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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
NASA NTRS1999

The Role of Calcium in the Response of Osteoblasts to Mechanical Stimulation

A major biomedical concern in the exploration and development of space is the rapid loss of bone associated with extended periods of spaceflight. Mineral content, bone formation, matrix protein production and total body calcium are all reduced during long-term periods of weightlessness. These effects of weightlessness appears to be due to decreases in the anabolic function of osteoblasts and osteocytes rather than changes in the resorptive activity of osteoclasts. Conversely, subjecting the skeleton to exogenous mechanical loading increases matrix protein synthesis and bone formation rate, a process which also appears mediated through osteogenic cells. Osteoblasts have been shown to respond to a number of types of mechanical stimulation. However recently we have demonstrated that osteoblasts respond to fluid shear, but not physiologic levels of mechanical strain, with increases in expression of the matrix protein, osteopontin. We have also shown similar responses in other markers for the anabolic response in bone. The expression of the early response gene, c-fos, and the inducible-isoform of the prostaglandin synthetic enzyme, cyclooygenase-2 (COX-2), both increase rapidly in response to fluid shear, but not strain. How osteoblasts and osteocytes perceive mechanical stimuli and convert this stimulus into a biochemical event within the cell is still unknown. However, examination of the cellular events following mechanical stimulation indicate that two of the earliest responses are a rapid increase in intracellular calcium ([Ca(2+)](sub i)) and a reorganization of the actin cytoskeleton. The increase in [Ca(2+)](sub i) is dependent on the presence of extracellular Ca(2+), suggesting the activation of membrane Ca(2+) channel. We have previously characterized a mechanosensitive, cation-selective channel (MSCC) in osteoblast-like clonal cells, which we postulate is important in this early response to mechanical loading. Using an antisense oligodeoxynucleotide strategy, we have tentatively identified this channel as an isoform of the alc subunit of the dihydropyridine-sensitive, voltage sensitive Ca(2+) channel (VSCC). However, a major component in this mechanically induced rise in [Ca(2+)](sub i) is the release of Ca(2+) from intracellular stores. The actin cytoskeleton also rapidly responds to fluid shear with an increase in stress fiber formation and a realignment of the cell parallel to the direction of flow. To ascertain whether these two observations are related and how they effect shear-induced gene expression, we examined the role of Ca(2+) channels and intracellular Ca(2+) release on cytoskeletal reorganization and the resultant increases in the expression and production of c-fos and COX-2 in response to fluid shear.

Duncan, R. L.↗
NASA NTRS2000

Centrifugation of Cultured Osteoblasts And Macrophages as a Model To Study How Gravity Regulates The Function of Skeletal Cells

Mechanical loading helps define the architecture of weight-bearing bone via the tightly regulated process of skeletal turnover. Turnover occurs by the concerted activity of osteoblasts, responsible for bone formation. and osteoclasts, responsible for bone resorption. Osteoclasts are specialized megakaryon macrophages, which differentiate from monocytes in response to resorption stimuli, such as reduced weight-bearing. Habitation in space dramatically alters musculoskeletal loading, which modulates both cell function and bone structure. Our long-term objective is to define the molecular and cellular mechanisms that mediate skeletal adaptations to altered gravity environments. Our experimental approach is to apply hypergravity loads by centrifugation to rodents and cultured cells. As a first step, we examined the influence of centrifugation on the structure of cancellous bone in rats to test the ability of hypergravity to change skeletal architecture. Since cancellous bone undergoes rapid turnover we expected the most dramatic structural changes to occur in the shape of trabeculae of weight-bearing, cancellous bone. To define the cellular responses to hypergravity loads, we exposed cultured osteoblasts and macrophages to centrifugation. The intraosseous and intramedullary pressures within long bones in vivo reportedly range from 12-40 mm Hg, which would correspond to 18-59 gravity (g) in our cultures. We assumed that hydrostatic pressure from the medium above the cell layer is at least one major component of the mechanical load generated by centrifuging cultured cells. and therefore we exposed the cells to 10-50g. In osteoblasts, we examined the structure of their actin and microtubule networks, production of prostaglandin E2 (PGE2), and cell survival. Analysis of the shape of the cytoskeletal networks provides evidence for the ability of centrifugation to affect cell structure, while the production of PGE2 serves as a convenient marker for mechanical stimulation. We examined cell survival, reasoning that osteoblasts might mold skeletal structure in a hypergravity environment in part by regulating apoptosis and thus the duration of osteoblast productivity. Finally, we tested the influence of centrifugation on microbial activation of a macrophage cell line (RAW264.7). In response to the appropriate hormonal stimulation, this cell line is reportedly capable of undergoing differentiation to express osteoclast markers. In addition, a component of the cell wall of gram-negative bacteria, lipopolysaccaride (LPS), stimulates the formation of osteoclasts in vivo. Thus we tested the influence on centrifugation on RAW264.7 cells stimulated with LPS to provide an index of the function of osteoclast precursors.

