Absence Of Osteoblasts Research Articles

The role of secreted osteoclastogenic factor of activated T cells in bone remodeling.

The process of bone remodeling is connected with the regulated balance between bone cell populations (including bone-forming osteoblasts, bone-resorbing osteoclasts, and the osteocyte). And the mechanism of bone remodeling activity is related to the major pathway, receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) signaling axis. Recently, researchers have found a novel cytokine secreted by activated T cells, which is related to osteoclastogenesis in the absence of osteoblasts or RANKL, leading to bone destruction. They name it the secreted osteoclastogenic factor of activated T cells (SOFAT). SOFAT has been proven to play an essential role in bone remodeling, like mediating the bone resorption in rheumatoid arthritis (RA) and periodontitis. In this review, we outline the latest research concerning SOFAT and discuss the characteristics, location, and regulation of SOFAT. We also summarize the clinical progress of SOFAT and assume the future therapeutic target in some diseases related to bone remodeling.

Bioinspired Mineralization Using Chondrocyte Membrane Nanofragments.

Biomineralization involves complex processes and interactions between organic and inorganic matters, which are controlled in part by the cells. The objectives of this study were, first, to perform a systematic and ultrastructural investigation of the initial mineral formation during secondary ossification center of mouse femur based on material science and biology viewpoint, and then develop novel biomaterials for mineralization based on the in vivo findings. First, we identified the very initial mineral deposition at postnatal day 5 (P5) at the medial side of femur epiphysis by nanocomputed tomography. Initial minerals were found in the surroundings of hypertrophic chondrocytes. Interestingly, histological and immunohistochemical analyses showed that initial mineralization until P6 was based on chondrocyte activity only, i.e., it occurred in the absence of osteoblasts. Moreover, electron microscopy-based ultrastructural analysis showed that cell-secreted matrix vesicles were absent in the early steps of osteoblast-independent endochondral ossification. Instead, chondrocyte membrane nanofragments were found in the fibrous matrix surrounding the hypertrophic chondrocytes. EDS analysis and electron diffraction study indicated that cell membrane nanofragments were not mineralized material, and could be the nucleation site for the newly formed calcospherites. The phospholipids in the cell membrane nanofragments could be a source of phosphate for subsequent calcium phosphate formation, which initially was amorphous, and later transformed into apatite crystals. Finally, artificial cell nanofragments were synthesized from ATDC5 chondrogenic cells, and in vitro assays showed that these nanofragments could promote mineral formation. Taken together, these results indicated that cell membrane nanofragments were the nucleation site for mineral formation, and could potentially be used as material for manipulation of biomineralization.

Abstract 5930: Osteoblasts protect AML cells from cytarabine-induced death

Abstract The hematological malignancy acute myeloid leukemia (AML) interacts closely with osteoblasts within the protective bone marrow microenvironment. The bone marrow microenvironment protects tumor cells from chemotherapies, which can prevent sufficient eradication of tumor cells. To study the role of osteoblasts in the bone marrow microenvironment, our lab utilized a co-culture model of osteoblasts (MC3T3 osteoblast cell line) and acute myelogenous leukemia cells (KG1a AML cell line or AML patient samples from bone marrow aspirates). Osteoblasts were cultured with AML cells, or AML cells were cultured alone; AML cells were challenged with the standard chemotherapeutic agent cytarabine (Ara-C) at doses of 0µM, 0.1µM, 0.5µM, 1µM, 5µM, or 10µM in the presence or absence of osteoblasts; and AML cells were assayed by flow cytometry to assess cell death via annexin-V staining. Our lab has previously found that differentiating osteoblasts protect AML cells from an apoptosis inducing agent naturally present in the bone marrow. We now show that osteoblasts are also capable of protecting AML cells from the standard chemotherapeutic cytarabine. In addition, we have found that treatment of osteoblasts with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) prevents these treated osteoblasts from protecting AML cells from cytarabine treatment, which is consistent with our lab’s previous findings that HDACi treatment inhibits protection of AML cells from an apoptosis inducing agent naturally present in the bone marrow. We have preliminary data that indicates that TAZS89A over-expression, a constitutively active form of TAZ, which is a transcriptional modulator that regulates osteoblast differentiation, may be sufficient to inhibit osteoblast mediated protection of AML cells from cytarabine. This finding would be consistent with the HDACi manipulated Nherf1-protein phosphatase 1α-TAZ signaling pathway that we have previously found to be sufficient to inhibit protection of AML cells from an apoptosis inducing agent naturally present in the bone marrow. Overall, these studies have delivered insights into the role of osteoblasts in protecting AML cells from chemotherapy in the bone marrow microenvironment and begun the characterization of mechanisms and targets responsible for the protective effects of osteoblasts. Manipulating differentiating osteoblasts within the bone marrow microenvironment therapeutically could aid in more complete destruction of the tumor cell burden and improve patient survival. Citation Format: Rosalie M. Sterner, Kimberly N. Kremer, Amel Dudakovic, Jennifer J. Westendorf, Andre J. van Wijnen, Karen E. Hedin. Osteoblasts protect AML cells from cytarabine-induced death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5930. doi:10.1158/1538-7445.AM2017-5930

