View More View Less
  • 1 Slovak University of Technology Institute of Biochemistry, Nutrition and Health Protection, Faculty of Chemical and Food Technology Radlinského 9 SK-81237 Bratislava Slovak Republic
  • 2 Slovak University of Technology Institute of Inorganic Chemistry, Technology and Materials, Faculty of Chemical and Food Technology Radlinského 9 SK-81237 Bratislava Slovak Republic
Restricted access

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 G 0 /G 1 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 G 0 /G 1 arrest of NIH-3T3 cells. Fluoridation of hydroxyapatite and different FHA and FA structure caused different cell response of NIH-3T3 cells to biomaterials.

  • 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.

    LeBoeuf R. A. , 'Sodium fluoride-induced chromosome aberrations in different stages of the cell cycle: a proposed mechanism ' (1989 ) 223 Mutat. Res. : 191 -203.

    • Search Google Scholar
  • 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.

    Duursma S. A. , 'Cytogenetic effects on lymphocytes in osteoporotic patients on long-term fluoride therapy ' (1998 ) 20 Pharm. World Sci. : 214 -218.

    • Search Google Scholar
  • Barile, F. A. (1994) Introduction to in vitro cytotoxicology. Mechanism and methods . Boca Raton CRC Press Florida, USA.

    Barile F. A. , '', in Introduction to in vitro cytotoxicology. Mechanism and methods , (1994 ) -.

  • 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.

    Brown A. , 'Mutagenic activity of fluorides in mouse lymphoma cells ' (1987 ) 187 Mutat. Res. : 165 -180.

    • Search Google Scholar
  • 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.

    Penman B. W. , 'Sodium fluoride is a less efficient human cell mutagen at low concentration ' (1990 ) 15 Environ. Molec. Mutagen : 71 -77.

    • Search Google Scholar
  • 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.

    Delloye C. , 'In vitro evaluation of acute cytotoxicity of human chemically treated allografts ' (2001 ) 1 Europ. Cells Mater. J. : 52 -58.

    • Search Google Scholar
  • 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.

    Ambrosio L. , 'Hyaluronic acid based materials for intestine tissue engineering: A morphological and biochemical study of cell-material interaction ' (2006 ) 17 J. Mat. Sci. Mater. Med. : 1365 -1372.

    • Search Google Scholar
  • Gábelová, A., Slameňová, D., Ružeková, L’., Farkašová, T., Horváthová, 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.

    Horváthová E. , '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 ' (1997 ) 44 Neoplasma : 380 -388.

    • Search Google Scholar
  • 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.

    Frayssinet P. , 'Degradation of hydroxyapatite, fluorapatite and fluorhydroxyapatite coating of dental implants in dogs ' (1999 ) 48 J. Biomed. Mater. Res. : 224 -234.

    • Search Google Scholar
  • 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.

    Chen J. G. , 'DNA damage, apoptosis and cell cycle changes induced by fluoride in rat oral mucosal cells and hepatocytes ' (2006 ) 12 World J. Gastroenterol. : 1144 -1148.

    • Search Google Scholar
  • Hongslo, J. K., Holland, R. I., Jonsen, J. (1974) Effect of sodium fluoride on LS cells. J. Dent. Res. 53 , 410–413.

    Jonsen J. , 'Effect of sodium fluoride on LS cells ' (1974 ) 53 J. Dent. Res. : 410 -413.

  • Ignjatovič, N., Ninkov, P., Kojič, P., Bokurov, M., Srdič, V., Krnolelac, D., Selakovič, S., Uskokovič, 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.

    Uskokovič D. , 'Cytotoxicity and fibroblast properties dutiny in vitro test of biphasic calcium phosphate/poly-dl-lactide-co-glycolide biocomposites and different phosphate materials ' (2006 ) 69 Microsc. Res. Tech. : 976 -982.

