Asia Pacific Dental Student Association Journal - APDSJV3N1

Asia Pacific Dental Students Journal | Volume 3| Number 1| June 2012
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Comparison of Avian Eggshell and Prawn Skin

Powder as Bone Graft Material
Evan Hendra and Irene Nathania Koman
Trisakti University, Jakarta, Indonesia
Asia Pacific Dental Students Journal 2012
ABSTRACT
Background and Objectives: The ideal bone substitute should be biocompatible, low
cost and have satisfactory mechanical properties. The objective of this study was to
compare the physical properties of bone graft material from avian eggshell (AE) and
prawn skin (PS) powder using three analyzing physicochemical methods. Material and
methods: Discarded AE and PS wastes were collected from Indonesian Research and
Technology Center. These wastes were cleaned, purified and powdered into particle size
of 150 μm range. Quantitative AE and PS powder were immersed in Na2HPO4 solution
(200ml, 0.1 mol/L) for 5, 8, 12 and 24 hours, calcined at 1250°C for 3 hours, followed
by a mechanical activation and milled for 24 hours. The physicochemical characteristics
by fourier-transform infrared (FTIR), x-ray diffraction (XRD) and scanning electron
microscopy (SEM) were used to compare the physical properties of AE and PS. Two-
way ANOVA and post hoc analysis was performed to evaluate differences between
groups (p < 0.05). Results: The XRD patterns of AE and PS both in terms of intensity
and despairing was obtained at 32.54 and 29.31. This indicates that AE have more
crystallinity of the composite than PS. The SEM indicates irregular pores ranging from
50–150 μm which is favourable for bone ingrowth. The FTIR spectrum of AE exhibited
absorption peaks when comparing the AE to the PS. The statistics data also showed that
AE indicates better result in time of 24 hours immersed than PS (p < 0.05).
Conclusions: The essential findings of this study indicated that there was statistically
significant difference in the possibility of using avian eggshell as bone graft material
compare to prawn skin in all physicochemical methods and in time of 24 hours
immersed. This will not only reduce the cost of bone graft but also avian eggshell waste
can be utilized for this purpose.
Keywords: prawn skin, avian eggshell, bone graft
INTRODUCTION
Bone grafts have been used to augment osseous defects in the dental and orthopedic
fields[1]. In the field of modern dentistry, such as implant surgery, bone availability is
the key to successful placement of endosseous implants in posterior maxilla and
mandible[2]. When the thickness of the bone between sinus and alveolar crest is less than
5 mm, increasing the thickness of alveolar sinus floor through grafting is necessary to
support the required length of implants[3]. On the other hand, the distance from the
mandibular canal is a critical condition to avoid serious nerve injury during implant
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installation[4]. In a case with insufficient alveolar bone height, vertical ridge

augmentation through onlay grafting is needed to increase the alveolar bone height[5].
Autogenous bone has long been considered the ideal grafting material in bone
reconstructive surgery owing to its osteogenic, osteoinductive and osteoconductive
properties[6]. However, harvesting the autogenous bone requires an additional surgery
which increases morbidity at the donor site, prolonged operation times, and higher
costs[7]. Therefore, varieties of bone graft substitutes are used as alternatives to
autogenous grafts[8],[9]. Among them, natural bone substitute biomaterials from bovine
sources[10], [11] and bone-like minerals (calcium carbonate) derived from corals or avian
eggshells, have been preferred due to their biodegradability, abundance and lower price
in comparison with synthetic biomaterials[12]. The coralline calcium carbonate (calcite),
which is totally resorbable and biocompatible and shows good osteoconductivity, has
been used as an effective bone substitute in the natural form or converted to
hydroxyapatite (HA) in bone healing in dentistry and orthopedic[13], [14], [15].
Avian eggshell, with a mineral composition similar to corals, has been introduced as a
potential bone substitute in maxillofacial and craniofacial surgeriesas they could easily
be acquired and contain ions of Sr and F[16]. One of the crucial characteristics to be
considered when using a bone substitute graft is its degradation rate due to the fact that
it may have effects on the long-term results. The aim of the present study was therefore
to compare the in vivo physical properties of bone graft material from avian eggshell
(AE) and prawn skin (PS) powder using three analyzing physicochemical methods.
MATERIAL AND METHODS

Sample preparation
Fragmented prawn skin (PS) and avian eggshells (AE) were immersed in water to
remove the inner membrane and surface contaminants. After dried, they were grinded
into small particles. These PS and AE powders were processed to be HAp using wet
methods. First, quantitative AE and PS powders were immersed in Na2HPO4 solution
(200mL, 0.1 mol/L) for 5, 8, 12, and 24 hours and calcined in an oven furnace (THERM
160; Indonesia, 2011) for 3 hours in 1250oC. After all the mixtures were rested for 24
hours, they were homogenized in a 60oC water. Then they were mechanically activated
by stirring it on magnettic stirrer (PTBIN; Indonesia, 2009) with velocity of 300rpm
within 4-5 hours. These gels were calcined in an oven furnace (THERM 160; Indonesia,
2011) for 5 hours in 1000oC. Finally, the powders were milled (dry ball mill – PW 700i;
Indonesia, 2010) for 24 hours to pulverize into fine particles.
Sample characterization
The characteristics of the prepared samples were evaluated by Scanning Electron
Microscopy (SEM, S-4300; Hitachi, Japan), powder X-Ray Diffractometry (Shimadzu
XD-610; Japan), and Fourier Transform Infrared Spectroscopy (FTIR, Mattson Galaxy
7020A; Mattson Instruments, Madison,WI).
Statistical analysis
The mean grain size of HAP powders were determined using Debye-Scherrer formula.
Data were processed using two-way analysis of variance (ANOVA) with Tukey‘s
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multiple comparison tests was performed to evaluate differences between groups.

Values of p less than 0.05 were considered statistically significant.
RESULTS AND DISCUSSIONS

XRD
Figure 2 represents the XRD patterns of HAP from AE powders. The chemical phases
which identified are only Calcium Phosphate Hydroxide (Hydroxylapatite, syn)
Ca5(PO4)3(OH) and Calcium Phosphate (Whitlockite, syn) Ca3(PO4)2. Other phases nor
amorphous phases are not able to be identified (below 1% of amount). This indicates
that the methods were succeed to be HAP powder, although the result does not show
pure HAP. However Calcium Phosphate has been well-known as a good material for
bone-grafting to increase bone-healing activities.
Figure 2 – Diffraction Patterns of Identified Phases by XRD
FTIR
FTIR spectra of AE and PS are shown in figures 1A and B respectively. The FTIR
spectrum of AE (fig.1A) shows the absorption bands of carbonate at 871 cm−1 and a
broad peak at around 1428 cm−1. This data confirms the presence of CaCO3 in the egg
shell powder. In the case of PS (fig.1B), FTIR spectrum shows peaks at 9611044 cm−1
and 569606 cm−1 representing the characteristic well crystallized apatite phase. The
peak at 961 cm−1 corresponds to ν1 stretching mode and 1044 cm−1 represents ν3
vibration mode of phosphate groups. The sharp peaks at 569606 cm−1 are assigned to
the bending mode of phosphate. The absorption bands at 1422–1460 and 876 cm−1 are
due to the vibration mode of carbonate groups. FTIR spectrum of AE is similar to that
of HA which was also confirmed in the earlier studies (Noorjahan and Sastry 2005).
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Figure 1A – FTIR spectra of avian eggshells
Figure 1B – FTIR spectra of prawn skins
Scanning electron microscope

The surface morphology of AE–SP composite is shown in figure 4. The porous nature
of the composite is seen in lower magnification (300×) as well as in higher
magnification. SEM pictures indicate irregular pores ranging from 50–150 μm, which is
favorable for the bone ingrowth (Zang et al 2003). There is no distinct difference for the
pore size distribution in the implant.
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(A) (B)
Figure 3 – Morphological details of avian eggshells (A), and prawn skins (B)
The results of this study demonstrate the potential effectiveness of prawn skin and avian
eggshell as a bone substitute material. Surface-modified AS particles exhibited good
osteoconductive properties, with increased new bone formation and a high degree of
direct bone apposition on their surfaces.
Prawn skin and avian eggshells have the greatest amount of waste in the world. They
both are abandon and useless to the environment. However, they were known for their
high calcium content, which then make them used as avian‘s food. Regarding to today‘s
demand for bone grafting, any biosynthetic materials with rich content of calcium are
needed. Unfortunately, those biosynthetic materials are expensive. Therefore, prawn
skin and avian eggshells were chosen as experimental samples due to its
biodegradability, abundance and lower price in comparison with biosynthetic materials.
The FTIR spectrum of AE has exhibited absorption peaks comparable to those of
CaCO3, similar results were observed in the case of XRD studies too. Previous
studieshave shown that the avian eggshells powder is a safe bone substitute, which is
easily available[16]. FTIR spectrum and XRD of AE exhibited absorption peaks
comparable to those of hydroxyapatite[13] [14]. In earlier studies, calcined bone grafts gave
clinically good results when the same was used in rabbit mandible defects[5] [16].
CONCLUSIONS
In conclusion, this pilot study indicates the potential efficacy of surface-modified
particulated avian AS as a bone graft substitute for treating osseous defects in dental
field. Ideally a bone graft substitute should mimic the native bone in both mechanical
and osteogenic properties. The advent of avian eggshell as a bone graft substitutes and
biologically active factors moves us ever closer to this goal.
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REFERENCES
1. Alfaro FH. Bone Grafting in Oral Implantology: Techniques and Clinical
Applications. Quintessence Publishing Co.Ltd. 2006; 9, 14
2. Hupp JR, Ellis E, Tucker MR. Contemporary: Oral and Maxillofacial Surgery. 5th
Ed. Mosby, Inc. 2008; 241-242
3. Turunen T, Peltola J, Yli-Urpo A, Happonen RP. Bioactive glass granules as a bone

adjunctive material in maxillary sinus floor augmentation. Clin Oral Implants Res
2004;15:135–141.
4. Bosch C, Melsen B, Vargervik K. Importance of the criticalsize bone defect in testing

bone-regenerating materials. J Craniofac Surg 1998;9:310–316.
5. Clavero J, Lundgren S. Ramus or chin grafts for maxillary sinus inlay and local onlay
augmentation: Comparison of donor site morbidity and complications. Clin Implant
Dent Relat Res 2003;5:154–160.
6. Trisi P, Rebaudi A, Calvari F, Lazzara RJ. Sinus graft with Biogran, authogenous
bone, and PRP: A report of three cases with histology and micro-CT. Int J Perio Rest
Dent 2006;26:113–125.
7. Marx RE, Morales MI. Morbidity from bone harvest in major jaw reocnstruction: A
randomised trial comparing the lateral anterior and posterior approaches to the ilium. J
Oral Maxillofac Surg 1988;48:196–203.
8. Zitzmann NU, Scharer P, Marinello CP, Schupbach P, Berglundh T. Alveolar ridge

augmentation with Bio-Oss: A histologic study in humans. Int J Perio Rest Dent
2001;21:289–295.
9. Daculsi G, Laboux O, Malard O, Weiss P. Current state of the art of biphasic calcium
phosphate bioceramics. J Mater Sci Mater Med 2003;14:195–200.
10. Meijndert L, Raghoebar GM, Schupbach P, Meijer HJA, Vissink A. Bone quality at
the implant site after reconstruction of a local defect of the maxillary anterior ridge with
chin bone or deproteinised cancellous bovine bone. Int J Oral Maxillofac Surg
2005;34:877–884.
11. Artzi Z, Tal H, Dayan D. Porous bovine bone mineral in healing of human
extraction sockets. Part 1: histomorphometric evaluations at 9 months. J Periodontol
2000;71:1015–1023.
12. Park JW, Lee CH, Choi BJ, Suh JY. Evaluation of natural calcium carbonate with
surface modification as a bone graft substitutes. Key Eng Mater 2006;309–311:183–
186.
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13. Coughlin MJ, Grimes JS, Kennedy MP. Coralline hydroxyapatite bone graft
substitute in hindfoot surgery. Foot Ankle Int 2006;27:19–22.
14. Yukna RA, Yukna CN. A 5-year follow-up of 16 patients treated with coralline
calcium carbonate (BiocoralTM) bone replacement grafts in infrabony defects. J Clin
Periodontol 1998;25:1036–1040.
15. Vuola J, Goransson H, Bohling T, Asko-Seljavaara S. Bone marrow induced

osteogenesis in hydroxyapatite and calcium carbonate implants. Biomaterials
1996;17:1761–1766.
16. Dupoirieux L, Pourquier D, Souyris F. Powdered eggshell: A pilot study on a new

bone substitute for use in maxillofacial surgery. J Craniomaxillofac Surg 1995;23:187–
194.
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Effects of Tofu Liquid Waste on Mandibular Bone of

Ovariectomized Rats
Meilia Aquina1, Agustine Hanafi1, Nungky Devita1
1
Dentistry Brawijaya University Malang Indonesia.
ABSTRACT
The aim of this study was to determine the effect of isoflavones in the tofu liquid waste
by examining the microscopic structure, estrogen receptor expression and MDA levels
in mandibular bone of post-ovariectomy rats (Rattus Norvegicus). The method of this
research was using a true experimental laboratory with randomized, post-test only,
control-group design. A total of twenty four female rats were divided randomly into six
groups, each group consists of normal, not ovariectomized rats (G1), 4 weeks
ovariectomized rats (G2), 8 weeks ovariectomized rats (G3) not given tofu liquid waste,
and 8 weeks ovariectomized rats which were given tofu liquid waste at the end of the 4th
week of ovariectomy with three different doses (G4 = 1.2, G5 = 6; G6 = 12 ml/kgBW).
Histopathological slide with haematoxylin eosin colour used to see resulted changes on
number of osteoclasts, osteocytes, periodontal ligament width, alveolar bone height and
immunohistochemistry used to count the amount of estrogen receptor expression.
While lipid peroxide test used to see the MDA levels. Data obtained from this study
were analyzed using one-way Analysis of Variance, Multiple Comparison Test post hoc
Tukey by Equal Variance and Pearson Correlation Test. There are significant
differences in all parameters among six groups of rats (ANOVA, p <0.05). There is also
a close relationship between the dose of the tofu liquid waste in all parameters
(osteoclast, r = -0,432; osteocytes, r = 0,883; periodontal ligament width, r = -0,945;
alveolar bone height, r = 0,933; estrogen receptor expression, r = -0,940; MDA level, r
= -0,700). The conclusion of this study is tofu liquid waste could improve the
microscopic structure, estrogen receptor expression and MDA levels in mandibular
bone of post-ovariectomy rats (Rattus Norvegicus).
Keywords: Ovariectomy, Periodontitis, Isoflavones, Tofu Liquid Waste.
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INTRODUCTION
Estrogen is a steroid hormone which mainly produced by ovarian follicles. Estrogen
production would reduce because the lower number of follicles in the ovaries at the time
of menopause.[1]
As many as 30% of menopausal women suffer periodontitis.[2] Periodontitis is an oral

disease that most often encountered. Facts shown that at the time of menopause, women
lose the protective effects of estrogen thus increases the risk of periodontitis.[3]
According to American Society for Reproductive Medicine, ten million people had
periodontitis, and 14 million postmenopausal women with low bone mass has a high
risk of severe periodontitis.[4]
In 1970 scientist introduced a treatment for menopause by using (Hormonal

Replacement Therapy / HRT). However, until now hormonal replacement drugs
(hormonal replacement therapy / HRT) is still considered expensive, and has a various
risks such as endometrial cancer and breast cancer.[5]
The use of natural materials that contain phytohormones has been developed, among
them are phytoestrogens. Phytoestrogens are a substrate from a plant which has similar
activity with estrogen.[6] Natural phytoestrogens which are now starting to be developed
is from Leguminoceae class. This class contain of Isoflavones compound. Isoflavones
compound shown to have hormonal effects, especially estrogenic effect. Estrogenic
effect of Isoflavones is associated with a structure that can be transformed into equol,
which has a phenolic structure similar with estrogen. In other words, Isoflavones may
prevent the process of periodontitis in the bone so the bone remains solid and massive.[7]
In the process of making tofu, there are two kinds of waste products, namely solid waste
and liquid waste, solid waste used as livestock feed, while the liquid waste discharged
into the environment thus contaminating the environment. Research prove that the
liquid waste products still contains organic compounds such as fats, proteins,
carbohydrates, and also compounds of isoflavon.[8] Therefore, research is needed to
demonstrate the use of liquid waste as an alternative therapy to reduce the risk of
periodontitis in postmenopausal women.

Research Design. The methodology of this study is a true experimental laboratory
research that uses randomized post test control group design. Subjects were divided into
6 groups. G1 (not ovariectomized and not given tofu liquid waste), G2 (ovariectomized
4 weeks and were not given tofu liquid waste), G3 (ovariectomized 8 weeks and were
not given tofu liquid waste), G4 (ovariectomy + 1, 2 ml/kg of tofu liquid waste), G5
(ovariectomized + 6 ml/kg of tofu liquid waste), G6 (ovariectomized + 12 ml/kg of tofu
liquid waste). After that, the sixth group would performed mandibular bone and
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periodontal tissue surgery to see the microscopic structure, expression of estrogen

receptors and levels of MDA.
Ovariectomy Procedure. Weight of rats were measured, and then the rat were fixed in
a supine position. Performed anesthesia using ketamine im dose 40mg/kgBW then
carried out sterilization using 70% alcohol and betadine solution. Performed
transabdominal incision about the top of the uterus along the 1.5 - 2 cm layer by layer to
penetrate the wall of the peritoneum. Find uterine horn-oviduct-ovary uteru. Oviduct
and ovary tissue freed from surrounding fat and connective tissue. The distal of oviduct
and ovary was ligated. Then the oviduct and the ovaries are removed. Give the
basitrasin powder (Nebacetin). Use the same procedure for the right ovary. Then the
incision given Gentamycin i.m at a dose of 60-80 mg/kgBW 1 time per day for 3 days,
and Novalgin i.m at a dose of 0.3 ml for 1 day.[9]
Procedure of Making Tofu Liquid Waste. Choose a clean and washed soybeans. Soak
in water for 8 hours (at least 3 liters of water to 1 kg of soy). Wash repeatedly soaked
soybeans. Mash soybeans and add warm water to form slurry. Cook the pulp, not to
coagulate at a temperature of 700 ~ 800C. Strain pureed soybeans and water using 3 ml
of vinegar to 1 liter of soymilk. Pressed to remove the sediment water (tofu liquid
waste).[10] This liquid waste is used as a research variable. After that, ovariectomized
rats were given out through a sonde with a specific dose for 4 weeks.
Slide Processing. Decalcification process for 5 days by sediment the organ in oncalek
liquid. The process of fixation, dehydration, clearing and impregnation by dipping into a
solution of the tissue in sequence according to the specified time. Performed tissue
embedding with microtome then painted using haematoxylin eosin procedure and
immunohistochemistry. Microscopic structure and expression of estrogen receptor
observation using a microscope Olympus BX51 with the Photo Slide DP71 12
Megapixel camera with 1000x magnification in each tissue / slides from each rat by 4
field of view and then on the average.
Test Lipid Peroxides (MDA) Mandible Bone Powder. Weigh 50 mg of bone that has
been powdered, then mortal with phosphate buffer as much as 1 cc. Add TCA 100% of
250 microliters, 200 1 N HCl 200 microliters, Natio Barbiturates 1% of 200 microliters.
Heat the water to 100o C for 20-25 minutes. Centrifuged at 2000 rpm then take the
supernatant with 3 cc posphat aquabides or buffer. Read results using a
spectrophotometer with 532 waves.
Analysis. Observations on the control and treated rat were statistically analyzed using
SPSS program for Windows 16 with a significance level of 0.05 (p = 0.05) and 95%
confidence level (α = 0.05). With the method of data analysis using One-way ANOVA
test, Multiple Comparison Test post hoc Tukey by Equal Variance, and Pearson
Correlation Test.
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RESULTS AND DISCUSSION

Number of Osteoclast. Administration of Tofu liquid waste containing Isoflavones has
an estrogenic activity that can bind to estrogen receptors located in the bone.[11] So that
it can cause changes in the bone which can be seen microscopically.
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One of the changes that happened was in osteoclasts. Homogeneity test showed normal
distribution. ANOVA test shows significant value p = 0.001, then from post hoc test
showed there were significant differences in group G2, G3, G4, G5, with a significant
value p <0.05. The Pearson test showed that tofu liquid waste has a enough relationship
with the number of osteoclasts on mandibular bone (r = -0,432). This suggests that the
dosage of different Tofu liquid waste know can reduce the number of osteoclasts in
ovariectomized rats.
Number of Osteocyte. Effect of low estrogen in the body will lead to increased
expression of pro-apoptotic genes (genes Insp3Rs) and the activity of caspase 3/7 on the
osteoblast cell apoptosis.[12]
Increased apoptosis resulting in decreased osteoblast cell number. Mature osteoblasts

was called osteocytes which is a major component of adult bone constituent on
mandibular bone. If the number of osteoblast cells decreased, the osteocytes cell
numbers can also be confirmed decreased.
Reduced osteocytes cell resulted in a decrease of bone regeneration. Decreased structure

regeneration of bone structure can weaken bones and cause periodontitis structures.
Provision of Tofu liquid waste containing Isoflavones expected to act as phytoestrogens
to increase the number of osteocytes cells. Therefore, tofu liquid waste can prevent the
occurrence of periodontitis. ANOVA test shows significant value p = 0.000, then from
post hoc test showed there were significant differences in group G2, G3, G4, G5,G6
with a significant value p <0.05. The Pearson test showed that tofu liquid waste has a
very strong relationship with the number of osteocytes on mandibular bone (r = 0,883).
This suggests that different dosages tofu liquid waste will influence the number of
osteocytes in rats post-ovariectomy.
Alveolar Bone Height. Tofu liquid waste contain Isoflavones is proved to have
hormonal effects, especially the estrogenic effect. Isoflavones are able to influence
calcification process. Isoflavones have a role in preventing alveolar bone resorption that
occurs in hipoestrogenic condition.[13]
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ANOVA test shows that there are significant differences (p = 0.000), then from post hoc
test showed there were significant differences in group G1, G2, G3, G4, G5,G6 with a
significant value p <0.05. The pearson test showed that tofu liquid waste has a very
strong relationship with the number of osteocytes on mandibular bone (r = 0933). This
suggests that an increased dosage of tofu liquid waste will increase the height alveolar
bone ovariectomized rats.
Periodontal Ligament Space Width. Reduced estrogen levels could be a trigger in

inflammation and decreased synthesis of fibroblasts that making up collagen so it can
makes an increased of bone resorption of periodontal tissue. Tofu liquid waste treatment
inhibit the inflammatory cytokines that inhibit the expression of inflammation that
causes bone resorption, decrease of periodontal tissue fibroblasts as well as collagen
formation in periodontal tissues may also be hampered.[14] So there is no increase in
periodontal ligament space width.
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ANOVA test shows that there are significant differences (p = 0.001), then from post hoc
test showed there were significant differences in group G1, G2, G3, G4, G5, G6 with a
significant value p <0.05. The Pearson test showed that tofu liquid waste has a very
strong relationship with the width of ligament periodontal space on mandibular.bone (r
= -0,945). This suggests that a dosage of tofu liquid waste will decrease the width of
periodontal ligament space in ovariectomized rats.
Expression of Estrogen Receptor. Tofu liquid waste containing Isoflavones is known

to have estrogenic activity that can bind to estrogen receptors located in the bone. As
the number of estrogen decreased, the number of free estrogen receptors are increased.1
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ANOVA test shows significant value p = 0.000, then from post hoc test showed there
were significant differences in group G1, G2, G3, G4, G5, G6 with a significant value p
<0.05. The Pearson test showed that tofu liquid waste has a very strong relationship
with the number of estrogen receptor expression (r = -0,940). This suggests that a
dosage of tofu liquid waste will decrease the number of estrogen receptor expression in
ovariectomized rats.
MDA Level of Mandible Bone. MDA is the result of calculation by using

spectrophotometer. Administration of tofu liquid waste containing Isoflavones works
through two pathway mechanisms. The first Isoflavones have estrogenic activity that
can bind to estrogen receptors and produces cellular endogenous antioxidant (ex, SOD)
and the second through free radical scavenger antioxidant activity, so as to reduce levels
of free radicals in post-menopausal.[14] ANOVA test shows that there are significant
differences (p = 0.000) in the levels of MDA (Malodialdehid) which is one product of
free radicals. In the post hoc test showed a significant difference between G1, G2, G3,
G4, G5, G6. The Pearson test shows that tofu liquid waste has a strong relationship
with the expression of estrogen receptor (r = -0,700). So that the tofu liquid waste can
improve MDA levels nearly normal mandibular bone.
CONCLUSIONS
The tofu liquid waste is a by-product of tofu-making process. It is potential enough to
be used as a therapeutic in hypoestrogen conditions. The conclusion of this research that
tofu liquid waste can improve the microscopic structure, estrogen receptor expression
and MDA levels of mandibular bone post-ovariectomy in rats.
REFERENCES
[1] Metsa, Merja. 2001. Estrogen Replacement Therapy And Antiestrogen Treatment
In Postmenopausal Breast Cancer Patients – Effects On Recurrence,
Gynecological Organs, Vascular Endothelium, And Bone. Dissertation
Department of Obstetrics and Gynecology Helsinki University Central Hospital
University of Helsinki, Finland.
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[2] Melton, et al. 2002. How Many Women Have Osteoporosis? J.Bone 92 (7): 1005-
1010.
[3] Bradford, P.G, et al. 2010. Estrogen Regulation of apoptosis in Osteoblast.
Physiology & Behavior 99 (2): 181–185.
[4] Ana, et al. 2005. Periodontitis And Osteoporosis. Medicine and Biology 12 (2):
100 – 103.
[5] Lethaby AE, Brown J, Marjoribanks J, Kronenberg F, Roberts H, Eden J. 2007.
Phytoestrogens for vasomotor menopausal symptoms. Cochrane Database of
Systematic Reviews. Issue 4: CD001395.
[6] Glover A. and Assinder S.J. 2006. Acute Exposure Of Adult Male Rats To Dietary
Phytoestrogen Reduces Fecundity And Alters Epididymal Steroid Hormon
Receptor Expression. Jour. Endocrine 189: 565-573.
[7] Messina, M. J. 2002. Soy foods and soybean isoflavones and menopausal health.
Nutr Clin Care 5: 272 - 282.
[8] Ayuningtyas, Dewi. 2009. Makalah Kimia Dasar Isoflavon Dalam Kedelai
(Online). http://www.scribd.com/doc/15749320/MAKALAH-KIMIA-DASAR,
Accessed on October 18, 2010.
[9] Guyton, Arthur C. 2000. Buku Ajar Fisiologi Kedokteran. Edisi 9. Jakarta : EGC.
Hal 1283-1300.
[10] Syireen, Amalia. 2008. Pengaruh Pemberian Genistein (Fitoestrogen) Terhadap
Kadar Malodyaldehid (MDA) Otak Tikus Yang Diovariektomi. Final Assignment
of Medicine Faculty Brawijaya University, Indonesia.
[11] Petter, V.N, et al. 1998. Estrogen Receptor is Developmentally Regulated During
Osteoblast Differentiation and Contributes to Selective Responsiveness of Gene
Expression. Endocrinology 139 (4): 2048-2057.
[12] Clarke, B. 2008. Normal Bone and Anatomy Physiology. Clin J Am Soc Nephrol
3: 131–139.
[13] Kalu, D.N. 1991. Evaluation of the pathogenesis of skeletal changes in
ovariectomized rats Endocrinology 115(2): 507-512.
[14] Diel, P., Smolnikar, K., Schults, T, et al. 2001. Phytoestrogens And
Carcinogenesis Differential Effects Of Genistein In Experimental Models Of
Normal And Malignant Rat Endometrium. Hum. Reprod 16 (5): 997-1006.
20
Oil Pulling Therapy As An Alternative in Reducing

