Thermogenic preworkout supplement induces alveolar bone loss in a rat model of tooth movement via RANK/RANKL/OPG pathwayAbstract
The aim of this study was to investigate the effect of thermogenic supplementation on the bone tissue of rats subjected to orthodontic movement. A total of 38 male Wistar rats underwent orthodontic movement of the left permanent maxillary first molars for 21 days. The rats were assigned to three groups: Control group: water; Thermogenic 1: C4 Beta Pump thermogenic; or Thermogenic 2: PRE-HD/Pre-workout. Micro-computed tomography (micro-CT) was used to investigate the dynamic changes in the microstructure of alveolar bone during orthodontic tooth movement in rats. Histopathologic analysis was performed by hematoxylin and eosin (H&E) staining, whereas tartrate-resistant acid phosphatase (TRAP) was employed for osteoclast count. Maxillary tissue was collected and evaluated by immunohistochemistry for receptor activator of NF-κB (RANK), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG). The Thermogenic 2 group exhibited a significantly lower percentage of bone volume fraction (BV/TV) (68.21% ± 17.70%) compared to the control (86.84% + 12.91%) and Thermogenic 1 groups (86.84% + 15.94%) (p < 0.05). The control group had a significantly higher mean orthodontic movement in the mesial direction (0.2143 mm + 0.1513 mm) than the Thermogenic 2 group (0.0420 mm + 0.05215 mm) (p < 0.05). The Thermogenic 2 and Thermogenic 1 groups showed a stronger immunostaining for RANKL when compared to the control group (p < 0.05). The supplementation used in the Thermogenic 2 group (PRE-HD/Pre-workout) induced alveolar bone loss in rats subjected to orthodontic movement, which can be related to the regulation of the RANK/RANKL/OPG signaling pathway. This suggests the influence of thermogenic supplements on bone metabolism seems to depend on their composition.
Orthodontics; Jaw; Rats; Metabolism
Introduction
Thermogenic supplements have been shown to improve weight and weight loss management, increase fat oxidation, decrease appetite, and enhance performance during physical exercise.1
Several substances are used in the composition of thermogenic supplements, such as caffeine, taurine, gluconolactone, inositol, and vitamins.2 Studies have shown some of these substances can interfere with bone metabolism and, therefore, with orthodontic tooth movement. This process involves bone turnover, with bone apposition in the alveolar region where tension is applied to the periodontal ligament and with bone resorption on the opposite side where pressure is exerted.3,4
Taurine, for example, can promote osteoblast proliferation and differentiation and inhibit osteoclastogenesis, potentially delaying orthodontic movement.5 On the other hand, vitamin C increases osteoclast proliferation and activation,6 stimulates the remodeling of collagen fibers in the periodontal ligament,7 and can also accelerate orthodontic tooth movement.8 Moreover, in vivo studies have already demonstrated the stimulant properties of caffeine in behavioral and cognitive changes and its effect on bone metabolism.9,10
Considering the high consumption of thermogenic supplements by the young population nowadays,2,11 the influence many of their compounds exert on bone metabolism, and their known excitatory activity,2 this study aimed to investigate the effect of two different thermogenic supplements on the bone tissue of rats subjected to orthodontic movement.
Methods
This experimental, randomized, in vivo study was approved by the Ethics Committee on the Use of Animals (CEUA) of the Federal University of Rio Grande do Norte/UFRN, Protocol no. 028/2018, July 2018, Brazil. The study followed the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines.
The sample for this study consisted of 38 healthy male Wistar rats (Ratus norvegicus albinus), aged 7 to 12 weeks, and weighing 150 to 350 g throughout the study, all with permanent dentition. The rats were obtained from the animal facility of the Experimental Laboratory of Biophysics and Pharmacology (LAFINC/UFRN). The number of animals per group was determined by the sample size formula n = DF/k + 1, applicable to three common ANOVA designs in animal studies, where k = number of groups, n = number of subjects per group, and DF= degrees of freedom.12 The rats were maintained on a 12-hour light/ 12-hour dark cycle. They were randomly assigned to three groups by simple randomization:
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Control group: 13 rats subjected only to orthodontic movement and water intake;
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Thermogenic 1: 13 rats subjected to the same orthodontic movement and a daily dose of C4 Beta Pump thermogenic;
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Thermogenic 2: 12 rats subjected to the same orthodontic movement and a daily dose of PRE-HD/Pre-workout thermogenic.