Globus, Ruth K.↗
NASA NTRS2001

Structural Modeling of an Osteoblast Subjected to Hypergravity Loading

Osteoblasts, the bone-forming cells, are sensitive to altered mechanical loads such as those induced by fluid shear forces and cyclic uniaxial tension and compression. The cytoskeleton is thought to play an important role in a cell's response to mechanical loading and to coordinate cell shape changes. While some theoretical calculations indicate that a cell should not be able to respond to relatively small changes in gravity given the magnitude of forces exerted by intracellular components, osteoblasts have been shown to respond to microgravity. In this study, we investigated whether an osteoblast can sense a constant hypergravity acceleration of 10G. We asked if this load changes the shape of an osteoblast, and if engineering modeling accurately predicts the shape change. We modeled a cell by representing it as a series of axisymmetric shell structures. To test this model empirically, we performed experiments with cultured osteoblasts to measure changes in cell shape resulting from hypergravity loading.

Globus, Ruth K.↗
NASA NTRS2001

Hypergravity Loading the Cultured Osteoblasts: Modeling and Experimental Analysis of Cellular Morphology and the Cytoskeleton

Bone forming cells, osteoblasts, respond to various mechanical forces, including mechanical strain and fluid-induced shear stress. This study examined whether osteoblasts detect changes in gravity as a mechanical force, as assessed by cellular morphology and dimensions of the cytoskeletal network. We used modeling to evaluate how gravity influences cell morphology given theoretical differences in densities between the surrounding medium, cytoplasm, and nucleus. A mechanical model was built based on analysis of axisymmetric shell structures (Fast4 software) to study the effects of 10 times gravity (10G) on cell height. The model indicated 0.02% decrease in overall cell height when the medium was 10% denser than the nucleus or cytoplasm, 5.9 x 10(exp-5)% decrease when the nucleus was 10% denser than the cytoplasm or medium, and 1.3 x 10(exp-5)% decrease when the cell cytoplasm was 10% denser than the nucleus or medium. To experimentally evaluate the influence of gravity, cultured primary fetal rat osteoblasts were grown to near confluence and centrifuged at 10G for 3 hours. Actin, microtubules, and nuclei were fluorescently labeled and analyzed by confocal microscopy to determine overall microtubule and actin network height. Centrifugation led to an apparent reduction in height of both the microtubule (-16%) and the actin (-20%) networks relative to stationary controls. Thus, both modeling and experiments indicate that hypergravity reduces the height of the osteoblast cell layer and their microtubule and actin networks. This combination of modeling and experimental analyses will help us to better understand the mechanical loading of osteoblasts.