Gingivitis Severity
F.M. Prasetio1, A.S. Nurrachman1, R. Hudyono 2, and E.M. Setiawati2
1 Undergraduate Student,
2 Resident in Periodontics Department,
3 Lecturer of Periodontics Department,
Faculty of Dentistry, Airlangga University, Surabaya-Indonesia
ABSTRACT
Background: Oil pulling is an ancient medicine mentioned that is a widely
recommended procedure in Ayurveda. For many centuries, it has been used extensively
as a traditional folk remedy for strengthening teeth, gums, and the jaw and to prevent
decay, oral malodor, bleeding gums, dryness of the throat, cracked lips and fixed
loosened teeth. Purpose: The aim of this review is to discuss the mechanism of oil
pulling therapy in improving gingival health. Reviews: In recent years complementary
and alternative medicine is gaining popularity over conventional allopathic medicine
because the products and practices used are natural and safe. The oil pulling therapy
with olive oil may be the one of it. The olive oil, contains oleocanthal, may inhibits
COX-2 enzymes and protects cavity from infection and inflammation by its antioxidant
property. COX-2 is expressed in inflammatory cells and plays a key role in the tissue
repair process. The olive oil also contains a main phenolic compound called
Hydroxytyrosol (HT) that has antiatherogenic properties with powerful antioxidant
activity and may inhibit the reactive oxygen species (ROS). The oil pulling therapy may
significantly reduced plaque scores. The viscosity of the oil may inhibits bacterial
adhesion and plaque co-aggregation. The bacteriosid effect of oil was shown as it may
reduce the Porphyromonas gingivalis count in plaque and saliva after oil pulling
therapy. Conclusion: oil pulling therapy may be beneficial in non surgical phase of
periodontal therapy as it may modulate the detrimental host responses. We suggest to
conduct a further scientific research and evaluation of this ancient health practice as an
adjunct in periodontal therapy and host modulatory agent.
Keywords: oil pulling therapy, gingivitis, COX-2 enzymes
INTRODUCTION
Ayuverdic medicine, or Ayuverda, is one of the ancient medical system that originated
in India. The term ―Ayuverda‖ consists of the Sanskrit words ayur (life) and veda
(science/knowledge), then Ayurveda means ―the science of life‖ or ―the knowledge for
long life‖.1,2 In some countries, for example the United States, Ayuverda is considered
as a complimentary and alternative medicine that is offered as herbs, massage and
specialized diets.2 Furthermore, its treatments rely heavily on herbs and other plants,
including oils and common spices.2
21
Ayuverda traditionally uses certain oils for oil pulling, such as sesame oil, coconut oil,
almond oil, vegetable oil and other organic and cold-pressed oils for this purpose.
Numerous studies had revealed that oil pulling therapy has the ability to improve oral
hygiene and reduce plaque and gingivitis.3 Olive oil is also a kind of good quality oil
that can be used in oil pulling therapy in dentistry. In vitro microbiological experiments
were conducted to examine the effect of olive oil, it is resulted that there are significant
plaque inhibition and the decreasing of bacterial growth and adhesion.4
The aim of this literature review is to discuss about the Olive oil (Olea eropeae L.) as
alternative natural, safe and effective material in oil pulling therapy for reducing
gingival inflammation, increasing oral hygiene and avoiding of periodontal disease.
Oil Pulling Therapy

Many people believe that the therapeutic effects and the science behind oil pulling is the
effects caused by the absorption of toxins and chemicals through blood vessels in the
mouth and tongue, as well as sublingual/transmucosal absorption of the fatty acids in
the oils used for pulling.6 Oil pulling therapy is one of alternative medicine treatments
that is a widely recommended procedure in Ayurveda. In dentistry, it has been used
extensively as a traditional folk remedy for strengthening teeth, gums, and the jaw and
to prevent decay, oral malodor, bleeding gums, dryness of the throat, cracked lips and
fixed loosened teeth.7,8 Method of oil pulling consists of swishing, not gargling,
vegetable oil in the mouth. The oil is ―worked‖ in the mouth by pushing, pulling, and
drawing it through the teeth for a period of 15 to 20 minutes. It is done one to three
times a day on an empty stomach. The best time is in the morning before eating
breakfast, but can be done before any meal. The used oil is discarded and the mouth
rinsed out with water. The oil is never swallowed because it is loaded with bacteria,
toxins, pus, and mucous.6 The therapy is also claimed to cure about 30 systemic diseases
ranging from headache, diabetes, ashtma, skin conditions, allergies, oral bacterial
infections, gum problems and more.3
Oil pulling has a very powerful detoxifying effect. Mouths are the home to billions of
bacteria, viruses, fungi and other parasites and their toxins. Candida and Streptococcus
are common residents in the mouths. It is these types of germs and their toxic waste
products that cause gum disease and tooth decay and contribute to many other health
problems including arthritis and heart disease. Immune system is constantly fighting
these troublemakers. If the immune system becomes overloaded or burdened by
excessive stress, poor diet, environmental toxins and such, these organisms can spread
throughout the body causing secondary infections and chronic inflammation, leading to
any number of health problems.3
Olive Oil
Olive is native to Mediterranean basin, wild olives from the trees were collected by
Neolithic peoples in the early of 8th millenium BC.9 The wild olive tree originated in
Asia Minor in modern Greece.10 The earliest surviving olive oil amphorae date to 3500
BC (Early Minoan times), though the production of olive oil is assumed to have started
before 4000 BC. An alternative view retains that olives extracted into oil by 4500 BC
by Canaanites in present-day Israel.11
22
Olive oil is a fundamental ingredient that is used in Mediterranean diet. Over the past
few years, its diffusion and consumption have spread outside the Mediterranean basin.
The growing interest in olive oil lies on its taste and nutritional properties. Besides as
food, olive oil also has been used for religious rituals, as a fuel in oil lamps, soap-
making, skin care application and medicines. There is a wealth of epidemiological
evidence showing that Mediterranean populations who consume large volumes of olive
oil in their daily diets (about 25–50 ml/day) have reduced risk for certain chronic
diseases (such as atherosclerosis, cardiovascular disease, particular types of cancer, and
extended life expectancy compared with other geographic populations.12-14,39
Furthermore, studies (including human, animal, in vivo and in vitro) have shown that
olive oil phenolics have beneficial effects on certain physiological parameters, such as
plasma lipoproteins, oxidative damage, inflammatory markers, platelet and cellular
function, antimicrobial activity, and bone health.15,16
Olive oil is an extraction product from the fruit of Olea europeae L., and is composed of
about 90-99% glycerol fraction and 0,4-5% non-glycerol fraction.17 Olive oil is mainly
composed of triacylglycerides/tryglycerols and contains small quantities of free fatty
acids, glycerol, phosphatides, pigments, phenolic compounds, and the other minor
components (waxes, tocopherols, hydrocarbons).18,19,20 Oleic acid, a MUFA, represents
70-80% of the fatty acids present in olive oil.17 Triglycerides contain two components,
glycerol and fatty acids. Fatty acids are carboxylic acids with a hydrocarbon chain
varying in length between 12 and 24 carbons for olive oil.20
(a)
(b) (c)
Figure 1. Chemical structure of olive oil (a) tryglycerides (b) glycerol (c) fatty acids. 20
The beneficial effects of olive oil could be due to its components, such as phenolic
compounds, α-tocopherol, carotenoids and to the high unsaturated/ saturated fatty acid
ratio with oleic acid (MUFA).21,22 Monounsaturated fatty acids in olive oil showed a
healthy impact on plasma cholesterol level.23 Olive oil also has been shown to have
relation with a better quality of life, longevity, and a lower incidence of cardiovascular
disease, cancer, and cognitive degeneration.24 The oleic acid and other non-glycerol
fraction, such as phenol and tocopherols, exhibit a high nutritional status and biological
value.19 In various studies (in vivo and in vitro), the polyphenols in olive oil have been
23
described and demonstrated as the main components that attributed the anti-
inflammatory, anti-microbial and anti-oxidant properties.22,25,26
The olive oils also have a bactericidal activities against microorganisms. Various
experiments showed that olive oil can act as bacteriostatic in formulas of some products
for oral hygiene which contains an average content of olive oil (1-60% of the
formula).27 Most of foodborne pathogens did not survive after 1-hour contact with olive
oils.28 It also reduced the population of bacteria present in the buccal cavity and
bacterial plaque, both supra-and infra-gingival with a significant improvement of
periodontal health (reduction of cavities, gingivitis and improvement in periodontitis),
like Streptococcus mutans, Staphylococcus aureus, Porphyromonas gingivalis, and also
the other anaerobic and gram-negative species bacteria, microorganisms that mostly
cause the occurrence of dental diseases and halithosis.27
Figure 2. Overview of the antimicrobial, antioxidant and anti-inflammatory activities
of extra virgin olive oil (EVOO) phenolic compounds. 22
Up to 36 phenolic compounds have been identified in olive oil.15 Phenolic compounds

found in extra virgin olive oil, including the dialdehydic form of decarboxymethyl
oleuropein, aglycon, oleocanthal, hydroxytyrosol and tyrosol, have been shown to
possess potent activity against several strains of bacteria.22,29 Additional beneficial
effects on oxidation also have been demonstrated by olive oil phenolics in vitro. Olive
oil phenolics have been found to decrease reactive oxygen species (ROS) production
and elicit significant free-radical scavenging effects.30,31 In vivo and in vitro research
24
also has reported the phenolics may attenuate inflammatory responses in the body and
reduce the risk of chronic inflammatory disease development.22,32
OleochantL compound possesses an antiinflammatory ability, a relatively similar
chemical structure than ibuprofen, due to its dose-dependent ability to inhibit
cyclooxygenase (COX) enzymes, both COX-1 and COX-2, which are involved in the
prostaglandin biosynthesis (inflammatory) pathway.39
Figure 3. Arachidonate metabolism pathways for prostaglandin synthesis by cyclooxygenase enzyme,

COX1 and COX2. Oleocanthal in olive oil acts like the NSAIDs, inhibiting COX enzyme. 44
Besides, Oleocanthal has also been found to escape hydrolysis under stomach-simulated
conditions and aid in inhibiting the growth of Helicobacter pylori bacteria, which have
been associated with peptic ulcer and gastric cancer development. 42 The anti-
inflammatory actions of oleocanthal reported most recently, conclude that this
compound has potent pharmacological actions in attenuating inflammatory mediators
such as inducible nitric oxide synthase (iNOS) which plays a role in the pathogenesis of
joint degenerative disease.43
Recent in vitro has shown that oleuropein aglycone, one of the olive oil phenolics,
inhibits tumour necrosis factor alpha (TNFα) induced matrix metalloproteinase 9
(MMP-9) in a monocyte cell line, and this has implications for health as monocytes
along with the molecules they express play a significant role in inflammation-based
disease development.45
25
Figure 4. Much research has been conducted to investigate the health-benefiting properties of (a)
hydroxytyrosol, (b) tyrosol and (c) oleuropein aglycone. Oleocanthal (d) has fast become an olive oil
phenolic of much interest due to its potent anti-inflammatory activity.22
Tyrosol and Hydroxytyrosol are phenolic compounds obtained from olive extract with
antioxidant effects. Hydroxytyrosol differs from tyrosol only in an additional hydroxyl
group at position 3 on the phenol ring. Tyrosol has been shown to conduct protective
effects against oxidative injuries in cell systems. It showed a high protective effect in
GSH and reinforce intracellular antioxidant defences. 45 Hydroxytyrosol also shows
cardioprotective effects, preventing oxidative stress-induced endothelial dysfunction48,
inhibiting lipid and protein oxidation in human plasma49, a wide range of antitumor
effects, inhibiting proliferation and promoting apoptosis in several human tumour-cell
lines through several mechanisms.50
Gingivitis
Gingivitis is diagnosed by the presence of redness, swelling, and increased edema of the
gingival tissues. There may be increased pocket depth without attachment loss caused
by gingival enlargement, and bleeding on probing is a hallmark of gingivitis and
periodontitis. Epidemiological research show that there is a near relation between
ammount of supragingival plaque and chronic gingivitis, so that clinically research has
been proved that supragingival plaque as a main etiology of gingival inflammation33.
26
Figure 5. Typical generalized marginal and papillary gingivitis34
Gingival bleeding varies in severity, duration, and ease of provocation. Bleeding on

probing is easily detected clinically and therefore is of value for the early diagnosis and
prevention of more advanced gingivitis. It has been shown that bleeding on probing
appears earlier than a change in color or other visual signs of inflammation35; in
addition, the use of bleeding rather than color changes to diagnose early gingival
inflammation is advantageous in that bleeding is a more objective sign that requires less
subjective estimation by the examiner. In general, gingival bleeding on probing
indicates an inflammatory lesion both in the epithelium and in the connective tissue that
exhibits specific histologic differences compared with healthy gingiva. Even though
gingival bleeding on probing may not be a good diagnostic indicator for clinical
attachment loss, its absence is an excellent negative predictor of future attachment
loss36. Score and criteria for gingival index (GI) were as follows 37:
0 = Normal gingiva.
1 = Mild inflammation: slight change in color and slight edema no bleeding on
brobing.
2 = Moderate inflammation: redness, edema, and Glazing, bleeding on probing.
3 = Severe inflammation: Marked redness and edema; ulceration; tendency to
spontaneous bleeding.
The most common cause of abnormal gingival bleeding on probing is chronic
inflammation. The bleeding is chronic or recurrent and is provoked by mechanical
trauma (e.g., from toothbrushing, toothpicks, or food impaction) or by biting into solid
foods such as apples. 35
The severity of bleeding and the ease of its provocation depend on the intensity of the
inflammation. Subgingival plaque (plaque below the gum line) is associated with
periodontitis, an inflammatory disease characterized by the irreversible destruction of
the epithelium, connective tissue, and bone supporting the teeth. Gingivitis is associated
with a mixture of gram-positive (56%) and gram-negative species(44%), as well as
facultative(59%) and anaerobic species(41%).35 Dominant Gram-positive species
consist of S. Sanguins, S. Mitis, S. Intermedius, S oralis A. Viscosius, A. Naeslundii,
Peptostreptococcus micros. Dominant gram-negative species consist of F nucleatum,
Prevotella Intermedia, V. Parvula, Haemohilus influenzae, Captocyphaga,
Campylabacter.37
27
Gingivitis can be a preamble to periodontal diseases and involves anaerobic bacteria

commonly found in supragingival plaque, for example, Porphyromonas gingivalis,
Fusobacterium nucleatum and Prevotella intermedia37. The general view is that not all
gingivitis leads to periodontal disease but that infectious periodontal disease usually
follows gingivitis. Chronic gingivitis often results in mild bleeding from the gums
during tooth brushing, which is generally only a minor inconvenience unless underlying
blood dyscrasias or bleeding disorders exist.35
DISCUSSION
From over the years oil pulling users all over the world are giving enough evidence that
by oil pulling they were getting benefit. Mouth is normally teeming with bacteria and
saliva also is a key defense against bacteria and viruses. It contains enzymes that
destroy bacteria in different ways. But harmful bacteria can sometimes grow out of
control and lead to periodontitis, a serious gum infection.4
The phenolic compounds found in extra virgin olive oil, including the dialdehydic form
of decarboxymethyl oleuropein, aglycon, oleocanthal, hydroxytyrosol and tyrosol, have
been shown to possess potent activity against several strains of bacteria.2 Oleocanthal is
one of olive oil phenolic compounds that suggested to human health benefit properties.
This compound possesses an antiinflammatory ability, a relatively similar chemical
structure than ibuprofen, due to its dose-dependent ability to inhibit cyclooxygenase
(COX) enzymes, both COX-1 and COX-2, which are involved in the prostaglandin
biosynthesis (inflammatory) pathway.39 Some studies have demonstrated that
oleocanthal leads an effective inhibition of the proliferation, migration, and invasion of
human breast and prostate cancer lines.39 Therapeutic activities has also been shown by
oleocanthal so it has been used for the treatment of Alzheimer's disease.41
Tyrosol and Hydroxytyrosol are phenolic compounds obtained from olive extract with
antioxidant effects. Tyrosol penetrates and accumulates in macrophages and improves
the intracellular antioxidant defence systems. Besides, in vitro hydroxytyrosol has been
reported to attenuate the TNF-α, iNOS, and COX-2 in LPS-induced human monocytic
(THP-1) cells. Hydroxytyrosol also displays anti-inflammatory effect, it inhibited the
production of nitric oxide and prostaglandin E (PGE) and decreased secretion of
cytokines and chemokines, reduced the expression of genes, and inhibited PGE synthase
in murine macrophages.46,47 Hydroxytyrosol phenolic antioxidants reduced generation
of ROS (Reactive Oxygen Species), which plays key role in many physiological and
pathogenic processes, including signal transduction, inflammation, aging,
neurodegeneration and atherosclerosis.51 H2O2 is an ubiquitous ROS main product
which can activate signaling processes, induce cytotoxicity in many cells, then lead to
oxidative damage.52
Pathological changes on gingiva because of microorganism adhesion at the tooth
surface or surounding gingival sulcus is called gingivitis. This gingival inflammation is
happened because the present of microorganism product had activated monocytes and
macrophages then yields some inflammation mediators like PGE2, INF, TNF, and IL-2.
At this study, olive oil pulling functionate as therapeutic because it could decrease
plaque even reducing gingivitis. The mechanism of olive oil phenolics as antimicrobial
activities inhibit gingival bacteria for example, Streptococcus mutans, Streptococcus
28
aureus and Porphyromonas gingivalis. Oleocanthal and hydroxytyrosol possess potent

activities against several strains of bacteria and attenuate inflammatory responses in the
body and also decrease reactive oxygen species (ROS) production.22,29-32 Oleochantal
also inhibits cyclooxygenase (COX) enzymes, both COX-1 and COX-2, which are
involved in the prostaglandin biosynthesis (inflammatory) pathway in gingiva without
attachment.6
Another research is also conducted to evaluate the effectiveness of bacterial plaque
elimination and in the bleeding decrease in gingivitis. Sixty samples were devided into
three groups with 20 persons for each group. The first group being required to use water
for tooth-brushing, second group were to use olive oil and the third group were to use
sunflower oil. The result showed that the group who used olive oil obtained a smaller
bleeding index and greater bacterial plaque elimination than the others. It concluded that
olive oil is a suitable substance for achieving total elimination of plaque and combating
gingivitis as the first sign of periodontal pathology.27
The pathological events leading to the destruction of the periodontium during
inflammatory periodontal diseases are likely to represent complex interactions involving
an imbalance in enzymic and non-enzymic degradative mechanisms. This paper also to
review the increasing body of evidence implicating reactive oxygen species (ROS),
derived from many metabolic sources, in the pathogenesis of periodontal tissue
destruction. ROS are generated predominantly by polymorphonuclear leukocytes
(PMN) during an inflammatory response and are regarded as being highly destructive in
nature. The detection of ROS oxidation products, the elevation of iron and copper ions,
which catalyse the production of the most reactive radical species, and the identification
of an imbalance in the oxidant/antioxidant activity within periodontal pockets, suggests
a significant role for ROS in periodontal tissue destruction. In vitro studies have shown
that ROS are capable of degrading a number of extracellular matrix components
including proteoglycans, resulting in the modification of amino acid functional groups,
leading to fragmentation of the core protein, whilst the constituent glycosaminoglycan
chains undergo limited depolymerisation. The identification and characterisation of
connective tissue metabolites in gingival crevicular fluid (GCF) resulting from the
degradation of periodontal tissues, notably alveolar bone, provides further evidence for
a role for ROS in tissue destruction associated with inflammatory periodontal
diseases.53
It is concluded that oil pulling therapy may be beneficial in non surgical phase of
periodontal therapy as it may modulate the detrimental host responses. We suggest to
conduct a further scientific research and evaluation of this ancient health practice as an
adjunct in periodontal therapy and host modulatory agent.
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32
Apoptosis and Viability Analysis of Human Calvarial

Osteoblasts Under Cyclic Mechanical Strain
Soh Shean Han1, Alethea Foong Li Yen1, Liesl Khoo Yi Min1, Sabrina
Ng Livia1
1
Faculty of Dentistry, National University of Singapore, Singapore.
ABSTRACT
Mechanical loading is an important determinant of bone mass and remodelling. In vitro
studies have investigated the effects of mechanical stimuli on osteoblast differentiation
or proliferation, but the role of mechanical strain on osteoblast apoptosis remains
poorly understood. Yet apoptosis is critical to bone remodeling and closely linked with
cell proliferation. The aim of the present investigation therefore was to study the effect
of cyclic mechanical strain on osteoblast apoptosis and viability over a 24h time-course.
Human calvarial osteoblasts (HCOs) obtained from ScienCell Research Laboratories

(Carlsbad, CA, USA) were used in all experiments. Third passage cells were cultured
on 6-well Bioflex plates coated with type I collagen and subjected to a cyclic
equibiaxial in-plane deformation of 2% for 5 sec (0.2 Hertz) every 90 sec for 6h, 12h,
and 24h. Metabolic activity/ cell viability was quantified by the MTT assay (Sigma-
Aldrich, Singapore) which measures mitochondrial succinate dehydrogenase activity.
Apoptosis was quantified by the Caspase-Glo 3/7 assay (Promega Corporation,
Madison, WI) which measures two executioner caspases.
The MTT Assay showed that mechanical deformation resulted in a transient, but
statistically significant reduction in overall metabolic activity at 6h, 12, 24h (p<0.001).
There was also a reduction in caspase-3 and -7 activity by mechanically strained cells at
6h, 12h, 24h (p=0.004), suggesting that mechanical strain is transiently anti-apoptotic in
nature. Although the MTT Assay is commonly used to test cytotoxicity, these findings
do not indicate that mechanical strain is cytotoxic, but represents a transient reduction in
succinate dehydrogenase activity, in response to the altered biomechanical environment
of the cells, without a reduction in actual cell numbers.
Thus we conclude that a cyclic, equibiaxial, tensile mechanical strain significantly

reduces cell apoptotic and metabolic activity of human calvarial osteoblasts over a 24h
time-scale.
33
Keywords: Human calvarial osteoblasts, apoptosis, cell viability, mechanical strain,

bone remodelling
INTRODUCTION AND LITERATURE REVIEW

Bone remodelling is the continuous turnover of bone matrix by resorption (osteoclasts)
and formation (osteoblasts). The mechanical environment via loading of bone plays an
important role in its regulation. (Bergmann et al 2010) According to the Frost
Mechanostat theory, in the Disuse Window (absence of loading), there is significant
bone loss while in the Overuse Window (strains exceed the physiological limit), there is
bone formation. Bone cells can respond to physical stimuli in vitro, and several studies
with various mechanical stimuli have been done to study the varying effects of
mechanical forces on bone and the involvement of numerous signal transduction
pathways. However, the process of translating the physical stimulus into the biological
response i.e. mechanotransduction, is poorly understood. (Liedert et al 2005)
Bone cells can respond to various mechanical stimuli such as stretch, fluid flow and
hydrostatic pressure, in vitro (Jansen et al 2004). Osteoblasts and osteocytes are the key
cells involved in sensing and communicating the signals for changes in bone mass as a
result of loading. However these cells are affected by numerous influences, hence the
responses of bone to loading alter under different circumstances. (Skerry 2008)
Currently, most in vitro cell studies on osteoblast mechanotransduction have focused on

investigating the effect of mechanical strain on the differentiation of mesenchymal stem
cells into osteoblastic phenotypes, or on the proliferation/ synthetic function of
osteoblasts. (Li et al 2003, Li et al 2004). These studies use differentiation markers such
as alkaline phosphatase (ALP) activity (Kim et al 2010; Zhao et al 2009), bone
morphogenetic proteins (BMPs) (Kim et al 2009), osteocalcin, osterix (Osx),
osteopontin, core binding factor alpha 1 (Cbfa-1), runt- related transcription factor 2
(Runx2), receptor activator of nuclear factor kappa-B ligand (RANKL), as well as
synthetic function markers such as Type 1 collagen activity, and real time PCR/ mRNA
expression of osteogenic marker genes (Krishnan and Davidovitch 2009; Zhao et al
2009). (Kim et al 2010) Other studies investigate the role of several transduction
pathways such as estrogen receptor kinase (ERK) 1/2 (Kim et al 2010; Fan 2006; Jessop
et al 2004), mitogen activated protein kinase (MAPK), RAS protein, second messengers
and various molecules such as vascular endothelial growth factor (VEGF), nitric oxide
(NO), p38 kinase (Kim et al 2010), cortisol (Pereira et al 2001), cyclo-oxygenase 2
(COX2), fibroblast growth factor receptor 3 (FGFR3) (Jackson et al 2006), in
osteoblastic differentiation..
These in vitro bone cell studies study the effect of varying type (dynamic or static, and
compressive, tensile or shear) and temporal characteristics (cyclic, continuous,
intermittent, or interrupted), duration, magnitude (Zhu et al 2009), frequency, number of
applied load cycles of the strain on the osteoblastic/ pre-osteoblastic response. (Duncan
34
and Turner 1995; Liedert et al 2005; Skerry and Suva 2003; Vinod and Davidovitchb
2006) Reported responses however vary widely. While mechanical stimulation has been
found to increase bone cell proliferation and cell numbers in most studies (Cheng et al
1999; Zhuang et al 1996; Kaspar et al 2000; Turner et al 1998), some studies purport a
decrease in cell numbers (Mikuni- Takagaki et al 1996; Matsuda et al 1998). A possible
hypothesis by Burger and Veldhuijzen (Burger and Veldhuijzen 1993) attributes the
different responses to the characteristic of the strain applied.
Previously it was unusual to find morphologically apoptotic osteoblasts at active bone

forming seams. Hence experiments involving apoptotic analysis upon mechanical strain
were usually limited to osteocytes and osteoclasts. For example, studies have
investigated the release of a paracrine agent by osteocytes in response to disuse, perhaps
even the products of apoptotic cell death, that were a chemoattractant to osteoclasts,
leading to bone resorption (Skerry 2008; Skerry and Suva 2003), or the apoptosis of
osteoclasts in bone formation (Kobayashi et al 2000).
However apoptosis is a critical process in skeletal maturation and bone remodelling. A

close link exists between bone cell proliferation, differentiation, and apoptosis, resulting
in an adequate pool of osteoblasts and osteoclasts for bone homeostasis (Masellaa and
Meistern 2006). Genes such as the SOST Gene are shown to be capable of reducing
osteoblast numbers through apoptosis (Masellaa and Meistern 2006). Hence this has led
to growing research in the study of osteoblastic lineage apoptosis. A recent study (Goga
et al 2006) has shown the time and force dependent increase of cellular apoptosis in
osteoblast- like cells (MG-63 cells), via the increase in activity of caspase-3 enzymes, in
the presence of a compressive force. Yet, no definitive evidence has been found.
Another study demonstrated that the expansion of premaxillary suture in rats led to an
accumulation of osteoblasts along the sutures, which peaked in numbers after 2 days.
Upon expansion, cell apoptosis was identified in osteoblasts along the suture and the
osteocytes, especially on day 2 and day 3. Hence it was established that apoptotic
osteoblast cell death might regulate the growth modification of premaxillary suture
upon mechanical stretching. (Zhang L. et al 2009)
While there is increasing research into investigating the regulation of apoptosis in
osteoblast- like cells via growth factors and cytokines, due to the difficulty in
determining accurately the stage of the osteoblastic lineage at which the various
molecular factors come into play, these studies have little significance on osteoblast
function and pathogenesis (Hughes and Boyce 1997). Other studies have demonstrated
that the differentiation stage of osteoblasts determine their apoptotic and proliferative
response to mechanical stimuli (Weyts et al 2002) and physiologic stretch levels are
capable of inducing apoptosis in young osteoblast cultures (Jansen et al 2004).
Recent findings have suggested that orthodontic loading may trigger bone remodeling
by producing microdamage or by stimulating the induction of a regional acceleratory
phenomenon—a reaction to trauma, hence suggesting the role of programmed cell death
35
(apoptosis). Here, the rate of bone remodeling exceeds normal tissue activity. (Trudy et
al 2009) Some microgravity studies suggest that, in addition to reduced osteoblast
differentiation and function, osteoblast apoptosis may have contributed to osteopenia
(Bucaro et al, 2004) while others have indicated independence of apoptosis induction
from the effect of microgravity on osteoblasts (Bucaro et al, 2007).
However there is very limited research conducted to investigate the time-dependent

process of cell death of osteoblasts (both apoptosis and necrosis) in the presence of
tensional forces, and its role in bone remodeling. Yet the role of osteoblasts is important
in the process of bone remodeling. They sense changes in mechanical stimuli, secrete
bone matrix, promote mineralization and also regulate bone resorption by producing
bone resorption-inhibitory factors such as RANKL/ osteoprogeterin (OPG) and bone
resorption stimulating factors such as macrophage- colony stimulating factor (M-CSF)
which inhibit and activate osteoclasts respectively.
Current mechanotransduction mechanisms postulate that mechanical loading stimulates

osteocytes that sense the fluid flow. This does not account for the mechanism in
distraction osteogenesis which involves gradual stretching of soft tissues in gaps
between bone segments. Hence this in vitro static stretch culture model can provide a
cellular basis for the role of static stretching load during the initial stages of bone
remodelling (Kim et al 2010).
AIMS AND HYPOTHESIS
The aim of our study is to examine how mechanical stimuli regulate the metabolism of
bone cells by investigating the apoptosis and cell viability of Human Calvarial
Osteoblasts (HCO) under mechanical strain. We hypothesized that there is a mean
difference in the cellular apoptotic activity and cellular viability of human calvarial
osteoblast cells which have undergone mechanical strain and the control group. In
addition, we hypothesized that the duration of mechanical strain affects cellular viability
and apoptotic response. For this purpose, Human Calvarial Osteoblasts were cultured
with Osteoblast medium (ObM). After Passage 3, the cells were plated on Flexcell
plates, designed to deliver tensional forces of variable duration. The cultured cells were
harvested after 6h, 12h, and 24h of mechanical stimuli. To evaluate and differentiate the
change in cell growth due to necrosis, or apoptosis, and to determine the level of
caspase enzymes (apoptotic activity), MTT and Caspase- GLO assays were performed
respectively. This study will reveal the role of osteoblast apoptosis in bone remodelling
and the effects of time-dependent tensional mechanical stimuli on the osteoblastic
response. This study will advance our understanding of the role of mechanical stimuli in
the complex autocrine/ paracrine system of cellular function regulation in bone
remodeling. Embryology shows that Human Calvarial Osteoblast cells, similar to PDL
and alveolar bone, are neural crest derivatives. Thus the findings of this study will add
on to what is known in the field of craniofacial skeletal defects (Masellaa and Meister
36
2006). It may also have implications in the clinical application of tensional mechanical
stimuli in distraction osteogenesis (Isaksson et al 2007), Orthodontic therapy, and the
osseointegration of implants (Leucht et al 2007) in orthopaedic joint replacement
therapy. As ultimately, excellence in these fields is derived from a comprehensive
knowledge of both mechanics and biology.
MATERIALS AND METHODS

