Metabolic reaction of cells to changes in the physical and chemical state of the nitinol surface induced by ultraviolet laser radiation with a wavelength of 266 nm

Aim. MTT (methylthiazolyldiphenyl-tetrazolium bromide) viability assessment of the in vitro cellular response to changes in the physicochemical properties (zeta potential, wettability, topography) of the nitinol surface after treatment with ultraviolet (UV) laser radiation (UV-LR) with a wavelength of 266 nm.
Materials and methods. The MCF-7 cell culture was incubated with nitinol-based alloy plates for 24 hours, before and after exposure to UV light (wavelength 266 nm, energy density 0.1 J/cm²) with a Q-smart 850 pulsed Nd:YAG laser. The samples were then analyzed for roughness (Ra) (GOST 2789-73) and surface contact angle with water. Zeta (ζ) potentials were also measured for both nitinol-based alloy substrates (SurPASS 3) and MCF-7 cells (Litesizer DLS 500). Metabolic activity of the cells was assessed using the MTT test, as recommended by GOST ISO 10993-5-2023 for the in vitro assessment of the cytotoxicity of medical materials and devices. Bioinformatics in silico analysis of intracellular signaling and metabolic pathways was conducted using the WikiPathways database.
Results. Surface modification caused by UV-LR significantly reduced the toxic response (decrease in metabolic activity) of MCF-7 cells to the nitinol-based alloy substrate. UV-LR did not affect the zeta potential (±10 mV from the isoelectric point), but it significantly decreased the water contact angle (from 75 to 8.7°) and increased the Ra index of surface roughness (from 71.5 nm to 93.9 nm). In silico mechanotransduction analysis revealed that the roughness and hydrophilicity/hydrophobicity of nitinol can independently induce gene expression in MCF-7 cells, as well as through cross-talking molecular mechanisms. At the same time, several signaling pathways (WP5504, WP3680, WP5477, WP3941, WP5046, and WP2435) were found to be close to those involved in the reduction of the MTT reagent, which is used as a marker for the cell death program induced by metabolic imbalance in MCF-7 cells.
Conclusion. UV-LR promotes an increase in hydrophilicity and/or nanoroughness of the nitinol-based alloy surface, but it does not affect the zeta potential. This may initiate a cascade of molecular mechanisms regulating cellular and tissue responses to implanted materials. These events can include inflammation, hemostasis activation, angiospasm, vessel regeneration, and metabolic activity of target cells.

1. Усачева Е.В., Нелидова А.В., Куликова О.М., Флянку И.П. Смертность трудоспособного населения России от сердечно-сосудистых заболеваний. Гигиена и санитария. 2021;100(2):159–165. DOI: 10.47470/0016-9900-2021-100-2-159-165.

2. Camici G.G. What is an optimal stent? Biological requirements of drug eluting stents. Kardiovaskuläre Medizin. 2008;11(1):22–25.

3. Mani G., Feldman M.D., Patel D., Agrawal C.M. Coronary stents: a materials perspective. Biomaterials. 2007;28(9):1689–1710. DOI: 10.1016/j.biomaterials.2006.11.042.

4. U.S. Food and Drug Administration, Center for Devices and Radiological Health. Technical considerations for non-clinical assessment of medical devices containing Nitinol [draft guidance]. Silver Spring (MD): U.S. Food and Drug Administration; 2021 Jul 15 [updated 2021 Jul 15; cited 2024 Aug 10]. Available from: https://www.fda.gov/media/145294/download

5. Fukushima O., Yoneyama T., Doi H., Hanawa T. Corrosion resistance and surface characterization of electrolyzed Ti-Ni alloy. Dental Materials Journal. 2006;25(1):151–60. DOI: 10.4012/dmj.25.151.

6. Pequegnat A., Michael A., Wang J., Lian K., Zhou Y., Khan M.I. Surface characterizations of laser modified biomedical grade NiTi shape memory alloys. Materials Science and Engineering: C. 2015;50:367–378. DOI: 10.1016/j.msec.2015.01.085.

7. Chenrayan V., Vaishnav V., Shahapurkar K., Dhanabal P., Kalayarasan M., Raghunath S. et al. The effect of fs-laser micromachining parameters on surface roughness, bio-corrosion and biocompatibility of nitinol. Optics & Laser Technology. 2024;170:110200. DOI: 10.1016/j.optlastec.2023.110200.

8. Саблина Т.Ю., Панченко М.Ю., Зятиков И.А., Пучикин А.В., Коновалов И.Н., Панченко Ю.Н. Исследование гидрофильности поверхности металлических материалов, модифицированных ультрафиолетовым лазерным излучением. Обработка металлов (технология, оборудование, инструменты). 2024;26(4):218–233. DOI: 10.17212/1994-6309-2024-26.4-218-233.

9. Panchenko M.Yu., Sablina T.Yu., Zyatikov I.A., Puchikin A.V., Panchenko Yu.N. Effect of UV laser treatment on wetting properties of TiNi alloy. Russian Physics Journal. 2024;67(10):1575–1582. DOI: 10.1007/s11182-024-03284-0.

10. Grenadyorov A.S., Chebodaeva V.V., Khlusov I.А., Semin V.O., Madzhara N.E., Solovyev A.A. Effect of a-C:H:- SiOx coating thickness on corrosion resistance and zeta potential level of Ti-6Al-4V. Journal of Vacuum Science & Technology A. 2025;43(2):023401. DOI: 10.1116/6.0004085.

11. Evtushenko D.N., Fateev A.V., Khainovsky M.A., Polishchuk J., Kokorev O.V., Nasibov T.F. et al. Intermolecular interactions, regioselectivity, and biological activity of L-ascorbic acid, nicotinic acid and their cocrystal. CrystEngComm. 2024;26(46):6650–6666. DOI: 10.1039/d4ce00770k.

12. Nasibov T., Gorokhova A., Porokhova E., Shupletsova V., Yurova K., Avdeeva E. et al. A hypothesis of mesenchymal stem cell osteogenic differentiation mediated by chelidonic acid through the calcium import: original research and computer simulation. Histochemistry and Cell Biology. 2025;163(1):14. DOI: 10.1007/s00418-024-02342-5.

13. Nasibov T.F., Gorokhova A.V., Brazovsky K.S., Porokhova E.D., Avdeeva E.Yu., Belousov M.V. et al. The pipeline for in silico prediction of target genes and target metabolic pathways for small molecules, using chelidonic acid as an example. Advanced targets in Biomedicine. 2025;1(1):6–19. DOI: 10.5922/ATB-2025-1-1-1.

14. Korkmaz S., Goksuluk D., Zararsiz G. MVN: An R package for assessing multivariate normality. The R Journal. 2014;6(2):151–162.

15. PMCMRplus (version 1.9.12). Rdocumentation [Internet]. URL: https://www.rdocumentation.org/packages/PMCMRplus/versions/1.9.12.

16. brunnermunzel (version 2.0). Rdocumentation [Internet]. [cited 2024 Jul 21]. URL: https://www.rdocumentation.org/packages/brunnermunzel/versions/2.0

17. Royston P. Remark AS R94: A remark on algorithm AS 181: the W-test for normality. Journal of the Royal Statistical Society. Series C (Applied Statistics). 1995;44(4):547–551. DOI: 10.2307/2986146.

18. Moser B.K., Stevens G.R. Homogeneity of variance in the two-sample means test. The American Statistician. 1992;46(1):19–21. DOI: 10.1080/00031305.1992.10475839.

19. Coombs W.T., Algina J., Oltman D.O. Univariate and multivariate omnibus hypothesis tests selected to control type I error rates when population variances are not necessarily equal. Review of Educational Research. 1996;66(2):137–179. DOI: 10.3102/00346543066002137.

20. Markowski C.A., Markowski E.P. Conditions for the effectiveness of a preliminary test of variance. The American Statistician. 1990;44(4):322–326. DOI: 10.1080/00031305.1990.10475752

21. Smith H.F., The problem of comparing the results of two experiments with unequal errors. Journal of the Council for Scientific and Industrial Research. 1936;9:211–212.

22. Satterthwaite F.E. An approximate distribution of estimates of variance components. Biometrics Bulletin. 1946;2(6):110–114. DOI: 10.2307/3002019.

23. Welch B.L. The significance of the difference between two means when the population variances are unequal. Biometrika. 1938;29(3/4):350–362. DOI: 10.2307/2332010.

24. Moser B.K., Stevens G.R., Watts C.L. The two-sample t test versus Satterthwaite’s approximate F test. Communications in Statistics-Theory and Methods. 1989;18(11):3963–3975. DOI: 10.1080/03610928908830135.

25. Zimmerman D.W., Zumbo B.D. Rank transformations and the power of the Student t test and Welch t test for non-normal populations with unequal variances. Canadian Journal of Experimental Psychology / Revue canadienne de psychologie expérimentale. 1993;47(3):523. DOI: 10.1037/h0078850.

26. Ruxton G.D. The unequal variance t-test is an underused alternative to Student’s t-test and the Mann – Whitney U-test. Behavioral Ecology. 2006;17(4):688–690. DOI: 10.1093/beheco/ark016.

27. Munzel U., Brunner E. Nonparametric tests in the unbalanced multivariate one-way design. Biometrical Journal. 2000; 42(7):837–854. DOI: 10.1002/1521-4036(200011)42:7<837::AID-BIMJ837>3.0.CO;2-S.

28. Brunner E., Munzel U. The nonparametric Behrens – Fisher problem: asymptotic theory and a small-sample approximation. Biometrical Journal. 2000;42(1):17–25. DOI: 10.1002/(SICI)1521-4036(200001)42:1<17::AID-BIMJ17>3.0.CO;2-U.

29. Karch J.D. Psychologists should use Brunner – Munzel’s instead of Mann – Whitney’s U- test as the default nonparametric procedure. Advances in Methods and Practices in Psychological Science. 2021;4(2): 2515245921999602. DOI: 10.1177/2515245921999602.

30. Noguchi K., Konietschke F., Marmolejo-Ramos F., Pauly M. Permutation tests are robust and powerful at 0.5% and 5% significance levels. Behavior Research Methods. 2021;53(6):2712–2724. DOI: 10.3758/s13428-021-01595-5.

31. Celik N. Welch’s ANOVA: Heteroskedastic skew-t error terms. Communications in Statistics-Theory and Methods. 2022;51(9):3065–3076. DOI:10.1080/03610926.2020.1788084.

32. Delacre M., Lakens D., Leys C. Why psychologists should by default use Welch’s t-test instead of Student’s t-test. International Review of Social Psychology. 2017;30(1):92–101. DOI: 10.5334/irsp.82.

33. Tomarken A.J., Serlin R.C. Comparison of ANOVA alternatives under variance heterogeneity and specific noncentrality structures. Psychological Bulletin. 1986;99(1):90. DOI: 10.1037/0033-2909.99.1.90.

34. Levy K.J. An empirical comparison of the ANOVA F-test with alternatives which are more robust against heterogeneity of variance. Journal of Statistical Computation and Simulation. 1978;8(1):49–57. DOI: 10.1080/00949657808810247.

35. Lix L.M., Keselman J.C., Keselman H.J. Consequences of assumption violations revisited: A quantitative review of alternatives to the one-way analysis of variance F test. Review of Educational Research. 1996;66(4):579–619. DOI: 10.3102/00346543066004579.

36. Tamhane A.C. A comparison of procedures for multiple comparisons of means with unequal variances. Journal of the American Statistical Association. 1979:74(366a):471–480. DOI: 10.1080/01621459.1979.10482541.

37. Shingala M.C., Rajyaguru A. Comparison of post hoc tests for unequal variance. International Journal of New Technologies in Science and Engineering. 2015;2(5):22–33.

38. Berridge M.V., Herst P.M., Tan A.S. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review. 2005;11:127–152. DOI: 10.1016/S1387-2656(05)11004-7.

39. Ghasemi M., Turnbull T., Sebastian S., Kempson I. The MTT Assay: utility, limitations, pitfalls, and Interpretation in bulk and single-cell analysis. International Journal of Molecular Sciences. 2021;22(23):12827. DOI: 10.3390/ijms222312827.

40. Ren Q., Zhu X., Li W., Wu M., Cui S., Ling Y. et al. Fabrication of super-hydrophilic and highly open-porous poly (lactic acid) scaffolds using supercritical carbon dioxide foaming. International Journal of Biological Macromolecules. 2022;205:740–748. DOI: 10.1016/j.ijbiomac.2022.03.107.

41. Long J., Zhong M., Zhang H., Fan P. Superhydrophilicity to superhydrophobicity transition of picosecond laser microstructured aluminum in ambient air. Journal of Colloid and Interface Science. 2015;441:1–9. DOI: 10.1016/j.jcis.2014.11.015.

42. Razi S., Mollabashi M., Madanipour K. Improving the hydrophilicity of metallic surfaces by nanosecond pulsed laser surface modification. Journal of Laser Applications. 2015;27(4):042006-1–042006-9. DOI: 10.2351/1.4928290.

43. Zhu H.Z., Zhang H.C., Ni X.W., Shen Z.H., Lu J. Fabrication of superhydrophilic surface on metallic nickel by sub-nanosecond laser-induced ablation. AIP Advances. 2019;9(8):085308. DOI: 10.1063/1.5111069.

44. Ganash M.A. Anticancer potential of ascorbic acid and inorganic selenium on human breast cancer cell line MCF-7 and colon carcinoma HCT-116. Journal of Cancer Research and Therapeutics. 2021;17(1):122–129. DOI: 10.4103/jcrt.JCRT_989_17.

45. Mobasher M.A. Apoptotic activity and cytotoxic effect of convolvulus spicatus on human breast cancer cell line (MCF-7). Pakistan Journal of Biological Sciences. 2021;24(6):646–655. DOI: 10.3923/pjbs.2021.646.655.

46. Gambardella G., Viscido G., Tumaini B., Isacchi A., Bosotti R., di Bernardo D. A single-cell analysis of breast cancer cell lines to study tumour heterogeneity and drug response. Nature Communications. 2022;13(1):1714. DOI: 10.1038/s41467-022-29358-6.

47. Fayyad R.J., Ali A.N.M., Saeed N.A.A.A.H., Hamzah I.H., Dwaish A.S. Phycosynthesis of silver nanoparticles using Cladophora glomerata and evaluation of their ability to inhibit the proliferation of MCF-7 and L20B cell lines. Asian Pacific Journal of Cancer Prevention: APJCP. 2022;23(10):3563–3569. DOI: 10.31557/APJCP.2022.23.10.3563.

48. Komarova E.G., Akimova E.B., Kondranova A.M., Porokhova E.D., Nasibov T.F., Syrokvashin M.M. et al. Laser interference microscopy study of the morphometric features of MCF-7 cancer cells contacted with 5-Fluorouracil-loaded calcium phosphate coatings on titanium implants. Materials Chemistry and Physics. 2025;333:130299. DOI: 10.1016/j.matchemphys.2024.130299.

49. Zohrabi T., Habibi N., Zarrabi A., Fanaei M., Lee L.Y. Diphenylalanine peptide nanotubes self-assembled on functionalized metal surfaces for potential application in drug-eluting stent. Journal of Biomedical Materials Research. Part A. 2016;104(9):2280–2290. DOI: 10.1002/jbm.a.35764.

50. Pintor A.V.B., Queiroz L.D., Barcelos R., Primo L.S.G., Maia L.C., Alves G.G. MTT versus other cell viability assays to evaluate the biocompatibility of root canal filling materials: a systematic review. International Endodontic Journal. 2020;53(10):1348–1373. DOI: 10.1111/iej.13353.

51. Ohashi K., Fujiwara S., Mizuno K. Roles of the cytoskeleton, cell adhesion and rho signalling in mechanosensing and mechanotransduction. The Journal of Biochemistry. 2017;161(3):245–254. DOI: 10.1093/jb/mvw082.

52. Burridge K., Monaghan-Benson E., Graham D.M. Mechanotransduction: from the cell surface to the nucleus via RhoA. Philosophical Transactions of the Royal Society B. 2019;374(1779):20180229. DOI: 10.1098/rstb.2018.0229.

53. Janota C.S., Calero-Cuenca F.J., Gomes E.R. The role of the cell nucleus in mechanotransduction. Current Opinion in Cell Biology. 2020;63:204–211. DOI: 10.1016/j.ceb.2020.03.001.

54. Khlusov I.A., Litvinova L.S., Yurova K.A., Khlusova M.Y. Precise tissue bioengineering and niches of mesenchymal stem cells: their size and hierarchy matter. Biocell. 2022;46(6):1365–1373. DOI: 10.32604/biocell.2022.018917.

55. Khlusov I.A., Khlusova M.Yu., Litvinova L.S. Native cell domains as stem cell regulatory microterritories for precise tissue engineering. Next Materials. 2023;1(3):100021. DOI: 10.1016/j.nxmate.2023.100021.

56. Spriano S., Sarath Chandra V., Cochis A., Uberti F., Rimondini L., Bertone E. How do wettability, zeta potential and hydroxylation degree affect the biological response of biomaterials? Materials Science and Engineering: C. 2017;74:542–555. DOI: 10.1016/j.msec.2016.12.107.

57. Song J., Gerecht S. Hydrogels to recapture extracellular matrix cues that regulate vascularization. Arteriosclerosis, Thrombosis, and Vascular Biology. 2023;43(8):e291–e302. DOI: 10.1161/ATVBAHA.122.318235.

58. Khlusov I.A., Grenadyorov A.S., Solovyev A.A., Semenov V.A., Zhulkov M.O., Sirota D.A. et al. Endothelial cell behavior and nitric oxide production on a-C:H:SiOx-coated Ti-6Al-4V substrate. International Journal of Molecular Sciences. 2023;24(7):6675. DOI: 10.3390/ijms24076675.

59. Zhang M., Zhao F., Zhu Y., Brouwer L.A., Van der Veen H., Burgess J.K. et al. Physical properties and biochemical composition of extracellular matrix-derived hydrogels dictate vascularization potential in an organ-dependent fashion. ACS Applied Materials & Interfaces. 2024;16(23):29930–29945. DOI: 10.1021/acsami.4c05864.

60. Grenadyorov A.S., Solovyev A.A., Oskomov K.V., Semenov V.A., Zhulkov M.O., Sirota D.A. et al. Morphofunctional reaction of leukocytes and platelets in in vitro contact with a-C:H:SiOx-coated Ti-6Al-4V substrate. Journal of Biomedical Materials Research. Part A. 2023;111(3):309–321. DOI: 10.1002/jbm.a.37470.

61. Belosludtsev K.N., Belosludtseva N.V., Agafonov A.V., Penkov N.V., Samartsev V.N., Lemasters J.J. et al. Effect of surface-potential modulators on the opening of lipid pores in liposomal and mitochondrial inner membranes induced by palmitate and calcium ions. Biochimica et Biophysica Acta (BBA) – Biomembranes. 2015;1848(10):2200–2205. DOI: 10.1016/j.bbamem.2015.05.013.

62. Oberleithner H. Quantifying salt sensitivity. Biological Chemistry. 2021;402(12):1597–1602. DOI: 10.1515/hsz-2021-0206.

63. Ghitescu L., Fixman A. Surface charge distribution on the endothelial cell of liver sinusoids. The Journal of Cell Biology. 1984;99(2):639–647. DOI: 10.1083/jcb.99.2.639.

64. Lee K.S., Lee J., Kim H.K., Yeom S.H., Woo C.H., Jung Y.J. et al. Extracellular vesicles from adipose tissue-derived stem cells alleviate osteoporosis through osteoprotegerin and miR- 21-5p. Journal of Extracellular Vesicles. 2021;10(12):e12152. DOI: 10.1002/jev2.12152.

65. Ouyang L., Shaik R., Xu R., Zhang G., Zhe J. Mapping surface charge distribution of single-cell via charged nanoparticle. Cells. 2021;10(6):1519. DOI: 10.3390/cells10061519.

66. Mao A.W., Jiang T.H., Sun X.J., Peng J. Application of chemokine receptor antagonist with stents reduces local inflammation and suppresses cancer growth. Tumor Biology. 2015;36(11):8637–8643. DOI: 10.1007/s13277-015-3557-1.

67. Venault A., Hsu K.J., Yeh L.C., Chinnathambi A., Ho H.T., Chang Y. Surface charge-bias impact of amine-contained pseudozwitterionic biointerfaces on the human blood compatibility. Colloids and Surfaces B: Biointerfaces. 2017;151:372–383. DOI: 10.1016/j.colsurfb.2016.12.040.

68. Choi S.Y., Habimana O., Flood P., Reynaud E.G., Rodriguez B.J., Zhang N. et al. Material- and feature-dependent effects on cell adhesion to micro injection moulded medical polymers. Colloids and Surfaces B: Biointerfaces. 2016;145:46–54. DOI: 10.1016/j.colsurfb.2016.04.032.

69. Mirzadeh H., Moghadam E.V., Mivehchi H. Laser-modified nanostructures of PET films and cell behavior. Journal of Biomedical Materials Research. Part A. 2011;98(1):63–71. DOI: 10.1002/jbm.a.33097.

70. Anderson C.R., Gambinossi F., DiLillo K.M., Laschewsky A., Wischerhoff E., Ferri J.K. et al. Tuning reversible cell adhesion to methacrylate-based thermoresponsive polymers: effects of composition on substrate hydrophobicity and cellular responses. Journal of Biomedical Materials Research Part A. 2017;105(9):2416–2428. DOI: 10.1002/jbm.a.36100.

71. Lamichhane S., Anderson J.A., Remund T., Sun H., Larson M.K., Kelly P. et al. Responses of endothelial cells, smooth muscle cells, and platelets dependent on the surface topography of polytetrafluoroethylene. Journal of Biomedical Materials Research. Part A. 2016;104(9):2291–304. DOI: 10.1002/jbm.a.35763.

72. Tan L., Xie Q., Jia X., Guo M., Zhang Y., Tang H. et al. Dynamic measurement of the surface stress induced by the attachment and growth of cells on Au electrode with a quartz crystal microbalance. Biosensors and Bioelectronics. 2009;24(6):1603–1609. DOI: 10.1016/j.bios.2008.08.021.

73. Xie Y., Sproule T., Li Y., Powell H., Lannutti J.J., Kniss D.A. Nanoscale modifications of PET polymer surfaces via oxygen-plasma discharge yield minimal changes in attachment and growth of mammalian epithelial and mesenchymal cells in vitro. Journal of Biomedical Materials Research: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials. 2002;61(2):234–245. DOI: 10.1002/jbm.10141.

74. Khorsandi L., Orazizadeh M., Niazvand F., Abbaspour M.R., Mansouri E., Khodadadi A. Quercetin induces apoptosis and necroptosis in MCF-7 breast cancer cells. Bratislavske Lekarske Listy. 2017;118(2):123–128. DOI: 10.4149/BLL_2017_025.

75. Zhu T., Wu J., Zhao N., Cai C., Qian Z., Si F. et al. Superhydrophobic / superhydrophilic Janus fabrics reducing blood loss. Advanced Healthcare Materials. 2018;7(7):e1701086. DOI: 10.1002/adhm.201701086.

76. Wu X.H., Liew Y.K., Mai C.W., Then Y.Y. Potential of superhydrophobic surface for bood-contacting medical devices. International Journal of Molecular Sciences. 2021;22(7):3341. DOI: 10.3390/ijms22073341.

77. Li H., Wang Y., Wu X., Zhou L., Liu L., Fang Y. Blood pump surface roughness: hemocompatibility and machining optimization. ASAIO Journal. 2025;10.1097. DOI: 10.1097/MAT.0000000000002501.