Searby, N. D.↗
NASA NTRS2002

How Osteoblasts Sense their Environment: Integrin-Extracellular Matrix Interactions and Mechanical Loading of Bone

Osteoblasts are the cells responsible for forming and replacing bone throughout life. We know that mechanical stimulation through weight-bearing at I gravity on Earth is needed to maintain healthy bone, and that osteoblasts play a critical role in that process. Over the last 9 years in my laboratory at NASA ARC, we have studied the regulation of osteoblast function by interactions between the extracellular matrix and die cell. Using a cell culture approach, we defined the repertoire of adhesion receptors, called integrins, which are expressed on the osteoblast surface, as well as specific extracellular matrix proteins, which are needed for cellular differentiation and survival. We are now extending these observations to determine if integrin signaling is involved in the skeletal responses to disuse and recovery from disuse using the rodent model of hindlimb unloading by tail suspension. Together, our cell culture and animal studies are providing new insight into the regulation of osteoblast function in bone.

Globus, Ruth K.↗
NASA NTRS2001

Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.

NASA Discipline Regulatory Physiology↗
NASA NTRS2000

Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

NASA Center ARC↗
NASA NTRS1998

Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.

Non-NASA Center↗
NASA NTRS1996

Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice

Our previous studies have shown that the 49-base pair region of promoter DNA between -1719 and -1670 base pairs is necessary for transcription of the rat COL1A1 gene in transgenic mouse calvariae. In this study, we further define this element to the 13-base pair region between -1683 and -1670. This element contains a TAAT motif that binds homeodomain-containing proteins. Site-directed mutagenesis of this element in the context of a COL1A1-chloramphenicol acetyltransferase construct extending to -3518 base pairs decreased the ratio of reporter gene activity in calvariae to tendon from 3:1 to 1:1, suggesting a preferential effect on activity in calvariae. Moreover, chloramphenicol acetyltransferase-specific immunofluorescence microscopy of transgenic calvariae showed that the mutation preferentially reduced levels of chloramphenicol acetyltransferase protein in differentiated osteoblasts. Gel mobility shift assays demonstrate that differentiated osteoblasts contain a nuclear factor that binds to this site. This binding activity is not present in undifferentiated osteoblasts. We show that Msx2, a homeodomain protein, binds to this motif; however, Northern blot analysis revealed that Msx2 mRNA is present in undifferentiated bone cells but not in fully differentiated osteoblasts. In addition, cotransfection studies in ROS 17/2.8 osteosarcoma cells using an Msx2 expression vector showed that Msx2 inhibits a COL1A1 promoter-chloramphenicol acetyltransferase construct. Our results suggest that high COL1A1 expression in bone is mediated by a protein that is induced during osteoblast differentiation. This protein may contain a homeodomain; however, it is distinct from homeodomain proteins reported previously to be present in bone.

NASA Discipline Cell Biology↗
NASA NTRS1996

Effects of microgravity on osteoblast growth activation

Space flight is an environmental condition where astronauts can lose up to 19% of weight-bearing bone during long duration missions. We used the MC3T3-E1 osteoblast to investigate bone cell growth in microgravity (10(-6) to 10(-9)g). Osteoblasts were launched on the STS-56 shuttle flight in a quiescent state with 0.5% fetal calf serum (FCS) medium and growth activation was initiated by adding fresh medium with 10% FCS during microgravity exposure. Four days after serum activation, the cells were fixed before return to normal Earth gravity. Ground controls were treated in parallel with the flight samples in identical equipment. On landing, cell number, cell cytoskeleton, glucose utilization, and prostaglandin synthesis in flight (n = 4) and ground controls (n = 4) were examined. The flown osteoblasts grew slowly in microgravity with total cell number significantly reduced (55 +/- 6 vs 141 +/- 8 cells per microscopic field). The cytoskeleton of the flight osteoblasts had a reduced number of stress fibers and a unique abnormal morphology. Nuclei in the ground controls were large and round with punctate Hoechst staining of the DNA nucleosomes. The flight nuclei were 30% smaller than the controls (P < 0.0001) and oblong in shape, with fewer punctate areas. Due to their reduced numbers, the cells activated in microgravity used significantly less glucose than ground controls (80.2 +/- 0.7 vs 50.3 +/- 3.7 mg of glucose/dl remaining in the medium) and had reduced prostaglandin E2 (PGE2) synthesis when compared to controls (57.3 +/- 17 vs 138.3 +/- 41 pmol/ml). Cell viability was normal since, on a per-cell basis, glucose use and prostaglandin synthesis were comparable for flight and ground samples. Taken together, these data suggest that growth activation in microgravity results in reduced growth, causing reduced glucose utilization and reduced prostaglandin synthesis, with significantly altered actin cytoskeleton in osteoblasts.

