Jack J.W.A. van Loon, Dirk-Jan Bervoets, Elisabeth H. Burger, Suzanne C. Dieudonné, Jan-Willem Hagen, Cor M. Semeins, Behrouz Zandieh Doulabi, J. Paul Veldhuijzen

Academic Centre for Dentistry Amsterdam (ACTA), Dept. of Oral Cell Biology, Amsterdam, The Netherlands.

Mechanical loading plays an important role in the development and maintenance of skeletal tissues. Sub-normal mechanical stress as a result of bed rest, immobilization, but also in spaceflight, results in a decreased bone mass and disuse osteoporosis, whereas supra-normal loads upon extremities result in an increased bone mass.

In this first in vitro experiment with complete fetal mouse cartilaginous long bones, cultured under microgravity conditions, we studied growth, glucose utilization, collagen synthesis, and mineral metabolism, during a 4 day culture period in space. There was no change in %length increase and collagen synthesis under microgravity compared to in-flight 1gravity. Glucose utilization and mineralization were decreased under microgravity. In addition mineral resorption, as measured by ⁴⁵calcium release, was increased.

These data suggest that weightlessness has modulating effects on skeletal tissue cells. Loss of bone during spaceflight could be the result of both impaired mineralization as well as increased resorption.

Key words: bone, microgravity, mineralization, resorption, mechanical loading.

Accepted for publication in the Journal of Bone and Mineral Research, April, 1995.

It has been well documented that bone is sensitive to its mechanical environment. A unique condition of skeletal unloading occurs during spaceflight, where the 1gravity weightbearing stimulation of the skeleton is absent. From one of the first spaceflight studies with humans^(1,2) and rats^(3,4) it has become clear that osteoblastic bone formation is retarded compared to Earth controls. Most spaceflight studies show no effect with respect to bone resorption by osteoclasts,^(3,4) but some have indicated an increased resorption activity under near weightlessness conditions in vivo, although the various recovery times, as in most spaceflight studies, must be kept in mind.^(5-8) It is not clear, however, whether these results on bone metabolism in vivo are a result of the lack of weightbearing, which acts locally, or whether systemic factors are also involved.

Previous experiments have shown that isolated fetal mouse metatarsal long bones respond to changes in mechanical loading. An increase of mineralization has been reported when these metatarsals were cultured under intermittent hydrostatic pressure⁽⁹⁾ or in a hypergravity field.⁽¹⁰⁾ In addition, mineral resorption was decreased in metatarsals cultured under intermittent compression for four days.⁽¹¹⁾

In the present study we used similar organ cultures to study growth, glucose utilization, collagen synthesis and mineral metabolism under spaceflight conditions during a 4 day culture period in microgravity. Using these parameters the hypothesis was tested that weightlessness influences the metabolic activity of skeletal tissues in vitro.

This microgravity experiment was performed during the first International Microgravity Laboratory mission (IML-1) of the Space Shuttle program in the Biorack facility of SpaceLab.

Fig. 3.1 ED16 mouse metatarsal long bones. A: immediately after dissection B: cultured under microgravity conditions C: cultured in the in-flight 1g centrifuge. Bar= 0.5 mm. During the 4 day culture period of an ED16 fetal mouse long bone, mineralization starts in the ossifying center of the rudiment (diaphysis). The mineralized diaphysis is clearly visible as an opaque part in the center of the rudiment (B and C). TL is total length of the metatarsal, D is length of the diaphysis.

3.2.1 Biorack

This experiment was performed in the Biorack facility of SpaceLab. Biorack is a multi user facility and consists of incubators, a cooler, a freezer and a sealed workbench, the glovebox.⁽¹²⁾ Two identical Biorack models were used; one was flown in SpaceLab (Flight Model), the other remained on Earth (Ground Model) as reference.

Biorack has an important advantage over other microgravity experiment facilities in that it provides a small radius (78 mm), slow rotating (107.0±0.5 rpm) centrifuge. This centrifugal force resulted for the Flight Model in a 1g acceleration, generating the unit gravity as present on Earth. Due to the geometry of the centrifuge and the location of samples this g-level ranges from 0.890 to 1.151g. The biological material of the microgravity group as well as the in-flight 1g group are both exposed to the same launch vibrations, accelerations, cosmic radiation or other environmental conditions. The only difference between the two flight groups is the gravity parameter. Centrifugation in the Ground Model results in a 2g (1.338-1.525g) load; the resultant of 1g from the Earth and 1g from the centrifuge. Also other experiments were accommodated on this 1g centrifuge. Due to handling of other experiments the centrifuge was stopped 10 times during the four day culture period. Each stop lasted no longer then 1.5 minutes. The g-vector acting on the metatarsals was oriented perpendicular to the rudiments long axis.