CELLS
In this experiment, we used Human Calvarial Osteoblast Cells (from ScienCell

Research Laboratories, Carlsbad, CA) that were kept frozen as primary cells. Human
Calvarial Osteoblast cells are able to better mimic the response of the alveolar and basal
bone in both the maxilla and mandible to mechanical strain, as they share a similar
origin (neural crest); as compared to Human Femoral Osteoblast Cells, which originated
from the lateral plate mesoderm, and thus have a higher strain threshold.
To recover the Human Osteoblast cells, the cells were placed in the Osteoblast Medium
(ObM, from ScienCell Research Laboratories, Carlsbad, CA) and centrifuged
(Centrifuge 5810 R, Eppendorf) after the cells have thawed.
TRYPSINIZATION AND CELL CULTURE
The 75ml Falcon flask containing the Human Osteoblast cells was washed twice with
Phosphate Buffered Saline (PBS, from 1st Base). 5ml of Trypsin (GIBCOBKL) was
added into flask and incubated for 5 min, at 37oC, 5% C02 (Hera Cell 150 Incubator,
Thermo Scientific). The trypsin and cells were removed, and an equal volume (5 ml) of
ObM (to neutralize the trypsin) was added to the cells in a 15ml tube. The cells were
centrifuged for 5min at 1500rpm, at room temperature 20-25°C. The supernatant was
discarded, and the cells were remixed with 10 ml of ObM. The cells were divided into
two equal 75ml Falcon flasks which contained 30ml of ObM each. The cells were
allowed to grow to confluence, as determined visually under a light microscope. This
was equivalent to one passage, and the processes of trypsinisation and cell culture were
repeated until the third passage.
The cell numbers were counted manually using a light microscope on a

haemocytometer.
The cells were plated on 6-well Bioflex Collagen I plates (Flexcell International
Corporation, Hillsborough, NC, USA) with 2ml of ObM per well and an appropriate
amount of cell culture to achieve 250000 cells/ well. The cells were cultured to
confluence in a 37oc, 5 % CO2 incubator, for approximately 3- 4 days.
37
APPLICATION OF CYCLIC MECHANICAL STRAIN
After the cells had reached confluence, the silicon rubber membranes were subjected to
an in-plane deformation of 2 % for 5 sec (0.2 Hertz) every 90 sec using a square wave-
form, around rectangular ArcTangle™ loading posts (required to correctly apply
uniaxial tensile strain to the cells) in a standard Bioflex base plate linked to a Flexercell
FX-4000 Strain Unit (Flexcell International Corporation, Hillsborough, NC, USA). The
loading unit was used inside a 37oc, 5% CO2 incubator (Nuaire DH Autoflow, C02 Air-
Jacketed Incubator, HEPA). Vacuum is applied to the area under 6 well plates, and
generated through a Venturi valve, using an airflow regulated by a computer drive valve
(using Flexercell software).
The characteristics of the strain were decided based on the minimal strain levels that
have shown to be capable of inducing cellular changes within similar batches of Human
Calvarial Osteoblast Cells, which were previously subjected to mechanical stretch in
vitro. At 6h, 12h, 24h time intervals, the cells were harvested. Apoptosis and vitality
analyses via Caspase- Glo 3/7 Assay and MTT 98% TLC Assay respectively were
conducted on the cells.
CELLULAR APOPTOTIC ACTIVITY
Caspase-Glo® 3/7 Assay
Caspases, a family of cysteine proteinases that specifically cleave proteins following

aspartate residues play key roles in mammalian apoptosis. Two ‗executioner‘ caspases
(3 and 7) were measured by the Caspase-Glo 3/7 Assay (Promega Corporation,
Madison, WI) which provides a homogenous luminescent Caspase-3/7 substrate
containing the tetrapeptide sequence (Asp-Glu-Val-Asp) in a reagent optimized for
caspase activity, luciferase activity and cell lysis.
Adding a single Caspase-Glo® 3/7 Reagent in an ―add-mix-measure‖ format results in

cell lysis, followed by caspase cleavage of the substrate and generation of a ―glow-type‖
luminescent signal produced by luciferase (Figure 1). Luminescence is proportional to

the amount of caspase activity present. The level of caspase enzymes activity indirectly
reflects the cellular apoptotic activity. The Caspase-Glo® 3/7 Reagent relies on the
properties of a proprietary thermostable luciferase (Ultra-Glo™ Recombinant
Luciferase), which is formulated to generate a stable ―glow-type‖ luminescent signal
and improve performance across a wide range of assay conditions.
At the end of each time point, the collagen coated rectangular area was cut and treated
with the Caspase-Glo® 3/7 reagent for 1 hour in the dark, at room temperature,
according to the manufacturer‘s instructions. Each sample was aliquoted into triplicate
tubes and luminescence, expressed as RLU (relative light units), was measured with a
Sirius Single Tube Luminometer (Berthold Detection Systems GmbH, Pforzheim,
Germany).
38
CELL VIABILITY
FIGURE 1. CASPASE-3/7
cleavage of the luminogenic substrate containing the DEVD sequence. Following caspase cleavage, a
substrate for luciferase (aminoluciferin) is released, resulting in the luciferase reaction and the
production of light.
Controls were also used to determine the basal caspase activity of the Human Calvarial
Osteoblast cells. It comprised the Caspase-Glo 3/7 reagent and the same batch of third
passage Human Calvarial Osteoblast cells, which were not subjected to mechanical
strain, but plated on the Bioflex Plates in the same medium.
CELL VIABILITY
Methylyhiazolyldiphenyl-tetrazolium bromide (MTT, approx 98% TLC) Assay
MTT has been used as a histochemical/ cytochemical reagent. Cell activation was
measured by the MTT assay (Sigma-Aldrich, Singapore), a colorimetric assay for
39
estimating mammalian cell survival, proliferation, and activation based on the ability of
viable cells to reduce yellow 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium
bromide (MTT) by mitochondrial succinate dehydrogenase (20, 21) to the water-
insoluble MTT formazan, which chelates with nickel, copper, and cobalt. The cobalt
chelate is used in oxidative systems. MTT has been used for detection of NAD.
The blue crystals are solubilised with acidified isopropanol and the intensity is
measured colorimetrically at a wavelength of 570 nm (absorbance A570nm).
At the end of each time point the cells were harvested by trypsinization and resuspended
in fresh media; 50 µl of this cells suspension was added to 96-well plates and spun at
4000 rpm for 5 mins. The supernatant was removed and 50 µl of the MTT solution
(4mg/ml in DMEM) was added to each well. The cells were incubated at 37oC for 2h in
the dark. Following incubation, the plate was centrifuged and the supernatant discarded.
The resulting formazan crystals were dissolved by the addition of 200µl of
dimethylsulfoxide (DMSO; Sigma-Aldrich) and absorbance measured at 570 nm in a
microplate reader (Tecan SpectrophorPlus; Singapore).
Controls were also used to determine the basal mitochondrial succinate dehydrogenase
activity of the Human Calvarial Osteoblast cells in the same medium.
CONTROLS
The controls used in this experiment are P3 passage of Human Osteoblast Cells similar
to those used in the experimental group. The cells were plated on similar 6- well Bioflex
Collagen-I coated plates, and placed in a 37oc, 5% CO2 incubator but were not subjected
to mechanical strain. The cells were harvested at 6h, 12h, and 24h respectively and
subjected to the same tests as the sample cells.
STATISTICAL ANALYSIS
All data are presented as mean ± SEM. Each experiment was performed once. Data is
presented as means from at least 6 wells for each condition.
The data were analyzed using the SPSS software (IBM SPSS 19).
Results from the Shaporo-Wilk test showed that the data collected from the Caspase-
Glo 3/7 and MTT assays were not normally distributed.
Hence, comparisons were made between the Human Calvarial Osteoblast Cells that had
undergone mechanical stretching and the controls using the (non parametric and
independent) Mann-Whitney t-test at 6h, 12h, 24h intervals. Correlations were also
investigated between the cellular responses and the duration they were subjected to
mechanical strain. The data were analysed using the Spearman‘s Correlation test.
Significance was determined as p< 0.05 for all tests.
40
HYPOTHESES AND VARIABLES
Null hypothesis 1
There is no difference in the mean cellular apoptotic activity of the Human Calvarial
Osteoblast Cells that have undergone mechanical strain for 6h, 12h, 24h and the control
group.
Independent variable: Whether mechanical strain was applied (Experimental vs.

Control) [nominal variable]
Dependent variable: Cell Viability Fluorescence (RFU) [continuous variable]
Test: Mann-Whitney t- test
Three separate t tests were conducted for each time point.
Null Hypothesis 2
There is no difference in the mean cell viability of the Human Calvarial Osteoblast
Cells that have undergone mechanical strain for 6h, 12h, 24h and the control group.
Independent variable: Whether mechanical strain was applied (Experimental vs.

Control) [nominal variable]
Dependent variable: A570nm [continuous variable]
Test: Mann-Whitney t- test
Three separate t tests were conducted for each time point.
Null Hypothesis 3
There is no correlation between the cellular apoptotic activity of the Human Calvarial
Osteoblast Cells and the length of time the mechanical strain was applied.
Variable: Length of time the mechanical strain was applied [ordinal variable]
Variable: Cell Viability Fluorescence (RFU) [continuous variable]
Test: Spearman‘s Correlation Test
Null Hypothesis 4
There is no correlation between the cell viability levels of the Human Calvarial
Osteoblast Cells and the length of time the mechanical strain was applied.
Variable: Length of time the mechanical strain was applied [ordinal variable]
41
Variable: A570nm [continuous variable]
Test: Spearman‘s Correlation Test
RESULTS
700000
(6)
(6)
600000
Mean Cell Viability Fluorescence
500000 (6) **
400000 **
** Control
300000 Treated
200000
100000
(RFU)
0
6 Hrs 12 Hrs 24 Hrs
Time (hours)
FIGURE 3. Mean Cell Viability Fluorescence (RFU) of HCO cells, as a measure of cellular
apoptotic activity, recorded at 6h, 12h, and 24h time points, using the Caspase- Glo 3/7
Assay.
Treatment: HCO cells subjected to 2%, 0.2Hz, cyclic, uniaxial, tensile strain
n=6 plates per experimental and control group
** p<0.01
As seen from Figure 3, the mean cell viability fluorescence level of the treated group at
6h is (37.91 ± 0.7897) x 104 RFU as compared to (42.59 ± 0.6405) x 104 RFU for the
control group. The fluorescence level of the treated group at 12 h is (44.74 ± 0.2168) x
104 as compared to (61.36 ± 0.5191) x 104 RFU for the control group. At 24h, the
42
fluorescence level for the treated group is (32.58 ± 0.2454) x 104 RFU as compared to
(55.81 ± 0.2284) x 104 RFU for the control group.
At all 3 time points, the mean cell viability fluorescence level (cellular apoptosis
activity) of the control group were higher than the treated group.
However, both groups showed a similar trend in the change in apoptotic activity. Figure
1 demonstrates an increase in the mean cell viability fluorescence level (apoptotic
activity) from 6h to 12h, followed by a drop in the mean cell viability fluorescence level
(apoptotic activity) from 12h to 24h, for both groups.
For the control group, the maximum and minimum cellular apoptotic activity was
demonstrated at 12h and 6h respectively, while for the treated group, there was
maximum and minimum apoptotic activity at 12h and 24h respectively.
1.8
1.6
1.4
Mean Absorbance570nm Value
1.2
1
control
0.8
treated
0.6
0.4
0.2
0
6hrs 12hrs 24hrs
Time (hours)
FIGURE 4. Mean Absorbance570nm value of HCO cells, as a measure of cell vitality, recorded
at 6h, 12h, and 24h time points, using the MTT 98% TLC Assay.
Treatment: HCO cells subjected to 2%, 0.2Hz, cyclic, uniaxial, tensile strain
n=6 plates per experimental and control group
*** p<0.001
43
As seen from Figure 4, the mean A570nm of the treated group at 6h is 1.359 ± 0.0327 as
compared to 1.527 ± 0.0331 for the control group. The mean A570nm at 12h for the
treated group is 0.933 ± 0.0316 as compared to 1.530 ± 0.0352 for the control group. At
24h, the mean A570nm for the treated group is 1.014 ± 0.0727 as compared to 1.249 ±
0.0713 for the control group.
At all three time points, the control group showed higher cell viability as demonstrated
by a higher mean absorbance value than the treated group.
However, both groups demonstrated a difference in the trend of cell viability across the
three time points. The control group shows a slight increase in cell viability from 6h to
12h, followed by a drop in cell viability from 12h to 24h. On the other hand, the treated
group shows a drop in cellular viability from 6h to 12h, followed by an increase in
cellular viability from 12h to 24h.
For the control group, the maximum and minimum cellular viability were demonstrated
at 12h and 24h respectively, while the maximum and minimum cellular viability were at
6h and 12h respectively for the treated group.
EFFECT OF MECHANICAL STRAIN ON CELLULAR APOPTOTIC

ACTIVITY
Hypothesis 1
The Mann-Whitney Test was used to compare the mean cellular apoptotic activity of the
treated and control group at 6h, 12h and 24h. (Figure 5)
At 6h, the p value= 0.004 which is < 0.05, hence the null hypothesis is rejected and
there is a difference between the mean cellular apoptotic activity of the human calvarial
osteoblast cells which had undergone mechanical strain and the control group.
44
FIGURE 5. Summary: Statistical results of Independent- Samples Mann-Whitney U Tests
comparing the effect of 2%, 0.2Hz, cyclic, uniaxial, tensile strain on Mean Cell Viability
Fluorescence (RFU) [Cell Apoptotic Activity] of HCO cells recorded using the Caspase- Glo 3/7
Assay at 6h, 12h, 24h time points
EFFECT OF MECHANICAL STRAIN ON CELLULAR VIABILITY
Hypothesis 2
The Mann-Whitney Test was used to compare the mean cell viability of the treated and
control group at 6h, 12h and 24h. (Figure 6)
At 6h, the p value <0.001 which is <0.05, hence the null hypothesis is rejected and there
is a difference between the mean cell viability of the human calvarial osteoblast cells
which had undergone mechanical strain and the control group.
At 12h, the p value <0.001 which is <0.05, hence the null hypothesis is rejected and
there is a difference between the mean cell viability of the human calvarial osteoblast
cells which had undergone mechanical strain and the control group.
45
At 24h, the p value <0.001 which is <0.05, hence the null hypothesis is rejected and
there is a difference between the mean cell viability of the human calvarial osteoblast
cells which had undergone mechanical strain and the control group.
FIGURE 6. Summary: Statistical results of Independent- Samples Mann-Whitney U
Tests comparing the effect of 2%, 0.2Hz, cyclic, uniaxial, tensile strain on Mean
Absorbance 570nm Values [Cell Viability] of HCO cells recorded using the MTT 98% TLC
Assay at 6h, 12h, 24h intervals
EFFECT OF DURATION OF MECHANICAL STRAIN ON CELLULAR

APOPTOTIC ACTIVITY
Hypothesis 3
The Spearman‘s Correlation Test was used to test if there was any significant
correlation between cellular apoptotic activity and duration of mechanical strain within
the 24h timeframe. (Figure 7)
46
As p value=0.064, which is >0.05, the null hypothesis is accepted and there is no

correlation between the cellular apoptotic activity of the human calvarial osteoblast cells
and the duration of mechanical strain.
However given the small sample size (n=6), we cannot exclude the possibility of a
significant correlation (p value approaches 0.05) with a larger sample size. While this
phenomenon warrants further investigation, a significant negative correlation may still
possibly exist between the 2 variables.
The Correlation Coefficient is -0.446. This suggests a moderately strong, negative

correlation between the cellular apoptotic activity and duration of mechanical strain,
within the 24h timeframe. In other words, an increase in the duration of mechanical
strain will lead to a reduction in cellular apoptotic activity and vice versa.
FIGURE 7. Statistical result of the Spearman’s Correlation Test studying the Correlation
between the Duration of Mechanical Strain (Hours) and the Mean Cell Viability
Fluorescence (RFU) [Cell Apoptotic Activity] of HCO cells recorded using the Caspase-
Glo 3/7 Assay
Significance Level p<0.05
EFFECT OF DURATION OF MECHANICAL STRAIN ON CELLULAR

VIALBILITY
Hypothesis 4
The Spearman‘s Correlation Test was used to test if there was any significant
correlation between cell viability and the duration of the mechanical strain within the
24h timeframe. (Figure 8)
As p value= 0.003, which is <0.05, the null hypothesis is rejected and there is a
correlation between cell viability and the duration of the mechanical strain.
47
The Correlation Coefficient is -0.553. This suggests that there is a moderately strong,
negative correlation between cell viability and duration of mechanical strain within the
24 timeframe. In other words, a reduction in mechanical strain will lead to an increase
in cell viability, and vice versa.
FIGURE 8. Statistical result of the Spearman’s Correlation Test studying the Correlation
between the Duration of Mechanical Strain (Hours) and the Mean Absorbance 570nm Level
[Cell Viability] of HCO cells recorded using the MTT 98% TLC Assay
Significance Level p<0.05
DISCUSSION
Human Calvarial Osteoblast cells which have undergone mechanical strain experience
an increase in apoptotic activity (measured by caspase enzymes levels) from 6h to 12h,
and the apoptotic activity is on a decline after the 12h time point (Figure 1).
Osteoblasts which have undergone mechanical strain undergo a drop in cell viability
between the 6h to 12 h time point, and the cell viability levels increase after the 12h
time point (Figure 2). This phenomenon corresponds with the changes in cellular
apoptotic activity within the 24 timeframe.
The above results also correspond with current literature which demonstrates cell death
within the initial hours of force application, followed by a gradual increase in viable cell
numbers as proliferation/ differentiation> apoptosis/ necrosis as time progresses (Proffit
et al 2007).
We had hypothesized that there is a mean difference in the cellular apoptotic activity
and cellular viability of human calvarial osteoblast cells which have undergone
mechanical strain and the control group. From the results above, we have shown that the
application of mechanical strain has led to a reduced Caspase 3 and 7 enzymes levels
cellular in the human calvarial osteoblast cells regardless of the duration in which the
48
mechanical strain was applied within the 24h timeframe (Figures 1 and 3). This
however, corresponded to overall reduced cell viability. It is thus possible that cell death
could thus have occurred through a cell death pathway that was independent of Caspase
3 and 7. Supraphysiological strains may obscure physiologically relevant responses or
damage cells (Weyts et al 2003). This is relevant taking into account the calvarial origin
of our osteoblasts as opposed to a femoral origin (Skerry 2008).
A study has shown that the optimal strain (for proliferation) is 10000 microstrain (1%
deformation), 1 Hz and 1800 load cycles for bone cells (Liedert et al 2005). Some
studies have shown that increasing the strain or load cycles beyond this level will not
stimulate proliferation (Skerry and Suva 2003) such that overloading may lead to
necrosis and reduced tooth movement in vivo (Schwarz 1932, Krishnana and
Davidovitchb 2006). On the other hand, other studies have demonstrated that direct
mechanical loading with strain levels similar to those measured in vivo evoke no
cellular reaction in human osteocytes in vitro (Henneman et al 2008; Owan et al 1997;
You et al 2000).
However, current research has yet to come to a consensus with regards to the optimal
strain to use in vitro- while one study demonstrated no proliferation at 10% strain (Kang
et al 2011), another showed increased expression of osteocalcin mRNA at 3%, but not
at 1%, or 10% strains (Zhao et al 2009). It has also been demonstrated that proliferation
exists up to 1.6% strain, with only synthetic function present at 3% strain (Zhu et al
2009). A similar trend was shown in another study where synthetic function without
proliferation was present at higher strain levels (Li et al 2004).
In our model, 2% deformation was used as it was shown to be the minimal strain level
capable of inducing cellular changes within similar batches of Human Calvarial
Osteoblast Cells, which were previously subjected to mechanical stretch in vitro.
To determine if our force is non physiological for calvarial cells, a similar experiment
has to be conducted on femoral cells and elicit if the latter respond in a similar manner.
Secondly, the force applied in this experiment was within the physiological window and
hence there could have been minimal apoptosis as loading stimulates bone formation.
This is accordance with Wolff‘s Law or the Mechanostat theory by Frost 1987 (Liedert
et al 2005). To determine if our control cells were in the Disuse state, the osteogenic
response could be measured by studying the gene expression of various osteogenic
markers such as osteocalcin, parathyroid hormone receptor, bone sialoprotein,
osteopontin, cbfa1, and collagen 1.
Thirdly, as there was greater cell viability/ proliferation/ metabolism in the control
group as shown by the MTT assay results, this could have led to a corresponding greater
caspase enzymes activity in the control group.
49
Lastly, this could be attributed to the fact that the application of mechanical stimuli has
led to the reorientation of the cells along the membrane, with a possible transient
reduction in cellular metabolism (as shown by the MTT results) that would translate
into a transient reduction in caspase enzyme activity.
In addition we have shown that regardless of the duration in which the mechanical
strain was applied within 24h, the mechanical stimulus has led to reduced cell viability
(Figures 2 and 4). Similarly, we had expected a reduction in the number of viable cells
through apoptosis/ necrosis within the first 24h of mechanical stimulation due to the
presence of aseptic acute inflammatory reaction (suggesting cell death) within the first
few days of mechanical stimulation (via the activation of an orthodontic appliance)
(Krishnan and Davidovitch 2009). However, while cell apoptosis was lower in the
experimental group, cell viability was also lower, which may appear to be contradictory.
Yet, besides attributing the lower cell apoptotic activity as a direct effect of reduced cell
proliferation and viability, this finding could also be explained by the fact that MTT
Assay measures the activity of mitochondrial succinate dehydrogenase activity, and
may be a reflection of cell metabolism instead of solely cell numbers. Mechanical
forces have been shown to be capable of altering the metabolic state of osteoblasts and
osteocytes (Krishnan and Davidovitch 2009). The reduced apoptotic activity in the
experimental group could thus be a direct effect of this reduction in cell metabolism
(perhaps due to cell reorientation in response to the force) instead of a direct effect of
mechanical stimulation.
In addition, Human Calvarial Osteoblasts which originate from the neural crest could
exhibit more resistance to the absence of strain than Human Femoral Osteoblasts (from
the lateral plate mesoderm), thus allowing greater cell vitality in the control group even
in the absence of strain. To confirm this, other tests on cell metabolic byproducts will
enable us to determine the true metabolic activity of the cells while PCR (via DNA
count) or a Cell- Count Kit will enable us to determine the cell numbers.
This phenomenon could also be attributed to the mechanics of mechanical stimulation

in our experiment- the Flexercell loading unit uses tensile force in one plane
deformation. But the regions at the peripheries experience more force than the centre.
Moreover there might be confounding shear and compressive forces. Programmed/
unprogrammed cell death can occur via many different pathways (Eastman 1993). If the
cells in the centre of the well are pulled very far apart they will undergo contraction
(compression), and under compression/shear forces, the cells undergo necrosis, leading
to a reduction in cell proliferation.
To further confirm if reduction in cell viability was due to necrosis, staining using
fluorescein isothiocyanate (FITC)-coupled Annexin V/ Propidium Iodide (PI) or tests
that detect pro-inflammatory cytokines such as IL-1 and TNF-alpha could be conducted.
50
In addition, we hypothesized that the duration of mechanical strain affects cellular

viability and apoptotic response.
Based on our results above, the p value (0.064>0.05) of the Spearman‘s Correlation
Test appears to demonstrate that there is no correlation between the duration of
mechanical stimulation within the 24h timeframe and cellular apoptotic activity (Figure
5). There is minimal current literature that studies the relationship of time-dependent
progression of osteoblastic apoptosis with mechanical stimulation within the first 24h.
However current literature does show an increase in cellular proliferation and
differentiation into osteoblasts when mechanical forces are applied to mesenchymal
stem cells or pre-osteoblast cells (Cheng et al 1999; Zhuang et al 1996; Kaspar et al
2000; Turner et al 1998). Moreover, current literature also demonstrates an effect of
force duration on the proliferation on osteoblasts and pre-osteoblasts (Duncan and
Turner 1995; Liedert et al 2005; Skerry and Suva 2003; Vinod and Davidovitchb 2006).
Although the p value has not reached statistical significance threshold, p=0.064 and is
reasonably close to 0.05, hence there still lies a possibility that the p value will approach
0.05 as the sample size or number of test replicates increases. Thus, based on the small
sample size and the proximity of the p value to 0.05, we can conclude that a moderately
strong, negative correlation or a trend may still exist between the duration of
mechanical strain and cellular apoptotic activity. But the exact phenomenon will
warrant further investigations.
Our results (p=0.03 <0.05) have also demonstrated a moderately strong, and negative
correlation between osteoblast cell viability and duration of mechanical stimulation
(Figure 6). Although this did not correspond with our expectations, it could be possible
if the force used in our experiment is considered supraphysiological for human calvarial
osteoblast cells. Current literature has shown that forces of excessive magnitude can
induce cellular necrosis and reduced cell proliferation, hence the application of a
supraphysiological force for an extended time may induce necrosis and hence reduced
cellular proliferation (Schwarz 1932, Skerry and Suva 2003, Krishnana and
Davidovitchb 2006). For bone formation to occur, the strength of a force applied needs
to be greater than the maximum force to which the bone is likely to be exposed to
during normal activity. This threshold varies in cells of different species, different
regions of the skeleton, and under different circumstances. In humans, talking, chewing
and other normal functions are associated with strains in the dome of the cranium of up
to 50 microstrain while more extreme activities such as striking a rubber mallet on the
head only induced strains around 200 microstrain This shows that the threshold for the
human skull is much lower than the limb bones (Skerry 2008) and they are adapted to
withstand physiological forces of lower magnitude.
This finding and explanation also support the results of our Mann-Whitney t-test which
demonstrated a reduction in cellular apoptotic activity in the treated as compared to the
control group (Figure 3).
51
Alternatively, this could be explained by a densensitization mechanism at longer

