7.1 GENERAL DISCUSSION AND SUMMARY

From the very beginning of the manned spaceflight era it was speculated that the microgravity environment in space would impose various stresses upon the human body. Besides increased cosmic radiation and body fluid shifts, one of the hypothesized changes would be the superfluity of the skeletons weightbearing function while in space. A relation between mechanical loading and shape of the skeleton was already suggested by Galilei (1564-1642).(1) Much later, in 1836, Ward(6) recognized the spatial distribution of bone trabeculae in the human femur head and suggested this to be related to its function of supporting mechanical loads. More then half a century later, in 1892, Wolff published his book 'Das Gezetz der Transformation der Knochen', postulating a 'law' saying that the shape and inner structure of bone is a reflection of its mechanical loading history.(7) Since then many in vivo studies have demonstrated the relation between bone structure or strength and mechanical loading. Only few experiments have been devoted to the effect of skeletal tissues and cells in vitro, in response to applied loads. Glucksmann, some 50 years ago, was one of the first who demonstrated the ability of skeletal tissues to respond to mechanical forces, while in culture.(2) Klein-Nulend et al. applied more controllable and defined forces onto fetal skeletal tissues in culture.(3,4) They studied the influence of mechanical forces, applied though intermittent hydrostatic compression (ICF), on fetal mouse long bone and calvaria development. Using this system, they showed that, in mouse long bones, hydrostatic compression resulted in an increased mineralization and a decreased mineral resorption compared to control, non-compressed cultures. Based on these results we adapted this tissue culture system to be used for microgravity experiments.

Why microgravity experiments?

On Earth it is only possible to increase mechanical stresses in tissue culture, compared to control conditions. The system used by Klein-Nulend et al. also imposed increased stresses upon long bone rudiments.(3,4) In spaceflight, at near weightlessness conditions, however, there are virtually no mechanical forces acting upon matter. By culturing the fetal mouse long bones in near weightlessness, or microgravity, in space we should see the impact of a very low stress environment on skeletal growth and differentiation. By using bones at two stages of development, 16 and 17 day old fetal mouse metatarsal long bones, we may also discriminate between the two main processes in bone, mineral formation and mineral resorption. In addition normal longitudinal growth could be observed.

Since this is a tissue culture experiment, it would be possible to clarify a still standing enigma resulting from the first data on bone in association to spaceflight as discussed in Chapter 1: Is the reduced bone density and / or strength found after spaceflight the result of a reduced weight bearing function of the skeleton or, is this bone loss resulting from an increased release of stress hormones or other perturbations in the systems homeostasis as a consequence of spaceflight per se? By using the model systems of these long bones, we could test the hypothesis that the effects seen in bones after spaceflight are, at least partly, due to the lack of mechanical loading.

Testing a hypothesis means conducting experiments, but especially in space research the expression 'easier said than done' applies. After the euphoria of being offered the opportunity to conduct a spaceflight experiment, comes a long and winding road of adapting normal, every days’ practice or tools to 'space qualified procedures and hardware'.

When thinking of outer space and spaceflight three things come to ones mind: weightlessness, vacuum and freezing temperatures. The latter two phenomena were not applicable, however, for the experiments discussed in this thesis were performed inside a spacecraft, the interior atmosphere of which is comparable to an ambient environment. The first property, weightlessness, or better near weightlessness (see Appendix B), is also present inside an orbiting spacecraft and is just the reason for many researchers to go into space. And just because of this near weightless environment, the majority of things so common in our unit gravity world have to be redesigned to be used in the near weightlessness or microgravity of space. Besides microgravity, there are also the launch and time-line constrains associated with spaceflight experiments. The hardware and the biological samples used should be such that they can withstand the harsh launch conditions and delay in experiment initiation times imposed upon the experimental protocols. In this respect it is surprising that it is mandatory only for the used hardware and not the biological material, to demonstrate that it withstands these kind of constrains.