Fig. 3.2. ED17.5 mouse metatarsal long bones. A: immediately after dissection B: cultured under microgravity conditions C: cultured in the in-flight 1g centrifuge. Bar= 0.5 mm.

3.2.2 Tissue preparation / culture procedure

Isolated embryonic mouse cartilaginous long bones (metatarsals) were cultured for 4 days under near weightlessness, or control conditions. The culture procedure and hardware are described in detail elsewhere.⁽¹³⁾ Briefly, per fetus the middle 3 metatarsals in each foot of 16 days embryonic (ED16) (Fig. 3.1) and 17.5 days embryonic (ED17.5) (Fig. 3.2) Swiss random mice (Harlan Sprague-Dawley, Indianapolis) were used. ED16 metatarsals had not yet calcified and start to calcify during culture (Fig. 3.1). ED17.5 rudiments had calcified in utero and start to resorb during culture (Fig. 3.2). Metatarsals were aseptically harvested and individually cultured in gas permeable polyethylene bags. The experiment was accommodated in eight small standard Biorack Type-I containers (204080 mm),⁽¹²⁾ inside Biorack; four flight and four ground containers. Each container held 16 bags. Each bag contained one metatarsal in 670 l culture medium and one 25 ml glass ampoule. For metatarsals used for biochemical studies this ampoule was filled with radioactive labels. For the ED16 biochemical samples, ampoules contained ⁴⁵Ca, ³²P and ³H-Pro. Ampoules used for the ED17.5 samples contained only ³H-Pro. The ampoules for an additional set of containers, used for histological evaluations, contained only formaldehyde.

The metatarsals were kept in a metabolically inactive state during a 24 hour lag period,⁽¹³⁾ used for transportation and launch, by storage at room temperature (see timeline for more detail). When the spacecraft was in orbit, all containers were transferred from stowage in the middeck locker inserts to the Biorack glovebox. For the ED16 and ED17.5 biochemical samples, the ampoules were broken by the facility operators at the beginning of the final 4 days of culture, to release the radioactive labels into the medium. For the histological samples the ampoules, containing fixative, were broken at the end of the experiment. Still in the glovebox, all containers were flushed with a 5% CO₂/in air gas mixture. They were then placed in a 36°C incubator which initiated cell activity. After 2 hours, pressure increase built up in the containers due to the temperature increase, was released. For logistic reasons all ground operations lagged two hours behind the flight experiment.

3.2.3 Timeline

L = launch of STS-42

Containers were transported in 4°C and -10°C Passive Thermal Conditioning Units (PTCU) from California to Kennedy Space Center (KSC), and again from KSC to our laboratory in Amsterdam, the Netherlands.

3.2.4 ED16 metatarsals

Medium used for ED16 cultures consisted of bicarbonate buffered MEM without nucleosides supplemented with 50 mg/l gentamicin and 0.5% v/v fungizone (Gibco), 1% heat inactivated rat serum (TNO, The Netherlands), 50 mg/l L-ascorbic acid, 300 mg/l Glutamine (Merck) and 1.0 mM Na--glycerophosphate (Sigma). The 16 day old metatarsals also received, 1 Ci/ml ⁴⁵Ca (specific activity; 1 Ci/mmol, with an average of 6.6910⁵ cpm per culture bag), 1 Ci/ml ³²P (carrier free, with an average of 1.1810⁶ cpm per culture bag) and 1 Ci ³H-proline/ml (24 Ci/mmole) (Amersham) by means of breaking the glass ampoule at the start of the final culture period (Day 0). ⁴⁵Ca and ³²P were added to the medium to be incorporated into the calcifying diaphysis of ED16 metatarsals only during the final 4 day culture period (Fig. 3.1B and 3.1C). This part of the experiment was stopped by placing the samples in a -10°C freezer until processing (see timeline). Finally, mineral was extracted, using 10% TCA, and radioactivity in the TCA fraction as well as in the medium was determined by liquid scintillation counting.

Other ED16 metatarsals were cultured without radioactive labels, for histological evaluations (see below).

