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MEDICAL-BIOLOGICAL SCIENCES
УДК 538.971:577.359:57.043
THE INFLUENCE OF SURFACE ALLOYING WITH TANTALUM OR SILICON ON CYTOCOMPATIBILITY OF TITANIUM NIKELID
Andrey Leonidovich MATVEEV1, Lyudmila Vladimirovna ARTEM'EVA1, Stanislav Nikolaevich MEISNER23, Vera Aleksandrovna MATVEEVA1, Lyudmila Leovidovna MEISNER2,3
1 Institute of Chemical Biology and Fundamental Medicine of SB RAS 630090, Novosibirsk, Lavrent'ev's ave., 8
2 Institute of Strength Physics and Materials Science of SB RAS 634055, Tomsk, Akademicheskiy ave., 2/4
National Research Tomsk State University
3 634050, Tomsk, Leniv ave., 36
The influence of physicochemical properties of titanium nikelid with the surface layers modified with silicon or tantalum ions was studied on in vitro cultured mesenchymal stem cells of the rats' bone marrow. It was shown by the methods of laser scanning microscopy, light microscopy, MTT test that the ion-plasma modification of the titanium nikelid surface layers with silicon or tantalum ions improves the cytocompatibility of metal alloy.
Keywords: titanium nikelid, ion-plasma modification, silicon, tantalum, cytocompatibility, mesenchymal stem cell,, viability, proliferation, mitochondrial tetrazolium test.
Titanium nikelid (TiNi) alloys are known for their unique properties of shape memory, superelas-tic [1] that allows their usage in medicine. However, there is a potential risk of toxic, allergic and nickel carcinogenic effect on cells and tissues during the washout from alloy [4]. An effective method of bio-compatibility improvement of products from nickel and titanium alloys limiting the washout of nickel from alloy and improving the integration of the implant with the surrounding tissue are the methods of ion-beam modification of the alloy surface [5].
For screening of materials after various kind of treatments the transformed cell lines and mesen-chymal stem cells (MSCs) are used. The selection of MSCs is due to the fact that they are cells of normal tissues, differentiate in vivo into cells of various organs and the viability, morphology, adhe-
sion, proliferation, directed differentiation of these cells can be studied in vitro during the evaluation of the influence of the physicochemical and morphological properties of the metal implant surface onto cells [2, 2].
Thereby, the aim of the work was to study the cytotoxicity of NiTi samples after modification of their surface with silicon or tantalum ion beams on the culture of mesenchymal stem cells of rat bone marrow in vitro.
MATERIAL AND METHODS
Materials
The samples of TH1 alloy were tested, which surface was successively exposed to chemical etching, mechanical polishing and electropolishing
Matveev A.L. - junior researcher of laboratory for molecular microbiology, e-mail: vam@niboch.nsc.ru Artem'eva L.V. - leading engineer of laboratory for stem cells
Meisner S.N. - candidate of technical sciences, junior researcher of laboratory for material sciences of shape memory allow, junior researcher of laboratory for medical material sciences Matveeva V.A. - candidate of biological sciences, researcher of laboratory for stem cell
Meisner L.L. - doctor ofphysico-mathematical sciences, professor, chief researcher of laboratory for material sciences of shape memory allow, professor of the chair for metal physics, leading researcher for medical material sciences
(TiNi samples) and ion-beam modification with mo-nocomponent ion beams of chemically pure tantalum (samples TiNi_Ta) or silicon (samples TiNi_Si). The samples TiNi were comparison samples. For biological experiments the samples were washed with water and sterilized for 90 minutes at 180 °C.
Cells cultivation
Rat bone marrow MSCs were used for experiments. The cells were cultivated in a-MEM medium with 10 % fetal calf serum, 4 mM GlutaMax, 1 % antibiotic/antimycotics (Gibco) at 37 °C in an atmosphere of 5 % CO2 at saturated humidity conditions. To determine the proliferative activity of the cells in the presence of samples the suspension of MSCs with the density of 5 x 103 cells/cm2 was placed on sterile samples in wells of 12-well plate. After 14 days of cultivation the cell viability in the wells was investigated by MTT method, the samples were washed, transferred to the new wells and cultivated for another 3 days to estimate the efficiency of colonies formation. After 3 days the samples were removed for MSCs visualization on the samples surface, the cells cultivation was continued and after 11 days the viability of the cells in MTT assay, the number and size of cell colonies on the surface of the wells were determined.
