Cartilage Tissue Engineering

Preface
Contents
Contributors
Chapter 1: Cartilage Tissue Engineering: An Introduction
1 Introduction
References
Chapter 2: Chondrocyte Isolation and Expansion
1 Introduction
2 Materials
2.1 Research Grade Mechanical and Enzymatic Extraction
2.2 Research Grade Chondrocyte Expansion
2.3 GMP Mechanical and Enzymatic Extraction
2.4 GMP Chondrocyte Expansion
3 Methods
3.1 Research Grade Chondrocyte Isolation
3.2 Research Grade Chondrocyte Expansion
3.3 GMP Chondrocyte Isolation
3.4 GMP Chondrocyte Expansion in Autologous Serum Supplemented Medium
3.4.1 Autologous Serum Preparation
3.4.2 GMP Chondrocyte Expansion
3.4.3 Harvesting, Concentrating, and Releasing Cells for Autologous Transplantation
4 Notes
References
Chapter 3: Isolation of Chondrons from Hyaline Cartilage
1 Introduction
2 Materials
2.1 Chemicals and Materials
2.2 Chondron Isolation Medium
3 Methods
3.1 Processing Hyaline Cartilage
3.2 Chondron Isolation Procedure
3.3 Next Day
4 Notes
References
Chapter 4: Articular Cartilage Chondroprogenitors: Isolation and Directed Differentiation
1 Introduction
2 Materials
3 Methods
3.1 Chondroprogenitor Isolation
3.2 Cellular Differentiation of Chondroprogenitors
4 Notes
References
Chapter 5: Physioxic Culture of Chondrogenic Cells
1 Introduction
2 Materials
2.1 Hypoxia/Physioxia-Controlled Work Environment
2.2 Chondrocyte Isolation and Culture
2.3 MSC Isolation and Culture
2.4 Chondrogenic Differentiation
2.5 Histological and Immunohistological Analysis
2.6 DNA and sGAG Assay
2.7 Gene Expression Analysis
3 Methods
3.1 Isolation and Seeding of Human Chondrocytes
3.2 Isolation and Seeding of Human Mesenchymal Stromal Cells (Bone Marrow MSCs)
3.3 Physioxic Culture of Chondrogenic Cells
3.4 Physioxia-Mediated Induction of Chondrogenesis
3.5 Qualitative Evaluation of Chondrogenic Differentiation: Collagen I and II Staining
3.6 Qualitative Evaluation of Chondrogenic Differentiation: Collagen X Staining
3.7 Qualitative Evaluation of Chondrogenic Differentiation: DMMB Staining
3.8 Quantitative Evaluation of Chondrogenic Differentiation
3.9 Gene Expression Analysis
4 Notes
References
Chapter 6: Isolation and In Vitro Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells
1 Introduction
2 Materials
2.1 Cell Isolation and Expansion
2.2 Chondrogenic Differentiation
3 Methods
3.1 Isolation of MSC from Bone Marrow Aspirate
3.2 Chondrogenesis in Pellet Culture
4 Notes
References
Chapter 7: Adipose-Derived Stromal Cells: Isolation, Expansion, and Differentiation
1 Introduction
2 Materials
2.1 Tissue
2.2 Media
2.3 Buffers
2.4 Other Reagents
2.5 Utensils
2.6 Equipment
3 Methods
3.1 ASC Isolation
3.2 Chondrogenic Differentiation via Pellet Culture
3.3 Osteogenic and Adipogenic Differentiation of ASC
4 Notes
References
Chapter 8: Chondrogenic Differentiation of Human-Induced Pluripotent Stem Cells
1 Introduction
1.1 Development of the Protocol
1.2 Applications of the Protocol
1.3 Comparison with Other Methods
1.4 Experimental Design
1.4.1 Cell Source
1.4.2 Mesodermal Differentiation
1.4.3 Chondrogenic Differentiation
1.4.4 Validation of Chondrogenic Differentiation
2 Materials
2.1 hiPSC Culture
2.2 Mesodermal Differentiation
2.3 Chondrogenic Differentiation
2.4 Chondrogenic Validation
2.4.1 Digestion of Chondrogenic Pellets
2.4.2 Histology Preparation
2.4.3 Safranin-O Staining
2.4.4 Immunohistochemistry
2.4.5 Biochemical Analysis Preparation
2.4.6 RT-qPCR Preparation
3 Methods
3.1 hiPSC Culture
3.2 Mesodermal Differentiation
3.3 Chondrogenic Differentiation
3.4 Chondrogenic Validation
3.4.1 Digestion of Chondrogenic Pellets
3.4.2 Histology Preparation
3.4.3 Safranin-O and Hematoxylin
3.4.4 Immunohistochemistry
3.4.5 Biochemical Assay Preparation
3.4.6 RT-qPCR Preparation
3.5 Anticipated Results
4 Notes
References
Chapter 9: The Dimethylmethylene Blue Assay (DMMB) for the Quantification of Sulfated Glycosaminoglycans
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 10: Extracellular Vesicle Isolation and Characterization for Applications in Cartilage Tissue Engineering and Osteoart...
