CALCIUM PHOSPHATE BASED ANTIBACTERIAL CERAMIC POWDER CONTAINING ENAMEL
Aydin Dogan1, Aslan Gencer1, Ceren Peksen1, Savas Koparal2 , Filiz Bayrakci2, Ayhan Çavusoglu3 and Salih Paytuncu3
1Anadolu University, Department of Materials Science and Engineering, Eskisehir/Turkey
2Anadolu University, Department of Environmental Engineering, Eskisehir/Turkey
3Ege Kimya San. Tic. A.S., Istanbul/Turkey

Abstract
Risk of epidemic diseases pushes scientist to create new products for healthier living environments. Recent epidemic of SARS and Bird flue killed many people and caused a lot of economical losses. In the last decade there have been many studies conducted on metal ion based especially metal cation consisting antibacterial systems. In this study, metal cation consisting calcium phosphate based antibacterial ceramic powders were synthesized with wet chemical technique and were input to the enamel composition to create antibacterial enamels.
Contact tests were performed to investigate the antibacterial activity of antibacterial enamel surface against harmful pathogenic microorganisms. For the antibacterial test Escherichia-coli bacteria which are the mostly encountered bacteria type for human beings in daily life were chosen as the indicator bacteria to show the antibacterial power.

Introduction
Bacteria are small living organism; their length change between 0.5-10 μm and their diameters are between 0.2-10 μm. Some bacteria types have polysaccharide-based capsule on their cell wall.
They don’t have nucleus membrane, mitochondria, endoplasmic reticulum and golgi complex. Most of the information about bacteria have been obtained from studies on Escherichia coli (E-coli), which are relatively less harmful parasite living in human and animal intestine [1].
E-coli is a bacteria type that is 2.0-6.0 μm in length and 1.0-1.5 μm in width. It has flat surface and in some kinds, they can appear as Y-shaped. Although they can move using their microvilli, their motion is very slow; even one can assume this motionless.
Some metal ions such as Ag+2, Zn+2 and Cu+2 can interact with bacteria’s metabolism and neutralize their enzymes. With the increase of their bactericide properties, silver and some other metal ions are re-emerging as a modern medicine because pathological organisms have failed to develop immunity to it. Silver, zinc and copper exhibit antibacterial effect at various levels [2].
Amorphous silicate, calcium-aluminum-silicate and calcium phosphate based structures are used as carrier body for antibacterial metal cations. Among them, calcium phosphate based antibacterial ceramics have highest bio-compatibility and nontoxic properties [3]. Besides, kinetic of cation exchange with metals is rather fast causing to increase in antibacterial efficiency of metal ions in system [3-4]. The enameled surfaces of the product have an image of cleanness. The enamel is very suitable for water tanks, boilers, washing machines, dishwashers and exhaust pipes. It shows high resistance to alkali, boiling water, water vapour and acids. The aim of this study was to make antibacterial enamel for home and industrial appliances for cleaner environments.

Experimental procedure
Synthesis of Metal Ion Doped Antibacterial Ceramic Powder: In this investigation in collaboration with Iltekser Seramik Ltd. a special composition for enamel applications was synthesized using wet chemical methods. After the reaction, solution was filtered and dried at 120 °C. Synthesized material basically had the calcium phosphate based structures with metal cations, leading to antibacterial effect. Crystal structure of the synthesized powder was characterized with a Rigaku Xray diffractometer.
Preparation of Antibacterial Enamel: The prepared antibacterial powder (named as PAK), was added to the 14100 series white colour enamel compositions given in the Table 1. Antibacterial powder was added to the enamel composition in 1, 3, and 5 weight percent based on the frit content in the enamel composition. White colour enamel was deliberately chosen because of its abundant usage and to investigate the effect of antibacterial agent addition on the optical properties of the enamel.

Table 1. The compositions of the antibacterial enamel samples
Sample
Name
Frit (g)
PAK (g)
Clay (g)
NaAlO2 (g)
Water
A
100
-
5
0.35
45
A1
100
1
5
0.35
45
A3
100
3
5
0.35
45
A5
100
5
5
0.35
45
A*: Reference enamel

the surfaces of the steel sheets were prepared by using cleaning solution and undercoat layer was applied on. Then, samples were dried at 150 oC for 30 minutes and fired at 840 oC for 5 minutes.
The thicknesses of undercoats were tried to keep in between 130-140 μm. The second layer of enamel with various compositions (A, A1, A3, and A5) of antibacterial agent was prepared and applied by using the route. The firing temperature of the second layers was 820 oC and the thickness was in between 250-260 μm.
Antibacterial Activity Test: There are three methods commonly used to investigate the antibacterial activity of materials; Halo test, contact film test and shake flask test. Contact test is the more accurate method for the dense coating surfaces. For this reason it was applied to investigate the bactericidal activity of the enameled surfaces. All glass equipments were sterilized in an autoclave at 145 °C for 45 min before bacteria test. Enameled samples were sterilized at 200 °C for 2 hr. Ecoli colonies were cultivated in nutrient broth at 37 °C for 24 hours in shaking incubator. Formed colonies were diluted using dilution solutions e.g. saline at the end of that period. Diluted E-coli bacteria consisting solutions were dropped to bottom of the Petri dishes and the enameled surface of the samples were faced down into them. It was assured that a tiny layer of bacteria consisting diluted solution has been in contact with the enameled surface. Then, the samples were left in incubators at 25 °C and 37 °C for 24 hours. After incubation period, the samples were removed from the Petri dishes and the remaining solutions beneath the samples were cultivated to supplemented medium in different Petri dishes. Then, the Petri dishes were set in incubator and finally bacteria growth was observed at various time intervals.
Optical Test of The Enamel Surface: From the customer point of view, color of the surface is also very important parameter. Optical properties of the enameled surface with antibacterial agent were tested with a Minolta-spectrophotometer CM-3600.