Experimental model
The rats were anesthetized with an anesthetic solution of 10% ketamine (80 mg/kg) and 2% xylazine (10 mg/kg). The experimental model was conducted by a properly calibrated orthodontist, as previously described by Klein et al.13
Each animal was placed in the supine position on a surgical table (Multifunctional Surgical Platform SurgiSuite, Kent Scientific®, Torrington, USA). The rats’ mouths were opened using two rubber bands (Yellow Elastic, n.18, Mercur®, São Paulo, SP, Brazil), stretched over the upper and lower incisors to allow for the attachment of the orthodontic device (Figure 1A). Additionally, two 7.5 mm retractor tips (Kent Scientific®, Torrington, USA) were placed in the right and left cheek mucosa to facilitate access to the rats’ oral cavity during the experimental procedures (Figure 1A). The oral cavity was then disinfected with flexible swabs with cotton tips (Cotonetes, Johnson & Johnson® Ltd, São Paulo, Brazil) soaked in 0.12% chlorhexidine gluconate (Clinexidin, Dental Clean®, Londrina, Brazil).
Placement of the orthodontic appliance: (A) Placement of the brace in the posterior region. (B) Adaptation of the ligature wire-spring set. (C) Light-curing of the resin composite applied to the posterior region. (D) Hole drilled in the anterior region. (E) Ligature wire applied to the anterior region. (F) Application of the resin composite in the anterior region.
Subsequently, a set consisting of two sections of 0.008” ligature wire (Morelli®, Sorocaba, SP, Brazil) was prepared and fixed at both ends of a 7 mm Ni-Ti closed helical spring (Morelli®, Sorocaba, Brazil). With the aid of a 11 cm Mathieu needle holder (Quinelato®, São Paulo, Brazil), one side of the set (spring and ligature wires – Morelli®, Figure 1B) was positioned in the cervical region of the left maxillary first molar, passing beneath the distal embrasure and braiding the wire around the tooth (Figure 1C). A small amount of resin composite (Transbond XT 3M® orthodontic adhesive, São Paulo, Brazil) was applied to the ligature-tooth set after etching with 37% phosphoric acid (Condac®, São Paulo, Brazil), for 12 s, application of the adhesive (primer Transbond XT 3M®, São Paulo, SP, Brazil), and light-curing with LEDX-T 2400 (Orthometric®, Marília,Brazil), to improve the retention and stability of the spring. Shortly thereafter, the remaining 0.008” ligature wire section was braided with the aid of a Mathieu needle holder to secure it in the anterior region (Figure 1D).
The 0.008” ligature wire in the anterior region was inserted after drilling a hole between the maxillary incisors, at the middle third, as performed in the study by Vieira et al.18 (Figures 1E and 1F). This hole was made with a ¼ spherical drill (KG Sorensen®, Cotia, SP, Brazil), coupled to a low-speed electric motor (Smart surgical electric motor, Driller®, São Paulo, Brazil). For spring activation, the mesial end of the ligature wire was attached to a calibrated dynamometer (Federwaage 25–250 gf – Dentaurum®, São Paulo, Brazil), set to a standard force of 50 cN. For the sake of measurement accuracy, this end of the spring was attached to the maxillary incisor with 0.008” ligature wire in the previously drilled hole.
After attachment of the 0.008” ligature, retention was reinforced in the anterior region by applying 37% phosphoric acid, adhesive, and light-cured resin composite (Transbond XT 3M® – São Paulo, Brazil) in the ligature wire-tooth set.
The rats were then kept in the animal facility for 21 consecutive days, the minimum time needed to assess tooth movement. They were weighed once a week to observe any changes in weight related to feeding and/or to the experimental conditions. During the placement of the orthodontic appliance, one animal from the Thermogenic 2 group died from anesthesia-related complications, resulting in a total of 12 animals in this group.
Preparation and administration of the solutions
The following thermogenic supplements were used: C4 Beta Pump (New Millen®, Cajamar, Brazil) and PRE HD/Pre-Workout (Body Action/Sport Nutricion®, São Paulo, SP, Brazil), at a dose of 0.4 g/kg and 0.68 g/kg, respectively. The composition of these products are provided in Table 1.
