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--> 6 Bone Physiology Introduction One of the best-documented pathophysiological changes associated with microgravity and the spaceflight environment is bone loss. The reduction in bone mass and its effect following reentry could substantially limit long-term human exploration of space. The development of effective countermeasures through better scientific understanding of this phenomenon is therefore essential for future crewed flights. This chapter very briefly reviews the functions of bone, bone growth and development, the process of bone remodeling (which underlies its physiological function), and the effects of hormones on this process. It summarizes current information on mechanical effects on bone, effects that may be the basis for the changes observed in microgravity or space environment conditions, briefly reviewing clinical observations; experiments on humans, animals, and cells; and putative mechanisms. It also summarizes spaceflight effects on the skeleton in humans and animals and gives caveats for these data. It then presents open questions and directions for future research aimed at continuing to characterize and understand, at a fundamental level, microgravity or space-environment-related bone loss and at developing effective countermeasures. Bone Functions, Growth And Development, And Remodeling Functions of Bone Bone has four major functions: (1) mechanical, including support of soft tissues and locomotion; (2) storage of ions and ion homeostasis; (3) housing of the bone marrow and support of hemopoiesis; and (4) protection of the central nervous system. By fulfilling these functions, the mineralized skeleton played a central role in the evolution of terrestrial vertebrates.
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--> Bones act as levers for muscles, and all aspects of locomotion (walking, running, climbing, flying) were tied to the evolution of the skeleton. Moreover, breathing (which requires expansion of the rib cage), brachiation, erect posture, and the use of tools are all skeletal functions. Maintenance of a healthy musculoskeletal system is essential for well-being, and its deterioration is often associated with aging, manifesting itself as a reduction in muscle mass and osteoporosis, probably due in part to reduced usage. Calcium is a major ion recruited from bone for homeostatic needs. The marine environment provides a constant adequate supply of calcium; however, on land, organisms rely on dietary calcium supplied intermittently during feeding. Between meals, the organism maintains a stringently controlled steady-state calcium concentration in the extracellular fluid by withdrawing it from bone through bone resorption (degradation). Steady calcium levels are needed for normal neural, muscular, and endocrine functions, as well as for blood clotting and for cellular adhesion, migration, and proliferation. Other ions stored in the skeleton are phosphate and hydrogen, both of which may affect the rate of mineralization and demineralization. In addition, the skeleton stores potassium and magnesium, for which no homeostatic mechanisms have been documented. Important in the context of this discussion is the fact that calcium can only be withdrawn by destroying the skeletal structure that contains it, weakening the skeleton at that site. Furthermore, calcium homeostasis seems to have priority over other skeletal functions, so that calcium deprivation leads to thinning and weakening of the skeleton. However, the bones exposed to maximum mechanical loads are destroyed least and last. Adult hemopoiesis in humans is totally confined to the bone marrow. Bone marrow cells with osteogenic potential may support hemopoiesis that, at least in tissue culture, requires a feeder layer of supportive cells. The bone-resorbing cells, the osteoclasts, are of hemopoietic origin. They are related to macrophages and originate from GM-CFUs (granulocyte macrophage colony forming units). In hypoxia, due to high altitude, for example, which causes active erythropoiesis, there is an expansion of the bone marrow cavity at the expense of bone. Whenever bone is formed, as at an ectopic site in muscle, it is always populated by bone marrow. With aging, active bone marrow is replaced by fatty marrow. It is presumed that stromal cells with dual capability (osteogenic and adipogenic) become adipocytes.1 It has been suggested that there is a gradual exhaustion with age of osteogenic cells in the marrow; however, fracture repair occurs at all ages. Housing of the brain and spinal column has survival advantages. Interestingly, unlike the rest of the skeleton, the skull does not seem to be subject to estrogen deficiency or to immobilization or microgravity-related bone loss. It is important to recognize that to fulfill most of its functions, bone has to be destroyed and eventually rebuilt. For mechanical function, bone is continuously rebuilding itself to optimize its structure and architecture. For calcium mobilization, bone packets are destroyed, potentially to be rebuilt when calcium is again available. There are fewer fluctuations in bone mass related to the other two functions, except during very active physiological or pathological hemopoiesis, when the marrow cavity expands at the expense of bone. The process of bone destruction and rebuilding is called remodeling. Bone Growth and Development Embryologically there are two types of bone: the membranous bone of the face and skull and endochondral bone. For flat membranous bone, bone develops directly in connective tissue, formed by osteoblasts (bone-forming cells) that differentiate locally from mesenchymal cells. The rest of the
