NOTE: Extended to 8/23/2015 per HRP and NSSC information (Ed., 10/21/2014)

Most ground-based microgravity models utilize rodent hindlimb suspension to simulate how reduced loading affects isolated physiologic systems. Unfortunately, results derived from these studies are difficult to directly translate to the human condition due to major anatomic and physiologic differences between rodents and humans. Specifically, the differences in rodent and human bone structures become increasingly important when studying orthopaedic issues such as bone maintenance and healing during spaceflight. For example, the basic microstructure of rodent bone, known as “plexiform” bone, lacks the osteons (Haversion systems) that are the main micro-architectural feature of human cortical bone. Furthermore, it is known that the osteogenic and healing potential of rodent bone far exceeds that of adult human tissue.

Due to these limitations in current ground-based microgravity models, there exists a need to develop a ground-based, large animal model of fracture healing in simulated weightlessness that more closely approximates the human condition as has been done in the first year of this study. This animal model should be capable of simulating a wide spectrum of microgravity and able to investigate exercise protocols that may aid in the optimization of the fracture healing cascade. Four specific aims were defined to meet these goals: 1) Develop a ground-based large animal model of bone unloading in order to simulate full weightlessness; 2) interrogate the effects of a simulated microgravity environment on bone fracture healing in a large animal model; 3) develop a computational model of weightbearing in ovine bone under different experimental conditions in order to characterize the loads experienced by the fracture site; and 4) investigate possible countermeasures to the deleterious effects of weightlessness on fracture healing.

The ground-based experiments utilizing this large animal (ovine) model directly address the need to know how varying microgravity environments affect fracture healing, as well as determining the applied loads at the fracture healing site through inverse dynamics and finite element simulations. The fracture rehabilitation protocols explored within this study will also aid in determining which mechanical environment leads to enhanced bone healing under microgravity conditions. The data produced during this study will significantly advance the basic mechanobiology of fracture healing by discerning which mechanical signals and environments facilitate enhanced bone healing.

Finally, in Specific Aim 4, two therapeutic countermeasures to the inhibited fracture healing of simulated microgravity unloading were investigated. The methodology of Specific Aim 2 was replicated, and shock wave therapy and low-intensity pulsed ultrasound were administered to animals healing in simulated microgravity and Earth gravitational loading environments. While fracture mechanical competency was not significantly altered following either countermeasure, both treatments significantly elevated osteoblast numbers and bone formation rates in simulated microgravity animals. The outcome of this study suggests that shock wave therapy and low-intensity pulsed ultrasound may be beneficial in situations involving aberrant fracture healing but elicit minimal modifications to the normal healing sequelae.

NOTE: Extended to 8/23/2015 per HRP and NSSC information (Ed., 10/21/2014)

Most ground-based microgravity models utilize rodent hindlimb suspension to simulate how reduced loading affects isolated physiologic systems. Unfortunately, results derived from these studies are difficult to directly translate to the human condition due to major anatomic and physiologic differences between rodents and humans. Specifically, the differences in rodent and human bone structures become increasingly important when studying orthopaedic issues such as bone maintenance and healing during spaceflight. For example, the basic microstructure of rodent bone, known as “plexiform” bone, lacks the osteons (Haversion systems) that are the main micro-architectural feature of human cortical bone. Furthermore, it is known that the osteogenic and healing potential of rodent bone far exceeds that of adult human tissue.

Due to these limitations in current ground-based microgravity models, there exists a need to develop a ground-based, large animal model of fracture healing in simulated weightlessness that more closely approximates the human condition as has been done in the first year of this study. This animal model should be capable of simulating a wide spectrum of microgravity and able to investigate exercise protocols that may aid in the optimization of the fracture healing cascade. Four specific aims were defined to meet these goals: 1) Develop a ground-based large animal model of bone unloading in order to simulate full weightlessness; 2) interrogate the effects of a simulated microgravity environment on bone fracture healing in a large animal model; 3) develop a computational model of weightbearing in ovine bone under different experimental conditions in order to characterize the loads experienced by the fracture site; and 4) investigate possible countermeasures to the deleterious effects of weightlessness on fracture healing.

The ground-based experiments utilizing this large animal (ovine) model directly address the need to know how varying microgravity environments affect fracture healing, as well as determining the applied loads at the fracture healing site through inverse dynamics and finite element simulations. The fracture rehabilitation protocols explored within this study will also aid in determining which mechanical environment leads to enhanced bone healing under microgravity conditions. The data produced during this study will significantly advance the basic mechanobiology of fracture healing by discerning which mechanical signals and environments facilitate enhanced bone healing.

