STEM Today

Special Edition on Long Duration Spaceflight

STEM TODAY May 2016, No.8

STEM TODAY May 2016 , No. 8

CONTENTS Medical Requirements Integration Document (MRID) Signaling Pathways in Osteoblast SOLO 足 SOdium LOading in Microgravity (Sodium retention in microgravity) Bisphosphonates as a Countermeasure to Spaceflight足Induced Bone Loss Melatonin and Bone Effects of Sex and Gender on Adaptation to Space: Bone Calcium Metabolism and Its Regulation in Cosmonauts during 30 to 438 days Mission Analysis of Polymorphism of Bone Metabolism Genes and Evaluation of the Risk of Osteopenia in Cosmonauts Comparative Analysis of Changes in the Skeleton of Cosmonauts during space flights on board the Mir Orbital Station (OS) and International Space Station (ISS)

Editorial Editor: Mr. Abhishek Kumar Sinha Advisor: Mr. Martin Cabaniss

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Cover Page Viking 1 Composite Image of Mars Viking 1 images composite of Mars by USGS University of Arizona. The Viking 1 Mission was flown in June of 1976.

Image Credit: NASA Background Hubble and a Stellar Fingerprint Showcased at the center of this NASA/ESA Hubble Space Telescope image is an emission-line star known as IRAS 12196-6300. Located just under 2,300 light-years from Earth, this star displays prominent emission lines, meaning that the star’s light, dispersed into a spectrum, shows up as a rainbow of colors marked with a characteristic pattern of dark and bright lines. The characteristics of these lines, when compared to the "fingerprints" left by particular atoms and molecules, can be used to reveal IRAS 12196-6300’s chemical composition.

Under 10 million years old and not yet burning hydrogen at its core, unlike the sun, this star is still in its infancy. Further evidence of IRAS 12196-6300’s youth is provided by the presence of reflection nebulae. These hazy clouds, pictured floating above and below IRAS 12196-6300, are created when light from a star reflects off a high concentration of nearby dust, such as the dusty material still remaining from IRAS 12196-6300’s formation.

Text Credit: European Space Agency Image Credit: ESA/Hubble and NASA, Acknowledgement: Judy Schmidt Back Cover Chasma Boreale and the North Polar Ice Cap LAYER CAKE. Climatic cycles of ice and dust built the Martian polar caps, season by season, year by year - and then whittled down their size when the climate changed. Here we are looking at the head of Chasma Boreale, a canyon that reaches 570 kilometers (350 miles) into the north polar cap. Canyon walls rise about 1,400 meters (4,600 feet) above the floor. Where the edge of the ice cap has retreated, sheets of sand are emerging that accumulated during earlier ice-free climatic cycles. Winds blowing off the ice have pushed loose sand into dunes, then driven them down-canyon in a westward direction, toward our viewpoint.

Image Credit: NASA/JPL/Arizona State University, R. Luk (vertical exaggeration 2.5x).

STEM Today , May 2016

Special Edition on Long Duration Spaceflight

Effect of long-duration spaceflight on Skeletal System Bone loss is one of the primary medical complications associated with the prolonged skeletal unloading in long-duration spaceflight. Bones are generally classified into four types according to shape: long, short, flat, or irregular. They range in size from the all-powerful leg bone the femur- about 20 inches long, more than an inch across at midshaft to the pisiform, the smallest of the wrist bones, shaped like a split pea; this bone lies at the base of the little finger, familiarly known as the pinkie. But whatever their size or shape, almost every bone in the body is designed to fit a particular need. The most notable exception is the coccyx, our tailbone.

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The arrangement of the individual bones is as precise, orderly and purposeful as the full skeletal system itself, and their distribution from top to bottom is extremely balanced. Most of the bones in our body are structured in a symmetrical fashion. That is, many of our bones are matched on each side of the body. This matched design allows us to balance and stabilize ourselves in the face of the various forces that act on our bodies. Although we will be discussing only the skeletal system, keep in mind that the sensory and balance organs of the nervous system, the muscles, and the bones work together to help achieve this stability.

The skull, the "top" of the skeletal system, has 29 bones that are fused together to form the cranium, or brain case, the face, and the ear bones. The only part of the skull that can move freely is the jawbone. The spine, to which are attached the pectoral (shoulder) girdle, rib cage and the pelvic (hip) girdle, has 26 vertebrae. The ribs number 24, 12 on each side. The two girdles, so named because of their shape, mark the upper and lower limits of the body’s trunk, or central area. From them, respectively, stem the bones of the upper and lower limbs, the arms and the legs, respectively. Each limb has 30 bones apiece. Of the 60 bones in the two upper limbs, all but 6 are concentrated in the hands and wrists; of the 60 bones in the 2 lower limbs, all but 8 are concentrated in the ankles and feet. Thus, appropriately, more than half of all the bones in the body support those parts of our bodies that maintain the busiest daily work schedule - our extremities.

There is a distinct difference, however, in the kinds of daily work that the upper extremities perform compared with the lower extremities. The skeletal components of the lower extremities are primarily involved in opposing gravity. They are considered our anti-gravity bones. The femur, for example, must withstand great weight and pressure. Its shaft is shaped like a hollow cylinder - an excellent design, as any engineer knows, for maximum strength with a minimum of material. Because of this special construction, the thigh bone can take enormous pressures, depending upon the weight of the person and the activity at the moment. In a 125-pound woman who is simply taking a walk, for example, some points of the femur withstand a pressure of 1200 pounds per square inch (psi).

The femur is not, by any means, the only anti-gravity bone in our bodies but it is the largest. The spine, pelvic girdle, tibia (lower leg), and the bones of the foot (particularly, the talus and calcaneus bones) are all important in our day-to-day "struggle" to stand and move against gravity.

Another superb example of how each bone was designed with a purpose is the vertebrae of the spinal column. To help bear the weight of the body, it is formed like a solid cylinder, but it actually consists of alternating layers of bone and cartilage. These compressible cartilage disks between the vertebrae absorb shock and keep the vertebrae from grinding together when the spine bends. At the back of the bony cylinder, a ring permits passage of the spinal nerve cord, and also serves to protect it. At the back of the ring are three sharp projections, or spurs, which join with the ribs and anchor the muscles of the back. The flexibility of the spine and its ability to stretch and compress contribute to the actual height changes that occur to all of us during the day.

After about age 25, a person’s height can go only one way - and that is down. A man or woman might lose an eighth of an inch between ages 25 and 40 as the spongy disks between the vertebrae in the spine shrink, causing the bones to move closer together. The back begins to bend forward after age 40. From age 20 to age 70, a woman may shrink about 2 inches, while a man might lose about an inch. In space, however, there is a height increase as the human vertebral column lengthens and straightens, probably because gravity does not compress the body. In fact, on past U.S. space flights, more than two-thirds of astronauts reported back pain.

This back pain may be associated with the stretching of the spine.

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Medical requirements Integration Document (MRID) MEDB 5.4 Calf Volume Measurement

IN-FLIGHT HARDWARE Calf Volume Measurement Device (EZOG)

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MR006L Exercise Treadmill Test

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MEDB 6.3 Arm Ergometry Test

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IN-FLIGHT HARDWARE ISS Ergometer CEVIS Accessory Bag Isolator Kit Assembly On-Orbit Mounting Frame IVIS Box, Blue IVIS Box, Red Heart Rate Monitor Kit Medical Equipment Computer Russian Velo Ergometer Russian Gamma Complex

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MEDB 1.11 Bone Densitometry

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Cycle Ergometer Test/ Aerobic Functional Capacity

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IN-FLIGHT HARDWARE Russian Ergometer (operational) Russian Ergometer (transport) ISS Ergometer CEVIS Accessory Bag Isolator Kit Assembly On-Orbit Mounting Frame IVIS Box, Blue IVIS Box, Red Station Support Computer Kit 1 USB Blood Pressure / Electrocardiograph Monitor (BP/ECG) Kit

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BP/ECG Resupply Kit Heart Rate Monitor Kit

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Special Edition on Long Duration Spaceflight NOTE No max exercise 24 hrs prior to testing; no regular exercise 8 hrs prior to testing.

Wear workout clothing (shorts, t-shirt, sneakers).

No large meals 2 hrs prior to test.

A light meal permitted up to 60 minutes before test.

Limit caffeine intake to one cup (8 oz) of regular coffee or equivalent 60 minutes before test.

No alcohol, or nicotine 8 hrs prior to test.

Do not apply lotion to the torso on the day of testing (pretest).

Contraindications: previous musculoskeletal injury which would prevent cycle exercise to maximal levels.

No Neutral Buoyancy training 48 hours prior to test; prefer 72 hours.

A "frontal plane only" (6 lead) ECG recording for rhythm monitoring purposes is required.

The metabolic gas analysis system used for post-flight testing shall be an identical make and model to that used for the L-3 to L-1 testing.

MEDB 4.1 will be not conducted with the crewmembers within 72 hours of returning from overseas travel or within 48 hours of domestic travel unless approved by the Crew Surgeon. This requirement may be waived if the normal direct crew return to the U.S. is delayed.

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MR019L Heart Rate Monitoring

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MEDB 5.3 Isokinetic Testing

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MEDB 5.2 On-Orbit Strength and Conditioning Monitoring

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MEDB 5.1 Functional Fitness Assessments

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MR078L Physical Fitness Evaluation: Functional Fitness

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MR026L Postflight Reconditioning

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MR081L Physical Fitness Evaluation: Handgrip Dynamometry Testing

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IN-FLIGHT HARDWARE Pinch Force Dynamometer Handgrip Dynamometer Medical Equipment Computer (MEC)

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SIGNALING PATHWAYS IN OSTEOBLAST The principal concern in extended spaceflight is the rapid and continous loss of bone mass during flight. Reduction in bone formation , mineral content and loss of bone density characterize the physiological response of human weight bearing skeleton to microgravity. 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 .

Analysis of in-flight urine, fecal, and plasma samples from Skylab missions revealed changes in urinary output of hydroxyproline indicating degradation of the collagenous matrix substance of weight bearing bones. Elevated concentrations of urinary calcium were noted in the early studies of Skylab astronauts starting during the first days of flight. In many of the astronauts urinary calcium concentrations remained at elevated levels throughout the mission.

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In Earth based studies, similar catabolic effects on bone are observed in human and animals subjected to immobilization. At the cellular level, weightlessness and skeletal unloading seem to modulate bone by changes in the osteoblasts . Osteoclasts increase in short duration flights, however they return to normal during long duration missions . Studies in rats have shown a marked reduction in the periosteal bone suggesting alteration in the osteoblasts metabolism . Finally, researchers demonstrated that microgravity inhibited differentiation of osteoblasts in microgravity . From animal and human studies investigators found that loss of weight bearing bone is as high as 1% per month and this loss is primarily due to lack of new osteoblast growth in spaceflight.

