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Jaw and long bone marrow derived osteoclasts differ in shape and their response to bone and dentin
... Остеокласти походять із гематопоетичних стовбурових клітин і нагадують макрофаги. Це високоміграційні багатоядерні поляризовані клітини, що несуть у собі необхідний арсенал лізосомальних ферментів [42]. Вони високоспеціалізовані й містять кілька унікальних ультраструктурних характеристик, таких як плеоморфні мітохондрії, вакуолі та лізосоми. ...
... Між остеокластами й кістковою поверхнею, особливо якщо остеокласт перебуває в лакуні Хаушипа, можна бачити численні прямі мікроворсинки, що утворять гофровану облямівку. Активізовані остеокласти здатні резорбувати 200 000 мкм кістки в день, сформованої десятьма поколіннями остеобластів із середньою тривалістю життя 15-20 днів [41,42]. ...
... Основний колаген кістки -це коллаген І типу, вміст якого становить близько 95 %. Інші типи колагену, такі як типи ІІІ і V [42], перебувають на низьких рівнях і, очевидно, модулюють діаметр колагенових фібрил першого типу. Мінеральний компонент і фібрилярний колаген І типу тісно зв'язані один з одним; останній функціонує як тривимірний шаблон, що організує відкладення й ріст першого [43,44]. ...
The review considers the features of morphogenesis of the lower jaw, its diff erences in comparison with tubular bones. Its embryogistogenesis, remodeling processes, osteogenic potential of mesenchymal stem cells are described. The features of the cytological structure of the cellular osteoblastic, osteocyte and osteoclast clusters are shown, their functional role is cleared. Its posttranslating modifi cations are presented as a result of intermolecular stitching and that hydroxylation of lysine, which are the most important determinant of the knocking of cogagen, determining the strength and elasticity of the lower jaw.
... 66 In addition, osteoclasts from the jawbone and the long bone exhibit distinctive cell morphology and response to substrates. 67 For example, bone marrow cells from the long bone generate more osteoclasts than those from the jawbone when cultured on the bone slice, while bone marrow cells from the jawbone generate more osteoclasts than those from the long bone when cultured on the dentin slice. 67 Since composition of ECM differs between the jawbone and the long bone, 48 such differences in ECM can provide an explanation for the differences in osteoclasts and their activity at different skeletal sites. ...
... 67 For example, bone marrow cells from the long bone generate more osteoclasts than those from the jawbone when cultured on the bone slice, while bone marrow cells from the jawbone generate more osteoclasts than those from the long bone when cultured on the dentin slice. 67 Since composition of ECM differs between the jawbone and the long bone, 48 such differences in ECM can provide an explanation for the differences in osteoclasts and their activity at different skeletal sites. It is reported that TRAP activity is approximately 30-fold higher in calvarial osteoclasts compared to long bone osteoclasts. ...
The jawbone is a unique structure as it serves multiple functions in mastication. Given the fact that the jawbone is remodeled faster than other skeletal bones, bone cells in the jawbone may respond differently to local and systemic cues to regulate bone remodeling and adaptation. Osteoclasts are bone cells responsible for removing old bone, playing an essential role in bone remodeling. Although bone resorption by osteoclasts is required for dental tissue development, homeostasis and repair, excessive osteoclast activity is associated with oral skeletal diseases such as periodontitis. In addition, antiresorptive medications used to prevent bone homeostasis of tumors can cause osteonecrosis of the jaws that is a major concern to the dentist. Therefore, understanding of the role of osteoclasts in oral homeostasis under physiological and pathological conditions leads to better targeted therapeutic options for skeletal diseases to maintain patients’ oral health. Here, we highlight the unique features of the jawbone compared to the long bone and the involvement of osteoclasts in the jawbone‐specific diseases.
... Here we examined bones from adseverin conditional null mice and background-matched wild-type mice by light microscopy, dual-energy X-ray absorptiometry (DEXA), and transmission electron microscopy (TEM). Since osteoclast function may differ in different skeletal sites [19][20][21], we compared osteoclast structure in alveolar bone with long bone and in healthy and inflamed sites. ...
... The structure and function of osteoclasts from different skeletal sites can be quite different [19]. In vitro studies showed that osteoclasts generated from precursors derived from alveolar bone and from long bone exhibit marked differences in cell shape [20]. Here we found no measureable differences in osteoclasts at long bone and alveolar bone sites in WT and adseverin null conditional mice. ...
Adseverin is an actin-severing/capping protein that may contribute to osteoclast differentiation in vitro but its role in bone remodeling of healthy animals is not defined. We analyzed bone and osteoclast structure in adseverin conditional null mice at alveolar and long bone sites. In wild-type and adseverin null mice, as measured by dual-energy X-ray absorptiometry, there were no differences of bone mineral content or bone mineral density, indicating no change of bone metabolism. In tibiae, TRAcP(+) osteoclasts were formed in comparable numbers in adseverin null and wild-type mice. Ultrastructural analysis showed normal and similar abundance of ruffled borders, sealing zones, and mitochondria, and with no difference of osteoclast nuclear numbers. In contrast, analyses of long bone showed that in the absence of adseverin osteoclasts were smaller (120 ± 13 vs. 274 ± 19 µm(2); p < 0.05), as were nuclear size and the surface area of cytoplasm. The nuclei of adseverin null osteoclasts exhibited more heterochromatin (31 ± 3%) than wild-type cells (8 ± 1%), suggesting that adseverin affects cell differentiation. The data indicate that in healthy, developing tissues, adseverin contributes to the regulation of osteoclast structure but not to bone metabolism in vivo.
... Two cytokines have been found to be necessary for osteoclastogenesis: M-CSF, which can stimulate osteoclast precursors by regulating their survival and proliferation [11], and RANKL, which promotes osteoclast differentiation and activity [12][13][14]. Some studies have shown that osteoclasts at different bone sites are not always identical [15][16][17][18][19][20]. Everts et al. [17] found that both matrix metalloproteinases and cathepsin K are involved in bone degradation by calvaria osteoclasts, whereas resorption by long-bone osteoclasts primarily depends on the activity of cathepsin K. Jansen et al. [18] showed Ae2 ab (Na + -independent chloride/bicarbonate anion exchanger 2) to be relevant to the activity of long-bone osteoclasts but not for those in calvaria. ...
... Studies have shown that the number of nuclei per osteoclast is proportional to the volume of the bone resorbed by the osteoclasts [45]. In addition, the number of actin rings formed by an osteoclast on bone or dentin was shown to increase with size [16]. Because it has previously been shown that IL-1b can stimulate bone resorption by large mature osteoclasts [46], we speculate that the increased number of large osteoclasts induced by IL-1b contributes to increased bone resorption. ...
Osteoclasts are bone-resorbing cells and targets for treating bone diseases. Previously, we reported that distinct murine osteoclast precursor subsets, such as early blasts (CD31(hi) Ly-6C(-)), myeloid blasts (CD31(+) Ly-6C(+)), and monocytes (CD31(-) Ly-6C(hi)), respond differently to the osteoclastogenesis-inducing cytokines, macrophage colony-stimulating factor, and receptor activator for nuclear factor κB ligand. It is unknown, however, how these cell types respond to the osteoclast-stimulating inflammatory cytokine interleukin 1β. This study aims to investigate the effect of interleukin 1β on osteoclastogenesis derived from different mouse bone marrow precursors. Early blasts, myeloid blasts, and monocytes were sorted from mouse bone marrow cells using flow cytometry. Cells were cultured on plastic or on bone slices in the presence of macrophage colony-stimulating factor and receptor activator for nuclear factor κB ligand, without or with interleukin 1β (0.1-10 ng/ml). We found that interleukin 1β stimulated multinucleation and bone resorption of osteoclasts derived from the 3 precursors at different rates. The most large osteoclasts (>20 nuclei) and highest level of bone resorption (16.3%) was by myeloid blast-derived osteoclasts. Interleukin 1β particularly accelerated proliferation of early blasts and the most small osteoclasts (3-5 nuclei) formed on plastic. Life span varied among osteoclasts derived from different precursors: large osteoclasts (>2400 µm(2)) formed most rapidly (75 h) from myeloid blasts but had a short life span (30 h). Monocytes needed the longest time (95 h) for the generation of such large osteoclasts, but these cells had a longer life span (50 h). Our results indicate that the different bone marrow osteoclast precursors are differently stimulated by interleukin 1β with respect to proliferation, multinucleation, life span, and bone resorption.
... Vice versa, bone impairs osteoclastogenesis compared to dentin. Azari et al. [17] described a similar observation, where different mineralized tissues evoked different effects on osteoclast formation and mainly affected their formation rather than their resorption. Nevertheless, the resorption process itself is likely to be the same on bone and dentin. ...
... One possible explanation for the divergence in the observed resorption behavior on both materials may come from a difference in absolute amount of noncollagenous matrix proteins, e.g., osteopontin. This protein is present in various concentrations in diverse bone types and is speculated to influence cell behavior [17]. In addition, osteocytic proteins, which are present in bone but are missing in dentin, may act on osteoclasts in the bone remodeling processes [20][21][22][23]. ...
... Vice versa, bone impairs osteoclastogenesis compared to dentin. Azari et al. [17] described a similar observation, where different mineralized tissues evoked different effects on osteoclast formation and mainly affected their formation rather than their resorption. Nevertheless, the resorption process itself is likely to be the same on bone and dentin. ...
... One possible explanation for the divergence in the observed resorption behavior on both materials may come from a difference in absolute amount of noncollagenous matrix proteins, e.g., osteopontin. This protein is present in various concentrations in diverse bone types and is speculated to influence cell behavior [17]. In addition, osteocytic proteins, which are present in bone but are missing in dentin, may act on osteoclasts in the bone remodeling processes [20][21][22][23]. ...
The main function of osteoclasts in vivo is the resorption of bone matrix, leaving behind typical resorption traces consisting of pits and trails. The mechanism of pit formation is well described, but less is known about trail formation. Pit-forming osteoclasts possess round actin rings. In this study we show that trail-forming osteoclasts have crescent-shaped actin rings and provide a model that describes the detailed mechanism. To generate a trail, the actin ring of the resorption organelle attaches with one side outside the existing trail margin. The other side of the ring attaches to the wall inside the trail, thus sealing that narrow part to be resorbed next (3-21 μm). This 3D configuration allows vertical resorption layer-by-layer from the surface to a depth in combination with horizontal cell movement. Thus, trails are not just traces of a horizontal translation of osteoclasts during resorption. Additionally, we compared osteoclastic resorption on bone and dentin since the latter is the most frequently used in vitro model and data are extrapolated to bone. Histomorphometric analyses revealed a material-dependent effect reflected by an 11-fold higher resorption area and a sevenfold higher number of pits per square centimeter on dentin compared to bone. An important material-independent aspect was reflected by comparable mean pit area (μm(2)) and podosome patterns. Hence, dentin promotes the generation of resorbing osteoclasts, but once resorption has started, it proceeds independently of material properties. Thus, dentin is a suitable model substrate for data acquisition as long as osteoclast generation is not part of the analyses.
