Download In-vitro study: Biodegradation of orthodontic brackets in artificial saliva

In Vitro - Evaluation of Biodegradation of Different Metallic Orthodontic Brackets
Authors:
Afifah Adilah Asshaari: Faculty of Dentistry, Universiti Teknologi MARA (UiTM), 40450 Shah
Alam, Selangor Darul Ehsan, Malaysia. E-mail: [email protected]
Author contribution:
Bibi Aisiah Babu Osman: Faculty of Dentistry, Universiti Teknologi MARA (UiTM), 40450
Shah Alam, Selangor Darul Ehsan, Malaysia. E-mail: [email protected]
Author contribution:
Saba F. Hussain: Faculty of Dentistry, Universiti Teknologi MARA (UiTM), 40450 Shah Alam,
Selangor Darul Ehsan, Malaysia. E-mail: [email protected]
Author contribution:
Fouad Hussain AL-Bayaty: Faculty of Dentistry, Universiti Teknologi MARA (UiTM), 40450
Shah Alam, Selangor Darul Ehsan, Malaysia. [email protected]
Author contribution:
Amalina bt Amir: Faculty of Dentistry, Universiti Teknologi MARA (UiTM), 40450 Shah Alam,
Selangor Darul Ehsan, Malaysia. E-mail: [email protected]
Corresponding author
Dr. Saba F. Hussain:
E-Mail:[email protected]
Address: Center of Study of Paediatric Dentistry and Orthodontics, Faculty of Dentistry, University
Technology MARA, (UiTM), 40450 Shah Alam, Selangor Darul Ehsan, Malaysia.
Tel.: +603-5543 5834; Fax: +603-5543 5803
1
ABSTRACT
This study was to evaluate the chemical and structural changes of different orthodontic metallic brackets in
biodegradation process. A total of 240 brackets of different manufacturers were used (nickel-
titanium and stainless steel brackets). Each control group and experimental group consisted of 120
brackets of both manufacturers were immersed in distilled water, artificial saliva respectively for
a period of 28 days at 37OC under mechanical agitation. The release of ions was tested using
inductively coupled plasma mass (ICP) spectrometry at 1, 7 and 28 days. Surface roughness was
tested using scanning electron microscope (SEM) and profilometer. Energy-dispersive X-ray
(EDX) spectroscopy was performed to detect changes in chemical compositions. In general, The
study showed a significant difference in biodegradation between different metallic orthodontic
brackets. Stainless steel brackets showed more aggressive chemical and structural changes
compared to nickel- titanium brackets as well as significant increase in surface roughness after
immersion in artificial saliva.
Keywords: Biodegradation, Biocompatibility, Corrosion, Artificial saliva, Orthodontic brackets
.
2
INTRODUCTION
In the oral environment, biodegradation of orthodontic brackets occurs usually by
electrochemical reaction, which is corrosion. Corrosion is an electrochemical reaction of a metal
or alloy with different components of surrounding environment (6). The biodegradation process
of stainless steel on titanium-based brackets in oral rehabilitation systems commences by corrosion
(7). These wear processes take place simultaneously and are influenced by many factors due to the
presence of ions, minerals, pH of saliva and solutions (8). The effect of corrosion, which is the
release of ions into the oral cavity, is discussed because of its biological effects. Biocompatibility
of orthodontic bracket material has been investigated over the past 20 years. However, these
studies have given rise to questions without scientific answers. Thus, this confirms the need for
more studies to look into the biocompatibility of these materials. Since the biodegradation process
is not fully clear and explained. On the other hand orthodontists may be confused in selection of
biologically safe appliance for the patient.
The introduction of metal ions into human body is an additional hazard to health. Since these ions
could be released from different metallic orthodontic brackets at different level in oral environment
resulting from biodegradation process. Depending on characteristic of metal ions and their
solubility of the product containing them, the biological function is affected and leads to systemic
and local effects like allergy (9)
Orthodontic metal brackets are subjected to biodegradation process in the oral cavity.
Biodegradation is defined as the series of processes by which living systems render chemicals less
noxious to the environment. Living systems in the oral environment, which are microorganisms
and enzymatic phenomena, can cause corrosion and other associated problems during long time
exposure (10).
