3
Bone Physiology

INTRODUCTION

Loss of bone constitutes one of the best-documented and oft-studied pathological consequences of exposure to microgravity. Despite more than a decade of research aimed at understanding this phenomenon, elucidation of its cellular and molecular basis remains incomplete, and effective preventive interventions remain elusive. In general terms, bone loss must reflect a disruption of the usual tightly coupled process of bone remodeling. Multiple lines of evidence suggest that exposure to microgravity may lead both to an increased activation of osteoclast-mediated bone resorption and to an inhibition of either the proliferation or the activity of bone-forming osteoblasts. The participation of various stress-and inflammatory-related cytokines, as well as alterations in the normal endocrine regulation of bone cell function, is highly likely. Thus, one hope for effective countermeasures may lie in the use of antiresorptive medications, and another might involve interventions aimed at stimulating bone formation activity. However, whereas several potent inhibitors of bone resorption are approved for clinical use, no approved skeletally anabolic medication is available, leaving mechanical interventions as the only means at present for this purpose.

The Strategy report (NRC, 1998) called for a series of both animal and human studies related to microgravity effects on bone loss and recommended several areas of particular emphasis. These included (a) a comprehensive description of the phenomenon in humans, including the development of a careful record of skeletal changes occurring during microgravity and postflight and the time course and rate of these changes; (b) a determination of whether changes produced by microgravity in animal bones mimic human changes and whether they have a similar mechanistic basis, and a comparison of pertinent animal models in spaceflight to ground-based models (such as hindlimb unloading); (c) an investigation of the mechanism of bone loss, including identification of the responsive cells and evaluation of the extent to which bone loss is secondary to systemic effects; and (d) the development of countermeasures for spaceflight and human pathology.



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Review of NASA’s Biomedical Research Program 3 Bone Physiology INTRODUCTION Loss of bone constitutes one of the best-documented and oft-studied pathological consequences of exposure to microgravity. Despite more than a decade of research aimed at understanding this phenomenon, elucidation of its cellular and molecular basis remains incomplete, and effective preventive interventions remain elusive. In general terms, bone loss must reflect a disruption of the usual tightly coupled process of bone remodeling. Multiple lines of evidence suggest that exposure to microgravity may lead both to an increased activation of osteoclast-mediated bone resorption and to an inhibition of either the proliferation or the activity of bone-forming osteoblasts. The participation of various stress-and inflammatory-related cytokines, as well as alterations in the normal endocrine regulation of bone cell function, is highly likely. Thus, one hope for effective countermeasures may lie in the use of antiresorptive medications, and another might involve interventions aimed at stimulating bone formation activity. However, whereas several potent inhibitors of bone resorption are approved for clinical use, no approved skeletally anabolic medication is available, leaving mechanical interventions as the only means at present for this purpose. The Strategy report (NRC, 1998) called for a series of both animal and human studies related to microgravity effects on bone loss and recommended several areas of particular emphasis. These included (a) a comprehensive description of the phenomenon in humans, including the development of a careful record of skeletal changes occurring during microgravity and postflight and the time course and rate of these changes; (b) a determination of whether changes produced by microgravity in animal bones mimic human changes and whether they have a similar mechanistic basis, and a comparison of pertinent animal models in spaceflight to ground-based models (such as hindlimb unloading); (c) an investigation of the mechanism of bone loss, including identification of the responsive cells and evaluation of the extent to which bone loss is secondary to systemic effects; and (d) the development of countermeasures for spaceflight and human pathology.