... OCs from calvariae versus LB show differential usage of proteinases 14,15 and expression levels of the enzyme tartrate resistant acid phosphatases (TRAP) 15,16 . In addition, murine jaw and LB marrows have different osteoclastic potential in the presence of M-CSF and RANKL stimulation and exhibit distinctive shape and response to culturing substrates 17,18 . ...
... Our results suggest a bone-site specific regulation of osteoclast activation. Combined with other published data that show bone-site specific differences in osteoclastic function 13,14,17 , potential specific inhibitors of alveolar bone osteoclast activation and/or function could be developed. Such pharmacologic interventions would be important in regulating periodontal bone loss, while sparing bone homeostasis in the remaining of the skeleton. ...
Background:
Mandibles (MB) and maxillae possess unique metabolic and functional properties and demonstrate discrete responses to homeostatic, mechanical, hormonal, and developmental stimuli. Osteogenic potential of bone marrow stromal cells (BMSCs) differs between MB versus long bones (LB). Furthermore, MB- versus LB-derived osteoclasts (OCs) have disparate functional properties. This study explores the osteoclastogenic potential of rat MB versus LB marrow in vitro and in vivo under basal and stimulated conditions.
Methods:
Bone marrow from rat MB and LB was cultured in osteoblastic or osteoclastic differentiation media. Tartrate-resistant acid phosphatase (TRAP) staining, resorption pit assays, and real-time polymerase chain reaction were performed. Additionally, osmotic mini-pumps were implanted in animals, mandibles and tibiae were isolated, and multinucleated cells (MNCs) were measured.
Results:
MB versus LB marrow cultures that were differentiated with receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor produced more TRAP(+) MNCs and greater resorptive area. To explore MB versus LB BMSC-supported osteoclastogenesis, confluent BMSCs were cultured with parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3 (1,25D3), or PTH+1,25D3. 1,25D3- or PTH+1,25D3-treated LB BMSCs expressed significantly higher RANKL and lower osteoprotegerin (OPG) mRNA and increased RANKL:OPG ratio. When whole marrow was cultured with PTH+1,25D3, more TRAP(+) MNCs were seen in LB versus MB cultures. Ultimately, rats were infused with PTH+1,25D3, and MB versus tibia MNCs were measured. Hormonal stimulation increased osteoclastogenesis in both MB and tibiae. However, higher TRAP(+) MNC numbers were observed in tibiae versus MB under basal and hormonal stimulation.
Conclusion:
Collectively, these data illustrate differences of both osteoclastogenic potential and OC numbers of MB versus LB marrow.
... Since marrow cells from different skeletal locations such as long bone and jaw respond differently to osteoclastogenesis-inducing cytokines M-CSF and RANKL, [29,32], it is possible that these marrow cells also respond differently to magnesium-deprived conditions. As the number of cells with more than six nuclei is higher in jaw cultures [29], only cells with 6-10 and N10 nuclei were counted. ...
... Several studies have suggested functional and phenotypic differences among osteoclast subpopulations [26,27,29,32]. Yet, deficiency of Mg 2+ had a similar effect on precursors obtained from long bone or from the jaw. ...
Magnesium (Mg(2+)) deficiency is a frequently occurring disorder that leads to loss of bone mass, abnormal bone growth and skeletal weakness. It is not clear whether Mg(2+) deficiency affects the formation and/or activity of osteoclasts. We evaluated the effect of Mg(2+) restriction on these parameters. Bone marrow cells from long bone and jaw of mice were seeded on plastic and on bone in medium containing different concentrations of Mg(2+) (0.8 mM which is 100% of the normal value, 0.4, 0.08 and 0 mM). The effect of Mg(2+) deficiency was evaluated on osteoclast precursors for their viability after 3 days and proliferation rate after 3 and 6 days, as was mRNA expression of osteoclastogenesis-related genes and Mg(2+)-related genes. After 6 days of incubation, the number of tartrate resistant acid phosphatase-positive (TRACP(+)) multinucleated cells was determined, and the TRACP activity of the medium was measured. Osteoclastic activity was assessed at 8 days by resorption pit analysis. Mg(2+) deficiency resulted in increased numbers of osteoclast-like cells, a phenomenon found for both types of marrow. Mg(2+) deficiency had no effect on cell viability and proliferation. Increased osteoclastogenesis due to Mg(2+) deficiency was reflected in higher expression of osteoclast-related genes. However, resorption per osteoclast and TRACP activity were lower in the absence of Mg(2+). In conclusion, Mg(2+) deficiency augmented osteoclastogenesis but appeared to inhibit the activity of these cells. Together, our in vitro data suggest that altered osteoclast numbers and activity may contribute to the skeletal phenotype as seen in Mg(2+) deficient patients.
... Through the cultivation on apatite-containing materials, osteoclastogenesis is enhanced and more robust sealing zones are formed compared to the cultivation on plastic [57]. Further, resorption pits, as results from osteoclast activity, can be used to make assumptions about the resorption activity [57][58][59]. ...
Bone is a highly dynamic tissue characterized mainly by the interactions of osteoblasts and osteoclasts. When the healing ability of bone regeneration is disturbed, targeted biophysical stimulations such as electrical stimulation are applied. In this study the indirect effects of electrically stimulated human osteoblasts on osteoclastogenesis were investigated to better understand detailed cellular interactions. Therefore, two different cell developmental stages were examined: peripheral blood mononuclear cells (PBMCs) as precursors and pre-osteoclasts as differentiated cells. Previously, over a 21-day period, human osteoblasts were stimulated with a low-frequency alternating electric field. The supernatants were collected and used for an indirect co-culture of PBMCs and pre-osteoclasts. The cellular viability and the induction of differentiation and activity were analyzed. Further, the secretion of relevant osteoclastic markers was examined. Supernatants of 7 d and 14 d stimulated osteoblasts led to a decrease in the viability of PBMCs and an increased number of cells containing actin ring structures. Supernatants from osteoblasts stimulated over 7 d induced PBMC differentiation and pre-osteoclastic activation. Furthermore, pre-osteoclasts showed varying mRNA transcripts of MCP-1, ACP5, CA2, and CASP8 when cultivated with media from osteoblasts. Supernatants from day 21 did not influence PBMCs at all but increased the viability of pre-osteoclasts. We could show that different time points of stimulated osteoblasts have varying effects on the cells and that changes can be observed due to the differentiation stages of the cells. Through the effects of the indirect stimulation, it was possible to underline the importance of studying not only osteoblastic differentiation and mineralization behavior under electric stimulation but also analyzing changes in osteoclastogenesis and the activity of osteoclasts.
... Another possible explanation is that Acvr1 is differentially expressed among different bones. In addition, the mandible is different from other bones in terms of embryonic origins, bone development, collagen compositions (Matsuura et al., 2014), cell autonomous characteristics (Akintoye et al., 2006;Azari et al., 2011), as well as mechanical loading (de Jong et al., 2010;Ehrlich & Lanyon, 2002). The sitespecificity of ACVR1 are supported by studies on another BMP receptor, BMPR1A, which also exhibits differential bone phenotypes depending on anatomical locations (H. . ...
Bone morphogenetic protein (BMP) signaling is well known in bone homeostasis. However, the physiological effects of BMP signaling on mandibles are largely unknown, as the mandible has distinct functions and characteristics from other bones. In this study, we investigated the roles of BMP signaling in bone homeostasis of the mandibles by deleting BMP type I receptor Acvr1 in osteoblast lineage cells with Osterix‐Cre. We found mandibular bone loss in conditional knockout mice at the ages of postnatal day 21 and 42 in an age‐dependent manner. The decreased bone mass was related to compromised osteoblast differentiation together with enhanced osteoclastogenesis, which was secondary to the changes in osteoblasts in vivo. In vitro study revealed that deletion of Acvr1 in the mandibular bone marrow stromal cells (BMSCs) significantly compromised osteoblast differentiation. When wild type bone marrow macrophages were cocultured with BMSCs lacking Acvr1 both directly and indirectly, both proliferation and differentiation of osteoclasts were induced as evidenced by an increase of multinucleated cells, compared with cocultured with control BMSCs. Furthermore, we demonstrated that the increased osteoclastogenesis in vitro was at least partially due to the secretion of soluble receptor activator of nuclear factor‐κB ligand (sRANKL), which is probably the reason for the mandibular bone loss in vivo. Overall, our results proposed that ACVR1 played essential roles in maintaining mandibular bone homeostasis through osteoblast differentiation and osteoblast‐osteoclast communication via sRANKL.
... A positive correlation between size and activity has been described by Piper et al., who correlated activity to the number of nuclei per osteoclast (40). We could later confirm this in another way, by correlating osteoclast area to actin ring surface (41). We showed that osteoclast size is proportional to the number of actin rings per osteoclast and that the percentage of actin ring area per osteoclast is relatively constant, being ∼20%. ...
Fibrodysplasia Ossificans Progressiva (FOP) is a rare genetic disease characterized by heterotopic ossification (HO) that occurs in muscle tissue, tendons, and ligaments. The disease is caused by mutations in the Activin receptor type I (ACVR1) gene resulting in enhanced responsiveness to Activin-A. Binding of this molecule to the mutated receptor induces HO. Bone metabolism normally requires the coupled action of osteoblasts and osteoclasts, which seems to be disturbed during HO. We hypothesize that Activin-A may also counteract the formation of osteoclasts in FOP patients. In this study we investigated the effect of Activin-A on osteoclast differentiation of CD14+ monocytes from FOP patients and healthy controls. The lymphocytic and monocytic cell populations were determined by FACS analysis. Expression of the mutated R206H receptor was assessed and confirmed by allele specific PCR. The effect of Activin-A on osteoclastogenesis was assessed by counting the number and size of multinucleated cells. Osteoclast activity was determined by culturing the cells on Osteo Assay plates. The influence of Activin-A on expression of various osteoclast related genes was studied with QPCR. Blood from FOP patients contained similar percentages of classical, intermediate, or non-classical monocytes as healthy controls. Addition of Activin-A to the osteoclastogenesis cultures resulted in fewer osteoclasts in both control and FOP cultures. The osteoclasts formed in the presence of Activin-A were, however, much larger and more active compared to the cultures without Activin-A. This effect was tempered when the Activin-A inhibitor follistatin was added to the Activin-A containing cultures. Expression of osteoclast specific genes Cathepsin K and TRAcP was upregulated, gene expression of osteoclastogenesis related genes M-CSF and DC-STAMP was downregulated by Activin-A. Since Activin-A is a promising target for inhibiting the formation of HO in FOP, it is important to know its effects on both osteoblasts and osteoclasts. Our study shows that Activin-A induces fewer, but larger and more active osteoclasts independent of the presence of the mutated ACVR1 receptor. When considering FOP as an Activin-A driven disease that acts locally, our findings suggest that Activin-A could cause a more pronounced local resorption by larger osteoclasts. Thus, when targeting Activin-A in patients with neutralizing antibodies, HO formation could potentially be inhibited, and osteoclastic activity could be slightly reduced, but then performed dispersedly by more and smaller osteoclasts.
