Caffeine induces alveolar bone loss in rats submitted to orthodontic movement via activation of receptor activator of nuclear factor ҡB, receptor activator of nuclear factor ҡB ligand, and osteoprotegerin pathway

Introduction

Caffeine is a widely consumed substance with several effects on bone metabolism. This study aimed to investigate the effect of caffeine on the bone tissue of rats submitted to orthodontic movement.

Methods

Twenty-five male Wistar rats underwent orthodontic movement (21 days) of the first permanent maxillary molars on the left side. The experimental group (caffeine; n = 13) and control group (n = 12) received caffeine and water, respectively, by gavage. Microcomputed tomography was performed to analyze orthodontic movement. Histologic analysis of the inflammatory infiltrate and osteoclast count by tartrate-resistant acid phosphatase were conducted. Maxilla tissue was evaluated for receptor activator of nuclear factor ҡB (RANK), RANK ligand (RANKL), and osteoprotegerin by immunohistochemistry.

Results

Caffeine exhibited a lower bone volume/tissue volume ratio (78.09% ± 5.83%) than the control (86.84% ± 4.89%; P <0.05). Inflammatory infiltrate was increased in the caffeine group compared with the control group ( P <0.05). A higher number of tartrate-resistant acid phosphatase-positive cells was observed in the caffeine (9.67 ± 1.73) than in the control group (2.66 ± 0.76; P <0.01). Immunoexpression of RANK and RANKL in the caffeine group was greater than the control ( P <0.05).

Conclusions

The use of caffeine thermogenic induces alveolar bone loss in rats submitted to orthodontic movement via activation of RANK, RANKL, and osteoprotegerin signaling pathways.

Highlights

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The effects of caffeine on the bone tissue of rats submitted to orthodontic movement were studied.
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Caffeine combined with tooth movement induced alveolar bone loss in rats.
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Caffeine exposure enhanced inflammation and osteoclastogenesis in rats.
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Caffeine altered the expression of receptor activator of nuclear factor ҡB, receptor activator of nuclear factor ҡB ligand, and osteoprotegerin in rats.

Orthodontic tooth movement resulting from the application of orthodontic force is dependent on the remodeling process in the periodontal ligament and alveolar bone, characterized by bone deposition and resorption in places of tension and pressure, respectively. Orthodontic forces can modify the periodontal space by promoting changes in blood flow and the localized electrochemical environment.

Several substances have been studied to investigate their effect on orthodontic movement in animals. Caffeine, one of the most consumed substances in the world, is a chemical compound of the xanthines group, psychoactive, found in several food sources (eg, coffee, tea, chocolate, cocoa, soft drinks containing cola) and in some medications. It promotes multiple actions involving phosphodiesterases and adenosine receptors.

Previous studies showed controversial results about the effects of caffeine on bone metabolism. Some have suggested that caffeine intake compromises bone metabolism in some way. The impact of caffeine consumption on bone metabolism, density, and healing has been studied with conflicting results. In rats with critical-size surgical defects created in the right tibia, high daily caffeine intake disrupted the early stages of bone healing but did not alter bone density after 56 days of administration. Efficient tooth movement with minimal side effects is the major goal of orthodontic treatments. According to findings, root resorption decreased after caffeine administration, but a simultaneous reduction in orthodontic tooth movement was also noted. In vivo, immunohistochemical staining showed that the caffeine group had significantly more tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and higher expression of the receptor activator of nuclear factor κB (RANK) ligand (RANKL) in the compressed periodontium. We only found 1 previous study using the model of orthodontic movement in rats, which linked osteoclastogenesis to an increase in the activity of osteoclasts in the RANKL pathway.

The RANKL, RANK, and osteoprotegerin (OPG) are known as key regulators of osteoclastogenesis and osteoclast function and are thought to play an important role in orthodontic tooth movement. ^, Increase in the osteoclast numbers and activity can occur because of a change in the RANKL/OPG ratio. Either an increase in the RANKL, a decrease in OPG, or an alteration in both may lead to a ratio in favor of RANKL.

