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1.
PLoS One ; 16(4): e0250081, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33878141

RESUMO

Dormancy of hematopoietic stem cells and formation of progenitors are directed by signals that come from the bone marrow microenvironment. Considerable knowledge has been gained on the murine hematopoietic stem cell microenvironment, while less so on the murine progenitor microenvironment and even less so on these microenvironments in humans. Characterization of these microenvironments is decisive for understanding hematopoiesis and finding new treatment modalities against bone marrow malignancies in the clinic. However, it is equally challenging, because hematopoietic stem cells are difficult to detect in the complex bone marrow landscape. In the present study we are characterizing the human hematopoietic stem cell and progenitor microenvironment. We obtained three adjacent bone marrow sections from ten healthy volunteers. One was used to identify a population of CD34+/CD38- "hematopoietic stem cells and multipotent progenitors" and a population of CD34+/CD38+ "progenitors" based on immunofluorescence pattern/intensity and cellular morphology. The other two were immunostained respectively for CD34/CD56 and for CD34/SMA. Using the combined information we performed a non-computer-assisted quantification of nine bone marrow components (adipocytes, megakaryocytes, bone surfaces, four different vessel types (arteries, capillaries, sinusoids and collecting sinuses), other "hematopoietic stem cells and multipotent progenitors" and other "progenitors") within 30 µm of "hematopoietic stem cells and multipotent progenitors", "progenitors", and "random cell profiles". We show that the microenvironment of the "hematopoietic stem cells and multipotent progenitors" is significantly enriched in sinusoids and megakaryocytes, while the microenvironment of the "progenitors" is significantly enriched in capillaries, other "progenitors", bone surfaces and arteries.


Assuntos
Células da Medula Óssea/citologia , Células-Tronco Hematopoéticas/citologia , Nicho de Células-Tronco/fisiologia , Adipócitos , Adulto , Idoso , Antígenos CD34 , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Diferenciação Celular , Separação Celular , Células Cultivadas , Feminino , Citometria de Fluxo , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Imunofenotipagem , Megacariócitos , Glicoproteínas de Membrana , Pessoa de Meia-Idade
2.
Front Cell Dev Biol ; 9: 644503, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33859985

RESUMO

The current models of osteoclastic bone resorption focus on immobile osteoclasts sitting on the bone surface and drilling a pit into the bone matrix. It recently appeared that many osteoclasts also enlarge their pit by moving across the bone surface while resorbing. Drilling a pit thus represents only the start of a resorption event of much larger amplitude. This prolonged resorption activity significantly contributes to pathological bone destruction, but the mechanism whereby the osteoclast engages in this process does not have an answer within the standard bone resorption models. Herein, we review observations that lead to envision how prolonged resorption is possible through simultaneous resorption and migration. According to the standard pit model, the "sealing zone" which surrounds the ruffled border (i.e., the actual resorption apparatus), "anchors" the ruffled border against the bone surface to be resorbed. Herein, we highlight that continuation of resorption demands that the sealing zone "glides" inside the cavity. Thereby, the sealing zone emerges as the structure responsible for orienting and displacing the ruffled border, e.g., directing resorption against the cavity wall. Importantly, sealing zone displacement stringently requires thorough collagen removal from the cavity wall - which renders strong cathepsin K collagenolysis indispensable for engagement of osteoclasts in cavity-enlargement. Furthermore, the sealing zone is associated with generation of new ruffled border at the leading edge, thereby allowing the ruffled border to move ahead. The sealing zone and ruffled border displacements are coordinated with the migration of the cell body, shown to be under control of lamellipodia at the leading edge and of the release of resorption products at the rear. We propose that bone resorption demands more attention to osteoclastic models integrating resorption and migration activities into just one cell phenotype.

