Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture

A method is presented for isolating osteoblasts from newborn mouse calvaria without the use of digestive enzymes. The procedure is based on the ability of osteoblasts to migrate from bone onto small glass fragments (Jones, S.J., and A. Boyde, 1977, Cell Tissue Res., 184:179- 193). The isolated cells were cultured for up to 14 d in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 micrograms/ml of ascorbic acid. 7-d cultures were incubated for 24 h with [3H]proline. High levels of collagen synthesis relative to total protein were found, as measured by collagenase digestion of medium and cell layer proteins. Analysis of pepsin-digested proteins from the same cultures by SDS PAGE showed that type I collagen was predominantly produced with small amounts of type III and V (alpha 1 chains) collagens. Osteoblasts grown in the presence of beta-glycerophosphate were able to initiate mineral deposition in culture. Electron microscopic analysis of the cultures revealed the presence of needle- shaped apatite-like crystals associated with collagen fibrils and vesicles in the extracellular space. Mouse skin fibroblasts cultured under identical conditions failed to initiate mineralization. Electron histochemical studies revealed the presence of alkaline phosphatase activity, associated with osteoblast membranes, matrix vesicles and on or near collagen fibrils. Thus these isolated osteoblasts retained in culture their unique property of initiating mineralization and therefore represent a model of value for studying the mineralization process in vitro.     

Demonstration of the capacity of nacre to induce bone formation by human osteoblasts maintained in vitro.

Nacre implanted in vivo in bone is osteogenic suggesting that it may possess factor(s) which stimulate bone formation. The present study was undertaken to test the hypothesis that nacre can induce mineralization by human osteoblasts in vitro. Nacre chips were placed on a layer of first passage human osteoblasts. None of the chemical inducers generally required to obtain bone formation in vitro was added to the cultures. Osteoblasts proliferated and were clearly attracted by nacre chips to which they attached. Induction of mineralization appeared preferentially in bundles of osteoblasts surrounding the nacre chips. Three-dimensional nodules were formed by a dense osteoid matrix with cuboidal osteoblasts at the periphery and osteocytic-like cells in the center. These nodules contained foci with features of mineralized structures and bone-like structures, both radiodense to X-ray. Active osteoblasts (e.m.) with abundant rough endoplasmic reticulum, extrusion of collagen fibrils and budding of vesicles were observed. Matrix vesicles induced mineral deposition. Extracellular collagen fibrils appeared cross-banded and electrodense indicating mineralization. These results demonstrate that a complete sequence of bone formation is reproduced when human osteoblasts are cultured in the presence of nacre. This model provides a new approach to study the steps of osteoblastic differentiation and the mechanisms of induction of mineralization.     

Metastatic breast cancer induces an osteoblast inflammatory response

Breast cancer preferentially metastasizes to the skeleton, a hospitable environment that attracts and allows breast cancer cells to thrive. Growth factors released as bone is degraded support tumor cell growth, and establish a cycle favoring continued bone degradation. While the osteoclasts are the direct effectors of bone degradation, we found that osteoblasts also contribute to bone loss. Osteoblasts are more than intermediaries between tumor cells and osteoclasts. We have presented evidence that osteoblasts contribute through loss of function induced by metastatic breast cancer cells. Metastatic breast cancer cells suppress osteoblast differentiation, alter morphology, and increase apoptosis. In this study we show that osteoblasts undergo an inflammatory stress response in the presence of human metastatic breast cancer cells. When conditioned medium from cancer cells was added to human osteoblasts, the osteoblasts were induced to express increased levels of IL-6, IL-8, and MCP-1; cytokines known to attract, differentiate, and activate osteoclasts. Similar findings were seen with murine osteoblasts and primary murine calvarial osteoblasts. Osteoblasts are co-opted into creating a microenvironment that exacerbates bone loss and are prevented from producing matrix proteins for mineralization. This is the first study implicating osteoblast produced IL-6, IL-8 (human; MIP-2 and KC mouse), and MCP-1 as key mediators in the osteoblast response to metastatic breast cancer cells.     

