Expression of TNF-α and TGF-β after the Administration of Cacao Bean Extract on Alveolar Bone

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12/7/23, 3:04 AM Editorial Board - PJMD

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Ziauddin University, Karachi, Pakistan

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Islamabad medical and dental college, Islamabad, Pakistan

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Dow University of Health Sciences, Karachi, Pakistan

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PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2023, VOL. 12 (04)

Alveolar Bone

Depi Praharani, Rina Sutjiati, Sulistiyani, Atik Kurniawati, Dessy Rachmawati, Dwi Prijatmoko, Rudy Joelijanto, Shierin Velly Fiolita, Firda Qurrotul Aini Rasyid, Lilis Nurhalifah, Syafika Nuring Fadiyah, Millenieo Martin

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05 PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2023, VOL. 12 (04) DOI: https://doi.org/10.36283/PJMD12-4/003

ORIGINAL ARTICLE OPEN ACCESS

Expression of TNF-α and TGF- β after the Administration of Cacao Bean Extract on Alveolar Bone

Depi Praharani¹, Rina Sutjiati², Sulistiyani³, Atik Kurniawati⁴, Dessy Rachmawati⁵, Dwi Prijatmoko², Rudy Joelijanto², Shierin Velly Fiolita⁶, Firda Qurrotul Aini Rasyid⁶, Lilis Nurhalifah⁶, Syafika Nuring Fadiyah⁶, Millenieo Martin⁶

1Periodontics Department, ²Orthodontics Department, ³Pedodontics Department, ⁴Oral Biology Department, ⁵Biomedical Science Department, ⁶General Dentistry Department, Faculty of Dentistry, University of Jember, Jember, Indonesia.

ABSTRACT

Background: Teeth that are given orthodontic forces can relapse because of excessive resorption on the pressure side. Cocoa bean extract enhances bone apposition during bone remodeling by increasing osteoblast proliferation. This research aimed to ascertain molecular mechanisms of alveolar bone during orthodontic tooth movement applied with cacao bean extract (Theobroma cacao L.) through TNF-α and TGF-β, expressions.

Methods: This study utilized an experimental laboratory posttest-only control group design. The NiTi closed coil spring was braced between the right upper first molar and the upper incisor, a strength of 10gf was estimated utilizing a tension gauge to mesially move the upper molars. The total 36 were divided into six groups, Wistar rats were beheaded on days 7 and 14 of treatment.

Immunohistochemical staining was utilized to show TNF-α and TGF-β expression. Results were assessed using the one-way ANOVA analysis.

Results: The immunohistochemistry findings in osteoclast cells that demonstrate positive results for TGF-β and TNF-α, expression by immunohistochemical staining calculated for each group (control, positive control, treatment). The treatment group (Q14) showed reduced expression (2±0.957) of TNF-α in osteoclast cells. TGF-β, on the other hand, was found with diminished expression in osteoblast cells C-7 and C-14 (3.75±0.816, 3.25±0.957) and increases in the treatment group (Q14) (11.25±0.957)^.significantly (p=0.000).

Conclusion: The administration of cacao bean extract on days 7 and 14 could decrease the expression of TNF-α and increase TGF-β of the treatment group. Therefore, with an increased TNF-α apposition process, relapse after orthodontic treatment can be prevented to accelerate the orthodontic treatment.

Keywords: Osteoclast, Osteoblast, Estrogen, Epicatechin, Cytokine.

Corresponding Author:

Dr. Rina Sutjiati

Orthodontic Department, University of Jember, Jember, Indonesia.

Email: [email protected]

doi: https://doi.org/10.36283/PJMD12-4/003

How to cite: Praharami D, Sutjiati R, Sulistiyani, Kurniawati A, Rachmawati D, Prijatmoko D, Joelijanto R, Fiolita SV, Rasyid FQA, Nurhalifah L, Fadiyah SN, Martin M. Expression of TNF-α and TGF-β after the Administration of Cacao Bean Extract on Alveolar Bone. 2023;12(4): 5-10. Doi: 10.36283/PJMD12-4/-003

This is an open-access article distributed under the terms of the CreativeCommons Attribution License (CC BY) 4.0 https://creativecommons.org/licenses/by/4.0/