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According to Yip et al, (11), corrosion is an electrochemical reaction of metals with their
surrounding environment, leading to a loss of metals or conversion of metal into oxide. Corrosion
can be divided into seven types which are pitting corrosion, crevice corrosion, galvanic corrosion,
inter-granular corrosion, fretting corrosion, stress corrosion and microbiologically influenced
corrosion. Pitting and crevice type of corrosion form on the metallic orthodontic brackets surfaces
exhibit many pits and crevices and it is thought to increase the susceptibility of corrosion because
of their ability to harbor plaque forming microorganism.
In the oral cavity, corrosion takes place by the release of metals ions from orthodontic alloys to
form more stable compounds (12). Thus, corrosion behavior of orthodontic metal brackets also
affects their biocompatibility. Biocompatibility is defined as “the ability of a material to elicit an
appropriate biological response in a given application” (12). It is the metallic corrosion products
entering the body, such as nickel, titanium, iron and chromium that are being subjects of interest
due to their allergenic and cytotoxic effects (13).
Allergic reaction is an acquired condition resulting in an overreaction upon contact with foreign
substance, and arises from a genetic predisposition and previous sensitization from exposure to the
substance. (14). Allergic was divided into four type (type I to IV), immediate reaction (type I) and
delayed reaction (type IV) have relevance to orthodontic biomaterials. Nickel and chromium
induce Type-IV hypersensitivity reaction in the body. These metals also cause several cytotoxic
responses including decrease in some enzyme activities, interference with biochemical pathways,
carcinogenicity, and mutagenicity. Nickel has attracted the most attention as a potential sensitizer
in the odontological setting (14) because nickel atoms are not strongly bonded to form some
4
intermetallic compound, the likelihood of in vivo slow nickel ion release from the alloy surface is
increased, which may have implications for the biocompatibility of these alloys (12).
In dentistry, a factor to determine the biocompatibility of the alloys used is their resistance to
corrosion. Austenitic stainless steel metallic orthodontic brackets have a high stiffness and
formability that contains of chromium, carbon and nickel. The chromium helps the brackets
become more resistance toward corrosion by forming a passive layer on the metal surface, but this
film can be damaged by aggressive attack by ions such as fluoride and chloride. The same goes to
nickel titanium type of orthodontic metallic brackets, where it forms a passivation titanium-oxide
layer that provides a protection against corrosion but 1% lactic solution causing this layer is
insufficient in reducing the dissolution of metal ions (11).
The observation of the surface structure and morphology in order to detect the corrosion of the
metallic orthodontic brackets is straightforward method to evaluate the biodegradation process of
the orthodontic brackets. Chappard et al found a positive relationship between roughness analyzed
by microscopic images (SEM) and level of roughness measured by contact profilometer. The
objectives of this study are to evaluate the effects of artificial saliva on chemical and structural
changes of orthodontic metallic brackets and to compare between the changes of different metallic
brackets in biodegradation process.
5
MATERIAL AND METHODS
General
Two hundred and forty metallic orthodontic brackets of two different manufacturer which are
American orthodontic (Stainless-Steel) and Dentarum (Nickel-Titanium) were used for this study.
All orthodontic brackets were disinfected using alchohol spray and allowed to dry before testing
to avoid any contamination during the procedure. The weight of each subgroup of orthodontic
brackets was assessed using electrical balance to insure standardization before testing
Control group
One hundred and twenty metallic orthodontic brackets of different manufacturer were immersed
in distilled water for period of 1, 7 and 28 days. Each bracket was mechanically agitate in a salt
spray tester machine set at constant temperature of 37°C in individual 15ml plastic-capped vials
containing 15ml solution.
Experimental group
One hundred and twenty metallic orthodontic brackets of different manufacturer were immersed
into Modified Fusayama artificial saliva under same condition for 1, 7, and 28 days.
Preparation of artificial saliva
Artificial saliva was formulated according to Fusayama with modification for the biodegradation
test. The prepared solution is frequently used in previous researches and it generates similar
electrochemical behavior of metallic material as in human saliva. The artificial saliva was prepared
according to the Table 1(15)
Microscopic brackets analysis (SEM)
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Scanning electron microscopy (SEM) was used to analyze the surface and structural changes of
randomly selected 72 metallic orthodontic brackets at T0, T1, T2, and T3 on the frontal, base and
wing surfaces. The micrographs obtained are presented at x200, x500, x1000 and x2000
magnification. A single examiner examined all the micrograph.