Osteoblasts: novel roles in orchestration of skeletal architecture

Osteoblasts are located on bone surfaces and are the cells responsible for bone formation through secretion of the organic components of bone matrix. Osteoblasts are derived from mesenchymal osteoprogenitor cells found in bone marrow and periosteum. Following a period of secretory activity, osteoblasts undergo either apoptosis or terminal differentiation to form osteocytes surrounded by bone matrix. Osteoblasts secrete a characteristic mixture of extracellular matrix proteins including type I collagen as the major component as well as proteoglycans, glycoproteins and gamma-carboxylated proteins. Cells of the osteoblast lineage also provide factors essential for differentiation of osteoclasts (bone-resorbing cells). By regulating osteoclast differentiation and activity in response to systemic influences, osteoblasts not only play a central role in regulation of skeletal architecture, but also in calcium homeostasis. Inadequate osteoblastic bone formation in relation to osteoclastic resorption results in osteoporosis, a disease characterised by enhanced skeletal fragility. Cellfacts: Osteoblasts are the cells responsible for bone formation. Osteoblasts indirectly control levels of bone resorption. Osteoblasts play a key role in the pathophysiology of osteoporosis and the resulting fractures, which constitute a major public health burden in developed countries.     

Morphological changes of osteoblasts in vitro

Summary The appearance of neonate rat endocranial osteoblasts exposed in situ and fixed immediately was compared with that of similar osteoblasts organ cultured for short periods of up to 48 hours in control medium and serum alone, or with added parathyroid extract (PTE). Normal osteoblasts showed a range of variation in size and shape, degree of elongation and orientation. Culturing the osteoblasts resulted in an overall loss of elongation and ordering of the cells, and the production of dorsal ruffles which were more complex and larger in the longer culture times. PTE added to the culture medium caused an increase in cell elongation and a striking recordering of the osteoblasts into domains of parallel cells. The swirling patterns made by these domains were similar to those of the underlying bone collagen. Ruffles, where present, were small and more often peripheral than dorsal.The results indicate that fully differentiated osteoblasts are able to adapt very rapidly to survival and function in culture conditions, and that the endocytosis necessary for synthetic activity is suppressed by PTE although some cell movement may continue.This work has been supported by grants from the Medical Research Council and the Science Research Council.We would like to thank Elaine Bailey and Philip Reynolds for technical assistance, and Dr. Martin Evans for helpfully providing the facilities of his laboratory.     

In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria

We investigated the capacity of a clonal osteogenic cell line MC3T3-E1, established from newborn mouse calvaria and selected on the basis of high alkaline phosphatase (ALP) activity in the confluent state, to differentiate into osteoblasts and mineralize in vitro. The cells in the growing state showed a fibroblastic morphology and grew to form multiple layers. On day 21, clusters of cells exhibiting typical osteoblastic morphology were found in osmiophilic nodular regions. Such nodules increased in number and size with incubation time and became easily identifiable with the naked eye by day 40-50. In the central part of well-developed nodules, osteocytes were embedded in heavily mineralized bone matrix. Osteoblasts were arranged at the periphery of the bone spicules and were surrounded by lysosome-rich cells and a fibroblastic cell layer. Numerous matrix vesicles were scattered around the osteoblasts and young osteocytes. Matrix vesicles and plasma membranes of osteoblasts, young osteocytes, and lysosome-rich cells showed strong reaction to cytochemical stainings for ALP activity and calcium ions. Minerals were initially localized in the matrix vesicles and then deposited on well-banded collagen fibrils. Deposited minerals consisted exclusively of calcium and phosphorus, and some of the crystals had matured into hydroxyapatite crystals. These results indicate that MC3T3-E1 cells have the capacity to differentiate into osteoblasts and osteocytes and to form calcified bone tissue in vitro.     

Experimental study of changes in osteoblastic shape induced by calcitonin and parathyroid extract in an organ culture system

Summary Neonate rat endocranial osteoblasts were cultured on their bone surfaces in control medium (CC) or medium to which either parathyroid extract (PTE) or calcitonin (CT) had been added for 2, 4, 8 or 24 h. Some were cultured for 24 h in CC, then for 2, 4, 8 or 24 h in either CT or PTE medium; or for 24 h in PTE, then for 2, 4, 8 or 24 h in either CC or CT; or 24 h in CT and 2, 4, 8 or 24 h in CC. The dorsal ruffling of the cells in CC was found to be suppressed by later culturing with PTE and the disoriented cells reorganized to form arrays of parallel cells. The effects of PTE were also reversed by CC or CT: the osteoblasts in the second culture (CC) lost elongation and order, and proceeded through a proliferative phase before exhibiting the ruffling form similar to a single CC 24 h culture. PTE-cultured osteoblasts showed an increase in cell overlap and contact so that a more competent barrier was formed separating the bone from the medium. In control or CT medium, however, intercellular gaps were greater than in vivo.We are grateful for the expert technical assistance of Elaine Bailey, for laboratory facilities kindly provided by Dr. Martin Evans, and for financial support from the Medical Research Council     

