Cytotoxicity of orthodontic temporary anchorage devices on human periodontal ligament fibroblasts in vitro

Abstract Objectives The objective of this study is to test cytotoxicity of four brands of commercially available orthodontic temporary anchorage devices (TADs). Setting and sample population Twenty‐four (six for each brand, i.e., Aarhus [AO]; Dual top [RMO]; Vector TAS [ORMCO]; and Unitek TAD [3M UNITEK]) TADs were tested. Materials and methods Twenty‐four (six for each brand, i.e., Aarhus [AO]; Dual top [RMO]; Vector TAS [ORMCO]; and Unitek TAD [3M UNITEK]) TADs were individually incubated in complete cell culture medium and shaken at a rate of 1.5 rpm at 37°C for 30 days to extract possible toxic substances in conditioned media (CM). To test cytotoxicity, human periodontal ligament fibroblasts were cultured and exposed to the CM for 24 hr, followed by the examinations of morphological changes, cell viability (MTT assay), and cell damage (lactate dehydrogenase [LDH] assay). Results No morphological changes were observed in any of the four brands of TADs compared with the negative control. LDH assay showed that none of the four brands of TADs caused significant cell damage after CM treatment compared with the negative control (P > .05). No significant differences were found between any of the four brands of TADs (P > .05). MTT assay showed similar results as did the LDH assay, except for a statistically significant difference found in the TADs from 3M UNITEK compared with the negative control (P = .047). Conclusions According to the International Standard Organization standards, except for the TAD from 3M, none of the other three brands of commercially available TADs (from AO, RMO, and ORMCO) exhibited significant cytotoxicity, suggesting their safe clinical applications.

A successful material to be used as for TADs should be biocompatible and have good mechanical properties and corrosion resistance.
Titanium has the property of oxidizing in the presence of air and aqueous electrolytes to form a passive titanium dioxide film that contributes to its biocompatibility and corrosion resistance(Velasco-Ortega, Jos, Cameán, Pato-Mourelo, & Segura-Egea, 2010) but needs to be alloyed to improve its strength and fatigue resistance (Eliades, Zinelis, Papadopoulos, & Eliades, 2009). The titanium alloy is composed of a fusion of two phases, alpha (6% aluminum) and beta (4% vanadium).
Both phases in equilibrium contribute towards advantages of mechanical resilience (alpha phase), good formability, and fatigue resistance (beta phase) (Cotrim-Ferreira, Quaglio, Peralta, Carvalho, & Siqueira, 2010). However, this leads to a decrease in the corrosion resistance of the Ti alloy in body fluids. Ti-6Al-4V alloys used in orthopedics for joint replacements have shown to be susceptible to bio-corrosion in the physiological environment of the human body (Cadosch et al., 2010;Cadosch, Chan, Gautschi, & Filgueira, 2009;David & Lobner, 2004;Knutson & Berzins, 2013). By the same rationale, TADs composed of Ti-6Al-4V alloys would be susceptible to bio-corrosion despite their shorter duration of use. De Morais et al. proposed that TADs are a potential source of metallic ions to the human body because of the corrosion of titanium (Ti) alloy in body fluids. They evaluated the systemic levels of metallic ions specifically the concentrations of titanium (Ti), vanadium (V), and aluminum (Al) in rabbits.
Low amounts of Ti, Al, and V were detectable in the 1-, 4-, and 12-week groups of the rabbits, confirming that release of these metals from the mini implants occurs, with diffusion and accumulation in remote organs such as kidneys, liver, and lungs. However, despite the tendency of ion release when using the Ti alloy as TADs, the amounts of metals detected were significantly below the average intake of these elements through food and drink and did not reach toxic con- Dual Top, RMO) were used in this study. All the TADs were tested directly from their sterile surgical packages as received, except for the product from AO that was autoclaved prior to use due to its unspecified sterile condition. According to the ISO standards (International Standard Organization, 2009), cytotoxicity can be tested by direct contact or extraction means. Due to the complicated surface topography of TADs and our purpose of stringently challenging the release of the possible toxic substances, we chose to use the extraction method, that is, to incubate TADs in cell culture medium for certain time to extract possible toxic substances and collect the CM for cytotoxicity test. Another advantage of using extraction method is to be able to extract the possible toxic substances for a long period of time as reported previously (Malkoç et al., 2012).
Twenty-four TADs (six for each brand) were individually submerged in 8-ml alpha-minimal essential medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (10,000 units of penicillin and 10 mg of streptomycin in 0.9% NaCl) and sealed in 15-ml volume test tubes. To mimic the clinical intraoral environment where the TADs are placed and exposed to the flow of saliva (Dawes, 2008), the test tubes were constantly shaken at a rate of 1.5 round per minute (rpm) (Zhou, Liu, You, & Wang, 2010)  LDH is an enzyme located in the cytosol and is released into culture medium upon cell damage or lysis. LDH activity in the culture medium can thus be used as an indicator of cell membrane integrity and hence of cytotoxicity (David & Lobner, 2004;Haslam, Wyatt, & Kitos, 2000;Wolterbeek & van der Meer, 2005). In this study, the quantity of LDH release after treating the cells with the CM for 24 hr was determined following the assay protocol of Cayman LDH Cytotoxicity Assay Kit (Cayman chemical company, Ann Arbor, MI). The absorbance was read at 490-nm wavelength with a plate reader (Bio-Tek power wave XS2, Winooski, VT). Blank LDH levels were subtracted from insult LDH values.
The number of viable cells correlates with the color intensity determined by photometric measurements (Scudiero et al., 1988;Sjögren, Sletten, & Dahl, 2000). The reduction of MTT is thought to occur mainly in the mitochondria through the action of succinate dehydrogenase, therefore providing a measurement of mitochondrial function. All the MTT and LDH assay data were expressed as means ± standard deviation (n = 6) in the graphs. Statistically, one-way analysis of variance was used to determine the difference between all the experimental groups as a whole and the controls, whereas Bonferroni post hoc adjustment was applied to find out the difference between any two of the four TAD groups. A P value less than.05 was considered significant (SPSS, version 11.10, Chicago, IL). The MTT cytotoxicity test quantitatively measures the cell viability. Our positive control group (exposed to 0.1% Triton X-100) had the lowest MTT value (0.16 ± 0.039), whereas the negative control (treated with CM without TADs) had the highest MTT level (0.47 ± 0.023). There was a highly significant difference between the positive control group and all the other groups (P = .000). There was no significant difference among the different brands of TADs (P > .05), except that the 3M Unitek product showed a statistically significant cytotoxicity (lost cell viability by about 30%) when compared with the negative control group (P = .047; Figure 3).

Morphologically
When the cellular plasma membrane is damaged or upon cell lysis, LDH is released from the cells, which is used as an indicator of cell F I G U R E 1 Experimental setup. (a) Sample pictures of the TADs tested in this study and (b) the experimental setup for incubating the TADs in cell culture media to extract potential toxic substances on shaker (1.5 rpm) at 37 C for 30 days. TADs, temporary anchorage devices membrane integrity and a measurement of cytotoxicity (David & Lobner, 2004). In our study, the positive control group cells treated with 0.1% Triton X-100 showed the highest release of LDH (0.40 ± 0.024) and thus the highest cytotoxicity, whereas the negative control resulted in the lowest LDH value (0.17 ± 0.012). All the four TADs groups together with the negative control showed significantly low LDH levels than that in the positive control (P = .000). There was no significant difference in LDH levels among the four different groups of TADs (P > .05; Figure 4).

| DISCUSSION
Biosafety and biocompatibility are of main concern to the clinical application of dental materials including TADs. in vitro cytotoxicity tests are advised by ISO to evaluate acute cytotoxicity of a material(International Standard Organization, 2009) and also aid in better understanding the pathogenicity of subacute effects. In contrast to animal experiments, cell cultures commonly used for dental material biosafety tests are generally simple, inexpensive, and can be performed under well-controlled conditions (Mockers, Deroze, & Camps, 2002;Samara et al., 2011). Ideally, cytotoxicity tests should be done on the same type of tissue that the tested compounds will be exposed to, and efforts should be made to simulate in vivo conditions as much as possible. Although many types of cells (primary cells vs. In this study, we used the commercially available TADs rather than Ti-6Al-4V discs (Watanabe et al., 2004), because it is more clinically relevant to use the products that are used intraorally. In addition, we submerged the TADs in the complete cell culture medium for 720 hr F I G U R E 2 Morphological changes of hPDLF cells after exposure to CM for 24 hr. In comparison with the positive control, none of the TADs groups showed significant changes of cell shape, size, and orientation, which is similar to the negative control (Bar = 40 μm). α-MEM, alphaminimal essential medium; CM, conditioned media; hPDLF, human periodontal ligament fibroblasts; TADs, temporary anchorage devices (30 days) on a flip-flop shaker (at a rate of 1.5 rpm) to mimic in vivo conditions favoring bio-corrosion and stringently stimulate the release of possible toxic substances (mainly metal ions) over a long period of time. In accordance with ISO guidelines, cytotoxicity test can be done in two ways-contact (direct) and extract (indirect). Due to the complex surface of the TADs and the limitations of direct method (long time culture cannot be performed), we chose to use the extract means. We incubated the TADs in cell culture medium for 30 days, much longer than the 24-72 hr as recommended in ISO standards in an attempt to make a stringent extraction of the possible toxic substances from the TADs. If long time extracts are not cytotoxic, it thereby follows that the clinical application in shorter time period should be safe.
In this study, no significant cytotoxic effects were found among the tested TADs for the morphological changes of the cells and the LDH assay analysis. This is not surprising as all these TADs have similar composition (Ti-6Al-4V). MTT assay analysis showed that among the four TAD groups, the 3M Unitek TADs had a 30% reduction of cell viability when compared with the negative control (P = .047). The explanation to this may lie in further evaluating bio-corrosion. A recent study examined the corrosion of TADs from 3M Unitek, ORMCO, and AO in artificial saliva and found subtle but not significant differences in the passivity of all the TADs tested. The 3M Unitek TADs had a comparatively less