MKGS Vol 44 No 1 Jan-Mar 2011.indd 7 Vol. 44. No. 1 March 2011 Research Report Cytotoxicity difference of 316L stainless steel and titanium reconstruction plate Ni Putu Mira Sumarta1, Coen Pramono Danudiningrat1, Ester Arijani Rachmat2, and Pratiwi Soesilawati2 1Department of Oral and Maxillofacial Surgery 2Department of Oral Biology Faculty of Dentistry, Airlangga University Surabaya - Indonesia ABSTRACT Background: Pure titanium is the most biocompatible material today and used as a gold standard for metallic implants. However, stainless steel is still being used as implants because of its strength, ductility, lower price, corrosion resistant and biocompatibility. Purpose: This study was done to revealed the cytotoxicity difference between reconstruction plate made of 316L stainless steel and of commercially pure (CP) titanium in baby hamster kidney-21 (BHK-21) fibroblast culture through MTT assay. Methods: Eight samples were prepared from reconstruction plates made of stainless steel type 316L grade 2 (Coen’s reconstruction plate®) that had been cut into cylindrical form of 2 mm in diameter and 3 mm long. The other one were made of CP titanium (STEMA Gmbh®)) of 2 mm in diameter and 2,2 mm long; and had been cleaned with silica paper and ultrasonic cleaner, and sterilized in autoclave at 121° C for 20 minutes.9 Both samples were bathed into microplate well containing 50 μl of fibroblast cells with 2 x 105 density in Rosewell Park Memorial Institute-1640 (RPMI-1640) media, spinned at 30 rpm for 5 minutes. Microplate well was incubated for 24 and 48 hours in 37° C. After 24 hours, each well that will be read at 24 hour were added with 50 μl solution containing 5mg/ml MTT reagent in phosphate buffer saline (PBS) solutions, then reincubated for 4 hours in CO2 10% and 37° C. Colorometric assay with MTT was used to evaluate viability of the cells population after 24 hours. Then, each well were added with 50 μl dimethyl sulfoxide (DMSO) and reincubated for 5 minutes in 37° C. the wells were read using Elisa reader in 620 nm wave length. Same steps were done for the wells that will be read in 48 hours. Each data were tabulated and analyzed using independent T-test with significance of 5%. Results: This study showed that the percentage of living fibroblast after exposure to 316L stainless steel reconstruction plate was 61.58% after 24 hours and 62.33% after 48 hours. And after exposure to titanium reconstruction plate, the percentage of living fibroblast was 98.69% after 24 hours and 82.24% after 48 hours. Based on cytotoxicity parameter (CD50%), both reconstruction plate made of 316L stainless steel or titanium showed as a non-toxic materials to fibroblast. Conclusion: Both reconstruction plate made of stainless steel and CP titanium were non-toxic to fibroblast, although the stainless steel plate showed lower cytotoxicity level compared to titanium. Therefore a reconstruction plate made from stainless steel type 316L can be used as a safe material for mandibular reconstruction. Key words: 316L Stainless steel plate, titanium plate, cyototoxicity, MTT assay ABSTRAK Latar belakang: Titanium murni adalah bahan yang paling biokompatibel saat ini dan digunakan sebagai standar emas implan logam. Saat ini stainless steel masih digunakan karena kekuatan, ductility, harganya yang murah, tahan terhadap korosi dan cukup biokompatibel. Tujuan: Penelitian ini dilakukan untuk mengetahui perbedaan sitotoksisitas antara plat rekonstruksi yang terbuat dari titanium murni komersial dan plat rekonstruksi yang terbuat dari stainless steel pada kultur sel fibroblas baby hamster kidney- 21 (BHK-21) menggunakan MTT assay. Metode: Delapan sampel yang masing-masing tipe 316L terbuat dari stainless steel 316L grade 2 (Coen’s reconstruction plate®) yang dipotong berbentuk silinder diameter 2 mm dan panjang 3 mm, serta yang terbuat dari titanium murni komersial (STEMA Gmbh®) diameter 2 mm dan panjang 2,2 mm; dan dibersihkan dengan kertas silika dan pembersih ultrasonik serta disterilkan dengan autoclave pada suhu 121° C selama 20 menit. Kedua sampel dimasukkan ke dalam sumur mikroplat yang mengandung 50 μl sel fibroblas dengan kepadatan 2 × 105 dalam media Rosewell Park Memorial Institute-1640 (RPMI-1640), diputar dengan kecepatan 30 rpm selama 5 menit. Sumur mikroplat diinkubasi selama 24 dan 48 jam pada suhu 37° C. Setelah 24 8 Dent. J. (Maj. Ked. Gigi), Vol. 44. No. 1 March 2011: 7–11 jam, pada tiap sumur yang akan dibaca pada jam ke 24 ditambahkan 50 μl cairan yang mengandung 5mg/ml MTT dalam phosphat buffer saline (PBS), kemudian diinkubasi kembali selama 4 jam dalam CO2 10% pada suhu 37° C. Assay kolorimetri dengan MTT