These molecular findings indicating the implications of caffeine on orthodontic tooth movement need to be corroborated by new studies. In this sense, this study aimed to evaluate the caffeine effect in an experimental model of orthodontic tooth movement in rats in the RANKL, RANK, and OPG signaling pathways.

Material and methods

This is an experimental, in vivo, and randomized study. The experiment was conducted by the guidelines of the Animal Research Reporting In Vivo Experiments, approved by the Ethics Committee on Animal Use of the Federal University of Rio Grande do Norte (UFRN) under protocol no. 028/2018, certificate no. 107,028/2018, and performed in the Pharmacology Laboratory of the UFRN Biosciences Center.

The sample consisted of 25 healthy male Wistar rats ( Rattus norvegicus albinus ), aged 7-12 weeks and weighing 200-300 g, from the vivarium of the UFRN Health Sciences Center.

The rats were kept in the animal house of the Department of Biophysics and Pharmacology at UFRN in propylene boxes measuring 41 × 34 × 16 cm, in the proportion of 3-4 rats per box. These animals were subjected to a 12:12 h light-dark regime, as recommended by the Ethical Principles of Animal Experimentation (Brazilian College of Animal Experimentation, 1991). They were fed a soft diet during the whole experiment, an appropriate standard chow (Nuvilab CR-1 autoclavable; QUIMTIA S/A, Colombo, Paraná, Brazil). In addition, the rats received food and water ad libitum. Before starting the experiment, the rats were acclimated to the breeding facility for 1 week. After this period, they were randomly distributed, through simple randomization, in a 1:1 ratio, into 2 groups: (1) control group, 12 rats submitted to orthodontic movement and water ingestion and (2) experimental group, 13 rats submitted to orthodontic movement and a daily dose of caffeine (Alpha Axcell, Pinhais, Paraná, Brazil).

The experimental model was performed for 21 consecutive days. Orthodontic tooth movement was exclusively conducted on the left side of the upper jaw, whereas the opposite side remained intact and was used as the control. Therefore, the rats were anesthetized by intraperitoneal administration of 10% ketamine (240 mg/kg) (Agener; União Química Farmacêutica Nacional, São Paulo, Brazil) and 2% xylazine (30 mg/kg) (Syntec do Brasil Ltd, Cotia, São Paulo, Brazil).

A device model proposed by King et al, modified for insertion into the maxillary incisors, to ensure greater retention. This model was performed by a properly calibrated orthodontist. Each animal was placed on a surgical intervention table (Multifunctional Surgical Platform SurgiSuite, Kent Scientific, Torrington, Conn) in the supine position. Initially, a closed nickel-titanium coil (7 mm) of 50 cN was bonded between the maxillary left first molar and the incisors. Metallic ligatures were attached around those teeth and fixed with fluid composite to reinforce the retention and stability. To standardize a force of 50 cN, a calibrated dynamometer was employed. After the attachment of the device, the mandibular incisors of each animal were ground to approximately 1.5 mm to avoid damage to the orthodontic appliance during the experiment. The final appearance after the installation of the closed spring is demonstrated in Figure 1 . The rats were then kept for 21 consecutive days, the minimum time needed to check for any tooth movement, and weighed once a week to observe any changes in weight related to feeding and experimental conditions.

Caffeine (Alpha Axcell) and water were administered by gavage (orally), as performed in the study by Yi et al, to ensure complete caffeine and water intake. For this purpose, a 3 mL disposable syringe containing 1 mL of each substance per animal was used. Both substances were administered with the aid of a 14 gauge curved metallic cannula (Kent Scientific).

Calculation of the dosage of caffeine was based on the average consumption of caffeine in humans—450 mg caffeine/60 kg body weight/day, according to the study by Yi et al. Caffeine (Alpha Axcell) was prepared daily for the experimental group at a concentration of 3 g/L, with the aid of an automatic single-channel pipette (Eppendorf Research Plus, Alto da Lapa, São Paulo, Brazil) of 1000 μL and 250 μL. Initially, caffeine (210 mg) was diluted in 7 mL of distilled water (solution 1) and, soon after, inserted in an ultrasonic washer (Schuster, Santa Maria, Rio Grande do Sul, Brazil) containing approximately 80 mL of distilled water, for 40 minutes, at a temperature of 600°C, for better dilution of caffeine. Then, a second solution was prepared with 750 μL of solution 1, combined with 1250 μL of distilled water (solution 2). Finally, 1080 μL of solution 2 to 3960 μL of distilled water was added, thus forming the ideal concentration solution proportional to the daily caffeine consumption for animals.

