Bone Research Protocols (Methods in Molecular Biology, v816, 2nd Ed.)

MMB_v816_9781617794148_1617794147......Page 1
front-matter......Page 2
Bone Research Protocols......Page 4
Preface......Page 6
Contents......Page 8
Contributors......Page 12
Part 1 Culture of osteoblasts and osteocytes......Page 16
1. Introduction......Page 17
1.2. Osteosarcoma Cell Lines......Page 18
1.3. Osteoblasts from MSCs......Page 19
2.2. Cell Isolation and Culture......Page 21
3.1. Establishing Primary Explant Cultures......Page 22
3.2. Secondary Explant Cultures......Page 23
3.3. Passaging Explant Cultures......Page 25
3.5. Mineralising Cultures......Page 26
4. Notes......Page 28
References......Page 31
1. Introduction......Page 33
2.3. Media and Solutions......Page 35
3.1. Isolation and Culture of Primary Bone Cells from Adult Mouse Long Bones......Page 36
3.2. Isolation and Culture of Primary Bone Cells from Adult Mouse Calvaria......Page 37
3.3. Isolation and Culture of Bone Cells from Neonatal Mouse Calvaria......Page 39
3.4. Isolation and Culture of Bone Cells from Neonatal Mouse Calvaria (Alternative)......Page 40
4. Notes......Page 41
References......Page 42
1. Introduction......Page 45
2. Materials......Page 47
3.2. Isolation and Culture of Primary Osteoblasts from the Long Bones of Neonatal Rats......Page 48
3.3. Bone Formation Assay......Page 49
3.6. Statistics......Page 51
4. Notes......Page 52
References......Page 55
1. Introduction......Page 57
2.2. Media and Solutions......Page 58
2.3. Monoclonal Antibody OB7.3......Page 59
3.1. Tissue Dissection......Page 60
3.2. Isolation of OBmix......Page 62
3.3. Isolation of Osteocytes......Page 63
4. Notes......Page 64
References......Page 66
1. Introduction......Page 69
2.1.2. Instruments, Material......Page 70
2.1.3. Media and Solutions......Page 71
3.1.1. Isolation of Newborn Mouse Calvaria......Page 72
3.1.2. Fractionation of Newborn Mouse Calvaria......Page 73
3.1.3. Fractionation of Adult Mouse Long Bone......Page 74
3.1.4. FACS Sorting of Newborn DMP1-GFP Mouse Calvaria Fractions......Page 75
3.1.6. Comparison of Gene Expression in the Osteocytic and Osteoblastic Fractions......Page 76
4. Notes......Page 78
References......Page 79
1. Introduction......Page 81
2.2. Fluid Flow Reagents......Page 83
3.1. General Maintenance of MLOY4 Osteocyte-Like Cell-Line......Page 84
3.2. Collagen Coating of Tissue Culture Treated Dishes......Page 85
3.3.2. To Set Up the Flow Loop......Page 86
3.4. Staining and Quantitation of Dendrite Length......Page 88
3.5. E11 siRNA Transfection of MLO-Y4......Page 90
3.7. Mineralization of MLO-A5......Page 91
4. Notes......Page 92
References......Page 94
1. Introduction......Page 97
2.3. Stromal Cell Isolation and Culture......Page 98
2.8. Von Kossa Staining......Page 99
2.10. Immunostaining for Type II Collagen and SOX-9......Page 100
3.1. Isolation of Bone Marrow Mononuclear Cells......Page 101
3.2. Antibody Labelling of STRO-1 Positive Cells......Page 103
3.3. Magnetic Separation of STRO-1 Positive Cells......Page 104
3.6. Adipogenic Differentiation of STRO-1 Positive Cells......Page 105
3.9. Measurement of Alkaline Phosphatase Activity......Page 106
3.12. Safranin O Staining of Chondrocytes Cultures......Page 107
3.13. Immunoloca- lisation of SOX-9 and Type II Collagen in Chondrocyte Cultures......Page 108
3.14. Oil Red O Staining......Page 109
3.15. Subcutaneous Implant Assay and Diffusion Chamber Assay......Page 110
4. Notes......Page 111
References......Page 113
Part 2  Culture of osteoclasts......Page 116
1. Introduction......Page 117
2. Materials......Page 118
3.1. Mouse Osteoclast Formation Assay......Page 120
