Establishment Of Experiment Conditions For Weightlessness Bone Loss Ground-based Simulation And Biological Evaluation

With the development of space technology, space biology is widely researched. There were a lot of studies have been carried out in the space environment. The research could not only ensure the health of astronauts, but also benefit to the people on the ground. Therefore, the study of space biology was gradually regarded highly by researchers.The present study aimed to establish the experimental conditions for ground-based simulation of weightlessness bone loss, and developed the biological evaluation on the basis. The experimental contents mainly included that random positioning, diamagnetic levitation, and hind-limb unloading.Firstly, we designed a set of cell culture container for bone loss research under random positioning for simulation of weightlessness. There were box, gas-permeable membrane,silica gel and container lid in the container. The container was more simple to operation,recycle, lower culture medium requirement, and easier to draw the bubble. We also evaluated the biological compatibility of container by experiments. Meanwhile, we designed a co-culture framework which included inner framework, outer framework and co-culture membrane. It could be used in the condition of random positioning for cell co-culture.Secondly, we developed a set of environment control system for bone loss research under diamagnetic levitation for simulation of weightlessness. The system consisted of CO2 concentration control unit, gas temperature and humidity control unit and gas circulating water heat preservation unit. The system could realize the CO2 concentration adjustment,environment temperature and humidity adjustment in the bore of Large Gradient High Magnetic Field, and ensure cell culture in vitro under the condition of strict environment control. And we also established a method of cell culture adhered to micro-carrier for diamagnetic levitation on the basis of environment control. The cells adhered to micro-carrier oriented the levitation position（μ g） precisely by themselves in Large Gradient High Magnetic Field and realized cell culture in the state of diamagnetic levitation. We also studied the effect of diamagnetic levitation on the cellular morphology and function based on this method. The murine calvarial osteoblast cell line MC3T3-E1 adhered to Cytodex-1micro-carrier was subjected to an apparent gravity of μ g（12 T）, 1 g（16 T） and 2 g（12 T）,respectively. The cell morphology, proliferation, apoptosis and cycle were detected. The results showed that high magnetic gravity environment induced changes of cellular morphology, and promoted the proliferation of MC3T3-E1. Whereas the gravity environment of 2 g inhibited the cell proliferation, the cell cycle was arrested at G0/G1 phase. The gravity environment of μ g and 2 g improved the activity of caspase-3 as compared with the normal gravity environment. Meanwhile, on the basis of cell culture method adhered to micro-carrier for diamagnetic levitation, we prepared the hydroxyapatite micro-carrier and evaluated the biological compatibility aimed to make the substrate of cell growth closer to the real growth substrate in vivo for bone tissue cells. Firstly, we prepared the hydroxyapatite powder using liquid phase precipitation method, and detected the phase composition by X-ray diffraction analyzer. Secondly, we prepared the hydroxyapatite micro-carrier by the spray-drying method using the prepared powder as raw material, and detected the morphology and particle size by scanning electron microscopy. Finally, we evaluated the biological compatibility about the HA micro-carrier by detecting the effects of micro-carrier extract on Ca²⁺concentration,cytotoxicity, cell areas and cell proliferation. The results showed that there were significant differences between experimental group and control group to Ca²⁺ concentration, cytotoxicity,cell areas and cell proliferation for micro-carrier first extract. But the secondary extract had no significant difference compared with control group to Ca²⁺ concentration, cytotoxicity, cell areas and cell proliferation.Finally, we developed a portable principle prototype for bone loss analysis under hind-limb unloading for simulation of weightlessness, and simultaneously we evaluated the biological characteristics. The hind-limb unloading was served as the experimental model to simulate the weightlessness bone loss. Ca²⁺ concentration in urine was regarded as a major research indicator, and the feasibility of Ca²⁺ concentration in urine as an early evaluation indicator for weightlessness bone loss was discussed preliminarily. The hind-limb unloading effect on rat weight, bone mineral density for femur and tibia, bone dry mass and bone ash mass for femur,and Ca²⁺ concentration in urine and serum were studied. The results showed that rat weight had obvious downward trend after a week of hind-limb unload. There was significant difference for bone mineral density of femur and tibia from the second week, and the bone dry mass and bone ash mass for femur significantly reduced after three weeks. The Ca²⁺concentration in urine was obvious higher than control group from the third day, and reached peak at the seventh day, and then steady decline, but still higher than control group. According to the correlation analysis between bone mineral density and Ca²⁺ concentration in urine, there was good negative correlation relation, and the Ca²⁺ concentration in urine was more timely and sensitively.In conclusion, this work has established and evaluated the bone loss experimental conditions for simulation of weightlessness which included random positioning, diamagnetic levitation,and hind-limb unloading. It will lay a solid foundation for further study on simulation of weightlessness.