Mitochondrial tetrazolium test (MTT)
The viability of cells cultivated on the surface of the wells was determined using a soluble form of formazan WST1 (Roche, USA) according to manufacturer's instructions. The estimation of MTT assay results was carried out by comparing of the optical density of the solution from control and test wells at
wavelength X = 450 nm and reference X = 655 nm on a plate spectrophotometer BioRad 680 (BioRad, USA).
Visualization of cells on the samples surface
The membranes and nucleus of MSCs cultivated on the surface of the samples were stained with fluorescent dyes Vybrant-CM-Dil and Hoechst (Life Thechnology, USA) according to the manufacturer's instructions. The nuclei and cell membranes visualization were performed on microscope LSM 780 (C. Zeiss, Germany).
Visualization of cells on the surface of wells
MSCs cultivated on the surface of wells were stained with Giemsa (Panreac) solution according to the manufacturer's instructions, then watched in the light microscope «Stemi 2000C» (C. Zeiss, Germany); the number and size of colonies were counted with «Axiovision» software.
Processing of measurement results
The statistical analysis was performed with standard Microsoft Excel software package. The statistical significance of differences between two groups of data was estimated with nonparametric Mann-Whitney U-test at the selected significance level p < 0,05 for n = 3 in three independent experiments.
RESULTS AND DISCUSSION
The influence of surface properties of titanium nikelid samples modified with silicon or tantalum ions on MSCs
Fig. 1. The pictures of mesenchymal stem cells of the rat bone marrow on the surface of TiNi samples before and after modification with silicon or tantalum ions; a — micrographs of cells; b — The pictures of cells structures and samples surface in the fluorescence channels; c — 3-D
Table 1
The influence of titanium nikelid samples on proliferation of rat bone marrow MSCs
The measure unit Sample
TiNi TiNi_Ta TiNi_Si
The average value of optical density 0,882 ± 0,231 0,926 ± 0,187 0,956 ± 0,162
The relative optical density in % 100,00 ± 7,06 104,99 ± 5,30 108,39 ± 4,62
The optical density in wells with cells after MSCs application to the surface of samples with surface-unmodified TiNi after 14 days of cultivation was taken as 100 %. For n = 3, the most representative data of 3 independent experiments are presented.
According to results of the laser scanning microscopy method (Fig. 1) with fluorescent dyes Vybrant-CM-Dil and Hoechst, the rat bone marrow MSCs were found on the surface of TiNi samples after 17 days of cultivation. The physico-chemical properties of the surface of samples TiNi_Si, TiNi_Ta and TiNi had no an acute toxic effect on cells cultured on their surfaces. The cells colonized the surface of all NiTi samples regardless of the surface modification options.
The influence of titanium nikelid samples modified with silicon or tantalum ions on proliferation of MSCs cultivated in the presence of the samples.
According to MTT assay results no significant differences were found in mitochondrial respiration index of cells colonized the surface of the wells after their application to the surface of NiTi samples and cultivation for 14 days (Table 1).
The samples TiNi_Si, TiNi-Ta and TiNi did not have acute toxic effects on mesenchymal stem cells. During the cells cultivation in the presence of TiNi samples, MSC on the surface of culture wells retained the proliferative activity. The effectiveness of the cells proliferation on the wells surface was not dependent of the variant of alloy surface modification.
According to the results of the light microscopy (Fig. 2), cells migrated from the surface
of TiNi_Si, TiNi-Ta and TiNi samples preserved the clonal activity in vitro, forming the colonies and colonizing the surface of the plastic cultural wells.
According to MTT assay (Table 2), the relative amount of viable cells migrated from TiNi-Ta and TiNi_Si samples surface did not significantly differ and was nearly 2 times higher in comparison with the number of viable cells migrated from TiNi samples surface.
The average number of colonies (Table 3) formed by cells migrated from TiNi samples surface modified by silicon or tantalum ions did not significantly differ. At the same time, the average number of colonies formed by cells migrated from TiNi samples was more than 2-fold smaller than the number of colonies formed by cells from TiNi_Si and TiNi_Ta samples. The colonies formed by cells migrated from TiNi_Ta or TiNi_Si samples took in average about 50-60 % of the area of the cultural well surface. While the total area of the cells colonies migrated from TiNi samples was in average about 30 % of the area of the wells surface and was approximately 2-fold less in comparison with the cells from TiNi_Ta or TiNi_Si samples.
This cells behavior could be possibly connected with both a decrease of the total number of viable cells and/or decrease of the population of fast pro-
Fig. 2. Micrograph of mesenchymal stem cells colonies formed on the surface of the plastic cultural wells by cells migrated from TiNi samples; increase x 4
Table 2
The influence of TiNi sample surface treatment with silicon or tantalum ions on the viability of cells migrated from TiNi samples surface and formed colonies on the cultural wells surface
The measure unit Sample
TiNi TiNi_Ta TiNi_Si
The average value of optical density 0,342 ± 0,127 0,786 ± 0,153 0,910 ± 0,134
The relative optical density in % 100,00 ± 6,04 229,82 ± 6,92 266,08 ± 7,30
The content of cells in wells migrated from the surface of unmodified TiNi samples after 14 days of cultivation was taken as 100 %. For n = 3, the most representative data of 3 independent experiments are presented.
Table 3
The influence of TiNi samples surface treatment with silicon or tantalum ions on the formation of cells colonies migrated from TiNi samples surface to the surface of cultural plastic wells
The measure unit Sample
TiNi TiNi_Ta TiNi_Si
The average number of colonies * 79 ± 10 163 ± 23 154 ± 15
The relative number of colonies, % * 100,00 ± 12,66 206,33 ± 14,11 194,937 ± 9,74
The relative area occupied by colonies on the culture wells surface, % ** 26,17 ± 7,54 50,88 ± 13,50 56,29 ± 9,55
* The number of colonies per well containing the cells migrated from unmodified TiNi sample surface after 14 days of cultivation was taken as 100 %.
** The area of the well was taken as 100 %. For n = 3, the most representative data of 3 independent experiments are presented.
liferating cells due to cell death because of chemical elements toxicity and/or TiNi samples surface morphology possibly affecting the attachment and/or differentiation of mesenchymal stem cells.
CONCLUSION
Based on the results of laser scanning microscopy, light microscopy and MTT assay the silicon or tantalum ion implantation is not toxic for mesen-chymal stem cells and enhances the cytocompatibil-ity of electropolished TiNi alloy.
ACKNOWLEDGMENTS
The authors thank the leading expert in laser scanning microscopy OOO OPTEK Barinov A.A. for the assistance provided under the work on microscope LSM 780 (C. Zeiss, Germany) in the CCU ICG SB RAS (Head of division, Bayborodin S.I., Ph.D.). The study was funded by Russian Science Foundation grant (project N15-13-0023 from 18.05.2015).
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ВЛИЯНИЕ ПОВЕРХНОСТНОГО ЛЕГИРОВАНИЯ С ИСПОЛЬЗОВАНИЕМ ТАНТАЛА ИЛИ КРЕМНИЯ НА ЦИТОСОВМЕСТИМОСТЬ НИКЕЛИДА ТИТАНА
Андрей Леонидович МАТВЕЕВ1, Людмила Владимировна АРТЕМЬЕВА1, Станислав Николаевич МЕЙСНЕР23, Вера Александровна МАТВЕЕВА1, Людмила Леонидовна МЕЙСНЕР2,3
1 ФГБУНИнститут химической биологии и фундаментальной медицины СО РАН 630090, г. Новосибирск, пр. Лаврентьева, 8
2 ФГБУН Институт физики прочности и материаловедения СО РАН 634055, г. Томск, пр. Академический, 2/4
3 Национальный исследовательский Томский государственный университет 634050, г. Томск, пр. Ленина, 36
Влияние физико-химических свойств никелида титана с приповерхностными слоями, модифицированными ионами кремния или тантала, изучали на культивируемых in vitro мезенхимальных стволовых клетках костного мозга крысы. Методами лазерной сканирующей микроскопии, световой микроскопии, теста МТТ показано, что ионно-плазменная модификация приповерхностных слоев никелида титана ионами кремния или тантала улучшает цитосовместимость металического сплава.
Ключевые слова: никелид титана, ионно-плазменная модификация, кремний, тантал, цитосовместимость.
Матвеев А.Л. - м.н.с. лаборатории молекулярной микробиологии, е-шаП: vam@niboch.nsc.ru Артемьева Л.В. - ведущий инженер лаборатории стволовой клетки Матвеева В.А. - к.б.н., научный сотрудник лаборатории стволовой клетки
Мейснер Л.Л. - д.ф.-м.н., профессор, главный научный сотрудник лаборатории материаловедения сплавов с памятью формы; профессор кафедры физики металлов, ведущий научный сотрудник лаборатории медицинского материаловедения
Мейснер С.Н. - к.т.н., м.н.с. лаборатории материаловедения сплавов с памятью формы; м.н.с. лаборатории медицинского материаловедения
