Shared oxidative pathways in response to gravity-dependent loading and gamma-irradiation of bone marrow-derived skeletal cell progenitors.
Hisataka KondoCharles L. LimoliN. D. SearbyEduardo AlmeidaDavid J. LoftusW. VercoutereEmily Morey‐HoltonErich GiedzinskiRose MojarrabDouglas J. HiltonRuth K. Globus
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Astronauts are exposed to radiation during space travel under conditions of dramatically reduced weightbearing activity. However, we know little about how gravity-dependent loading affects tissue sensitivity to radiation. We hypothesize gravity-dependent loading and irradiation share common molecular signaling pathways in bone cell progenitors that are sensitive to stress-induced reactive oxygen species (ROS), species capable of impacting skeletal health. To address this, progenitor cells with potential to differentiate into bone-forming osteoblasts were extracted from bone marrow, then cells were centrifuged (from 5-gravity (g) to 50-g for 5-180 min) on day 2 in culture, or were exposed to a single dose (1-5 Gy) of irradiation (137Cs 1 Gy/min) on day 3 or 4. Production of ROS was measured via fluorescence-activated cell sorting (FACS) using an oxidation-sensitive dye. Cell numbers were assessed by measurement of DNA content (CyQUANT). Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (Alizarin Red staining). Transient centrifugation was a potent stimulus to bone marrow stromal cells, increasing production of ROS (1.2-fold), cell number (1.5-fold to 2.2-fold), and ALP activity (2.7-fold). Radiation also caused dose- and time-dependent increases in ROS production (1.1-fold to 1.4-fold) by bone marrow stromal cells, but inhibited subsequent osteoblast differentiation. In summary, gravity-dependent loading by centrifugation stimulated ROS production and increased numbers of osteoblasts. Although radiation increased production of ROS by bone marrow stromal cells, cell number and differentiation of osteoprogenitors appeared reduced. We conclude gravity-dependent loading and radiation both stimulate production of ROS and affect critical bone cell functions including growth and differentiation.Keywords:
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Exposure of astronauts in space to radiation during weightlessness may contribute to subsequent bone loss. Gamma irradiation of postpubertal mice rapidly increases the number of bone-resorbing osteoclasts and causes bone loss in cancellous tissue; similar changes occur in skeletal diseases associated with oxidative stress. Therefore, we hypothesized that increased oxidative stress mediates radiation-induced bone loss and that musculoskeletal disuse changes the sensitivity of cancellous tissue to radiation exposure. Musculoskeletal disuse by hindlimb unloading (1 or 2 wk) or total body gamma irradiation (1 or 2 Gy of (137)Cs) of 4-mo-old, male C57BL/6 mice each decreased cancellous bone volume fraction in the proximal tibiae and lumbar vertebrae. The extent of radiation-induced acute cancellous bone loss in tibiae and lumbar vertebrae was similar in normally loaded and hindlimb-unloaded mice. Similarly, osteoclast surface in the tibiae increased 46% as a result of irradiation, 47% as a result of hindlimb unloading, and 64% as a result of irradiation + hindlimb unloading compared with normally loaded mice. Irradiation, but not hindlimb unloading, reduced viability and increased apoptosis of marrow cells and caused oxidative damage to lipids within mineralized tissue. Irradiation also stimulated generation of reactive oxygen species in marrow cells. Furthermore, injection of alpha-lipoic acid, an antioxidant, mitigated the acute bone loss caused by irradiation. Together, these results showed that disuse and gamma irradiation, alone or in combination, caused a similar degree of acute cancellous bone loss and shared a common cellular mechanism of increased bone resorption. Furthermore, irradiation, but not disuse, may increase the number of osteoclasts and the extent of acute bone loss via increased reactive oxygen species production and ensuing oxidative damage, implying different molecular mechanisms. The finding that alpha-lipoic acid protected cancellous tissue from the detrimental effects of irradiation has potential relevance to astronauts and radiotherapy patients.
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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.
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Objective:To study the effect of estrogen on proliferation and differentiation of rat bone marrow stromal cells and to explore the mechanism of stimulation of bone formation by estrogen. Methods:Bone marrow stromal cells were isolated from four-week-old female Sprague-Dawley rats and cultured in DMEM medium to be induced into osteoblasts. They were cultured in medium with estrogen at different concentrations. The prolifereation, ALP activity and type-I pro-collagen of osteoblasts were examined by MTT, PNPP and assaying the level of hydroxyproline. The capacity of mineralization were measured by counting bone nodules. Results:The cell quantity, the alkaline phosphatase activity and the level of hydroxyproline of osteoblasts were increased , and the number of nodules was significantly higher. The peak value of all these stimulating effects on osteoblasts was in 10-7mol/L group. Conclusion:Estrogen can increase proliferation, differentiation and the capability of mineralization of osteoblast and this may contribute to bone formation.
