Denosumab is a monoclonal anti-RANKL antibody that inhibits bone resorption, increases bone mass, and reduces fracture risk. Denosumab discontinuation causes an extensive wave of rebound resorption, but the cellular mechanisms remain poorly characterized. We utilized in situ hybridization (ISH) as a direct approach to identify the cells that activate osteoclastogenesis through the RANKL/OPG pathway. ISH was performed across species, skeletal sites, and following recombinant OPG (OPG:Fc) and parathyroid hormone 1–34 (PTH) treatment of mice. OPG:Fc treatment in mice induced an increased expression of RANKL mRNA mainly in trabecular, but not endocortical bone surface cells. Additionally, a decreased expression of OPG mRNA was detected in bone surface cells and osteocytes of both compartments. A similar but more pronounced effect on RANKL and OPG expression was seen one hour after PTH treatment. These findings suggest that bone surface cells and osteocytes conjointly regulate the activation of osteoclastogenesis, and that OPG:Fc treatment induces a local accumulation of osteoclastogenic activation sites, ready to recruit and activate osteoclasts upon treatment discontinuation. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data from murine bone marrow stromal cells revealed that Tnfsf11⁺ cells expressed high levels of Mmp13, Limch1, and Wif1, confirming their osteoprogenitor status. ISH confirmed co-expression of Mmp13 and Tnfsf11 in bone surface cells of both vehicle- and OPG:Fc-treated mice. Under physiological conditions of human/mouse bone, RANKL is expressed mainly by osteoprogenitors proximate to the osteoclasts, while OPG is expressed mainly by osteocytes and bone-forming osteoblasts.

Until recently, it was well-accepted that osteoclasts resorb bone according to the resorption cycle model. This model is based on the assumption that osteoclasts are immobile during bone erosion, allowing the actin ring to be firmly attached and thereby provide an effective seal encircling the resorptive compartment. However, through time-lapse, it was recently documented that osteoclasts making elongated resorption cavities and trenches move across the bone surface while efficiently resorbing bone. However, it was also shown that osteoclasts making rounded cavities and pits indeed resorb bone while they are immobile. Only little is known about what distinguishes these two different resorption modes. This is of both basic and clinical interest because these resorption modes are differently sensitive to drugs and are affected by the gender as well as age of the donor. In the present manuscript we show that: 1. levels of active cathepsin K determine the switch from pit to trench mode; 2. pit and trench mode depend on clathrin-mediated endocytosis; and 3. a mechanism integrating release of resorption products and membrane/integrin recycling is required for prolongation of trench mode. Our study therefore contributes to an improved understanding of the molecular and cellular determinants for the two osteoclastic bone resorption modes.

The strictly regulated bone remodeling process ensures that osteoblastic bone formation is coupled to osteoclastic bone resorption. This coupling is regulated by a panel of coupling factors, including clastokines promoting the recruitment, expansion, and differentiation of osteoprogenitor cells within the eroded cavity. The osteoprogenitor cells on eroded surfaces are called reversal cells. They are intermixed with osteoclasts and become bone-forming osteoblast when reaching a critical density and maturity. Several coupling factors have been proposed in the literature, but their effects and expression pattern vary between studies depending on species and experimental setup. In this study, we investigated the mRNA levels of proposed secreted and membrane-bound coupling factors and their receptors in cortical bone remodeling events within the femur of healthy adolescent human controls using high-sensitivity RNA in situ hybridization. Of the proposed coupling factors, human osteoclasts showed mRNA-presence of LIF, PDGFB, SEMA4D, but no presence of EFNB2, and OSM. On the other hand, the osteoblastic reversal cells proximate to osteoclasts presented with LIFR, PDGFRA and PLXNB1, but not PDGFRB, which are all known receptors of the proposed coupling factors. Although EFNB2 was not present in mature osteoclasts, the mRNA of the ligand-receptor pair EFNB2:EPHB4 were abundant near the central blood vessels within intracortical pores with active remodeling. EPHB4 and SEMA4D were also abundant in mature bone-forming osteoblasts. This study highlights that especially LIF:LIFR, PDGFB:PDGFRA, SEMA4D:PLXNB1 may play a critical role in the osteoclast-osteoblast coupling in human remodeling events, as they are expressed within the critical cells.

Septins are known to play key roles in supporting cytoskeletal stability, vesicular transport, endo-/exocytosis, stabilizing cellular membranes and forming diffusion barriers. Their function in mammalian cells is poorly investigated. The osteoclast offers an interesting tool to investigate septins because all cellular activities septins were reported to be involved in are critical for osteoclasts. However, the existence of septins in osteoclasts has not even been reported. Here we show that theSEPT9gene and Septin 9 (SEPT9) protein are expressed and synthesized during differentiation of human osteoclasts. Pharmacological stabilization of septin filaments dose dependently inhibits bone resorption of human osteoclastsin vitrosuggesting a role for septins in bone resorption. Attesting to this, conditional deletion ofSept9in mice leads to elevated levels of trabecular bone and diminished femoral growthin vivo. Finally, systematic interrogation of the spatial organization of SEPT9 by confocal microscopy reveals that SEPT9 is closely associated to the structures known to be critical for osteoclast activity. We propose that septins in general and SEPT9 in particular play a previously unappreciated role in osteoclastic bone resorption.

Legalized use of cannabis for medical or recreational use is becoming more and more common. With respect to potential side-effects on bone health only few clinical trials have been conducted - and with opposing results. Therefore, it seems that there is a need for more knowledge on the potential effects of cannabinoids on human bone cells. We studied the effect of cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) (dose range from 0.3 to 30 μM) on human osteoclasts in mono- as well as in co-cultures with human osteoblast lineage cells. We have used CD14⁺ monocytes from anonymous blood donors to differentiate into osteoclasts, and human osteoblast lineage cells from outgrowths of human trabecular bone. Our results show that THC and CBD have dose-dependent effects on both human osteoclast fusion and bone resorption. In the lower dose ranges of THC and CBD, osteoclast fusion was unaffected while bone resorption was increased. At higher doses, both osteoclast fusion and bone resorption were inhibited. In co-cultures, both osteoclastic bone resorption and alkaline phosphatase activity of the osteoblast lineage cells were inhibited. Finally, we observed that the cannabinoid receptor CNR2 is more highly expressed than CNR1 in CD14⁺ monocytes and pre-osteoclasts, but also that differentiation to osteoclasts was coupled to a reduced expression of CNR2, in particular. Interestingly, under co-culture conditions, we only detected the expression of CNR2 but not CNR1 for both osteoclast as well as osteoblast lineage nuclei. In line with the existing literature on the effect of cannabinoids on bone cells, our current study shows both stimulatory and inhibitory effects. This highlights that potential unfavorable effects of cannabinoids on bone cells and bone health is a complex matter. The contradictory and lacking documentation for such potential unfavorable effects on bone health as well as other potential effects, should be taken into consideration when considering the use of cannabinoids for both medical and recreational use.