Results and discussions
Structural Analysis: Result of the X-ray diffractometer showed that the crystal structure of synthesized powder was a mixture of calcium phosphates. After drying process agglomeration on the powder has been observed. Dry milling process was employed to decrease the particle size to micrometer levels. Particle size of the used antibacterial ceramic powder was measured around 1 μm with Malvern Mastersizer 2000 particle size analyzer.
Color Test: Optical L*a*b* values of the enamel samples were measured by using Minoltaspectrophotometer CM-3600 d and the results are listed in the Table 2. Moreover, the surfaces of the enamel were also visually inspected. For the colour measurement; L* is used for the indication of darkness-brightness, a* is used for greenness-redness and b* is used for yellowness-blueness in spectral explanation of color. The brightness of colour expressed with h. As it is seen on the table, addition of 1 % antibacterial (A1) agent to the white color enamel does not cause any major changes on the optical properties of the enamel. On the visual inspection, no difference has been observed in between the surfaces of the reference enamel and A1. However, as a general trend increasing antibacterial content of the enamel makes the enamel surface slightly darker and causes slight decrease on brightness. It was also visually observed that even tough there are not considerable colour changes; the bright surface of the enamel gets matt with increasing antibacterial agent content. In all compositional variation no defects have been observed on enamel surfaces.

Table 2. The coloring parameters of the samples
L*
DL
a*
D a*
b*
D b*
C*
h
D h
A1
93.01
0
-1.06
0
1.62
1.93
123.18
0
A3
93.75
0.74
-0.97
+0.09
1.39
-0.23
1.69
124.99
1.81
A5
93.64
0.63
-1.05
+0.01
2.09
+0.47
2.34
111.66
-11.52

Antibacterial Activity Test: The reduction ratio on the growth rate of bacteria shows the antibacterial activity. E-coli bacteria have been used for the antibacterial activity test. Coli form Unit (CFU) and colony numbers on the Petri dishes were counted and following formula were used or the antibacterial activity.

Results of the antibacterial activity test of the samples are listed on the Table 3. Values on the table are the average of five to six samples. The reference enamel sample shows no antibacterial activity. Very high antibacterial activity level has been observed for all compositions of the antibacterial agent added enamel composition.

Table 3. Antibacterial activity test results
Sample
CFU/ml at 0 hr
CFU/ml after 24 hr
Antibacterial
Activity %
A
~2x103
~2.3x103
0
A1
~2x103
~2
99.91
A3
~2x103
~2
99.91
A5
~2x103
~3
99.91

The images of the Petri dishes are show in the Figures 1-4 for various compositions after 24 hours.
As it is mentioned on the test method, E-coli bacteria in saline solution were contacted on enamel surfaces for 24 hrs incubation period, afterwards remaining solution beneath the samples were cultivated to supplemented medium in different Petri dishes. Therefore the Petri dishes with supplemented medium and bacteria colonies are shown on the pictures. As it is clearly seen there is no antibacterial activity of the reference enamel. White spots on the Petri dishes are the E-coli bacteria colonies. Only a few bacteria colonies have been observed for the antibacterial enamels for various compositions. As a result of this investigation, it is possible to say that addition of even % 1 antibacterial agent (PAK) to the enamel composition can create antibacterial activity.

Fig. 1 E-coli bacteria colonies on Petri dishes for reference enamel after 24 hours
Fig. 2 Petri dishes for 1% PAK added enamel after 24 hours, no colony formation

Fig. 3 Petri dishes for 3 % PAK added enamel after 24 hours, no colony formation
Fig. 4 Petri dishes for 5 % PAK added enamel after 24 hours, no colony formation

References
[1] Geo.F. Brooks, Janet S. Butel, L. Nicholas Ornston, Ernest Jawetz, Joseph L. Melnick and Edward A. Adelberg: Medical Microbiology (Prentice-Hall International Inc. 1991),
[2] Zhao G., Stevens S.E., Department of Microbiology and Molecular Cells Sciences, The University of Memphis, USA, 1997.
[3] L.L. Hench and J. Wilson: An Introduction to Bioceramics (World Scientific Publications, 1993),
[4] A. Dogan, E. Uzgur, S. Koparal and F. Bayrakci: Ceramic: Art, Science & Technology Turkish Ceramic Society Vol. 17 (2001), p.20 (in Turkish).

 

 

 

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