Bone Research Protocols

The last decade has seen a tremendous advance in our understanding of bone biology. The genes responsible for the majority of rare inherited bone disorders have been identified and much progress has been made in the identification of genes in polygenic disorders such as Paget’s disease and complex multigene diseases such as osteoporosis. Transgenic technology has identified further genes, sometimes unexpectedly, with profound effects on bone. This wealth of new genetic information will undoubtedly lead to extensive cell biological studies to understand the mechanisms by which these gene products affect bone mass and bone strength. In Bone Research Protocols a catalogue of protocols has been assembled to perform such mechanistic studies. In the tradition of the Methods in Molecular Medicine series, the chapters are practical laboratory protocols that should enable the reader to carry out the techniques from scratch. To our knowledge this is the first time such a truly practical manual on well-established bone methods has been assembled, and this volume aims to be complementary to and follow on from the more theoretical Methods in Bone Biology, edited by Arnett and Henderson (1).

Table of Contents:
Culture of Osteolblasts and Osteocytes.- Human Osteoblast Culture.- Osteoblast Isolation from Murine Calvariae and Long Bones.- Mineralizing Fibroblast-Colony-Forming Assays.- Osteocyte Isolation and Culture.- Culture of Osteoclasts.- Isolated Osteoclast Cultures.- Primary Isolation and Culture of Chicken Osteoclasts.- Isolation and Purification of Rabbit Osteoclasts.- Generating Human Osteoclasts from Peripheral Blood.- Generating Human Osteoclasts In Vitro from Bone Marrow and Peripheral Blood.- Generating Murine Osteoclasts from Bone Marrow.- Osteoclast Formation in the Mouse Coculture Assay.- RANKL-Mediated Osteoclast Formation from Murine RAW 264.7 Cells.- Bone Organ Culture.- Analysis of Osteoclast Function in Mouse Calvarial Cultures.- Assessing Bone Formation Using Mouse Calvarial Organ Cultures.- Histological and Ultrastructural Techniques.- In Situ Hybridization and In Situ Reverse Transcription Polymerase Chain Reaction in Human Bone Sections.- Techniques for the Study of Apoptosis in Bone.- Protein Localization in Wax-Embedded and Frozen Sections of Bone Using Immunohistochemistry.- Detection of Noncollagenous Bone Proteins in Methylmethacrylate-Embedded Human Bone Sections.- Fluorescence Imaging of Bone-Resorbing Osteoclasts by Confocal Microscopy.- Bone Histomorphometry.- Transmission Electron Microscopy of Bone.- Scanning Electron Microscopy of Bone.- Imaging Techniques.- Bone Measurements by Peripheral Quantitative Computed Tomography in Rodents.- In Vivo Techniques.- Studies of Local Bone Remodeling.- Inflammation-Induced Osteoporosis.- Ovariectomy and Estrogen Replacement in Rodents.- Mechanical Loading Techniques.- Mechanical Testing of Bone Ex Vivo.- Bone Cell Responses to Fluid Flow.- Methods for Analyzing Bone Cell Responses to Mechanical Loading Using In Vitro Monolayer and Organ Culture Models.- Molecular Analysis.- Extraction of Nucleic Acids from Bone.- Analysis of Gene Expression in Bone by Quantitative RT-PCR.