[Show abstract][Hide abstract] ABSTRACT: Osteoblast recruitment during bone remodeling is obligatory to re-construct the bone resorbed by the osteoclast. This recruitment is believed to be triggered by osteoclast products and is therefore likely to start early during the remodeling cycle. Several osteoclast products with osteoblast recruitment potential are already known. Here we draw the attention on the osteoblast recruitment potential of the collagen that is freshly demineralized by the osteoclast. Our evidence is based on observations on adult human cancellous bone, combined with in vitro assays. First, freshly eroded surfaces where osteoblasts have to be recruited show the presence of non-degraded demineralized collagen and close cell-collagen interactions, as revealed by electron microscopy, while surface-bound collagen strongly attracts osteoblast lineage cells in a transmembrane migration assay. Compared with other extracellular matrix molecules, collagen's potency was superior and only equaled by fibronectin. Next, the majority of the newly recruited osteoblast lineage cells positioned immediately next to the osteoclasts, exhibit uPARAP/Endo180, an endocytic collagen receptor reported to be involved in collagen internalization and cell migration in various cell types, and whose inactivation is reported to lead to lack of bone formation and skeletal deformities. In the present study, an antibody directed against this receptor inhibits collagen internalization in osteoblast lineage cells and decreases to some extent their migration to surface-bound collagen in the transmembrane migration assay. These complementary observations lead to a model where collagen demineralized by osteoclasts attracts surrounding osteoprogenitors onto eroded surfaces, and where the endocytic collagen receptor uPARAP/Endo180 contributes to this migration, probably together with other collagen receptors. This model fits recent knowledge on the position of osteoprogenitor cells immediately next to remodeling sites in adult human cancellous bone.
[Show abstract][Hide abstract] ABSTRACT: Bone-remodeling compartments (BRCs) were recently recognized to be present in patients with primary hyperparathyroidism and critical for bone reconstruction in multiple myeloma and endogenous Cushing's syndrome. The BRCs are outlined by a cellular canopy separating the bone-remodeling events on the bone surface from the marrow cavity. The present study on human iliac crest biopsy specimens reveals that BRC canopies appear frequently absent above both eroded and formative surfaces in post-menopausal osteoporosis patients, and that this absence was associated with bone loss in these patients. The absence of BRC canopies above the eroded surfaces was furthermore associated with the accumulation of arrested reversal surfaces and a reduced extent of formative surfaces, which both reflect an increased incidence of aborted remodeling cycles. Moreover, the absence of BRC canopies above formative surfaces was associated with a shift in the osteoblast morphological characteristics, from cuboidal to flattened. Collectively, this study shows that the BRCs are unique anatomical structures implicated in bone remodeling in a widespread disease, such as post-menopausal osteoporosis. Furthermore, it particularly highlights the role of the BRC canopies to make the reversal phase progressing toward initiation of matrix deposition, thereby preventing bone loss.
American Journal Of Pathology 02/2014; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bone-forming osteoblasts recruited during bone remodeling might originate from bone marrow perivascular cells, bone-remodeling compartment canopy cells, or bone-lining cells. However, an assessment of osteoblast recruitment during adult human cancellous bone remodeling is lacking. We addressed this question by quantifying cell densities, cell proliferation, osteoblast differentiation markers, and capillaries in human iliac crest biopsy specimens. We found that recruitment occurs on both reversal and bone-forming surfaces, as shown by the cell density and osterix levels on these respective surfaces, and bone formation occurs only above a given cell density. Next, canopies represent an important source of osteoprogenitors, because canopy cells proved to be more proliferative and less differentiated than bone surface cells, as shown by the inverse levels of Ki-67 and procollagen-3 N-terminal peptide versus osterix; and canopy cell densities, which decline with age, and canopy-capillary contacts above eroded surfaces correlated positively with osteoblast density on bone-forming surfaces. Bone-remodeling compartment canopies also arise from a mesenchymal envelope surrounding the red bone marrow, which is lifted and hypertrophied on initiation of bone resorption. This study, together with earlier reports, led to a model in which canopies and nearby capillaries are critical for reaching the osteoblast density required for bone formation.
