Our lab has shown over the past several years that interactions between the (relatively) ubiquitously expressed molecule, CD200, and its receptor(s), expressed mainly on myeloid-derived cells, can control activation of cells of the monocyte/macrophage lineage. Under simulated microgravity conditions, using exogenous CD200 (in the form of the fusion protein, CD200Fc) or cross-linking mAbs to CD200R1, we reported that CD200:CD200R1 interactions could suppress osteoclastogenesis at the expense of osteoblastogenesis. We now report data from studies on a recent FOTON mission, using an eOSTEO cell culture system developed by Systems Technologies Inc., showing that in space overexpression of CD200 (using cells derived from transgenic mice expressing CD200 under control of a doxycycline-inducible promoter) is associated with an attenuation in the suppression of mRNA markers of osteoblastogeneis (including BSP, OPG) with concomitant loss of the preferential increased osteoclastogenesis seen in the absence of CD200. In addition, cells from cytokine-receptor KO mice were used to explore a role for inflammatory cytokines (TNF, IL-1) in the modulation of bone development in flight.

OCp and OBp were isolated from 7d precultures of mouse marrow or calavaria respectively, and grown on 2D osteologic slides using feeder cells derived from normal or transgenic mice.Cells were fed automatically in an enclosed eOSTEO module for 11d in space (or under control ground conditions), having been "held" in low serum culture conditions for a 5d period prior to launch during the time period necessary for module loading followed by flight to Baikanur and payload integration. The payload was monitored for temperature fluctuations throughout the mission. Immediately prior to recovery the 2D slides were fixed and cooled to 14degrees, and on recovery samples from individual wells were harvested, mRNA extracted (TRizol), and RT-PCR analysis performed for mRNA expression in various groups. In selected groups microarray analysis was performed to explore additional changes in gene expression associated both with flight and manipulation of CD200:CD200R1 interaction.

Validating the methodology used to assess bone differentiation, cells in flight showed diminished mRNA expression for BSP and OPG, with increased mRNA expression for TRAP and RANKL, the latter both acknowledged as markers of osteoclastogenesis. Using either OBp/OCp from CD200 transgenic animals, or by including such cells as feeders in culture, increased expression of CD200 in space attenuated the decreased expression of BSP/OPG mRNAs observed in control cultures under the same conditions, with concomitant decreased expression of mRNAs for RANKL and TRAP. Importantly, if cells from CD200R1KO mice were used, the relative suppression of osteoclastogenesis seen in the context of increased CD200 expression in cultures in space was lost, consistent with our hypothesis that the suppression of osteoclastogenesis by CD200 is dependent upon delivery of signals through CD200R1 expressed on OCp. Finally, preliminary microarray analysis of control vs CD200-expressing cultures in flight revealed altered expression of a number of other genes implicated in regulation of bone differentiation in space.

In summary we have documented the value of a novel closed culture system (eOSTEO) for studying bone development and shown in this system the importance of the CD200:CD200R1 axis in control of osteoclastogenesis in space flight.

Stato- or otoliths are calcified structures in the organ of balance and equilibrium of vertebrates, the inner ear, where they enhance its sensitivity to gravity. The compact otoliths of fish are composed of the calcium carbonate polymorph aragonite and a small fraction of organic molecules. The latter form a protein skeleton, which determines the morphology of an otolith as well as its crystal lattice structure.

This review will address findings, according to which the brain obviously plays a prominent role in regulating the mineralization of fish otoliths, and that this regulation depends on the gravity vector. Overall, otolith mineralization has thus been identified to be a unique, neuronally guided biomineralization process. Efferent vestibular neurons are supposed to act on the mineralization process in a negative feed forward fashion. The following bullet points address the main factors that are hypothesized to regulate calcification on site:

Spaceflight causes profound changes in the skeleton, in particular, in the weight-loading bones. Stalled cell division in precursor bone cells and uncoupling of bone remodeling equilibrium between bone formation and resorption are considered responsible for the microgravity-induced bone loss. The weakened and brittle bones are prone to fracture on re-entry and accelerated osteoporosis, making bone deterioration as a major problem obstructing the prospects of long-duration manned space flight. However, the underlying molecular mechanism remains unraveled. Osteoblasts and osteocytes are known to be mechano-sensor, short-exposure of osteoblasts to simulated microgravity ensnarled cell adhesion and cytoskeleton.

We developed a novel 3D in vitro culture system by seeding cells onto porous bioceramics, mimicking the physiological niche of bone turn-over and enhancing cellular differentiation respective to conventional 2D Petri Dish cultures. Its application in testing anti-osteoporosis drugs is being evaluated. Having overcome several technological difficulties, in a series of STROMA spaceflight experiments 3D cultures of bone marrow derived mesenchymal stem cells (BMSC) and co-cultures of osteoblasts and osteoclast precursors were maintained and conserved in automated bioreactors on orbit. Genechip analysis revealed an inhibition of cell proliferation in microgravity. Unexpectedly, genes related to various processes of neural development were significantly upregulated in microgravity, raising the question on the lineage restriction in BMSC.

