e78916c89b25a9ea1e16403e8448b944 pmc_35008860.pdf 671325888727765e1651305da5095158e253ad4b pmc_35008860.pdf 4b50530a70f9973266e8ff725be81398a786e2c2a939b629c5ad201a231941fb pmc_35008860.pdf Title: Biocompatibility and Electrical Stimulation of Skeletal and Smooth Muscle Cells Cultured on Piezoelectric Nanogenerators Subject: Nanogenerators are interesting for biomedical applications, with a great potential for electrical stimulation of excitable cells. Piezoelectric ZnO nanosheets present unique properties for tissue engineering. In this study, nanogenerator arrays based on ZnO nanosheets are fabricated on transparent coverslips to analyse the biocompatibility and the electromechanical interaction with two types of muscle cells, smooth and skeletal. Both cell types adhere, proliferate and differentiate on the ZnO nanogenerators. Interestingly, the amount of Zn ions released over time from the nanogenerators does not interfere with cell viability and does not trigger the associated inflammatory response, which is not triggered by the nanogenerators themselves either. The local electric field generated by the electromechanical nanogenerator–cell interaction stimulates smooth muscle cells by increasing cytosolic calcium ions, whereas no stimulation effect is observed on skeletal muscle cells. The random orientation of the ZnO nanogenerators, avoiding an overall action potential aligned along the muscle fibre, is hypothesised to be the cause of the cell-type dependent response. This demonstrates the need of optimizing the nanogenerator morphology, orientation and distribution according to the potential biomedical use. Thus, this study demonstrates the cell-scale stimulation triggered by biocompatible piezoelectric nanogenerators without using an external source on smooth muscle cells, although it remarks the cell type-dependent response. Keywords: stimulation; nanogenerators; muscle cells; piezoelectric; ZnO; electroceutical; bioelectronics; nanosheets; biomedical; biocompatibility Author: Andreu Blanquer, Oriol Careta, Laura Anido-Varela, Aida Aranda, Elena Ibáñez, Jaume Esteve, Carme Nogués and Gonzalo Murillo Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Dec 31 11:11:29 2021 CET ModDate: Fri Dec 31 12:00:38 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5987979 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- MWHIBA+VnURWPalladioL Type 1 Custom yes yes no 10 0 JBJVQF+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 PKJJGF+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 ESQMDW+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 OJYQXC+CMSY10 Type 1 Builtin yes yes no 28 0 GIFXFT+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 40 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 44 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 48 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 60 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 63 0 IUFFFR+EURM10 Type 1 Builtin yes yes no 88 0 GLKEAI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 101 0 GLKEAJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 104 0 GLKEAK+PalatinoLinotype TrueType WinAnsi yes yes no 110 0 GLKEBM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 113 0 GLKEAI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 128 0 GLKEAJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 131 0 GLKEAK+PalatinoLinotype TrueType WinAnsi yes yes no 137 0 GLKEBM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 140 0 GLKEAI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 155 0 GLKEAJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 158 0 GLKEAK+PalatinoLinotype TrueType WinAnsi yes yes no 164 0 GLKEBM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 167 0 GLKEAI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 181 0 GLKEAJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 184 0 GLKEAK+PalatinoLinotype TrueType WinAnsi yes yes no 190 0 GLKEBM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 193 0 GLKEAI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 207 0 GLKEAJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 210 0 GLKEAK+PalatinoLinotype TrueType WinAnsi yes yes no 216 0 GLKEBM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 219 0 GLKEAI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 230 0 GLKEAJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 233 0 GLKEAK+PalatinoLinotype TrueType WinAnsi yes yes no 239 0 GLKEBM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 242 0 IOLAWT+PazoMath-Italic Type 1 Builtin yes yes no 263 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-03-10 02:52:33 CET RepresentationInformation: pmc_35008860.