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\title{Protective Effect of Different Extracts of Celtis Australis and Syzygium Aromaticum on Tetrahymena for the Cytotoxicity of Nickel-Titanium-Based Orthodontic Wires}
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             \author[1]{Nassiba  Fatene}

             \author[2]{Mar Papa  Daouda}

             \author[3]{Rachida  Cadi}

             \author[4]{Abdelaziz  Soukri}

             \author[5]{Khadija  Mounaji}

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\date{\small \em Received: 9 September 2021 Accepted: 5 October 2021 Published: 15 October 2021}

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\begin{abstract}
        


Introduction:The present work aims to study the toxicity of orthodontic archwires based on Nickel-Titanium and to evaluate the protective effect of different types of extracts of Celtis australis and Syzygium aromaticum, considered as natural corrosion inhibitors, on Tetrahymena thermophila and Tetrahymena pyriformis.Methods: Tetrahymena thermophila and Tetrahymena pyriformis were cultured in artificial saliva previously incubated in the presence of NiTi or CuNiTi wires with or without the addition of different types of plant extracts (extract, hydrosols, essential oils). The effect of wires and plant extracts on Tetrahymena was evaluated after 2, 4 and 7 days of growth, by the protozoan viability test and the microscopic observation of the shape.Results: For the two Tetrahymena species, NiTi and CuNiTi cause a decrease in Tetrahymena growth by about 50%.

\end{abstract}


\keywords{orthodontic wires, nickel-titanium, cytotoxicity, aromatic plants, tetrahymena.}

