the solution was stored at -80^ountil the osteoprotegerin expression test was carried out.

The evaluation of OPG expression was carried out at the Molecular Biology Laboratory, Faculty of Medicine, Public Health and Nursing, UGM by employing the ELISA method. The data obtained in the study were recorded, collected, and statistically analyzed in SPSS software, version 21.0 (SPSS Inc., Chicago, IL, USA), the three-way ANOVA test to determine the interaction among the factors (olive oil consumption, age of experimental animals, and duration of olive oil administration), and the Post Hoc Test to identify which group had a significant difference.

RESULTS:

The measurement of OPG expression on GCF from the tension side involved the ELISA method. The measurement results among the four groups provided in Table 1 show the lowest OPG expression in the old control group (OC) and the highest in the young group given olive oil (YO).

Table 1. The mean and standard deviation (SD) values of OPG expression in the YC, YO, OC, OO groups

Group	Mean ± SD (pg/ml)
Group	D-0	D-7	D-14
YC	22.33 ± 1.22	32.11 ± 1.72	42.65 ± 0.69
YO	25.88 ± 1.43	41.60 ± 0.81	68.27 ± 0.88
OC	18.52 ± 1.01	23.10 ± 1.80	31.78 ± 1.57
OO	19.02 ± 0.33	30.42 ± 1.12	46.48 ± 0.90

Note:

D-0: Day 0

D-7: Day 7

D-14: Day 14

YC: Young, control

YO: Young, olive oil

OC: Old, control

OO: Old, olive oil

Figure 2. Diagram of mean and standard deviation in the controland treatment groups

The OPG expression among all groups increased from day 0 to 14 (Figure 2). The highest OPG expression in young guinea pigs receiving olive oil treatment was identified on day 14. On average, the OPG expression of young guinea pigs with olive oil treatment on day 14 increased by 42.39pg/ml, while the average OPG expression of young guinea pigs in the control group increased by 20.32pg/ml.The highest OPG expression in old guinea pigs treated with olive oil was seen on day 14. The mean OPG expression of the old guinea pig group given olive oil on the 14^th day increased by 27.46 (pg/ml) from day 0, while the mean OPG expression of the old guinea pig control group on day 14increased by 13.26 (pg/ml) from day 0.

Table 2. Average OPG expression by three-way ANOVA test from old and young age groups given olive oil and as controls

Variable	Significance
Age	0,000 *
Olive oil	0,000 *
Duration of giving	0,000 *
Age - Olive oil	0,000 *
Age - Duration of administration	0,000 *
Olive - Duration of administration	0,000 *
Age - Olive oil - Duration of administration	0,000 *

*Significant differences between groups (p< 0.05).

ANOVA: Analysis of variance.

The results of the three-way ANOVA test (Table 2) showed that there were significant differences in the OPG expression between age groups, between groups given olive oil and control groups, and between groups with different duration of administration (p<0.05). The ANOVA test results also showed that there was a correlation between age and the factor of olive oil administration, between age and duration of administration, between olive oil administration and duration of administration, and between the age given olive oil and the duration of olive oil administration (p <0.05).

Table 3: Mean OPG expression by post hoc test on the tension side of old and young age groups

MO D0

MO D7

MO D14

MK D0

MK D7

MK D14

TO D0

TO D7

TO D14

TKD0

TKD7

TKD14

MO D0

MO D7

0,000 *

MO D14

0,000 *

MK D0

0.001 *

0,000 *

MK D7

0.009 *

0,000 *

MK D14

0,000 *

0.298

0,000 *

TO D0

0,000 *

0.003 *

0,000 *

TO D7

0,000 *

0.099

0,000 *

TO D14

0,000 *

0.001 *

0,000 *

TK D0

0,000 *

0.001 *

0,000 *

0.612

0,000 *

TK D7

0.009 *

0,000 *

0.442

0,000 *

TK D14

0,000 *

0.744

0,000 *

0.178

0,000 *

Note:

significant differences between groups p <0.05

D-0: Day 0

D-7: Day 7

D-14: Day 14

YC: Young, control

YO: Young, olive oil

OC: Old, control

OO: Old, olive oil

The post hoc test results (Table 3) indicated that the expression of OPG significantly increased in the group of old guinea pigs receiving olive oil (p<0.05), but no significant difference was identified in the intervention group compared to the control group on day 0, and no significant difference was found between the old guinea pig intervention group on day 7 and old guinea pig control group on day 14(p>0.05). The OPG expression significantly increased in the young intervention group (p<0.05), but no significant difference was identified between the young intervention group on day 7 and the control group on day 14(p>0.05). The OPG expression was significantly higher in the young control group compared to the old control group (p<0.05), but no significant difference was identified between the young control group on day 0 and old control group on day 7, while no significant difference was reported between the young control group on day 7 and old control group on day 14(p>0.05).

The OPG expression was significantly higher in the young group given olive oil compared to the older control group (p<0.05). The OPG expression was also significantly higher in the young control group compared to the older group given olive oil (p<0.05), but no difference was found in the young control group on day 7 compared to the older group given olive oil (p>0.05).

DISCUSSION:

The study shows that OPG expression significantly increases in young and old guinea pigs treated with olive oil (p <0.05). A study indicated that olive oil as a food source contains phytoestrogens, a compound similar to mammalian estrogens ^24,31. Estrogen has a positive effect through the regulation of PACE4 proprotein which activates BMP and osteocalcin, thereby increasing osteoblast differentiation and increasing OPG expression ³².

Other studies stated that olive oil also has antioxidant benefits, which can prevent oxidative stress due to overproduction of ROS^20,33,34. The antioxidants in olive oil have an opposite effect to ROS, maintain vital osteocytes that contribute to osteoblast activity and osteogenesis, and reduce osteoclast differentiation and activation^35,36,37. Previous studies also stated that osteoblasts and osteocytes in the production of RANKL and OPG are highly sensitive to increased oxidative status^36,37.

The results of the study in Table 3 indicate that OPG expression was significantly higher in the young control group than in the elderly control group (p<0.05), in the young group given olive oil than in the old group given olive oil (p<0.05), and in the young group given olive oil than in the control group (p<0.05). The results of this study prove that OPG expression decreases with aging ³⁷. This result supports the finding of a previous study in which aging is associated with phenotypic changes in osteoblasts, increased RANKL expression, and decreased OPG expression³⁹.

The study results also showed that the OPG expression significantly increased during the observation from day 0 to day 14 in the old and young guinea pig control groups (p<0.05), thus indicating that there was an increase in OPG expression after the activation of orthodontic appliance. The results of this study are supported by previous research in which the expression of OPG increases after 48 hours of fixed orthodontic appliance installation, then decreases after 2 weeks, and increases again after 5 weeks⁸. This study also reported that an extended period of intervention contributes to the higher OPG expression (p<0.05). The results are relevant to the findings of Suparwitri et al. (2019) with wheat seed (a natural producer of phytoestrogens) supplementation to mice. Another study suggested that OPG level on the orthodontic tooth movement of the tension side increases from day 0 to day 14, reaching a maximum level on day 14 among the intervention and control groups⁹.

The post hoc test results showed some insignificant differences since there was no significant difference in the OPG expression of the old group given olive oil on day 0 compared to that in the control group on day 0 (p>0.05). The GCF samples were taken 1 hour after olive oil treatment as a baseline before orthodontic device installation. The olive oil administration does not affect GCF biomarkers. The hydroxytyrosol compound in olive oil takes 30-50 minutes to reach the maximum concentration in plasma, and another study reported that the postprandial evaluation after 100g olive oil intake demonstrates that the tyrosol and hydroxytyrosol compounds in plasma LDL reach peak concentrations in between 60 and 120 minutes^40,41.

