Spaceflight increases risk of bone loss: The continuous and progressive loss of calcium and weight bearing bone noted in space flight crews is one of the most serious impediments to long duration manned space flight. Skeletal changes and loss of total body calcium have been noted in both humans and animals exposed to microgravity from 7 to 237 days. During the Apollo and Skylab missions, photon absorptiometry was used to assess pre- and post-flight bone mineral mass. For the 12 crew members of Gemini 4, 5, and 7, and Apollo 7 and 8, the average post-flight loss from the os calcis (heel) was 3.2 percent over an average of 8.5 days (1-3) . Analysis of in-flight urine, fecal, and plasma samples from Skylab missions revealed changes in urinary output of hydroxy-proline indicating degradation of the collagenous matrix substance of weight bearing bones. Nitrogen output also increased with a resultant muscle atrophy occurring. Elevated concentrations of urinary calcium were noted in Skylab astronauts starting within the first days of flight; in many of the astronauts urinary calcium concentrations remained at elevated levels throughout the mission.
A direct effect of microgravity is the loss of mechanical stress on the skeletal system. Although in-flight exercise is a helpful countermeasure used by astronauts, the greatest losses in the US flight program occurred in the 84-day Skylab 4 mission even though exercise was regularly performed. Crew members using exercise as a countermeasure still lost an average of 4 percent of bone over the 84 day mission period (4). In the 237-day Soviet Soyuz T-10 mission, the Cosmonauts lost bone in spite of 2-4 hours of daily exercise. Both compact and trabecular bone was lost from the os calcis during this mission. Bone loss appears to increase in general proportion to mission length, from 4 percent to 19.8 percent over an 84 to 184 day period (4, 13) . The bone loss suffered in flight is not fully recovered after return to gravity, which will be a problem on long term missions. Paradoxically, excessive exercise may be part of the problem; Stein et al have reported a negative energy balance in some of the longer term missions strongly suggesting the need for a more efficient exercise programs that would not result in a negative energy balance.
The loss of bone in the presence of consistent exercise suggests additional molecular mechanisms in spaceflight bone loss. Various lines of evidence from human and animal studies suggest that bone loss in space flight is due to a decrease in bone formation. Rat studies aboard Spacelab 3 and animal studies flown on the Cosmos Biosatellites provided evidence for significant skeletal changes including bone mineral content, even on flights of short duration. It is probable that the decrease in bone formation is caused by the direct lack of gravity in microgravity. Previous studies performed by this laboratory have shown that there is a direct gravitational effect on osteoblasts, which alters the extracellular matrix, nuclear morphology and gene expression. First, on STS-56 we demonstrated a change in cell and nuclear shape with slowing of osteoblast growth after 4 days of spaceflight, even though glucose metabolism per cell was unchanged (18) . More recently, we have demonstrated that gene expression, cytoskeleton and nuclear structure is changed when compared to ground controls and to on board 1-g controls.
The consequences of bone loss in space are:
1) the possibility of irreversible bone loss in astronauts after long-term flight;
2) the potential toxic effects of high serum levels of calcium and phosphate with the possibility of renal stone formation in long-term flight and
3) the normal functional hazards of a diminished and weakened skeleton which could result in multiple fractures upon return to earth's gravity.
Taken together, these data predict that bone loss has serious implications for long-term inhabitants of the space station and for future long-term space exploration, e.g., a Mars Mission.
Current countermeasures to bone loss in space flight: To date, all countermeasures used to stop bone demineralization during spaceflight or bed-rest have had limited success. Exercise, dietary supplementation with calcium or phosphorous, and pharmacological treatment with salmon calcitonin and diphosphonates have been tried either in flight or in bed-rest studies. The most promising countermeasures are exercise and diphosphonate treatment. However, exercise is only effective in bed-rest studies when there is at least 4 hours of activity. The older diphosphonates have side effects which include a potential for causing tumors (8) . The newer diphosphonate compounds show promise and may be useful in future flights, however, they do not stimulate new osteoblast growth and their action merely slows the remodeling breakdown by the osteoclast. Moreover, there is currently little information on bone quality after long term use of diphosphonates. Here on earth it has been demonstrated that bone growth is supported by hormone replacement therapy (HRT) (33) and by stress exercise. (10-12) . Normal exercise can result in forces from 1.5xg to 12xg, however, we found no studies that defined the absolute time and duration of gravity force that is required to stimulate bone gene expression and growth.
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