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Updated: May 22, 2026
A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro
Published on: June 16, 2022
A 3D scanning confocal imaging method measures pit volume and captures the role of Rac in osteoclast function
Stephanie R Goldberg1, John Georgiou, Michael Glogauer
1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Abstract:
Modulation of Rho GTPases Rac1 and Rac2 impacts bone development, remodeling, and disease. In addition, GTPases are considered treatment targets for dysplastic and erosive bone diseases including Neurofibromatosis type 1. While it is important to understand the effects of Rac modulation on osteoclast function, two-dimensional resorption pit area measurements fall short in elucidating the volume aspect of bone resorption activity. Bone marrow from wild-type, Rac1 and Rac2 null mice was isolated from femora. Osteoclastogenesis was induced by adding M-CSF and RANKL in culture plates containing dentin slices and later stained with Picro Sirius Red to image resorption lacunae. Osteoclasts were also plated on glass cover slips and stained with phalloidin and DAPI to measure their surface area and the number of nuclei. Volumetric images were collected on a laser-scanning confocal system. Sirius Red confocal imaging provided an unambiguous, continuous definition of the pit boundary compared to reflected and transmitted light imaging. Rac1- and Rac2-deficient osteoclasts had fewer nuclei in comparison to wild-type counterparts. Rac1-deficient osteoclasts showed reduced resorption pit volume and surface area. Lacunae made by single Rac2 null osteoclasts had reduced volume but surprisingly surface area was unaffected. Surface area measures are deceiving since volume changed independently in resorption pits made by individual Rac2 null osteoclasts. Our innovative confocal imaging technique allows us to derive novel conclusions about Rac1 and Rac2 in osteoclast function. The data and method can be applied to study effects of genes and drugs including Rho GTPase modulators on osteoclast function and to develop pharmacotherapeutics to treat bone lytic disorders.
Insights
Rac1 and Rac2 GTPases are crucial for osteoclast function and bone resorption. This study reveals Rac1 deficiency reduces bone resorption volume and surface area, while Rac2 deficiency impacts resorption volume but not surface area.
Area of Science:
- Cell Biology
- Bone Biology
- Biochemistry
Background:
- Rho GTPases, specifically Rac1 and Rac2, play significant roles in bone development, remodeling, and disease.
- Dysplastic and erosive bone diseases, such as Neurofibromatosis type 1, are potential targets for GTPase modulation therapies.
- Traditional 2D resorption pit area measurements do not fully capture the volumetric aspect of osteoclast bone resorption.
Purpose of the Study:
- To investigate the specific roles of Rac1 and Rac2 in osteoclast function and bone resorption using a novel 3D imaging approach.
- To compare the effects of Rac1 and Rac2 deficiency on osteoclast morphology, proliferation, and resorptive activity.
- To establish an innovative confocal imaging technique for precise volumetric analysis of bone resorption.
Main Methods:
- Osteoclast differentiation was induced from bone marrow of wild-type, Rac1 null, and Rac2 null mice.
- Osteoclasts were cultured on dentin slices for resorption assays and on glass coverslips for cellular analysis.
- Picro Sirius Red staining and laser-scanning confocal microscopy were employed for volumetric imaging of resorption lacunae.
Main Results:
- Rac1- and Rac2-deficient osteoclasts exhibited fewer nuclei compared to wild-type osteoclasts.
- Rac1 deficiency led to reduced resorption pit volume and surface area.
- Rac2 deficiency resulted in reduced resorption pit volume, while surface area remained unaffected, highlighting the limitations of 2D measurements.
Conclusions:
- Rac1 and Rac2 GTPases are essential for osteoclast-mediated bone resorption, with distinct roles in regulating resorption volume and surface area.
- The developed confocal imaging technique provides a more accurate assessment of bone resorption compared to traditional methods.
- This study offers insights into potential therapeutic strategies targeting Rho GTPase pathways for treating bone lytic disorders.