It is likely that studies of the osteoblast in microgravity will give us new target molecules for development of pharmalogical agents that will stimulate bone growth. A projected bone loss of 20-30% in astronauts is one of the major physiological problems in the proposed 30-month manned mission to Mars.

Studies have noted that in some astronauts bone is not recovered even after 6 months return to earth . Exercise has long been utilized as a countermeasure to weightlessness and to increase bone mass here on Earth.

Bone mineral density (BMD) at the distal radius and tibia in 15 cosmonauts who spent 1, 2 or 6 months on the Russian MIR space station. They found that neither the cancellous nor cortical bone of the radius was significantly changed at any of the time points. In contrast, at the weight-bearing tibial site, cancellous BMD loss was seen after 2 months of microgravity. After 6-months, loss of cancellous bone was more pronounced than cortical bone. In some individuals, the tibial deterioration was great and BMD loss did not seem to depend on previous exposure to microgravity. Moreover, tibial bone loss persisted after return to earth and was not recovered during the post flight study period, suggesting the recovery time is greater than the time spent in microgravity . Since microgravity-induced bone loss is not fully recovered after return to gravity this is a significant complication of long-term missions and must be addressed before a Mars Mission.

Exercise has been used as a countermeasure for bone loss for decades. However, exercise alone is not a total solution for the countermeasures, since paradoxically, excessive exercise aggravates bone loss. Stein et al. reported a negative energy balance in longer-term missions . This strongly suggests the need for a programmatic and efficient exercise program that would not result in a catabolic state. The cause of bone loss in response to reduction of mechanical stress is not yet known, but several ground studies have demonstrated that eicosanoids may be involved. Prostaglandins are released with exercise and are key regulators in exercise-induced bone growth in vivo . The specific cyclooxygenase-2 inhibitor, NS-389, completely blocked mechanical stress induced bone formation in vivo. Other studies have demonstrated that PGE2 augments bone growth in vivo and in vitro .

On the cellular level, numerous studies have demonstrated that mechanical force (fluid shear, flexing, bending and compression) can induce osteoblasts proliferation . The mechanisms by which these various mechanical strains induce growth may involve multiple pathways. The possible mechanism of action and signal transduction of gravity perception by the osteoblasts are discussed. How the mammalian cell perceives gravity is of upmost importance since it will determine the proliferative state of the osteoblasts.

Cell Proliferation in Microgravity Cell proliferation in microgravity is a key issue in cell biology that may determine if multiple generations of gravity evolved life can adapt to a novel evolutionary existence in microgravity. Although flight opportunities have been limited and control experiments sometimes inadequate, the volume of data about microgravity effects on cell proliferation has been accumulating. Specifically, it has been demonstrated that cell growth of lymphocytes and osteoblasts is inhibited or altered in a microgravity environment. Cytoskeletal changes in actin, intermediate filament and microtubule networks have been found in microgravity as well . In addition, several investigators have noted that specific gene expression is modulated by the lack of gravity . These data point to a possible inhibition of anabolic stimuli in the absence of Earth’s gravity. There are several anabolic signals that are regulated by mechanical stress and these same signals may be downregulated in microgravity. The transducers are tyrosine kinase and serine/threonine kinase growth factors, receptors and mechanical stress and all depend

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Special Edition on Long Duration Spaceflight on the central controlling point, MAPK for signal transduction. In addition, integrins, actin cytoskeleton, and G proteins can act through the MAPK pathway. Inhibition of any one of these steps may inhibit activation of nuclear transcription factors and/or induction of early immediate genes. Taken together, the data suggest that the inhibition of cell proliferation is due to changes in early signal transduction in microgravity, which lead to alterations in downstream events that effect gene expression and cell cycle in spaceflight.

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Effects of Simulated Microgravity on Proliferation and Chemosensitivity Simulated microgravity induces growth inhibition through reduction of mitochondrial activity, and enhances the chemosensitivity of malignant glioma cells to CDDP. MECHANO-SENSING RESPONSE PATHWAYS Mechanical force is an important regulator of cell morphology and function especially in osteoblasts. Mechanical stress is known to activate cox-2 both in vitro and in vivo leading to bone growth . Mechanical stress causes induction of cox-2 and c-fos in osteoblasts . It has recently been shown that mechanical stress (FlexcellT M or 50rpm clinorotation) in ROS 17/2.8 osteoblasts cause increase in erg-1 and NFkB nuclear translocation . The promoter regions of both cox-2 and c-fos are driven by both erg-1 and NFkB, it is interesting to note that both of these transcription factors are upregulated by MAPK .

Mechanical loading of gravity increased induction of c-fos and cox-2. It is also found that gravity induces MAPK activity and upregulates c-fos within 30 minutes of stress. This activation is inhibited by addition of U0126 (MEK kinase inhibitor) but not SB203580 (p38 inhibitor). ERK 1/2 phosphorlation most probably causes translocation of activated ERK to the nucleus in the osteoblast within minutes of sera activation. Translocation of ERK 1/2 to the nucleus is critical since activation of erg-1 and NFkB by intra-nuclear ERK causes induction of immediate early gene c-fos (as well other genes regulated by NFkβ and erg-1) since addition of MAPK inhibitors inhibits c-fos induction. Taken together, these data suggest that anabolic signal transduction is regulated, at least in part, by the MAPK phosphorylation pathway. It is not yet known if MAPK plays a role in the downregulation of signal transduction in spaceflight.

Intra-Nuclear action of MAPK signaling pathway Many of the signaling processes leading to induction of gene expression by fetal calf serum are mediated through MAPK pathway. Signal transduction generated by stress, growth factors or sera have the same approximate timed response, with ERK1/2 activation occurring within minutes of the stimulus, reaching a maximum signal at about 30 minutes. Phosphorylation of ERK1/2 occurs within minutes and these data show that translocation to the nucleus occurs during this time period. As seen below, translocation of activated ERK1/2 to the nucleus occurs within 30 minutes of sera activation. Phosphorylation of ERK and translocation to the nucleus occurs in gravity stimulation over the same timecourse. This translocation is mandatory for proper signal transduction in the osteoblast. As seen below, addition of sera to quiescent osteoblasts can cause phosphorylation of ERK and translocation to the nucleus.

Osteocytes, mechano-sensation and mechano-transduction How does the skeleton perceive mechanical forces?

Mechanotransduction, i.e." (the) process of converting physical forces into biochemical signals and integrating these signals into (a) cellular response" is the prerequisite for a functional and healthy skeleton . Because mechanical stimuli regulate various cellular functions, including gene expression, protein synthesis, as well as cell proliferation and differentiation, understanding mechano-transduction at the cellular level is key to understanding basic bone biology and designing new treatments for bone diseases such as osteoporosis .

Osteocytes, the bone cells deeply embedded in the mineralized matrix are thought to be the mechanosensor of bone. Their location, deep within the mineralized matrix and their structural organization of a cellular network, make them ideal to sense mechanical stimuli and to transfer them to the surrounding cells. They derive from osteoblasts that, during the process of bone formation, assume a more differentiated morphology and become entrapped in the matrix that they are actively synthesizing . Three types of mechanical stimuli

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Special Edition on Long Duration Spaceflight have been proposed: (a) direct deformation of the cells via bending or compression of the matrix to which they are attached, (b) fluid flowinduced shear stress and (c) electric fields resulting from electrokinetic effects linked to fluid.

The leading theory is that shear stress induced by fluid flow in the lacuno-canalicular system, is indeed the mechanical signal perceived by the cells.

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The most convincing evidence that osteocytes are indeed the mechanosensors of bone, comes from a work of Tatsumi et al. who engineered a mouse model in which osteocytes could be selectively ablated upon diphtheria toxin administration. Using this model, the authors demonstrated not only that osteocytes are key regulators of skeletal homeostasis (these mice have severe osteopenia), but that they are resistant to unloading induced bone loss, as achieved by tailsuspension.

IMPORTANT Osteocytes respond to mechanical forces (or lack of them) by secreting several molecules such as nitric oxide (NO), prostaglandin E2 (PEG2 ) and Sclerostin , to name a few. Sclerostin, the product of the SOST gene, is an osteocyte-specific protein and recently has emerged as an important therapeutic target for bone diseases such as osteoporosis and osteopenia. This osteocyte-specific protein inhibits bone formation, both in vitro and in vivo, by directly reducing proliferation and differentiation of osteoblasts via the canonical Wnt signaling pathway. It has been shown that Sclerostin acts by binding the low-density lipoprotein receptor 5 and 6 (LRP5 and 6) and inhibit Wnt-βcatenin signaling pathway and that both the mRNA and the protein are regulated by mechanical forces. Recent studies in both animal models and patients have shown that Sclerostin synthesis and expression is highly regulated by mechanical forces. Several studies have shown that the protein is elevated during reduced loading as achieved by bed rest , paralysis or hind-limb unloading and it is reduced during increased loading.

Regulation of SOST/Sclerostin Expression Osteocytes have been long implicated in mechanosensing and initiation of the bone anabolic response to mechanical load . In support of this, specific ablation of osteocytes in mice resulted in fragile bone, and these mice did not respond with bone loss to unloading. Wnt signaling may play an important role in the anabolic response to deformation and loading since increased Wnt signaling has been found after loading of osteoblastic cells in vitro and of tibiae in vivo and the Wnt coreceptor LRP5 was found to be essential for the increase in bone mass after loading . Since sclerostin is produced by osteocytes in bone and inhibits bone formation by antagonizing canonical Wnt signaling, it may play a role in regulating Wnt signaling in response to mechanical loading. Consistent with this hypothesis, loading decreased SOST mRNA and sclerostin levels, while unloading increased SOST mRNA expression in vivo (Fig.). Interestingly, reduction of sclerostin staining intensity was most pronounced in areas with the highest strain, indicating a response to local loading conditions. Furthermore, SOST knockout mice do not exhibit bone loss after unloading nor is canonical Wnt signaling altered.

Several systemic and local factors have been suggested as possible regulators of SOST/sclerostin expression by osteocytes. Recombinant human PTH and active fragments of this protein are used in the treatment of osteoporosis . In contrast to the bone resorption-stimulating effect of continuous elevation of endogenous PTH, as is seen in patients with hyperparathyroidism, intermittent increases of PTH provided by daily injections are associated with distinct anabolic effects. The mechanisms by which PTH mediates this bone anabolic effect are not completely understood. Part of it may be mediated via sclerostin as PTH has been shown to inhibit its expression both in vitro and in vivo (Fig.).

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Special Edition on Long Duration Spaceflight In vitro, PTH decreased SOST transcription by osteoblastic and osteocytic cells within 4 h. This was not affected by the protein synthesis inhibitor cyclohexamide but was decreased by the cAMP inducer forskolin .

