ISSN 00125008, Doklady Chemistry, 2015, Vol. 461, Part 1, pp. 86–88. © Pleiades Publishing, Ltd., 2015. Original Russian Text © E.O. Nasakina, A.S. Baikin, K.V. Sergienko, M.A. Sevost’yanov, A.G. Kolmakov, B.A. Goncharenko, V.T. Zabolotnyi, R.S. Fadeev, I.S. Fadeeva, S.V. Gudkov, K.A. Solntsev, 2015, published in Doklady Akademii Nauk, 2015, Vol. 461, No. 1, pp. 49–52.
CHEMICAL TECHNOLOGY
Biocompatibility of Nanostructured Nitinol with Titanium or Tantalum Surface Composite Layers Formed by Magnetron Sputtering E. O. Nasakinaa, A. S. Baikina, K. V. Sergienkoa, M. A. Sevost’yanova, A. G. Kolmakova, B. A. Goncharenkoa, V. T. Zabolotnyia, R. S. Fadeevb, c, I. S. Fadeevab, c, S. V. Gudkovb, c, d, and Academician K. A. Solntseva Presented by Academician A. Yu. Tsivadze September 30, 2014 Received September 2, 2014
DOI: 10.1134/S0012500815030027
of toxic responses and protect the implant made of this material from damage.
Despite the fact that the search for materials for medical implants has been pursued for more than two hundred years, the existing materials, especially met als, are not inert to the human body [1]. The implan tation of these materials often induces adverse response from the patient’s body such as development of metallosis, implant rejection or fibrosis of the adja cent tissues. Simultaneously, the lack of a biologically inert barrier at the contact between the metallic parts of the implant and active biological body fluids may cause metal surface corrosion and, hence, the loss of the fundamental implant properties such as strength and plasticity [2]. Owing to the high corrosion resis tance, fast passivation, and easy formation of stable oxide, titanium and its alloys were among the first metals to be used for the manufacture of medical implants [3]. Currently, nitinol (titanium nickelide NiTi) is the most popular titanium alloy for medicine. Despite the superelasticity and the shape memory inherent in nitinol, high percentage of toxic nickel has long held up the extensive use of the alloy in medicine. In view of the above, it is obvious that the presence of a stable bioinert barrier layer on the nitinol surface that would restrict the penetration of nickel into the body interior, would substantially mitigate the potential risk
Now a lot of technologies are known for modifica tion of the surface layers of nitinol by carbon, silicon, their various compounds, and metals (Ti, Ta, Au, Ag, Pt, etc.) [4]. Most of these modifications substantially improved the biocompatibility; however, the stability of these coatings left much to be desired. Currently, the problem of nitinol biostability has been solved to a considerable extent by using magnetron sputtering of chemically pure titanium and tantalum [5]; however, the biocompatibility of these composites with human tissues is unknown. The purpose of this work was to study the biocom patibility of nanostructured nitinol [NiTi (55.91 wt % Ni–44.03 wt % Ti] with the titanium (Ti@NiTi) or tantalum (Ta@NiTi) surface composite layers formed by magnetron sputtering. As noted above, the presence of thin nickelfree barrier layers formed on the nitinol surface consider ably enhances its corrosion resistance [4] and titanium and tantalum are much less toxic than nickel [6, 7]. The composite “nitinol substrate–surface layer” was manufactured by magnetron sputtering using a Torr International facility (United States) [5] in an argon medium at a residual pressure of less than 0.5 mPa and operating pressure of ≈0.4 Pa. The magnetron with a target made of chemically pure tantalum or titanium operated on a 860 mA d.c. at a sputtering distance of 150 mm from the substrate, a voltage of 400 V, and a sputtering time of 3 h. Prior to sputtering, the sub strates were subjected to preliminary ionic etching (surface cleaning, activation, and polishing upon argon ion bombardment). The temperature of the sub strate surface did not reach 150°C.
