Dental Students' Research Journal, Issue 2 by International Association of Dental Students

March, 2014

Issue

#02

DSR DENTAL STUDENTS’ RESEARCH Switzerland, ISSN: 2296 - 1216

OFFICIAL RESEARCH PUBLICATION OF INTERNATIONAL ASSOCIATION OF DENTAL STUDENTS, IN COLLABORATION WITH ASIA PACIFIC DENTAL STUDENTS’ ASSOCIATION

EXPLORE, PERCIPIO, INVENIO

DENTAL STUDENTS’ RESEARCH

Scientific Content 32 COMPARISON OF ORAL HEALTH KNOWLEDGE, ATTITUDE, PRACTICES AND ORAL HYGIENE STATUS OF CRPF OFFICIALS IN SRINAGAR, KASHMIR, (INDIA)

36 A CASE REPORT OF KERATOCYST ODONTOGENIC TUMOR MIMICKING A LATERAL PERIODONTAL CYST (IRAN)

06 DEGRADATION OF FIBERGLASS POSTS TREATED UNDER DIFFERENT DISINFECTANTS: AN IN VITRO STUDY (CHILE) 10 EFFECT OF SPINACH(AMARANTHUS HIBRIDUS L.) LEAVES EXTRACT SOLUTION AND MILK TO LEVEL OF TOOTH DISCOLORATION DUE TO COFFEE (INDONESIA) 16 EFFECT OF GREEN TEA TOOTHPASTE ON PLAQUE INDEX (INDONESIA)

16 20 EFFECT OF CACAO SEED ETHANOL EXTRACT (Theobroma cacao L.) ON OSTEOCYTE COUNT OF ALVEOLAR MANDIBLE BONE GIVEN TO POSTOVARIECTOMIZED RAT (Indonesia) 26 CORONECTOMY OF

MANDIBULAR WISDOM TEETH: A CASE SERIES AND BRIEF (Cambodia)

20 26

DENTAL STUDENTS’ RESEARCH

PUBLISHER

from the editors

International Association of Dental Students

EDITORIAL LEADERSHIP Editor-in-chief - Pavel Scarlat (Romania/R. of Moldova) Scientific Associate Editor - Hossein Basir (USA/Iran) Communication Associate Editor - Esti Riyanda (Indonesia)

Dear Reader,

ADVISORY HONORARY BOARD Dr. Irina Dragan (USA) Dr. Jukka H. Meurman (Finland) Dr. Mauricio Gonzalez Balut (Mexico) Dr. Octavian Fagaras (Romania) Dr. Onur Kadioglu (USA) Dr. Derek Mahony (Australia) Dr. Latifa Berrezouga (Tunisia) Dr. Joao Malta Barbosa (Portugal) Dr. Mohamed Salman (Saudi Arabia) Dr. Ionut Luchian (Romania)

EDITORIAL BOARD

t is our pleasure to have worked on the making of the 2nd Issue of the Dental Students’ Research Journal and assure the continuity of the leading international research publication of it’s kind. We have received many articles during the last months, them undergoing a strict revisory process and due to the large number, some of them will be published within the next issue. The Editorial Board of the journal has expended it’s borders currently having in it’s team Editorial Officers from 60 countries worldwide and new collaborations have been established in order to assure that our publication grows and serves well the research interests of dental students worldwide. As we rely on the support and advise of our Advisory Board, we were very glad to receive the acceptance of more experienced doctors from all over the world with whom together we will make sure to raise the level of our standards. Therefore, we would like to express our deep gratitude towards everyone who has contributed to the making of the current issue, it being a living proof of our ambition and desire to serve the betterment of dental student research activities.

Yours sincerely, Editorial Leadership 2012 - 2014 editor.dsr@iads-web.org scientific.editor.dsr@iads-web.org communication.editor.dsr@iads-web.org

Jessica Zachar (Australia) Petronela Buiga (Romania) Dinesh Rokaya (Thailand) Swapnil Bumb (India) Anka Koskova (Slovakia) Diana Buturca(Romania) Nicolas Cohn (Chile) Ahmed El- Sayed (United Arab Emirates) Victor Vega Morales (Puerto Rico/USA) Eva Sinic (Slovenia) Fairuz Atig (Tunisia) Flavia Joarza (Romania) Giacomo Armani (Romania/Italy) Davina Peh (Malaysia) Gorkem Sengun (Turkey) Jana Nakladova (Czech Republic) Karin Pokoma(Czech Republic) Jana Nakladova(Czech Republic) Magdalena Wiczak (Poland) Victor Palumbo (Italy) Monica Ferran (Slovenia) Murad Alrsheedi (Saudi Arabia) Petra Horakova (Czech Republic) Petronela Buiga (Romania) Pradeep Sharma (India) Ricardo Fillipe Mendes (Portugal) Rifqi Aulia Destiansyah (Indonesia) Sajjad Ashnagar (Iran) Sara Ehsani (Iran) Tarek Omran (United Arab Emirates) Andrei Baltaev (Russian Federation) Tigran Gyokchyan (Armenia) Juan Guttierez Quintero (Columbia) Veronika Piskova (Czech Republic) Wika Ardianti Putri (Indonesia) Yana Sadykova (Kazakhstan) Zen Feng Chong (Malaysia) Ahmad Salah (Sudan) Kiki Saputri (Indonesia) Tsai, Chi - Hasuan (Republic of China Taiwan) Patrick Bannerman-Agbesi (Ghana) Taher Elkowiery (Egypt) Augusto Elias (Puerto Rico/USA) Abdulla A Alajmi (Sudan) Mustafa Gurkan (Turkey) Resat Batuhan Cetiner (Turkey) Busra Zengin (Turkey) Nur Oztoprak (Turkey) Ferdiye KÜÇÜK (North Cyprus) Melike Atalar (Turkey) Omar Al Bairat (Morocco) Faniran Felix (Nigeria) Sina Saygili (Turkey) Esat Bugrahan Toksoz (Turkey) Augusto Elias (Puerto Rico - USA) Mert Kalak (Turkey) Mustafa Ozcan (Turkey) Beley Ostap (Ukraine) Gregory Tour (Sweden) Ferdiye Küçük (Cyprus - North Cyprus) Dimitri Mcgregor (Jamaica)

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11th IADS Lecture Contest KIKI SAPUTRI ESTI RIYANDA

he IADS Standing Committee on Research and Education (SCORE) will organize the 11th edition of the IADS Lecture Contest which will be hold during the IADS 61st Annual Congress ( 29th August- 3rd september , 2014) in Yogyakarta, Indonesia.

This event will gather international dental students and young researchers who will enter in a scientific competition. They will prepare presentations which should reflect the research work and a Jury composed of renowned doctors will assess their scientific importance and the studentsâ&#x20AC;&#x2122; work. If you are a young scientist and organized/assisted a dental scientific project, the IADS Lecture Contest is the chance for you to show the entire world that you can make a difference within your dental community.

Eligibility: Undergraduate dental students and young graduates Deadline for abstract submission: 1st July, 2014 Notification of selection: 21st July 2014

Important submission elements: Title: abstract titles are limited to 12 words Authors/ Coauthors: the name of the Authors and Co-authors should be capitalized. Abstract text: all abstracts should be 350 words or less. Tables are permitted but should be simple and concise.

DENTAL STUDENTSâ&#x20AC;&#x2122; RESEARCH

Winners of the 10th IADS Lectue Contest - Tunisia 1. Matej Hafo Magura (Slovakia/Czech Republic) 2. Amina Ben Salem(Tunisia) 3. Ayu Jembar Sari (Indonesia)

Yogyakarta , Indonesia September 2nd, 2014 The dental student research leaders from all over the world will compete in it. Will you?

Content of the abstract: 1. The objectives of the investigation 2. Experimental methods used 3. Essential results, including data and eventual statistics 4. Conclusion Keywords: up to 5 keywords may be selected

For more information and abstract submission, please contact : IADS International Scientific Officer Gorkem Sengun iso@iads-web.org

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DEGRADATION OF FIBERGLASS POSTS TREATED UNDER DIFFERENT DISINFECTANTS: AN IN VITRO STUDY Joel Andrés Morales Parra 1, Luis Felipe Barría Pérez 2 Email address: joel.morales.p@gmail.com, luisfelipebarria@gmail.com

1,2

School of Dentistry, Universidad Austral de Chile, Valdivia, Chile

No conflict of interest with the company named in this article for any of the authors.

Abstract: Purpose: Manufacturers’ instructions of fiberglass posts recommend disinfestation of a post by the use of solvents before the cementation. Nevertheless, this procedure does specify neither the time of exposition, nor the graduation of that disinfectant. Although there are studies that have shown the decrease of the adhesion post-duct, the possible damage of the disinfestation in the ultra-structure of the post, and the characterization or the quantification of the release of the organic matrix have not been investigated. Therefore, the present study aimed to quantify and compare the released material from fiberglass post after disinfection. Methods: For the quantitative analysis, posts were immersed at different times in: sodium hypochlorite 5%, ethanol 70%, ethanol 95%, or distilled water (control). The subsequent samples were collected. Then, samples were subdued to molecular absorption spectrophotometry (UV-visible ThermoSpectronic - Heλios Gamma), getting the corresponding absorbance of each of these, which is characterized as a loss of material. This was compared to the absorbance of the molecule of Bis-GMA, which has its peak at 270 mm of wavelength. The data was analyzed with the software Vision 32. The qualitative analysis was done with scanning electron microscope (LEO 420). Results: The concentration substances from the fiberglass posts were directly proportional with respect to the time of exposition to the three types of disinfectants. At 20 minutes, the concentrations of loss in the fiberglass post were significantly higher in 5% sodium hypochlorite (1,22E-2 grams) compared to 70% ethanol (0,23E-2 grams) and 95% ethanol (0,05E-02 grams). There was not a loss in the control group subdued to distilled water.

Conclusion: The present data suggests use 95% of ethanol for disinfection of fiberglass posts due to the lower degradation of fiberglass posts after disinfection when this solvent was used. KEYWORDS: Damage, Degradation, Disinfection, Fiberglass posts, Scanning electron microscopy, Spectrophotometry.

Introduction The resistance of the teeth under an endodontic treatment has been thoroughly studied, and many of its concepts are in continuous revision. The fiberglass posts (FP) are used in intraradicular restorations of teeth receving endodontic treatment. Fiberglass posts give a solid base to increase the mooring to get the prosthetic restoration, prevent the fracture of the teeth under endodontic treatment, and proportionate support and internal resistance (1). The oldest references of prosthetic restorations in severely destroyed teeth date from the period of Tokugawa (1603-1867) in Japan. They created a dental Crown with black boxwood, which was very aesthetic in that time. During 1728, Pierre Fauchard inserted wood spikes inside the radicular channels of the teeth in order to improve the retention, but he failed due to the absence of resistance and absorption of the humidity in the mouth. In 1746, Claude Hounton published his design of the gold crown with an endopost made of the same material that was put in the radicular channel. Then, in 1747, Pierre Fauchard used gold and silver endoposts covered with an adhesive soften by

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heat, a process called “mastic”. In 1970, a new era of endoposts was born; the prefabricated metal endoposts that had different forms and length were introduced. Later, in 1987 in France, the first post made of carbon fiber was introduced, which was commercialized in 1990 in USA. Carbon fiber post offered less elasticity than metals or conventional alloys. It had characteristics similar to the dentin that gave more resistance to the fracture. It was demonstrated that the carbon fiber was more resistant than the prefabricated metallic posts and the post-core units. Subsequently, in order to improve the aesthetic, the fiberglass posts were created. The post-core posts are used more scarcely due to the cost in comparison to the prefabricated ones. Besides, they demand more time to build, and they can cause damage to the dental structure since they can provoke more corrosion. However, they have a close relation with the dental structure and with the conformation of the radicular channel (2, 3). On the other hand, the prefabricated posts have an easier implementation, and they demand less time and less cost. At the beginning, prefabricated posts were just made of titanium and stainless steel. Nowadays, with the creation of different fibers, the structure and properties of prefabricated posts are similar to the natural dental structure, offering more success during restorations (2, 3). The indicated protocol in the technical datasheet of the fiberglass includes the disinfestation of the post through different solvents. Nevertheless, this protocol does determine neither the time of exposition, nor the graduation of this disinfectant. It has been demonstrated that this disinfestation affects the adhesion of the fiberglass post during the restoration process (4). In spite of the fact that there are studies about this situation, the majority of them are focused in the capacity of adhesion post-restoration (5), and they do not consider neither the possible damage that the disinfestation provoke in the ultrastructure of the fiberglass post, nor the characterization and quantification of the release of the organic matrix.

Therefore, the present study aimed to quantify and compare the released material from fiberglass post after disinfection by 5% sodium hyplochlorite, 70% and 90% ethanol. For this purpose, molecular absorption spectrophotometry (MAS) was used.

Materials and Methods Quantitative characterization In the present study, 78 RTD fiberglass posts (RTD, St. Egreve, France) of 19 mm length were used. 72 posts were divided in the three equal experimental groups of 24 posts. Each of these groups were subdued to a specific solvent: 5% sodium hypochlorite, 70% ethanol, and 95% ethanol, respectively. In the control group, which consisted of 6 samples, posts were processed in distilled water. All posts were immersed in Eppendorf tubes in the described solvents. The measurements were made at the following times: 1, 3, 5, 7, 10 y 20 minutes. Four measurements were made at each time in order to obtain average values. After filtration, 2 ml. of each solvent were collected using a micropipette in order to develop an absorbance of the released substances by comparing them to the calibration curve of the molecule Bis-GMA, which absorbance peak was 290 nm of length wave. This last procedure was done through a spectrophotometer (UV-visible ThermoSpectronic - Heλios Gamma). The measurements were analyzed with the software Vision 32. Two-way ANOVA test was used to analyzed the data by the software PRISM 6 TRIAL (GraphPad). Qualitative characterization After this procedure, four posts (one from each group) were taken and were analyzed through scanning electron microscopy, using the SEM model LEO42.

There are still several questions regarding the loss of material in the post when disinfesting and also about the variation of this loss of material between different disinfectants in different clinical times of exposition.