durations of stretching, after taking into account the variations in force applied. A study
(Jansen et al 2004) has demonstrated a rapid increase in ERK 1/ 2 phosphorylation
induced by a cyclic stretch with a maximum between 5 and 15min. Prolonged stretching
for 60min led to a decrease in phosphorylated ERK 1/ 2 towards baseline level.
Thirdly, this phenomenon this could be attributed to the limitations of the Flexercell
loading unit. Excessive tensional forces may generate a region of compressive/ shear
strain in the centre of the well due to the contraction in response to the stretch. Thus, the
Flexercell loading machine may have unequal distribution of the magnitude and type of
force throughout the well and as osteoblasts are affected by numerous influences, hence
the responses of bone to loading will alter under different circumstance (Skerry 2008).
However, in practice, bones also experience complex strain patterns that include
different proportions of compressive, tensile and shear components (Skerry 2008).
Lastly, our study was conducted within a timeframe of 24h, which may not be truly
indicative of the expected osteogenic response to mechanical strain under an extended
period of time (e.g. 3 weeks). Increasing the duration of the experiment will enable a
more conclusive determination of this trend.
LIMITATIONS AND WEAKNESSES
1. The duration of our study (24h) was too short to have a conclusive
understanding of the trend in cellular apoptotic activity and viability upon
mechanical stimulation. For instance while our study showed a reduction in
apoptotic activity and increase in viability between the 12h and 24h time point,
we were unable to determine if there will be a continuation of this trend beyond
24h. Extending our experiment to a week, or even 3 weeks, will advance our
understanding of the trend in the osteogenic response to mechanical strain with
sustained forces.
2. With regards to mechanical strain, our study only investigated one variable that
could affect the response of osteoblasts to mechanical stimulation, i.e. the
duration of the mechanical strain (and at only 3 time points). Hence this could
have a negative impact on the internal and external validity of our data, as in
reality many other factors affect the response of osteoblasts such as the force
magnitude, etc.
3. Although our data was collected from six different wells, the entire experiment
was conducted only once. This could have a negative impact on the reliability of
our findings. Replicating the experiment will enable us to eliminate erroneous
results or results due to chance.
4. The limitations of the Flexercell loading unit in restricting the type and
magnitude of strain would have led to confounding results. This can only be
overcome by in vivo experiments and even in true function, cells are constantly
being subjected to such complex force combinations.
52
5. The limitation of the MTT Assay was its inherent inability to distinguish
between cell numbers and cell metabolism. Hence further tests could be done to
distinguish if the absence of response was due to a down regulation in cell
numbers or metabolism.
6. While the Caspase Assay measures the caspase enzyme levels, this does not
necessarily translate to apoptosis. Hence a second apoptosis test could have been
conducted as a confirmation, for example via detection of cytomorphologic
alterations using a Transmission Electron microscopy (TEM) (Elmore 2007).
7. Our experiment was conducted on Calvarial Osteoblasts, hence this
phenomenon may not be applicable to limb bones. Further investigations will be
needed to confirm if Human Femoral Osteoblasts will respond in a similar
manner. Moreover, future experiments will need to confirm the characteristics of
the strain within the physiological window for Calvarial Osteoblasts.
8. Tests on pro-inflammatory cytokines (Krishnan and Davidovitch 2009) or FITC-
coupled Annexin V and Propidium Iodide tests could be carried out to confirm
the presence of necrosis. (Weyts et al 2003). And osteogenic response of the
control HCO cells could be measured by studying the gene expression of various
osteogenic markers.
9. Further investigations will be needed to confirm the negative correlation
between cellular apoptotic activity and duration of mechanical strain within 24h
as the p value in our experiment failed to reach statistical significance threshold.
FUTURE RESEARCH
In future, other variables affecting the effect of mechanical strain on the apoptotic and
proliferative response should be incorporated into our experiment design. These
variables include the type of force, (compressive/tensile/ shear; equiaxial/
uniaxial/biaxial; cyclic/ intermittent/ continuous/ interrupted and static/ dynamic), the
magnitude, the frequency, the number of load cycles, as well as the duration of
mechanical strain, the type and composition of the cells, ageing and presence of local
hormones (Duncan and Turner 1995).
In reality, there are many variables that affect the osteogenic response of osteoblasts to
mechanical strain. After conducting investigations on the above variables, a regression
analysis (linear or non-linear) could be done on the data collected to have a better
understanding of the importance of each variable as well as the effect of the
combination of variables on the osteoblastic response.
More importantly, the duration of the study should be extended to obtain more
conclusive findings of the trend in the cellular apoptotic activity and viability under
mechanical strain.
Further investigations varying the magnitude of strain/ types of cells will allow a better
understanding of the clinical effect of mechanical strain on different cell origins.
53
In the study of mechanotransduction and its clinical applications, orthodontic force has
been identified as a force applied for the purpose of solely tooth movement and has a
magnitude lower than orthopaedic forces that attempt to modify craniofacial bones
(Krishnana and Davidovitchb 2006). Calvarial osteoblasts cells also demonstrate a
lower threshold than femoral osteoblasts (Skerry 2008), hence the characteristics of the
strain optimal to the different areas of the skeleton will have to be determined.
The next step in our research would be to include markers of the osteoblastic phenotype
such as ALP, BMP, Cbfa-1/ Runx2 gene to study the effect of mechanical strain on
osteoblastic function. In addition to proliferative, apoptotic and differentiation markers,
future research could possibly include markers of its synthetic activity such as type I
collagen production, various proteoglycans, and cell adhesion molecules. To have a
better understanding of the mechanotranduction pathway, the effect of mechanical strain
on growth factors, interferons, tumour necrosis factor, and colony stimulating factors
can be further studied.
To study the role of mechanical stimulation on the autocrine/paracrine system of

osteoblasts in bone remodelling, especially with regards to its effect on osteoclasts,
various osteoclastic factors secreted by osteoblasts such as RANKL and M-CSF can
also be included in our study.
IMPLICATIONS
This is the first in vitro cell study that investigates the systematic real-time progression
of apoptosis of human calvarial osteoblasts in response to tensile forces. It lays the
foundation for further investigation into the role of osteoblast apoptosis in bone
remodeling, how this relates to cell viability in the presence of mechanical strain and the
effects of time-dependent tensional mechanical stimuli on the early stages of the
osteoblastic response. This study will advance our understanding of the role of
mechanical stimuli in the complex autocrine/ paracrine system of cellular function
regulation in bone remodeling.
In addition, coupled with more extensive research, our study may have potential
implications in the clinical application of tensional mechanical stimuli in distraction
osteogenesis (Isaksson et al 2007), tooth movement and active functional appliances in
Orthodontic therapy, as well as the osseointegration of implants (Leucht et al 2007) in
orthopaedic joint replacement therapy. There will also be potential implications in
craniofacial orthopaedic devices which deliver macro-scale mechanical forces that
produce micro-structural suture bone strain and induce cellular growth response in
sutures (Krishnana and Davidovitchb 2006). It will advance our understanding on how
iatrogenic mechanical stimulation may impact clinical bone remodeling on a cellular
level, especially on tissues of the neural crest origin.
54
CONCLUSION
In conclusion, we have established that the changes in apoptotic activity over 24h
corresponds with the changes in cell viability over 24h amongst mechanically
stimulated Human Calvarial Osteoblast cells.
The presence of a cyclic, uniaxial, tensile mechanical stimulation (2%, 0.2Hz, applied
every 90sec) also significantly reduces cellular apoptotic activity and cellular viability
within the first 24h. This reduction in cellular viability could be attributed to a reduction
in cellular proliferation (caused by cell death), or sheer reduction in cellular
metabolism.
With increasing duration of mechanical stimulation, there is a reduction in cellular

viability and apoptotic activity within the first 24h. However, the latter phenomenon
will warrant further investigations for more conclusive results.
These findings will advance our understanding of the role of mechanical stimuli in the
complex autocrine/ paracrine system of cellular function regulation, specifically
osteoblast apoptosis, in the bone remodeling process.
ACKNOWLEDGEMENTS
We would like to thank Dr Samuel Li Xiaobing, Dr Vinoth Kumar (our Project
Supervisors) and Prof. Murray Clyde Meikle for their invaluable advice and assistance.
We would also like to thank the Faculty of Dentistry, National University of Singapore,
for generously funding our project.
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59
Improving Lung Quality of Atopic Asthma Patient

with Assisted Drainage Theraphy, Scaling Root
Planing and Toothpick Technique
Kamadjaja SK1, Djohan DS1, Hudyono R2, Endah HS2, Setiawatie
EM3, Wibisono PA3
1
Student of Dentistry Faculty, Airlangga University, Surabaya, Indonesia.
2
Graduate Student of Dentistry Faculty, Airlangga University, Surabaya, Indonesia
3
Periodontology Department, Dentistry Faculty, Airlangga University, Surabaya,
Indonesia
ABSTRACT
Objectives : Asthma was a chronic respiratory disease affecting individual of all ages.
Although it was based on immunogenic origin, neural mechanism may contribute in its
pathology, in which focal infections from the oral cavity may trigger it. Assisted
Drainage Therapy, originated from bloodletting technique, was cleaning the inner part
of the gingival sulcus that may induce Hsp70 as an anti-inflamatory agent and
signifcantly result in improving lung quality. Toothpick technique was a new brushing
technique which was able to stimulate the gingival sulcus to increase Hsp70 secretion.
Methods : The experiment was conducted by observing the lung quality of juvenile
asthma patients in two hospitals. A preliminary screening of gingivitis and oral hygiene
was conducted using the Gingival Index and Oral Hygiene Index. Subjects were treated
with SRP, ADT, and toothpick technique. The lung quality was then assessed after
each treatment. The lung quality was evaluated 2 weeks after the intial treatment.
Paired t-test was used for the statistical analysis of this research. Results: The result
of this experiment showed that was there was no siginificant difference between
patients treated using toothpick technique and SRP but the ADT showed significant
increase of lung quality. The patients treated using ADT also maintained lung quality 2
weeks after the initial treatment. Conclusion : The conclusion of this research was that
the ADT, SRP, and toothpick technique effectively and rapidly improve lung quality
but ADT maintains the lung quality.
Keywords: Assisted Drainage, Atopic Asthma, Oral Hygiene, Toothpick, Scalling Root
Planning
INTRODUCTION
Asthma was a compilation of clinical symptoms marked by respiratory tract
obstruction dan has reversible properties. 1 Asthma can attack any individual of any
age and was marked by recurrent episodes of wheezing, breathlessness, chest
tightness, and cough, particularly at night and in the early morning. 2
60
So far the pathology of asthma was caused by fluid blockage and exudate obstructing
the respiratory tract causing hyperinflation but excludes parenchym damage. 3 Many
cells and cellular elements play a role in asthma pathology, in particular, mast cells,
eosinophils, T lymphocytes, neutrophils, and epithelial cells which was associated
with the occurrence of clinical symptoms.
Although asthma was a chronic inflammatory disease of the respiratory tract, neural
mechanism also contributes in its pathology. 4 Asthma can be caused by neurogenic
inflamation triggered by neuropeptides. Certain proteins such as tachykinin especially
substance P (SP) 5, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP)
and persistence of tumour necrosis factor α (TNF-α) plays a part as a trigger 6, 7, 8.
Where those neuropeptides will activate Th2 receptors which will produce an
interleukin sequence and trigger asthma symptoms in the respiratory tract. 9
Based on the focal infection concept, infection inside the oral cavity can cause
systemic inflamation including the respiratory tract. 8, 10 According to those concept,
elimination of focal dental plaque should decrease asthma severity. Nevertheless that
concept contradicts with the hygiene hypothesis proposed by Strachan (1989). 11 The
hypothesis states that lack of infectious agents during juvenile period increases the
childrens susceptibility towards allergy because the lack of development of the
natural immune system.
On the oral mucosa, stimulus induction such as LPS from Porphyromonas gingivalis
on unsheated sensory nerves induces neuropeptide release such as SP and CGRP can
trigger neurogenic inflamation and cause sensitization of afferent and parasympathic
nerve tissue. 12, 13, 14 Sensitization of the maxillary nerve, sphenopalatine ganglion,
and the nose parasympathic ganglion causes an elevated sensitivity towards asthma
triggers which are allergent exposure, thermal and chemical changes; those conditions
causes a speed increase in allergic asthma symptoms although with less amount of
triggers. 15
Assisted drainage therapy (ADT) was a therapy that massages the inner wall of the
gingival sulcus with the aim to stimulate Hsp70 which will decrease the immunogenic
inflamation of the gingiva. The working principal of this therapy was similar to
bloodletting technique, which was useful to stimulate discharge of certain protein to
expel exudate and decrease inflamation. 16 Assisted drainage therapy was proposed
to be a breakthrough in the field of periodontal therapy and may function to improve
lung quality (FEV1) measured in minutes . 17
The mechanism of assisted drainage therapy which was done was the gingival sulcus
of the upper molar as asthma therapy has followed valid principle by lowering local
gingival inflamation, there will be repair of inflamation occuring in the nasal passage
(rhinitis) which was served by the maxillary nerve. This concept was in accordance
to the ‖one airway-one disease‖ 18 and axon-reflex concept 19 which was embraced
by doctors generally stating that the lowering of nasal passage inflamation will lower
asthma symptoms.
From a medical point of view asthma therapy with assisted drainage therapy has very
minimal effect. This therapy also does not use chemical substances if consumed long
61
term will cause adverse effect. A special note was that for the symptoms not reappear
was that the patient must maintain an optimal degree of oral hygiene health 16.
Tooth picks was commonly known as a hygiene instrument to clean food stuck
between teeth it has recently been used to show significant usage to clean dental plaque.
While Toothpick technique was a new brushing technique which was able to stimulate
the gingival sulcus to increase Hsp70 secretion and aid in increasing breathing quality
as well as preserving it.

In this research the test subjects qualify based on these criterias; asthma patients age 12
to 17; suffers from seldom or frequent episcodic asthma attacks varying from degree of
juvenile asthma classification based on GINA; qualified reversibility criteria with
increase of FEV1 score > 12% pre and post bronchodilator; patiets suffers chronic
gingivitis with a gingival index score >1; willing to participate in the research and
signed an informed consent contract; patients do not suffer from acute irreversible
pulpitis; patients do not suffer from CLP, uses prosthetic nor orthodontic appliances; no
systemic diseases or abnormality which may affect the outcome of this procedure;
patients are not undergoing desensitizing treatment.; patients are not consuming
antibiotics and antihistamines during the research; patients are not either active or
passive smokers. This research utilizes simple random sampling for sample gathering.
Implementation Procedures: All asthma patients which are registered in pulmonary
outpatients department at Mardi Rahayu Christian Hospital Kudus and Kartini Hospital
Mojosari Mojokerto Indonesia; parents of subjects who fits the inclusion criteria are
then given informed consents; interview was conducted, alongside completing medical
documents, physical examination, and supporting examinations for primary data of the
subjects; gingivitis and oral hygiene examination was conducted using the GI and OHI
as refference; all visible caries lesions on the dentition was closed using temporary
filling; subjects from the 1st group was treated using SRP; subjects from the 2nd
group was treated using ADT; subjects from the 3rd group was treated using
Toothpick technique; spyrometry examination was done post treatment; lung function
quality was assessed by spyrometry examination. SRP implementation methods:
subjects from the treated group was given SRP therapy with dental education; areas of
the 1st molar was then covered with chlorhexidine digluconate as an antiseptic; the
SRP therapy was done; lung function quality was assessed by spirometry
examination. ADT implementation methods: subjects from the treated group was
given ADT with dental education; areas of the 1st molar was then covered with
chlorhexidine digluconate as an antiseptic; the ADT was done using a small scaler
sickle on the gingival sulcus of the 1st molar for 3 minutes until there was passive
bleeding visible. Pressure on the gingival wall must not induce pain; lung function
quality was assessed by spirometry examination pre and post ADT. Toothpick
technique implementation methods: subjects from the treated group was given
toothpick technique therapy with dental education; areas of the 1st molar was then
covered with chlorhexidine digluconate as an antiseptic; the ADT was done using
small and thin sickle scaler; lung function quality was assessed by spirometry
examination pre and post ADT. Statistical analysis was done using paired T-test.
62
RESULTS
Table 1. Group Average and Standard Deviation
Groups Number Mean SD

of
samples
OHI Before DI 12 1.36 0.82

CI 12 0.39 0.40
Total 12 0.88 0.59
GI before 12 1.39 0.37
Before 12 64.83 5.69
After SRP 12 67.25 7.63
After ADT 12 81.58 12.52
After T. Pick 12 83.75 9.72
OHI After DI 12 0.62 0.59

CI 12 0.00 0.00
Total 12 0.31 0.30
GI After 12 0.78 0.43
2 Weeks Post 12 81.17 10.57
OHI
2.5
DI BEFORE
2
CI BEFORE
SCORE
TOTAL BEFORE
1.5
DI AFTER
CI AFTER
1
TOTAL AFTER
0.5
0
1 2 3 4 5 6 7 8 9 10 11 12
SAMPLE
63
Image 1. Visual OHI score per sample graph
Image 2. Visual Spyrometer percentage per sample graph
Image 3. Visual GI score per sample graph
64
Image 4. Visual OHI score average graph
Image 5. Visual Spyrometer percentage average graph
Image 6. Visual GI score average graph
The data above shows the average OHI and GI score pre and post SRP; the breathing
passage percentage pre and post SRP, ADT, and toothpick technique; the breathing
passage percentage 2 weeks after SRP, ADT, and toothpick technique from each group.
From these data we can see the average of each group. The total sample of this group
was 12 mild persistent asthma patients. Average DI score pre treatment was 1.36
(SD=0.82); average CI score pre treatment 0.39 (SD=0.40); DI and CI average total
0.88 (SD=0.59); GI average score pre treatment 1.39 (SD=0.37). Average DI score post
treatment 0.62 (SD=0.59); average CI score post treatment 0.00 (SD=0.00); DI and CI
average total 0.31 (SD=0.30); average GI score post treatment 0.78 (SD=0.43)
Spyrometer measurement result showed FEV1 score average pre treatment 64.83%
(SD=5.69); group post SRP treatment 67.25% (SD=7.63); group post ADT treatment
81.58% (SD=12.52); group post toothpick technique 83.75% (SD=9.72);group post
treatment and 2 week post treatment control 81.17% (SD=10.57).
To determine if there was variety in each group a paired t-test was used predeced by a
normality data test. Method used for the data normality test was Kolmogorov Smirnov.
If a data has a signifance score >0.05, then the data has a normal distribution and if the
score was <0.05 then the data was not distributed normally berarti data tersebut tidak
terdistribusi normal.
From the data above, it can be concluded that all groups have a normal distribution (p
>0.05). because all the data have a normal distribution then to determine the difference
65
between the treated and control group a paired t-test was utilized. On the paired t test
there was a signifance score of 0.001 which was lower than 0.05 signifying there was
difference in each group.
DISCUSSION
In this research it was found that FEV1 of juvenile alergic asthma patients will
increase significantly after ADT was done. From the paired t test a p=0.001 score was
obtained suggesting there was significant difference between FEV1 scores pre and
post ADT. This result suggest there was improvement in lung quality in less than 1
hour after the ADT was executed. The elevated FEV1 score effect was similar with if
the patient was adiministered with bronchodilator. 20
The result of this research supports the previous research conducted by Wiyarni
(2008)21 with subjects being children age 5 to 12 who suffers from mild persistent
asthma. This research also proves that gingivitis can trigger asthma and ADT can
improve lung quality in a matter of minutes.
Stensson et al (2011) compared oral hygiene of 20 asthma patients age 18 to 24 with
similar control groups. The result of that research suggests that asthma patients has
higher prevelance in caries and gingivitis accompanied with decrease in saliva flow.22
Another research also involving juvenile asthma patients also showed similar results.
McDerra (1998) observed 100 asthma patients age 4 to 16 and found that children that
suffers from asthma has higher caries, plaque, calculus, and gingivitis score than of
those who are healthy.23 Stensson et al (2010) compared oral hygiene of asthma patients
age 3 to 6 and showed that juvenile asthma patients has a tendency to have a higher
caries and gingiva severity index due to a higher intake in sugar and that asthma patients
tend to breath from the mouth.24
According to del Rio Navarro et al (2001), the correlation between asthma and
gingivitis severity was caused by medications consumed by the patient. 25 This research
uses pre-post controlled study design to compare the effect of salmeterol and
beclomethasone with saliva flow lowering, IgA level in saliva, and total amount of
protein found in saliva of the test subjects being children age 6 to 16. The study showed
evidence that asthma medication was the primary factor that causes gingivitis in asthma
patients.
Various research also showed that oral hygiene has no correlation with asthma. Eloot et
al (2004) conducted a cross-sectional study research on 140 juvenile asthma patients.
The result of this research showed asthma severity nor asthma medication caused any
difference in caries, gingival bleeding, and plaque index.26 This results are much alike
with a previous research done by Bjerkeborn (1987) on 61 children age 5 to 18, where
in this research there was no difference in caries and periodontal status to both asthma
and healthy patients. 27
The activation of inflamation triggers a neurogenic inflamation reaction stated by Van
Der Kleij (2002)28 and Meggs (1995) 29, whereas the immunogenic inflamation reaction
can play a role in triggering a neurogenic inflamation. The activity of the inflamation
66
can be transduced from the central nervous system to other sites where in the new site
inflamation will appear eventhough there was no immunogenic inflamation present.
This research was also a clinical confirmation that a research previously done by
Utomo (2009), proved that there was an involvement between LPS from PG that causes
gingivitis towards changes in the tracheal tissue. The research used rats as animal test
subjects which was injected with PgLPS 1435/1450. The research mentioned that
there was an increased expression of LTC4 and ECP in gingival,nasal, and tracheal
tissue. 16
ADT effect towards Alergic Asthma

Stress from enviroment for living organisms are commonly thermal, osmotic variety,
antibiotics, and various solution or chemical substances these stressors not only disturb
the transcription and translation but also often change the structure or 3 dimentional
protein. In sudden thermal changes, organisms release various proteins called heat-
shock protein. Including molecular chaperone and protease to cope with the damage
caused by thermal increase. 30
According to other researches, Hsp70 lowers the sensitivity of immunocompetent cells
such macrophages 31, 32 and tissue 33 towards LPS. Utomo (2009) proved that Hsp70
expression increased significantly compared to allergy. 16 Although not always linier
with the dosage increase of PgLPS 1435/1450 injection, except the tracheal tissue.
ADT was thought to stimulate the release of intracellular Hsp 70 to the extracellular
tissue, also inhibits the neurogenic switching mechanism; also functions to aid the
release of pro-inflamatory mediators, pro-inflamtory neuropeptides, prostaglandin,
toxins amd other chemical mediators. Even so ADT mechanism can quickly have an
effect on immune response and allergic reactions was still undetermined.
According to some literatures, mechanical stimulus can increase tissue temperature that
there was Hsp70 release, 34, 35 pH lowering and extracellular Ca2+ level increase
ekstraseluler. 35 Those mechanical stimulation causes stimulation to afferent nerves
because there was pressure on the area. According to Tai and Baraniuk (2002), various
stimulation will cause the afferent nerve fiber to release SP and CGRP. 36
Utomo and Harsono (2009) reported that within 5 minutes after ADT there was
improvement in breathing quality based on FEV1 measurement. 17 The event where
there was improvement in breathing quality within 5 minutes was not caused by the
lowering of immunogenic inflamation alone, neurogenic inflamations role in involving
sensory afferent and parasympathic nerve tissue should play a larger role.
Utomo (2009) also found TNFR1 expression (immunogenic inflamation indicator), SP
and CGRP (sensory nerve activity indicator) and VIP (parasympathic nerve activity
indicator) was significantly lower on OVA inhalation with ADT compared to that
without ADT in the nasal ,tracheal breathing passage tissue and TNF-α level in the
serum from the rats which was euthanized ±60 minutes post ADT. 16 This finding
was a verification of the ADT effect on the lowering of immunogenic and neurogenic
breathing passage tissue also systemically faster (within minutes) on juvenile alergic
asthma patients. Eventhough in this research after OVA inhalation on rats with allergy
67
was not followed with breathing quality test, significant lowering of LTC4 and ECP
expression which are bronchocontrictors after ADT also supports the verification the
event of breathing quality improvement within minutes.
This research supports the breathing quality improvement concept with ADT stated by
Utomo (2009) as a mild persistent allergic asthma therapy with an NNT=1.09 score (CI
95%; 0,9-1,3)16, meaning that achieving recovery in one patient one subject was
required (Wen et al, 2005). 37 Wiyarni et al(2007) the event where there was breathing
quality improvement after ADT and plaque control therapy showed an NNT = 1,25
score (CI 95%, 0,25-0,78);21 Utomo (2009) stated an NNT= 1,04± 0,4 score (CI 95%).
16
Based on the almost similar NNT score, an empiric evidenced-based fact ADT has been
done for years on asthma patients 16, 17, 38 then a randomized controlled trial research
was done on a juvenile mild persistent allergic asthma patient also verified with rats
with allergy can be applied to human, especially children and teenagers with mild
persistent allergic asthma patients up to 17 years old. This research was also part of a 4
step clinical application research on humans 16 so that the result of this research can be
widely applied for asthma patients to lessen the dependency on corticosteroid drugs.
Related to the hygiene hypothesis concepts which was proposed by Strachan in 1990.
Other research involving Th1 and Th2 in allergic reactions should be done since the
results are still inconsistent. The result of the research found that Th2 cytokine profile
was connected with the significant lowering of TLR4/MD-2-mediated LPS uptake,
TLR ligand-induced 1 B phosphorylation and IL-8.39 According to Utomo (2009) it
was most likely that LPS with low bioactivity and low dosage such as PgLPS
1435/1450 can trigger allergy because Th2 cytokine (IL-4 and IL-13)causes TLR4/MD-
2 complex less effective in pushing the immune response towards Th1,16 not like the
exposure of LPS with high levels of bioactivity such as Toxoplasma gondii and
Helicobacter pylori.
Discrepansies of LPS elimination concept inside the oral cavity with the hygiene
hypothesis was because that hygiene hypothesis was only based on the exposure of
endotoxins with various immunogenicity and only consider that the connection of LPS
dosage and and allergic reaction was linear. Utomo showed that the pressence of non
linear patterns maybe was not the same for each LPS depends on bioactivity. 16
The result if this research alos suggests that there was immunlogical connection
between dental plaque and the immune system of the oral and breathing passage
mucosa. The oral mucosa was a mucosa potential immune system because majority of
the epithelial tissue contains a large amount of Langerhans as well as dendritic cells
which are a part of the hosts innate immune system. 40 Other epidimiology research
supports that the presence of oral cavity bacteria colonization and periodontitis infact
has protective effects towards allergic diseases. 41, 42
An epidimiologic research done by Laurikanen (2002) proved that oral hygiene of
asthma patients was lower than those inside the control group.43 The role of oral
infection in allergy was proved by Kato et al., 2006 which suggests a low dosage
(20ng) subcutaneous injection of LPS Prophyromonas gingivalis ATCC 33277 on
68
Balb/c neonates rats and given OVA intraperitoneal injection at the age 180 and 187
days showed a sigificant elevated level of IgE serum compared to the control group.
Immunologic and neurologic influence was proven from the increase of SP expression
consistent with the elevetad level of IFN-y serum. 16 Herberth et al (2006) found that
this event was caused by the lowering of GATA3 and SOCS3 expression causing the
increase of SP giving an protective for the allergic reaction. 44
According to Aneja et al, 2006 and Cuschieri et al, 2006 Hsp70 release causes
immunocompetent cells such as macrophages and tissue (suganuma et al, 2002) to be
more resistent towads subsequent LPS stimulation. 31, 32, 33 Eventhough Hsp70 was
obtained from a normal indivuals there can be a significant increase in Hsp70 level a
few minutes after the lesion occurs. 16, 31 Also in Wang et al (2007)supports the role of
VIP in lowering allergic reaction towards ovalbumin by increasing the oral tolerance
induction effect towards ovalbumin. 45 VIP was capable of performing oral tolerance
regulation by inhibiting both cellular and hormonal response.
Compared to classic neurotransmitter (acetylcolin and adrenalin), neuropeptide
modulation effect was slower but lasts longer. Besides that neuropeptides can interact
with cells and inflamation mediators that correlates with more complex allergic
reactions from classic neurotransmitters. 46 The role of a neuropeptide within an allergic
reaction especially allergic asthma reaction has several differences. According to
Mendoca and Dolci, 2005, SP and NKA triggers bronchoconstriction towards allergic
asthma reaction while according to Herberth et al (2006), SP has a protective effect
because it increases Th1s immune response.44, 46
Herberth et al (2006) found that several neuropeptides such as VIP, somastostatin
(SOM) and SP plays a role in modulating Th1/Th2‘s immune responseand allergic
reaction.44 High level of VIP serum was connected with the lowering of T Cell
transcription factor expression (Tibet dan SOCS3) which leans more to Th2 immune
response. This happens because there was inhibition of SOCS3 molecule on IL-12-
mediated STAT4 activation. On the other hand the elevated level of SP was connected
with lowering of GATA3,SOS3 expression and increase of IFN-γ level causing a
protective effect on allergic reaction. Wang et al (2007) supports VIP in lowering
allergic reaction towards ovalbumin by increasing tolerance induction effect towards
ovalbumin and capable of performing oral tolerance regulation by inhibiting cellular
response.45
This research after executing the toothpick techique on asthma patients selected as test
subjects, it was found that there was an elevated number in the breathing passage
obstruction percentage. Meaning that the toothpick technique has sufficient effect in
lowering asthma symptoms.
Toothpick technique was done to preserve oral hygiene but also the aim of this method
was to control the OHI and GI score. OHI was measured by the degree of cleanliness of
the oral cavity and GI was used to measure the degree of gingivitis severity depending
on the degree of the oral hygiene. As explained before that oral hygiene affects the
severity of asthma.
69
CONCLUSION
This research suggest that asthma patients treated with SRP, ADT, and SRP showed
significant improvement in lung quality. Observing the OHI it was visible that there
was a lowering before and after the patient was treated using SRP, and Toothpick
Techique alongside the GI there was also lowering of the score as well. The percentage
of breathing passage obstruction all treatment showed improvement of lung quality
having all percentage climbing up either slightly or vastly. ADT showed more promise
in maintaining lung quality post treatment after being recalled. Comparisson of the GI
with the breathing obstruction percentage results was undetermined to show
correlation between the height of the GI score and lung quality.
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73
Methacrylate and Silorane Based Composite Hardness