In Chapter 2 a few of these constrains are addressed, namely adapting standard laboratory tissue culture practice and tools to space qualified procedures and hardware. In addition it is shown how we could overcome the delayed initiation time of an experiment. One important factor in tissue culture is the atmosphere in which tissues and cells grow. Most mammalian cells need 37°C temperature and 5% CO2 in air gas phase. The former prerequisite was provided for by the Biorack facility on board the Space Shuttle in which the experiment had to be performed. The 5% CO2 in air gas phase, however, was not provided by the on board facility. Only after several alternative culture techniques, not based on a 5% CO2 atmosphere had been disqualified, it was decided to introduce a 5% CO2 in air gas bottle for this experiment. An other problem was the time gap between the final preparation of an experiment and the actual initiation while in orbit. This time is needed for hand-over procedures, transportation to launch site, integration, launch, and final start-up while in orbit. Luckily, simply by keeping the tissues at room temperature it was possible to overcome a 24 hours lag-period between experiment integration and actual performance. In addition, the 24 well tissue culture plates, in which metatarsals are normally cultured, could be replaced by polyethylene culture bags, without changing the in vitro developmental characteristics of the rudiments.

By increasing the gas phase pressure inside the Type-I containers the long bone rudiments are subjected to hydrostatic stress. It has been shown previously by Klein-Nulend et al.(3) that increasing the pressure above the culture medium produced a mechanical stress referred to as continuous compressive force (CCF). By using mouse metatarsal long bones it was shown that CCF had an anabolic effect on mineralization. An additional question we also asked ourselves was that if microgravity produces effects on fetal mouse long bones comparable to effects seen in disuse osteoporosis (decreased mineral content), would it be possible to counteract these effects by applying a mechanical stress through an increase of the gas phase pressure inside the containers while under microgravity. Unfortunately, although the hardware functioned perfectly in flight, for reasons unknown, this part of the experiment failed. We could not duplicate the CCF effect in control cultures, hence, no conclusions related to the hypothesis could be made.

Although hardware development is crucial and can be an interesting part in preparing for a spaceflight experiment, the main goal are the final microgravity results. As mentioned before, the reason for us for going into space and experiment under microgravity conditions was to test the hypothesis that part of the deteriorating effects found in vivo in human and rat bone after spaceflight are due to loss of mechanical stress applied on the skeleton. To test this, we used two slightly different in vitro skeletal model systems. First, 16 day old (ED16) fetal mouse long bones (metatarsals) were used in which we studied mainly matrix mineralization. In the second model, one and a half day older, 17.5 day old (ED17.5) rudiment, we assayed osteoclastic matrix resorption, the procedures of which are described more in detail in Chapter 3. In additions to the data on mineral metabolism, also results on growth, glucose utilization and collagen synthesis could be obtained. By using ED16 and ED17.5 long bones for the experiment 'Bones' in the Biorack facility of ESA during the first International Microgravity Laboratory (IML-1, STS-42, launch 22 January 1992) Shuttle flight, we have shown that microgravity indeed has an impact on mineral metabolism in skeletal rudiments while in culture. Near weightlessness resulted in reduced mineralization and reduced glucose utilization. In addition, osteoclastic mineral resorption was increased. Overall growth, as measured by increase in total length, was not or only marginally decreased under microgravity, while collagen synthesis was not changed. All the microgravity effects were compared to the data from the on board 1×g centrifuge samples. It is also argued in Chapter 3 why the on board 1×g centrifuge is the best control for such spaceflight experiments.

Part of the outcome of the IML-1 flight was verified during a Russian mission. Chapter 4 describes experiments testing the hardware for this unmanned flight. Chapter 5 describes the setup and results of this Cosmos-2224 mission (Bion-10) which was launched on 29 December 1992. The ED17 metatarsals used for this experiment were cultured in completely automated tissue culture devices, so called plunger boxes. In a slightly different experiment protocol as compared to the IML-1 setup, ED17 metatarsals were used to study mineralization and longitudinal growth. This experiment also showed that mineralization under microgravity conditions was decreased in the long bone rudiments bones. In both microgravity studies, although using two completely different sets of hardware and timeline constrains, fetal mouse metatarsal long bones reacted with a significantly decreased matrix mineralization to the near weightlessness environment.