3.2. Isolation of Mature Osteoclasts from Neonatal Rat Long Bones......Page 121
3.4. Quantification of Resorption......Page 122
4. Notes......Page 123
References......Page 130
1. Introduction......Page 133
2.1. Tissue Culture Medium, Solutions, and Supplies......Page 134
2.4. Fixation and Immunostaining......Page 136
2.5. Preparation of Devitalized Bone or Ivory Discs for Bone Pit Resorption Studies......Page 137
2.6. Preparation of Gold-Coated Glass Coverslips for Phagokinetic Motility Studies ( see Note 4)......Page 138
3.1. Isolation of Osteoclasts from Calcium-Deficient Chicks ( see Note 5)......Page 139
3.1.1. Percoll Purification of Osteoclasts......Page 141
3.1.2. Immunomagnetic Purification of Osteoclasts ( see Note 8)......Page 143
3.2.2. Immunomagnetically Purified OC ( see Note 8)......Page 144
3.3.1. Morphology and Ultrastructure......Page 145
3.3.2. Cytochemical Staining......Page 146
3.3.3. Antigenic Profile......Page 148
3.3.5. Motility ( see Note 4)......Page 151
3.3.6. Bone Resorption......Page 152
4. Notes......Page 153
References......Page 157
1. Introduction......Page 159
2.5. Staining for Tartrate Resistant Acid Phosphatase......Page 163
3.1. Isolation and Culture of Rabbit Osteoclasts......Page 164
3.2. Enrichment of Osteoclasts Using Foetal Calf Serum Gradients......Page 166
3.5. Isolation of Rabbit Osteoclasts Using Immunomagnetic Beads......Page 167
3.6.3. Staining for Tartrate Resistant Acid Phosphatase ( see Note 11)......Page 168
4. Notes......Page 169
References......Page 171
1. Introduction......Page 173
2. Materials......Page 176
2.2. TRAcP Activity Reagents......Page 177
3.1. Isolation of PBMCs from Peripheral Blood or Buffy Coats......Page 178
3.2. Purification of CD14 + Osteoclast Precursor Cells......Page 179
3.3. Expansion of Macrophages/Osteoclast Precursors......Page 180
3.5. Generation of Osteoclasts for Assessment of Osteoclastogenesis......Page 181
3.6. Generation of Osteoclasts for Functional Studies......Page 182
3.7.1. Assessment of Vitronectin Receptor-Positive Cells......Page 183
3.7.3. Alternative Measurements of Osteoclast Formation......Page 184
3.8.2. Measurement of Calcium or Collagen Fragments in the Supernatant......Page 185
4. Notes......Page 186
References......Page 187
1. Introduction......Page 191
2.2. Tissue Culture Reagents......Page 192
3.2. Isolation of Bone Marrow Cells......Page 193
3.4. Collagen Gel Culture......Page 194
3.6. Tartrate Resistant Acid Phosphatase Staining......Page 195
3.7. Quantification of the Resorption Area......Page 196
4. Notes......Page 198
References......Page 199
1. Introduction......Page 201
2.1. Tissue Culture Medium, Solutions, and Supplies......Page 203
3.1. Preparation of Devitalized Bone or Dentine Slices......Page 204
3.2. RAW 264.7 Cell Culture......Page 205
3.4. Serum Gradient Purification of RAW-OC......Page 206
3.5. Phenotypic and Functional Characterization of RAW-OCs......Page 208
4. Notes......Page 211
References......Page 215
Part 3  Biochemical and molecular analysis of bone cells......Page 217
1.1. Amaxa™ Nucleofector\r......Page 219
1.2. Lentiviral Vectors......Page 221
1.3. Adenoviral Vectors......Page 222
2.1. Primary Cells......Page 223
2.4. Culture Media and Buffers......Page 224
3.1. Transfecting Human Osteoclast Precursors Using the Amaxa™ Nucleofector......Page 225
3.2. Generating Lentivirus......Page 226
3.4. Generating Recombinant Adenoviruses......Page 227
3.6. Amplification of Adenoviral Stocks ( See Note 16)......Page 229
3.7. Adenovirus Transduction of Mature Human Osteoclasts......Page 230
3.9. Assessment of Cell Viability......Page 231