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--> skeleton appears first as a cartilagenous anlage. Starting at ossification centers, the cartilage is calcified and is invaded by blood vessels. Osteoprogenitor cells start depositing bone on the calcified cartilage that is subsequently removed by osteoclast-like cells. This mixture of calcified cartilage and early bone is subsequently replaced by lamellar bone, the dense type found in the mature organism. At the periphery of all bones, there is bone deposition in the periosteum, which resembles membranous bone formation. This process of bone growth according to the genetic template is called modeling, whereas the process of replacement of existing bone (for carrying out its functions) is remodeling. Bones, as well as other structures, are apparently unaffected when embryonic development of chicks occurs during microgravity in shuttle flights.2 Bones grow in length only at the cartilagenous epiphyses, where chondrocytes proliferate, differentiate, undergo hypertrophy, and mineralize the surrounding matrix on which the initial bone deposits, as described above for endochondral bone formation. To maintain its shape, the new bone is actively remodeled. Bones grow in diameter by deposition of external bone at the periosteum, a process that continues throughout life and is subject to mechanical regulation. Most of the load in long bones is sustained by the cylindrical bone of the shaft, which is formed in that manner by the periosteum. It should be noted for experimental studies in rodents (especially mice) that the epiphyses never close and there is continuous longitudinal growth. In rats, the growth rate is significantly attenuated at about 12 to 14 months of age. In humans, epiphyses close around the age of 18 and longitudinal growth ceases. However, peak bone mass (the maximum amount of bone before age-related loss starts) is only reached toward the middle of the third decade. Multiple humoral, local, and systemic factors affect skeletal growth and development and remodeling in the adult. Bone Remodeling: Hormonal Effects As mentioned above, bone remodeling is the process of replacement and rebuilding of packets of bone as part of bone's normal functions. Architecturally, bone structure is either cancellous (trabecular), shaped like honeycomb plates in the interior of each bone; or cortical (compact), denser bone at the boundary. The skeleton contains 80 percent cortical bone (by mass, not volume) and 20 percent cancellous bone. In humans, the turnover rate is 20 to 30 percent per year for cancellous bone, depending on the site, and about 3 percent per year for cortical bone. The cancellous bone is thus more readily affected by conditions that increase bone remodeling. This section is a summary of the action of hormones that affect the skeleton by playing a role in calcium homeostasis or bone metabolism. Changes in the level of these hormones due to gravity and/or the space environment could play a role in the bone loss observed. Systemic hormonal changes that stimulate bone resorption (destruction) include a rise in parathyroid hormone (from calcium deficiency, for example), a rise in thyroid hormone, or a decrease in sex steroids (estrogens in women and androgens in men). Both sex steroids have profound effects on the skeleton in both genders. For instance, males usually have a larger skeleton than females. Congenital mutations in the estrogen or androgen receptor in humans suggest that estrogen is required for the closure of the epiphyses and both hormones are needed to produce a skeleton with normal density. In androgen-receptor-deficient individuals, who therefore have a female phenotype despite their XY chromosomes, there is reduced bone density that could not be corrected by estrogen administration. In a male patient who was estrogen-receptor deficient, the epiphyses were not closed at the age of 28 and the bone density was significantly decreased. In mature females, estrogen deficiency causes an increase in bone resorption leading to osteoporosis. However, androgens seem to be required for normal bone as well, since the addition of androgens to
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--> estrogen in hormone replacement therapy further increased bone mineral density.3 In males, testoster-one deficiency has effects similar to those of estrogen deficiency in women (increasing bone resorption and causing bone loss), which can be reversed by testosterone administration. These hormonal effects could be important during prolonged spaceflights, which could affect the level of sex steroids due to stress or other causes.4 A reduction in estrogen (along with amenorrhea) can be induced by sustained stress, vigorous exercise (in marathon runners and gymnasts), or dietary deprivations (in people with anorexia). Exogenous administration of glucocorticoids ("steroids") also suppresses sex hormone production and has additional effects that reduce bone mass: inhibition of calcium absorption that causes (via feedback) elevations in parathyroid hormone (PTH), and direct suppression of bone formation. However, these effects are seen at relatively high glucocorticoid levels (e.g., 7.5 mg prednisolone/day) that are not reached during physiological stress. Bone resorption as well as defective mineralization is also caused by deficiency of 1,25(OH)2 vitamin D3. The major action of 1,25(OH)2D3 is to facilitate calcium absorption in the gut. Its absence reduces circulating calcium levels and raises PTH. High doses of vitamin D, reached by exogenous administration, stimulate bone resorption. Endogenous 1,25(OH)2D3 levels are controlled by PTH through its induction of the 1-hydroxylase in the kidney, part of the calcium homeostasis feedback loop.5 The only systemic hormone that inhibits bone resorption is calcitonin, a 32-amino-acid peptide produced by the thyroid clear cells. Calcitonin is released in response to elevated calcium concentrations.6 A systemic hormone needed for skeletal development is growth hormone (GH). It is released by the pituitary and increases liver production