Effect of low-intensity pulsed ultrasound on the activity of osteoclasts: An in vitro study

ObjectiveThe objective of this in vitro study was to evaluate the effect of low-intensity pulsed ultrasound on the resorption activity of osteoclast cell cultures. DesignRAW 264.7 cells were cultured and seeded over plates that were pre-coated with a synthetic carbonate apatite, and marked with fluoresceinamine-labeled sodium chondroitin polysulfate. Plates were randomly divided into 4 groups according to the treatment assigned to each one of them: NO RANKL (no RANK-L addition and no ultrasound application), NO LIPUS (addition of RANK-L and no ultrasound application), LIPUS 10 (addition of RANK-L and 10min of ultrasound application per day), and LIPUS 20 (addition of RANK-L and 20min of ultrasound application per day). The ultrasound device produced 1.5MHz pulses with a repetition rate of 1kHz and intensity of 30mW/cm2. The experiment extended for one week and afterwards, resorption activity was evaluated according to the fluorescence intensity analysis and pit resorption measurements (number of pits and mean area). ResultsOur experiment consistently demonstrated that low-intensity pulsed ultrasound application enhanced osteoclasts resorptive activity. In addition, it was demonstrated that when daily ultrasound application lasted longer (20min) the resorption was the highest. Results obtained from both evaluation methods were reasonably coherent. ConclusionsLow-intensity pulsed ultrasound increases osteoclast resorptive activity in the absence of osteoblasts. This effect seems to be influenced by ultrasound treatment time. Future research might be directed to investigate osteoclast response to different ultrasound application protocols (frequencies and intensities) and potential cellular mechanisms.

Modulation of hematopoietic progenitor cell fate in vitro by varying collagen oligomer matrix stiffness in the presence or absence of osteoblasts

To recreate the in vivo hematopoietic cell microenvironment or niche and to study the impact of extracellular matrix (ECM) biophysical properties on hematopoietic progenitor cell (HPC) proliferation and function, mouse bone-marrow derived HPC (Lin-Sca1+cKit+/(LSK) were cultured within three-dimensional (3D) type I collagen oligomer matrices. To generate a more physiologic milieu, 3D cultures were established in both the presence and absence of calvariae-derived osteoblasts (OB). Collagen oligomers were polymerized at varying concentration to give rise to matrices of different fibril densities and therefore matrix stiffness (shear storage modulus, 50–800Pa). Decreased proliferation and increased clonogenicity of LSK cells was associated with increase of matrix stiffness regardless of whether OB were present or absent from the 3D culture system. Also, regardless of whether OB were or were not added to the 3D co-culture system, LSK within 800Pa collagen oligomer matrices maintained the highest percentage of Lin-Sca1+ cells as well as higher percentage of cells in quiescent state (G0/G1) compared to 50Pa or 200Pa matrices. Collectively, these data illustrate that biophysical features of collagen oligomer matrices, specifically fibril density-induced modulation of matrix stiffness, provide important guidance cues in terms of LSK expansion and differentiation and therefore maintenance of progenitor cell function.

AB0097 Th17 Cytokines Regulate Osteoclastogenesis in Rheumatoid Arthritis

BackgroundTh17 cells, which are defined by a selective interleukin (IL)-17 secretion, are involved in the bony destruction of rheumatoid arthritis (RA).ObjectivesThis study aimed to determine the regulatory effect of Th17...

