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DOE OSTI2021

Phenotypical modifications of immune cells are enhanced by extracellular matrix

Immune cells not only constitute tumour microenvironment but they may even affect disease prognosis as a result of dual functional roles that they may play in tumour tissues. Two frequently used established immune cell lines (lymphocytic Jurkat and monocytic THP-1) were used to test whether microenvironmental factors, especially molecular components of extracellular matrix, can shape the phenotype of immune cells. Proliferation, morphological and phenotypical analyses were applied to compare behaviour of the immune cells, typically cultured as suspensions in culture medium, with their behaviour in collagen type I-based and Matrigel-based 3D cultures. Density of both immune cell types in routine suspension cultures affected their subsequent proliferation in extracellular matrices. THP-1 cells appeared to be more sensitive to their surrounding microenvironment as judged from extracellular matrix type-dependent changes in their cell doubling times and from slight increase in their diameters in both extracellular matrix-containing cell cultures. Moreover, even chemically uninduced monocytic THP-1 cells were present in a minor fraction as CD68 positive cell population in collagen type I matrix indicating their partial differentiation to macrophages. Observed modifications of immune cells by microenvironmental factors may have profound implications for their roles in healthy and pathological tissues.

60 APPLIED LIFE SCIENCES↗

NASA NTRS1961

Photosynthetic Gas Exchange in the Closed Ecosystem for Space. Phase II, Part II. Studies on the Growth of Thermophilic Chlorella 71105

Bench-scale studies with Chlorella pyrenoidosa 71105 were conducted in four- and eight-liter culture vessels, with and without recycling. It was established that the algal strain could be maintained for periods up to 72 days with supplemented re-cycled medium. In a factorial series of experiments, the highest yields were obtained with a 2.0% concentration of carbon dioxide, a 0.07 dilution factor (nutrient dilution rate/culture volume), and an 8.5 ft(exp -1) ratio of light surface to liquid culture volume. Optimum pH and concentrations of the constituents of the growth medium were determined in corollary test-tube studies. Other test-tube studies showed that the nutrient medium used for mass culture could be stored up to five weeks at 25 C or at refrigerator temperature (4 C) with little change in nitrogen-urea level. Algal suspensions stored at 4 C remained viable for periods of at least 12 weeks; growth resumed after a lag period when stored suspensions were cultured at 39 C. Streptomycin was found suitable for controlling a blue-green containment.

ALGAE↗

NASA NTRS2006

Immune Response in Microgravity: Genetic Basis and Countermeasure Development Implications

Impairment of the immunity in astronauts and cosmonauts even in shortterm flights is a recognized risk. Longterm orbital space missions and anticipated interplanetary flights increase the concern for more pronounced effects on the immune system with potential clinical consequences. Studies in true and modeled microgravity (MG) have demonstrated that MG directly affects numerous lymphocyte functions. The purpose of this study was to screen for genes involved in lymphocytes response to modeled microgravity (MMG) that could explain the functional and structural changes observed earlier. The microgravity-induced changes in gene expression were analyzed by microarray DNA chip technology. CD3and IL2activated Tcells were cultured in 1g (static) and modeled microgravity (NASA Rotating Wall Vessel bioreactor) conditions for 24 hours. Total RNA was extracted using the RNeasy isolation kit (Qiagen, Valencia, CA). Microarray experiments were performed utilizing Affymetrix Gene Chips (U133A), allowing testing for 18,400 human genes. To decrease the biological variation and aid in detecting microgravity-associated changes, experiments were performed in triplicate using cells obtained from three different donors. Exposure to modeled microgravity resulted in alteration of 89 genes, 10 of which were upregulated and 79 down-regulated. Altered genes were categorized by their function, structural role and by association with metabolic and regulatory pathways. A large proportion was found to be involved in fundamental cellular processes: signal transduction, DNA repair, apoptosis, and multiple metabolic pathways. There was a group of genes directly related to immune and inflammatory responses (IL7R, granulysin, proteasome activator subunit 2, peroxiredoxin 4, HLADRA, lymphocyte antigen 75, IL18R and DOCK2 genes). Among these genes only one (IL7R) was upregulated, the rest were downregulated. The upregulation of the IL7 receptor gene was confirmed by RT PCR. Three genes with altered expression were identified in the apoptosis related group (Granzyme B, APO2 ligand and Beta3endonexin). All of them were downregulated. Gene expression changes in MG might appear pivotal in identifying potential molecular targets for countermeasure development. (Supported by NRA OLMSA02 and NSCORT NAG54072 grants).