After Application of 40% Hydrogen Peroxide
Amalina Putri, Opik Taofik Hidayat and Rahmi Alma Farah Adang
Padjajaran University, Indonesia.
ABSTRACT
Objective: To observe the difference of composite surface hardness number between
methacrylate and silorane based composite resin after application of 40% hydrogen
peroxide. Methods : This true experiment involved 36 specimens from two different
disc-shaped composite resins based from methacrylate and silorane. Every specimen
groups were soaked in artificial saliva solution and divided into two groups; The first
group is consisted of 9 specimens of control which were tested directly using
microvickers hardness tester and another group is consisted of 9 specimens which had
been added by 40% hydrogen peroxide for microvickers hardness test. Data were
analyzed with T test. Result : There was a different average numbers of composite resin
hardness from metachrylate and silorane based after the application of 40% Hydrogen
Peroxide. The hardness of methacrylate based composite resins without application of
hydrogen peroxide was 44,1 and with application was 41.8 VHN. The hardness of
silorane based composite without application was 33,9 and with application was 33.7
VHN. The data were analyzed with T test, and the result is p>0,05, with significance
p<0,05. Conclusion : There was a difference between methacrylate and silorane based
composite after application of 40% Hidrogen Peroxide, but with no significant result.
The differences of hardness number between methacrylate based composite without
application and with application had a bigger number than silorane based composite
without application and with application.
Keywords : 40% hydrogen peroxide, hardness surface number, methacrylate composite
resin, silorane composite resin.
INTRODUCTION AND LITERATURE REVIEW

Bleaching is used more than century ago, with bleaching agents applied directly on
tooth surface or packed inside a nonvital tooth. The earliest agents used reportedly was
oxalic acid, described by Chappel in 1877 and then Harlan claimed the first used of
hydrogen peroxide as bleaching agent. 1
Bleaching is a chemical process for whitening materials, work by oxidation. Oxidation
is the chemical process by which organic materials are eventually converted into carbon
dioxide and water. In dental bleaching, hydrogen peroxide diffuses through the organic
matrix of enamel and dentin. Hydrogen peroxide is an oxidizing agent and has the
ability to produce free radicals, HO2• + O2•, which are very reactive. Because these free
radicals have unpaired elecetrons, they are extremely electrophhilic and unstable so will
74
attact most other organic molecules to achieve stability, generating other radicals. These
radicals can react with most unsaturated bonds, resulting in disruption of electron
conjugation and a change in the absorption energy of the organic molecules in toot
enamel. Simpler molecules that reflect less light are formed, creating a successful
whitening action. This process occurs when the oxidizing agent (hydrogen peroxide)
reacts with organic material in the spaces between the inorganic salts in tooth enamel. 1
There may be composite restorations on the teeth to be bleached 2. Composite resin is
tooth-colored restorative materials that has been used for nearly 50 years as a restorative
materials(pinar yilmaz atali). Use if this materials has recently increased because of
consumer demands for esthetic restoration2. Composite resin has four major component,
filler which is nonorganic component, matrix which is organic component, coupling
agent, and initiator-accelerator system3 . In the last decade some tooth-colored
restoration has improved, including composite resins. Example of this improvement was
monomer ―ring opening‖ silorane. Silorane is cationic, ring-opening, hybrid, monomer
system that contains both siloxane and oxiranes. The silorane-based resin composite
have shown low polymerization shrinkage and stress and good stability and insolubility
in biological fluid stimulants. 4,5
Recently, some study has been reviewed the effect of bleaching on dental restorative
materials 6 . Bleaching agents may change the surface morphology, as well as the
chemical and physical properties of composite resins. Chemical softening from
bleaching may affect the cilinical longevity of the composite restoration. 7
Sevil Gurgan and Filiz Yalcin claimed that bleaching agent can affect the surface of
restorative materials, and showed a surface hardness decrease after bleaching agent
application 8. Wattanapayungkul and Yap reported that hydrogen peroxide and its free
radical can affected the matrix-filler debonding which can make water absorbtion and
corotion.2
The purpose of this study is to observe the difference of composite surface hardness
number between methacrylate and silorane based composite resin after application of
40% hydrogen peroxide.
MATERIALS AND METHOD

The composite resins selected for this experiment are methacrylate based composite
(Filtek Z350 from 3M ESPE) and silorane based composite (Filtek P90 from 3M
ESPE). All materials were of the A2 shade. The composite resins were placed in the
disc-shaped mold with 6mm of diameter and 3mm of thickeness. Before placing the
composite, matrix mylar were placed on the base of the mold and also on the surface of
the composite after placed the composite. The composite were light polymerized in 20
seconds with a Elipar S10 Light Cure from 3M ESPE and intensity of the light source
600mW/cm2.
Thirty six specimens were fabricated and divided into two group. Group one is consist
of eighteen specimens of methacrylate resin and Group 2 is consist of eighteen
specimens of silorane composite resin. Specimens in all groups were soaked into
artificial saliva solution for twenty four hours, and then each group were divided again
into two groups, control group with no application, and treatment group with
75
application of 40% Hydrogen Peroxide from Ultradent ( Opalescence Boost 40%). The
control group was tested directly with microvickers hardness tester ( 0,1 kg load and
dwell time of 15 seconds). The treatment groups were applied with 40% hydrogen
peroxide in twenty minutes, and rinsed with distilled water for going through the test
with microvickers hardness tester. All statistical analysis was conducted at a
significance level of p<0.05. The unpaired T test analysis was used to see the difference
of composite surface hardness number between methacrylate and silorane based
composite resin after application of 40% hydrogen peroxide.
RESULTS
Table 1 shows the average VHN of methacrylate and silorane based composite without
(control group) and with application of 40% hydrogen peroxide (treatment group). The
result showed that average control group has lower VHN than treatment group for both
materials. And the larger difference average VHN showed in methacrylate based
composite resin.
Table 1. average VHN of Methacrylate and Silorane composite without and with
application of 40% Hydrogen Peroxide
Group Methacrylate Silorane
Control (Artificial Saliva) 44,1 (3,6) 33,9 (2,0)
Treatment (40% Hydrogen Peroxide) 41,8 (3,8) 33,7 (4,2)
Table 2 shows statistical analysis using unpaired T test that showed that no significant
difference between mehtacrylate and silorane based composite after application of 40%
hydrogen peroxide with p<0.05
Table 2. statistical analysis using unpaired T test
Materials Group T test p
Control treatment
Methacrylate 44.1 (3.6) 41.8 (3.8) 1.308 0.209
Silorane 33.9 (2,6) 33.7(4.2) 0.122 0.905
76
DISCUSSION
Hydrogen peroxide lightned discolored tooth structure trough the chemical
reaction, the decomposition of peroxide into free radicals 9 . In composite restoration,
Bailey and Hannig explained that bleaching agent can decrease hardness number of
composite due to hydrogen peroxide that oxidize and generate free radicals10,11. This
hydrogen peroxide will break down into H+ ions which are unstable free radicals. These
free radicals will interact with organic component in composite (matrix) and break the
cyclic carbon bond contained in Bis-GMA.10,1,11,12
Reaction happened in composite resins is same as in enamel tooth when
hydrogen peroxide applied. Double bond in cyclic carbon bond will break become
single bond. This process still continue until oxidation is complete. The condition leads
the Bis-GMA cyclic carbon became weaker and degradated so that the hardness of
composite decreased 13,11,14. Beside attack the carbon bond in matrix resin, free radicals
also attact bond in coupling agent. Perhydroxyl which weak acid will break the siloxane
bond between coupling agent and filler into silanol bond , so it makes the filler apart
from coupling agent.10,11,12
Some study explained effect of in-office bleaching on surface hardness of new
composite resin 11,15,16 .Polydorou reported that effect of bleaching agent on surface
texture composite was material and time dependent17. Other study claimed that
bleaching agent not only soften the surface restoration, but also the deeper layer. 11
CONCLUSION
There was a difference of surface hardness number between methacrylate based
composite resin and silorane based composite resin after application 40% hydrogen
peroxide but not significant. The treatment group which applied with hydrogen peroxide
had the lower vickers hardness number than the control group, with the bigger
difference is methacrylate based composite and the lower difference (almost no
difference) is silorane based composite. So that the silorane based composite is better
than methacrylate based composite after bleaching.
REFERENCES
1. Goldstain R.E; garber D.A. Complete Dental Bleaching. Chicago: Quintessence
Books; 1995.p. 25-34,57-60.
2. Wattanapayungkul, P.; Yap, A.U.J. Effects of in-office tooth whiteners on
hardness of tooth-colored restoratives. J Oper Dent. 2002 (28): 15-19.
3. Powers, John M.; Sakaguchi, Ronald L. Craig‘s Restorative Dental. India
:Mosby Elsevier; 2006. p. 191-194.
4. Weinmann, W., et al. Siloranes in dental composites. Siloranes in dental
composites. J Dent Mater. 2005 (21) 68-74.
5. Eick JD, Smith RE, Pinzino CS, Kostoryz EL . Stability of silorane dental
monomers in aqueous systems. J. Dent. 2006( 34): 405–410.
6. Attin T, Hannig C, Wiegand A, Attin R. Effect of bleaching on restorative
materials and restorations—a systematic review. J Dent Mater. 2004 (20): 852–
861.
77
7. Stein PS, Sullivan J, Haubenreich JE, Osborne PB. Composite resin in medicine
and dentistry. J. Long Term Eff. Med. Implants. 2005 (15): 641-654.
8. Gurgan, S.; Yalcin, F.. The effect of 2 different bleaching regimens on the
suraface roughness and hardness of tooth-colored restorative materials.
Quintessence International. 2007 (38):90.e83–87
9. Taher, N.M.. The effect of bleaching agents on the surface hardness of tooth
colored restorative materials. J Contemp Dent Pract. 2005 (6): 18-26.
10. Bailey, S.J.; Swift, E.J. Effects of home bleaching products on composite resins.
Quintessence Int. 1992 23(7): 489-94.
11. Hannig, C.; Duong, S.; Becker, K.; Brunner, E.; Kahler E.; Attin T. Effect of
bleaching on subsurface micro-hardness of composite and polyacid modified
composite. Dent Mater. 2006(23):198-203.
12. Polydorou, O.; Hellwig, E.; Auschill T.M. The effect of at-home bleaching on
the microhardness of six esthetic restorative materials. JADA. 2007: 138.
13. Ferrance, J.L. 2006. Hygroscopic and hydrolitic effects in dental polymer
networks. Dental Mat 22:211-222.
14. Callister, W.D.. Material Science and Engineering. An Introduction. St Louis,
Missouri: Jhon Willey and Sons, Inc; 1994. p.130
15. Polydorou, O.; Beiter, J.; Konig, A.; Hellwig, W.; Kummerer, K. Effect of
bleaching on the elution of monomers from modern dental composite materials.
J Dent Mater. 2009: 254-260.
16. Wattanapayungkul, P.; Yap, A.U.J. Effects of in-office bleaching products on
surface finish of tooth-colored restorations. J Oper Dent. 2003(28):15-19.
17. Polydorou O.; Hellwig E.; Auschill T.M. The effect of different bleaching agents
on the surface texture of restorative materials. J Oper Dent. 2006 (31): 473-480.
78
Carica papaya Latex Extract As Accelerating Agent

for Oral Mucosa Lesion Healing
Priska Angelia, Yovita Kusnadi, Nathania
Trisakti university, Indonesia
ABSTRACT
Background : Papaya is one of Indonesian famous fruits. However,Currently, people
only consume the fruit without using its latex. Whereas, papaya latex contains a lot of
beneficial nutrients such as saponin, flavonoid, and several kinds of vitamin that could
accelerate tissue healing process. oral mucosa lesions which is not treated immediately
often causes inflammation, clinically characterized by swelling and discoloration of the
mucosa and serologically increase the levels of IL-1 β. Objective: The aim of this study
is to determine the effect of carica papaya latex in accelerating the healing process of
oral mucosa lesion, by observing the clinical symptoms and IL-1β.Material and
methods : The method which is used is experimental laboratory. Papaya latex was
made into powder by using centrifuge tube and freeze dry machine. Twelve Spraque
dawleys had their labial mucosa rubbed by the hydrogen peroxide (H2O2) 3% and
divided equally into two groups, treatment and negative control group. The papaya latex
extract powder was applied twice a day on the treatment group. Each subject from each
group were executed on the second, fifth, and seventh day to make specimen for healing
process evaluation. Followed score the colour of mucosa : 1. Dark red 2. Red 3. Light
red 4. Normal gingivaResults : On the second day, subjects of both group‘s oral lesion
scored 1, while on the fifth day, treatment group scored 3, negative control group scored
2. On the seventh day, both groups scored 4. The interleukin (IL) measurement showed
that after one day, the level of IL-1β in blood of treatment group and negative control
group are 3.12 pg/ mL and 1.19 pg/mL respectively, and on the seventh day are 9.07
pg/mL and 14.11 pg/mL respectively.Conclusion : This study concludes that Carica
papaya latex extract may accelerate the healing of oral mucosa lesion.
Keywords : healing of oral mucosa lesion, carica papaya extract, IL-1β
INTRODUCTION AND LITERATURE VIEW

Papaya is one of Indonesian most favourite fruits. Papaya (Carica papaya) is a fruit
that originated from southern Mexico and northern parts of South America1,
Currently, papaya is well widespread and widely grown in tropical. Commonly, people
only consume the meat of the fruit. 2
In tropical country such as Indonesia, this fruit can be easily grown either in
lowland and highland with a simple treatment and low cost maintanance. Papaya has
79
many benefits for the human body, whereas papaya latex contains a lot of beneficial
nutrients such as alkaloid, saponin, flavonoid, and several kinds of vitamin.
Papaya also has an antibiotics function which can be used for some treatments without
any side effects.3 It helps digestion process, prevention of ulcer and short-
sighted. The seeds are used as an helmintic 4 Since ancient times, the
root of papaya gives a good effect in treatment of kidney, bladder and
soreness. The leaves have the benefit of treating malaria, stomach cramps 4 Stems,
leaves and fruit of papaya contain white latex. This latex contains protein-breaking
enzyme or proteolytic enzyme called papain (Moehd,1999) . 6
Component Amount
Calories 46 cal
Protein 0.5 gr
Fat 0 gr
Carbohydrate 12.2 gr
Calsium 23 mg
Phosphor 12 mg
Iron / Fe 17 mg
Vitamin A 365 SI
Vitamin C 78 mg
Table 1. Content of Carica papaya (100g) 5
Papaya (Carica papaya) are classified as:

Kingdom : Plantae
Divisio : Spermatophyta
Subdivisio : Angiospermae
Class : Dicotyledonae
Ordo : Caricales
Familia : Caricaceae
Genus : Carica
Species : Carica papaya L.
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Papain in papaya latex contains a proteolytic enzyme. Commonly it is used for meat
tenderizer, silk cloth washing material, beer purifying beverages, and milk coagulant .
Papain is well stabilized in pH 5.00 solution. Papain has a synthetic activity and the
characteristic of heat resistant is higher than any other enzymes. Besides to break the
activity of proteins, papain has also an ability to form new protein called plastein or
hydrolized protein. 8 Papain is a protease enzyme that can destroy protein primary
structure 9 and catalyze the reaction of peptide hydrolisis.10
The latex also contains alkaloids, saponins, flavonoids and ascorbic acid. Papaya latex
has a benefit effect to tissues especially the soft tissue that found in the human oral
cavity.
Saponins are found in various animals and plants. Saponins‘ character is similar to soap
because of its surfactant characteristic . Saponins‘ biological activities that can
accelerate wound healing process are hemolytic activity, anti – inflammation,
and immune system stimulation . Moreover, it showed anti-microbial character, not
only against fungi but also bacteria and protozoa. The chemical structure of saponin is
a complex compound consist of saccharide, attached to a steroid or triterpene. 11.
Flavonoids (or bioflavonoids), also known as Vitamin P and Citrin, is a class of
secondary metabolites plant 12. Flavonoid, a type of phenolic compounds, is a subtitude
benzene with-OH groups. Flavonoids mostly derived from plants, its colours
are usually red, purple, blue, and yellow. Flavonoids basic structure consist of two
benzene rings which are bonded to three carbon atoms (propane). Based on its basic
structure, flavonoids can be divided into three type,
namely flavonoids, isoflavonoids, and neoflavonoids.
Flavonoids are phenolic compounds derivied from various types of vascular
plants, with more than 8,000 individual compounds known. In plant, flavonoids act as
antioxidants, antimicrobials, photoreceptors, visual attractor,repellants
feeder, and for light filter. Many studies have shown that flavonoids has
biological activity, including anti-allergenic, antiviral, anti-
inflammatory, and vasodilator. 13
Vitamin C or ascorbic acid is a vitamin with molecular weight 178 and molecular
formula C6O8H8 14. Vitamin C is more stable at low pH compared to high pH. It
oxidized easily , especially if it catalyzed by Fe, Cu, ascorbate
oxidase enzyme, light, high temperatures. Vitamin C aqueous solutions at pH <7.5 is
still stable when there is no catalysts. Oksidated Vitamin C will
15
form dehydroascorbic acid . Vitamin C deficiency can cause gum bleeding, canker
sores, muscle pain or neurological disorders. Further deficiencies may cause
anemia, frequent infections and rough skin. While the excess vitamin C can cause
diarrhea. Excess vitamin C due to the use of supplements in a long time can cause
kidney stones, whereas the excess vitamin C derived from fruits generally do not cause
side effects 16. The following table shows content of vitamin C in fruits every
100 grams 17.
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Fruits Vitamin C content

of fruit (mg/100 g)
Guava 183
Kiwi 100
Longan 84
Papaya 62
Mandarins orange 31
Melon 42
Mango 28
Pineapple 15
Banana 9
Avocado 8
Breadfruit 29
Wine 32
Tables 2. Content of vitamin C in fruits

One of the most important function of Vitamin C is associated with collagen synthesis.
Collagent is the main protein of connective tissue, bone, cartilago, dentin, and the layer
of vascular endothelium. Vitamin C defieciency intake causing the scurvy. Spontan
bleeding is the manifestation of scurvy as the result of gingivitis and systemic disease
such as anemia, which may be related to the specific functions of ascorbic acid in the
synthesis of hemoglobin. Scurvy is associated with imperfect synthesis of collagen
which manifested by poor healing wounds imperfect the formation of teeth,
capillaries rupture 18
Based on the theory, we conduct a research that papaya latex can heal the damaged
tissue in the oral mucosa. The aim of this study is to determine the effect of carica
papaya latex in accelerating the healing process of oral mucosa lesion, by observing the
clinical symptoms and IL-1β.

Sample Selection
The study was approved by ethical committee of the Animal Hospital of Bogor
Agricultural University. Twelve rats (Spraque dawleys) divided equally into two
82
groups, treatment group and negative control group. All rats were 2 months old, with
average weiight 200 gr.
Study Protocol
The way to collect the carica papaya latex
The equipments which is used are:

- Knife is used to strike the papaya to get it‘s latex. It should be sharp, and stainless
- Bowl is used to collect all the latex from the papaya. The bowl must be made from
stainless steel, light, and unbreakable. The diameter is 6-7 cm, heigh 4-5cm
- Holder is used to place the bowl when the latex taking process.
After all the equipments are well prepaired, the taking process can be started. the most
suitable time to collect the latex is 5.00am before the sun rises. The skin of the papaya is
stroken, the depth is about 0,5cm from the top to the bottom. Each papaya get 4 times
stroken. While doing this process, avoid latex get contact to the skin, it can make the
skin irittated. Papayin which is contained in the latex can get the skin itchy and irritated.
Gloves is needed during the process.
From the striking process, the drop of the latex is put in the bowl. The taking process is
done in 2 or 3 days. The important things must be remembered is new stroke process
must be spaced 2cm from the previous stroke. Usually the latex drop will be stopped in
1 hour.
The Way to Purify The Carica Papaya Latex Extract

The latex which is already collected is mixed with 27mL natrium carbonate devided
into six centrifuge tube 1,5mL (volume ratio of the natrium carbonate and papaya latex
is 3:1)
Place the centrifuge tube in a laboratory refrigerated centrifuge with 6000rpm (the
cooling type is used to avoid the stuctural damage which is causing by the centrifuge
process)
The latex will be separated, the dense part will be at the bottom and the liquid will be at
the top. Throw away the liquid by using the pipet to reduce the water. The dense part is
dried by the freeze dry in -40C in about 24 hours until it perfectly dried.
Six rats (Spraque dawleys) had their labial mucosa rubbed by the hidrogen peroxide
(H2O2) 3% and divided equally into two groups, treatment group and negative control
group. The papaya latex extract powder was applied twice a day on the treatment group.
One sprague dawley from each group were executed on the second, third, fifth, and
seventh day to make specimen for healing process evaluation.
Evaluation of serology
The steps of "indirect" ELISA follows the mechanism below:
83
 A buffered solution of the antigen to be tested for is added to each well of

a microtiter plate, where it is given time to adhere to the plastic through charge
interactions.
 A solution of non-reacting protein, such as bovine serum albumin or casein, is

added to block any plastic surface in the well that remains uncoated by the
antigen.
 Next the primary antibody is added, which binds specifically to the test antigen
that is coating the well. This primary antibody could also be in the serum of a
donor to be tested for reactivity towards the antigen.
 Afterwards, a secondary antibody is added, which will bind the primary

antibody. This secondary antibody often has an enzyme attached to it, which has
a negligible effect on the binding properties of the antibody.
 A substrate for this enzyme is then added. Often, this substrate changes color
upon reaction with the enzyme. The color change shows that secondary antibody
has bound to primary antibody, which strongly implies that the donor has had an
immune reaction to the test antigen. This can be helpful in a clinical setting, and
in R&D.
 The higher the concentration of the primary antibody that was present in the
serum, the stronger the color change. Often a spectrometer is used to give
quantitative values for color strength.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain
bound, the enzyme molecules will produce many signal molecules. Within common-
sense limitations, the enzyme can go on producing color indefinitely, but the more
primary antibody is present in the donor serum the more secondary antibody + enzyme
will bind, and the faster color will develop. A major disadvantage of the indirect ELISA
is that the method of antigen immobilization is non-specific; when serum is used as the
source of test antigen, all proteins in the sample may stick to the microtiter plate well,
so small concentrations of analyte in serum must compete with other serum proteins
when binding to the well surface. The sandwich or direct ELISA provides a solution to
this problem, by using a "capture" antibody specific for the test antigen to pull it out of
the serum's molecular mixture.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a
simple positive or negative result (yes or no) for a sample. The cutoff between positive
and negative is determined by the analyst and may be statistical. Two or three times the
standard deviation (error inherent in a test) is often used to distinguish positive from
negative samples. In quantitative ELISA, the optical density (OD) of the sample is
compared to a standard curve, which is typically a serial dilution of a known-
concentration solution of the target molecule. For example, if a test sample returns an
OD of 1.0, the point on the standard curve that gave OD = 1.0 must be of the same
analyte concentration as your sample.
84
Phytochemical test
Flavonoid test
A little amount of water added into 4 grams of papaya latex extract powder, boiled for 5
minutes. The filtrate mixed together with a little Mg powder and 1cc concentrated
HCL. The positive result shown by the formation of red, yellow, or orange colour.
Saponin test
4 grams of papaya latex extract added with hot water and concentrated HCl was boiled
for 5 minutes. 10 mL of the Fitrate was taken and then being shake for 10 seconds. The
positive results shown by formation of foam.
RESULTS
Clinical
Negative
Days Treatment Group Score control Score
2 P1 1 N1 1
P2 1 N2 1
5 P3 3 N3 1
P4 3 N4 2
7 P5 4 N5 4
P6 3 N6 3
Day 2
85
Day 3
Day 7
Day 5
86
The clinical symptom was evaluated by observing the colour of mucosa with score as
followed: 1. Dark red ; 2. Red ; 3. Light red ; 4. Normal gingiva
On the second day, subjects of both group‘s oral lesion had score 1, while on the fifth
day, treatment group had score 3, negative control group had score 2. On the seventh
day, both groups had score 4. On the second day, subjects of both group‘s oral lesion
scored 1, while on the fifth day, treatment group scored 3, negative control group scored
2. On the seventh day, both groups scored 4.
Interleukin -1β
Days Treatment Group Negative Controp Group
1 3,12 pg/mL 1,19 pg/mL
7 9,07 pg/mL 14,11 pg/mL
The interleukin (IL) measurement showed that after one day, the level of IL-1β in blood
of treatment group and negative control group are 3.12 pg/ mL and 1.19 pg/mL
respectively, and on the seventh day are 9.07 pg/mL and 14.11 pg/mL respectively.
Fitochemical examination results
Alkaloid +++
Saponin +
Tanin -
87
Fenolik +
Flavonoid ++
Triterpenoid -
Steroid -
Glikosida +
- : negative
+ : positive
++ : strong positive
+++ : very strong positive
DISCUSSION
Although based on the statistic data there isn‘t any significant differences between the
treatment group and control group, based on the scoring , the conclusion and discussion
should be:
Clinical observation = on the fifth day,the healing process of the treatment group is
more significant than the control group (score 3 for the treatment group and score 1,2
for the control group)
The observation of the inflammatory cells with the interleukin 1 beta showed on the
first day, the treatment group showed more inflammatory response compared to control
group (3,12 pg/mL compared to 1,19pg/ mL). It means the healing process is
significantly more active on the treatment group. It can be seen from the release of the
interleukin 1 beta
In contrary, on the 7 days, the IL-1 beta score on the control group is higher than on the
treatment group (14.11 pg / mL compared to 9:07 pg / mL). This means the healing
process is faster on the treatment group compared to the control group so that the
amount of chemical inflammatory mediator released by the control group was less than
the treatment group‘s
CONCLUSION
This study concludes that Carica papaya latex extract may accelerate the healing of oral
mucosa lesion which is observed by clinical and serology.
REFERENCES
1. http://www.kesehatan123.com/2111/manfaat-pepaya/
2. Kalie, Moehd Baga, Bertanam Pepaya (Revisi) .2008. Jakarta : Penebar Swadaya
88
3. Suprapti, M. Lies. 2005. Teknologi Pengolahan Pangan ANEKA OLAHAN

PEPAYA MENTAH. Kanisius : Yogyakarta
5. Direktorat Gizi Departemen kesehatan RI. 1996 .Daftar Analisis Bahan Makanan.
Jakarta
6. Kalie, Moehd Baga, Bertanam Pepaya (Revisi) .2008. Jakarta : Penebar Swadaya
8. Begot, Santoso. 2007. Pelajaran Biologi untuk SMA , Jakarta : Interplus
9. Hasbullah, 2004. Teknologi Tepat Guna Industri Kecil Sumatera Barat.
Dewan Ilmu Pengetahuan, Teknologi dan Industri.
10. Muchtadi, 1992. Enzim dalam Industri Pangan. Departemen Pendidikan
dan Kebudayaan Direktorat Jenderal Pendidikan Tinggi, Pusat
Antar Universitas Pangan dan Gizi Institut Pertanian Bogor.
11. Academic Press.‖Toxic constituents of plant foodstuffs‖. 1969, New York
12. http://dictionary.reference.com/browse/vitamin+p)
13 (Pietta PG, 2000) http://www.ncbi.nlm.nih.gov/pubmed/10924197
14. deMann, John M. 1989. Principles of Food Chemistry. Wadsworth,Inc : Canada
15. Sudarmadji, S, dkk. 2003. Analisa Bahan Makanan dan Pertanian. Liberty:
Yogyakarta.
16. http://www.anneahira.com/vitamin-c.htm
17. http://herball.net/kandungan-vitamin-c-pada-buah/
18. Ltd Michael J. Gibney, dkk. 2005, Public health nutrition, Blackwell publishing Ltd :
Oxford
89
Anti Bacterial Activity and Cytotoxicity of Guava