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\let\tabcellsep& 	 	 		 
\section[{Introduction}]{Introduction}\par
he biocompatibility of orthodontic materials has been widely studied due to the importance of this property for patient safety. Most orthodontic materials contain metals, which can be toxic and produce allergic reactions  {\ref 1,}\hyperref[b1]{2} . It has been proven that in the oral environment, orthodontic archwires undergo chemical corrosion leading to the release of ions in saliva \hyperref[b2]{3} . Nickel-Titanium wires are an important part of the therapeutic arsenal during fixed orthodontic treatment. They contain about 47-50\% of Nickel and are the richest source of this metal in the oral cavity of most patients with orthodontic appliances. Furthermore, Nickel and Titanium are known for their toxic and carcinogenic effects \hyperref[b3]{[4]}\hyperref[b4]{[5]}\hyperref[b5]{[6]} .\par
In the literature, cell culture is the most widely used method to assess the toxicity of orthodontic materials in the oral environment. Several other models for studying toxicity were described. Among them, Saccharomyces cerevisiae have been used to study orthodontic material cytotoxicity \hyperref[b7]{7} . Other microorganisms have also been used in toxicology, like the ciliated protozoan Tetrahymena \hyperref[b8]{8} .\par
Unlike other single-cell microorganisms that are widely used as models, this protozoan has the advantage of having several genes found in several eukaryotes, including humans \hyperref[b9]{9} . More than 800 human genes have orthologs in Tetrahymena thermophila, but not in S. cerevisiae, 58 of them are associated with human diseases \hyperref[b10]{10} . This characteristic suggests that Tetrahymena can be used as a model to improve the understanding of the molecular mechanisms involved in the toxicity of orthodontic materials \hyperref[b11]{11} .\par
On the other hand, to stop alloy'scorrosion, researchers tested several methods. Among them, the use of natural corrosion inhibitors or green corrosion inhibitors extracted from aromatic plants has been widely studied in industry \hyperref[b12]{12} . Indeed, the use of different types of aromatic plant extracts (essential oils, hydrosols and extracts) has a protective effect against the corrosion of metals in an acid environment avoiding by the way the use of chemical substances \hyperref[b13]{13} . In addition, it has been described that some aromatic plants, such as Artemisia and Syzygium aromaticum, have anti-corrosive properties \hyperref[b14]{[14]}\hyperref[b15]{[15]}\hyperref[b16]{[16]} .\par
The aim of this work is to assess the cytotoxicity of Nickel-Titanium-based orthodontic archwires and to study the protective effect of different types of aromatic plant extracts, considered as natural corrosion inhibitors, using Tetrahymena thermophila and Tetrahymena pyriformis as study models. 
\section[{II.}]{II.} 
\section[{Material and Methods}]{Material and Methods} 
\section[{a) Culture of Tetrahymena}]{a) Culture of Tetrahymena}\par
Tetrahymena thermophila SB 1969 and Tetrahymena pyriformis SE, ATCC30005 were used for this study. Both species were kept growing in the PPYE medium containing 0.5\% (w/v) of Proteose Peptone and 0.2\% (w/v) of yeast extract. Artificial saliva was prepared by adding to the PPYE medium 0.035\% (w/v) of Sodium Chloride (NaCl), 0.2\% (w/v) of Calcium Chloride (CaCl2) and 0.2\% (w/v) Potassium Chloride (KCl). Then, in this culture medium was added 1\% (v/v) of a pre-culture of Tetrahymena thermophila (1.5×105 cells/ml) and incubated at 32°C. or of Tetrahymena pyriformis (104 cells/ml) and incubated at 28°C. In order to check the growth and adaptation of the protozoan to artificial saliva, pre-cultures were carried out and monitored for 3 months. Then, during one year, a transplanting was carried out once a week. 
\section[{b) Preparation of wires and plant extracts}]{b) Preparation of wires and plant extracts}\par
NiTi (3M) and CuNiTi (ORMODENT, California) orthodontic arch-wires were cut into 10mm pieces and then sterilized. The different types of extracts were prepared from Syzygium aromaticum (Clove) and Celtis australis. The essential oil and the hydrosol were obtained by hydrodistillation using a Clevenger type device (2 liter reactor), for a period of five hours. These extracts were then stored inamber glass bottles at a temperature of 4°C.\par
The extract was obtained by macerating the powder of the leaves of Celtis australis in distilled watermethanol (2V/3V) for 48 hours at 25°C.\par
The essential oil and hydrosol of Syzygium aromaticum, the extract and the essential oil of Celtis australis were chosen for this study (the choice of plant extracts and concentrations used was based on the results obtained by our team; results being published). 
\section[{c) Assessment of the effect of orthodontic archwires and the anti-corrosion potential of different types of plant extracts on the growth of Tetrahymena}]{c) Assessment of the effect of orthodontic archwires and the anti-corrosion potential of different types of plant extracts on the growth of Tetrahymena}\par
Each piece of orthodontic archwire was incubated in 20ml of artificial saliva with or without the addition of the extract, hydrosol or essential oils, as shown in detail in Figure \hyperref[fig_0]{1}. These media were incubated at 37°C for 15 days with agitation to simulate the oral conditions. Then, these media were distributed in 4 tubes, of 5 ml each, then inoculated with a pre-culture of Tetrahymena thermophila (1.5x105 cells/ml) or Tetrahymena pyriformis (104 cells/ml).\par
Protozoan growth was monitored during 7 days of culture by measuring the optical density at 600 nm using the spectrophotometer.  
\section[{d) Evaluation of cell viability and morphology of Tetrahymena}]{d) Evaluation of cell viability and morphology of Tetrahymena}\par
In order to calculate the percentage of living cells and to analyse the shape of the protozoan, a sample of 20 ?l of each culture medium was taken after 48 h, 96 h and 169 h of growth of Tetrahymena. These samples were stained with Trypan blue (2\%), fixed with Formaldehyde (4\%) and then placed in a Malassez cell for observation under the microscope.  