3.2.5 ED17.5 metatarsals

To study mineral resorption ED17.5 metatarsals were used. Pregnant mice were injected intraperitoneally at day 16.5 of gestation with 100 Ci ⁴⁵CaCl₂, to label the rapid mineralizing bones of the embryos. One day later, ⁴⁵Ca prelabeled metatarsals were dissected and precultured at 37°C for one day to remove the freely exchangeable ⁴⁵Ca. Subsequent culture conditions were identical to ED16 rudiments but no ⁴⁵Ca, ³²P and Na--glycerophosphate were added. The absence of Na--glycerophosphate prevented additional growth of the mineralized diaphysis (Fig. 3.2). The glass ampoule only contained ³H-proline at a final concentration of 1 Ci ³H-proline/ml. This part of the experiment was stopped by placing the samples in a -10°C freezer until processing. Percentage release of calcium was calculated by counting mobilized ⁴⁵Ca in the culture medium, and measuring the remaining ⁴⁵Ca in the metatarsals after extraction with 10% TCA. Radioactivity was measured using liquid scintillation counting.

Other ED17.5 metatarsals were cultured without radioactive labels, for histological evaluations (see below).

3.2.6 Lengths

Data on lengths were assessed from photomicrographs taken with an inverted microscope directly after dissection of the metatarsals, and again after return of the samples. Total length and length of the mineralized zone were measured directly from photographic negatives, after enlarging the image using a professional enlarger. The lengths were measured with a standard ruler with a one millimeter accuracy. The final magnifications were 177 and 145 for ED16 samples, and 177 and 91 for ED17.5 samples, for start and end point of the culture, respectively. Lengths are also indicated in Fig. 3.1.

3.2.7 Glucose utilization

For glucose utilization, the remaining D-glucose in the medium per metatarsal after culture was measured enzymatically. The NADPH end-product was measured spectrophotometrically at 366 nm (Boehringer-Mannheim, kit #716251). Fresh medium contained 1015 mg/l D-glucose.

3.2.8 Collagen synthesis

Relative collagen synthesis was assayed according to Peterkofsky and Diegelmann, and Kream et al.^(14,15) The collagen values were corrected for the relative abundance of proline in collagen compared to noncollagen proteins.⁽¹⁶⁾ Specificity of the batch collagenase (Worthington, New Jersey) was tested by digestion of ³H-labeled non collagenous protein of tooth enamel (amelogenins, courtesy dr. ALJJ Bronckers), which was found to be negative. ³H-proline was introduced in the media by breaking ampoules at the beginning of the final 4 day culture period.

3.2.9 Histology

At day 4 of the final culture period the samples used for histological evaluations were transferred from the incubator to the glovebox. Fixative ampoules were broken and formaldehyde was released into the culture bags at a final concentration of 1.26%. After fixation these containers were placed in a 4°C cooler. At return in our laboratory in Amsterdam the samples were dehydrated and embedded in Lowicryl K4M. 1 m sections were made and stained with 0.2% toluidine blue without borax, pH=5.2, for one minute at room temperature.

3.2.10 Groups and statistics

Samples were divided into 4 groups. Microgravity (-g), in-flight 1g centrifuge, ground 1g, and ground centrifuge 2g. Metatarsals were contralateral paired; -g with in-flight 1g and ground 1g with ground 2g.

All measurements; lengths, glucose utilization, relative collagen synthesis, mineral uptake (ED16), and mineral resorption (ED17.5) were performed on the same metatarsal, or its culture medium.

Statistics were calculated using an analysis of variance with the centrifuge position as a within subject factor and the ground versus flight location as a between subject factor. All data are expressed as mean ± SEM, n=8 for all groups unless indicated otherwise.

Table 3.I Growth parameter. Length of ED16 and ED17.5 metatarsal long bones before and after the 4 day culture period. Rudiments were contralateral paired: -g with in-flight 1g and ground 1g with ground 2g. %Length increase is determined as length (T₄-T₀)/T₀100%. Data are mean ± SEM, n=8, except for 16 day old microgravity, n=7.

Details of an ED16 and ED17.5 metatarsals cultured for four days under microgravity conditions are shown in Fig. 3.3 and 3.4. The cells and surrounding matrix appeared quite healthy. No adverse effects on cell integrity could be observed in the microgravity group as well as in the other groups (data not shown).

