¹: Academic Centre for Dentistry Amsterdam (ACTA), Dept. of Oral Cell Biology, Amsterdam, The Netherlands.
²: I.I. Shmalgauzen Institute of Zoology of the Academy of Sciences of the Ukraine, Kiev, The Ukraine.

Skeletal tissues are sensitive for their mechanical environment. Mechanical forces due to every day weight bearing ambulatory activities, are necessary to maintain skeletal integrity. An exceptional situation of a reduced mechanical environment is near weightlessness as a consequence of orbital spaceflight.

Several papers have indicated already that the near weightlessness environment of space results in detrimental effects on bone matrix and/or mechanical strength. From these in vivo studies it is not clear, however, whether the effects are the results of a lack of load bearing on the skeleton or whether they are, due to hormone changes or body fluid shifts as a result of entering microgravity.

For the present study we used organ cultures of mouse long bones as were also flown in a previous manned Space Shuttle flight. During this unmanned Russian Cosmos-2224 mission, however, the rudiments were cultured in completely automated tissue culture devices for a period of 4 days.

The results contribute to our earlier observations and show that also in this completely automated experiment, fetal long bone growth was not affected by microgravity, while matrix mineralization was decreased compared to the 1g control conditions.

Key words: spaceflight, bone, mineralization, in vitro.

Future spaceflights shall be characterized by its long durations. This is mainly due to occupation of space station Mir and the upcoming international space station or even to missions to Mars, lasting about two years.

One of the biophysical complications of spaceflight is the reduction of bone mass which occurs during flight. This phenomenon has already been demonstrated in flight crews after a flight of only 7 days during Gemini⁽¹⁰⁾ as well as after the longer duration (28-84 days) SkyLab missions.⁽¹¹⁾ More detailed studies on bone morphology and histology in rats⁽⁴⁾ and monkeys⁽¹³⁾ revealed similar results of bone loss. Skeletal unloading in the near weightlessness environment of space could be the prime possible cause for this phenomenon. It is also possible, however, that part of the effects reported are secondary, resulting from body fluid shifts or alterations in hormone levels as a consequence of spaceflight.

The aim of the present in vitro microgravity experiment was to study the effect of near weightlessness in the absence of complications such as changes in systemic factors like hormonal levels. This experiment was also a verification of a comparable study performed during the Space Shuttle mission STS-42 (the first International Microgravity Laboratory, IML-1).

We have used 17 day old fetal mouse metatarsal long bones, which were cultured in completely automated hardware on board the unmanned Russian satellite, Bion-10, to study in vitro growth and mineralization under microgravity conditions.

The central three cartilaginous metatarsal long bone rudiments of 17 day old fetal (ED17) Swiss mice (University Leiden, The Netherlands) were used. Rudiments were aseptically harvested and individually precultured in 24 wells plates, in 300 l culture medium per well in a 5% CO₂ incubator for an overnight (o/n) period. The following day the metatarsals were transferred into the hardware, the plunger boxes (PBs) (Fig. 5.1). Every PB accommodated eight metatarsals, 4 metatarsals in each culture compartment filed with 1.0 ml medium. After integration the PBs were handed over for transport (see timeline). All these pre-flight preparations were carried out in the ESA (European Space Agency) Moslab facility, at the Institute of Biomedical Problems (IBMP) in Moscow, Russia.

By retaining the samples at room temperature (20 ± 1.5°C), the rudiments were kept in a metabolically relative inactive state during a 100 hours lag period, used for transportation (form Moscow to Plesetsk), integration and launch. When the spacecraft was in orbit, the temperature was increased to 37 ± 1.5C (culture day 0). To replenish the culture medium, the first plunger of each culture compartment was activated after 60 min in flight. A second plunger, also releasing fresh culture medium, was activated 48 hours later. The experiment was terminated after 96 hours by activating a third plunger, containing formaldehyde fixative at a final concentration in the culture compartment of 0.5% (v/v). The gas phase during the whole culture period was 5% CO₂ in air.

A schematic drawing of a 17 day old fetal mouse metatarsal long bone (average length at start of the experiment is 1.5 mm) is presented in Fig. 5.2. In the 17 day old metatarsals the central part, also referred to as the diaphysis, has already mineralized, in vivo (see also Fig. 5.3). Under favorable conditions in vitro, these bones increase in total length for more than 25% during 4 days culture. In parallel with this increased growth, also mineralization advances. This can be seen in the increased length of the diaphysis after culture (Fig. 5.3, 5.5c and 5.5d).