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06 PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2023, VOL. 12 (04) DOI: https://doi.org/10.36283/PJMD12-4/003 INTRODUCTION

Orthodontic treatment is a way to move a malposition tooth to an appropriate position. Giving external mechanical forces in orthodontic tooth movement influences the remodeling of the periodontal ligament and alveolar bone^1,2. These external mechanical forces induce neurotransmitters, cytokines, and growth factors, which send signals to pressure-related osteoclasts, causing bone resorption^3-5. When an external mechanical force is applied to the pressure area of the periodontal membrane, osteoclasts are induced to resorb the alveolar bone. As a result, the tooth moves ⁶. Excessive bone resorption is an unwanted side effect of orthodontic treatment ^7,8. The expression of the tumor necrosis factor (TNF) α will be induced by mechanical force in orthodontic tooth movement along with IL-1 to stimulate the activity of mature osteoclasts and withdraw other monocytes^9-11. Additionally, lymphocyte and endothelial cell production of systemic RANKL are aided by TNF-α. Prostaglandin and TNF-α play a significant role in the maturation of osteoclasts^12-14. One of the most important factors in bone formation is the transforming growth factor beta TGF-β, which balances the dynamic processes of bone formation and resorption ¹⁵. By limiting the lifespan of osteoclasts through the promotion of apoptosis mediated by TGF-β, estrogen replacement may prevent excessive bone loss ^7-16. Osteoblast and other cell production of TGF-β are effectively regulated by estrogen. By adjusting the levels of TGF-β in adults during OTM, natural remedies for the promotion of this mechanism could be a novel and useful therapeutic strategy for enhancing bone remodeling ^4,6,17.

Phytoestrogen, a plant substrate with estrogen-like activity, is a form of estrogen replacement from plants ^18,19. Epicatechin is one type of catechin, a polyphenolic compound in the flavonoid family ^20-21. A few subsidiaries of cacao beans have been perceived to emphatically affect bone remodeling without promoting any side effects ^22,23. It has previously been demonstrated that epicatechin improves osteoblast proliferation ^4,23.

The aforementioned description raises a problem of whether cacao bean extract (Theobroma cacao L.) can affect the expression of TNF-α and TGF-β on orthodontic tooth movement at the pressure side of the alveolar bone. This study aims to determine the role of cacao bean extract (Theobroma cacao L.) on the expression of TNF-α and TGF-β on the movement of orthodontic teeth in the alveolar bone. The previously mentioned description raises an issue of whether cacao bean extract (Theobroma cacao L.) can influence the expression of TNF-α and TGF-β on orthodontic tooth movement

at the pressure side of the alveolar bone. This study expects to decide the role of cacao bean extract (Theobroma cacao L.) on the expression of TNF-α and TGF-β of orthodontic tooth movement in the alveolar bone.

METHODS

The sample size was 36 male Wistar rats aged 12-16 weeks and weights from 200-300 grams were partitioned into the experimental and control categories. Healthy rats without abnormalities were separated into six categories and marked with names and characteristics, as follows.

Group 1: a) Category with a 7-day control consisting of 6 rats (C7), which were forfeited following seven days, getting orthodontic movement or not, and not getting cacao bean extract administration. b) Category with a 14-day control consisting of 6 rats (C14), which were forfeited following seven days, getting orthodontic movement or not, and not getting cacao bean extract administration.

Group 2: a) Category with a 7-day control consisting of 6 rats (C-7), which were forfeited following seven days, getting orthodontic movement, and not getting cacao bean extract administration. b) Category with a 14-day control consisting of 6 rats (C-14), which were forfeited following seven days, getting orthodontic movement, and not getting cacao bean extract administration.

Group 3: a) Category with a 7-day control consisting of 6 rats (Q7), which were forfeited following seven days, getting orthodontic movement, and getting cacao bean extract administration. b) Category with a 14-day consisting of 6 rats (Q14), which were forfeited following fourteen days, getting orthodontic movement, and getting cacao bean extract administration.

A tension gauge was utilized to measure the distal heading of 10 grams of orthodontic power to the maxillary molar of male Wistar rats utilizing a prefabricated closed stainless steel coil spring (Ormco® Glendora, USA). The experiments were approved by the Research Ethics Committee (No.1728/UN25.8/KEPK/DL/2022).