These observations suggest a direct and cAMP-dependent regulatory effect of PTH on the expression of SOST. Within the 52-kb genomic region deleted in van Buchem disease, an MEF2 response element has been identified that is essential for the PTH-induced downregulation of SOST expression . In vivo, PTH administration resulted in a decrease in SOST mRNA and sclerostin expression in mice and rats . In addition, a constitutively active PTH receptor 1 (caPTHR1) exclusively expressed in osteocytes resulted in increased remodeling with decreased osteoblast apoptosis and suppression of SOST expression . This effect was blunted in mice lacking LRP5, suggesting that the effect of caPTHR1 was mediated by increased Wnt signaling due to suppression of SOST. The importance of SOST regulation by PTH is further supported by the observations that the anabolic effect of PTH is blunted in SOST-deficient mice as well as in mice overexpressing SOST using a constitutive active promoter.

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Two other systemic factors have also been shown to affect SOST/sclerostin expression. 1,25-Dihydroxyvitamin D3 alone or in combination with retinoic acid increased SOST expression in human osteoblastic cells in vitro . The specific effect of glucocorticoids on SOST expression depends on the experimental conditions. In vitro, dexamethasone suppressed SOST expression in osteoblastic cells , while in vivo treatment of mice with prednisolone increased SOST expression in tibiae, suggesting that suppression of Wnt signaling by the upregulation of sclerostin may account for the glucocorticoid-induced suppression in bone formation (Fig.).

BMP2, -4, and -6 are local growth factors shown to stimulate SOST expression in osteoblastic cells in vitro ; and putative BMP responsive elements are present within the SOST promoter region . Decreased BMP signaling due to osteoblast-specific knockout of BMPR1A decreased SOST mRNA and sclerostin protein expression in embryonic mice calvariae and was associated with increased bone mass . In these mice, however, both bone formation and resorption were inhibited. The authors proposed that the decrease in bone formation was independent of sclerostin expression and a direct result of decreased BMP signaling. The decrease in bone resorption, however, may be an effect of increased Wnt signaling due to the decrease in sclerostin expression. This in turn may be due to upregulation of osteoprotegerin in mature osteoblasts by Wnts and, thereby, inhibition of RANKLinduced osteoclastogenesis.

Despite the rapid progress in our understanding of the regulation of the production and function of sclerostin, there are still important questions that need to be addressed in future research. These include the identification of factors that regulate sclerostin/SOST expression and determine its highly restricted expression pattern.

Furthermore, the mechanism by which sclerostin binding to LRP5/6 interferes with canonical Wnt signaling as well as potential additional functions of sclerostin, besides antagonizing canonical Wnt signaling, need to be elucidated. More detailed and structured analysis of bone metabolism in patients with sclerosteosis and van Buchem disease, sclerostin expression in pathological conditions, and a genotype-phenotype characterization of SOST are required to better understand its function and regulation in humans.

Osteocytes and microgravity What are the effects of microgravity (or reduced loading) on osteocytes?

Research preformed by Krempein et al. first demostrated the effects of reduced mechanical loading on osteocyte morphology. Rats were immobilized by spinal cord severing, plaster cast, or nerve dissection. Three weeks of immobilzation caused a significant decrease percentage of small metabolitcally inactive osteocytes (spinal cord severing, -20%, plaster cast, -15.4%) and a corresponding increase percentatge of mature enlarged osteocytes (spinal cord severing, +12.6%, +14.6%). These morphology observations support the theory of immobolization leading to an increased number of mature osteocytes secreting sclerostin, thereby inhibiting bone formation. In addition, an increase percentage of empty lacunae was observed supporting the theory of immobilized induced osteocyte apoptosis as a initiator of osteoclast activity.

Twenty five years after the Krempein study, a Russian "Bion-11" biosatellite launched (2001) carrying two Macaca mullatta monkeys for a fourteen day mission. Examination of iliac crest biopsies of the flight animals showed that mature osteocytes had increased specific volume of the Golgi complex and increased osteolytic activity. However, a definitive correlation between immobilized morphological changes and underlying gene/protein expression changes currently cannot be established as both the Krempein et al. and Russian flights occurred prior to the discovery of osteocyte specific genes over the past several years.

Simulated microgravity inhibits L-type calcium channel currents partially by the up-regulation of miR103 in MC3T3-E1 osteoblasts Simulated microgravity substantially inhibits L-type voltage-sensitive calcium channels (LTCCs) in MC3T3E1 osteoblast-like cells by suppressing Cav1.2 expression. Furthermore, authors demonstrated that the upregulation of miR-103 that is induced by simulated microgravity is involved in the down-regulation of Cav1.2

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Special Edition on Long Duration Spaceflight expression and in the inhibition of LTCCs in MC3T3-E1 cells. Orbital spaceflight has clearly demonstrated that the absence or the reduction of gravity has significant detrimental effects on astronauts.

Health hazards in astronauts are represented by cardiovascular deconditioning and bone loss. Skeletal deconditioning, such as reduced bone mass, altered mineralization patterns and decreased bone matrix gene expression, has been described in astronauts and in rat models of simulated microgravity. The skeletal system impairment that is induced by mechanical unloading, which is one of the main limitations of long-term spaceflight, has received general attention by researchers. LTCCs are involved in the production and release of paracrine/autocrine factors and in changes in gene expression in response to mechanical stimulation.

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Li et al. reported that LTCC inhibition significantly attenuates the bone formation that is associated with mechanical loading in rats and mice. These findings suggest that LTCCs play important roles in the regulation of osteoblast function and bone metabolism. In the study, authors investigated the effects of simulated microgravity on LTCC currents in cultured MC3T3-E1 cells using whole-cell patch clamp recordings. By measuring inward currents, authors found that simulated microgravity significantly reduced LTCC currents. This finding was also confirmed by calcium imaging, which showed that simulated microgravity significantly reduced Bay K8644-induced intracellular calcium increases. These observations are consistent with previous studies. Numerous bone anabolic regulatory factors, including parathyroid hormone , vitamin D3, and mechanical stimuli are able to activate and enhance LTCC currents. Therefore, microgravity, which is a form of mechanical unloading, may reduce LTCC currents in osteoblasts .

Many factors can regulate LTCCs. The major LTCC subunit in osteoblasts is Cav1.2 . Recent studies have shown that amyloid precursor protein (APP) inhibits LTCCs by down-regulating Cav1.2 expression in GABAergic inhibitory neurons. Ronkainen et al. reported that LTCC currents in cardiomyocytes are suppressed by calciumcalmodulin-dependent protein kinase II (CaMKII) through the down-regulation of Cav1.2 expression. Considering the inhibition of LTCC currents in MC3T3-E1 cells under simulated microgravity condition, authors investigated Cav1.2 expression in these cells. The findings showed that simulated microgravity markedly suppresses the expression of Cav1.2 in MC3T3-E1 cells. Then, authors examined these currents following the knockdown of Cav1.2 expression to confirm that the reduction of Cav1.2 was involved in the alteration of LTCC currents in MC3T3-E1 cells. The results demonstrated that the down-regulation of Cav1.2 expression notably reduces LTCC currents in MC3T3-E1 cells. These data suggested that the decreased activity of LTCCs in MC3T3-E1 cells under simulated microgravity condition could be attributed to a decreased amount of Cav1.2 channel proteins.

In addition to the APP and CaMKII studies mentioned above, other reports have investigating the regulation of the Cav1.2 channel protein. For example, selenium deficiency increases oxidative stress levels in the mouse myocardium, which is positively related to the up-regulation of Cav1.2 genes and proteins.

Wang et al. demonstrated that Cav1.2 mRNA and protein levels increase in ROS cells following a 24-h incubation with a permeable analog of cAMP. These experiments suggested that changes in Cav1.2 expression that are induced by different factors coincide with altered Cav1.2 mRNA expression. However, the findings indicated that increased Cav1.2 mRNA expression is not consistent with decreased Cav1.2 protein expression in MC3T3-E1 cells under simulated microgravity conditions. Therefore, this result suggested that a mechanism of posttranscriptional regulation might participate in regulating Cav1.2 protein expression.

In conclusion, simulated microgravity inhibits LTCCs in MC3T3-E1 cells via the suppression of Cav1.2 expression. Moreover, the down-regulation of Cav1.2 expression and the inhibition of LTCCs are partially related to the up-regulation of miR-103 induced by simulated microgravity.

SOLO - SOdium LOading in Microgravity (Sodium retention in microgravity) SOdium LOading in Microgravity (SOLO - later renamed: Sodium retention in microgravity) is a continuation of extensive research into the mechanisms of fluid and salt retention in the body during bed rest and spaceflights.

It is a metabolically-controlled study, conducted during long-term space missions. Astronauts participated in two study phases, 5 days each. The subjects follow a diet of constant, either low or normal, sodium intake, fairly high fluid consumption and isocaloric nutrition.

The hypothesis of an increased urine flow as the main cause for body mass decrease has been questioned in several recently flown missions. The idea had been that in microgravity blood is redistributed from the caudal to the cephalic parts of the body so that the heart is distended. This distension would stimulate volume and pressure receptors, which in turn augment the renal output of fluid and electrolytes through neuroendocrine pathways. In contrast to this, data from several missions (MIR 92, D-2, Euromir 94, MIR 97) as well as data from the American SLS1/2 mission, show that urine flow during the first days in space is most likely unchanged.

Concomitantly to our studies, Norsk et al. performed repeatedly oral water load tests to examine urine output

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Special Edition on Long Duration Spaceflight after this stimulus. Surprisingly, urine output following the water load was always low in space compared to the urine volume following the water load in supine position.

STEM Today, May 2016, No.8

Hormonal data from several missions meanwhile show consistent results. The cardiac member of the natriuretic peptide family, atrial natriuretic peptide (ANP) is reduced after some days in space thus indicating that intravascular volume and pressure compared to Earth are reduced. The renin-aldosterone system is activated in space as shown in several missions (MIR 92, D-2, MIR 97). However, one may argue that this hormone pattern - reduced ANP concentration and activated rennin-aldosterone system - may be caused by variations in sodium intake. This was taken into account during the MIR 97 mission. In this mission we kept sodium intake of the examined astronaut constant during 15 days in space as well as during the baseline data collection periods. Surprisingly, in this astronaut the metabolic sodium balances (intake minus urinary output) were positive summing up to sodium storage of 750 mEq after 15 days. This amount of retained sodium should have led to fluid retention of about 5 liters which we did not see in the MIR 97 mission. As mentioned above the metabolic sodium balances were calculated from sodium intake minus urinary output. Sodium may also have been excreted via skin by sweating or via feces. But, the skin water loss during the mission was less than during the baseline data collection. Fecal sodium excretion does not need to be considered because: 1. fecal sodium excretion is very low and 2. does not change with increasing sodium intake.