of insulin-like growth factor (IGF-1), which acts on the epiphyses to stimulate longitudinal growth in endochondral bone formation. GH may also act directly on cartilage or bone, which has been shown to contain GH receptors. In adults, GH or IGF-1 stimulate both bone formation and bone resorption without increasing bone mass. In individuals with a GH deficiency, bone and muscle mass are reduced and have been reported to increase following GH administration.7 IGF-1 and IGF-2 have been extracted from bone, are made by bone cells, and were reported in experimental studies to stimulate osteoblast proliferation and collagen synthesis in vitro. In rat bone explants and cells, osteogenic agents, such as PTH and prostaglandin E, stimulate IGF-1 production. Taken together, these observations suggest that IGF-1 could be important for maintaining normal bone. The effects of IGF-1 on its target tissues depend on the level of the IGF binding proteins, both present in the circulation and locally produced.8 Changes in GH have been reported in rats exposed to the space environment (see Chapter 9, "Endocrinology"). Thyroid hormones also play a role in the development and maintenance of the skeleton, either indirectly, through effects on GH production, or through direct action on osteoblasts. Thyroid hormone deficiency during development causes short stature and skeletal malformations, known as cretinism. Thyroid hormone excess in adults, usually caused by hormone replacement therapy, increases bone turnover and cases bone loss. Insulin also acts on the skeleton; anomalies in bone metabolism have been associated with diabetes, but the link is not clearly established. 9 A large number of local factors increase bone resorption and/or formation, and some have been shown to participate in the mechanical effects on bone. Skeletal remodeling resembles inflammation, and most inflammatory cytokines have been shown to affect osteoblasts and/or osteoclasts. Prostaglandin E (PGE), like PTH, stimulates both bone resorption and bone formation. Prostaglandins E and I2 have been implicated as mediators of mechanical effects on bone remodeling (see below), as well as in the bone loss associated with inflammation in periodontal disease and rheumatoid arthritis. Prostaglandins are produced by many cell types, including osteoblast lineage cells and macrophages.
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--> Potent local stimulators of bone resorption include Interleukin-1 (IL-1) and tumor necrosis factor α (TNFα), which are produced by macrophages.10 Other cytokines are IL-4, reported to suppress osteoclast formation in vitro, and IL-6, implicated in osteoclast formation and estrogen-deficiency bone loss, at least in mice. IL-11 is also a potent stimulator of osteoclast formation in vitro.11 Locally produced growth factors that may play a role in skeletal remodeling are the bone morphogenetic proteins (BMPs), a large family of growth and differentiation factors related to transforming growth factor β (TGFβ). Initially identified in bone extracts found to induce bone formation when injected into muscle or dermis, BMPs also play a role in limb development and stimulate differentiation of pluripotent mesenchymal cells into the osteogenic lineage. They have therefore been implicated in osteogenesis and possibly in bone remodeling. BMPs also play a role in the development of other organs. Evidence for the involvement of specific BMPs in physiological or pathological bone remodeling or bone formation in the adult or in response to mechanical or gravitation changes is not yet available. TGFβ like BMPs, stimulates bone formation when applied directly to bone. In vitro, it inhibits osteoclast formation. TGFβ is produced by platelets, as well as osteoblasts and other cells, and is abundant in bone in the precursor form that is activated by proteolytic cleavage. 12 There are at least two growth factors that may participate in the link between blood flow or vascular changes and bone changes and thus play a role in the microgravity-induced pathophysiology. Fibroblast growth factor-2 (basic FGF) is produced by osteoblasts as well as endothelial cells and stimulates bone formation when administered to rats in vivo, either locally or systemically.13 Vascular endothelial growth factor is produced in bone and by osteoblasts in vitro in response to PGE. It is a potent stimulator of angiogenesis, which is required for osteogenesis, and may participate in a positive feedback loop that connects the two processes.14 Bone cells have receptors for many other factors and hormones, such as endothelin, enkephalin, thrombin, epidermal growth factor, epinephrine, and norepinephrine, but their role in bone metabolism is uncertain. Bone metabolism is thus subject to a large number of systemic and local factors, many of which are part of other physiological processes that could be affected by microgravity, stress, nutrition, fluid, and electrolyte balance. Mechanical Effects On Bone Remodeling Clinical Observations and Human Experimentation Clinical observations and human experimentation show that human bone mass and structure are ideally suited to sustain the loads exerted upon them. There is well-documented bone loss after the removal of mechanical loading and somewhat more limited documentation of increased bone mass in response to mechanical stimulation. This information is highly relevant to gravitational effects on the skeleton. The effect of mechanical loads on the architecture of the human skeleton has been recognized for some time and was scientifically described at the end of the 19th century by Roux and Wolff. The ability of bone to adapt to mechanical forces has been extensively used in orthopedics and orthodontics. Briefly stated, bone will change its mass and architecture to adapt to the forces exerted upon it, providing maximum strength for minimum material. This is achieved through an intricate structure where appropriately oriented collagen fibers provide tensile strength and the minerals embedded in the matrix provide compressive strength. 15 This mechanical adaptation is