digunakan untuk mengetahui viabilitas populasi sel setelah 24 jam. Setiap sumur ditambahkan pelarut dimetil sulfoksida (DMSO) dan diinkubasi kembali selama 5 menit pada suhu 37° C. sumur-sumur tersebut kemudian dibaca dengan Elisa reader dengan panjang gelombang 620 nm. Langkah yang sama dilakukan pada sumur-sumur yang akan dibaca pada jam ke 48. Data kemudian ditabulasi dan dianalisis dengan menggunakan independent T-test dengan signifikansi 5%. Hasil: Penelitian ini menunjukkan presentase fibroblas hidup setelah terpapar plat rekonstruksi yang terbuat dari stainless steel adalah 61,58% setelah 24 jam dan 62,33% setelah 48 jam. Dan setelah paparan dengan plat rekonstruksi yang terbuat dari titanium murni adalah 98,69% setelah 24 jam dan 82,24% setelah 48 jam. Berdasarkan pada parameter sitotoksisitas (CD50%) kedua plat rekonstruksi baik yang terbuat dari titanium murni maupun yang terbuat dari stainless steel tipe 316L merupakan bahan yang tidak bersifat toksik terhadap fibroblas. Kesimpulan: Kedua plat rekonstruksi baik yang terbuat dari stainless steel maupun CP titanium tidak bersifat toksik terhadap fibroblas, walaupun plat stainless steel menunjukkan level sitotoksisitas yang lebih rendah daripada titanium murni. Dengan demikian plat rekonstruksi yang terbuat dari stainless steel 316 L aman digunakan sebagai bahan untuk rekonstruksi mandibula. Kata kunci: Plat stainless steel, plat titanium, sitotoksisitas, MTT assay Correspondence: Ni Putu Mira Sumarta, c/o: Departemen Bedah Mulut dan Maksilofasial, Fakultas Kedokteran Gigi Universitas Airlangga. Jl. Mayjend. Prof. Dr. Moestopo 47 Surabaya 60132, Indonesia. E-mail: putumira_omfs@yahoo.co.id INTRODUCTION Implant materials can be classified into biotolerant, bioinert, and bioactive materials. Stainless steel is a biotolerant implant material, that characterized by the presence of a thin fibrous layer overlying implant surface in contact to bone. Titanium is a bioinert material with a characteristic of direct contact to bone or osseointegration, osseointegration can be achieved because there is no chemical reaction between material surface to surrounding tissue or to body fluid.3 metallic materials implanted in the human body rarely induce serious conditions. Metallic materials conventionally used in medicine and dentistry does not show toxicity. However, some elements of the alloys show toxicity. The toxicity of a metallic material is governed not only by the elements content of the material but also by its corrosion and wear resistance.4 Before implantation, every materials should pass compatibility study. In vitro citotoxicity study is the primary study to determined a material or material component’s biocompatibility. In this study, an unprocessed material or material component’s are placed directly into tissue cell culture.5,6 Cell culture can be used to evaluate material’s citotoxicity through microscopic examination or quantitatif analysis. Cell morphology and characteristics in the adhesion process are evaluated to determine the effect of cellular citotoxicity, adhesion mechanism changes, cell atypia, or cell damage. Fibroblast and osteoblast are the common cell type used in the study of implant biocompatibility.7 One of the citotoxicity study that commonly used is the MTT assay or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. This study are based on the reduction of yellow tetrazolium salt into purple formazan crystal by hidrogenase enzyme secreted from the mitochondria of the metabolically active cells. Amounts of purple formazan crystal define amounts of living cells.8 Until these days, stainless steel and titanium alloys commonly used as implant material in orthopedics and dentistry. Since 2003 until 2008, there were 51 patients with benign mandibular tumor at the department of oral and maxillofacial surgery, Airlangga University/Dr. Soetomo hospital Surabaya were being treated with mandibular resection and immediate reconstruction wether using stainless steel reconstruction plate, bone graft, and combined bone graft and stainless steel plate as stabilizer. Airlangga University Dental Hospital use stainless steel plate because it’s cheaper than titanium plate but have a good strength, ductility, resistant to corrosion, and compatible. Whereas pure titanium is considered as the most biocompatible material, so commonly used as a gold standard in metallic implant. Inexpensive reconstruction material are still needed in Indonesia because of the social economic level of the Indonesian people which mayoritas could not afford expensive materials. The aim of this study was to evaluate the cytotoxicity of mandibular reconstruction