NASA Program Flight↗
NASA NTRS1994

Signal transduction pathways mediating parathyroid hormone regulation of osteoblastic gene expression

Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism. For example, excessive or inappropriate production of PTH or the related hormone, parathyroid hormone related protein (PTHrP), accounts for the majority of the causes of hypercalcemia. Both hormones act through the same receptor on the osteoblast to elicit enhanced bone resorption by the osteoclast. Thus, the osteoblast mediates the effect of PTH in the resorption process. In this process, PTH causes a change in the function and phenotype of the osteoblast from a cell involved in bone formation to one directing the process of bone resorption. In response to PTH, the osteoblast decreases collagen, alkaline phosphatase, and osteopontin expression and increases production of osteocalcin, cytokines, and neutral proteases. Many of these changes have been shown to be due to effects on mRNA abundance through either transcriptional or post-transcriptional mechanisms. However, the signal transduction pathway for the hormone to cause these changes is not completely elucidated in any case. Binding of PTH and PTHrP to their common receptor has been shown to result in activation of protein kinases A and C and increases in intracellular calcium. The latter has not been implicated in any changes in mRNA of osteoblastic genes. On the other hand activation of PKA can mimic all the effects of PTH; protein kinase C may be involved in some responses. We will discuss possible mechanisms linking PKA and PKC activation to changes in gene expression, particularly at the nuclear level.

Review↗
NASA NTRS1982

Nuclear size as a cell-kinetic marker for osteoblast differentiation

A nuclear morphometric assay for preosteoblasts is introduced as a cell-kinetic technique, applicable to routine histological preparations of mineralized tissue. Because this method is a morphological marker for osteoblast precursor cell differentiation, it provides a new dimension for determining the mechanism of osteoblast histogenesis. Osteoblast precursors of the periodontal ligament are a mixed population of progenitors, kinetically separable into two distinct groups according to nuclear size. Preosteoblasts, the immediate proliferating precursors of osteoblasts, have large nuclei (greater than 170 micrometers3) and are derived from relatively undifferentiated fibroblastlike cells, which have smaller nuclei (less than 80 micrometers3). Increase in nuclear volume, during G1 phase of the cell cycle, is apparently a morphological manifestation of change in genomic expression. This key event in preosteoblast differentiation is related to mechanical stress/strain and may be an important rate-limiting step in osteoblast histogenesis.

Non-NASA Center↗
NASA NTRS2004

Experiments with osteoblasts cultured under hypergravity conditions

To understand further the role of gravity in osteoblast attachment, osteoblasts were subjected to hypergravity conditions in vitro. Scanning electron microscopy of all confluent coverslips from FPA units show that the number of attached osteoblasts was similar among gravitational levels and growth durations (~90 cells/microscopic field). Specifically, confluent 1.0 G control cultures contained an average of 91 +/- 8 cells/field, 3.3 G samples had 88 +/- 8 cells/field, and 4.0 G cultures averaged 90 +/- 7 cells/field. The sparsely plated cultures assessed by immunohistochemistry also had similar numbers of cells at each time point (l.0 G was similar to 3.3 and 4.0 G), but cell number changed from one time point to the next as those cells proliferated. Immunohistochemistry of centrifuged samples showed an increase in number (up to 160% increase) and thickness (up to 49% increase) of actin fibers, a decrease in intensity of fibronectin fluorescence (18-23% decrease) and an increase in number of vinculin bulbs (202-374% increase in number of vinculin bulbs/area). While hypergravity exposure did not alter the number of attached osteoblasts, it did result in altered actin, fibronectin, and vinculin elements, changing some aspects of osteoblast- substrate adhesion.