... Moreover, dentin has an absolute number of NCPs, including dentin sialoprotein, osteopontin, dentin matrix protein-1, and matrix extracellular phosphoglycoprotein, 18,19 which are thought to influence cell behavior. 39 For example, osteocytic proteins, which are expressed in bone but not in dentin, may suppress osteoclast formation during the bone remodeling process. 40,41 As a result, quan-tification of osteoclastic resorption behaviors revealed 11fold higher resorption on dentin compared with the bone. ...
In 2007, recombinant human bone morphogenetic protein-2 (rhBMP-2) was approved for use in humans at a concentration of 1.5 mg/mL with absorbable collagen sponges as an alternative to autogenous bone grafts for alveolar ridge augmentation, defects associated with extraction sockets, and sinus augmentation. However, the use of supraphysiological doses and the insufficient retention of rhBMP-2, when delivered through collagen sponge, result in dose-dependent side effects related to off-label use. Demineralized dentin matrix (DDM), an osteoinducing bone substrate, has been used as an rhBMP-2 carrier since 1998. In addition, DDM has both microparticle and nanoparticle structures, which do not undergo remodeling, unlike bone. In vitro, DDM is a suitable carrier for BMP-2, with the continued release over 30 days at concentrations sufficient to stimulate osteogenic differentiation. In this review, we discuss the histological outcomes of DDM loaded with rhBMP-2 to highlight the biological functions of exogenous rhBMP-2 associated with the DDM carrier in clinical applications in implant dentistry.
Impact Statement
Demineralized dentin matrix (DDM) has been used as an recombinant human bone morphogenetic protein (rhBMP-2) carrier and osteo-inducing bone substrate to facilitate continued release and stimulate osteogenic differentiation. In this review, we discuss the histological outcomes of DDM loaded with rhBMP-2 in order to highlight the biological functions of exogenous rhBMP-2 associated with the DDM carrier in clinical applications in implant dentistry.
... Cells that can differentiate into osteoclasts are widely distributed in the body, including bone marrow precursors, peritoneal macrophages, splenocytes, peripheral blood-borne monocytes and dendritic cells (Quinn et al., 1996;Marks and Walker, 1981;Scheven et al., 1986;Rivollier et al., 2004). Several studies have shown that osteoclast precursors isolated from different skeletal sites are not always identical in terms of osteoclastogenesis Azari et al., 2011;De Souza Faloni et al., 2011). Even within the same site, different precursor subsets were shown to differ in their capacity to form osteoclasts de Vries et al., 2015;Jacquin et al., 2006;Sprangers et al., 2016;Cao et al., 2016). ...
Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF-α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31(hi) Ly-6C(-) ), myeloid blasts (CD31(+) Ly-6C(+) ) and monocytes (CD31(-) Ly-6C(hi) ) were sorted from mouse bone marrow using flow cytometry and cultured with M-CSF and RANKL, with or without TNF-α. Surprisingly, TNF-α prevented the differentiation of TRAcP(+) osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL-1β or IL-6. When monocytes were pre-cultured with M-CSF and RANKL followed by exposure to TNF-α, a stimulatory effect was found. TNF-α also stimulated monocytes' osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF-α was added to monocytes cultured on plastic, RANK, NFATc1 and TRAcP were significantly down-regulated while TNF-αR1 and TNF-αR2 were up-regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF-α, indicating an altered ratio of bound-RANK/unbound-RANK. Our findings suggest a diverse role of TNF-α on monocytes' osteoclastogenesis: it affects the RANK-signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre-cultured with M-CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte-derived osteoclast formation away from the bone. This article is protected by copyright. All rights reserved.
... These data from in vitro experiments evidence that the dentin substrate shows much greater potential than bone for inducing the genesis and maturation of new clastic cells. A possible explanation for this different appetite and resorption behavior is that dentin contains more matrix proteins of noncollagenous origin, such as osteopontin, when compared with bone (Azari et al. 2011). The absence of regulatory osteocytes and osteocyte proteins in dentin may also account for the quantitative differences (Cabahug-Zuckerman et al. 2016). ...
External apical root resorption during orthodontic treatment implicates specific molecular pathways that orchestrate nonphysiologic cellular activation. To date, a substantial number of in vitro and in vivo molecular, genomic, and proteomic studies have supplied data that provide new insights into root resorption. Recent mechanisms and developments reviewed here include the role of the cellular component—specifically, the balance of CD68⁺, iNOS⁺ M1- and CD68⁺, CD163⁺ M2-like macrophages associated with root resorption and root surface repair processes linked to the expression of the M1-associated proinflammatory cytokine tumor necrosis factor, inducible nitric oxide synthase, the M1 activator interferon γ, the M2 activator interleukin 4, and M2-associated anti-inflammatory interleukin 10 and arginase I. Insights into the role of mesenchymal dental pulp cells in attenuating dentin resorption in homeostasis are also reviewed. Data on recently deciphered molecular pathways are reviewed at the level of (1) clastic cell adhesion in the external apical root resorption process and the specific role of α/β integrins, osteopontin, and related extracellular matrix proteins; (2) clastic cell fusion and activation by the RANKL/RANK/OPG and ATP-P2RX7-IL1 pathways; and (3) regulatory mechanisms of root resorption repair by cementum at the proteomic and transcriptomic levels.
... Although not considered traditional immune cells, a growing body of evidence suggests that osteoclasts contribute to inflammation and immune responses via the release of cytokines and via antigen presentation [77]. Functional differences between subsets of osteoclasts have been reported in homeostasis and include differences in size and proteolytic enzymes used for bone matrix digestion [78]. Although it was long assumed that monocytes are an important source of osteoclast precursors, this was not proven in situ until recently when fluorescently labeled monocytes were recruited from the circulation to the bone surface and differentiated locally into osteoclasts [79]. ...
Blood monocytes are precursors of dendritic cells, macrophages, and osteoclasts. They are a heterogeneous cell population with differences in size, phenotype, and function. Although monocytes maintain several tissue-specific populations of immune cells in homeostasis, their contribution to populations of dendritic cells, macrophages, and osteoclasts is significantly increased in inflammation. Identification of a growing number of functionally different subsets of cells within populations of monocyte-derived immune cells has recently put monocyte heterogeneity into sharp focus. Here, we summarize recent findings in monocyte heterogeneity and their differentiation into dendritic cells, macrophages, and osteoclasts. We also discuss these advances in the context of the formation of functionally different monocyte-derived subsets of dendritic cells, macrophages, and osteoclasts.
... During the past decade, several studies have demonstrated the existence of bone-site-specific osteoclasts. By comparing osteoclasts from calvaria with those from long bone or from the jaw, it was shown that these different bone sites contain osteoclasts that differ (i) in the enzymes they use for bone matrix digestion (4,5), (ii) in the amount of TRACP expressed by the cells (6), (iii) in the proton pumps employed to modulate the intraand extracellular pH (7), (iv) in the effects of cytokines or hormones, such as PTH and 1,25-dihydroxyvitamin D3 (VitD3) on their precursors (8), and (v) in their response to different substrates such as bone and dentin (9). It is, however, unclear how the occurrence of such heterogeneity can be explained. ...
Several studies have demonstrated the existence of functional differences between osteoclasts harbored in different bones. The mechanisms involved in the occurrence of such a heterogeneity are not yet understood. Since cells of the osteoblast lineage play a critical role in osteoclastogenesis, osteoclast heterogeneity may be due to osteoblasts that differ at the different bone sites. In the present study we evaluated possible differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. Osteoblasts were isolated from calvaria and long bone of mice and co-cultured with osteoclast precursors obtained from bone marrow of both types of bone, spleen and peripheral blood. Irrespective of the source of the precursors, a significantly higher number of TRACP-positive multinucleated cells were formed with calvaria osteoblasts. The expression of osteoclastogenesis related genes was analyzed by qPCR. OPG was significantly higher expressed by long bone osteoblasts. The RANKL/OPG ratio and TNF-α gene expression were significantly higher in calvaria osteoblast cultures. OPG added to the culture system inhibited osteoclastogenesis in both groups. Blocking TNF-α had no effect on osteoclastogenesis. Calvaria and long bone osteoblasts were pre-stimulated with VitD3 for 5days. Subsequently, osteoclast precursors were added to these cultures. After a co-culture of 6days, it was shown that VitD3 pre-stimulation of long bone osteoblasts strongly improved their capacity to induce osteoclast formation. This coincided with an increased ratio of RANKL/OPG. Taken together, the data demonstrated differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. This appeared to be due to differences in the expression of RANKL and OPG. VitD3 pre-stimulation improved the ability of long bone osteoblasts to induce osteoclast formation. Our findings demonstrate bone-site specific differences in osteoblast-mediated formation of osteoclasts. The data may suggest that the heterogeneity of osteoclasts is partially due to the way the osteoblasts induce their formation.
... However, although the distribution and possibly the priming of the monocytes is affected in inflammation, the influence of inflammatory factors such as IL-17A on specific osteoclast precursor populations is unknown. Previous studies have identified characteristic differences between mature osteoclasts and their precursors derived from various bone sites (Everts et al. 1999;Zenger et al. 2010;Jansen et al. 2009;Azari et al. 2011). Given the heterogeneity at the level of osteoclast precursors, we hypothesized that: (1) each given subset of blood monocytes is committed to developing into a unique type of mature osteoclast and (2) this process is affected by inflammatory factors, such as IL-17A. ...
Osteoclasts are bone-degrading cells that are formed through fusion of their monocytic precursors. Three distinct subsets of monocytes have been identified in human peripheral blood: classical, intermediate and non-classical monocytes. They are known to play different roles in physiology and pathology, but their capacity to differentiate into osteoclasts and whether inflammatory cytokines influence this differentiation is unknown. We hypothesized that classical, intermediate and non-classical monocytes generate functionally different osteoclasts and that they respond in different ways to the inflammatory cytokine interleukin-17A (IL-17A). To investigate this, the different monocyte subsets were isolated from human peripheral blood and osteoclastogenesis was induced with the cytokines M-CSF and RANKL, with or without IL-17A. We found that all subsets are able to differentiate into osteoclasts in vitro, and that both osteoclastogenesis and subsequent bone resorption was distinctly affected by IL-17A. Osteoclastogenesis and bone resorption by osteoclasts derived from classical monocytes remained unaffected by IL-17A, while osteoclast formation from intermediate monocytes was inhibited by the cytokine. Surprisingly, bone resorption by osteoclasts derived from intermediate monocytes remained at similar levels as control cultures, indicating an increased bone resorbing activity by these osteoclasts. Limited numbers of osteoclasts were formed from non-classical monocytes on bone and no bone resorption was detected, which suggest that these cells belong to a cell lineage different from the osteoclast. By providing more insight into osteoclast formation from human blood monocytes, this study contributes to the possible targeting of specific osteoclast precursors as a therapeutic approach for diseases associated with inflammatory bone loss. This article is protected by copyright. All rights reserved.