Osteoporosis and adynamic bone in chronic kidney disease

Among the chronic kidney disease-mineral bone disease (CKD-MBD) disorders, osteoporosis and adynamic bone are highly prevalent, and they have been consistently associated with low bone mass, bone fractures, vascular calcifications and greater mortality in general and CKD populations. Despite the fact that osteoporosis and adynamic bone have similar clinical outcomes, they have different pathogeneses and clinical management. In osteoporosis, there is a lack of balance between bone formation and bone resorption, and less new bone is formed to replace bone losses. Osteoporosis is defined by the World Health Organization as "a disease characterized by low bone mineral density and micro architectural deterioration leading to low bone strength and increased risk of fractures." In the general population, there is a good correlation between dual-energy X-ray absorptiometry measurements and bone fractures, but this is not the case with CKD patients. Despite the fact that we have a great number of active antiosteoporotic drugs, the experience in CKD patients is limited. Adynamic bone is suspected based on biochemical parameters, mainly parathyroid hormone (PTH) and bone alkaline phosphatase, but it needs to be proven using a bone biopsy, where a low or zero bone formation rate and a reduction or absence of osteoblasts and osteoclasts should be found. The clinical management of adynamic bone has important limitations and currently does not allow taking many active measures. Treatment is mainly based on the prevention of risk factors known to induce PTH oversuppression, such as aluminium and calcium load and very high doses of vitamin D receptor activators. Due to the limitations in the treatment of both conditions, prevention plays a key role in the management of these disorders.

Microcrack density and nanomechanical properties in the subchondral region of the immature piglet femoral head following ischemic osteonecrosis

Development of a subchondral fracture is one of the earliest signs of structural failure of the immature femoral head following ischemic osteonecrosis, and this eventually leads to a flattening deformity of the femoral head. The mechanical and mineralization changes in the femoral head preceding subchondral fracture have not been elucidated. We hypothesized that ischemic osteonecrosis leads to early material and mechanical alterations in the bone of the subchondral region. The purpose of this investigation was to assess the bone of the subchondral region for changes in the histology of bone cells, microcrack density, mineral content, and nanoindentation properties at an early stage of ischemic osteonecrosis in a piglet model. This large animal model has been shown to develop a subchondral fracture and femoral head deformity resembling juvenile femoral head osteonecrosis. The unoperated, left femoral head of each piglet (n=8) was used as a normal control, while the right side had a surgical ischemia induced by disrupting the femoral neck vessels with a ligature. Hematoxylin and eosin (H&E) staining and TUNEL assay were performed on femoral heads from 3 piglets. Quantitative backscattered electron imaging, nanoindentation, and microcrack assessments were performed on the subchondral region of both control and ischemic femoral heads from 5 piglets. H&E staining and TUNEL assay showed extensive cell death and an absence of osteoblasts in the ischemic side compared to the normal control. Microcrack density in the ischemic side (3.2±0.79 cracks/mm2) was significantly higher compared to the normal side (0.27±0.27 cracks/mm2) in the subchondral region (p<0.05). The weighted mean of the weight percent distribution of calcium (CaMean) also was significantly higher in the ischemic subchondral region (p<0.05). Furthermore, the nanoindentation modulus within localized areas of subchondral bone was significantly increased in the ischemic side (16.8±2.7GPa) compared to the normal control (13.3±3.2GPa) (p<0.05). Taken together, these results support the hypothesis that the nanoindentation modulus of the subchondral trabecular bone is increased in the early stage of ischemic osteonecrosis of the immature femoral head and makes it more susceptible to microcrack formation. We postulate that continued loading of the hip joint when there is a lack of bone cells to repair the microcracks due to ischemic osteonecrosis leads to microcrack accumulation and subsequent subchondral fracture.