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--> responsible for the reshaping of bone during fracture healing to resume the prefracture anatomy (if properly set). Bone loss, caused by paralysis and immobilization, is well documented and selectively affects the immobilized bones (for example, lower extremities in paraplegia, or a single arm kept in a cast). This bone loss is always associated with the loss of muscle mass and strength and is a result (at least in part) of the loss of muscular tension continuously exerted on bones under 1 g.16 Bone degradation can be evaluated by monitoring bone collagen degradation products in urine and blood (C-terminal or N-terminal pyridinoline peptides), for which commercial ELISA assays are available. Bone formation rates can be estimated by measuring the osteoblast products, bone alkaline phosphatase, or osteocalcin in the plasma. In human volunteers under forced bed-rest conditions, calcium balance and the excretion of bone collagen degradation products showed that immobilization causes bone destruction that was detectable within days.17 The bone loss caused by microgravity (see below) is consistent with these observations. There is no question that the lack of mechanical stimulation causes bone loss. The increase in bone mass in response to mechanical stimulation is less well documented. Extensive controlled exercise programs in postmenopausal women who are estrogen deficient produced inconclusive results or had limited effects.18 19 It had been noted that the bone mass is much larger in the dominant arm of professional tennis players. Data from bone mass measurements collected over the last couple of years on gymnasts and other athletes show that mechanical stimulation produced by high-impact loading increases bone mass.20 21 Weight lifters and other athletes also had higher bone density in their extremities and vertebral column during the years they practiced the sport, but bone mass returned to average levels later in life.22 Mechanical stimulation seems to be more effective in increasing bone mass before peak bone mass is reached at approximately age 25.23 24 Animal Studies Observations regarding bone remodeling in humans have all been reproduced in animal studies, in which some of the mechanisms have been elucidated at the tissue level. Hindlimb paralysis caused in rats by severance of the sciatic nerve or of the knee tendons leads to significant bone loss in the affected limb, due to an initial increase in bone resorption and a sustained decrease in bone formation.25 When immobilization is produced in rats by putting a limb in a cast, most of the bone loss is reversible after remobilization. An extensively used model that aims to mimic microgravity is hindlimb unloading, often by tail suspension or a body sling in rats. Within 2 weeks, these animals lose about 25 percent of their cancellous bone in the proximal tibia and show a 30 percent reduction in the mechanical strength of the shaft.26 27 Interestingly, recent studies have shown that hindlimb unloading causes a 40 percent reduction in blood flow in the unloaded extremities.28 Immobilization experiments conducted in dogs, whose cortical bone is more similar to that in humans, also showed bone loss, including significant reduction in the cancellous bone of the proximal radius of the immobilized dogs. There was significant recovery of bone following remobilization, except for the bone mineral density in the central radius.29 An interesting model is the disarticulated ulna of a turkey, which maintains its vascular supply but is totally unloaded.30 This bone is undergoing rapid resorption, which can be prevented with the amazingly limited mechanical stimulation of four loading cycles per day. Further mechanical loading stimulates periosteal bone formation that depends on loading strain and frequency.31 32
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--> Other experimental protocols have shown that mechanical stimulation increases bone formation in the rat caudal vertebra or the rat tibia. Short stimulation cycles (30 per day) with loads that produce strains approximately equivalent to those generated by walking were effective (700 µε; a strain of 1 ε produces a deformation of 0.1 percent).33 There is also evidence of interaction between hormonal factors and mechanical stimuli on bone. It was recently shown that bone loss caused by estrogen deficiency in rats is influenced by running treadmill exercises and functional unloading.34 Whether these findings extrapolate to humans remains to be seen. Putative Mechanisms There is no question that bone cells respond to and therefore perceive mechanical changes that cause deformation of the matrix and shear stress due to fluid flow. The proposed sensors (whose function has yet to be proven in bone) include ion channels and integrins. Initial stimuli are followed by signal transduction and amplification, as well as secondary responses that include secretion of prostaglandins and possibly nitrous oxide and growth factors, which act in an autocrine and paracrine fashion. However, many details of this general scheme have not yet been elucidated. To explain the adaptation of the skeleton and its response to mechanical loads, Frost proposed the existence of a mechanostat or sensor, set at about 1,200 to 1,400 µε, that would initiate bone loss if the strain in the tissue is below that and bone gain if it is above.35 Such a mechanism is consistent with the physiological evidence and predicts a system of feedback-controlled bone mass homeostasis responsible for adjusting the amount of bone to its mechanical function.36 The proposed cellular and molecular basis of this feedback-regulated system