Risin, Diana↗

NASA NTRS2007

Contrasting Perspectives Of Junior versus Senior NASA ISS Flight Controllers On Leadership And Cultural Issues

NASA flight controllers have always worked in a very demanding environment, but the International Space Station (ISS) poses even more challenges than prior missions. A recent NASA/Ames survey by Parke and Orasanu of NASA/Johnson flight controllers uncovered concerns about communications problems between American personnel and their international counterparts. To better understand these problems, we interviewed 14 senior and 12 junior ISS flight controllers at NASA/Johnson about leadership and cultural challenges they face and strategies for addressing these challenges. The qualitative interview data were coded and tabulated. Here we present quantitative analyses testing for differences between junior and senior controllers. Based on nonparametric statistical tests comparing responses across groups, the senior controllers were significantly more aware of the impact of working in dispersed teams, the context of constant change, and the upcoming multilateral challenges, while junior controllers were more aware of language and cultural issues. We consider our findings in light of other studies of controllers and other known differences between senior and junior controllers. For example, the fact that senior controllers had their formative early experience controlling pre-ISS short-duration Shuttle missions seems to have both positive and negative aspects, which are supported by our data. Our findings may also reflect gender differences, but we cannot unconfound this effect in our data because all the senior respondents were males. Many of the junior-senior differences are not only due to elapsed time on the job, but also due to a cohort effect. The findings of this study should be used for training curricula tailored differently for junior and senior controllers.

Clement, James L.↗

NASA NTRS2013

Challenges of Integrating NASAs Space Communication Networks

The transition to new technology, innovative ideas, and resistance to change is something that every industry experiences. Recent examples of this shift are changing to using robots in the assembly line construction of automobiles or the increasing use of robotics for medical procedures. Most often this is done with cost-reduction in mind, though ease of use for the customer is also a driver. All industries experience the push to increase efficiency of their systems; National Aeronautics and Space Administration (NASA) and the commercial space industry are no different. NASA space communication services are provided by three separately designed, developed, maintained, and operated communications networks known as the Deep Space Network (DSN), Near Earth Network (NEN) and Space Network (SN). The Space Communications and Navigation (SCaN) Program is pursuing integration of these networks and has performed a variety of architecture trade studies to determine what integration options would be the most effective in achieving a unified user mission support organization, and increase the use of common operational equipment and processes. The integration of multiple, legacy organizations and existing systems has challenges ranging from technical to cultural. The existing networks are the progeny of the very first communication and tracking capabilities implemented by NASA and the Jet Propulsion Laboratory (JPL) more than 50 years ago and have been customized to the needs of their respective user mission base. The technical challenges to integrating the networks are many, though not impossible to overcome. The three distinct networks provide the same types of services, with customizable data rates, bandwidth, frequencies, and so forth. The differences across the networks have occurred in effort to satisfy their user missions' needs. Each new requirement has made the networks more unique and harder to integrate. The cultural challenges, however, have proven to be a significant obstacle for integration. Over the past few decades of use, user missions and network personnel alike have grown accustomed to the processes by which services are provided by the NASA communications and navigation networks. The culture established by each network has created several challenges that need to be overcome in order to effectively integrate the networks. As with any change, there has been resistance, an apprehension to explore automation of existing processes, and a working environment that attempts to indirectly influence change without mandating compliance. Overcoming technical and cultural challenges is essential to successfully integrating the networks and although the challenges are numerous, the integration of the networks promises a more efficient space communications network for NASA and its customers, as well as potential long-term cost savings to the agency. This paper, Challenges of Integrating NASA Legacy Communications Networks, will provide a brief overview of the current NASA space communications networks as well as the an overview of the process implemented while performing the SCaN Trade Studies and an introduction to the requirements driving integration of the SCaN Networks. This paper will describe in detail the challenges experienced, both technical and cultural, while working with NASA space communications network-specific personnel. The paper will also cover lessons learned during the performance of architecture trade studies and provide recommendations for ways to improve the process.

