Nutr Bras. 2024;23(4):1097-1107
doi:10.62827/nb.v23i4.3034

ORIGINAL ARTICLE

Radiographic evaluation of the effects of a high-fat-diet on mandibular bone in animals

Avaliação radiográfica dos efeitos da dieta hiperlipídica no osso mandibular em animais

Karina M.M.C. Pinto1, Vitor M. Encarnação2, Denise G.P. Dantas2, Moises J. Oliveira2, Camila S. Sodré2, Marco Orsini3, Carlos A. S. Costa4, Rachel M. M. Santos5, Sabrina C. Brasil6, Luciana Armada1

1Estácio de Sá University, Rio de Janeiro, RJ, Brasil

2Uninassau University, Rio de Janeiro, RJ, Brasil

3Iguaçu University (UNIG), Nova Iguaçu, Brasil

4Federal University of Rio de Janeiro (UFRJ), Macaé, RJ, Brasil

5Fluminense Federal University (UFF), Niterói, RJ, Brasil

6Grande Rio University (Unigranrio Afya), Duque de Caxias, RJ, Brasil

Received: october 10, 2024; Accepted: october 11, 2024.

Correspondence: Luciana Armada, luadias@hotmail.com

Como citar

Pinto KMMC, Encarnação VM, Dantas DGP, Oliveira MJ, Sodré CS, Orsini M, Costa CAS, Santos RMM, Brasil SC, Armada L. Radiographic evaluation of the effects of a high-fat-diet on mandibular bone in animals. Nutr Bras. 2024;23(4):1097-1107. doi:10.62827/nb.v23i4.3034

Abstract

Introduction: Considering the pathophysiology of apical periodontitis (AP), an inflammatory disease characterized by bone resorption, it is likely that bone changes promoted by high-fat- diet (HFD) are also able to influence the mechanism of AP lesions development. Objective: To evaluate by periapical radiography the main effects of a high-fat- diet on mandibular bone in animal models. Methods: Isogenic Wistar rats (n=16), 8 weeks old, were used. Half of the animals were submitted to HFD and the other half to control diet (CD). AP lesions were induced 8 weeks later by creating pulp exposure of the mandibular left first molars. After 40 days, the animals were euthanized. Blood was collected for biochemical analysis (total cholesterol, HDL, VLDL and serum triglycerides) and mandible for radiographic analysis (periodontal ligament space, condyle diameter and mandibular thickness). Results: The HFD promoted significant increase in serum triglyceride (p<0.05) and VLDL (p<0.05) concentrations in the HFD group. The HFD group animals showed a reduction in both condyle diameter (p<0.05) and mandibular thickness (p<0.05), and increased periodontal ligament space (p<0.05) when compared to the CD group. Conclusion: The HFD produced significant changes in the mandibular bone, modifying the periradicular response to endodontic infection.

Keywords: Bone; Diet high-fat; Mandible.

Resumo

Introdução: Considerando a fisiopatologia da periodontite apical (PA), uma doença inflamatória caracterizada pela reabsorção óssea, é provável que as alterações ósseas promovidas pela dieta hiperlipídica (DH) também sejam capazes de influenciar o mecanismo de desenvolvimento das lesões de PA. Objetivo: Avaliar através de radiografia periapical os principais efeitos da dieta hiperlipídica no osso mandibular em modelos animais. Métodos: Foram utilizados ratos Wistar (n=16), isogênicos, com 8 semanas de idade. Metade dos animais foi submetida à DH e a outra metade à dieta normal (C). Após 8 semanas, foi estimulado o desenvolvimento de lesão perirradicular (LP) nos primeiros molares inferiores esquerdos através da exposição pulpar. Após 40 dias, os animais foram eutanasiados. O sangue foi coletado para a realização da análise bioquímica (colesterol total, HDL, VLDL e triglicerídeos séricos) e mandíbula para análise radiográfica (espaço do ligamento periodontal, diâmetro do côndilo e espessura mandibular). Resultados: A DH promoveu aumento significativo nas concentrações séricas de triglicerídeos (p<0.05) e VLDL (p<0.05) no grupo DH. Os animais do grupo DH apresentaram redução tanto no diâmetro do côndilo (p<0.05) como na espessura mandibular (p<0.05), e aumento no espaço do ligamento periodontal (p<0.05) quando comparados ao grupo C. Conclusão: A DH provocou alterações significativas no osso mandibular, influenciando inclusive na resposta perirradicular à infecção endodôntica.