While preparing for the Russian unmanned Bion-10 flight, some modifications to the hardware had to be made, which are described in Chapter 4. Special tissue culture devices, already developed for earlier spaceflight applications, had to be adapted to our needs. Some engineering had to be performed in addition to biological testing. It appeared that the initially chosen material, polysulphone (PSU), did not allow mineralization of bone rudiments as well as bone marrow cultures, which were planned to be used for the spaceflight study. Both studies at the Flemish Institute for Technological Research (VITO) in Belgium, as well as in our laboratory showed the inability of skeletal tissues and cells to differentiate in contact with PSU leacheble factors. Typical markers in bone growth and development like alkaline phosphatase, collagen formation and matrix mineralization were strongly reduced or completely absent in cultures with polysulphone plastics. Surprisingly, there was no effect on proliferation, measured by 3H-thymidine incorporation into osteoblast like cells or increase in total length of metatarsal long bones. Eventually, instead of polysulphone, polyethyleneterephthalate (PETP) was chosen as prime material for the construction of the tissue culture module to be used for the Bion-10 experiment.

Polysulphone polymers are also used in orthopedic implants. Therefore the experiments prepared for this spaceflight experiment appeared of interest for the orthopedic practice. The results of the biocompatibility tests argue against the use of polysulphone in contact with bony tissue, as used in bone replacement therapy.

To complement experiments performed under microgravity, Chapter 6 describes experiments performed with fetal mouse long bones cultured at increased gravitational stresses. Under increased accelerations, or hypergravity, growth and mineral metabolism were studied in ED16 and ED17 metatarsals. Most of the experiments were performed using a self-made prototype tissue culture centrifuge, while for the pilot experiments performed at the University of Texas in Houston, a more advanced tissue culture centrifuge was used.

In both experimental setups it was demonstrated that increased gravity ranging from 2.2 to 3.1×g resulted in increased matrix mineralization in ED16 metatarsals. Total length, as an indicator for general growth, was also increased in 2.2×g ED16 bones compared to 1.0×g controls. 17 Day old 45Ca prelabeled rudiments, used for mineral removal studies, showed that under 2.2×g calcium release was increased, indicating increased osteoclastic activity.

Fig. 7.1. Schematic representation of various parameters of skeletal growth and differentiation, the data of which is based on the experiments described in this thesis. Under microgravity there is a decrease in glucose utilization and mineralization, and probably also retarded longitudinal growth. Mineral resorption is increased under microgravity. Under hypergravity conditions, however, longitudinal growth is increased and there is an augmented mineral resorption, compared to 1×g controls.

The results of Chapter 3,5, and 6, may be combined to show the effects of microgravity and access gravity on fetal mouse long bone development. Fig. 7.1 shows a schematic representation of the microgravity and hypergravity results. Mineralization and growth show a continuum in response to gravity through the 1×g reference point. They are both decreased under microgravity and augmented under increased gravity. This in contrast to osteoclastic mineral resorption, which is increased both under micro- as well as hypergravity conditions. For collagen synthesis and glucose utilization only two values are available, microgravity and 1×g. Hypergravity experiments are needed to extend these lines.

The non-linear response on mineral resorption could mean that there are two different processes involved in this response. Under microgravity mineral resorption may be increased due to lack of mechanical forces, which causes bone loss. In case of hypergravity, the increase in mineral resorption may be a result of a continuous stress applied onto these fetal mouse long bones.

Parameters like glucose utilization and collagen synthesis still have to be measured in hypergravity cultures. In order to facilitate future hypergravity experiments a new centrifuge has been build. A study by van Loon et al. describes the requirement study and subsequent construction of the Medium sized Centrifuge for Acceleration Research (MidiCAR).(Appendix A and ref. 5) The realization of this project was a close collaboration of various gravitational scientists, industry, and the national Dutch Organization for Space Research (SRON). This apparatus provides future experimenters with a well controlled and convenient apparatus to conduct hypergravity experiments. Although the results of these hypergravity studies do not necessarily have to be mirror images of microgravity data (see Fig. 7.1), they contribute to our knowledge of cellular processes in relation to accelerations i.e. mechanical forces, applied upon skeletal as well as various other tissues and cells. On the other hand, the microgravity environment provides a unique place to survey the effects of nearly zero mechanical force on fetal bone growth and differentiation in vitro. In this way both microgravity as well as hypergravity experiments contribute to the identification of the mechanotransduction system in skeletal cells and tissues. Hence these gravitational studies indeed contribute to our understanding of Wolffs' law at the cell- and molecular level.

Under microgravity several physical phenomena are changed compared to on Earth 1×g or centrifuge hypergravity conditions (Appendix B). Surprisingly, still very little is known, even within the field of gravitational science, about the impact of various physical phenomena on biological systems. It has been documented for a long time already that events like convection, hydrostatic pressure or Coriolis forces are influenced by accelerations, i.e. gravity. Several experiments have now been performed on the impact of reduced gravity in cells and tissues, but usually these studies have not addressed the question of primary or secondary effects of gravity. It should be realized that effects seen in a spaceflight experiment may be indirect resulting from changes in the tissues' environmental conditions, in contrast to direct effects of microgravity on cells. Therefore, Appendix B provides a brief introduction to physical phenomena which can be of value to microgravity of hypergravity experimenters. Future experiments could systematically screen the impact of these processes in biological systems. Most of these experiments can be done on Earth with occasional checks under microgravity conditions. Only than, direct (micro-)gravity effects can be distinguished from secondary, environmental causes. These questions urge for further cooperation and integration of the two disciplines of biology and physics. Spaceflight experimentation in particular provides common grounds for both kinds of science and this could be synergistic for future understanding of the behavior of cells in space, the field of gravitational biophysics.

7.2 CONCLUSION

In conclusion this thesis reports, for the first time, a comprehensive study of the effect of various accelerations, ranging from near weightlessness to an access gravity of more than 3×g on mineral metabolism on bone organ cultures. The bone rudiments respond to the lack of gravity by a decreased mineralization and glucose utilization, and increased mineral resorption, while general growth and collagen synthesis are, statistically, not affected. Increasing accelerations to 2.2×g, result in increased growth, increased mineralization as well as increased mineral resorption. Decreasing the mechanical load leads to decreased bone mineral content while increasing the stress resulted in increased mineralization. These results show that the ideas of Wolff also pertain to fetal mouse long bones cultured under various gravity conditions.

Future ground based hypergravity in conjunction with spaceflight hypogravity experiments, should be devoted to the search for the mechanosensor system in skeletal tissues and cells. More research is needed in the area of second messengers, signal transduction, stretch activated ion channels, membrane potentials and the role of the cytoskeleton. In the long run, studies on the cellular behavior in response to mechanical stresses contribute to the knowledge and understanding of the pathogenesis and possible treatment of immobilization osteoporosis and, maybe, other biomechanical related disorders.

7.3 REFERENCES

1 Galileo G. Two new sciences. In; "The second day". Translated by Stillman Drake, The University of Wisconsin Press 109-146, 1974.
2 Glucksmann A. Studies on bone mechanics in vitro. II. The role of tension and pressure in chondrogenesis. Anat. Rec. 73, 39-56, 1942.
3 Klein Nulend J., Veldhuijzen J.P., Burger E.H. Increased calcification of growth plate cartilage as a result of compressive force in vitro. Arthritis Rheum. 29, 1-9, 1986.
4 Klein Nulend J., Veldhuijzen J.P., Strien M.E. van, Jong M. de, Burger E.H. Inhibition of osteoclast bone resorption by mechanical stimulation in vitro. Arthritis Rheum. 33, 66-72, 1990.
5 van Loon J.J.W.A., van den Bergh L.C., Schelling R., Veldhijzen J.P., Huijser R.H. Development of a centrifuge for acceleration research in cell and developmental biology. 44th International Astronautical Congress, IAF/IAA-93-G.4-166, 39, Graz, Austria, 16-22 October 1993.
6 Ward F.O. Outlines of human osteology. 3rd edition, Henry Renshaw, London, 1836.
7 Wolff J. Das Gezetz der Transformation der Knochen, Berlin, 1892.