Osteoblasts can induce dental pulp stem cells to undergo osteogenic differentiation

Recent studies have shown that, in numerous species, systemically administered bone marrow-derived mesenchymal stem cells undergo site-specific differentiation. This suggests that osteoblasts, by means of cytokine secretion, may promote dental pulp stem cells (DPSCs) to undergo osteogenesis. The objective of this study was to assess the potential synergistic interaction effect of osteoblasts on DPSCs for promotion of osteogenesis. Stem cells, derived from dental pulp of healthy human donors, were co-cultured with calvaria osteoblasts using a culture insert system. The proliferation rate, calcium deposition, osteogenic-related gene expression of induced DPSCs, including Runx-2, bone sialoprotein, osteocalcin and collagen-1, were assayed using MTT, Alizarin Red S staining and reverse transcriptase polymerase chain reaction, respectively. Co-cultured DPSCs had the highest rate of proliferation compared with those cultured in absence of osteoblasts. The morphology and ultrastructure of DPSCs in the co-cultures showed improvement, with co-cultured DPSCs becoming more osteoblast-like as compared with DPSCs cultured alone, and the mineralization potential of co-cultured DPSCs was enhanced compared with DPSCs cultured alone. Furthermore, osteogenic-related genes were significantly over-expressed in co-cultured DPSCs after osteogenic induction. The results demonstrate that DPSCs successfully differentiate towards osteoblasts and that the paracrine interaction of osteoblasts is likely to contribute to DPSC differentiation. It is believed that this study demonstrates certain useful applications for DPSCs in bone tissue engineering.

Short-Term Effects of Whole-Body Exposure to56Fe Ions in Combination with Musculoskeletal Disuse on Bone Cells

Space travel and prolonged bed rest cause bone loss due to musculoskeletal disuse. In space, radiation fields may also have detrimental consequences because charged particles traversing the tissues of the body can elicit a wide range of cytotoxic and genotoxic lesions. The effects of heavy-ion radiation exposure in combination with musculoskeletal disuse on bone cells and tissue are not known. To explore this, normally loaded 16-week-old male C57BL/6 mice were exposed to (56)Fe ions (1 GeV/nucleon) at doses of 0 cGy (sham), 10 cGy, 50 cGy or 2 Gy 3 days before tissue harvest. Additional mice were hindlimb unloaded by tail traction continuously for 1 week to simulate weightlessness and exposed to (56)Fe-ion radiation (0 cGy, 50 cGy, 2 Gy) 3 days before tissue harvest. Despite the short duration of this study, low-dose (10, 50 cGy) irradiation of normally loaded mice reduced trabecular volume fraction (BV/TV) in the proximal tibiae by 18% relative to sham-irradiated controls. Hindlimb unloading together with 50 cGy radiation caused a 126% increase in the number of TRAP(+) osteoclasts on cancellous bone surfaces relative to normally loaded, sham-irradiated controls. Together, radiation and hindlimb unloading had a greater effect on suppressing osteoblastogenesis ex vivo than either treatment alone. In sum, low-dose exposure to heavy ions (50 cGy) caused rapid cancellous bone loss in normally loaded mice and increased osteoclast numbers in hindlimb unloaded mice. In vitro irradiation also was more detrimental to osteoblastogenesis in bone marrow cells that were recovered from hindlimb unloaded mice compared to cells from normally loaded mice. Furthermore, irradiation in vitro stimulated osteoclast formation in a macrophage cell line (RAW264.7) in the presence of RANKL (25 ng/ml), showing that heavy-ion radiation can stimulate osteoclast differentiation even in the absence of osteoblasts. Thus heavy-ion radiation can acutely increase osteoclast numbers in cancellous tissue and, under conditions of musculoskeletal disuse, can enhance the sensitivity of bone cells, in particular osteoprogenitors, to the effects of radiation.

A novel T cell cytokine, secreted osteoclastogenic factor of activated T cells, induces osteoclast formation in a RANKL-independent manner.