In Specific Aim 1, a ground-based, ovine model of skeletal unloading was developed in order to simulate a microgravity loading condition. The external fixation unloading technique utilized in this model was able to induce mechanical unloading of the metatarsus and significant alterations in the relevant radiographical, biomechanical, and histomorphometric parameters characteristic of spaceflight. Specifically, the newly developed ovine model captured the characteristic decrease in osteoblast numbers and increase in osteoclast activity associated with human spaceflight. The unloading methodology developed in Specific Aim 1 was extended to the investigation of fracture healing in a simulated microgravity loading environment in Specific Aim 2. The findings of this study revealed that the mechanical loading environment dramatically affects the fracture healing cascade and resultant mineralized tissue strength, and that animals that healed in a reduced loading environment demonstrated significant reductions in healing rate and callus mechanical competency as compared to animals healing in a 1G Earth gravitational environment.

Specific Aim 3 outlined the development of a finite element of the ovine hindlimb in order to characterize the localized mechanical environment of a healing fracture in simulated microgravity and Earth gravitational environments. External fixation componentry was modeled to mimic the experimental methodology of Specific Aim 2 and correlate model predictions to experimental outcomes. The findings indicate that simulated microgravity unloading decreases hydrostatic stress and principal strain within the callus and fracture gap, resulting in primarily intramembranous bone formation rather than the endochondral bone formation pathway characteristic of Earth-based fracture healing.

Finally, in Specific Aim 4, two therapeutic countermeasures to the inhibited fracture healing of simulated microgravity unloading were investigated. The methodology of Specific Aim 2 was replicated, and shock wave therapy and low-intensity pulsed ultrasound were administered to animals healing in simulated microgravity and Earth gravitational loading environments. While fracture mechanical competency was not significantly altered following either countermeasure, both treatments significantly elevated osteoblast numbers and bone formation rates in simulated microgravity animals. The outcome of this study suggests that shock wave therapy and low-intensity pulsed ultrasound may be beneficial in situations involving aberrant fracture healing but elicit minimal modifications to the normal healing sequelae.

While the results reported in this dissertation work provide an initial foundation to the understanding of fracture healing in reduced gravitational loading environments and possible countermeasures to the negative effects of reduced mechanical loading, additional investigations are warranted. Further investigation of the dose-dependent relationship and long-term healing characteristics of shock wave therapy and low-intensity pulsed will provide valuable information regarding their efficacy as a countermeasure during long-duration spaceflight. These efforts, as well as the investigation of other countermeasures, should be part of future simulated microgravity fracture healing work.

NOTE: Extended to 8/23/2015 per HRP and NSSC information (Ed., 10/21/2014)

Most ground-based microgravity models utilize rodent hindlimb suspension to simulate how reduced loading affects isolated physiologic systems. Unfortunately, results derived from these studies are difficult to directly translate to the human condition due to major anatomic and physiologic differences between rodents and humans. Specifically, the differences in rodent and human bone structures become increasingly important when studying orthopaedic issues such as bone maintenance and healing during spaceflight. For example, the basic microstructure of rodent bone, known as “plexiform” bone, lacks the osteons (Haversion systems) that are the main micro-architectural feature of human cortical bone. Furthermore, it is known that the osteogenic and healing potential of rodent bone far exceeds that of adult human tissue.

Due to these limitations in current ground-based microgravity models, there exists a need to develop a ground-based, large animal model of fracture healing in simulated weightlessness that more closely approximates the human condition as has been done in the first year of this study. This animal model should be capable of simulating a wide spectrum of microgravity and able to investigate exercise protocols that may aid in the optimization of the fracture healing cascade. Four specific aims were defined to meet these goals: 1) Develop a ground-based large animal model of bone unloading in order to simulate full weightlessness; 2) interrogate the effects of a simulated microgravity environment on bone fracture healing in a large animal model; 3) develop a computational model of weightbearing in ovine bone under different experimental conditions in order to characterize the loads experienced by the fracture site; and 4) investigate possible countermeasures to the deleterious effects of weightlessness on fracture healing.

The ground-based experiments utilizing this large animal (ovine) model directly address the need to know how varying microgravity environments affect fracture healing, as well as determining the applied loads at the fracture healing site through inverse dynamics and finite element simulations. The fracture rehabilitation protocols explored within this study will also aid in determining which mechanical environment leads to enhanced bone healing under microgravity conditions. The data produced during this study will significantly advance the basic mechanobiology of fracture healing by discerning which mechanical signals and environments facilitate enhanced bone healing.

Aim 2: To date, the work for Specific Aim 2 is 100% complete. The findings of Specific Aim 2 have been presented at the 2014 NASA Human Research Program Investigators’ Workshop, and have been submitted to the Journal of Biomechanics.

