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An In Vitro Model of Murine Osteoclast-Mediated Bone Resorption

小鼠破骨细胞介导的骨吸收体外模型

XS Xiaoyue Sun
ZW Zijun Wang
YT Yi Tang
SW Stephen J. Weiss
LZ Lingxin Zhu

发布: 2024年11月05日第14卷第21期 DOI: 10.21769/BioProtoc.5100 浏览次数: 2203

评审: Valérian DORMOYJaira Ferreira de VasconcellosNona Farbehi

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Abstract

Osteoclasts are terminally differentiated multinucleated giant cells that mediate bone resorption and regulate skeletal homeostasis under physiological and pathological states. Excessive osteoclast activity will give rise to enhanced bone resorption, being responsible for a wide range of metabolic skeletal diseases, ranging from osteoporosis and rheumatoid arthritis to tumor-induced osteolysis. Therefore, the construction of in vitro models of osteoclast-mediated bone resorption is helpful to better understand the functional status of osteoclasts under (patho)physiological conditions. Notably, it is essential to provide an in vivo–relevant bone substrate that induces osteoclasts to generate authentic resorption lacunae and excavate bone. Here, we summarize the experimental design of a reproducible and cost-effective method, which is suitable for evaluating the regulatory mechanisms and influence of molecular agonists and antagonists as well as therapeutics on osteoclast-mediated bone-resorbing activity.

Key features

• Experiments are performed using bovine cortical bone slices to simulate bone substrate resorption by murine osteoclasts in vivo.

• The method allows for quantification of bone resorption in vitro.

• The method is suitable for evaluating the regulatory mechanisms that control osteoclast-mediated bone-resorbing activity.

Keywords: Osteoclast (破骨细胞) search Bone resorption (骨吸收) search Bone slices (骨切片) search Resorption pits (吸收孔) search Cell culture (细胞培养) search

Graphical overview


Schematic diagram of the protocol for assessing osteoclast-mediated bone-resorbing activity in vitro. Step 1: Euthanizing 8–12-week-old male mice to separate tibias and femurs. Step 2: Flushing bone marrow cells by injector and obtaining the cells. Step 3: Preparation of BMDMs with complete medium containing 20 ng/mL M-CSF in 10 cm Petri dishes. Step 4: Cultivation of osteoclasts with complete medium containing 20 ng/mL M-CSF and 30 ng/mL RANKL atop bovine cortical bone slices in a 96-well plate. Step 5: Evaluating bone-resorbing activity of osteoclasts.

Background

Osteoclasts are exclusive and specialized multinucleated bone-resorbing cells derived from both embryonic and hematopoietic stem cell precursors of erythromyeloid and myeloid lineages [1]. An imbalance of skeletal remodeling as a consequence of increased osteoclast-mediated bone resorption is responsible for a wide range of metabolic skeletal diseases, ranging from osteoporosis and rheumatoid arthritis to tumor-induced osteolysis. Therefore, the construction of in vitro osteoclast-mediated bone resorption models is helpful to better characterize the functional status of osteoclasts under both physiological and pathological conditions.

The transition of cells of the monocyte/macrophage lineage into osteoclasts in vitro requires two essential cytokines—macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). The presence of M-CSF promotes the proliferation of osteoclast precursor cells, and RANKL further induces precursor cells to express an osteoclast phenotype [2]. Mature osteoclasts at the bone surfaces undergo a polarization process marked by extensive morphologic changes, including the formation of an actin ring at their basal surface, termed the sealing zone. The sealing zone surrounds a differentiated and intricately folded region of the plasma membrane called the ruffled border, where protons, chloride ions, and proteases are secreted into the underlying resorption lacunae where bone demineralization and extracellular matrix degradation proceed [3]. As the organic phase of bone is dominated by type I collagen, the proteolytic release of C-terminal telopeptide fragments of type collagen (CTX-1) is a characteristic biochemical marker of osteoclast-mediated bone resorption [4]. When cultured on bone or dentin substrate, osteoclasts assemble resorption lacunae, thereby generating resorption pit structures similar to those formed during bone resorption in vivo [5]. Notably, bovine cortical bone slices are superior to synthetic materials or dentine-derived matrix as they better mimic bone substrates encountered in vivo [6]. When combined with an appropriate detection assay, osteoclast-mediated bone-resorbing activity can be quantified under a variety of experimental conditions [7,8].

