Antifungal of Acrylic Resin Denture Base Plate Incorporate Nano Particle

High Density Chitosan

Titik Ismiyati 1, Siswa Setyahadi 2

*E-mail:ismiyati.titik@yahoo.co.idProsthodontic Departement, Faculty of Dentistry, Gadjah
Mada University, Jl. Denta, Sekip, Yogyakarta, Indonesia.
2
Biocatalyst Production Technology Division, Center for BioindustrialTechnology, Agency for
the Assessment and Application of Technology.

Abstract
95% acrylic resin is used as a denture material, because of several advantages that are
not toxic, does not irritate the tissue, the physical and aesthetic properties of both, the price is
relatively cheap, easy to manufacture and easy manipulation and repair work.
Microbial plaque on the denture surface facing the mucosa is the cause of denture stomatitis
factor, which has been reported that 65% of denture wearers suffer from denture stomatitis.
The buildup of plaque and food residue will lead to increasing the number Candida albicans.
The aim of this study was to investigatethe effect of incorporate of nano particles
made using high-density chitosan with Acrylic Resin Denture Base Plate. Different
concentrations of nanoparticles were applied to optimize the performance of the Acrylic
Resin Denture Base Plate to inhibit growth Candida albicans.
All sampels were incubated with a suspension of Candida albicansfor 24 h at 37 C
and disc containing Candida albicansput into sterilized aquadest. 0.01 ml of liquid was
incubated for 72 h at 37 C in Saboraud media and count the total number of Candida
albicans. The data analyzed by analysis of variants and the Least Significant Difference.
The conclude of this study was showed that acrylic resin incorporate with chitosan
nano can inhibit the growth of Candida albicans on the base plate denture acrylic resin.
Acrylic resin incorporate with nano chitosan concentration of 1% had the highest antifungal
against Candida albicans growth in the base plate denture acrylic resin.

Key words: nano chitosan, high molecular weight, Candida albicans, acrylic resin denture

Introduction
Prosthesis that replaces part or all natural teeth are missing, and the surrounding tissue
is called denture. Manufacture of denture aims to restore the masticatory function, aesthetics,
comfort and health are compromised due to the loss of teeth. 95% reported denture is made of
acrylic resin materials, because these materials have the advantage that is not toxic, does not
irritate the tissues, physical and aesthetic nature a good, the price is relatively cheap, easy
method manufacture as well as easy manipulation and repaired, but the disadvantage acrylic
resin can cause allergies , porous, absorbs water and is less resistant to abrasion1. Material
composed of acrylic resin powder (polymer) and liquid (monomer), the use of polymer
powder mixed with liquid monomer.
The use of denture plaque accumulation will occur, especially in the denture base
plate in direct contact with the oral mucosa. Plaque is a soft deposit which is attached to the
denture surface that contains many microorganismsmainly Candida albicans is an important
factor that causes inflammation known as denture stomatitis2.3. Shape of the surface plate or
denture base acrylic resin affect adhesion plaque-forming microorganisms. Surface roughness
and porosity of acrylic resin denture base allows microorganisms penetrate into it. Denture
surface that deal directly with the mucosal surface is not polished, so the texture is rough, and
therefore will affect more attachment plaque4. Denture stomatitis prevalence between 25%
-65% depending At the population, but it makes conditions ekstrims. This situation is
worsened by the incidence of stroke and lack of effectively cleaning the denture. Frequency
of less severity of infection if the denture hygiene in continuous use.