NASA Program Fundamental Space Biology↗
NASA NTRS2016

Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture.

Cell and animal studies conducted onboard the International Space Station and formerly on Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. However the intercellular communicative mechanisms associated with the regulation of bone synthesis and bone resorption cells are still largely unknown. Connexin-43 (CX43), a gap junction protein, is hypothesized to play a significant role in osteoblast and osteocyte signaling. The purpose of this investigation was to evaluate within a novel three-dimensional microenvironment how the osteocyte-osteoblast gap-junction expression changes when cultures are exposed to exaggerated mechanical load. MLO-Y4 osteocyte-like cells were cultured on a 3D-Biotek polystyrene insert and placed in direct contact with an MC3T3-E1 pre-osteoblast co-cultured monolayer and exposed to 48 h of mechanical stimulation (pulsatile fluid flow (PFF) or monolayer cyclic stretch (MCS)) then evaluated for viability, proliferation, metabolism, and CX43 expression. Mono-cultured MLO-Y4 and MC3T3-E1 control experiments were conducted under PFF and MCS stimulation to observe how strain application stimuli (PFF cell membrane shear or MCS cell focal adhesion/attachment loading) initiates different signaling pathways or downstream regulatory controls. TotalLive cell count, viability and metabolic reduction (Trypan Blue, LIVEDead and Alamar Blue analysis respectively) indicate that mechanical activation of MC3T3-E1 cells inhibits proliferation while maintaining an average 1.04E4 reductioncell metabolic rate, *p0.05 n4. MLO-Y4s in monolayer culture increase in number when exposed to MCS loading but the percent of live cells within the population is low (46.3 total count, *p0.05 n4), these results may indicate an apoptotic signaling cascade. PFF stimulation of the three-dimensional co-cultures elicits a universal increase in CX43 in MLO-Y4 and MC3T3-E1 cells, illustrated by immunohistological observation. Increased CX43 expression is also observed with the three-dimensional co-cultures with MC3T3-E1 MCS stimulation but the increased gap-junction protein presence was limited to the osteoblast-osteocyte interface region. Previously reported PCR evaluation of osteogenic markers further corroborate that the co-cultured populations communicative networks play a role in translating mechanical signals to molecular messaging. These findings suggests an osteocyte-osteoblast gap-junction signaling feedback mechanism may regulate mechanotransduction of apoptosis initiation and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed.

mechanical loading↗
NASA NTRS2016

Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture

Cell and animal studies conducted onboard the International Space Station and formerly on Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. However the intercellular communicative mechanisms associated with the regulation of bone synthesis and bone resorption cells are still largely unknown. Connexin-43 (CX43), a gap junction protein, is hypothesized to play a significant role in osteoblast and osteocyte signaling. The purpose of this investigation was to evaluate within a novel three-dimensional microenvironment how the osteocyte-osteoblast gap-junction expression changes when cultures are exposed to exaggerated mechanical load. MLO-Y4 osteocyte-like cells were cultured on a 3D-Biotek polystyrene insert and placed in direct contact with an MC3T3-E1 pre-osteoblast co-cultured monolayer and exposed to 48 h of mechanical stimulation (pulsatile fluid flow (PFF) or monolayer cyclic stretch (MCS)) then evaluated for viability, proliferation, metabolism, and CX43 expression. Mono-cultured MLO-Y4 and MC3T3-E1 control experiments were conducted under PFF and MCS stimulation to observe how strain application stimuli (PFF cell membrane shear or MCS cell focal adhesionattachment loading) initiates different signaling pathways or downstream regulatory controls. TotalLive cell count, viability and metabolic reduction (Trypan Blue, LIVEDead and Alamar Blue analysis respectively) indicate that mechanical activation of MC3T3-E1 cells inhibits proliferation while maintaining an average 1.04E4 reductioncell metabolic rate, *p0.05 n4. MLO-Y4s in monolayer culture increase in number when exposed to MCS loading but the percent of live cells within the population is low (46.3 total count, *p0.05 n4), these results may indicate an apoptotic signaling cascade. PFF stimulation of the three-dimensional co-cultures elicits a universal increase in CX43 in MLO-Y4 and MC3T3-E1 cells, illustrated by immunohistological observation. Increased CX43 expression is also observed with the three-dimensional co-cultures with MC3T3-E1 MCS stimulation but the increased gap-junction protein presence was limited to the osteoblast-osteocyte interface region. Previously reported PCR evaluation of osteogenic markers further corroborate that the co-cultured populations communicative networks play a role in translating mechanical signals to molecular messaging. These findings suggests an osteocyte-osteoblast gap-junction signaling feedback mechanism may regulate mechanotransduction of apoptosis initiation and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed.