After preparation, caffeine and water were administered to the experimental and control groups, respectively. Gavages started 1 day after installation of the device, totaling 21 consecutive days of administration and experimentation. Gavage was performed by 2 properly trained handlers (M.C.M. and G.R.G.C.), 1 of which performed the containment of the animal and the other the administration of substances, blinded to the compound administered to each animal.

Open field test consists of a field monitor (Insight Ribeirão Preto, São Paulo, Brazil) to observe the behavior of the rats, equipped with a detachable acrylic monitoring box measuring 500 × 480 × 50 mm and weighing 17 kg, and performed 4 times (baseline, after the installation of the orthodontic device, after the second gavage, and after 21 days). Before starting the test, there was a 3 h habituation time for the rats (13 animals/control group and 11 animals/caffeine group).

The patterns observed were as follows: distance traveled (cm), average speed (cm/s), time spent in the central zone, and number of rearing.

Some predetermined desired settings were required in the “Activities Monitor.” The duration of the block was defined as 6 minutes, with 1 minute being the animal’s habituation time in the cubic arena and 5 minutes being the duration of the experiment. The Activity Monitor was set in a silent environment under a 15 W, 50-60 Hz red light.

To start the open field test, 1 animal was inserted at a time in the center of the cubic arena, and the box was closed. During the predetermined time, the movements performed by each animal were selected and recorded through a report in a data sheet. After the expiry of this time, remove the animal from the cubic arena and place it in a box different from the one in which it does not influence the animal’s behavior. After each experiment, the square arena was properly sanitized after each experiment with 70% alcohol.

Rats were killed on the 22nd day, after blood collection, through an overdose of 10% ketamine (80 mg/kg) (Agener; União Química Farmacêutica Nacional, São Paulo, Brazil) and 2% xylazine (10 mg/kg), 3 times higher than the dose required for general anesthesia.

The amount of orthodontic movement was assessed using microcomputed tomography (micro-CT). After dissection, the maxillae (7 animals/control group and 6 animals/caffeine group) were fixed with 4% paraformaldehyde in a saline solution buffered with 0.1 M phosphate for 24 hours and then in 70% alcohol. The samples were digitized in a high-resolution microtomography (model 1172; Skyscan, Kontich, Belgium; resolution, 15 μm/pixel; rotation, 0.4; average frames, 10; scanning time, 30 minutes; and 0.5 mm aluminum filter) allocated at the University of California, Los Angeles. Skyscan’s volumetric Nrecon reconstruction software was used to reconstruct cross-section slices from acquired micro-CT angular projections through the object. Reconstructed slices can be saved as bitmap file format and evaluated using the DataViewer software (version 1.5.0.0; Bruker, Belgium). Next, volumetric measurements regarding bone volume/total volume (BV/TV) were obtained using the CTAn software program (Skyscan) by selecting the 3 regions of interest among all the roots of the maxillary left first molar ( Fig 2 ).

Tooth movement was measured as the distance between the first and second molars at the end of the experimental period. Images were reconstructed, converted to DTM file format, and reconstructed using Dolphin Imaging software (Dolphin Imaging and Management Solutions, Chatsworth, Calif). The measurement was done with a software tool designed to identify and measure the closest distance between 2 parallel or near-parallel surfaces or lines. Tooth movement distance was measured in the axial sections and then in the sagittal sections. The mean of the 2 sections was taken to overcome any variation that might arise because of variations in the sample angulations on the scanner. The linear measurements in the sagittal view were: (1) the mesial movement, defined as the distance between the crowns of the first and second molars from the points of contact; (2) tension (mm, periodontal ligament thickness); and (3) pression (mm, periodontal ligament thickness).