Reinert, Jessica M.↗

NASA NTRS2013

Challenges of Integrating NASA's Space Communications Networks

Systems Integration↗

DOE OSTI2024

An expedited screening platform for the discovery of anti-ageing compounds in vitro and in vivo

Background: Restraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan. Methods: Using Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture. Results: We developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo. Conclusions: Our method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2016

Optic Nerve Sheath Mechanics in VIIP Syndrome

Visual Impairment Intracranial Pressure (VIIP) syndrome is a major concern in current space medicine research. While the exact pathology of VIIP is not yet known, it is hypothesized that the microgravity-induced cephalad fluid shift increases intracranial pressure (ICP) and drives remodeling of the optic nerve sheath. To investigate this possibility, we are culturing optic nerve sheath dura mater samples under different pressures and investigating changes in tissue composition. To interpret results from this work, it is essential to first understand the biomechanical response of the optic nerve sheath dura mater to loading. Here, we investigated the effects of mechanical loading on the porcine optic nerve sheath.Porcine optic nerves (number: 6) were obtained immediately after death from a local abattoir. The optic nerve sheath (dura mater) was isolated from the optic nerve proper, leaving a hollow cylinder of connective tissue that was used for biomechanical characterization. We developed a custom mechanical testing system that allowed for unconfined lengthening, twisting, and circumferential distension of the dura mater during inflation and under fixed axial loading. To determine the effects of variations in ICP, the sample was inflated (0-60 millimeters Hg) and circumferential distension was simultaneously recorded. These tests were performed under variable axial loads (0.6 grams - 5.6 grams at increments of 1 gram) by attaching different weights to one end of the dura mater. Results and Conclusions: The samples demonstrated nonlinear behavior, similar to other soft connective tissue (Figure 1). Large increases in diameter were observed at lower transmural pressures (approximately 0 to 5 millimeters Hg), whereas only small diameter changes were observed at higher pressures. Particularly interesting was the existence of a cross-over point at a pressure of approximately 11 millimeters Hg. At this pressure, the same diameter is obtained for all axial loads applied to the tissue; i.e., as the axial load is varied, the diameter of the dura mater remains constant. This cross-over in the pressure-diameter curves occurred in all optic nerve sheaths that were tested, and may correspond with in vivo ICP levels for pigs. These data suggest that diameter of the dura mater of the optic nerve remains nearly constant in vivo despite being stretched axially. This may be a homeostatic mechanism aimed at maintaining target stresses/strains on the cells in the dura mater, and deviations from these stresses may play an important role in optic nerve sheath remodeling. Future studies will involve subjecting the dura mater to varying pressures and axial tensions for extended periods of time, while monitoring changes in the biomechanical properties. The data can then be used to study the effects of changes in ICP on the remodeling of the dura mater.

biomechanics↗

NASA NTRS2016

Instrumentation for Examining Microbial Response to Changes In Environmental Pressures