Chronic T cell activation is central to the etiology of rheumatoid arthritis (RA), an inflammatory autoimmune disease that leads to severe focal bone erosions and generalized systemic osteoporosis. Previous studies have shown novel cytokine-like activities in medium containing activated T cells, characterized by potent induction of the osteoblastic production of interleukin-6 (IL-6), an inflammatory cytokine and stimulator of osteoclastogenesis, as well as induction of an activity that directly stimulates osteoclast formation in a manner independent of the key osteoclastogenic cytokine RANKL. This study was undertaken to identify the factors secreted by T cells that are responsible for these activities. Human T cells were activated using anti-human CD3 and anti-human CD28 antibodies for 72 hours in AIM V serum-free medium to obtain T cell-conditioned medium, followed by concentration and fractionation of the medium by fast-protein liquid chromatography. Biologically active fractions were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major bands were analyzed by mass spectrometry, and a major candidate protein was identified. This novel cytokine was cloned, and its expression was analyzed using recombinant DNA technologies. A single novel cytokine that could induce both osteoblastic IL-6 production and functional osteoclast formation in the absence of osteoblasts or RANKL and that was insensitive to the effects of the RANKL inhibitor osteoprotegerin was identified in the activated T cell-conditioned medium; this cytokine was designated secreted osteoclastogenic factor of activated T cells (SOFAT). Further analysis of SOFAT revealed that it was derived from an unusual messenger RNA splice variant coded by the threonine synthase-like 2 gene homolog, which is a conserved gene remnant coding for threonine synthase, an enzyme that functions only in microorganisms and plants. SOFAT may act to exacerbate inflammation and/or bone turnover under inflammatory conditions such as RA or periodontitis and in conditions of estrogen deficiency.

IL‐17 induces osteoclastogenesis from human monocytes alone in the absence of osteoblasts, which is potently inhibited by anti‐TNF‐α antibody: A novel mechanism of osteoclastogenesis by IL‐17

IL-17 is a proinflammatory cytokine crucial for osteoclastic bone resorption in the presence of osteoblasts or synoviocytes in rheumatoid arthritis. However, the role of IL-17 in osteoclastogenesis from human monocytes alone remains unclear. Here, we investigated the role of IL-17 in osteoclastogenesis from human monocytes alone and the direct effect of infliximab on the osteoclastogenesis induced by IL-17. Human peripheral blood mononuclear cells (PBMC) were cultured for 3 days with M-CSF. After non-adherent cells were removed, IL-17 was added with either infliximab or osteoprotegerin (OPG). Seven days later, adherent cells were stained for vitronectin receptor. On the other hand, CD11b-positive monocytes purified from PBMC were also cultured and stained as described above. CD11b-positive cells were cultured with TNF-alpha and receptor activator of NF-kappaB ligand (RANKL). In the cultures of both adherent cells and CD11b-positive cells, IL-17 dose-dependently induced osteoclastogenesis in the absence of soluble-RANKL. OPG or infliximab inhibited IL-17-induced osteoclastogenesis. Interestingly, in the culture of CD11b-positive cells, the osteoclastogenesis was more potently inhibited by infliximab than by OPG. TNF-alpha and RANKL synergistically induced osteoclastogenesis. The present study clearly demonstrated the novel mechanism by which IL-17 directly induces osteoclastogenesis from human monocytes alone. In addition, infliximab potently inhibits the osteoclastogenesis directly induced by IL-17.

The Interaction Between Burn Injury and Vitamin D Metabolism and Consequences for the Patient

The stress and inflammatory responses to burn injury are associated with bone loss. The stress response entails production of large amounts of endogenous glucocorticoids that decrease osteoblasts on the mineralization surface of bone and decreases differentiation of marrow stromal cells into osteoblasts, thereby decreasing the amount of bone formation. Deficiency of osteoblasts also blocks osteoclastogenesis thus leading to low bone turnover and bone loss. The inflammatory response generates cytokines such as interleukin 1-beta and interleukin-6, which normally increase osteoclastogenic bone resorption via stimulation of osteoblast production of RANK ligand. However, in the absence of osteoblasts as a target we postulate that they attack the parathyroid gland chief cells and up-regulate the calcium-sensing receptor. The consequence of this upregulation is the lowering of the circulating calcium necessary to suppress parathyroid hormone production and the development of hypocalcemia and urinary calcium wasting. It is the parathyroid hormone suppression that causes us to postulate acute deficiency of 1,25-dihydroxyvitamin D and the consequence of this for post-burn metabolism could include derepression of the gene that controls renin production, leading to elevated levels of angiotensin II, which can contribute to insulin resistance, as can vitamin D deficiency itself. Moreover, the skin from burned patients cannot synthesize vitamin D normally. Thus vitamin D supplementation is the only means by which to ensure vitamin D sufficiency for burn victims. The proper requirement for vitamin D in acutely burned patients remains unknown.