The tissue culture medium consisted of bicarbonate buffered a-MEM without nucleosides supplemented with 50 mg/l gentamicin, 0.5% v/v fungizone (Gibco), 0.2% BSA factor V, 3.0 mM Na--glycerophosphate ⁽⁵⁾ (Sigma), 50 mg/l L-ascorbic acid and 300 mg/l L-glutamine (Merck). The same medium was used for the overnight preculture in the 5% CO₂ incubator except for the addition of Na--glycerophosphate.

Data on total lengths of the metatarsal long bones and the increase length of the mineralized zone (diaphysis) were assessed from photomicrographs taken directly before loading the samples in the PBs and again after return of the samples in Moscow (see Figs. 5.2-5.5).

The lengths were measured from photo's which were enlarged and digitized on a Nikon dissection microscope fitted with a Sony XC-77CE b/w CCD camera in combination with Videoplan software (Zeiss). %Length increase was determined as length (L_day4-L_day0)/L_day0100%.

The concept of the tissue culture modules has previously been used for microgravity experiments.⁽⁶⁾ The primary material used for the tissue culture modules, or plunger boxes (PBs) was polyethyleneterephthalate (PETP). The PBs (Fig. 5.1) were manufactured by CCM^*. Before use, the PBs were thoroughly cleaned. All PETP parts were rinsed o/n in 10% nitric acid, changed once, followed by 24 hrs in running aqua dest, acetone for 2 hrs, o/n in 70% ethanol, and finally air dried in a laminar flow hood. All other PB parts were rinsed in aqua dest, ethanol 96% and air dried.

After the bones were placed in the PBs, the PBs were integrated in CIS-boxes (Cells In Space; CCM and FSS^**).⁽¹⁾ The CIS-box provides a sealed gas tight environment containing all electronics for time programmed plunger activation and housekeeping data acquisition. The CIS-box was integrated in the Biobox (Dornier^***). Biobox provides a temperature controlled environment and supplies the interface with the spacecraft. Biobox also accommodates a small radius, 1g reference centrifuge (radius: 71.4 mm at position of the samples). Two PBs, mounted in standard ESA Type-I containers, were placed on the 1g in-flight centrifuge in Biobox. These two containers were slightly modified to increase the inner volume used for the gas phase (5% CO₂ in air). The modification was to increase the gas volume inside the Type-I containers to equal the gas volume per PB in the CIS box. After integrating the PBs, the CIS box as well as the Type-I containers were flushed with 5% CO₂ in air. Both units remained closed until after the mission, at return in Moscow.

A duplicate Biobox, with an identical configuration as the flight model, remained on the ground. The centrifuge on this ground model contained no biological samples. The in-fight 1g gravity field remained within 1.5% accuracy during the first 52 hours of flight. However, the speed of the centrifuge gradually increased up to a g-level of 1.4-1.5g for the last 12 hours of the experiment.

The Bion-10 or Cosmos 2224 mission was launched by a SL-4/A2 Soyuz rocket booster on December 29, 1992 at 16:30 hrs Moscow time (MT) from the Cosmodrome launch facility in Plesetsk, Russia. After its orbital flight the Bion spacecraft landed approximately 100 km North of Karaganda, Kazakhstan, January 10, 1993, 07:16 hrs (MT). Recovery of the spacecraft was 12 hrs after landing. Total flight time was 11 days, 14 hrs and 46 min.

After return of the PBs in Moscow they were unloaded and the bone rudiments were temporarily transferred to a fixative solution containing 0.5% formaldehyde, 1% sucrose in 0.1 M phosphate buffer, pH=7.6. After photomicrographs were taken, the metatarsals were rinsed in 0.1 M phosphate buffer containing 50 mM NH₄Cl and 1% sucrose, and stored in 70% ethanol during transport. Part of the samples were processed in Amsterdam The Netherlands, the other part in Kiev, Ukraine.

After return at the home laboratories, the samples were further dehydrated in ethanol and embedded in either plastic (Historesin, Reichert-Jung) or paraffin (Histoplast, Shandon). Only serial sections of 3 m were made for light microscopic evaluation. Sections were stained with 0.2% toluidine blue pH=4.54 for 1 min.