It has also be shown several times in rats and humans that increased sodium intake is paralleled by increased urinary calcium losses and extracellular pH decreases. Calcium is the main mineral in bone. Astronauts loose calcium when they are in space most likely because of immobilization of the weight-bearing bones. Concomitantly, bone density decreases during microgravity. Since normal sodium intake on Earth leads to a balanced situation while under identical conditions in space sodium is retained, the question remains if already an average sodium intake in space exacerbates the existing increased calcium excretion. If this holds true bone density decrease in microgravity may exacerbate due to sodium retention and thereby further weaken bone.

The main questions in this experiment to be asked are: - What is the effect of the stored sodium?

- Does this have an impact and if which on health of astronauts or patients?

Therefore, another goal of the experiment is to evaluate the operationally adequate sodium intake for long-term missions without any negative effect on the astronautst’ health.

The second aim of this proposal is to examine calcium excretion and bone resorption markers following a normal and low sodium intake in space and on Earth.

PRELIMINARY RESULTS Salt intake was investigated in a series of studies, in ground-based simulations and in space, and it was found that not only is sodium retained (probably in the skin), but it also binds to certain sugar-protein molecules and affects the acid balance of the body and bone metabolism. High salt intake increases acidity in the body, which can accelerate bone loss. ESA’s recent SOdium LOad in microgravity experiment, or SOLO, focused on this question.

Nine crew members, during their long-duration flights in 2010 and 2011, followed low- and high-salt diets. The expected results may show that additional negative effects can be avoided either by reducing sodium intake or by using a simple alkalizing agent like bicarbonate to counter the acid imbalance.

Preliminary results show that high sodium chloride intake leads to higher calcium excretion, which may lead to bone loss in the long run. As expected bone formation is not different between the two salt regimes.

Markers of bone resorption show high individual differences and need to be further investigated in combination with sodium retention patterns.

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Bisphosphonates as a Countermeasure to Spaceflight-Induced Bone Loss Bisphosphonate is a therapeutic agent that has been used to treat osteoporosis patients for more than a decade, with a proven efficacy to increase bone mass and decrease the occurrence of bone fracture. Through 90-day bed rest research on Earth, researchers confirmed that this agent has a preventive effect on the loss of bone mass. Based on these results as well as studies conducted by others, JAXA and NASA decided to collaborate on a space biomedical experiment to prevent bone loss during space flight. Dr. Leblanc, USRA, and Dr. Matsumoto, Tokushima University, are the two principal investigators of this study.

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Data reported from the seven astronauts (six males and one female; mean age, 49.6Âą4.1 year) who completed the pre- and in-flight protocol without incident. Six of the seven took 70 mg/week (Fosamax) starting 3 weeks before flight and continuing throughout the flight. The seventh crewmember experienced a launch delay of 4 weeks after starting the preflight treatments, and therefore took seven weekly preflight doses instead of the nominal three doses. The mission lengths varied between 4.5 and 6.2 months with a mean mission length of 5.5 months. Subjects were instructed to take the pill (Fosamax, 70 mg) after an overnight fast with 236 ml of water and to refrain from eating or drinking for 30 min after ingestion.

Results Figure 1 presents the mean DXA changes for the three groups of subjects. Results are graphed as percentage change from preflight for the femur neck, trochanter, total hip, pelvis and lumbar spine. Our analyses revealed significant group by pre/post-interaction effects for the bisphosphonate group vs. pre-ARED group in the femoral neck (p<0.001), trochanter (p<0.001), total hip (p<0.001), pelvis (p<0.005), and lumbar spine (p<0.001).

The models revealed significant group by pre/post-interaction effect comparing the bisphosphonate group vs.

ARED alone in the total hip (p<0.02) and lumbar spine (p<0.01), with weaker effects trending in the same direction but not reaching statistical significance in the pelvis (p=0.15) and trochanter (p=0.16). A-priori contrasts indicate that pre-ARED users lost significant bone density in all regions as a result of spaceflight (all sites p<0.001), while the bisphosphonate treatment group showed no significant changes during flight. The ARED alone group showed postflight decreases in the total hip (p<0.001) and femoral neck (p<0.01) with trochanter trending in the same direction but not reaching statistical significance (p<0.08). Overall these results suggest that the ARED device improved bone balance compared with the pre-ARED group while statistical analysis did not demonstrate a BMD loss in the group that received bisphosphonate treatment plus ARED.

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Table 1 gives the body composition for all 3 groups showing an overall decrease in whole body total mass (p<0.01) and fat (p<0.001) after flight (there were no interaction effects or group differences). Analysis of lean body mass (LBM) revealed no main effects or group interactions.

Table 2 shows the femur neck, trochanter, and total hip QCT trabecular, cortical and integral BMD and BMC, and the nonlinear fall and standing strength data for the bisphosphonate treatment group and the 18 pre-ARED controls. The analyses of the trabecular bone in all three subregions revealed significant group (pre-ARED, bisphosphonate) by pre/post-interaction effects for both BMD and BMC (p<0.05). A-priori contrasts showed significant pre/post-losses of trabecular and cortical bone in all three subregions (p<0.001) for BMD and BMC in the pre-ARED group but not in the bisphosphonate group.

Authors did not find significant group interaction effect in the BMD cortical bone compartments (p=0.07-0.21), while the BMC cortical compartments revealed significant group (pre-ARED vs. bisphosphonate) by pre/postinteraction effects (p<0.05) in all three regions. QCT integral BMD and BMC analyses revealed significant treatment benefits in all three regions (BMD, p<0.001, for all three bone sites; BMC, p<0.05 for all three bone sites). Similarly, the femoral strength indices show clear treatment benefits for both the standing and fall strength (p<0.001).

Table 3 gives the urine and blood data before, during and after flight for the bisphosphonate treatment group.

These data show that compared with preflight, there were no significant in-flight changes in urinary Ca, although the data did suggest an initial decline from preflight to early-flight that was not statistically significant (p=0.10). Compared with preflight, NTX levels did not show significant changes at the early, mid- or late-flight observations; however, the data trended downwards throughout the course of the study, with differences at R+0

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Special Edition on Long Duration Spaceflight from preflight nearing significance (p=0.07), and subsequent R+14 and R+30 observations being significantly lower than preflight (p<0.05 and 0.01, respectively). Similarly, there were no changes in the resorption marker serum CTX-β during flight with decreases at R+0 and R+14 (p<0.001) returning to baseline levels by R+30 (p>0.60). Pairwise comparisons of preflight BSAP levels to all in- and post-flight observations were nonsignificant (p>0.12). Total serum Ca levels decreased at the early-flight time point (p<0.01); however, subsequent in- and post-flight comparisons to preflight were not significant.

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Compared with preflight, ionized Ca was decreased at R+14 and R+30 but not at R+0. Serum 25(OH) vitamin D levels declined during and after flight; however, all values remained within normal limits (>50 nmol/l). In contrast, the 1, 25-dihydroxyvitamin D levels were unchanged during flight compared with baseline. There was a decrease on R+0 (p<0.03) but not at R+14 or R+30. Authors observed no significant differences in osteocalcin levels during flight relative to preflight, although R+0 data were significantly decreased (p<.01). Intact PTH did not show changes from preflight. All pre- and postlight ultrasound scans were negative for renal stones (data not presented).

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RESULTS Loss of BMD has been a consistent finding after longduration spaceflight, in spite of the exercise regimens on board Mir and early ISS missions. The DXA, QCT, and bone marker results of this study show that a bisphosphonate in combination with newer exercise prescriptions will reduce the loss of bone from multiple sites, reduce compartmental loss in trabecular and cortical bone, maintain hip strength, and prevent spaceflight increases in bone resorption and elevated levels of urinary Ca. The latter will help reduce the risk of kidney stone formation during and possibly after spaceflight. Preservation of bone during spaceflight may protect bone from the persistent geometrical and trabecular bone mass changes observed 2 to 4.5 years following 6 months on board the ISS.

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The DXA data presented in this research suggest that increased resistance exercise using ARED has a significant mitigating effect on BMD loss in microgravity compared with pre-ARED crewmembers using the iRED or other devices. The ARED provides significantly increased loading capability with ease of use, promoting increased exercise effectiveness compared with the previous devices. The peak concentric resistance is 2,668 N compared with 1,334 N for the iRED device. Secondly, the eccentric-concentric ratio is about 90 % compared with 70 % for the iRED. Also, in contrast to the iRED which generated forces that increased with excursion when the device was set at the higher loads necessary for lower extremity exercise, the ARED generates forces that vary with acceleration of the exercise movement when the flywheels are engaged. Inertial flywheels were incorporated to simulate free weight exercise. However, this mitigating effect on DXA BMD is accompanied by elevated urinary Ca excretion (≈50 % during flight) and increased resorption markers (e.g., urinary NTX at ≈100-200 % above preflight) with a nonsignificant trend for increased bone formation markers suggesting elevated and possibly uncoupled bone remodeling.

Large number of minor back injuries that have been experienced in the course of using exercise equipment on ISS; 12 of 14 musculoskeletal injuries occurring on the ISS were attributable to use of aerobic and/or resistance exercise equipment.

A significant disadvantage of oral treatment with bisphosphonate is the potential for GI intolerance either before or during flight as shown in two of the ten crewmembers participating in this experiment. A single zoledronic acid infusion given preflight would suffice for at least a 6-month flight and would have many advantages over a weekly oral prescription. However, the risk of other adverse events, such as osteonecrosis of the jaw, is less for oral treatment.

FDA: Possible increased risk of thigh bone fracture with bisphosphonates The U.S. Food and Drug Administration warned patients and health care providers about the possible risk of atypical thigh bone (femoral) fracture in patients who take bisphosphonates, a class of drugs used to prevent and treat osteoporosis. A labeling change and Medication Guide will reflect this risk.

Bisphosphonates inhibit the loss of bone mass in people with osteoporosis. Bisphosphonates have been shown to reduce the rate of osteoporotic fractures – fractures that can result in pain, hospitalization, and surgery– in people with osteoporosis. While it is not clear whether bisphosphonates are the cause, atypical femur fractures, a rare but serious type of thigh bone fracture, have been predominantly reported in patients taking bisphosphonates. The optimal duration of bisphosphonate use for osteoporosis is unknown, and the FDA is highlighting this uncertainty because these fractures may be related to use of bisphosphonates for longer than five years.

The labeling changes and Medication Guide will affect only those bisphosphonates approved for osteoporosis, including oral bisphosphonates such as Fosamax, Fosamax Plus D, Actonel, Actonel with Calcium, Boniva, Atelvia, and their generic products, as well as injectable bisphosphonates such as Reclast and Boniva.

Zoledronic acid is the most potent third-generation aminobisphosphonate. It is able to decrease bone resorption quickly and, consequently, bone formation markers, due to the normal coupling of bone resorption to formation.