Thermogenic ingredients: C4 Beta Pump (New Millen®, Cajamar, Brazil) and PRE HD/Pre-Workout (Body Action/Sport Nutricion®, São Paulo, Brazil).
Thermogenic supplements and water were administered by gavage. A 3 mL disposable syringe containing 1 mL of each substance per rat was used. All substances were administered with a 14-gauge curved metal cannula (Kent Scientific®, Torrington, USA). Oral gavages started one day after placement of the appliance and continued for 20 days, totaling 21 days of administration and experimentation.
Behavioral assessment through open field testing
The open field test was employed to observe the behavior of the animals in four time points: T0 (before orthodontic movement and gavage), T1 (one day after placement of the orthodontic appliance), T2 (one day after the first gavage), and T3 (21 days after orthodontic movement). A crystal acrylic monitoring box (Insight/SP/Brazil) was used to conduct the test. The following behaviors were analyzed: number of ambulatory movements, number of standing movements, number of jumps, number of rests, distance traveled in cm, average speed in cm/s, and movements on the edges and at the center.
Euthanasia
Euthanasia was performed on the 22nd day after blood sample collection through an overdose of an anesthetic solution containing 10% ketamine (240 mg/kg) and 2% xylazine (130 mg/kg).
Computed microtomography
Orthodontic movement was assessed using computed microtomography (micro-CT). After dissection, the maxillae were fixed in 4% paraformaldehyde buffered with 0.1 M phosphate saline for 24 h and then immersed in 70% alcohol. The samples were digitized in a high-resolution microtomography scanner (SkyScan 1172, Kontich, Belgium) (pixel size of 15 µm, 70 kV, 148 µA x-ray source, and 0.5 mm aluminum filter) at the University of California, Los Angeles, USA. Tooth movement was evaluated using the Data Viewer software (version 1.5.0.0). 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 format, and analyzed using DOLPHIN® software (Chatsworth, USA). A software tool specifically designed to determine and measure the shortest distance between two parallel or nearly parallel surfaces or lines was used for the measurements. Tooth movement was measured first in the axial sections and then in the sagittal sections. To address any variations in sample angulations on the scanner, the average of the two sections was calculated. Linear measurements in the sagittal view were used to assess mesial movement, defined as the distance between the crowns of the first and second molars from the contact points.
Interradicular alveolar bone volume fraction measurements (BV/TV %) were obtained using the CTAn software (CT analyser version 1.13.11.0, Bruker, Kontich, Belgium). The region of interest (ROI) was defined as the area containing all the roots from the furcation and extending 1.5 mm in the apical direction.
Histologic analysis
After euthanasia and assessment of tooth movement in each group, the left hemimaxillae were removed, dissected, fixed in a 10% formalin solution for 48 h, washed in running water for 24 h, and decalcified in ethylenediaminetetraacetic acid (EDTA) solution at 4.8% for 3 months.20 The hemimaxillae were then dehydrated, cleaned, and embedded in paraffin. Five-μm-thick sections were obtained from the paraffin blocks using a microtome, and these sections were subsequently mounted on glass slides for hematoxylin and eosin (H&E) staining and tartrate-resistant acid phosphatase (TRAP) staining.
The H&E slides were examined under light microscopy by an experienced pathologist, who was blinded to the experimental groups. The inflammatory infiltrate in the alveolar bone crest area between the roots of the teeth subjected to orthodontic movement was assessed using the following scoring system:14 (0) absent (no inflammatory cells); (a) mild (slight inflammation, characterized by a few inflammatory cells); (b) moderate (numerous inflammatory cells dispersed throughout the connective tissue above the alveolar crest); and (c) severe inflammation (predominance of inflammatory cells).
TRAP staining was performed using the Acid Phosphatase, Leukocyte (TRAP) Kit (387A, Sigma-Aldrich, St. Louis, USA) according to the manufacturer’s instructions. Five images at 400× magnification were obtained from the mesial region of the mesiobuccal root of the first molar, using an Olympus BX-50 microscope (Olympus, Tokyo, Japan) equipped with a Dinolite AM and a 423X microcamera (AmMo Electronics Corporation, New Taipei, Taiwan). TRAP-positive multinucleated cells (with three or more nuclei) located near the bone tissue were identified as osteoclasts, which were counted using the Image Pro-Plus 4.5 morphometric software (Media Cybernetics, Silver Spring, USA).
