NOTE: New end date is 5/19/2012 per NSSC information (Ed., 5/31/2011)

The cross-cutting area, or element, of the Bioastronautics Critical Path Roadmap (CRP) that this research project addresses is Human Health & Countermeasures (HHC). The specific health risks are the Risk of Bone Fracture and the Risk of Accelerated Osteoporosis as identified in the Bioastronautics Roadmap and the Human Research Program (HRP) Integrated Research Plan. The Gaps addressed, as defined in the HRP-IRP, are: Gap B1: a) Is there an increased lifetime risk of fragility fractures/osteoporosis in astronauts; b) is bone strength completely recovered post-flight, and does BMD reflect it; c) what are the risk factors for poor recovery of BMD/bone strength? Gap B10: How can skeletal adaptation be monitored to a) determine whether there is a plateau in bone loss, b) describe gender effects, and c) reflect changes in bone turnover/calcium kinetics?

The 2007 NASA Research Announcement (NNJ07ZSA002N) to which the proposal for this project responded included the following specific solicitation wording related to Gap B1: "There are preliminary indications that overall bone quality/strength does not recover at the same rate that bone mineral density recovers after spaceflight. It is not known if there is a long term health effect related to this discordant recovery dynamic." {emphasis added} Research proposals are solicited that directly address this relationship. The specific topic solicited is: Novel research that defines the precise relationship between long term recovery of bone mineral density and bone strength/quality, including the effects of multiple spaceflights." {emphasis added}

Specific Aim 1. To determine the precise relationships between bone mass, BMD, and bone strength during recovery from 28 days of HU. Recovery periods of 28, 56, and 84 days will be studied, representing 1, 2, and 3 times the period of HU.

Hypothesis 1A. Bone mass will recover completely by 28 to 56 days.

Hypothesis 1B. BMD will not recover as fast, or extensively, as bone mass.

Hypothesis 1C. Bone strength will not recover as fast, or extensively, as bone mass or BMD.

Hypothesis 1D. The strongest predictors of bone strength at the end of HU, and also after recovery from HU, will be a combination of bone mass, BMD, and bone organic matrix (collagen) parameters.

Specific Aim 2. To determine the precise relationships between bone mass, BMD, and bone strength after a second exposure to 28 days of HU, following an initial 28 days of HU plus a recovery period. Two recovery periods interposed between HU exposures will be examined, 28 and 56 days. The recovery periods may be modified, however, based upon results from the experiments for Specific Aim 1.

Hypothesis 2A. The initial HU exposure plus recovery will have minimal effect on decrements in bone mass for the second HU exposure. That is, reductions in bone mass for the second HU will be approximately the same as for the first HU exposure.

Hypothesis 2B. Incomplete recovery from the initial HU exposure for BMD and bone strength will cause compounding decrements in these parameters due to the second HU exposure. That is, values for these parameters will be lower after the second HU than at the end of the first HU.

Hypothesis 2C. The strongest predictors of bone strength after the second period of HU will be a combination of bone mass, BMD, and bone organic matrix (collagen) parameters.

Specific Aim 3. To characterize and compare the effects of resistance training and treadmill running during recovery from 28 days of HU on the relationships between bone mass, BMD, and bone strength.

Hypothesis 3A. Resistance training during recovery will cause BMD and bone strength to recover at a rate and to an extent similar to the recovery of bone mass, but treadmill running will not be as effective in improving the recovery of BMD and bone strength.

Hypothesis 3B. Resistance training during recovery (from an initial 28 days of HU) will significantly improve the status of BMD and bone strength, relative to bone mass, after a second exposure to 28 days of HU. Treadmill running will not be as effective in improving BMD and bone strength for the same scenario.

Three sets of experiments were conducted to address the three specific aims of the project. In all cases, adult male Sprague-Dawley rats (6-mos.-old) were used, and the period of initial HU was 28 days. Recovery can be characterized in three ways: (a) by comparing to age-matched, ambulatory cage control animals (no HU, but same age); (b) by comparing to values at the end of the initial 28 days of HU (day 0 of recovery); and (c) by comparing to values at baseline (day 0 before HU). The major outcome variables examined are bone mass (size, geometry, BMC), bone mineral density (total, cortical, cancellous BMD), and bone quality (strength, plus measures of tissue-level organic matrix). Tissue-level organic matrix assays quantify collagen content, cross-link maturity, and gene expression. These were being assessed for: (i) cortical bone in the mid-diaphysis (tibia and femur); (ii) mixed cortical and cancellous bone in the metaphysis (proximal tibia and distal femur); (iii) mixed cortical and cancellous bone in the femoral neck. Using these anatomic sites allowed evaluation of the response of both cortical and cancellous bone, both separately and combined (integrally).