The influence of elemental composition and surface topography on adhesion,proliferation and differentiation of osteoblasts

The influence of elemental composition and surface topography of substrates on the adhesion, proliferation and early differentiation stages of mouse osteoblasts, line MC3T3-E1, cultured on the surface of titanium and multicomponent bioactive nanostructured films (MuBiNaFs) Ti-Ca-(P)-C-O-N and Ti-Ca-Si-C-O-N has been studied. Osteoblasts spread both on the surface of uncoated titanium samples and on the samples coated with the films and had more elongated and irregular shape than the cells cultured on control glass substrate. Immunofluorescence study has shown that osteoblasts formed straight actin bundles associated with focal contacts. Osteoblasts on titanium films formed fewer focal contacts than the cells cultured on Ti-Ca-(P)-C-O-N films. The sandblast treatment did not affect the distribution of the focal contacts and actin structures within the cells. Osteoblasts actively divided on the surface of Ti-Ca-(P)-C-O-N and Ti-Ca-Si-C-O-N films and alteration of surface topography did not affect their growth rate. The quantitative colorimetrical test on alkaline phosphatase activity has shown that modification of surface topography does not affect the level of osteoblastic differentiation. Thus, the elemental composition plays an important role in the interaction of osteoblasts with the surface of films, while the modification of the surface topography in the range of S _q from 0.4 to 1 μm does not influence adhesion, proliferation, and differentiation of osteoblasts.     

Alignment and proliferation of MC3T3-E1 osteoblasts in microgrooved silicone substrata subjected to cyclic stretching

Previous studies have shown that many types of cells align in microgrooves in static cultures. However, whether cells remain aligned and also proliferate in microgrooves under stretching conditions has not been determined. We grew MC3T3-E1 osteoblasts in deformable silicone dishes containing microgrooves oriented in the stretch direction. We found that with or without 4% stretching, cells aligned in microgrooves of all sizes, with the groove and ridge widths ranged from 1 to 6microm, but the same groove depth of about 1.6microm. In addition, actin cytoskeleton and nuclei became highly aligned in the microgrooves with and without 4% cyclic stretching. To further examine whether MC3T3-E1 osteoblasts proliferate in microgrooves with cyclic stretching, we grew the cells in six-well silicone dishes containing microgrooves in three wells and smooth surfaces in other three wells. After 4% cyclic stretching for 3, 4, and 7 days, we found that cell numbers in the microgrooves were not significantly different (p>0.05) from those on the smooth surface (p>0.05). Taken together, these results show that MC3T3-E1 osteoblasts can align and proliferate in microgrooves with 4% cyclic stretching. We suggest that the silicone microgrooves can be a useful tool to study the phenotype of MC3T3-E1 osteoblasts under controlled substrate strains. The silicone microgrooves can also be useful for delivering defined substrate strains to other adherent cells in cultures.     

Synthesis of colony-stimulating factor (CSF) and differentiation-inducing factor (D-factor) by osteoblastic cells, clone MC3T3-E1

The role of osteoblasts in inducing the proliferation and differentiation of bone marrow cells was examined. Conditioned medium obtained from mouse osteoblastic cell (MC3T3-E1) cultures stimulated colony formation of mouse bone marrow cells (CSF) and differentiation of mouse myeloid leukemia cells (M1) into macrophage-like cells (D-factor). The CSF activity increased time dependently in parallel with the increase of alkaline phosphatase activity during the culturing of the MC3T3-E1 cells. The activity of the D-factor attained a maximum on days 12 - 15 and decreased thereafter. Both the CSF and the D-factor were eluted in a range of 25,000 to 67,000 daltons on gel filtration. The fraction containing both factors exhibited bone-resorbing activity. These results suggest that osteoblasts are involved in bone resorption at least in part by enhancing the proliferation and differentiation of osteoclast progenitors.     

