Thomas Levin Andersen

A Physical Mechanism for Coupling Bone Resorption and Formation in Adult Human Bone

During skeletal remodeling, pre-osteoclasts and pre-osteoblasts are targeted to critical sites of the bone to resorb and reconstruct bone matrix, respectively. Coordination of site-specific recruitment of these two cell types is a prerequisite to maintain the specific architecture of each bone within strict limits throughout adult life. Here, we determined that the bone marrow microanatomy adjacent to remodeling areas is a central player in this process. By using histomorphometry and multiple immunostainings, we demonstrated in biopsies exhibiting coupled bone resorption and formation that osteoclasts and osteoblasts on the bone surface were always covered by a canopy of flat cells expressing osteoblast markers. In contrast, in biopsies in which this canopy was disrupted, bone formation was deficient. Three-dimensional visualizations revealed that this canopy covered the entire remodeling site and was associated with capillaries, thereby forming a previously unrecognized microanatomical entity. Furthermore, pre-osteoclasts were positioned along these capillaries. These findings led to a model that implicates vasculature in the site-specific recruitment of osteoclasts and osteoblasts and embraces the current knowledge on the molecular mechanism of bone remodeling.

Ovariectomized rats as a model of postmenopausal osteoarthritis: validation and application.

We aimed to assess the effect of ovariectomy on cartilage turnover and degradation, to evaluate whether ovariectomized (OVX) rats could form an experimental model of postmenopausal osteoarthritis. The effect of ovariectomy on cartilage was studied using two cohorts of female Sprague–Dawley rats, aged 5 and 7 months. In a third cohort, the effect of exogenous estrogen and a selective estrogen receptor modulator was analyzed. Knee joints were assessed by histological analysis of the articular cartilage after 9 weeks. Cartilage turnover was measured in urine by an immunoassay specific for collagen type II degradation products (CTX-II), and bone resorption was quantified in serum using an assay for bone collagen type I fragments (CTX-I). Surface erosion in the cartilage of the knee was more severe in OVX rats than in sham-operated animals, particularly in the 7-month-old cohort (P = 0.008). Ovariectomy also significant increased CTX-I and CTX-II. Both the absolute levels of CTX-II and the relative changes from baseline seen at week 4 correlated strongly with the severity of cartilage surface erosion at termination (r = 0.74, P < 0.01). Both estrogen and the selective estrogen receptor modulator inhibited the ovariectomy-induced acceleration of cartilage and bone turnover and significantly suppressed cartilage degradation and erosion seen in vehicle-treated OVX rats. The study indicates that estrogen deficiency accelerates cartilage turnover and increases cartilage surface erosion. OVX rats provide a useful experimental model for the evaluation of the chondroprotective effects of estrogens and estrogen-like substances and the model may be an in vivo representation of osteoarthritis in postmenopausal women.

The Chloride Channel Inhibitor NS3736 Prevents Bone Resorption in Ovariectomized Rats Without Changing Bone Formation

Chloride channel activity is essential for osteoclast function. Consequently, inhibition of the osteoclastic chloride channel should prevent bone resorption. Accordingly, we tested a chloride channel inhibitor on bone turnover and found that it inhibits bone resorption without affecting bone formation. This study indicates that chloride channel inhibitors are highly promising for treatment of osteoporosis.

OSCAR is a collagen receptor that costimulates osteoclastogenesis in DAP12-deficient humans and mice

Osteoclasts are terminally differentiated leukocytes that erode the mineralized bone matrix. Osteoclastogenesis requires costimulatory receptor signaling through adaptors containing immunoreceptor tyrosine-based activation motifs (ITAMs), such as Fc receptor common γ (FcRγ) and DNAX-activating protein of 12 kDa. Identification of these ITAM-containing receptors and their ligands remains a high research priority, since the stimuli for osteoclastogenesis are only partly defined. Osteoclast-associated receptor (OSCAR) was proposed to be a potent FcRγ-associated costimulatory receptor expressed by preosteoclasts in vitro, but OSCAR lacks a cognate ligand and its role in vivo has been unclear. Using samples from mice and patients deficient in various ITAM signaling pathways, we show here that OSCAR costimulates one of the major FcRγ-associated pathways required for osteoclastogenesis in vivo. Furthermore, we found that OSCAR binds to specific motifs within fibrillar collagens in the ECM that become revealed on nonquiescent bone surfaces in which osteoclasts undergo maturation and terminal differentiation in vivo. OSCAR promoted osteoclastogenesis in vivo, and OSCAR binding to its collagen motif led to signaling that increased numbers of osteoclasts in culture. Thus, our results suggest that ITAM-containing receptors can respond to exposed ligands in collagen, leading to the functional differentiation of leukocytes, which provides what we believe to be a new concept for ITAM regulation of cytokine receptors in different tissue microenvironments.