a
Baikov Institute of Metallurgy and Materials Science, Russian Academy of Sciences, Leninskii pr. 49, Moscow, 119991 Russia b Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow oblast, 142292 Russia c Pushchino State Institute of Natural Sciences, pr. Nauki 5, Pushchino, Moscow oblast, 142292 Russia d Prokhorov General Physics Institute, Russian Academy of Sciences, ul. Vavilova 38, Moscow, 117942 Russia email:
[email protected] 86
BIOCOMPATIBILITY OF NANOSTRUCTURED NITINOL
It is known that the toxic effect of nickel and other transition metal ions is realized at the molecular level via the effect of these metal ions on the generation of reactive oxygen species and via interaction with pro teins and nucleic acids [8]. Therefore, we studied the effect of nanostructured nitinol and the nitinolbased composites with titanium or tantalum surface layers on the formation of H2O2 in the phosphate buffer (pH 6.8) on heating (37°C) for 200 min in the enhanced chemiluminescence system (luminol–p iodophenol–peroxidase), as described previously [9, 10]. As can be seen from the table, nanostructured nit inol and the composites affect the H2O2 formation in different ways. Sputtering of titanium or tantalum decreases the concentration of hydrogen peroxide formed, particularly, the tantalum layer decreases the H2O2 concentration by approximately 60%, whereas the titanium layer reduces it by only about 40%. By using a fluorescence probe specific to the OH radicals, coumarin3carboxylic acid (Aldrich, USA) [11, 12], it was found that all types of barrier coatings decrease the amounts of these radicals formed in a 20 mM phosphate buffer solution (pH 6.8) on heating (80°C) for 2 h (table). Titanium and tantalum coatings decreased the amount of the hydroxyl radicals by about 70 and 80%, respectively. The test systems we used showed that the titanium or tantalum surface composite layers prevent the excessive generation of reactive oxygen species such as hydroxyl radicals and hydrogen peroxide. The biocompatibility of the nanostructured nitinol and the composites in question with titanium or tanta lum surface layers was measured in vitro using stan dard test systems. The cultures of myofibroblasts from human peripheral vessels and human bone marrow mesenchymal stromal cells (MSC) were used as stan dard cell models. The myofibroblasts were isolated from cut peripheral veins by a procedure reported pre viously [13] and grown in the DMEM medium (Bio lot, Russia) with addition of 10% fetal calf serum (Gibco, United States), 40 µg/mL of gentamicin at 37°С, and 5% carbon dioxide in a CO2 incubator (Binder, Germany). The marrow mesenchymal stro mal cells (Biolot, Russia) were grown in the alpha MEM medium (Sigma, United States) under the same conditions. Fragment samples of materials (25 × 25 mm) were placed into the wells of a 6well plate (Greiner, Ger many), one sample per well. Then cells were inocu lated on the sample surface (5 × 103 cells per cm2) and cultured for 5 days. To determine the numbers of vital and dead cells, the cells growing on the material sur faces were stained with fluorescent dyes—acridine orange (Sigma, USA), 1 µg/mL, and propidium iodide (Sigma, USA), 1 µg/mL. Acridine orange DOKLADY CHEMISTRY
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Effect of nitinol and nitinol with surface composite layers based on tantalum or titanium on the heatinduced formation of hydrogen peroxide (40°C, 200 min) and hydroxyl radicals (80°C, 120 min) in aqueous solutions Treatment
.
Δ [Н2О2]
Δ [ OH ] nM
Reference NiTi Ta@NiTi Ti@NiTi
3.2 ± 0.2 * 10.7 ± 0.8 4.5 ± 0.3 * 6.5 ± 0.5 *
14.3 ± 0.9 * 120.7 ± 4.9 30.9 ± 2.0 * 26.1 ± 1.3 *
* The difference from the NiTi group is statistically significant, p < 0.05 (Student ttest).