L = launch
R = recovery
L-5 days: -Filling reservoirs with culture medium and fixative.
-Dissecting metatarsals.
-Pre-incubation of metatarsals in 24 well plates (Moscow, Russia).
- L-4 days: -Photomicrographs of samples.
-Mounting metatarsals and further integrate the PBs.
-Handover PBs to ESA officials.
-Mounting PBs in CIS-box and Biobox (20°C).
-Transport to launch site.
-Integration into the satellite.
Launch: -From Plesetsk, Russia.
L+10 min. -Start of warming up of Biobox to 37°C.
-60 min after launch, first medium change.
L+2 days: -Second medium change.
L+4 days: -Termination of experiment by fixation of the metatarsals.
L+9 days: -Lowering of the temperature in Biobox from 37 to 14°C.
R: -Recovery at Karaganda, Kazakhstan (at landing + 12 hrs).
R+2 days: -Unloading the PBs (Moscow, Russia).
-Photomicrographs of samples.
-Ethanol 70% for transport.
R+9 days: -Start histological preparations (Amsterdam, The Netherlands).

After dissection the rudiments were divided into four groups. Three groups were cultured in the specific PB hardware, namely, the actual microgravity group (F), the in-flight 1g group cultured on the on board centrifuge (FC) and the ground control group (G). The fourth group consisted of metatarsals cultured in standard laboratory multiwell plates (MW). The study was divided into two parts. One part, the F and FC groups, was flown on board the Bion-10 mission, the other part, the G and MW groups, remained on the ground.

The number of samples used for final analysis was: flight microgravity (F) n=6, in-flight 1g (FC), n=12, ground (G), n=9 and the multiwell group (MW), n=7.

Statistics were calculated using the two tailed Student t-test. Data are expressed as mean ± SEM, unless indicated otherwise.
^* Center for Construction and Mechatronics, Nuenen, The Netherlands)
^** Dutch Space (former Fokker Space) and Systems (Leiden, The Netherlands)
^*** Dornier GmbH (Friedrichshafen, Germany)

A total of four groups of metatarsal long bones were used. 1: The actual flight groups; the in-flight microgravity (F). 2: The in-flight 1g control (FC). Both other groups remained on ground; one group in plunger boxes inside a replica of the Biobox (G) and the other group was cultured in normal 24 multiwell plates in a standard tissue culture incubator (MW).

After histological observations, it appeared that a large number of metatarsals had not increased in length and showed a significant amount of necrotic tissue. Based on criteria of longitudinal growth and central necrosis, ambiguous samples were excluded. Only metatarsals which had increased in length and showed no or only limited signs of necrosis (see Fig. 5.5e and 5.5f) were included in the final analysis. As a result, the number of samples used for evaluation was: flight microgravity (F), n=6 (21% of total number of samples), in-flight 1g (FC), n=12 (100% of total number), ground (G), n=9 (56% of total number) and the multiwell group (MW), n=7 (100% of total number).

The metatarsals showed a moderate percentual increase in length ranging from 11.5% (FC) to 27.0% (MW) (Fig. 5.4). The increase in total length of the microgravity group did not differ significantly from the in-flight 1g samples (p=0.07). However, the in-flight 1g group differed significantly from the samples grown in ground PBs (G) (p<0.005). The multiwell group increased more in length than any other group after the 4 days culture at 37C (Fig. 5.4).

Fig. 5.5 shows a macroscopic picture of ED17 metatarsals before (5.5a and 5.5b) and after (5.5c and 5.5d) culture, under microgravity or on the in-flight 1g centrifuge, respectively. A small increase in length of the diaphysis can be seen in 5.5c as compared to 5.5a, which was characteristic

for all flight microgravity samples. The increase in length of diaphysis was less in the microgravity group compared to the in-flight 1g group (Fig. 5.5c versus 5.5d). Histological observations of these fetal long bones revealed no or only minor signs of cell death (Fig. 5.5e and 5.5f).

Fig. 5.6 shows that the increase in the length of the diaphysis ranged from 76.7% (flight microgravity, F) to 221.9% (ground multiwell, MW). Under conditions of near weightlessness there was significantly less mineral apposition compared to the in-flight 1g group (p<0.05), this can also be seen in Fig. 5.5c and 5.5d. Delta increase in length of diaphysis in the in-flight 1g was also significantly different from the ground 1g (p<0.005). In analogy with increase in total length, also the increase in length of the mineralized zone was most pronounced in the multiwell group. Apposition of mineral in the multiwell group was significantly increased compared to any other group.

In this in vitro experiment with fetal mouse long bones, using completely automated hardware, we studied growth and mineralization under conditions of near weightlessness for a period of four days.