3.
Bone ; 145: 115850, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33465485

RESUMO

Bisphosphonates are widely used anti-osteoporotic drugs targeting osteoclasts. They strongly inhibit bone resorption, but also strongly reduce bone formation. This reduced formation is commonly ascribed to the mechanism maintaining the resorption/formation balance during remodeling. The present study provides evidence for an additional mechanism where bisphosphonates actually impair the onset of bone formation after resorption. The evidence is based on morphometric parameters recently developed to assess the activities reversing resorption to formation. Herein, we compare these parameters in cancellous bone of alendronate- and placebo-treated postmenopausal osteoporotic patients. Alendronate increases the prevalence of eroded surfaces characterized by reversal cells/osteoprogenitors at low cell density and remote from active bone surfaces. This indicates deficient cell expansion on eroded surfaces - an event that is indispensable to start formation. Furthermore, alendronate decreases the coverage of these eroded surfaces by remodeling compartment canopies, a putative source of reversal cells/osteoprogenitors. Finally, alendronate strongly decreases the activation frequency of bone formation, and decreases more the formative compared to the eroded surfaces. All these parameters correlate with each other. These observations lead to a model where bisphosphonates hamper the osteoprogenitor recruitment required to initiate bone formation. This effect results in a larger eroded surface, thereby explaining the well-known paradox that bisphosphonates strongly inhibit bone resorption without strongly decreasing eroded surfaces. The possible mechanism for hampered osteoprogenitor recruitment is discussed: bisphosphonates may decrease the release of osteogenic factors by the osteoclasts, and/or bisphosphonates released by osteoclasts may act directly on neighboring osteoprogenitor cells as reported in preclinical studies.


Assuntos
Reabsorção Óssea , Difosfonatos , Alendronato/farmacologia , Remodelação Óssea , Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/tratamento farmacológico , Difosfonatos/farmacologia , Humanos , Osteoclastos , Osteogênese
4.
Semin Cell Dev Biol ; 112: 8-15, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32563679

RESUMO

Osteoclasts, the multinucleated cells responsible for bone resorption, have an enormous destructive power which demands to be kept under tight control. Accordingly, the identification of molecular signals directing osteoclastogenesis and switching on their resorptive activity have received much attention. Mandatory factors were identified, but a very essential aspect of the control mechanism of osteoclastic resorption, i.e. its spatial control, remains poorly understood. Under physiological conditions, multinucleated osteoclasts are only detected on the bone surface, while their mono-nucleated precursors are only in the bone marrow. How are pre-osteoclasts targeted to the bone surface? How is their progressive differentiation coordinated with their approach to the bone surface sites to be resorbed, which is where they finally fuse? Here we review the information on the bone marrow distribution of differentiating pre-osteoclasts relative to the position of the mandatory factors for their differentiation as well as relative to physical entities that may affect their access to the remodelling sites. This info allows recognizing an "osteoclastogenesis route" through the bone marrow and leading to the coincident fusion/resorption site - but also points to what still remains to be clarified regarding this route and regarding the restriction of fusion at the resorption site. Finally, we discuss the mechanism responsible for the start of resorption and its spatial extension. This review underscores that fully understanding the control of bone resorption requires to consider it in both space and time - which demands taking into account the context of bone tissue.

5.
JBMR Plus ; 4(11): e10412, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33210064

RESUMO

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.

6.
Bone ; 141: 115628, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32919109

RESUMO

Proper bone remodeling necessarily requires that osteoblasts reconstruct the bone that osteoclasts have resorbed. However, the cellular events connecting resorption to reconstruction have remained poorly known. The consequence is a fragmentary understanding of the remodeling cycle where only the resorption and formation steps are taken into account. New tools have recently made possible to elucidate how resorption shifts to formation, thereby allowing to comprehend the remodeling cycle as a whole. This new knowledge is reviewed herein. It shows how teams of osteoclasts and osteoblast lineage cells are progressively established and how they are subjected therein to reciprocal interactions. Contrary to the common view, osteoclasts and osteoprogenitors are intermingled on the eroded surfaces. The analysis of the resorption and cell population dynamics shows that osteoprogenitor cell expansion and resorption proceed as an integrated mechanism; that a threshold cell density of osteoprogenitors on the eroded surface is mandatory for onset of bone formation; that the cell initiating osteoprogenitor cell expansion is the osteoclast; and that the osteoclast therefore triggers putative osteoprogenitor reservoirs positioned at proximity of the eroded bone surface (bone lining cells, canopy cells, pericytes). The interplay between magnitude of resorption and rate of cell expansion governs how soon bone reconstruction is initiated and may determine uncoupling and permanent bone loss if a threshold cell density is not reached. The clinical perspectives opened by these findings are discussed.


