Skeletal Development and Repair Methods and Protocols

This second edition provides a comprehensive laboratory manual on skeletal development and skeletal repair research utilizing mouse models. Chapters provide methods and protocols on the most current and cutting-edge techniques in skeletal development and repair, histological, cellular, and molecular techniques. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Skeletal Development and Repair: Methods and Protocols-Second Edition aims to ensure successful results in the further study of this vital field.

Part I.              Murine Skeletal Development, Repair, and Genetic Models

 

1. Overview of Skeletal Development

Tatsuya Kobayashi and Henry M. Kronenberg

 

2. Overview of Skeletal Repair (Fracture Healing and Its Assessment)

Elise F. Morgan, Anthony De Giacomo, and Louis C. Gerstenfeld

 

3. Advantages and Limitations of Cre Mouse Lines Used in Skeletal Research

Florent Elefteriou and Greig Couasnay

 

 

Part II.             Skeletal Repair, Parabiosis, Transplantations, and Organ Cultures

 

4. Generation of Closed Transverse Fractures in Small Animals

Anthony De Giacomo, Elise F. Morgan, and Louis C. Gerstenfeld

 

5. A Mouse Femoral Ostectomy Model to Assess Bone Graft Substitutes

Ryan Trombetta, Emma K. Knapp, and Hani A. Awad

 

6. Surgical Induction of Posttraumatic Osteoarthritis in the Mouse

Robert D. Maynard, David A. Villani, William G. Schroeder, Douglas J. Adams, and Michael J. Zuscik

 

7. Parabiosis: Assessing the Effects of Circulating Cells and Factors on the Skeleton

Benjamin Aaron Alman and Gurpreet Singh Baht

 

8. Murine Limb Bud Organ Cultures for Studying Musculoskeletal Development

Martin Arostegui and T. Michael Underhill

 

9. Murine Limb Explant Cultures to Assess Cartilage Development

Manuela Wuelling and Andrea Vortkamp

 

10. Renal Capsule Transplantation to Assay Angiogenesis in Skeletal Development and Repair

Anais Julien, Simon Perrin, Rana Abou-Khalil, and Céline Colnot

 

 

 

Part III.           Structural, Histological, and Molecular Analyses on Skeletal Tissues and Tissue Sections

 

11. MicroCT for Scanning and Analysis of Mouse Bones

Yung Kim, Michael D. Brodt, Simon Y. Tang, and Matthew J. Silva

 

12. Four-Point Bending Testing for Mechanical Assessment of Mouse Bone Structural Properties

Hattie C. Cutcliffe and Louis E. DeFrate

 

13. Phenotyping Intact Mouse Bones using Bone CLARITY

Jennifer B. Treweek, Aidan Beres, Nathan Johnson, and Alon Greenbaum

 

14. Processing and Sectioning Undecalcified Murine Bone Specimens

Thomas B. Bemenderfer, Jonathan S. Harris, Keith W. Condon, Jiliang Li, and Melissa A. Kacena

 

15. Preparation of Thin Frozen Sections from Nonfixed And Undecalcified Hard Tissues Using Kawamoto's Film Method (2020)

Tadafumi Kawamoto and Komei Kawamoto

 

16. Demineralized Murine Skeletal Histology

Anthony J. Mirando and Matthew J. Hilton

 

17. Assessment of Osteocytes: Techniques for Studying Morphological and Molecular Changes Associated With Perilacunar/ Canalicular Remodeling Of the Bone Matrix

Neha S. Dole, Cristal S. Yee, Charles A. Schurman, Sarah L. Dallas, and Tamara Alliston

 

18. Cell Lineage Tracing: Co-localization of Cell Lineage Markers with a Fluorescent Reporter

Yan Jing, Patricia Simmer, and Jian Q. Feng

 

19. Immunofluorescent Staining of Adult Murine Paraffin-embedded Skeletal Tissue

Neta Felsenthal and Elazar Zelzer

 

20. Detection of Hypoxic Regions in the Bone Microenvironment

Wendi Guo and Colleen Wu

 

21. EdU-based Assay of Cell Proliferation and Stem Cell Quiescence in Skeletal Tissue Sections

Marco Angelozzi, Charles R. de Charleroy, and Véronique Lefebvre

 

22. Whole-mount In Situ Hybridization in Murine Tissues

Deepika Sharma, Matthew J. Hilton, and Courtney M. Karner

 

 

Part IV.           Primary Cell Isolations, Cultures, and Assays

 

23. Bone Marrow Stromal Cell Assays – In Vitro and In Vivo

Pamela Gehron Robey, Sergei A. Kuznetsov, Paolo Bianco, and Mara Riminucci

 

24. Isolation and Culture of Periosteum Derived Progenitor Cells from Mice

Chinedu C. Ude, Girdhar G. Sharma, Jie Shen, and Regis J. O’Keefe

 

25. Isolation and Culture of Murine Primary Chondrocytes: Costal and Growth Plate Cartilage

Yihan Liao, Jason T. Long, Christopher J.R. Gallo, Anthony J. Mirando, and Matthew J. Hilton

 

26. Isolation and Culture of Neonatal Mouse Calvarial Osteoblasts

Madison L. Doolittle, Cheryl L. Ackert-Bicknell, and Jennifer H. Jonason

 

27. Mitochondrial Function and Metabolism of Cultured Skeletal Cells

Li Tian, Clifford J Rosen, and Anyonya R Guntur

 

28. Radiolabeled Amino Acid Uptake Assays in Primary Bone Cells and Bone Explants

Leyao Shen and Courtney M. Karner

 

29. RANKL-based Osteoclastogenic Assay from Murine Bone Marrow Cells

Zhenqiang Yao, Lianping Xing, and Brendan F. Boyce

 

30. Hematopoietic Stem Cell Cultures and Assays

Benjamin J. Frisch