Effect of olive oil administration on the level of transforming growth factor β1 during orthodontic tooth movement in old and young guinea pigs

Introduction

Orthodontic treatment aims to correct malposition, malocclusion, and malrelation of dentoskeletal abnormality to get harmony, balance in aesthetics, face and head structure. Orthodontic tooth movement is a result of the remodeling of periodontal ligaments and alveolar bone¹. Bone remodeling during orthodontic tooth movement is described as a continuous and balanced process². The process of bone remodeling is regulated by osteoblasts and osteoclasts³. Osteoclasts in pressure areas will resorb the alveolar bone so that tooth movement occurs, and this phenomenon will be balanced by osteoblasts in the tension area⁴. Transforming growth factor-β1 (TGF-β1) is known as a multifunctional cytokine that plays an important role in bone formation. Moreover, TGF-β1 activity increases with bone formation⁵.

The need for orthodontic treatment in adulthood increases with age, but the balance of bone resorption and remodeling processes decreases with increasing age⁶. Therefore, cellular activity in periodontal tissues of young and old individuals needs to be further studied. This may greatly influence the success of orthodontic treatment⁷. The process of aging is caused by free radicals, hormones, genetics, lifestyle, diet, pollution, and socioeconomic conditions. If these causative factors can be avoided, then the aging process can be prevented, slowed down, and may even be inhibited⁸.

Food ingredients that contain antioxidants can inhibit the oxidation process in the aging process. The oxidation process can increase intracellular reactive oxygen activity so that it can inhibit bone remodeling⁹. Olive oil is a food ingredient that rich of antioxidants and is often consumed by the public. Olive oil which extracted from fresh olives have been shown to slow the aging process¹⁰. According to Liu et al., olive oil acts effectively as a substitute for estrogen to prevent bone loss¹¹. Melguizo-Rodríguez et al., proved the phenolic compound in virgin olive oil extracts can increase the proliferation and differentiation of osteoblasts. Phenolic compounds (caffeic acid, ferulic acid, coumaric acid, apigenin, and luteolin) in olive oil extracts can increase TGF-β1 and its receptor¹².

Therefore, olive oil is expected to reduce osteoclastogenesis so as to reduce tooth movement orthodontics, especially for cases to prevent loss of anchorage, periodontitis and tooth retention. The purpose of this study is to study the differences in TGF-β1 levels between old and young ages, using guinea pig models, and the effect of olive oil administration on TGF-β1 levels in old and young guinea pigs with orthodontic tooth movement. The guinea pig was chosen as an animal model as it has been previously used to study the effect of medications on orthodontic tooth movement and has the potential to simulate the response of human tissue¹³.

Methods

Calculation of olive oil dosage and administration

We used extra virgin olive oil (Bertolli®, USA). Olive oil consumed by humans per day is 30–50 grams so the conversion of a human dose (70 kg) to guinea pig (400 grams) is 0.031. The dose was converted from human olive oil dose (weight, 70 kg) to a guinea pig dose (weight, 400g): 0.031 × 30 ml = 0.93 ml¹⁵. Based on body weight, the dose of olive oil given to young guinea pigs (300–400 g) was 0.7 ml and for old guinea pigs (800–1000 g) was 1.86 ml. Olive oil treatment groups (see below) were given olive oil daily from the beginning of the installation of orthodontic devices until day 14, while control groups received mock oil. Administration of oil was carried out orally with injection syringes that have been cut off (Figure 1) and were given at 9am every morning.

Figure 1. Administration of olive oil to a guinea pig.

Orthodontic installation

Guinea pigs were first anesthetized with ketamine (160095, Kepro™, Netherlands; 35mg/kgBW) and xylazine (160096, Xyla™, Netherlands; 5mg/kgBW) intramuscularly on the thigh, then a separator was placed between the two lower incisors (Figure 2). Next, the teeth were cleaned with rubber cups and pumice (22257, Kerr, USA) and an incisivus Roth bracket 0.022” (American Orthodontics, USA) was bonded on both lower guinea pig’s incisor using an orthodontic adhesive (Transbond XT, 3M Unitek, USA), followed by light-curing for 40 s according to the manufacturer (10 s for each side: mesial, occlusal, gingival, and distal) using a quartz-tungsten-halogen (QTH) light-curing unit (Litex 680A, Dentamerica, USA) with a light intensity of 450 mW/cm². The wire arch mounted was 0.018” stainless steel wire arch, which was ligated using power O (American orthodontics, USA). Open coil spring NiTi (American Orthodontics, USA) was installed between the two brackets to move the teeth distally with a force of 0.35 N (Figure 3). The magnitude of the open coil spring force was measured using a tension gauge. The length of the stainless-steel wire was cut 4 mm longer than the open coil spring as a tolerance for orthodontic tooth movement.

Figure 2. Separator installation on a guinea pig to facilitate the installation of the bracket.

Figure 3. Installation of bracket and gingival crevicular fluid isolation.

Results

All the experimental procedures were well-tolerated. Furthermore, olive oil administration at the used dosage did not induce any general toxicity, including edema. The results of measurements of TGF-β1 levels of GCF before and after tooth movement can be seen in Table 1. The mean TGF-β1 levels in the older age groups on days 0, 7 and 14 were higher than in the younger age groups, both in the olive oil groups and control groups. The mean TGF-β1 levels in the older and younger age groups increased on day 7 and decreased on day 14 (Table 1).