Figure 1. the denture inserted in mouth Figure 2. Denture stomatitis

Candida albicans multiply by forming buds that will continue psedohifa elongated
shape, can grow at pH variation, but growth will be better at pH 4.5 to 6.5 and at a
temperature of 28oC - 37oC. White-cream-colored colonies, flat round or half round with a 2-
5 mm diameter smell like beer or yeast 5. Candida albicans cell wall membrane sterols have an
important role as a cell protection and the workings of enzymes that play a role in cell wall
synthesis. The sterol membrane is the target of several antifungal6. Cell wall serves to give
shape to the cell, protects yeast cells from the outside environment and play a role in the
process of attachment and colonization are antigenic7.
 Fugure 2. Candida albicans

On treatment, dentures should be cleaned of flora in the mouth and the accumulation
of plaque and food debris, such as by soaking the dentures in disinfectant solution3. Denture
cleaning methods can generally be done in two ways, namely mechanically and chemically.
Mechanically cleansing carried out by using a toothbrush or ultrasonic instrument, whereas
chemically cleaning carried out by soaking the dentures in a solution of anti-organism. that
denture cleaning agents should have the characteristics of non-toxic, easily removed and do
not leave residual material which is irritating, has the ability to destroy or dissolve inorganic
and organic material accumulation contained in the denture, do not damage the materials used
in the manufacture of artificial teeth, stable in storage and should be bactericidal and
fungicidal. Mechanism of action of fungicide material that damage the cell wall by inhibiting
the formation or change the structure after completion form, changing the permeability of the
cell membrane thus inhibiting the cell growth subsequent cell death, inhibit the action of
enzymes that lead to disruption of metabolism or cell death, inhibition of the synthesis of
nucleic acids and proteins. Determination of the activity carried out by the method
antifungisid dilution or diffusion.
used as an acrylic resin denture material, because it has the advantage that it is not
toxic, does not irritate the tissues, physical and aesthetic properties of a good, the price is
relatively cheap, easy way of making as well as easy manipulation and repaired, but the loss
of acrylic resin can cause allergies, porous, absorbing water and less resistant to abrasion1
Composed of acrylic resin powder (polymer) and liquid (monomer), the use of polymer
powder mixed with liquid monomer. Acrylic resin polymerization process can be divided by
a heat curing acrylic resin and a curing cold8.
 On use of denture plaque accumulation will occur, especially on the part of the
denture base plate. Denture base plate is part of a denture that contact with the oral mucosa,
where sticking and support elements denture, distribute occlusal forces to the supporting
tissues and provide retention and stability of the denture9. Shape of the surface plate or
denture base acrylic resin affect adhesion plaque-forming microorganisms. Surface roughness
and porosity of acrylic resin denture base allows microorganisms penetrate into it. Denture
surface that deal directly with the mucosal surface is not polished, so the texture is coarse,
and therefore will affect more attachment plaque4. Plaque is a soft deposit which is attached to
the denture surface that contains many microorganisms Denture plaque is an important factor
that can cause inflammation of the palatal mucosa3.Microbial plaque on the denture surface
facing the mucosa is the cause of denture stomatitis factor, most microbial inherent dominant
is Candida albicans3. Candida albicans multiply by forming buds that will continue psedohifa
elongated shape, can grow at pH variation, but growth will be better at pH 4.5 to 6.5 and at a
temperature of 28oC - 37oC. White-cream-colored colonies, flat round or half round with a
diameter of 2-5 mm aroma smells like beer or yeast5. Candida albicans cell wall membrane
sterols have an important role as a cell protection and the workings of enzymes that play a
role in cell wall synthesis. The sterol membrane is the target of several antifungal
(Samaranayake, 2002). Cell wall serves to give shape to the cell, protects yeast cells from the
outside environment and play a role in the process of attachment and colonization are
antigenic7.
Indonesia is rich in biodiversity are potentially useful as an industrial development
area of medicine and food. Chitosan is the result of deacetylation of chitin which is a natural
polysaccharide shell crustasea. Chitosan is composed by units of D-Glucasamine
(deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit)10. Chitosan with a molecular
weight <50 kDa were categorized as low-molecular weight chitosan, molecular weight 50-
150 kDa molecular weight chitosan is moderate, and> 150 kDa is a high molecular weight
chitosan11. High molecular weight chitosan has a high viscosity and low absorsinya level,
with the chitosan enzymatic method can be obtained with shorter chain length, water soluble
and low viscosity so that the lighter molecular weight chitosan oligomers12. The use of
chitosan as an antimicrobial should be required high concentration..
Mechanisms of antimicrobial chitosan against bacteria and fungi have the same
mechanism, namely through the interaction between the charge positive group NH3 + on the
unit glucosamine chitosan and the negative charge on the cell membrane of microbes
resulting in electrostatic interactions that lead to changes in the permeability of the membrane
walls of microbes that alter the osmotic balance internally that can inhibit the growth of
microbes, hydrolysis and microbial peptidoglycan in walls resulting in loss of intracellular
electrolytes, proteins, nucleic acids and glucose on microbial11. Research nylon thermoplastic
denture cleaning using high molecular weight chitosan as an antifungal by immersion. At the
immersion method, a fungus that infiltration into the pores of thermoplastic nylon can not be
affordable by the soaking material anti fungal, so fungus inherent in the pores can still
developing. It is therefore necessary to find methods of inhibiting mold in other ways13. This
study aimed to determine the effect of acrylic resin incorporate with chitosan nano to the antifungal .