American Journal Of Pathology 01/2014; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The bone matrix is maintained functional through the combined action of bone resorbing osteoclasts and bone forming osteoblasts, in so-called bone remodeling units. The coupling of these two activities is critical for securing bone replenishment and involves osteogenic factors released by the osteoclasts. However, the osteoclasts are separated from the mature bone forming osteoblasts in time and space. Therefore the target cell of these osteoclastic factors has remained unknown. Recent explorations of the physical microenvironment of osteoclasts revealed a cell layer lining the bone marrow and forming a canopy over the whole remodeling surface, spanning from the osteoclasts to the bone forming osteoblasts. Several observations show that these canopy cells are a source of osteoblast progenitors, and we hypothesized therefore that they are the likely cells targeted by the osteogenic factors of the osteoclasts. Here we provide evidence supporting this hypothesis, by comparing the osteoclast-canopy interface in response to two types of bone resorption inhibitors in rabbit lumbar vertebrae. The bisphosphonate alendronate, an inhibitor leading to low bone formation levels, reduces the extent of canopy coverage above osteoclasts. This effect is in accordance with its toxic action on periosteoclastic cells. In contrast, odanacatib, an inhibitor preserving bone formation, increases the extent of the osteoclast-canopy interface. Interestingly, these distinct effects correlate with how fast bone formation follows resorption during these respective treatments. Furthermore, canopy cells exhibit uPARAP/Endo180, a receptor able to bind the collagen made available by osteoclasts, and reported to mediate osteoblast recruitment. Overall these observations support a mechanism where the recruitment of bone forming osteoblasts from the canopy is induced by osteoclastic factors, thereby favoring initiation of bone formation. They lead to a model where the osteoclast-canopy interface is the physical site where coupling of bone resorption to bone formation occurs.
Biochemical and Biophysical Research Communications 12/2013; · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Odanacatib (ODN) is a bone resorption inhibitor which differs from standard antiresorptives by its ability to reduce bone resorption without decreasing bone formation. What is the reason for this difference? In contrast with other antiresorptives, such as alendronate (ALN), ODN targets only the very last step of the resorption process. We hypothesize that ODN may therefore modify the remodeling events immediately following osteoclastic resorption. These events belong to the reversal phase and include recruitment of osteoblasts, which is critical for connecting bone resorption to formation. We performed a histomorphometric study of trabecular remodeling in vertebrae of estrogen-deficient rabbits treated or not with ODN or ALN, a model where ODN, but not ALN, was previously shown to preserve bone formation. In line with our hypothesis, we found that ODN treatment compared to ALN results in a shorter reversal phase, faster initiation of osteoid deposition on the eroded surfaces, and higher osteoblast recruitment. The latter is reflected by higher densities of mature bone forming osteoblasts and an increased subpopulation of cuboidal osteoblasts. Furthermore, we found an increase in the interface between osteoclasts and surrounding osteoblast-lineage cells. This increase is expected to favor the osteoclast-osteoblast interactions required for bone formation. Regarding bone resorption itself, we show that ODN, but not ALN, treatment results in shallower resorption lacunae, a geometry favoring bone stiffness. We conclude that, compared to standard antiresorptives, ODN shows distinctive effects on resorption geometry and on reversal phase activities which positively affect osteoblast recruitment and may therefore favor bone formation.
Calcified Tissue International 10/2013; · 2.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
American Journal Of Pathology 06/2013; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Telomere shortening is associated with a number of common age-related diseases. A role of telomere shortening in osteoarthritis (OA) has been suggested, mainly based on the assessment of mean telomere length in ex vivo expanded chondrocytes. We addressed this role directly in vivo by using a newly developed assay, which measures specifically the load of ultra-short single telomeres (below 1,500 base pairs), that is, the telomere subpopulation believed to promote cellular senescence.
Samples were obtained from human OA knees at two distances from the central lesion site. Each sample was split into three: one was used for quantification of ultra-short single telomeres through the Universal single telomere length assay (STELA), one for histological Mankin grading of OA, and one for mean telomere length measurement through quantitative fluorescence in situ hybridization (Q-FISH) as well as for assessment of senescence through quantification of senescence-associated heterochromatin foci (SAHF).
The load of ultra-short telomeres as well as mean telomere length was significantly associated with proximity to lesions, OA severity, and senescence level. The degree of significance was higher when assessed through load of ultra-short telomeres per cell compared with mean telomere length.
These in vivo data, especially the quantification of ultra-short telomeres, stress a role of telomere shortening in human OA.
Arthritis research & therapy 01/2012; 14(1):R12. · 4.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A remarkable property of bone remodeling is that osteoblasts form bone matrix exactly where and when osteoclasts have removed it. The bone remodeling compartment (BRC) canopies that cover bone surfaces undergoing remodeling were proposed to be critical players in this mechanism. Here, we provide support to this hypothesis by analyzing the changes in prevalence of BRC canopies during the progress of the remodeling cycle in a cohort of healthy individuals and in patients with endogenous Cushing's syndrome (CS), and by relating these changes in prevalence with the extent of bone forming surfaces. Both cohorts showed almost 100% canopy coverage above resorbing osteoclasts, and only about 76% above bone forming surfaces. This indicates that BRC canopies are invariably associated with the early stage of the remodeling cycle, but may disappear later. Interestingly, in control and two-thirds of the CS patients, a significant decline in canopy coverage occurred only once bone formation was initiated, but in the remaining third of the CS patients the prevalence of canopies already decreased before bone formation. This canopy loss before initiation of bone formation coincided with significantly less bone-forming surface compared with canopy loss at a later stage. These observations support a model where bone restitution is compromised in the absence of BRC canopies, and apparently does not start when the BRC canopy is lost before initiation of the bone formation step. This model is discussed in the context of possible biological roles of BRC canopies. It suggests that BRC canopies could be privileged targets for treating patients suffering from a negative bone formation-resorption balance.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 12/2011; 27(4):770-80. · 6.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of clinical investigation 08/2011; 121(9):3505-16. · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Increased matrix metalloproteinase (MMP) activity has been implicated in the pathogenesis of colorectal anastomotic leakage. Tumor necrosis factor-α (TNF-α) induces MMPs and may influence anastomosis repair.