Specific Aim 1. The basic design was to determine the time course of recovery in bone outcomes following an initial period of HU (28 days). The experiments were conducted in three cohorts of 45 animals each, constituting 5 animals in each of the 9 groups. Animals were acclimated for two weeks and singly housed in a temperature-controlled room with a 12:12 hour light/dark cycle. Body weights and total vBMD of the proximal tibia metaphysis were used to assign animals to groups with the goal of having equal mean values in all groups for these two variables. All HU animals were allowed access to food and water ad libitum. During the first week of the HU period, control animals were pair-fed to the HU animals to account for the typically observed reduced food intake during the transition to HU. All animal procedures were in compliance with the Texas A&M University Institutional Animal Care and Use Committee rules and regulations.

The in vivo animal portion of Experiment 2 was completed in April 2010. Based on results from Experiment 1, and also considering the need to accelerate the overall time schedule of the project, the detailed protocol for Experiment 2 was modified from that originally proposed. The 28d recovery groups were eliminated because results from Experiment 1 indicated that the vast majority of pQCT-based parameters did not recover at that point, and this was deemed inappropriate with respect to realistic crew member scenarios. A new group was added to allow assessment of recovery following the second HU exposure.

Research review meetings were held in Houston with JSC personnel in both November 2010 and November 2011. Several modifications were agreed upon to the scope and nature of the research plan as a result of these meetings. The major items were as follows:

(1) A new collaboration was agreed upon with Dr. Stefan Judex of Stony Brook University. Ex vivo specimens from the proximal tibia would be shipped to Dr. Judex for him to conduct microCT scans and analysis of the proximal tibia metaphysis.

(2) An internal collaboration was also established with muscle biologist Dr. James Fluckey of the Health & Kinesiology Department at Texas A&M University. As a result, Dr. Fluckey and/or one of his students participated in all subsequent necropsy sessions and harvested muscle specimens. The two main outcome variables from this collaboration are wet muscle weights and muscle protein fractional synthesis rates (FSR). These measures are being made on the whole posterior crural muscle complex, as well as the separate muscles (gastrocnemius, plantaris, soleus).

(3) Only one type of exercise would be used for Experiment 3. Several factors precipitated this decision. These included persisting scheduling issues, increased animal costs, additional time delays associated with the new collaborations, and logistical details involved with the exercise protocols and integrating these within a double-HU experimental scenario. The single exercise to be used would be the resistance training approach.

Unfunded Synergy Activity continued during Year 3. Specifically, Co-Investigator Dr. Martinez continued to participate in all tissue collection necropsy sessions, and not only did he collect tissues to be analyzed as prescribed for this project, but he also collected the following additional tissues for potential future analysis: • Knee Ligaments ~ both medial and lateral collateral ligaments ; • Tendons ~ both the patellar tendon and the Achilles tendon

Experiment 3 animal procedures started during Year 3 and were completed during Year 4. The goal of this phase was to characterize the effects of resistance exercise during the recovery period on the nature and extent of recovery following the initial HU exposure, and also on the response to the second HU exposure (both loss and recovery following HU).

Major Findings for Specific Aim 1.

In Vivo pQCT at the Proximal Tibia Metaphysis and Tibia Diaphysis

• Both vBMD and BMC (total, integral) at the proximal tibia metaphysis exhibit loss and recovery patterns better matching crewmember data than do similar parameters for the femoral neck.

• Cortical bone in the cortical shell of the proximal tibia metaphysis was lost primarily endocortically and recovered mainly periosteally.

• Calculated strength indices suggested a loss in strength at the tibia diaphysis, which was not confirmed with direct testing of mechanical properties. HU had no effect on maximum fracture force at mid-tibia diaphysis.

Femoral Neck Ex Vivo pQCT and Mechanical Testing

• Bone response to disuse and reloading is site-specific, as both mass and density at the femoral neck in the rat recover after twice the duration of unloading, whereas maximum force reaches age-matched control levels after only one recovery period.

• The femoral neck experienced a significant loss of maximum force due to unloading that fully recovered after 28 days. Estimated strength indices for the femoral neck indicated a recovery period of 56 days in contrast to the 28-day recovery that was observed with mechanical testing.

• Strength values are not substantially different for axial loading versus lateral loading in the adult rat HU animal model.

• Losses due to HU in strength for axial loading were greater than those for lateral loading, however.

• Losses due to HU in strength were generally greater than losses in vBMD or BMC especially for axial loading.

• NCSI was a better predictor of strength then NBSI.

Major Findings for Specific Aim 2.

In Vivo pQCT at the Proximal Tibia Metaphysis for Double-HU

• Both vBMD and BMC (total, integral) exhibit milder losses for the second HU compared to the first HU or the age–matched single HU. This suggests a possible protective effect of the initial HU.

• Bone in the cortical shell was lost primarily endocortically and recovered mainly periosteally.

• Recovery was not complete following the initial unloading cycle, and losses due to the second unloading cycle were not significant and smaller in magnitude than those due to initial exposure to hindlimb unloading.