The Automated Adaptive Directed Evolution Chamber (AADEC) is a device that allows operators to generate a micro-scale analog of real world systems that can be used to model the local-scale effects of climate change on microbial ecosystems. The AADEC uses an artificial environment to expose cultures of micro-organisms to environmental pressures, such as UV-C radiation, chemical toxins, and temperature. The AADEC autonomously exposes micro-organisms to selection pressures. This improves upon standard manual laboratory techniques: the process can take place over a longer period of time, involve more stressors, implement real-time adjustments based on the state of the population, and minimize the risk of contamination. We currently use UV-C radiation as the main selection pressure, UV-C is well studied both for its cell and DNA damaging effects as a type of selection pressure and for its related effectiveness as a mutagen; having these functions united makes it a good choice for a proof of concept. The AADEC roadmap includes expansion to different selection pressures, including heavy metal toxicity, temperature, and other forms of radiation.The AADEC uses closed-loop control to feedback the current state of the culture to the AADEC controller that modifies selection pressure intensity during experimentation, in this case culture density and growth rate. Culture density and growth rate are determined by measuring the optical density of the culture using 600 nm light. An array of 600 nm LEDs illuminate the culture and photodiodes are used to measure the shadow on the opposite side of the chamber.Previous experiments showed that we can produce a million fold increase to UV-C radiation over seven iterations. The most recent implements a microfluidic system that can expose cultures to multiple different selection pressures, perform non-survival based selection, and autonomously perform hundreds of exposure cycles. A scalable pump system gives the ability to pump in various different growth media to individual cultures and introduce chemical toxins during experimentation; AADEC can perform freeze and thaw cycles. We improved our baseline characterization by building a custom UV-C exposure hood, a shutter operates on a preset timer allowing the user to set exposure intensity consistently for multiple iterations.

Environment↗

NASA NTRSDate not supplied

Enabling Space Biology Knowledge Discovery Through Biospecimen Sharing: The NASA Biological Institutional Scientific Collection

NASA and international partners have conducted experiments in space to understand the biological impacts and address hazards to health. The resulting basic and applied science is imperative to enabling humanity to venture back to the Moon and then to Mars and beyond. Sending organisms into space is a costly endeavor. All biospecimens not required by spaceflight-relevant Principal Investigators are harvested, preserved, and archived in the NASA Biological Institutional Scientific Collection (NBISC) to maximize the scientific return. The NASA Biological and Physical Sciences (BPS) Division has an ‘Open Science’ endeavor which includes NASA Genelab, the Space Biology Program’s Biospecimen Sharing Program, Physical Sciences Informatics, the Ames Life Sciences Data Archive, and NBISC. Its purpose is to integrate extensive data and biospecimen resources from spaceflight and/or ground-based analog experiments. NBISC biospecimens are collected and preserved according to well-established standard operating procedures to maintain scientific quality and are available on-request by the international scientific community. NBISC currently stores over 32,000 biospecimens from Shuttle, International Space Station, and ground-based space analog investigations. Tissue sharing has resulted in at least 33 publications since 2011 and 48 requests since 2016. Many requests for NBISC biospecimen come from first-time investigators who subsequently submit grants as the port-of-entry into the field of space biology. Some NBISC biospecimens have been awarded to NASA Genelab, who then generate various ‘Open Science’ -omics data sets on their platform for bioinformatics. Other NBISC biospecimen awards have led to multiple studies such as fecal microbiome analysis, DNA damage analysis using single-cell DNA sequencing, enzymatic-pathway identification involved in spaceflight muscle atrophy, and characterization of ocular morphological changes. Of note, NBISC has expanded to include a new Space Microbial Culture Collection (SMCC) for the collection, identification, documentation, long-term preservation, and distribution of space-related microbial isolates.

Ryan T. Scott↗

NASA NTRSDate not supplied

Enabling Biological Discovery Through Biospecimen Sharing: The Nasa Biological Institutional Scientific Collection

Understanding biological impacts from spaceflight hazards and the subsequent development of countermeasures are a high priority to enable humanity to venture back to the Moon, and then to Mars and beyond. Experiments have been conducted with model organisms flown to space and analogous investigations terrestrially, to identify biological mechanistic impacts from spaceflight hazards and to develop mitigation countermeasures, thus contributing towards basic and applied science goals. However, sending organisms into space is a costly endeavor. To maximize scientific return, all biospecimens not required by spaceflight-relevant Principal Investigators are harvested, preserved, and archived in the NASA Biological Institutional Scientific Collection (NBISC). Biospecimens are collected and preserved according to well-established standard operating procedures to maintain scientific quality and are available on-request by the international scientific community. NBISC currently stores over 32,000 biospecimens from Shuttle, International Space Station, and ground-based space analog investigations. Tissue sharing has resulted in at least 33 publications since 2011 and 51 requests since 2016. Many requests for NBISC biospecimens come from first-time investigators who subsequently submit grants as their point-of-entry into the field of spaceflight biology and health. The NBISC biorepository is part of the NASA ‘Open Science for Life in Space’ collaborative group of projects, which includes NASA Genelab, the Space Biology Program’s Biospecimen Sharing Program, Physical Sciences Informatics, and the Ames Life Sciences Data Archive. NBISC biospecimens have been awarded to NASA Genelab, who then generated various open access science ‘omics datasets through the GeneLab Sample Processing laboratory, with resulting data widely used for biological study. Other NBISC biospecimen awards have led to studies on fecal microbiome analysis, DNA damage analysis using single-cell DNA sequencing, enzymatic-pathway identification involved in spaceflight muscle atrophy, and characterization of ocular morphological changes. Of note, NBISC is expanded to include a new Space Microbial Culture Collection (SMCC) for the collection, identification, documentation, long-term preservation, and distribution of space-related microbial isolates.

