Osteoclasts and their roles

Osteoclast (OC) is the only multi-nucleated cell with bone resorption activity in vivo, with 2-50 nuclei and a diameter of about 100μm. Osteoclasts are differentiated from mononuclear/macrophage cell lines in blood and bone marrow, and the differentiation process goes through osteoclast precursor cells, OPCs, Also known as preosteoclasts, fused polykaryons (non-functional polykaryons), mature osteoclasts (also known as polarized multinuclear osteoclasts) and other stages. Bone is a dynamic tissue that is constantly broken down and reorganized under the influence of structural stresses and the body's need for calcium. Osteoclasts are the mediators of continuous bone destruction and play an important role in bone development, growth, repair and reconstruction. Abnormal activation of osteoclasts leads to bone loss and is commonly seen in autoimmune diseases such as postmenopausal osteoporosis or rheumatoid arthritis.

Osteoclast markers

Osteoclasts occupy small depressions on the surface of bone called Howship lacunae; Such cavities are thought to be caused by the erosion of bone by enzymes of osteoclasts. Osteoclasts produce many enzymes, most of which are tartrate-resistant acidic phosphatase (Trap). Trap is specifically distributed in osteoclasts and is unique to osteoclasts, and is usually used as an important marker to identify osteoclasts。

The acquisition of osteoclasts

In the past, osteoclasts were usually isolated from bone fragments of newborn animals or were obtained by co-culturing osteoclast precursors with osteoblasts in an osteoclast environment. In the last decade or so, a major breakthrough in bone biology has been the identification of macrophage colony-stimulating factor (M-CSF) and nuclear factor κB ligand receptor activator (RANKL) as essential cytokines for osteoclast differentiation and activation. Therefore, experimental protocols have been developed to induce the production of mature and functional osteoclasts from bone marrow monocyte/macrophage lineage cells using recombinant M-CSF and RANKL. (The following scheme is for reference)

Formation of bone marrow osteoclasts in mice

1. Mice aged 6-17 weeks were killed by CO2 inhalation or cervical dislocation. Mouse fur was disinfected with 70% alcohol.

2. Carefully remove the femur and tibia and place them in a 10cm petri dish.

3. Prepare femur and tibia under sterile conditions:
a. Use tweezers and scissors to remove all skin, tendons and muscles whenever possible.
b. Use a scalpel or scissors to cut off both ends of all bones.
c. Use a 5ml syringe and a 23-G needle to flush marrow from all bones with 10ml osteoclast medium in a fresh dish. Discard washed bones.
d. Using a 1ml syringe and a 27-G needle, isolate cell aggregates as far as possible by inhaling cell suspensions into and out of the syringe. The cell suspension was then flushed through a 70µm cell filter and placed into a 50 ml conical centrifuge tube.
e. Rinse the nylon mesh of the cell filter with 2ml medium

4. The obtained cell suspension was 300×g, centrifuged at 4°C for 7min, and the supernatant was discarded.

5. The cells were suspended with 5ml of red cell lysate. Ice cracking 10min.

6. Centrifuge and discard supernatant. Then 5ml α-MEM medium containing 10%FBS was re-suspended, centrifuged, and the supernatant was discarded.

7. The cells were re-suspended with 5ml α-MEM medium containing M-CSF (25 ng/ml) and 10%FBS. They were inoculated in a 6-well cell culture plate (one well per mouse) and placed overnight (14 to 18 hours) in a 5%CO2 cell incubator at 37°C.

8. On the second day, absorb the medium and collect the unadhered cells, taking care not to scratch the bottom of the plate. Transfer medium into 50ml conical centrifuge tube. 300×g, 4℃, centrifuge for 7min, discard supernatant.

9. The cells were re-suspended with 5ml α-MEM medium containing 10%FBS, and the live cells were counted using a cell counting device.

10. The cells were inoculated on the culture plate at a density of 1×106cells/ml, and M-CSF (50ng/ml) and RANKL (25-100 ng/ml) were added.

11. Replace the medium after 3 days. Polykaryotic mature osteoclasts are generally observed around the fifth day.

12. TRAP staining was used to identify osteoclasts.

Attention:

a. The concentrations of RANKL and M-CSF used may vary depending on mice and laboratory conditions.

b. Observe the cells at least once a day from day 4 after the addition of RANKL and M-CSF, as mouse osteoclasts are very unstable after full differentiation. At the beginning of osteoclast formation, the cells are spindle shaped, and after maturity, the cells become large, multinucleated, and round. In contrast to human osteoclasts, mouse osteoclasts die within 24 hours of maturity.

Osteoclasts were induced by macrophage cell line RAW264.7

1. RAW264.7 cells were prepared into cell suspension using α-MEM medium containing 10%FBS and inoculated into 24-well plates at a density of 3×10^4 cells/ well.

2. 12h after cell inoculation, RANKL (20-100ng/ml) was added.

3. Replace the medium every 3 days and add RANKL (20-100ng/ml) at the same time.

4. More obvious multinucleated osteoclasts will begin to appear after 4 days of differentiation and culture, and gradually become abundant from day 5 to day 6.

5. TRAP staining was performed to identify osteoclast formation.

Attention:

a. RAW264.7 cells could proliferate and retain their potency to differentiate into osteoclasts in RPMI-1640 media with 10% FBS, while it will be cultured in α-MEM with 10% FBS for the osteoclast differentiation assay.

b. RAW 264.7 cells express both M-CSF and its receptor c-fms.Therefore, the addition of RANKL alone is sufficient to induce osteoclast differentiation, and no treatment with M-CSF is necessary.

Related Cytokines

References

1. Marino S, Logan JG, Mellis D, Capulli M. Generation and culture of osteoclasts.Bonekey Rep. 2014 Sep 10;3:570.

2. Arnett TR, Dempster DW. A comparative study of disaggregated chick and rat osteoclasts in vitro: effects of calcitonin and prostaglandins.Endocrinology. 1987 Feb;120(2):602-8.

3. Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin osteoclastogenesisinhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3597-602.

4. Takahashi N, Udagawa N, Suda T. A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun. 1999 Mar 24;256(3):449-55.

5. Jingxuan Yang, Xiaohong Bi, and Min Li. Osteoclast Differentiation Assay. Methods Mol Biol. 2019;1882:143-148.

6. Iris Boraschi-Diaz , Svetlana V Komarova. The protocol for the isolation and cryopreservation of osteoclast precursors from mouse bone marrow and spleen. Cytotechnology. 2016 Jan;68(1):105-114.

7. Ulrike Steffen, Fabian T Andes and Georg Schett.Generation and Analysis of Human and Murine Osteoclasts. Curr Protoc Immunol. 2019 Jun;125(1):e74.