Assuntos
Remodelação Óssea , Reabsorção Óssea , Osso e Ossos , Humanos , Osteoblastos , Osteoclastos , Osteogênese
7.
Int J Mol Sci ; 21(17)2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32887359

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Reabsorção Óssea/patologia , Diferenciação Celular , Fusão Celular , Proteínas de Membrana/metabolismo , Menopausa , Osteoclastos/citologia , Doadores de Tecidos/estatística & dados numéricos , Adulto , Fatores Etários , Idoso , Reabsorção Óssea/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Osteoclastos/metabolismo
8.
Int J Mol Sci ; 21(16)2020 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-32824687

RESUMO

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.


Assuntos
Reabsorção Óssea/metabolismo , Catepsina K/metabolismo , Osteoclastos/metabolismo , Adulto , Idoso , Reabsorção Óssea/patologia , Células Cultivadas , Endocitose , Feminino , Humanos , Integrinas/metabolismo , Lisossomos/metabolismo , Masculino , Pessoa de Meia-Idade
9.
Bone Res ; 8: 27, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32637185

RESUMO

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.

10.
Bone ; 130: 115127, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31689525

RESUMO

The gradual conversion of cortical bone into trabecular bone on the endocortical surface contributes substantially to thinning of the cortical bone. The purpose of the present study was to characterize the intracortical canals (3D) and pores (2D) in human fibular bone, to identify the intracortical remodeling events leading to this endocortical trabecularization. The analysis was conducted in fibular diaphyseal bone specimens obtained from 20 patients (6 women and 14 men, age range 41-75 years). µCT revealed that endosteal bone had a higher cortical porosity (p< 0.05) and canals with a larger diameter (p< 0.05) than periosteal bone, while the canal spacing and number were similar in the endosteal and periosteal half. Histological analysis showed that the endosteal half versus the periosteal half: (i) had a higher likelihood of being non-quiescent type 2 pores (i.e. remodeling of existing pores) than other pore types (OR = 1.6, p< 0.01); (ii) that the non-quiescent type 2 pores contributed to a higher porosity (p< 0.001); and that (iii) amongst these pores especially eroded type 2 pores contributed to the elevated cortical porosity (p< 0.001). In conclusion, we propose that endocortical trabecularization results from the accumulation of eroded cavities upon existing intracortical canals, favored by delayed initiation of bone formation.


Assuntos
Remodelação Óssea , Osso Cortical , Adulto , Idoso , Densidade Óssea , Osso e Ossos , Osso Cortical/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteogênese , Porosidade
11.
Bone Res ; 8(1): 27, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34193811

RESUMO

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.

12.
Calcif Tissue Int ; 105(4): 430-445, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31236622

RESUMO

Fusion is the final osteoclast differentiation step leading to bone resorption. In healthy trabecular bone, osteoclast fusion is restricted to bone surfaces undergoing resorption, and necessarily requires site-specific recruitment of mononucleated pre-osteoclasts originating from the bone marrow. However, the spatiotemporal mechanism coordinating recruitment and fusion is poorly investigated. Herein we identify a collagen/vascular network as a likely structure supporting this mechanism. We therefore used multiplex immunohistochemistry and electron microscopy on human iliac crest bone samples, in combination with functional assays performed in vitro with osteoclasts generated from healthy blood donors. First, we found that putative pre-osteoclasts are in close vicinity of a network of collagen fibers associated with vessels and bone remodeling compartment canopies. Based on 3D-reconstructions of serial sections, we propose that this network may serve as roads leading pre-osteoclasts to resorption sites, as reported for cell migration in other tissues. Importantly, almost all these bone marrow pre-osteoclasts, but only some osteoclasts, express the collagen receptor OSCAR, which is reported to induce fusion competence. Furthermore, differentiating osteoclasts cultured on collagen compared to mineral show higher fusion rates, higher expression of fusogenic cytokines, and a CD47 plasma membrane distribution pattern reported to be typical of a pre-fusion state-thus collectively supporting collagen-induced fusion competence. Finally, these in vitro assays show that collagen induces high cell mobility. The present data lead to a model where collagen fibers/vasculature support the coordination between traffic and fusion of pre-osteoclasts, by serving as a physical road and inducing fusion competence as well as cell mobility.