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Diagnostic radiation for immediate post-surgical assessment of osseointegrated dental implants has been discouraged, due to the possibility of detrimental effects of ionizing radiation on healing and remodeling of bone. To assess this possibility, we investigated the effects of ionizing radiation on proliferation and differentiation of osteoblasts using osteoblast-like cells isolated from the calvariae of newborn rats (ROB) and a clonal osteoblastic cell line (MC3T3-E1). The cells were exposed on day 3 to a single dose of x-rays at either 40, 100, 400, or 4000 mGy, respectively, from a linear accelerator radiotherapeutic machine (Linac) or a 40-mGy dose from a diagnostic chest x-ray machine. The effects of radiation on cell growth and alkaline-phosphatase-specific (ALP) activity were evaluated at three-day intervals after irradiation up to day 12 in ROB cells, and evaluated at day 12 in MC3T3-E1 cells. At the culture end-point, the effects on formation of bone-like nodules were also evaluated in both ROB and MC3T3-E1 cells. Exposure of 4000 mGy differentially affected the two cell types. It inhibited cell growth and alkaline phosphatase activity in ROB cells, slightly increased alkaline phosphatase activity, and inhibited DNA content in MC3T3-E1 cells. This irradiation also strongly inhibited the formation of bone-like nodules in ROB cells. On the other hand, exposure of 40-, 100-, and 400-mGy (Linac) and 40-mGy (diagnostic quality) irradiation induced no significant changes in cell growth, alkaline phosphatase activity, and formation of bone-like nodules in ROB cells. These doses also induced no significant changes in DNA content and ALP activity in MC3T3-E1 cells. These results indicate that ionizing radiation at a single dose of up to 400 mGy induces no significant changes in cell growth and differentiation of osteoblast-like cells, at least in vitro. Higher radiation doses (4000 mGy) may exert different effects on cell proliferation and cell differentiation of osteoblasts, depending on the cell types affected. Thus, diagnostic radiation seems to have less effect on proliferation and differentiation of osteoblasts.
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Adult vertebrates require a continuous supply of osteoblasts for both bone remodeling and regeneration during fracture repair. This implies the existence of a reservoir of cells in the body capable of osteogenesis. One source of these osteoprogenitors is the stem cells within the fibroblastic component of bone marrow stroma. Mature osteoblasts are characterized by high alkaline phosphatase and osteopontin levels, combined with expression of the bone-specific matrix proteins osteocalcin and bone sialoprotein and the capacity for matrix mineralization. We have used these markers to define the conditions permitting rapid osteoblast differentiation from cultured bone marrow stromal cells. Osteoblastic differentiation was induced by continuous culture with 10-5M dexamethasone (dex) which stimulated alkaline phosphatase (AP) activity and mRNA levels as well us osteopontin, bone sialoprotein, and osteocalcin mRNA by Day 8 of culture; coaddition of 10-5M 1,25-dihydroxyvitamin D3 (vitamin D) with dex was essential for high osteocalcin mRNA expression. Recombinant bone morphogenetic protein-2 (BMP-2) exerted similar effects to dex and acted in synergy with dex to yield greatly elevated AP activity as well as increased levels of osteoblastic mRNAs. Using in situ hybridization to detect the presence of mRNAs in individual cells, it was shown that appearance of osteopontin mRNA preceded AP mRNA, and was expressed in dex-treated cell colonies as early as Day 4. Quantitation of cell surface AP protein by flow cytometry indicated that culture with dex or BMP-2 produced a mixed population of cells with low AP (dim cells) and cells with high AP levels, while the combination of dex + BMP-2 yielded very few dim cells and a population of cells containing higher AP levels than with either inducer alone. When the dim population from dex-treated cells was sorted and recultured with inducers, these cultures developed high AP levels and were able to deposit a mineralized matrix. Thus, treatment of marrow stromal cells with inducer results in a population of mature osteoblasts as well as a population of undifferentiated cells which retains the capacity for osteoblastic differentiation with further exposure to inducers. These data demonstrate that stem cells within the stromal compartment of bone marrow are capable of rapidly acquiring osteoblast features and suggest a potential role for glucocorticoids in combination with BMP-2 and vitamin D in stages of osteogenic development.