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Review of NASA’s Biomedical Research Program NASA’S CURRENT RESEARCH PROGRAM IN BONE PHYSIOLOGY In the National Aeronautics and Space Administration’s (NASA’s) biomedical and countermeasure research budget in FY 1999, about $1.7 million was allocated for grants in the NRA program for studies of bone physiology. An additional $959,000 for bone research was funded through the National Space Biomedical Research Institute (NSBRI) (see Table 3.1). More than a dozen projects in the bone program are being conducted or have been completed recently in laboratories at the Ames Research Center (ARC) and Johnson Space Center (JSC), in four university-based laboratories funded through NSBRI, and in several other university laboratories through extramural grants. Studies range in scope from fundamental cellular biology of bone cells, to biomechanics, to the use of modern technology to assess bone status, to intervention research that could be incorporated into countermeasure programs. Studies at NASA centers and within NSBRI are aimed at understanding the skeletal response to mechanical loading and its deprivation and the development of countermeasures to ameliorate space-flight-related bone loss. The more basic of these projects are carried out at ARC and NSBRI, whereas interventional research is done at ARC, JSC, and NSBRI. A few service functions of the bone program related to NASA missions are handled exclusively at JSC. Basic Research Several basic research initiatives are under way. A system has been developed to study the effects of well-standardized, quantifiable loads applied to cultured bone cells. A program has been initiated to evaluate the histomorphometric and other characteristics of bone loss in hindlimb-unloaded rats. In addition, the assessment of a clinical trial of a combined intervention with exercise loading and a potent bisphosphonate should provide complementarity to ongoing bed rest studies in humans. Basic programs located at ARC constitute a major component of the overall bone program that should provide valuable information bearing directly on problems identified in the Strategy report as being of high priority. These programs include studies of musculoskeletal biomechanics as well as of fundamental bone cell biology. Current projects in bone biology involve the use of contemporary molecular biological techniques to assess the effects of mechanical loads on osteoblastic cells of varying maturity and phenotype. Recently initiated studies involve the application of mechanical loads with complete control and characterization of cycle number, rate, peak intensity, and rate of strain, in order to document changes in osteoblast regulatory genes and gene products. In accord with Strategy report recommendations, one of the NSBRI programs explores the effects of a variety of pharmacologic agonists known to interact with the estradiol, vitamin D, or calcium-sensing receptors on mature bone cells and their precursors. Another addresses the effects of unloading on bone TABLE 3.1 Summary of FY 1999 Funding for Bone Physiology   NRA   NSBRI   Subdiscipline Total ($ thousands) No. of Projects Total ($ thousands) No. of Projects Molecular and cellular 625 6 675 3 Countermeasures 1,102 6 284 1 Total 1,727 12a 959 4 aThis number may include projects that were still active but were no longer receiving funding in 1999.

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Review of NASA’s Biomedical Research Program blood flow, incorporating pharmacologic agents to define the possible roles of adrenergic and nitric oxide systems in the regulation of bone circulation. A third program concerns the development and application of newly developed computer algorithms to predict bone strength and fracture risk from noninvasive densitometric data. These algorithms can be applied to both astronaut and animal studies. Animal Studies Several animal models for microgravity-associated bone loss have been proposed, with particular attention given in recent years to a rat model involving hindlimb unloading. Although many insights have been obtained using this and other antiorthostatic models of skeletal unloading, concerns remain about their ability to provide a true reflection of what may be experienced by the human skeleton during spaceflight. In particular, it must be recognized that the number and variety of hormonal, nutrient, and other stresses applicable to the human situation may differ fundamentally from those of the hindlimb-unloaded rat. In accord with Strategy report recommendations, work at ARC continues to validate the hindlimb-unloading model and to delineate the mechanistic role of various hormones and cytokines. This work should continue to provide valuable information, particularly if molecularly based studies can be incorporated. Also in accord with Strategy report recommendations, some studies are exploring the effects of unloading on bone content of bone-specific proteins, and others are developing densitometric methods that should allow accurate descriptions of the nature and distribution of skeletal changes during unloading and spaceflight. New animal work funded in FY 1999 includes a study of skeletal development in embryonic quail. This study might eventuate in follow-up studies on the International Space Station. Also funded for FY 1999 are an assessment of the musculoskeletal effects of a growth hormone-releasing hormone agonist in unloaded mice and a study of the prevention of oxidative damage to bone during microgravity by vitamin E. Human Studies For human studies, the Strategy report recommended the development of a comprehensive description of microgravity-induced bone loss, using state-of-the art noninvasive methods entailing a careful record of skeletal changes postflight for each astronaut. It recommended initiation of a comprehensive database for the purposes of correlating skeletal changes with age, gender, muscle changes, diet, and genetic factors. To facilitate countermeasure development it recommended that the mechanisms of bone loss be defined using contemporary biochemical markers of bone turnover. Finally, it recommended that both loading and pharmacological interventions be pursued as potential countermeasures. To date, essentially no research has been conducted in accord with the first three of these recommendations. However, the opportunity to address the first two has been severely inhibited by operational constraints imposed by International Space Station (ISS) construction and by the limitation of missions to brief flights. In human studies not included in Strategy report recommendations, ARC biomechanics program scientists have developed, validated, and initiated evaluation of a shoe insert that provides quantitative information about load cycles and cycle intensity over several consecutive days. This information should permit further elucidation of the relationships between habitual mechanical loading and bone mass and architecture. In addition, limited work at ARC continues with the human head-tilt bed rest model, but the high cost of this type of study may require external collaborations in the future. Several new projects were funded for FY 1999. These include an evaluation of a skin patch for monitoring the loss of calcium in sweat and bone turnover. Validation of such a technique would greatly simplify the collection of in-flight data on mineral metabolism. Another project involves development