... Mouse bone marrow was obtained as described earlier from 7 week old male mice [31]. Permission for animal experiments was obtained from the Animal Welfare committee of the VU University Amsterdam, according to guideline EC Directive 86/609/EEC. ...
Strontium Ranelate (SrRan) is used to decrease the risk of bone fractures. Any factor that alters the release of paracrine signals by osteocytes in response to mechanical stimuli potentially affects bone mass and structure, and thus fracture resistance. We hypothesized that SrRan affects paracrine signaling from mechanically-stimulated osteocytes towards osteoclast-precursors and osteoblasts. MLO-Y4 osteocytes were cultured for 24 h with SrRan (0.1–3 mM) and either or not mechanically stimulated by pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) for 60 min. Nitric oxide (NO) and prostaglandin E 2 (PGE 2) release, and expression of mechanoresponsive genes were quantified. Conditioned medium (CM) from oste-ocytes was added to mouse bone marrow cells for 7 days to assess osteoclastogenesis, or MC3T3-E1 osteo-blasts for 4–16 days to measure osteogenic gene expression. SrRan (3 mM) enhanced NO and PGE 2 release to the same extent in static osteocytes (NO: 1.6-fold; PGE 2 : 2.8-fold) and PFF-stimulated osteocytes (NO: 1.3-fold; PGE 2 : 2.6-fold). CM from PFF-treated osteocytes without SrRan enhanced Ki67 expression but reduced Runx2 and Ocn expression in osteoblasts. This effect on gene expression was not observed with CM obtained from osteocytes treated with the combination of PFF and 3 mM SrRan. CM from PFF-treated osteocytes inhibited osteoclastogenesis by 1.9-fold. The combination of PFF and 3 mM SrRan reduced osteocyte-stimulated osteoclastogenesis even more strongly (4.3-fold). In conclusion, SrRan affects paracrine signaling between mechanically-stimulated MLO-Y4 osteocytes and both osteoblasts and osteoclast precursors. The positive effects of SrRan on bone fracture resistance may thus be partly explained by altered paracrine signaling by osteocytes.
... The mandible appears similar to calvaria in total amount of collagen and mature crosslinks. Although the proteinases used for resorption in the mandible are not clear, the osteoclasts do exhibit a number of different properties from those in the long bones [58][59][60]. ...
... The mandible appears similar to calvaria in total amount of collagen and mature crosslinks. Although the proteinases used for resorption in the mandible are not clear, the osteoclasts do exhibit a number of different properties from those in the long bones [58][59][60]. ...
Bone undergoes constant remodeling throughout life. The cellular and biochemical mechanisms of bone remodeling vary in a region-specific manner. There are a number of notable differences between the mandible and long bones, including developmental origin, osteogenic potential of mesenchymal stem cells, and the rate of bone turnover. Collagen, the most abundant matrix protein in bone, is responsible for determining the relative strength of particular bones. Posttranslational modifications of collagen, such as intermolecular crosslinking and lysine hydroxylation, are the most essential determinants of bone strength, although the amount of collagen is also important. In comparison to long bones, the mandible has greater collagen content, a lower amount of mature crosslinks, and a lower extent of lysine hydroxylation. The great abundance of immature crosslinks in mandibular collagen suggests that there is a lower rate of cross-link maturation. This means that mandibular collagen is relatively immature and thus more readily undergoes degradation and turnover. The greater rate of remodeling in mandibular collagen likely renders more flexibility to the bone and leaves it more suited to constant exercise. As reviewed here, it is important in clinical dentistry to understand the distinctive features of the bones of the jaw.
... For a cell-mediated release, bone marrow cells (BMC) were harvested from femurs and tibias of 6-week-old male mice and 1 9 10 6 BMCs were seeded on the tablets. The tablets were cultured in duplicate in a-MEM (Gibco BRL) containing 10% foetal calf serum (FCS) and 1% penicillin/streptomycin (100 U/ml and 100 lg/ml, respectively) with M-CSF (25 ng/ml, R&D Systems, Minneapolis, MN, USA) and RANKL (40 ng/ml, PreProtech, Rocky Hill, NJ, USA) (Azari et al. 2011). Bio-CaP tablets without BMCs were incubated in a-MEM culture media to investigate the passive (spontaneous) release of BSAs. ...
Objectives:
In this study, a novel biomimetic calcium phosphate bone substitute (BioCaP) is introduced as a dual-drug release system with two drug/protein delivery modes: protein is incorporated into (i) the interior of BioCaP (an internal depot); and (ii) a superficial calcium phosphate coating on BioCaP (a surface-coated depot). Our aim is to investigate each of the two delivery modes of BioCaP. Our hypotheses are that (i) both of the drug delivery modes, in in vitro as well as in vivo environment, can achieve a sustained cell-mediated protein release; and (ii) BioCaP with these two delivery modes with incorporated bone morphogenetic protein-2 (BMP-2) promotes bone formation.
Materials and methods:
Tablets of BioCaP were prepared with different carrying modes using bovine serum albumin (BSA) as model protein. The release of this protein was analysed (n = 6 per group). Granules of BioCaP with different carrying modes of BMP-2 were implanted subcutaneously in rats (n = 6 animals per group). Samples were collected after 5 weeks for histomorphometric analysis.
Results:
In vitro data showed that the internal and surface-coated depots of BSA resulted in a sustained osteoclast-mediated release, while the adsorbed BSA was rapidly released, and this release was not affected by osteoclasts. In vivo data showed that the volume densities of bone, bone marrow, and blood vessels were significantly higher in samples where BMP-2 was incorporated internally or in the coating compared with granules with adsorbed growth factor. Osteoclast-like cells were associated with the granules, and resorption lacunae were frequently observed.
Conclusion:
It is shown that different modes of incorporation of BMP-2 on and in BioCaP granules have a beneficial effect on the formation of ectopic bone. This dual-drug release system makes BioCaP granule a promising tool for delivering multiple therapeutic agents for different clinical applications.
... Thus, in the present study, we have verified that, during tooth eruption, osteoclastogenesis in the occlusal crypt bone does not depend only on RANKL expression. Moreover, the bone marrow from jaws, which have an ectomesenchymal embryonic origin , possesses peculiar osteoclastogenic properties when compared with that of long bones (de Souza Azari et al. 2011). Despite alendronate having an effect on osteoclasts, osteoclast recruitment, differentiation and activation result from a signaling cascade that involves molecules expressed by bone stromal cells and osteoblasts ( Chavez and Bradaschia-Correa 2009) and in particular, dental follicle and stellate reticulum cells in the tooth eruption process (Wise 2009). ...
The creation of the eruption pathway requires the resorption of the occlusal alveolar bone by osteoclasts and signaling events between bone and dental follicle are necessary. The aim of the present study has been to evaluate the effect of alendronate on osteoclastogenesis and the expression of the regulator proteins of osteoclast activation, namely RANK, RANKL and OPG, in the bone that covers the first molar germ. Newborn Wistar rats were treated daily with 2.5 mg/kg alendronate for 4, 8, 14, 21 and 28 days, whereas controls received sterile saline solution. At the time points cited, maxillae were fixed, decalcified and processed for light and electron microscopic analysis. TRAP histochemistry was performed on semi-serial sections and the osteoclasts in the occlusal half of the bony crypt surface were counted. TUNEL analysis was carried out on paraffin sections. The occlusal bone that covers the upper first molar was removed in additional 4- and 8-day-old alendronate-treated and control rats in which the expression of RANK, RANKL and OPG was analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. TRAP-positive osteoclasts were more numerous in the alendronate group at all time points, despite their unactivated phenotype and the presence of apoptotic cells. RANKL expression in the alendronate specimens was inhibited at all time points, unlike in controls. Our findings indicate that the expression of RANKL in the occlusal portion of the bony crypt is unrelated to osteoclast recruitment and differentiation but is crucial to their activation during the creation of the eruption pathway.
The mandible (lower jaw) bone is aesthetically responsible for shaping the lower face, physiologically in charge of the masticatory movements, and phonetically accountable for the articulation of different phonemes. Thus, pathologies that result in great damage to the mandible severely impact the lives of patients. Mandibular reconstruction techniques are mainly based on the use of flaps, most notably free vascularized fibula flaps. However, the mandible is a craniofacial bone with unique characteristics. Its morphogenesis, morphology, physiology, biomechanics, genetic profile, and osteoimmune environment are different from any other non-craniofacial bone. This fact is especially important to consider during mandibular reconstruction, as all these differences result in unique clinical traits of the mandible that can impact the results of jaw reconstructions. Furthermore, overall changes in the mandible and the flap post-reconstruction may be dissimilar, and the replacement process of the bone graft tissue during healing can take years, which in some cases can result in postsurgical complications. Therefore, the present review highlights the uniqueness of the jaw and how this factor can influence the outcome of its reconstruction while using an exemplary clinical case of pseudoarthrosis in a free vascularized fibula flap.
Resorption of mineralized tissues like bone is accomplished by a unique cell type: the osteoclast. This multinucleated cell has the capacity to dissolve the mineral by lowering the pH. Subsequently, the release of certain proteolytic enzymes, in particular cathepsin K, results in the digestion of the organic matrix mainly consisting of collagen. In this short review we will describe the unique features of the osteoclast making it possible for the cell to resorb mineralized tissues.
Osteoclasts are large multinucleated cells from hematopoietic origin and are responsible for bone resorption. A balance between osteoclastic bone resorption and osteoblastic bone formation is critical to maintain bone homeostasis. The alveolar bone, also called the alveolar process, is the part of the jawbone that holds the teeth and supports oral functions. It differs from other skeletal bones in several aspects: its embryonic cellular origin, the form of ossification, and the presence of teeth and periodontal tissues; hence, understanding the unique characteristic of the alveolar bone remodeling is important to maintain oral homeostasis. Excessive osteoclastic bone resorption is one of the prominent features of bone diseases in the jaw such as periodontitis. Therefore, inhibiting osteoclast formation and bone resorptive process has been the target of therapeutic intervention. Understanding the mechanisms of osteoclastic bone resorption is critical for the effective treatment of bone diseases in the jaw. In this review, we discuss basic principles of alveolar bone remodeling with a specific focus on the osteoclastic bone resorptive process and its unique functions in the alveolar bone. Lastly, we provide perspectives on osteoclast‐targeted therapies and regenerative approaches associated with bone diseases in the jaw.