Palavras-chave: Osso; Dieta hiperlipídica; Mandíbula.

Introduction

Lipids are organic molecules that have physiological, structural and nutritional functions. They act as hormone precursors, promote the transport of vitamins and serve as an energy reserve [1]. The association between lifestyle, diet, and genetic factors may be responsible for the development of a hyperlipidemic state, which is considered a risk factor for the development and progression of some diseases [2-5], grouped in the so-called metabolic syndromes: hypercholesterolemia; hyperglycemia; cardiovascular diseases; atherosclerosis and hepatic steatosis [6,7].

The hyperlipidemia is associated with a pro-inflammatory state, characterized by an expressive increase in cytokines, such as interleukins (IL) and tumor necrosis factor alpha (TNF-α) [6-8] . And the inflammatory process, associated with hyperlipidemia, is also negatively correlated with bone architecture, reducing bone quantity and quality due to inhibition of osteoblast differentiation by bioactive lipids and increased functional activity of osteoclasts [4,9,13].

Considering the pathophysiology of apical periodontitis (AP), an inflammatory disease characterized by bone resorption, it is likely that bone changes promoted by high-fat- diet (HFD) are also able to influence the mechanism of AP lesions development [10,12]. There are indications that bone resorption associated with AP occurs by the involvement and interaction between pro-inflammatory cytokines and the triad Nuclear factor kappa B activating receptor (RANK)/ Nuclear factor kappa B activating receptor ligand (RANK-L) / Osteoprotegerin (OPG) [14,15].

Therefore, the aim of this study was to perform a radiographic evaluation of the main effects of an HFD on mandibular bone in animal models. The research hypotheses were: (1) The HFD has an inflammatory potential that may interfere with bone microarchitecture, reducing mandibular bone quantity and quality; and (2) It is believed that animals in hyperlipidemic state will show greater bone changes when compared to control groups.

Methods

Animals were cared for in accordance with the “Guide for the Care and Use of Laboratory Animals”, National Academy of Sciences. Washington DC and have the approval of an institutional committee (594).

Sample Selection and Diet

For this study 16 adult male Wistar rats were used, which, after weaning, at 8 weeks of age, were kept in individual cages, with controlled room temperature (25 to 27ºC), constant humidity, and a 12-hour light/dark cycle (6:00 am to 6:00 pm). After an acclimatization period of 1 week, the animals were randomly divided into 2 groups control diet (CD) group, with normal diet and HFD group, with high-fat diet). Water was provided ad libitum and the feed was weighed and quantified at each feeding for a period of 8 weeks.

The formulation of the high-fat diet followed the protocol established by AIN- 93G [16] (Table 1). The animals were kept in the animal house of the Experimental Nutrition Department of the Universidade Federal Fluminense (UFF).

Table 1 - Nutritional Composition of high-fat diet (AIN 93-G).

Ingredient

Control diet (g/kg)

High-fat diet (g/kg)

Casein

20

20

Maize starch

52.9

35.9

Sucrose

10

10

Soy oil

7

7

lard

0

17

Cellulose

5

5

AIN-93G Mineral Mix

3.5

3.5

AIN-93G Vitamin Mix

1

1

L-Cystine

0.3

0.3

Choline Bitartrate

0.25

0.25

BHT (mg)

14

14

Quantity of ingredients used in the preparation of 100g of feed.

Apical periodontitis induction

After the 8-week feeding period [11], all animals were anesthetized with Thiopenthal (0.1 mL/100 g body weight), and the enamel and dentin of the left mandibular first molars were worn with a ½ spherical carbide bur (KG Sorensen, São Paulo) with a low rotation motor (Dentec, CS 421, Brazil). The opening was performed in the mesial fossula of the occlusal surface until pulpexposure by a single operator previously calibrated. After 40 days, the animals were euthanized by exsanguination under anesthesia with Thiopenthal (0.2 mL/100 g body weight) and blood and mandible were collected.