• Rates of recovery for total BMC, vBMD, and cortical area were slower in older animals exposed to single or double HU cycles compared to recovery of younger animals exposed to a single HU bout.

Ex Vivo Mechanical Properties and Micro-CT at the Proximal Tibia Metaphysis for Double-HU

• Losses in densitometric properties were most dramatic for the first HU and for the cancellous bone compartment.

• Trabecular number (Tb.N) was minimally affected, with the greatest effects being reductions in trabecular thickness (Tb.Th).

• Mechanical properties derived from reduced platen compression (RPC) mechanical testing of machined proximal tibia metaphysis samples followed trends generally similar to densitometric measures for the trabecular compartment but the magnitudes of the effects were greater for mechanical properties. Specifically, ultimate stress showed age-related declines for age-matched controls, along with a general decrease for pre-to-post HU. However, the only significant difference from age-matched controls (-36%) was noted after the 1st HU exposure at a young age (6 months old).

• Trabecular vBMD following the initial HU cycle at a young age was only 11% lower than control values, which is much less than the 36% decline in ultimate stress, and thus demonstrates how BMD is not always an accurate predictor of mechanical strength.

Overall and taken together, these findings indicate that initial exposure to mechanical unloading does not exacerbate bone loss during a subsequent unloading period and two cycles of unloading followed by recovery does not have a cumulative net negative effect on total bone mass and density at the proximal tibia metaphysis.

Ex Vivo Results at the Distal Femur Metaphysis for Double-HU

• Losses in densitometric properties were most dramatic for the first HU and for the cancellous bone compartment.

• Mechanical properties of the cancellous bone, as estimated by RPC testing, were affected quite dramatically by the initial HU exposure. However, the effects of both HU exposures at the older age were essentially indistinguishable from age–related declines and properties.

Major Findings for Specific Aim 3.

Adding exercise during recovery between HU exposures revealed impressive and powerful benefits for the vast majority of variables measured.

• For in vivo pQCT results, both total BMC and total vBMD showed significantly enhanced recovery with the exercise added. Values not only recovered completely to control levels, but the exercise also engendered an apparent "protective" effect, as the losses for the 2nd HU were milder.

• At the femoral neck, however, the results were slightly different. Specifically, exercise produced benefits for total BMC only, with no appreciable effect on total vBMD. For total BMC, however, the benefits were much more dramatic, as the exercise produced "super-recovery," which is defined to indicate that mean values actually exceeded control animal values at the end of the exercise+recovery period.

• As was true at the proximal tibia, both BMC and vBMD exhibited a protective effect for the 2nd HU at the femoral neck.

• Perhaps the most dramatic effects of exercise were reflected by the mechanical strength of the FN. Both axial and lateral loading cases yielded super-recovery, with the maximum force for axial loading 35% higher than age-matched controls, and the maximum force for lateral loading 20% higher. A protective effect was also generated for maximum force at the FN, as the 2nd HU had no significant effect (no statistically significant changes pre- to post-HU).

• Trabecular vBMD for the non-exercise (2HU) group showed losses after the second HU exposure, and there was also a very significant age-related decline in trabecular vBMD in the weightbearing control animals. With exercise, however, trabecular vBMD increased during the 2nd recovery period to be higher than both non-exercise 2HU and AC groups (n.s.) and, in fact, restored to essentially the same level as baseline (day 0).

• As revealed by the microCT results, the enhanced trabecular vBMD was mainly the result of significant increases in trabecular thickness (Tb.Th) the following the 1st HU. Tb.Th was significantly higher than both 2HU (24.5%) and AC9 (26.3%) at the end of exercise period, and remained higher after the second HU period (21.8% and 27.6%, respectively). Rats exposed to resistance training did not exhibit increased trabecular BV/TV, however, presumably because of the significant decrease in trabecular number (Tb.N), which contributed to an increase in trabecular separation (Tb.Sp).

NOTE: New end date is 5/19/2012 per NSSC information (Ed., 5/31/2011)

The cross-cutting area, or element, of the Bioastronautics Critical Path Roadmap (CRP) that this research project addresses is Human Health & Countermeasures (HHC). The specific health risk is the Risk of Accelerated Osteoporosis as identified in the Bioastronautics Roadmap (Risk No. 1, Bone Loss, p. 19 of NASA/SP–2004–6113) and the Human Research Program (HRP) Integrated Research Plan (Risk 14.0). The Gaps addressed, as defined in the HRP-IRP, are:

B1 (Is bone strength completely recovered with recovery of BMD)

B10 (Time-course of bone degradation during missions)

The 2007 NASA Research Announcement (NNJ07ZSA002N) to which the proposal for this project responded included the following specific solicitation wording for Gap B1: "There are preliminary indications that overall bone quality/strength does not recover at the same rate that bone mineral density recovers after spaceflight. It is not known if there is a long term health effect related to this discordant recovery dynamic." {emphasis added} Research proposals are solicited that directly address this relationship. The specific topic solicited is: Novel research that defines the precise relationship between long term recovery of bone mineral density and bone strength/quality, including the effects of multiple spaceflights." {emphasis added} The research conducted as part of this project will provide unique data addressing these issues through well-controlled animal studies. The wide range of outcome variables will provide a comprehensive set of results that will give rise to new insights at the basic and applied levels.