The most common way of making cacao bean extract begins with unfermented mass (link) which is then extracted using the maceration method. A total of 1 kg of cocoa beans was extracted using 96% ethanol solvent with a ratio of 1:4 material and solvent which was carried out for 3 days and covered with aluminum foil with occasional stirring.

The results of the macerate were concentrated using a rotary evaporator for 2 hours at a temperature of 40-50. The final result was 10.3 grams of cocoa bean extract. The dose of cocoa bean

extract given to each rat was 50 mg diluted in 2 ml of aqua.

The dose of gel from cacao bean extract is 80mg/ml. It was given in the sulcus gingiva of the Wistar rat.

Formalin was employed to fix the dissected maxillaries, and EDTA was employed to decalcify them. Paraffin-embedded and dehydrated samples were employed. TNF-α and TGF-β were stained on tissue areas under 180 µm from the distal root’s furcation of the upper molar. After dewaxing, the sections were treated with antigen retrieval and blocked for endogenous hydrogen peroxidase.

Then, TNF-α and TGF-β of secondary antibodies were incubated.

The expression of TNF-α and TGF-β in the pressure side area was distinct from that of other groups. To

show that the data were normally distributed, the Shapiro-Wilk normality test was conducted (p>0.05). Meanwhile, to demonstrate that the data were homogeneous, the Levene test was conducted. One-way ANOVA analysis was conducted to determine whether the expression was significantly affected; p<0.05 indicates a significant result. This study was also examined immunohistochemically. Meantime, a digital camera (Optilab Advance Plus) was utilized to take photos of the area.

RESULTS

This experimental study observed 36 male Wistar rats aged 12-16. Their classification is as follows. (C) with an absence of treatment and movement of the maxillary molar tooth. (C-) with maxillary molar tooth movement however not getting treatment. (Q) with maxillary molar tooth movement and treatment utilizing cacao bean extract. On days 7 and 14, the results of each category were examined.

DISCUSSION

Most infectious diseases that colonize the oral cavity and respiratory tract, including the novel coronavi- rus, can be transmitted in a dental clinic. A High percentage of affected people worldwide visit dental clinics, which, in turn, poses a risk of exposure and disease-contraction by dentists and other staff members¹⁷.This study assessed the knowledge of students and their corresponding practicing behav- ior concerning cross-infection control in government and private hospital settings. The overall knowledge of cross-infection control was satisfactory among dental students and similar findings were reported by other studies in Pakistan^12,13. The contributing factors could be curriculum emphasis and educational seminars conducted during their graduate program. Also, professional guidelines are set by the institute and regulatory bodies. Dental students are typically taught to adhere to these guidelines, ensuring they are well-informed about the best practices

in infection control.

Upon comparing our findings regarding the use of personal protective gear with other studies, both similarities and differences were observed. In our study, the percentage of participants wearing gloves was 98%, which aligns with a study conducted in Saudi Arabia¹⁷. However, variations were noted in the utilization of masks, gowns, and eyewear. In our study, the respective percentages were 79%, 46%, and 13%, whereas in a study by Hala- vani et al, they were 93%, 98%, and 60.7% ¹⁸. These discrepancies indicate a lack of sufficient under- standing regarding the importance of eye protection, considering the potential transmission of diseases through aerosols and blood. Nevertheless, other studies conducted in Pakistan demonstrated similar compliance with personal protection against infections^12,13. This underscores the necessity to enforce rules and regulations, with educational institutions

ensuring strict adherence to protocols by students.

Hands serve as the primary reservoir for numerous pathogens, highlighting the utmost significance of hand hygiene in preventing infections. Handwash- ing is considered a fundamental and effective method for infection prevention¹⁹. In response to the COVID-19 pandemic, the World Health Organiza- tion (WHO) has recommended a comprehensive handwashing approach, involving washing hands with soap and water for a minimum of 20 seconds, followed by the use of alcohol-based hand rub (ABHR) for visibly soiled hands ²⁰.

