Astronauts exposed to long periods of unloading due to extended spaceflight experience on average a decrease in bone strength at 2.0 - 2.5% per month. This sharp decline in bone strength can predispose astronauts to fragility fractures, especially when re-entering a gravity-based loading environment due to extra-vehicular activities, extraterrestrial exploration, off-nominal spacecraft landings, and or upon return to Earth. While emerging evidence suggests that unloading, as would occur in microgravity during spaceflight, impairs fracture healing, the cellular and molecular mechanisms by which this occurs largely remains unknown due to a lack of ground-based rodent analog models mimicking spaceflight conditions. Understanding the mechanisms underlying the microgravity-induced impairment in bone regeneration following fracture will lead to the development of new countermeasure targets. One potential countermeasure target is Connexin 43 (Cx43), the primary gap junction protein in bone. Gap junctions facilitate intercellular communication between neighboring bone cells such as osteoblasts and osteocytes and have been strongly implicated in bone fracture healing and bone adaptation to the mechanical environment.

In order to study how the duration of microgravity and Cx43 affect fracture healing outcomes, a novel murine-healing model undergoing different periods of unloading before and during fracture healing, will be developed and characterized. This model will be created by combining the ground-based microgravity analog, hindlimb tail unloading, in conjunction with an established mouse endochondral bone healing model, the stabilized open surgical femoral fracture model. Bone healing outcomes via molecular, histological, mechanical and cellular techniques, will be evaluated in wildtype and Cx43 transgenic mice. Biomarker characterization of healing progression will be evaluated. The outcomes of this research will provide better mechanistic insight into how microgravity and gravitational reloading such as that found during spaceflight and terrestrial exploration, respectively, affects bone healing. Furthermore, this proposal will identify whether possible treatment strategies targeting Cx43, and or other biological targets, is an efficacious approach to augment bone healing during microgravity.

This is the first project to look at mechanical loading interventions and targeted gene deletions using transgenic mice to increase fracture healing during simulated microgravity. If successful, we can combat multiple musculoskeletal health risks (bone, muscle) of spaceflight using a single countermeasure (mechanical loading, Cx43 deletion, and or exercise).

Our Aim 2 result demonstrated that constitutive DMP1 Cre Cx43 deficient mice, have noticeable GJA1 recombination in skeletal muscle and bone. We observed no protection with Cx43 deficiency against HLU induced intact contralateral bone loss or improvements in callus mineralization by microCT. However, our results did show protection against HLU induced muscle atrophy based on muscle force generation in vivo.

These findings support our Aim 1 hypotheses and mirror the poor/delayed hard and soft callus formation seen in rodent models of spaceflight and the ground based analog hindlimb suspension. Our Aim 2 results using the noninducible DMP1-Cre Cx43 fl/fl mice, which show a lack of a protective effect of DMP1 Cx43 deficiency on bone and fracture healing during HLU refutes our initial hypothesis but does suggest it may have potential benefits on muscle strength. This project supports the collective consensus that musculoskeletal health and bone fracture healing is extremely altered by long-duration microgravity exposure and that gravitational reloading and Cx43 deficiency may alleviate some but not all these of these impairments. These results also have clinical parallels to fracture during extended bedrest and paralysis and suggest that skeletal fracture and altered healing is imminent in any scenario with prolonged unloading. Additional countermeasure testing looking at acute mechanical loading of fractures during simulated microgravity exposure are forthcoming. Additional countermeasure testing looking at partial weight-bearing and galactic cosmic radiation exposure during healing are necessary to understand longterm risks of impaired fracture healing on the Moon or Mars.

Astronauts exposed to long periods of unloading due to extended spaceflight experience on average a decrease in bone strength at 2.0 - 2.5% per month. This sharp decline in bone strength can predispose astronauts to fragility fractures, especially when re-entering a gravity-based loading environment due to extra-vehicular activities, extraterrestrial exploration, off-nominal spacecraft landings, and or upon return to Earth. While emerging evidence suggests that unloading, as would occur in microgravity during spaceflight, impairs fracture healing, the cellular and molecular mechanisms by which this occurs largely remains unknown due to a lack of ground-based rodent analog models mimicking spaceflight conditions. Understanding the mechanisms underlying the microgravity-induced impairment in bone regeneration following fracture will lead to the development of new countermeasure targets. One potential countermeasure target is Connexin 43 (Cx43), the primary gap junction protein in bone. Gap junctions facilitate intercellular communication between neighboring bone cells such as osteoblasts and osteocytes and have been strongly implicated in bone fracture healing and bone adaptation to the mechanical environment.