All metatarsals showed a considerable increase in length, as is shown in Table 3.I. There was an increase in %length of about 75% in ED16 and 55% in ED17.5 bones. No consistent differences were found between the -g and in-flight 1g groups.

Glucose utilization is shown in Fig. 3.5. The microgravity groups of both ED16 and ED17.5 metatarsals utilized significantly less glucose compared to most other groups. The ED17.5 samples utilized more than 3 times as much glucose compared to ED16 metatarsals.

Matrix formation, as measured by collagen synthesis is shown in Fig. 3.6. Collagen synthesis was unchanged in the microgravity groups of ED16 as well as ED17.5 metatarsals compared to any other group. There is, however, a difference between both flight groups combined compared to both ground groups combined in ED16 as well as ED17.5 metatarsals, p=0.064 and p=0.012, respectively.

Mineralization was studied in the ED16 bones by measuring the length of the calcified diaphysis, which at this stage of development (ED16 plus 4 day culture) consists of a core of mineralized cartilage surrounded by a primitive bone collar (Fig. 3.3). A 31.3% reduction in length of the diaphysis in the microgravity group was found versus in-flight 1g (Figs. 3.1 and 3.7). There were no differences between microgravity and both ground groups. The lengths of the diaphysis of both flight groups combined were significantly larger compared to both ground groups combined (p=0.045).

Mineralization was also studied by measuring the radioactive calcium and phosphate uptake in TCA extracted mineral of ED16 metatarsals (Fig. 3.8). A decrease of 46.4% in calcium uptake was found under microgravity compared to the in-flight 1g. There was also a difference in calcium uptake between the in-flight 1g and ground 1g groups. Phosphorus followed the same pattern as calcium; a decreased uptake of 40.4% under microgravity versus in-flight 1g.

Finally, mineral resorption was studied in ED17.5 ⁴⁵Ca prelabeled bones (Fig. 3.9). In contrast to the decreased mineral formation there was a pronounced increase of 42.7% in mineral resorption under microgravity conditions versus in-flight 1g. No significant differences were found between flight groups and either ground group.

Fig. 3.3 Photomicrograph of the central part of an ED16 metatarsal cultured in microgravity for 4 days. CC= calcified cartilage, HC= hypertrophic chondrocytes, PO= periosteum. Bar= 50 mm.

In this first experiment with fetal mouse long bones cultured under microgravity conditions, we studied growth, glucose utilization, collagen synthesis and mineral metabolism.

Four days of microgravity revealed no significant differences in %length increase between -g and in-flight 1g groups. There was, however, a slight tendency with ED17.5 metatarsals (p=0.064), towards a reduced %length increase under microgravity compared to in-flight 1g as well as for ground 1g compared to 2g. Duke et al. reported a significant reduction in length of the reserve and hypertrophic/calcification zone in tibiae of rats flown for 12.5 days on the Cosmos 1887,⁽¹⁷⁾ although the more than 48 hours recovery time before sacrificing the animals after this flight should be noted. Also, longitudinal growth was impaired in the humerus⁽¹⁸⁾ and proximal tibiae⁽⁴⁾ of rats flown for 7 days. The considerable increase in length in all groups during the 4 day culture does emphasize favorable growth conditions in the culture method used.

Fig. 3.4 Photomicrograph of the central part of an ED17.5 metatarsal cultured in microgravity for 4 days. CC= calcified cartilage, HC= hypertrophic chondrocytes, PO= periosteum, BC= primitive mineralized bone collar. Note the excavation of the primitive marrow cavity (MC). Bar= 50 mm.

We found a profound reduction of glucose utilization under -g compared to in-flight 1g group in ED16 as well as ED17.5 metatarsals. An impaired glucose utilization was also found in femoral-diaphyseal fragment explants from rats grown under simulated near weightlessness.⁽¹⁹⁾ On the other hand, an increased energy utilization after cyclic loading was found by Skerry et al. and Dodds et al. in vivo, and by El-Haj et al. in vitro. They reported an increase in glucose-6-phosphate dehydrogenase activity in osteoblasts and osteocytes after loading turkey and rat long bones.^(20-22) The increased glucose utilization by ED17.5 versus ED16 metatarsals is probably due to the larger number of cells in the bigger ED17.5 rudiments.