(Psidium Guajava L.) Leaves Extract Againsts
Streptococcus mutans
Reza Dony Hendrawan, N.A.R. Putranti, M.N.Falah
Student of Faculty of Dentistry, Airlangga University, Indonesia
Email : dony_world@yahoo.com
ABSTRACT
Objective : This study aimed to examine cytotoxicity and antibacterial activity of guava
leaf ethanol extract (GLEE) against Streptococcus mutans. Guava leaves contain tannin
9,1%, alkaloids 6%, phenyl propanoate 2.2% and flavonoids 2%. Tannins contained in
guava leaves can cause spasmolitik effect that can inhibit or kill bacteria cell's life cycle.
Methods: This experiment uses of guava leaves fresh that has been dried and ground.
Simplicia processed into a thick maserat filtrate with a rotary evaporator and evaporated
in vacuo. Anti bacterial determined by Minimal Inhibitory Concentration (MIC) and
Minimum bactericidal Consentration (MBC) test conducted by the serial dilution
method. S. mutans optical density compared to control McFarland 1 (3 X 108 UCF /
mL). GLEE was serial diluted from 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78%,
and 0.39% concentration. The result concentration of the dilution series that has been
obtained is used to assess the presence of antibacterial activity by using disc diffusion
method. To determine the level of GLEE toxicity, test performed by the method of
MTT assay. Mesencimal stem cells placed in 96-well plate and then treated with
concentration GLEE (12.5%, 6.25%, 3.12%, 1.5%, 0.78%, and 0.39 %) followed by
readings by ELISA reader at 595 nm after incubation 24 hours. Data were analyzed with
a one-way ANOVA with significance level 0.05. Results: The results of this
experiment showed that the antibacterial activity of GLEE 6,25% concentration could
inhibit the growth of S. Mutans. Cytotoxic test using MTT assay showed no toxic power
of GLEE at 12.5%, 6.25%, and 3.125% concentration. Statistical analysis showed
significant differences between the concentrations group. Conclusion: The extract of
guava leaves has antibacterial activity against S. Mutans and does not have the power
toxicity. In the future, the application of GLEE are expected to be used as a herbal
alternative non-invasive caries preventive measures.
Keywords : Guava Leaves Extract, Anti-Bacterial Activity, Streptococcus Mutans,
Cytotoxicity
INTRODUCTION
Studies of the herbal ingredients beneficial to human health have been widely carried
out recently. The World Health Organization (WHO) has recorded that more than
20,000 species of plants with medicinal properties may provide remedies for such
90
complaints as pneumonia, gastritis, diarrhea, bronchitis, colds and respiratory tract

diseases. Researches of the antimicrobial activity of medicinal plants should be
published to encourage the awareness of alternative medicines that promote
biotechnological developments.1 Medicinal plants are increasingly recognized by
scientists as a low-cost, important alternative.2 Various parts of plants usable as
natural remedies include flowers, leaves, seeds, barks, and roots and so on. One
method for use of medicinal plants is to extract and consume plants efficacious as
medicines.3
Indonesia abounds with sources of traditional medicinal plants that have been used for
some generations by most Indonesian people. Advantages of using traditional
medicines in Indonesia are partly due to abundant availability of herbal materials with
inexpensive cost. Currently, Indonesia has ± 237,641,326 population, 50.21% of them
live in rural areas. Those rural areas are generally difficult to reach by modern
medicines and medical personnel due to distribution, communication and
transportation problems. In addition, the relatively low purchasing power of rural
people makes them less able to afford modern medicines, so that they tend to prefer
traditional ones. Traditional medicines are of great significance since they are
affordable over the counter.
In Indonesia, medicinal plants are found abundantly both in quantities and types. In
addition, an extract of the plant may contain one or more types of antimicrobial
compounds. Active components that act as medicines are those chemical substances
contained in herbal mixtures. In terms of chemotherapy, these components can act,
among others, as absorbents, astringents, spasmolytics, antibacterials, adjuvants and
so on.5
Guava plant (Psidium guajava L.) is one of medicinal plants efficacious as traditional
medicine and often used by Indonesian people. It is known that the extract of guava
leaves has anti-diarrheal properties. Research has shown that guava leaves have
analgesic and anti-inflammatory properties in animal experiments with mice. 6
Guava leaves have anti-diarrhoeal effects and it been demonstrated that the ethanol
extract of guava leaves was capable of inhibiting growth of Shigella dysenteriae,
Shigella flexneri, Escherichia coli and Salmonella typhi that are bacteria causing
diarrhoea in humans. 7 In addition to having anti-diarrhoeal, analgesic, and anti-
inflammatory effects, guava leaves have potential anti-oxidant activities derived from
its polyphenolic contents so that completing a balanced diet with extract leaf of guava
can provide the body with health effects.
Oral cavity represents the habitat of Streptococcus mutans by colonizing the tooth
surfaces and becoming the predominant bacterium that cause dental caries.
Streptococcus mutans has characteristics of inactive motility, non-formation of spores,
having two or more chain compositions, ± 0.51 μm in cellular diameter, somewhat
oval in shape, facultative anaerobe and belonging to gram-negative group. 8
Extract of guava leaves is expected to inhibit growth of Streptococcus mutans as the
predominant cause of dental caries. Toxicity testing of ethanol extracts of guava
leaves needs to be performed as a safety requirement of materials for application to
human oral cavity as a natural non-invasive prevention of dental caries.
91

Preparation of extract of Guava leaves
Fresh and young leaves of the white-flesh P. guajava were taken from the Flora
Garden of Surabaya. Extracts of guava leaves were prepared in the Pharmacognosy
Laboratory, Pharmacy Faculty of Airlangga University. A total of 400 g of white-flesh
guava leaves was collected and dry-aired at room temperature for 2 weeks. The
resulting 200 g of dried leaves was crushed into fine powders. The simplicia was
macerated in ethanol solvent at room temperature for 3 days with daily stirring.
After 3 days of maceration, the macerates were then filtered. The filtrates were
separated and the residues were re-macerated in new ethanol solvent. This maceration
procedure was performed five times until the final crystal-clear macerates were
obtained. Filtrates obtained were concentrated by a rotary evaporator at a temperature
not exceeding 50C and evaporated in a vacuum to separate ethanol solvent from the
concentrated plant extracts. Concentrated extracts of 20 g were then put into sterile
vials and stored in a refrigerator at 5C.
Antimicrobial testing of guava leaf extracts
Antimicrobial tests were carried out in the Microbiology Laboratory of Dentistry
Faculty of Airlangga University. It included Minimum inhibitory Concentration
(MIC) and Minimum Bactericidal Concentration (MBC) tests in order to determine
extracts‘ minimum concentration capable of causing inhibitory and bactericidal effects
on Streptococcus mutans.
1. Preparation of test bacteria

Streptococcus mutans is taken from the stock of Streptococcus mutans isolated from
patients. One loop (oese) of Streptococcus mutans was taken from culture of tryptone
yeast cysteine (TYC) and added into a liquid medium of Brain Heart Infusion Broth
(BHIB) and incubated in anaerobic jar at 37°C for 24 h. BHIB that had contained the
bacteria (Streptococcus mutans) turbidity visually comparable with the standard Mc
Farland 1. In case of similarity, bacterial cell count in 1 ml of culture medium was
estimated to be 3 X 108.
2. Determination of the MIC and MBC

Antibacterial activity of guava leaf extracts against Streptococcus mutans could be
determined by conducting the MIC and MBC tests. MIC was the lowest concentration
of an antimicrobial capable of inhibiting bacterial growth in their standard state. The
MIC of an antimicrobial could be used to determine bacterial sensitivity to
antimicrobials. 12 tube set consists of 9 treatment tube and 2 tubes of control. Each
tube is filled media treatment of bacterial growth (BHIB) of 5ml. 1 tube of a negative
control, a positive control. Positive control is a tube containing BHIB media plus
Streptococcus mutans and a negative control tube containing BHIB media.
Manufacture of 100% concentration, by weighing 5 grams of guava extract (paste
form) in 5 ml BHIB, then performed with serial dilution method (Serial Dilution
Method) so obtained concentration, 50%, 25%, 12.5%, 6.25% , 3,%, 1.56%, 0.78%,
92
0.39%, after dilution series, incubated in anaerobic jar at 37 ° C for 24 hours.

Subsequent to incubation, each treatment and control was inoculated into TYC agar
plates in order to observe bacterial growth. Plates were re-incubated anaerobically at
37°C for 2 X 24 h.
Cytotoxic testing of guava leaf extracts

Cytotoxic tests of the ethanol extract of guava leaves were performed at the Stem Cell
Laboratory of Tropical Disease Institute of Airlangga University by using method of
MTT assay.
Mesenchymal stem cells were used in the tests due to their high sensitivity to toxic
agents. Harvest was carried out by removing the cells out of the CO 2 incubator when
the cells were under standard condition (80% confluence) to be harvested. Cell harvest
was conducted in accordance with the cell harvest protocol.
The harvested cells were then washed 5 times with PBS in order to remove residual
serum. Cell cultures were divided into 96-well tissue culture (TS) plate according to
the number of samples and controls. Each well plate was filled with 50.000 cells. Cell
dilutions were performed by using the RPMI culture medium as required in
accordance with cell count protocol. Of the 53 well plates used, 50 were for treatment
and 3 for control. Controls used were those for cells, Phosphate Buffer Saline (PBS)
and cell media. Cells fulfilling the standards were transferred into their respective
wells and incubated for 12 h.
Concentrations of ethanol extract of guava leaves used were 12.5%, 6.25%, and
3.125%. A 50 μL of each concentration was put into the well plate and added with 150
μL of cell medium (RPMI). The plate was incubated in a CO 2 incubator at 37°C for 24
to 48 h. Subsequently, medium was removed and cells were washed with PBS once.
Each well plate was added with 25 μL of MTT reagent (5 mg/mL) per well plate
including media controls (without cells). Plates were re-incubated for 2 to 4 h in a
CO2 incubator. If formazan was formed clearly, the absolute stopper DMSO was
added to the well plate. Plate was wrapped with aluminum foils and placed in the dark
room at 37°C for 12 h.
After 12 hours, the cover of the plate was opened and the cell absorbance was read by
means of ELISA READER (λ = 595 nm). To determine percentage of viable cells,
data derived from ELISA READER was converted by using formula:
% Viable Cells : (absorbance of treated cells – media control absorbance ) X 100%

( solvent control absorbance – media control absorbance)
The formula was used if the absorbance of the solvent control was lower than that of
cell control.9
Data Analysis
The collected data were tabulated and statistical tests performed using the SPSS Data
Editor for Windows 17.00 . The test for normally distribution use the Kolmogorof-
93
Smirnov test with a significance level of 5% and then analyzed by one-way ANOVA
and Tukey HSD (significance level = 0.005)
RESULTS
Results of MIC and MBC tests showed that the 6.25% concentration of the guava leaf
extract was capable of inhibiting the growth of Streptococcus mutans. The bacteria
were killed when the 12.5% concentration of the extract was administered.
Table 1: Data on the growth of Streptococcus mutans colonies at each

concentration of the guava leaf extract
Concentration of guava leaves extract
Replicatio 100 50 25 12.5 6.25 3.125 1.56 0.78 0.39
n % % % % % % % % %
I - - - - - + + + +
II - - - - - + + + +
III - - - - - + + + +
IV - - - - - + + + +
V - - - - + + + +
+: Bacterial growth present
-: Bacterial growth not present
The 6.25% concentration of guava leaves extract of could inhibit the growth of
Streptococcus mutans on agar medium (TYC), while the 12.5% concentration was the
lowest concentration bactericidal to Streptococcus mutans in vitro. The results of disk
diffusion guava leaves extracts showed that the presence of anti-bacterial
effectiveness at a concentration 6.25%, 12.5%, 25%, 50%, 100%. (data not shown)
Cytotoxic test results guava leaves extract to stem cells mecensimal

Tabel 2.1 : The result of control cell (Mecensimal stem cells) and media control
absorbance
Cell control Mean of Cell Media Control Mean of Media Cell control
absorbance control Absorbance Control absorbance minus
absorbance absorbance media control
absorbance
1.807 1.401
1.822 1.425
1.812 1.820 1.415 1.414 0.406
1.825 1.420
1.824 1.413
94
Tabel 2.2 : The cytotoxicity result of guava leaves extract (Psidium Guajava)
Consentratio Absorbance* Mean of cell absorbance % viable cells

n (%)
12,5% 1.715 1.733 1.703 1.716 1.713 81.4 %

1.716
6,25% 1.763 1,759 1.766 1.765 1.773 88.4 %

1.742
3,125% 1.792 1.780 1,7793 1.781 1.790 1.787 100.5 %

*5 replication
Tabel 2.3 : Solvent control
Solvent control Mean

absorbance
1.783
1.791
1.785
1.781
1.780
1.792
Tabel 2.4 : Significance test differences between groups using one way Annova
test
Consentration Consentrasi Consentration Cell Media Solvent
12.5% 6.25% 3.125% Control Control control
Consentration - 0.456 0.001* 0.000* 0.000* 0.001*
12.5%
Consentration - 0.044 0.000* 0.000* 0.058
6.25%
Consentration - 0.233 0.000* 1.000
3.125%
- 0.000* 0.187
Cell Control
Media - 0.000*
Control
Solvent -
Control
* Significant level at 0.005
95
The results of cytotoxic test using the MTT assay method showed that the percentage
concentration 12.5% of guava leaves extract reaching 81.4% of living cells. At a
concentration of 6.25% indicates the percentage of living cells by 88.4% while the
concentration of 3.125% indicates the percentage of living cells by 100.5%.
ANOVA test showed significant differences in several groups:
1. Group concentration of 12.5% to 3125% concentration
2. Group of cell Control to consentration 12.5%
3. Cell control to 6.25% concentration group
4. Media Control group to concentration 12.5%
5. Media control to the control group 6.25%
6. Media control to the concentration of 3125%
7. Media control to cell control
8. Solvent control to concentration 12.5%
9. Solvent control to media control
DISCUSSION
Antibacterial activity of ethanol extract of guava leaves (Psidium guajava) was
determined by observing the growth of colonies on TYC plates. Growth of
Streptococcus mutans was characterized by formation of colonies on TYC agar plates.
Colonies of Streptococci mutans had characteristics of 15 mm in diameter, rough
surface with a hard adhesive consistency, opaque in color (yellowish white),
irregularly oval rounded edges, and hemolytic on blood agar. 10
Guava (Psidium guajava L.) has varieties of white flesh and red flesh, among others.
Guava leaves contain tannins (9.1%), alkaloids (6%), phenyl propanoate (2.2%),
flavonoids (2%), essential oils (2.05%) and malic acid (1.88%). Most of the
therapeutic activities of guava are affected by flavonoids. Flavonoids have been
shown to have antibacterial activities. Quercetin is thought to contribute to the anti-
diarrheal effects of guava, has an ability to relax intestinal smooth muscles and inhibit
intestinal contractions. In addition, other flavonoids and triterpenes of guava leaves
show antispasmodic activities.11 Essential oils contained in extract of guava may
leaves also inhibit bacterial growth or kill them by disrupting formation processes of
membranes or cell walls, leading to an absent or defective formation of them. 12
Results of in vitro studies using serial dilution method indicated that the minimum
concentration of ethanol extract of guava leaves capable of inhibiting the growth of
Streptococcus mutans was 6.25%. The concentration was used for cytotoxic tests by
MTT assay. Concentrations of 12.5%, 6.25%, and 3.125% were used to assess toxic
effects of ethanol extract of guava leaves.
96
Culture of mesenchymal stem cells was used for cytotoxic tests of ethanol extract of
guava leaves. Cytotoxic tests were performed by inoculating the guava leaf extracts
into culture of mesenchymal stem cells and, after 24 hours, evaluating percentage of
viable cells in the culture by MTT assay. MTT assay was a parameter used as the
basis for colorimetric studies of the metabolic activity of viable cells. For example,
the microtiter plate using MTT tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] is widely used to measure the quantity of cell
proliferation and cytotoxicity. Yellow MTT is converted into purple formazan by a
mitochondrial enzyme dehydrogenase within viable cells.
MTT assay is a method for determining cell viability by colorimetric assessment of
viable cells‘ metabolic activity. Results of experimental research on cytotoxic effects
of guava leaf extract on mesenchymal cell cultures are obtained quantitatively by
looking at percentage of viable cells in the cultures. Percentage of viable cells can be
detected by optical density measurements of mesenchymal stem cell cultures in each
group. Quantitative examination using MTT assay is based on viable cells‘ ability to
reduce yellow MTT salts into purple tetrazolium salt formazan. Production of
formazan can be determined by measuring the optical density of the resulting solution.
Purplish reaction is used as a measure of viable cell count. The more intense the
purplish color is, the higher value of absorbance will be, showing an increasing count
of viable cells.13
Results of cytotoxic studies of guava leaf extract indicated that concentration of
12.5% did not cause toxic effects on mesenchymal stem cells. Percentage of viable
cells at concentration of 12.5% was 81.4%. toxic properties of the extract to sell new
mesensimal known if the percentage of living cells is less than 50%.
MTT assay as a method for determining viability of mesenchymal stem cells been
widely applied.14 Toxicity of administration of guava leaf extract on mesenchymal
stem cells was determined by comparing their viability with controls considered as
having 100% viability. Data obtained from the readings of ELISA READER were
converted by a formula to obtain the percentage of viable cells. 15,16
CONCLUSION
Ethanol extracts of guava leaves inhibited growth of the dominant caries-
causing bacteria, Streptococcus mutans, at the lowest concentration of 6.25% and
killed the life cycle of Streptococcus mutans at the minimum bactericidal
concentration of 12.5%. Anti bacterial activity of guajava leaves extract show
consentration at 12.5% can kills Streptococcus mutans. Cytotoxic tests showed no
toxic effect of concentrations of 12.5%, 6.25% and 3.125% of guava leaves ethanol
extracts on mesenchymal stem cells.
REFERENCES
1. Goncalves, flaviae.(2008) Antibacterial Activity Of Guava, Psidium Guajava

Linnaeus, Leaf Extracts On Diarrhea-Causing Enteric Bacteria Isolated From
Seabob Shrimp, Xiphopenaeus Kroyeri (Heller). Rev. Inst. Med. trop. S. Paulo
50(1):11-15,
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2. CÁCERES, A.; FLETES, L.; AGUILAR, L. et al. - Plants used in Guatemala

for the treatment of gastrointestinal disorders. Confirmation of activity against
enterobacteria of 16 plants.In : Goncalves, flaviae.(2008) Antibacterial
Activity Of Guava, Psidium Guajava Linnaeus, Leaf Extracts On Diarrhea-
Causing Enteric Bacteria Isolated From Seabob Shrimp, Xiphopenaeus Kroyeri
(Heller). Rev. Inst. Med. trop. S. Paulo 50(1):11-15
3. Goncalves, flaviae.(2008) Antibacterial Activity Of Guava, Psidium Guajava
Linnaeus, Leaf Extracts On Diarrhea-Causing Enteric Bacteria Isolated From
Seabob Shrimp, Xiphopenaeus Kroyeri (Heller). Rev. Inst. Med. trop. S. Paulo
50(1):11-15
4. http://sp2010.bps.go.id. Accesed April,28 2012
5. Sinaga,Lusiana. 2008 Uji Antimikrobial Ekstrak Metanol Daun Jambu Biji
Daging Putih Dan Jambu Biji Daging Merah (Psidium Guajava L.) Terhadap
Beberapa Spesies Bakteri Patogen.Skripsi, Univesitas Sumatra utara
6. Ojewole.(2006) Antiinflammatory and analgesic effects of Psidium guajava
Linn. (Myrtaceae) leaf aqueous extract in rats and mice. Methods Find Exp
Clin Pharmacol. Sep;28(7):441-6.
7. Adnyana K,Yulinah E, Sigit J,fisheri K, insanu M. 2004 Efek ekstrak Daun
Jambu biji daging buah putih dan daun jambu biji daging buah merah sebagai
anti diare.Acta Pharmaceutica indonesia, Vol.29
8. Sherris,John C.(1984) Medical microbiology an introduction to infectious
disese.Oxford:Elsevier
9. Junedi, sendy. Prosedur Tetap Uji Sitotoksik Metode MTT. Cancer
Chemoprevention Research Center,Fakultas Farmasi UGM.
10. Cappucino, james G, natalie s.(2001) Microbiology. 6th edition. San fransisco:a
laboratory manual
11. http://www.rain-tree.com/plants.Accesed April 29, 2012 [online]
12. Ajizah, A. 2004. Sensitivitas Salmonella typhimurium Terhadap Ekstrak Daun
Psidium guajava.In : Sinaga,Lusiana. 2008 Uji Antimikrobial Ekstrak Metanol
Daun Jambu Biji Daging Putih Dan Jambu Biji Daging Merah (Psidium
Guajava L.) Terhadap Beberapa Spesies Bakteri Patogen.Skripsi, Univesitas
Sumatra utara
13. Sananta, panji.(2010) Uji Toksisitas Fresh Frozen tendon graft dan Freeze
dried tendongraft terhadap mesensimal stem sell. Tesis, Universitas Airlangga
14. Ahmadian S,Barar & Amir,omidi.(2009) Cellular toxicity of nano
genomedicine in MCF-7 cell line:MTT Assay. Journal of visualized
Experiment 1191
15. Shetty SS, Kaushik S.(2006) Viability testing of homograf valves using methyl
thiazol tetrazolium assay.JPGM volume 42
16. Vannet BV & hansens JL. (2007) The use of three dimensional oral mukosa
cell cultures to asses toxicity of soldered and welded wires.The european
Journal of Orthodontics
98
Biofilm Formation Ability and Effect of

Immunoglobulin-Y on Streptococcus mutans Protein
Expression
Rizky Aditiya Irwandi a, Rachel E. Anastasya a, Sonia Margareth a,
Felicia Paramita a, Putri Annika a, Endang Winiati Bachtiar b,*,
Mindya Yuniastuti b
a
Dental Student, Faculty of Dentistry, University of Indonesia, Jakarta 10430,
Indonesia
b
Department of Oral Biology, Faculty of Dentistry, University of Indonesia, Jakarta
10430, Indonesia
*
Corresponding author at: Faculty of Dentistry, University of Indonesia, Jakarta 11430,
Indonesia. Tel.: +62813 1957 1866
E-mail address: endang04@ui.ac.id
ABSTRACT
Objective: The aim of this study was to evaluate the correlation between biofilm
formation ability and the effect of IgY anti Streptococcus mutans (S.mutans) on 40 kDa
S.mutans protein isolated from caries and caries-free subjects. Methods: Plaque
samples were obtained from caries and caries-free subjects. Plaque samples were
cultured on TYS20B for three days. The selected colonies were then picked and
cultured on tryptic soy broth for three days. After colony counting, biofilm assay was
conducted and inoculated for one day. The biofilm was tested using crystal violet
binding assay and quantified by measuring the absorbance at 655 nm optical density.
Meanwhile, each collected bacteria (whether from caries or caries-free subjects) were
grouped into control and exposure group. In the exposure group, S.mutans was exposed
to a pre-incubated IgY anti S.mutans for one hour at 37°C. Protein expression of
S.mutans was analyzed with SDS PAGE after the preparation of its antigen and
Bradford protein assay. Result: There was slightly increased biofilm formation in caries
subject compared to free-caries subject (p = 0,950). In addition, the expression of 40
kDa protein of S.mutans in caries subject was increased after the incubation of IgY anti
S.mutans. Conclusion: It can be suggested that the increased in biofilm formation
ability is in line with the increased of 40 kDa protein after exposure to IgY anti
S.mutans in caries.
Keywords: Streptococcus mutans, biofilm formation ability, 40 kDa protein,
immunoglobulin-Y anti S.mutans
99
1. INTRODUCTION AND LITERATURE REVIEW

Dental caries is one of the most widespread disease in human1, which is caused by loss
of mineral ion from the enamel or root surface of the tooth that is stimulated by certain
bacterial flora and their metabolic products.2 Streptococcus mutans (S.mutans) is an
important microbial agent in the pathogenesis of dental caries.3 However, S.mutans is
widely distributed not only in populations with high caries prevalences, but also in
populations with low or even no caries experiences. The virulence factors of S.mutans
may be involved in subjects with low caries experience.4 These virulence factors are
associated with S.mutans cariogenicity including adhesion, acidogenicity, and acid
tolerance.5, 6 One of the virulence factors that can induce dental caries is protein antigen
located on the cell surface, as these proteins involved in S.mutans pathogenicity.7
Anastasya previously reported that protein expression of S.mutans isolated from dental
caries and caries-free subjects was different.8
Biofilm is a microbial community found on the tooth surface embedded in extracellular
matrix of polymers of bacterial and salivary origin.9 According to ecological plaque
hypothesis, the dynamic of environmental factor will trigger a shift in the balance of the
plaque microflora and promote the emergence of more cariogenic bacteria followed by
dental demineralization.10 A point to understand how S.mutans colonies form in the oral
cavity concerns the mechanisms of how molecular components on the bacterial cell-
surface interact with acquired dental pellicle. S.mutans possesses cell surface
substances, including antigen I/II (AgI/II), glucosyltransferase (gtf), and glucan-binding
protein (gbp). These cell-surface molecules are thought to play important roles in the
interaction between organism and its host, and have been given much attention as
vaccine candidates against dental caries.11 Sucrose-independent adherence is thought to
be influenced by Antigen I/II, a 185 kDa surface protein whereas the sucrose-dependent
adhesion is influenced by the action of gtf. The glucan-binding proteins are served as
the receptor for glucans synthesized by gtf.5
Immunoglobulin-Y (IgY) is an antibody largely found in chicken eggs. The use of
chicken egg yolk as a source for antibody production results in a reduction of animal
use, as chicken produces larger amounts of antibodies than laboratory rodents.12 The
success of IgY anti S.mutans as an agent of passive immunization in eliminating
S.mutans has been previously reported. Otake et al. reported that specific pathogen-free
rats infected with S.mutans MT8148 (c) and fed with a cariogenic powder containing
more than 2% IgY anti S.mutans developed significantly lower caries scores in
comparison to rats infected with the same strain and fed with a diet containing IgY
obtained from non-immunized hens.13 Then almost two decade later, Anggraeni
reported that swabbed gel containing IgY anti S.mutans to rats‘ teeth could reduce
biofilm formation of S.mutans. The studies were not only conducted in animal, but also
in human using mouthwash and toothpaste containing IgY anti S.mutans.14 Hatta et al.
reported that in the short-term (4-hours) test using a mouthwash containing 10%
sucrose, IgY anti S.mutans decreased the percentage ratio of S.mutans in saliva.15
Meanwhile, Paau et al. reported that experimental group in which using toothpaste
containing IgY anti S.mutans twice a day showed significant percentage reduction of
S.mutans level in saliva and plaque in comparison to the control group that was using
conventional toothpaste.16_ENREF_16_ENREF_16_ENREF_16
100
Since S.mutans possesses proteins on cell wall that construct biofilm, its protein
expression that represents the virulence factors was apparently different between
S.mutans isolated from caries and caries-free subjects. Moreover, the efficacy of IgY
anti S.mutans in reducing dental caries development by eliminating S.mutans factor was
clinically proven. Therefore it would be useful to evaluate the correlation between
biofilm formation ability and the effect of IgY anti S.mutans on protein expression of
S.mutans isolated from caries and caries-free subjects.
2. MATERIAL AND METHODS

This study was approved by the Research Ethical Committee of Faculty of Dentistry,
University of Indonesia No. 51/Ethical Clearance/FKGUI/XI/2011 (Fakultas
Kedokteran Gigi Universitas Indonesia).
2.1 Subjects
This study was conducted on 30 volunteers in both sexes between 17 and 23 years of
age. The inclusion criteria were good oral health, has no extensive caries in the first
mandibular molar (buccal surface remains undamaged), Decay Missing Filled Teeth
(DMFT) score are 4-12 for 15 caries subjects and 0 for 15 caries-free subjects, capable
of giving informed consent. The exclusion criteria were no in possession of the first
mandibular molar, is currently using fixed orthodontic appliances, consuming
medication that reduces immune reaction and has oral manifestation of systemic
disease. All subjects were asked to brush their teeth in the morning and not allowed to
drink and eat at least 3 hours before sampling.
2.2 Sampling and bacterial culture procedure

Each dental plaque was collected by swabbing the buccal surface of the first lower
permanent molar of caries and caries-free subjects. The swabbed dental plaque was
mixed in 1 mL sterile phosphate buffer saline (PBS) solution. 10 µL of the solution was
then cultured at 37°C for three days on TYS20B agar in anaerobic jar containing NO2,
CO2, and H2O. From TYS20B cultures, 3-5 colonies of S.mutans were picked for
further cultures in a 5 mL of tryptic soy broth and incubated for another three days as
above.
2.3 Colony counting and biofilm formation assay by crystal binding violet
The counting of bacteria in tryptic soy broth was conducted using microplate reader by
measuring absorbance at 655 nm optical density and the value of Optical Density (OD)
were convert to Colony Forming Unit (CFU). Bacteria were diluted up to 10 6 CFU/ml
priors to use. Biofilm formation was assayed using a flat bottom 96-well plate
polystyrene (Costar 3595; Corning Inc., Corning, NY). Briefly, 200 μl of 106 CFU/ml
bacterial suspensions were added into 96-well plate. After inoculation, the plates were
incubated at 37oC for 24 hours. The culture medium was then discarded and the plates
were gently washed twice with 200 μL of sterile PBS to remove planktonic and loosely
bound cells. The adherent bacteria were stained with 100 μL of 0.1% crystal violet for
15 min. After rinsing once with 200 μL of sterile PBS, the bound dye was extracted
from the stained cells using 200 μL of 99% ethanol. Biofilm formation was then
101
quantified by measuring the absorbance of the solution at 655 nm in a

spectrophotometer.
2.4 Exposure of IgY anti S.mutans

Each cultured bacteria in tryptic soy broth whether from caries or caries-free subjects
was grouped into control and exposure groups after its concentration had been adjusted
to 109 CFU/mL. In the exposure group, 5 mL of S.mutans 109 CFU/mL was exposed to
1 mL pre-incubated IgY anti S.mutans 120 µg/mL for 1 h at 37°C. On the other hand, 5
ml of S.mutans 109 CFU/ml without any exposure was prepared as the control group.
2.5 Antigen preparation of S.mutans and protein expression analysis