Cell orientation and cytoskeleton organisation on ground titanium surfaces

A stable connection between the biomaterial surface and the surrounding tissue is one of the most important prerequisites for the long-term success of implants. Therefore, a strong adhesion of the cells on the biomaterial surface is required. Beside the surface composition the surface topography influences the properties of the adherent cells. The quality of the connection between the cell and the biomaterial is-among other factors-determined by the dimensions of the surface topography. Osteoblasts and fibroblast-like cells in contact with a ground biomaterial surface spread in the direction of the surface structures. These aligned cells provide a more favourable adhesion behaviour than a spherically shaped cell. To determine the influence of the surface structure on the cell alignment and cytoskeleton organisation or arrangement, substrate discs of cp-titanium were ground, producing different roughness of the substrates. The oriented cells had a higher density of focal contacts when they were in contact with the edges of the grooves and showed a better organisation of the cytoskeleton and stronger actin fibres. These changes of the aligned cells depend on the peak to valley height of the surface structures.     

Human osteoblasts from younger normal and osteoporotic donors show differences in proliferation and TGFβ-release in response to cyclic strain

Mechanical stimulation of bone tissue by physical activity stimulates bone formation in normal bone and may attenuate bone loss of osteoporotic patients. However, altered responsiveness of osteoblasts in osteoporotic bone to mechanical stimuli may contribute to osteoporotic bone involution. The purpose of the present study was to investigate whether osteoblasts from osteoporotic patients and normal donors show differences in proliferation and TGFβ production in responses to cyclic strain. Human osteoblasts isolated from collagenase-treated bone explants of 10 osteoporotic patients (average age 70 ± 6 yr) and 8 normal donors (average age 54 ± 10 yr) were plated into elastic rectangular silicone dishes. Subconfluent cultures were stimulated by cyclic strain (1%, 1 Hz) in an electromechanical cell stretching apparatus at three consecutive days for each 30 min. The cultures were assayed for proliferation, alkaline phosphatase activity and TGFβ release in each three parallel cultures. In all experiments, osteoblasts grown in the same elastic dishes but without mechanical stimulation served as controls. Significant differences between stimulated cultures and unstimulated controls were determined by a paired two-tailed Wilcoxon test. In comparison to the unstimulated controls, osteoblasts from normal donors significantly increased proliferation (p = 0.025) and TGFβ secretion (p = 0.009) into the conditioned culture medium. In contrast, osteoblasts from osteoporotic donors failed to increase both proliferation (p > 0.05) and TGFβ release (p > 0.05) in response to cyclic strain. Alkaline phosphatase activity was not significantly affected (p > 0.05) in normal as well as osteoporotic bone derived osteoblasts.

These findings suggest a different responsiveness to 1% cyclic strain of osteoblasts isolated from normal and osteoporotic bone that could be influenced by both the disease of osteoporosis and the higher average age of the osteoporotic patient group. While osteoblasts from osteoporotic donors failed to increase proliferation and TGFβ release under the chosen mechanical strain regimen that stimulated both parameters in normal osteoblasts, it is possible that some other strain regimen would provide more effective stimulation of osteoporotic cells.     

Growth and differentiation of alveolar bone cells in tissue-engineered constructs and monolayer cultures

The ability to enhance bone regeneration by implanting autologous osteoblasts in combination with an appropriate scaffold would be of great clinical interest. The aim of our study was to compare the growth and differentiation of alveolar bone cells in tissue-engineered constructs and in monolayer cultures, as the basis for developing procedures for routine preparation of bone-like tissue constructs. Alveolar bone tissue was obtained from four human donors and explant cultures of the cells were established. Expanded cells were seeded on macroporous hydroxyapatite granules, and cultured in medium supplemented with osteogenic differentiation factors for up to 3 weeks. Control monolayer cultures were established in parallel, and cultured in media with or without osteogenic supplements. Cell proliferation, alkaline phosphatase (AP) activity and gene expression of AP, osteopontin and osteocalcin were determined under different culture conditions at weekly intervals. Cells in tissue constructs exhibited growth patterns similar to those in control monolayer cultures: enhanced proliferation was noted during the first 2 weeks of cultivation, followed by a decrease in cell numbers. AP activity at 3 weeks was higher in all cultures in osteogenic medium than in control medium. Gene expression levels were stable in monolayer cultures in both types of media whereas, in tissue constructs, they exhibited patterns of osteogenic differentiation. Light and scanning electron microscopy examination of the cell-seeded constructs showed uniform cell distribution, as well as cell attachment and growth into the interior region of the hydroxyapatite granules. Our results show that bone-like constructs with viable cells exhibiting differentiated phenotype can be prepared by cultivation of alveolar-bone cells on the tested hydroxyapatite granules.     

