Physiologic Archives - Research on StationResearch on Station

Modeled microgravity inhibits osteogenic differentiation of human mesenchymal stem cells and increases adipogenesis

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Space flight-induced bone loss has been attributed to a decrease in osteoblast function, without a significant change in bone resorption. To determine the effect of microgravity (MG) on bone, we used the Rotary Cell Culture System [developed by the National Aeronautics and Space Administration (NASA)] to model MG. Cultured mouse calvariae demonstrated a 3-fold decrease in alkaline phosphatase (ALP) activity and failed to mineralize after 7 d of MG. ALP and osteocalcin gene expression were also decreased. To determine the effects of MG on osteoblastogenesis, we cultured human mesenchymal stem cells (hMSC) on plastic microcarriers, and osteogenic differentiation was induced immediately before the initiation of modeled MG. A marked suppression of hMSC differentiation into osteoblasts was observed because the cells failed to express ALP, collagen 1, and osteonectin. The expression of runt-related transcription factor 2 was also inhibited. Interestingly, we found that peroxisome proliferator-activated receptor gamma (PPARgamma2), which is known to be important for adipocyte differentiation, adipsin, leptin, and glucose transporter-4 are highly expressed in response to MG. These changes were not corrected after 35 d of readaptation to normal gravity. In addition, MG decreased ERK- and increased p38-phosphorylation. These pathways are known to regulate the activity of runt-related transcription factor 2 and PPARgamma2, respectively. Taken together, our findings indicate that modeled MG inhibits the osteoblastic differentiation of hMSC and induces the development of an adipocytic lineage phenotype. This work will increase understanding and aid in the prevention of bone loss, not only in MG but also potentially in age-and disuse-related osteoporosis.

Impaired osteoblastogenesis potential of progenitor cells in skeletal unloading is associated with alterations in angiogenic and energy metabolism profile

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Skeletal unloading provokes bone loss. These bone alterations have been shown to be associated with impairment of osteoblastic activity. In the present study, we evaluated the effect of skeletal unloading on bone marrow progenitor cells, for exploration of the underlying mechanism. Wistar rats were randomized to be either hindlimb unloaded for 9 days or to act as controls. Micro-CT was used to detect tibial trabecular architecture changes in response to skeletal unloading. Microgravity conditions for 9 days resulted in a decreased number and an increased spacing of the bone trabeculae in the proximal tibia. The proliferative capacity of the femoral bone marrow samples was assessed (fibroblast-colony-forming assay). By using qPCR, the expression of selected markers of vascularization (Vegfa; Hif1a; Angpt1), energy metabolism (Prkaa2; Mtor), bone formation (Runx2; Alp; Bglap; Bmp2; Bmp4; Bmp7) and bone resorption (Acp5; Tnfsf11; Tnfrsf11b) in these bone marrow suspensions was measured. We demonstrated a striking decrease in the number of fibroblastic progenitors in response to hindlimb unloading. This deficit in proliferation was shown to be accompanied by altered hindlimb perfusion and cellular energy homeostasis. Ex vivo culture assays of the bone marrow-derived progenitor cells screened for osteogenic (Runx2; Alp; Bglap) and adipogenic (Pparg; Fabp4) differentiation alterations in response to microgravity. Induced progenitor cells from unloaded rats showed a delay in osteogenic differentiation and impaired adipogenic differentiation compared to control. The data of this multi-level approach demonstrate that skeletal unloading significantly affects the bone tissue and its metabolism at the progenitor stage. The molecular expressions of the bone marrow population support a role of cellular metabolic stresses in skeletal alterations induced by inactivity.

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The ALTEA/ALTEINO projects: studying functional effects of microgravity and cosmic radiation

by cfynanon 9 June 2015in Biology & Biotechnology No comment

The ALTEA project investigates the risks of functional brain damage induced by particle radiation in space. A modular facility (the ALTEA facility) is being implemented and will be operated in the International Space Station (ISS) to record electrophysiological and behavioral descriptors of brain function and to monitor their time dynamics and correlation with particles and space environment. The focus of the program will be on abnormal visual perceptions (often reported as "light flashes" by astronauts) and the impact on retinal and brain visual structures of particle in microgravity conditions. The facility will be made available to the international scientific community for human neurophysiological, electrophysiological and psychophysics experiments, studies on particle fluxes, and dosimetry. A precursor of ALTEA (the 'Alteino' project) helps set the experimental baseline for the ALTEA experiments, while providing novel information on the radiation environment onboard the ISS and on the brain electrophysiology of the astronauts during orbital flights. Alteino was flown to the ISS on the Soyuz TM34 as part of mission Marco Polo. Controlled ground experiments using mice and accelerator beams complete the experimental strategy of ALTEA. We present here the status of progress of the ALTEA project and preliminary results of the Alteino study on brain dynamics, particle fluxes and abnormal visual perceptions.

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.

Three-dimensional culture environments enhance osteoblast differentiation

by cfynanon 9 June 2015in Biology & Biotechnology No comment

PURPOSE: In previous work from our laboratory, we demonstrated that the three-dimensional (3D) cell cultures developed in simulated microgravity environments enhanced osseous-like aggregate formation and accelerated preosteoblast cell differentiation. Thus, as described here, we hypothesize that aggregate formation and mineralization would occur with fewer than 10 x 10(6) cells as previously described. MATERIALS AND METHODS: Human preosteoblastic cells were cultured at different concentrations in a rotary wall vessel to simulate microgravity for 7 days. Aggregate size was assessed, and mineralization and collagen expression detected using Von Kossa and Masson Trichrome staining. Scanning electron microscopy was used for structural and elemental analysis. Immunohistochemistry was used to detect expression of the osteogenic markers BSPII and osteopontin (OP). RESULTS: Size and calcium expression were dependent upon cultured cell number (p < 0.01). Calcium and collagen expression were detected throughout the aggregate, but organization was independent of cell number. Aggregates had similar microscopic structural patterns demonstrating organized development. Presence of BSPII and OP showed that the aggregates share common differentiation proteins with in vivo bone formation. CONCLUSIONS: These results may lead to novel bone engineering techniques associated with dental treatment.

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Research Containing: Physiologic

Modeled microgravity inhibits osteogenic differentiation of human mesenchymal stem cells and increases adipogenesis

Impaired osteoblastogenesis potential of progenitor cells in skeletal unloading is associated with alterations in angiogenic and energy metabolism profile

The ALTEA/ALTEINO projects: studying functional effects of microgravity and cosmic radiation

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

Three-dimensional culture environments enhance osteoblast differentiation