Metatarsals increased in length after a four days lag period followed by four days culture at 37°C, but microgravity conditions had no consistent effect on longitudinal growth. Matrix mineralization, on the other hand, was markedly reduced in microgravity compared to in-flight 1g controls. These data are in agreement with previous experiments performed on the Space Shuttle as part of the first International Microgravity Laboratory Mission (IML-1).⁽⁹⁾ In that study the authors showed, in a different but closely related experiment, a decrease of more then 40% in mineralization measured by ⁴⁵Ca incorporation and more than 30% in length of diaphysis in 16 day old metatarsals cultured for four days in microgravity compared to the in-flight 1g controls. Similar fetal mouse long bones have been used by other experimenters.⁽³⁾ They confirmed the possibility to culture metatarsal long bones under microgravity conditions, although effects on matrix mineralization were not studied.

In ground based studies it has already been demonstrated that mouse metatarsals are responsive to changes in gravitational forces.⁽⁷⁾ Growth of comparable long bones under 2.2g or 3.1g resulted in an increased mineralization. In addition, the application of intermittent hydrostatic pressure (an alternative way to apply mechanical loads upon these bones) upon ED16 metatarsals also resulted in increased matrix mineralization.⁽²⁾ Both the present and previous experiments show that metatarsal long bones respond to spaceflight, hypogravity, as well as to centrifuge, hypergravity, conditions while in culture. Since the present study comprises an in vitro experiment, the response of skeletal tissue to near weightlessness was not mediated by spaceflight induced changes in levels of systemic factors such as calcium regulating hormones or stress hormones. This implies that skeletal tissue by itself is sensitive to decreased loads due to weightlessness as well as increased loads due to hypergravity and hydrostatic pressure.

After morphological inspection, it appeared that a large number of metatarsals showed a significant amount of cell death and that growth was often impaired. For reasons unknown, this phenomenon was most pronounced in the microgravity (F) samples. There might be several possible causes for this phenomenon. As has been shown by van Loon et al., the biological activity of these tissues can be slowed down for a 24 hrs period by lowering the incubation temperature to ambient.⁽⁸⁾ Although pilot experiments demonstrated that bones could survive a 4 days at 20C lag period needed for transport and integration, such treatments are not very beneficial to these tissues and may have increased the risk of cell death. Also, one reservoir for each culture compartment was filled with 1% formaldehyde fixative. It is possible that minute amounts of the fixative started to contaminate the culture compartment before the end of the incubation. This may have caused some of the metatarsals not to increase in length and develop signs of necrosis. It is surprising that the negative effects were absent in FC samples. The only difference in F and G compared to FC was that the former two groups were cultured inside a CIS container, while the latter was cultured in a slightly modified Type-I container.

Longitudinal growth as well as mineralization were more pronounced in the multiwell control group compared to all others (Figs. 5.4 and 5.6). A slightly diminished gas exchange in the PBs compared to the open multiwell trays may have attributed to this difference. The PBs were covered with a gas permeable foil while multiwell cultures were directly exposed to the 5% CO₂ in air atmosphere. Also the ratio of the surface area for gas exchange compared to the culture medium volume was less favorable in the PBs due to their mechanical requirements and construction. Finally, the use of different materials, PETP for the PBs and polystyrene for the multiwell plates may also have influenced growth and mineralization. This should be tested in a separate study.

There is a significant difference in mineralization between in-flight 1g (FC) and ground (G). A difference in calcification was also shown between the same two groups in a previous microgravity experiment (IML-1) using similar metatarsal long bones.⁽⁹⁾ The differences between in-flight 1g and ground 1g are launch accelerations and vibrations, and in-flight cosmic radiation experienced by the in-flight 1g samples. These factors could be responsible for the differences between in-flight 1g and ground 1g. However, the mineralization in the in-flight 1g was diminished in the present experiment, but augmented in the previous microgravity study.⁽⁹⁾ Although the flight 1g and ground 1g data varied in the two experiments, the microgravity effect of reduced mineralization compared to in-flight 1g remains. These discrepancies plead, again, for the use of an on board 1g centrifuge as the best control for microgravity experiments.

In the previous microgravity experiment⁽⁹⁾ it was argued by the authors that the in-flight 1g samples might have been sensitized to the increase in gravity load during the 6 hours start-up time between reaching orbit and the actual start of the experiment, in which microgravity was present even in the in-flight 1g samples. Since in the Bion-10 protocol the lag time under microgravity was only 10 minutes before starting the centrifuges, the possibility of sensitizing seems unlikely. Also the repetitive stop and start events, characteristic for the Biorack centrifuge during the IML-1 experiment, were not applicable to this study. The centrifuges on board the Bion-10 operated non-stop for the full four day period of the experiment.