plates made from stainless steel type 316L and the one made from pure titanium in BHK-21 cell culture through MTT assay in 24 and 28 hours measurements. MATERIALS AND METHODS This was an experimental laboratory study conducted in December 2009 at Pusat Veterenaria Farma (PUSVETMA) Surabaya. Eight samples were prepared from reconstruction plates made stainless steel type 316L grade 2 (Coen’s reconstruction plate®) that had been cut with wire cutter into cyllindrical form of 2 mm in diameter and 3 mm long; and from one made of commercially pure (CP) titanium (STEMA Gmbh®)) that had been cut into cylindrical form of 2 mm in diameter and 2.2 mm long. After cutting, all 9Sumarta, et al.: Cytotoxicity difference of 316L stainless steel samples were cleaned with silica paper to remove cutting debris, soaked into ethanol 70% in ultrasonic cleaner for 60 minutes, and sterilized in autoclave at 121° C for 20 minutes.9 Fibroblast cells from BHK-21 cell line were cultivated in a roux bottle until confluent, and harvested with trypsine versene solution. Harvested fibroblast were cultured in Rosewell Park Memorial Institute-1640 (RPMI-1640) media which contain 10% fetal bovine serum albumin, incubated for 24 hours in 37° C, cells were transported into small roux botle dan cultured in the density of 2 × 105 into each 96 well microplate until confluent. Each microplate well contain 50 μl of cells with 2 × 105 density in RPMI media, spanned at 30 rpm for 5 minutes. There was also cell control contains cells in culture media as a positive control which assumed as 100% living cells, and media control contains culture media without cells which assumed as 0% living cells. Each microplate well was examined under light microscope to ensure that the incubation time was enough to form crystals. Both samples made from stainless steel and titanium bathed into the well containing fibroblast cells and RPMI. Microplate well was incubated for 24 and 48 hours in 37° C. after 24 hours, each well that will be read at 24 hour were added with 50 μl solution containing 5 mg/ml MTT reagent in PBS, then reincubated for 4 hours in CO2 10% and 37° C. Colorimetric assay with MTT was used to evaluate viability of the cells population after 24 hours. Then, each well were added with 50 μl DMSO and reincubated for 5 minutes in 37° C. the wells were read at Elisa reader in 620 nm wave length. Same steps were done for the wells that will be read in 48 hours. Each data were tabulated and analyzed using independent T-test with significance of 95%. Percentage of the living fibroblast cells were calculated with the following formula, according to experimental study by Meizarini et al:10 Percentage of the living cells = treatment + media × 100% cell + media RESULTS Cell in control group had the highest mean value of optical density and media control group had the lowest mean value of optical density (Table 1). Normality study with nonparametric test using Kolmogorof Smirnov test, showed that all study groups had p > 0.05 which mean that all study groups had a normal distribution that mean values of the data lies between standard deviation and significance test was done with Independent t-test, showed in table 2 and 3. Table 1. Average of optical density and standard deviation of each study groups in 24 and 48 hours measurements Groups n X + SD 24 hours 48 hours Titanium 8 0.42 ± 0.10 0.55 ± 0.05 Stainless steel 8 0.23 ± 0.05 0.39 ± 0.05 Cell control 8 0.42 ± 0.14 0.69 ± 0.12 Media control 8 0.09 ± 0.02 0.10 ± 0.02 Table 2. Difference test of the optical density from each study groups in 24 hours measurement using Independent t-test Titanium Stainless steel Cell control Media control Titanium - 0.001** 0.915 0.001** Stainless steel - - 0.002** 0.001** Cell control - - - 0.001** Media control - - - - Table 3. Difference test of the optical density from each study groups in 48 hours measurement using Independent t-test Titanium Stainless steel Cell control Media control Titanium - 0,001** 0,013** 0,001** Stainless steel - - 0,001** 0,001** Cell control - - - 0,001** Media control - - - - There was a significant difference of the formazan density in the 24 and 48 hours measurement, showing there was a significant increased number of living fibroblast following exposure time to titanium and stainless steel plate (Table 2 and 3). There was no significant difference between the titanium group and the cell control group in the 24 hours measurement with p > 0.05 (Table 2). Table 4. Percentage of the living fibroblast cells in each study groups in 24 and 48 hours measurement Cell group n 24 hours (%) 48 hours (%) Titanium 8 98.69 82.24 Stainless steel 8 61.58 62.33 10 Dent. J. (Maj. Ked. Gigi), Vol. 44. No. 1 March 2011: 7–11 The percentage of the living fibroblast cells in each study groups were more than 50% (Table 4). This results showed both plate made of stainless steel and CP titanium were non-toxic to fibroblast based on CD50%. 