The results and findings from Year 4 can be summarized in terms of several highlights. The microCT of the proximal tibia metaphysis results for the double-HU study follow similar trends as densitometric results from pQCT. Namely, BV/TV shows an age-related decline for control animals, a significant drop due to the 1st HU, but little effect of the 2nd HU. In contrast, both trabecular thickness and cortical shell thickness show significant decrements due to both the 1st and 2nd HU exposures. At the femoral neck (FN), both total BMC and total vBMD were negatively affected by both HU exposures. For total vBMD, however, values recovered faster after HU exposures. Biomechanical strength of the femoral neck also showed significant reductions due to both HU exposures. Recovery was even faster than vBMD though, particularly for the FN strength under axial loading.

Adding exercise during recovery between HU exposures revealed impressive and powerful benefits for the vast majority of variables measured. For in vivo pQCT results, both total BMC and total vBMD showed significantly enhanced recovery with the exercise added. Values not only recovered completely to control levels, but the exercise also engendered an apparent "protective" effect, as the losses for the 2nd HU were milder. At the femoral neck, however, the results were slightly different. Specifically, exercise produced benefits for total BMC only, with no appreciable effect on total vBMD.

For total BMC, however, the benefits were much more dramatic, as the exercise produced "super-recovery," which is defined to indicate that mean values actually exceeded control animal values at the end of the exercise+recovery period. As was true at the proximal tibia, both BMC and vBMD exhibited a protective effect for the 2nd HU at the femoral neck. Perhaps the most dramatic effects of exercise were reflected by the mechanical strength of the FN. Both axial and lateral loading cases yielded super-recovery, with the maximum force for axial loading 35% higher than age-matched controls, and the maximum force for lateral loading 20% higher. A protective effect was also generated for maximum force at the FN, as the 2nd HU had not significant effect (no statistically significant changes pre- to post-HU).

These results effectively illustrate the value and usefulness of this ground–based animal model, particularly for studying repeated exposures to simulated microgravity in a controlled fashion. Results for the initial HU exposure, and recovery there from, compare quite favorably to recent results on ISS crew members.

NOTE: New end date is 5/19/2012 per NSSC information (Ed., 5/31/2011)

The cross-cutting area, or element, of the Bioastronautics Critical Path Roadmap (CRP) that this research project addresses is Human Health & Countermeasures (HHC). The specific health risk is the Risk of Accelerated Osteoporosis as identified in the Bioastronautics Roadmap (Risk No. 1, Bone Loss, p. 19 of NASA/SP–2004–6113) and the Human Research Program (HRP) Integrated Research Plan (Risk 14.0). The Gaps addressed, as defined in the HRP-IRP, are:

B1 (Is bone strength completely recovered with recovery of BMD)

B10 (Time-course of bone degradation during missions)

The 2007 NASA Research Announcement (NNJ07ZSA002N) to which the proposal for this project responded included the following specific solicitation wording for Gap B1: "There are preliminary indications that overall bone quality/strength does not recover at the same rate that bone mineral density recovers after spaceflight. It is not known if there is a long term health effect related to this discordant recovery dynamic." {emphasis added} Research proposals are solicited that directly address this relationship. The specific topic solicited is: Novel research that defines the precise relationship between long term recovery of bone mineral density and bone strength/quality, including the effects of multiple spaceflights." {emphasis added} The research conducted as part of this project will provide unique data addressing these issues through well-controlled animal studies. The wide range of outcome variables will provide a comprehensive set of results that will give rise to new insights at the basic and applied levels.

Numerous new results were generated for a variety of tests, variables, and scenarios. This includes additional results for the loss and recovery animal experiments conducted during year 1. Because of the sequential nature of the procedures for many of the analyses, assays, and tests, results must necessarily be spread over a protracted time period following the conclusion of the animal experiment portion for each phase. The following new results were generated for the year 1 (experiment 1) phase of the project:

• Mechanical testing of the femoral neck. This included testing using both axial and lateral loading configurations, and also permitted direct comparison of mechanical strength results with pQCT results. Results showed a much more dramatic affect upon strength than bone mineral density and very little difference between axial and lateral loading cases.

• Mechanical testing of the distal femur metaphysis. Reduced platen compression (RPC) testing was used for this, and the protocols required careful and meticulous procedures for specimen preparation as well as mechanical testing. Results showed dramatic losses in trabecular bone density and strength, with the greatest losses in strength.

• Collagen biochemistry for the tibia midshaft. Results showed that HU had a strong and consistent negative affect on collagen content as well as cross-links.