Table 1. Composition of the fiberglass posts used in this study.

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Figure 1: Degradation graphic of the organic material in mass concentration of grams (MEDIA+SD) v/s time in minutes of different solvents. Results Quantitative results: The released substances were found to be directly proportional with the time in the three types of disinfectants (Figure 1). At the end, at 20 minutes, the final concentrations of the released substances were significantly higher in 5% sodium hypochlorite (1,22E-2 grams) compared to 70% ethanol (0,23E-2 grams) and 95% ethanol (0,05E-02 grams). In each of the four measurements per experimental groups the standard deviation produced results minor to 0.001. Qualitative results: The qualitative results are demonstrated in figure 2. .

Discussions The clinical success of a resin post-core restoration depends not only on the type of composed resin, but also on the quality of the interface of the post-core, where the different materials are in minimal contact (6). The Longevity of a restoration is affected by the formation of a strong union between the composed resin and the fiber post, which can present variation according to the previous treatment in the post (7, 8, 9, 10). The nature of Bis-GMA molecule (main component of the epoxy resin in fiberglass posts), composed by two rings that are highly hydrophobic; therefore, Bis-GMA molecule is soluble in polar solvents like the ethanol. The present data shows that the degradation of the fiber post in 70 % ethanol was directly proportional to the time. This could be attributable to the fluency of the 70 % ethanol (product of greater concentration of water, and phenomenon of transportation that creates this at the moment of being combined with alcohol), which can produce an inflammation in the fiberglass. This inflammation facilitates the appearance of solvents, which can react when mixed with the Bis-GMA molecule. This would be the responsible mechanism of extraction in the loss

of resin that has not reacted in ethanol 70%, process that is more evident in NaCIO. Therefore, the molecule of Bis-GMA is affected in concentrations of dependent alcohol, and not the fiberglass pole. The degradation of the organic matrix of fiberglass posts could affect the adhesion of the fiber post in the radicular channel. This is an area that has not been explored, since there are not studies that consider this loss of material as a cause of failures in the strength and adhesion properties. It is important to note that, in the present study, only the amount of release Bis-GMA from fiberglass posts were evaluated. Therefore, further studies are required to investigate the amount of other released substances from fiberglass posts.

Conclusion The present data suggests superiority of 95% ethanol over 70% ethanol or 5% sodium hypochlorite for disinfection of fiberglass posts due to the lower degradation of fiberglass posts during disinfection when 95% ethanol was used.

ACKNOWLEDGEMENT School of Dentistry, Faculty of Medicine, Universidad Austral de Chile, Valdivia, Chile. *Dr. Pedro Gainza, Odontostomatology Institute, Universidad Austral de Chile. *Dr. Pedro Aravena, Faculty of Medicine, Universidad Austral de Chile. *Nicolas Cohn, Laboratory of Polymer Chemistry Sciences Institute. Faculty of Science, Universidad Austral de Chile. *Professor Ricardo Silva Electron Microscopy Laboratory, Institute of Pharmacy, Faculty of Science, Universidad Austral de Chile.

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a) Control fiber post without modifications in its original state. 500x a smooth surface. 3000x there are not fibers exposed, since they are covered with epoxic resins.

b) Fiber post in ethanol 95%. 500x it is observed little irregularities in the organic matrix. 3000x crystal structure slightly affected (raising).

c) Fiber post in ethanol 70%. 500x The raising of the matrix is more noticeable. 3000x major damage.

d) Fiber post in sodium hypochlorite 5%. 500% it is seen more degradation. 3000x the structural damage is noticeable.

Figure 2. Photographs of scanning electron microscopy SEM (500x and 3000x) in three minutes of exposition. References (1) Ring, ME. Dentistry an illustrated history C.V. St. Louis: Mosby; 1985. (2) Schwartz RS, Robbins JW. Post placement and restoration of endodontically treated teeth: A literature review. J Endod. 2004;30(5);289-301. (3) Christensen JG. Post concepts are changing. J Am Dent Assoc. 2004;135(9):1308-10. (4) Alava M, Mena N, Sandoval F. Adhesion-cohesion inter-phase among fiberglass posts, dual cement and dentine before irrigation with two disinfectant materials. Revista odontológica Mexicana. 2012;16(3):182-7. (5) Cecchin D, de Almeida JF, Gomes BP, Zaia AA, Ferraz CC. Influence of chlorhexidine and ethanol on the bond strength and durability of the adhesion of the fiber posts to root dentin using a total etching adhesive system. J Endod. 2011;37(9):1310–15. (6) Park SJ, Jin JS. Effect of silane coupling agent

on interphase and performance of glass fibers/ unsaturated polyester composites. Journal of Colloid and Interface Science. 200; 242 (1):174–9. (7) Aksornmuang J, Foxton RM, Nakajima M, Tagami J. Microtensile bond strength of a dual cure resin core material to glass and quartz fibreposts. J Dent. 2004;32(6):443-50. (8) Cekic-Nagas I, Sukuroglu E, Canay S. Does the surface treatment affect the bond strength of various fibre-post systems to resin-core materials? J Dent. 2011;39(2):171-9. (9) Yenisey M, KulunkS. Effects of chemical surface treatments of quartz and glass fiber posts on the retention of a composite resin. J Prosthet Dent. 2008 Jan;99(1):38-45. (10) Monticelli F, Toledano M, Tay FR, Cury AH, Goracci C, Ferrari M. Post-surface conditioning improves interfacial adhesion in post/core restorations. Dent Mater. 2006;22(7):6029

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EFFECT OF SPINACH (AMARANTHUS HIBRIDUS L.) LEAVES EXTRACT SOLUTION AND MILK TO LEVEL OF TOOTH DISCOLORATION DUE TO COFFEE Lilis Iskandar 1 1

Faculty of Dentistry, Universitas Indonesia, Indonesia

email address: lilis_iskandar@hotmail.com

No conflict of interest

Abstract Purpose: The present study aimed to analyze the effect of combination of spinach leaves extract solution and milk on the level of tooth discoloration caused by coffee. Methods: In the present in vitro study, 24 teeth were color-measured with VITA Easyshade® and divided into four groups, which are control group and three other groups that were immersed in spinach leaves extract solution of 10%, 20%, and 30% plus milk for 60 minutes. Then, specimens were then immersed in coffee for 24 hours. Afterwards, the teeth color change was measured. Results: Kruskal-Wallis test showed significant difference in ΔL* (brightness) among each group. Compared to control group, two-Tailed Independent T-Test and Wilcoxon Test showed significant difference in ΔL*of all extract group, Δa* (degree of green-red color) of group 10% and 20%, Δb* (degree of blue-yellow color) of none group, and ΔE (value of color change) of group 20%. Pearson Correlation Test did not show any significant correlation between extract concentration and ΔL*, Δa*, Δb*, or ΔE. Conclusion: Within the limitations of the present study, it can be concluded that the combination of Spinach (Amaranthus hybridus L.) leaves extract solution and milk can reduce level of tooth discoloration caused by coffee. However, there is no significant correlation between the increasing concentrations of spinach leaves extract solution and level of tooth discoloration due to coffee. Keywords: calcium oxalate, coffee, milk, spinach leaves extract, tooth discoloration

Introduction Coffee is a very popular drink all over the world (1). It can cause teeth discoloration by changing the color of teeth biofilm (2). Coffee is rich with bioactive substances, such as nicotinic acid, trigonelline, quinolinic acid, tannic acid, pyrogallic acid and caffeine (3). Tannin or tannic acid is

responsible for making the teeth become darker. Tannin also makes the pH of coffee become acidic (3,4). Acidic condition will weaken the enamel and make it more vulnerable to get infiltrated by stain (2). Caffeine makes people get addicted to coffee so it will become daily habit to consume coffee (5). Teeth color change is a big esthetic problem. Bright teeth color makes individual feel more confident in public. Treatment options to fix discoloration of teeth are quite varied, such as porcelain or composite veneer, bleaching, and full veneer crown. However, all of the treatment options are relatively expensive and invasive (6). Color is described in terms of hue, value and chroma (7). Hue is term to differentiate color into red, blue or green (7,8). Value is the brightness of a color from black to white (7). Chroma is saturation level, intensity of a color with same hue, like from pink to dark red (7,8). Tooth color is valued from three aspect described by Commission Internationale d’Eclairage (CIE), the L* (brightness) value where it shows the degree of blackwhite (0 for black and 100 for white), a* (degree of greenred) where a*<0 means greener and a*>0 is more reddish, and b* (degree of blue-yellow) where b*<0 means more blue and b*>0 is more yellowish (9). The value of color change is generally represented by ∆E, formula to calculate it (10). There is a unique phenomenon happens when people eat spinach. Some people are wondering why their teeth feel weird, a bit gritty or chalky after they eat spinach salad. It is called spinach teeth sensation (11). This phenomenon is caused by the oxalic acid content in spinach that reacts with calcium ions from saliva and the spinach itself forming calcium oxalate crystal. Since calcium oxalate is insoluble, it deposits on teeth surface. Drinking milk will exaggerate spinach teeth sensation, because milk gives additional calcium (12). Calcium content in saliva is only1 mmol/L, while milk contains higher calcium which is 1200 mg/L (13,14). Milk contains the highest calcium compared to other foods containing calcium like sardines, almond, broccoli or cabbage (15).

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Spinach (Amaranthus hybridus L.) contains carbohydrate, fat, vitamin, sodium, calcium, phosphor, zinc, potassium, thiamin, riboflavin, and niacin. Spinach is consisted of 91% water (16), and it contains high amount of oxalate acid content (17). Oxalate is soluble in water (18), and the highest oxalate content is in the leaves (19). Based on research conducted by Hartini on five different vegetables frequently consumed, calcium oxalate content in spinach leaves is the highest, which is 39% (20). Oxalate content varies depending on water content, age of the plant, soil type, climate, and genetic factor (17,19,21). Oxalate in spinach increases at initial growth, decreases as the plant grows older, and reduced by the heat (18,21). Spinach leaves also have high calcium content of 99 mg/100 g (22). The ratio of oxalate acid: calcium in spinach is about 4:1 (18).

rotavapor until becoming the dense extract (54.1 g).

In normal and alkalic condition, calcium and oxalate acid will form calcium oxalate crystal (23). Calcium oxalate reaction is acid and base reaction as seen below (18). Calcium oxalate on tooth can cover dentinal tubules, forming a layer than can reduce dentine permeability. Phytocomplex of spinach 1.5% spray solution has been used topically to prevent dissolving smear layer and exposure of dentinal tubules after periodontal instrumentation. The similar solution can also be used to prevent dentine hypersensitivity due to scaling and root planing procedure (24).

Spinach Leaves Extract Solution Making: Spinach leaves extract was made into varying concentration by weighing the extract and water using digital weighingmachine. 10% extract means 10 g of spinach leaves extract dissolved in 100 ml aquadest, 20% extract means 20 g of spinach leaves extract dissolved in 100 ml aquadest, 30% extract means 30 g of spinach leaves extract dissolved in 100 ml aquadest. Using the result of measuring water content in the spinach leaves dense extract, the weight of dense extract and aquadest needed to make different concentration were able to calculate.

The present study aimed to evaluate the effect of spinach leaves extract solution and milk on level of tooth discoloration caused by coffee. It was hypothesized that oxalate film will be formed on enamel surface, similar to the spinach teeth phenomenon, which will act as a protection from tannic acid of coffee that darken the teeth. Furthermore, a secondary aim of this study was to assess correlation between concentration of spinach leaves extract solution and level of tooth discoloration in order to know the optimal concentration for the application.

Tooth Protection with Calcium Oxalate: Teeth specimens were immersed in spinach leaves’ extract solution with concentration of 10%, 20%, and 30% which each was mixed with 1 ml of high calcium milk, left for 60 seconds. Calcium content of the milk (brand Ultra) is 20% AKG which means 20% of daily need. Calcium daily need is about 1 gram (15). It means that the milk contains 0.2 gram/125 ml or 1.6 mg/ml.

Materials and Methods Spinach Leaves Maceration: 5 kg of fresh green spinach was cleaned and sorted only for its leaves. Spinach leaves were then dried into 500 g simplicia and ready to be macerated. Maceration was done by putting simplicia into a pot immersed in aquadest solvent with comparison of 1:4 (500 g of simplicia in 2 liters of solvent). The simplicia was then stirred for 3 hours and left for 24 hours. Filtrate was then filtered with lint and put into a glass pot. Afterwards, the residue was remacerated with the same method once more. The filtrate was collected and filtered. The solvent was vapored with

Phytochemical Screening Test: Spinach leaves dense extract sample was examined for its calcium, oxalate acid, calcium oxalate, tannin, and water content. Technique used to examine calcium content was AAS (Atomic Absorption Spectrophotometry), for oxalic acid and calcium oxalate was HPLC (High-Performance Liquid Chromatography), for tannin was spectrophotometry, and for water content was using gravimetry method. Specimen Preparation: Freshly extracted human premolar teeth were collected and kept in NaCl solution 0.9%. All surfaces besides enamel were covered by nail paint so that they would not get any contact with the materials.

Coffee Staining: Specimens were immersed in coffee made from Nescafe Classic coffee powder and boiled drinking water (brand Aqua) with comparison of 1 gram of coffee powder in 10 ml of water. The coffee was stirred 50 times with spoon and cooled until room temperature. The pH was examined with litmus paper. Teeth were soaked in 4 ml coffee per vial and kept in 37°C incubator for 24 hours.

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Tooth Color Change Test: Tooth color was examined twice with color description of CIE L*a*b* using VITA Easyshade®. First measurement was done before given any action, and the second measurement was performed after immersing in coffee. Both data were compared and analyzed. All changes of L*, a*, and b* (ΔL*, Δa*, and Δb*) values were obtained from tooth color difference after coffee contact and the initial color of the tooth (L0, a0, and b0). Color change (ΔE*) was calculated using the formula. Statistical Analysis: To analyze difference between effects of each extract concentration (10%, 20%, and 30%), comparative tests towards color change value (ΔE*) was done using One-Way ANOVA or Kruskal-Wallis (depends on normality of data distribution). Comparative test was also done using Independent T-Test or Mann-Whitney/Wilcoxon (depends on normality of data distribution). To see the correlation between extract concentration and tooth discoloration, Two-Tailed Pearson correlation test was conducted. All analysis was done using Windows SPSS 17.0 program.

c. Two-Tailed Pearson Correlation Test Significance value of Two-Tailed Pearson correlation test<0.05 means it is significantly correlated. If there is a significant correlation, it can be tested for the correlation strength. Positive value means direct correlation, negative value means inverse correlation. Correlation value > 0.5 means both variables have a strong correlation, while value< 0.5 means weak correlation (Table 7).