Osteoblasts develop from isolated fetal mouse chondrocytes when co-cultured in high density with brain tissue

Summary Chondrocytes from the hypertrophic and proliferative zones of 16-day-old fetal murine metatarsal bones were enzymatically dissociated and cultured in a high-density type of culture, exposed to the gas phase. We ascertained that no cells of the perichondrium were included in the cell suspension. Control cultures formed a solid cartilaginous mass, of which all the chondrocytes were alkaline phosphatase positive and the matrix started to calcify after 4 days. After 6 days, nearly the entire matrix was calcified. When co-cultured with pieces of cerebral tissue, some chondrocytes had transdifferentiated into osteoblasts after 4 days. They had started to form osteoid. After 6 and 11 days part of the cartilage had been replaced by bone, especially in the periphery of the cultures, but also in areas in the center. The bone matrix was partly calcified. Osteoblasts and bone matrix were identified as such electron microscopically. The nature of the bone matrix was also confirmed by immunohistochemical demonstration of collagen type I and osteocalcin. These results show that enzymatically isolated chondrocytes are able to become osteoblasts when properly stimulated. This supports the concept of chondrocytes being responsible for (part of) the endochondral bone formation in the marrow cavity of long bones.     

Epirubicin induces apoptosis in osteoblasts through death-receptor and mitochondrial pathways

Epirubicin is an anthracycline and is widely used in tumor treatment, but has toxic and undesirable side effects on wide range of cells and hematopoietic stem cells (HSC). Osteoblasts play important roles in bone development and in supporting HSC differentiation and maturation. It remains unknown whether epirubicin-induced bone loss and hematological toxicity are associated with its effect on osteoblasts. In primary osteoblast cell cultures, epirubicin inhibited cell growth and decreased mineralization. Moreover, epirubicin arrested osteoblasts in the G2/M phase, and this arrest was followed by apoptosis in which both the extrinsic (death receptor-mediated) and intrinsic (mitochondrial-mediated) apoptotic pathways were evoked. The factors involved in the extrinsic apoptotic pathway were increased FasL and FADD as well as activated caspase-8. Those involved in the intrinsic apoptotic pathway were decreased Bcl-2; increased reactive oxygen species, Bax, cytochrome c; and activated caspase-9 and caspase-3. These results demonstrate that epirubicin induced osteoblast apoptosis through the extrinsic and intrinsic apoptotic pathways, leading to the destruction of osteoblasts and consequent lessening of their functions in maintaining bone density and supporting hematopoietic stem cell differentiation and maturation.     

Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin

While mammals have a limited capacity to repair bone defects, zebrafish can completely regenerate amputated bony structures of their fins. Fin regeneration is dependent on formation of a blastema, a progenitor cell pool accumulating at the amputation plane. It is unclear which cells the blastema is derived from, whether it forms by dedifferentiation of mature cells, and whether blastema cells are multipotent. We show that mature osteoblasts dedifferentiate and form part of the blastema. Osteoblasts downregulate expression of intermediate and late bone differentiation markers and induce genes expressed by bone progenitors. Dedifferentiated osteoblasts proliferate in a FGF-dependent manner and migrate to form part of the blastema. Genetic fate mapping shows that osteoblasts only give rise to osteoblasts in the regenerate, indicating that dedifferentiation is not associated with the attainment of multipotency. Thus, bone can regenerate from mature osteoblasts via dedifferentiation, a finding with potential implications for human bone repair.     

Nucleostemin在大鼠骨髓基质干细胞向成骨细胞诱导分化中的研究

目的探讨Nucleostemin（NS）在大鼠骨髓基质干细胞向成骨细胞诱导分化中的机制及作用。方法取4～6周龄雄性SD大鼠两侧股骨、胫骨骨髓基质细胞,原代及传代培养。取第3代骨髓间充质干细胞分别用普通和矿化培养基培养,通过相差倒置显微镜观察细胞生长、MTT法检测细胞增殖、碱性磷酸酶和茜素红钙染色了解成骨活性。免疫组化SABC法及免疫荧光法检测NS在细胞中的表达情况,比较NS分别在普通培养基和成骨细胞诱导培养基作用下的表达情况。结果大鼠骨髓间充质干细胞在矿化培养基诱导下其NS的表达较普通培养基培养下的表达明显减弱,且呈时间依赖性衰减。成骨细胞的NS表达则为阴性。在矿化液作用下,骨髓间充质干细胞可诱导为成骨细胞。MTT显示矿化液培养细胞生长潜伏期长,对数生长期较对照组延长。结论NS作为核干细胞因子,在干细胞中和某些肿瘤细胞中表达丰富。细胞分化后,其表达明显减少。因此,NS很可能作为大鼠基质干细胞是否具有潜在分化能力的标志性因子。     