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-01-17 03:36:34 CET Size: 5987979 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 433 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: NG Characterization and Zn Ions Release over Time Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: ZnO Cytocompatibility for Smooth Muscle Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Cell Morphology Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Immunological Response Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Intracellular Calcium Changes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Finite Element Modelling Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: NG Synthesis and Optimization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: NG Characterization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: COMSOL Simulation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Zinc Ions Released from NGs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Cell Lines Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Cell Viability Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Initial Cell Adhesion and Spreading Area of Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Cell Morphology Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Immunofluorescence Detection of Smooth Muscle Actin on A7r5 Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Immunofluorescence Detection of Sarcomeric -Actinin on C2C12 Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Quantification of Inflammatory Cytokines Secretion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Intracellular Calcium Quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Statistical Analysis 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: Biocompatibility and Electrical Stimulation of Skeletal and Smooth Muscle Cells Cultured on Piezoelectric Nanogenerators Author: Andreu Blanquer, Oriol Careta, Laura Anido-Varela, Aida Aranda, Elena Ibáñez, Jaume Esteve, Carme Nogués and Gonzalo Murillo Subject: Nanogenerators are interesting for biomedical applications, with a great potential for electrical stimulation of excitable cells. Piezoelectric ZnO nanosheets present unique properties for tissue engineering. In this study, nanogenerator arrays based on ZnO nanosheets are fabricated on transparent coverslips to analyse the biocompatibility and the electromechanical interaction with two types of muscle cells, smooth and skeletal. Both cell types adhere, proliferate and differentiate on the ZnO nanogenerators. Interestingly, the amount of Zn ions released over time from the nanogenerators does not interfere with cell viability and does not trigger the associated inflammatory response, which is not triggered by the nanogenerators themselves either. The local electric field generated by the electromechanical nanogenerator cell interaction stimulates smooth muscle cells by increasing cytosolic calcium ions, whereas no stimulation effect is observed on skeletal muscle cells. The random orientation of the ZnO nanogenerators, avoiding an overall action potential aligned along the muscle fibre, is hypothesised to be the cause of the cell-type dependent response. This demonstrates the need of optimizing the nanogenerator morphology, orientation and distribution according to the potential biomedical use. Thus, this study demonstrates the cell-scale stimulation triggered by biocompatible piezoelectric nanogenerators without using an external source on smooth muscle cells, although it remarks the cell type-dependent response. Keywords: stimulation; nanogenerators; muscle cells; piezoelectric; ZnO; electroceutical; bioelectronics; nanosheets; biomedical; biocompatibility Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Dec 31 11:11:29 CET 2021 ModDate: Fri Dec 31 12:00:38 CET 2021 ID: 0x3e27c0453e46a0e3ebfe238fa0d49c64, 0x3e27c0453e46a0e3ebfe238fa0d49c64 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: GIFXFT+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 