Table 1. Phytochemical Screening Test Results(private or public)

Results 1. Phytochemicaland PH test results Pytochemical screening test results are presented in Table 1. PH test results are shown in Table 2.

Table 2. PH Test Results

2. Teeth Color Test Results Teeth color test results are demonstrated in Figure 1 and Table 3. 3. Statistic Test Results a. One Way ANOVA and Kruskal-Wallis: Because the sample is less than 50, data distribution normality test was done using Saphiro-Wilk test. If the significance value<0.05, it means that the data is not normally distributed. In One Way ANOVA and Kruskal-Wallis, significance value <0.05 means significantly different (Table 4).

Table 3. Average Value of Color Change

b. Independent T-Test and Mann-Whitney/Wilcoxon Test: These tests were done to compare change in every group independently

each color to control.

- Normality of Data Distribution (Saphiro-Wilk): Because the sample is less than 50, Saphiro-Wilk testwas used to know data distribution normality. Significance value <0.05 means the data is not normally distributed (Table 5). - Independent T-Test and Mann-Whitney/Wilcoxon Test Results: Normally distributed data was tested with Independent T-Test, while abnormally distributed data was tested with nonparametric test that is Mann-Whitney/Wilcoxon. Significance value<0.05 means it is significantly different (Table 6).

Table 4. Data Normality, One Way ANOVA, and Kruskal-Wallis Test Results

Table 5. Data Distribution Normality Test Results

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Results: Kruskal-Wallis test showed significant difference in ΔL* (brightness) among each group. Compared to control group, twoTailed Independent T-Test and Wilcoxon Test showed significant difference in ΔL*of all extract group, Δa* (degree of green-red color) of group 10% and 20%, Δb* (degree of blue-yellow color) of none group, and ΔE (value of color change) of group 20%. Pearson Correlation Test did not show any significant correlation between extract concentration and ΔL*, Δa*, Δb*, or ΔE.

Discussion Tannin is one of the factors that can cause teeth extrinsic discoloration (7). From tannin test, spinach leaves dense extract contains tannin with small percentage (2.57%) if compared to coffee which has 19.5% to 23.1% tannin (25). Moreover, in the present study, spinach leaves extract has been diluted to 10%, 20%, and 30% concentration. Therefore, tannin content in spinach leaves extract would not give any significant discoloration to teeth. Calcium content in spinach leaves extract is very small, which is only 211.38 mg/100g or 0.0021138%. This shows that free calcium ion in spinach leaves extract had reacted with existing oxalic acid. Calcium oxalate content (2.43%) is the result of the reaction. Because the content of oxalic acid is higher than calcium ion in spinach leaves extract, some oxalic acid remains unreacted (3.46%). Thus, spinach leaves extract is still potential to form calcium oxalate deposition on tooth surface when it met calcium from milk. From pH test, it was seen that 30% spinach leaves extract solution and coffee are acidic (pH=5). Critical pH of enamel hydroxiapatite to demineralize is 5.5 and for fluorapatite is 4.5 (26). If the soaking solution is acidic, it can demineralize enamel, tannin can infiltrate and causing stain. Even though the 30% spinach leaves extract solution is acidic, the pH will increase when mixed with milk which pH is 7. Calcium is alkalic, acidbase reaction of oxalic acid and calcium will produce calcium oxalate salt that is neutral which pH is around 6.65-6.75 (27,28). Calcium oxalate can be formed if the pH is neutral or alkali; therefore, it is very important to keep the pH not acidic (23). When compared simultaneously, Kruskal-Wallis test shows significant difference at ΔL* (brightness) in each group. But when comparing one by one versus control, it can be seen that group immersed in 10% spinach leaves extract solution has significant difference at ΔL* and Δa*, group 20% has significant difference at ΔL*, Δa*, and ΔE*, while group 30% is only significantly different at ΔL*.It can be concluded that tooth brightness change (ΔL*) decreased significantly when the tooth was protected by the calcium oxalate before stained with coffee. Furthermore, spinach leaves extract of 10% and 20% concentration also decreased Δa* (less reddish) compared to control group. While the whole color change (ΔE*) is only significantly different when tooth immersed in 20% spinach leaves extract. Coffee with its acidity can demineralize tooth enamel so that staining material like tannin can penetrate into deeper enamel or even into dentine, deposited there, and cause discoloration (5).

Discoloration will become permanent and cannot be removed directly because when the oral pH is back to neutral, enamel is remineralized, and the stain is trapped inside. Nevertheless, with blocking from calcium oxalate crystal on tooth surface, tooth enamel does not directly contact with coffee. Thus, no demineralization happens, and tannin is just deposited on the surface of the calcium oxalate, which can be removed easily from tooth surface with mechanical action. Eventually, the discoloration does not happen permanently. Calcium oxalate tends to deposite on the rough surface of tooth because the crystal needs mechanical retention to attach. Due to the different roughness of each tooth surface, calcium oxalate deposition was not evenly distributed. It makes agap for tannin to penetrate and has a direct contact to enamel surface; thus, the tooth still have slight color change. However, the presence of calcium oxalate has lowered the effect of coffee discoloration. Pearson correlation test shows no significant correlation between spinach leaves extract concentration and level of tooth color change (ΔL*,Δa*, Δb*, and ΔE*). Increasing extract concentration does not significantly lowering the color change. Therefore, the ability of protecting the tooth from discoloration due to coffee is not far different among 10%, 20% or 30% extract.

Conclusion Within the limitations of the present study, it can be concluded that the combination of Spinach (Amaranthus hybridus L.) leaves extract solution and milk can reduce level of tooth discoloration caused by coffee. This effect seems to be due to formation of calcium oxalate layer on tooth enamel surface. However, there is no significant correlation between the increasing concentrations of spinach leaves extract solution and level of tooth discoloration due to coffee. It seems that spinach leaves extract and milk have a great potency to be developed into preventive products of teeth discoloration due to extrinsic stain from foods and drinks.

Table 6. Independent T-Test and Mann Whitney/Wilcoxon Test Results

Table 7. Two-Tailed Pearson Correlation Test Results

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Figure 1. Diagram of Color Change Average Acknoledgement

10. Burkinshaw RM. Colour in relation to dentistry. Fundamental of colour science. British Dental Journal. 2004;196(1): 33-41.

I would like to say thank you to:

11. What Causes ”Spinach Teeth”. [cited on August 2012]. Available at http://www.fitsugar.com/WhatCauses-Spinach-Teeth-551249

• drg.Andi Soufyan,M.Kes and drg.Niti Matram as my supervisors in this research • Dr. Drg. Yosi Kusuma Eriwati, M.Si as head of Dental Material Department Faculty of Dentistry Universitas Indonesia, Mr. Dudy Suryadi Soebawi, ST as the laboratorian, Mr. Slamet and Miss Maryamah as the department staffs who allowed and helped me to use the laboratory • Mr. Dedi Rosyadi and Mr. Dedi Kustiwa from Balittro Bogor who helped me to extract the spinach leaves • Dr.Berna Elya, MS., Apt. as the lecturer of Pharmacology Faculty Universitas Indonesia who taught me about the extraction techniques

References

12. Webber R. Why Does Spinach Leave a Film on Your Teeth? [cited on August 2012]. Available at http://www.chow.com/food-news/55264/why-does-spinach-leave-a-film-on-your-teeth/ 13. García-Godoy F, Hicks MJ. Maintaining the integrity of the enamel surface: The role of dental biofilm, saliva and preventive agents in enamel demineralization and remineralization. JADA. 2008;139(suppl 2):25-34. 14. Calcium from Milk. Copyright © IDF 2008. All rights reserved from www.idfdairynutrition.org 15. Calcium. 2005. [cited on December 2012]. Available at http://www.moh.gov. my/images/gallery/ rni/14_chat.pdf 16. Mortati S.Spinach: Scientific Classification and Entymology. College Seminar 235 Food for Thought: The Science, Culture, & Politics of Food. 2008. 17. Mou B. Evaluation of Oxalate Concentration in the U.S. Spinach Germplasm Collection. Hortscience. 2008;43(6): 1690-3. 18. Noonan SC, Savage GP. Oxalate content of foods and its effect on humans. Asia Pacific J Clin Nutr. 1999;8(1): 64-74. 19. Ahmed J, Ojha K, Vaidya S, Ganguli J, Ganguli AK. Formation of Calcium Oxalate Nanoparticles in

1. Wang CC, Chou YY, Sheu SR, Jang MJ, Chen TH. Application of ultrasound thermal process on extracting flavor and caffeine or coffee. Thermal Science. 2011;1(1): 69-74. 2. Stained Teeth, our favourite foods are hard on our pearly whites. Canadian Dental Association. 2012 [cited on August 2012]. Available at http://www.canadian-health.ca/7_3/20_e.pdf 3. Hečimović I, Belščak-Cvitanović A, Horžić D, Komes D. Comparative study of polyphenols

Leaves: Significant Role of Water Content and Age of Leaves. Current Science. 2012;103(3): 293-8. 20. Hartini, Yustina Sri. Penetapan kadar kalsium oksalat pada lima macam sayuran yang dikonsumsi daunnya. Jurnal Ilmiah Nasional 1999;5:51. 21. Caliskan M. The Metabolism of Oxalic Acid. Turk J Zool.2000;24:103-6. 22. USDA Nutrient Database [cited on September 2012]. Available athttp://ndb.nal.usda.gov/ndb/

and caffeine in different coffee varieties affected by the degree of roasting. Food Chemistry.

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ridged&new=

4. Caffeine Addiction Becomes a National Issue [cited on August 2012]. Available at http://www. caffeineawareness.org/caffeineawarness4page.pdf 5. O’Meara C. Changing Lives, Changing Habits. 2011 [cited on August 2012]. Available at http:// www.bmd.com.au/GoodHealth/_upload/20110616144731 394.pdf 6. Shenoi P, Kandhari A, Gunwal M. Esthetic Enhancement of Discolored Teeth by Macroabrasion; Microabrasion and its psychological impact on patients - A case series. Indian Journal of Multidisciplinary Dentistry. 2012;2(1): 388-92. 7. Watts A, Addy M. Tooth Discolouration and Staining: A Review of the Literature. British Dental Journal. 2001;190(6): 309-316. 8. Fondriest J. Shade Matching in Restorative Dentistry: The Science and Strategies. Int J Periodontics Restorative Dent. 2003;23(5):467-79. 9. JJL Technologies, LLC. Vita Easyshade®. The principles of use of a spectrophotometer and its application in the measurement of dental shades. 2003. [cited on September 2012]. Available at http://vident.com/files/ 2009/01/easyshade_technology_paper.pdf

23. Sauro S, et al. The Influence of Soft Acidic Drinks in Exposing Dentinal Tubules after NonSurgical Periodontal Treatment: A SEM Investigationon the Protective Effects of Oxalate-Containing Phytocomplex. Med Oral Patol Oral Cir Bucal. 2007;12(7):542-8. 24. Taha ST. Enamel Paste in the Treatment of Dentin Hypersensitivity. The University of Michigan, 2010. 25. Harvey W. Beverages and Their Adulteration Origin, Composition, Manufacture, Natural, Artificial, Fermented, Distilled, Alkaloidal And Fruit Juices. [cited on December 2012] Available at http:// chestofbooks.com/food/ beverages/Adulteration-Origin/Composition-Of-coffee.html#.UL7lEORJ4gt# ixzz2E9gcjYKh 26. Nieves AM. Enhancing Nature Through Science™: Recognize, Rejuvenate and Restore The Three “R”s In Minimum Intervention Dentistry. [cited on December 2012] Available at http://wcdental.org/ wcd_professionals/2010_annual_meeting/pdf/Nieves Handouts.pdf 27. Acids, Bases, and Salts. [cited on December 2012] Available at http://alex.state.al.us 28. Grohe B, Rogers KA, Goldberg HA, Hunter GK. Crystallization Kinetics of Calcium Oxalate Hydrates Studied by Scanning Confocal Interference Microscopy. Journal of Crystal Growth.2006;295(2):148-57.

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EFFECT OF GREEN TEA TOOTHPASTE ON PLAQUE INDEX Palmira V. Mumpuni1, Laila Novpriati2, Arbi Wijaya, Decky J. Indrani, Siti Triaminingsih3

Graduate student, 2,3Undergraduate student

Faculty of Dentistry â&#x20AC;&#x201C; University of Indonesia email address: palmiravidya@gmail.com Authors declare no conflict of interest

Abstract Purpose: The objective of the research was to analyze the effect of toothpaste containing green tea extract on dental plaque index. Methods: Green tea extract was dissolved in distillled water. Toothpastes containg green tea extract with concentration of 5, 10, and 15% were made. 20 subjects were included in the study. The study was done in five different days. In each day, subjects were asked to rinse with one of the following agents: toothpaste solution of 5%, 10 % or 15 % concentration of the green tea extract, toothpaste solution without green tea extract (control 1) or drinking water (control 2). The application was done in a crossover method with 3-4 days of wash-out periods. Prior to the application, the teeth of the students were cleansed. Following the cleaning, each student was given a carbohydrate meal. After five hours, plaque index was measured and categorized into 4 categories of very good, good, moderate and bad. Results: Toothpaste containing green tea with concentration of 5, 10, or 15 % showed higher incidence of good plaque index compared to the two control groups (p < 0.05). Increasing the concentration of green tea in the toothpaste from 5% to 10 % or 15% increased good plaque index incidence significantly (p < 0.05). However, there was no difference in good plaque index incidence between the concentration of 10% and 15% (p>0.05). Conclusion: the use of toothpaste with concentration of 5, 10, and 15 % of green tea extract can increase the incidence of good plaque index score.