The left hemimaxillae (6 animals/group) were dissected, fixed in 10% buffered formalin for 2 days, washed in running water for 24 h, and decalcified in ethylenediaminetetraacetic acid solution at 4.8% for 3 months. The specimens were then dehydrated, cleaned, and embedded in paraffin. All specimens were subjected to routine histologic processing, followed by a direct mesiodistal cut to obtain two 5 μm-thick cross sections: 1 for staining with hematoxylin and eosin and the other for TRAP.

All slides were scanned into high-resolution images using a digital slide scanner system (Pannoramic MIDI II; 3DHISTECH, Budapest, Hungary), and the images were visualized by the Panoramic Viewer software (version 1.15.2; 3DHISTECH). Three fields were digitally photographed (10× magnification) from the area around the first molar adjacent to the site of orthodontic device placement to evaluate the inflammatory infiltrate, according to the following scores: (0) absent; (1) mild; (2) moderate; and (3) intense. The analysis of inflammation scores was performed in a semiquantitative manner through direct visual observation by a blinded pathologist at 2 distinct time points.

TRAP technique using the TRAP 387 kit (Sigma-Aldrich, St. Louis, Mo) was performed (6 animals/group) according to the manufacturer’s instructions. TRAP enzyme is considered an osteoclast marker and can be used to determine bone resorption quantitatively. TRAP-positive multinucleated cells located near bone tissue were considered osteoclasts. In the bone crest area between the distal roots of the first molar and the mesial roots of the second molar, left superior (orthodontic movement side), the area with the highest positivity for TRAP was identified at a magnification of 40×. From the selected area, 3 fields were digitally photographed at a magnification of 400× to quantify the absolute number of osteoclasts, with each field corresponding to an area of 0.0998 mm ² .

The tissue slices (5 animals/group) were washed with 0.3% Triton X-100 in phosphate buffer and quenched with endogenous peroxidase (3% hydrogen peroxide). Next, incubation with the primary antibodies RANKL, RANK, and OPG (dilution of 1:400; Santa Cruz Biotechnology; INTERPRISE, Paulinia, São Paulo, Brazil) overnight at 4°C was performed. Specimens were washed with phosphate buffer and incubated for 30 min with streptavidin-horseradish peroxidase-conjugated secondary antibodies (Biocare Medical, Concord, Calif). Colorimetric detection kit TrekAvi- din- horseradish peroxidase label + Kit Biocare Medical (Dako Corporation, Carpinteria, Calif) was used according to the manufacturer’s instructions to visualize the immunoreactivity. Immunohistochemical analysis was performed by a blinded pathologist, under light microscopy, in the region between the distal roots of the first molar and the mesial roots of the second molar, left superior (orthodontic movement side). The following scores were assigned to evaluate the immunostaining: score 0 (no staining), 1 (<50% of positive cells), and 2 (>50% of positive cells).

Statistical analysis

Data were analyzed using SPSS software (version 20.0; IBM, Armonk, NY). The Shapiro-Wilk test was used to test the normality of data distribution, and normality was not rejected. Comparisons of the quantitative independent variables according to the dependent categorical variables were performed using the Student t test for inferential analysis. Semiquantitative data from variables of an ordinal scale nature (scores) were evaluated by a nonparametric Mann-Whitney test. Results obtained from the open field test were analyzed using repeated measures analysis of variance. A significance level of 5% was adopted for all statistical analyses.

Results

Blood biochemical analysis of control and caffeine rats is demonstrated in Table I , with values compatible with normal activity. The total distance traveled in the open field was not changed by treatment (F[1,16] = 0.68; P = 0.421) and trial vs treatment effect (F[3,48] = 0.67; P = 0.574) ( Table II ). In addition, there was neither effect of treatment (F[1,16] = 0.07; P = 0.788) nor interaction between treatment and trials (F[3,48] = 1.99; P = 0.129] when average speed was analyzed. In the same direction, additional behaviors observed during the 5 minutes of the behavioral test were not modified by caffeine.

Table I

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