Osteoclast nuclei of myeloma patients show chromosome translocations specific for the myeloma cell clone: a new type of cancer–host partnership?

A major clinical manifestation of bone cancers is bone destruction. It is widely accepted that this destruction is not caused by the malignant cells themselves, but by osteoclasts, multinucleated cells of monocytic origin that are considered to be the only cells able to degrade bone. The present study demonstrates that bone‐resorbing osteoclasts from myeloma patients contain nuclei with translocated chromosomes of myeloma B‐cell clone origin, in addition to nuclei without these translocations, by using combined FISH and immunohistochemistry on bone sections. These nuclei of malignant origin are transcriptionally active and appear fully integrated amongst the other nuclei. The contribution of malignant nuclei to the osteoclast population analysed in this study was greater than 30%. Osteoclast–myeloma clone hybrids contained more nuclei than normal osteoclasts and their occurrence correlated with the proximity of myeloma cells. Similar hybrid cells were generated in myeloma cell–osteoclast co‐cultures, as revealed by tracing myeloma nuclei using translocations, bromo‐deoxyuridine, or the Y chromosome of male myeloma cells in female osteoclasts. These observations indicate that hybrid cells can originate through fusion between myeloma cells and osteoclasts. In conclusion, malignant cells contribute significantly to the formation of bone‐resorbing osteoclasts in multiple myeloma. Osteoclast–myeloma clone hybrids reflect a previously unrecognized mechanism of bone destruction in which malignant cells participate directly. The possibility that malignant cells corrupt host cells by the transfer of malignant DNA may have been underestimated to date in cancer research. Copyright

Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion

Generation of osteoclasts through fusion of mono-nucleated precursors is a key event of bone physiology and bone resorption is inefficient without osteoclast fusion. Several factors playing a critical role in the fusion process have already been recognized, but the factors involved in the actual fusion of the lipid bilayers of their cell membranes are still unknown. Syncytin-1 is a protein encoded by a human endogenous retroviral gene which was stably integrated into the human ancestor genome more than 24 million years ago. Upon activation, syncytin-1 is able to destabilize the lipid bilayer of the target cell and to force the merging of plasma membranes. This protein is a key player in the fusion of cytotrophoblasts. In the present study, syncytin-1 as well as its putative receptor ASCT2 was found to be expressed in differentiating osteoclasts in vitro, both on mRNA and protein level. This was documented through Q-PCR, Western blot and immunofluorescence analyses. These in vitro findings were confirmed by immunohistochemical stainings in human iliac crest biopsies. A syncytin-1 inhibitory peptide reduced the number of nuclei per osteoclast by 30%, as well as TRACP activity. From a mechanistic point of view, it is interesting that the distribution of syncytin-1 immunoreactivity on the cell surface parallels that of actin, another important player in cell fusion, and that cell-cell proximity induces particular patterns of distribution of syncytin-1 and actin in the respective cells. These complementary observations support a critical role of syncytin-1 in osteoclast fusion, which is of special interest in view of its well-known ability to force the merging of plasma membranes.

Pulse treatment with the proteasome inhibitor bortezomib inhibits osteoclast resorptive activity in clinically relevant conditions

Myeloma bone disease is due to bone degradation by osteoclasts, and absence of repair by bone forming osteoblasts. Recent observations suggest that the anti-myeloma drug bortezomib, a proteasome inhibitor, stimulates bone formation and may inhibit bone resorption. Here, we tested bortezomib on cultured osteoclasts in conditions mimicking the pulse treatment used in the clinic, thereby avoiding continuous proteasome inhibition and unselective toxicity. A 3 h pulse with 25 nM bortezomib followed by a 3-day culture in its absence markedly inhibited osteoclast activity as evaluated through bone resorption, TRAcP release, and RANKL-induced NF-kappaB translocation into nuclei, an event dependent on proteasomes and critical for osteoclast function. The effect on TRAcP was maximal during the first 24 h post-pulse, and then tended to subside. Importantly, applying this pulse treatment to cultured myeloma cells drastically reduced their survival. We measured next the levels of two bone resorption markers in patients during the 3 days following five and seven therapeutic bortezomib administrations, respectively. These levels decreased significantly already 1-2 days after injection, and then increased, showing temporary inhibition of osteoclast activity and paralleling the in vitro effect on TRAcP. Our study demonstrates a direct inhibition of osteoclasts by bortezomib in conditions relevant to treatment of myeloma.

