Women gradually lose bone from the age of ~35 years, but around menopause, the rate of bone loss escalates due to increasing bone resorption and decreasing bone formation levels, rendering these individuals more prone to developing osteoporosis. The increased osteoclast activity has been linked to a reduced estrogen level and other hormonal changes. However, it is unclear whether intrinsic changes in osteoclast precursors around menopause can also explain the increased osteoclast activity. Therefore, we set up a protocol in which CD14⁺blood monocytes were isolated from 49 female donors (40–66 years old). Cells were differentiated into osteoclasts, and data on differentiation and resorption activity were collected. Using multiple linear regression analyses combining in vitro and in vivo data, we found the following: (1) age and menopausal status correlate with aggressive osteoclastic bone resorption in vitro; (2) the type I procollagen N-terminal propeptide level in vivo inversely correlates with osteoclast resorption activity in vitro; (3) the protein level of mature cathepsin K in osteoclasts in vitro increases with age and menopause; and (4) the promoter of the gene encoding the dendritic cell-specific transmembrane protein is less methylated with age. We conclude that monocytes are “reprogrammed” in vivo, allowing them to “remember” age, the menopausal status, and the bone formation status in vitro, resulting in more aggressive osteoclasts. Our discovery suggests that this may be mediated through DNA methylation. We suggest that this may have clinical implications and could contribute to understanding individual differences in age- and menopause-induced bone loss.

Osteoclasts (OCs) seeded on bone slices either drill round pits or dig long trenches. Whereas pits correspond to intermittent resorption, trenches correspond to continuous and faster resorption and require a distinct assembly of the resorption apparatus. It is unknown whether the distinction between pits and trenches has any biological relevance. Using OCs prepared from different blood donors, we found that female OCs achieved increased resorption mainly through pit formation, whereas male OCs did so through trench formation. Trench formation went along with high collagenolytic activity and high cathepsin K (CatK) expression, thereby allowing deeper demineralization. A specific CatK inhibitor abrogated the generation of trenches, while still allowing the generation of pits. OCs obtained from bone marrow were more prone to generate trenches than those obtained from blood. Scanning electron microscopy of bone surfaces eroded in vivo showed trenches and pits of similar size as those made by OCs in culture. We conclude that the distinction between trench- and pit-forming OCs is relevant to the differences among OCs from different skeletal sites, different individuals, including gender, and results from differences in collagenolytic power. This indicates a biological relevance and highlights the importance of discriminating between pits and trenches when assessing resorption.

Investigations addressing the molecular keys of osteoclast fusion are primarily based on end‐point analyses. No matter if investigations are performed in vivo or in vitro the impact of a given factor is predominantly analyzed by counting the number of multi‐nucleated cells, the number of nuclei per multinucleated cell or TRAcP activity. But end‐point analyses do not show how the fusion came about. This would not be a problem if fusion of osteoclasts was a random process and occurred by the same molecular mechanism from beginning to end. However, we and others have in the recent period published data suggesting that fusion partners may specifically select each other and that heterogeneity between the partners seems to play a role. Therefore, we set out to directly test the hypothesis that fusion factors have a heterogenic involvement at different stages of nuclearity. Therefore, we have analyzed individual fusion events using time‐lapse and antagonists of CD47 and syncytin‐1. All time‐lapse recordings have been studied by two independent observers. A total of 1808 fusion events were analyzed. The present study shows that CD47 and syncytin‐1 have different roles in osteoclast fusion depending on the nuclearity of fusion partners. While CD47 promotes cell fusions involving mono‐nucleated pre‐osteoclasts, syncytin‐1 promotes fusion of two multi‐nucleated osteoclasts, but also reduces the number of fusions between mono‐nucleated pre‐osteoclasts. Furthermore, CD47 seems to mediate fusion mostly through broad contact surfaces between the partners’ cell membrane while syncytin‐1 mediate fusion through phagocytic‐cup like structure. J. Cell. Physiol. 232: 1396–1403, 2017.

It is well established that multinucleation is central for osteoclastic bone resorption. However, our knowledge on the mechanisms regulating how many nuclei an osteoclast will have is limited. The objective of this study was to investigate donor-related variations in the fusion potential of in vitro-generated osteoclasts. Therefore, CD14⁺ monocytes were isolated from 49 healthy female donors. Donor demographics were compared to the in vivo bone biomarker levels and their monocytes’ ability to differentiate into osteoclasts, showing that: (1) C-terminal telopeptide of type I collagen (CTX) and procollagen type I N-terminal propeptide (PINP) levels increase with age, (2) the number of nuclei per osteoclast in vitro increases with age, and (3) there is a positive correlation between the number of nuclei per osteoclast in vitro and CTX levels in vivo. Furthermore, the expression levels of the gene encoding dendritic cell-specific transmembrane protein (DCSTAMP) of osteoclasts in vitro correlated positively with the number of nuclei per osteoclast, CTX levels in vivo, and donor age. Our results furthermore suggest that these changes in gene expression may be mediated through age-related changes in DNA methylation levels. We conclude that both intrinsic factors and age-induced increase in fusion potential of osteoclasts could be contributing factors for the enhanced bone resorption in vivo, possibly caused by increased expression levels of DCSTAMP.

Zoledronic acid is a bisphosphonate commonly used to treat bone diseases such as osteoporosis and cancer‐induced bone disease. Patients exhibit a variable sensitivity to zoledronic acid; the underlying explanation for this remains unclear. The objective of this study was to obtain more knowledge in this regard. We hypothesized that osteoclasts generated from different individuals would show a variable sensitivity to zoledronic acid in vitro. Osteoclasts were generated using monocytes from 46 healthy female blood donors (40 to 66 years). Matured osteoclasts were reseeded onto bone slices precoated with different concentrations of zoledronic acid. IC50 values were determined based on total eroded bone surface after 3 days of resorption. The IC50 for inhibition of osteoclastic bone resorption varied from 0.06 to 12.57μM zoledronic acid; thus, a more than 200‐fold difference in sensitivity to zoledronic acid among osteoclasts from different individuals was observed. Multiple linear regression analyses showed that the determined IC50 correlated with smoking status, and the average number of nuclei per osteoclast in vitro. Further analyses showed that: (i) increasing protein levels of mature cathepsin K in osteoclast cultures rendered the osteoclasts less sensitive to zoledronic acid; (ii) surprisingly, neither the gene nor the protein expression of farnesyl diphosphate synthase was found to correlate with the IC50; and (iii) trench‐forming osteoclasts were found to be more sensitive to zoledronic acid than pit‐forming osteoclasts within the same cell culture. Thus, we conclude that there indeed is a high degree of variation in the potency of zoledronic acid on osteoclasts when generated from different individuals. We propose that our findings can explain some of the varying clinical efficacy of zoledronic acid therapy observed in patients, and may therefore be of clinical importance, which should be investigated in a clinical trial combining in vitro and in vivo investigations. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

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.