OSTEOPOROSIS AND OSTEOARTHRITIS.

Preface
Contents
Contributors
Part I: Cellular and Molecular Biology of Osteoarthritis and Osteoporosis
	Chapter 1: Isolation of Murine and Human Osteocytes
		1 Introduction
		2 Materials
			2.1 Isolation of Osteocytes from Skeletally Mature Mouse  Bone
			2.2 Isolation of Osteocytes from Human Trabecular Bone
		3 Methods
			3.1 Isolation of Osteocytes from Skeletally Mature Mouse Bone
			3.2 Isolation of Osteocytes from Human Trabecular Bone
		4 Notes
		References
	Chapter 2: Expansion and Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells
		1 Introduction
		2 Materials
			2.1 Expansion of BM-MSCs (See Table 1)
			2.2 Chondrogenic Differentiation of BM-MSCs (See Table 2)
		3 Methods
			3.1 Expansion and Passaging of BM-MSCs
			3.2 Chondrogenic Differentiation of BM-MSCs
			3.3 Evaluation of Chondrogenic Differentiation Capacity of BM-MSC
				3.3.1 Histological Analysis
				3.3.2 Transcript Analysis (See Note 13)
		4 Notes
		References
	Chapter 3: A Novel Enzymatic Digestion Approach for Isolation and Culture of Rodent Bone Marrow Mesenchymal Progenitors
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Instruments
			2.3 Reagents and Media
		3 Methods
			3.1 Harvest of Rodent Hind Long Bones
			3.2 Isolation of Central and Endosteal Bone marrow Cells
			3.3 CFU-F Assays of Endosteal Mesenchymal Progenitors
			3.4 Culture and Differentiation of Endosteal Mesenchymal Progenitors
		4 Notes
		References
	Chapter 4: Isolation of Nucleus Pulposus and Annulus Fibrosus Cells from the Intervertebral Disc
		1 Introduction
		2 Materials
			2.1 Medical Ethical Approval for Human Tissues
			2.2 Cell Culture
			2.3 Cell Counting
			2.4 IVD Marker Gene Analyses by RT-qPCR
		3 Methods
			3.1 Tissue Dissection and Cell Isolation
			3.2 Cell Counting
			3.3 Expansion of Cells in Monolayer
			3.4 Passaging IVD Cells in Monolayer
			3.5 Freezing Down IVD Cells
			3.6 Thawing IVD Cells from the Liquid Nitrogen
			3.7 RT-qPCR Analyses of NP and AF Marker Genes
		4 Notes
		References
	Chapter 5: Engineering Cartilage Tissue by Co-culturing of Chondrocytes and Mesenchymal Stromal Cells
		1 Introduction
		2 Materials
			2.1 Cell Sources
			2.2 Media, Solutions, Chemicals, and Kits
			2.3 Equipment
		3 Methods
			3.1 Isolation of Human Articular Chondrocytes
			3.2 Isolation of Human Bone marrow Mesenchymal Stromal Cells
			3.3 Cell Tracking of Cell Populations in Pellet Co-cultures with Organic Fluorescent Dyes CM-DiI
			3.4 Co-culture of bPCs and hMSCs in Pellets
			3.5 Co-culture of bPCs and hMSCs in Injectable Hydrogels
			3.6 Co-culture of bPCs and hMSCs in Micro-aggregates
			3.7 Examination of Cell Proliferation in Pellets by EdU Labeling and Staining
			3.8 Image Acquisition and Analysis by Fluorescent Microscopy
			3.9 Quantitative GAG and DNA Assay
			3.10 Cell Tracking with Species-Specific PCR
			3.11 Rheological Analysis
			3.12 Compression Test
			3.13 RNA-Sequencing Data Analysis
			3.14 Short Tandem Repeats (STR) Analysis
		4 Notes
		References
	Chapter 6: Generation of Induced Pluripotent Stem Cells
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Equipment