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Melatonin and Bone Remolding Bone is a dynamic tissue undergoing remodeling throughout life, and this remodeling requires a balance between deposition of new bone by osteoblasts and resorption of old bone by osteoclasts . Bone modeling requires the interaction between multiple bone cells (osteoblasts/osteoclasts/osteocytes) to renew, maintain, or adjust bone strength and/or mineral homeostasis in response to changing environmental influences . There are four distinct phases to this process: activation, resorption, reversal, and formation with resorption and formation taking place via osteoclasts and osteoblasts, respectively . Initially, osteoclast precursors are attracted to a particular area of bone surface, then differentiation into osteoclast, which is responsible to bone resorption by acidification and proteolytic digestion. In the reversal phase, bone resorption transitions into bone formation, and osteoblast precursors are recruited to proliferate and differentiate into osteoblasts that invade the resorption area and begin to form new bone by secreting osteoid, which is eventually mineralized .

Bone remolding processes are mediated by hormones, cytokines, growth factors and other molecules . One of the hormones modulating bone formation and resorption is melatonin. It is hypothesized that melatonin, perhaps through three principle actions, modulates bone metabolism.

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Firstly, melatonin directly affects the actions of osteoblast and osteoclast. Numerous studies documented that melatonin increases pre-osteoblast/osteoblast/osteoblast-like cell proliferation, promotes the expression of type I collagen and bone marker proteins (e.g., alkaline phosphatase, osteopontin, bone sialoprotein and osteocalcin), and stimulates the formation of a mineralized matrix in these cells . Besides, melatonin inhibits the differentiation of osteoclasts via decreases in the expression of RANK mRNA and increases in both the mRNA and protein levels of osteo-protegerin.

Secondly, melatonin indirectly regulates bone metabolism through the interaction with systemic hormones (e.g., PTH, calcitonin, and estrogen) or other moleculars. Ladizesky et al. revealed that estradiol treatment could prolong the effect of melatonin to augment bone remodeling in ovariectomized rats; it indicates that appropriate circulating estradiol levels might be needed for melatonin effects on bone.

Thirdly, osteoclasts generate high levels of superoxide anions during bone resorption that contribute to the degradative process. Melatonin is a significant free-radical scavenger and antioxidant. It can clear up the free radicals generated by osteoclast during the bone resorption process and protect bone cells from oxidative attacks.

Melatonin and Bone Repair Bone fracture and bone defect are the common bone disease which originate from trauma, neoplasm invasiveness, surgery, or as a secondary effect from some bone diseases. The repair of bone fracture and bone defect is an important process to maintain the integrity and function of the bone.

Bone repair is a complex and continuous process. Biologically, it takes place in three stages: inflammatory, proliferative and remodeling phases . During these stages, a set of complex biochemical events take place, including inflammatory cell infiltration, angiogenesis, cell proliferation and differentiation, collagen deposition, granulation tissue formation and mineral matrix deposition, etc. . Previous studies have indicated that melatonin may play an important role in the bone-healing process due to its antioxidant properties, regulation of bone cells, and promotion of angiogenesis actions . In the early stage of bone fracture/defect healing, the inflammatory phase is characterized by clot formation, ischemia and reperfusion injury and inflammatory cells (leukocytes, macrophage and mast cells) infiltration . During this period, neutrophils produce free oxygen radicals that initiate a chain reaction leading to cell membrane damage via lipid peroxidation . It has a negative effect on fracture/defect healing. The pineal hormone melatonin is a significant free radical scavenger and antioxidant at both physiological and pharmacological concentrations.

Halici et al. carried out biochemical and histopathologic observation of the outcomes of intraperitoneal applications of melatonin (30 mg/kg/day) for accelerating bone fracture healing in a rat model. The authors found that malondialdehyde (MDA) levels (indicator of free-radical concentration), superoxide dismutase (SOD) activity and myeloperoxidase ((MPO) plays a fundamental role in oxidant production) in the melatonin group decreased at the early stage of fracture healing compare to the control group, and SOD activity returned to the first-day value after 28 days in the melatonin group . These findings indicate that the administration of melatonin maybe beneficial in suppressing the effects of free oxygen radicals and regulating antioxidant enzyme (SOD) activity, thereby accelerating bone formation in the fracture-healing process.

In the proliferative phase, angiogenesis, osteoblast and fibroblast differentiation, collagen deposition and for-

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Special Edition on Long Duration Spaceflight mation of granulation tissue take place in this phase. Melatonin promotes the osteoblast proliferation and differentiation and enhances the type I collagen deposition . Additionally, a recent study revealed that melatonin promotes angiogenesis during repair of bone defect in rabbit . They observed the commencement of neovascularization and a significant increase in the number of vessels in the melatonin group in the first two weeks, which were also accompanied by an increase in the length of cortical formation. A similar outcome was also found by Soybir et al. who reported an increase in the number of blood vessels resulting from melatonin applications to wounds in rats. Angiogenesis is an important physiological process in bone wound healing. Yamada et al. suggest that angiogenesis precedes osteogenesis. The regeneration of new bone was dependent on blood vessels for the supply of mineral elements and the migration of angiogenic and osteogenic cells into secluded spaces.

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Melatonin and Osteoporosis Osteoporosis was defined as "a systemic skeletal disease characterised by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture" by the World Health Organization. Recent therapies include targeting bone-resorbing osteoclasts by use of bisphosphonates, estrogen, selective estrogen receptor modulators (SERM) and calcitonin to prevent further bone breakdown, and stimulating bone-forming osteoblasts by anabolic drugs (e.g., teriparatide) to increase bone mass . However, these therapies are limited because of their negative side effects or high costs. In this sense, melatonin as a complementary therapy for the prevention and treatment of osteoporosis should be considered, because of its modulating bone metabolism function, lack of side effects, and economical advantages.

Some studies revealed the possible etiologic role of melatonin in osteoporosis. Nocturnal plasma melatonin levels decline with age. It has also been reported that melatonin secretion decreases sharply during menopause, which is associated with post-menopausal osteoporosis . A correlation between decreased plasma melatonin levels and an increased incidence of bone deterioration as seen in post-menopausal women has been examined . Furthermore, Ostrowska et al. found that a pinealectomy in rats promotes the induction of bone metabolism biomarkers. In addition, Feskanich et al. reported that twenty or more years of nightshift work significantly increased the risk of wrist and hip fractures in post-menopausal women. Nightshift work leads to disturbances of melatonin secretion as well as severe circadian rhythm disruption. These observations taken together suggest that melatonin may be involved in the pathogenesis of osteoporosis.

At present, few clinical trials have focused on the possible therapeutic value of melatonin in the prevention/treatment of osteoporosis. Most experimental studies were performed in ovariectomized rats, as a model for menopause. Uslu et al. described that melatonin treatment increased trabecular thickness and the trabecular area of vertebra and femur and cortical thickness of femur, which decreased after ovariectomy in rats. Another similar study reported that melatonin significantly reduced the number of apoptotic cells in nucleus pulposus and epiphyseal cartilage of the spinal column and the expression of inducible nitric oxide synthase (iNOS), which increased after ovariectomy . iNOS plays a pivotal role in the pathogenesis of osteoporosis. It generates nitric oxide, a free radical contributing to the imbalance between bone resorption and formation caused by estrogen depletion .

Recently, a randomized, double-blind, placebo-controlled clinical trial was carried out by Kotlarczyk et al.. In this study, 18 perimenopausal women (ages 45-54) were randomized to receive melatonin (3 mg, per. os. n = 13) or placebo (n = 5) nightly for six months. The results showed no significant change in bone density, Type-I collagen cross-linked N-telopeptide (NTX), or osteocalcin (OC) between groups; however, the ratio of NTX:OC trended downward over time toward a ratio of 1:1 in the melatonin group, while the trend was not seen in the placebo group. NTX and OC are the bone turnover markers: NTX for bone resorption, and OC for bone formation. The ratio of NTX:OC trending downward to 1:1 in the melatonin group indicates that melatonin supplementation may restore imbalances in bone remodeling to prevent bone loss in perimenopausal women.

From these studies, we found that melatonin application markedly influenced the bone microenviroment and bone metabolism after ovariectomy or menopausal, suggesting its potential use in the prevention/treatment of osteoporosis.

Effects of Sex and Gender on Adaptation to Space: Bone Sex differences in bone mineral density (BMD) are well documented; since bone mass scales to body mass, men on average have a larger skeletal mass. There is little evidence, however, on whether there is a sex difference in rates of bone loss with unloading or in the rate or magnitude of recovery therefrom. While the effects of bed rest on BMD and/or bone metabolism have been examined separately in men and in women, there have been no human studies that have been statistically powered to make direct sex comparisons. In one 17-week bed rest study that included both men (n = 13) and women (n = 5) at one of 10 sites measured (calcaneus), bone loss was markedly less in women than men. However, the dual energy x-ray absorptiometry-assessed total hip BMD

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Special Edition on Long Duration Spaceflight for women in a 60-day bed rest study revealed a substantial loss at that site, whereas men in a similar study did not have a decrease in total hip bone mineral content.

Volumetric BMD and bone geometry of tibial cancellous and cortical compartments have been evaluated after prolonged bed rest using peripheral quantitative computed tomography (pQCT) in men and high-resolution pQCT in women. The small gender differences observed in the bone loss rates at those tibial sites are within the reported precision for these pQCT variables. Side-by-side investigations using rodent hindlimb unloading (a commonly used surrogate for microgravity) reveal greater cancellous bone loss in skeletally mature female mice18 and a distinct effect of starting values (mice with greater bone volume at the start lost less bone). However, in mature rats few differences between genders are apparent.

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There is little definitive evidence showing sex-specific differences in the rate of bone loss. Certainly, some of the individual differences may be related to sex-specific hormonal factors. As is the case with muscle, the individual variability within gender in response to unloading is large and should be better understood.

Sex-based differences have been identified in the incidence of osteoarthritis (OA), with OA of the knee, in particular, significantly more common in women. Sex-based risk factors explaining this include the loss of esË? such trogenâ&#x20AC;&#x2122;s anabolic effect on cartilage after menopause, a higher incidence of predisposing knee injuriesU as anterior cruciate ligament tears-in women, and increased joint laxity. There is clear evidence from animal studies that regular mechanical loading is essential to cartilage health. In humans, 6 or more weeks of nonweight-bearing can produce changes in magnetic resonance imaging images of knee cartilage that resemble OA. However, sex-based differences in the response to joint unloading have not been elucidated. Because articular cartilage health is impacted by the quality of the underlying bone as well as the strength of muscles around the joint, assessment of the potential risk for articular cartilage injury imposed by unloading needs to include evaluation of all three tissues: bone, muscle, and cartilage. There is some evidence to suggest that osteopenia of subchondral bone underlying articular cartilage contributes to cartilage degeneration. Conversely, damaged cartilage releases receptor activator of nuclear factor kappa-B ligand (RANKL) and other inflammatory components, which can lead to the loss of adjacent bone. Since muscles serve to stabilize and dampen forces across joints, loss of muscle mass and strength after a prolonged unloading can contribute to joint injury risk and early degenerative joint changes, especially in the knee. However, sex-based differences in the relative impact of bone and muscle loss on joint health have not been defined. Specific interventions to increase loadbearing or strengthening activities in space will be indicated. They may also identify the need for progressive strengthening and joint loading upon arrival on a planetary surface after extended microgravity exposure, after return from space or after prolonged period of non-weight-bearing on Earth.

