8912a65a2b4837217406fec29f61c67c intjoumol_a2020v22n2p528.pdf 54282e67671e7360a37171459d39a3462e35d7c8 intjoumol_a2020v22n2p528.pdf c5a29d910fd61a20a3601ba1d584b67f375a5448ac96808947c5f502295938d8 intjoumol_a2020v22n2p528.pdf Title: Electroless Palladium-Coated Polymer Scaffolds for Electrical Stimulation of Osteoblast-Like Saos-2 Cells Subject: Three-dimensional porous scaffolds offer some advantages over conventional treatments for bone tissue engineering. Amongst all non-bioresorbable scaffolds, biocompatible metallic scaffolds are preferred over ceramic and polymeric scaffolds, as they can be used as electrodes with different electric field intensities (or voltages) for electric stimulation (ES). In the present work we have used a palladium-coated polymeric scaffold, generated by electroless deposition, as a bipolar electrode to electrically stimulate human osteoblast-like Saos-2 cells. Cells grown on palladium-coated polyurethane foams under ES presented higher proliferation than cells grown on foams without ES for up to 14 days. In addition, cells grown in both conditions were well adhered, with a flat appearance and a typical actin cytoskeleton distribution. However, after 28 days in culture, cells without ES were filling the entire structure, while cells under ES appeared rounded and not well adhered, a sign of cell death onset. Regarding osteoblast differentiation, ES seems to enhance the expression of early expressed genes. The results suggest that palladium-coated polyurethane foams may be good candidates for osteoblast scaffolds and demonstrate that ES enhances osteoblast proliferation up to 14 days and upregulate expression genes related to extracellular matrix formation. Keywords: electrical stimulation; osteoblast; Pd-coated polyurethane scaffold; gene expression; differentiation Author: Oriol Careta, Asier Salicio-Paz, Eva Pellicer, Elena Ibáñez, Jordina Fornell, Eva García-Lecina, Jordi Sort and Carme Nogués Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Jan 7 08:48:19 2021 CET ModDate: Thu Jan 7 08:55:18 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2404508 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- ETMLXX+VnURWPalladioL Type 1 Custom yes yes no 10 0 JZVTJZ+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 YMELZN+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 ACKVXH+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 VBSGJL+CMSY10 Type 1 Builtin yes yes no 28 0 IOJCNK+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 40 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 43 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 65 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 69 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 73 0 PMTDSU+EURM10 Type 1 Builtin yes yes no 88 0 OMDDIE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 108 0 OMDDIG+PalatinoLinotype TrueType WinAnsi yes yes no 111 0 OMDDII+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 114 0 OMDDIE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 130 0 OMDDIG+PalatinoLinotype TrueType WinAnsi yes yes no 133 0 OMDDII+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 136 0 OMDDIE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 150 0 OMDDIG+PalatinoLinotype TrueType WinAnsi yes yes no 153 0 OMDDII+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 156 0 OMDDIE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 170 0 OMDDIG+PalatinoLinotype TrueType WinAnsi yes yes no 173 0 OMDDII+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 176 0 OMDDIE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 195 0 OMDDIG+PalatinoLinotype TrueType WinAnsi yes yes no 198 0 OMDDII+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 201 0 OMDDIE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 218 0 OMDDIG+PalatinoLinotype TrueType WinAnsi yes yes no 221 0 OMDDII+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 224 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2021-01-23 02:55:46 CET RepresentationInformation: intjoumol_a2020v22n2p528.