Methylsulfonylmethane (MSM) is a naturally occurring anti-inflammatory compound that effectively treats multiple degenerative diseases such as osteoarthritis and acute pancreatitis. Our previous studies have demonstrated the ability of MSM to differentiate stem cells from human exfoliated deciduous (SHED) teeth into osteoblast-like cells. This study examined the systemic effect of MSM in 36-week-old aging C57BL/6 female mice in vivo by injecting MSM for 13 weeks. Serum analyses showed an increase in expression levels of bone formation markers [osteocalcin (OCN) and procollagen type 1 intact N-terminal propeptide (P1NP)] and a reduction in bone resorption markers [tartrate-resistant acid phosphatase (TRAP) and C-terminal telopeptide of type I collag (CTX-I)] in MSM-injected animals. Micro-computed tomographic images demonstrated an increase in trabecular bone density in mandibles. The trabecular bone density tended to be higher in the femur, although the increase was not significantly different between the MSM- and phosphate-buffered saline (PBS)-injected mice. In mandibles, an increase in bone density with a corresponding decrease in the marrow cavity was observed in the MSM-injected mice. Furthermore, immunohistochemical analyses of the mandibles for the osteoblast-specific marker – OCN, and the mesenchymal stem cell-specific marker – CD105 showed a significant increase and decrease in OCN and CD105 positive cells, respectively. Areas of bone loss were observed in the inter-radicular region of mandibles in control mice. However, this loss was considerably decreased due to stimulation of bone formation in response to MSM injection. In conclusion, our study has demonstrated the ability of MSM to induce osteoblast formation and function in vivo, resulting in increased bone formation in the mandible. Hence, the application of MSM and stem cells of interest may be the right combination in alveolar bone regeneration under periodontal or other related diseases that demonstrate bone loss.
It is known that estrogen deficiency increases osteoclast formation and activity. Autophagy, a cell survival pathway, has been shown to be crucial for osteoclast function. However, little is known about the effects of estrogen depletion on osteoclast autophagy. Here, we evaluated the effects of estrogen deficiency in the immunoexpression of autophagy mediators in alveolar bone osteoclasts of ovariectomized rats. Twelve adult female rats were ovariectomized (OVX-group) or SHAM-operated (SHAM-group). After three weeks, the rats were euthanized and maxillary fragments containing alveolar bone of the first molars were processed for light microscopy or transmission electron microscopy (TEM). Paraffin-sections were subjected to the TRAP method (osteoclast marker) or to the immunohistochemical detections of beclin-1, LC3α, and p62 (autophagy mediators); araldite-sections were processed for TEM. The number of TRAP-positive osteoclasts and the number of immunolabeled-multinucleated cells (MNCs) along the alveolar bone surface of the first molar were computed. The number of TRAP-positive osteoclasts and the number of beclin-1-, LC3α- and p62-immunolabelled osteoclasts were significantly higher in OVX-group than the SHAM-group. MNCs were frequently located juxtaposed to Howship lacunae along the alveolar bone surface, indicating that these cells are osteoclasts. TEM revealed osteoclasts exhibiting autophagosomes. Our data indicate that autophagy plays an important role during estrogen deficiency-induced osteoclastogenesis. Thus, our results contribute to a better understanding on the role of autophagy on osteoclasts under estrogenic deficiency, and reinforce the idea that modulation of autophagy may be a useful tool to inhibit excessive oral bone resorption in post-menopausal women.
Ineffective oral wound healing is detrimental to patients’ oral health–related quality of life. Delineating the cellular mechanisms involved in optimal healing will elicit better approaches to treating patients with compromised healing. Osteal macrophages have recently emerged as important positive regulators of bone turnover. The contributions of macrophages to long bone healing have been studied, but their role in oral osseous wound healing following tooth extraction is less clear. Clodronate-loaded liposomes were used as a tool to deplete macrophages in C57BL/6J mice and assess oral osseous bone fill after extraction. In addition to macrophage ablation, osteoclast ablation occurred. Interestingly, depletion of macrophages and osteoclasts via clodronate treatment had differential effects based on skeletal location. In the nonwounded tibiae, clodronate treatment significantly increased CD68+ cells and decreased F4/80+ cells in the marrow, which correlated with increased trabecular bone volume fraction after 7 and 14 d. Serum formation and resorptive markers P1NP and TRAcP 5b were decreased as were tibial TRAP+ osteoclasts. In healing extraction sockets, clodronate treatment increased extraction socket trabecular bone thickness at 14 d, which correlated with decreased TRAP+ osteoclasts and F4/80+ macrophages. Conversely, nonwounded maxillary interseptal bone was unaffected by clodronate treatment. Furthermore, the increase in extraction socket bone fill with clodronate was less than the large increase in trabecular bone observed in a nonwounded long bone. These data suggest a temporal and spatial specificity in the roles of macrophages and osteoclasts in normal turnover and healing. © International & American Associations for Dental Research 2019.
Bisphosphonates (BPs) are widely used in the treatment of several bone diseases, such as osteoporosis and cancers that have metastasized to bone, by virtue of their ability to inhibit osteoclastic bone resorption. Previously, it was shown that osteoclasts present at different bone sites have different characteristics. We hypothesized that BPs could have distinct effects on different populations of osteoclasts and their precursors, for example as a result of a different capacity to endocytose the drugs. To investigate this, bone marrow cells were isolated from jaw and long bone from mice and the cells were primed to differentiate into osteoclasts with the cytokines M-CSF and RANKL. Before fusion occurred, cells were incubated with fluorescein-risedronate (FAM-RIS) for 4 or 24hours and uptake was determined by flow cytometry. We found that cultures obtained from the jaw internalized 1.7 to 2.5 times more FAM-RIS than long-bone cultures, both after 4 and 24hours, and accordingly jaw osteoclasts were more susceptible to inhibition of prenylation of Rap1a after treatment with BPs for 24hours. Surprisingly, differences in BP uptake did not differentially affect osteoclastogenesis. This suggests that jaw osteoclast precursors are less sensitive to bisphosphonates after internalization. This was supported by the finding that gene expression of the anti-apoptotic genes Bcl-2 and Bcl-xL was higher in jaw cells than long bone cells, suggesting that the jaw cells might be more resistant to BP-induced apoptosis. Our findings suggest that bisphosphonates have distinct effects on both populations of osteoclast precursors and support previous findings that osteoclasts and precursors are bone-site specific. This study may help to provide more insights into bone-site-specific responses to bisphosphonates.
Strontium ranelate (SrRan) is used to decrease the risk of bone fractures. Any factor that alters the release of paracrine signals by osteocytes in response to mechanical stimuli potentially affects bone mass and structure, and thus fracture resistance. We hypothesized that SrRan affects paracrine signaling from mechanically-stimulated osteocytes towards osteoclast-precursors and osteoblasts. MLO-Y4 osteocytes were cultured for 24h with SrRan (0.1-3mM) and either or not mechanically stimulated by pulsating fluid flow (PFF; 0.7±0.3Pa, 5Hz) for 60min. Nitric oxide (NO) and prostaglandin E(2) (PGE(2)) release, and expression of mechanoresponsive genes were quantified. Conditioned medium (CM) from osteocytes was added to mouse bone marrow cells for 7days to assess osteoclastogenesis, or MC3T3-E1 osteoblasts for 4-16days to measure osteogenic gene expression. SrRan (3mM) enhanced NO and PGE(2) release to the same extent in static osteocytes (NO: 1.6-fold; PGE(2): 2.8-fold) and PFF-stimulated osteocytes (NO: 1.3-fold; PGE(2): 2.6-fold). CM from PFF-treated osteocytes without SrRan enhanced Ki67 expression but reduced Runx2 and Ocn expression in osteoblasts. This effect on gene expression was not observed with CM obtained from osteocytes treated with the combination of PFF and 3mM SrRan. CM from PFF-treated osteocytes inhibited osteoclastogenesis by 1.9-fold. The combination of PFF and 3mM SrRan reduced osteocyte-stimulated osteoclastogenesis even more strongly (4.3-fold). In conclusion, SrRan affects paracrine signaling between mechanically-stimulated MLO-Y4 osteocytes and both osteoblasts and osteoclast precursors. The positive effects of SrRan on bone fracture resistance may thus be partly explained by altered paracrine signaling by osteocytes.
Osteoclasts are known to be important for the coupling process between bone resorption and formation. The aim of this study was to address when osteoclasts are anabolically active. Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium was collected from macrophages, pre-osteoclasts, and mature functional or non-resorbing osteopetrotic osteoclasts on either bone, plastic, decalcified bone or dentine with or without diphyllin, E64 or GM6001. Osteoclasts numbers were measured by TRACP activity. Bone resorption was evaluated by CTX-I and calcium release. The osteoblastic cell line 2T3 was treated with 50% of CM or non-CM for 12days. Bone formation was assessed by Alizarin Red extraction. CM from mature osteoclasts induced bone formation, while CM from macrophages did not. Non-resorbing osteoclasts generated from osteopetrosis patients showed little resorption, but still an induction of bone formation by osteoblasts. Mimicking the reduction in bone resorption using the V-ATPase inhibitor Diphyllin, the cysteine proteinase inhibitor E64 and the MMP-inhibitor GM6001 showed that CM from diphyllin and E64 treated osteoclasts showed reduced ability to induce bone formation compared to CM from vehicle treated osteoclasts, while CM from GM6001 treated osteoclasts equaled vehicle CM. Osteoclasts on either dentine or decalcified bone showed strongly attenuated anabolic capacities. In conclusion, we present evidence that osteoclasts, both dependent and independent of their resorptive activity, secrete factors stimulating osteoblastic bone formation.
Background and objective:
Increasing evidence has shown the presence of transient receptor potential vanilloid-1 (TRPV1) in a variety of nonneuronal tissues; however, the function of TRPV1 in these cells is not well understood. In this study, we aimed to investigate the expression and function of TRPV1 in human periodontal ligament (HPDL) cells. As HPDL cells are known to play an important role in the bone-remodeling process, we hypothesized that TRPV1 might be implicated in the regulation of osteoprotegerin (OPG) and RANKL expression.
Material and methods:
TRPV1 expression was examined by western blot analysis. The function of TRPV1 was studied using capsaicin, a well-known TRPV1 agonist. RT-PCR was performed to study the expression of OPG and RANKL mRNAs. The expression of OPG and RANKL proteins was analyzed by ELISA and western blotting, respectively. The mechanisms of capsaicin-induced OPG expression in HPDL cells were studied using inhibitors.
Results:
In this study we found that TRPV1 was present in HPDL cells. Treatment with capsaicin induced OPG expression in a dose-dependent manner but did not affect the expression of RANKL. The increase of the OPG/RANKL ratio was also found in human osteoblasts, but not in MC3T3-E1 cells, a mouse osteoblastic cell line, suggesting species specificity. Capsazepine, the competitive TRPV1 antagonist, significantly abolished the effect of capsaicin on OPG expression in HPDL cells. In addition, studies investigating the effects of a calcium chelator and a phospholipase C inhibitor indicated that calcium ions and phospholipase C were required for the induction. Interestingly, capsaicin was able to increase the OPG/RANKL ratio, even in the presence of prostaglandin E2, a potent inducer of RANKL.