Musculoskeletal injuries have been reported in-flight at a rate of 0.021 per flight day for men and 0.015 per flight day for women; hand injuries are the most common, with abrasions and small lacerations the most common manifestations. There are few data on the recovery of the musculoskeletal system following spaceflight and even less data on sex differences in recovery rates. Generally, international space station crew have substantial recovery ofmuscle strengthwithin a month following flight. The time course of recovery of bone mineral density has been evaluated but not specifically for sex differences. In general, half-lives for recovery of bone mineral density are â&#x2030;&#x2C6;150-200 days depending on site.

Calcium Metabolism and Its Regulation in Cosmonauts during 30 to 438 days Mission The studies were performed before and after SF of 30 to 438 day duration on the Russian orbital stations. In total, 44 cosmonauts were examined, 18 of them participated twice in long-term space expeditions (altogether, 62 pre- and postflight determinations were carded out, and seven were carried out during SF at different terms after the start).

Samples of blood and 24-h urine were taken. In all cases, blood samples were analyzed 45 to 60 days before SF and also on the 1st, 6 to 7th, and 14th days after landing. The urine was collected five to seven days before the start and also for several days after landing (the "0"-day). Moreover, calcium kinetics (CK) was studied using stable isotopes in three cosmonauts before, during, and after a 115-day SE . The experiment procedure was approved by the Biomedical Ethics Commission. The cyclogram and the CK determination were described in detail . The whole blood level of Ca++ was analyzed directly during SF with ion-selective electrodes (i-STAT Corporation, Princeton,NJ).

Results Pre- and posflight findings It was shown that the total calcium content in the blood serum was increased after SF of a medium (3 to 6 months) and long (6 to 14 months) duration mainly at the expense of the ionized fraction. Thus, on the first

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postflight day, the ionized calcium level was increased by 15.3 ±1.5%, compared to its individual background value, whereas the total calcium level was increased significantly less, by 4.3 ± 1.3%. However, in many cases the blood potassium content was decreased. These changes in the blood ionogram were significant on the first day after the landing, and only in some cases were they recorded on the sixth to seventh day.

The kidney is an effector organ of the mineralotropic hormone (MTH) system and influences the organism’s mineral metabolism by changing ion reabsorption and excretion. On the first day after long-term SF, renal excretion of electrolytes was characterized by the increased urinary excretion of calcium along with its significantly increased urine concentration. A tendency for hyperkaliuria was found a little later, on the second day after the landing.

To analyze the mechanism of development and maintenance of changes in calcium metabolism, the regulatory systems were studied. Concentrations of hormones involved in the system regulation of calcium metabolism were analyzed by the matched-pair comparison in percent to the individual preflight levels, and it was found that the blood content of the c-PTH was significantly increased and that of CT decreased on the first day after the long-term exposure to microgravitation. On the seventh day, the average group value of c-PTH was unchanged but scattering of individual results was significantly increased. Thus, the individual postfiight values of hormonal regulation parameters varied markedly and often not unidirectionally even in members of the same

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crew. Nevertheless, on the first day after the long-term SF, the MTH system had manifestations of tension that became less pronounced by the seventh day. In addition to changes in the MTH parameters, the level of gastrin was increased on the first postflight day, which was normalized by the seventh day. It was suggested to be due to the activated secretion of gastrointestinal hormones in response to the compensatory increased absorption of alimentary calcium. The blood level of cortisol was also increased in the postflight period, especially notable on the first day, and by the seventh day it decreased. These changes could be, probably, induced by factors of the final stage of SF. Studies of the post flight dynamics of osteocalcin, which is a marker peptide of the bone de novo formation , provided important information. The content of osteocalcin was increased in all test subjects and continued to increase up to 180% of the preflight level on the 15th postflight day, and this seems to be associated with the stimulation of osteogenesis.

Studies during SF The level of ionized calcium was significantly decreased on the 14th day of flight in two of three subjects and the total calcium level was also decreased. On the fourth month of flight (on the 93rd and 110th days), the level of Ca++ was increased in all cosmonauts. However, the ratio of ionized and total calcium was always increased compared to its preflight value. The levels of renal excretion of electrolytes and fluid were determined during certain "metabolic" days of every flight session along with the recording of consumption of food products and drinks (Table 3). Table 3 presents only those data on the consumption which were recorded concurrently with collection of blood samples in the crew members.

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In two of three examined subjects, the fluid excretion was lower than the mean preflight level (Table 2) (this parameter was calculated as the arithmetic mean of findings during 15 preflight "metabolic" days) and the classical pattern of increased fluid excretion during early adaptation with subsequent stabilization on the new level was observed only in one case (no. 1). Paradoxically, the increased fluid excretion in the third cosmonaut was recorded from the third month of SF with a tendency for an increase in this parameter in the early period.

However, in all urine samples collected during SF, the content of calcium was increased, and in two of three subjects this increase grew as the flight duration increased and this always occurred due to an increase in calcium concentration in the urine and not due to an increase in the volume of the excreted fluid that was changed either oppositely or less markedly.

Except separate determinations, the rate of potassium excretion was decreased in the third cosmonaut. It seems

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that the specific features of fluid and ion excretion were somewhat associated with changes in their consumption (Table 3) and really, two cosmonauts (nos. 1 and 2) had a sharply decreased consumption of fluid, sodium, potassium, and calcium during SF, whereas in the third cosmonaut it was virtually the same as before the flight, and this seems to partially explain the absence of changes in fluid excretion during early adaptation and its subsequent increase. It seems that just the increased sodium consumption, compared to the preflight level, was one of the causes of increased excretion (in the third subject) of this electrolyte on the 93rd, 94th, and 110th day of SF However, a comparison of calcium consumption and excretion indicated that its urinary excretion was always (except the 14th day in the first subject) increased by 10-70% compared to the preflight one. Note that the same effect was observed when consumption of the mineral exceeded that typical of the ground conditions.

According to the data presented in Tables 2 and 3, the partial calcium balance during SF was negative (except the 94th day in the third cosmonaut) due to both the decreased consumption and increased urinary excretion.

But this is only a preliminary conclusion because gastrointestinal excretion of the mineral, which was not taken into account, significantly contributed to the calcium balance.

The contents of mineralotropic hormones were analyzed in the blood samples taken during SF (Table 4), and, except for samples taken on the 14th and 76th days, the c-PTH level was decreased (the values with 100 Âą 5% difference of the background ones were considered as the changed ones). Individual dynamics of the c-PTH content was studied in three cosmonauts, and this parameter was found to decrease in one and to increase in two

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of them as the duration of weightlessness increased. The calcitonin concentration decreased in four, increased in three, and remained unchanged in four samples. No individual dynamics was found.

Data on the individual dynamics of MTH response during SF were scarce; therefore, it was impossible to make reliable conclusions about the specific features of changes in secretion of mineral metabolism hormones as the SF duration increased. However, the same (and not the opposite) direction of changes in the PTH and CT dynamics in two subjects indicated that the groundspecific response was changed during SF. It was already noted that the total calcium content during SF was decreased in only two cases and no changes were recorded in most samples. On the contrary, the ionized calcium concentration tended to increase, and the ionization index (the ratio Ca++ /CaT was increased with the pH parameter being maintained at the background level. Such a shift of the homeostatic constant, Ca++ , under ground conditions would have occurred along with suppressed PTH secretion and with CT stimulated secretion. But such changes were recorded in only nine of eleven and in four of eleven blood samples taken during SF (Table 4). Thus, it was suggested that adaptation of the regulatory system to changes found in the homeostasis parameters occurred not only on the level of the MTH basal secretion.

Specific changes were found during and after the three-month SF in the contents of vitamin D3 (Table 5).

The contents of both the transport and hormonal forms of vitamin D3 were decreased in most blood samples collected before and during SF. At the early stage of microgravitation influence (on the 14th day of SF), synthesis of the transport form of vitamin D3 (and of the basic stored form) was suppressed; as a result, production of the hormonal form of this vitamin in the kidneys decreased. This suppression intensified after three months of SF but was not accompanied by an adequate decrease in the level of dihydroxycholecalciferol.

Hydroxycholecalciferol production was also suppressed in the beginning of the postflight recovery period, but the ratio of the vitamin forms established during the flight was not changed, i.e., a more pronounced decrease in the production of the transport form synthesis of the hormonal form was less suppressed. Moreover, later the postflight synthesis of 1.25 (OH)2 D3 was even increased compared to its preflight condition, although the rate of production of the transport form was slightly increased or this form maintained a decreased level (in subject no. 1 on the 74th day and in subject no. 3 on the 9th day). These findings seem to show changes in susceptibility of 1.25 (OH)2 D3 synthesis to the substrate provision during SF. In addition to determination of the c-PTH level, the content of the middle ram-fragment of the polypeptide (mm-PTH), as of the hormone secretion marker, was determined in the blood of ten cosmonauts. The individual mm-PTH background levels varied during preflight examination carded out in terms remote and close to SF. The blood level of this fragment varied in the range of 79 to 260 pg/ml. On the first postflight day the mm-PTH content increased by l0 to 46% compared to the individual background values in half of the subjects, was virtually unchanged in four cosmonauts, and decreased in one. The statistical treatment revealed that at the average 107% increase in the mm-PTH concentration on the first postflight day this parameter continued to increase up to 125% of individual background values by the fourteenth day. Pronounced positive dynamics was found in eight of ten cosmonauts.

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The mm-PTH level in the blood samples taken from two cosmonauts on the 76th and in two others on the 97th day of SF had differently directed changes: the decrease had a different degree (10 to 17% in two subjects to 92% in the third one); in one subject the level of mm-PTH was increased by 15.7%. Study of the c-PTH/mm-PTH ratio in the blood of cosmonauts (Table 6) showed that secretion and metabolism of this mineral metabolism regulator was assessed more reliably. The c-PTH/mmPTH ratio varied in the range of 5 to 29% in the background samples and was unstable even in the same subject. During SF the ratio of molar concentrations of c-PTH and the mmPTH was not changed monophasically. Thus, on the 76th day of SF, its increase varied compared to the corresponding individual preflight values. But on the 96th day of SF, the cPTH and mm-PTH levels underwent a twofold decrease compared to the background values.

During the postflight recovery, an increase in the cPTH concentration compared to the preflight value was significantly greater than the increase in the mmPTH content; therefore, the ratio of the c-PTH and mm-PTH fragments increased. Thus, on the first day after landing, the ratio of these fragments was increased in five of ten subjects, and the positive postflight dynamics with a progressive increase in this parameter was found in eight subjects. Note that such changes in the fragment ratio indicated that the mm-PTH fraction decreased in the total content of PTH determined by the radioimmunoassay technique. Consequently, after SF, a relatively greater fraction of the PTH molecules in the circulation was inactive compared to the preflight period. The above-described findings showed that this effect was caused by specific and/or nonspecific changes in parathormone metabolism but not by its decreased secretion under the influence of space flight factors.

Evaluation of the parameters of calcium kinetics and absorption during SF It has long been suggested that a decrease in the intestinal absorption of calcium plays a certain role in the development and maintenance of negative calcium balance during SF . At present, studies performed with the participation of three cosmonauts during the 115-day expedition on the Mir orbital station provide the most complete report about the kinetics and absorption of calcium.

Findings with two stable isotopes (Table 7) indicated that, despite individual variations in the calculated parameters of calcium kinetics, its intestinal absorption and gastrointestinal excretion were decreased during SF along with its increased urinary excretion. In the early postflight period, intestinal absorption was, on average, lower than during SF and its excretion through the intestine was increased. It is difficult to explain the data on intestinal excretion of calcium because changes in this parameter were caused by at least three main factors: changes in the portion of alimentary calcium consumption (i.e, in the exogenous calcium "entrance" into the organism), changes in the portion of alimentary calcium absorption, and changes in its endogenous excretion.

Calcium consumption studied during SF in the "metabolic" days was decreased except one day in subject no. 3, i.e., concurrently with studies of its kinetics and absorption. Note that along with the decreased calcium consumption its intestinal absorption was also significantly decreased.

It is known that a certain amount of calcium entering into the gastrointestinal tract with bile acids and digestive juices is produced not only due to its absorption but also due to its presence in the circulating blood. Figures 1 and 2 show that excretion of calcium with feces (in %), considering its endogenous excretion, decreased mainly due to its more decreased consumption as compared to its decreased absorption. Calcium absorption decreased by 40%, on average, during SF and immediately after landing. This parameter returned to its preflight value only two to three months after SF. The urinary excretion of calcium was significantly decreased in two of the three subjects by the 110th day

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of SF (Table 1 ). In the recovery period, the rate of calcium excretion decreased rather rapidly to its preflight level. Thus, contributors to the negative calcium balance on the organismâ&#x20AC;&#x2122;s level were clearly seen. However, study of calcium balance was especially interesting for the bone tissue, which could be calculated using a mathematical model (Fig. 3).

The calcium balance for the bone system during SF determined with the mathematical model and calculated as a difference between calcium entrance into the bone tissue and its release as the result of bone resorption was negative and amounted to 250 mg per day. Changes in the processes contributing to bone calcium balance were as follows. During SF, the amount of calcium entering bone tissue decreased by 16% and remained at this level up to the ninth day of SF. The tendency for recovery to normal values was found only two to three months after landing. Calcium release from the bone tissue as a result of its resorption was increased by 49% during SF and decreased after it. The increased resorption of bone tissue was also manifested by increased excretion of markers of bone exchange with the urine (of n-telopeptide, as well as of pyridine and dioxypyridinoline) (Table 8).

These changes, in essence, determined the value of negative calcium balance of about 250 mg/day in the examination period during SF .The positive bone calcium balance was found on the ninth day after SF, which persisted two to three months after the landing. These findings indicate that the negative balance of calcium under conditions of microgravitation was mainly determined by its decreased gastrointestinal absorption and increased mobilization from the bone stores due to increased resorption of bone tissue with the concurrent decrease in the bone tissue formation during SF.

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After a long-term SF, processes responsible for calcium metabolism on both the organism and tissue (bone) level proceed at different rates, i.e., asynchronously. Thus, despite the postflight stimulation of osteogenesis under the effect of the increased weight-bearing and functional load onto the bone, the negative calcium balance in the bone was maintained in the early postflight period because bone resorption was stimulated along with its de novo formation, and in this case, unlike microgravitation conditions, the increased secretion of parathormone and its increased specific effects might be especially important. Despite changes in the nutrition regimen, calcium absorption in the intestine was normalized in later terms, and this also promoted maintenance of a negative postflight calcium balance. Note that calcium absorption significantly depends on the production (and under SF conditions on its entrance with food) of vitamin D and its metabolism to be converted to forms with hormonal activity. It seems that the preflight provision with vitamin D3 and its modification to active metabolites are important.

The rate of calcium accumulation in the bone during SF that was calculated on the basis of the rates of main calcium flows was decreased in one subject and unchanged in two others. However, all three subjects had increased resorption of bone tissue which caused the negative bone balance during SF. The activity of resorption processes in the bone attained the initial level only on the ninth day of the recovery period. Thus, the direct data on the decreased rate of bone tissue remodeling and its enhanced resorption obtained in man during long-term SF indicated that the flightinduced loss of bone tissue depended on many factors. This conclusion is supported by the findings of studies of the biochemical and endocrine markers and also by mathematical models. It should be noted that, on the 15th and 110th days of this SF, the urinary excretion of collagen degradation products (in nmol6 * mmol6 /creatinine â&#x2C6;&#x2019;1 ) was increased: elimination of deoxypyridinolinic bridges was, on average, 4.6 compared to 2.8 in the preflight period; elimination of pyridine bridges was 29.1 compared to 16.8 (Table 8). Renal excretion of calcium on the 14th, 93th, and 110th days of SF exceeded the average individual level by 13.4, 27.9, and 43.2%, respectively. After the flight, renal excretion of calcium decreased compared to the background during the periods of 10 to 15 and of 74 to 80 days.

Analysis of Polymorphism of Bone Metabolism Genes and Evaluation of the Risk of Osteopenia in Cosmonauts The examination of 63 cosmonauts-the crew of the orbital station Mir and the International Space Station (ISS)was performed. For genetic studies, blood samples of certain cosmonauts were taken repeatedly. For vital bone assessment, various modern noninvasive osteodensitometry technologies were used.

Results It was found that the direction and degree of changes in BMD in different segments of the skeleton depend on their position relative to the gravity vector, the functional biomechanical profile of the tissue structure, and individual characteristics. The decrease in the bone mass in the trabecular structure of bones of the lower half of the skeleton-the lumbar spine, the proximal femoral epiphysis (BMD), and the pelvis (BMC)-is natural. The rate of losses in these segments of the skeleton was 0.94, 1.36, and 2% per month, respectively. On average for a group, the losses for a 5- to 7-month flight were 6, 8, and 11%, respectively.

In general, the bone mass decrease in different skeleton segments significantly (r = 0.904) correlated with their weight load under the Earthâ&#x20AC;&#x2122;s conditions. The dynamics of changes in the BMD after spaceflights allows, with allowance for the regulations of WHO , interpreting them as a rapidly developing, yet reversible osteopenia and, therefore, as a manifestation of the expression of functional adaptation to changing mechanical requirements, at least for flights lasting not more than 6 months .

Note the high individual variability of the detected changes. Individual changes in BMD after flights lasting 5-7 months varied in the following range: from +1.6 to -15.9% for the lumbar vertebrae, from 0 to -22.7% for the proximal femur, and from +6.4 to -22.8% for the pelvis (BMC) . Authors also identified a pattern consisting of the fact that the ratio of changes in the bone mass in different segments of the skeleton of cosmonauts is individual and remains constant regardless of the type of spacecraft (OS Mir, ISS).

To perform a molecular genetic study, blood samples were taken from 49 subjects. Of these, 36 people had a history of densitometric observations (surveys). Two cosmonauts had data on the dynamics of bone mass after the flight, and six cosmonauts had data only for only one densitometry session, which allows their BMD to be estimated. Five cosmonauts had a long history of osteodensitometry, including information about the loss of BMD during repeated flights and its subsequent recovery. For eight subjects that did not participate in space missions (veteran cosmonauts), data of osteodensitometry (one session) and genotypic studies (samples TsP007, TsP-008, TsP-009, TsP-012, TsP-013, TsP-016,TsP-017, and TsP-021) were available.

Authors identified the following types of reactions of bone tissue in microgravity:

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• Type 1: high BMD-low loss and effective recovery(sample TsP-001).

• Type 2: average BMD-more substantial loss and effective recovery (samples TsP-004 and TsP-041).

• Type 3: low BMD (osteopenia, below -1SD)- more substantial loss and partial recovery (sample TsP-029).

• Type 4: low BMD (osteopenia, below -1SD)-more substantial loss and complete though slow recovery (sample TsP-028).

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In the studied sample, authors revealed the following genotypes and determined, with allowance for the results of the previous genotyping studies, the odds ratio (OR) for a high bone loss, multiply exceeding the population odds ratio (Table 1). A comprehensive analysis of the data obtained was of the greatest interest. Authors performed pairwise comparisons of the groups of test subjects by the values and changes in the BMD depending on their genotypes using the Mann-Whitney U test. The value of p <0.05 was accepted as statistically significant, and the value of p < 0.1 was regarded as a trend. Statistical analysis of the results was performed using the STATISTICA v.6.0 software (Statsoft Inc., Tulsa, United States). However, no significant differences have been revealed. However, it is worth noting that for test subjects with genotype Ss(ss) for the Col1a1 gene, Tt(tt) for the VDR gene and ST(CC) for the CALCR gene had higher BMD values compared to subjects with other genotypes.

This can be clearly seen in Fig. 1, especially for the Col1a1 gene. The data are somewhat contrary to the wide-spread opinion on the high BMD values in individuals with genotypes TT and SS, but confirm the thesis that the peak bone mass only slightly depends on the genotype. As is seen in Fig. 2, the most important loss of BMD was observed in the subjects with the TT genotype compared to the subjects with genotypes Tt and tt (p =0.0528).

For the Col1a1 gene, only a trend for the loss of BMD was revealed. Interestingly, for the VDR gene, the dependence of the genotype on the initial BMD values was retained by the BMD change as well; for the Col1a1 gene, the opposite dependence was observed. In other words, subjects with the TT genotype for the VDR gene had the lowest BMD values and higher BMD loss during the experiment.

The results of individual studies were somewhat contradictory. For example, the greatest risk of bone mass loss was detected in sample TsP-001, the carrier of which showed type 1 response of bone tissue (high BMD-low loss and effective recovery) in the bone densitometry study. However, for the carrier of sample TsP-028, who showed type 4 response of bone tissue in the densitometry study, no genetic risk of the loss of BMD was identified. It should be noted that the significance of the genetic risk in the subsequent development of osteopenia is preliminary, because it was determined on the basis of the results obtained in the analysis of Col1a1, VDR, and CALCR genes according to the materials of the Ott Research Institute of Obstetrics and Gynecology, Russian Academy of Medical Sciences. In further studies, the genetic risk of an accelerated BMD loss will be corrected by comparison with the history, clinical, and densitometric data.

