The effects of seeding density and osteoclastic supplement concentration on osteoclastic differentiation and resorption

Remmers, Stefan; Heijden, Freek C. van der; Ito, Keita; Hofmann, Sandra

doi:10.1016/j.bonr.2022.101651

Cited by 2 publications

(5 citation statements)

References 56 publications

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“…In line with recent results from Remmers et al . [ 13 ], we observed that the cell density has a major effect on the efficiency of osteoclastogenesis and measured the most efficient differentiation at an intermediate cell number. The fact that seeding density significantly impacts osteoclast differentiation has not only been observed for osteoclasts of human origin, but also for differentiation protocols based on murine/rat origin [ 26 , 27 ], thus indicating that cell density is a critical parameter in determining efficient osteoclastogenesis across various culture models.…”

Section: Discussionmentioning

confidence: 99%

“…One possibility is to use the human monocytic cell line THP-1 for osteoclastogenesis [ 12 ]. However, these cells were isolated from a patient with acute leukemia, and as such, the monocytes have undergone malignant transformation and hence might not reflect the physiological behavior of healthy osteoclasts [ 13 ]. A more promising alternative is to isolate human peripheral blood mononuclear cells (PBMCs) as the basis for osteoclast differentiation and cultivation.…”

Section: Introductionmentioning

confidence: 99%

“…A more promising alternative is to isolate human peripheral blood mononuclear cells (PBMCs) as the basis for osteoclast differentiation and cultivation. Only few studies have used human PBMCs as osteoclast precursors, although they harbor the advantage of easy harvest from blood and enable a syngeneic in vitro culture model for studies in the human setting [ 13 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

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A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes

Chandrabalan,

Dang,

Hansen

et al. 2024

Biol Proced Online

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Background Osteoclasts are the tissue-specific macrophage population of the bone and unique in their bone-resorbing activity. Hence, they are fundamental for bone physiology in health and disease. However, efficient protocols for the isolation and study of primary human osteoclasts are scarce. In this study, we aimed to establish a protocol, which enables the efficient differentiation of functional human osteoclasts from monocytes. Results Human monocytes were isolated through a double-density gradient from donor blood. Compared to standard differentiation schemes in polystyrene cell culture dishes, the yield of multinuclear osteoclasts was significantly increased upon initial differentiation of monocytes to macrophages in fluorinated ethylene propylene (FEP) Teflon bags. This initial differentiation phase was then followed by the development of terminal osteoclasts by addition of Receptor Activator of NF-κB Ligand (RANKL). High concentrations of RANKL and Macrophage colony-stimulating factor (M-CSF) as well as an intermediate cell density further supported efficient cell differentiation. The generated cells were highly positive for CD45, CD14 as well as the osteoclast markers CD51/ITGAV and Cathepsin K/CTSK, thus identifying them as osteoclasts. The bone resorption of the osteoclasts was significantly increased when the cells were differentiated from macrophages derived from Teflon bags compared to macrophages derived from conventional cell culture plates. Conclusion Our study has established a novel protocol for the isolation of primary human osteoclasts that improves osteoclastogenesis in comparison to the conventionally used cultivation approach.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes

Chandrabalan,

Dang,

Hansen

et al. 2024

Biol Proced Online

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“…Moreover, we identified b3-integrin as an upstream regulator of this pathway. This marker of mature osteoclasts 85,86 mediates their ability to polarize, spread and degrade bone 9,[87][88][89] . We propose that activation of b3-integrin limits the phosphorylation of moesin through the inhibition of the RhoA/SLK axis and in this way controls the number/architecture of the sealing zones.…”

Section: Discussionmentioning

confidence: 99%

“…Finally, to determine the signal that could trigger RhoA/SLK-dependent ERM regulation, we tested the importance of αvβ3 integrin. Indeed, this marker of mature osteoclasts 85,86 mediates their ability to polarize, spread and degrade bone 9,[87][88][89] . We showed that b3-integrin depletion (Figure 5C) affects the formation of the sealing zones confirming previous observations 9 , with a decrease in their width (Figure 5E-F).…”

Section: Moesin Depletion Boosts Bone Degradation and The Formation O...mentioning

confidence: 99%

Moesin activation controls bone resorption and tunneling nanotube-dependent osteoclast fusion

Dufrancais,

Verdys,

Métais

et al. 2024

Preprint

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Osteoclasts are multinucleated cells unique in their ability to resorb bone. Osteoclastogenesis involves several steps of actin-driven rearrangements that participate not only in the cell-cell fusion process, but also in the formation of the sealing zone, the adhesive structure determining the resorption area. Despite the importance of these actin cytoskeleton-based processes, their precise mechanisms of regulation are still poorly characterized. Here, we found that moesin, a member of the Ezrin/Radixin/Moesin (ERM) protein family, is activated during osteoclast maturation and plays an instrumental role for both osteoclast fusion and function. In mouse and human osteoclast precursors, moesin is negatively regulated to potentiate their ability to fuse and degrade bone. Accordingly, we demonstrated that moesin depletion decreases membrane-to-cortex attachment and enhances formation of tunneling nanotubes (TNTs), F-actin-containing intercellular bridges that we revealed to trigger osteoclast fusion. In addition, via Beta3-integrin/RhoA/SLK pathway and independently of its role in fusion, moesin regulates the number and organization of sealing zones in mature osteoclast, and thus participates in the control of bone resorption. Supporting these findings, we found that moesin-deficient mice are osteopenic with a reduced density of trabecular bones and increased osteoclast abundance and activity. These findings provide a better understanding of the regulation of osteoclast biology, and open new opportunities to specifically target osteoclast activity in bone disease therapy.

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The effects of seeding density and osteoclastic supplement concentration on osteoclastic differentiation and resorption

Cited by 2 publications

References 56 publications

A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes

A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes

Moesin activation controls bone resorption and tunneling nanotube-dependent osteoclast fusion

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