11 DISCUSSION Biocompatibility refers to the ability of a biomaterial to perform its desired function with respect to a medical therapy, without eliciting any undesirable local and systemic effects in the recipient or beneficiary of that therapy, but generating the most appropriate beneficial cellular or tissue response in that specific situation, and optimizing the clinically relevant performance of that therapy.12 Biocompatibility is important because implant surface in contact with the tissue can undergo corrosion in vivo. Implant corrosion can cause lost of load bearing strength and can undergo degradation into toxic substances in the tissue.13 Main criteria in choosing metallic implants is its biocompatibility.14 Materials can be classified as a biocompatible material if culture cells remain living and metabolically active in long term culture. There are 2 quick and simple quantitative assay to test biocompatibility in vitro, these are cell viability based on physical uptake of neutral red (NR) and based on cell’s metabolic activity through MTT assay, which based on cellular enzyme activity. Both test are widely accepted as a biocompatibility and cytotoxicity study to evaluate cell viability and growth.15 In this study MTT assay was performed to evaluate the biocompatibility of stainless steel 316L and titanium reconstruction plates, because this method is a simple, accurate measure, and can be done in a large scale study.16–18 This study showed that there was a significant optical density difference of treatment group exposed to stainless steel in 24 and 48 hours measurement, and in treatment group exposed to titanium in 48 hours measurement. Whereas in 24 hours measurement, there was no significant optical density difference between group exposed to titanium and cell control group. This was consistent with the reference that stated titanium is a bioinert material because there is no chemical reaction between material surface to surrounding tissue or to body fluid.3 There were increased optical density of the cell control group from 0.4223 in 24 hours into 0.6860 in 48 hours measurement, optical density of the group exposed to titanium only increased from 0.4156 in 24 hours into 0.5470 in 48 hours measurement. This study showed that reconstruction plates made from stainless steel 316L and CP titanium had a good in vitro biocompatibility to fibroblast cell which indicated from there was no cytotoxic effect to BHK-21 cells. That was concluded from the percentage of living fibroblast cell after exposure to stainless steel 316L in 24 hours was 61.58%; after 48 hours was 62.33%; after exposure to CP titanium was 98.69% in 24 hours; and 82.24% after 48 hours. All measures showed there was no cytotoxicity based on CD50%, the cytotoxicity parameter.11 The difference caused by increasing number of living fibroblast cell from both exposure time, showing increasing living fibroblast cell, because fibroblast were able to anchor and adapt accordingly to metal particle,9 so that cells can replicate. This study showed that CP titanium plates had a higher biocompatibility level compared to stainless steel 316L plates, this consistent with the classification of material which categorize CP titanium into bioinert material, whereas stainless steel 316L is a biotolerant material.19 Biocompatibility of implant material also affected by their resistance and their alloy to corrosion process in body fluid that was known as electrolyte. After implantation there was changes in neutral pH that depend on implantation time and appropriate healing process, tissue pH were between 6.8–7.4.20 Titanium and its alloy are materials with the best corrosion resistance, because of their passive nature and an unreactive passive film formed on titanium surface. Stainless steel 316L had an acceptable corrosion resistance. Stainless steel 316L implant was mostly manufactured in India to be used for orthopedic application because of its lower price, easier welding and processing compared to cobalt-chromium alloy and titanium or titanium alloy. Other properties of stainless steel 316L are biocompatible, good tensile strength, fatigue resistance, with appropriate density for weight bearing, made it a preferable surgical implant material. The disadvantage of titanium are expensive, easily wear off, and brittle.21 It can be concluded that both reconstruction plate made of stainless steel and CP titanium were non-toxic to fibroblast, although the stainless steel plate showed lower cytotoxicity level compared to titanium. Therefore a reconstruction plate made from stainless steel type 316L can be used as a safe material for mandibular reconstruction. REFERENCES 1. Disa JJ, Hidalgo DA. Mandibular reconstruction In: Thorne CA, Beasley RW, Aston SJ, Bartlett SP, Gurtner GC, Spear SL, editors. Grabb and Smith’s plastic surgery. 