Exposed S.mutans isolated from both caries and caries-free subjects were collected by
centrifugation (13000 rpm, 1 min). Each of the collected pellets was washed once with
200 µL of PBS and homogenized by ultrasonic homogenizer (Omni-Ruptor 250, OMNI
International Inc.), six cycles (one cycle was conducted alternately with 30 sec of pulse
period and 30 sec of rest period). Protein expression of S.mutans was analyzed with
sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) after antigen
protein concentration of the homogenized samples had been minimally adjusted to 2000
µg/mL with Bradford protein assay.
2.6 Data Analysis

The data of OD from biofilm assay that define biofilm formation ability between
S.mutans isolated from caries and caries-free subject was analyzed using non-parametric
statistical analyses. Saphiro-Wilk normality test was the first to be performed, and
showed non-normal distribution of the data. Thus, Kruskal-Wallis test was conducted to
test for significance. The critical value for statistical significance was considered to be p
< 0.05.
The data of band appearance frequency from SDS PAGE that define protein expression
of S.mutans was analyzed by comparing the control group and the exposure group.
Correlation of the effect of IgY anti S.mutans on biofilm formation ability and the
protein expression were evaluated in caries and caries-free subjects.
3. RESULTS
3.1 Biofilm formation ability
Biofilm formation ability between caries subject and caries-free subjects indicated by
the OD was shown in table 1. Mean OD was different between caries-free subject and
caries subject. However, the difference was not statistically significant.
Table 1 - Mean optical density (OD) from biofilm assay

Group N Mean±SD
Caries-free subjects 15 0.1696±0.1426
Caries subjects 15 0.2024±0.1573
p = 0.950*
102
*Statistically not significant difference (p>0.05) between mean OD of caries-free

subjects versus caries subjects.
3.2 Effect of IgY anti S.mutans on protein expression of S.mutans

The result of our study showed that proteins band of around 41.3 (± 2) kDa were
predominantly emerged (Fig. 1). There are S.mutans ± 41.3 kDa protein expressions
from 8 out of 15 samples in the control group of caries subjects and 10 out of 15
samples in the control group of caries-free subjects. In the exposure group of caries
subjects, there are S.mutans ± 41.3 kDa protein expressions from 12 out of 15 samples
whereas in the exposure group of caries-free subjects, there are S.mutans ± 41.3 kDa
protein expressions from 9 out of 15 samples (Fig. 2).
A B
Fig. 1 –Coomassie blue stained gel (A). Protein profile of S.mutans identified as
molecular weight from Gel-doc Bio-Rad (B). Lane 1: Caries-free subject (control),
Lane 2: Caries subject with IgY anti S.mutans, Lane 3: Marker, Lane 4: Caries-
free subject with IgY anti S.mutans, Lane 5: Caries subject (control).
103
Fig. 2 - Effect of IgY anti S.mutans on S.mutans ± 41.3 kDa protein expression.
Black bars, caries subjects; Grey bars, caries-free subjects.
4. DISCUSSION
4.1 Biofilm formation ability
A number of investigations have been carried out, comparing S.mutans isolated from
caries and caries-free subjects.4, 17-21 These result of our study shows that there is a
difference in biofilm formation ability of S.mutans isolated from caries and caries-free
subjects although it was not statistically significant. A study by Khoo et al. (2005)
showed that S.mutans isolated from the caries subjects produced more water-insoluble
glucans than the caries-free subjects. Water-insoluble glucans is the primary
extracellular matrix component of dental plaque.17
Other study by Napimoga et al. (2004) also showed that there is a difference between
water-insoluble glucans production of S.mutans isolated from caries and caries-free
subjects although the adherence to the glass surface was not significant. The study also
observed a statistically significant positive correlation between the level of water-
insoluble glucans synthesis and the high proportion of adherent cells in the presence of
sucrose in caries-active subjects but not in caries-free subjects.4 This suggests that
S.mutans isolated from the caries subjects have more ability to colonize and to
accumulate on teeth and consequently induce caries. However, a single genotype of
caries-free subjects S.mutans showed low synthesis of water-insoluble glucans but high
adherence.4 This may cause the statistical insignificance in biofilm forming ability test
in our study.
Anastasya also reported that in S.mutans, the expression of glucan-binding protein
(Gbp) C playing role in the initiation of biofilm formation and this was statistically
different between caries subjects and caries-free subject. S.mutans isolated from caries
104
subjects are likely to express GbpC five times higher than S.mutans isolated from
caries-free subjects.8
4.2 Effect of IgY anti S.mutans on protein expression of S.mutans

The result of this study suggests that change in protein expression of S.mutans are
caused by the effect of IgY anti S.mutans, especially a protein of around 41.3 kDa. One
distinct protein that is secreted by S.mutans is the glucan binding protein B (GbpB). It is
more immunogenic than another Gbp. Saliva of young children often contains
Immunoglobulin-A (IgA) antibody to S.mutans GbpB. Furthermore, active and passive
antibody to S. mutans GbpB could have had a protective effect against cariogenic S.
mutansinfection and disease.22 Mattos-Granner et al. reported that a calculated
molecular weight of 41.3 kDa is the GbpB protein.1
GbpB protein expression of the exposure group in caries subjects was up-regulated
compared to the control group. However GbpB protein expression of exposure group in
caries-free subjects was down-regulated. This present study provides an information
that there is a different response to IgY anti S.mutans in S.mutans isolated from dental
caries and caries-free subjects, especially in expression of GbpB. Smith et al.
demonstrated that IgY anti S.mutans GBP-B developed reduction of S.mutans colonies
and caries index relative to the control.22 In the present study, GbpB expression in the
exposure group of caries-free subjects is down-regulated compared to the control group.
According to Smith et al., down-regulation of GbpB expression can cause the reduction
of S.mutans colonies. This is consistent with the role of GbpB in growth and
construction of cell wall of S.mutans.23
However, in the exposure group of caries subjects, GbpB protein expressions were up-
regulated compared to the control group. This is probably because of the IgY anti
S.mutans that is derived from whole cell mutans or not GbpB specific, which could
have caused the IgY anti S.mutans to binds with another protein epitop.
The result suggests that biofilm formation ability of S.mutans isolated from caries
subjects is higher than those isolated from caries-free subjects. On the other hand, the
expression of around 41.3 kDa protein in S.mutans isolated from caries subjects is
increased after the exposure of IgY anti S.mutans compared to the control group. In
addition in caries-free subject, the expression of around 41.3 kDa protein is decreased
compared to the control group. The result are summarized in table 2.
Table 2 – Result Summary of biofilm formation ability and effect of IgY anti
S.mutans
Result Caries-free subjects Caries subjects
Biofilm formation ability 0.1696±0.1426 0.2024±0.1573

(OD)
Effect of IgY anti S.mutans
(frequency of expression)
105
 Control group 10 8
 Exposure group 9 (↓) 12 (↑)
This study also found that the ability of GbpB in S.mutans is different between caries
subjects and caries-free subjects. The ability of GbpB in S.mutans isolated from caries
subjects is higher than in S.mutans isolated from caries free subjects, since our
investigation shows that in the control group, frequency of GbpB expression in
S.mutans isolated in caries subjects is more frequent than those isolated from caries-free
subjects. Moreover, the exposure of IgY anti S.mutans confirms that the ability of GbpB
in S.mutans isolated from caries subjects is higher than those isolated from caries-free
subjects and it is shown in our investigation that the expression of GbpB in S.mutans
isolated from caries-free subjects was decreased whereas in caries subjects, it was
increased compared to the control group.
5. CONCLUSION
It can be concluded that increased biofilm formation ability is in line with increased 40
kDa protein after exposure of IgY anti S. mutans in caries.
6. ACKNOWLEDGEMENT
The author would like to thank Prof. drg. Boy M. Bachtiar, M.S, PhD and Dr. drg.
Anggraeni, SpKG for their valuable assistance in this study.
REFERENCES
1. Smith DJ. Dental Caries Vaccines: Prospects and Concerns. Crit Rev Oral Biol
Med. 2002;13(4):335-49.
2. McIntyre JM. Dental Caries - The Major Cause of Tooth Damage. In: Mount GJ,
Hume WR, editors. Preservation and Restoration of Tooth Structure. 2 ed.
Queensland: Knowledge Books and Software; 2005. p. 21-33.
3. Bowen WH. Vaccine Against Dental Caries - A Personal View. J Dent Res.
1996;75(8):1530-3.
4. Napimoga MH, Kamiya RU, Rosa RT, Rosa EAR, Hofling JF, Mattos-Graner
RdO, et al. Genotypic Diversity and Virulence Traits of Streptococcus mutans in
Caries-Free and Caries Active Individuals. J Med Microbiol. 2004;53:697-703.
5. Banas JA. Virulence Properties of Streptococcus mutans. Front Biosci.
2004;9:1267-77.
6. Napimoga MH, Hofling JF, Klein MI, Kamiya RU, Goncalves RB.
Transmission, Diversity, and Virulence Factors of Streptococcus mutans
Genotypes. J Oral Sci. 2005;47(2):59-64.
106
7. Matsumoto-Nakano M, Fujita K, Ooshima T. Comparison of Glucan-Binding

Proteins in Cariogenicity of Streptococcus mutans. Oral Microbiol Immunol.
2007;22:30-5.
8. Anastasya RE. Analisis Profil Protein Streptococcus mutans yang Diisolasi dari
Subjek Karies dan Bebas Karies. Jakarta: Universitas Indonesia; 2011.
9. Marsh PD, Martin MV. Dental Plaque. Oral Microbiology. 5 ed. Edinburgh:
Elsevier; 2009. p. 75-102.
10. Marsh PD, Martin MV. Plaque-mediated Disease - Dental Caries and
Periodontal Disease. Oral Microbiology. Edinburgh: Elsevier; 2009. p. 104-17.
11. Kruger C. Passive Immunization Against Oral Pathogens. Stockholm:
Karolinska Institutet; 2004.
12. Carlender D. Avian IgY Antibody: In vitro and In vivo: Upsala University;
2002.
13. Otake S, Nishihara Y, Makimura M, Hatta H, Kim M, Yamamoto T. Protection
of Rats Against Dental Caries by Passive Immunization with Henn-egg-yolk
Antibody (IgY). J Dent Res. 1991;70(3):162-6.
14. Anggraeni. Prospek Gel Imunoglobulin-Y Anti Streptococcus mutans Sebagai
Bahan Imunisasi Pasif Anti Karies: Analisis Sifat Biologis Gel Imunoglobulin-Y
Anti Streptococcus mutans dan Pengaruhnya terhadap Proses Karies pada Tikus
Sprague Dawley. Jakarta: University of Indonesia; 2010.
15. Hatta H, Tsuda K, Ozeki M, Kim M, Otake S, Hirasawa M. Passive
Immunization Against Dental Plaque Formation in Humans: Effect of a Mouth
Rinse Containing Egg Yolk Antibodies (IgY) Specific to Streptococcus mutans.
Caries Res. 1997;31(4):268-74.
16. Paau S, Yang R-J, inventors; Preparation Method of IgY for Preventing and
Cure Mouth Disease and the Toothpaste Base on the IgY. US patent
0198849A1. 2006.
17. Khoo G, Zhan L, Hoover C, Featherstone JDB. Cariogenic Virulence
Characteristics of Mutans Streptococci Isolated from Caries-Active and Caries-
Free Adults. CDA Journal. 2005;33(12):973-80.
18. Doifode D, Damle SG. Comparison of Salivary IgA Levels in Caries-Free and
Caries-Active Children. Int J Clin Dent Sci. 2011;2(1):10-4.
19. Cowman RA, Schaefer SJ, Fitzgerald RJ, Rosner D, Shklair IL, Walter RG.
Differential Utilization of Proteins in Saliva from Caries-active and Caries-free
Subjects as Growth Substrates by Plaque-forming Streptococci. J Dent Res.
1979;58(10):2019-27.
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20. Pieralisi FJS, Rodrigues MR, Segura VG, Maciel SM, Ferreira FBA, Garcia JE,
et al. Genotypic Diversity of Streptococcus mutans in Caries-Free and Caries-
Active Preschool Children. Int J Dent. 2009;2010:1-5.
21. Walter RG, Shklair IL. Streptococcus mutans in Caries-free and Caries-active
Naval Recruits. J Dent Res. 1982;61(11):1229-32.
22. Smith DJ. Passive Transfer of Immunoglobulin-Y Antibody to Streptococcus
mutans Glucan-binding Protein B Can Confer Protection Against Experimental
Dental Caries. Infect Immun. 2001;69(5):3135-42.
23. Fujita K, Takashima Y, Inagaki S, Nagayama K, Nomura R, Ardin AC.
Correlation of Biological Properties with Glucan-binding Protein B Expression
Profile in Streptococcus mutans Clinical Isolates. Arch Oral Biol. 2011;56:258-
63.
108
Combined Effects of Curcuma Xanthorrhiza Extract

With Ketoconazole Against C. Albicans in HIV/AIDS
Patients
K.N. Firsty1, D. M. Larasati1, A. Rahmadi1, R. P. Rahayu2
1
Undergraduated Student of Faculty of Dentistry, Airlangga University Indonesia
2
Lecturer of Oral Biology Department, Faculty of Dentistry, Airlangga University Indonesia
ABSTRACT
Background: C. albicans is one of the manifestations of infection are most commonly
found in HIV/AIDS patients. Candidiasis caused by C. albicans can be overcome by
various types of anti-fungal. Recently, there are many cases of C. albicans‘s resistance
to the antifungal like ketoconazole. In several studies, the effectiveness of ketoconazole
is weaker than the other. Xanthorrhizol, isolated from methanol extract of Curcuma
xanthorrhiza, was investigated for its antifungal activity using C. albicans. This study is
to find out the effectiveness of combination of ketoconazole and Curcuma Xanthorrhiza
extract (xanthorrhizol), for oral candidiasis therapy. Objective: To study the combined
effect of xanthorrhizol with ketoconazole against C. albicans in the oral mucosal non
ARV HIV/AIDS patients. Methods: The study used a sample of C. albicans which has
been incubated in the Institute of Tropical Disease Airlangga University. C. albicans
was recultured and divided into 3 groups: single ketoconazole (Group 1), Curcuma
xanthorrhiza extract (Group 2), and combination (Group 3). In each group was
performed serial dilution. In this serial dilution process, ketoconazole diluted with initial
concentration of 4 mg/mL until 0.0625 mg/mL, whereas is given constant in each tube =
50%. Tubes were incubated for 24h and 370C. After that, the colony were cross-checked
and re-planted on SDA media. Serial dilution also conducted for Curcuma xanthorrhiza
extract by diluted 2mL-1.25 mL. MIC of the ketoconazole was 2 mg/mL and then were
combined with extract. Colonies‘ calculation was conducted and the data was analyzed
with SPSS. Results: From the calculation of the colony, we found the difference
between the number of colony in group 1, group 2 and group 3. Conclusion: It can be
concluded that administration of ketoconazole combination of Curcuma xanthorrhiza
extract-ketoconazole is more effective than single.
Keywords: ketoconazole, Curcuma xanthorrhiza, C. albicans, HIV/AIDS
109
INTRODUCTION
Candida albicans is an opportunistic fungal pathogen causing life-threatening mucosal
and systemic infections in immunocompromised humans. In healthy individuals,
Candida spp. have low pathogenicity; however, with depressed host immunity or when
changes occur in oral microbial flora, overgrowth can occur, resulting in oropharyngeal
candidiasis (also known as thrush) or denture stomatitis.1 Oropharyngeal candidiasis
(OPC) is the most frequent opportunistic fungal infection among human
immunodeficiency virus (HIV)-infected patients.2
This condition can be treated with either systemic or topical antifungal agents, such as
amphotericin B, fluconazole, flucytosine, nystatin, and ketoconazole. The topical
antifungal were tested according to the methodology of microdilution in the Clinical
and Laboratory Standards Institute (CLSI) results the values of minimal inhibitory
concentration (MIC).3
Indonesia face an increasing problem with HIV/AIDS given fact that 2682 AIDS
subjects were identified in 2004, while up to 16.110 cases have been reported at the end
of 2008. The HIV referral clinics are also faced with patients that often present in late
stage disease and with low CD4 cell counts (median CD4: 47/mm3; range 11-276). This
case indicates the barriers for HIV testing is high and/ or adequate testing facilities are
lacking. Indeed, in the present facilities, more than 50% of the HIV tests yield a positive
result.4
AIDS caused by retroviral virus that has an affinity for CD4-bearing T helper
lymphocytes that has been named Human Immunodeficiency Virus (HIV).5 T helper 17
(TH17) cells have well-described roles in autoimmune disease.6 Th17 cells and their
production of interleukin-17 (IL-17) play a pivotal role in mucosal host defense against
candidiasis in mice. In the lack of Th17 cells and their cytokines were critical for the
pathogenesis of mucosal candidiasis, one could speculate that in patients with a low
total CD4 count, such as in the low-CD4 syndrome, another rare primary
immunodeficiency, or in patients with the acquired immunodeficiency syndrome, the
lack of CD4 differentiation into Th17 cells is critical for maintaining the mucosal host
defense against candida.7 HIV invades the susceptible lymphocytes and renders it
nonfunctional, thereby interfering with a large number of important immunologic
functions. Such interference can result in opportunistic infections and in the occurrence
of a variety of normally rare neoplasms.5
Patients with clinical AIDS, left untreated, have a median survival of only 6.4 months.
With current treatment and the prompt and aggressive management of opportunistic
infection, the median survival is estimated at 3.8 years.5
110
The antifungal agents were effectively proved can reduce the growth and colonization
of Candida sp. Ketoconazole is the first drug of the azole class of agent capable of
achieving therapeutic blood levels when administrated orally. While ketoconazole is
used to treat immunocompromised patients, its adverse side effect, including nausea and
hepatotoxicity, has restricted its use. However, despite the toxicity, it shows several
adverse drugs interactions, disabling its use in association with some drugs, including
antiretrovirals (ARV).3 The other group azole, such as fluconazole, itraconazole, and
voriconazole were also have the same adverse effect, hepatotoxicity to the patients
treated by ARV. 8
The antifungal effect of essential oils of many plants and their specific anticandidal
activity are well established. Curcuma xanthorrhiza Roxb., commonly known as
Javanese turmeric, has been traditionally used in South-East Asian countries for food
and medicinal purposes. Xanthorrhizol, isolated from Curcuma xanthorrhiza, has been
reported to possess anticariogenic activity against Streptococcus mutans. It exhibited
antifungal activity against C. albicans, C. glabarata, C, guiliermondii, C. krusei, C.
parapsilosis and C. tropicalis.9
Rukayadi et al have found that xanthorrhizol has the ability to inhibit and kill C.
albicans.11 Xanthorrhizol may be potentially valuable as a natural compound for fungal
infection. However, the clinical applications of xanthorrhizol are used in the study to
interpret a lack of pharmacokinetics and safety studies. 9
The combination of ketoconazole with Curcuma xanthorrhiza extract may prove to be

a promising strategy for the therapy of disseminated candidiasis. Considering this
background, the aim of this study was to find out the effect of combination against C.
albicans in patients with HIV/AIDS.

Candida isolates
The study used a sample of Candida albicans which has been incubated in the Institute
of Tropical Disease (ITD) UNAIR, Surabaya, Indonesia. The C. albicans used in this
study were obtained from sputum of the patients with HIV/AIDS in Dr. Soetomo
Hospital. Stock of C. albicans was taken from refrigerator with -800C temperature. We
did a minute of vortex after 45 minutes in a room temperature. The C. albicans were
cultured in 2 tubes contains Sabouraud Dextrose Broth (SDB) for 24h and 370C.10 A
standardized inoculums (a McFarland standard) for each isolate was 5 x 106 colony-
forming units (cfu) /mL.11
111
Anticandidal agents
Xanthorrhizhol was isolated from methanol extract of Curcuma xanthorrhiza. Curcuma

xanthorrhiza was weighed, 110.741 g of it mixed with 400 mL methanol. Sonication
was conducted to obtain nano-particles, so that the Curcuma xanthorriza can be mixed
and easily filtrated. The methods above was repeated 3 times. After all was conducted
methanol evaporation. Ketoconazole was weighed, 4 mg/mL was dissolved in in 0,2
mL dimethylsulfoxide (DMSO) and 10 mL aquadest to obtain 100% of concentrations.
Single Dilution of Ketoconazole
Ketoconazole would be divided into 9 tubes. The first tube was filled 10 mL of
ketoconazole with 100% concentrations. The second until seventh tubes was filled with
5 mL of SDB. Single dilution were did from the first tube to the second tube and so
forth until the seveth tube to obtain the variation concentration. Every single tube
contain 5 mL liquid. The concentrations of ketoconazole in the tube 1, 2, 3, 4, 5, 6 and 7
were 100% - 1.5625% (4mg/mL-0.0625mg/mL). 0,1 ml of inoculums with McFarland
standard was 5 x 106 cfu/mL were isolated in every tube. The eight and ninth tubes
were used to be the control. Control positive contained SDB and inoculums, and control
negative contained only SDB. The tubes were incubated for 24h and 370C.10 Replanted
inoculums from the SDB to the Sabouraud Dextrose Agar (SDA) and re-incubated to
cross check the result and acquired the minimum inhibitory concentrations (MIC). MIC
is the lowest concentration with no visible growth of organism.11
Inhibition examination of Curcuma xanthorrhiza extract
The stock solution of Curcuma xanthorrhiza extract were divided into 6 tubes with 6
kinds of concentration, 100%, 90%, 80%, 70%, 60%, 50% (2.5mL-1.25mL) and 2 tubes
for control. Tube 2-6 were contained 2,5 mL Sabouraud Dextrose Broth and 0,1
inoculums. The seventh and eight tube used as control positive and negative. After
incubation, all inoculums were replanted in SDA and incubated again for 24h and 370C
to assessed potency of inhibition.
Inhibition examination of combination
Five type of Curcuma xanthorrhiza extract concentrations were loaded into 5 tubes, 2,5
mL for each tube. 2 mg/mL (MIC of Ketoconazole as a result of single dilution) were
used to be combined with extract. 5 mL Sabouraud Dextrosa Broth (SDB) as media and
0,1 inoculums were planted on each. The others 2 tubes for control, one contained SDB,
inoculums, and ketoconazole, another contained SDB and inoculums. The tubes were
incubated for 24h and 370C, then replanted on SDA and incubated for the same hours
and temperature until growth was seen in the growth control plates to determine the
numbers of the colonies.
112
RESULTS
The Curcuma xanthorrhiza Roxb. extract was active against C. albicans. Ketoconazole
and mixed or combination of both were also conducted to the C. albicans. Results
shows he MIC of the treatments in different numbers. Ketoconazole shows 50% of its
concentration as MIC (2 mg/mL), Curcuma xanthorrhiza extract shows MIC in 70%
(1.75 mL). Combination of both was conducted, 100%-60% (plates 1-5) of xanthorrhiza
for 5 plates and 50% of ketoconazole for each plate. The plates of combination show
that there is no growth of C. albicans in plates 1-4.
Fig 1. Colony of C. albicans after treating by combination of extract and ketoconazole
The graphic of colonies‘ calculation of ketoconazole, xanthorrhizol, and combine of

xanthorrhizol and ketoconazole. These treatments was given to count the colonization
of C. albicans in different kinds of antifungal.
113
Graphic 1. The results of colony‘s calculation by giving single ketoconazole, single Curcuma
xanthorrhiza extract, and combination of Curcuma xanthorrhiza extract-ketoconazole to C.
albicans (laboratorium of microbiology Airlangga University, Faculty of Dentistry).
Independent Samples Test was conducted to determine effectiveness of combination.

Values that were obtained in this test p> 0.05, in conclusion that there was no
significant difference between ketoconazole and combination, and between
xanthorrhizol and combination.
DISCUSSION
Candidiasis has a wide variety of manifestations. Patients may be totally asymptomatic,
or feel pain or burning in the mouth. Patients have raised, curdy, white areas often of the
palate and tongue. The white areas which are necrotic, can usually be scraped off with a
tongue blade, leaving a raw, bleeding surface. Candidiasis usually responds well to
topical therapy if there‘s no significant underlying pathology. Systemic medications for
oral mucosal candidiasis include ketooconazole tablets and fluconazole.5
Xanthorrhizol has an ability as a trigger of protein‘s denaturation in cell wall of

Candida which can lead releasing of protein. The cell wall will be wrinkled and dead. 12
The wrinkled and dead cells cause inhibiting the affinity of Candida to the host‘s
superficial. Affinity was found as one of requirements of infection‘s growth. Protein
denaturation that triggered by xanthorrhizol inhibit the infection‘s growth of the cell
host. 12
Ketoconazole inhibits adrenal steroid synthesis by reversible, dose-dependent inhibition

of the cytochromes P450-dependent 11-β-hydroxylation of steroids.11 Ketoconazole was
found as adrenal blocking drugs that suppress adrenal cortisol production via inhibition
of steroidogenic enzymes.13 C. albicans contains transcriptional activator of sterol
synthesis, called Upc2. Upc2 has been described for both C. albicans as being important
114
for the hypoxic induction of ergosterol biosynthetic enzymes.14 By inhibiting the sterol
synthesis, C. albicans also can be indirectly inhibited.
Chemical laboratory test results was shown that Curcuma xanthorrhiza containing
essential oil (1.05%), tannin (6.91%), curcumol (1.36%), curcumin (1.82%), and
xanthorrhizol (2,64%). Curcumin was used to further explore its ability to prevent the
adhesion of Candida species to BEC (Buccal Epithelial Cells). These experiments were
performed with curcumin at its MIC values. Curcumin was able to inhibit the adhesion
to BEC of all the Candida.15 The activity of essential oil was found on Candida with the
mechanism of action: cytoplasmic membrane lesion.16 Tannin is polyphenol
compounds also showed antifungal activity. 17 Several studies have shown that tannin
has an ability to binding ferri and which Ca(OH)2 is a basic compound.18
Tannin can bind ketoconazole which is a basic compound. In conclusion, tannin can
reduce the activity of ketoconazole. The less activity of ketoconazole might be maintain
the lack of combination‘s ability to inhibit the colonization. It shows a relevant relation
with statistics, that there are no significant results between ketoconazole and
combination, and between Curcuma xanthorrhiza with combination.
CONCLUSION
Ketoconazole and Curcuma xanthorrhiza extract were found as potential antifungals.
Xanthorrhizol isolated from Curcuma xanthorrhiza extract has an ability to inhibit the affinity
of C. albicans to the superficial of the host. The other substances that contained in Curcuma
xanthorrhiza extract, such as tannin and curcumin were also have antifungal activity that can
predispose the effectiveness of xanthorrhizol. By the calculation of the colonies we can found
compared with single
the decreasing numbers of the colonies from the combination
ketoconazole and single Curcuma xanthorrhiza extract.
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11. DiPiro JT, Talbert RL, Yee GC, et al. Pharmacotherapy: A Pathophysiologic
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117
Effect of Mangosteen (garciamangostana L.) pericarp

extracts on biofilm formation of Streptococcus mutans
on orthodontic wire ( in-vitro)
Nurul A. R. Putranti1 , Reza D. Hendrawan2, Markus B. Rahardjo3
1,2
3
Lecturer of Oral Biology Department, Faculty of Dentistry, Airlangga University
Indonesia
ABSTRACT
Background: Hard and soft surfaces in the oral cavity are coated with a dental biofilms.
Orthodontic wire are considered to be a clinical risk factor in terms enamel integrity
because of biofilm accumulation on these surface. Objective : To examine the effect of
Mangosteen (Garcia Mangostana L.) pericarp extracts on the biofilm formation of S.
mutans on orthodontic wire. Methods : The minimum inhibitory concentration (MIC)
was first determined. The stainless steel orthodontic wire was incubated in BHI broth
containing S. mutans and 0,39% concentration of mangosteen (garciamangostana L.)
pericarp extract. The colony forming unit (CFU) of S.mutans on orthodontic wire was
counted after 48 hours incubation on TYC agar. The statistical analysis used paired ―t‖
test with 0,05 significance degree level. Result : The minimum inhibitory concentration
(MIC) of Mangosteen (garciamangostana L.) pericarp extracts on S.mutans growth was
determined at 0.39%. There were significant differences (p=0.003 ; p < 0,05) on biofilm
formation of S. mutans between sample groups and control. Conclusion : These finding
suggest that mangosteen (garcia mangostana L.) pericarp extracts had antibacterial
activity toward S. mutans and decreased biofilm formation of S.mutans on orthodontic
wires.
Keywords : Mangosteen (garcia mangostana L.) pericarp , Orthodontic wire, S. mutans
adhesion
INTRODUCTION
Dental plaque is a biofilm of oral microorganisms on the tooth surface that plays an
important part in the development of dental caries. Among bacteria in dental plaque,
Streptococus mutans is considered the most significant cariogenic bacteria.1 Bacterial
adhesion to biomaterials and it ability to form biofilm on bodies are well-known as
steps in the pathogenesis of oral infections.2
Orthodontic appliances are considered to be a clinical risk factor in terms of enamel
integrity because of biofilm accumulation on these surfaces.3 Indeed, increased levels of
mutans streptococi and lactobacilli were detected in the oral cavity folowing
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orthodontic treaments.4 Orthodontic wire which is used for long time during
orthodontic treatment tends to create new surfaces available for biofilm formation an
therefore to increase the level of microorganism in the oral cavity. It has long been
suggested that orthodontic wires lead to increased plaque accumulation and elevated
levels of streptococci and lactobacilli. In addition, orthodontic patients with fixed
appliances frequently present and abundance of Streptococcus mutans in plaque
compared with untreated orthodontic patients. Therefore, prevention of bacterial
attachment to orthodontic wires is a critical concern for orthodontists.5
Garcia Mangostana L. commonly known as ―mangosteen‖, is a tropical evergreen tree ,
presumed to have a combination of appealing subjective characteristics, such as taste,
fragrance and visual qualities, nutrient richness, antioxidant strength and potential
impact for lowering risk of human diseases.6 Its pericarp contains a veriety of
xanthones, such as α-, β-, γ-mangosteen which have remarkable biological
activities.Among these, α-mangosteen has the most antibacterial activity.7 Extract from
its pericarp has demonstrated antibacterial activity againts a wide variety of
microorganism including Stphylococcus aereus (both normal and methicilin-resistant),
Staphylococus epidermidis, Pseudomonas aeruginosa, Salmonella typhimurium,
Enterococcus species, Mycobacterium tuberculosis and Propionibacterium acnes.8-11
Some researchers carried out the effect mangosteen pericarp extracts. for medicine.
However, they mostly concentrated on the effect of Mangosteen pericarp extract for
medicine. The effect a mangosteen pericarp extract as a prevention on dental bioflm
formation has not been well studied. Since S.mutans exists almost exclusively in oral
bioffilms and is considered the primary etiologic agent of human dental caries, we
evaluated the effect of Mangosteen pericarp extract on biofilm formation by S.mutans
on orthodontic wire in vitro.