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2021-01-08 11:24:59 CET Size: 2404508 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 432 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Children: Item: Title: Pd-Coated PU Foam Synthesis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Scanning Electron Microscopy Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: X-ray Diffraction Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Cell Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Construction and Use of an Electrical Stimulation Chamber Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Cell Proliferation Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: SEM Analysis of Cell Morphology Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Actin Staining Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Real-Time Quantitative PCR Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Mineralization Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Children: Item: Title: Structural Characterization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Cell Proliferation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Cell Morphology and Adhesion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Expression of Osteogenic Markers Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Calcium Deposition Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Info: Title: Electroless Palladium-Coated Polymer Scaffolds for Electrical Stimulation of Osteoblast-Like Saos-2 Cells Author: Oriol Careta, Asier Salicio-Paz, Eva Pellicer, Elena Ibáñez, Jordina Fornell, Eva García-Lecina, Jordi Sort and Carme Nogués Subject: Three-dimensional porous scaffolds offer some advantages over conventional treatments for bone tissue engineering. Amongst all non-bioresorbable scaffolds, biocompatible metallic scaffolds are preferred over ceramic and polymeric scaffolds, as they can be used as electrodes with different electric field intensities (or voltages) for electric stimulation (ES). In the present work we have used a palladium-coated polymeric scaffold, generated by electroless deposition, as a bipolar electrode to electrically stimulate human osteoblast-like Saos-2 cells. Cells grown on palladium-coated polyurethane foams under ES presented higher proliferation than cells grown on foams without ES for up to 14 days. In addition, cells grown in both conditions were well adhered, with a flat appearance and a typical actin cytoskeleton distribution. However, after 28 days in culture, cells without ES were filling the entire structure, while cells under ES appeared rounded and not well adhered, a sign of cell death onset. Regarding osteoblast differentiation, ES seems to enhance the expression of early expressed genes. The results suggest that palladium-coated polyurethane foams may be good candidates for osteoblast scaffolds and demonstrate that ES enhances osteoblast proliferation up to 14 days and upregulate expression genes related to extracellular matrix formation. Keywords: electrical stimulation; osteoblast; Pd-coated polyurethane scaffold; gene expression; differentiation Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Jan 07 08:48:19 CET 2021 ModDate: Thu Jan 07 08:55:18 CET 2021 ID: 0xa445a50690ac21e99bd753cba8eedc03, 0xa445a50690ac21e99bd753cba8eedc03 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: IOJCNK+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 189 FontDescriptor: FontName: IOJCNK+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: YMELZN+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 241 FontDescriptor: FontName: YMELZN+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ACKVXH+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 246 FontDescriptor: FontName: ACKVXH+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: PMTDSU+EURM10 FontSubset: true FirstChar: 12 LastChar: 33 FontDescriptor: FontName: PMTDSU+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: ETMLXX+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: ETMLXX+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VBSGJL+CMSY10 FontSubset: true FirstChar: 0 LastChar: 52 FontDescriptor: FontName: VBSGJL+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: JZVTJZ+URWPalladioL-Ital FontSubset: true FirstChar: 45 LastChar: 122 FontDescriptor: FontName: JZVTJZ+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 181, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 97, 662, 109, 675 Annotation: Subtype: Link Rect: 261, 662, 272, 675 Annotation: Subtype: Link Rect: 428, 662, 439, 675 Annotation: Subtype: Link Rect: 531, 662, 542, 675 Annotation: Subtype: Link Rect: 92, 649, 103, 663 Annotation: Subtype: Link Rect: 204, 649, 215, 663 Annotation: Subtype: Link Rect: 146, 313, 154, 322 Annotation: Subtype: Link Rect: 35, 301, 156, 310 Annotation: Subtype: Link Rect: 36, 162, 98, 184 Annotation: Subtype: Link Rect: 123, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 154, 86 Annotation: Subtype: Link Rect: 35, 66, 49, 74 Annotation: Subtype: Link Rect: 213, 188, 220, 197 ActionDest: 235 Annotation: Subtype: Link Rect: 223, 188, 230, 197 ActionDest: 235 Annotation: Subtype: Link Rect: 289, 125, 295, 134 ActionDest: 235 Annotation: Subtype: Link Rect: 296, 125, 303, 134 ActionDest: 235 Annotation: Subtype: Link Rect: 303, 125, 310, 134 ActionDest: 235 Annotation: Subtype: Link Rect: 549, 100, 556, 109 ActionDest: 235 Annotation: Subtype: Link Rect: 135, 17, 274, 28 Annotation: Subtype: Link Rect: 420, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 401, 87, 420 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 464, 712, 471, 721 ActionDest: 235 Annotation: Subtype: Link Rect: 334, 686, 341, 695 ActionDest: 235 Annotation: Subtype: Link Rect: 406, 548, 413, 557 ActionDest: 235 Annotation: Subtype: Link Rect: 502, 523, 509, 532 ActionDest: 235 Annotation: Subtype: Link Rect: 484, 486, 491, 495 ActionDest: 235 Annotation: Subtype: Link Rect: 491, 485, 503, 495 ActionDest: 235 Annotation: Subtype: Link Rect: 506, 486, 518, 495 ActionDest: 238 Annotation: Subtype: Link Rect: 350, 460, 362, 469 ActionDest: 238 Annotation: Subtype: Link Rect: 365, 461, 377, 469 ActionDest: 238 Annotation: Subtype: Link Rect: 435, 461, 447, 469 ActionDest: 238 Annotation: Subtype: Link Rect: 345, 435, 357, 444 ActionDest: 238 Annotation: Subtype: Link Rect: 198, 373, 205, 382 ActionDest: 235 Annotation: Subtype: Link Rect: 386, 347, 398, 356 ActionDest: 238 Annotation: Subtype: Link Rect: 401, 348, 413, 356 ActionDest: 238 Annotation: Subtype: Link Rect: 355, 272, 366, 281 ActionDest: 238 Annotation: Subtype: Link Rect: 282, 222, 294, 231 ActionDest: 238 Annotation: Subtype: Link Rect: 342, 184, 354, 193 ActionDest: 238 Annotation: Subtype: Link Rect: 538, 134, 550, 143 ActionDest: 238 Annotation: Subtype: Link Rect: 449, 71, 461, 80 ActionDest: 238 Annotation: Subtype: Link Rect: 461, 71, 473, 80 ActionDest: 238 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 351, 722, 363, 731 ActionDest: 238 Annotation: Subtype: Link Rect: 366, 722, 378, 731 ActionDest: 238 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 503, 609, 515, 618 ActionDest: 238 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 325, 77, 332, 89 ActionDest: 98 Annotation: Subtype: Link Rect: 253, 65, 260, 77 ActionDest: 98 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 448, 694, 456, 706 ActionDest: 98 Annotation: Subtype: Link Rect: 341, 669, 348, 681 ActionDest: 98 Annotation: Subtype: Link Rect: 304, 647, 316, 656 ActionDest: 238 Annotation: Subtype: Link Rect: 505, 634, 517, 643 ActionDest: 238 Annotation: Subtype: Link Rect: 218, 240, 225, 252 ActionDest: 98 Annotation: Subtype: Link Rect: 269, 177, 276, 189 ActionDest: 98 Annotation: Subtype: Link Rect: 208, 114, 215, 126 ActionDest: 118 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 340, 360, 347, 372 ActionDest: 140 Annotation: Subtype: Link Rect: 522, 348, 529, 360 ActionDest: 140 Annotation: Subtype: Link Rect: 231, 310, 238, 322 ActionDest: 140 Annotation: Subtype: Link Rect: 546, 310, 553, 322 ActionDest: 140 Annotation: Subtype: Link Rect: 496, 272, 503, 284 ActionDest: 140 Annotation: Subtype: Link Rect: 496, 222, 503, 234 ActionDest: 140 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 389, 378, 396, 390 ActionDest: 160 Annotation: Subtype: Link Rect: 484, 315, 491, 327 ActionDest: 160 Annotation: Subtype: Link Rect: 437, 302, 444, 314 ActionDest: 160 Annotation: Subtype: Link Rect: 442, 290, 449, 302 ActionDest: 160 Annotation: Subtype: Link Rect: 490, 265, 497, 277 ActionDest: 160 Annotation: Subtype: Link Rect: 514, 252, 521, 264 ActionDest: 160 Annotation: Subtype: Link Rect: 232, 227, 239, 239 ActionDest: 160 Annotation: Subtype: Link Rect: 300, 214, 307, 226 ActionDest: 160 Annotation: Subtype: Link Rect: 232, 202, 239, 214 ActionDest: 160 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 405, 208, 412, 220 ActionDest: 181 Page: Sequence: 10 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 375, 641, 382, 653 ActionDest: 206 Annotation: Subtype: Link Rect: 406, 216, 418, 226 ActionDest: 238 Annotation: Subtype: Link Rect: 256, 154, 268, 163 ActionDest: 238 Annotation: Subtype: Link Rect: 467, 103, 479, 113 ActionDest: 238 Annotation: Subtype: Link Rect: 235, 78, 247, 88 ActionDest: 238 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 257, 659, 269, 669 ActionDest: 238 Annotation: Subtype: Link Rect: 401, 634, 408, 643 ActionDest: 235 Annotation: Subtype: Link Rect: 409, 634, 421, 643 ActionDest: 238 Annotation: Subtype: Link Rect: 423, 634, 435, 643 ActionDest: 238 Annotation: Subtype: Link Rect: 389, 559, 396, 568 ActionDest: 235 Annotation: Subtype: Link Rect: 222, 521, 234, 530 ActionDest: 241 Annotation: Subtype: Link Rect: 290, 396, 302, 405 ActionDest: 241 Annotation: Subtype: Link Rect: 358, 283, 370, 292 ActionDest: 241 Annotation: Subtype: Link Rect: 373, 283, 385, 292 ActionDest: 241 Annotation: Subtype: Link Rect: 529, 258, 541, 267 ActionDest: 241 Annotation: Subtype: Link Rect: 544, 258, 556, 267 ActionDest: 241 Annotation: Subtype: Link Rect: 265, 170, 277, 179 ActionDest: 241 Annotation: Subtype: Link Rect: 277, 170, 289, 179 ActionDest: 241 Annotation: Subtype: Link Rect: 230, 157, 242, 166 ActionDest: 241 Annotation: Subtype: Link Rect: 392, 107, 399, 116 ActionDest: 235 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 516, 735, 528, 744 ActionDest: 241 Annotation: Subtype: Link Rect: 529, 735, 541, 744 ActionDest: 241 Annotation: Subtype: Link Rect: 192, 697, 204, 706 ActionDest: 241 Annotation: Subtype: Link Rect: 369, 660, 381, 669 ActionDest: 241 Annotation: Subtype: Link Rect: 282, 534, 294, 543 ActionDest: 241 Annotation: Subtype: Link Rect: 295, 534, 307, 543 ActionDest: 241 Annotation: Subtype: Link Rect: 530, 446, 542, 455 ActionDest: 241 Annotation: Subtype: Link Rect: 421, 409, 433, 417 ActionDest: 238 Annotation: Subtype: Link Rect: 410, 333, 421, 342 ActionDest: 241 Annotation: Subtype: Link Rect: 251, 308, 263, 317 ActionDest: 238 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 305, 378, 343, 389 Annotation: Subtype: Link Rect: 354, 355, 391, 366 Annotation: Subtype: Link Rect: 183, 332, 220, 343 Annotation: Subtype: Link Rect: 226, 332, 262, 343 Annotation: Subtype: Link Rect: 264, 309, 301, 320 Annotation: Subtype: Link Rect: 175, 286, 213, 297 Annotation: Subtype: Link Rect: 219, 286, 255, 297 Annotation: Subtype: Link Rect: 187, 252, 224, 262 Annotation: Subtype: Link Rect: 247, 228, 285, 239 Annotation: Subtype: Link Rect: 207, 206, 245, 216 Annotation: Subtype: Link Rect: 520, 182, 557, 193 Annotation: Subtype: Link Rect: 372, 160, 410, 170 Annotation: Subtype: Link Rect: 385, 124, 422, 135 Annotation: Subtype: Link Rect: 428, 124, 464, 135 Annotation: Subtype: Link Rect: 203, 90, 240, 101 Annotation: Subtype: Link Rect: 246, 90, 283, 101 Annotation: Subtype: Link Rect: 189, 56, 227, 66 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 363, 721, 401, 732 Annotation: Subtype: Link Rect: 388, 698, 426, 709 Annotation: Subtype: Link Rect: 432, 698, 468, 709 Annotation: Subtype: Link Rect: 95, 664, 133, 674 Annotation: Subtype: Link Rect: 520, 640, 557, 651 Annotation: Subtype: Link Rect: 59, 618, 97, 628 Annotation: Subtype: Link Rect: 322, 594, 360, 605 Annotation: Subtype: Link Rect: 118, 572, 155, 582 Annotation: Subtype: Link Rect: 113, 526, 151, 536 Annotation: Subtype: Link Rect: 157, 526, 193, 536 Annotation: Subtype: Link Rect: 213, 479, 251, 490 Annotation: Subtype: Link Rect: 257, 479, 293, 490 Annotation: Subtype: Link Rect: 350, 456, 388, 467 Annotation: Subtype: Link Rect: 394, 456, 430, 467 Annotation: Subtype: Link Rect: 424, 433, 461, 444 Annotation: Subtype: Link Rect: 59, 411, 97, 421 Annotation: Subtype: Link Rect: 421, 387, 458, 398 Annotation: Subtype: Link Rect: 473, 376, 510, 387 Annotation: Subtype: Link Rect: 59, 354, 97, 364 Annotation: Subtype: Link Rect: 300, 330, 338, 341 Annotation: Subtype: Link Rect: 176, 295, 560, 306 Annotation: Subtype: Link Rect: 56, 284, 99, 295 Annotation: Subtype: Link Rect: 220, 284, 258, 295 Annotation: Subtype: Link Rect: 189, 261, 364, 272 Annotation: Subtype: Link Rect: 500, 261, 538, 272 Annotation: Subtype: Link Rect: 274, 226, 311, 237 Annotation: Subtype: Link Rect: 519, 203, 557, 214 Annotation: Subtype: Link Rect: 59, 192, 95, 203 Annotation: Subtype: Link Rect: 455, 169, 492, 180 Annotation: Subtype: Link Rect: 145, 146, 182, 157 Annotation: Subtype: Link Rect: 376, 123, 414, 134 Annotation: Subtype: Link Rect: 442, 100, 479, 111 Annotation: Subtype: Link Rect: 104, 66, 142, 76 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 59, 722, 97, 732 Annotation: Subtype: Link Rect: 505, 709, 560, 720 Annotation: Subtype: Link Rect: 56, 698, 289, 709 Annotation: Subtype: Link Rect: 410, 698, 448, 709 Annotation: Subtype: Link Rect: 339, 676, 377, 686 Annotation: Subtype: Link Rect: 383, 676, 419, 686 Annotation: Subtype: Link Rect: 95, 653, 133, 663 Annotation: Subtype: Link Rect: 295, 618, 332, 628 Annotation: Subtype: Link Rect: 339, 618, 375, 628 Annotation: Subtype: Link Rect: 345, 594, 382, 605 Checksum: 34fb8aa5 Type: CRC32 Checksum: 8912a65a2b4837217406fec29f61c67c Type: MD5 Checksum: 54282e67671e7360a37171459d39a3462e35d7c8 Type: SHA-1