Effect of spaceflight and hypergravity on mineral metabolism in organ cultures of fetal mouse long bones.
Jack J.W.A. van Loon
Free University Amsterdam, 23 March 1995.

Promotoren: prof.dr. E.H. Burger
Copromotor: dr. J.P. Veldhuijzen
Referent: prof.dr.ir R. Huijskes


Chapter 1
Bone and Spaceflight: An Overview

Published in: Jack J.W.A. van Loon, J. Paul Veldhuijzen, Elizabeth H. Burger. Bone and space flight: an overview. in Biological and Medical Research in Space, Edt. D. Moore, P. Bie and H. Oser. Springer-Verlag Berlin Heidelberg, Chapter 5, 259-299, 1996.

Chapter 2
Development of Tissue Culture Techniques and Hardware to Study Mineralization under Microgravity Conditions.

J.J.W.A. van Loon1, J.P. Veldhuijzen1, E.J. Windgassen2, T. Brouwer3, K. Wattel3, M. van Vilsteren3 and P. Maas4. Academic Centre for Dentistry Amsterdam (ACTA), Dept. of Oral Cell Biology1 and Dept. of Engineering2 and Vrije Universiteit, Fac. of Biology, Dept. of Electronics3, and of Engineering4. Amsterdam, The Netherlands.

To study the effects of weightlessness on fetal mouse long bone rudiment growth and mineralization we have developed a tissue culture system for the Biorack facility of Spacelab. The technique uses standard liquid tissue culture medium, supplemented with Na-b-glycerophosphate, confined in gas permeable polyethylene bags mounted inside ESA Biorack Type-I experiment containers. The containers can be flushed with an air/5% CO2 gas mixture necessary for the physiological bicarbonate buffer used. Small amounts of fluid can be introduced at the beginning (e.g. radioactive labels for incorporation studies) or at the end of the experiment (fixatives). A certain form of mechanical stimulation (continuous compression) can be used to counteract the, possibly, adverse effect of microgravity. Using 16 day old metatarsals the in vitro calcification process under microgravity conditions can be studied for a 4 day period.

Published in: Adv. Space Res. 14(8), 289-298, 1994.

Chapter 3
Decreased Mineralization and Increased Calcium Release in Isolated Fetal Mouse Long Bones under Near Weightlessness.

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 VeldhuijzenAcademic 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 1gravity. 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.

Published in: J Bone Min Res, 10(4), 1995.

Chapter 4
Polysulphone Inhibits Final Differentiation Steps of Osteogenesis In Vitro.

Jack J.W.A. van Loon1, Johan Bierkens2, Jef Maes2, Greet E.R. Schoeters2, Daniella Ooms2, Behrouz Zandieh Doulabi1, J.Paul Veldhuijzen1. 1 Academic Centre for Dentistry Amsterdam (ACTA), Dept. of Oral Cell Biology, Amsterdam, The Netherlands. 2 Flemish Institute for Technological Research (VITO), Environmental Division, Mol, Belgium.

Biocompatibility is an important factor in the development of orthopaedic implants as well as in the development of new tissue culture devices. Polysulphone has been used for orthopaedic implants because of its mechanical properties, ease of sterilization, its molding capacity and its biocompatibility. Therefore polysulphone has been chosen as the prime material for the construction of tissue culture devices to be used for cultivation of osteogenic cells (pre-osteoblast-like MN7 cells and primary bone marrow fragments) as well as complete fetal long bone explants under space flight conditions. Whereas polysulphone did not interfere with the proliferation in early stages of bone forming cells, we show that leacheble factors within the polysulphone polymer prevented the final steps of matrix formation as measured by collagen synthesis and matrix mineralization. These data argue against polysulphone is a material for orthopaedic implants.

Published in: J. Biomed. Mat. Res. Vol 29, pp 1155-1163, 1995.

Chapter 5
Reduced Mineralization in Isolated Fetal Mouse Long Bones Flown on Board the Russian Bion-10 Satellite.

Jack J.W.A. van Loon1, Olga Berezovska2, Behrouz Zandieh Doulabi1, Cor M. Semeins1, Natalia V. Rodionova2, J. Paul Veldhuijzen1. 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 1g control conditions.

Chapter 6
Increased Mineralization and Calcium Release in Fetal Mouse Long Bones Cultured under Hypergravity Conditions.

Jack J.W.A. van Loon, J. Paul Veldhuijzen. Academic Centre for Dentistry Amsterdam (ACTA), Dept. of Oral Cell Biology, Amsterdam, The Netherlands.

It is now well documented that skeletal tissues and cells are sensitive to their mechanical environment. It has been shown previously that fetal mouse metatarsal long bone rudiments respond with an increased mineralization and reduced mineral resorption when subjected to an intermittent (1/3 Hz) hydrostatic compression of 13 kPa above ambient, while in culture.(11,12) In the present study we applied gravitational forces, or accelerations, of 2.2g on bone rudiments for four to five days in organ culture, to study the effect of a vectorial force similar long on longitudinal growth and mineral metabolism. In the mineralization model, using 16 day old bones, 2.2g hypergravity stimulated cartilage growth by 28% and increased mineralization, as indicated by a more than 4 times longer calcified diaphysis as well as more than 7 times higher 45Ca incorporation. In addition, in the resorption model, using 17 day old bones, hypergravity increased mineral resorption by 37% compared to 1g controls. This data suggests that both growth and mineral metabolism in fetal bones are sensitive to mechanical stress resulting from hypergravity during organ culture.

Go to the INDEX page