Immunohistochemistry
Three-micrometer-thick sections were obtained from the paraffin-embedded blocks. The sections were washed with 0.3% Triton X-100 in phosphate buffer, quenched with endogenous peroxidase (3% hydrogen peroxide), and incubated overnight at 4ºC with the following primary antibodies (Santa Cruz Biotechnology, INTERPRISE, Brazil): receptor activator of NF-κB (RANK; 1:400), receptor activator of NF-κB ligand (RANKL; 1:400), and osteoprotegerin (OPG; 1:400). The tissues were washed with phosphate buffer and incubated for 30 min with streptavidin-HRP (horseradish peroxidase)-conjugated secondary antibodies (Biocare Medical, Concord, USA). Immunoreactivity to RANK, RANKL. and OPG was detected using a colorimetric detection kit according to the manufacturer’s instructions (TrekAvi- din-HRP Label + Kit, Biocare Medical, Dako, USA). The following scoring system was used: 0 for no staining; 1 for <50% of positive cells, and 2 for >50% of positive cells.
Statistical analysis
Micro-CT and TRAP variables were compared statistically by ANOVA, followed by Tukey’s post-hoc test. Inflammation and immunohistochemical scores were assessed by the nonparametric Kruskal-Wallis test, followed by Dunn’s multiple comparison. Repeated-measures ANOVA, followed by Dunnett’s multiple comparison test, where applicable, was carried out to analyze behavioral data. The significance level was set at 5% (p < 0.05) for all statistical analyses.
Results
Behavioral assessment through open field testing
Data on behavioral assessment are presented in Table 2. Repeated-measures ANOVA revealed no treatment effect on the total distance traveled in the open field test [F(2,14) = 0.1446, p = 0.1446]. However, interaction [F(6,42) = 2.687, p = 0.0268] and time [F(3,42) = 5.053, p = 0.0044] effects were observed. Dunnett’s multiple comparison test indicated the Thermogenic 1 group increased the distance traveled at T2 when compared to the control group (p = 0.05). The control group showed a reduction in distance traveled at T1 (p = 0.0334), T2 (p = 0.0054), and T3 (p = 0.0228) compared to T0. In addition, a decrease in open field exploration was observed for the Thermogenic 1 group between T3 (21 days after orthodontic movement) and T0 (before orthodontic movement and gavage) (p = 0.0321).
Distance traveled and average speed (mean and standard deviation) in the open field test performed at four times: T0 (before orthodontic movement and gavages), T1 (one day after installing the orthodontic device), T2 (one day after performing the first gavage) and T3 (After 21 days of orthodontic movement).
Considering the other behavioral indices evaluated, neither the number of rearings nor the average speed in the open field were affected by the treatment [F(2,14) = 0.04745, p = 0.9538]. No interaction effect was found [F(6,42) = 1.783, p = 0.1260], but a time effect was observed [F(3,42) = 4.849, p = 0.0055. In the Thermogenic 1 group, the average speed decreased at T3 compared to T0 (p = 0.0167).
Micro-CT
Micro-CT showed a significantly lower (p < 0.05) percentage of BV/TV (68.21% ± 17.70%) in the Thermogenic 2 group than in the control (86.84% + 12.91%) and Thermogenic 1 groups (86.84% + 15.94%; Figure 2).
Micro-CT images showing coronal, sagittal, and axial views of the control, Thermogenic 1, and Thermogenic 2 groups. Bar charts displaying data on BV/TV (%) and mesial movement (mm) expressed as mean and standard deviation. Blue arrow: mesial movement. Yellow arrow: bone loss. ANOVA t followed by Tukey’s post-hoc test. *p < 0.05.
A higher average of orthodontic movement in the mesial direction (0.2143 ± 0.1513 mm) was noted in the control group compared to the Thermogenic 2 group (0.0420 ± 0.0521 mm) (p < 0.05; Figure 2).
Histologic analysis
H&E slides (Figure 3) revealed no statistically significant differences (p = 0.492) in inflammatory infiltrate between the Thermogenic 2 (median score: 2; Q25–Q75: 2–3), the Thermogenic 1 (median score: 2; Q25–Q75: 1.5–2), and the control (median score: 2; Q25–Q75: 1.25–2) Groups.