We assessed the efficacies of the nonselective hydroxamate MMP inhibitor GM6001, the selective hydroxamate MMP inhibitor AG3340 and a TNF-α antagonist with respect to anastomotic breaking strength of left-sided colon anastomoses in male Sprague-Dawley rats.
Systemic GM6001 treatment effectively blocked MMP activity and maintained the initial breaking strength day 0 of the anastomoses when administered subcutaneously as daily depositions (100 mg/kg) or continuously (10 mg/kg/day). In contrast, the anastomotic biomechanic strength was lowered by 55% (p < 0.001) in vehicle-treated rats on postoperative day 3. GM6001 treatment increased breaking strength by 88% (p < 0.0005) compared with vehicle-treated rats day 3 and reduced (p = 0.003) the occurrence of spontaneous anastomotic dehiscence. Histologically, the anastomotic wound was narrower (p < 0.05) in the longitudinal direction in GM6001-treated animals whereas GM6001 had no significant effect on inflammatory cell infiltration or epithelialization. AG3340 (10 mg/kg) increased (p < 0.012) breaking strength by 47% compared with vehicle on day 3 but did not significantly prevent the reduction of the initial breaking strength on day 0. Although the increased TNF-α levels in the wound were attenuated, the anastomotic breaking strength was not improved (p = 0.62) by the TNF-α (10 mg/kg) inhibitor given systemically.
Pharmacological nonselective MMP inhibition ought to be explored as a prophylactic regimen to reduce anastomotic complications following colorectal resection. The involvement of TNF-α was insignificant in anastomotic wound healing in an experimental model.
International Journal of Colorectal Disease 03/2011; 26(3):329-37. · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The plant phytoalexin resveratrol was previously demonstrated to inhibit the differentiation and bone resorbing activity of osteoclasts, to promote the formation of osteoblasts from mesenchymal precursors in cultures, and inhibit myeloma cell proliferation, when used at high concentrations. In the current study, we screened five structurally modified resveratrol analogues for their ability to modify the differentiation of osteoclasts and osteoblasts and proliferation of myeloma cells. Compared to resveratrol, analogues showed an up to 5,000-fold increased potency to inhibit osteoclast differentiation. To a lesser extent, resveratrol analogues also promoted osteoblast maturation. However, they did not antagonize the proliferation of myeloma cells. The potency of the best-performing candidate in vitro was tested in vivo in an ovariectomy-induced model of osteoporosis, but an effect on bone loss could not be detected. Based on their powerful antiresorptive activity in vitro, resveratrol analogues might be attractive modulators of bone remodeling. However, further studies are required to establish their efficacy in vivo.
Calcified Tissue International 11/2010; 87(5):437-49. · 2.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Monitoring of bone disease in multiple myeloma is becoming increasingly important because bone-protecting treatment with bisphosphonate is becoming restricted after the awareness of osteonecrosis of the jaw. Despite the potential of biochemical markers of bone remodeling to monitor dynamic bone turnover, they are not used in everyday practice. Here, we investigate their usefulness to detect imminent progressive osteolysis in relapsing patients with multiple myeloma.
In an unselected cohort of 93 patients, we measured the bone resorption markers C-terminal telopeptide of collagen type I (CTX-I), C-terminal cross-linked telopeptide of type-I collagen generated by MMPs (ICTP), N-terminal cross-linked telopeptide of type-I collagen (NTX-I), and the bone formation marker bone-specific alkaline phosphatase (bALP) monthly for 2 yr. Retrospectively, we identified 40 cases where patients had progressive disease. We investigated how the bone markers developed prior to disease progression.
We observed that CTX-I and bALP changed significantly before progressive disease were recognized. More interestingly, these changes differed depending on whether concurrent progressive osteolysis was present. In patients with progressive osteolysis, there was a large increase in bone resorption which was not compensated by increased bone formation. In contrasts, patients with stable bone disease had only a slight increase in bone resorption which was compensated by concurrent increased bone formation. By calculating a patient-specific CTX-I/bALP ratio, we quantified the risk a patient experiences if the ratio increases.
By analyzing patient-specific changes in the ratio of CTX-I/bALP, we might tailor treatment with bone-protecting agents in the individual patient.
European Journal Of Haematology 05/2010; 84(5):412-20. · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
British Journal of Haematology 11/2009; 148(4):551-61. · 4.94 Impact Factor