is a controlling effect of mechanical strain on osteoblasts (the bone-forming cells) and osteoclasts (the bone-resorbing cells). Strong experimental evidence points to a decrease in osteoblastic bone formation during immobilization (lack of mechanical load), primarily in rats. In several species, there is fairly good evidence for increased osteoblastic bone formation with increase in mechanical loads. There is less evidence for mechanical regulation of osteoclast activity, which is usually indirectly controlled through osteoblast-lineage and other cells. Several types of mechanical perturbations are believed to be perceived by the cells. Mechanical loading increases the strain in the matrix, and that strain could be transmitted to the cells through their attachments. In addition, deformation of bone or cartilage causes the displacement of fluid, which produces shear stress. The current hypothesis for mechanical sensors is that specific structures in bone cells act as mechanochemical transducers that generate biochemical signals, which are part of the known signal transduction pathways. One of the outcomes is the production of cytokines that act in an autocrine and paracrine manner to propagate the signal to neighboring cells. Based on information from other systems, three types of sensors have been proposed, with functions that are not mutually exclusive. Stretch-activated cation channels show increased calcium permeability in membranes stretched in patch clamp experiments and were proposed as mechanosensors.37 Such channels have also been described in osteoblasts. 38 Similar ion channels act as mechanosensors in C. elegans.39 It is not clear if the strain in the bone matrix (1,500 µε to 5,000 µε) can cause sufficient distortion of the membranes in attached cells to activate these channels. Calcium channel inhibitors were shown to block mechanical responses in rat bone.40 A recent mathematical model attempted to quantify the relationship between fluid shear stress on the extracellular surface and the increased calcium permeability of stress-gated ion channels in endothelial cells.41
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--> The second class of possible sensors are the integrins through which cells attach to the extracellular matrix. Integrin ligation by antibodies or ligand occupancy activates the sodium proton exchanger or increases intracellular calcium. In addition, integrins can activate signal transduction pathways that involve kinases, similar to those activated by growth factors and other extracellular signals.42 Mechanical effects on smooth muscle cells can be blocked by arginine-glycine-aspartic acid peptides and antibodies against β3 and αvβ5 integrins (cell adhesion receptors).43 Integrins were shown to be mechanically connected to cytoskeletal filaments and the nucleoplasm.44 Cyclical strain (10 percent, 60 Hz) of human umbilical vein endothelial cells caused redistribution of β1 integrins and tyrosine phosphorylation of focal adhesion kinase (pp125FAK).45 Several secondary responses can participate in the propagation of mechanical effects to neighboring cells. The best documented is up regulation of prostaglandin release.46 47 Inhibitors of prostaglandin synthesis block responses to mechanical stimulation;48 49 prostaglandin E2 and I2 have been implicated.50 Other possible secondary responses include TGFβ and IGF1, which increase bone formation and bone mass in unloaded bones in rats.51 52 Mechanical strain promotes release of FGF2 from vascular smooth muscle cells,53 and shear stress induces cyclooxygenase 2, as well as endothelial cell nitric oxide synthase and manganese superoxide dismutase in endothelial cells.54 In osteoblasts, shear stress also causes nitric oxide release.55 In addition, pulsating fluid flow was shown to stimulate prostaglandin release and cyclooxygenase 2 in mouse bone cells.56 G proteins and nitric oxide were also shown to mediate the response of bovine articular chondrocytes to shear stress.57 Another recently reported secondary response is the induction of the gene for the glutamate transporter,58 which is of interest because the glutamate receptor in C. elegans has been implicated in tactile responses.59 Microgravity Effects On The Skeleton Caveats More than 30 years of microgravity research has clearly established that the skeleton is one of the organs at risk (for a recent review see van Loon et al., 199360). However, the available data have several limitations. For human studies, the sample size is always small and data collection often incomplete. Collection of urine and feces is difficult in outer space. The high-precision bone density measurement technology (with a 1 to 2 percent coefficient of variation) is relatively new and was too bulky in the past to be used on spacecrafts to monitor the rate of bone loss during flight. Countermeasures were not always rigorously observed. Confounding variables, which may affect the skeleton in addition to microgravity, have not always been recorded. Animal studies have similar limitations to those enumerated for human studies. The sample sizes are small. The most frequently used species are rodents. These animals grow throughout their lives, and their cortical bone is not similar to that of humans. Because of payload limitations, animals were often very young and actively growing. Flights were mostly short (less than 2 weeks). During spaceflight, animals were usually unattended, and no samples were collected or experiments conducted. Some, but not all, experiments had 1-g controls on board; otherwise, the references were "synchronous" ground-based and vivarium controls. Confounding conditions included the acceleration and stress associated with launching and landing, and occasionally flight conditions not precisely mimicked on land, such as higher temperature, crowding, and so on, plus delays between landing and sample collection.
