Microgravity and bone cell mechanosensitivity: FLOW experiment during the DELTA mission

The catabolic effects of microgravity on mineral metabolism in bone organ cultures might be explained as resulting from an exceptional form of disuse. It is possible that the mechanosensitivity of bone cells is altered under near weightlessness conditions, which likely contributes to disturbed bone metabolism observed in astronauts. In the experiment "FLOW", we tested whether the production of early signaling molecules that are involved in the mechanical load-induced osteogenic response by bone cells is changed under microgravity conditions. FLOW was one of the Biological experiment entries to the Dutch Soyuz Mission "DELTA" (Dutch Expedition for Life Science, Technology and Atmospheric Research). FLOW was flown by the Soyuz craft, launched on April 19, 2004, on its way to the International Space Station. Primary osteocytes, osteoblasts, and periosteal fibroblasts were incubated in plunger boxes, developed by Centre for Concepts in Mechatronics, using plunger activation events for single pulse fluid shear stress stimulations. Due to unforeseen hardware complications, results from in-flight cultures are considered lost. Ground control experiments showed an accumulative increase of NO in medium for osteocytes (as well as for osteoblasts and periosteal fibroblasts). Data from the online-NO sensor showed that the NO produced in medium by osteocytes increased sharply after pulse shear stress stimulations. COX-2 mRNA expression revealed high levels in osteoblasts compared to the other cell types tested. In conclusion, preparations for the FLOW experiment and preliminary ground results indicate that the FLOW setup is viable for a future flight opportunity

The Importance of Storage Time for Human Dental Pulp Cells Isolation

Objective: To compare the importance of storage time and the tooth type for isolation of dental pulp cells (DPCs) from extracted human teeth. Methods: 35 human teeth were used in this study. The teeth were stored in phosphate buffered saline (PBS) after extraction and divided into two groups randomly according to the time elapsed between extraction and isolation. In group one, the isolation was performed within 2 hours and in the other group it was performed 24 hours after extraction. Results: No significant differences between isolation time and total cell counts (p=0.483) and between isolation time and viable cells (p=0.341). No significant differences between the first molar and the premolar related cell counts and viable cells, but both teeth groups showed significant higher viability and had higher total cell amounts than third molars after isolation. Statistically significant correlations were found between age of donors and viable cells and viability after 24 hours isolation time. Conclusion: The immediate isolation of DPCs is not necessary after the tooth extraction. The tooth can be stored in PBS at room temperature up to twenty four hours after the extraction without a significant reduction in cell viability and counts. The cells obtained from younger donors might have more chance for more viability even if storage time was extended. Premolars and first molars were better donors than the third molars for DPCs isolations and the high number of success revascularization rate in premolars with necrotic immature premolars might be because of their high cell viability potentials

Decreased mineralization and increased calcium release in isolated fetal mouse long bones under near weightlessness

Mechanical loading plays an important role in the development and maintenance of skeletal tissues. Subnormal mechanical stress as a result of bed rest, immobilization, but also in spaceflight, results in a decreased bone mass and disuse osteoporosis, whereas supranormal loads upon extremities result in an increased bone mass. In this first in vitro experiment with complete fetal mouse cartilaginous long bones, cultured under microgravity conditions, we studied growth, glucose utilization, collagen synthesis, and mineral metabolism, during a 4‐day culture period in space. There was no change in percent length increase and collagen synthesis under microgravity compared with in‐flight 1× gravity. Glucose utilization and mineralization were decreased under microgravity. In addition, mineral resorption, as measured by 45Ca release, was increased. These data suggest that weightlessness has modulating effects on skeletal tissue cells. Loss of bone during spaceflight could be the result of both impaired mineralization as well as increased resorption

PLS3 Mutations in X-Linked Osteoporosis with Fractures

<p>Plastin 3 (PLS3), a protein involved in the formation of filamentous actin (F-actin) bundles, appears to be important in human bone health, on the basis of pathogenic variants in PLS3 in five families with X-linked osteoporosis and osteoporotic fractures that we report here. The bone-regulatory properties of PLS3 were supported by in vivo analyses in zebrafish. Furthermore, in an additional five families (described in less detail) referred for diagnosis or ruling out of osteogenesis imperfecta type I, a rare variant (rs140121121) in PLS3 was found. This variant was also associated with a risk of fracture among elderly heterozygous women that was two times as high as that among noncarriers, which indicates that genetic variation in PLS3 is a novel etiologic factor involved in common, multi-factorial osteoporosis.</p>

Fluid shear stress-induced TGF-β/ALK5 signaling in renal epithelial cells is modulated by MEK1/2

Renal tubular epithelial cells are exposed to mechanical forces due to fluid flow shear stress within the lumen of the nephron. These cells respond by activation of mechano-sensors located at the plasma membrane or the primary cilium, having crucial roles in maintenance of cellular homeostasis and signaling. In this paper, we applied fluid shear stress to study TGF-β signaling in renal epithelial cells with and without expression of the Pkd1-gene, encoding a mechano-sensor mutated in polycystic kidney disease. TGF-β signaling modulates cell proliferation, differentiation, apoptosis, and fibrotic deposition, cellular programs that are altered in renal cystic epithelia. SMAD2/3-mediated signaling was activated by fluid flow, both in wild-type and Pkd1−/− cells. This was characterized by phosphorylation and nuclear accumulation of p-SMAD2/3, as well as altered expression of downstream target genes and epithelial-to-mesenchymal transition markers. This response was still present after cilia ablation. An inhibitor of upstream type-I-receptors, ALK4/ALK5/ALK7, as well as TGF-β-neutralizing antibodies effectively blocked SMAD2/3 activity. In contrast, an activin-ligand trap was ineffective, indicating that increased autocrine TGF-β signaling is involved. To study potential involvement of MAPK/ERK signaling, cells were treated with a MEK1/2 inhibitor. Surprisingly, fluid flow-induced expression of most SMAD2/3 targets was further enhanced upon MEK inhibition. We conclude that fluid shear stress induces autocrine TGF-β/ALK5-induced target gene expression in renal epithelial cells, which is partially restrained by MEK1/2-mediated signaling.Multi Phase System