Introduction Dental caries and periodontal disease are instrumental issues within the area of Oral Health in Indonesia. Based on the Basic Health Research (Riskesdas) in 2007 by Health Research and Development (Litbangkes Depkes RI), only 7.3% of Indonesians brush their teeth correctly, after having breakfast and before sleeping, of which only 5.5 % check their oral health to the dentist routinely (1). This condition has caused so many dental problems in Indonesia, such as dental caries, periodontal disease, oral soft tissue disease, and poor oral hygiene. A morbidity study on National Household Health Survey (Survei Kesehatan Rumah Tangga; SKRT) in 2001 states that, among 10 groups of diseases about which Indonesians complain, tooth and oral disease tops the list (60%) (2). Another SKRT in 2004 states that 39 % of people in Indonesia have teeth and oral disease. The research further finds that the prevalence of caries for age group of 10 years and above is 71.2%, with a note that the caries severity level is positively correlated with higher age, lower education, and lower economic status. 46% of people above 10 years and have periodontal disease, with more prevalence in the higher age (3). One cause of caries and periodontal disease is the existence of plaque on the tooth surface. Efforts to inhibit the formation of dental plaque that can cause periodontal disease and dental caries have been promoted and carried out (4). These include chewing gums with certain active substances, mouthwashing, tooth brushing, and gel using. However, the use of the toothbrush remains to be the popular method in maintaining oral health.

Keywords: Green Tea Extract, Plaque index; Toothpaste Studies have shown that toothpastes used in Indonesia are not yet quite effective to inhibit the formation of plaque on the tooth surfaces which causes dental caries and periodontal disease. This argument can be supported by the high prevalence of dental caries and periodontal disease in Indonesia (2,3). Therefore, there is a necessity to improve the effectiveness of inhibition of the formation of dental plaque. This can be possibly done by adding certain substances to the toothpaste that can potentially inhibit the formation of dental plaque. Extracts from plants are already used globally in the form of topical or application in the oral cavity. It has been shown that green

DENTAL STUDENTS’ RESEARCH

tea from Camellia sinensis species, which is not fermented during the process, has several advantages in oral health due to its antibacterial and antioxidant properties (5). Substance closely associated with antibacterial and antioxidant in Green tea extract is a collection of polyphenol components called catechins (6). Polyphenols (catechins) in green tea extract can inhibit Glycosyltransferases enzyme activity, which allows the inhibition of the formation of glucans from sucrose that has a fundamental role in sucrose-dependent adhesion and is contributive to the formation of plaque (7). Polyphenols (catechins) are also able to prevent the growth and activity of tooth decay bacteria which then causes dental caries and periodontal disease (5,6). It has been shown that subjects consuming green tea have lower dental caries and plaque than subjects who only drink mineral water (8). The effectiveness of green tea extract in reducing formation of dental plaque has been investigated. It has been reported that a mouthwash congaing green tea extracts significantly significantly inhibit plaque deposition in oral cavity (9). However, there in very limited evidence on the effectiveness of toothpaste containing green tea extract. Green tea is a beverage that is often consumed by Indonesians. Content of green tea extract has substance that can reduce the formation of dental plaque. In addition, green tea is relatively inexpensive, easy to process, and can be found easily in Indonesia. In the other hand, the use of toothpaste is the commonly material that used to protect the oral health. With regular brushing, the formation of plaque in a person will decline, raising one’s plaque index. Combination of toothpaste with green tea extract is expected to have a positive influence on the plaque index. Therefore, the purpose of this study is to evaluate the effectiveness of using of green tea extract in toothpaste on plaque control, which is measured by plaque index.

Materials and Methods Study Design: The present study is a clinical experimental research. The study was done in five different days. In each day, subjects must rinse with one of the following agents: (1) toothpaste solution of 5% concentration of the green tea extract, (2) toothpaste solution with 10% green tea extract, (3) toothpaste solution with 15% of green tea extract, (4) toothpaste solution without green tea extract (0%), or (5) drinking water (Aqua ®, Golden Mississippi, Indonesia). Then, in each day, all subjects had same breakfast, and plaque index was measure 5 hour later. The plaque index measurements in different days were compared to each others. The independent variable is toothpaste with green tea extract (5%, 10%, 15%), toothpaste without green tea extract as the control and drinking water (Aqua ®, Golden Mississippi, Indonesia) as comparison. The dependent variable is the dental plaque index. Toothpaste preparation: Toothpaste with green tea extract (5%, 10%, 15%) made from sapo medicates, glycerin, calcium carbonate and contain green tea extract, green tea extract in 5%, 10% or 15% concentrations which were made in Tea and Quinine Research

Center, Gambung. Green tea is extracted with 1: 50 (gr/gr) ratio between green tea and water. Firstly, green tea must be mashed and then heated with temperature up to 70°C for 15 minutes. Afterwards, extraction was gradually done until the color of the solution disappear. Next, the solution was evaporated with rotary evaporator machine that produced viscous green tea extract. Toothpastes were made with 5%, 10% and 15% concentrations of the green tea extract. Toothpaste without green tea extract was made with mix 90 % calcium carbonate, 5 % of sapo medicates and glycerin from the total weight. Clinical phase: 20 subjects were included in this study. The experiment protocol was explained to the subjects, and all subjects signed informed consent form. All subjects received full mouth debridement, and oral hygiene instructions were given to them. Subjects were asked to rinse with 5 different agents at five different times. Subject were randomized depend on the order of the treatment. In the first treatment, subjects were asked to rinse with toothpaste solution without green tea extract (0) %; in the second treatment, subjects were asked to rinse with toothpaste solution with 5% green tea extract; In the third treatment, subjects were asked to rinse with 10% green tea extract; In the fourth treatment, subject subjects were asked to rinse with 15% green tea extract; and in the last treatment, subjects were asked to rinse with Aqua drinking water. For each treatment, subjects used 5 ml toothpaste solution for 5 minutes. Afterwards, subject had same menu and portion for breakfast. Then, subjects were requested to not eat, rinse, or toothbrush for 5 hours. Subject only could drink mineral water during that period. Consideration of five hours is based on the average of plaque formation on teeth (+- 3 - 8 hours). Furthermore, it is also based on consideration of the subjects’ schedule. After the 5 hours period, plaque index evaluation was conducted through a thorough observation of the dental plaque distribution. We gave subjects disclosing agent and examined each subject with mouth mirror and halfmoon probe. Labial/ buccal and palatal/ lingual surfaces of teeth number 16, 12, 24, 44, 32, 36 were examined and then amount of plaque was converted into the categories of very good, good, moderate, or bad. Between the first, second, third, fourth and fifth treatment, there was a span of 3-4 days where the subject did not not undergo any treatment. It aimed to eliminate the effect of the previous treatment so as it would not effect to the results of subsequent treatment. This period is also called the washout time, where the subject brushed their teeth twice a day with toothpaste without green tea extract (0%) at home. Statistical analysis: The differences between the plaque index measurements in the first, second, third, fourth and fifth treatment were analyzed. Data processing was done using the computer program SPSS 17.0. The data obtained in this study are ordinal data. To find out the distribution of research data distribution, descriptive test is presented in a bar chart. Then, to test the hypothesis, Friedman test was used for the subject of research for more than two groups and pairs. Friedman test was followed with the Post Hoe Wilcoxon test to determine the most effective concentration of the green tea extract against plaque index.

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Results The present data shows that there was no subject with very good plaque index score in any group. In the treatment group without green tea extract toothpaste (0%), only one subject had good plaque index score, while the number of subjects with moderate plaque index score are 19. In treatment group of toothpaste with 5% of green tea extract concentrations, the subjects with moderate plaque index score are in majority, and 6 subjects had good plaque index score. The majority of subjects in the groups with toothpaste containing green tea extract 10% and 15% had good plaque index score (Table 1). In the treatment group of drinking water, a bad plaque index score was reported in 1 subject, while the majority showed moderate plaque index (Table 1). Table 2 presents the statistical comparison of plaque index measurements in different groups. There are significant differences in plaque score measurements between groups with and without green tea extract toothpaste. Plaque index after 5% toothpaste green tea extract concentration treatment was compared to the treatment of (0%) toothpaste without green tea extract and it is significantly different (P <0.05). Plaque index after the 10% green tea extract toothpaste treatment was compared with 5% green tea extract toothpaste treatment and the result was significantly different concentrations of 5% (p <0.05). Whereas plaque index after treatment of 15% green tea extract toothpaste compared to 10% green tea extract toothpaste treatment did not differ significantly (p> 0.05). As a comparison, plaque index in the treatment group of drinking water compared to toothpaste treatment without green tea extract (0%) did not differ significantly (p> 0.05).

Discussion This study shows that toothpaste containing green tea extract affects the plaque index. There was a significant trend of having good plaque index when toothpaste that contains green tea extract concentrations were used. The present results showed that good plaque index score was seen more frequently when subject were given green tea extract toothpaste concentration on 5%, 10% and 15%. This finding can be explained by presence of catechin in green tea. The present results are inline with those of a study conducted by Muin and Munandar in 2008 who found a significant difference of dental plaque index score between control groups, which were not given green tea, and treatment group, which were given green tea (10). According to Sakanaka (1990), the catechin compound is the most responsible agent to inhibit the activity of glucosyltransferase enzyme (7). The compounds which

are containing catechin are epicatechin gallate (ECg) and epigallocatechin gallate (EGCg). According to of Naim (2004), the amount of phenolyc hydroxyl group has a correlation with increasing relative toxicity rate of catechin on bacteria, so that the increase of hydroxyl group can increase the inhibition activity of catechin (11). Based on a research by Skobeleva and Bokuchava, it is known that the rate of catechin in ECG is 3-6% of tea dry weight. While the rate of catechin in EGCg is 7-13% of tea dry weight (12). Sakanaka (1997) in his research showed that the minimum amount inhibition concentrate (MIC) of cathecin that is used to inhibit the formation of glucosyltransferase enzyme is 0,025-0,030 mg/ml. In the other hand, Sakanaka also showed that the ideal MIC of cathecin amount to kill S. mutans is 0,5-1 mg/ml (13). From the calculation of 50% concentration of green tea, it contains 0,65-1,265 mg/ml catechin. So, the 5% concentration of green tea extract toothpaste contains 0,05-0,1265 mg/ml, 10% concentration contains 0,13-0,253 ml/ml, and 15% concentration contains 0,2-0,38 mg/ml catechin. The 5%, 10%, and 15% concentration of green tea extract toothpaste showed a higher value than MIC on Sakanakaâ&#x20AC;&#x2122;s research on inhibiting the formation of glucan but not kill the bacteria. Therefore, it can be assumed that using toothpaste containing green tea extract in concentration 5%, 10% and 15% can inhibit the formation of dental plaque through inhibit glucan formation but not kill the bacteria. Results of this study show the no significant differences between toothpaste containing green tea extract treatment in concentration of 10% and 15% on the index plaque. This is potentially due to the hypothesis that the rate or concentration of catechin in toothpaste containing green tea extract in concentration of 10% and 15% is much higher than MIC catechin, so that any effective difference is not too clearly seen. Furthermore, moderate and good, but not bad, plaque index scores were reported when using toothpaste application without green tea extract (0%). It might be due to the availability of calcium carbonate in the toothpaste, which can affect the inhibition of dental plaque after tooth brushing. Drinking water treatment does not provide any additional benefits compared to toothpaste treatment without green tea extract (0%). However, drinking water treatment can also help in reducing the formation of dental plaque. Reduction of dental plaque by drinking water can be explained by study of Seymour and Heasman (1992) who found the interaction between pellicle and bacteria is hydrophobic, so that the presence of water could inhibit the adhesion of bacteria to the pellicle (14).

Conclusion Within the limitations of the present study, it can be concluded

DENTAL STUDENTS’ RESEARCH

Table 1. The number of subjects with plaque index due to the treatment of toothpaste without green tea extract (0%) and the concentration of 5%, 10%, 15% also Aqua ®, Golden Mississippi, Indonesia drinking water, as a comparison. (N=20)

Table 2 Significance of each concentration in green tea extract that the use of toothpaste with concentration of 5, 10, and 15 % of green tea extract can increase the incidence of good plaque index score. Among the three concentrations, concentrations of 10% or 15% green tea extract in toothpaste are the optimum concentrations. ACKNOWLEDGEMENT Special thanks is given to Dr.drg Decky J. Indriani and drg. Siti Triaminingsih, MT for helping and supporting us to finish this research. REFFERENCES 1. Badan Penelitian dan Pengembangan Kesehatan, Departemen Kesehatan R.I. Hasil Riset Kesehatan Dasar (Riskesdas): Menggosok Gigi dengan Benar. Tahun 2007. 2. Badan Penelitian dan Pengembangan Kesehatan, Departemen Kesehatan R.I. Laporan SKRT 2001: Studi Morbiditas dan Disabilitas. Tahun 2002. 3. Badan Penelitian dan Pengembangan Kesehatan, Departemen Kesehatan R.I. Laporan SKRT 2004: Prevalensi Karies dan Penyakit Periodontal. Tahun 2004. 4. Rubinstein DL, Dean MC. Strategies for Oral Health Promotion and Disease Prevention and Control. In: Michele DL. Mosby’s Comprehensive Review of Dental Hygiene. 5th ed. Missouri:Mosby; 2002.P. 633-84 5. Rasheed A, Haider M. Antibacterial activity of Camelia sinensis extract against dental caries. Arch Pharm Res. 1998:21(3):348-52.