Surface characterization analysis (Profilometer)
In order to compare the surface roughness of metallic orthodontic brackets of different
manufacturer included in this study, profilometer is used to quantify and compare surface
roughness (Ra) for all groups at T0, T1, T2 and T3.
Analysis of the chemical composition of the brackets (EDX)
An Energy Dispersive X-Ray (EDX), which is a SEM resource that allow for evaluation of the
chemical composition of the brackets. The procedure was standardizing by performed on 6
brackets for each group on different surface buccal, gingival, base and wing surfaces. In order to
quantify the Nickel, Copper, Ferrous, Cobalt and Chromium ions in metallic orthodontic brackets
at T0, T1, T2, and T3.
Analysis of the chemical composition of the solution (ICP)
The different immersion solution including distilled water and artificial saliva were tested with an
inductively coupled plasma (ICP) spectrometer at T0, T1, T2 and T3. Each solution was analyzed
for Nickel, Copper, Ferrous, Cobalt and Chromium ions.
Statistical analysis
7
The result of present study was subjected to statistical analysis to interpret the differences and the
significance between surface roughness and chemical composition values in each group. Two-way
analysis of variance (ANOVA) was used in this study. ANOVA was used to study the overall
variance within groups.
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RESULT
The result obtained from the control and experimental group demonstrated in terms of
surface changes on the brackets viewed from scanning electron microscope (SEM), values of
surface roughness on the brackets surface measured by profilometer, chemical composition of
metal ions of the brackets measured with EDX and amount of metal ion release inside each solution
quantified by ICP.
After 28 days, it was clearly shown there were changes in all of the brackets; in terms of surface
roughness and their chemical composition, and significant amount of ion release into the solutions.
AO brackets reviled more structural changes compared to Dentaurum brackets in terms of more
surface roughness obtained from SEM. These findings was obviously supported by profilometer.
Besides that the results showed that artificial saliva caused more obvious corrosion compared to
distilled water and this is evident from the analysis of all four methods. The time interval also
played an important role as the evidence of biodegradation was increased proportionally with
duration of immersion of brackets.
Microscopic bracket analysis (SEM)
The SEM analysis at T0 and T3 indicated that alterations were found on the surfaces of the brackets
after the immersion period. In the frontal images, products of corrosion were identified in both
brands. When comparing Dentarum brackets with AO brackets immersed in artificial saliva at T3,
the corrosion pattern in seen more obviously with AO brackets, as the corrosion effect is deeper.
The type of corrosion is also different between each brand. Evidence of corrosion was also seen at
the solder junction of the bracket. Analysis of the side images indicated that the region most
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significantly affected in were the solder junction, especially the angle formed between the wing
and the bracket base. (Fig 1)
Surface characterization analysis (Profilometer)
The descriptive statistic for the surface roughness data of metallic orthodontic bracket after the
immersion into different solution are shown in Table 2. The result of two ways ANOVA showed
that total mean value of surface roughness in AO (mean = 0.57) is higher than Dentarum (mean =
0.45) metallic orthodontic brackets. (Table 2, Fig. 2)
The two ways ANOVA test showed there was statistically different between surface roughness
value and different immersion solution (P = 0.07) and there was no statistically difference between
surface roughness value and different brands (P = 0.369).
Mean level and standard deviation of chemical composition of metallic orthodontic brackets in the
groups are shown in the Table 3. The result of two ways ANOVA showed that the chemical
composition of metallic orthodontic brackets of different brands in different immersion solution
was significantly difference (P = 0.01). The chemical composition of ferrous between different
brands was significantly different (P = 0.025) but chromium and copper are not significantly
different (P = 0.067, P = 0.162). Also, composition of copper in different immersion solution was
significantly difference (P = 0.024) with no significant difference of nickel, ferrous and chromium
composition in different immersion solution (P = 0.185, P = 0.376, P = 0.873). Chemical
composition of chromium and nickel in AO metallic orthodontic brackets showed lower mean
value compare to Dentarum but copper and ferrous showed higher mean value in AO compare to
Dentarum bracket. (Table 3, Fig 3).