In order to study how the duration of microgravity and Cx43 affect fracture healing outcomes, a novel murine-healing model undergoing different periods of unloading before and during fracture healing, will be developed and characterized. This model will be created by combining the ground-based microgravity analog, hindlimb tail unloading, in conjunction with an established mouse endochondral bone healing model, the stabilized open surgical femoral fracture model. Bone healing outcomes via molecular, histological, mechanical and cellular techniques, will be evaluated in wildtype and Cx43 transgenic mice. Biomarker characterization of healing progression will be evaluated. The outcomes of this research will provide better mechanistic insight into how microgravity and gravitational reloading such as that found during spaceflight and terrestrial exploration, respectively, affects bone healing. Furthermore, this proposal will identify whether possible treatment strategies targeting Cx43, and or other biological targets, is an efficacious approach to augment bone healing during microgravity.

This is the first project to look at mechanical loading interventions and targeted gene deletions using transgenic mice to increase fracture healing during simulated microgravity. If successful, we can combat multiple musculoskeletal health risks (bone, muscle) of spaceflight using a single countermeasure (mechanical loading, Cx43 deletion, and or exercise).

2. Characterization of bone muscle and callus formation in a novel murine healing model undergoing different periods of unloading before and during fracture healing in male and female wildtype mice has yielded key insights. First, we demonstrated that gravitational reloading for 2 weeks during fracture healing following spaceflight-like conditions resulted in similar gastrocnemius (hybrid - mainly fast twitch) but not soleus (slow twitch) muscle mass to control levels. Second, following these two weeks post-fracture, diaphyseal (cortical) and epiphyseal (cancellous) bone volume fraction increased from HLS but didn't reach control levels. Looking at the injured limb, semi-automated micro-CT analysis showed significantly reduced callus size and mineralized femoral callus bone formation with continued HLS compared to ground controls after 14 days of fracture healing. In addition, even with a period of HLS for 3 weeks, normal reambulation during bone healing partially restored callus bone formation and fully restored callus volume to control levels. Automated histological analysis using a proprietary machine learning algorithm from these same femurs reinforced micro-CT results by showing significantly reduced callus cross-sectional area and bone formation accompanied by increased callus osteoclast activity and reduced cartilage formation in HLS versus reambulated and ground control groups. Trends suggest that DMP1 Cx43 may protect from metaphyseal but not cortical bone loss or muscle atrophy with HLS. Trends suggest that DMP1 Cx43 may decrease fracture healing on ground but lead to improved healing during HLS.

3. These findings support our hypotheses and mirror the poor/delayed hard and soft callus formation seen in rodent models of spaceflight and the ground based analog hindlimb suspension. The improved osteochondral callus formation with gravitational reloading, despite a prior history of simulated microgravity exposure, suggests that normal gravitational loading immediately following fracture can overcome some of the negative aspects of extended unloading on bone healing. This demonstrates that small amounts of artificial loading at the fracture sight during prolonged unloading may be beneficial to fracture healing. This knowledge has broadened the scope of the project to now include direct mechanical loading and or exercise as potential countermeasures for impaired fracture healing as well during spaceflight. Furthermore, Cx43 deficiency in DMP1 cells (osteocytes) may preserve bone mass and improve fracture healing at certain anatomical sites during simulated microgravity but more data is needed for validation before Cx43's role as a countermeasure target would increase in readiness level.