Serum Analysis

Blood samples were collected by cardiac puncture and total cholesterol, VLDL- cholesterol, HDL- cholesterol, and triglycerides values were analyzed using BT Plus 3000 (Biotecnica Instruments, Rome, Italy) equipment and Wiener Lab Group (Rosario, Argentina) kits (Colestat enzimatico AA líquida, HDL Colesterol monofase AA plus, TG Color GPO/PAP AA líquida).

Radiographic evaluation

The left hemi-mandibles were washed and immersed in buffered formalin solution. After this process, the radiographs were taken in a digital radiographic device (Radioesfera model - Siemens - SP, Brazil), and the positioning of the pieces on the radiographic film was standardized in order to avoid image distortion.

Image J software (National Institute of Mental Health, Bethesda, USA) was used to measure: the periodontal ligament space near the mesial roots of the left mandibular first molars, the diameter of the condyle and the thickness of the mandible. Image analysis was performed separately by two calibrated evaluators who were blinded to the groups analyzed. After image analysis, the evaluators compared the results. The few cases of disagreement between them were resolved by joint discussion.

Statistical Analysis

The comparative analysis of the data obtained was performed through non- parametric Kruskal-Wallis and Dunn’s Multiple Comparison tests using the GraphPad Prism 6 program (GraphPad Software, Inc, California, USA). The statistical significance considered was p<0.05.

Results

Biochemical evaluation

The numerical values of the mean serum levels of total cholesterol, HDL, VLDL and triglycerides per group can be seen in Table 2. The serum levels of total cholesterol and HDL were similar in all HFD and CD groups, showing no statistically significant difference (p>0.05). VLDL and triglyceride values were higher in the HFD group, with statistical significance (p<0.05).

Table 2 - Biochemical evaluation per group

Groups

Cholesterol

total (mg/dL)

HDL

(mg/dL)

VLDL

(mg/dL)

Triglycerides

(mg/dL)

CD

28.4 ± 6.1

14.5 ± 2.8

5.8 ± 2.1

29.0 ± 11.6

HFD

27.1 ± 3.0

11.0 ± 2.8

15.6 ± 6.2*

79.5 ± 32.8*

Results are expressed by mean ± DPM. * p<0.05

Radiographic evaluation

Periodontal ligament space

All animals in the HFD and CD groups showed increased periodontal ligament space (Table 3), since both developed AP lesions. However, it was possible to observe that the thickening was significantly greater in the HFD group (p=0,0015). Some of the images obtained through digital radiography can be seen in Figure 1.

Table 3 - Radiographic evaluation per group

Groups

Ligament space periodontal (pixel)

Head diameter

of the condyle (pixel)

Thickness mandibular (pixel)

CD

303.8 ± 24.1

46.6 ± 10.2

1453 ± 55.49

HFD

323.7 ± 32.2*

32.3 ± 8.6*

1278 ± 106.6 *

Results are expressed by mean ± DPM. * p<0.05

Fig. 1 - Radiographs of rat mandibles. A: CD group, B: HFD group. Arrows indicate increased periodontal ligament space in the mesial roots of the left mandibular first molars

Condylar head diameter

The condylar head diameter (Table 3) of the HFD group was significantly smaller (p<0.05) when compared to the CD group. Some of the images obtained by digital radiography can be seen in Figure 2.

Fig. 2 - Radiographs of rat mandibles. A: CD group, B: HFD group. Arrows indicate the left condyles.

Thickness of the left hemi-mandibles

The mandibular thickness (Table 3) of the HFD group was significantly lower (p<0.05) when compared to the CD group. Some of the images obtained through digital radiography can be seen in Figure 3.

Fig. 3 - Radiographs of rat mandibles. A: CD group B: HFD group. Arrows indicate mandibular thickness

Discussion

In the present study, the formulation of the hyperlipidic diet used followed the protocol established by the American Institute for Nutrition for Animal Experimentation [16], using lard in the composition. The animals of the HFD group were subjected to this diet for a feeding period of 8 weeks, which according to a previous study [11] is considered the proper time for the establishment of changes in the lipidogram.

The hypercaloric diet was effective, as the biochemical evaluation performed showed higher serum levels of VLDL-cholesterol and triglycerides in the HFD group. However, serum concentrations of total and HDL-cholesterol were similar between the groups. A previous work [11] employed a hyperlipidic diet similar to the present study to assess the correlation of hyperlipidemia with the development of periodontal disease in mice, but with 12- week supply and found considerably higher levels of triglycerides, total cholesterol and HDL.