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We investigated the effects of the time course of addition of osteogenic supplements dexamethasone, β-glycerolphosphate, and L-ascorbic acid to rat marrow stromal cells, and the exposure time on the proliferation and differentiation of the cells. It was the goal of these experiments to determine the time point for supplement addition to optimize marrow stromal cell proliferation and osteoblastic differentiation. To determine this, two studies were performed; one study was based on the age of the cells from harvest, and the other study was based on the duration of exposure to supplemented medium. Cells were seen to proliferate rapidly at early time points in the presence and absence of osteogenic supplements as determined by 3H-thymidine incorporation into the DNA of replicating cells. These results were supported by cell counts ascertained through total DNA analysis. Alkaline phosphatase (ALP) activity and osteocalcin production at 21 days were highest for both experimental designs when the cells were exposed to supplemented medium immediately upon harvest. The ALP levels at 21 days were six times greater for cells maintained in supplements throughout than for control cells cultured in the absence of supplements for both studies, reaching an absolute value of 75 × 10−7 μmole/min/cell. Osteocalcin production reached 20 × 10−6 ng/cell at 21 days in both studies for cells maintained in supplemented medium throughout the study, whereas the control cells produced an insignificant amount of osteocalcin. These results suggest that the addition of osteogenic supplements to marrow-derived cells early in the culture period did not inhibit proliferation and greatly enhanced the osteoblastic phenotype of cells in a rat model. J. Cell. Biochem. 71:55–62, 1998. © 1998 Wiley-Liss, Inc.
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Human bone marrow stromal cells were examined for their osteogenic potential in an in vitro cell culture system. Dexamethasone (Dex) treatment induced morphological transformation of these cells from an elongated to a more cuboidal shape, increased their alkaline phosphatase activity and cAMP responses to PTH and prostaglandin E2, and was essential for mineralization of the extracellular matrix. Dex-induced differentiation of human bone marrow stromal cells was apparent after 2-3 days of treatment and reached a maximum at 7-14 days, as judged by alkaline phosphatase activity, although induction of osteocalcin by 1,25-dihydroxyvitamin D3 was attenuated by Dex. Withdrawal of Dex resulted in an enhancement of the 1,25-dihydroxyvitamin D3-induced secretion of osteocalcin, whereas alkaline phosphatase activity and the cAMP response to PTH remained at prewithdrawal levels. The steady state mRNA level of osteonectin was not affected by Dex. Our results, which demonstrate that Dex conditions the differentiation of human bone marrow osteogenic stromal cells into osteoblast-like cells, support the hypothesis of a permissive effect of glucocorticoids in ensuring an adequate supply of mature osteoblast populations. Furthermore, the established human bone marrow stromal cell culture provides a good model of an in vitro system to study the regulation of differentiation of human bone osteoprogenitor cells.
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Willey, J. S., Lloyd, S. A. J., Robbins, M. E., Bourland, J. D., Smith-Sielicki, H., Bowman, L. C., Norrdin, R. W. and Bateman, T. A. Early Increase in Osteoclast Number in Mice after Whole-Body Irradiation with 2 Gy X Rays. Radiat. Res. 170, 388–392 (2008).Bone loss is a consequence of exposure to high-dose radiotherapy. While damage to bone vasculature and reduced proliferation of bone-forming osteoblasts has been implicated in this process, the effect of radiation on the number and activity of bone-resorbing osteoclasts has not been characterized. In this study, we exposed mice to a whole-body dose of 2 Gy of X rays to quantify the early effects of radiation on osteoclasts and bone structural properties. Female C57BL/6 mice (13 weeks old) were divided into two groups: irradiated and nonirradiated controls. Animals were killed humanely 3 days after radiation exposure. Analysis of serum chemistry revealed a 14% increase in the concentration of tartrate resistant acid phosphatase (TRAP)-5b, a marker of osteoclast activity, in irradiated mice (P < 0.05). Osteoclast number ( 44%; P < 0.05) and osteoclast surface ( 213%; P < 0.001) were elevated in TRAP-stained histological sections of tibial metaphyses. No significant change was observed in osteoblast surface or osteocalcin concentration or in trabecular microarchitecture (i.e. bone volume fraction) as measured through microcomputed tomography (P > 0.05). This study provides definitive, quantitative evidence of an early, radiation-induced increase in osteoclast activity and number. Osteoclastic bone resorption may represent a contributor to bone atrophy observed after therapeutic irradiation.
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