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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Abstract

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Introduction

Bone homeostasis is a complex physiological process that is regulated by bone-resorbing osteoclasts and bone-forming osteoblasts1. A balance between osteoblastic and osteoclastic activity mediated through osteoblasts and osteoclasts, respectively, is highly essential for maintaining bone health and homeostasis, because perturbations in bone homeostasis might lead to bone diseases, such as abnormal bone growth or loss of bone density. As unique bone-resorption cells, osteoclasts are important in diseases related to abnormal bone destruction, including osteoporosis, periodontitis, and periprosthetic osteolysis2,....

Protocol

All the methods involving the animals described here are approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing University of Chinese Medicine.

1. Setup

  1. Prepare several longitudinally cut 1 mL pipette tips (1 cm) and several 1.5 mL microcentrifuge tubes. Put the pipette tips and microcentrifuge tubes at 103 kPa and 121 °C for 20 min and ensure that they are sterile.
  2. Prepare a box of ice and several sterile dishes to preserve the isolated tissu.......

Representative Results

The purpose of the protocol was to isolate and purify large numbers of osteoclast precursors conveniently and induce osteoclasts successfully. By supplementing with M-CSF and RANKL, giant osteoclasts were seen on days 5–6. The formation of osteoclasts was successfully identified by TRAP staining (Figure 1A). Large and purple cells were regarded as TRAP-positive cells with multiple nuclei (typically ≥ three nuclei). Through this method, it was typi.......

Discussion

The ability to obtain and study osteoclasts in vitro is a critical and fundamental skill for any researcher wishing to study bone metabolism, which may help understand the mechanisms of bone-absorbing diseases and develop novel therapeutic agents. The present study described a protocol with some modifications based on previous methods.

By using pipette tips and microcentrifuge tubes to obtain bone marrow, it largely reduced the operation time of obtaining bone marrow and the workload of labora.......

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81473692) to Yong Ma. The authors thank all the staff of the Medical Research Center of the First College of Clinical Medicine in Nanjing University of Chinese Medicine.

....

Materials

NameCompanyCatalog NumberComments
Acid Phosphatase, Lekocyte (TRAP) kitSigma-Aldrich387A
Automatic Hard Tissue SlicerLeciaRM2265
Bovine femoral bonePurchased by ourselves
Cell IncubatorHerausBB16/BB5060
Fetal Bovine SerumSaranas-fbs-au-015
Goat Anti-Rabbit IgG H&L (Alexa Fluor 488)Abcamab150077
Hard Tissue GrindersLeciaSP-2600
Histopaque KitTianJing HaoyangTBD2013DRSilicified centrifugal tube amd cell separation solution
Hochest33342Sigma-AldrichB2261
Inverted Phase Contrast MicroscopeOlympusCKX31
Inverted Fluorescence MicroscopeLeciaDMI-3000
MEM, no GlutamineGibco11090-081
Penicillin-Streptomycin, LiquidGibco15140122
Rabbit Anti-Calcitonin receptorBiossbs-0124R
Recombinant Rat M-CSFPeproTech400-28
Recombinant Rat sRANK LigandPeproTech400-30
Red Blood Cell Lysis BufferAbsinabs47014932
Scanning Electron MicroscopyFEIQuanta 200
Toluidine BlueSigma-Aldrich89649

References

  1. Boyle, W. J., Simonet, W. S., Lacey, D. L. Osteoclast differentiation and activation. Nature. 423 (6937), 337-342 (2003).
  2. Kular, J., Tickner, J., Chim, S. M., Xu, J. K. An overview of the regulation of bone remodelling at the cellular leve....

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