Conclusion:
Our study demonstrates that activation of TRPV1 leads to an increase of the OPG/RANKL ratio in HPDL cells. These findings suggest the novel function of TRPV1 in periodontal tissues, at least, as the regulator of the OPG/RANKL axis.
Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is not completely understood and difficult to treat. Even though the occurrence of BRONJ is mainly related to the therapy with intravenous bisphosphonates (BPs), this article reports a case of long-term oral BP use and BRONJ occurrence. In addition, a literature review provides some additional information about BPs, BRONJ, and also a guideline for the prevention and treatment of BRONJ stages. A 79-year-old patient presented intense and persistent pain, purulent secretion, and exposed bone at the right side of the lower jaw. Thus, she was submitted to anamnesis, radiologic and tomographic examinations, and bacterial culture and sensitivity tests. These procedures were followed by surgical debridement of the bone and surrounding tissues/cyst and antibiotic prescription and histopathologic analysis of the fragments. Together, the examinations performed showed the occurrence of stage 2 BRONJ. Moreover, the antibiotic prescription, discontinuation of oral BP, and surgical procedures ensure that the patient had no more symptoms. Therefore, considering the presented case, we believe that an accurate approach is promising to diagnose and treat stage 2 BRONJ and other associated pathologic findings.
Osteoclasts, the multinucleated bone-resorbing cells, arise through fusion of precursors from the myeloid lineage. However, not all osteoclasts are alike; osteoclasts at different bone sites appear to differ in numerous respects. We investigated whether bone marrow cells obtained from jaw and long bone differed in their osteoclastogenic potential. Bone marrow cells from murine mandible and tibiae were isolated and cultured for 4 and 6 days on plastic or 6 and 10 days on dentin. Osteoclastogenesis was assessed by counting the number of TRAP(+) multinucleated cells. Bone marrow cell composition was analyzed by FACS. The expression of osteoclast- and osteoclastogenesis-related genes was studied by qPCR. TRAP activity and resorptive activity of osteoclasts were measured by absorbance and morphometric analyses, respectively. At day 4 more osteoclasts were formed in long bone cultures than in jaw cultures. At day 6 the difference in number was no longer observed. The jaw cultures, however, contained more large osteoclasts on plastic and on dentin. Long bone marrow contained more osteoclast precursors, in particular the myeloid blasts, and qPCR revealed that the RANKL:OPG ratio was higher in long bone cultures. TRAP expression was higher for the long bone cultures on dentin. Although jaw osteoclasts were larger than long bone osteoclasts, no differences were found between their resorptive activities. In conclusion, bone marrow cells from different skeletal locations (jaw and long bone) have different dynamics of osteoclastogenesis. We propose that this is primarily due to differences in the cellular composition of the bone site-specific marrow.
Bone resorption by osteoclasts depends on the assembly of a specialized, actin-rich adhesive 'sealing zone' that delimits the area designed for degradation. In this study, we show that the level of roughness of the underlying adhesive surface has a profound effect on the formation and stability of the sealing zone and the associated F-actin. As our primary model substrate, we use 'smooth' and 'rough' calcite crystals with average topography values of 12 nm and 530 nm, respectively. We show that the smooth surfaces induce the formation of small and unstable actin rings with a typical lifespan of approximately 8 minutes, whereas the sealing zones formed on the rough calcite surfaces are considerably larger, and remain stable for more than 6 hours. It was further observed that steps or sub-micrometer cracks on the smooth surface stimulate local ring formation, raising the possibility that similar imperfections on bone surfaces may stimulate local osteoclast resorptive activity. The mechanisms whereby the physical properties of the substrate influence osteoclast behavior and their involvement in osteoclast function are discussed.
Tumor cells in the bone microenvironment are able to initiate a vicious cycle of bone degradation by mobilizing osteoclasts, multinucleated cells specialized in bone degradation. c-Src is highly expressed both in tumors and in osteoclasts. Therefore, drugs like AZD0530, designed to inhibit Src activity, could selectively interfere with both tumor and osteoclast activity. Here we explored the effects of AZD0530 on human osteoclast differentiation and activity. The effect on osteoclasts formed in vivo was assessed in mouse fetal calvarial explants and in isolated rabbit osteoclasts, where it dose-dependently inhibited osteoclast activity. Its effect on formation and activity of human osteoclasts in vitro was determined in cocultures of human osteoblasts and peripheral blood mononuclear cells. AZD0530 was most effective in inhibiting osteoclast-like cell formation when present at the onset of osteoclastogenesis, suggesting that Src activity is important during the initial phase of osteoclast formation. Formation of active phosphorylated c-Src, which was highly present in osteoclast-like cells in cocultures and in peripheral blood mononuclear cell monocultures, was significantly reduced by AZD0530. Furthermore, it reversibly prevented osteoclast precursor migration from the osteoblast layer to the bone surface and subsequent formation of actin rings and resorption pits. These data suggest that Src is pivotal for the formation and activity of human osteoclasts, probably through its effect on the distribution of the actin microfilament system. The reversible effect of AZD0530 on osteoclast formation and activity makes it a promising candidate to temper osteoclastic bone degradation in bone diseases with enhanced osteoclast activity such as osteolytic metastatic bone disease.
Cells of the myeloid lineage at various stages of maturity can differentiate into multinucleated osteoclasts. Yet, it is unclear which developmental stages of this lineage are more prone to become osteoclasts than others. We investigated the osteoclastogenic potential of three successive stages of myeloid development isolated from mouse bone marrow. Early blasts (CD31hi/Ly-6C-), myeloid blasts (CD31+/Ly-6C+), and monocytes (CD31-/Ly-6Chi), as well as unfractionated marrow cells, were cultured in the presence of M-CSF and receptor activator of NF-B ligand (RANKL), and the differentiation toward multinucleated cells and their capacity to resorb bone was assessed. Myeloid blasts developed rapidly into multinucleated cells; in only 4 days, maximal numbers were reached, whereas the other fractions required 8 days to reach maximal numbers. Bone resorption was observed after 6 (myeloid blasts and monocyte-derived osteoclasts) and 8 (early blast-derived osteoclasts) days. This difference in kinetics in osteoclast-forming capacity was confirmed by the analysis of osteoclast-related genes. In addition, the myeloid blast fraction proved to be most sensitive to M-CSF and RANKL, as assessed with a colony-forming assay. Our results show that osteoclasts can develop from all stages of myeloid differentiation, but myeloid blasts are equipped to do so within a short period of time.
Data in the literature suggest that site-specific differences exist in the skeleton with respect to digestion of bone by osteoclasts. Therefore, we investigated whether bone resorption by calvarial osteoclasts (intramembranous bone) differs from resorption by long bone osteoclasts (endochondral bone). The involvement of two major classes of proteolytic enzymes, the cysteine proteinases (CPs) and matrix metalloproteinases (MMPs), was studied by analyzing the effects of selective low molecular weight inhibitors of these enzymes on bone resorption. Mouse tissue explants (calvariae and long bones) as well as rabbit osteoclasts, which had been isolated from both skeletal sites and subsequently seeded on bone slices, were cultured in the presence of inhibitors and resorption was analyzed. The activity of the CP cathepsins B and K and of MMPs was determined biochemically (CPs and MMPs) and enzyme histochemically (CPs) in explants and isolated osteoclasts. We show that osteoclastic resorption of calvarial bone depends on activity of both CPs and MMPs, whereas long bone resorption depends on CPs, but not on the activity of MMPs. Furthermore, significantly higher levels of cathepsin B and cathepsin K activities were expressed by long bone osteoclasts than by calvarial osteoclasts. Resorption of slices of bovine skull or cortical bone by osteoclasts isolated from long bones was not affected by MMP inhibitors, whereas resorption by calvarial osteoclasts was inhibited. Inhibition of CP activity affected the resorption by the two populations of osteoclasts in a similar way. We conclude that this is the first report to show that significant differences exist between osteoclasts of calvariae and long bones with respect to their bone resorbing activities. Resorption by calvarial osteoclasts depends on the activity of CPs and MMPs, whereas resorption by long bone osteoclasts depends primarily on the activity of CPs. We hypothesize that functionally different subpopulations of osteoclasts, such as those described here, originate from different sets of progenitors.
Interleukin-1 (IL-1) and tumor necrosis factor (TNF) mediate bone resorption in a variety of diseases affecting bone. Like TNF, IL-1 is secreted by osteoclast precursors (OCPs), but unlike TNF, it does not induce osteoclast formation directly from OCPs in vitro. TNF induces IL-1 expression and activates c-Fos, a transcription factor required in OCPs for osteoclast formation. Here, we examined whether IL-1 can induce osteoclast formation directly from OCPs overexpressing c-Fos and whether interaction with bone matrix affects OCP cytokine expression. We infected OCPs with c-Fos or green fluorescent protein retrovirus, cultured them with macrophage colony-stimulating factor and IL-1 on bone slices or plastic dishes, and assessed osteoclast and resorption pit formation and expression of IL-1 by OCPs. We used a Transwell assay to determine whether OCPs secrete IL-1 when they interact with bone matrix. IL-1 induced osteoclast formation directly from c-Fos-expressing OCPs on plastic. c-Fos-expressing OCPs formed osteoclasts spontaneously on bone slices without addition of cytokines. OCPs on bone secreted IL-1, which induced osteoclast formation from c-Fos-expressing OCPs in the lower Transwell dishes. The bone matrix proteins dentin sialoprotein and osteopontin, but not transforming growth factor-beta, stimulated OCP expression of IL-1 and induced c-Fos-expressing OCP differentiation into osteoclasts. Osteoclasts eroding inflamed joints have higher c-Fos expression compared with osteoclasts inside bone. We conclude that OCPs expressing c-Fos may induce their differentiation directly into osteoclasts by an autocrine mechanism in which they produce IL-1 through interaction with bone matrix. TNF could induce c-Fos expression in OCPs at sites of inflammation in bone to promote this autocrine mechanism and thus amplify bone loss.
The multinucleated osteoclast has a unique function: degradation of mineralized tissues. It is generally taken that all osteoclasts are alike, independent of the skeletal site where they exert their activity. Recent data, however, question this view as they show that osteoclasts at different bony sites appear to differ, for example in the machinery responsible for resorption. Support for the notion that there may be heterogeneity in osteoclasts is obtained from studies in which osteoclast activity is inhibited and from observations in osteopetrosis and inflammatory bone conditions. In this review we discuss the available evidence and propose the existence of bone-site-specific osteoclast heterogeneity.