\section[{e) Statistical analysis}]{e) Statistical analysis}\par
Three replicates were made for each experiment and the mean and standard deviation were calculated. Statistical analysis was performed using Student's T-test and the differences were considered statistically significant if p<0.05. 
\section[{III.}]{III.} 
\section[{Results}]{Results} 
\section[{a) Growth of Tetrahymena thermophila and Tetrahymena pyriformis in artificial saliva}]{a) Growth of Tetrahymena thermophila and Tetrahymena pyriformis in artificial saliva}\par
Results show that in artificial saliva, the growth curves of Tetrahymena thermophila and Tetrahymena pyriformis are not modified in comparison with the PPYE medium (Figure \hyperref[fig_2]{2}).  Similarly, observation under the microscope does not show any change in the shape of the two species of Tetrahymena in artificial saliva compared to the PPYE medium (Figure \hyperref[fig_5]{4}).  In the presence of NiTi or CuNiTi orthodontic archwires, the growth of Tetrahymena thermophila is significantly reduced by 50\% and 60\% respectively compared to controls (Artificial saliva) (p <0.01) (Figure \hyperref[fig_4]{5}).\par
The same results are noted in Tetrahymena pyriformis the growth of Tetrahymena pyriformis is reduced by 72\% and 60\% respectively compared to the controls (artificial saliva) (p <0.01) (Figure \hyperref[fig_4]{5}).Results of the morphology analysis show that in the presence of orthodontic archwires, the majority of the protozoan appears in 2 shapes; elongated and rounded with a blue color after Trypan blue test compared to control (Figure \hyperref[fig_6]{6}).  
\section[{Year}]{Year} 
\section[{Global}]{Global} 
\section[{i. Effect of Celtis australis extracts on the growth of Tetrahymena}]{i. Effect of Celtis australis extracts on the growth of Tetrahymena}\par
During the protozoan growth kinetics, the number of living cells was counted during the 3 essential phases of the normal growth cycle of Tetrahymena: latency phase (24h), exponential phase (72h) and stationary phase (168h).\par
When the protozoan was cultured in the presence of the extract of Celtis australis and NiTi or CuNiTi, a remarkable increase in the number of living cells was noted for the two species compared to control (artificial saliva+arch)(figure \hyperref[fig_7]{7}). This increase was about 40\% during the first phase of protozoan growth and the growth continues to increase during the second and third phase. The growth curves of Tetrahymena thermophila and Tetrahymena pyriformis almost align with those of control (artificial saliva alone). However, Celtis australis essential oil did not show any protective effect on the growth of Tetraymena in the presence of wires (Figure \hyperref[fig_7]{7}).\par
Extract of Celtis australis protects the two species of Tetrahymena against the effect of NiTi and CuNiTi; the majority of cells presents a pear shape, which characterizes the normal shape of the protozoan, at the end of the latency phase (figure \hyperref[fig_8]{8}).\par
In solutions containing the extract of Celtis australis alone, there is no statistically significant difference in the growth of Tetrahymena thermophila and Tetrahymena pyriformis compared to the control (artificial saliva alone) (Figure \hyperref[fig_7]{7}).   
\section[{Effect of Syzygium aromaticum extracts on the growth of Tetrahymena}]{Effect of Syzygium aromaticum extracts on the growth of Tetrahymena}\par
In the presence of Syzygium aromaticum hydrosol alone with Tetrahymena, growth is approximately 80\% (p <0.05). Also, in the presence of the essential oil of Syzygium aromaticum alone, the growth is around 70\% (p <0.05).\par
In the solutions containing the wires and the hydrosol of Syzygium aromaticum, there is an increase in the rate of living cells by 50\% during the 1st phase of ii.\par
growth compared to the control (p<0.05). This growth decreases during the second (-20\%) and the third (-60\%) phase for the two Tetrahymena species. In addition, no growth was noted in the presence of the essential oil of Syzygium aromaticum for the two wires (Figure \hyperref[fig_8]{8}). All the differences are statistically significant except for the hydrosol of Syzygium aromaticum at the end of protozoan growth (Figure \hyperref[fig_10]{9}).\par
Regarding the morphology, in the solutions containing the hydrosol of Syzygium aromaticum, the two species of Tetrahymena show a pear shape at the end of the first phase of growth. In addition, from the second phase, the shape becomes rounded and the number and the mobility of cells decrease (Figure \hyperref[fig_12]{10}).     IV. 
\section[{Discussion}]{Discussion}\par
Fixed orthodontic appliances must guarantee absolute safety and biocompatibility \hyperref[b17]{17} . These qualities are of paramount importance in the oral cavity because this one constitutes a hostile chemical microenvironment that requires a high mechanical resistance of orthodontic alloys \hyperref[b18]{18} . In the presence of saliva that acts as an electrolyte, the orthodontic archwires undergo corrosion that causes the release of metal ions in the environment \hyperref[b19]{19} . To combat this corrosion, certain aromatic plants have proven their effectiveness as inhibitors of alloy corrosion \hyperref[b12]{12} .\par
The aim of this study was to assess the toxicity of Nickel-Titanium-based orthodontic archwires and to study the protective effect of different types of aromatic plant extracts, using Tetrahymena as a study model. Indeed, this protozoan constitutes a choice model for studies of environmental and industrial pollutants and of toxicity \hyperref[b21]{20} and several studies has shown that Tetrahymena can constitute a reliable and effective biomarker for the estimation of toxic effects from several chemical wastes \hyperref[b22]{21,}\hyperref[b23]{22} .\par
In addition, studies have reported that this unicellular organism has similar genes to those of humans \hyperref[b10]{10} and that it may also be useful in understanding the molecular mechanisms of toxicity in humans \hyperref[b24]{23} , this was the reason of its use in our study.\par