6. Otake S, Makimura M, Kuroki T, Nishihara Y, Hiraswa M. Anticaries effects of polyphenolic compounds from Japanese green tea. Caries Res. 1991;25(6):438-43. 7. Sakanaka S, Sato T, Kim M, Yamamoto T. Inhibitory Effects of Green Tea Polyphenols on Glucan Synthesis and Cellular Adherence of Cariogenic Streptococci. Agric Biol Chem. 1990; 54(11):2925-9. 8. Paula M, Paul EP. Diet, nutrition, and the prevention of dental disease. Public Health Nutr. 2004;7(1A) 201-26. 9. Ooshima T, Minami T, Aono W, et al. Reduction of dental plaque deposition in humans by oolong tea extract. Caries Res. 1994;28(3):146-9. 10. Muin AI, Munandar S. Pengaruh Pemberian Teh Hijau (Camellia sinensis) Terhadap Pembentukan Plak Gigi. Universitas Diponegoro. 31 May 2008. Available in http://111.m3undip.org/ ed2. 11. Naim R. Senyawa Antimikroba dari Tanaman. 15 September 2004. Available in http://www2.kompas.com/kompascetak/ 0409/15/sorotan/1265264.htm. 12. Bokuchava MA, Skobeleva NI. The biochemistry and technology of tea manufacture. Crit Rev Food Sci Nutr. 1980;12(4):303-70. 13. Sakanaka, S. Green tea polyphenols for prevention of dental caries. Chemistry and Application of Green Tea. 1997: 87-101. 14. Seymour, RA, Heasman PA, Gregor, ID. Drugs, Diseases and the Periodontium. Oxford University Press. 1992.

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EFFECT OF CACAO SEED ETHANOL EXTRACT (THEOBROMA CACAO L.) ON OSTEOCYTE COUNT OF ALVEOLAR MANDIBLE BONE GIVEN TO POST-OVARIECTOMIZED RAT Muhammad Rizky Radliya Maulana, 2Amanda Andika Sari, 3Nurita Zahra, 4Muhammad Bayu Indratomo

Student of Dentistry Major, Medical Faculty of Universitas Brawijaya,

1,2,3,4

Malang Indonesia Email address: boeboehoets@gmail.com Authors declare no conflict of interest

Abstract Purpose: Estrogen is an essential hormone in human, especially on female that can keep the physiologic balance. Women with menopause are lack of estrogen that can lead post-menopausal osteoporosis and loss of bones. Bone loss occurs due to increase of osteoclast level and apopotosis of osteocyte that lead to bone resorption. Cacao seed contains polifenol substances such as flavonoid, catechin and procyanidins. The aim of this study is to evaluate the effect of cacao seed ethanol extract on ostecyte of mandibular alveolar bone in postmenopausal osteoporosis using the ovariectomized rat model. Methods: A Post-Control Group Design Experiment using 25 randomized female rats (Rattus novergicus) divided into 5 different groups which is: normal group (negative control); 60 day old post-ovariectomy group (positive control); 60 day old post-ovariectomy group given cacao ethanol seed extract at the end of the day 30th with different dose respectively (p1 = 125 mg/kgBW, p2 = 250 mg/kgBW, p3 = 500 mg/kgBW) for another 30 day. A histologic view was used to count osteocytes of alveolar mandible bone. Results: The result showed that there are significant difference between positive control, P2 with P3 and a strong correlation between extract of cacao given to rat and a increasing level of osteocytes (p < 0.05, correlation coefficient = 0.555) on mandibular bone after ovariectomy surgery. Conclusion: This study concludes that cacao seed ethanol extract can increase mandibular osteocytes count of a postovariectomy rat. Keywords: Alveolar mandible; Cacao seed; estrogen; osteocyte; ovariectomy

Introduction During menopause, which usually occurs to women at the age of forty, there is a steady decrease in the size of ovary that can lead to some health problems (1). The problems that could arise from menopause include post-menopausal osteoporosis and the loss of periodontal tissues which function is to support the teeth (1,2). Post-menopausal osteoporosis is a disease that causes a decrease in the bone mass and usually occurs on vertebrae and long bones because of estrogenic deficiency. At the process of post-menopausal osteoporosis, there is an imbalance in bones remodeling in which the creation of osteocytes by osteoblasts is lower than the tearing down of osteocytes by osteoclasts. If the imbalance goes on for long, it will lead to a bone loss (3). During menopause, women also lose the protective effects of estrogen in jawbones (4). Research has shown that during menopause and post-menopausal osteoporosis, there is a decrease in the density of jawbones which is followed by an increase in progressive resorption of residual ridge and alveolar bone (5). This fact can be seen from ovariectomized rat in which a disappearing of alveolar bone is (6). However, we still cannot verify the mechanism of relationship between post-menopausal osteoporosis and the process of alveolar bone loss, which interests many researchers in the field of dental medical knowledge. Cacao seeds are rich with phenolic compounds such as catechins (around 37%), proanthocyanidines (around 58%), phenolate acid, theanine, and other flavanoids (7). It is known that the rate of polyphenol per cacao serving size is higher compared with green tea which is due to the fact that people consume more serving size of cacao rather than green tea (8). Polyphenol is known to not only help in preventing heart diseases and cancer, but also osteoporosis since its potency as antioxidant and abilities in regulating cytokines in inflammation responses (8). Therefore, the present study aimed to evaluate the effect of cacao seed ethanol extract on ostecyte of mandibular alveolar bone in postmenopausal osteoporosis using the ovariectomized rat model.

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Materials and Methods This research was done at the Laboratory of Pharmacology, Laboratory of Anatomy-Histology, and Laboratory of Pathology-Anatomy in Medical Faculty, Universitas Brawijaya, Malang, Indonesia from February until May 2012.

with another povidone-iodine and neomycin-bacitracin powder (Nebacetin, Pharos, Indonesia) and dressed with sterile gauze. Then, rats were injected intramuscullary with Metamizole Na (Novalgin, Sanofi Aventis, Indonesia) (20mg/kgBW) directly after surgical procedure, and were injected with gentamicin sulfate IM (Gentamicin, B. Braun, Germany) (60mg/kgBW) for the following 3 days after surgery.

Dose of polyphenol for repairing alveolar mandibular bone is not yet known, thus we are using serving size of human consumption in hope that per serving size of cacao for human can cause therapy effect. The administration of dosage is based on the amount of polyphenol in cacao serving size of 7.3 gram, which is around 1166 ppm (8). Serving of 7.3 gram cacao in the usual conversion of Indonesian diet is 4 gram (9). After that, we calculate t per serving with a conversion to rat by using NutriSurvey formula (10).

After completing 60 days of experiment, all rats were sacrificed using ether and mandibular bone of each rats were resected, fixed in 10% neutral buffer formalin, decalcified with HCl 6% for 1 week, embedded in paraffin, sectioned at 4 Îźm thickness. The sections were stained with Hematoxylin-Eosin and photos of each section were taken under modified light microscopy (Olympus Photo Slide BX51, Olympus, Japan). Quantitative count of osteocyte was performed using Cell Count for Windows (freeware).

4000 mg cacao x 0.0128 = 51.2 mg cacao (rounded to 50 mg)

Data analysis for the amount of osteocytes in alveolar mandibular bone of rats post-ovariectomy was done using SPSS19 for Windows (SPSS Inc.). Tests which were used consists of One Way ANOVA, Post-Hoc LSD, and Pearson correlation test.

From the calculation above we take the lowest serving which is 25 mg/200 grBW as serving I; middle serving at 50 mg/200 grBW; and highest serving at 100 mg/200 grBW. The format is then converted to mg/kgBW, thus, serving I at 125 mg/kgBW, serving II at 250 mg/kgBW, and serving III at 500 mg/kgBW.

Results 1. Osteocytes Calculation Result

25 randomized female Wistar Rats (Rattus novergicus), aged 3 months, weight 150-200 gram, were divided into 5 groups, each consisting of 5 rats. Group K(-) is the negative control group which has not received any treatment and is the normal control group in this research. Group K(+) is the positive control group where ovariectomy is done on the rats and waited for 30 days. During 30 days, the rats were not given ethanol extracts of cacao seeds. Group P1 is the group of rats with ovariectomy and waited for 30 days. After that, rats are given ethanol extracts of cacao seeds at 125 mg/kgBW for 30 days. Group P2 is the group of rats that have been ovariectomized for 30 days, and then given ethanol extracts of cacao seeds at 250 mg/kgBW for 30 days. Group P3 is the group of rats that have been previously ovariectomized for 30 days and given ethanol extract of cacao seed 500mg/kgBW for 30 days. Cacao seeds were obtained from PT. PTPN XII Blitar, East Java, Indonesia, in the form of coarse dry cocoa powder and re-milled for a finer powder. Ethanol extraction was done using maceration method, filtered using ethanol and concentrated using rotary evaporator. Results of ethanolic extract are in the form of pasta which should be mixed with aqua dest to make 3 different dose. Delivery of ethanol extract of cacao using a modified syringe which can be mounted sonde at the end of it, and can be inserted through the mouth to the stomach of rats. Ovariectomy of rats was done under anesthesia, the rats were injected with ketamine (Ketamine Hydrochloride Injection, Bioniche Pharma, Ireland) (20 mg/BW), the furs that covering surgical site were shaved approximately 1 cm above the ovary imaginary line. Surgical site were sterilized using povidone-iodine (Betadine, Mahakam Beta Farma, Indonesia). Incision was done using sharp scalpel and both right and left ovaries were taken. Incision was sutured using catgut thread (Catgut Chrom, B. Braun, Germany), smeared

The calculation of osteocytes is done under microscope with 400x enlargement from four views, supported with Cell Count for Windows application, to count the total and average of osteocytes per view. The calculation result of osteocytes in rat mandible (Rattus norvegicus) control and measurement can be seen in Table 1. The post hoc statistics test showed a significant difference between K(-) and K(+) groups (p = 0.006). There was no significant difference between P1 and K(+) groups (p = 0.059). Post-hoc test showed a significant difference between P2 and K(+) groups (p = 0.027). A significant difference was found between P3 and K(+) groups (p = 0.007). The correlation analysis result shows that there is a strong (r=0.555) and significant (p = 0.006) correlation regarding the increased dosage of ethanol extract of cacao seed with the number of osteocytes in rats. The correlation is positive, which means the bigger the serving or dosage of ethanol extract of cacao seed given, the more osteocytes that the rats will have. 2. Histologic Observation Result Histologic observation result in group K(-) shows osteocytes seen inside lacuna and osteocytes are spread evenly in microscope slides. This can be seen in Figure 1. In group K(+), (Figure 2) the number of osteocytes can be seen sparse and less than that in K(-). The intercellular tissue of group K(+) is darker than group K(-). This is an effect of the ovariectomy on group K(+). Group P1, in figure 3, shows the histologic view of the jawbone 60 days after it was ovariectomized and given ethanol extract of cacao

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Figure 1. microscopic view on alveolar bones of group K(-) with 400x enlargement, Hematoxylin Eosin staining

Figure 2. microscopic view on alveolar bones of group K(+) with 400x enlargement, Hematoxylin Eosin staining

Figure 3. microscopic view on alveolar bones of group P1 with 400x enlargement, Hematoxylin Eosin staining

Figure 4. microscopic view on alveolar bones of group P2 with 400x enlargement, Hematoxylin Eosin staining

Group P1, in figure 3, shows the histologic view of the jawbone 60 days after it was ovariectomized and given ethanol extract of cacao seed. Compared with group K(+), the number of osteocytes is higher and are spread evenly. The intercellular tissue of group P1 looks better than that of group K(+). In Figure 4, group P2 is proved to have a higher number of osteocytes and an even spread. The intercellular tissue of group P2 looks better thanthat of K(+). This happens because group P2 has been given higher dose (250 mg/kgBW) of ethanol extract of cacao seed than what was given to group P1. In figure 5, group P3 has more osteocytes and are spread more evenly compared to group K(-). The intercellular tissue is also better than group K(-). This is because group P3 has been given cacao seed therapy of 500mg/kgBW.

Discussion This research is conducted with an aim to acknowledge the effects of ethanol extracts of cacao seed towards the number of osteocytes in alveolar mandibular bones of ovariectomized rats. According to the research, the average count of osteocytes of group K(-) stands at 99.6 and has become a normal standard of the numbers of osteocytes in alveolar bones in this research. The histologic observation of

group K(-) shows that osteocytes are seen inside the lacuna, and they are spread evenly on the microscope slides as seen on Figure 1. This suits with Harjana, which argues that matured osteocytes are located in the lacuna (11). The alteration of osteocytes are seen clearly in group K(+), which is the group of rats that were ovariectomized and 60 days later their alveolar bones were observed under microscopes. The result of an average of 57.85 osteocytes shows a decline compared to the average of group K(-). Histologic observation shows a less number of osteocytes compared to group K(-) and a sparse spread. Intercellular tissues of group K(+) looks darker than group K(-) (Figure 2). The post hoc statistics test between K(-) and K(+) showed a significant difference between those groups. This situation matches with the research of Ejiri and Yang, where ovariectomized rats go through a decrease in jawbones osteocytes, like post-menopause (5,6). In addition, both Tanaka and Hekimsoy also mentioned that the loss of estrogen after ovariectomy causes the bone resorption surpasses the bone creation (12,13). The loss of estrogen inducted osteoclastogenesis and inducted an apoptosis of osteocytes. The decrease of estrogen causes increase in oxidative stress so that osteoblasts fall, which in turn fastforwards bone destruction (14). Women who have gone through menopause also show an increase in proinflammatory cytokines concentration such as TNF- Îą, IL-1, and IL-6 (15).

This of

increase osteoclasts,

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stimulates differentiation and maturation increasing bone resorption (16).