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--> Human Studies The comprehensive experience of spaceflight effects on the human skeleton has recently been reviewed by van Loon et al.61 and is summarized here. Major recurrent findings were a negative calcium balance and a decrease in bone mineral density (BMD). BMD estimates the amount of bone by measuring the attenuation of two narrow beams of x rays of different wavelengths, by the calcium in the x-ray path, with correction for soft tissue. A precision of 1 to 2 percent coefficient of variation can be achieved with this instrumentation, which is relatively bulky (the size of a large table plus console). However, portable smaller-size equipment for measuring bone mineral density in some bones is currently available and is being adapted for in-flight measurements. Following are several illustrations. A reduction of 3 to 9 percent in the bone mineral content of os calcis was observed following a 4- to 14-day orbital spaceflight.62 A loss of 0.2 percent total body calcium was observed in astronauts on Apollo 17 after a 14-day flight.63 A reduction in the BMD of os calcis and radius was also observed in astronauts on the Skylab 3 and 4 missions, and there was no return to preflight levels after 97 days.64 65 A negative calcium balance was recorded in the Skylab 2 crew (50 mg/day).66 The most compelling data have recently been compiled from the experience on the Mir space station. BMD was measured on various bones in 18 crew members who spent 4 to 14.4 months on the station. The data show an average loss of up to 1 percent BMD per month (Table 6.1). For comparison, the rapid bone loss in some women in early menopause is 2 to 4 percent per year. The in-flight exercise program was clearly not sufficient to prevent the bone loss caused by weightlessness and flight condition. Other changes include a relative reduction in lean mass (primarily muscle) and relative increase in fat mass. It should be emphasized (see caveats) that the flight database is still very small (by comparison, the natural history of age-related and postmenopausal bone loss is based on thousands of subjects). There was significant variability in spaceflight-related bone loss among subjects as well as in postflight recovery, on which there is relatively limited information. Different bones were affected to varying degrees. There have been few female astronauts so far, and there are no reported data on the effect of microgravity on skeletal changes, specifically in women. Some women astronauts could also be estrogen deficient, an additional risk for bone loss. Evaluating the effect of microgravity on the female skeleton and the possible interaction (additive, synergistic, and so on) between microgravity and hormonal TABLE 6.1 Bone Loss on Mir Space Station (percent bone mineral density lost/month) Variable Number of Crew Members Mean Loss (percent) Standard Deviation Spine 18 1.07* 0.63 Neck of femur 18 1.16* 0.85 Trochanter 18 1.58* 0.98 Total body 17 0.35* 0.25 Pelvis 17 1.35* 0.54 Arm 17 0.04 0.88 Leg 16 0.34* 0.33 *p < 0.01. SOURCE: LeBlanc, A., Schneider, V., Shackelford, L., West, S., Ogavov, V., Bakulin, A., and Veronin, L. 1996. Bone mineral and lean tissue loss after long duration spaceflight. J. Bone Miner. Res. 11: S323.