Assuntos
Medula Óssea/metabolismo , Movimento Celular/fisiologia , Colágeno/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Remodelação Óssea/fisiologia , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoblastos/patologia , Osteoclastos/patologia , Células-Tronco/metabolismo
13.
J Cell Sci ; 132(10)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30975918

RESUMO

Osteoblast lineage cells in human bone were recently shown to colonize eroded bone surfaces and to closely interact with osteoclasts. They proved to be identical to reversal cells and are believed to differentiate into bone-forming osteoblasts thereby coupling resorption and formation. However, they also exert catabolic activity that contributes to osteoclastic bone resorption, but this has not received much attention. Herein, we used co-cultures of primary human osteoblast lineage cells and human osteoclasts derived from peripheral blood monocytes to investigate whether a catabolic activity of osteoblast lineage cells could impact on osteoclastic bone resorption. Through a combination of immunofluorescence, in situ hybridization and time-lapse experiments, we show that MMP-13-expressing osteoblast lineage cells are attracted to and closely interact with bone-resorbing osteoclasts. This close interaction results in a strong and significant increase in the bone resorptive activity of osteoclasts - especially those making trenches. Importantly, we show that osteoclastic bone resorption becomes sensitive to inhibition of matrix metalloproteinases in the presence, but not in the absence, of osteoblast lineage cells. We propose that this may be due to the direct action of osteoblast-lineage-derived MMP-13 on bone resorption.


Assuntos
Reabsorção Óssea/metabolismo , Metaloproteinases da Matriz/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Humanos , Técnicas In Vitro
15.
Sci Rep ; 9(1): 5361, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926835

RESUMO

Mounting evidence from animal studies suggests a role of the nervous system in bone physiology. However, little is known about the nerve fiber localization to human bone compartments and bone surface events. This study reveals the density and distribution of nerves in human bone and the association of nerve profiles to bone remodeling events and vascular structures in iliac crest biopsies isolated from patients diagnosed with primary hyperparathyroidism (PHPT). Bone sections were sequentially double-immunostained for tyrosine hydroxylase (TH), a marker for sympathetic nerves, followed by protein gene product 9.5 (PGP9.5), a pan-neuronal marker, or double-immunostained for either PGP9.5 or TH in combination with CD34, an endothelial marker. In the bone marrow, the nerve profile density was significantly higher above remodeling surfaces as compared to quiescent bone surfaces. Ninety-five percentages of all nerve profiles were associated with vascular structures with the highest association to capillaries and arterioles. Moreover, vasculature with innervation was denser above bone remodeling surfaces. Finally, the nerve profiles density was 5-fold higher in the intracortical pores compared to bone marrow and periosteum. In conclusion, the study shows an anatomical link between innervation and bone remodeling in human bone.


Assuntos
Remodelação Óssea , Osso e Ossos/inervação , Idoso , Medula Óssea/irrigação sanguínea , Medula Óssea/inervação , Osso e Ossos/irrigação sanguínea , Feminino , Humanos , Hiperparatireoidismo Primário/etiologia , Hiperparatireoidismo Primário/metabolismo , Hiperparatireoidismo Primário/patologia , Masculino , Pessoa de Meia-Idade , Fibras Nervosas/metabolismo , Periósteo/inervação
16.
Calcif Tissue Int ; 104(1): 92-101, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30194476