Osteoclasts are bone-resorbing cells formed by fusion of mononuclear precursors. The matrix proteins, fibronectin (FN), vitronectin (VN), and osteopontin (OPN) are implicated in joint destruction and interact with osteoclasts mainly through integrins. To assess the effects of these matrix proteins on osteoclast formation and activity, we used RAW 264.7 (RAW) cells and mouse splenocytes differentiated into osteoclasts on tissue culture polystyrene (TCP) or osteologic™ slides pre-coated with 0.01-20 µg/ml FN, VN, and OPN. At 96 h, osteoclast number and multinucleation were decreased on VN and FN compared to OPN and TCP in both RAW and splenocytes cell cultures. When early differentiation was assessed, VN but not FN decreased cytoplasmic tartrate-resistant acid phosphatase activity and pre-osteoclast number at 48 h. OPN had the opposite effect to FN on osteoclast formation. When RAW cells were differentiated on OPN and treated by FN and OPN, osteoclast number only in the FN treated group was 40-60% lower than the control, while the total number of nuclei was unchanged, suggesting that FN delays osteoclast fusion. In contrast to its inhibitory effect on osteoclastogenesis, FN increased resorption by increasing both osteoclast activity and the percentage of resorbing osteoclasts. This was accompanied by an increase in nitric oxide (NO) levels and interleukin-1β (IL-1β). IL-1β production was inhibited using the NO-synthase inhibitor only on FN indicating a FN-specific cross-talk between NO and IL-1β signaling pathways. We conclude that FN upregulates osteoclast activity despite inhibiting osteoclast formation and that these effects involve NO and IL-1β signaling.
Mice lacking HIP/RPL29, a component of the ribosomal machinery, display increased bone fragility. To understand the effect of sub-efficient protein synthetic rates on mineralized tissue quality, we performed dynamic and static histomorphometry and examined the mineral properties of both bones and teeth in HIP/RPL29 knock-out mice using Fourier transform infrared imaging (FTIRI). While loss of HIP/RPL29 consistently reduced total bone size, decreased mineral apposition rates were not significant, indicating that short stature is not primarily due to impaired osteoblast function. Interestingly, our microspectroscopic studies showed that a significant decrease in collagen crosslinking during maturation of HIP/RPL29-null bone precedes an overall enhancement in the relative extent of mineralization of both trabecular and cortical adult bones. This report provides strong genetic evidence that ribosomal insufficiency induces subtle organic matrix deficiencies which elevates calcification. Consistent with the HIP/RPL29-null bone phenotype, HIP/RPL29-deficient teeth also showed reduced geometric properties accompanied with relative increased mineral densities of both dentin and enamel. Increased mineralization associated with enhanced tissue fragility related to imperfection in organic phase microstructure evokes defects seen in matrix protein-related bone and tooth diseases. Thus, HIP/RPL29 mice constitute a new genetic model for studying the contribution of global protein synthesis in the establishment of organic and inorganic phases in mineral tissues.
Osteoclasts are multinucleated cells specialized in degrading bone and characterized by high expression of the enzymes tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CtsK). Recent studies show that osteoclasts exhibit phenotypic differences depending on their anatomical site of action. Using immunohistochemistry, RT-qPCR, FPLC chromatography and immunoblotting, we compared TRAP expression in calvaria and long bone. TRAP protein and enzyme activity levels were higher in long bones compared to calvaria. In addition, proteolytic processing of TRAP was more extensive in long bones than calvaria which correlated with higher cysteine proteinase activity and protein expression of CtsK. These two types of bones also exhibited a differential expression of monomeric TRAP and CtsK isoforms. Analysis of CtsK(-/-) mice revealed that CtsK is involved in proteolytic processing of TRAP in calvaria. Moreover, long bone osteoclasts exhibited higher expression of not only TRAP and CtsK but also of the membrane markers CD68 and CD163. The results suggest that long bone osteoclasts display an augmented osteoclastic phenotype with stronger expression of both membranous and secreted osteoclast proteins.
Extracellular acidification by osteoclasts is essential to bone resorption. During proton pumping, intracellular pH (pH(i)) is thought to be kept at a near-neutral level by chloride/bicarbonate exchange. Here we show that the Na(+)-independent chloride/bicarbonate anion exchanger 2 (Ae2) is relevant for this process in the osteoclasts from the long bones of Ae2(a,b)(-/-) mice (deficient in the main isoforms Ae2a, Ae2b(1), and Ae2b(2)). Although the long bones of these mice had normal numbers of multinucleated osteoclasts, these cells lacked a ruffled border and displayed impaired bone resorption activity, resulting in an osteopetrotic phenotype of long bones. Moreover, in vitro osteoclastogenesis assays using long-bone marrow cells from Ae2(a,b)(-/-) mice suggested a role for Ae2 in osteoclast formation, as fusion of preosteoclasts for the generation of active multinucleated osteoclasts was found to be slightly delayed. In contrast to the abnormalities observed in the long bones, the skull of Ae2(a,b)(-/-) mice showed no alterations, indicating that calvaria osteoclasts may display normal resorptive activity. Microfluorimetric analysis of osteoclasts from normal mice showed that, in addition to Ae2 activity, calvaria osteoclasts--but not long-bone osteoclasts--possess a sodium-dependent bicarbonate transporting activity. Possibly, this might compensate for the absence of Ae2 in calvaria osteoclasts of Ae2(a,b)(-/-) mice.
Osteoclasts of the peripheral portions of the endocranial aspect of young rat parietal and frontal bones were studied by scanning electron microscopy of glutaraldehyde fixed, critical point dried specimens. These studies show osteoclasts to have a much more complicated form than has previously been realised. Extensively branching, elongated, smooth-surfaced cells, which are for the most part elevated above the level of the surrounding bone matrix surface and sometimes above portions of osteoblasts or other osteoclasts, were identified as motile non-resorbing cells. Portions of the former and other entire cells may be embowered in Howship's lacunae, have microvilli on their dorsal surface, and are surrounded by a serrated border of microprojections which have an apparently firm attachment to the matrix surface. Osteoclasts in short term culture show additional free surface ruffles which are not encountered in specimens taken fresh from the animal. No evidence of recruitment of osteoblasts or osteocytes into osteoclasts was found. Disinterred osteocytes retained an ability to migrate from their lacunae on to surrounding bone matrix surface.
The cell-cell contact-induced behaviour of osteoclasts and osteoblasts in vitro was investigated by time-lapse videomicroscopy. Contact interactions of osteoclasts with autologous cells, derived mostly from chick but also from rat bones, included contact inhibition, failure of contact inhibition, contact guidance along stabilised edges of other cells, and underlapping of other cells. Message-mediated contact behaviour (MMCB) between osteoclasts and autologous osteoblastic cells resulted, after a time delay, in zeiosis of the osteoblast-like cell which could continue, or even begin, after the osteoclast broke contact, leading to retraction of the cell and occupation of its position by the osteoclast. MMCB may play a part in the breaching of the osteoblastic sheet by osteoclasts and, in general, in the malignant spread of neoplastic cells. Two or more osteoclasts were often joined by connecting and coordinating tubules (CCTs) of varied, and varying, lengths and widths. Osteoclasts could travel along the CCTs in both directions, or send nuclei through them. The CCTs became temporarily attached to the surface of other cells, or to the substrate, then acting as a temporary anchorage for orientation and for the return of the cell to the same spot. The dynamics of osteoclastic behaviour suggest that such a networking of osteoclasts is valuable for the 3D coordination of their role in bone turnover.
Recently, tumour necrosis factor-related activation-induced cytokine (TRANCE) was shown to be necessary for osteoclast formation. We now report that TGF(beta), a cytokine enriched in bone matrix, is also required. TGF(beta) not only powerfully synergized with TRANCE for induction of osteoclast-like cells (OCL) from bone marrow precursors and monocytes, but OCL formation was abolished by recombinant soluble TGF(beta) receptor II (TGF(beta)sRII). Preincubation in TGF(beta) was as effective as simultaneous incubation with TRANCE. TGF(beta)-preincubation enhanced OCL formation at least partly by preventing the development of resistance to OCL-induction that otherwise occurs when precursors are incubated in M-CSF. OCL formed in TRANCE also showed more rapid apoptosis than OCL in TRANCE plus TGF(beta). Like TGF(beta), incubation on bone matrix prolonged and enhanced the sensitivity of precursors to OCL-induction by TRANCE, and this was reversed by TGF(beta)sRII. Taken together, this data is compelling evidence for a model in which TGF(beta) in matrix or released from bone-lining or other cells maintains and enhances the osteoclast-forming potential of precursors as they migrate towards sites of cell-bound TRANCE. Thus, the specific circumstances necessary for osteoclast formation and survival are TRANCE expression on osteoblastic cells and TGF(beta) in bone.
Precursors of osteoclasts seeded on top of a confluent layer of osteoblasts/bone lining cells induced retraction of the latter cells. The (pre)osteoclasts then migrated in the formed cell-free areas and fused to form osteoclast-like cells. Retraction of the osteoblasts/bone lining cells proved to depend on activity of matrix metalloproteinases, and TGF-beta1 prevented the retraction.
It is well known that osteoblasts have a profound effect on (pre)osteoclasts in inducing the formation of bone-resorbing osteoclasts. Whether, on the other hand, (pre)osteoclasts also modulate osteoblast activity is largely unknown. Because osteoblasts/bone lining cells have to retract from the surface before resorption of bone by osteoclasts, we addressed the question of whether (pre)osteoclasts have the capacity to induce such an activity.
Rabbit calvarial osteoblasts/bone lining cells or periosteal fibroblasts were cultured until confluency, after which rabbit peripheral blood mononuclear cells (PBMCs) were seeded on top of them. The co-cultures were maintained for up to 15 days in the presence or absence of the cytokines transforming growth factor (TGF)-beta1 and TNF-alpha and selective inhibitors of matrix metalloproteinases and serine proteinases. The formation of cell-free areas and the number of TRACP+ multinucleated osteoclast-like cells were analyzed. In addition, formation of cell-free areas was analyzed in co-cultures of osteoblasts with mature osteoclasts.
The seeding of PBMCs on a confluent layer of osteoblasts/bone lining cells resulted in the following sequence of events. (1) A low number of PBMCs strongly attached to osteoblasts. 2) At these sites of contact, the osteoblasts retracted, thus forming cell-free areas. (3) The PBMCs invaded these areas and attached to the surface of the well, after which they fused and formed multinucleated TRACP+ osteoclast-like cells. Retraction was only seen if the cells were in direct contact; conditioned media from cultured PBMCs added to osteoblasts had no effect. Mature osteoclasts seeded on osteoblasts similarly induced retraction, but this retraction occurred at a much faster rate (within 2 days) than the retraction effectuated by the osteoclast precursors (after 8 days in co-culture). Inhibition of matrix metalloproteinase activity, but not of serine proteinases, strongly reduced retraction of the osteoblasts, thus indicating that this type of cell movement depends on the activity of matrix metalloproteinases. A similar inhibitory effect was found with TGF-beta1. TNF-alpha had no effect on osteoblast retraction but enhanced the formation of multinucleated osteoclast-like cells. Addition of PBMCs to confluent layers of periosteal fibroblasts resulted in similar phenomena as observed in co-cultures with osteoblasts. However, the cell-free areas proved to be significantly smaller, and the number of multinucleated cells formed within cell-free areas was three to four times lower.