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--> status will be essential. This knowledge could be used to help develop appropriate preventive countermeasures. Genetic Variability Usually the number of subjects in a given database is small and there is considerable variation in individual responses. Some of this might be the result of experimental "noise," but it is likely that humans will show considerable individual variability in how they respond to microgravity. With the prospect of relatively large numbers of individuals residing in space for extended periods, it is time to begin investigating the genetic variability in the response to spaceflight for those situations where ground-based studies suggest a genetic component. From the perspective of spaceflight, one of the most important effects on human health is bone loss. It is well established from identical twin studies that osteoporosis has a strong genetic component, accounting for about 60 percent of the variance in bone mineral density.67 However, no single gene has been convincingly proven to be a risk factor for osteoporosis. The genetic component has been attributed instead to the cumulative effects of a number of genes with small individual effects. There is some evidence from a twins study suggesting that the vitamin D receptor gene may correlate with bone mineral density. 68 69 Other polymorphisms that reportedly correlate with bone density or osteoporosis but have not been as extensively studied include collagen type I, estrogen receptor, and interleukin 1. There is also a report on familial high bone density, localized by linkage analysis to chromosome 11. Osteoporosis, like atherosclerosis and other similar diseases, is complex and multifactorial, dependent on several genes that act as risk factors. Identification of such genes is currently under way. The point is that there is human polymorphism with respect to osteoporosis and bone mineral density; this may imply that the degree of bone loss found during spaceflight may involve a genetic component. Locating and identifying the genes involved in osteoporosis is currently being pursued. To quote a recent review on the topic, this goal is "certainly attainable within the foreseeable future, given proper application of newer advances in molecular genetics and genetic epidemiology."70 Although NASA should not be involved in this task, the information and methodology for identifying those individuals most vulnerable to osteoporosis is extremely important to NASA. Countermeasures for bone mineral loss are important; selecting crew persons for long-term missions with consideration of their susceptibility to osteoporosis would be a complementary approach. Several effective therapies are currently available for the treatment and/or prevention of osteoporosis. These include hormone replacement therapy for estrogen-deficient women, bisphosphonates, and calcitonin. Other therapies are currently being developed. The efficacy of these agents in preventing microgravity bone loss should be explored. Well-designed studies (including randomization, placebo groups, and/or crossover) should be conducted. Although the number of astronauts is small, the magnitude of the bone loss (about 1 percent per month) may make it possible to obtain conclusive data for therapeutic effects and identify pharmacological means for the prevention of microgravity bone loss. Studies in space should be preceded by evaluation of therapeutic intervention in the bed-rest model. Renal Stone Formation One of the consequences of bone calcium loss is an increase in susceptibility to renal stone formation. Microgravity changes urine composition to favor supersaturation of stone-forming salts.71 Specifically, the combination of increased calcium excretion in the urine secondary to the bone calcium loss, with decreased fluid intake, high salt intake,72 and a reduction in urine pH, will predispose a person
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--> to renal stone formation. This is a potentially serious problem. Dietary factors (especially fluid intake and pharmacological interventions) can significantly influence urinary chemical composition, and their use as countermeasures should be further explored. Recommendations To address the questions related to microgravity effects on bone loss in humans, the following studies should be conducted: Obtain a comprehensive and detailed description of the phenomenon. A careful record of skeletal changes occurring during microgravity and postflight should be generated for each astronaut using dual-beam x-ray absorptiometry (DXA). Monitoring should compare weight-bearing bones of the lower extremities and vertebral column to those of the upper extremities and the cranium. A database should be established to correlate skeletal changes with age and gender, muscle changes, hormonal changes during flight, diet, and genetic factors (e.g., susceptibility to osteoporosis) if and when these genetic factors become known. The course of microgravity-related bone loss should be thoroughly documented, with attention paid to the rate of bone loss as a function of flight duration. Researchers should establish if bone loss levels off and bone mass stabilizes at a new steady-state level and to what extent the bone loss is reversible after reentry. The mechanism of bone loss should be determined, with emphasis on useful information for developing countermeasures. Determine if bone loss is the result of increased bone destruction (resorption), decreased bone formation, or both. Markers of bone formation and resorption are currently in clinical use. Given the variability of these parameters and the small sample size, astronauts should serve as their own controls. Further validate the bed-rest model as representative for microgravity-induced bone loss and use it to test countermeasures. Effective countermeasures for preventing bone loss should be developed. Instrumentation that can be used for different types of mechanical stimulation should be developed and different types of exercise should be evaluated. Ground experiments suggest that impact loading is most effective in maintaining or increasing bone mass, and animal experiments suggest that short-duration mechanical stimulation may be sufficient to maintain bone mass in immobilized bones. The use of pharmacological means should be examined. Animal Studies Findings on the skeletal changes observed in rats exposed to microgravity have been recently reviewed73 and are briefly summarized here. It should be reiterated that the applicability of the rat data to the human skeleton is limited due to the use of young animals, the continuous growth of the rodent skeleton, and the differences in cortical bone, which lacks vascular canals in rodents. However, there is a vast database on rodent bone biology, and gravitation-related changes in rats or mice could be explored for mechanistic studies. In the Cosmos flights, the major positive finding was a reduction in