RESUMO

Cathepsin K (CatK) inhibition allows reducing bone resorption with specific advantages compared to the existing anti-osteoporosis drugs. Its clinical use appears even more promising with the recent development of ectosteric inhibitors. A confusing observation, however, is that a low dose of the active site CatK inhibitor odanacatib (ODN) was reported to decrease bone mineral density and increase serum levels of the bone resorption marker carboxy-terminal collagen crosslinks (CTX). The present study provides a possible explanation for this paradox. The resorptive activity of human osteoclasts seeded on bone slices was inhibited when subjected to ODN at doses of 20 nM, but about 100-fold lower doses induced a significant increase in CTX levels and in eroded surface (12 repeats). This low-dose-induced stimulation was prevented by inhibition of non-CatK cysteine proteinases, thereby indicating that the stimulation results from an interplay between CatK and other cysteine proteinases. Effective interplay between these proteinases was also shown in enzymatic assays where the CatK-mediated degradation of collagen was enhanced upon addition of cathepsins B or L. Furthermore, extracts of osteoclasts subjected to a low dose of ODN showed higher levels of cathepsin B compared with extracts of control osteoclasts. In conclusion, the low-dose-induced stimulation of resorption observed in the clinical study can be reproduced in osteoclasts cultured in the absence of any other cell. Our data support an osteoclast-intrinsic mechanism where a mild inhibition of CatK results in increased levels of other proteinases contributing to the collagen degradation process.


Assuntos
Compostos de Bifenilo/farmacologia , Reabsorção Óssea/metabolismo , Catepsina K/antagonistas & inibidores , Osteoclastos/metabolismo , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/tratamento farmacológico , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Catepsina K/metabolismo , Humanos , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo
17.
Bone ; 117: 70-82, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30240959

RESUMO

Cortical bone is remodeled by intracortical basic multicellular units (BMUs), whose end result can be observed as quiescent osteons in histological sections. These osteons offer a unique opportunity to investigate the BMU balance between the magnitude of bone resorption and subsequent bone formation at the BMU level. Our main objective was to investigate whether the latter parameters change between defined categories of osteons and with age, and to which extend these changes contribute to age-induced cortical porosity. Cortices of iliac bone specimens from 35 women (aged 16-78 years) with a higher porosity with age were investigated. A total of 3084 quiescent osteons reflecting 75% of the intracortical pores were histological examined. The osteons diameter, pore diameter, wall thickness, prevalence and contribution to the porosity were highly variable, but unchanged with age. Next, the osteons were categorized according to whether they reflected the remodeling of existing canals (type 2Q osteons) or the generation of new canals (type 1Q osteons). Type 2Q osteons versus type 1Q osteons: (i) had more frequently a pore diameter > 75 µm (7.4 vs. 1.3%; p < 0.001); (ii) had a larger mean pore diameter (40 ±â€¯10 vs. 25 ±â€¯4 µm; p < 0.001), osteon diameter (120 ±â€¯21 vs. 94 ±â€¯21 µm; p < 0.001) and wall thickness (40 ±â€¯10 vs. 35 ±â€¯9; p < 0.05); (iii) had a larger contribution to the cortical porosity (29 ±â€¯18 vs. 8 ±â€¯8%; p < 0.001); (iv) were more prevalent (44 ±â€¯10 vs. 31 ±â€¯11%; p < 0.001); and (v) were more prevalent with age. Collectively, this study demonstrates that quiescent osteons with age more frequently result from remodeling of existing canals, which in some cases had a more negative BMU balance. Still, the osteons showed no overall age-related change in their pore diameter i.e. BMU balance. In contrast to conventional wisdom, these data show that non-quiescent pores, not pores of quiescent osteons, were the main contributor to a higher cortical porosity.


Assuntos
Envelhecimento/fisiologia , Osso Cortical/fisiologia , Osteon/fisiologia , Adolescente , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Porosidade , Adulto Jovem
18.
Sci Rep ; 8(1): 13016, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158637

RESUMO

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 the SEPT9 gene 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 osteoclasts in vitro suggesting a role for septins in bone resorption. Attesting to this, conditional deletion of Sept9 in mice leads to elevated levels of trabecular bone and diminished femoral growth in 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.