    • Search Google Scholar
  • Jantová, S., Theiszová, M., Letašiová, S., Birošová, L., Palou, T. M. (2008) In vitro effect of fluorhydroxyapatite, fluorapatite and hydroxyapatite on colony formation, DNA damage and mutagenicity. Mutat. Res. 652 , 139–144.

    Palou T. M. , 'In vitro effect of fluorhydroxyapatite, fluorapatite and hydroxyapatite on colony formation, DNA damage and mutagenicity ' (2008 ) 652 Mutat. Res. : 139 -144.

    • Search Google Scholar
  • Jarcho, M., Kay, J. F., Gumaer, K. I., Doremus, R. H., Drobeck, H. P. (1977) Tissue, cellular and sub-cellular events at a bone-ceramic hydroxylapatite interface. J. Bioeng. 1 , 79–92.

    Drobeck H. P. , 'Tissue, cellular and sub-cellular events at a bone-ceramic hydroxylapatite interface ' (1977 ) 1 J. Bioeng. : 79 -92.

    • Search Google Scholar
  • Jeng, J. H., Hsieh, C. C., Lan, W. H., Chang, M. C., Lin, S. K., Hahn, L. J., Kuo, M. Y. (1998) Cytotoxicity of sodium fluoride on human oral mucosal fibroblasts and its mechanism. Cell Biol. Toxicol. 14 , 383–389.

    Kuo M. Y. , 'Cytotoxicity of sodium fluoride on human oral mucosal fibroblasts and its mechanism ' (1998 ) 14 Cell Biol. Toxicol. : 383 -389.

    • Search Google Scholar
  • Jones, C. A., Callaham, M. F., Huberman, E. (1988) Sodium fluoride promotes morphological transformation of Syrian hamster cells. Carcinogenesis 9 , 2279–2284.

    Huberman E. , 'Sodium fluoride promotes morphological transformation of Syrian hamster cells ' (1988 ) 9 Carcinogenesis : 2279 -2284.

    • Search Google Scholar
  • 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.

    Kim H. E. , 'Enhanced performance of fluorine substituted hydroxyapatite composites for hard tissue engineering ' (2003 ) 10 J. Mater. Sci. Mater. Med. : 899 -904.

    • Search Google Scholar
  • 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.

    Knowles J. C. , 'Dissolution control and cellular responses of calcium phosphate coating on zirconia porous scaffold ' (2004 ) A3 J. Biomed. Mater. Res. : 522 -530.

    • Search Google Scholar
  • 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.

    Kim H. E. , 'Mechanical performance and osteoblast-like cell responses of fluorine-substituted hydroxyapatite and zirconia dense composite ' (2005 ) A3 J. Biomed. Mater. Res. : 258 -268.

    • Search Google Scholar
  • Kirkpatrick, C. J., Bittinger, F., 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.

    Otto M. , 'Current trends in biocompatibility testing ' (1998 ) 212 Proc. Inst. Mech. Engrs. : 75 -84.

    • Search Google Scholar
  • 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.

    Harreus U. A. , 'Cytotoxicity and genotoxicity of fluorides in human mucosa and lymphocytes ' (2001 ) 80 Laryngohinootologie : 187 -190.

    • Search Google Scholar
  • Lee, E. J., 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.

    Kim H. E. , 'Fluoridated apatite coating on titanium obtained by electron-bean deposition ' (2005 ) 18 Biomaterials : 3843 -3851.

    • Search Google Scholar
  • LeGeros, R. Z. (1993) Biodegradation and bioresorption of calcium phosphate ceramics. Clinic. Mater. 14 , 65–88.

    LeGeros R. Z. , 'Biodegradation and bioresorption of calcium phosphate ceramics ' (1993 ) 14 Clinic. Mater. : 65 -88.

    • Search Google Scholar
  • 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.

    LeGeros J. P. , 'Biphasic calcium phosphate bioceramics: preparation, properties and applications ' (2003 ) 4 J. Mat. Sci. Mater. Med. : 201 -209.