Idiopathic Calvarial Thinning -Case Report-

An 82-year-old diabetic female had been aware of gradually enlarging, painless scalp depressions in the bilateral parietal regions for more than 6 years. She had no history of head injury, or inflammatory or malignant disease. Her family history was unremarkable for hereditary bone disease. She had diabetes mellitus which had been well controlled with orally administered drugs. Blood examination showed normal renal and liver functions with normal serum calcium, magnesium, and phosphorus concentrations, in addition to normal parathyroid hormone and calcitonin levels. Neuroimaging including skull radiography, head computed tomography, and magnetic resonance imaging demonstrated symmetric thinning in the bilateral parietal bones attributed to loss of diploe and thinning of the outer table. The inner table was intact without associated soft tissue mass or vascular lesions. Technetium-99m methylene diphosphate systemic bone scintigraphy showed mild hypoaccumulation in the affected calvarium on the left. No other pathological findings were found by systemic examination. She underwent open biopsy for histological verification which revealed homogeneous membranous bone tissue with regressed diploe, absence of osteoblasts, absence of osteoclastic appearance, and absence of findings of underlying malignancy. There was no markedly fibrovascular connective tissue typical of Gorham-Stout disease. Calvarial thinning in the present case may have been caused by an undetermined complex mechanism.

IL-23 induces human osteoclastogenesis via IL-17 in vitro, and anti-IL-23 antibody attenuates collagen-induced arthritis in rats

This study demonstrates that IL-23 stimulates the differentiation of human osteoclasts from peripheral blood mononuclear cells (PBMC). Furthermore, in vivo blockade of endogenous IL-23 activity by treatment with anti-IL-23 antibody attenuates collagen-induced arthritis in rats by preventing both inflammation and bone destruction. IL-23 induced human osteoclastogenesis in cultures of PBMC in the absence of osteoblasts or exogenous soluble-receptor activator of NF-kappaB ligand (RANKL). This IL-23-induced osteoclastogenesis was inhibited by osteoprotegerin, anti-IL-17 antibody, and etanercept, suggesting that RANKL, IL-17, and TNF-alpha are involved. In addition, we found the ratio of production levels of IL-17 to those of IFN-gamma from activated human T cells was elevated at 1 to 10 ng/ml IL-23. The inductive effect of IL-17 and the inhibitory effect of IFN-gamma on osteoclastogenesis indicate that the balance of these two cytokines is particularly important. We also demonstrated that IL-23 administered at a later stage significantly reduced paw volume in rats with collagen-induced arthritis, in a dose-dependent manner. Furthermore, anti-IL-23 antibody reduced synovial tissue inflammation and bone destruction in these rats. These findings suggest that IL-23 is important in human osteoclastogenesis and that neutralizing IL-23 after onset of collagen-induced arthritis has therapeutic potential. Thus, controlling IL-23 production and function could be a strategy for preventing inflammation and bone destruction in patients with rheumatoid arthritis.

Absence of osteoblasts and osteoids in rabbit and human calcifying aortas

Whether osteogenesis is involved in the calcification process during atherosclerosis remains an important issue. In rabbits fed 0.5% cholesterol and 2% peanut oil for 3–4 months, mineral deposition initiates at the deeper zones of lesions adjacent to the smooth muscle layer and progresses along the interface after the intima is extensively thickened. In advanced human atherosclerotic aortas, calcification occurs both in the media and thickened intima. Contrary to the concept of osteogenesis as a major cause of vascular calcification, neither bone cells nor osteoids were observed in the mineralized areas in any of atherosclerotic rabbit and human aortas studied. Furthermore, tissue non-specific alkaline phosphatase (ALP), an essential biomarker of cartilage and bone was not detected in frozen sections of atherosclerotic rabbit and human aortas using a chromogenic ALP activity procedure. Unlike ALP-abundant matrix vesicles in bone or cartilage, calcifying vesicles from human or rabbit atherosclerotic aortas do not exhibit ALP activity. However, both types of vesicles are rich in ATPase and AMPase, which may play a role in providing Pi from nucleotides for calcification. These observations strongly suggest that aortic calcification induced by dietary cholesterol represents non-osseous dystrophic calcification, similar to the one frequently occurring in various diseased and injured tissues.

Myeloma Cells Cultivate Megakaryocytes To Promote Lytic Bone Disease.