6th ed. Philadelphia: Lippincott Williams and Wilkins; 2007. p. 428. 2. Doty JM, Pienkowski D, Goltz M, Haug RH, Valentino J, Arosarena OA. Biomechanical evaluation of fixation techniques for bridging segmental mandibular defects. Arch Otolaryngol Head and Neck Surg 2004; 130: 1388–92. 3. Gupta R, Caiozzo VJ, Skinner HB. Basic science in orthopedic surgery. In: Skinner HB, editor. Current diagnosis and treatment in orthopedics. 4th ed. New York: The McGraw-Hill companies, Inc; 2006. p. 31–3. 4. Hanawa T. Evaluation techniques of metallic biomaterials in vitro. Sci and Tech of Adv Mater 2002; 3: 289–95. 5. Wataha JC. Biocompatibility of dental materials. In: Anusavice KJ, editor. Science of dental materials. 11st ed. Philadelphia: Elsevier Science; 2003. p. 173–92. 6. Kao CT, Ding SJ, Min Y, Hsu TC, Chou MY, Huang TH. The cytotoxicity of orthodontic metal bracket immersion media. Europ J Orthodont 2007; 29: 198–203. 7. An YH, Martin KL, editors. Handbook of histology methods for bone and cartilage. New Jersey: Humana Press Inc; 2003. p. 36, 443–5. 8. Vannet VB, Hanssens JL, Wehrbein H. The use of three-dimensional oral mucosa cell cultures to assess the toxicity of soldered and welded wires. European J Orthodont 2007; 29: 60–6. 11Sumarta, et al.: Cytotoxicity difference of 316L stainless steel 9. Cheung S, Gauthier M, Lefebvre LP, Dunbar M, Filliaggi M. Fibroblastic interactions with high porosity Ti-6Al-4V metal foam. J Biomed Mater Res Part B: Apll Biomater 2007; 82B: 440–9. 10. Meizarini A, Munadziroh E, Rachmadi P. Sitotoksisitas bahan restorasi cyanoacrylate dengan variasi perbandingan powder dan liquid menggunakan MTT assay. Jurnal Penelitian Medika Eksakta 2005; 6(1): 16–25. 11. Telli C, Serper A, Dogan AL, Guc D. Evaluation of the cytotoxicity of calcium phosphate root canal sealers by MTT assay. J Endodon 1999; 25: 811–3. 12. Williams DF. On the mechanisms of biocompatibility. Biomaterials 2008; 30: 1–13. 13. Desai S, Bidanda B, Bártolo P. Metallic and ceramic biomaterials: current and future developments. In: Bártolo P, Bidanda B, editors. Bio-materials and prototyping applications in medicine. New York: Springer-Science; 2008: 1–2. 14. Mudali UK, Sridhar TM, Raj B. Corrosion of bio implants. Sadhana 2003; 28(3–4): 601–37. 15. Schmutz P, Quach-Vu NC, Gerber I. Metallic medical implants: electrochemical characterization of corrosion processes. The Electrochemical Society Interface. Summer 2008; 35–40. 16. Wutticharoenmongkol P, Sanchavanakit V, Pavasant P, Supaphol P. Preparation and characterization of novel bone scafolds based on electrospun polycaprolactone fibers filled with nanoparticles. Macromol Biosci 2006; 6: 70–7. 17. Sjogren G, Sletten G, Dahl JE. Cytotoxicity of dental alloys metals, and ceramics assessed by millipore filter, agar overlay, and MTT tests. J Prosthet Dent 2000; 84: 229–36. 18. Tomadiki P. Assessment of acute cyto- and genotoxicity of corrosion eluates obtained from orthodontic materials using monolayer cultures of immortalized human gingival keratinocytes. J Orofac Orthop 2000; 61: 2–19. 19. Beloti MM, Rollo JMDA, Filho AI, Rosa AL. In vitro biocompatibility of duplex stainless steel with and without 0.2% niobium. J App Biomat & Biomech 2004; 2: 162–8. 20. Kiel M, Marciniak AK. Corrosion resistance of metallic implants used in bone surgery. Arch Mater Sci and Engin 2008; 30(2): 77–80. 21. Mudali UK, Sridhar TM, Raj B. Corrosion of bio implants. Sadhana 2003; 28(3-4): 601–37. << /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. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /PTB /SUO /SVE /ENU (Use these settings to create Adobe PDF documents for quality printing on desktop printers and proofers. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames true /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /ConvertColors /NoConversion /DestinationProfileName () /DestinationProfileSelector /NA /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure true /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles true /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /LeaveUntagged /UseDocumentBleed false >> ] >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice