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Acta Biologica Hungarica

, Volume 60, Issue 1, pp 89–107 | Cite as

Comparison of Murine Fibroblast Cell Response to Fluor-Hydroxyapatite Composite, Fluorapatite And Hydroxyapatite By Eluate Assay

  • Soňa Jantová
  • Silvia LetašiováEmail author
  • Marica Theiszová
  • M. Palou
Article

Abstract

Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetic composite that contains the same molecular concentration of OH groups and F ions. The aim of this experiment was to evaluate the cellular responses of murine fibroblast NIH-3T3 cells in vitro to solid solutions of FHA and FA and to compare them with the effect of hydroxyapatite (HA). We studied 24, 48 and 72 h effects of biomaterials on cell morphology, proliferation and cell cycle of NIH-3T3 cells by eluate assay. Furthermore, we examined the ability of FHA, FA and HA to induce cell death and DNA damage. Our cytotoxic/antiproliferative studies indicated that any of tested biomaterials did not cause the total inhibition of cell division. Biomaterials induced different antiproliferative effects increasing in the order HA < FHA < FA which were time- and concentration-dependent. None of the tested biomaterials induced necrotic/apoptotic death of NIH-3T3 cells. On the other hand, after 72 h we found that FHA and FA induced G0/G1 arrest of NIH-3T3 cells, while HA did not affect any cell cycle phases. Comet assay showed that while HA demonstrated weaker genotoxicity, DNA damage induced by FHA and FA caused G0/G1 arrest of NIH-3T3 cells. Fluoridation of hydroxyapatite and different FHA and FA structure caused different cell response of NIH-3T3 cells to biomaterials.

Keywords

Fluor-hydroxyapatite composite fluorapatite cytotoxicity/genotoxicity cell death cell cycle 

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Notes

Acknowledgements

This work was supported by Technology Assistance Agency under the contract No. APVT 20-015904. The authors would like to thank Dr. Mira Horváthová for the opportunity to measure cell cycle profiles.

References

  1. 1.
    Aardema, M. J., Gibson, D. P., LeBoeuf, R. A. (1989) Sodium fluoride-induced chromosome aberrations in different stages of the cell cycle: a. proposed mechanism. Mutat. Res. 223, 191–203.CrossRefGoogle Scholar
  2. 2.
    Astern, P., Darroudi, F., Natarajan, A. T., Terpstra, I. J., Duursma, S. A. (1998) Cytogenetic effects on lymphocytes in osteoporotic patients on long-term fluoride therapy. Pharm. World Sci. 20, 214–218.CrossRefGoogle Scholar
  3. 3.
    Barile, F. A. (1994) Introduction to in vitro cytotoxicology. Mechanism and methods. Boca Raton CRC Press Florida, USA.Google Scholar
  4. 4.
    Caspary, W. J., Myhr, B., Bowers, L., McGregor, D., Riach, C., Brown, A. (1987) Mutagenic activity of fluorides in mouse lymphoma cells. Mutat. Res. 187, 165–180.CrossRefGoogle Scholar
  5. 5.
    Crespi, C. L., Seixas, G. M., Turner, T., Penman, B. W. (1990) Sodium fluoride is a. less efficient human cell mutagen at low concentration. Environ. Molec. Mutagen 15, 71–77.CrossRefGoogle Scholar
  6. 6.
    Dufrane, D., Cornu, O., Verraes, T., Schecroun, N., Banse, X., Schneider, Y. J., Delloye, C. (2001) In vitro evaluation of acute cytotoxicity of human chemically treated allografts. Europ. Cells Mater. J. 1, 52–58.CrossRefGoogle Scholar
  7. 7.
    Esposito, A., Mezzogiorno, A., Sannino, A., De Rosa, A., Menditti, D., Esposito, V., Ambrosio, L. (2006) Hyaluronic acid based materials for intestine tissue engineering: A. morphological and biochemical study of cell-material interaction. J. Mat. Sci. Mater. Med. 17, 1365–1372.CrossRefGoogle Scholar
  8. 8.
    Gabelova, A., Slamenova, D., Ruzekova, E., Farkasova, T., Horvathova, E. (1997) Measurement of DNA strand breakage and DNA repair induced with hydrogen peroxide using single cell gel electrophoresis, alkaline DNA unwinding and alkaline elution of DNA. Neoplasma 44, 380–388.PubMedGoogle Scholar
  9. 9.
    Gineste, I., Gineste, M., Ranz, X., Ellefterion, A., Guilhem, A., Rouquet, N., Frayssinet, P. (1999) Degradation of hydroxyapatite, fluorapatite and fluorhydroxyapatite coating of dental implants in dogs. J. Biomed. Mater Res. 48, 224–234.CrossRefGoogle Scholar
  10. 10.
    He, L. F., Chen, J. G. (2006) DNA damage, apoptosis and cell cycle changes induced by fluoride in rat oral mucosal cells and hepatocytes. World J. Gastroenterol. 12, 1144–1148.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Hongslo, J. K., Holland, R. I., Jonsen, J. (1974) Effect of sodium fluoride on LS cells. J. Dent. Res. 53, 410–413.CrossRefGoogle Scholar
  12. 12.
    Ignjatovic, N., Ninkov, P., Kojic, P., Bokurov, M., Srdic, V., Krnolelac, D., Selakovic, S., Uskokovic, D. (2006) Cytotoxicity and fibroblast properties dutiny in vitro test of biphasic calcium phosphate/poly-dl-lactide-co-glycolide biocomposites and different phosphate materials. Microsc. Res. Tech. 69, 976–982.CrossRefGoogle Scholar
  13. 13.
    Jantova, S., Theiszova, M., Letasiova, S., Birosova, L., Palou, T. M. (2008) In vitro effect of fluor-hydroxyapatite, fluorapatite and hydroxyapatite on colony formation, DNA damage and mutagenicity. Mutat. Res. 652, 139–144.CrossRefGoogle Scholar
  14. 14.
    Jarcho, M., Kay, J. F., Gumaer, K. I., Doremus, R. H., Drobeck, H. P. (1977) Tissue, cellular and subcellular events at a. bone-ceramic hydroxylapatite interface. J. Bioeng. 1, 79–92.PubMedGoogle Scholar
  15. 15.
    Jeng, J. H., Hsieh, C. C., Lan, W. H., Chang, M. C., Lin, S. K., Hahn, L. I., Kuo, M. Y. (1998) Cytotoxicity of sodium fluoride on human oral mucosal fibroblasts and its mechanism. Cell Biol. Toxicol. 14, 383–389.CrossRefGoogle Scholar
  16. 16.
    Jones, C. A., Callaham, M. R., Huberman, E. (1988) Sodium fluoride promotes morphological transformation of Syrian hamster cells. Carcinogenesis 9, 2279–2284.CrossRefGoogle Scholar
  17. 17.
    Kim, H. W., Noh, Y. J., Koh, Y. H., Kim, H. E. (2003) Enhanced performance of fluorine substituted hydroxyapatite composites for hard tissue engineering. J. Mater. Sci. Mater. Med. 10, 899–904.CrossRefGoogle Scholar
  18. 18.
    Kim, H. W., Kim, H. E., Salih, V., Knowles, J. C. (2004) Dissolution control and cellular responses of calcium phosphate coating on zirconia porous scaffold. J. Biomed. Mater. Res. A3, 522–530.Google Scholar
  19. 19.
    Kim, H. W., Knowles, J. C., Li, L. H., Kim, H. E. (2005) Mechanical performance and osteoblast-like cell responses of fluorine-substituted hydroxyapatite and zirconia dense composite. J. Biomed. Mater Res. A3, 258–268.Google Scholar
  20. 20.
    Kirkpatrick, C. I., Bittinger, P., Wagner, M., Kohler, H., van Kooten, T. G., Klien, C. L., Otto, M. (1998) Current trends in biocompatibility testing. Proc. Inst. Mech. Engrs. 212, 75–84.CrossRefGoogle Scholar
  21. 21.
    Kleinsasser, N. H., Weissacher, H., Wallner, B. C., Kastenbauer, E. R., Harreus, U. A. (2001) Cytotoxicity and genotoxicity of fluorides in human mucosa and lymphocytes. Laryngohinootologie 80, 187–190.CrossRefGoogle Scholar
  22. 22.
    Lee, E. I., Lee, S. H., Kim, H. W., Kong, Y. M., Kim, H. E. (2005) Fluoridated apatite coating on titanium obtained by electron-bean deposition. Biomaterials 18, 3843–3851.CrossRefGoogle Scholar
  23. 23.
    LeGeros, R. Z. (1993) Biodegradation and bioresorption of calcium phosphate ceramics. Clinic. Mater. 14, 65–88.CrossRefGoogle Scholar
  24. 24.
    LeGeros, R. Z., Lin, S., Rohanizadeh, R., Mijares, D., LeGeros, J. P. (2003) Biphasic calcium phosphate bioceramics: preparation, properties and applications. J. Mat. Sci. Mater. Med. 4, 201–209.CrossRefGoogle Scholar
  25. 25.
    Li, Y. M., Zhang, W., Noblitt, T. W., Dunipace, A. I., Stookey, G. K. (1989) Genotoxic evaluation of chronic fluoride exposition: sisterchromatid exchange study. Mutat. Res. 227, 159–165.CrossRefGoogle Scholar
  26. 26.
    Li, Y., Liang, C. K., Katz, B. P., Brizendine, E. J., Stookey, G. K. (1995) Long-term exposure to fluoride in drinking water and sister chromatid exchange frequency in human blood lymphocytes. J. Dent. Res. 74, 1468–1474.CrossRefGoogle Scholar
  27. 27.
    Lickorish, D., Ramshaw, J. A., Werkmeister, J. A., Glattauer, V., Hewlett, C. R. (2004) Collagen-hydroxyapatite composite prepared by biomimetic process. J. Biomed. Mater. Res. Part A. 68A, 19–27.Google Scholar
  28. 28.
    Marois, Y., Guidoin, R., Roy, R., Vidovsky, T., Jakubiec, B., Sigot-Luizard, M. F., Braybrook, J., Mehri, Y., Laroche, G., King, M. (1996) Selecting valid in vitro biocompatibility tests that predict the in vivo healing response of synthetic vascular prostheses. Biomaterials 17, 1835–1842.CrossRefGoogle Scholar
  29. 29.
    McKelvey-Martin, V. I., Green, M. H. L., Schmezer, P., Pool-Zobel, B. L., de Meo, M. P., Collins, R. A. (1993) The single cell electrophoresis assay (comet assay): A. European review. Mutat. Res. 288, 47–63.CrossRefGoogle Scholar
  30. 30.
    Nablo, B., Schoenfisch, M. H. (2005) In vitro cytotoxicity of nitric oxide-releasing sol-gel derived materials. Biomaterials 26, 4405–4415.CrossRefGoogle Scholar
  31. 31.
    Wang, W. (2003) Developing bioactive composite materials for tissue replacement. Biomaterials 24, 2133–2151.CrossRefGoogle Scholar
  32. 32.
    Oguro, A., Koizumi, K., Horii, A. (1983) Effect of fluoride ion on proliferation of Vero cell line cells: growth acceleration by sodium fluoride. J. Dent. Health 31, 453–460.CrossRefGoogle Scholar
  33. 33.
    Otsuki, S., Marshed, S. R. M., Chowdhury, S. A., Takayama, R., Satoh, T., Hashimoto, K., Sugiyama, K., Amano, O., Yasui, T., Yokate, Y., Akahane, K., Sakogami, H. (2005) Possible link between glycolysis and apoptosis induced by sodium fluoride. J. Dent. Res. 84, 919–923.CrossRefGoogle Scholar
  34. 34.
    Rajab, N. F., Yaakob, T. A., Ong, B. Y., Hamid, M., Ali, A. M., Aimuar, B. O., Inayat-Hussain, S. H. (2004) DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay. Med. J. Malaysia 59 (Suppl B), 170–171.Google Scholar
  35. 35.
    Ribeiro, D. A., Alves de Lima, P. L., Marques, M. E. A., Salvadori, D. M. F. (2006) Lack of DNA damage induced by fluoride on mouse lymphoma and human fibroblast cells by single cell gel (Comet) assay. Braz. Dent. J. 17, 91–94.CrossRefGoogle Scholar
  36. 36.
    Rice, R. M., Hegyeli, A. F., Gourlay, S. L., Wade, C. W., Dillon, J. G., Jaffe, H., Kulkarni, R. K. (1978) Biocompatibility testing of polymers: in vitro studies with in vivo correlation. J. Biomed. Mater. Res. 12, 43–54.CrossRefGoogle Scholar
  37. 37.
    Shan, K. R., Qi, X. L., Long, Y. G., Nordberg, A., Guan, Z. Z. (2004) Decreased nicotinic receptors in PC 12 cells and rat brains influenced by fluoride toxicity-a mechanism relating to a. damage at the level in post-transcription of the receptor genes. Toxicology 200, 169–177.CrossRefGoogle Scholar
  38. 38.
    Singh, N. P., McCoy, M. T., Tice, R. R., Schneider, E. L. (1988) A. simple technique for quantification of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191.CrossRefGoogle Scholar
  39. 39.
    Slamenova, D., Gabelova, A., Ruppova, K. (1992) Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells. Mutat. Res. 16, 109–115.CrossRefGoogle Scholar
  40. 40.
    Slamenova, D., Gabelova, A., Ruzekova, L., Chalupa, I., Horvathova, E., Farkasova, T., Bozsakyova, E., Stetina, R. (1997) Detection of MNNG-induced DNA lesions in mammalian cells; validation of comet assay against DNA unwinding technique, alkaline elution of DNA and chromosomal aberrations. Mutat. Res. 383, 243–252.CrossRefGoogle Scholar
  41. 41.
    Theiszova, M., Jantova, S., Letasiova, S., Palou, M., Cipak, L. (2008) Cytotoxicity of hydroxyapatite, fluorapatite and fluor-hydroxyapatite: a. comparative in vitro study. Neoplasma 55, 312–316.PubMedGoogle Scholar
  42. 42.
    Theiszova, M., Jantova, S., Letasiova S., Valik, L., Palou, M. (2008) Comparative study of a. new composite biomaterial fluor-hydroxyapatite on fibroblast cell line NIH-3T3 by direct test. Biologia 63, 1–9.CrossRefGoogle Scholar
  43. 43.
    Tsutsui, T., Suzuki, N., Ohmori, M., Maizumi, H. (1984) Cytotoxicity, chromosome aberations and unscheduled DNA synthesis in cultured human diploid fibroblast induced by sodium fluoride. Mutat. Res. 139, 193–198.CrossRefGoogle Scholar
  44. 44.
    Vallittu, P. K., Ekstrand, K. (1999) In vitro cytotoxicity of fibre-polymethyl methacrylate composite used in dentures. J. Oral. Rehabil. 28, 666–671.CrossRefGoogle Scholar
  45. 45.
    Verne, E., Bosetti, M., Brovarone, C. V., Moisescu, C., Lupo, F., Spriano, S., Carinas, M. (2002) Fluorapatite glass-ceramic coating on alumina: structural, mechanical and biological characterisation. Biomaterials 16, 3395–3403.CrossRefGoogle Scholar
  46. 46.
    Wang, A. G., Xia, T., Chu, Q. L., Zhang, M., Liu, F., Chen, X. M., Yang, K. D. (2004) Effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocytes. Biomed. Environ. Sci. 17, 217–222.PubMedGoogle Scholar
  47. 47.
    Yoon, B. H., Kim, H. W., Lee, S. H., Bae, C. H. J., Koh, Y. H., Kong, Y. M., Kim, H. E. (2005) Stability and cellular responses to fluorapatite-collagen composites. Biomaterials 26, 2957–2963.CrossRefGoogle Scholar

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© Akadémiai Kiadó, Budapest 2009

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  • Soňa Jantová
    • 1
  • Silvia Letašiová
    • 1
    Email author
  • Marica Theiszová
    • 1
  • M. Palou
    • 2
  1. 1.Institute of Biochemistry, Nutrition and Health Protection, Faculty of Chemical and Food TechnologySlovak University of TechnologyBratislavaSlovakia
  2. 2.Institute of Inorganic Chemistry, Technology and Materials, Faculty of Chemical and Food TechnologySlovak University of TechnologyBratislavaSlovakia

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