The results also showed that there was no significant difference in the young control group on day 0 compared to the old control group on day 7, and in the young control group on day 7 compared to the old control group on day 14(p>0.05). This study confirms that orthodontic movement gives an effect on increasing OPG expression over time. This is supported by a previous study which showed that on the tension side, there is an increase in OPG expression starting from the initial phase (0-3 days) then increasing in the linear phase (day 7 to day 14)⁴². Then, the results showed that there was no significant difference in the old group given olive oil on day 7 compared to the control group on day 14, and in the young group given olive oil on day 7 compared to the control group on day 7. These results prove that the duration of olive oil administration increases OPG expression in proportion to the increase in OPG expression due to orthodontic movement. The results also showed that there was no significant difference in the young control group on day 7 compared to the old group that was given olive oil on day 7 (p>0.05). This proves that giving olive oil increases OPG expression in the old group compared to the control group on day 7.

In addition, the results showed that there was an interaction among ages, olive oil application, and time of observation on the OPG expression of the tension side in gingival groove fluid attracted to orthodontic tooth movement (p<0.05). Giving olive oil increases OPG expression in both old and young ages but still depends on the time of observation related to the orthodontic tooth movement phase and the effect of the duration of olive oil administration, so that OPG expression increases over time.

CONCLUSION:

Administration of olive oil to guinea pigs in the young group at a dose of 0.7 ml and the old group at a dose of 1.86 ml can increase the expression of OPG. The longer the duration of olive oil administration, the higher the OPG expression.

ACKNOWLEDGMENTS:

We would like to thank Dana Masyarakat UGM 2019 for funding this research.

CONFLICTS OF INTEREST:

No competing interests were disclosed.

CONTRIBUTION OF AUTHORS:

Dina Listyowati: Conceptualization, Data Curation, Funding Acquisition, Investigation, Methodology, Validation, Visualization, Writing – Original Draft Preparation. Sri Suparwitri: Supervision, Conceptualization, Data Curation, writing – Review and Editing. Cendrawasih Andusyana Farmasyanti: Supervision, Conceptualization, Data Curation, writing – Review and Editing.

REFERENCES:

1. Wise GE, King GJ, Mechanisms of tooth eruption and orthodontic tooth movement, J Dent Res,2008;87: 414–21.

2. Alhasyimi AA, Pudyani PS, Asmara W, Ana ID, Enhancement of post-orthodontic tooth stability by carbonated hydroxyapatite-incorporated advanced platelet-rich fibrin in rabbits, Orthod Craniofac Res, 2018;21(2):112–8.

3. Hofbauer LC, Heufelder AE, Role of receptor activators of nuclear factor-kappaB ligand and osteoprotegerin in bone cell biology, J Mol Med, 2001;79(5):243–53.

4. Alhasyimi AA, Pudyani PS, Asmara W, Ana ID, Locally inhibition of orthodontic relapse by injection of carbonated hydroxy apatite-advanced platelet rich fibrin in a rabbit model, Key Eng Mater 2017;758:255–63.

5. Boyce BF, Xing L, Function of RANKL / RANK / OPG in bone modeling and remodeling, Arch Biochem Biophys. 2008;473(2):139–46.

6. Khrisnan V, Davidovitch Z, Cellular, Molecular, and Tissue Level Reactions to Orthodontic Force. Am J Orthod Dentofacial Orthop, 2006;129 (4): 460-68.

7. King GJ, Keeling SD, 1995, Orthodontic bone remodeling in relation to appliance decay, Angle Orthod, 1995;65 (2): 129-40.

8. Mukherjee, Nayak K, Nayak A, Adarsh, Variations of salivary level of osteoprotegerin during osthodontic tooth movement, J Indians Orthod, 2019; 53(1): 10-13.

9. Suparwitri S, Niswati FR, Alhasyimi AA, Wheat seeds can delay orthodontic tooth movement by blocking osteogenesis in rats, Clin Cosmet Investig Dent, 2019; 11: 243–249.

