Differentiation of Cells Isolated from Human Femoral Heads into Functional Osteoclasts

Halloran, Daniel; Heubel, Brian; MacMurray, Connor; Root, Denise; Eskander, Mark S.; McTague, Sean; Pelkey, Heather; Nohe, Anja

doi:10.3390/jdb10010006

Cited by 7 publications

(6 citation statements)

References 65 publications

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“…Another critical factor is the concentration of the two cytokines that reprogram monocytes towards osteoclasts, M-CSF and RANKL. Confirming previously published data, our results demonstrate that high concentrations of these two factors are clearly superior in inducing efficient osteoclast differentiation [ 26 , 28 – 30 ]. Some studies have applied an even higher concentration of RANKL (100 ng/ml) and observed a further increase in the number of TRAP-positive cells.…”

Section: Discussionsupporting

confidence: 91%

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: Discussionsupporting

confidence: 91%

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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“…The activation of osteogenic signaling pathways in osteoblasts is mediated by BMP-2, which was approved by the FDA in 2002 to be used as a therapeutic [84]. However, several side-effects were reported after usage of BMP-2, such as hematoma formation, radiculitis, and increased osteolysis, as BMP-2 also enhances osteoclastogenesis [67,68,85,86]. Further, it was demonstrated that primary osteoblasts isolated from OP patients and BMSCs obtained from aged (15-and 20-month) B6 mice are unresponsive to BMP-2 stimulation [58,67].…”

Section: Discussionmentioning

confidence: 99%

Methyl-Beta-Cyclodextrin Restores Aberrant Bone Morphogenetic Protein 2-Signaling in Bone Marrow Stromal Cells Obtained from Aged C57BL/6 Mice

Halloran,

Pandit,

Chukwuocha

et al. 2023

Preprint

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As humans age, aberrancies in several signaling pathways occur. In the aging population, some patients display abnormal bone morphogenetic protein (BMP) signaling. This can lead to osteoporosis (OP), a debilitating bone disorder caused by an imbalance between osteoblasts and osteoclasts. In 2002, the Food and Drug Administration (FDA) approved usage of recombinant human BMP-2 (rhBMP-2) during spinal fusion surgery as it is crucial for bone formation. However, complications have been reported with rhBMP-2 and primary osteoblasts isolated from OP patients are unresponsive to BMP-2. While patient samples are available, previous medical history may alter results. Therefore, C57BL/6 mice, an aging model that displays aberrant BMP-signaling, can be utilized to investigate OP and aging. We show that BMP receptor type Ia (BMPRIa) is upregulated in the bone marrow stromal cells (BMSCs) of 15-month mice similar to previous data. We show that a conjugation between BMP-2 and Quantum Dots (QDot®s) effectively binds to BMPRIa and can investigate BMP-2 activity. After incubating BMSCs with methyl-β-cyclodextrin (MβCD), BMP-signaling is restored in 15-month mice as shown by western blotting and the von Kossa assay. MβCD may be used to rescue BMPRIa and the BMP-signaling pathway can be utilized by future therapeutics to treat OP.

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“…As the bone tissue is obtained from donors aged above 60y, the bone is likely to contain yellow marrow, rich in adipocytes and with reduced hematopoietic activity, but the marrow content was not specifically characterized in the explants [55]- [57]. On the other hand, previous research demonstrated that bone marrow cells isolated from human femoral heads of osteoarthritic patients were able to differentiate into viable and functional osteoclasts in vitro [58]. It was found that only optimal culture conditions, 25 ng/ml M-CSF for 3 days followed by 50 ng/ml RANKL and 25 ng/ml M-CSF for 11 days, generated large amounts of osteoclasts [58].…”

Section: Discussionmentioning

confidence: 99%

Integration of Osteoclastogenesis through addition of PBMCs in Human Osteochondral Explants culturedEx vivo

Cramer,

de Wildt,

Hendriks

et al. 2023

Preprint

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The preservation of tissue specific cells in their native 3D extracellular matrix in bone explants provides a unique platform to study remodeling. Thus far, studies involving bone explant cultures showed a clear focus on achieving bone formation and neglected osteoclast activity and resorption. To simulate the homeostatic bone environment ex vivo, both key elements of bone remodeling need to be represented. This study aimed to assess and include osteoclastogenesis in human osteochondral explants through medium supplementation with RANKL and M-CSF and addition of peripheral blood mononuclear cells (PBMCs), providing osteoclast precursors. Osteochondral explants were freshly harvested from human femoral heads obtained from hip surgeries and cultured for 20 days in a two-compartment culture system. Osteochondral explants preserved viability and cellular abundance over the culture period, but histology demonstrated that resident osteoclasts were no longer present after 4 days of culture. Quantitative extracellular tartrate resistant acid phosphatase (TRAP) analysis confirmed depletion of osteoclast activity on day 4 even when stimulated with RANKL and M-CSF. Upon addition of PBMCs, a significant upregulation of TRAP activity was measured from day 10 onwards. Evaluation of bone loss trough µCT registration and measurement of extracellular cathepsin K activity revealed indications of enhanced resorption upon addition of PBMCs. Based on the results we suggest that an external source of osteoclast precursors, such as PBMCs, needs to be added in long-term bone explant cultures to maintain osteoclastic activity, and bone remodeling.

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Differentiation of Cells Isolated from Human Femoral Heads into Functional Osteoclasts

Cited by 7 publications

References 65 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

Methyl-Beta-Cyclodextrin Restores Aberrant Bone Morphogenetic Protein 2-Signaling in Bone Marrow Stromal Cells Obtained from Aged C57BL/6 Mice

Integration of Osteoclastogenesis through addition of PBMCs in Human Osteochondral Explants culturedEx vivo

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