4. Notes......Page 232
References......Page 235
1. Introduction......Page 237
2.2. Western Blotting......Page 240
3.1.2. Immunoprecipitation......Page 241
3.2. Western Blotting......Page 242
3.2.3. Electrophoresis......Page 243
3.2.5. Protein Detection......Page 244
4. Notes......Page 245
References......Page 246
1. Introduction......Page 247
2.2. Nonradioactive Electrophoretic Mobility Shift Assay......Page 248
3.1. Dual Luciferase Reporter Assay......Page 249
3.2. Electrophoretic Mobility Shift Assay......Page 251
3.2.4. Electrophoresis and Transfer from Gel to Membrane......Page 252
3.3.1. Cross-Link Chromatin......Page 255
3.3.2. Examination of the Sheared Chromatin......Page 256
3.3.3. Immunoprecipitation of Chromatin Bound to Protein......Page 257
3.3.4. Recover DNA Bound to Protein of Interest......Page 259
4. Notes......Page 260
References......Page 261
1. Introduction......Page 263
2.2. For DNA Extraction......Page 264
3.1. DNA Extraction from Fresh Bone......Page 265
3.3. DNA Extraction from Dried or Embedded Bone......Page 266
3.4. RNA Extraction from Fresh Bone......Page 267
3.6. Extraction of RNA from Adherent Cultured Cells......Page 268
3.8. Quantification of Nucleic Acids by Spectrophotometry......Page 269
3.9. Evaluation of RNA Integrity by Agarose Gel Electrophoresis......Page 270
4. Notes......Page 271
References......Page 273
1.1. Assay Choice for Quantitative PCR......Page 275
1.4. Preparation of Input RNA......Page 277
2.1. Reverse Transcription......Page 278
3.1. Reverse Transcription......Page 279
3.3. Design of Primers for SYBR Green qPCR Assays......Page 280
3.4. Preparation of an Exogenous Control......Page 281
3.6. Preparation of cDNA Reference Curve for Relative Quantification......Page 282
3.8. Performing Quantitative RT-PCR Using SYBR Green Assays......Page 283
3.9. Analysis of q-RT-PCR Data......Page 284
4. Notes......Page 285
References......Page 288
Part 4 Microscopical techniques......Page 292
1. Introduction......Page 293
2.1. Sectioning, Fixation and Embedding......Page 294
2.4. Tartrate Resistant Acid Phosphatase Stain......Page 296
3.1. Fixation, Dehydration and Infiltration of Bone Samples......Page 297
3.4. Embedding for Histochemistry and Immunohisto\rchemistry......Page 298
3.5. Preparation of Micromilled Cross-Sections for Routine Histological Analysis......Page 299
3.8. Von Kossa/McNeal Stain......Page 300
3.10. Tartrate Resistant Acid Phosphatase Staining......Page 301
3.12. Cancellous Bone Histomorphometry......Page 302
3.13. Assessment of Bone Structural Parameters......Page 304
3.14. Analysis of Dynamic Histomorphometry in Mice......Page 305
3.15. Assessment of Osteoclast Numbers and Bone Resorption in Mice......Page 307
3.16. Assessment of Bone Mineralization in Mice......Page 308
3.18. Assessment of Static Histomorphometry in Rats......Page 310
3.19. Assessment of Remodeling-Based Parameters in Rats......Page 311
3.20. Cortical Bone Histomorphometry......Page 312
3.21. Periosteal and Endocortical Bone Formation......Page 313
4. Notes......Page 314
References......Page 316
1. Introduction......Page 319
2.3. Generation of DIG-Labeled Riboprobes......Page 320
2.4. Hybridization, Washes, Probe Detection, and Signal Development......Page 321
3.1. Tissue Collection, Fixation, Decalcification, and Embedding......Page 322
3.3. Linearization of the Plasmid Containing the Probe of Interest......Page 324
3.4. Generation of DIG-Labeled Riboprobes......Page 325
3.5. Hybridization......Page 326
3.6. Washes, Probe Detection, and Signal Development......Page 328
4. Notes......Page 329
References......Page 334
1. Introduction......Page 335
2. Materials......Page 337
3.1. Preparation of Mouse Knee Joint Paraffin Blocks......Page 339
3.2. Immuno-histochemical Staining Using Enzyme-Conjugated Antibodies\r......Page 340
3.3. Immuno-fluorescence Staining Using Fluorochrome-Conjugated Antibodies\r......Page 341
4. Notes......Page 342
References......Page 348
1. Introduction......Page 349
1.3. Nick Translation......Page 350
1.4. DNA Laddering......Page 351
2.3. LDH Assay......Page 352
2.6. Caspase 3-7 Detection......Page 353
3.2. Assessment of Cell Viability Using the LDH Assay......Page 354
3.4. Detecting of DNA Laddering in Cells and Tissue Sections......Page 356
3.4.1. Preparation of Cells for Analysis......Page 357
3.4.3. Isolation of DNA......Page 358
3.5.2. Preparing the Labelling Reagent......Page 359
4. Notes......Page 361
References......Page 362
2. Materials......Page 365
2.3. Osmium and Cacodylate Postfixative......Page 366
2.6. Methylene Blue......Page 367
3.1. Perfusion Fixation of Animal Bones......Page 368
3.4. Immersion Fixation of Cultured Mineralized Tissues......Page 369
3.7. Embedding of Small Tissue Samples......Page 370
3.10. Methylene Blue Staining of Semi-Thin Sections......Page 371
3.11. Modified Goldner’s Masson Stain......Page 372
3.13. Staining of Ultrathin Sections with Uranyl Acetate......Page 373
4. Notes......Page 374
References......Page 377
1. Introduction......Page 379
1.1. Instrumentation and Imaging Modes......Page 380
1.2.1. Unembedded Bone, with 3D Surface Detail......Page 382
1.2.3. Resin Replicas or Internal Casts of Space in Bone Matrix......Page 385
2.2. Instrumentation......Page 386
3.1.2. Small Bones......Page 387
3.3.1. Freeze Drying......Page 388
3.3.3. Air Drying from Volatile Solvents......Page 389
3.5. Morphological Imaging of Cells on Bone in 3D......Page 390
3.7.1. Removing Cells and Soft Tissue and/or Uncalcified Bone Matrix with Tergazyme......Page 391
3.7.4. Making Samples Anorganic Using Hypochlorite......Page 393
3.7.6. Making Samples Anorganic Using Sodium or Potassium Hydroxide......Page 394
3.8. Bone and Dentine Slices Etched by Osteoclasts In Vitro......Page 395
3.8.3. Compositional A+B BSE SEM Imaging of Carbon-Coated Samples\r......Page 396
3.9.2. Mechanical Tilting......Page 397
3.9.3. Changing the BSE Detector Location (Discotheque Illumination)......Page 399
3.9.4. Changing the BSE Detector Location (In, Off, and Far Illumination)......Page 400
3.10.1. Creating a Polished Surface ( see Fig.  7)......Page 401
3.10.2. Making Casts from PMMA or Other Resin-Embedded Samples......Page 403
3.11. Correlation of SEM with Other Imaging Means......Page 404
3.11.1. Correlated Confocal (or Other LM) with SEM Imaging of PMMA (or Other Resin)-Embedded Bone......Page 405
3.11.2. X-Ray Microtomography (XMT or m CT)......Page 406
3.11.3. Faxitron Point Projection Microradiography......Page 407
3.12. Shipping Bone Samples......Page 408
4. Notes......Page 409
References......Page 413
1. Introduction......Page 415
1.1. Principles of Confocal Microscopy......Page 417
1.2. The Use of Confocal Microscopy in Osteoclasts......Page 419
2.1. Equipment......Page 420
2.2. General Reagents......Page 421
2.4. Useful Probes for Staining Osteoclast Structures......Page 422
3.1. Synthesis of Fluorescently Labelled Alendronate......Page 423
3.2. Culture of Osteoclasts for Confocal Microscopy......Page 424
3.2.2. Culture of Osteoclasts on Dentine......Page 425
3.3. Staining of Osteoclasts......Page 426
3.4. Confocal Microscopy of Osteoclasts......Page 427
3.5. Visualisation of the Data Post-acquisition......Page 428
3.6. Generating Three-Dimensional Data Sets......Page 430
3.7. Visualising Three-Dimensional Data Sets......Page 431
3.8. Spectral Imaging......Page 432
3.10. Advanced Uses and Probes for Confocal Microscopy......Page 433
4. Notes......Page 434
References......Page 437
1. Introduction......Page 439
1.2. General Considerations About Equipment......Page 440
1.3. Image Quality Versus Phototoxicity......Page 443
1.4. Selection of Imaging Fields......Page 444
1.6. Types of Probes and Their Limitations......Page 445
1.7. Focal Drift......Page 446
2.1. Animals, Cells, and Tissues......Page 447
2.3. Other Buffers, Solutions, and Reagents......Page 448
2.4. Equipment......Page 449
3.2. Preparation of Neonatal Mouse Calvaria......Page 450
3.3.1. Time Lapse Imaging of Mineralization Dynamics and Osteocyte Transition in Primary Osteoblasts from Dmp1-GFP Transgenic Mice......Page 452
3.3.2. Time-Lapse Imaging of Fibronectin Assembly in 2T3 Osteoblast-Like Cells......Page 456
3.3.3. Time-Lapse Imaging of Osteocytes in Calvaria from Dmp1-GFP Transgenic Mice......Page 459
3.4.1. Generation of an In-Focus Image Stack from Multi-dimensional Imaging Datasets......Page 461
3.4.2. Scaling, Contrasting, and Background Subtraction......Page 462
3.4.4. Pseudocolouring and Merging RGB and DIC-Fluorescence Images......Page 463
3.4.6. Quantitation of Dynamic Events......Page 464
4. Notes......Page 466
References......Page 470
Part 5 Imaging techniques......Page 474
1. Introduction......Page 475
1.4. Analysis of the 3D Image Stack......Page 476
2.2. Computing Equipment......Page 477
3.1.1. Voltage......Page 478
3.1.2. Resolution......Page 479
3.1.3. Rotation Step......Page 481
3.1.4. Sample Preparation......Page 482
3.1.6. Analysis......Page 483
3.3. In Vivo m CT Analysis......Page 487
References......Page 490
1. Introduction......Page 491
3.1.1. Group Size \rand Scanner Precision In Vivo and In Vitro......Page 493
3.1.2. Age of Animals......Page 494
3.1.3. Study Design......Page 495
3.2.1. Preferred Sites to Study Cancellous and Cortical Bone Parameters In Vivo......Page 496
3.2.2. Preferred Sites to Study Cortical Bone Parameters In Vivo......Page 498
3.4. Limb Positioning......Page 499
3.5.1. Detection of Cortical and Trabecular Compartments......Page 500
3.6. Data Analysis and Interpretation......Page 502
3.7.2. Estimation of Bone Bending Strength......Page 504
3.8.1. Analysis of Bone Size......Page 505
3.8.2. Analysis of Bone Mineral Content......Page 506
3.8.3. Analysis of Changes in Cortical Bone......Page 508
3.8.6. Summarising the Results......Page 509
4. Notes......Page 510
References......Page 511
1. Introduction......Page 513
3.1. Dissection and Preparation of Skeletal Elements......Page 514
3.3. Image Processing......Page 515
3.7. Determination of Relative Mineral Content......Page 517
4. Notes......Page 519
Reference......Page 520
1.1. Optical Imaging......Page 521
1.4. Models of Skeletal Metastases......Page 522
2. Materials......Page 523
3.2. Induction of Bone Metastases by Direct Intra-Osseous Inocculation of Cancer Cells......Page 524
3.3. Bioluminescence Imaging Using the IVIS Spectrum System......Page 526
4. Notes......Page 527
References......Page 528
1. Introduction......Page 531
3.1. Preparation of Section......Page 532
3.3. Select the Area to be Analyzed......Page 533
3.5.2. Analysis of Carbonate/Phosphate Ratio......Page 534
3.6.1. Correction for PMMA......Page 535
3.6.3. Ratio of Carbonate to Phosphate......Page 536
4. Notes......Page 537
References......Page 538
1. Introduction......Page 540
2. Materials......Page 541
3.3. Acquiring Spectra......Page 542
3.5. Analysis......Page 543
3.6. Principal Component Analysis......Page 544
3.7. Polarisation/Orientation......Page 545
References......Page 546
Part 6 In vivo techniques......Page 549
1. Introduction......Page 551
2.4. Goldner’s Trichrome Stain......Page 552
3.2. Tissue Processing......Page 553
3.3. TRAcP/Von Kossa/Light Green Staining of Mouse Calvariae......Page 554
3.5. Analysis of Results......Page 555
4. Notes......Page 556
References......Page 558
1. Introduction......Page 559
3.1. Animal Husbandry......Page 560
3.4. Operative Technique for Ovariectomy......Page 561
3.5. Operative Technique for Orchidectomy......Page 562
3.6. Post-operative Care......Page 563
References......Page 564
Part 7 Mechanical loading techniques......Page 567
1. Introduction......Page 569
2. Materials......Page 571
3.2. Preparing Prisms of Cortical Bone......Page 572
3.4. Preparation of Mouse Bones......Page 573
3.5. General Considerations for Mechanical Testing......Page 575
3.7. Types of Mechanical Testing......Page 576
3.8. Tension Testing......Page 577
3.9. Analysing Data from Tension Testing......Page 578
3.11. Bending......Page 579
3.13. Density of Bone by Archimedes’ Principle......Page 580
3.14. Speed of Sound Measured Using Ultrasound......Page 581
4. Notes......Page 582
References......Page 584
1. Introduction......Page 587
2.1. Tissue Culture Media and Solutions......Page 589
2.2. Instruments......Page 590
2.3.2. Peristaltic Pump Modified to Create Oscillating Flow......Page 591
2.4.2. FlexFlow™ Shear Stress Device (Flexcell, Hillsborough, NC)......Page 592
2.4.3. Aberdeen Live Imaging Fluid Flow Chamber......Page 593
3.1. Preparation of Cells......Page 594
3.2.2. Seeding of Cells on Glass Slides (FlexFlow™ Chamber/ALIFF Chamber/Amsterdam Fluid Flow Chamber)......Page 595
3.3.1. Pulsatile Flow, Online Monitoring of Cell Responses......Page 596
3.3.2. Oscillating Fluid Flow, Online Monitoring of Cell Responses......Page 598
3.3.3. Pulsatile Fluid Flow, Isolation of RNA or Protein......Page 599
3.3.4. Steady Fluid Flow......Page 600
3.4. Analysis of the Cellular Response to Fluid Flow......Page 601
3.4.1. Online Monitoring of NO Production......Page 602
4. Notes......Page 603
References......Page 605
1. Introduction......Page 607
1.1. Limitations of In Vitro Organ and Cell Culture Strain Application Models......Page 609
1.2. The Effects of Loading: Mechanical Strain, Fluid Shear, and Streaming Potentials......Page 610
1.3. In Vitro Cell Culture Loading Models......Page 612
1.4. Biaxial Straining......Page 613
1.5.1. Stretching of Substrates......Page 615
1.5.3. Alternative Methods for Applying Strain......Page 616
2.1. Tissue Culture Media......Page 617
3.2. In Vitro Organ Culture Loading Models......Page 618
3.2.1. Loading of Adult Canine Cancellous Bone Cores ( see Note 5)......Page 621
3.3. Rat Ulnae Organ Cultures ( see Notes 5 and 6)......Page 623
3.4. Loadable Rat Calvaria Organ Cultures ( see Notes 5 and 6)......Page 624
3.6. Longer-Term Perfusion Loading Models......Page 625
4. Notes......Page 626
References......Page 629
1.1. Mechano-Adaptation......Page 635
1.2. Loading Models......Page 636
1.3. A ssessment of Adaptive Modelling and Remodelling In Vivo......Page 638
1.4. Determination of the In Vivo Mechanical Environment of Bone......Page 639
2.1. Measurement of Bone Strains In Vivo......Page 640
3.1. Strain Gauge Preparation......Page 641
3.4. Attach the Gauge......Page 642
3.5. Measure Strains......Page 643
3.6.1. Setting up the Dartec......Page 644
3.7. Mechanical Loading of the Tibia/Ulna......Page 646
4. Notes......Page 647
References......Page 649
INDEX......Page 651