Preparation of Mangosteen pericarp extract
A fresh mangosteen pericarp was cleansed from dirt. Then washed it into hot water, cut
into pieces 0,5cm and the mangosteen pericarp dried in the air flow for 2 hours of heat.
After the drying process, 300gr of mangosteen pericarp was inserted in to extractors
machine, then 96% ethanol solvent was added and shaken for 6 hours. The next
process was filtered using filter paper and put in a vacuum evaporator for 2-3 hours
until all the alcohol solvent separately, in order to obtain viscous red-colored
mangosteen pericarp extracts.
Minimum inhibitory Concentration (MIC) Determination

MIC was determined by a broth dilution method. Thirty-six well microtiter plates were
used to identified minimum inhibitory concentration (MIC), each mangosteen pericarp
extract concentration being tested in triplicate at serial dilutions of mangosteen extract
from 50%, 25%, 12.5%, 6.25%, 3.13% , 1.56%, 0.78%, 0.39% , 0.19%. Colums 1 and 2
were used for mangosteen pericarp extract as a negative control, and colomns 11 and 12
were used for positive controls. Each well was filled with 5ml BHI broth containing 5ml
Mangosteen extract and S.mutans, incubated overnight at 37˚C.
119
Microbial suspension in sterile water containing 108 CFU/ml of bacteria was adjusted to
McFarland no 0,5 standard turbidity. After incubation 24 hours, a 20µml of bacterial
suspension was applied and spread on the TYCagar and incubated for 24 hours.
S. mutans attachment on orthodontic wire

To evaluate the effect of mangosteen pericarp extract on biofilm formation, we used
sterile orthodontic wire (Ortho Organizer Inc. Aston Avenue; stainless steel, round,
0.016) for biofilm formation. The 2-cm orthodontic wires were coated with freshly
collected 1 ml of the unstimulate clarified whole saliva. Following 1 hour of incubation
at 37˚C, the saliva-coated orthodontic wires were moved to S.mutans containing sterille
BHI broth. Samples were then incubated for 24 hours at anaerob condition. The
orthodontic wire was removed from BHI broth, coated with 0.39% of concentration of
mangosteen pericarp extract for 4 hours and washed with phosphate buffered saline
without calcium and magnesium (PBS(-)). The orthodontic wire was vortex for 10
seconds. A solution was plated on TYC agar by spreading technique. The colony
forming unit (CFU) of S.mutans on orthodontic wire was counted on TYC agar after 48
hours incubation at 37˚C
Statistical analysis
All statistical computations were performed by SPSS for Windows (version 13.0;SPSS,
Inc.,Chicago, IL,USA). Data from bacterial attachment on orthodontic wire were
analyzed by used paired ―t‖ test. Statistical significance was defined as P<0.05.
RESULT
The mangosteen pericarp extract was active againts Sterptococcus mutans. The result
shows, that minimum inhibitory concentration (MIC) of mangosteen pericarp extract
againts S.mutans was at concentartion 0,39%.
(a) (
b
)
Figure 1. Test result for MIC after serial dilution of mangosteen extract from 100%,
50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78% (a) , until 0.39% , 0.19% (b)
120
Figure 2. Colony of S.mutans on orthodontic wire with and without mangosteen

pericarp extract
Figure 3. Colony of S.mutans on TYC agar

In this study, the pericarp extract of Garcinia Mangostana L. shown inhibit S. mutans
attachment on the surface of orthodontic wire (p=0.003 ; p < 0,05) (Table 2)
121
Paired Samples Test
Paired Differences
Std. Std. Error

Mean Deviation Mean
Control - Sample
Pair 1 21.83333 9.57949 3.91081
group
Paired Samples Test
Paired Differences
95% Confidence Interval of

the Difference
Sig. (2-
Lower Upper t df tailed)
Control - Sample
Pair 1 11.78027 31.88639 5.583 5 .003
group
Table 2. Statistical analysis of S. mutans attachment on orthodontic wire shown

significant differences (p=0.003 ; p < 0,05)
DISCUSSION
The pericarp extract of garcinia mangostana linn has a wide spectrum of antibacterial
against several gram positive and gram negative bacteria such as; staphylococcus
aureus, staphylococcus epidermidis, Bacillus subtilis, Propionibacterium acnes,
Pseudomonas aeruginosa, Salmonella enteritidis, and eschercia colli.8,11 Torrunruang
K,et al suggest that pericarp extract of Garcinia mangostana was effective againts
cariogenic streptococcus mutans. 12
In the present study, we examined the effect of pericarp extract of Garcinia mangostana
linn on dental biofilm formation using Streptococus mutans on the surface of
orthodontic wire. Biofilm formation begins with pellicle formation. The pellicle is a
thin coating of salivary proteins that attach to the tooth surface or material surface
within minutes after a professional cleaning. The pellicle acts like double-sided
122
adhesive tape, adhering to the tooth surface or material surface on one side and on the
other side, providing a sticky surface that facilitate bacterial attachment to the tooth
surface or on the surface of materials. 13-15 The purpose using saliva in this study was to
cover the specimens with a pellicle.2,16, The initial conditioning salivary coat plays and
important role in bacterial adsorption to surfaces as the absorbed proteins can
manipulate bacterial adhesion to the conditioning film. Albumin , a protein found in the
saliva, is an inhibitor of hydrophobic interactions, and was implicated in bacterial
adhesion mediated by hydrophobic interactions. Amylase, another salivary protein, has
been shown to promote bacterial adhesion by inducing specific interactions with several
types of Streptococci.17
Firtsly, we identified the minimum bacteria concentration (MIC) and founded that the
MIC was at concentration 0,39%. Another studies reports that the MIC of the pericarp
extract for streptococcus mutans was 0.625 µg/ml. 12
The biofilm formation on the surface of orthodontic wire was examined by using viable
counting method (CFU/ml) and the result shown significance different between the
samples and control (p<0.003). The result suggested that pericarp extract of Garcinia
mangostana linn can inhibit biofilm formation on the surface of orthodontic stainless
steel wire.
Mangosteen pericarp extract containing several xanthones such as α, β and γ
mangosteen, gartinin and iso mangosteen.7 Chemical laboratory test result in this study
was shown the pericarp extract mangosteen containing xhanton (4.91%) , saponin
(3,82%) , tanin (9,24%) , mangostin (8,74%) and mangostanin (11,88%). The chemical
components of the extract often vary depending upon the extraction protocol. When
using 40% ethanol as solvent , the extract containing 10% α mangostin and 12% γ
mangostin. Another study using ethyl acetate as solvent reported that the extract was
composed of 77,8% α-mangostin and 15,9% γ-mangosteen. Among xanthone derivates
from mangosteen extract, α-mangostin has been shown by several study to exert the
most potent antibacterial activity.12 The possible explanation from this study is the α
mangostin contained the pericarp extract of garcinia mangostana might be plays an
important role to inhibit biofilm formation on the surface of orthodontic wire. Further
studies are still required to clarify the mechanism inhibition of biofilm formation on the
surface of orthodontic wire by using pericarp extracts of Garcinia mangostana.
CONCLUSION
In conclusion, this study showed that extract from Garcinia Mangostana was effective
againts antibacterial activity of Streptococcus mutans and it also decreased biofilm
formation of S.mutans on orthodontic wires.
REFERENCES
1. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev.
1986 ; 50 : 353-80.
2. Yuehuei H.An, Richard J. Friedman, 1997. Study literature : Concise review of
mechanism of bacterial adhesion to biomaterial surfaces.
123
3. D. Steinberg , S.Eyal. Initial biofilm formation of Streptococcus cabrinus on various

orthodontic appliances. Journal of Oral Rehabilitation, 2004. 31;1041-1045
4. Scheie AA, Anneberg P,Krogstad O. Effect of orthodontic treatment on prevalence
of Streptococcus mutans in plaque and saliva. Scand J Dent Res. 1984;92:211-217
5. Heon-Jin Lee, Hyo-Sang Park, Kyo-Han Kim, Tae-Yub Kwon, Su-Hyung Hong,
Effect of Garlic on bacterial bioflm formation on orthodontic wire. Journal of Angle
Orthodontist, 2011. Vol 81, No 5.
6. Vishnu Priya et al. Antimicrobial activity of pericarp extract of Garcinia
Mangostana Linn. International journal of Pharma Sciences an Research, 2010 Vol.
1 (8) ;278-281
7. Sarin Tadtong, Antityrosinase and antibacterial activities of Mangosteen pericarp
extract. J Health Res, 2009 , 23(2) : 98-102
8. Linuma M, Tosa H, Tanaka T, Asai F, Kobayashi Y, Shimano K, et al. Antibacterial
activity of xanthones from gutti feraeous plants againts methicillin-resistant
Staphylococcus aureus. J Pharm Pharmacol. 1996;48:861-5
9. Sunaram BM, Gopalakhrishnan C, Subramanian S, Shankaranarayanan D,
Kameswaran L. Antimicrobial activities of Garcinia mangostana Planta Med.
1983;48:59-60.
10. Mahabusarakam W, Wiriyachcitra P, Phongpaichit S. Antimicrobial acyivities of
chemical constituents from Garcinia mangostana Linn. J sci Soc Thailan.
1986;12:239-42
11. Suksamrarn S, Suwannapoch N, Phakhodee W, Thanuhiranlert J, Ratananukul P,
Chimnoi N, et al. Antimycrobacterial activity of prenylated xanthones from the
fruits of Garcinia mangostana. Chem Pharm Bull (Tokyo). 2003;51:857-9
12. Torrungruang K, Vichienroj P, Chutimawarapan S. Antibacterial activity of
mangosteen pericarp extract againts cariogenic Streptococcus mutans. CU Dent J.
2007;30:1-10
13. Kroes I, Lepp PW, Reiman DA Bacterial diversity within the human subgingival
crevice. Proc Natl Acad Sci USA 1999;96(25):14547-14552
14. Elder MJ,Stapelton F,Evans E,Dart JK. Biofilm-related infections in ophthalmology
Eye 1995;9(Pt.1):102-109
15. Nield –Gehrig JS and Willmann DE. Foundations of Perodontitics for Denal
Hygenist. Philadhelpia: Lippincott Williams&Wilkins 2003:67-73
16. S.Eick, E. Glockmann, B.Brandl, W.Pfister. Adherence of Streptococcus mutans to
various restorative materials in a continunious flow system. Journal of Oral
Rehabilitation 2004; 31: 278-285.
124
Analgesic Effect Liquid Smoke of Coconut Shell

Induced by Acetic Acid 0,6%
Antonius Lucky Arnando1, Meircurius Dwi Condro Surboyo2,
Tantiana3, Ira Arundina4
1,2
3,4
Lecturer of Oral Biology Department, Faculty of Dentistry, Airlangga
University, Indonesia
ABSTRACT
Background: One mechanism of drug can be used to eliminate pain is to inhibit activity
of conversing arachidonic acid into prostaglandin, which can cause pain. The chemical
composition of coconut shell are cellulose, pentosan, lignin, solvent extraction, uronat
anhydrous, nitrogen, and water. One active ingredient in coconut shell is phenyl
propanoid which consisting lignin structure and guaicol. Phenyl propanoid and guaicol
are phenolic compounds that can be used as an antioxidant, antiseptic, anti-
inflammatory, anesthetic and analgesic. Liquid smoke of coconut (Cocos nucifera L)
contains phenolic compound which believed to bind a component that conversing
arachidonic acid into prostaglandin. Purpose: To prove liquid smoke of coconut shell
(Cocos nucifera L) has analgesic effect. Method: The analgesic effect is determined by
its peripheral pharmacological actions using acetic acid-induced writhing reflex on
mice. The type of this research is the experimental laboratories research, conducted on
2-3 month age and 20-30 grams weight male Mus musculus. The Mus musculus divided
into 4 groups, each group consist of 7. Control group was given 0.01 ml/weight aquades
per-oral and after 30 minutes, 10 ml/weight acetic acid 0,6% delivered via
intraperitoneal injection. Other groups were given liquid smoke of coconut shell (Cocos
nucifera L) by the concentrations of 100%, 50% and 25%. The recording of writhing
reflex was done every 5 minutes during 30 minutes. Result: There are differences of
writhing reflexes which given liquid smoke of coconut shell with significancy 0.000 (p
< 0.05) by the concentration of 100%, 50% and 25%. Conclusion: Liquid smoke of
coconut shell (Cocos nucifera L) has analgesic effect on Mus musculus which induced
by acetic acid 0,6%.
Keywords : analgesic effect, liquid smoke of coconut shell, acetic acid.
INTRODUCTION
Nociception is a response of neural tissue in response to noxious stimuli. Pain is the
perception that arises as a result of nociception.1 Inflammatory is a complex biological
response including vascular tissues response to a tissue damage as well as some
125
irritants.2 Inflammatory posed on a tissue that is exposed to an excessive stimulus will

cause pain.1
Pain is a sensory stimulus which is unpleasant and uncomfortable, associated with tissue
damage and causes a person to relieve soreness.3 Management of pain is done by
reducing the variety of factors that cause pain, both in central and peripheral. According
to Hargreaves there are three classes of medications that can be used to reduce pain, i.e.
non-opioid analgesics, opioid analgesics, and a local anaesthetic.4
The drug frequently used in cases of acute pain is non-opioid drugs such as NSAID
(Non-steroidal Anti Inflamtion Drug). This drug has fast onset and duration of action.
Mild side effects that can arise as a result of consuming this drug are dizziness, nausea
and vomiting. In dentistry, these drugs are effective as analgesics for mild to moderate
pain relief, as in pulpitis, third molar tooth extraction and gingival curettage.5
Coconuts are produced in Indonesia around 15,5 billion grains/year.6 Coconut fruit
processing industry is still largely focused on processing meat as the primary results,
whereas the by-product processing industry, such as coconut fiber and shell, are still
traditionally done.7 Coconut shell is one part of the agricultural products which have
high economic and functional value.8 Chemical composition of coconut shell composed
of cellulose, pentosan, lignin, ash, extractive solvent, anhydrous uronic acid, nitrogen,
and water.9 One of the active components in coconut shell is a compound phenyl
propanoid contained in the lignin. Phenyl propanoid compounds is a compound of
phenols that can be used as an antiseptic, antioxidant, anti-inflammatory, anesthetic, and
analgesic.10,11
Knowledge about nutritious drugs plant based on the experience and skills that are
walled has passed down from one generation to the next. The role of medicinal plants in
medicine remains to be ascertained through analysis and scientific experiments which
can be accountable. Therefore, efforts toward the scientific substantiation rationally
should remain committed, inter alia through the analysis of the substances contained in
natural materials, as well as investigating their therapeutic effect (Lucia, 2006).12
Therefore, it is need to be done on the utilization of by-product produced by coconut-
processing industry of which is coconut shell, which can be utilized to produce liquid
smoke shell coconut (Cocos nucifera L.), that can be used as an analgesic.

Material used in this research is the coconut shell. Coconuts (Cocos nucifera L.) used is
the fruit of coconut which was 6-8 months, which acquired and identified in the House
Plant Conservation and Botanic Garden of Purwodadi Pasuruan.
Liquid smoke of coconut shell (Cocos nucifera L.) obtained by process of pyrolysis.
Pyrolysis is the thermochemistry decomposition of organic materials on over 4300C
temperature with or without oxygen.13 The manufacture of liquid smoke of coconut
shell (Cocos nucifera L.) performed at the Hall of Research and Consulting Industry,
Research Laboratories and consulting Industry Surabaya.
126
Liquid smoke is obtained from pyrolysis is liquid smoke with concentration of 100%.
To obtain a solution of liquid smoke coconut shell with lower concentration of 50% and
25%, dilution is done using aquades to the pure substance. This result in the addition of
the aquades, the concentration is changed and volume is enlarged but the amount of
moles of dissolved substances remain.14
On this research, the animals used are the male mice (Mus musculus), aged between 2-3
months, with a weight of 20—30 grams. Animals are obtained from Test Animal Unit,
Biochemical Laboratory, Faculty of Medicine, Airlangga University.
Analgesic effect of coconut shell liquid smoke (Cocos nucifera L.) is determined using
the methode acetic acid induced writhing reflex. This method is used for either
screening peripherally acting analgesic, local peritoneal cell response and
prostaglandins pathway.15 Mus musculus which are used as test animal are divided into
4 groups each contained 7 (n=7) and were given treatment as follows, the first group
(the control group) was given aquades 0,01ml/gr bw (po), group I was given the liquid
smoke of coconut shell 100% 0,01ml/gr bw (po), group II was given liquid smoke of
coconut shell 50% 0,01ml/gr bw (po) and group III was given liquid smoke of coconut
shell 25% 0,01ml/gr bw (po). After 30 minutes, those test-animals are given the acetic
acid 0,6% with doses of 0,01ml/gr weight (ip) to induced the pain. After 5 minutes
counting is done by observing the cumulative amount of writhing reflex in test-animals
every 5 minutes for 30 minutes,16,17 then calculated the percentage analgesic power with
formula as follows:18
The amount of bend (writhing reflex) obtained is analyzed using one-way Analysis of
Variance (ANOVA) with significance level of 95%. If the result of one-way ANOVA
showed difference, then continued with the LSD (Least Significant Difference) test
RESULT
Table 1: The mean result of test-animals‘ bend or writhing reflex, standard deviation and percentage of
inhibition (analgesic power)
Group Treatment N X SD Percentage of
inhibition
Control Aquades 7 48.57 4.79 -
1 Liquid smoke of coconut 7 29.00 4.55 40.29%
shell 100%
127
shell 50%
shell 25%
Table 2: Results of analysis of one-way ANOVA on test-animals‘ bend or writhing reflex in all treatment.
Sum of Square Df Mean Square F Sig.
Between Groups 1375.571 3 458.524 29.651 .000
Within Groups 371.143 24 15.464
Total 1746.714 27
The data is analyzed using the Kolmogorov Smirnov Test and noted that the whole
group has a probability value greater than 0.05 (p > 0.05), which means that the data are
normally distributed, Levene test is also done and noted that the value significance of
0.608 (p > 0.05) which means that the data is homogeneous. Data which are normally
distributed and homogeneous can be analyzed using one-way ANOVA, which results
can be seen in table 2.
From table 2 it is known that the value significance is greater (p < 0.05) which is equal
to 0.000 which means that there is a difference of bend or writhing reflex upon test-
animals between the groups. LSD (Least Significant Different) test is done to tell the
difference between each group. There is a significant difference between aquades group
(the control group), compared to a group of liquid smoke 100%, the group of liquid
smoke 50% and 25% of group liquid smoke; Group liquid smoke 100% compared to the
control group, the group liquid smoke 50% and 25%; Group of liquid smoke 50%
compared to the control group and group of liquid smoke 100%; Group liquid smoke
25% compared to the control group and the Group liquid smoke 100% of (p < 0.05)
which means that there is a reduction of bend or writhing reflects on test-animals .
DISCUSSION
This research was conducted to prove the analgesic effect of liquid smoke of coconut
shell (Cocos nucifera L.) by looking at the writhing reflex (bend) of test-animals.
Writhing reflex in this research occurred by giving acetic acid 0.6% intraperitoneal on
test-animals which can stimulate the chemo-sensitive-nociceptor.15 Acetic acid that
raises the writhing reflex significantly decrease (p < 0.05) on the group of test-animals
given liquid smoke of coconut shell in 3 different concentrations (100%, 50% and 25%)
when compared with the control group. This is because, the content of the active
128
ingredient contained in the liquid smoke of coconut shell which is phenolics compound
is capable of inhibiting pain caused by writhing reflex due to acetic acid.
Administering liquid smoke of coconut shell (Cocos nucifera L.) will reduce the pain
response. This is because, the content of the active ingredient contained in the liquid
smoke of coconut shell which is phenolics compound is capable of inhibiting pain.
Phenolics compound contained in liquid smoke of coconut shell is a compound of
phenol and guaiacol which are substances that catch potential radical. This compound is
a compound that has the ability of antioxidant compounds as redox, which serves as a
reducing agent, converting hydrogen and can inhibit the initiation stage on lipid
oxidation.19,20 Antioxidant is an agent of anti-inflammatory that works through arresting
free radicals of oxygen released by peroxide. Liquid smoke of coconut shell inhibit pain
response which is the role of phenolics compound that inhibits cyclooxygenase on the
tissue by reducing the synthesis of prostaglandins E2 (PGE2).21 The decreasing amount
of prostaglandins E2 (PGE2) due to the binding compounds of prostaglandins G2
(PGG2) and prostaglandins H2 (PGH2) when arachidonic acid conversion into
prostaglandins E2 (PGE2) by phenolics compound. Both of these compounds are
endoperoxyde compounds produced during the conversion of arachidonic acid into
prostaglandins.A compound that can cause irritation in the tissue (the irritant substance)
for example acetic acid, can stimulate the release of prostaglandins through nociceptive
neurons which are sensitive to non-steroidal anti inflammatory drug. Irritant substance
causes the release of endogenous substance such as prostaglandins, which can stimulate
peripheral nociceptor neurons and non-steroidal anti-inflamatory drug sensitive
neurons.22 Administration of liquid smoke of coconut shell (Cocos nucifera L.) related
to the inhibiting mechanism of cyclooxygenase in peripheral tissues, thus reducing the
synthesis of prostaglandins and interfere with the primary transduction mechanism in
the afferent nociceptor.23 The analgesic effect mechanism generated by liquid smoke of
coconut shell works by inhibiting the production and function of prostaglandins. Thus it
can be said that liquid smoke of coconut shell is a non-steroidal anti inflammatory like
drug.
Percentage of inhibition (analgesic power) is the ability of liquid smoke of coconut shell
(%) in lowering the number of bend or writhing reflex on test-animals due to acetic
acid. Percentage of inhibition (analgesic power) from liquid smoke of coconut shell
increases proportional to the increase in the concentration of liquid smoke shell given to
test-animals. There is a significant difference (p < 0.05) percentage of inhibition
(analgesic power) between liquid smoke shell coconut 100% with liquid smoke coconut
shell 50% and 25%. This is probably because the concentration of phenolics compound
contained in each is proportional to the amount concentration liquid smoke coconut
shell. There were no significant differences (p < 0.05) in the percentage of inhibition
(analgesic power) between liquid smoke of coconut shell 50% and 25%. This is possible
because, a decrease amount of phenolics compound contained in liquid smoke of
coconut shell, leading to a decrease in percentage of inhibition (analgesic power).
Difference percentage of inhibition (analgesic power) produced by liquid smoke of
coconut shell with concentration of 100%, 50% and 25%, made possible because of the
differences in the concentration of phenolics compound. The concentration of phenolics
compound in each liquid smoke concentration is proportional to the concentration of the
liquid smoke itself.
129
CONCLUSION
From the results of this research, can be inferred that the liquid smoke shell
coconut (Cocos nucifera L.) has an analgesic effect comparable with the increasing
concentration of liquid smoke. Further research is needed to determine the effect of
analgesic liquid smoke of coconut shell (Cocos nucifera L.) as an analgesic in case of
pulpitis.
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Asia Pacific Dental Students Journal | Volume 3| Number 1| June 2012

Comparison of Avian Eggshell and Prawn Skin

Asia Pacific Dental Students Journal 2012

installation[4]. In a case with insufficient alveolar bone height, vertical ridge

MATERIAL AND METHODS

multiple comparison tests was performed to evaluate differences between groups.

RESULTS AND DISCUSSIONS

Figure 2 – Diffraction Patterns of Identified Phases by XRD

Figure 1A – FTIR spectra of avian eggshells

Figure 1B – FTIR spectra of prawn skins

Scanning electron microscope

3. Turunen T, Peltola J, Yli-Urpo A, Happonen RP. Bioactive glass granules as a bone

4. Bosch C, Melsen B, Vargervik K. Importance of the criticalsize bone defect in testing

8. Zitzmann NU, Scharer P, Marinello CP, Schupbach P, Berglundh T. Alveolar ridge

15. Vuola J, Goransson H, Bohling T, Asko-Seljavaara S. Bone marrow induced

16. Dupoirieux L, Pourquier D, Souyris F. Powdered eggshell: A pilot study on a new

Effects of Tofu Liquid Waste on Mandibular Bone of

Asia Pacific Dental Students Journal 2012

Keywords: Ovariectomy, Periodontitis, Isoflavones, Tofu Liquid Waste.

As many as 30% of menopausal women suffer periodontitis.[2] Periodontitis is an oral

In 1970 scientist introduced a treatment for menopause by using (Hormonal

MATERIAL AND METHODS

periodontal tissue surgery to see the microscopic structure, expression of estrogen

RESULTS AND DISCUSSION

Increased apoptosis resulting in decreased osteoblast cell number. Mature osteoblasts

Reduced osteocytes cell resulted in a decrease of bone regeneration. Decreased structure

Periodontal Ligament Space Width. Reduced estrogen levels could be a trigger in

Expression of Estrogen Receptor. Tofu liquid waste containing Isoflavones is known

MDA Level of Mandible Bone. MDA is the result of calculation by using

Oil Pulling Therapy As An Alternative in Reducing

Asia Pacific Dental Students Journal 2012

Oil Pulling Therapy

Figure 2. Overview of the antimicrobial, antioxidant and anti-inflammatory activities

of extra virgin olive oil (EVOO) phenolic compounds. 22

Up to 36 phenolic compounds have been identified in olive oil.15 Phenolic compounds

Figure 3. Arachidonate metabolism pathways for prostaglandin synthesis by cyclooxygenase enzyme,

Figure 5. Typical generalized marginal and papillary gingivitis34

Gingival bleeding varies in severity, duration, and ease of provocation. Bleeding on

Gingivitis can be a preamble to periodontal diseases and involves anaerobic bacteria

aureus and Porphyromonas gingivalis. Oleocanthal and hydroxytyrosol possess potent

Apoptosis and Viability Analysis of Human Calvarial

Asia Pacific Dental Students Journal 2012

Human calvarial osteoblasts (HCOs) obtained from ScienCell Research Laboratories

Thus we conclude that a cyclic, equibiaxial, tensile mechanical strain significantly

Keywords: Human calvarial osteoblasts, apoptosis, cell viability, mechanical strain,

INTRODUCTION AND LITERATURE REVIEW

Currently, most in vitro cell studies on osteoblast mechanotransduction have focused on

Previously it was unusual to find morphologically apoptotic osteoblasts at active bone

However apoptosis is a critical process in skeletal maturation and bone remodelling. A

However there is very limited research conducted to investigate the time-dependent

Current mechanotransduction mechanisms postulate that mechanical loading stimulates

AIMS AND HYPOTHESIS

MATERIALS AND METHODS

In this experiment, we used Human Calvarial Osteoblast Cells (from ScienCell

TRYPSINIZATION AND CELL CULTURE

The cell numbers were counted manually using a light microscope on a

APPLICATION OF CYCLIC MECHANICAL STRAIN

CELLULAR APOPTOTIC ACTIVITY

Caspase-Glo® 3/7 Assay

Caspases, a family of cysteine proteinases that specifically cleave proteins following

Adding a single Caspase-Glo® 3/7 Reagent in an ―add-mix-measure‖ format results in

luminescent signal produced by luciferase (Figure 1). Luminescence is proportional to

Methylyhiazolyldiphenyl-tetrazolium bromide (MTT, approx 98% TLC) Assay

Significance was determined as p< 0.05 for all tests.