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--> the rate of periosteal bone formation, which returned to control levels approximately 4 weeks postflight. Decreases in the mechanical properties of weight-bearing bones were also observed.74 75 76 77 78 79 80 81 These findings were not always reproducible.82 83 84 85 86 In Spacelab 3, defective mineralization was observed.87 88 89 90 In some experiments, there was evidence of increased bone resorption. 91 There were no differences in the biochemistry of collagen or proteoglycans. Thus, examination of animal bones, primarily from rats exposed to microgravity for short durations (4 to 15 days) showed either no effect or a reduction in bone formation, relative to controls that were not always perfectly matched. A limited number of experiments have been conducted using bone cells cultured under microgravity conditions. Considering the difficulty in controlling experimental variables in space, such as convection, media changes, partial pressure of gases, and the like, no definitive conclusions could yet be reached regarding microgravity's effects on isolated cells in petri dishes. However, similar experiments in the space station, conducted side-by-side with controls in a 1-g centrifuge, could generate relevant data on cultured cells. Reproducible changes, if observed, should provide a suitable system for exploring the mechanism for microgravity perception by, and effects on, isolated bone cells. Well-documented ground-based in vitro models for studying the interaction between osteoblasts and osteoclasts are currently available. Most recently, RANK ligand (TRANCE), which is expressed on membranes of osteoblast lineage cells, was shown to promote osteoclast differentiation in macrophage or osteoclast precursors. Other agents active in this coculture system include prostaglandins, interleukins, osteotropic hormones, and hematopoietic growth factors. This experimental system could be used under microgravity conditions with 1-g centrifuge controls. If differences are observed, their basis can be investigated further. Recommendations The two crucial questions are, What is the validity of animal models for mimicking the changes produced by microgravity in the human skeleton? and, What experiments can be conducted under ground-based conditions? Both in space and on the ground, mostly young rodents with an actively growing skeleton have been used. When an effect was observed, it was primarily a decrease in bone formation. The following recommendations are listed in order of priority. Determine if changes produced by microgravity in animal bones are similar to those in humans, and if they have a similar basis—that is, increased bone resorption and/or decreased formation. Older animals (rats or mice) should be used (the growth in rats is significantly attenuated at about 12 to 14 months). Mice are preferable, because they are smaller and include genetically homogenous strains, mutants, and transgenic animals ("knockouts," and so on). Dual x-ray absorptiometry, histomorphometry, and biochemical markers (described under human studies) should be used. When an animal model is identified that mimics human changes in spaceflight, it should be compared to ground-based models, such as hindlimb unloading. If appropriate, it should be used to study mechanisms that can then be corroborated in space. Emphasis should be on investigations that can help develop countermeasures and shed light on human pathology (e.g., osteoporosis). Studies should be conducted that will do the following: Evaluate the contribution of changes in muscle function and blood flow. Muscle, bone, and blood flow, which strongly affect both, should be studied side by side. Given the dynamic nature of
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--> the vascular system, in-flight experiments should include rapid tissue fixation to best address this question. Evaluate to what extent the bone loss is secondary to systemic effects on hormones, growth factors, and cytokines, which should be measured in the same experiments. Identify putative mediators involved in the tissue response: prostaglandins (PGE2, PGI2), growth factors (FGF, TGFβ), and so on. Elegant pharmacological studies were used in the past. Recombinant proteins and mutated cells and animals can supplement the pharmacological approach. Identify the cells responsive to gravity changes (osteoblasts, osteocytes, osteoclasts) and determine at what level the cellular processes are altered: cell recruitment, cell activity, cell survival (apoptosis). These questions can be addressed by the combination of in vivo (histology) and in vitro (cell biology) methods. Evaluate the interaction between osteoblast lineage cells and osteoclast precursors under microgravity conditions with 1-g centrifuge controls, using the well-established ground-based experimental models. Examine if production of factors known to mediate the communication between osteoblasts and osteoclasts (such as prostaglandins, inflammatory cytokines, hemopoietic growth factors, matrix molecules, and others) are altered under microgravity conditions. Examine whether bone cells, or cells that convey the information to bone cells, respond to (a) strain in the matrix to which they are attached, (b) shear stress produced by fluid flow, and/or (c) electrical fields produced by deformation or fluid flow. This should be investigated using primarily ground-based experiments, taking advantage of the potential of current cellular and molecular biology methods (transgenic animals and the like). Identify the sensors for these perturbations (e.g., integrins, ion channels), and determine the pathways for their signal transduction and amplification. Determine if there are molecular alterations in the structure of bone matrix or bone mineral. Examine the composition of the matrix and the presence of posttranslational modifications (e.g., collagen cross-links). The discovery of new proteins and their secondary modifications warrants reexamination of this question in the context of mechanical and gravitational changes. Determine if there are changes in mineral structure (crystal size and composition), the degree of mineralization, or relationship of the bone mineral to the bone matrix. Many of these issues have been addressed in the past, but few conclusive answers are available at this point. It is also clear from this list that many experiments could be conducted in ground-based research and that solid results obtained from such investigations, especially regarding mechanisms, could increase the payoff from experiments conducted in microgravity. Equipment Needs Specific Bone mineral density measurement instrument suited for use in-flight, for humans and animals. Exercise instrumentation that provides different types of mechanical stimulation (impact-loading, muscle-mediated bone strain) for humans and animals.
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Representative terms from entire chapter: