ON THE CYTOTOXICITY OF ORTHODONTIC ARCHES IN HUMAN FIBROBLAST CULTURES

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1 Oral biology ON THE CYTOTOXICITY OF ORTHODONTIC ARCHES IN HUMAN FIBROBLAST CULTURES Liliana-Gabriela HALIŢCHI 1, Oana-Maria DARABĂ 1, Irina-Paula MERLUŞCĂ 2, Codruta ILIESCU 3, Ionuţ CHIRAP 4, Vasile BURLUI 5 1 Assist. Prof., PhD, Faculty of Medical Dentistry, Apollonia University of Iaşi 2 Univ. Assist., Faculty of Medical Dentistry, Apollonia University of Iaşi; PhD student, Faculty of Chemical Engineering and Environmental Protection, Gheorghe Asachi Technical University of Iaşi 3 Univ. Assist., PhD, Faculty of Medicine, Gr. T. Popa University of Medicine and Pharmacy, Iasi 4 PhD Student, Faculty of Physics, Alexandru Ioan Cuza University of Iasi 5 Prof. PhD, Faculty of Medical Dentistry, Apollonia University of Iaşi Corresponding author: oana_daraba@yahoo.com Abstract The present study analyzes the orthodontic nickeltitanium alloy with orthodontic shape memory (Niti GAC arches, Nitinol 3M arches, Beta Titanium 3M arches) from the viewpoint of its cytotoxicity. Apart from the intrinsic toxicity of the metals upon cells, corrosion may dramatically alter the behaviour of alloys, as evidenced by the in vitro and in vivo studies developed in the field. Due to the high and variable nickel content, its is possible that, at least theoretically, the released ions should produce as a result of intraoral corrosion secondary effects, which makes necessary to assert the cytotoxicity of NiTi alloys prior to their safe utilizationor in the oral cavity of children and young patients. Evaluation of cell morphology and determination of cell viability, following exposure to the 3 types of orthodontic materials, evidenced no toxic reactions. Several problems are still to be elucidated, related to the consequences of the surface conditions, of the dissolution and in vivo release of the nickel ions after a longer treatment, of the accumulations of ion traces, of the response of the dental-periodontal tissues, of the effects manifested at cell and molecular level. Apart from the intrisic toxicity of metals upon cells, corrosion may dramatically influence the behaviour of alloys under in vitro conditions, comparatively with the in vivo ones, the nitinol samples being biologically safe. Keywords: orthodontic alloys, nitinol, cytotoxicity, cell cultures, corrosion. 1. INTRODUCTION The Niti alloys, discovered by W. F. Buecker, started to be used in orthodontic treatments by Andreasen since 1972, due to their remarkable superelasticity, shape memory, limited formability, increased bending capacity. The nitinol arches are more difficult to deform when arranged in slots, being useful in the alignment and levelling phases, which leads to fewer treatment stages and their prolongued application without modifications [1,2]. However, they may produce expansion of the dental-alveolar arch, and insufficient stability in the end of the treatment. As demonstrated by scientists [3], the level of nickel in the saliva increases significantly after insertion of fixed devices, only 30 ppm nickel being necessary for producing a cytotoxic effect [4], which may lead to nephrotoxicity, carcinogenetic effects or immune modifications accompanied by alveolar bone loss. Intraoral reactions, such as inflammation of the oral, gingival and/or labial mucosa, colour modifications and painful sensitivity have been associated with the metallic brackets, vestibular arches, combined procedures and acrylic orthodontic devices [ 5,6]. Primary cytotoxicity and the ratio of NiTi alloy corrosion have been frequently evaluated on cell cultures of human osteoblasts and fibroblasts, so that one may assert that the alloy does not induce toxic effects, determining reduction of cell proliferation rate and inhibiting the growth of the cells in contact with the metal surface [7-9], which assures a good biocompatibility of the nitinol alloy. Due to the high and variable nickel content, the ions released as a result of intraoral corrosion may produce at least theoretically secondary effects, which may characterize the differential diagnosis of bacterial gingivitis, so that the biocompatibility of NiTi alloys should be confirmed prior to their International Journal of Medical Dentistry 277

2 Liliana-Gabriela Haliţchi, Oana-Maria Darabă, Irina-Paula Merluşcă, Codruta ILIESCU, Ionuţ CHIRAP, Vasile Burlui safe application in the oral cavity of children and young patients. Estimation of cytotoxicity is part of the initial evaluation of biocompatibility. The technique of cell cultures is useful for evaluating the cytotoxic effects of solid xenobiotic materials [10-12], being frequently applied for testing the biological effects of dental materials [13]. Such an alternative method to the experiments made on animals is relevant in the biomedical investigations aimed at testing the potential toxicity of xenobiotics in children and adolescents. The quality and specificity of the data obtained from in vitro models depend on the following factors: - utilisation of a biological system which reproduces, to a large extent, the metabolic behaviour of the organ tested as to the toxic effects of xenobiotics; - a correct selection of parameters for evaluating the toxic effects in vitro; - a correct design of the experiment, so that the data obtained in vitro should predict possible in vivo effects; - the model of cell cultures provides a constant, stable, measurable and reproducible in vitro method for the study of xenobiotic orthodontic materials [14]. Nowadays, the existing literature of the domain does not offer sufficient and reliable information on the biocompatibility of NiTi arches during the orthodontic treatment, even if numerous studies assert that no biocompatibility issues are to be faced. However, several aspects are still to be elucidated, related to the consequences of the surface conditions, dissolution and in vivo release of the nickel ions after a longer period of treatment, to the accumulations of ion traces, response of the dento-periodontal tissues, or possible effects at both cellular and molecular level. Scope of the study. The scope of the study was to evaluate the cytotoxicity of the arches made of orthodontic wires with various compositions, by in vitro testing on a cell line of HDFa (Human Dermal Fibroblasts, adult) human fibroblasts put into contact with 3 types of orthodontic materials (Niti GAC arches, Nitinol 3M arches, Beta Titanium 3M arches). The three types of arches the patients had to wear for 1 month were put in cultures of human fibroblasts, while the samples were checked at intervals of 24 h after cell cultivation, for 5 days. The effects of cytotoxicity and the loss of cell viability in vitro were quantified in a similar manner with that applied in the studies of Mohebbian [15,16]. 2. MATERIALS AND METHOD The experiment was performed in the Laboratory of Cell Cultures of Apollonia University of Iaşi, equipped according to international standards, on a cell line of human fibroblasts provided by Antisel. The HDFa (Human Dermal Fibroblasts, adult) were delievered in carbonic ice. The working protocol involved the following stages: preparation of the nutritive medium, melting of the cell line, cultivation of fibroblasts, changing of the nutritive medium, passage of the fibroblasts cells, introduction of the orthodontic materials in the cultures of fibroblasts, numbering of cells, calculation of density and of cell viability. For the experiments, 4 samples were analyzed, namely: the control sample, the sample with Niti GAC arches, the sample with Nitinol 3M arches, the sample with Beta Titanium 3M arches. For the preparation of the nutritive medium, 350 ml DMEM medium were poured into a 500 ml vial over which: 40 ml phoetal bovine serum, 4 ml solution of penicillin with streptomycin, and 4 ml non-essential aminoacids were added. The thus prepared medium was distributed in 2 smaller vials, one of them being introduced in the bath of water warmed at 37 C, for 5 min, whereas the other vial was preserved in the refrigerator at 4 o C. For cell line melting and fibroblasts cultivation, 5ml medium were pipetted in a centrifugal tube with laminary flow, while the cryotube containing the cell line of fibroblasts was melt in the water bath at a temperature of 37 C. The content of the cryotube was poured into 5 ml of an already prepared medium, followed by the centrifugation stage, for 5 min, at 1,000 rpm. After supernatant aspiration, 1 ml medium was added over the remaining sediment and the whole amount was introduced into a culture flask (T 75 ml) already containing 10 ml fresh medium. The obtained 278 Volume 5 Issue 4 October / December 2015

3 ON THE CYTOTOXICITY OF ORTHODONTIC ARCHES IN HUMAN FIBROBLAST CULTURES sample was analyzed microscopically and introduced in an incubator at 37 C, at a concentration of 5% CO 2. One day after the incubation, the culture medium was changed, the culture medium from the flask containing the sample was aspired with a vacuum pump and other 10 ml of fresh medium were introduced (Fig.1). Trypsinization and passage of cells involved aspiration of the medium from the vial of cell culture, the fibroblasts attached to the flask wall were washed with 8 ml PBS (Phosphate Buffer Saline) for a few seconds, after which the PBS was aspired and 3 ml trypsin were added into the flask. After incubation for 2-3 min, ended with fibroblasts detachment, 10 ml medium heated at 37 C were added over the culture and trypsin flask. The whole content was centrifuged for 5 min, at 1,000 rpm. After centrifugation and elimination of supernatant, 5 ml of fresh medium were added in the flask containing the remaining sediment. The content was distributed in 4 flasks (T25) containing 5 ml medium (a flask for the control sample, the other ones for the materials to be tested). Following microscopic analyses, written down on the flasks were: data, culture medium and passage. The fibroblast-containing samples from the three flasks, obtained through trypsinization, were put into contact with the 3 types of materials, and following microscopic analysis introduced in the incubator. Cell numbering was done on a hemocytometer, with an invertoscope (Figs. 7-8). Cell viability in the culture was calculated as the number of living cells: total number of cells ratio (number of living cells/ total (living and dead) cells x 100 = %). [17] Fig.1. Working protocol: preparation of the nutritive medium, passage, investigated arches 3. RESULTS AND DISCUSSION Evaluation of cell morphology and determination of cell viability, following exposure to the 3 types of orthodontic materials, evidenced no toxic reactions (Figs. 2 to 6). Calculation of cell viability comparatively with the control sample (viability ratio = 98.15%) showed that all orthodontic materials under investigation may be employed in dental medicine, the ratios of cell viability recording only small variations: the sample with Nitinol 3M = 95.2 %, the one with Niti GAC = %, the sample with Beta Titanium 3M= % (Fig.10). The study of Wever [18,19] asserts, thus agreeing with the present analysis, that nitinol allays are biologically safe, and may be applied in the oral cavity with no risk. Similarly with the studies of Grosgogeat, Oh or Yonekura [20-22], which took advantage of International Journal of Medical Dentistry 279

4 Liliana-Gabriela Haliţchi, Oana-Maria Darabă, Irina-Paula Merluşcă, Codruta ILIESCU, Ionuţ CHIRAP, Vasile Burlui the orthodontic materials testing on the cell cultures, no acute toxicity was recorded in the orthodontic arches subjected to intra-oral corrosion during wearing. The different external exposures to the alimentary input, intraoral temperature or mechanical factors derived from the orthodontic treatment as such could not be tested and evaluated. Fig.2. Control sample (attached fibroblasts) Fig.5. Beta 3 Titanium 3M sample Fig.3. Control sample (after trypsinization) Fig.6. Niti GAC sample Fig.4. Nitimol 3M sample Fig.7. Morphological analysis invertoscope 280 Volume 5 Issue 4 October / December 2015

5 ON THE CYTOTOXICITY OF ORTHODONTIC ARCHES IN HUMAN FIBROBLAST CULTURES Fig.8. Cell numbering hemocytometer A relative comparison among the 3 types of arches shows that the NiTi 3M arches induce a lower cytotoxicity than the GAC and Beta Titanium 3M ones (Fig. 9). The nitinol arches studied in vitro by Castleman and Motzkin [18] on human fibroblasts showed a more reduced cell growth and considerable morphological modifications. According to Putters, [19] nickel produces a significant inhibition of mytosis in human fibroblasts. Nitimol 3M Niti GAC Beta 3 Titanium 3M Fig.9. Comparative morphological analysis of the working orthodontic arches Some studies evidenced that the metallic compounding elements of the alloys employed in orthodontics may be toxic through corrosion in oral fluids [23-25]. Any metal exerts its own toxicity upon cells, however corrosion may dramatically modify the behaviour of alloys in in vitro studies, comparatively with in vivo investigations. The presence of nickel in the superficial layer of the NiTi alloys may lead to different results as to the different formulae of nitinol orthodontic arches, as also evidenced in the present study. In vitro investigations on the cell response to nitinol are quite few, and provide quite contradictory results, the differences being caused by the testing protocold, selected cell type, analyzed factors, surface treatments applied by producers to the arches, processing degree. Fig.10. Graphical representation of cell viability International Journal of Medical Dentistry 281

6 Liliana-Gabriela Haliţchi, Oana-Maria Darabă, Irina-Paula Merluşcă, Codruta ILIESCU, Ionuţ CHIRAP, Vasile Burlui 4. CONCLUSIONS 1. Determination of cell viability, for the three types of arches made of orthodontic wire here under study showed no significant toxicity for either of them. 2. A relative comparison among the 3 types of arches evidenced that the NiTi 3M arches induce a lower cytotoxicity than the GAC and Beta Titanium 3M ones, due to the surface treatments applied upon them by producers, and also to their processing degree. 3. Several aspects are still to be elucidated, related to the consequences of the surface conditions, in vivo dissolution and release of nickel ions after a longer treatment period, accumulations of ion traces, response of the dental-periodontal tissues, effects at cell and molecular level. 4. Estimation of cytotoxicity is part of the initial evaluation of biocompatibility. The technique of cell cultures is useful for evaluating the cytotoxic effects of solid xenobiotic orthodontic materials, being frequently applied for testing the biological effects of dental materials. Such a method, alternative to the experiments on animals, is particularly relevant in biomedical investigations aimed at testing the potential toxicity of orthodontic xenobiotics in children. References 1. Andreasen GF, Fahl JL. Alloys, Shape Memory. In: Webster JG, editor. Encyclopedia of medical devices and instrumentation. Volume 2. New York. Wiley;1987. p Hanawa T. Titanium and its oxide film: A substrate for Formation of Apatite. In: Davies JE, editor. The bone-biomaterial interface. Toronto. University of Toronto Press;1991. p Anderson JM, Gristine AG, Hanson SR. Host reactions to biomaterials and their evaluation. In: Ratner BD, Hoffman AS, Schoen FJ, Lemons JE, editors. Biomaterials science; an introduction to materials in medicine. San Diego. Academic Press;1996; p Ryhänen J, Kallioinen M, Tuukkanen J, Junila J, Niemelä E, Sandvik P, Serlo W. In vivo biocompatibility evaluation of nickel-titanium shape memory metal alloy: muscle and perineural tissue responses and encapsule membrane thickness. J Biomed Mater Res. 1998;41(3): Ryhänen J, Niemi E, Serlo W, Niemelä E, Sandvik P, Pernu H, Salo T. Biocompatibilityof nickel-titanium shape memory metal and its corrosion behavior inhuman cell cultures. J Biomed Mater Res. 1997;35(4): Ryhänen J, Kallioinen M, Tuukkanen J, Lehenkari P, Junila J, Niemelä E, Sandvik P, Serlo W. Bone modeling and cell-material interface responses induced by nickel titanium shape memory alloy after periosteal implantation. Biomaterials. 1999; 20(14): Ryhänen J, Kallioinen M, Serlo W, Perämäki P, Junila J, Sandvik P, Niemelä E, Tuukkanen J. Bone healing and mineralization, implant corrosion and trace metals after nickel-titanium shape memory metal intramedullary fixation. J Biomed Mater Res. 1999;15;47(4): Jacobsen N, Hensten-Pettersen A. Occupational health problems and adverse patient reactions in orthodontics. Eur. J Orthod. 1989;11(3): Zachrisson BU, Zachrisson S. Gingival condition associated with orthodontic treatment. Acta Odontol Scand. 1972;30(1): Bernard F-X, Pedretti N, Rosdy M, Deguercy A. Comparison of gene expression profiles in human keratinocyte mono-layer cultures,reconstituted epidermis and normal human skin; transcriptional effects of retinoid treatments in reconstituted human epidermis. Exp Dermatol. 2002; 11(1): Vannet BV, Mohebian N,Wehrbein H. Toxicity of used orthodontic archwires assessed by threedimensional cell culture. Eur J Orthod. 2006;28(5): De Wever B, Charbonnier V. Using tissue engineered skin to evaluate the irritation potential of skin care products. Cosmetics and Toiletries Magazine. 2002;10: Locci P, Lilli C, Marinucci L, Calvitti M, Belcastro S, Bellocchio S, Staffolani N, Guerra M, Becchetti E. In vitro cytotoxic effects of orthodontic appliances. J Biomed Mater Res. 2000; 53(5): SR EN Biological evaluation of medical devices. Part 5: Tests for cytotoxicity: in vitro methods Mohebbian N. Cell cultures to assess cytotoxicity [PhD thesis]. Belgium: Free University of Brussels; Mohebbian N, Bottenberg P. Importance of cell cultures in biocompatible dental materials research. Belgisch Tijdschrift voor Tandheelkunde. 2003; 58: Wever DJ, Veldhuizen AG, Sanders MM, Schakenraad JM van Horn JR. Cytotoxic, allergic and genotoxic activity of a nickel-titanium alloy. Biomaterials. 1997;18(16): Volume 5 Issue 4 October / December 2015

7 ON THE CYTOTOXICITY OF ORTHODONTIC ARCHES IN HUMAN FIBROBLAST CULTURES 18. Castleman LS, Motzkin SM, Alicandri FP, Bonawit VL. Biocompatibility of nitinol alloy asan implant material. J Biomed Mater Res. 1976; 10(5): Putters JL, Kaulesar SD, De ZG, Bijma A, Besselink PA. Comparative cell culture effects of shape memory metal (Nitinol), nickel and titanium: a biocompatibility estimation. Eur Surg Res. 1992;24: Grosgogeat B, Pernier C, Schiff N, Comte V, Huet A. Biocompability and resistance to corrosion of orthodontic wires. Orthodontie Française. 2003; 74(1): Oh TK, Kim YS, Park YS, Kim KN. Properties of super stainless steels for orthodontic applications. J Biomed Mater Res B Appl Biomater. 2004;69(2): Yonekura Y, Endo K, Iijima M, Ohno H, Mizoguchi I. In vitro corrosion characteristics of commercially available orthodontic wires. Dent Mater J. 2004;23(2): Poehler OEM. Degradation of metallic orthopedic implants. In: Rubin L., editor. Biomaterials in reconstructive surgery. St. Louis: The C.V. Mosby Co.;1983. p Hanawa T, Asami K, Asaoka K. Repassivation of titanium and surface oxide film regeneration in simulated bioliquid. J Biomed Mater Res. 1998;40(4): Hanawa T, Ota M. Calcium phosphate naturally formed on titanium in electrolyte solution. Biomaterials. 1991;12(8): International Journal of Medical Dentistry 283