Photomicrographs showing representative H&E-stained specimens of the control, Thermogenic 1, and Thermogenic 2 groups. Scale bar: 200 μm. Black arrow: inflammatory cells. Data on the inflammation scores are expressed as median with 95% confidence interval in the bar chart. Non-parametric Kruskal-Wallis test revealed non-significant differences (p > 0.05).
TRAP staining did not show significant differences in osteoclast count between the groups. The control group had a mean of 1.60 ± 0.92 TRAP-positive multinucleated cells, whereas the Thermogenic 1 group yielded a mean of 3.25 ± 0.85, and the Thermogenic 2 group had a mean of 4.50 ± 0.50 (p > 0.05; Figure 4).
Representative images of TRAP staining in the control, Thermogenic 1, and Thermogenic 2 groups. Black arrow: Osteoclast. 40× and 400× magnification. Data are expressed as mean osteoclast count with standard deviation (error bars) in the bar chart. ANOVA revealed non-significant differences (p > 0.05).
Immunohistochemistry
Immunohistochemical results are illustrated in Figure 5. RANKL immunostaining was higher in the Thermogenic 2 group (median score: 2; Q25–Q75: 2–2) and in the Thermogenic 1 (median score: 2; Q25–Q75: 2–2), when compared to the control group (median score: 1; Q25–Q75: 1-1) (p = 0.0429). Comparison of RANK and OPG expression scores between the three groups revealed no significant differences (p > 0.05).
Immunoexpression of RANK, RANKL, and OPG in the control, Thermogenic 1, and Thermogenic 2 groups. Scale bar: 300 μm. Data on the immunohistochemical (IHC) scores are expressed as median with 95% confidence interval in the bar chart. Non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons. *p < 0.05.
Discussion
The present study consists of a preclinical evaluation of the effects of thermogenic supplements on the orthodontic movement of maxillary molars in rats. Male rats were used to rule out any potential influence of estrogen on tooth movement.15 The consumption of Thermogenic 2 supplement was found to increase RANKL expression and reduce bone fraction in the region of movement, associated with a reduction in the amount of mesial movement by the orthodontic appliance. Investigations on the impact of specific substances on periodontal tissues mainly aim to predict the response of a specific population to orthodontic treatment over time, contributing to the development of personalized treatment approaches.
The structural changes detected through volumetric and linear analyses via micro-CT result from molecular and cellular events.16 The reduced bone fraction and orthodontic tooth movement may be associated, in this study, with an increase in RANKL immunoexpression, which was verified in the animals of the experimental groups. Despite the lack of statistical significance, there was a tendency towards higher counts of TRAP-positive cells. The RANK-RANKL-OPG system is an important signaling pathway that regulates bone tissue metabolism.17 RANKL, by binding to RANK, promotes osteoclast activation and differentiation.18 Although an increase in RANKL does not necessarily imply bone loss, such increase, coupled with the lack of significant differences in OPG, alters the RANKL/OPG ratio, suggesting bone reabsorption outweighs bone formation.19 The reduction in the BV/TV parameter associated with an increase in RANKL immunostaining, but not with a decrease in OPG, has also been observed in previous experimental data on bone dynamics.20
Caffeine, one of the major components of the thermogenic supplements investigated in this study, is well-documented in the literature for its effects on orthodontic tooth movement when administered in isolation.2,21Moreno et al. (2024), in a recent study on the impact of caffeine in an experimental model of orthodontic movement, found a reduction in the BV/TV ratio in rats subjected to oral caffeine gavage compared to the control group.20 This finding is in line with those observed for the Thermogenic 2 group when compared to the control group. As documented in the literature, caffeine acts as an antagonist to the adenosine receptor, affecting bone metabolism and leading to osteoclast differentiation and higher bone resorption.22
Although the Thermogenic 2 group showed an increase in RANKL expression and a reduction in the BV/TV fraction, it exhibited a lower rate of orthodontic movement compared to the control group. Increased bone resorption is often associated with a tendency for larger orthodontic movement or with a reduction in the time needed to achieve the same distance between the teeth. Nevertheless, bone apposition is fundamental for effective orthodontic movement.23 Therefore, the observed outcome may be due to impaired bone apposition on the side where tension is applied, possibly caused by excessive suppression of osteoblast activity.
