Abstract
ESA’s Biobox, an automated incubator
for space biological experiments, completed three missions on Bion/Foton
whereby 10 experiments were executed. All experiments were aimed at elucidating
how mammalian cells, cultured in isolation from the body, react to the absence
of gravity. In most experiments, osteoblast-like cells were used to test the
theory that microgravity might have a direct, negative effect on the
bone-forming activity of these cells. Although the results obtained so far are
definitely in support of that theory, final proof is still missing.
The
investigations on in-vitro cultures of bone cells in space, pioneered by
Biobox, have recently seized the attention of the pharmaceutical industry.
Since the loss of bone mass in space as experienced by space travellers can be
regarded as an accelerated version of osteoporosis in older people, Biobox-like
experiments may contribute to find a remedy for this medical problem on
Earth.
Biobox experiments on
Bion/Foton
ESA-affiliated life scientists have
been involved in nine Bion/Foton missions, whereby 24 biological microgravity
experiments were completed, all aimed at detecting if, and how, living
organisms are influenced by the absence of gravity. The test objects varied
from small organisms (fruit flies, beetles, plantlets, algae) down to isolated
organs (foetal mouse bones) and in vitro cultures of mammalian cells. For the
last categories, ESA developed an automated incubator called Biobox, which flew
three missions on Bion/Foton (Table 1).
Most cultures in Biobox contained
osteoblasts and osteoblast-like cells. The scientific objective was to
investigate whether bone-forming cells, isolated from the body, would display a
suppressed level of activity under weightlessness. The results could help
clarifying the frequently reported, but insufficiently understood phenomenon of
bone mass loss in space in cosmonauts and astronauts. The research teams
involved in ESA's Biobox experiments were those of Prof. Alexandre (F), Prof.
Bouillon (B), Prof. Lapière (B), Dr. Rodionova (UKR), Dr. Schoeters (B), Prof.
Tairbekov (RU) and Dr. Veldhuijzen (NL).
Table 1: List
of experiments flown in ESA’s Biobox on Bion/Foton
|
mission |
flight |
experiment name |
cell/organ type |
|
Biobox-1 |
Bion-10 (1992/93) |
Bones Fibro-1 Marrow-1 Oblast-1 |
embryonic bones (mouse) fibroblasts (mouse) pre-osteoblastic cells (mouse) osteosarcoma cells (rat) |
|
Biobox-2 |
Foton-10 (1995) |
Fibro-2 Marrow-2 Oblast-2 |
fibroblasts (human) osteosarcoma cells (human) osteosarcoma cells (rat) |
|
Biobox-3 |
Foton-11 (1997) |
Fibro-3 Marrow-3 Oblast-3 |
fibroblasts (human)
osteosarcoma cells (human) osteosarcoma cells (rat) |
Culturing
conditions and control experiment philosophy in Biobox
The incubator
of Biobox holds 30 experiment units, small culturing devices about the size of
a packet of cigarettes. In a single unit, one or two 1-ml cell cultures can be
grown. The cultures are automatically presented with growth media, biochemical
stimulants and fixatives, according to a pre-programmed timeline.
From the 30
experiment units, six are placed on a centrifuge which generates 1g during
flight (artificial terrestrial gravity). For reference, a duplicate model of
Biobox is operated on ground during flight (Figure 1). After flight, the
results obtained in microgravity are compared with those obtained at 1g (both
from the in-flight centrifuge and from ground) to identify effects which are
specifically linked to weightlessness.
All 24 + 6 experiment
units are subjected to the same temperature profile. The temperature in Biobox
is kept at 20 °C before flight to suppress the growth and development of the
cultures before microgravity has been reached. Immediately after launch the
temperature is raised to a value close to 37 °C, which is the optimal culturing
temperature for mammalian cells. By the end of the flight, when all cultures
are fixed, the temperature is lowered to prevent the fixed material from
decaying.
Overview
of the results of the Biobox experiments on Bion/Foton
BONES
investigators: J.P. Veldhuijzen et al.
(NL), N.V. Rodionova et al. (UA)
The influence of
weightlessness on bone development was tested. Embryonic long bones, dissected
from mouse foetuses, were cultured in vitro over a period of four days. The
mineralisation of the bone matrix was suppressed in space, in particular in the
microgravity-exposed series, whereas the length growth of the bones was not
affected.