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Analysis of the chemical composition of immersion solution (ICP)
The descriptive statistic of mean level and standard deviation of ions released in the groups are
shown in the table (Table 4). The result of two ways ANOVA showed that the release of ions in
different solution and brands was significantly different. (P = 0.04). The total mean value of ions
release in AO (mean = 31.04) is higher than in Dentarum (mean = 2.704). Also, the mean value of
nickel release in artificial saliva is higher than and distilled water. On other hand, the mean value
of all ions released in artificial saliva was higher than the amount of ions released in distilled water.
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DISCUSSION
Over the years, studies have shown alarming reports on the corrosion potential of AISI Type 316L
austenitic stainless steel alloy currently used for stainless steel manufacturing. Corrosion resistance
of stainless steel is formed by the formation of a chromium oxide layer at its surface; meanwhile
titanium and its alloy are made corrosion resistant by formation of titanium oxide layer. The subject
that gains interest is the release of nickel ions during corrosion process. Nickel is incorporated into
austenitic stainless steel alloys to stabilize its formation process. Then recently, titanium brackets
are making its way into the orthodontic clinical application. Their mechanical properties are
equivalent and even better from stainless steel brackets and more importantly; they are more
resistant to corrosion and less potential for nickel leaching.
Huang et al stated that metal ions are released from an orthodontic metal bracket at pH 4, which is
an acidic environment (16). It is also stated that metal brackets showed higher corrosion tendencies
in the pH 4 (17). Artificial saliva is used in this study to demonstrate their effects to the structural
and biological integrity of metallic brackets. Not many studies have been done up to this date
studying the topic of corrosion in artificial saliva in different type of brackets. We find that this is
an important topic to be studied on because some orthodontic patients are highly susceptibility to
allergy due to metal ions release from certain brackets especially stainless steel. Artificial saliva is
included in this study to best simulate the process of corrosion in the oral cavity. The samples were
also mechanically agitated. Mechanical agitation is achieved by the salt spray test machine which
is a standardized test method used to check corrosion resistance of samples. It works by production
of electrical currents due to the dynamic movement of water inside the machine’s test chamber.
The electrical currents will further enhance in the corrosion process of the metallic brackets.
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Power analysis can be used to calculate the minimum sample size required so that one can be
reasonably likely to detect an effect of a given size. In our study, to meet the required power of
sample, we have a total of 240 brackets altogether.
Microscopic analyses can provide only a visualization of surface morphology but cannot actually
quantitatively measure it (18). The surface finish of the metal alloy is an important factor to prevent
corrosion pits and cracks. Surfaces that are rough and irregular predispose to corrosion process
and increase the area of metal dissolution. (19) It was stated that the solder used in bracket is the
significant factor for the onset of the corrosion process (20). AO brackets seem to be more
susceptible to corrosion because they displayed a marked increase in formation of corrosion
products compared to Dentarum brackets. This is due to the fact that the composition of alloys
used in the two different brands where Dentarum ia a nickel- titanium based alloy meanwhile AO
is a stainless-steel based alloy. Moreover, we observed at T3 that the brackets often showed the
formation of superficial corrosion layers, which we can assume to be a dynamic stage in the
corrosion process.
Profilometry is a widely accepted method to assess surface condition and a common method to
analyze surface configuration with a non-invasive approach (21). Plus, it provides a quantitative
scale and can be used to compare surface roughness of the treated surfaces, which SEM cannot.
Comparing between the two solutions, surface roughness was significantly different, for example
AO bracket immersed in artificial saliva showing higher value (Ra =0.609987) compared to
distilled water (Ra = 0.174853). On the other hand, AO brackets showed the higher value of
corrosion compared to Dentarum brackets in all immersion solution. This is in correlation to the
SEM results that artificial saliva produced more corrosion and AO brackets are more prone to
corrosion compared to Dentarum brackets.
13
The EDX is a SEM tool that detects and quantifies the metals comprised in an alloy, and this
enables us to measure the release of metallic ions in an indirect fashion. This method according to
Eliades et al has clinical relevance and produces results with high reliability (22&23). From our
study, we can see that the chemical compositions of AO brackets are much less compared to
Dentarum brackets. This shows that AO brackets are less corrosion resistant where the metallic
ions from the brackets are released into the solution. The chemical composition of nickel and
ferrous between different brand was significantly different. The mean value of nickel composition
of metallic orthodontic bracket immersed in artificial saliva was higher compare to distilled water.