In this study collagen synthesis was not affected by microgravity. However, an increase in hydroxyproline concentration in vertebrae has been found in rats after a 7 day spaceflight,⁽²³⁾ and in rat intervertebral disks after a 14 day mission.⁽²⁴⁾ Duke et al. reported wider collagen fibrils in the growth plate of rat tibia flown aboard Cosmos 1887.⁽¹⁷⁾ These changes in collagen fibers were probably the result of changes in matrix proteoglycans, which were not studied in the present investigation.

Our in vitro data on a reduced mineral formation under microgravity are in agreement with earlier in vivo studies under near weightlessness conditions.^(1-5,7,8,25) The reduction in length of the diaphysis, and the reduced calcium and phosphorus uptake indicate an impairment of mineralization resulting from lack of the 1gravity mechanical stimulation. The decreased total length of the ED16 microgravity group at day 4 (0.067 mm, data not shown) can not account for the 0.124 mm reduction in diaphyseal length (Fig. 3.7).

It has been suggested that mineralization of metatarsals in vitro may be accelerated by shear stresses at the mineral/cartilage interface.⁽²⁶⁾ Using a finite element analysis model it was, retrospectively, concluded that these shear stresses are probably responsible for the increased mineralization found in embryonic mouse long bones treated with intermittent hydrostatic pressure.⁽⁹⁾ It is obvious that in microgravity shear stresses will not occur, since there are no mechanical forces acting. It will be interesting to evaluate whether shear stresses play a role in the increased mineralization of the in-flight 1g samples, compared to metatarsals cultured under microgravity conditions.

Mineral uptake studies in skeletal tissues are very complex because of the bone turnover. In the ED16 model, used for these experiment, one starts with a non-mineralized metatarsal. Considering the fact that the in vitro development always leaps behind in vivo growth, the developmental stage for ED16 metatarsals plus 4 days culture is comparable to an ED17 in vivo stage. This means that no osteoclasts or their precursors have entered the mineralized diaphysis yet.^(27,28) Histological observations failed to show any osteoclasts in the mineralized zone of ED16 metatarsals after 4 days culture (Fig. 3.3). Also, the primitive marrow cavity as can be seen in ED17.5 metatarsals (Fig. 3.4) is not observed in ED16 samples (Fig. 3.3). Consequently, besides the always present physicochemical mineral exchange, there is only de novo mineralization in these ED16 metatarsals, and no mineral resorption.

Fig. 3.5 D-Glucose utilization of ED16 and ED17.5 metatarsals, per rudiment during the final 4 days of culture. Significance between groups is indicated by the same letter. a: p<0.05; b: p<0.001; c,d,e: p<0.005.

Fig. 3.6 Relative collagen synthesis (%) during the final 4 days in culture. N=7 for ED17.5 in-flight 1g group.

In vivo spaceflight studies are not equivocal with respect to bone resorption related parameters. Some found no or a decrease ^(3,4), while others reported an increase ^(5-8) in osteoclastic activity. Our in vitro results show that osteoclastic mineral resorption is increased under microgravity versus in-flight 1g. This is in agreement with extensive in vivo studies by Whedon et al. and Leach et al., where an increased calcium, phosphate and hydroxylysine excretion in astronauts during several Skylab missions was reported, indicating an increased bone resorption.^(29,30) The clearly increased resorption found in the present study resulted from using a more sensitive biological system and/or a more sensitive assay for osteoclastic activity. And, since this was an in vitro organ culture, direct effects were not obscured by systemic factors.

In the ED17.5 metatarsals, besides physicochemical mineral exchange, probably only calcium release prevails in this model. What is measured in this assay is only labeled calcium. The released ⁴⁵Ca in the medium, after culture, only derives from the in utero prelabeled mineral. Cell mediated mineralization in this system hardly occurs since no sodium--glycerophosphate was added to this medium. This is reflected by the lack of increase in length of the mineralized diaphysis (Fig. 3.2). When cell mediated re-uptake would take place, the calcium ions derive from the culture medium pool which contains no ⁴⁵Ca in the beginning, and only minute amounts later in culture. The chance for a released ⁴⁵Ca ion to be incorporated again is insignificant since its concentration in the medium is negligible compared to non-labeled calcium. Also, the amount of calcium incorporated in the very actively mineralizing ED16 metatarsals is only ±1.1% of the total medium calcium. Therefore, the maximum amount of released ⁴⁵Ca to be incorporated again probably would only have been a fraction of this 1.1%.

Fig. 3.7 Length of the calcified zone (diaphysis) of ED16 metatarsals after the final 4 days of culture. Significance between groups is indicated by the same letter. a: p<0.01.