Unlike the Thermogenic 2 group, the Thermogenic 1 group showed bone fraction and tooth orthodontic movement measurements similar to those of the control group. Notably, RANKL staining was consistently high for both thermogenic supplements. Although caffeine levels were proportional across the experimental groups, the observed differences may be attributed to the ingredients rather than to the amount of caffeine in each thermogenic supplement, which could influence alveolar bone metabolism in different ways.
One of the key ingredients in Thermogenic 1 supplement, not present in Thermogenic 2, is grape seed extract (GSE). GSE has been extensively studied for its effects on bone metabolism.24-26Wahyuningtyas et al. (2024), in a scoping review evaluating the potential of GSE on bone remodeling and healing, found that GSE affects the alveolar bone by stimulating bone formation, increasing density and mineral deposition and suppressing apoptosis and osteoclastogenesis.27 These effects are primarily due to its high content of polyphenols and flavonoids.28 Thus, the effects of caffeine observed in the Termogenic 2 group might have been offset by GSE in the Thermogenic 1 group, resulting in a more balanced bone turnover.
Inflammatory response is an essential precursor to bone remodeling processes, which facilitate orthodontic tooth movement.29 In orthodontic treatment, the initial inflammatory response typically peaks within the first few days after force application. This acute phase involves the release of pro-inflammatory cytokines and the recruitment of inflammatory cells, serving as the primary trigger for bone remodeling mediated by the RANKL-RANK-OPG pathway.29,30 As the treatment advances, the inflammation generally shifts to a more chronic phase, characterized by a steady-state inflammatory response with fewer inflammatory cells and cytokines.31 In line with this rationale, all studied groups demonstrated a comparable level of moderate inflammation. The analysis at a single time point, i.e., on the 22nd day (euthanasia), may have influenced the absence of differences in inflammation across groups, which may have coincided with a reduction in inflammation.
In addition to the analyses of periodontal tissues, the effect of thermogenic supplements on the behavior of rats subjected to orthodontic movement was evaluated by the open field behavior test. This is particularly relevant because of the presence of caffeine, given that energy drinks can affect the central nervous system by increasing neurotransmitter release,2 thus leading to behavioral symptoms such as anxiety, restlessness, and irritability.32 Therefore, the effect on the neurochemical cascade may interfere with tooth movement, considering that the bone remodeling response to orthodontic forces is closely related to neural feedback from the periodontal ligament.20
Behavioral data showed the supplement used in the Thermogenic 1 group increased the distance traveled in the open field when compared to the control group at T2. Note that tests using novelty as a motivational factor for exploration may be affected by a natural reduction in exploratory behavior because prolonged or repeated exposure to the same environment can reduce the novelty effect.33 In line with previous data,33 the present study demonstrated that rats in the control group traveled shorter distances at T1, T2, and T3, when compared to T0. This shorter distance traveled by the control group may have contributed to the difference between the Thermogenic 1 and control groups at T2. Importantly, this reduction in exploratory behavior during trials should not be attributed to stress caused by orthodontic appliance placement.
The main limitation of this study was the lack of control over metabolic rate, caloric intake, and intestinal motility, important factors that may have influenced bone metabolism in rats treated with thermogenic supplements. Future studies should address these factors and evaluate other signaling pathways, such as cytokine networks and oxidative stress markers, to gain a more comprehensive understanding of how different ingredients of these supplements can affect orthodontic tooth movement.
Conclusion
The use of Thermogenic 2 (PRE-HD/Pre-workout) supplement induced alveolar bone loss in rats subjected to orthodontic movement via activation of the RANK/RANKL/OPG signaling pathway. These findings highlight the importance of evaluating the composition of thermogenic supplements because their ingredients can interfere with bone loss and osteoclastogenesis during orthodontic movement.
Acknowledgments
Graduate Program in Oral Science/UFRN and Ucla/School of Dentistry, Department of Periodontics. This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ process no: 304382/2020-5); Capes/PRINT/UFRN (process no: 88887.363681/2019-00). Capes/Brazil—Finance Code 001. The authors would also like to thank the National Institute of Science and Technology of the Health Economic-Industrial Complex - iCEIS for their support in conducting this research.
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