FIBRO-1
investigators: M.G. Tairbekov et al. (RUS)
Monolayer cultures
and three-dimensional tissue blocks of mouse fibroblasts were flown in space to
investigate the influence of microgravity on the morphology, locomotion and
proliferation. In the monolayers, the cells acquired a more rotund shape and
the direction of locomotion became more irregular. In both culture types, the
nuclei became significantly smaller and rounder. The rate of cell proliferation
(thymidine incorporation) was not affected.
FIBRO-2
investigators: M.G. Tairbekov et al.
(RUS), C. Lapière et al. (B)
A repetition and
extension of Fibro-1. No usable results were obtained due to technical problems
(leakage and bacterial infections) in the experiment units.
FIBRO-3
investigators: M.G. Tairbekov et al.
(RUS), C. Lapière et al. (B)
Repetition
and extension of Fibro-1 and -2. No results due to an electrical failure in
Biobox.
MARROW-1
investigators:
G. Schoeters et al.(B), N.V. Rodionova et al. (UA)
Cultures of
pre-osteoblastic cells (MN7) were grown for nine days under weightlessness in a
three-dimensional matrix of collagen sponge. The osteogenic activity, as judged
by the production of alkaline phosphatase and collagen type I, was suppressed
in microgravity. In contrast, if the cells were presented with IL-1
(interleukin-1) or PTH (parathyroid hormone), the synthesis of alkaline
phosphatase and collagen type I was enhanced in microgravity. The rate of cell
proliferation (protein and DNA content) was not affected.
MARROW-2
investigators: R. Bouillon et al.(B)
A
repetition of Marrow-1 with a different cell type (MG-63). The activity and
differentiation of the cells was measured by the production of matrix materials
as well as by gene expression. In microgravity, the expression of the genes
responsible for osteoblast-specific proteins (collagen type I, alkaline
phosphatase and osteocalcin) was reduced.
MARROW-3
investigators: R. Bouillon et al.(B)
Follow-on
to Marrow-2. Results in line with those from Marrow-2. The mRNA levels of
collagen type I, alkaline phosphatase and osteocalcin were reduced in
microgravity.
OBLAST-1
investigators: C. Alexandre et al. (F)
Monolayers of
osteosarcoma cells (ROS 17/2.8) were grown in space from pre-confluence to
confluence. After four days the shape of the cells began to change, culminating
after six days in a mixture of morphologies including contracted cells with
cytoplasmatic extensions and rotund cells layered on top of each other. A
normal cell morphology was displayed in the 1g controls, both on ground and in
flight. From the six-day cultures, two times more alkaline phosphatase activity
was extracted than from the corresponding ground controls. The rate of cell
proliferation (cell number, total protein content) was not affected.
OBLAST-2
investigators: C. Alexandre et al. (F)
A repetition and
extension of Oblast-1. No results were obtained, as the container with the
Oblast-2 experiment units was not recovered from the crash site of the Foton-10
capsule.
OBLAST-3
investigators: C. Alexandre et al. (F)
Repetition of
Oblast-1, but with cultures on glass (instead of plastic) coverslips to allow
for immunofluorescence. Whereas the morphological changes as observed in
Oblast-1 were reproduced, the amount of extractable alkaline phosphatase
activity was this time decreased in space. A new finding was that the
organization of the focal adhesion plaques was changed in microgravity, as
judged by the immunofluorescence images. The investigators have named this
phenomenon the “anti-adhesive effect” of microgravity.
Common
trends in the results of the Biobox experiments
A remarkable aspect of the results
listed in the previous paragraph is that the data from the different
experiments fit together well. It is even possible to identify a set of
recurring tendencies:
I In all experiments the cultures appeared
to have been influenced by the absence of
gravity;
II In no experiment a difference in
proliferation rate was found;
III The reported effects were all related to a
changed activity of the cells;
IV In the osteogenic cultures (fetal mouse
bones, MN7, MG-63, ROS 17/2.8) the changed
activity always pointed in the same
direction: the bone-forming activity was reduced;
V The effects did not seem to depend on the
level of biological organization (they were
found in organs, in three-dimensional
and in two-dimensional cell cultures).