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Review of NASA’s Biomedical Research Program of a dual energy X-ray system for bone mineral density and diagnostic radiography. At JSC, a project has been initiated to estimate the calcium kinetics and bone turnover on spaceflight missions. This project represents a continuation of work done in association with the Mir program. To date, progress congruent with many of the Strategy report recommendations has been made in basic, animal, and human studies. Lack of progress in recommended areas appears to be largely due to limited opportunity for conducting flight experiments. PROGRAMMATIC BALANCE Balance of Subdiscipline Areas The program as a whole contains an appropriate mixture of both basic and applied studies having clear relevance to Strategy report recommendations. Among current bone-related research projects, three (two human, one animal) specifically test potential countermeasures, two assess fundamental aspects of drug and hormone action on bone that could lead to new countermeasure development, two explore innovative technologies aimed at improved countermeasures, and one provides additional validation of an animal model. Balance of Ground and Flight Investigations The Strategy report recommended a series of studies to characterize the magnitude and regional distribution of bone loss during flight, as well as postflight changes. It also recommended the establishment of a systematic database to permit correlations of skeletal changes with a number of factors, including age, gender, muscle and hormonal changes, and diet. In addition, a series of mechanistic studies were recommended to give insight into the relative contributions of bone resorption and formation to the skeletal changes of microgravity. However, the paucity of flight opportunities currently requires the bone program to be entirely ground based, and except for continuation of a calcium turnover project, no work currently addresses these recommendations. Successful completion of several of the current studies would lead logically to flight-based investigations in the future. Emphasis Given to Fundamental Mechanisms The Strategy report recommended a series of experiments designed to understand fundamental mechanisms of bone loss. At ARC, an independent program of basic cellular and molecular biology has been initiated. In addition, work with hindlimb-unloaded rodent models has components addressing fundamental hormonal responses. One of four NSBRI-funded laboratory groups is conducting an extensive cell and molecular biology program addressing skeletal aspects of reproductive and vitamin D steroid receptor physiology. Several of the extramurally funded programs also explore changes in bone cell regulation, signal transduction, and the biochemistry of bone-specific proteins during unloading. Utilization and Validation of Animal Models The primary animal model used in investigations of bone loss and its mechanisms has been the hindlimb-unloaded rat. Over the past decade a large number of studies have been published that define and refine this model as well as demonstrate its value as a surrogate for space-based bone loss. The Strategy report recommended that the distribution and characteristics of bone loss throughout the

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Review of NASA’s Biomedical Research Program skeleton be defined more completely and also that this model be the standard for comparing new models as they are introduced. The report specified that additional validational studies should involve bone histomorphometry, biomechanical and biochemical characteristics of unloaded bone, regulation of calciotropic hormones during hindlimb unloading, and features of cultured osteoprogenitor cells from unloaded animals. At present, little work has been done on these recommendations, but studies of this type are currently being initiated. Overall, the bone program shows a reasonable distribution of effort among basic cellular, animal, and human studies. The paucity of flight opportunities currently requires the bone program to be entirely ground based. However, successful completion of several of the current studies would lead logically to flight-based investigations. In consonance with Strategy report recommendations, a strong effort is being conducted to address cellular and physiological mechanisms. Studies to address recommendations regarding animal model validation are being initiated. By permitting the conduct of transgene and gene knockout experiments, extension of hindlimb-unloading studies to mice will permit studies to assess genetic contributions to skeletal response. Further validation of the hindlimb-unloaded mouse should be conducted. DEVELOPMENT AND VALIDATION OF COUNTERMEASURES Most skeletal countermeasures that have been employed to date have involved some form of mechanical loading, such as treadmill exercise, with or without bungee cords, or resistance exercise. These generally have been unsuccessful. Probably the best hope for a successful intervention lies in a program that replicates or at least approaches normal gravitational force. Compatible with a Strategy report recommendation, two countermeasure projects have been conducted. An ARC program has developed a prototype positive pressure device that permits establishment of regional areas of increased “gravitational” loading for individuals in a microgravity environment. Results of a recent JSC study show that a combination of heavy resistance exercise plus a bisphosphonate can attenuate bone loss in humans subjected to bed rest. Effective collaborations are maintained with local universities to continue this work. An additional approach could involve administration of newly developed drugs and biological agents that regulate osteoblast or osteoclast function as a countermeasure against bone loss. Despite the fact that countermeasure studies are in progress at ARC, JSC, and NSBRI laboratories, there is little evidence of coordination of these programs or, for that matter, of a system-wide coordinated process for testing and validating potential skeletal countermeasures as recommended by the Strategy report. NASA personnel at JSC seemed relatively unaware of ARC programs, and no formal mechanism exists to encourage bringing the results of these programs to program leaders at JSC. EPIDEMIOLOGY AND MONITORING Comprehensive monitoring of bone mineral density and turnover in flight crews was instituted in the past. However, despite success in obtaining sequential bone mass measurements and specimens for turnover measurements, the results of these studies are fragmentary and not generally available. The current Astronaut Medical Evaluation Requirements Document (AMERD) for missions exceeding 30 days mandates dual energy x-ray absorptiometry (DXA) assessment of body composition and bone mineral density one month prior to, and about one week after, completing the mission. The Strategy report clearly recommended the collection and maintenance of a database adequate to document and characterize the nature and distribution of bone loss during spaceflight missions. However, it does not