Increased presence of capillaries next to remodeling sites in adult human cancellous bone.

Vascularization is a prerequisite for osteogenesis in a number of situations, including bone development, fracture healing, and cortical bone remodeling. It is unknown whether a similar link exists between cancellous bone remodeling and vascularization. Here, we show an association between remodeling sites, capillaries, proliferative cells, and putative osteoblast progenitors. Iliac crest biopsies from normal human individuals were subjected to histomorphometry and immunohistochemistry to identify the respective positions of bone remodeling sites, CD34‐positive capillaries, smooth muscle actin (SMA)‐positive putative osteoblast progenitors, including pericytes, Ki67‐positive proliferative cells, and bone remodeling compartment (BRC) canopies. The BRC canopy is a recently described structure separating remodeling sites from the bone marrow, consisting of CD56‐positive osteoblasts at an early differentiation stage. We found that bone remodeling sites were associated with a significantly increased presence of capillaries, putative osteoblast progenitors, and proliferative cells in a region within 50 µm of the bone or the canopy surface. The increases were the highest above eroded surfaces and at the level of the light‐microscopically assessed contact of these three entities with the bone or canopy surfaces. Between 51 and 100 µm, their densities leveled to that found above quiescent surfaces. Electron microscopy asserted the close proximity between BRC canopies and capillaries lined by pericytes. Furthermore, the BRC canopy cells were found to express SMA. These ordered distributions support the existence of an osteogenic‐vascular interface in adult human cancellous bone. The organization of this interface fits the current knowledge on the mode of action of vasculature on osteogenesis, and points to the BRC canopy as a central player in this mechanism. We propose a model where initiation of bone remodeling coincides with the induction of proximity of the vasculature to endosteal surfaces, thereby allowing capillary‐BRC canopy interactions that activate marrow events, including recruitment of osteoblast progenitors to bone remodeling sites.

Understanding Coupling between Bone Resorption and Formation: Are Reversal Cells the Missing Link?

Bone remodeling requires bone resorption by osteoclasts, bone formation by osteoblasts, and a poorly investigated reversal phase coupling resorption to formation. Likely players of the reversal phase are the cells recruited into the lacunae vacated by the osteoclasts and presumably preparing these lacunae for bone formation. These cells, called herein reversal cells, cover >80% of the eroded surfaces, but their nature is not identified, and it is not known whether malfunction of these cells may contribute to bone loss in diseases such as postmenopausal osteoporosis. Herein, we combined histomorphometry and IHC on human iliac biopsy specimens, and showed that reversal cells are immunoreactive for factors typically expressed by osteoblasts, but not for monocytic markers. Furthermore, a subpopulation of reversal cells showed several distinctive characteristics suggestive of an arrested physiological status. Their prevalence correlated with decreased trabecular bone volume and osteoid and osteoblast surfaces in postmenopausal osteoporosis. They were, however, virtually absent in primary hyperparathyroidism, in which the transition between bone resorption and formation occurs optimally. Collectively, our observations suggest that arrested reversal cells reflect aborted remodeling cycles that did not progress to the bone formation step. We, therefore, propose that bone loss in postmenopausal osteoporosis does not only result from a failure of the bone formation step, as commonly believed, but also from a failure at the reversal step.

Myeloma cell-induced disruption of bone remodelling compartments leads to osteolytic lesions and generation of osteoclast-myeloma hybrid cells

Osteolytic lesions are a hallmark of multiple myeloma. They are due to the hyperactivity of bone resorbing osteoclasts and hypoactivity of bone forming osteoblasts, in response to neighbouring myeloma cells. This study identified a structure that deeply affects this response, because of its impact on the physical organisation of the myeloma cell microenvironment. The proximity between myeloma cells and osteoclasts or osteoblasts was shown to be conditioned by the recently discovered layer of flat cells that separates the osteoclasts and osteoblasts from the bone marrow, by forming a canopy over bone remodelling compartment (BRC). These canopies are frequently disrupted in myeloma, and this disruption correlates with increased proximity and density of myeloma cells. In vitro evidence indicates that this disruption may be due to direct contact between myeloma and BRC canopy cells. Importantly, this disruption and increased proximity and density of myeloma cells coincides with key myeloma‐induced bone events, such as osteolytic lesions, impaired bone formation despite increased bone resorption, and fusion of myeloma cells with osteoclasts thereby forming myeloma‐osteoclast hybrid cells. These findings strongly support a critical role of BRC canopies in myeloma‐induced bone disease. BRC canopies could therefore be considered as a new therapeutic target.