In our present study, we found that students were well aware of the importance of hand hygiene, with an impressive awareness rate of 94%. Their adherence to the standard hand hygiene protocol was 72% in private schools and 55% in government schools, as observed in the study by Waheed Tahir et al. conducted in a government setup, which reported a compliance rate of 74%¹³. In contrast, Marium et al. reported a higher compliance rate of 94% with hand hygiene in a private institute¹⁹. Similarly, a study conducted in India by Bommireddy et al. demonstrated a compliance rate of 59% in handwashing practices among dental practitioners²¹.While the majority of students in our study changed gloves between patients, it was noted that not all consis- tently followed proper handwashing protocols, as also reported by Noura A. et al¹⁷.This indicates a lack of institutional emphasis and guidelines regarding hand hygiene. To address this concern, we recom- mend reinforcing proper handwashing techniques among students and considering placing educational posters in handwashing areas, which would serve as useful reminders.

Although the participants in our study believed that environmental barriers such as plastic wrapping for dental units were important the practice was not very consistent, with only 33% in government and 39% in private. Similar results were seen for the disinfection of the working surface. This finding also concurs with past studies12,13, 19, 22.However, studies by Halawani et al. and Chang HC et al. show significantly improved compliance with the environment disinfection protocol post-COVID-19^18,23. This suggests that the basics of infection control should be overemphasized by continuous lectures and training sessions.

Our study revealed excellent knowledge and attitudes of students towards handling and dispos- ing of needles. Also, a thorough medical history and patients with communicable diseases were treated according to OSHA guidelines. This finding is similar to previous studies^13,15,24. However, the immunization status of students was found to be 72% and 88% in government and private institutes respectively.

Similar results were seen in previous studies in the subcontinent^12,13,25.A study conducted by Elagib MFA et al. in Sudan and Saudi also reported a low percentage of post-HBV serology²⁶. This finding suggests that the institute should make hep B vacci- nation proof mandatory for all students and a boost- er dose before starting clinical practice/rotations. A study by Alharbi et al. found that 93.1% of their undergraduate students were vaccinated due to this regulation followed by the college ²⁷.

Nonetheless, this study involved both government and private colleges therefore it displays more variability. The dissimilarity in the attitude of participants may be due to a person’s own beliefs, thoughts, and behavioral aspects. The lack of resources and adherence to strict institutional policy, particularly in the government sector could also play a role.

One of the limitations of this study was the method used to assess the practice of infection control guidelines which is based on students’ subjective self-assessment. Also, the generalizability of the findings is constrained by the convenience sample of dental students and the limited sample size.

Another limitation was the absence of qualitative data due to the limited time available during data gathering. This was a multi-center study (being carried out in private and public sector dental colleges of Karachi), which will help us elucidate the level of knowledge and practice regarding cross-infection control procedures among undergraduate dental students. It will help the institutes identify the awareness and practice of cross-infection protocols being practiced at their institutes and will also help to learn and share information with other institutes for the best interest of their students and patients' safety. It will also allow the conduct of regular work- shops to enhance and improve awareness among future dentists.

CONCLUSION

The students from both public and private institutes reported good knowledge indicating that cross-infection control guidelines are outlined by OSHA.

Lack of adherence to the guidelines at certain levels spotlights the need for an evaluation program as a means of assuring compliance with recognized policy in clinical practice. This will help create a safe environment for both the practitioners and the patients and help build patients' trust in doctors and healthcare facilities.

ACKNOWLEDGMENTS

We would like to acknowledge the honest participa- tion of all the participants and the public and private institutes’ faculty who were part of this research study for their immense cooperation.

CONFLICT OF INTEREST

Authors declare no conflict of interest ETHICAL APPROVAL

The study was approved by the Ethics Review Com- mittee of BUMDC reference # ERC 13/2020.

PATIENT CONSENT

Written and verbal consent was taken by all participants before handing out the questionnaire.

AUTHORS CONTRIBUTION

AS was responsible for the literature research, concept, and design of the study. AS and RB performed data collection and manuscript writing.

RB conducted a statistical analysis and reviewed the final manuscript. KFR supervised the research

work and provided final proofreading.

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07 PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2023, VOL. 12 (04) DOI: https://doi.org/10.36283/PJMD12-4/003 Expression of TNF-α and TGF-β after the Administration of Cacao Bean Extract on Alveolar Bone

INTRODUCTION