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Review of NASA’s Biomedical Research Program appear that a strategy for systematic analysis and interpretation of these measurements has emerged. Similarly, repository specimens of urine and blood from previous missions could be used to generate important information regarding the time course and characteristics of altered bone remodeling during flight, but plans to use these specimens for such purposes are not described in the resource documents. One problem created by the focus of current missions on Space Station construction is that the duration in microgravity for any given crew member will not exceed 30 days. Such flights would neither be covered by the requirement for DXA measurements nor be likely to provide insights relevant to long-term spaceflight. Thus, epidemiology and monitoring are currently at a standstill. However, there remains an opportunity to derive useful research information from even these short-term missions. Current practice includes collection of multiple urine and blood specimens pre- and postflight. Inclusion of in-flight data collections would provide substantial benefit, but this is not currently part of routine flight procedure. Assessment of bone turnover markers using pre-, intra-, and postflight specimens should afford valuable insights regarding the skeletal effects of flight and of countermeasure intervention. In this regard, by the terms of the ISS Medical Operations Requirements Document (MORD), in-flight health and fitness evaluations are routinely mandated to assess crew health and also to validate and adjust countermeasures (Section 3.6.5). Also mandated (Section 6.2.4.1) are individualized in-flight programs of resistive and aerobic exercise. Access to specimens and data remains problematic. Analysis of in-flight specimens for markers of bone resorption and formation would offer a unique opportunity to determine the relative efficacy of these various exercise programs. The Strategy report recommended the collection of a comprehensive database concerning changes in bone mass and turnover during spaceflight. However, no strategy for analyzing, interpreting, and disseminating the results of flight measurements currently exists. Biological specimens from previous and current missions could be used to generate important information, but no plans are described for their use. Skeletal epidemiology and monitoring are currently dormant, but opportunities exist to derive useful information even from current short-term missions. SUPPORT OF ADVANCED TECHNOLOGIES The Strategy report recommended development of both general and bone-specific equipment. General equipment concerned modifications of animal facilities, animal centrifuges, and animal handling equipment to be used during spaceflight. Bone-specific equipment included modified bone densitometers for use in humans and animals during flight and exercise instruments to apply different types of mechanical stimulation (e.g., low- and high-impact loading). No studies currently address general instrumentation needs. Bone-specific instrumentation work is summarized below. State-of-the art methodology is being applied in new ways. Application of contemporary molecular biology techniques to mechanically loaded cells, the load-sensing shoe insert, new computer algorithms for resolving bone geometry from DXA scans, and the miniature bone densitometer described above are examples of such advanced technology. In accord with Strategy report recommendations and as part of an NSBRI Technology Development Program, an innovative miniature bone densitometer is currently under development that will theoretically be sufficiently portable for skeletal monitoring to be accomplished during the course of an extended mission. Moreover, it should be possible for such an instrument to measure body composition in addition to bone mass. In accord with Strategy report recommendations for bone-specific instrumentation, the bone program appears to be providing an appropriate level of support for advanced technologies. General instrumentation needs are not currently being addressed.

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Review of NASA’s Biomedical Research Program SUMMARY The current bone program is broad in scope and includes projects that could generate countermeasure interventions. The program appears to be appropriately balanced in terms of basic and applied science. Many of the specific projects are highly consistent with goals of the Strategy report recommendations. Other specific recommendations for both animal and human research have not yet been addressed. With respect to countermeasure development, one identified problem is the apparent lack of a formal mechanism to bring potential countermeasures to formal assessment, as well as an apparently haphazard and unfocused epidemiology and monitoring component that fails to take advantage of unique and potentially valuable research data. BIBLIOGRAPHY National Aeronautics and Space Administration (NASA). 1997. Task Force Report on Countermeasures: Final Report. Washington, D.C.: NASA. NASA. 1998a. Life Sciences Program Tasks and Bibliography for FY 1998. Washington, D.C.: NASA. NASA. 1998b. International Space Station Medical Operations Requirements Document (ISS MORD), Baseline SSP 50260. Houston, Tex.: NASA. NASA and Universities Space Research Association (USRA). 1999. Proceedings of First Biennial Biomedical Investigators’ Workshop, January 11-13, 1999, League City, Texas. Houston, Tex.: NASA and USRA. National Research Council (NRC), Space Studies Board. 1998. A Strategy for Research in Space Biology and Medicine in the New Century. Washington, D.C.: National Academy Press.