			2.3 Special Equipment/Supplies for Freezing iPSCs in Straws
			2.4 Supplies
			2.5 Working Solutions
		3 Methods
			3.1 Expansion of Passage 2 DR4 MEFs
			3.2 Production of MEF-Conditioned Medium
			3.3 Preparation of Irradiated MEF Stocks in Cryovials
			3.4 Preparing Irradiated-MEF Plates for iPSCs
			3.5 Generating iPSCs from Fibroblasts/Mesenchymal Stem Cells Using Lenti and Foamy Viral Reprogramming Vectors
			3.6 Generating iPSCs from Fibroblasts/Mesenchymal Stem Cells Using Nonintegrating Sendai Viral Reprogramming Vectors
			3.7 Passaging iPSCs with Dispase
			3.8 Freezing iPSCs in Straws
			3.9 Freezing iPSCs in Cryovials
			3.10 Thawing iPSCs from Straw
			3.11 Thawing iPSCs from Cryovial
		4 Notes
		References
	Chapter 7: Specimen Preparation for Single-Cell Sequencing Analysis of Skeletal Cells
		1 Introduction
		2 Materials
			2.1 Reagents
		3 Methods
			3.1 Tissue Sample Preparation
			3.2 FACS Staining
			3.3 Sample Collection for Single-Cell Sequencing
		4 Notes
		References
	Chapter 8: Mapping 5-Hydroxymethylcytosine (5hmC) Modifications in Skeletal Tissues Using High-Throughput Sequencing
		1 Introduction
		2 Materials
			2.1 DNA Shearing and Glucosylation Reaction
			2.2 DNA Fragment Enrichment
			2.3 DNA Library Preparation and Next-Generation Sequencing
		3 Methods
			3.1 DNA Shearing and Glucosylation Reaction
			3.2 DNA Fragment Enrichment
			3.3 DNA Library Preparation and Next-Generation Sequencing
			3.4 Data Analysis (See Note 11)
		4 Notes
		References
	Chapter 9: Using FRAP to Quantify Changes in Transcription Factor Dynamics After Cell Stimulation: Cell Culture, FRAP, Data An...
		1 Introduction
			1.1 Different Methods for Measuring Protein Dynamics
			1.2 When to Use FRAP
			1.3 FRAP Principle
			1.4 Mapping Signal Transduction Pathways Regulating Transcription Factor Mobility
			1.5 Quantitation of Protein Mobility
			1.6 Analyze FRAP Data
			1.7 Explanation of FRAP Parameters
		2 Materials
			2.1 Materials for Cell Culture and Transfection
			2.2 Materials for FRAP
				2.2.1 Imaging Buffer (Tyrode´s Solution, See Note 1)
				2.2.2 Confocal Laser Scanning Microscope
			2.3 Materials for FRAP Analysis
			2.4 Materials for Data Visualization and Statistical Analysis
		3 Methods
			3.1 Cell Culture and Transfection
				3.1.1 Cell Culture
				3.1.2 Choice of the Fluorophore
				3.1.3 Transfection
				3.1.4 Cell Stimulation
				3.1.5 Preparing Cells for FRAP
			3.2 FRAP
				3.2.1 Optimal FRAP Parameters
				3.2.2 Performing FRAP Using a Nikon A1 Confocal Laser Scanning Microscope
			3.3 Data Analysis
				3.3.1 FRAP Data Validation Before Analysis
				3.3.2 FRAP Data Analysis in MATLAB
			3.4 Data Visualization and Statistics
				3.4.1 Data Visualization
				3.4.2 Statistics
		4 Notes
		References
	Chapter 10: Quantitative Molecular Models for Biological Processes: Modeling of Signal Transduction Networks with ANIMO
		1 Introduction
			1.1 The Need of Modeling for Biological Networks
			1.2 Building a Model
			1.3 Precision of Models
			1.4 Comparison of some Modeling Formalisms and Tools
		2 Materials
			2.1 Computational Materials
			2.2 Wet-Lab Materials
		3 Methods
			3.1 Preliminary Model and Hypotheses
			3.2 Wet-Lab Experiments
			3.3 Validation and Adjustment of Model
		4 Notes
		References
Part II: In Vivo Models of Skeletal Tissue Injury, Degeneration, and Repair
	Chapter 11: Generation and Characterization of Mouse Models for Skeletal Disease
		1 Introduction
		2 Materials
			2.1 Surgical Destabilization of the Medial Meniscus (DMM)
			2.2 Osteoarthritis (OA) Histology
			2.3 Microcomputed Tomography
			2.4 Bone Mineral Density Calibration
			2.5 Trabecular Analysis of Microcomputed Tomography Images
			2.6 Cortical Analysis of Microcomputed Tomography Images
			2.7 Bone Modeling
			2.8 Fluorochrome Labeling of Bone
			2.9 Fixation
			2.10 Infiltration
			2.11 Embedding and Cross-Sectioning
			2.12 MMA Embedding and Coronal Sectioning
			2.13 Golder´s Trichrome Stain
			2.14 Paraffin Embedding and Sectioning
			2.15 TRAP Stain
			2.16 Slide Imaging
			2.17 Histomorphometry
		3 Methods
			3.1 DMM Surgery
			3.2 Osteoarthritis (OA) Histological Grading
			3.3 Imaging by Microcomputed Tomography
			3.4 Bone Mineral Density Calibration
			3.5 Trabecular Analysis of Microcomputed Tomography Images
			3.6 Cortical Analysis of Microcomputed Tomography Images
			3.7 Modeling Skeletal Phenotypes
			3.8 Mouse Fluorochrome Injections
			3.9 Infiltration
			3.10 Femoral Embedding and Cross-Sectioning
			3.11 MMA Embedding and Coronal Sectioning of Femurs
			3.12 Goldner´s Trichrome Staining
			3.13 Paraffin Embedding and Sectioning of Femurs
			3.14 TRAP Staining
			3.15 Imaging Slides
			3.16 Histomorphometry
		4 Notes
		References
	Chapter 12: Drill Hole Models to Investigate Bone Repair
		1 Introduction
		2 Materials
			2.1 Instrument Preparation
			2.2 Anesthetic Agents
			2.3 Analgesic Agents
			2.4 Anti-Infective Agents
		3 Methods
			3.1 Animal Preparation
			3.2 Anesthetic
				3.2.1 Isoflurane Anesthesia
				3.2.2 Injectable Anesthesia
			3.3 Long Bone Cortical Hole Drilling
			3.4 Calvaria Cortical Bone Hole Drilling
			3.5 Parameters Monitored After Surgery
			3.6 Early Euthanasia Criteria
		4 Notes
		References
	Chapter 13: Generation and Experimental Outcomes of Closed Femoral Fracture in Mice
		1 Introduction
		2 Materials
			2.1 Closed Fracture Model
			2.2 Microcomputed Tomography (Micro-CT)
			2.3 Torsional Testing
			2.4 Radiographic Scoring
			2.5 Histology
			2.6 RNA Isolation and RT-qPCR
				2.6.1 RNA Isolation from Histological Sections
				2.6.2 Reverse Transcription and RT-qPCR
		3 Methods
			3.1 Closed Femoral Fracture Model
			3.2 Micro-CT
			3.3 Torsion Testing
			3.4 Radiographic Scoring
			3.5 Histology
				3.5.1 Paraffin Embedding
				3.5.2 Paraffin Sectioning
				3.5.3 Histological/IHC Staining
				3.5.4 Histomorphometric Quantification
			3.6 Gene Expression
				3.6.1 RNA Isolation from Whole Fracture Callus
				3.6.2 RNA Isolation from Histological Sections
				3.6.3 Reverse Transcription
				3.6.4 Quantification of Gene Expression
		4 Notes
		References
	Chapter 14: Mouse Models of Osteoarthritis: Surgical Model of Post-traumatic Osteoarthritis Induced by Destabilization of the ...
		1 Introduction
		2 Materials
			2.1 Conditional Deletion or Induction of Transgene Expression in Genetically Modified  Mice
				2.1.1 Tamoxifen Treatment for Deletion of Floxed Alleles
				2.1.2 Doxycycline Treatment for Control of Tetracycline-Regulated Promoters
			2.2 Anesthesia Induction and Maintenance
			2.3 Preparation of Surgical  Site
			2.4 Surgical Reagents and Equipment
			2.5 Mouse Housing
			2.6 Sample Fixation, Decalcification, and Processing
				2.6.1 Tissue Fixation
				2.6.2 Tissue Decalcification
				2.6.3 Tissue Processing
				2.6.4 Sectioning
			2.7 Histological Staining
			2.8 Immunohistochemistry (IHC) and Immunofluorescence (IF)
			2.9 RNA and DNA Extraction for Gene Expression and DNA Methylation Analyses of Mouse Articular Cartilage
				2.9.1 Isolation of Articular Cartilage
				2.9.2 Total RNA Isolation from Cartilage Using a Modified mirVana Protocol
				2.9.3 Total RNA isolation from Cartilage Using a Modified Rneasy Mini Protocol
				2.9.4 DNA Isolation from Cartilage Using a Modified Gentra Puregene DNA Isolation Protocol
		3 Methods
			3.1 Conditional Deletion or Induction of Transgene Expression in Genetically Modified  Mice
				3.1.1 Tamoxifen Treatment of Mice for Conditional Gene Ablation
				3.1.2 Tamoxifen Administration by Intraperitoneal Injection
				3.1.3 Doxycycline Treatment for Tetracycline-Inducible Transgene Expression
				3.1.4 Doxycycline Administration (See Note 2)
			3.2 Surgical Resection of Mouse Knee Joints
			3.3 Histological Assessment of OA Pathology
				3.3.1 Fixation
				3.3.2 Decalcification
				3.3.3 Processing and Embedding
				3.3.4 Sectioning
				3.3.5 Histological Staining
				3.3.6 Histological Scoring
				3.3.7 Statistical Analysis
				3.3.8 Osteophyte Scoring
			3.4 Immunohistochemistry
				3.4.1 Immunoperoxidase Staining
				3.4.2 Immunofluorescence Staining Protocol
			3.5 Cartilage Microdissection for RNA and DNA Extraction for Gene Expression and DNA Methylation Analyses
			3.6 RNA Isolation from Cartilage Using a Modified mirVana miRNA Isolation Kit (Ambion) Protocol
				3.6.1 Ethanol Precipitation
			3.7 RNA Isolation from Cartilage Using a Modified RNeasy Mini RNA Isolation Kit (Qiagen) Protocol
			3.8 DNA Isolation from Cartilage Using a Modified Gentra Puregene DNA Isolation Kit (Qiagen) Protocol
		4 Notes
		References
	Chapter 15: Immunostaining of Skeletal Tissues
		1 Introduction
			1.1 Fixation
			1.2 Decalcification
			1.3 Antigen Retrieval
			1.4 Data Analysis
		2 Materials
			2.1 IHC on Paraffin Sections
			2.2 IF on Frozen Sections
		3 Methods
			3.1 IHC on Paraffin Sections
				3.1.1 Tissue Preparation
				3.1.2 Antibody-Based Staining
			3.2 IF on Frozen Sections
				3.2.1 Tissue Preparation
				3.2.2 Antibody-Based Staining
		4 Notes
		References
	Chaptert 16: Mapping Regional Cortical Bone Responses to Local Changes in Loading and Systemic Stimuli
		1 Introduction
		2 Materials
			2.1 Strain Gauging and In Vivo Axial Tibial Loading
			2.2 Ex Vivo μCT Imaging
			2.3 Site Specificity and Statistical Analysis
		3 Methods
			3.1 Strain Gauging and In Vivo Axial Tibial Loading
				3.1.1 Preparation of the GAUGES
				3.1.2 Attach the Strain Gauge to the Tibia of a Recently Euthanized Mouse to Pre-gauge (See Fig. 3)
				3.1.3 Attachment of the Gauge to the Mouse Tibia
				3.1.4 Pre-gauging Protocol
				3.1.5 Axially Load the Tibia of Anesthetized Mice
			3.2 Ex Vivo μCT Imaging
				3.2.1 Dissect the Left (Control) and Right (Exogenously Loaded) Tibia
				3.2.2 Fix and Dehydrate the Tibia
				3.2.3 μCT Scan Each Bone
				3.2.4 Reconstruction
			3.3 Site Specificity and Statistical Analysis
		4 Notes
		References
	Chapter 17: Pain and Activity Measurements
		1 Introduction
		2 Materials
			2.1 von Frey Filament Assays
			2.2 Open-Field Activity Measurements
			2.3 Static Weight Bearing Incapacitance Assays
		3 Method
			3.1 von Frey Filament Assays
				3.1.1 Acclimation Period
				3.1.2 On Day of Testing
			3.2 Open-Field Activity Measurements
			3.3 Static Weight Bearing Incapacitance Assays
		4 Notes
		References
Index