# Fileset [14TOBIOTJ.pdf](https://mdr.nims.go.jp/filesets/f539ea5e-9396-47b1-a876-6f35e88a7447/download) ## Creator Junko Okuda-Shimazaki, [Akiko Yamamoto](https://orcid.org/0000-0002-9182-4886), Daisuke Kuroda, Takao Hanawa, [Akiyoshi Taniguchi](https://orcid.org/0000-0001-7835-2522) ## Rights [Creative Commons BY Attribution 4.0 International](https://creativecommons.org/licenses/by/4.0/) ## Other metadata [The Effect of Metal Materials on Heat Shock Protein 70B’ Gene Expression](https://mdr.nims.go.jp/datasets/d766d33f-52fd-406b-815b-9b694e795951) ## Fulltext Microsoft Word - Taniguchi_OBTJ14 The Open Biotechnology Journal, 2007, 1, 14-17 1874-0707/07 2007 Bentham Science Publishers Ltd. The Effect of Metal Materials on Heat Shock Protein 70B’ Gene Expression Junko Okuda-Shimazaki1, Akiko Yamamoto2, Daisuke Kuroda3, Takao Hanawa4 and Akiyoshi Taniguchi*,1 1Cell-Sensing Group, Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan 2Biometal Group, Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan 3Department of Materials Science and Engineering, Suzuka National College of Technology, Shiroko, Suzuka, Mie 510-0294, Japan 4Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 164-0011, Japan Abstract: To avoid the toxic effect of released nickel ions and compounds from conventional stainless steels, nickel-free austenitic stainless steels have been developed. We previously established a new manufacturing process to produce nickel-free austenitic stainless steel that involves nitrogen adsorption treatment. Although the cytocompatibility of nickel-free austenitic stainless steel produced using this method has been evaluated using two viability assay, molecular level analysis, such as gene expression analysis, has not been previously performed. In the present study, the cytotoxicity of our nickel-free austenitic stainless steel, as well as of commercially available metal materials, was evaluated by analysis of heat shock protein 70B’ (HSP70B’) gene expression as a stress response marker. Furthermore, to investigate the effect of metal materials on cytotoxicity, HSP70B’ gene expression was quantified using human osteoblast-like SaOS-2 cells, hu-man monocyte THP-1 cells and the mouse macrophage cell line J774A.1. We found no significant differences in HSP70B’ expression among the various metal materials, including the nickel-free austenitic stainless steel, indicating that the nickel-free austenitic stainless steel produced using our nitrogen adsorption method has the same cytocompatibility as commercially available metal materials. INTRODUCTION In order to avoid the toxic effect of released nickel ions and compounds from conventional stainless steels, nickel-free austenitic stainless steels were developed [1-4]. Gener-ally, nickel-free austenitic stainless steels are difficult to work into fine foils and wires. To overcome this, we devel-oped a new manufacturing process to produce nickel-free austenitic stainless steel by nitrogen adsorption treatment [5]. The cytocompatibility of nickel-free austenitic stainless steel produced using this nitrogen adsorption treatment has been evaluated by two different cytotoxicity tests, which indicated that this nickel-free austenitic stainless steel has higher cell growth and cytocompatibility than other nickel-free austeni-tic stainless steels [6]. However, a molecular level analysis, such as gene expression analysis, has never been performed. The induction of heat shock protein (HSP) genes is a potential marker of general cytotoxicity, due to the fact that the expression of HSP genes can be substantially induced by a wide range of stimuli [7,8]. The most prominent inducible HSP is the 7,000 kDa protein, HSP70, and the expression of HSP70 mRNA is a valuable tool for estimation of biomate-rial cytotoxicity [9-14]. In humans, at least eleven HSP70 genes have been previously identified [15]. HSP70B' was *Address correspondence to this author at the Cell-Sensing Group, Biomate-rials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan; Phone: +81-29-860-4505; Fax: +81-29-860-4714; E-mail: TANIGUCHI.Akiyoshi@nims.go.jp cloned by Leung et al. [16] as a novel HSP70 gene, and stud-ies have suggested that the mRNA expression of this gene is greatly induced by cellular stresses and that its basal mRNA expression level is lower than that of other HSP70 genes [17,18]. Thus, HSP70B' may provide advantages in high-sensitivity detection of cytotoxicity compared to HSP70. In the present study, the cytotoxicity of nickel-free aus-tenitic stainless steel produced by nitrogen adsorption treat-ment was evaluated by analysis of HSP70B’ gene expression as a stress response marker. MATERIALS AND METHODS Materials Nickel-free stainless steel Fe-24Cr-2Mo-1N, 18Cr-12Ni-2Mo-0.02C, SUS316L, cpTi (JIS-2), titanium alloy Ti-6Al-4V and Co-Cr-Mo alloy (ASTM F75-92) discs (diameter: 25 mm, thickness: 2 mm) were prepared by metallographically polished. The prepared disc surfaces were then ultrasonically cleaned with acetone (three x 1 min washes). Prior to seed-ing for culture, all discs were ultrasonically cleaned in 70% ethanol (20 min), dried, and exposed to ultraviolet light for 3 min on each side. Cells and Cell Culture Human osteoblast-like SaOS-2 cells, derived from an osteosarcoma (American Type Culture Collection ATCC HTB 85) were routinely grown in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS) and 2 mM The Effect of Metal Materials on Heat Shock Protein 70B’ Gene Expression The Open Biotechnology Journal, 2007, Volume 1 15 glutamine. Human monocyte THP-1 cells, derived from pe-ripheral blood (ATCC TIB 202) were routinely grown in RPMI1640 medium supplemented with 10% FBS and 2 mM glutamine containing 0.05 mM 2-mercaptoethanol. Before seeding on the metal samples, THP-1 cells were differenti-ated with 50 nM 12-myristate 13-acetate (PMA) for 72 h. The differentiated THP-1 cells were adherent to the tissue culture polystyrene. Mouse macrophage cell line J774A.1 (ATCC TIB 67) was cultured in Dulbecco’s Modified Ea-gle’s Medium (DMEM) supplemented with 10% FBS. Each cell was maintained in a fully humidified atmosphere con-sisting of 95% air/ 5% CO2 at 37°C, and passaged every third or forth day. For analysis, SaOS-2 cells, THP-1 and J774A.1 cells were seeded onto each disc in 6-well plates at a density of 2 x 105 cells/well, 6 x 105 cells/well and 1 x 106 cells/well respectively, as well as onto tissue culture polysty-rene without discs as the negative control. Cells were proc-essed for RNA extraction as indicated below. mRNA Isolation, cDNA Synthesis and Real Time PCR Each cell line was cultured on the various metals for 24 h. To quantitatively assess the cellular expression level of HSP70B’, total RNA was isolated from each cell using ISOGEN reagent (Nippongene, Japan) according to the manufacturer’s instructions. Duplicate or triplicate samples were combined to ensure sufficient amounts of RNA. Total RNA was reversibly transcribed with a random hexamer primer using the SuperScript III First-Strand Synthesis Sys-tem for RT-PCR (Invitrogen Life Technologies, USA) ac-cording to the manufacturer’s instructions. Real time PCR was performed using the ABI PRISM® 7000 sequence detec-tion system (Applied Biosystems, USA). A 1 μl aliquot of cDNA was mixed with 10 μl of 2x Master Mix from the qPCRTM Mastermix Plus for SYBR® Green I QuickGold-Star kit (Eurogentec) and 1 μl of specific primer for human HSP70B’ or human GAPDH. The primers for amplification of HSP70B’ or GAPDH were 5’-CCGGCCCCATCATTGA G-3’ and 5’-CCCATAGCATAGCCCTGACAGT-3’ or 5’-CCCCCACCACACTGAATCTC-3’ and 5’-GCCCCTCCCC TCTTCAAG-3’, respectively. The PCR procedure was as previously described [19-22]. The results from four inde-pendent tests were evaluated using the Dunnet type multiple comparison test. RESULTS AND DISCUSSION In order to investigate the effect of various metal materi-als on cytotoxicity at the gene expression level, HSP70B’ gene expression was quantified using human osteoblast-like SaOS-2 cells, human monocyte THP-1 cells and a mouse macrophage cell line J774A.1. Human osteoblast-like SaOS-2 cells were seeded on our nickel-free stainless Fe-24Cr-2Mo-1N and the commercially available metal materials SUS316L, cpTi, Titanium alloy Ti-6Al-4V and Co-Cr-Mo alloy. No differences were observed in the initial adherent number among the metal materials (this term includes our nickel-free stainless Fe-24Cr-2Mo-1N unless otherwise noted) (data not shown). Analysis of cell proliferation also revealed no differences among the metal materials (data not shown). In addition, the morphol-ogy of seeded SaOS-2 cells, as determined by differential interference contrast microscopy, did not differ among the metal materials (Fig. 1). Fig. (1). Differential interference contrast microscopy pictures of SaOS-2 cells seeded on Fe-24Cr-2Mo-1N (A), SUS316L (B), Co-Cr-Mo (C), c.p.Ti (D) and Ti-6Al-4V (E). Bar = 50 μm. To investigate the effect of the metal materials on cyto-toxicity, HSP70B’ gene expression was quantified by real-time PCR after 24 hour exposure to the metal materials. No significant increase in HSP70B’ expression by the cells was noted among any of the various metal materials or on tissue culture polystyrene alone (negative control) whereas 1 mM of NiCl2 induced HSP70B’ mRNA expression in SaOS-2 (Fig. 2). Fig. (2). HSP70B’ mRNA levels of SaOS-2 cells seeded on Fe-24Cr-2Mo-1N (1), SUS316L (2), Co-Cr-Mo (3) c.p.Ti (4), Ti-6Al-4V (5), tissue culture polystyrene (TCPS) (6, as a negative control) and TCPS + 1 mM NiCl2 (7, as a positive control). The levels of HSP70B’ mRNA were determined by quantitative real-time PCR and normalized against GAPDH. Results are representative of four experiments. Error bars indicate the standard deviation of mean changes. *p < 0.01 versus TCPS. The seeding experiments were also reproduced with the mouse macrophages cell line J774A.1 and human monocyte THP-1 cells. Similar to SaOS-2 cells, the morphology of seeded J774A.1 and THP-1 cells, as determined by differen-tial interference contrast microscopy, did not differ among the metal materials or tissue culture polystyrene alone (Fig. 3 for J774A.1 cells and Fig. 5 for THP-1 cells). On the other hand, a significant increase in HSP70B’ expression by SaOS-2 and J774A.1 cells (Fig. 4), but not THP-1 cells (Fig. 6), was observed for all the metal materials and tissue culture polystyrene alone. Thus, these metal mate-rials show little cytotoxicity to J774A.1 cells suggesting that metal materials have different effects on various cell lines. In a previous study using two different cytotoxicity tests we found that nickel-free austenitic stainless steel produced 00.10.20.30.41 2 3 4 5 6 7% GAPDH16 The Open Biotechnology Journal, 2007, Volume 1 Okuda-Shimazaki et al. by nitrogen adsorption treatment has higher cell growth and cytocompatibility than other nickel-free austenitic stainless steel [6]. In the present study, we found no significant differ-ence in HSP70B’ expression among various metal materials, including our nickel-free austenitic stainless steel, indicating that nickel-free austenitic stainless steel produced by nitro-gen adsorption treatment has the same cytocompatibility as other commercially available metal materials. Fig. (3). Differential interference contrast microscopy pictures of J774A cells seeded on Fe-24Cr-2Mo-1N (A), SUS316L (B), Co-Cr-Mo (C), c.p.Ti (D) and Ti-6Al-4V (E). Bar = 10 μm. Fig. (4). HSP70B’ mRNA levels of J774A cells seeded on Fe-24Cr-2Mo-1N (1), SUS316L (2), Co-Cr-Mo (3) c.p.Ti (4), Ti-6Al-4V (5), TCPS (6, as a negative control) and TCPS + 1 mM NiCl2 (7, as a positive control). The levels of HSP70B’ mRNA were determined by quantitative real-time PCR and normalized against GAPDH. Results are representative of four experiments. Error bars indicate the standard deviation of mean changes. *p < 0.01 versus TCPS. Fig. (5). Differential interference contrast microscopy pictures of THP-1 cells seeded on Fe-24Cr-2Mo-1N (A), SUS316L (B), Co-Cr-Mo (C), c.p.Ti (D) and Ti-6Al-4V (E). Bar = 10 μm. Fig. (6). HSP70B’ mRNA levels of THP-1 cells seeded on Fe-24Cr-2Mo-1N (1), SUS316L (2), Co-Cr-Mo (3) c.p.Ti (4), Ti-6Al-4V (5), TCPS (6, as a negative control) and TCPS + 1 mM NiCl2 (7, as a positive control). The levels of HSP70B’ mRNA were de-termined by quantitative real-time PCR and normalized against GAPDH. Results are representative of four experiments. Error bars indicate the standard deviation of mean changes. *p < 0.01 versus TCPS. REFERENCES [1] Menzel J, Kirschner W, Stein G. High nitrogen containing Ni-free austenitic steels for medical applications. ISIJ Int 1996; 36: 893-900. [2] Uggowitzer PJ, Magdowski R, Speidel MO. Nickel free high nitro-gen austenitic steels. ISIJ Int 1996; 36: 901-8. [3] Gebeau RC, Brown RS. Biomedical implant alloy. Adv Mater Processes 2001; 159: 46-8. 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Received: May 25, 2007 Revised: June 7, 2007 Accepted: June 7, 2007 << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJDFFile false /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /Description << /CHS /CHT /DAN /DEU /ESP /FRA /ITA /JPN /KOR /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken voor kwaliteitsafdrukken op desktopprinters en proofers. 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