Biospecimens↗

DOE OSTI2022

Dose-dependent consequences of sub-chronic fentanyl exposure on neuron and glial co-cultures

Fentanyl is one of the most common opioid analgesics administered to patients undergoing surgery or for chronic pain management. While the side effects of chronic fentanyl abuse are recognized (e.g., addiction, tolerance, impairment of cognitive functions, and inhibit nociception, arousal, and respiration), it remains poorly understood what and how changes in brain activity from chronic fentanyl use influences the respective behavioral outcome. Here, we examined the functional and molecular changes to cortical neural network activity following sub-chronic exposure to two fentanyl concentrations, a low (0.01 μM) and high (10 μM) dose. Primary rat co-cultures, containing cortical neurons, astrocytes, and oligodendrocyte precursor cells, were seeded in wells on either a 6-well multi-electrode array (MEA, for electrophysiology) or a 96-well tissue culture plate (for serial endpoint bulk RNA sequencing analysis). Once networks matured (at 28 days in vitro ), co-cultures were treated with 0.01 or 10 μM of fentanyl for 4 days and monitored daily. Only high dose exposure to fentanyl resulted in a decline in features of spiking and bursting activity as early as 30 min post-exposure and sustained for 4 days in cultures. Transcriptomic analysis of the complex cultures after 4 days of fentanyl exposure revealed that both the low and high dose induced gene expression changes involved in synaptic transmission, inflammation, and organization of the extracellular matrix. Collectively, the findings of this in vitro study suggest that while neuroadaptive changes to neural network activity at a systems level was detected only at the high dose of fentanyl, transcriptomic changes were also detected at the low dose conditions, suggesting that fentanyl rapidly elicits changes in plasticity.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1985

Control of arachidonic acid release in chick muscle cultures

Cultures from thigh muscles of 12 day old embryonic chicks are utilized to examine arachidonic release, prostaglandin (PG) biosynthesis, and protein synthesis. The preparation of the cultures is described. It is observed that exogenous arachidonic acid is formed into photsphatidylethanolamine and phosphatidylcholine, is released by a calcium ionosphere or phospholiphase simulator, and is the substrate for the biosynthesis of PG; the epidermal growth factor and PGF do not stimulate protein synthesis over the basal levels. The relationship between arachidonate release and melittin is studied. The data reveal that a change in intracellular calcium stimulates phospholiphase activity, arachidonate release, and PG synthesis in chick muscle culture.

Templeton, G. H.↗

NASA NTRS1985

Mature phenotype in Hemerocallis plantlets fortuitously generated in vitro

Daylily plantlets generated on semi-solid media from morphogenetically competent cells or morphogenetically competent cells regenerated from protoplasts can give rise in aseptic culture to plantlets with a mature phenotype. The individual leaves of these plantlets open to the extreme base so that no encircling leaf sheath is present. This permits the overlapping bases and leaves to assume an open fan-like arrangement. The occurrence of fans correlates with exceptionally tightly sealed culture vessels and experiments to date suggest a gaseous component is associated with this change of growth form. It has not been possible to fix the mature growth mode, however, and new leaf growth assumes the more normal juvenile phenotype when the gaseous environment is altered by admitting or exposure to room air.