178 FontDescriptor: FontName: GIFXFT+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: PKJJGF+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: PKJJGF+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IOLAWT+PazoMath-Italic FontSubset: true FirstChar: 97 LastChar: 97 FontDescriptor: FontName: IOLAWT+PazoMath-Italic Flags: Symbolic FontBBox: -70, -277, 902, 733 FontFile: true Font: BaseFont: ESQMDW+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: ESQMDW+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IUFFFR+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: IUFFFR+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: MWHIBA+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: MWHIBA+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OJYQXC+CMSY10 FontSubset: true FirstChar: 0 LastChar: 14 FontDescriptor: FontName: OJYQXC+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: JBJVQF+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 241 FontDescriptor: FontName: JBJVQF+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 107, 323, 152, 332 Annotation: Subtype: Link Rect: 34, 311, 130, 320 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 180, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 120, 661, 131, 674 Annotation: Subtype: Link Rect: 197, 661, 208, 674 Annotation: Subtype: Link Rect: 115, 648, 126, 662 Annotation: Subtype: Link Rect: 237, 648, 248, 662 Annotation: Subtype: Link Rect: 195, 193, 202, 202 ActionDest: 270 Annotation: Subtype: Link Rect: 205, 193, 212, 202 ActionDest: 270 Annotation: Subtype: Link Rect: 514, 155, 521, 164 ActionDest: 270 Annotation: Subtype: Link Rect: 524, 155, 531, 164 ActionDest: 270 Annotation: Subtype: Link Rect: 290, 118, 297, 127 ActionDest: 270 Annotation: Subtype: Link Rect: 135, 17, 274, 28 Annotation: Subtype: Link Rect: 422, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 420, 86, 439 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 200, 698, 207, 707 ActionDest: 270 Annotation: Subtype: Link Rect: 460, 660, 472, 669 ActionDest: 270 Annotation: Subtype: Link Rect: 282, 547, 294, 556 ActionDest: 270 Annotation: Subtype: Link Rect: 294, 547, 306, 556 ActionDest: 270 Annotation: Subtype: Link Rect: 424, 497, 436, 506 ActionDest: 270 Annotation: Subtype: Link Rect: 214, 459, 226, 468 ActionDest: 270 Annotation: Subtype: Link Rect: 227, 459, 239, 468 ActionDest: 270 Annotation: Subtype: Link Rect: 464, 434, 476, 443 ActionDest: 270 Annotation: Subtype: Link Rect: 190, 321, 202, 330 ActionDest: 270 Annotation: Subtype: Link Rect: 544, 283, 556, 293 ActionDest: 270 Annotation: Subtype: Link Rect: 398, 221, 410, 230 ActionDest: 270 Annotation: Subtype: Link Rect: 411, 221, 423, 230 ActionDest: 270 Annotation: Subtype: Link Rect: 204, 66, 211, 78 ActionDest: 85 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 199, 695, 206, 707 ActionDest: 85 Annotation: Subtype: Link Rect: 522, 683, 529, 695 ActionDest: 85 Annotation: Subtype: Link Rect: 240, 657, 247, 669 ActionDest: 85 Annotation: Subtype: Link Rect: 219, 175, 226, 187 ActionDest: 118 Annotation: Subtype: Link Rect: 199, 150, 205, 162 ActionDest: 118 Annotation: Subtype: Link Rect: 415, 124, 422, 136 ActionDest: 118 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 424, 222, 431, 234 ActionDest: 118 Annotation: Subtype: Link Rect: 494, 134, 501, 146 ActionDest: 118 Annotation: Subtype: Link Rect: 295, 122, 302, 134 ActionDest: 118 Annotation: Subtype: Link Rect: 454, 59, 461, 71 ActionDest: 145 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 512, 733, 519, 745 ActionDest: 145 Annotation: Subtype: Link Rect: 318, 695, 325, 709 ActionDest: 145 Annotation: Subtype: Link Rect: 422, 670, 429, 684 ActionDest: 145 Annotation: Subtype: Link Rect: 404, 233, 411, 245 ActionDest: 145 Annotation: Subtype: Link Rect: 242, 195, 249, 207 ActionDest: 171 Annotation: Subtype: Link Rect: 280, 157, 287, 169 ActionDest: 171 Annotation: Subtype: Link Rect: 200, 69, 207, 81 ActionDest: 171 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 514, 135, 521, 147 ActionDest: 197 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 464, 410, 471, 422 ActionDest: 197 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 426, 708, 433, 720 ActionDest: 197 Annotation: Subtype: Link Rect: 481, 632, 488, 644 ActionDest: 197 Annotation: Subtype: Link Rect: 515, 622, 527, 631 ActionDest: 270 Annotation: Subtype: Link Rect: 530, 622, 542, 631 ActionDest: 270 Annotation: Subtype: Link Rect: 453, 610, 465, 619 ActionDest: 273 Annotation: Subtype: Link Rect: 546, 481, 554, 493 ActionDest: 197 Annotation: Subtype: Link Rect: 493, 394, 500, 406 ActionDest: 197 Annotation: Subtype: Link Rect: 211, 145, 223, 154 ActionDest: 270 Annotation: Subtype: Link Rect: 228, 95, 240, 104 ActionDest: 273 Annotation: Subtype: Link Rect: 384, 69, 396, 79 ActionDest: 273 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 544, 710, 556, 719 ActionDest: 273 Annotation: Subtype: Link Rect: 544, 622, 556, 631 ActionDest: 270 Annotation: Subtype: Link Rect: 223, 535, 235, 544 ActionDest: 273 Annotation: Subtype: Link Rect: 255, 258, 267, 267 ActionDest: 273 Annotation: Subtype: Link Rect: 544, 233, 556, 242 ActionDest: 273 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 430, 698, 442, 707 ActionDest: 270 Annotation: Subtype: Link Rect: 403, 685, 415, 694 ActionDest: 273 Annotation: Subtype: Link Rect: 466, 535, 478, 544 ActionDest: 270 Annotation: Subtype: Link Rect: 478, 535, 490, 544 ActionDest: 273 Annotation: Subtype: Link Rect: 544, 522, 556, 531 ActionDest: 273 Annotation: Subtype: Link Rect: 250, 447, 262, 456 ActionDest: 273 Annotation: Subtype: Link Rect: 204, 309, 216, 318 ActionDest: 273 Annotation: Subtype: Link Rect: 217, 309, 229, 318 ActionDest: 273 Page: Sequence: 11 Page: Sequence: 12 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 419, 219, 560, 230 Annotation: Subtype: Link Rect: 165, 207, 259, 218 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 395, 565, 432, 576 Annotation: Subtype: Link Rect: 439, 565, 475, 576 Annotation: Subtype: Link Rect: 479, 553, 516, 564 Annotation: Subtype: Link Rect: 521, 553, 557, 564 Annotation: Subtype: Link Rect: 422, 530, 460, 541 Annotation: Subtype: Link Rect: 483, 507, 520, 518 Annotation: Subtype: Link Rect: 356, 484, 393, 495 Annotation: Subtype: Link Rect: 373, 461, 411, 472 Annotation: Subtype: Link Rect: 415, 438, 452, 449 Annotation: Subtype: Link Rect: 479, 427, 516, 438 Annotation: Subtype: Link Rect: 521, 427, 557, 438 Annotation: Subtype: Link Rect: 59, 405, 96, 415 Annotation: Subtype: Link Rect: 102, 405, 138, 415 Annotation: Subtype: Link Rect: 200, 382, 237, 392 Annotation: Subtype: Link Rect: 59, 347, 96, 357 Annotation: Subtype: Link Rect: 283, 324, 321, 334 Annotation: Subtype: Link Rect: 266, 300, 304, 311 Annotation: Subtype: Link Rect: 310, 300, 346, 311 Annotation: Subtype: Link Rect: 376, 277, 413, 288 Annotation: Subtype: Link Rect: 419, 277, 455, 288 Annotation: Subtype: Link Rect: 462, 254, 499, 265 Annotation: Subtype: Link Rect: 506, 254, 542, 265 Annotation: Subtype: Link Rect: 306, 231, 343, 242 Annotation: Subtype: Link Rect: 59, 209, 96, 219 Annotation: Subtype: Link Rect: 102, 209, 138, 219 Annotation: Subtype: Link Rect: 322, 185, 359, 196 Annotation: Subtype: Link Rect: 387, 162, 425, 173 Annotation: Subtype: Link Rect: 304, 139, 341, 150 Annotation: Subtype: Link Rect: 204, 117, 242, 127 Annotation: Subtype: Link Rect: 295, 93, 332, 104 Annotation: Subtype: Link Rect: 338, 93, 374, 104 Annotation: Subtype: Link Rect: 86, 59, 123, 70 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 420, 722, 458, 733 Annotation: Subtype: Link Rect: 291, 699, 328, 710 Annotation: Subtype: Link Rect: 334, 699, 370, 710 Annotation: Subtype: Link Rect: 118, 676, 155, 687 Annotation: Subtype: Link Rect: 492, 664, 529, 675 Annotation: Subtype: Link Rect: 458, 652, 495, 664 Annotation: Subtype: Link Rect: 244, 630, 282, 640 Annotation: Subtype: Link Rect: 139, 607, 176, 617 Annotation: Subtype: Link Rect: 140, 573, 177, 583 Annotation: Subtype: Link Rect: 184, 573, 220, 583 Annotation: Subtype: Link Rect: 519, 549, 557, 560 Annotation: Subtype: Link Rect: 59, 538, 95, 548 Annotation: Subtype: Link Rect: 131, 504, 168, 514 Annotation: Subtype: Link Rect: 175, 504, 211, 514 Annotation: Subtype: Link Rect: 59, 469, 96, 479 Annotation: Subtype: Link Rect: 59, 446, 96, 456 Annotation: Subtype: Link Rect: 102, 446, 138, 456 Annotation: Subtype: Link Rect: 437, 422, 475, 433 Annotation: Subtype: Link Rect: 481, 422, 517, 433 Checksum: 27760f67 Type: CRC32 Checksum: e78916c89b25a9ea1e16403e8448b944 Type: MD5 Checksum: 671325888727765e1651305da5095158e253ad4b Type: SHA-1