    • Search Google Scholar
  • Li, Y. M., Zhang, W., Noblitt, T. W., Dunipace, A. J., Stookey, G. K. (1989) Genotoxic evaluation of chronic fluoride exposition: sisterchromatid exchange study. Mutat. Res. 227 , 159–165.

    Stookey G. K. , 'Genotoxic evaluation of chronic fluoride exposition: sisterchromatid exchange study ' (1989 ) 227 Mutat. Res. : 159 -165.

    • Search Google Scholar
  • 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.

    Stookey G. K. , 'Long-term exposure to fluoride in drinking water and sister chromatid exchange frequency in human blood lymphocytes ' (1995 ) 74 J. Dent. Res. : 1468 -1474.

    • Search Google Scholar
  • Lickorish, D., Ramshaw, J. A., Werkmeister, J. A., Glattauer, V., Hewlett, C. R. (2004) Collagenhydroxyapatite composite prepared by biomimetic process. J. Biomed. Mater. Res. Part A 68A , 19–27.

    Hewlett C. R. , 'Collagenhydroxyapatite composite prepared by biomimetic process ' (2004 ) 68A J. Biomed. Mater. Res. Part A : 19 -27.

    • Search Google Scholar
  • 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.

    King M. , 'Selecting valid in vitro biocompatibility tests that predict the in vivo healing response of synthetic vascular prostheses ' (1996 ) 17 Biomaterials : 1835 -1842.

    • Search Google Scholar
  • McKelvey-Martin, V. J., Green, M. H. L., Schmezer, P., Pool-Zobel, B. L., de Méo, M. P., Collins, R. A. (1993) The single cell electrophoresis assay (comet assay): A European review. Mutat. Res. 288 , 47–63.

    Collins R. A. , 'The single cell electrophoresis assay (comet assay): A European review ' (1993 ) 288 Mutat. Res. : 47 -63.

    • Search Google Scholar
  • Nablo, B., Schoenfisch, M. H. (2005) In vitro cytotoxicity of nitric oxide-releasing sol-gel derived materials. Biomaterials 26 , 4405–4415.

    Schoenfisch M. H. , 'In vitro cytotoxicity of nitric oxide-releasing sol-gel derived materials ' (2005 ) 26 Biomaterials : 4405 -4415.

    • Search Google Scholar
  • Wang, W. (2003) Developing bioactive composite materials for tissue replacement. Biomaterials 24 , 2133–2151.

    Wang W. , 'Developing bioactive composite materials for tissue replacement ' (2003 ) 24 Biomaterials : 2133 -2151.

    • Search Google Scholar
  • 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.

    Horii A. , 'Effect of fluoride ion on proliferation of Vero cell line cells: growth acceleration by sodium fluoride ' (1983 ) 31 J. Dent. Health : 453 -460.

    • Search Google Scholar
  • Otsuki, S., Marshed, S. R. M., Chowdhury, S. A., Takayama, F., 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.

    Sakogami H. , 'Possible link between glycolysis and apoptosis induced by sodium fluoride ' (2005 ) 84 J. Dent. Res. : 919 -923.

    • Search Google Scholar
  • Rajab, N. F., Yaakob, T. A., Ong, B. Y., Hamid, M., Ali, A. M., Annuar, 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.

    Inayat-Hussain S. H. , 'DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay ' (2004 ) 59 Med. J. Malaysia : 170 -171.

    • Search Google Scholar
  • 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.

    Salvadori D. M. F. , 'Lack of DNA damage induced by fluoride on mouse lymphoma and human fibroblast cells by single cell gel (Comet) assay ' (2006 ) 17 Braz. Dent. J. : 91 -94.

    • Search Google Scholar
  • Rice, R. M., Hegyeli, A. F., Gourlay, S. J., 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.

    Kulkarni R. K. , 'Biocompatibility testing of polymers: in vitro studies with in vivo correlation ' (1978 ) 12 J. Biomed. Mater. Res. : 43 -54.

    • Search Google Scholar
  • Shan, K. R., Qi, X. L., Long, Y. G., Nordberg, A., Guan, Z. Z. (2004) Decreased nicotinic receptors in PC12 cells and rat brains influenced by fluoride toxicity-α mechanism relating to a damage at the level in post-transcription of the receptor genes. Toxicology 200 , 169–177.

    Guan Z. Z. , 'Decreased nicotinic receptors in PC12 cells and rat brains influenced by fluoride toxicity-α mechanism relating to a damage at the level in post-transcription of the receptor genes ' (2004 ) 200 Toxicology : 169 -177.

    • Search Google Scholar
  • 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.

    Schneider E. L. , 'A simple technique for quantification of low levels of DNA damage in individual cells ' (1988 ) 175 Exp. Cell Res. : 184 -191.

    • Search Google Scholar
  • Slameňová, D., Gábelová, A., Ruppová, K. (1992) Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells. Mutat. Res. 16 , 109–115.

    Ruppová K. , 'Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells ' (1992 ) 16 Mutat. Res. : 109 -115.

    • Search Google Scholar
  • Slameňová, D., Gábelová, A., Ružeková, L’., Chalupa, I., Horváthová, E., Farkašová, T., Bozsakyová, E., Štětina, 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.

    Štětina R. , 'Detection of MNNG-induced DNA lesions in mammalian cells; validation of comet assay against DNA unwinding technique, alkaline elution of DNA and chromosomal aberrations ' (1997 ) 383 Mutat. Res. : 243 -252.

    • Search Google Scholar
  • Theiszová, M., Jantová, S., Letašiová, S., Palou, M., Čipák, L’. (2008) Cytotoxicity of hydroxyapatite, fluorapatite and fluor-hydroxyapatite: a comparative in vitro study. Neoplasma 55 , 312–316.

    Čipák L.’. , 'Cytotoxicity of hydroxyapatite, fluorapatite and fluor-hydroxyapatite: a comparative in vitro study ' (2008 ) 55 Neoplasma : 312 -316.

    • Search Google Scholar
  • Theiszová, M., Jantová, S., Letašiová S., Valík, 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.

    Palou M. , 'Comparative study of a new composite biomaterial fluor-hydroxyapatite on fibroblast cell line NIH-3T3 by direct test ' (2008 ) 63 Biologia : 1 -9.

    • Search Google Scholar
  • 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.

    Maizumi H. , 'Cytotoxicity, chromosome aberations and unscheduled DNA synthesis in cultured human diploid fibroblast induced by sodium fluoride ' (1984 ) 139 Mutat. Res. : 193 -198.

    • Search Google Scholar
  • Vallittu, P. K., Ekstrand, K. (1999) In vitro cytotoxicity of fibre-polymethyl methacrylate composite used in dentures. J. Oral. Rehabil. 28 , 666–671.

    Ekstrand K. , 'In vitro cytotoxicity of fibre-polymethyl methacrylate composite used in dentures ' (1999 ) 28 J. Oral. Rehabil. : 666 -671.

    • Search Google Scholar
  • Verne, E., Bosetti, M., Brovarone, C. V., Moisescu, C., Lupo, F., Spriano, S., Cannas, M. (2002) Fluorapatite glass-ceramic coating on alumina: structural, mechanical and biological characterisation. Biomaterials 16 , 3395–3403.

    Cannas M. , 'Fluorapatite glass-ceramic coating on alumina: structural, mechanical and biological characterisation ' (2002 ) 16 Biomaterials : 3395 -3403.

    • Search Google Scholar
  • 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.

    Yang K. D. , 'Effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocytes ' (2004 ) 17 Biomed. Environ. Sci. : 217 -222.

    • Search Google Scholar
  • 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.

    Kim H. E. , 'Stability and cellular responses to fluorapatite-collagen composites ' (2005 ) 26 Biomaterials : 2957 -2963.

    • Search Google Scholar