The total RNA was extracted from 2T3 pre-osteoblast cells exposed to static or simulated microgravity (Rotating Wall Vessel) conditions. The RNA was then sent to Affymetrix microarray core facility at Baylor College of Medicine (Houston TX) for microarray analysis.

The below table includes a smaller list of data that was analyzed by dChip and filtered by pvalue such that a file with about 4600 genes was obtained which allowed for ease of use from 40,000 genes. Experiment Overall Design: The total RNA was extracted from 2T3 pre-osteoblast cells exposed to static or simulated microgravity (Rotating Wall Vessel) conditions. The RNA was then sent to Affymetrix microarray core facility at Baylor College of Medicine (Houston TX) for microarray analysis.

Background Although the role of the osteoclast in bone resorption is becoming better understood, much remains to be learned about osteoclastogenesis and the exact mechanism of action of anti-resorbing agents such as 17β-estradiol. This study investigated bone and morphologic osteoclast alterations following long-term estrogen administration to the B6D2F1 mouse. B6D2F1 mice aged 4-5 weeks were exposed to high levels of estrogen via implanted silastic tubing for at least 12 weeks; controls received empty tubing. Femurs of control and treated mice were assessed with radiology, quantitative histomorphometry and transmission electron microscopy. Results After 8 weeks of treatment, there was radiologic evidence of severe osteosclerosis and 86% of femoral marrow space was replaced with bone. After 12 weeks histologic studies of treated animals revealed that osteoclasts were positive for tartrate-resistant acid phosphatase but showed markedly abnormal ultrastructure which prevented successful bone resorption. Conclusions Findings extend our understanding of osteoclast structure and function in the mouse exposed in vivo to high doses of estrogen. Ultrastructural examination showed that osteoclasts from estrogen-treated mice were unable to seal against the bone surface and were unable to form ruffled borders.

In March 2006 murine Bone Marrow Stromal Cells (BMSC) were flown in the Soyuz 12S to the International Space Station to investigate the effects of microgravity on their osteogenic potential in a three-dimensional environment. BMSC were grown in porous bioceramic Skelite disks (dia 9 mm x T 1.2 mm). The constructs were exposed to microgravity for ca. 8 days then fixed for RNA extraction. While the flight experiment was performed in fully automated hardware inside the KUBIK incubator one group of control samples were incubated inside manually operated hardwares (flight control) and the other control group was incubated under routine laboratory conditions (lab control). The altered gene expression profile was analyzed by Mouse Gene 1.0 ST array (Affymetrix) representing whole-transcript coverage. Each one of the 28853 genes is represented on the array by approximately 26 probes spread across the full length of the gene providing a more complete and more accurate picture of gene expression than the 3 based expression array design. A few days of microgravity were sufficient to determinate at least at the molecular level an effect in the BMSC; this response expressed a stress condition able to determinate consequences on several compartments and cellular functions. In particular it seems to promote a gene expression known to be associated with neurogenic activity (e.g. axon guidance) perhaps promoting the BMSC capability to be committed in that direction. The osteo-induction by dexamethasone-based medium due to the short duration of stimulation did not have the possibility to manifest itself at the phenotypic level but only partially at the molecular level.

In March 2006 murine Bone Marrow Stromal Cells (BMSC) were flown in the Soyuz 12S to the International Space Station to investigate the effects of microgravity on their osteogenic potential in a three-dimensional environment. BMSC were grown in porous bioceramic Skelite disks (? 9 mm x T 1.2 mm). The constructs were exposed to microgravity for ca. 8 days then fixed for RNA extraction. While the flight experiment was performed in fully automated hardware inside the KUBIK incubator one group of control samples were incubated inside manually operated hardwares (flight control) and the other control group was incubated under routine laboratory conditions (lab control). The altered gene expression profile was analyzed by Mouse Gene 1.0 ST array (Affymetrix) representing whole-transcript coverage. Each one of the 28853 genes is represented on the array by approximately 26 probes spread across the full length of the gene providing a more complete and more accurate picture of gene expression than the 3 xc3 x94 xc3 xb8 xce xa9 based expression array design. A few days of microgravity were sufficient to determinate at least at the molecular level an effect in the BMSC; this response expressed a stress condition able to determinate consequences on several compartments and cellular functions. In particular it seems to promote a gene expression known to be associated with neurogenic activity (e.g. axon guidance) perhaps promoting the BMSC capability to be committed in that direction. The osteo-induction by dexamethasone-based medium due to the short duration of stimulation did not have the possibility to manifest itself at the phenotypic level but only partially at the molecular level.