Group P1 is the group of rats that had been ovariectomized for 30 days and were given ethanol extracts of cacao seeds at 125 mg/ kgBW for 30 days. The average osteocytes count at 85.2 is higher compared to that of the positive control group. Result of histologic observation shows the osteocytes rate to be higher than the negative control. Intercellular tissues of P1 look better than K(+) (Figure 3). Post-hoc statistics test between P1 and K(+) showed no significant difference between the two groups even though the average osteocytes of P1 is higher than that of K(-). This shows that the administration of ethanol extracts from cacao seeds on this group is not yet significant. In group P2, which is the group of rats that have been ovariectomized for 30 days and then given ethanol extracts of cacao seeds at 250 mg/kgBW for 30 days. The average osteocytes of P2 at 90.5 shows significant increase compared to that of K(-). Result of histologic observation shows higher osteocytes than P1. Intercellular tissues of P2 looks better than K(+) (Figure 4). Post-hoc test between P2 and K(+) showed significant difference between those groups and goes towards the normal state. This matches the theory that says ethanol extract of cacao seeds contain polyphenol substances that are known to prevent bone destruction caused by post-ovariectomy osteoporosis because the potential antioxidants and anti-inflammatory activities (8). Flavonoid (procyanidins) in cacao is also known able to regulate proinflammatory cytokines in inflammation responses (8). Catechin types of polyphenol also prevents the production of TNF-α, IL-1 and IL-6 (proinflammatory cytokine) through the process of intracellular persecution from level ERK1/2 and NF-κB activation as well as reduction of oxidative stress with a persecuting activation mechanism of Nuclear Factor-κB (NF-κB), which is a transcription factor that is used to induce iNOS (inducible Nitric Oxide synthase) (17). An optimal result was observed in the group P3, which is the group of rats that have been previously ovariectomized for 30 days and given ethanol extract of cacao seed 500mg/kgBW for 30 days. The calculation result of P3 osteocytes of 98.9 shows that there was a significant increase compared to group K(-) and almost levels up with the average osteocyte number on normal state. Histologic observation shows that the number of osteocytes is higher than that of group P2. Intercellular tissues of group P3 looks better than that of group K(+) (Figure 5). Post hoc statistic test between P3 and K(+) showed that there is a significant difference between those two groups. The correlation analysis showed that the bigger the serving or dosage of ethanol extract of cacao seed given, the more osteocytes that the rats will have. This result strengthens the hypothesis of the research which says that the ethanol extract of cacao seed servings can increase the count of osteocytes. In the serving of ethanol extract of cacao seed at 250mg/kgBW, the cacao extract has shown an increase in the number of osteocytes. In the serving of ethanol extract of cacao seed at 500mg/kgBW, the cacao extract has been able to increase the number of osteocytes up until it almost reaches the normal state. Other factor that should be taken into account in the present study is that the microscope slides results that are rather unsatisfying.

This is due to the decalcification method that used HCI 6% which acts as an aggressive decalcification. Further studies are required to focus on cacao processing method that can be used in human diet to eliminate ther contents that are not needed or the ones that influence the effect of cacao therapy. Based on the explanation above, it seems that the serving of ethanol extract of cacao seed can increase the number of osteocytes in alveolar mandible bones in post-ovariectomized rats which can be due to the polyphenol’s mechanism that works as an antiinflammatory as well as oxidative stress reducing agent. This mechanism might increase the number of osteocytes in the reaction of the destruction of alveolar mandible bones. In other words, it can be said that cacao polyphenol can cause an increase in the number of osteocytes.

Conclusion According to the present results it can be concluded that: 1. There is a decreased number of osteocytes in alveolar mandible bones in post-ovariectomy wistar rats (Rattus novergicus) 2. There is an increase of the number of osteocytes in alveolar mandible bones in post-ovariectomy rats (Rattus novergicus) after given ethanol extract of cacao seed. 3. The dose of ethanol extract of cacao seed that can increase the number of osteocytes in post-ovariectomy wistar rats (Rattus novergicus) to the almost normal state is at 500mg/kgBW. 4. The correlation between the dose of ethanol extract of cacao seed (Theobroma cacao L) with the number of osteocytes in wistar rats is positive, quite strong and significant which means the more dose of ethanol cacao seed extract is given, the more number of osteocytes is alveolar mandible bones the rats will present.

Acknowledgements This work was part of a final paper submitted for bachelor degree (to Muhammad Rizky Radliya Maulana) at Universitas Brawijaya, Malang, Indonesia.

Figure 5. microscopic view on alveolar bones of group P3 with 400x enlargement, Hematoxylin - Eosin staining

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Table 1. Calculation results of the number of osteocytes in alveolar mandible bones in rats

References 1. Joelijanto, R. Pengaruh Ovariektomi Terhadap Jaringan Periodontal Pada Tikus Wistar. Tesis Kekhususan Biologi Kedokteran Program Studi Ilmu Biomedik. Program Pascasarjana. Fakultas Kedokteran Universitas Indonesia; 2004. 2. Buencamino MC, Palomo L, Thacker HL. How Menopause Affect Oral Health and What We Can Do About It. Cleve Clin J Med. 2009;76(8):467-75 3. Reginster JY, Burlet N. Osteoporosis: A still increasing prevalence. Bone. 2006;38(2 Suppl 1):S4-9. 4. Cho, Eunyoung. 2010. Periodontitis in Estrogen hormones-Deficient Women. (online). http://www.naturalhormones.net/estrogen/researc h/periodontitis-estrogenhormones-deficient- women.htm 5. Ejiri S, Tanaka M, Watanabe N, et al. Estrogen deficiency and its effect on the jaw bones. J Bone Miner Metab. 2008;26(5):409-15. 6. Yang J, Pham SM, Crabbe DL. Effect of esterogen deficiency on rat mandibular and tibial microarcitcture. Dentomaxillofac Radiol. 2003;32(4):247-51. 7. Rusconi M, Conti A. Theobroma cacao L., the Food of the Gods: A scientific approach beyond myths and claims. Pharmacol Res. 2010;61(1):5-13. 8.Subhashini R, Rao UM, Sumathi P, Gunalan G. A Comparative Phytochemical Analysis of Cocoa and Green Tea. Indian Journal of Science and Technology. 2010;3(2):188-92. 9. Zairisman, SZ. Potensi Imunomodilator Ekstrak Bubuk Kakao Bebas Lemak sebagai produk Substandar secara in vitro pada sel Limfosit Manusia. Skripsi. IPB. Bogor; 2006. 10. Erhardt J, Gross R. Nutrition Survey & Calculations – Nutrisurvey Software; 2010.

11. Harjana, Tri. Buku Ajar Histologi. FMIPA UNY; 2011. 12. Tanaka M, Tooyoka E, Kohno S, Ozawa H, Ejri S. Longterm changes in trabecular structure of aged rat alveolar bone after ovariectomy. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2003;95(4):495-502. 13. Hekimsoy, Zeliha. Osteocytes-The Known and Unknown. Turk Jem. 2008;12(1):23-7. 14. Almeida M. Estrogens Attenuate Oxidative Stress and the Differentiation and Apoptosis of Osteoblasts by DNABinding-Independent Actions of the ERα. J Bone Miner Res. 2009;25(4):769–81. 15. Sudoyo AW, Setiyohadi B, Alwi I, Simadibrata KM, Setiati. Buku Ajar Ilmu Penyakit Dalam. Jakarta: Interna Publishing; 2007. 16. Pfeilschifter J, Koditz R, Pfohl M, Schatz H. Changes in proinflammatory cytokine activity after menopause. Endocr Rev. 2002;23(1):90-119. 17. Shin HY, Kim SH, Jeong HJ, et al. Epigallocatechin-3gallate inhibits secretion of TNF-alpha, IL-6 and IL-8 through the attenuation of ERK and NF- kappaB in HMC-1 cells. Int Arch Allergy Immunol. 2007;142(4):335-44.

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CORONECTOMY OF MANDIBULAR WISDOM TEETH: A CASE SERIES AND BRIEF REVIEW OF LITERATURE Rasha Alafaleg1 SAN DANETH

Bachelor of Dental Surgery, University of Health Sciences, Phnom Penh, Cambodia

Email: drsandaneth@gmail.com

Authors declare no conflict of interest

Abstract Purpose: This study aims to collect data related to radiographic assessment, surgical procedures, and postoperative complications following coronectomy.

Methods: A literature reviews related to coronectomy of mandibular wisdom teeth was done. Moreover, the present case series reports five patients underwent coronectomy with follow-up examinations at one week, 1 month, 3 months, and 6 months. All roots were left at least 3 mm below the buccal and lingual plates of bone. Radiographic examination was done for all patients preoperatively to identify the intimate relationship between the root and inferior alveolar nerve canal.

Results: Among the five cases of coronectomy, there were no evidence of inferior alveolar nerve damage in this study.

Conclusion: Within the limitations of the present study, it can be concluded that coronectomy is a safe technique to avoid IAN injury where there is an intimate relationship between tooth roots and IAN. Key words: Coronectomy, Inferior Alveolar Nerve (IAN), Nerve Injury

Introduction Third molar surgery related inferior alveolar nerve injury (IANI) is reported to occur in up to 3.6% of cases permanently and 8% of cases temporarily (1,2). Age, difficulty of surgery and proximity to the inferior alveolar nerve canal are the associated factors leading to IAN injury. The radiographs are the mainstay for evaluation when surgical removal of mandibular wisdom teeth is required. Radiographic signs indicative of possible IAN risk include (3,4): - Diversion of the canal - Darkening of the root - Narrowing the root/ canal - Interruption of the canal lamina dura

- Interruption of the juxta-apical area If there is any evidence among these radiographic signs, frequently two of the above signs, total removal of the third molar will result in increased risk of IANI up to 2% permanently and 20% temporarily (5,6,7). Coronectomy reduces the possibility of nerve injury by securing retention of the vital roots when the intimate relationship between inferior alveolar canal and roots is estimated on panoramic radiographs or CBCTs. This study aims to collect data related to radiographic assessment, surgical procedures, and postoperative complications following coronectomy.

Literature Review: 1. Radiographic assessment In order to estimate the risk factor of IAN injury, radiograph is very essential. Risk is up to 1-4% permanent injury and 20% temporary IAN injury of the patients who radiographically present the intimate relationship between root of the teeth and IAN canal (3). Three radiological signs were found to be significantly related to nerve injury: (a) diversion of the inferior alveolar canal (b) darkening of the third molar root at the site of over-projection and (c) an interruption of the white line of the mandibular canal (8). Frafjord & Renton (2010) stated that in the present of one or more radiological signs of warning the prospect of nerve injury must be discussed with the patient and surgery may be postponed until the advent of absolute indication. Moreover, Renton et al. (2005) identified one preventive measure might be coronectomy with intentional root retention. According to the study taken by Blaeser et al (2003) showed that panoramic findings of diversion of the inferior alveolar canal, darkening of the third molar root, and interruption of the cortical with line are statistically associated with IAN injury. In addition, the absence of predictive radiographic signs, the risk of IAN injury is negligible (9). 2. Surgical procedures Different surgical techniques have used among each surgeon. For instance, Renton and Leung & Cheung sectioned through the crown was partial whereas complete section of the

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crown from the roots was undertaken by Dolanmaz and Pogrel (4,5,6,7). This may explain why there were root mobilisations in in the fully sectioned groups. One considerable point is that complete sectioning of the crown from the root may place the lingual nerve at risk, so Dolanmaz and al. suggested that complete sectioning may not be vital (6). For antibiotic, Renton and Leung & Cheung did not prescribe any antibiotic and recommended only pre- and post-operative chlorhexidine mouth wash. 3. Postoperative complications Short-term postoperative complications Postoperative complications in relation to mandibular wisdom tooth removal have been reported to occur in 4.6-30.9% of cases (8). The most commonly reported complications are localised alveolar osteitis, infection, bleeding, and paresthesia (9). Coronectomy reduces the incidence of paresthesia, but rated for the others are similar (10). Long-term postoperative complications Root migration and root eruption are the longterm complications. The remnants of root seem to move the most during the first 6 months according to Dolanmaz et al (6). Rarely, continued migration of the root(s) may result in eruption into the oral cavity (10).

Materials and Methods: Five patients who attended Faculty of Dentistry in Cambodia approved to get coronectomy. Those patients were judged to be included criteria group because they all were high risk of Inferior alveolar nerve injury based on radiographic features in routine preoperative dental radiographs. These features included the morphology of the root and proximity of the mandibular wisdom teeth to the nerve canal. Patients who were more susceptible to the local infection (from diabetes, immune compromise such as HIV, chemotherapy, osteoradiotherapy, and osteosclerosis), and who had systemic infections were excluded. The anaesthetic injection technique was the same among five patients in which Inferior Dental Block (IDB), Buccal Block, and infiltration with Lidocaine 1:100,000 Adrenaline were injected. One of the five patients got sedation with Triazolem 0.25mg via oral administration for one hour before operation due to apprehension. Regarding surgical technique, long envelope flap was raised; tooth crown was sectioned by leaving the root and vital pulp in-situ. The socket was then irrigated with saline, and flap was closed with silk suture 3.0. Antibiotic (Amoxicilline 500mg + Clavulanic acid 125mg), analgesic drug (Ibuprofen 400 + Paracetamol 500mg + Codeine 15mg), Dexamethazone 0.5mg and chlorhexidine mouthwash were prescribed. Moreover, all patients had preoperative chlorhexidine mouthwash. Questionnaires were filled before surgical procedure and at the time of follow-up (Box 1). The follow up examinations

were done at one week, one month, three months and 6 months after surgery. All the patients were contacted by having a phone call. Post-operative radiographs were taken at three months and six months after surgery.

Results: Among the five cases presented, no complication of paresthesia was found. There was not any evidence of numbness on the gingiva, lip, or chin. Root migration has not yet encounter.

Discussion: The result of this study showed that there was not any complication of paresthesia among the five cases. This study seems to highlight the success of coroncetomy. The present results are in line with those reported in the randomized controlled clinical trial of Retnon and colleagues who compared the incidence of injury to the inferior alveolar nerve as a result of coronectomy and removal of mandibular third molars (2). In that study, 128 patients who needed operations on mandibular third molars were randomized to receive either extraction or coronectomy. 102 teeth were extracted, while coronectomy was performed for 94 teeth. Among all cases, no nerve damaged was found in the coroncetomy group. However, nerve damage was observed in 19 patients in the extraction group. Some study did not rely on about coronectomy. Instead of leaving the root with vital pulp in the bone, Metin and colleagues performed a study to assess whether root canal treatment is necessary for coronectomy (11). Ten patients with 16 lower third molars, which were in close relationship with IAN, were divided into two groups. Coronectomy with endodontic treatment was performed for patients in the test group, while in the control group, patients underwent coronectomy without endodontic treatment. The result of that study indicated that infection was observed in the 8 patients in study group that may be due to the prolonged time of flap opening. On the other hand, no infection was occurred in control group. It is important to mention that the results of the present study should be interrupted with caution since the sample size of the study is small and the follow up period is relatively short. Therefore, future studies with larger sample size and longer follow-up period are necessary to make a clear conclusion on the success and long-tem complications of the coronectomy procedure.