10. Rivas A, Romero A, Mariscal M, Monteagudo C, Herna'ndez J, Olea-Serrano F, Validation of questionnaire for the study of food habits and bone mass, Nutr Hosp,2009;24 (5): 521-28.

11. Bonjour JP, Protein intake and bone health, Int J Vitam Nutr Res, 2011;81 (2): 134-42.

12. Marwaha RK, PuriS, Tandon N, Dhir S, Agarwal N, Bhadra K, Saini N, Effects of sports training and nutrition on bone mineral density in young indian healthy female, IndiaJ Med Re,2011;134 (3): 307-13.

13. Keiler AM, Zierau O, Bernhardt R, Scharnweber D, Lemonakis N, Ternetzi A. et al, Impact of A Functionalized Olive oil Extract on The Uterus and The Bone in A Model of Postmenopausal Osteoporosis, Eur JNutr, 2014; 53 (4): 1073-81.

14. Zerzour A, Hadding NE, Derouiche S, Analysis of osteoporosis risk factors in menopausal women’s of Algeria population, Asian J. Res. Pharm. Sci. 2020; 10(2):79-84.

15. Patel VK, Kpatel C, Patel HU, Patel CN, Vitamins, minerals and carotenoids as a antioxidants, Asian J. Research Chem. 3(2): April- June 2010; Page 255-260.

16. Jaydeokar AV, Bandawane DD, Nipate SS, Chaudhari PD, Natural antioxidants: a review on therapeutic applications, Research J. Pharmacology and Pharmacodynamics. 4(1): Jan. - Feb., 2012, 55-61

17. Tiwari P, Patel RK, Estimation of total phenolics and flavonoids and antioxidant potential of ashwagandharishta prepared by traditional and modern methods, Asian J. Pharm. Ana. 3(4): Oct. - Dec. 2013; Page 147-152.

18. Ashfaq MH, Siddique A, Shahid S, Antioxidant activity of cinnamon zeylanicum: (A review), Asian Journal of Pharmaceutical Research. 2021; 11(2):106-116.

19. Pérez-Jiménez F, Ruano J, Perez-Martinez P, Lopez-Segura F, Lopez-Miranda J, 2007, The influence of olive oil on human health: Not a question of fat alone, Mol Nutr Food Res, 2007; 51, 1199-206.

20. Brown L, Poudyal H, Panchal, SK, Functional food as a potential therapeutic options for metabolic syndrome, Obes. Rev, 2015:16 (11): 914-41.

21. Harshitha C, Murthykumar K, Deepak A, Beneficial effect of olive oil on human health – A review, Research J. Pharm. and Tech. 2016; 9(5): 593-595.

22. Khobragade CN, Bodade RG, Manwar AV, Synthesis and antioxidant activity of some flavone derivatives, Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 139-141

23. Thompson L, Boucher B, Lui Z, Cotterchio M, Kreiger N, Phytoestrogen content of foods consumed in Canada, including isoflavones, ligans and coumestan, Nutrition and cancer, 2006;54 (2): 184-201.

24. LiuH, Huang H, Li B, Wu D, Wang F, Zheng XH et al, Olive oil in the prevention and treatment of osteoporosis after artificial menopouse, Clin Interv Aging, 2014; 9, 2087-95.

25. OstrowskaE, Gabler NK, Ridley D, Suster D, Eangling DR, DunsheaFR, Extra virgin and refined olive oils decrease plasma triglyceride, moderately affect lipoprotein oxidation susceptibility and increase bone density in growing pigs, JSci FoodAgric, 2006; 86 (22): 1955-62.

26. Ren Y, Maltha JC, Van't Hof MA, Kuijpers-Jagtman AM, Age effect on orthodontic tooth movement in rats, J Dent Res, 2003; 82 (1): 38-42.