Prolonged skeletal unloading through bedrest results in bone loss similar to that observed in elderly osteoporotic patients but with an accelerated timeframe. This rapid effect on weight-bearing bones is also observed in astronauts who lose up to 2% of their bone mass per month spent in Space. Despite important implications for Spaceflight travellers and bedridden patients on Earth the exact mechanisms involved in disuse osteoporosis have not been elucidated. Parathyroid hormone-related protein (PTHrP) regulates many physiological processes including skeletal development and has been proposed as a gravisensor. To investigate the role of PTHrP in microgravity-induced bone loss trabecular osteoblasts (TOs) from Pthrp+/+ and -/- mice were exposed to simulated microgravity for 6 days. Viability of TOs decreased in inverse proportion to PTHrP expression levels. Microarray analysis of Pthrp+/+ TOs after 6 days at 0g revealed expression changes in genes encoding prolactins,apoptosis and survival molecules bone metabolism and extra-cellular matrix composition proteins chemokines IGF family and Wnt-related signalling molecules. Importantly 88% of 0g-induced expression changes in Pthrp+/+ cells overlap those observed in Pthrp-/- cells in normal gravity. Pulsatile treatment with PTHrP1-36 peptide during microgravity exposure reversed a large proportion of 0g-induced changes in Pthrp+/+ TOs. Our results confirm PTHrP efficacy as an anabolic agent to prevent microgravity-induced cell death in TOs. Total RNA samples extracted from Pthrp+/+and -/- trabecular osteoblasts (TOs) exposed for 6 days to simulated 0g in Synthecon rotating cell or left 6 days in culture at 1g. Cells had either been treated with a pulsatile treatment (2 h/day) of PTHrP1-36 peptide (10-8M) or received a change in growth medium. In total: 8 different conditions with 2 replicates each i.e. Pthrp+/+ TOs at 0g or 1g with or without PTHrP1-36 treatment and Pthrp-/- TOs at 0g or 1 g,with or without PTHrP1-36 treatment.

Prolonged skeletal unloading through bedrest results in bone loss similar to that observed in elderly osteoporotic patients but with an accelerated timeframe. This rapid effect on weight-bearing bones is also observed in astronauts who lose up to 2% of their bone mass per month spent in Space. Despite important implications for Spaceflight travellers and bedridden patients on Earth the exact mechanisms involved in disuse osteoporosis have not been elucidated. Parathyroid hormone-related protein (PTHrP) regulates many physiological processes including skeletal development and has been proposed as a gravisensor. To investigate the role of PTHrP in microgravity-induced bone loss trabecular osteoblasts (TOs) from Pthrp+/+ and -/- mice were exposed to simulated microgravity for 6 days. Viability of TOs decreased in inverse proportion to PTHrP expression levels. Microarray analysis of Pthrp+/+ TOs after 6 days at 0g revealed expression changes in genes encoding prolactins,apoptosis and survival molecules bone metabolism and extra-cellular matrix composition proteins chemokines IGF family and Wnt-related signalling molecules. Importantly 88% of 0g-induced expression changes in Pthrp+/+ cells overlap those observed in Pthrp-/- cells in normal gravity. Pulsatile treatment with PTHrP1-36 peptide during microgravity exposure reversed a large proportion of 0g-induced changes in Pthrp+/+ TOs. Our results confirm PTHrP efficacy as an anabolic agent to prevent microgravity-induced cell death in TOs. Total RNA samples extracted from Pthrp+/+and -/- trabecular osteoblasts (TOs) exposed for 6 days to simulated 0g in Synthecon rotating cell or left 6 days in culture at 1g. Cells had either been treated with a pulsatile treatment (2 h/day) of PTHrP1-36 peptide (10-8M) or received a change in growth medium. In total: 8 different conditions with 2 replicates each i.e. Pthrp+/+ TOs at 0g or 1g with or without PTHrP1-36 treatment and Pthrp-/- TOs at 0g or 1 g,with or without PTHrP1-36 treatment.

During space flight bone mineral density is decreased by the influence of osteoclast activation which molecular mechanism is expectantly investigated. In the study of medaka bone development we investigated the system of vertebra formation and firstly identified the presence of osteoclasts in medaka. Moreover osteoclast rsorbing activity was affected by hypergravity indicating the possibility that we can investigate the effect of microgravity on osteoclasts in space. To find this effect we examine the alteration of osteoclast activity under microgravity with the histological analysis or the expression analysis by RNA in-situ hybridization. Furthermore since we have succeeded the establishment of the medaka osteoclast-specific transgenic lines we perform the in-vivo imaging analyses for gene expression and cell mobility. Finally to examine the gravity sensing system we employ tooth and bone as the high density organs which are highly sensitive to gravity and perform the histological analysis and the gene expression analysis of such gravity-sensitive tissues at surrounding pharyngeal teeth and supporting bone.

During space flight bone mineral density is decreased by the influence of osteoclast activation which molecular mechanism is expectantly investigated. In the study of medaka bone development we investigated the system of vertebra formation and firstly identified the presence of osteoclasts in medaka. Moreover osteoclast rsorbing activity was affected by hypergravity indicating the possibility that we can investigate the effect of microgravity on osteoclasts in space. To find this effect we examine the alteration of osteoclast activity under microgravity with the histological analysis or the expression analysis by RNA in-situ hybridization. Furthermore since we have succeeded the establishment of the medaka osteoclast-specific transgenic lines we perform the in-vivo imaging analyses for gene expression and cell mobility. Finally to examine the gravity sensing system we employ tooth and bone as the high density organs which are highly sensitive to gravity and perform the histological analysis and the gene expression analysis of such gravity-sensitive tissues at surrounding pharyngeal teeth and supporting bone.