Collagen processing, crosslinking, and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic acid

Human foreskin fibroblasts were cultured for up to 6 weeks in medium supplemented with ascorbic acid. During this time, the cells produced an extensive new connective tissue matrix in which the accumulated collagen (mostly type I) amounted to about 0.25 mg/10(6) cells. The matrix was highly differentiated as shown by complete processing of procollagen to collagen alpha-chains and covalent crosslinking of the collagen. Alignment of collagen fibrils occurred as the fibrils were deposited between cells, and binding of adjacent fibrils to the cell surface appeared to hold the fibrils in register. Groups of aligned fibrils were subdivided into bundles by cell-surface folds. If beta-aminopropionitrile was added to the medium, collagen crosslinking was inhibited, but not collagen synthesis or fibril bundle organization. If ascorbic acid was omitted from the culture medium, the extensive new connective tissue matrix was not produced. Our results indicate that fibroblasts in long-term cultures supplemented with ascorbic acid produce a connective tissue matrix with many in vivo-like properties including supermolecular organization of collagen.     

IL-1 alpha stimulates the formation of osteoclast-like cells by increasing M-CSF and PGE2 production and decreasing OPG production by osteoblasts

Interleukin-1alpha (IL-1alpha) is one of the most potent bone-resorbing factors involved in the bone loss that is associated with inflammation. We examined the effect of the inflammatory mediator IL-1alpha on the expression of macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and prostaglandin E2 (PGE2) in rat osteoblasts, and the indirect effect of IL-1alpha on the formation of osteoclast-like cells. Osteoblasts were cultured in alpha-minimum essential medium containing 10% fetal bovine serum with or without 100 units/ml of IL-1alpha for up to 14 days. The gene and protein expression of M-CSF and OPG were estimated by determining mRNA levels using the real-time polymerase chain reaction and protein levels using Western blot analysis. PGE2 expression was determined using an enzyme-linked immunosorbent assay. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase (TRAP) staining of osteoclast precursors in culture with conditioned medium from IL-1alpha-treated osteoblasts and the soluble receptor activator of NF-kappaB ligand (RANKL). M-CSF and PGE2 expression in osteoblasts increased markedly in cells cultured with IL-1alpha, whereas OPG expression decreased. The conditioned medium containing M-CSF and PGE2 produced by IL-1alpha-treated osteoblasts and soluble RANKL increased the TRAP staining of osteoclast precursors. These results suggest that IL-1alpha stimulated the formation of osteoclast-like cells via an increase in M-CSF and PGE2 production, and a decrease in OPG production by osteoblasts.     

Effect of dexamethasone on proliferating osteoblasts: inhibition of prostaglandin E2 synthesis, DNA synthesis, and alterations in actin cytoskeleton.

Elevated levels of glucocorticoids caused by disease (Cushing's syndrome) or therapeutic treatment of asthma are known to cause osteoporosis. Space flight, an environmental condition, is known to cause a rise in endogenous cortisols accompanied by a significant loss of bone and calcium. Long-term space inhabitants have lost up to 18% of weight bearing bone during long-term flight. This study demonstrates that elevated concentrations of glucocorticoids lower the endogenous production of PGE2 and interfere with osteoblast proliferation. Osteoblasts grown with dexamethasone had significantly lower DNA synthesis and endogenous synthesis of PGE2. Addition of exogenous dmPGE2 to the dexamethasone growth-inhibited cells stimulated DNA synthesis over twofold. In synchronous control cultures, we found that endogenous prostaglandin synthesis increased in late G1, preceding S-phase DNA synthesis by several hours. The addition of exogenous dexamethasone to synchronous cultures resulted in a significant decrease in the prostaglandin synthesis followed by a significant decrease in DNA synthesis in parallel cultures. Further, dexamethasone caused the actin cytoskeleton to collapse and the cell morphology to become rounded and spindle shaped. Addition of exogenous PGE2 to the dexamethasone-treated osteoblasts caused recovery of the actin architecture and phenotype. These data support the hypothesis that the glucocorticoid-mediated decrease in prostaglandin synthesis may be a contributing factor in the reduced bone quality and trabecular bone formation seen in glucocorticoid-induced osteoporosis.