The perfect medium for Tetrahymena's growth is PPYE; a medium that contains all the nutrients that the protozoan needs for its growth \hyperref[b25]{24} . The use of this medium for toxicity tests of orthodontic archwires was not appropriate due to the absence of the elements constituting natural saliva. For this, our choice fell on artificial saliva; a culture medium which has already been described in the literature and adapted to the growth of Tetrahymena \hyperref[b26]{25} . During one year, several precultures of Tetrahymena were carried out, using artificial saliva, to have a generation perfectly adapted to this environment thus eliminating the specific stress due to artificial saliva. Our results have shown that the protozoan growth kinetic in artificial saliva is similar to the one of the PPYE medium.\par
In artificial saliva, Tetrahymena was cultured in the presence of NiTi or CuNiTi orthodontic wires to assess their cytotoxicity and the results showed a decrease in protozoan growth as well as a change in shape (elongated or rounded shape). Our results agree with those of Zhang and al. \hyperref[b27]{26} who also showed a decrease in protozoan growth in the presence of heavy metals.\par
In addition, other work has reported that the released nickel and copper ions penetrate inside Tetrahymena and stop its growth \hyperref[b28]{27,}\hyperref[b29]{28} . On the other hand, the released ions cause an unbalance between oxidants and antioxidants in the cell, inducing an oxidative stress that is involved in inflammation and in tumor pathology \hyperref[b30]{29} .\par
Our results showed that there is a protective effect of the extracts of Celtis australis and the hydrosol of Syzygium aromaticum against the toxicity of orthodontic archwires on Terahymena. Nilsson reported that the protozoan tolerates copper and nickel better in an organic solution than in a culture medium containing no nutrient \hyperref[b31]{30,}\hyperref[b32]{31} which may explain the protective effect of the two extracts on the protozoan. Other authors have confirmed the protective effect of these two aromatic plants, which is consistent with our results \hyperref[b33]{[32]}\hyperref[b34]{[33]}\hyperref[b35]{[34]} . These two plants are also known for their anticorrosive effect on metals that could have an indirect protective action on the protozoan by limiting the release of free radicals in the environment \hyperref[b36]{35,}\hyperref[b37]{36} . Indeed, in a later study conducted by our team \hyperref[b38]{37} , a high corrosion of NiTi and CuNiTi wires under the same conditions as the present study was noted.\par
The effect of aromatic plants on Tetrahymena has been the subject of several works in our laboratory \hyperref[b39]{[38]} {\ref [39]} {\ref [40]} and the protective effect of several essential oils (argan oil, sage and oregano) has been proven. However, the effect of essential oils and their corresponding extracts and hydrosols has never been studied on Tetrhaymena. The chemical composition of the extract and the hydrosol differs considerably from the corresponding essential oil 41 , they contain a good concentration of the main molecule of the plant without the toxic phenolic substances constituting the essential oils  {\ref 42} .The results of this study show that the essential oil of Syzygium aromaticum and Celtis autralis have no protective effect on Tetrahymena against the cytotoxicity of orthodontic archwires by indirect action causing chemical corrosion which would increase the rate of ions present in saliva. On the other hand, the use of the extract of Celtis australis and the hydrosol of Syzygium aromaticum would protect the protozoan against the cytotoxicity of ions released in saliva.\par
V. 
\section[{Conclusion}]{Conclusion}\par
This study has shown that Tetrahymena thermophila and Tetrahymena pyriformis can constitute a model for studying the cytotoxicity of orthodontic materials. These cell cultures are simple to carry out, reproducible and inexpensive. In addition, the extract of Celtis australis could constitute a protective compound against the cytotoxicity generated by the corrosion of orthodontic archwires.\begin{figure}[htbp]
\noindent\textbf{1}\includegraphics[]{image-2.png}
\caption{\label{fig_0}Figure 1 :}\end{figure}
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\noindent\textbf{}\includegraphics[]{image-3.png}
\caption{\label{fig_1}}\end{figure}
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\noindent\textbf{2}\includegraphics[]{image-4.png}
\caption{\label{fig_2}Figure 2 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{3}\includegraphics[]{image-5.png}
\caption{\label{fig_3}Figure 3 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{5}\includegraphics[]{image-6.png}
\caption{\label{fig_4}Figure 5 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{4}\includegraphics[]{image-7.png}
\caption{\label{fig_5}Figure 4 :}\end{figure}
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\noindent\textbf{6}\includegraphics[]{image-8.png}
\caption{\label{fig_6}Figure 6 :}\end{figure}
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\noindent\textbf{7}\includegraphics[]{image-9.png}
\caption{\label{fig_7}Figure 7 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{8}\includegraphics[]{image-10.png}
\caption{\label{fig_8}Figure 8 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{12}\includegraphics[]{image-11.png}
\caption{\label{fig_9}Figures 11 and 12 J}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{9}\includegraphics[]{image-12.png}
\caption{\label{fig_10}Figure 9 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{11}\includegraphics[]{image-13.png}
\caption{\label{fig_11}Figure 11 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{10}\includegraphics[]{image-14.png}
\caption{\label{fig_12}Figure 10 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{12}\includegraphics[]{image-15.png}
\caption{\label{fig_13}Figure 12 :}\end{figure}
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\end{bibitemlist}
 			 		 	 
\end{document}