• Proximal tibia micro-CT. Results were generated from our newly established collaboration with Dr. Stefan Judex at Stony Brook University. The data provide much higher resolution results and greater insight into the details of the effects of HU and recovery upon the proximal tibia metaphysis, including both trabecular bone and the cortical shell.

Additionally, numerous new results were generated for experiment 2, which consisted of two HU exposures plus recovery periods following each. Specifically, results were generated for the following:

• Longitudinal (in vivo) pQCT scans of the proximal tibia. These measurements allowed tracking of various densitometric parameters at 28-day intervals over a total of 168 days. Results indicated that previous HU exposure had no additional negative affect on the second HU, and may have even had a protective effect for some variables.

• Mechanical testing of the distal femur metaphysis. Reduced platen compression (RPC) testing was also conducted for bones harvested at each 28-day endpoint for the double–HU protocol. Once again, mechanical properties were much more dramatically affected by HU. Also, trabecular bone properties never fully recovered to baseline values although many did recover to aging control values. : The lack of recovery to baseline is consistent with reported crewmember data.

• Analysis of posterior crural (calf) muscles. Results showed that muscles were affected similarly for all three HU cases. Muscle properties recovered rapidly after each HU exposure.

Another major accomplishment during this project year was refinement and establishment of resistance exercise protocols to be used in the final set of experiments. Although the exercise protocols have been used previously other studies, there were several details that needed to be explored and updated for the current study. In particular, our goal was to exercise the animals for 7 to 8 weeks during the recovery period following the end of the first HU and until the beginning of the second HU. This entire recovery period was 8 weeks total. Thus, the procedures for operant conditioning of the animals had to be streamlined and shortened in order to allow a few days of recovery of the animals after removal from the first HU and early enough initiation of exercising in order to gain 6 to 7 weeks of meaningful exercise. This effort was enhanced significantly by our having direct interaction with Dr. James Fluckey and his graduate students here at Texas A&M University. Several technical and procedural challenges were encountered during this process but all were eventually overcome. The animal procedures for the last phase of the project (experiment 3) were initiated near the end of year 3 (in March 2011).

The cross-cutting area, or element, of the Bioastronautics Critical Path Roadmap (CRP) that this research project addresses is Human Health & Countermeasures (HHC). The specific health risk is the Risk of Accelerated Osteoporosis as identified in the Bioastronautics Roadmap (Risk No. 1, Bone Loss, p. 19 of NASA/SP–2004–6113) and the Human Research Program (HRP) Integrated Research Plan (Risk 14.0). The Gaps addressed, as defined in the HRP-IRP, are: B1 (Is bone strength completely recovered with recovery of BMD) ; B10 (Time-course of bone degradation during missions)

The 2007 NASA Research Announcement (NNJ07ZSA002N) to which the proposal for this project responded included the following specific solicitation wording for Gap B1: "There are preliminary indications that overall bone quality/strength does not recover at the same rate that bone mineral density recovers after spaceflight. It is not known if there is a long term health effect related to this discordant recovery dynamic." Research proposals are solicited that directly address this relationship. The specific topic solicited is: Novel research that defines the precise relationship between long term recovery of bone mineral density and bone strength/quality, including the effects of multiple spaceflights."

The research products to be generated from this project will mainly take the form of new knowledge about bone recovery from simulated microgravity and the response to a second exposure of simulated microgravity (following recovery from the first). In particular, insight will be gained into the interrelationships between three different, but crucial, types of bone parameters: bone mass, bone mineral density (BMD), and bone quality. Bone mass is characterized by size, shape, geometry, and bone mineral content (BMC) outcomes. Bone mineral density (BMD) has its typical meaning of the amount of mineral per unit volume, and it is thus a characteristic of bone properties at the tissue level (i.e., normalized for amount). The most basic and fundamental measure of bone quality is bone strength, and this will be measured directly with comprehensive mechanical testing of bone from multiple sites following each experiment. We will also quantify pertinent properties of the organic matrix (the unmineralized portion of bone), which is another important contributor to bone quality and forms the scaffold on which mineral nucleation occurs. Specifically, collagen content, gene expression, and cross-link maturity will be assessed. Very few of these outcome measures are available from human studies (bed-rest or flight-based). The results will define more completely the consequences of discordant recovery dynamics on long term skeletal health. The experiments will also quantify the risks of previous exposure to microgravity, plus recovery, on a second exposure to microgravity.