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A male 30-year-old patient presented problem with lower left partial erupted tooth (#38) and wanted to remove.

Pre-operative Radiograph & Procedure Photos

Post-operative Radiograph

A male 21-year-old patient with coronectomy. Coronectomy on lower right wisdom tooth (#48).

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Post-operative Radiograph A male 30-year-old patient had a coronectomy on lower left partially erupted tooth (#38).

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A male 38-year-old patient. Coronectomy on #38.

Conclusion If radiograph examination shows the close intimacy of mandibular molars to the IAN, using safe surgical techniques should be taken into account, proper radiographic assessment should be done, and the patient should be informed about possible postoperative complications. Within the limitations of the present study, it can be concluded that coronectomy is a safe technique to avoid IAN injury where there is an intimate relationship between tooth roots and IAN. Therefor, It is recommended that coronectomy be further studied and practiced among dental students and dentists.

Acknowledgement I really appreciate Prof. Dr Hong Someth who devoted both time and efforts to help me in generating this article. Also, I deeply thank Ping Bushra, Choa Sivkay, Oeng San Eak, Leng Daren, and other friends who encouraged me to accomplish this achievement. Finally, prettily thank to San Dana who taught me how to design for some necessary document.

References 1. Blaeser BF. August MA, Donoff RB, et al. Panoramic radiographic risk factors for inferior alveolar nerve injury after third molar extraction. J Oral Maxillofac Surg. 2003;61(4):417-21. 2. Renton T, Hankins M, Sproate C, McGurk MA. Randomised controlled clinical trial to compare the incidence of injury to the inferior alveolar nerve as a result of coronectomy and removal of mandibular third molars. Br J Oral Maxillofac Surg. 2005;43(1):7-12. 3. Renton T. Notes on coronectomy. Br Dent J. 2012;212(7):323-6. 4. Segaghatfar M, August MA, and Dodson TB. Panoramic Radiographic Findings as Predictors of Inferior Alveolar

Nerve Exposure Following Third Molar Extraction. J Oral Maxillofac Surg. 2005;63(1):3-7. 5. Leung YY, Cheung LK. Safety of coronectomy versus excision of wisdom teeth: a randomized controlled trial. Oral Surg Oral Med Oral Patol Oral Radiol Endodontol. 2009; 108(6):821-7. 6. Dolanmaz.D, Yildirim G, Isik K, Kucuk K, Ozturk A. A preferable technique for protecting the inferior alveolar nerve: coronectomy. J Oral Maxillofac Surg. 2009;67(6):1234-8. 7. Pogrel MA. An update on coronectomy. J Oral Maxillofac Surg. 2009;67(8):1782-3. 8. Bui CH, Seldin EB, Dodson TB. Types, frequencies, and risk factors for complications after third molar extraction. J Oral Maxillofac Surg. 2003;61(12):1379-89. 9. Bouloux GF, Steed MB, Perciaccante VJ. Complication of third molar surgery. Oral and Maxillofac Surg Clinics of North America. 2007; 19(1):117-28. 10. Patel V, Gleeson CF, Kwok J, Sproat C. Coronectomy practice. Paper 2: complications and long term management. Br J Oral Maxillofac Surg. 2013;51(4):347-52. 11. Sencimen M, Ortakaglu K, Aydin C, Aydintug YS, Ozyigit A, Ozen T: Is Endodontic Treatment Necessary During Coroncectomy Procedure? J Oral Maxillofac Surg. 2010 Oct;68(10):2385-90.

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COMPARISON OF ORAL HEALTH KNOWLEDGE, ATTITUDE, PRACTICES AND ORAL HYGIENE STATUS OF CRPF OFFICIALS IN SRINAGAR, KASHMIR, INDIA. Dr.Swapnil S. Bumb1, Dr. D J Bhaskar2, Dr. Himashu Punia3 , Dr. Vikas Singh4 Dr.Safalya S. Kadtane5, Dr. Chaitanya Dev Jain6 Post Graduate Student, Department Of Public Health Dentistry, Teerthanker Mahaveer Dental College & Research Centre, Moradabad Professor & Head, Department Of Public Health Dentistry, Teerthanker Mahaveer Dental College & Research Centre, Delhi- Moradabad 3 Assistant Professor, Department Of Public Health Dentistry, Teerthanker Mahaveer Dental College & Research Centre Moradabad 6 Post Graduate Student, Department Of Oral Medicine and Radiology, Teerthanker Mahaveer Dental College & Research Centre, Moradabad,

1,4,5 2

India Email address: swapnil_bumb@yahoo.com, drswapnilbumb@gmail.com Authors declare no conflict of interest

Abstract Purpose: The objective of this study is to determine Oral health Knowledge, Attitude, Practices and Oral hygiene status of CRPF officials in Srinagar, Kashmir, India. Methods: A cross sectional questionnaire survey was done among 321 Central Reserve Police Force (CRPF) officials who voluntary participated in Srinagar, Kashmir. A questionnaire consisting of 21 questions was used to check knowledge, attitude and practices. Clinical examination was done to assess the oral hygiene status (Simplified Oral Hygiene Index). Results: Out of 321officials majority of people (57.63%) were aware that tooth brushing helps in preventing caries and gum diseases, only 26.48% people were aware about the role of fluorides and fluoridated tooth paste in prevention of dental caries. 80.37% of people thinks that regular visit to dentist is necessary. 84.42% people uses tooth paste with brush for cleaning of teeth, 4.98% uses toothpowder with fingers and 10.59% uses tooth powder with brush. 62.62% people changes there brush due to fraying of bristles. 27.73% of people has never visited dentist so far.

Conclusion: OCRPF official’s knowledge about gum diseases, use of fluoridated toothpaste and oral hygiene aids was found to be low. Among the total number of officials recorded, 82% of them had poor and 18% had fair oral hygiene status. None of them had good oral hygiene status. Most of the CRPF officials did not have knowledge about the causes and the prevention of dental diseases. Key words: CRPF trainees, Correlation, Oral health knowledge, attitude.

Introduction Health is a theme in most cultures and is fundamental human right without distinction of race, religion and political belief, economic, and social condition (1). The work environment constituent an important part of man’s total environment, so health to large extent is affected by working condition. Though several types of environment exist, it is the physical environment that plays an important bearing on health (2). Oral health is an integral part of general health, and it is one of the determinants of the quality

of life. Oral health and general health are determined by various factors such as life style, dietary habits, socioeconomic conditions, occupational environment, and etc (3). Oral health is concerned with maintaining the health of craniofacial complex, the teeth, and gums as well as the tissue of face and head that surrounds the mouth. It has been found that loss of teeth and deterioration of oral tissue substantially reduces the quality of life (1). Military is to defend the country from external threats as well as to maintain the internal peace of community (4). Police personnel are law enforces with busy work schedule. The nature of job is such that they are subjected to physical, mental and emotional stress. The irregular shifts in their work schedule leads to neglecting (skipping) of their regular diet and indulging into regular habits. The work shifts also deprive them of their routine sleeping pattern and social activities (5,6). The Central Reserve Police Force (CRPF) is the largest of India’s Central Armed Police Forces. It functions under the aegis of Ministry of Home Affairs (MHA) of the Government of India. The CRPF’s primary role lies in assisting the State/Union Territories in police operations to maintain law and order and contain insurgency. It came into existence as the Crown Representative’s Police on 27 July 1939. After Indian Independence, it became the Central Reserve Police Force on enactment of the CRPF Act on 28 December 1949 (7). The rigour of discipline –physical, mental and social- insisted upon the military personnel is also true in case of police. One of the aims of the military training given to soldiers is to achieve the required physical and mental fitness for the mission to be carried out. This requires sufficient general and oral health for training and taking part in exercises, maneuvers and deployments (8). For years, the police profession has been ranked among the top five of most stressful occupations. The constant risk of uncertainty and tension which are inherent in law enforcement, the vast amounts of human suffering, and violence can lead susceptible individuals to stress, anxiety, depression and alcoholism. Many studies have shown relationship of stress factors to periodontal diseases (9). All the above factors might have an effect on general as well

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as oral health. It is the responsibility of society to safeguard the health of their defenders. Literatures regarding the oral health situation of these police people are not available. A clear picture of oral diseases will help us organize preventive and curative programs in containment of oral disease (10). This information is very important for establishing priority and determining the type and quantity of prevention and treatment services required. The target population on which we conducted this study was police of Central Reserve Police Force, Srinagar, Kashmir. This study was thereby conducted to determine Oral health Knowledge, Attitude, Practices and Oral hygiene status of CRPF officials in Srinagar, Kashmir, India.

Materials and Methods: The present study was conducted among 321 CRPF officials who voluntarily participated in the study. Convenience sampling was use to collect the sample based on subject present during the conduction of study. The subjects were in the age group of 20-60 years. Ethical clearance was obtained from Institutional Review Board of Teerthanker Mahaveer University. Permission to conduct the study was obtained from commanding officer as well as medical officer of CRPF Srinagar unit. A cross sectional questionnaire survey consisting of 21 questions was used to check knowledge, attitude, and practices. Clinical examination was done to assess the oral hygiene status of subjects using Simplified Oral Hygiene Index. Following instruments were used to conduct clinical examination: Plane mouth mirror, Explorer, Tweezers, Kidney trays, Chip blowers, Cotton holders, Disposable mouth masks, Disposable gloves, Sterilized cotton and gauze pieces, Towels, and Soap. A single trained examiner who was calibrated conducted all the examinations. A well-trained assistant recorded the data. The questionnaires were distributed and ask to be completed within half an hour. Knowledge, attitude and practices scores were calculated separately. The significance level was set at P < 0.05. Statistical analysis was performed using the Statistical Package for Social Sciences software version 18. â&#x20AC;&#x192;

Results: Oral health knowledge: Data on the oral health knowledge of participants is as it follows: he majority of participants (57.63%) were aware that tooth brushing helps in preventing caries and gum diseases. Only 26.48% of subjects were aware about the role of fluorides and fluoridated toothpaste in the prevention of dental caries. 54.83% of subjects were aware that tobacco consumption leads to oral cancer. Oral health attitude: 80.37% of participants think that regular visit to dentist is necessary

(Table 1). 54.83% of subjects believe immediate replacement of missing natural teeth is necessary. 90.34% of participants agreed that gutkha chewing and smoking is a bad habit. Oral health behavior: 52.34% of subjects brush once a day, and 44.86% of subjects brush twice a day. 52.02% of participants brush once (in morning only) and 42.06% of participants brush twice a day (morning before meals & night after meals). 84.42% of subjects use toothpaste with brush for cleaning of teeth, 4.98% of subjects use toothpowder with fingers, and 10.59% of subjects use tooth powder with brush. 62.62% of participants change their brush due to fraying of bristles. Data regarding the oral health status of subjects are presented in Table 2 and Table 3.

Discussions The present study was aimed to gather epidemiology data regarding oral health knowledge, attitude, and behavior among CRPF officials of Srinagar, Kashmir and to measure oral hygiene status. In present study, majority of officials were not aware whether they use fluoride-containing dentrifices, similar to findings of Abhinav Singh (11) who conducted a study to assess the oral health knowledge, attitude & practice among NCC cadets in South India. A majority of officials did not know whether dental floss help in preventing dental caries and gum disease. However, most of the officials (51.71%) knew that tooth brushing prevents dental caries. Lack of knowledge regarding dental floss and gum diseases can be attributed to the low level dental education of CRPF officials. Peterson et al, in a study conducted among 12-year-old urban and rural school children in southern Thailand, concluded that consumption of sweets is an important predicator of high caries experience (12). In present study 76% of officials reported consumption of sweets 1-3 times a day, 11.76% consumed 4-6 times a day, and only 12.77% do not consumed sweets at all. Similar findings were reported by Abhinav Singh, who conducted a study to assess the oral health knowledge, attitude & practice among National Cadet Corps in South India in which 49% of cadets reported consumption of sweets very often and 14.4% reported its consumption all the time (11). The present finding indicates lack of knowledge of the most of the officials about the causes and prevention of dental disease. The present findings regarding oral health knowledge and attitudes are in line with those reported by Al-Hussaini et al (13), Christensen et al (14) and El-Qaderi and Taani (15). The result of the present study showed that officials appeared to be interested in receiving more information on a substantial number of different dental issues. A significant number of officials wanted further information, so it can be argued that officials had a positive attitude towards oral health.

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Conclusion: Officials must be provided with oral health education along with physical and general health education. Prevention of oral disease is considered to be the most effective, acceptable and efficient method implicated to pave the way to oral health. Ideally, adults should be educated to prevent the initiation and progression of the spectrum of dental disease that they are likely to encounter throughout life.

CRPF official’s knowledge about gum diseases and use of fluoridated dentifrices was found to be low. Officials’ dental visits were frequent and their consumption of sweets was found to be very high. The present study recommends that proper oral health education is required to improve attitude and behaviors of CRPF officials toward oral health. Health authorities should implement oral disease prevention and health programs for CRPF officials.

Table 1. Oral health attitude of CRPF officials in Srinagar, Kashmir, India.

Table 2: Oral health status of CRPF officials in Srinagar, Kashmir, India.

Table 3. Oral hygiene index- simplified of CRPF officials in Srinagar, Kashmir, India.