The development of lytic bone lesions in myeloma (MM) is caused by the activation of osteoclasts through direct and indirect interactions with MM cells and by inactivation of osteoblasts by MM cells. Megakaryocytes have been shown to promote osteoblast differentiation and produce critical factors associated with osteoclast activity. Cells of megakaryocytic linage are often expanded in myelomatous bone. The aim of this study was to investigate the impact of myeloma cells on regulation of bone remodeling by megakaryocytes. Culture of megakaryocytes was prepared by incubating mobilized peripheral blood in IMDM media supplemented with BSA, thrombopoeitin (TPO), IL-6 and IL-3. Following removal of all non-adherent cells, the remaining adherent megakaryocytes (>90%) highly expressed c-MPL, CD41a and factor VIII, and had various degree of ploidy. Mesenchymal stem cells (MSCs), osteoclast precursors and CD138-selected MM cells were isolated as previously described (Pittenger et al., Science, 1999; Yaccoby et al., Cancer Res., 2004). Our study revealed that MSCs expressed TPO and that expression of TPO was upregulated 5 fold in MSCs after co-culture with MM cells, as determined by quantitive real time PCR (qRT-PCR). To study the effect on osteoblastogenesis, MSCs were co-cultured with megakaryocytes in the presence and absence of primary MM cells (n=5), in media lacking growth factors. Megakaryocytes promoted osteoblast differentiation as determined by elevated expression of alkaline phosphatase, an effect that was completely abrogated by adding MM cells to the cultures. To study the involvement of megakaryocytes on osteoclastogenesis, we initially examined expression of RANKL and OPG by these cells cultured with and without MM cells, using qRT-PCR. OPG and RANKL were expressed by megakaryocytes and gene expression of both factors was upregulated in co-cultured megakaryocytes. However, whereas OPG was insignificantly upregulated by 1.6±0.3 fold, RANKL expression was increased by 5.1±0.4 folds after co-culture with MM cells (p<0.01). RANKL:OPG expression ratio in cultures of megakaryocytes with and without MM cells were 10.2±2.8 and 2.9±1.6, respectively (p<0.03). This suggests that in the absence of osteoblasts, megakaryocytes are a substantial source of RANKL in myelomatous bone, whereas OPG may serve as a MM cell survival factor by neutralizing TRAIL (Shipman & Croucher, Cancer Res., 2003). The numbers of multinucleated osteoclasts counted after culturing of osteoclast precursors with MM+megakaryocytes, MM cells and megakaryocytes were 453±159, 273±95 and 291±104, respectively (n=3, p<0.04, co-culture vs. MM cells). We conclude that MM cells prevent osteoblast differentiation promoted by megakaryocytes and induce megakaryocytes to increase RANKL:OPG ratio. These effects contribute to the uncoupling processes of osteoclastic resorption and osteoblastic bone formation, induction of lytic bone disease and myeloma progression.

Conditional deletion of Indian hedgehog from collagen type 2α1-expressing cells results in abnormal endochondral bone formation

Indian hedgehog (Ihh) is actively involved in endochondral bone formation. Although expression of Ihh is mostly restricted to pre-hypertrophic chondrocytes, the role of chondrocyte-derived Ihh in endochondral bone formation is not completely understood. To address such unresolved issues, we used the Cre/loxP approach to generate mice (Col2alpha1Cre; Ihhd/Ihhd) in which the Ihh gene was selectively ablated from collagen type II expressing cells. Mutant mice were born with the expected ratio of Mendelian inheritance, but died shortly after birth and were smaller in size, exhibiting malformed and retarded growth of limbs with severe skeletal deformities. Alizarin red S staining showed abnormal mineralization of axial and appendicular bones. Histological analysis of mutant long bones revealed abnormal endochondral bone formation with loss of a normal growth plate. In addition, in vivo bromo-deoxyuridine (BrdU) labelling showed a marked decrease in chondrocyte proliferation. A delay in chondrocyte hypertrophy in Col2alpha1Cre; Ihhd/Ihhd mice was detected by the expression of collagen type X and osteopontin, using in situ hybridization. Furthermore, there was no expression of bone markers such as collagen type I, bone Gla protein, Runx2/Cbfa1 or PTH-R in the perichondrium of mutant mice, indicating the absence of osteoblasts from endochondral bones. Thus, selective loss of chondrocyte-derived Ihh recapitulated the defects in Ihh(-/-) animals, providing direct in vivo evidence that Ihh not only regulates chondrocyte proliferation and differentiation but also exerts effects on osteoblast differentiation. Understanding the exact functions of the molecules involved in endochondral bone formation will form the basis for further study to determine the molecular mechanisms of skeletal diseases involving various cellular components of bone.