1 Introduction
2 Materials
2.1 Devices and Software
2.2 Buffers, Reagents, and Media
2.3 Consumables
3 Methods
3.1 Isolation of EVs
3.2 Ultracentrifugation (UC)
3.3 Size-Exclusion Chromatography (SEC)
3.4 Combined UC and SEC
3.5 Characterization of EVs
3.5.1 Nanoparticle Tracking Analysis (NTA)
3.5.2 Western Blot Analysis
3.5.3 Cryo-electron Microscopy (cryoEM)
4 Notes
References
Chapter 11: Metabolomic Profiling to Understand Chondrocyte Metabolism
1 Introduction
2 Materials
2.1 Chondrocyte Harvest
2.2 Alginate Encapsulation and Culture
2.3 Alginate Release and Agarose Encapsulation
2.4 Metabolite Extraction
2.5 Liquid Chromatography-Mass Spectrometry
2.6 Data Analysis
3 Methods
3.1 Chondrocyte Harvest
3.2 Alginate Encapsulation
3.3 Alginate Release, Agarose Encapsulation, and Tissue Culture
3.4 Metabolite Extraction (Figs. 1 and 2)
3.4.1 Alginate Released Agarose-Embedded Chondrocytes
3.4.2 Cartilage and Other Soft Tissues
3.4.3 Synovial Fluid
3.5 Liquid Chromatography-Mass Spectrometry (LC-MS)
3.6 Metabolomics: Statistical Analysis (Fig. 3)
3.7 Metabolomics: Pathway Analysis (Volcano Plot, Fig. 5)
3.8 Targeted Metabolite Analysis
4 Notes
References
Chapter 12: Techniques for Visualization and Quantification of Primary Cilia in Chondrocytes
1 Introduction
1.1 Cilia Structure and Function
1.2 Primary Cilia in Cartilage and Chondrocytes
2 Materials
2.1 Materials for Chondrocyte Culture
2.2 Materials for Immunofluorescence of Primary Cilia
2.3 Materials for Microscopy
2.4 Materials for Measurement of Cilia
3 Methods
3.1 Chondrocyte Monolayer Culture for Imaging of Cilia
3.2 Chondrocyte Culture in 3D Agarose Gel
3.3 Immunofluorescence of Primary Cilia in Monolayer Culture
3.4 Immunofluorescence of Primary Cilia in 3D Cell-Seeded Gels
3.5 Immunofluorescence of Primary Cilia in Cartilage and Ossified Musculoskeletal Tissues
3.6 Epifluorescence Microscopy Imaging of Primary Cilia
3.7 Confocal Microscopy Imaging of Primary Cilia
3.8 Super Resolution Structure Illumination Microscopy (sr-SIM) Imaging of Primary Cilia
3.9 Measurement of Cilia Prevalence
3.10 Measurement of Cilia Length
3.11 Measurement of Ciliary Protein Distribution Along the Axoneme
4 Notes
References
Chapter 13: Co-culture of Human Articular Chondrocytes Seeded in Polyurethane Scaffolds and Human Mesenchymal Stromal Cells En...