This increase in serum triglyceride values and decrease in HDL, called the atherosclerotic profile, is considered a potential risk factor for the development of some systemic disorders that have been well reported in previous studies, such as insulin resistance [17,18]; cardiovascular diseases [3, 5, 6, 8, 19-21] and hepatic steatosis [22].

The hyperlipidemic state also correlates with some alterations in bone homeostasis. The association with alterations in alveolar bone metabolism has been proven in several previous studies, both in humans [2, 9, 12, 17, 20] and in animal models [8, 11, 12, 21, 24] that have linked the hyperlipidic diet with periodontal disease. Such results suggested that this diet has an inflammatory potential that may accelerate bone loss, contributing to the development and progression of periodontitis, as well as, to the failure of periodontal therapy. Through a quantitative radiographic evaluation it was possible to observe similar results in the present study showed that animals in hyperlipidemic conditions showed a significant reduction in condylar head diameter and mandibular bone thickness compared to the control group. These results also corroborate the findings of previous studies, which stated that hyperlipidemia compromises alveolar bone quantity and quality by inhibiting osteoblastic differentiation and stimulating osteoclastic activity [10, 13]. Being AP, an inflammatory disease characterized by bone destruction, they proposed that this condition is also associated with its etiopathogenesis.

The present work is similar to several previous studies [19, 25-30] that used animal models with AP lesions induced by pulpal exposure to the oral environment to elucidate the main mechanisms involved in the pathogenesis of periradicular diseases. The rat was the experimental model of choice because it has some advantages over other animals, such as ease of obtaining and low cost.

But the most relevant aspect is related to the fact that the kinetics of development, as well as the aspects of the main peri-radicular alterations observed in this study model are very similar to those in humans, thus allowing greater credibility to the results [31].

When assessing the periodontal ligament space, we observed that there was an increase in this structure in all groups due to inflammatory edema formation in response to microbial aggression from root canal infection [32]. However, this thickening was significantly greater in the HFD group. Additionally, we found that all components of the HFD group developed lesions in the furcation region and on the mesial and distal root apices, and these radiolucent areas were significantly larger when compared to those present in the CD group. These results were consistent with those observed by previous studies [4, 33, 34], reinforcing the hypothesis that a hyperlipidic diet increases the speed of progression of periradicular tissue destruction. Under these conditions, we can assume that pro-inflammatory cytokines secreted by immunocompetent cells are a linking factor. Therefore, it would be of great scientific relevance that further studies be performed to evaluate and quantify the main inflammatory mediators that mediate this correlation between hyperlipidemia and peri-radicular disease.

The results revealed that the hyperlipidemic state caused significant changes in the mandibular bone, including the influence of the periradicular response to endodontic infection.

Conflict of interest

The authors declare no potential conflicts of interest regarding the research, authorship, and/or publication of this article.

Funding

This study was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (E-26/ 202.917/2019).

Author contribution statement

Conceptualization: Pinto KMMC, Brasil SC, Armada L; Data Curation: Pinto KMMC, Encarnação VM, Dantas DGP, Oliveira MJ; Research: Pinto KMMC, Encarnação VM, Dantas DGP, Oliveira MJ; Resources: Costa CAS, Santos RMM; Project Administration: Pinto KMMC, Encarnação VM, Dantas DGP, Oliveira MJ, Costa CAS, Santos RMM, Brasil SC, Armada L; Visualization: Pinto KMMC, Encarnação VM, Dantas DGP, Oliveira MJ, Gonçalves CSS, Orsini M, Costa CAS, Santos RMM, Brasil SC, Armada L, Methodology: Gonçalves CSS, Orsini M, Costa CAS, Santos RMM, Brasil SC, Armada L; Validation: Gonçalves CSS, Orsini M, Costa CAS, Santos RMM, Brasil SC, Armada L, Supervision: Brasil SC, Armada L; Formal Analysis: Gonçalves CSS, Orsini M; Acquisition of Funding: Armada L; Writing original draft: Pinto KMMC, Encarnação VM, Dantas DGP, Oliveira MJ; Writing analysis and editing: Pinto KMMC, Encarnação VM, Dantas DGP, Oliveira MJ, Gonçalves CSS, Orsini M, Costa CAS, Santos RMM, Brasil SC, Armada L.

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