Aim 3: Substantial progress has been made in the development of the musculoskeletal and finite element models of Specific Aim 3. To date, the musculoskeletal model has been validated and muscle forces have been incorporated in the finite element model. Additionally, the finite element model has successfully passed an in vitro and an in vivo validation process. Currently, external fixation and sham finite element models with mid-diaphyseal metatarsal fractures are being finalized. The final phase of Specific Aim 3 is ongoing and consists of utilizing the finite element models to predict the forces, stresses, and strains that are experienced at a simulated diaphyseal fracture site under varying degrees of microgravity. These predictions will be directly correlated with the histological data derived in Specific Aim 2 in order to delineate what specific mechanical signals (e.g., deviatoric stress, hydrostatic stress) are directing the fracture healing cascade under different microgravity environments.

Aim 4: Work on Specific Aim 4 has commenced with the investigation of shock wave therapy as a countermeasure to the inhibited fracture healing of microgravity. The first experimental group is currently undergoing shock wave treatment, and the expected completion date for biomechanical, microCT, and histomorphometric analyses of this portion of Specific Aim 4 is no later than November, 2014. Additionally, the in vivo investigation of low-intensity pulsed ultrasound as a countermeasure to inhibited fracture healing will commence in September, 2014 with an anticipated completion date for all biomechanical, microCT, and histomorphometric analyses no later than May 2015.

Most ground-based microgravity models utilize rodent hindlimb suspension to simulate how reduced loading affects isolated physiologic systems. Unfortunately, results derived from these studies are difficult to directly translate to the human condition due to major anatomic and physiologic differences between rodents and humans. Specifically, the differences in rodent and human bone structures become increasingly important when studying orthopaedic issues such as bone maintenance and healing during spaceflight. For example, the basic microstructure of rodent bone, known as “plexiform” bone, lacks the osteons (Haversion systems) that are the main micro-architectural feature of human cortical bone. Furthermore, it is known that the osteogenic and healing potential of rodent bone far exceeds that of adult human tissue.

Due to these limitations in current ground-based microgravity models, there exists a need to develop a ground-based, large animal model of fracture healing in simulated weightlessness that more closely approximates the human condition as has been done in the first year of this study. This animal model should be capable of simulating a wide spectrum of microgravities and able to investigate exercise protocols that may aid in the optimization of the fracture healing cascade. Four specific aims were defined to meet these goals: 1) Develop a ground-based large animal model of bone unloading in order to simulate full weightlessness; 2) interrogate the effects of a simulated microgravity environment on bone fracture healing in a large animal model; 3) develop a computational model of weightbearing in ovine bone under different experimental conditions in order to characterize the loads experienced by the fracture site; and 4) develop treadmill protocols that enhance bone fracture healing in the presence of simulated microgravity.

The ground-based experiments utilizing this large animal (ovine) model directly addresses the need to know how varying microgravity environments affect fracture healing, as well as determining the applied loads at the fracture healing site through inverse dynamics and finite element simulations. The fracture rehabilitation protocols explored within this study will also aid in determining which mechanical environment leads to enhanced bone healing under microgravity conditions. The data produced during this study will significantly advance the basic mechanobiology of fracture healing by discerning which mechanical signals and environments facilitate enhanced bone healing.

Aim 2: Solid progress has been made in determining the effects of simulated microgravity on haversian bone healing in Specific Aim 2. Utilizing the previously characterized external fixation device, simulated microgravity was induced for a period of 3 weeks in an animal model resulting in a mean 18% loss in metatarsal bone mineral density. Following the 3-week simulated microgravity exposure period, a 3.0 mm ostectomy was created at the mid-diaphysis of the metatarsal bone and stabilized via an orthopaedic locking plate instrumented with a strain gage. Inhibited in vivo fracture healing occurred in the Microgravity Group as evidenced by an 18% percent increase in orthopaedic plate strain over the 4-week healing period versus a 98% decrease in orthopaedic plate strain in the Earth gravity control group. These findings were further substantiated by biomechanical four-point bending and micro-computed tomography results (µCT) which displayed a statistically significant 88% (p<0.01) decrease in 4-point bending stiffness between the Microgravity and Control Groups as well as an 11-fold (p<0.01) decrease in callus bone volume between the two groups.