Three sets of experiments will be conducted to address three specific aims. In all cases, adult male Sprague-Dawley rats (6-mos.-old) will be used, and the period of initial HU will be 28 days. Recovery will be characterized in two ways: (a) by comparing to age-matched, ambulatory cage control animals (no HU, but same age); and (b) by comparing to values at the end of the initial 28 days of HU (day 0 of recovery). The major outcome variables to be examined are bone mass (size, geometry, BMC), bone mineral density (total, cortical, cancellous BMD), and bone quality (strength, plus measures of tissue-level organic matrix). Tissue-level organic matrix assays will quantify collagen content, cross-link maturity, and gene expression. These will be assessed for: (i) cortical bone in the mid-diaphysis (tibia and femur); (ii) mixed cortical and cancellous bone in the metaphysis (proximal tibia and distal femur); (iii) mixed cortical and cancellous bone in the femoral neck. Using these anatomic sites permits evaluation of the response of both cortical and cancellous bone, both separately and combined (integrally).

The main goal of Experiment 1 is to determine the extent of loss and the time course of recovery in bone outcomes following an initial period of HU (28 days). Bone mass and density outcome variables are derived from pQCT (peripherel quantitative computed tomography) scanning, which is done both in vivo (longitudinally) and ex vivo (at each time endpoint). In vivo scans are limited to the tibia midshaft and proximal metaphysis, but ex vivo scans will be made for a full slate of anatomic sites to provide an even more thorough approach. Specifically, ex vivo pQCT results will be generated at the following locations:

* Left Tibia ~ midshaft (primarily cortical bone)

~ proximal metaphysis (below knee, mixed cortical & cancellous)

* Left Femur ~ midshaft (primarily cortical bone)

~ distal metaphysis (above knee, mixed cortical & cancellous)

~ femoral neck (mixed cortical & cancellous)

A distinct advantage of animal studies is that bone strength can be measured directly using harvested bones and machined specimens. The specific sites, type of test, and bone tissue are:

* Left Tibia ~ 3-point bending of midshaft (primarily cortical bone)

~ RPC or ICC of proximal metaphysis (primarily cancellous bone)

* Left Femur ~ 3-point bending of midshaft (primarily cortical bone)

~ RPC or ICC of proximal metaphysis (primarily cancellous bone)

~ femoral neck (mixed cortical & cancellous bone)

* Right Femur ~ femoral neck (mixed cortical & cancellous bone)

Bone quality and underlying mechanisms will be studied and characterized through biochemical assays and gene expression quantification. This work will be done by Dr. Martinez at the University of Houston. The properties of the organic matrix will be assessed using remnants from mechanical testing specimens. The following outcome measures are planned:

* Collagen Concentration ~ hydroxyproline (Hyp) by HPLC

* Collage Cross-Links ~ non-reducible hydroxylysylpyridinoline (HP) by HPLC. Six genes of interest have been identified for characterization using qRT-PCR (quantitative Reverse Transcription Polymerase Chain Reaction). These genes are:

* Col1a2 and Col3a1 ~ Extra Cellular Matrix (ECM) collagen genes

* MMP2 or 13 ~ ECM resorption marker genes

* Osteocalcin ~ mineral homeostasis and osteogenic marker

* Runx2 and Osterix ~ mechanical loading/unloading transcription factors

The aim of Experiment 2 is to determine the precise relationships between bone mass, BMD, and bone strength after a second exposure to 28 days of HU, following an initial 28 days of HU plus a recovery period. Two recovery periods interposed between HU exposures will be examined, 28 and 56 days. The recovery periods may be modified, however, based upon results from Experiment 1. Experiment 3 will also follow the two-exposure procotol but with exercise (both aerobic and resistive) added during the recovery period. Data will be analyzed to characterize and compare the effects of resistance training and treadmill running during recovery from 28 days of HU on the relationships between bone mass, BMD, and bone strength.

Bone losses observed in hindlimb unloaded adult rats parallel those in humans subjected to long-duration ISS or Mir flights; hence, this ground-based animal model is highly relevant to human astronauts. The new knowledge gained from the proposed studies will provide a better understanding of the factors affecting long term astronaut health in response to periods of exposure to microgravity. The results will provide direct, quantitative, and objective evidence for better defining the risks of space travel on long term crew member health. The results will also help define which factors are most critical to monitor in assessing recovery of bone health following single or multiple missions. From the last set of experiments, exercise countermeasure efficacy will be better understood by comparing resistance training and treadmill running when used during recovery from an initial exposure to microgravity. Results will characterize the relative effectiveness of these two types of exercise in reducing the risks of initial exposure on a second exposure to microgravity.

As Year 2 ends and Year 3 begins, data analysis and interpretation of results from animal Experiments 1 and 2 remains the highest priority and continues in earnest. The in vivo animal protocols for Experiment 3 will begin in late summer or early autumn.

Several trends and conclusions are apparent from results analyzed thus far:

1. The results that most closely match astronaut data for bone mass (BMC) and density (vBMD) outcome variables are from the in vivo scans of the proximal tibia metaphysis. Loss and recovery dynamics for ex vivo scans of the distal femur metaphysis and proximal tibia metaphysis do not match as well.