Fig. 3.8 Calcium and phosphorus uptake of the mineralized diaphysis of ED16 rudiments after the final 4 days of culture. Significance between groups is indicated by the same letter. a,c: p<0.001; b: p<0.05.

In some parameters measured, differences were found between the microgravity group and the in-flight 1g control, whereas no such differences were found between the microgravity and the ground 1g groups. This poses the problem, which has emerged with the use of on board 1g centrifuges, what to chose as the appropriate control group in microgravity experiments. As discussed above, we have chosen for comparing the microgravity and the in-flight 1g groups because these groups have been subjected to the same launch related events and cosmic radiation. For that purpose metatarsals of these groups were paired. For the ground 1g and the ground centrifuge (2g) groups, which were not subjected to the launch and flight related stimuli, we have used separate paired metatarsals. The conclusion that in this in vitro model microgravity decreased glucose utilization and matrix mineralization and increased mineral resorption is based on comparing microgravity and in-flight 1g groups. However, there is a possibility that exposure to microgravity of the in-flight 1g group prior to culture on the on board 1g centrifuge (which is about 6 hours in this experiment; see time-line) has sensitized the cells for the subsequent change to 1g acceleration, while being stored at room temperature. This means that the skeletal tissue cells indeed respond to microgravity, however only in terms of becoming more sensitive to the following increase in acceleration. Taken the ground 1g as control group it could be argued that microgravity per se, as an ongoing event for 4 days, is ineffective to change mineralization and resorption. It should be noted that this conclusion conflicts with many in vivo findings which have shown a decreased mineralization under microgravity.^(1-4) If true, it could mean that the reported in vivo effects of microgravity on skeletal tissues are indirect and the result of microgravity induced hormonal changes.

Significant differences were found between one or both flight groups and ground groups, in glucose utilization, relative collagen synthesis, length of diaphysis and calcium uptake. These effects must be due to launch characteristics, cosmic radiation and/or other local environmental conditions, since these are the only differences. This implies that the metatarsals are sensitive to these accelerations and vibrations during launch while stored at room temperature and/or respond to the cosmic radiation while in orbit. This emphasizes the importance of the use of an in-flight 1g centrifuge as the best control to study microgravity effects.

In general, the in vitro experimental approach to study the role of the mechanical environment in bone metabolism is to apply loads upon cells and tissues. This is done for example by stretching substrata,^(31,32) or applying hydrostatic pressure,^(9,11,22) fluid shear stresses⁽³³⁾ and hypergravity.^(10,34) The question can be raised whether the control, non-stressed, conditions on Earth are indeed totally unloaded (disuse), or whether the on Earth always present 1gravity acceleration is a situation of stress per se. The only possibility to decrease mechanical load in vitro is to reduce the unit gravity vector which acts upon cultures. Microgravity provides this unique condition, in which cells and tissues can be studied in the lowest possible mechanical stress environment. Since the differences found in this experiment are due to a decrease of the gravitational force by the reduction of 1g to microgravity, it is obvious that a 1g condition is a situation of loading. Therefore on Earth, all objects, and thus cells, are inherently subjected to this load. Some cells and tissues are even responding to this acceleration as we have shown in this paper.

Summarizing we conclude that fetal mouse metatarsal long bones in vitro are sensitive to the near absence of gravity. Metatarsals respond, during a 4 day culture period under microgravity, with a decreased glucose utilization, a decreased calcification and an increased mineral resorption, while overall growth and collagen synthesis are not affected. These are effects of microgravity on skeletal tissues only, and are not mediated by changes in systemic factors. The fetal mouse metatarsal long bone is a sensitive model for further studies of the cellular mechanisms involved in the changed metabolism of skeletal tissues under microgravity.

Fig. 3.9 Percentage release of ⁴⁵calcium from ED17.5 metatarsals after the final 4 days of culture. Significance between groups is indicated by the same letter. a: p<0.01.

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3.6 ACKNOWLEDGMENTS

We like to thank the whole Biorack-team of ESA, STS-42 crew, Bionetics and NASA-KSC for their excellent support. Statistical support by Mr. FC van Ginkel and HJ Ader is greatly acknowledged. This work was supported by the Space Research Organization of the Netherlands (SRON) grant MG-004, and by the Dutch Organization of Scientific Research (NWO) grant 900-541-133.