Research Methods
Materials and Instrument: high molecular weight chitosan nano with a concentration
of 0.25%, 0.5% and 0.75%, and 1%.Suspension of Candida albicans 108 CFU / ml, 0.05%
acetic acid, Brain heart infusion (BHI), artificial saliva, Saboraud order. Acrylic resin plate
model of disc shape with a diameter of 10 mm and a thickness of 2 mm. Petri dish, spreader,
incubators, colony counters, micropippet yellow tip, dappen dish, vortex mixer.
Chitosan nano is made to create a solution consisting of 0.2% chitosan solution
dissolved 0.1% acetic acid solution and filtered with Whatman filter paper no. 4. Solution B
was prepared by dissolving sodium tripolyphosphate 0.1% in distilled water and filtered
through Whatman filter paper no. 4. Add solution B into solution A gradual / be followed
dropwise while stirring with a speed of 5000-10000 rpm with a volume ratio of 1: 5 or 1: 3.
After the addition is complete, and to form a suspension, allow the suspension, stirring
constantly for 30 minutes. Suspension was then analyzed its particles and subsequently
entered into a spray drying to obtain chitosan nanoparticles.

The research subject were made discoid 10mm diameter 2mm thick amount of 50
pieces. Acrylic resin in the same proportion in each group that is powder: liquit = 10 g: 4 ml.
Group I acrylic resin without mixing with chitosan nano. Group II acrylic resin mixed with
0.25% chitosan nano. Group III acrylic resin mixed chitosan nano 0.5%. Group IV acrylic
resin mixed chitosan nano 0,75%. Group V acrylic resin mixed chitosan nano 1%.
Polymerization process is carried out by injection.

Candida albicans using sterile Ose taken and dissolved in 0.5 ml of BHI liquid
medium, and then incubated for 24 hours at a temperature of 370C to obtain a suspension of
Candida albicans. The suspension was diluted so as to achieve certain standards compliant
turbidity Brown III is 108 CFU / ml. Each of the study subjects were incubated in artificial
saliva for 24 hours, then incubated in a suspension of Candida albicans for 24 hours at a
temperature of 370C. Later incubated for 8 hours at room temperature, shaken with a vortex
mixer for 1 h. The next dilution series to 10-1. 0.01 ml of test solution were taken and planted
in order Saboraud media, incubated for 72 hours at a temperature of 370C. Performed
counting the number of colonies. The data obtained were calculated by ANOVA statistics and
followed by LSD test.