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The results of analysis of Col1a1 and VDR genes in the last group of cosmonauts (n = 14) are summarized in Table 2. On the basis of the analysis of Col1a1 and VDR genes involved in the bone tissue metabolism and the results of previous studies, high genetic risks of the BMD loss were classified as follows (Table 2). The highest genetic risk of the BMD loss, which exceeded the population risk by a factor of 8, was found in cosmonaut TsP-067, which was a homozygote (tt) for the VDR gene and a carrier of the heterozygous variant (Ss) for the collagen gene. In this cosmonaut, the loss of bone mass was 2.5 times higher than the average value for the group of cosmonauts. In seven cases, the genetic risk was triple the population value, and in the remaining six subjects it was equal to the population value.

In general, it can be seen that, in this group of test subjects, high bone loss is personified with the carriers of recessive VDR alleles (in the homozygous (tt) and heterozygous (Tt) state) and collagen gene heterozygote (Ss). Authors note that the results of this stage somewhat vary in the association of the bone mass loss with certain allelic variants of E-VDR and Col1a1 genes. However, importantly, the association of the BMD loss with an incomplete allele (s) of the collagen gene is common to the groups compared.

To personalize the analysis of the associated changes in the BMD and genetic markers of bone tissue metabolism, it is necessary to recall some well-known, though somewhat contradictory data, on the association of the polymorphism of one or another gene with susceptibility to osteoporosis. For the collagen gene Col1a1, represented by genotypes SS, ss, and Ss, the comparison of the presence of mutant allele s in postmenopausal women, who were and were not taking hormone replacement therapy (HRT), led to the conclusion that there is a strong association of the s allele with the rate of BMD reduction (up to 2% per year). The association of the VDR geneâ&#x20AC;&#x2122;s polymorphism with the bone tissueâ&#x20AC;&#x2122;s remodeling process is well established . The TT genotype of Taq I polymorphism for the VDR gene is associated with lower BMD values; at the same time, significantly higher levels of bone metabolism markers were revealed in subjects with the TT genotypes and low BMD values . However, in the majority of previous studies in European populations, subjects with the tt genotype for the VDR gene showed greater bone loss compared to the carriers of TT and Tt genotypes.

To determine the mechanism underlying the effect of mutant alleles of VDR and Col1a1 genes on the rate of BMD loss, it is appropriate to consider the molecular and biochemical mechanisms of the action of the genes of interest on the process of bone formation. Collagen type 1 is a major bone protein. Its amino acid structure

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Biochemical analyses of bone samples of Ss heterozygotes showed a decrease in the bone strength compared to the SS homozygotes. Possibly, the presence of the pathological homotrimeric type 1 collagen determines the changes in its quaternary structure, with the subsequent disturbance of the mineralization of the bone matrix.

It is now recognized that vitamin D and its active metabolites are the major components of the system that regulates the metabolism of calcium and phosphorus. They are involved in the mineralization of bone tissue and maintenance of calcium homeostasis and, through the nuclear vitamin D receptor, can influence the bone remodeling process. As a nuclear receptor, the protein product of the VDR gene functions as an intermediate in the transfer of the biological effect of 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3-cal-citriol), influencing the expression of different target genes. The mutation studied in this work leads to the T → C substitution in exon 9 (methionine for isoleucine) in the nuclear vitamin D receptor protein, resulting in the formation of a functionally inactive receptor, possibly leading to the disruption of the calcium homeostasis in the body.

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Indeed, the analysis of the 3’-end region of the VDR gene of two subjects, who were homozygous for the most common haplotypes Bat and baT, showed a few differences in the nucleotide sequence of this region. The experiment on the transfection of a bone cell line with the DNA fragment of the VDR gene containing two different haplotypes made it possible to identify functional differences in the VDR activity.

After a spaceflight (for as long as 6 or 7 months), an increase in the body fat of cosmonauts is observed.

This increase is especially pronounced in the legs, where a decrease in the lean mass, including muscles, is simultaneously observed . At the same time, the experimental data obtained onboard Cosmos biosatellites should be taken into account, which showed suppression of the differentiation of osteoblasts at the osteoprogenitorpreosteoblast stage , a decrease in the activity of the collagen-synthesizing activity of mature osteoblasts , and inhibition of osteogenic differons of human bone marrow mesenchymal stem cells under conditions of the randomized of the position of the cell culture relative to the vector of gravity in a 3D clinostat .

The results of the analysis showed that, in the majority of cosmonauts, a rapid BMD loss correlated with the TT genotype for the VDR gene, but not with Tt and tt genotypes, and was associated with the carriage of an incomplete s-allele in the Col1a1 gene. However, in several cases, high BMD loss rates were personified with the carriers of the VDR gene alleles (in homozygous and heterozygote states, tt and Tt) and the heterozygote for the Col1a1 gene (Ss).

Comparative Analysis of Changes in the Skeleton of Cosmonauts during space flights on board the Mir orbital station (OS) and international space station (ISS) Fifty-eight cosmonauts with ages varying between 33 and 53 years (Russian and foreign crew members of the Mir OS), including 20 cosmonauts that had performed two or more flights, were examined. The analysis included the data on 20 observations of Russian crew members of the ISS, many of which were made after repeated flights. The duration of most flights was five to seven months; some of them lasted less (20-30 days) or more (10-14.5 months). Some cosmonauts participated in several expeditions; therefore, the long-term monitoring of the state of their bone system was carried out. A total of 80 examinations, including several sessions of a compulsory or extended cyclogram, were performed.

During the flight, the cosmonauts received food, whose composition and amount was balanced and contained dietary calcium (700-1200 mg/day). In addition, they used a system of preventive measures (SPM). At different stages, the SPM included diuretics and pharmacotherapy to prevent space motion disease; physical exercises (running on a treadmill, bicycle exercise, and resistive exercises) 2 h daily in the form of a 4-day cycle with one day of rest; ultraviolet irradiation sessions, wearing a special Penguin loading suit; and training with exposure of the lower part of the body to negative pressure.

Results A space flight exerts a considerable influence on bone metabolism and mineralization and body content. The total mineral losses during the flight are not significant and are equal, on average, to 38.59 ± 9.11 g, or 1.36% of the skeletal minerals, with a total calcium excretion of 100 ± 20 mg/day during five- to seven- month flights.

These changes would not have deserved serious attention from either the biomechanical or metabolic point of view had we not observed an appreciable redistribution of minerals in the skeleton. The analysis observed both

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typical (expected) and extreme losses of bone mass values after flights lasting five to seven months. They are shown in the table.

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Authors established that the direction and degree of the BMD changes in different parts of the skeleton depended on their position in relation to the vector of gravity, the functional biomechanical profile, the tissue structure, and individual peculiarities. Bone mass reduction in the trabecular structures of the bones of the lower part of the body, namely, in the lumbar vertebrae, the proximal epiphysis of the femur (BMD) and the pelvic bones (BMC), is natural. The rate of losses in these skeletal segments was 0.94, 1.36%, and about 2% monthly respectively on average across the group during a five- to seven-month flight. On the whole, the bone mass losses in different segments of the skeleton was significantly correlated (r = 0.904) with their weight bearing under 1 g conditions (Fig. 1).

Figure 1 also shows the results of osteodensitometry of the lower third of the shin of 11 Russian cosmonauts on the Mir OS with the use of the peripheral computer tomograph (trabecular and compact structures). In the upper skeletal segments (skull, arms, and ribs), a distinct tendency towards an increase of the BMC was observed. The phenomenon is regarded as a secondary response to the redistribution of the body fluids and electrolytes in the cranial direction and, hence, may be connected with changes in the volume and ion regulation systems. The distribution of the BMC in the skeleton determined by this, despite the small total mineral losses, puts the skeletal segments that are critical from the biomechanical point of view at risk. An almost identical pattern of distribution of the BM losses along the vector of gravity was observed by authors when authors analyzed the results of examinations of Russian crew members of the ISS.

The data available give evidence of high variability of individual bone mass loss. The range of variations in the bone mass changes during the flight (five to seven months) was, e.g., +3 to -12% for the BMD of the lumbar vertebrae; 0 to -20% for the femur, and -1 to -23% for the BMC of the pelvic bones; i.e., the individual losses in some cases exceeded the groupâ&#x20AC;&#x2122;s average values by a factor of almost two. The averaged values with their statistical scatter may reflect the expected changes at the existing level of the means of prevention. At the same time, unexpectedly great losses are observed in a number of cases, with clinically significant osteopenia found upon return to Earth, which requires individual regulation of physical loads in the rehabilitation period and the use of pharmacological means of correction.

When the results obtained during the expeditions on the Mir OS and the ISS were compared, the amplitude of the changes in the mineral density in repeated flights could substantially differ, whereas the individual scatter

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in the data significantly exceeded the in-flight changes on board the two stations. This does not allow us to speak at present about statistically significant mineral density changes during the expeditions on board the two stations of comparable durations. No substantial differences in the changes in the mineral density, depending on the performance of the start and landing on the Soyuz spaceships or the United Statesâ&#x20AC;&#x2122; reversible space crafts, were observed either. However, comparative analysis of the data obtained from the Mir OS and the ISS, where cosmonauts performed repeated (sometimes multiple) flights, allowed a new consistent pattern to be observed: the ratio of losses in different segments of the skeleton retains its individuality and remains virtually constant in repeated flights, irrespective of the type of the orbital station (Fig. 2).

In addition, certain differences in changes in the height of the intervertebral disks were observed after the flights of the Mir OS and the ISS. First, they could be linked to different preferences of the SPM elements: on the ISS, these are resistive physical exercises; on the Mir OS, they are predominantly locomotion on the treadmill with a pulling force partly simulating the body weight. Second, it cannot be ruled out that the phenomenon might be connected with different durations of examinations after returning from the Mir OS (days 3-5) and from the ISS (days 18-21). The cosmonauts did not make serious complaints after landing; however, this fact may be of alerting prognostic importance and, hence, requires additional observations and analysis to be made in the readaptation period.

It was established that, in the initial period of recovery (from 1.5 up to 12 months or longer), a further reduction in the BMD of the critical skeletal regions may occur and normally does occur. This phenomenon of the secondary negative BMD trend is entirely explainable by the reversal of terrestrial mechanical loads and reflects the beginning of the recovery of the bone remodeling processes. With the normal balance of these processes, the resorption rate exceeds that of the bone formation (regeneration), which is reflected by densitometry at this stage of readaptation. In the typical cases, favorable changes were observed as early as the second month of rehabilitation.

It was shown that it took 2.5 to 3 years for the bone mass to recover adequately to attain the preflight level.

This is important, because after repeated flights, recovery of only the preflight level of the last flight is attained.

The combined analysis of the results of the postflight observations in the readaptation period of 45 Russian cosmonauts and Americal astronauts showed that the time period of the bone mass recovery in different skeletal segments is described by an exponential mathematical function. According to the model, recovery of 50% of the loss of the bone mass in different skeletal segments occurs within nine months after landing.

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