Figure 4 shows the administration of olive oil increases the average level of TGF-β1 in the young and old age groups. The mean TGF-β1 levels in the olive oil group were higher than the mean TGF-β1 in the group that were not given olive oil (control). The mean TGF-β1 levels in the old control and treatment groups and were higher than the young control and treatment on all days. Based on the time of observation, the highest TGF-β1 level was seen on day 7 and the lowest on day 0 in all groups. The highest TGF-β1 level was found in the young guinea pig group treated with olive oil at day 7 and the lowest TGF-β1 level was found in the old guinea pig group who were not given olive oil (young control) on day 0.

Figure 4. TGF-β1 levels (pg/ml) in gingival crevicular fluid of guinea pigs with tooth movement treated and untreated with olive oil.

Normality test (Saphiro-Wilk) and homogeneity test (Levene) showed normally distributed and homogeneous data. The research data were then analyzed using three-way ANOVA (Analysis of variance). The results of the three-way ANOVA (Table 2) showed that age, olive oil administration and observation time had a significant effect on TGF-β1 levels (p <0.05). There was an interaction between administration of olive oil and observation time, and also between observation time and age, but there was no interaction between administration of olive oil and age or administration of olive oil, observation time, and age (p> 0.05).

A post hoc test was performed after ANOVA to see groups that had significant differences (p <0.05). The post hoc test results showed that most of the TGF-β1 activity of the old and young age groups who were given olive oil and not given olive oil on days 0, 7, and 14 showed significant differences (Table 3).

Discussion

The process of bone remodeling is regulated by osteoblasts and osteoclasts. Osteoblasts and osteoclasts will appear 40–48 hours after being exposed with orthodontic forces¹⁶. Osteoclasts in depressed areas will absorb alveolar bone so that tooth movement occurs. This phenomenon will be offset by the apposition of osteoblasts in the tension area². The balance of bone resorption and remodeling processes decreases with increasing age, which greatly influences the success of orthodontic treatment^6,7. Olive oil has been shown to contains antioxidant compounds, namely phenolic compounds. Phenolic compounds are able to provide a strong protective effect against aging^17–19. The results of the current data analysis showed that the average TGF-β1 level in GCF in the olive oil groups was higher than the mean TGF-β1 in the groups that was not given olive oil (controls). The results of the three-way ANOVA (Table 2) showed that age, olive oil administration and observation time had a significant effect on TGF-β1 levels (p<0.05). TGF-β1 is a multifunctional cytokine that modulates proliferation, growth, differentiation, adhesion and cell survival, but it also plays a role in the production of extracellular matrix proteins⁵. The application of orthodontic forces can activate TGF-β1 transcription in these regions, thereby increasing TGF-β1 levels in GCF¹². TGF-β1 can recruit osteoblasts precursors in the area of bone formation and stimulate osteoblasts to differentiate to produce bone matrix²⁰. During tooth movement, latent TGF-β1 is activated in the periodontal tissue by plasmin, which is produced by retracted periodontal ligament cells and set in the cascade of plasminogen activator⁵. During bone formation, TGF-β1 inhibits the recruitment of osteoclast precursor cells and directly inhibits osteoclast activity in bone resorption¹⁸. Moreover, TGF-β1 in this area will stimulate osteoblastogenesis and new bone formation together with inhibition of osteoclastogenesis in this area^5,19. Previous study showed that TGF-β1 can stimulate osteoclast apoptosis in the area. The effect of TGF β proapoptosis is mediated by several factors such as osteoblasts and stromal cells under TGF-β1 control⁵.

The phenolic content in virgin olive oil extracts such as apigenin, luteolin, coumaric acid, ferulic acid and affeic acid can increase the proliferation capacity and differentiation of osteoblasts¹². Inhibition of osteoclastogenesis is also seen in bone resorption markers, such as OPG(osteoprotegerin) and Receptor Activator of Nuclear Factor-KappaB Ligand (RANKL)². The phenolic content in virgin olive oil extract can improve bone repair, and this is related to osteoblastogenesis and related factors, such as transcription factor 2 (RUNX-2), osterix (OSX), collagentype I (COL I) osteocalcin (OSC), and alkalinephosphatase (ALP). The phenolic compounds contained in virgin olive oil extract significantly increase the levels of TGFβ-1 and its receptors, and TGFβ-1 receptor expression is significantly increased in the presence of phenolic compounds¹².

Tooth movement in orthodontic treatment can be divided into three stages: initial phase, lag phase, and post lag phase. In the initial phase, the orthodontic force will produce 0.4-0.9 mm of tooth movement. This phase takes place within one week. Cellular reaction starts after the application of force, this is seen by the presence of osteoclasts, osteoblasts, osteoblast progenitors and inflammatory cells¹⁶. In the present study, TGFβ-1 levels of in the young and old guinea pigs reached the maximum value on observation day 7 indicating that the movement of the teeth is in the initial phase. Decreased levels of TGFβ-1 on day 14 indicate that tooth movement is in the lag phase. The lag phase is characterized by no or minimal movement of the teeth²¹.

Conclusion

Olive oil increases TGFβ-1 levels in both young and old guinea pigs during orthodontic tooth movement. Further studies are necessary at clinical levels to confirm the efficacy and potency of olive oil in increasing bone remodeling in orthodontic patients, especially in older patients.

Data availability

Figshare: Picture1.jpg, https://doi.org/10.6084/m9.figshare.10095101²²

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).