Here, we describe a step-by-step protocol for in vitro bone-resorbing activity using an osteoclast-cortical bone slice co-culture system. This protocol consists of three main steps: mouse bone marrow–derived macrophage (BMDM) preparation, osteoclast induction and characterization, and bone resorption pit quantification. This method can be universally utilized to evaluate the bone-resorbing activity of osteoclasts in response to both agonists and antagonists.

Materials and reagents

Biological materials

  1. 8–12-week-old male C57BL/6J mice (The Jackson Laboratory, catalog number: 000664)

  2. Bovine cortical bone slices (Immunodiagnostic Systems, catalog number: DT-1BON1000-96)


Reagents

  1. Alpha minimum essential medium (α-MEM) (Hyclone, Cytiva, catalog number: SH30265.01)

  2. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F0193)

  3. Penicillin-streptomycin solution (Hyclone, Cytiva, catalog number: SV30010)

  4. Red blood cell lysing buffer (Biosharp, catalog number: BL504A)

  5. Phosphate buffered saline (PBS) (Servicebio, catalog number: G0002)

  6. Ethylenediaminetetraacetic acid (EDTA) solution, 0.5 M (Sigma-Aldrich, catalog number: 03690)

  7. Recombinant murine M-CSF (R&D Systems, catalog number: 416-ML)

  8. Recombinant murine RANKL (R&D Systems, catalog number: 462-TEC)

  9. Bovine serum albumin (Sigma-Aldrich, catalog number: A1933)

  10. Paraformaldehyde powder (Sigma-Aldrich, catalog number: 158127)

  11. Glutaraldehyde solution, 50% (Sigma-Aldrich, catalog number: 49629)

  12. Tartrate-resistant acid phosphatase (TRAP) stain kit (Sigma-Aldrich, catalog number: 387A)

  13. Triton X-100 solution, 10% (Sigma-Aldrich, catalog number: 49629)

  14. Phalloidin-tetramethylrhodamine B isothiocyanate (Sigma-Aldrich, catalog number: P1951)

  15. α-Tubulin mouse mAb (Cell Signaling Technology, catalog number: 3873)

  16. Donkey anti-rat Alexa 488-conjugated secondary antibodies (Invitrogen Molecular Probes, catalog number: A-21202)

  17. Sodium hydroxide (NaOH) powder (Sigma-Aldrich, catalog number: 655104)

  18. Peroxidase-conjugated wheat germ agglutinin (WGA) (Sigma-Aldrich, catalog number: L3892)

  19. Diaminobenzidine (DAB) tablets (Sigma-Aldrich, catalog number: D4418)

  20. FITC conjugate WGA (Sigma-Aldrich, catalog number: L4895)

  21. CrossLaps ELISA kit for quantifying CTX-1 release (Immunodiagnostic Systems, catalog number: AC-07F1)


Solutions

  1. 5 mM EDTA solution (see Recipes)

  2. 0.1% Bovine serum albumin solution (see Recipes)

  3. Murine recombinant M-CSF solution (see Recipes)

  4. Murine recombinant RANKL solution (see Recipes)

  5. 4% Paraformaldehyde solution (see Recipes)

  6. 2.5% Glutaraldehyde solution (see Recipes)

  7. NaOH solution (see Recipes)

  8. 0.1% Triton X-100 solution (see Recipes)

  9. Peroxidase-conjugated WGA solution (see Recipes)

  10. FITC-conjugated WGA solution (see Recipes)


Recipes

  1. 5 mM EDTA solution

    ReagentFinal concentrationQuantity or VolumeRecommended storage conditionRecommended storage time
    0.5 M EDTA solution5 mM0.5 mLRoom temperature
    Sterile PBSNA49.5 mLRoom temperature
    Final volume50 mL41 week


  2. 0.1% Bovine serum albumin solution

    ReagentFinal concentrationQuantity or VolumeRecommended storage conditionRecommended storage time
    Bovine serum albumin0.1%0.02 g4
    Double-distilled waterNA20 mLRoom temperature
    Final volume20 mL-201 month


  3. Murine rM-CSF solution

    ReagentFinal concentrationQuantity or VolumeRecommended storage conditionRecommended storage time
    Murine rM-CSF50 μg/mL50 μg-80
    Sterile PBS containing 0.1% bovine serum albuminNA1 mL-20
    Final volume1 mL-801 year