Figure 4. The research sample

Results and Discussion

Calculation results Mean and Standard deviation number fungus Candida albicans on
acrylic resin incorporate with nano chitosan at various concentrations can be showed in
Table 1:
Table 1.Mean and Standard deviation number fungus candida albicans in resin
acrylic with different concentrations of chitosan nano (CFU / ml)

Group N Mean Standar Deviasi

I 10 2202 9.189
II 10 1962.500 6.346
III 10 1861 6.146
IV 10 1386 6.146
V 10 652.500 6.346
Note : Group I : control
II : Acrylic resin incorporate nano chitosan 0,25%
III : Acrylic resin incorparate nano chitosan 0,5%
IV : Acrylic resin incorporate nano chitosan 0,75%
V : Acrylic resin incorporate nano chitosan1 %
N : Number of sample
 Table 1 shows, the results of mean number of Candida albicans fungus is highest in
the control group. The highest rate in the treatment group was 0.25% concentration (1962.500
6.346 ). being the lowest mean value found in group V is the concentration of 1% (652.500
6.346). This suggests that of acrylic resin incorporate with nano chitosan concentration of
1% showed inhibition of fungal growth is higher than the group of acrylic resin incorporate
with chitosan nano concentrations of 0, 25%.

Figure 5. The growth of Candida albicans

This result is due to NH3 + chitosan has an anti fungal. Concentration of 1% chitosan
nano contains anti-fungal higher concentrations of chitosan nano 0.25%, 0.5% and 0.75%, so
the growth of Candida albicans more inhibited. These results are consistent with the notion
that the higher the concentration of a substance anti-microorganism would the more quickly
die or inhibited cell growth of microorganisms, the ability to inhibit and damage the fungal
life by anti-fungal properties due terapoetik14. To determine the effect of acrylic resin
incorporate with nano chitosan on the growth of the fungus Candida albicans in denture used
analysis of variance (p <0.05) which can be seen in Table 2:
Table 2. Results of the analysis of variance calculation lane number on the fungus Candida
albicans different concentrations of chitosan nano.

Sum of Df Mean Square F Siq

Squares
Between 1.5057 4 3761660.750 78186.944 000
Groups
Within 2165.000 45 48.111
Total 1.5057 49

Calculation results of the oneway analysis of variance, significant differences in

acrylic resin incorporate with chitosan nano with a concentration of 0.25%, 0.5%, 0.75% and
1% to the growth of candida albicans (p <0,05). To determine differences in the mean number
of colonies of Candida albicans in the treatment group with a control group tested LSD.
Calculation by LSD test showed significant difference between groups nano chitosan
concentration on the growth of Candida albicans. These results showed different inhibition of
the growth of the fungus Candida albicans NH3+ group contained by chitosan. Interaction
between chitosan and fungi can resist fungal growth biological membranes by fungal cell
destruction, so that chitosan can be used as anti-fungal agents potential16. Chitosan antifungal
mechanism through interactions between the positive charge on the unit group NH3 +
Glucosamine with a negative charge on the cell membrane of fungi. This electrostatic
interaction process causes changes in the permeability of fungal cell walls and change the
internal osmotic balance which can inhibit fungal growth, resulting in the hydrolysis of
peptidoglycan in walls of fungi intrselluler resulting in loss of electrolytes, proteins, nucleic
acids and glucose in fungi11. Denaturation of proteins can damage the cell harvester and can
not be repaired anymore14.

using the number of control fungi and number in each Candida albicans
concentrations obtained MIC group of acrylic resin with nano chitosan concentration of
0.25% is 84.57%, at a concentration of 0.05% by 85.35%, 0.75% concentration of 92.70%
and at 0.1% concentrations of chitosan nano 94.88%. The results showed that acrylic resin
incorporate with nano chitosan concentration of 1% is the concentration of the most effective
in inhibiting the growth of Candida albicans. This is due to the concentration of 0.1%
inhibition levels reaching 94.88% which means are fungistatic. A solution is a fungicide if
MIC reached 99.9% and fungistatic if the MIC less than 99.9%16.
 Based on the results of this study can be concluded: acrylic resin incorporate with
chitosan nano can inhibit the growth of Candida albicans on the base plate denture acrylic
resin. Acrylic resin incorporate with nano chitosan concentration of 1% had the highest
antifungal against Candida albicans growth in the base plate denture acrylic resin

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