2. For 'estimated' strengths, results from the ex vivo scans of the proximal femur metaphysis best match astronaut data.

3. Based on the limited results to date for mechanical testing of the femoral neck, the 'estimated' strength parameters Neck Compressive Strength Index (NCSI) and Neck Bending Strength Index (NBSI) do not predict actual measured strength values very well. Further, measured strength values to not mimick astronaut results for estimated strengths.

4. In analyzing gene expression in the tibia and femur, patterns are differentially expressed in different bones and within the same bone at different anatomic sites after HU. Also, initial reloading of 28d after HU demonstrated the greatest change in collective gene expression compared to longer durations of recovery from HU.

Another important development during Year 2 was consideration of additional analyses that could significantly leverage results from the studies. These possibilities arose from the Investigator Review Meeting held in November 2009, and also from the NASA HRP Workshop in February 2010. The specific idea is to have microCT analyses done for the metaphysis region of either the tibia or femur. This will be done on bones slated for mechanical testing, but no adverse effect is expected because microCT is non-invasive and non-destructive. Results will provide important insights into the microarchitecure of cancellous bone as well as additional density and mass outcomes.

The cross-cutting area, or element, of the Bioastronautics Critical Path Roadmap (CRP) that this research project addresses is Human Health & Countermeasures (HHC). The specific health risk is the Risk of Accelerated Osteoporosis as identified in the Bioastronautics Roadmap (Risk No. 1, Bone Loss, p. 19 of NASA/SP–2004–6113) and the Human Research Program (HRP) Integrated Research Plan (Risk 14.0). The Gaps addressed, as defined in the HRP-IRP, are: B1 (Is bone strength completely recovered with recovery of BMD) ; B10 (Time-course of bone degradation during missions)

The 2007 NASA Research Announcement (NNJ07ZSA002N) to which the proposal for this project responded included the following specific solicitation wording for Gap B1: "There are preliminary indications that overall bone quality/strength does not recover at the same rate that bone mineral density recovers after spaceflight. It is not known if there is a long term health effect related to this discordant recovery dynamic." {emphasis added} Research proposals are solicited that directly address this relationship. The specific topic solicited is: Novel research that defines the precise relationship between long term recovery of bone mineral density and bone strength/quality, including the effects of multiple spaceflights." {emphasis added}

The research products to be generated from this project will mainly take the form of new knowledge about bone recovery from simulated microgravity and the response to a second exposure of simulated microgravity (following recovery from the first). In particular, insight will be gained into the interrelationships between three different, but crucial, types of bone parameters: bone mass, bone mineral density (BMD), and bone quality. Bone mass is characterized by size, shape, geometry, and bone mineral content (BMC) outcomes. Bone mineral density (BMD) has its typical meaning of the amount of mineral per unit volume, and it is thus a characteristic of bone properties at the tissue level (i.e., normalized for amount). The most basic and fundamental measure of bone quality is bone strength, and this will be measured directly with comprehensive mechanical testing of bone from multiple sites following each experiment. We will also quantify pertinent properties of the organic matrix (the unmineralized portion of bone), which is another important contributor to bone quality and forms the scaffold on which mineral nucleation occurs. Specifically, collagen content, gene expression, and cross-link maturity will be assessed. Very few of these outcome measures are available from human studies (bed-rest or flight-based). The results will define more completely the consequences of discordant recovery dynamics on long term skeletal health. The experiments will also quantify the risks of previous exposure to microgravity, plus recovery, on a second exposure to microgravity.

Three sets of experiments will be conducted to address three specific aims. In all cases, adult male Sprague-Dawley rats (6-mos.-old) will be used, and the period of initial HU will be 28 days. Recovery will be characterized in two ways: (a) by comparing to age-matched, ambulatory cage control animals (no HU, but same age); and (b) by comparing to values at the end of the initial 28 days of HU (day 0 of recovery). The major outcome variables to be examined are bone mass (size, geometry, BMC), bone mineral density (total, cortical, cancellous BMD), and bone quality (strength, plus measures of tissue-level organic matrix). Tissue-level organic matrix assays will quantify collagen content, cross-link maturity, and gene expression. These will be assessed for: (i) cortical bone in the mid-diaphysis (tibia and femur); (ii) mixed cortical and cancellous bone in the metaphysis (proximal tibia and distal femur); (iii) mixed cortical and cancellous bone in the femoral neck. Using these anatomic sites permits evaluation of the response of both cortical and cancellous bone, both separately and combined (integrally).