27. Shobha S, Rao MD, Nitin, BMD, Ambreen A, Osteoporosis in men. Article, Am Fam Physician, 2010; 82 (5): 503-10.

28. Gruber CJ, Tschugguel W, Schneeberger C, Huber CJ, Production and actions of estrogens, N Engl J Med, 2002; 346 (5): 340-52.

29. Hofbauer LC, Lacey DL, Dunstan CR, Spelsberg TC, Riggs BL, Khosla S, Interleukin-1beta and tumor necrosis factor-alpha, but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblasic cells, J Bone, 1999; 25: 255–59.

30. Dr. Chitra V, Ali MA, Animal models for osteoporosis – A review, Research J. Pharm. and Tech 2020; 13(3):1543-1548.

31. Radhakrishna B, Ashok M, Harish PL, Joythsna M, Gowda SKP, Current and future trends of drugs used in osteoporosis, Research J. Pharmacology and Pharmacodynamics. 3(6): Nov.-Dec., 2011, 329-333.

32. Kim, Tabata A, Tomoyasu Tet al, Estrogen stimuli promote osteoblasic differentiation via the subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells, J Bone Miner Metab, 2015; 33 (1): 30-39.

33. Jiménez, Ruano J, Perez-Martinez P, Lopez-Segura F, Lopez-MirandaJ, The influence of olive oil on human health: Not a question of fat alone, Mole Nutr Food Res, 2007; 51 (10): 1199–208.

34. Irianti E, Ilyas S, Hutahaen S, Rosidah, The role of EVOO (extra virgin olive oil) in serum MDA concetration and blood pressure of pregnant mice (rattus norvegicus) with ple-eclampsia, Research J. Pharm. and Tech. 2018; 11(3):1053-1057.

35. BanfiG, Iorio EL, Corsi, MM, Oxidative stress, free radicals and bone remodeling, Clin Chem Lab Med, 2008; 46; 1550–64.

36. Romagnoli C, Marcucci G, Favilli F, Zonefrati R, Mavilia C, Galli G, Tanini A, Iantomasi T, Brandi ML, Vincenzini MT, Role of GSH / GSSG redox couple in osteogenic activity and osteoclastogenic markers of human osteoblast-like SaOS-2 cells, FEBS J 2013; 280: 867–79

37. Fontani F, Marcucci G, Iantomasi T, Brandi ML, Vincenzini MT, Glutathione, N-acetylcysteine and lipoic acid down-regulate starvation-induced apoptosis, RANKL / OPG ratio and sclerostin in osteocytes: involvement of JNK and ERK1 / 2 signaling, Calcif Tissue Int, 2015; 96: 335–46.

38. IkedaT, Utsuyama M, Hirokawa K, Expression profiles of receptor activator of nuclear factor kappaB ligand, receptor activator of nuclear factor kappa B, and osteoprotegerin messenger RNA in aged and ovariectomized rat bones, JBone Miner Res, 2001; 16 (8): 1416–25.

39. Cao, Venton L, Sakata T, Expression of RANKL and OPG correlates with age-related bone loss in male C57BL / 6 mice. J Bone Miner Res, 2003; 18 (2): 270-6.

40. Miro-Casas E, Albaladejo MF, Covas MI, Rodriguez JO, Colomer EM, Raventos RML, Hydroxitirosol disposition in humans, Clinical Chemistry, 2003; 49: 945-52.

41. KendallM, Batterham M, Prenzler PD, Ryan D, Robards K, Absorbtion, Metabolism and Excretion of Phenols Derived from Olive Products, Functional Plant Science and Biotechnology 2009; (3): 81-91.

42. Tan, Ren Y, Wang J, Jiang L, Cheng H, Sandham A, Zhao Z, Osteoprotegerin and Ligand of Receptor Activator of Nuclear Factor, Angle Orthod,2009;79(2): 292-99.

Received on 19.01.2021 Modified on 28.04.2021

Research J. Pharm. and Tech. 2022; 15(6):2621-2626.

DOI: 10.52711/0974-360X.2022.00438