Due to a software problem, the centrifuge speed of the on board 1g centrifuge began to fluctuate after the first two days in flight. For the last two days of the experiment, the gravitational force ranged from 1.0 to approximately 1.5g (119 to 140 rpm; nominal speed 112 rpm). It is not likely, however, that the generated increase in gravity had an influence on growth and mineralization. It has been shown earlier that an increase in acceleration from 1.0g to 1.3-1.5g, with comparable tissues on a comparable centrifuge, had no impact on percentual increase in length nor on various mineralization parameters.⁽⁹⁾ However, greater accelerations, from 2.2 to 3.1g, resulted in an increased growth and increased mineralization in ED16 metatarsal long bones.^{(Chapter 6 and ref. 9)}

In summary, in this in vitro experiment with mouse fetal long bones, using completely automated hardware, we have demonstrated that longitudinal growth was not affected by a near weightlessness environment in space, while matrix mineralization was greatly reduced under microgravity. These results confirm our hypothesis that bone mineral metabolism is affected by lack of gravity. They suggests that the response of fetal bone to near weightlessness represents an adaptive response to a condition of extreme unloading, which is in accordance with Wolff's law of functional adaptation.⁽¹²⁾ Future experiments should focus on the cellular mechanisms by which gravity changes bone cell metabolism.

1 Huijser R., Aartman L., Willemsen H. Cells In Space: sounding rocket facilities for cell biology and biotechnology in microgravity. Proc. Fourth Europ. Symp. Life Sci. Res. in Space, ESA SP-307, 455-466, 1990.
2 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.
3 Klement B.J., Spooner B.S. Pre-metatarsal skeletal development in tissue culture at unit- and microgravity. J. Exp. Zool. 269, 230-241, 1994.
4 Morey E.R., Baylink D.J. Inhibition of bone formation during space flight. Science 201, 1138-1141, 1978.
5 Tenenbaum H.C. Role of organic phosphate in mineralization of bone in vitro. J. Dent. Res. 60, 1586-1589, 1981.
6 Ubbels G.A. The role of gravity in the establishment of the dorso/ventral axis in the amphibian embryo. In: Longdon N. David V., eds. Biorack on Spacelab D1, ESA Publication Division, ESTEC, Noordwijk, The Netherlands, ESA SP-1091, 147-155, 1988.
7 van Loon J.J.W.A., Veldhuijzen J.P., Burger E.H. Hypergravity and bone mineralization. ESA SP-307; Proc. 4^th Europ. Symp. Life Sci. Res. in Space, Trieste, Italy, May 1990. edt. V. David ESA Publication Division, ESTEC, Noordwijk, The Netherlands. 393-396, 1990.
8 van Loon J.J.W.A., Veldhuijzen J.P., Windgassen E.J., Brouwer T., Wattel K., Vilsteren van M., Maas P. Development of tissue culture techniques and hardware to study mineralization under microgravity conditions. Adv. Space Res., 14, 289-298, 1994.
9 van Loon J.J.W.A., Bervoets D.J., Burger E.H., Dieudonné C.S., Hagen J.W., Semeins C.M., Zandieh Doulabi B., Veldhuijzen J.P. Decreased mineralization and increased calcium release in isolated fetal mouse long bones under near weightlessness. J. Bone Min. Res. 1995, accepted.
10 Vose G.P. Review of roentgenographic bone demineralization studies of the gemini space flights. Am. J. Roentgenol., Rad. Therapy & Nuclear Med. 121, 1-4, 1974.
11 Vogel J.M., Whittle M.W. Bone mineral content changes in the Skylab astronauts. Am. J. Roentgenol., Rad. Therapy & Nuclear Med. 126, 1296-1297, 1976.
12 Wolff J.D. Das Gezetz der Transformation der Knochen, Berlin, A. Hirschwald, 1892.
13 Zerath E., Holy X., Malouvier A., Caissard J-C., Nogues C. Rat and monkey bone study in the biocosmos 2044 space experiment. Physiologist. 34, S194-S195, 1991.

We like to thank the ESA-Moslab Bion-10 team and the IBMP-Bion-10 team for their excellent support. We also thank IBMP staff members, dr. M. Tairbekov in particular. This research was funded by the Dutch Organization for Space Research (SRON) grant MG-004 and the Dutch Organization of Scientific Research (NWO) grant 900-541-133.