One of the most crucial medical challenges for long-term space flights is the prevention of bone loss affecting astronauts and its dramatic consequences on their return to gravitational field. Related bone strength is the result of density, composition and internal micro-architecture. It is maintained by a continuous structural adaptation to the mechanical environment through the remodelling process. The result is a structure optimised to bear the loads applied on it. Under microgravity conditions the external stimulus is not sufficient to sustain the proper bone structure leading to a rapid bone loss thereby dramatically increasing the fracture risk. Recently, a new noise-induced phenomenon in bone formation has been reported experimentally. With this contribution we propose a model for this findings based on Stochastic Resonance. Our simulations suggest new countermeasures for bone degeneration during long space flights using the effect of Stochastic Resonance. Our results may also be a starting point for research aimed to quantify optimal Stochastic Resonance regimes in order to treat, on Earth, bone diseases such as osteoporosis.

Recent studies have shown that simulated microgravity (SMG) results in altered proliferation and differentiation not only osteoblasts but also affects on osteogenic capacity of mesenchymal stem cells (MSCs) from various sources. Earlier we have demonstrated delay in proliferation rate, phenotype changes and reduced matrix mineralization of mesenchymal stem cells derived from human bone marrow culturing in osteogenic conditions for 20-30 days at horizontal clinostat (6 rpm). For present study we developed cell culture system that simulates some effects of microgravity produced by a 3D-clinostat (RPM, manufactured by Dutch Space, The Netherlands). We examined the hypothesis that MSCs as less commited precursors' of osteoblasts also can change response to osteogenic stimuli by influence of SMG at the RPM. Cultured MCSs from human bone marrow and human osteoblasts (OBs) were exposed to SMG for 10-30 days. Induced osteogenic differentiation of these progenitor cells was compared with the appropriate static (1g) and dynamic (horizontal shaker) controls. The main culture period lasted 20 days. The level of alkaline phosphatase activity during differentiation of MSCs (ALP, naphtol-method or flow cytometry by Epics XL, Beckman Coulter, USA), extracellular matrix mineralization (Alizarin staining) and immunophenotype (MSCs only) were evaluated. To obtain sufficient mineralization of MSC-derived cultures at least 30 days of culturing in varied modifications was applied.

Results: Clinorotated OBs and MSCs showed proliferation rate lower than static and dynamic control groups of cells in the early terms of SMG. Significant reduction of ALP activity was detected after 10 days of clinorotation of MSCs. Interestingly, there was no such dramatic difference in ALP activity of MSCs-derived cells between SMG and control groups after 20 days of clinorotation but the expression of ALP was still reduced (1,5-2 fold). However, virtually no matrix mineralization was found in OBs cultured under SMG conditions in the presence of differentiation stimuli (dexamethasone, ascorbic acid and beta-glycerol-phosphate). At the same time there was abundant calcium mineralization in the control groups. The similar effect was observed when we assayed matrix calcification of MSCs-derived cultures that were subjected to osteogenic induction for 10 days before 20 days of SMG. The obtained results confirm low gravity mediated reduction of osteogenesis of precursors' cells with different stage of maturity and can clarify the mechanisms of bone loss during spaceflight.

Small fish models, mainly zebrafish (Danio rerio) and medaka (Oryzias latipes), have been used for many years as powerful model systems for vertebrate developmental biology. Moreover, these species are increasingly recognized as valuable systems to study vertebrate physiology, pathology, pharmacology and toxicology. In recent years, analysis of gene function by mutation or genetic manipulation has shown that the homologs of many genes previously described to be involved in bone development and homeostasis in mammals also play very similar roles in small fish species. The homologs of genes whose expression is affected by microgravity in mammals also respond to microgravity simulation in fish. Thus, small fish models represent a valuable tool to investigate physiological changes induced by space flight in vertebrates.

Zebrafish and medaka present many advantages for studying development, such as transparency of the embryos, external development, possibility for large scale mutagenesis screening, and rapid development. Further characteristics, e.g. large number of embryos from one single clutch, small size, easy containment in water tanks and usability of existing instrumentation, are particularly useful for space research. Many technologies for visualizing and characterizing bones, such as specific staining or fluorescent transgenic animals, have been adapted to these fish and can be routinely performed on large numbers of larvae. Furthermore, the genome sequencing and annotation is close to completion for both species, opening the possibilities for whole genome analysis.

Our aim is to investigate the changes induced by microgravity in small fish species by combining several whole genome approaches, such as micro array expression analysis, proteomics or Chromatin ImmunoPrecipitation (ChIP) with a special emphasis on bone related genes. Sample collection is being performed using various methods for micro- and hyper-gravity simulation on ground as well as drug treatments. These data provide a first hint to the physiological processes being affected and the data will be compared to the changes observed in space. More quantitative methods, such as quantitative RT-PCR or Western blotting,, are used to confirm specific results. Highly regulated genes will be studied further by generating transgenic fish lines carrying the gene coding or a fluorescent protein under the control of the regulatory sequences for this particular gene. These lines will allow the real time and continuous observation of living larvae (fluorescent in bones) in microgravity; they will also be used for drug screening experiments in order to identify new drugs for preventing osteoporosis.