Serious effects on human health are experienced after long duration space missions. Prolonged exposure to microgravity seems to affect several physiological systems. Bone loss is considerable, with losses of 1-2% of bone mass per month in flight, occurring predominantly in the load bearing regions of the legs and lumbar spine. Microgravity induces an uncoupling of bone remodeling between bone formation and resorption that could lead to bone loss. Both processes are probably involved, but their relative importance and how they are orchestrated remain unclear. In order to fully understand the mechanisms underlying this bone loss we participated to the FOTON M3 mission launched on September 2007 that carried three experiments OSTEO, OCLAST and PITS developed by our team. We studied for the first time in vitro the effect of microgravity on osteoclasts (OCs) and our preliminary results indicate that OCs are directly affected.

The OSTEO experiment was performed within a perfusion system of bioreactors, where the differentiation of precursors cells in mature OCs was tested on a synthetic 3D bone-like biomaterial, skelite, that partially reproduces the chemical composition and physical structure of natural bone. The aim was to analyze the gene expression pattern of osteoclasts differentiated in microgravity, compared with ground controls. RNA extracted from the cells has been examined by RT-PCR. The preliminary results indicate that genes involved in osteoclast final maturation and activity, as integrin beta3, cathepsin K, MMP9 were upregulated in microgravity compared to ground control, while other genes were substantially at the same level. In OCLAST and in PITS experiments, bone resoption by mature OCs was investigated and the cells were cultured on bovine bone slices. The experiments, started in orbit lasted 4 days. After landing the amount of collagen telopeptides, as index of bone resorption, was measured in OCLAST samples, while, for a genetic screening RNA was obtained from PITS samples. We found an increase in bone resorption after 4 days of space flight, indicated by higher telopeptide concentration and upregulation of genes related to osteoclast activity. These results are consistent with an increased osteoclastogenesis observed by our group in simulated microgravity conditions, and indicate osteoclasts precursors as direct target of microgravity.

Bone loss is still a critical issue for astronauts and dietary countermeasures would be a great benefit. We have recently shown that high salt intake led to decreases in blood pH and bicarbonate concentration accompanied by increases in bone resorption markers (Frings-Meuthen et al. JBMR, Epub 2007). Since pH decrease is a mandatory condition to activate osteoclasts we hypothesize that the increased bone resorption is initiated by a low-grade metabolic acidosis. Administration of an alkali salt may counteract the acidosis and thereby decrease the bone resorption induced by the usually high level of salt intake of astronauts.

In a metabolic ward study with 8 healthy male test subjects (mean age: 25.8 ± 3.9 years; body weight (BW) 75.4 ± 3.6 kg) we examined the ability of KHCO3 to prevent salt-induced bone loss. 4 days of adaptation with a normal salt intake (2.8 mmol/kgBW/d) were followed by 10 days of high salt intake (7.7 mmol/kgBW/d) combined in one trial with a supplementation of 3 x 30 mmol KHCO3 per day in a cross-over design. Urinary calcium (UCa) excretion and bone resorption markers (C- and N-terminal telopeptide of type I collagen (CTX, NTX)) were analyzed in all 24 hour urine collections. Fasting morning blood was analyzed for the bone formation markers, bone specific alkaline phosphatase (bAP), and N-terminal of propeptide of type I procollagen (PINP). Postprandial parathyroid hormone (ppPTH) measurements were used to provide an insight into the involvement of PTH in salt-induced bone loss. KHCO3 supplementation was able to reduce salt-induced calciuria by only 12 % (p = 0.04) and NTX excretion by only 8 % (p = 0.04); no significant decrease was observed in the bone resorption marker CTX (p = 0.18). Bone formation marker PINP (p = 0.92) and bAP (p = 0.95) remained unchanged. However, ppPTH levels were twofold higher 25 minutes after KHCO3 supplementation (p < 0.001). High salt intake itself did not affect ppPTH. The KHCO3-induced PTH increase might play an important role in the attenuated effectiveness of potassium bicarbonate to reduce salt-induced bone loss.

The aim of this on-going research project is to assess the influence of bone microarchitecture on the mechanical performance of bone and to develop an approach for quantification of its changes in microgravity condition as well as in osteoporotic patients on Earth. For this purpose a testing chain consisting of three steps was established:

Spaceflight conditions down-regulate the ribosomal RNA (rRNA) level in osteoblasts (Kumei et al., 2008). Rat osteoblasts were cultured aboard space shuttle STS-65 for 4 and 5 days. Cells were treated with 1ƒ¿,25 dihydroxyvitamin D3 during the last 20 hrs, then fixed by guanidine solution. The quantitative RT-PCR was used to determine the levels for 18S rRNA and the mRNA levels for RNA polymerase I transcription machinery: the upstream binding factor UBF and the TATA box-binding protein-associated factor TAFI48. The total amount ratio of RNA / DNA in the flight cultures was remarkably decreased to 30% ~ 10% (p<0.01) of the ground control cultures.

In the flight cultures, the TAFI48 mRNA levels were 50% decreased, whereas the UBF mRNA levels were 2-fold increased, as compared to the ground control cultures. Upon extra- and intra-cellular environmental change, living organisms act to retain homeostasis through concerted action of RNA polymerase I transcription machinery in the nucleolus. The rRNA synthesis is highly regulated by the cellular conditions. A variety of physical and chemical stresses are likely to disturb the concerted action of RNA polymerase I transcription machinery and eventually reduce rRNA synthesis. A limited number of studies have shown that the spaceflight-induced bone mass loss is attributed to the osteoblast dysfunction due to retarded differentiation or apoptotic events under microgravity. Reduced levels of rRNA during spaceflight may imply the pathological response to microgravity stress or cellular adaptation to a new gravitational environment. Further studies are needed to clarify the possible involvement of RNA polymerase I activity, post-transcriptional processing and degradation. Ribosomal RNA might be the global target of microgravity. Supported by grants from JAXA (Japan Aerospace Exploration Agency) to Y. Kumei.

Limb disuse is well recognized as a cause of bone loss in men and women. Generalized bone loss is a complication after para or tetraplegia, and localized bone loss occurs with regional disuse after fracture, unilateral amputation, stroke or poliomyelitis and reduced physical activity. In men, several risk factors commonly implicated in the pathogenesis of osteoporosis include hypogonadism, smoking, alcohol abuse, previous gastric surgery and glucocorticoid therapy. It has been proposed that these risk factors have additive and cumulative effects in male osteoporosis. Orchidectomy (ORX) in rat and unilateral hindlimb paralysis induced by botulinum neurotoxin (BTX) are respectively suitable models for hypogonadism and disuse induced osteoporosis. ORX and BTX models were combined to see if their effects were cumulative and if bone loss could be prevented by an antiresorptive agent (risedronate) or testosterone.

Four groups of 12 rats were examined for 1 month: SHAM operated; ORX and immobilized on the right hindlimb; ORX+BTX+risedronate (5µg/kg/d), ORX+BTX+testosterone (30µg/kg/day). The left hindlimb served as a non-immobilized control. Modifications of bone, lean and fat mass were examined by dual energy X-ray absorptiometry (DXA) on whole body, regional left and right hindlimbs and on excised tibia and femur. Modification of bone mass and trabecular bone microarchitecture were assessed by histomorphometry and X-ray microtomography. Static and 2D microarchitectural histomorphometric measurements were performed: Trabecular Volume (BV/TV), Trabecular Thickness (Tb.Th), Trabecular Number (Tb.N), Trabecular Separation (Tb.Sp). Trabecular 3D and cortical 2D measurements were assessed by X-ray microtomography.

Whole body bone mineral content (BMC) and lean mass were decreased in ORX-BTX rats. ORX and BTX had additive effects on bone loss since differences were maximized on the immobilized bone. The decrease in BMC on the tibial metaphysis reached -33.6% versus 11.3% in the non-immobilized limb. BV/TV and Tb.N decreased and Tb.Sp increased in both hindlimbs whereas Tb.Th was significantly lower only in the immobilized limb. Decrease of tibial cortical area and thickness was greater in the immobilized limb. Risedronate prevented BMC, BV/TV and architecture loss but not reduction in Tb.Th. Cortical bone was preserved only in the non-immobilized limb. Testosterone was unable to prevent trabecular and cortical bone loss, but it prevents loss of whole body lean mass.
In conclusion, ORX and BTX resulted in additive effects on bone loss. This model combining two methods for inducing bone loss is easy to handle and is suitable to evaluate pharmacological compounds since the bone loss is large and occurs rapidly. In conclusion, using this model we have shown that bisphosphonate risedronate has early protective effects against the loss of trabecular bone mass and bone architecture due to ORX and BTX but was less effective on cortical bone.