The main goal of Experiment 1 is to determine the extent of loss and the time course of recovery in bone outcomes following an initial period of HU (28 days). Bone mass and density outcome variables are derived from pQCT (peripherel quantitative computed tomography) scanning, which is done both in vivo (longitudinally) and ex vivo (at each time endpoint). In vivo scans are limited to the tibia midshaft and proximal metaphysis, but ex vivo scans will be made for a full slate of anatomic sites to provide an even more thorough approach. Specifically, ex vivo pQCT results will be generated at the following locations:

* Left Tibia ~ midshaft (primarily cortical bone)

~ proximal metaphysis (below knee, mixed cortical & cancellous)

* Left Femur ~ midshaft (primarily cortical bone)

~ distal metaphysis (above knee, mixed cortical & cancellous)

~ femoral neck (mixed cortical & cancellous)

A distinct advantage of animal studies is that bone strength can be measured directly using harvested bones and machined specimens. The specific sites, type of test, and bone tissue are: * Left Tibia ~ 3-point bending of midshaft (primarily cortical bone)

~ RPC or ICC of proximal metaphysis (primarily cancellous bone)

* Left Femur ~ 3-point bending of midshaft (primarily cortical bone)

~ RPC or ICC of proximal metaphysis (primarily cancellous bone)

~ femoral neck (mixed cortical & cancellous bone)

* Right Femur ~ femoral neck (mixed cortical & cancellous bone)

Bone quality and underlying mechanisms will be studied and characterized through biochemical assays and gene expression quantification. This work will be done by Dr. Martinez at the University of Houston. The properties of the organic matrix will be assessed using remnants from mechanical testing specimens. The following outcome measures are planned:

* Collagen Concentration ~ hydroxyproline (Hyp) by HPLC

* Collage Cross-Links ~ non-reducible hydroxylysylpyridinoline (HP) by HPLC Six genes of interest have been identified for characterization using qRT-PCR (quantitative Reverse Transcription Polymerase Chain Reaction). These genes are:

* Col1a2 and Col3a1 ~ Extra Cellular Matrix (ECM) collagen genes

* MMP2 or 13 ~ ECM resorption marker genes

* Osteocalcin ~ mineral homeostasis and osteogenic marker

* Runx2 and Osterix ~ mechanical loading/unloading transcription factors

The aim of Experiment 2 is to determine the precise relationships between bone mass, BMD, and bone strength after a second exposure to 28 days of HU, following an initial 28 days of HU plus a recovery period. Two recovery periods interposed between HU exposures will be examined, 28 and 56 days. The recovery periods may be modified, however, based upon results from Experiment 1. Experiment 3 will also follow the two-exposure procotol but with exercise (both aerobic and resistive) added during the recovery period. Data will be analyzed to characterize and compare the effects of resistance training and treadmill running during recovery from 28 days of HU on the relationships between bone mass, BMD, and bone strength.

Bone losses observed in hindlimb unloaded adult rats parallel those in humans subjected to long-duration ISS or Mir flights; hence, this ground-based animal model is highly relevant to human astronauts. The new knowledge gained from the proposed studies will provide a better understanding of the factors affecting long term astronaut health in response to periods of exposure to microgravity. The results will provide direct, quantitative, and objective evidence for better defining the risks of space travel on long term crew member health. The results will also help define which factors are most critical to monitor in assessing recovery of bone health following single or multiple missions. From the last set of experiments, exercise countermeasure efficacy will be better understood by comparing resistance training and treadmill running when used during recovery from an initial exposure to microgravity. Results will characterize the relative effectiveness of these two types of exercise in reducing the risks of initial exposure on a second exposure to microgravity.

A notable achievement during the first year of the project was development of a new technique for mechanical testing of cancellous bone tissue from rat bones such as the femur and tibia. The proximal metaphysis of the tibia (below the knee) and the distal methapysis of the femur (above the knee) both typically contain significant proportions of both cortical and cancellous bone tissue. Our lab has previously developed a method for testing cancellous bone in specimens from these anatomic locations. The technique is called Reduced Platen Compression (RPC) testing and involves compression testing of specimens that contain both cortical and cancellous bone. The loading platens are reduced in size so as to contact only the cancellous bone. This method has proved useful in quantifying changes in properties of cancellous bone, but the presence of the cortical shell as part of the speciment inherently gives rise to some degree of load sharing and a stiffer response than would be obtained with totally isolated cancellous bone specimens.

Determining mechanical properties of cancellous bone tissue for the rats undergoing HU and recovery in the current project is a critical element in providing a comprehensive and thorough overall approach. While RPC testing provides an effective way to do this, an effort was undertaken during year 1 to develop a way to create totally isolated cancellous bone specimens for mechanical testing. Testing isolated samples would avoid the artifacts in RPC testing due to the presence of the cortical bone shell. Such a method was indeed successfully developed and is called Isolated Cancellous Core (ICC) testing. The first step in the process is to cut a slice from the metaphsysis in a manner similar to the slices used for RPC testing. Next, a special fixture was designed and built to hold the slice piece for proper interfacing with a diamond wire saw (Well Diamond Wire Saws, Norcross, GA, USA). With proper placement and slow rotation of the coring fixture system, small cylindrical specimens were cut. The specimens were 2.3mm in diameter and 1.5 mm in height. Specimens were successfully created, and subsequently tested, for a bones having a wide range of densities. It was particularly encouraging that viable specimens were obtained from even the lowest density bones.