NASA Discipline Plant Biology↗

NASA NTRS1989

Ethylene-associated phase change from juvenile to mature phenotype of daylily (Hemerocallis) in vitro

Hemerocallis plantlets maintained in vitro for extended periods of time in tightly closed culture vessels frequently show a phenotype, albeit on a miniaturized scale, typical of more mature, field-grown plants. The positive relationship of elevated ethylene in the headspace of such vessels to the phase shift from juvenile to mature form is established. Rigorous restriction in air exchange with the external environment by means of silicone grease seals hastens the phase change and improves uniformity of response. Although some plantlets may take longer to accumulate enough ethylene in sealed jars to undergo change, added ethylene and ethylene-releasing agents promote it. Ethylene adsorbants (e.g. mercuric perchlorate) block the shift of juvenile to mature form. Critical ambient ethylene level for the shift is ca 1 microliter l-1. Levels up to 1000 microliters l-1 do not hasten the response but are not toxic. The phase change is fully reversible when air exchange permits ethylene to drop below 1 microliter l-1. At least 1 microliter l-1 ethylene is required to sustain the mature phenotype. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) prevents the phase change, while the ethylene biosynthesis intermediate 1-aminocyclopropanecarboxylic acid (ACC) improves it. KOH, as a CO2 absorbent, does not prevent the phase change. Histology sections demonstrate subtle changes in the form of shoot tips of plantlets undergoing phase change.

NASA Discipline Plant Biology↗

DOE OSTI2020

Karuk Climate Resiliency Plan

Statewide, California is the hottest and driest since modern record keeping has taken place. Among the most urgent of the local dimensions of climate change in Karuk Ancestral Territory is the increased frequency of high-severity fire. The 2016 Karuk Climate Vulnerability Assessment (CVA) outlines how these influences have created landscape conditions that hold the potential to be devastating in the face of future high-severity fire events. The project followed the format of our CVA and furthers information garnered to accomplish two primary objectives. The Karuk Tribe developed a Climate Adaptation Plan to address established vulnerabilities to Tribal traditional foods and cultural use species, Tribal program infrastructure, and Tribal management authority and political status resulting from changing conditions with a focus on increased frequency of high-intensity wildfire events within the Karuk Ancestral Territory.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗

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 NTRS2000

Effects of orbital spaceflight on human osteoblastic cell physiology and gene expression

During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation. It is not clear, however, whether the force imparted by gravity has direct effects on bone cells. To examine the response of bone forming cells to weightlessness, human fetal osteoblastic (hFOB) cells were cultured during the 17 day STS-80 space shuttle mission. Fractions of conditioned media were collected during flight and shortly after landing for analyses of glucose utilization and accumulation of type I collagen and prostaglandin E(2) (PGE(2)). Total cellular RNA was isolated from flight and ground control cultures after landing. Measurement of glucose levels in conditioned media indicated that glucose utilization occurred at a similar rate in flight and ground control cultures. Furthermore, the levels of type I collagen and PGE(2) accumulation in the flight and control conditioned media were indistinguishable. The steady-state levels of osteonectin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) were not significantly changed following spaceflight. Gene-specific reductions in mRNA levels for cytokines and skeletal growth factors were detected in the flight cultures using RNase protection assays. Steady-state mRNA levels for interleukin (IL)-1alpha and IL-6 were decreased 8 h following the flight and returned to control levels at 24 h postflight. Also, transforming growth factor (TGF)-beta(2) and TGF-beta(1) message levels were modestly reduced at 8 h and 24 h postflight, although the change was not statistically significant at 8 h. These data suggest that spaceflight did not significantly affect hFOB cell proliferation, expression of type I collagen, or PGE(2) production, further suggesting that the removal of osteoblastic cells from the context of the bone tissue results in a reduced ability to respond to weightlessness. However, spaceflight followed by return to earth significantly impacted the expression of cytokines and skeletal growth factors, which have been implicated as mediators of the bone remodeling cycle. It is not yet clear whether these latter changes were due to weightlessness or to the transient increase in loading resulting from reentry.

Non-NASA Center↗