1 Introduction
2 Materials
2.1 Cell Isolation, Culture, and Growth Media
2.2 PU-Scaffold Seeding
2.3 Alginate Beads
2.4 Harvest Cells from Alginate Beads
3 Methods
3.1 Isolation and Culture of Human Chondrocytes
3.2 PU Scaffold Seeding
3.3 Alginate Bead Preparation
3.4 Harvesting Cells from Alginate Beads
4 Notes
References
Chapter 14: Isolation of Murine Articular Chondrocytes for Single-Cell RNA or Bulk RNA Sequencing Analysis
1 Introduction
2 Materials
2.1 Appropriate Personal Protective Equipment (PPE)
2.2 Animal Euthanasia and Tissue Harvesting
2.3 Articular Cartilage Digest
2.4 Cell Preparation and Chondrocyte FACS Isolation
2.5 Library Preparation and Sequencing
3 Methods
3.1 Articular Cartilage Dissection from Long Bones
3.2 Articular Cartilage Dissociation and Digestion
3.3 Cell Preparation and Chondrocyte FACS Isolation
3.4 Bulk Library Preparation
3.5 Single-Cell Library Preparation and Sequencing
3.6 Sequencing
4 Notes
References
Chapter 15: Modulation of MicroRNA Expression During In Vitro Chondrogenesis
1 Introduction
2 Materials
2.1 Key Equipment and Tools
2.2 Reagents, Cell Culture Media, and Kits
2.2.1 Isolation and Culture of Human Cartilage Progenitor Cells (CPCs)
2.2.2 Chondrogenic Induction Media
2.2.3 Lentiviral-Induced Overexpression of microRNAs
2.2.4 RNA Isolation and Detection of miRNA Expression
3 Methods
3.1 Isolation of Cartilage Progenitor Cells from Human Articular Cartilage
3.2 Generation of Lentivirus Particles to Overexpress microRNAs
3.2.1 Lentivirus Packaging and Titering
3.3 Lentiviral-Induced Modulation of microRNAs During Chondrogenesis
3.3.1 Lentiviral Transduction of Human CPCs
3.3.2 Chondrogenic Induction of Lentiviral-Transduced CPCs
3.3.3 Isolation of RNA from Lentiviral-Transduced Pellet Cultures
3.3.4 Determining miRNA Expression Levels in Lentiviral-Transduced Cells
4 Notes
References
Chapter 16: Purification and Isolation of Proteins from Hyaline Cartilage
1 Introduction
2 Materials
2.1 Homogenization of Cartilage
2.2 Sequential Protein Extraction
2.3 Pepsin Digestion
2.4 Differential Salt Precipitation
2.5 Collagenase Digestion
2.6 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
2.7 In-Gel Trypsin Digestion for Mass Spectrometry Analysis
2.8 Immunoblot Identification of Proteins
3 Methods
3.1 Homogenization of Cartilage
3.2 Sequential Protein Extraction
3.3 Pepsin Treatment
3.4 Collagenase Treatment
3.5 Differential Salt Precipitation
3.6 In-Gel Trypsin Digestion
3.7 Immunoblot
4 Notes
References
Chapter 17: Histological Preparation and Evaluation of Cartilage Specimens
1 Introduction
2 Materials
3 Methods
3.1 Processing Non-decalcified Samples Embedded in Polymethylmethacrylate
3.1.1 Sample Preparation and Fixation
3.1.2 Dehydration, PMMA Embedding, and Sectioning
3.1.3 Staining Non-decalcified PMMA Sections
3.1.3.1 Toluidine Blue
3.1.3.2 Alcian Blue
3.1.3.3 Safranin O
3.2 Processing Decalcified Samples Embedded in Paraffin
3.2.1 Sample Preparation, Fixation, and Decalcification
3.2.2 Dehydration, Paraffin Embedding, and Sectioning
3.2.3 Staining Decalcified Paraffin Sections
3.2.3.1 Toluidine Blue
3.2.3.2 Alcian Blue
3.2.3.3 Safranin O
3.2.3.4 Immunohistochemistry
3.3 Evaluation of Cartilage Histological Sections
3.3.1 Scores for the Evaluation of Osteoarthritis
3.3.1.1 OARSI
3.3.1.2 Mankin
3.3.1.3 Little
3.3.2 Score Systems for Cartilage Repair
3.3.2.1 O´Driscoll
3.3.2.2 ICRS I and II
3.3.2.3 MSRU Subchondral Bone Cyst Score
4 Notes
Appendix: Score Scales for Cartilage Evaluation
References
Chapter 18: Articular Cartilage Imaging in the Context of the Superficial Chondrocyte Spatial Organization (SCSO) as a Surroga...