Disuse models have been extensively used to mimic the effects of microgravity on bone and to develop countermeasures. Disuse, as observed after a paraplegia or denervation, is known to induce a rapid bone loss in adults. It reduces mass and length of bones and can create macroanatomical changes in a growing skeleton. Howevers, these animals models also induce associated neurological and symphathetic changes. Hypoactivity has been found to decrease bone mass in adults but its effects on bone mass and bone quality have been seldom explored in young growing animals. The aim of the present study was to analyze the effects of a prolonged hypoactivity without nerve or muscle lesion in a group of growing chicken.

Animals, methods: Ten chicks of the rapidly growing strain 857K were grown in a large enclosure where they could walk freely; 10 others were kept in small cages with littlespace to move around. All chicks were given equal amount of normal feed. The chickens were sacrificed to 56 days by electronarcosis. Femur and tibia were excised and radiographied. They were analyzed by texture analysis of X-ray films with a fractal algorithm and also by DEXA (Dual energy X-ray absorptiometry - to measure the bone mineral density -BMD) and microcomputed X-ray tomography (microCT - to measure the bone volume and microarchitectural parameters in 3D).

Results: Hypoactivity had no effect on the length and diameter the bones. No statistical difference could be evidenced by any method on the tibia of hypodynamic chickens v.s. ambulatory ones. On the contrary, BMD, microCT (BV/TV and trabecular microarchitecture) and Fractal dimension were always found significantly reduced in the femurs of hypodynamic animals.

____________________hypodynamic____controls_____p
BMD metaphysis femur....0.137 ± 0.01........0.153 ± 0.01......0.008
BMD diaphysis femur.......0.232 ± 0.018......0.261 ± 0.034....0.02
Fractal D femur................2.422 ± 0.023......2.456 ± 0.017....0.001

Conclusion: Hypoactivity, by reducing the amount of muscular strains on certain bones, can induce bone loss during growth. Bone quality (microarchitecture of trabecular bone) is altered while the gross anatomy and sizes are not. The femur appeared the most sensitive bone to the effect of hypoactivity in this model.

Most of the skeleton forms via a cartilage model, and the use of cartilage to replace or repair bone is reasonable as (a) no scaffolding is required to form the implant which (b) disappears as bone is formed during the endochondral process. Previously, we have shown that cartilaginous spheroids, derived from embryonic mouse limb bud cells, and grown in a rotating bioreactor (Synthecon, Inc.) ossified when implanted adjacent to a bone defect (Ann. Biomed. Eng. 32:1-6). In these studies, to assess mineralization of the cartilage, the skull containing the implant was imaged using microCT scanning. The skull was then demineralized, and the defect region and implanted spheroid sectioned and stained with a alcian blue-tartrazine stain which differentially stains cartilage and bone. The micro CT scan showed that mineralization was occurring and this was confirmed by the histology which showed that the implant consisted of a mixture of cartilage and bone, and that the amount of cartilage diminished over time, as the amount of bone increased. Vascularization of the tissue occurred concomitant with the formation of bone. We also reported that cartilage spheroids, again grown in the rotating bioreactor, implanted into a skull defect, contributed to healing of the defect. In controls, which had a 2 mm defect, with no implant, some sections exhibited only a thickened periosteum on either side, or a layer of bone that was thinner than the surrounding region, but again with thickened periosteum. In contrast, the defect in implanted skulls was filled with a mass of bone.

In the current report, additional skulls with or without implants in the 2 mm defect were subjected to microCT scans, and sections from these scans were compared to histological sections of the defect region of demineralized skulls from the same experiment. The area of the defect that stained yellow with tartrazine in histological sections of demineralized skulls, indicating the presence of bone, was the same region that was shown to be mineralized in CT sections. Defects without implants were shown in serial CT sections, as in histological sections, to be incompletely healed. This study and the previous one demonstrate that cartilage spheroids, grown in the rotating bioreactor, can heal bone, and that microCT scans are an important corollary to histological studies evaluating the use of implants in healing of bony defects. Supported by: Supported in part by NIH/NIDCR Training Grant T35 DE07252 and by Cancer Center Support Grant (CA-16672).