stains both vital and dead cells, while propidium iodide stains only dead cells. After that, the samples were incubated for 10 min at 37°С [14]. Then the sam ples were examined under a DM 6000 fluorescence microscope (Leica, Germany). For the assay, at least 500 cells on the sample surface were counted. In the case of myofibroblasts from peripheral vessels, the per centages of vital cells for NiTi (reference), Ta@NiTi, and Ti@NiTi were 91 ± 3, 95 ± 2, and 97 ± 2%, respec tively. In the case of the MSC, the percentages of vital cells for NiTi, Ta@NiTi, and Ti@NiTi were 95 ± 1, 96 ± 3, and 96 ± 2%, respectively. Thus, none of the material surface samples used in the study had a short term toxic effect on the cells that overgrew these sur faces de novo. The mitotic activity of the cells was assessed con sidering the mitotic index of the cells in the logarith mic growth phase (the 3rd day after inoculation), Fig. 1. The number of mitotic cells was determined by fluo rescence microscopy using the vital staining with the Hoechst 33342 fluorescent dye (Sigma, USA). The mitotic cells were identified based the distribution of chromatin inherent in the prophase (P), meta phase (M), anaphase (A), and telophase (T). At least 500 cells on the sample surface were counted for the assay. The mitotic index (MI) was calculated as MI = (P + M + A + T)/N × 100%, where (P + M + A + T) are the numbers of cells occurring at the prophase, metaphase, anaphase, and telophase stages, respec tively, and N is the total number of counted cells [15]. The MI value for the cells growing on the NiTi (refer ence) surface was 3.1% for the myofibroblast culture and 1.8% for the MSC culture. In the case of Ta@NiTi, the MI was 6.1% for the myofibroblasts and 4.3% for the bone marrow MSC. For the myofibro blasts and MSC cultured on Ti@NiTi, the mitotic indices were 5.8% and 4.7%, respectively. We performed morphological analysis of the myo fibroblasts from peripheral vessels and bone marrow MSC on the surface of materials after 5 days of cultur ing. The surfaces of Ti@NiTi and Ta@NiTi were found
NASAKINA et al. 7
60
6
50
5
Free surface, %
Mitotic index, %
88
4 3 2
30 20 10
1 0
40
0 NiTi
Ta@NiTi
Ti@NiTi
NiTi
Ta@NiTi
Ti@NiTi
Fig. 1. Effect of nitinol and nitinol with surface composite layers based on tantalum or titanium on the change in the mitotic index of the myofibroblasts from human peripheral vessels (empty columns) and human bone marrow mesen chymal stromal cells (hatched columns). The averaged data from three independent experiments are given.* The difference from the NiTi group is statistically significant, p < 0.05 (Mann–Whitney U test).
Fig. 2. Effect of nitinol and nitinol with surface composite layers based on tantalum or titanium on the population of the substrate surface by the myofibroblasts of human peripheral vessels (empty columns) and human bone mar row mesenchymal stromal cells (hatched columns). The averaged data from three independent experiments are given.* The difference from the NiTi group is statistically significant, p < 0.05 (Mann–Whitney U test).
to be more suitable for cell attachment and spreading than the NiTi surface (Fig. 2). The cells growing on the NiTi surface (reference) occupied a smaller area, accessible for growth, than the cells on the Ti@NiTi and Ta@NiTi samples. After 5 days of culturing, both myofibroblasts and MSC form a merged monolayer on the Ti@NiTi and Ta@NiTi surfaces. On the NiTi sur face, no monolayer is formed for either myofibroblasts or MSC. Myofibroblasts occupy ≈75% of the NiTi sur face accessible for the growth, while MSC occupy ≈50% of the accessible NiTi surface.
REFERENCES
Thus, the higher mitotic activity as compared with the NiTi reference sample and formation of a merged cell monolayer of the myofibroblasts and the mesen chymal stromal cells growing on the surfaces of the Ti@NiTi and Ta@NiTi samples indicate that Ti@NiTi and Ta@NiTi exhibit higher biocompatibility than the NiTi reference. ACKNOWLEDGMENTS This work was supported by the Russian Founda tion for Basic Research (project no. 13–03–12218 ofi_m and partial support by projects no. 13–04– 00730a and 14–04–32191a), by the Ministry of Education and Science of the Russian Federation (State order), and the Council for Grants of the Pres ident of the Russian Federation for State Support of Young Russian Scientists and State Support of Leading Scientific Schools of the Russian Federa tion (grant no. SP6867.2013.4).
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Translated by Z. Svitanko DOKLADY CHEMISTRY
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