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 19: Unconfined Compression Experimental Protocol for Cartilage Explants and Hydrogel Constructs: From Sample Preparati...
1 Introduction
2 Materials
3 Methods
3.1 Hydrogel Materials and Preparation
3.2 Cartilage Explant Preparation
3.3 Sample Preservation
3.4 Test Setup Preparation
3.5 Compression Loading Protocol
3.6 Experimental Data Acquisition and Testing
3.7 Experimental Data and Inverse Mechanical Characterization
4 Notes
Supplementary Materials
References
Chapter 20: Viral Gene Delivery in Chondrocytes
1 Introduction
1.1 Salient Properties of Common Viral Vectors (Table 1)
1.1.1 Adenovirus Vectors
1.1.2 Adeno-Associated Virus (AAV) Vectors
1.1.3 Retrovirus Vectors
1.1.4 Lentivirus Vector
2 Materials
3 Methods
3.1 Overnight Transduction
3.2 Strategies to Enhance Transduction Efficiency
3.2.1 Spinoculation
3.2.2 Charge Neutralization
3.2.3 Magnetic Transduction
3.2.4 Other
4 Notes
References
Chapter 21: Decellularization of Articular Cartilage: A Hydrochloric Acid-Based Strategy
1 Introduction
2 Materials
3 Methods
3.1 Decellularization and GAG Depletion (``decell-deGAG´´ Procedure)
3.2 Alternative Protocol: Quick decell-deGAG
3.3 Evaluation of Treatment Success
4 Notes
References
Chapter 22: Self-Assembly Culture Model for Engineering Musculoskeletal Tissues
1 Introduction
2 Materials
2.1 Harvest Supplies
2.2 Chondrocyte Isolation and CTA Generation Supplies
3 Methods
3.1 Chondrocyte Harvest from Bovine Femoral Condyles
3.2 CTA Preparation
4 Notes
References
Chapter 23: Osteochondral Explant Isolation and Culture Under a Compression and Shear Bioreactor
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Working Space
3.2 Harvesting Osteochondral Plugs
3.3 Chondral or Osteochondral Defect Creation
3.4 Clean and Sterilize Tools
3.5 Fixation of Osteochondral Plugs in Bioreactor Loading Holders
4 Notes
References
Chapter 24: A Mouse Model of Acute Cartilage Injury and Repair
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 25: Small-Animal Compression Models of Osteoarthritis
1 Introduction
2 Materials
3 Methods
3.1 Mouse Cyclic Compression (See Notes 11-19)
3.2 Mouse ACL-R (See Notes 20-33)
3.3 Rat ACL-R (See Notes 34-44)
4 Notes
4.1 Cyclic Compression in Mice
4.2 ACL-R in Mice
4.3 ACL-R in Rats
References
Chapter 26: Modeling and Assessing Osteoarthritis in Mice by Destabilization of the Medial Meniscus (DMM)
1 Introduction
2 Materials
2.1 Surgical Equipment
2.2 Surgical Suite
2.3 Anesthesia and Analgesia
2.4 Post-surgery Recovery
2.5 Pain Measurement
2.6 Histology Sample Preparation
3 Methods
3.1 Surgery
3.2 Pain Assessment
3.3 Histology Sample Preparation
3.4 Embedding
3.5 Sectioning
3.6 Safranin O/Fast Green Staining of Sections
4 Notes
References
Chapter 27: Collagenase-Induced Osteoarthritis in Mice
1 Introduction
2 Materials
2.1 Solutions
2.2 Supplies
3 Methods
3.1 Preparation for Osteoarthritis Induction
3.2 Induction of Osteoarthritis
3.3 Osteoarthritis Induction Aftercare
3.4 Knee Joint Dissection
4 Notes
References
Chapter 28: Platelet-Rich Plasma for Intra-articular Injections: Preclinical and Clinical Evidence
1 Introduction
2 Preparation Method and Administration
3 In Vitro Evidence
4 Preclinical Evidence
5 Clinical Evidence
References
Index