This indicates that more nickel ions were lost from the brackets by corrosion in artificial saliva.
(24)
ICP analysis is a different method from AAS analysis where it has the advantage of extracting
each metal ions and detecting them without causing any interference to other ions. Therefore, ICP
analysis was selected to detect the level of ion released in the resulting solutions after mechanical
agitation. From our study, we found that the amount of ion released were higher in AO brackets
compared to Dentarum brackets regardless of its immersion solution. We can conclude that this is
due to the more corrosion resistant characteristic of Dentarum bracket that is composed of nickel
titanium alloy. It is also seen that the amount of nickel ion released is highly significant in artificial
saliva compared to distilled water. This shows the corrosiveness of the artificial saliva and its
effect, which is the release of harmful ions, which is nickel into the living systems.
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CONCLUSIONS
The orthodontic brackets, which released the most ions in this study, are those immersed in
artificial saliva. With the limitations of our study, we can recommend orthodontists to choose NiTi
orthodontic brackets, as they are biologically safer for the patient. NiTi brackets are more
biocompatible and have higher corrosion resistance compared to stainless steel brackets. However,
even a small amount of ions released in biological system may provoke sensitivity reaction when
orthodontic appliances and placed in oral cavity for several years.
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ACKNOWLEDGEMENTS
First and foremost, we would like to thank to our supervisor of this project, Dr Saba F. Hussein
for her valuable guidance and advice. Her support has contributed tremendously to our project.
Also, we would like to take this opportunity to thank Faculty of Applied Sciences, Universiti
Teknologi Mara. In addition, we would also like to thank Nano Molecular Lab, Institute of Science,
Universiti Teknologi Mara. Not to forget, we would like to thank Research Laboratory, Faculty of
Dentistry, Universiti Teknologi Mara.
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Table 1: Composition of the stock Fusayama’s artificial saliva solution (20)
Compounds
Mg/l
NaCl
400
KCl
400
CaCl2·2H2O
795
NaH2PO4·H2O
690
KSCN
300
Na2S·9H2O
5
Urea
1000
Table 2: Mean and standard deviations of surface roughness value (Ra) in different brand and solution
Brand
Solution
Surface roughness (Ra)
Dentarum
Distilled water
0.15±0.03
Artificial saliva
0.32±0.12
Distilled water
0.18±0.02
Artificial saliva
0.41± 0.13
AO*
* (American orthodontics).
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Table 3: Chemical composition (mg/l) in different solution and brand: Mean and standard deviations (EDX)
Solution
Brand
Chromium
Cobalt
Copper
Nickel
Ferrous
Distilled water
AO*
15.85±0.99
0
1.595±0.12
6.856±0.62
67.75±5.02
Dentarum
18.68±3.00
0
1.66±0.20
9.155±3.88
62.15±2.89
AO*
16.13±2.20
0
1.29±0.02
5.69±0.86
70.02±0.28
Dentarum
16.62±0.51
0
0.425±0.60
9.455±1.35
54.5±5.69
Artificial saliva
* (American orthodontics).
Table 4: Metal ion concentrations (mg/l) in different solution and brand: Mean and standard Deviations
(ICP)
Solution
Brand
Chromium
Cobalt
Copper
Nickel
Ferrous
Distilled water
AO*
0.29±0.00
0.04±0.00
0.02±0.00
0.04±0.00
0.008±0.00
Dentarum
0.27±0.00
0.04±0.00
0.001±0.06
0.2±0.04
0.02±0.05
AO*
0.006±0.06
0.003±0.07
0.09±0.11
0.12±0.28
0.029±0.02
Dentarum
0.005±0.06
0.002±0.07
0.05±0.11
0.01±0.08
0.05±0.06
Artificial saliva
*(American orthodontics).
21
A
B
Fig.1. Metallic orthodontic brackets under SEM with magnification 2000 x after immersion in artificial saliva (T3)
Showing fissure corrosion on the wings. A: NiTi orthodontic brackets (Dentarum). B: Stainless steel orthodontic
brackets (American orthodontics).
Fig 2: Surface roughness (Ra) in different brands and solutions
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Fig. 3: Chemical composition of AO (Stainless steel) metallic orthodontic brackets (Chromium, Copper, Nickel and
Ferrous) using EDX of all solutions.
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