  4. Murine rRANKL solution

    ReagentFinal concentrationQuantity or VolumeRecommended storage conditionRecommended storage time
    Murine rRANKL50 μg/mL50 μg-80
    Sterile PBS containing 0.1% bovine serum albuminNA1 mL-20
    Final volume1 mL-801 year


  5. 4% Paraformaldehyde solution

    ReagentFinal concentrationQuantity or VolumeRecommended storage conditionRecommended storage time
    Paraformaldehyde powder4% (w/v)0.4 g-4
    Sterile PBSNA10 mLRoom temperature
    Final volume10 mL-41 week


  6. 2.5% Glutaraldehyde solution

    ReagentFinal concentrationQuantity or VolumeRecommended storage conditionRecommended storage time
    50% glutaraldehyde solution2.5% (v/v)0.5 mLRoom temperature
    Sterile PBSNA9.5 mLRoom temperature
    Final volume10 mL-41 week


  7. NaOH solution

    ReagentFinal concentrationQuantity or VolumeRecommended storage conditionRecommended storage time
    NaOH powder0.5 N1 gRoom temperature
    Double-distilled waterNA50 mLRoom temperature
    Final volume50 mL-41 month


  8. 0.1% Triton X-100 solution

    ReagentFinal concentrationQuantity or VolumeRecommended storage conditionRecommended storage time
    10% Triton X-100 solution0.1%0.01 mLRoom temperature
    Double-distilled waterNA9.99 mLRoom temperature
    Final volume10 mL-41 month


  9. Peroxidase-conjugated WGA solution

    ReagentFinal concentrationQuantity or VolumeRecommended storage conditionRecommended storage time
    Peroxidase-conjugated WGA2 mg/mL1 mg-20
    Sterile PBSNA0.5 mLRoom temperature
    Final volume0.5 mL-201 year


  10. FITC-conjugated WGA solution

    ReagentFinal concentrationQuantity or VolumeRecommended storage conditionRecommended storage time
    FITC-conjugated WGA2 mg/mL1 mg-20
    Sterile PBSNA0.5 mLRoom temperature
    Final volume0.5 mL-201 year


Laboratory supplies

  1. 10 mL syringe with 25 G needle

  2. Sterile absorbent gauze

  3. 70 µm cell strainer (Corning, catalog number: 431751)

  4. Pipette

  5. 15 mL conical tube (Nest, catalog number: 601001)

  6. 50 mL conical tube (Nest, catalog number: 601002)

  7. 10 × 10 cm Petri dish with cover (Nest, catalog number: 752001)

  8. Cell lifter (Corning, catalog number: CLS3008)

  9. 96-well cell culture plates (Nest, catalog number: 713011)

Equipment

  1. Dissecting tweezers and scissors (Autoclaved)

  2. Cell counter (Thermo Fisher Scientific Inc, model: Countess 3)

  3. Cell culture incubator (37 °C, 5% CO2)

  4. Centrifuge (Eppendorf)

  5. Inverted microscope (Olympus)

  6. Confocal laser scanning microscope (CLSM) (Nikon A1)

  7. Critical point drying (Balzers Union)

  8. Au/Pg sputtered (Polaron Equipment Ltd, model: E-5100)

  9. Field emission scanning electron microscopy (FE-SEM) (AMRAY 1910)

Software and datasets

  1. ImageJ is a Java-based (runs on Mac OS X, Linux, and Windows) freeware available for download at: http://rsb.info.nih.gov/ij/

  2. GraphPad Prism 7

Procedure

English
中文翻译

文章信息

稿件历史记录

提交日期: Jun 20, 2024

接收日期: Sep 3, 2024

在线发布日期: Oct 13, 2024

出版日期: Nov 5, 2024

版权信息

© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Sun, X., Wang, Z., Tang, Y., Weiss, S. J. and Zhu, L. (2024). An In Vitro Model of Murine Osteoclast-Mediated Bone Resorption. Bio-protocol 14(21): e5100. DOI: 10.21769/BioProtoc.5100.
  2. Zhu, L., Tang, Y., Li, X. Y., Kerk, S. A., Lyssiotis, C. A., Sun, X., Wang, Z., Cho, J. S., Ma, J., Weiss, S. J., et al. (2023). Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption. J Cell Biol. 222(4): e202206121.

    3. ris ris Download Citation in RIS Format

分类

发育生物学 > 细胞生长和命运决定 > 分化

细胞生物学 > 细胞分离和培养 > 细胞分化

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