Aim 3: Computational models of the sheep hindlimb were created in order to delineate the specific mechanical signals responsible for directing the fracture healing cascade. A musculoskeletal model of the full ovine hindlimb was created from the phalanges to the hip complete with all relevant muscular structures. Physiologic muscle moment arms and attachment sites of the limb were experimentally determined, and proper anthropometric properties of the hindlimb were acquired via dual-energy x-ray absorptiometry (DEXA) scans. In order to validate the musculoskeletal model, a treadmill experiment was performed wherein in vivo hindlimb motion were quantified via three-dimensional stereophotogrammetry, ground-reaction forces of the limb were acquired via a force plate positioned beneath the treadmill belt, and muscle activation was measured via electromyography (EMG) for speeds ranging from 0.25m/s to 1.0 m/s. Muscle activation predictions from the musculoskeletal model were then compared to the experimentally-derived EMG measurements for the various gate speeds in order to verify the model.

A finite element model of the hindlimb was created consisting of the phalanges, metatarsus, hock joint, tibia, and relevant ligamentous structures. Transversely isotropic material properties were assigned to the cortical and cancellous bone constituents while the articular cartilage was modeled with a mooney-rivlin hyperelastic material definition. The finite element model was validated via comparison to experimentally-derived metatarsal surface strain readings as well as three-dimensional stereophotogrammetry motion tracking during simulated hindlimb compression. Metatarsal surface strain and joint rotation predictions of the finite element model were within one standard deviation of the experimental values indicating satisfactory validation of the model.

The remaining three months in Year 2 will be utilized to complete the sample size of the in vivo fracture healing study. Additionally, decalcified and undecalcified histomorphometric analysis will be performed to quantify bone volume, mineralizing surface, mineral apposition rate, bone formation rate, and osteoblast and osteoclast numbers in the fracture callus and within the fracture gap. Computationally, model validation will be completed, and muscle forces predicted by the musculoskeletal model will be incorporated into the finite element model to begin ascertaining which specific mechanical signals are responsible for driving the fracture healing cascade.

Based upon the data generated to date, it is expected that the additional specimens of Specific Aim 2 will conclusively and statistically demonstrate that the mechanical unloading associated with spaceflight significantly inhibits haversian bone healing. The findings of Specific Aim 2 will motivate Specific Aim 4 in which therapeutic interventions capable of increasing the fracture healing cascade during simulated microgravity will be investigated with the direct application to human spaceflight.

Most ground-based microgravity models utilize rodent hindlimb suspension to simulate how reduced loading affects isolated physiologic systems. Unfortunately, results derived from these studies are difficult to directly translate to the human condition due to major anatomic and physiologic differences between rodents and humans. Specifically, the differences in rodent and human bone structures become increasingly important when studying orthopaedic issues such as bone maintenance and healing during spaceflight. For example, the basic microstructure of rodent bone, known as “plexiform” bone, lacks the osteons (Haversion systems) that are the main micro-architectural feature of human cortical bone. Furthermore, it is known that the osteogenic and healing potential of rodent bone far exceeds that of adult human tissue.

Due to these limitations in current ground-based microgravity models, there exists a need to develop a ground-based, large animal model of fracture healing in simulated weightlessness that more closely approximates the human condition as has been done in the first year of this study. This animal model should be capable of simulating a wide spectrum of microgravities and able to investigate exercise protocols that may aid in the optimization of the fracture healing cascade. Four specific aims were defined to meet these goals: 1) Develop a ground-based large animal model of bone unloading in order to simulate full weightlessness; 2) interrogate the effects of a simulated microgravity environment on bone fracture healing in a large animal model; 3) develop a computational model of weightbearing in ovine bone under different experimental conditions in order to characterize the loads experienced by the fracture site; and 4) develop treadmill protocols that enhance bone fracture healing in the presence of simulated microgravity.

Computational models of the sheep hindlimb have also been created to assess the loading generated by the musculature and the stresses in the individual bones. A CT scan of a sheep hindlimb was utilized to construct the geometry of a musculoskeletal and finite element model. Appropriate muscles, tendons, and ligaments were attached to the musculoskeletal model, and validation will be performed via ground reaction forces and three-dimensional motion capture data acquired on a custom-built force measuring treadmill. The three-dimensional surfaces from the CT scan were then meshed and imported the finite element solver software. Material properties were assigned, and ligaments were attached. Mesh optimization and model validation will be performed via in vivo strain and kinematic measurements. The muscle and joint reaction forces from the musculoskeletal model will serve as inputs to the finite element model to quantify strain levels experienced in the metatarsal bone at various microgravity levels.

The remaining three months in Year 1 will complete the sample size of the in vivo animal model development, and computational model validation. Based upon the data generated to date, it is expected that these animal specimens will conclusively and statistically demonstrate that the current ovine microgravity model effectively simulates the decline in bone tissue (and associated mechanical properties) that accompanies long-term spaceflights. The architectural and physiological similarities between ovine and human bone will allow future results regarding bone fracture healing in various simulated microgravity environments using this model directly applicable to human spaceflight.