stable passive layer at open circuit potential above 0.3V (Knutson & Berzins, 2013), and a less stable passive oxide layer typically is associated with greater corrosion rate (Bohni, 2005). The authors also observed that the silver/grey colored Unitek TADs may suggest a thinner oxide layer in contrast to the AO and ORMCO TADs that were blue and pink colored. Different surface treatment may or may not have been performed on the TADs accounting for the variability in oxide layer. However, it is of value to consider that some manufacturers provide color-coded options to differentiate sizes and locations for use and so not all TADs from a particular manufacturer may perform the same (Knutson & Berzins, 2013).
In contrast to our study, both 3M Unitek and RMO products had similar color but 3M Unitek TADs still had a slightly less cell viability based on MTT assay analysis. The AO TADs with the blue color had a significantly more noble open circuit potential (P < .05) compared with the others (Knutson & Berzins, 2013). Although our TADs were submerged in culture medium instead of artificial saliva, the same medium was used for all test products, and hence, this additive variable was eliminated in our study. titanium and suggested that it was because of toxic effects of released F I G U R E 3 MTT assay-based cell viability of hPDLF cells after exposure to CM for 24 hr. In comparison with the negative control, three TADs groups (RMO, ORMCO, and AO) showed no significant reduction of cell viability (P > .05, n = 6), opposite to the positive control showing a 66% cell viability reduction (P < .05, n = 6). However, the TADs from 3M UNITEK exhibited a statistically significant 30% reduction of cell viability when compared with the negative control (*P = .047, n = 6). CM, conditioned media; hPDLF, human periodontal ligament fibroblasts; TADs, temporary anchorage devices F I G U R E 4 LDH release from hPDLF cells after exposure to CM for 24 hr. In comparison with the negative control, all four TADs groups (RMO, ORMCO, AO, and 3M) showed no statistically significant increase of LDH (P > .05, n = 6), opposite to the positive control showing a 1.35-fold increase of LDH (P < .05, n = 6). CM, conditioned media; hPDLF, human periodontal ligament fibroblasts; LDH, lactate dehydrogenase; TADs, temporary anchorage devices vanadium ions (Okazaki, Rao, Ito, & Tateishi, 1998). The reasons of different responsiveness of different types of cells (e.g., osteoblasts vs. fibroblasts) and of same type of cells but from different species (e.g., human vs. mouse) are unknown.
There were limitations in this study. It is essential to appreciate the factors associated with cytotoxicity results namely dose of the toxin, exposure time, cell type, and mechanism of action (necrosis vs. apoptosis). Even though the ISO standards were followed in this study as in others, special attention needs to be paid when attempting to interpret the results and relate them to clinical situations. Considering the variables of difference in methodology (discs vs. TADs, end point testing vs. real cell analysis, incubation, and testing duration) and different cell origins, it is expected to see variable outcomes. Further standardization of cytotoxicity tests needs to be considered in order to draw more reliable and reproducible results. The release of metal ions from TADs might directly affect their biocompatibility. There are no exhaustive data correlating metal ion release from TADs, their biocompatibility, and association with failure of orthodontic mini implants or TADs.
Considering the clinical importance of TADs in orthodontics, further investigations should be performed to better understand the biological effect of the TADs on oral tissues.

| CONCLUSIONS
Within the limitations of this in vitro study, according to the ISO standards, except for the TAD from 3M, none of the other three brands of commercially available TADs (from AO, RMO, and ORMCO) exhibited significant cytotoxicity, suggesting their safe clinical applications.