Our results indicate that osteoclast precursors and mature osteoclasts have the capacity to modulate the activity of osteoblasts and that, yet unknown, membrane-bound signaling molecules are essential in inducing retraction of osteoblasts and the subsequent formation of cell-free areas.
It has been suggested that functional heterogeneity exists between osteoclasts from different bone sites. This could be exploited to design therapeutics that would selectively inhibit bone resorption only at compromised sites. To further investigate the existence of functional differences between osteoclasts from different bone sites we assessed whether osteoclasts isolated from intramembranous bone differ from osteoclasts isolated from endochondral bone in the extent that they utilize cysteine proteinases and matrix metalloproteinases to degrade the organic matrix of bone. The differential involvement of the two classes of proteases was assessed by analyzing dose-dependent effects of the matrix metalloproteinase inhibitor, CT-1746, and of the cathepsin inhibitor, E64, on bone resorption. Osteoclasts isolated from the scapula (intramembranous) and long bones (endochondral) of newborn New Zealand white rabbits were seeded on cortical bovine bone slices in the presence or absence of inhibitors. Resorptive activity was evaluated by measuring the number and area of resorption pits and by measuring the release of collagen degradation products in the culture medium. In the absence of inhibitors, scapular osteoclasts and long bone osteoclasts had similar activity based on these criteria. The resorptive activity of scapular osteoclasts was inhibited to a greater extent by the MMP inhibitor CT-1746 than by the cysteine proteinase inhibitor E64. Conversely, resorption by osteoclasts derived from long bones was inhibited to a greater degree by the cysteine proteinase inhibitor. These results strongly suggest that there are functional differences between dispersed osteoclasts derived from the scapula and long bones, with scapular osteoclasts utilizing matrix metalloproteinases to a greater extent than cysteine proteinases and long bone osteoclasts using cysteine proteinases to a greater extent than matrix metalloproteinases.
In vitro studies have shown that CD44 is involved in the fusion process of osteoclast precursor cells. Yet, in vivo studies do not support this, since an osteopetrotic phenotype has not been described for CD44 knock-out (CD44 k.o.) mice. This discrepancy may suggest that the role of CD44 in fusion may depend on the microenvironment of osteoclast formation. We investigated osteoclast formation of CD44 k.o. and wild-type mice under three conditions: in vitro, both on plastic and on bone and in vivo by analyzing osteoclast number, and size in long bones from wild-type and CD44 k.o. mice. Bone marrow cells from wild-type and CD44 k.o. mice were analyzed for their capacity to form osteoclasts on plastic and on bone in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). On plastic, the number of multinucleated tartrate resistant acid phosphatase (TRAP) positive cells in CD44 k.o. cultures was twofold higher than in wild-type cultures. On bone, however, equal numbers of osteoclasts were formed. Interestingly, the total number of osteoclasts formed on bone proved to be higher than on plastic for both genotypes, strongly suggesting that osteoclastogenesis was stimulated by the bone surface, and that CD44 is not required for osteoclast formation on bone. Functional analyses showed that bone resorption was similar for both genotypes. We further studied the osteoclastogenic potential of wild-type bone marrow cells in the presence of CD44 blocking antibodies. Osteoclastogenesis was not affected by these antibodies, a further indication that CD44 is not required for the formation of multinucleated cells. Finally, we analyzed the in vivo formation of osteoclasts by analyzing long bones from wild-type and CD44 k.o. mice. Morphometric analysis revealed no difference in osteoclast number, nor in number of nuclei per osteoclasts or in osteoclast size. Our in vitro experiments on plastic showed an enhanced formation of osteoclasts in the absence of CD44, thus suggesting that CD44 has an inhibitory effect on osteoclastogenesis. However, when osteoclasts were generated on bone, no differences in number of multinucleated cells nor in bone resorption were seen. These observations are in agreement with in vivo osteoclast characteristics, where no differences between wild-type and CD44 k.o. bones were encountered. Therefore, the modulating role of CD44 in osteoclast formation appears to depend on the microenvironment.
Tooth eruption depends on the presence of osteoclasts to create an eruption pathway through the alveolar bone. In diseases where osteoclast formation, or function is reduced, such as the various types of osteopetrosis, tooth eruption is affected. Diseases in which osteoclast formation or activity is increased, such as familiar expansile osteolysis and Paget's disease, are associated with dental abnormalities such as root resorption and premature tooth loss. Less is known about the origin of the dental problems in these conditions as there are no rodent models of these diseases as yet. In this short review, the genes currently known to be mutated in human osteoclast diseases will be reviewed and, where known, the effect of osteoclast dysfunction on dental development described. It will focus on human conditions and only mention rodent disease where no clear data in the human are available.
Murine BM was fractionated using a series of hematopoietic markers to characterize its osteoclast progenitor populations. We found that the early osteoclastogenic activity in total BM was recapitulated by a population of cells contained within the CD11b(-/low) CD45R- CD3- CD115high fraction.
Osteoclasts are of hematopoietic origin and they have been shown to share the same lineage as macrophages. We further characterized the phenotype of osteoclast progenitor populations in murine bone marrow (BM) by analyzing their cell surface markers.
We used fluorescence-activated cell sorting (FACS) to identify the subsets of BM cells that contained osteoclast progenitors. We fractionated BM according to several markers and cultured the sorted populations for a period of 2-6 days with macrophage-colony stimulating factor (M-CSF) and RANKL. The numbers of multinucleated osteoclast-like cells (OCLs) that formed in the cultures were counted.
We found that the CD45R- CD11b(-/low) population recapitulated the early osteoclastogenic activity of total BM. In addition, although previous experiments indicated that osteoclastogenic activity was enriched within the CD45R+ population, we found that highly purified CD45R+ BM was incapable of differentiating into osteoclasts in vitro. We also found that CD45R- CD11b(high) BM cells were an inefficient source of osteoclast progenitors. However, CD11b was transiently upregulated by cells of the CD45R- CD11b(-/low) fraction early (within 24 h) during culture with M-CSF. Finally, further fractionation of BM using CD115 and CD117 showed that, as osteoclast precursor cells matured, they downregulate CD117 but remain CD115+. Curiously, pure populations of CD117- (CD115high) cells isolated fresh from BM have low osteoclastogenic activity in vitro.
We provided a refined analysis of the precise subpopulations of murine BM that are capable of differentiating into OCLs in vitro when treated with M-CSF and RANKL.
Osteoclastic bone degradation involves the activity of cathepsin K. We found that in addition to this enzyme other, yet unknown, cysteine proteinases participate in digestion. The results support the notion that osteoclasts from different bone sites use different enzymes to degrade the collagenous bone matrix.
The osteoclast resorbs bone by lowering the pH in the resorption lacuna, which is followed by secretion of proteolytic enzymes. One of the enzymes taken to be essential in resorption is the cysteine proteinase, cathepsin K. Some immunolabeling and enzyme inhibitor data, however, suggest that other cysteine proteinases and/or proteolytic enzymes belonging to the group of matrix metalloproteinases (MMPs) may participate in the degradation. In this study, we investigated whether, in addition to cathepsin K, other enzymes participate in osteoclastic bone degradation.
In bones obtained from mice deficient for cathepsin K, B, or L or a combination of K and L, the bone-resorbing activity of osteoclasts was analyzed at the electron microscopic level. In addition, bone explants were cultured in the presence of different selective cysteine proteinase inhibitors and an MMP inhibitor, and the effect on resorption was assessed. Because previous studies showed differences in resorption by calvarial osteoclasts compared with those present in long bones, in all experiments, the two types of bone were compared. Finally, bone extracts were analyzed for the level of activity of cysteine proteinases and the effect of inhibitors hereupon.
The analyses of the cathepsin-deficient bone explants showed that, in addition to cathepsin K, calvarial osteoclasts use other cysteine proteinases to degrade bone matrix. It was also shown that, in the absence of cathepsin K, long bone osteoclasts use MMPs for resorption. Cathepsin L proved to be involved in the MMP-mediated resorption of bone by calvarial osteoclasts; in the absence of this cathepsin, calvarial osteoclasts do not use MMPs for resorption. Selective inhibitors of cathepsin K and other cysteine proteinases showed a stronger effect on calvarial resorption than on long bone resorption.
Our findings suggest that (1) cathepsin K-deficient long bone osteoclasts compensate the lack of this enzyme by using MMPs in the resorption of bone matrix; (2) cathepsin L is involved in MMP-mediated resorption by calvarial osteoclasts; (3) in addition to cathepsin K, other, yet unknown, cysteine proteinases are likely to participate in skull bone degradation; and finally, (4) the data provide strong additional support for the existence of functionally different bone-site specific osteoclasts.
The mammalian skeleton consists of bones that are formed in two different ways: long bones via endochondral ossification and flat bones via intramembranous ossification. These different formation modes may result in differences in the composition of the two bone types. Using the 2D-difference in gel electrophoresis technique and mass spectrometry, we analyzed the composition of murine mineral-associated proteins of calvaria and long bone. Considerable differences in protein composition were observed. Flat bones (calvariae) contained more soluble collagen (8x), pigment epithelium derived factor (3x) and osteoglycin (4x); whereas long bones expressed more chondrocalcin (3x), thrombospondin- 1 (4x), fetuin (4x), secreted phosphoprotein 24 (3x), and thrombin (7x). Although cystatin motifs containing proteins, such as secreted phosphoprotein 24 and fetuin are highly expressed in long bone, they did not inhibit the activity of the cysteine proteinases cathepsin B and K. The solubility of collagen differed which coincided with differences in collagen crosslinking, long bone containing 3x more (hydroxylysine)-pyridinoline. The degradation of long bone collagen by MMP2 (but not by cathepsin K) was impaired. These differences in collagen crosslinking may explain the differences in the proteolytic pathways osteoclasts use to degrade bone. Our data demonstrate considerable differences in protein composition of flat and long bones and strongly suggest functional differences in formation, resorption, and mechanical properties of these bone types.
Osteoclast heterogeneity: lessons from osteopetrosis and inflammatory conditions
- V Everts
- T J De Vries
- M H Helfrich
V. Everts, T.J. de Vries, M.H. Helfrich, Osteoclast heterogeneity: lessons from
osteopetrosis and inflammatory conditions, Biochim. Biophys. Acta 1792
(2009) 757-765.
Osteoclast heterogeneity: lessons from osteopetrosis and inflammatory conditions
- Everts