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. 2012 Jun;90(6):515-22.
doi: 10.1007/s00223-012-9600-y. Epub 2012 Apr 25.

Osteoclast fusion and fission

Ineke D C Jansen  1 , Jenny A F Vermeer, Veerle Bloemen, Jan Stap, Vincent Everts
Affiliations

Osteoclast fusion and fission

Ineke D C Jansen et al. Calcif Tissue Int. 2012 Jun.

Abstract

Osteoclasts are specialized multinucleated cells with the unique capacity to resorb bone. Despite insight into the various steps of the interaction of osteoclast precursors leading to osteoclast formation, surprisingly little is known about what happens with the multinucleated cell itself after it has been formed. Is fusion limited to the short period of its formation, or do osteoclasts have the capacity to change their size and number of nuclei at a later stage? To visualize these processes we analyzed osteoclasts generated in vitro with M-CSF and RANKL from mouse bone marrow and native osteoclasts isolated from rabbit bones by live cell microscopy. We show that osteoclasts fuse not only with mononuclear cells but also with other multinucleated cells. The most intriguing finding was fission of the osteoclasts. Osteoclasts were shown to have the capacity to generate functional multinucleated compartments as well as compartments that contained apoptotic nuclei. These compartments were separated from each other, each giving rise to a novel functional osteoclast or to a compartment that contained apoptotic nuclei. Our findings suggest that osteoclasts have the capacity to regulate their own population in number and function, probably to adapt quickly to changing situations.

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Figures

Fig. 1
Fig. 1
Mouse bone marrow cells were precultured for 3 days in the presence of M-CSF and RANKL. Culture media were refreshed on day 3, and cells were cultured for another 68 h and simultaneously followed by live cell imaging. Fusion is seen of a multinucleated cell with another multinucleated osteoclast (OC). Before fusion the cells make contact with each other (arrow in a and b) as if to find the appropriate site to fuse. Cells are in close contact with each other (b). c Fusion has occurred
Fig. 2
Fig. 2
Mouse bone marrow cells (cultured in α-MEM with M-CSF and RANKL) were followed by live cell imaging for 68 h after a preculture period for 3 days. In the micrograph fusion (arrow) is shown of a mononuclear cell (mnc) with a multinucleated osteoclast (OC)
Fig. 3
Fig. 3
Mouse bone marrow cells cultured for 6 days with M-CSF and RANKL. After refreshment of the media at day 3, cells were followed by time lapse imaging. Fusion is shown of a large osteoclast (OC) with a smaller one. Note the two small mononuclear cells (smc) that are present in the direct vicinity of the site where fusion occurs
Fig. 4
Fig. 4
In vitro generated osteoclast from mouse bone marrow. a The osteoclast (OC) forms different compartments (C1, C2, C3; shown in b–e) that are connected to each other by thin, tubular structures (closed arrow in b, d, and e). Each compartment contains a number of nuclei. These tubular structures were not firmly attached to the bottom of the culture well because osteoblasts were able to move underneath (asterisks in c and d). Following elongation, the connections became very thin and often broke, resulting in the generation of two separate multinucleated osteoclasts (OC1, OC2) (e). Time scale of the micrographs: a was made after 13 h of culturing, 11 h later b was taken, and c–e were taken every 3 h thereafter
Fig. 5
Fig. 5
Mouse bone marrow cells precultured for 3 days in the presence of M-CSF and RANKL. Culture media were refreshed on day 3, and cells were cultured for another 68 h and simultaneously followed by live cell imaging. Tubular cytoplasmic structures (arrow) were formed between multinucleated compartments (C1, C2). Just prior to the breaking up of the connection between compartments small mononuclear cells (smc) moved across the bridging extensions, and at the site where these cells made contact the extension was broken. Two osteoclasts (OC1, OC2) were formed
Fig. 6
Fig. 6
Green fluorescent staining (Alexa-488) of the small mononuclear cell that could be involved in the separation of the osteoclast (OC) compartments. Cells were labeled with anti-ERMP20 (a), anti-MMP9 (b), and anti-ICAM1 (c). Nuclei stained with DAPI show up in blue. Arrow indicates the labeled mononuclear cell. Asterisk marks the site where the labeled cell is in close contact with the cytoplasmic extension that connects different osteoclast parts (Color figure online)
Fig. 7
Fig. 7
Fission of an osteoclast following the formation of two compartments (C1, C2) results in the formation of two “new” osteoclasts (OC1 and OC2, shown in a and b). Subsequently, OC2 fuses with another multinucleated cell (OC3). Time span between micrographs a and c is 3 h. The separation of the osteoclast starts 20 h after the start of visualization. Note the small mononuclear cells (smc) close to the thin, tubular structure in micrograph (a)
Fig. 8
Fig. 8
The formation of compartments (C1, C2, C3) connected by thin, tubular structures (thick arrows) was also noted with osteoclasts seeded on cortical bone slices. Actin rings (green, thin arrows) were present in these different osteoclast compartments, indicating bone resorption activity. The osteoclast membrane is stained for CD44 (blue). Nuclei are red. In osteoclast compartment 2 (C2) nuclei are reduced in size and appear apoptotic (arrowheads) (Color figure online)
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