Juran, C. M.↗
NASA NTRS1997

Effects of Hypogravity on Osteoblast Differentiation

Weightbearing is essential for normal skeletal function. Without weightbearing, the rate of bone formation by osteoblasts decreases in the growing rat. Defective formation may account for the decrease in the maturation, strength and mass of bone that is caused by spaceflight. These skeletal defects may be mediated by a combination of physiologic changes triggered by spaceflight, including skeletal unloading, fluid shifts, and stress-induced endocrine factors. The fundamental question of whether the defects in osteoblast function due to weightlessness are mediated by localized skeletal unloading or by systemic physiologic adaptations such as fluid shifts has not been answered. Furthermore, bone-forming activity of osteoblasts during unloading may be affected by paracrine signals from vascular, monocytic, and neural cells that also reside in skeletal tissue. Therefore we proposed to examine whether exposure of cultured rat osteoblasts to spaceflight inhibits cellular differentiation and impairs mineralization when isolated from the influence of both systemic factors and other skeletal cells.

Globus, Ruth↗
NASA NTRS1999

The Effects of Orbital Spaceflight on Human Osteoblastic Cell Physiology and Gene Expression

The purpose of the proposed study is to establish whether changes in gravitational loading have a direct effect on osteoblasts to regulate TGF-6 expression. The effects of spaceflight and reloading on TGF-B MRNA and peptide levels will be studied in a newly developed line of immortalized human fetal osteoblasts (HFOB) transfected with an SV-40 temperature dependent mutant to generate proliferating, undifferentiated hFOB cells at 33-34 C and a non-proliferating, differentiated HFOB cells at 37-39'C. Unlike previous cell culture models, HFOB cells have unlimited proliferative capacity yet can be precisely regulated to differentiate into mature cells which express mature osteoblast function. If isolated osteoblasts respond to changes in mechanical loading in a manner similar to their response in animals, the cell system could provide a powerful model to investigate the signal transduction pathway for gravitational loading.

Turner, R. T.↗
NASA NTRS2000

Role of Integrin in Mechanical Loading of Osteoblasts

Mechanical forces generated by gravity, weightbearing, and muscle contraction play a key role in the genesis and maintenance of skeletal structure. The molecular mechanisms that mediate changes in osteoblast activity in response to altered patterns of skeletal loading are not known, and a better understanding of these processes may be essential for developing effective treatment strategies to prevent disuse osteoporosis. We have elucidated specific integrin/ECM (extracellular matrix) interactions that are required for osteoblast differentiation and survival and have developed a useful loading system to further explore the molecular basis of mechano-sensitivity of osteoblasts. The long term goal of our collaborative research is to understand how the ECM and cell adhesion proteins and integrins interaction to mediate the response of osteoblasts and their progenitors to mechanical loading. We suggest that integrin/ECM interactions are crucial for basic cellular processes, including differentiation and survival, as well as to participate in detecting and mediating cellular responses to mechanical stimuli.

Globus, Ruth↗