References: 1. Bhardwaj, VK, Sharma KR, Jhingta, P, Luthra RP, Sharma D. Assessment of oral health status and treatment needs of police personnel in Shimla city, Himachal Pradesh: A crosssectional study.” International Journal of Health & Allied Sciences. 2012;1(1):20. 2. Satapathy DM, Behera TR, Tripathy RM. Health status of traffic police personnel in Brahmapur city. Indian journal of community medicine. 2009;34(1):71. 3. Sohi RK, Bansal V, Veeresha K, Gambhir RS. Assessment Of Oral Health Status And Treatment Needs Of Police Personnel Of Haryana, India. The Internet Journal of Epidemiology. 2010; 9(1). 4. Veera Badraiah. Karnataka Police Manual, Lions Law Book Bangalore. 5. Silbert MH. “Job stress and burn out of new police officers”. Police chief. 49(6): 46-8. 6. Ortho Gomer K et al. “ The police for public order in stokcholm15, Health Reporter francbraporiet for klinisk stress forskning, carlinska institute stockcholm 1981 May 14 7. Central Reserve Police Force. http://en.wikipedia.org/ wiki/Central_Reserve_Police_Force. Accessed on 13th July 2013. 8. Basavaraj P, Khuller N, Dadu M, Kumar P. Dental caries experience and periodontal status of police personnel in Ghaziabad City. Indian Association of Public Health Dentistry. 2011;17:44-51. 9. Mahendra L, Mahendra J, Austin RD, Rajasekhar S, Mythili R. Stress as an Aggravating Factor for Periodontal Diseases. Journal of Clinical and Diagnostic Research. 2011;5(4): 889-93. 10. Naveen N, Reddy CVK. Oral Health Status And Treatment Needs Of Police Personnel In Mysore City, Karnataka. SRM University Journal of Dental Science. 2010;1(2).156-160. 11. Abhinav singh – Oral Health Knowledge, Attitude And Practice Among NCC Cadets And Their Correlation With Oral Hygiene In South India. Oral Health Prev Dent. 2009;7(4):3637. 12. Petersen PE, Hoerup N, Poomviset N, Prommajan J,

13. Al-Hussaini R, Al-Kandari M, Hamadi T, Al-Mutawa A, Honkala S, Memon A. Dental health knowledge, attitudes and behaviour among students at the Kuwait University Health Sciences Centre. Medical Principles and Practice. 2003;12(4), 260-5. 14. Christensen LB, Petersen PE, Bhambal A. Oral health and oral health behaviour among 11–13-year-olds in Bhopal, India. Community Dent Health. 2003;20:153–8. 15. El-Qaderi SS, Taani DQ. Oral health knowledge, dental health practices among schoolchildren in Jerash district/ Jordan. Int J Dent Hyg. 2004;2:78–85.

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A CASE REPORT OF KERATOCYST ODONTOGENIC TUMOR MIMICKING A LATERAL PERIODONTAL CYST Mahsa Esfehani 1, Pantea Nazeman2 1

Assistant professor of Oral Medicine, department of Oral Medicine, School of Dentistry, Qazvin University of Medical Sciences, Qazvin, Iran

Research center, School of Dentistry, Qazvin University of Medical Sciences, Qazvin, Iran

Email: address pnazeman@yahoo.com

Abstract Keratocyst odontogenic tumor is a benign cyst usually affecting the posterior segments of the mandible in the second and third decades of life. This lesion is detected by a multi locular radiolucency in radiographs. This cyst is rare in anterior segments of the mandible and older ages. The aim of this case report is to report a 63-year-old male affected by keratocyst odontogenic in the anterior segment of the mandible. Key words: Biopsy, Diagnosis, Keratocyst odontogenic tumor, Lateral periodontal cyst, Mandible.

Introduction Odontogenic keratocyst is a benign however a locally aggressive cyst, which was introduced for the first time by Phillipsen in 1956. The most important consideration about this cyst is great tendency to recurrence and extension prior to exposure in oral cavity (1). It has been proposed that this cyst arises from rests of dental lamina in mandible or maxilla. However, some other studies have suggested that the proliferation of the basal layer of oral cavity lining cells may result in formation of this cyst (2). Odontogenic keratocyst is characterized by its unique histopathologic features and multiple recurrences. The most commonly affected sites are the posterior body of mandible. However, the lesion can occur in any region in mandible and maxilla. According to radiographic features, the lesion alters from an asymptomatic unilocular radiolucency to a multilocular radiolucency (3). keratocyst Odontogenic is more prevalent in males and the most commonly affected groups are in the 2nd and 3rd decades while it is less prevalent in older ages (4). The aim of this case report is to report a 63-yearold male affected by keratocyst odontogenic in the anterior segment of the mandible.

Case Description: A 63-year-old man with swelling in the anterior part of the mandible was referred to the Oral Medicine Department of Qazvin University of Medical Sciences, Iran. According to the patient’s history, the swelling was detected from 5 months earlier while it was gradually enlarged in the past month. There were no significant medical problems and he was not taking any medication. Extra oral examination revealed no lesions, masses, or swelling. In clinical intraoral examination, a firm and welldefined 1.5 × 2 cm swelling was observed in the left lateral and canine site of the mandible. The surface of the lesion was completely intact, and the color was similar to the normal mucosa with telangiectasia in some sites (figure 1). In radiologic examination, a well-defined radiolucency was detected in the lateral and canine region, which was not corticated (figure 2). An aspiration biopsy was performed in order to differentiate cyst and tumor. The result of aspiration was indicative of pus, which is a sign of infection superimposed on the site. In the microscopic sections, a cyst lined with a wavy parakeratinized stratified squamous cell epithelium was observed. The epithelium was composed of 4-8 layers of uniformly thin cells, which possesses a hyperchromatic palisading pattern. Detachment of the epithelium from the inferior connective tissue was evident in some sections. Interface of epithelium-connective tissue was flat and devoid of rete ridges. The connective tissue located beneath the epithelium was consisted of fibrocollagenous tissue among few congested blood vessels and some sites of hemorrhage (figure 3). According to histopathologic examinations, the diagnosis was made as keratocyst odontogenic.

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Discussions Keratocyst odontogenic is an inflammatory lesion comprising 4-12% of the jaw cysts. According to World Health Organization (WHO) category, this cyst is classified as developmental and non-inflammatory odontogenic cyst which arises from dental lamina rests (7). Recently, WHO regarded this lesion as keratocyst odontogenic tumor (KCOT) and defined it as “a benign uni- or multi-cystic, intraosseous tumor of odontogenic origin (8). Generally, this lesion is a solitary lesion unless associated with a hereditary condition (9, 10). The previous studies suggest that the cyst is prevalent in the 5th and the 6th decades, while most of the studies reveal the predilection of this cyst to the 2nd and 3rd decades and its association with an impacted tooth (5, 6, 11). Therefore, this cyst is not a prevalent finding in our patient’s age range. Men are more commonly affected by this lesion, (6, 11) and majority of keratocyst odontogenic are detected in mandible, especially ramus, posterior part of the mandible, and third molar region (6, 7, 11, 12). In this case, in accordance to the literature, mandible was the affected jaw, but on the contrary, the epicenter was in the anterior part of the mandible. The symptoms include pain, swelling, drainage and bone perforation; however, in half of the cases, the cyst is asymptomatic and is discovered in routine radiographs. In the present case, swelling was the chief complaint of the patient though this swelling was due to the infection superimposed on the lesion that was confirmed by the aspiration. The radiographic characteristics of a keratocyst odontogenic are as follow: (1) a multilocular radiolucency with a well defined and usually scalloped corticated border; (2) expansion in the medial side and extension towards length of the mandibular bone; (3) displacement of a developing

Figure 1- Swelling in the left lateral and canine segment of mandible

tooth, separation or resorption of the erupted tooth’s roots (5). Therefore, according to the radiologic features and the site of the lesion, the cyst mimicked a lateral periodontal cyst. Lateral periodontal cyst is a rare developmental cyst located interdentally. Lateral periodontal cyst is usually symptomless and possesses a predilection to men and fifth decade. It is commonly located in the mandibular canine- premolar area (13). To date, a great number of studies have aimed to reveal the pathogenesis of keratocyst odontogenic. Results of several researches have proposed a role for point mutation in PTCH gene in pathogenesis of nevoid basal cell carcinoma syndrome, a hereditary condition characterized by several recurrent keratocysts odontogenic, (14) and sporadic keratocysts odontogenic as well (15-18). On the other hand, other studies revealed over expression of some genes such as EGFR and ki67, cytokeratin 6B, epidermal growth factor receptor ERBB3, and glioma-associated oncogen homologue 1 (GLI1) (19, 20). Protein expression analysis of keratocyst odontogenic, have revealed that this specimen are positive for PCNA, Ki67 and p53 protein, markers routinely detected in neoplasms (21). However, specific etiology of this condition still remains unknown (22). Treatment of keratocyst odontogenic tumors is can be affected by several factors such as age, location, size, extension of the lesion, and soft tissue involvement (23). Based on these factors, the treatment falls into two categories: aggressive or conservative. Peripheral ostectomy, chemical curettage with Carnoy’s solution, cryotherapy, electrocautery and resection are considered as aggressive treatment approaches. Conservative treatments include enucleation, with or without curettage, or marsupialization (24,25). Consecutively, the ideal treatment must induce the least morbidity and decrease the risk of recurrence (23).

Figure 2- Radiographic examination revealed a corticated well defined radioluscency between

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Conclusion: According to the age, gender, site of the lesion, and other clinical manifestations in the present patient, keratocyst odontogenic wouldn’t be categorized in the first choices of differential diagnosis. This case report emphasizes that radiographic and clinical features do not provide sufficient information on cysts. Therefore, definite diagnosis of whole lesions must be based on histological examinations.

References: 1. Mahadesh J, Kokila, Laxmidevi B.L. Odontogenic keratocyst of maxilla involving the sinus- okc to be a cyst or tumor. Journal of Dental Science and Research. 2010;1(2): 83-90 2. Joseph A, Regezi, James J, Sciubba, Ricard CK. Oral pathology, clinical pathologic correlations. 5th ed. St Louis: Saunders; 2008. p. 288-322. 3. Green berg MS, Glick M.Ship. Burkets oral medicine, diagnosis and treatment. 11th ed. India: Bc Decker inc; 2008. p. 147-8. 4. Wood N, Goaz P. Differential diagnosis in oral and maxillofacial lesion. 5th ed. St Louis: Mosby; 1997; p. 33355. 5. Yonetsu K, Bianchi JG, Troulis MJ, Curtin HD. Unusual CT Appearance in an Odontogenic Keratocyst of the Mandible: Case Report. AJNR Am J Neuroradiol. 2001;22(10):1887-9. 6. Gransmuck EA, Nelson BL. Keratocyst odontogenic tumor. Head Neck Pathol. 2010;4(1):94-6. 7. Pace R, Cairo F, Giuliani V, Prato LP, Pagavino G. A diagnostic dilemma: endodontic lesion or odontogenic keratocyst? A case presentation. Int Endod J. 2008;41(9):800-6. Lapointe H, Madras J. Keratocyst Odontogenic Tumour: Reclassification of the Odontogenic Keratocyst from Cyst to Tumour. JCDA. 2008;74(2):165. 8. Payne TF. An analysis of the clinical and histologic parameters of odontogenic keratocyst. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1972; 33: 536–46 9. Chirapathomsakul D, Sastravaha P, Jansisyanont P. A review of odontogenic keratocysts and the behavior of recurrences. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101(1): 5–9. 10. Ortakoglu K, Suer BT, Sencimen M. A Large Odontogenic Keratocyst Containing A Third Molar Tooth in The Maxillary Antrum. Turk J Med Sci. 2005;35:341-46 11. Gryfe A, Gryfe JH. Isolated odontogenic keratocyst. CMA JOURNAL. 1977;117(17):1392. 12. Siponen M, Neville BW, Damm DD, Allen CM. Multifocal lateral periodontal cysts: a report of 4 cases and review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011;111(2):225-33. 13. Lo Muzio L. Nevoid basal cell carcinoma syndrome (Gorlin syndrome). Orphanet J Rare Dis. 2008. 25;3:32.

Figure 3- Microscopic appearance of the lesion indicative of keratocyst odontogenic tumor

14. Pan S, Xu LL, Sun LS, Li TJ. Identification of Known and Novel PTCH Mutations in Both Syndromic and Nonsyndromic Keratocystic Odontogenic Tumors. Int J Oral Sci. 2009;1(1): 34–8. 15. Gu XM, Zhao HS, Sun LS, Li TJ. PTCH mutations in sporadic and Gorlin-syndrome-related odontogenic keratocysts. J Dent Res. 2006; 85(9): 859–63. 16. Barreto DC, Bale AE, De Marco L, Gomez RS. PTCH gene mutations in odontogenic keratocysts. J Dent Res. 2000; 79(6): 1418–12. 17. Pan S, Dong Q, Sun LS, Li TJ. Mechanisms of Inactivation of PTCH1 Gene in Nevoid Basal Hypothesis. Cell Carcinoma Syndrome: Modification of the Two-Hit Hypothesis. Clin Cancer Res. 2010. 15;16(2):442-50. 18. de Oliveira MG, Lauxen Ida S, Chaves AC, Rados PV, Sant’Ana Filho M. Odontogenic epithelium: immunolabeling of Ki-67, EGFR and survivin in pericoronal follicles, dentigerous cysts and keratocystic odontogenic tumors. Head Neck Pathol. 2011;5(1):1-7. 19. Heikinheimo K, Jee KJ, Morgan PR, Nagy B, Knuutila S, Leivo I. Genetic changes in sporadic keratocystic odontogenic tumors (odontogenic keratocysts). J Dent Res. 2007; 86(6):544-9. 20. Shear M. The aggressive nature of the odontogenic keratocyst: is it a benign cystic neoplasm? Part 2. Proliferation and genetic studies. Oral Oncol. 2002; 38(4):323-31. 21. González-Moles MA, Mosqueda-Taylor A, DelgadoRodríguez M, Martínez-Mata G, Gil-Montoya JA, Díaz-Franco MA, et al. Analysis of p53 protein by PAb240, Ki-67 expression and human papillomavirus DNA detection in different types of odontogenic keratocyst. Anticancer Res. 2006; 26(1A):17581. 22. Abdullah WA. Surgical treatment of keratocystic odontogenic tumour: A review article. The Saudi Dental Journal. 2011;23(2): 61-5. 23. Blanas N, Freund B, Schwartz M, Furst IM. Systematic review of the treatment and prognosis of the odontogenic keratocyst. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000;90(5): 553-8. 24. Morgan TA, Burton CC, Qian F. A retrospective review of treatment of the odontogenic keratocyst. J Oral Maxillofac Surg. 2005;63(5):635-9.