Normal human osteoclasts formed from peripheral blood monocytes express PTH type 1 receptors and are stimulated by PTH in the absence of osteoblasts

The prevailing view for many years has been that osteoclasts do not express parathyroid hormone (PTH) receptors and that PTH's effects on osteoclasts are mediated indirectly via osteoblasts. However, several recent reports suggest that osteoclasts express PTH receptors. In this study, we tested the hypothesis that human osteoclasts formed in vitro express functional PTH type 1 receptors (PTH1R). Peripheral blood monocytes (PBMC) were cultured on bone slices or plastic culture dishes with human recombinant RANK ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) for 16-21 days. This resulted in a mixed population of mono- and multi-nucleated cells, all of which stained positively for the human calcitonin receptor. The cells actively resorbed bone, as assessed by release of C-terminal telopeptide of type I collagen and the formation of abundant resorption pits. We obtained evidence for the presence of PTH1R in these cells by four independent techniques. First, using immunocytochemistry, positive staining for PTH1R was observed in both mono- and multi-nucleated cells intimately associated with resorption cavities. Second, PTH1R protein expression was demonstrated by Western blot analysis. Third, the cells expressed PTH1R mRNA at 21 days and treatment with 10(-7) M hPTH (1-34) reduced PTH1R mRNA expression by 35%. Finally, bone resorption was reproducibly increased by two to threefold when PTH (1-34) was added to the cultures. These findings provide strong support for a direct stimulatory action of PTH on human osteoclasts mediated by PTH1R. This suggests a dual regulatory mechanism, whereby PTH acts both directly on osteoclasts and also, indirectly, via osteoblasts.

Thyroid hormone stimulates osteoclast differentiation by a mechanism independent of RANKL-RANK interaction.

It is well known that thyroid hormone excess causes bone loss. However, the precise mechanism of bone loss by thyroid hormone still remains unclear. When T(3) was added to unfractionated bone cells after degeneration of pre-existent osteoclasts, T(3) (1 pM-100 nM) dose-dependently stimulated osteoclast-like cell formation, irrespective of the presence of indomethacin and IL-6 Ab. T(3) increased the expression of osteoprotegerin (OPG) messenger RNA (mRNA), but not of receptor activator of nuclear factor kappaB ligand (RANKL) in unfractionated bone cells, suggesting that the stimulatory effect of T(3) on osteoclast formation was not mediated by the RANKL/OPG system. We next examined the direct effect of T(3) on osteoclast precursors in the absence of osteoblasts, using hemopoietic blast cells derived from spleen cells. T(3) (1 pM-100 nM) dose-dependently stimulated osteoclast-like cell formation from osteoclast precursors. OPG did not inhibit T(3)-induced osteoclast formation from osteoclast precursor cells. The polymerase chain reaction (PCR) product corresponding in size to the mouse T(3) receptor alpha1 cDNA was detected in osteoclast precursors from mouse hemopoietic blast cells as well as mouse heart and mouse osteoblastic cell line MC3T3-E1 cells, suggesting that T(3) directly stimulated osteoclast-like cell formation from osteoclast precursors in the absence of osteoblasts. Further, T(3) increased the expression of c-Fos mRNA at 15 min and 24 h and Fra-1 mRNA at 2 and 6 h in osteoclast precursors. Consistent with the increased expression of c-Fos mRNA observed by RT-PCR, the activation of c-Fos occurred in osteoclast precursor cells stimulated by T(3), while the activation of neither NF-kappaB nor MAPKs was observed by immunoblot analysis. Antisense oligodeoxynucleotides (as-ODN) complementary to c-Fos mRNA at 1 microM significantly inhibited T(3)-induced osteoclast-like cell formation from osteoclast precursors in the absence of stromal cells while sense-ODN did not affect T(3)-induced osteoclast-like cell formation. These results indicate that T(3) directly stimulates osteoclast differentiation at least in part by up-regulation of c-fos protein in osteoclast precursor cells.