Biobox results
under discussion
The
phenomenon of bone mass reduction in space is thought to originate at the
cellular level, where the bone mass budget is controlled by the antagonistic efforts
of osteoblasts (bone-forming cells) and osteoclasts (bone-degrading cells).
However, how these cells are triggered to change their activity in space is not
known: the osteoclast may feel the loss of tension in the weight-carrying bones
in space (“unloading of the skeleton”), stress hormones released in the body
could play a role, the re-distribution of body fluids in weightlessness could
have an impact and finally, it could be that bone-forming cells sense the
absence of gravity directly. To test the last hypothesis, in-vitro cultures
were used in Biobox, with cells that were literally cut off from any signals
generated by the body.
The results from the Biobox experiments
seem to support the idea that bone-forming cells may react autonomously to the absence
of gravity. Over the past years however, several arguments have been adduced by
the scientific community against the concept that mammalian cells would be
capable of sensing microgravity by themselves. These arguments are the
following:
1.) From a bio-physical
point of view, gravisensing is impossible at the small size of a single cell.
Within a cell, the forces generated by weight are considered too low to sort
any effect, unless supported by some intracellular amplifier/gravisensor (see
2.);
2.) No specialised
gravisensing organelles, nor candidates thereof, have ever been identified in a
mammalian cell (in contrast, such sensors have been found in plant root cells
and in some protists);
3.) The reported
effects could as well be indirect: the lack of convection in the culturing
fluids under microgravity may have an impact on the activity of the cells
(note: proof for this alternative explanation is still missing).
The criticism summarised above has been
raised since many years. Therefore, it becomes increasingly urgent to design
and perform new experiments which can bring clarity in this controversy.
Biobox on the US Space Shuttle
After
three missions on Bion/Foton, the Biobox project was transferred to the US
Space Shuttle. The fourth mission of Biobox was completed in 1998 on STS-95. A
fifth mission, again on the US Space Shuttle, is currently being prepared and
scheduled for launch on STS-107 in July 2002.
The relevance of the Biobox experiments in
relation to the osteoporosis problem
Biobox
not only carried the very first bone cell cultures into space (on Bion-10, see
Fig. 2) but more importantly, the Biobox experiments on Bion/Foton may be
considered as the first systematic set of investigations on the behaviour of
bone-forming cells in vitro under microgravity conditions. In later years,
similar and complementary work has been done on the US Space Shuttle (some in
Biobox) and Foton (Ibis) with results often in agreement with the trends
presented above.
The loss of bone mass in space is
nowadays thought to represent a useful test model for the loss of bone density
and mass in older people on Earth, a disorder known as osteoporosis.
Osteoporosis is considered as a major, world-wide medical problem. Future ESA
investigations about the behaviour of bone forming cells in space will partly
be supported by the pharmaceutical industry through a recently established
programme named MAP (Microgravity Applications Programme). As such, Biobox has
helped to lay the foundation for one of today’s most-acclaimed biomedical
research projects in space.
References
BONES
Is calcification of
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O.P. Berezovska, L.G.
Karmozina, N.V. Rodionova & J.P. Veldhuijzen
Cospar ‘94 Book of
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Razvitie
embrional'noj kosti in vitro v usloviyakh kosmicheskogo poleta
(Development of fetal
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N.V. Rodionova &
J.P. Veldhuijzen
Space Biology and
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Reduced
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J.J.W.A. van Loon, O.
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Veldhuijzen
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FIBRO
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(eksperiment
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culture (in vitro) under microgravity conditions
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Cospar ‘94 Book of
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Vliyanie
mikrogravitatsii na rost i podvizhnost' kletok fibroblastov v kul'ture (in
vitro)
(Microgravity effects
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MARROW
Decreased
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interleukin-1 and parathyroid hormone in pre-osteoblast like cells under
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J. Bierkens, J. Maes,
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Space Scientific Research in Belgium
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(Ultrastructural analysis of osteogenic MN7 cells cultured
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OBLAST
First
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Vol.
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C. Genty
PhD thesis, Université Jean Monnet, Saint-Etienne, pp
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Demonstration
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Bone
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Biobox
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R.
Demets
Microgravity
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Bone
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Effects
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L. Vico, M-H. Lafage-Proust & C. Alexandre
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Microgravity
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The effect of
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G. Carmeliet &
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The FASEB Journal Vol 13, Supplement pp S129-S134 (1999)
Osteobiology, strain,
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