Assuntos
Reabsorção Óssea , Diferenciação Celular , Osteoclastos/fisiologia , Septinas/metabolismo , Animais , Células Cultivadas , Fêmur/crescimento & desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Septinas/deficiência
19.
J Bone Miner Res ; 33(12): 2177-2185, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30048570

RESUMO

During aging and in osteoporosis, cortical bone becomes more porous, making it more fragile and susceptible to fractures. The aim of this study was to investigate the intracortical compression- induced strain energy distribution, and determine whether intracortical pores associated with high strain energy density (SED) in the surrounding bone matrix have a different morphology and distribution, as well as different remodeling characteristics than matrix with normal SED. Fibular diaphyseal specimens from 20 patients undergoing a jaw reconstruction (age range 41 to 75 years; 14 men and 6 women) were studied. Bone specimens were µCT-scanned, plastic embedded, and sectioned for histology. Three-dimensional microfinite element models of each specimen were tested in compression, and the SED of the bone immediately surrounding the intracortical pores was calculated within a plane of interest corresponding to the histological sections. The SED of a pore, relative to the distribution of the SED of all pores in each specimen, was used to classify pores as either a high or normal SED pore. Pores with high SED were larger, less circular, and were located closer to the endosteal surface of the cortex than normal SED pores (p < 0.001). Histological analysis of the remodeling events generating the pores revealed that the high SED pores compared with normal SED pores had 13.3-fold higher odds of being an erosive (70%) or formative (7%) pore versus a quiescent pore (p < 0.001), 5.9-fold higher odds of resulting from remodeling upon existing pores (type 2 pore) versus remodeling generating new pores (type 1 pore) (p < 0.001), and 3.2-fold higher odds of being a coalescing type 2 pore versus a noncoalescing type 2 pore (p < 0.001). Overall, the study demonstrates a strong relationship between cortical bone mechanics and pore morphology, distribution, and remodeling characteristics in human fibular bone. © 2018 American Society for Bone and Mineral Research.


Assuntos
Remodelação Óssea/fisiologia , Osso Cortical/fisiologia , Adulto , Idoso , Fenômenos Biomecânicos , Osso Cortical/diagnóstico por imagem , Feminino , Humanos , Imageamento Tridimensional , Masculino , Pessoa de Meia-Idade , Porosidade , Microtomografia por Raio-X
20.
J Bone Miner Res ; 33(4): 606-620, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29193312

RESUMO

Intracortical bone remodeling normally ensures maintenance of the cortical bone matrix and strength, but during aging, this remodeling generates excessive porosity. The mechanism behind the age-induced cortical porosity is poorly understood and addressed in the present study. This study consists of a histomorphometric analysis of sections of iliac bone specimens from 35 women (age 16-78 years). First, the study shows that the age-induced cortical porosity reflects an increased pore size rather than an increased pore density. Second, it establishes a novel histomorphometric classification of the pores, which is based on the characteristics of the remodeling sites to which each pore is associated. It takes into consideration (i) the stage of the remodeling event at the level where the pore is sectioned, (ii) whether the event corresponds with the generation of a new pore through penetrative tunneling (type 1 pores) or with remodeling of an existing pore (type 2 pores), and (iii) in the latter case, whether or not the new remodeling event leads to the coalescence of pores. Of note, the advantage of this classification is to relate porosity with its generation mechanism. Third, it demonstrates that aging and porosity are correlated with: a shift from type 1 to type 2 pores, reflecting that the remodeling of existing pores is higher; an accumulation of eroded type 2 pores, reflecting an extended resorption-reversal phase; and a coalescence of these eroded type 2 pores into enlarged coalescing type 2 cavities. Collectively, this study supports the notion, that age-related increase in cortical porosity is the result of intracortical remodeling sites upon existing pores, with an extended reversal-resorption phase (eroded type 2 pores) that may likely result in a delayed or absent initiation of the subsequent bone formation. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.


Assuntos
Envelhecimento/metabolismo , Remodelação Óssea , Osso Cortical/metabolismo , Adolescente , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Porosidade
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