4. Research for the upcoming year will be aimed at further interrogation of the mechanistic role of Cx43 in altering fracture repair due to simulated microgravity in mature osteoblasts and osteocytes using a noninducible transgenic mouse model (DMP1-Cre CX43 fl/fl) and now inducible model (DMP1-CreERT2 CX43 fl/fl). This will allow us to determine if our Cx43-deficient fracture phenotype during simulated microgravity is due to confounding bone developmental changes or rather changes in immediate mechanosensation during healing. We will also determine the optimal loading regimen for normal bone repair during HLS using direct bone loading and Optogenetics to stimulate muscle contraction during simulated microgravity exposure. This work will inform exercise interventions and the potential of optogenetics as effective countermeasures for improving muscle function and bone healing during spaceflight.

Astronauts exposed to long periods of unloading due to extended spaceflight experience on average a decrease in bone strength at 2.0 - 2.5% per month. This sharp decline in bone strength can predispose astronauts to fragility fractures, especially when re-entering a gravity-based loading environment due to extra-vehicular activities, extraterrestrial exploration, off-nominal spacecraft landings, and or upon return to Earth. While emerging evidence suggests that unloading, as would occur in microgravity during spaceflight, impairs fracture healing, the cellular and molecular mechanisms by which this occurs largely remains unknown due to a lack of ground-based rodent analog models mimicking spaceflight conditions. Understanding the mechanisms underlying the microgravity-induced impairment in bone regeneration following fracture will lead to the development of new countermeasure targets. One potential countermeasure target is Connexin 43 (Cx43), the primary gap junction protein in bone. Gap junctions facilitate intercellular communication between neighboring bone cells such as osteoblasts and osteocytes and have been strongly implicated in bone fracture healing and bone adaptation to the mechanical environment.

In order to study how the duration of microgravity and Cx43 affect fracture healing outcomes, a novel murine-healing model undergoing different periods of unloading before and during fracture healing, will be developed and characterized. This model will be created by combining the ground-based microgravity analog, hindlimb tail unloading, in conjunction with an established mouse endochondral bone healing model, the stabilized open surgical femoral fracture model. Bone healing outcomes via molecular, histological, mechanical and cellular techniques, will be evaluated in wildtype and Cx43 transgenic mice. Biomarker characterization of healing progression will be evaluated. The outcomes of this research will provide better mechanistic insight into how microgravity and gravitational reloading such as that found during spaceflight and terrestrial exploration, respectively, affects bone healing. Furthermore, this proposal will identify whether possible treatment strategies targeting Cx43, and or other biological targets, is an efficacious approach to augment bone healing during microgravity.

So far, we have demonstrated that simulated microgravity exposure leads to bone and muscle degradation and impairs fracture healing. However, weight-bearing reambulation (gravitational reloading) following simulated microgravity exposure can improve fracture healing by increasing cartilage and bone formation. Therefore, mechanical loading following disuse, as would occur with spaceflight, extended bedrest, and natural aging has the potential to improve bone repair.

However, many individuals suffering from disuse-induced bone and muscle loss, such as found during spaceflight, may have difficulties exercising the injured limb due to locomotor impairments from pain, paralysis, or cardiovascular complications. Therefore, this proposal will identify whether possible therapeutic treatment strategies targeting Cx43, and or other biological targets, is an efficacious approach to augment bone healing during spaceflight conditions.

2. Characterization of a novel murine healing model undergoing different periods of unloading before and during fracture healing in wildtype mice has yielded key insights. Semi-automated micro-computed tomography (micro-CT) analysis has shown significantly reduced callus size and mineralized callus bone formation with continued hindlimb suspension (HLS) compared to ground controls after 14 days of fracture healing. In contrast, even with a period of HLS for 3 weeks, normal reambulation during bone healing fully restored callus bone formation and partially restored callus volume to control levels. Automated histological analysis using a proprietary machine-learning algorithm from these same femurs reinforced micro-CT results by showing trends toward reduced callus bone content, increased callus osteoclast activity, and reduced cartilage formation in HLS versus reambulated and ground control groups.

3. These findings support our hypotheses and mirror the poor/delayed hard and soft callus formation seen in rodent models of spaceflight and the ground-based analog hindlimb unloading. The normal osteochondral callus formation with reambulation, despite a prior history of unloading, suggests that normal gravitational loading immediately following fracture can overcome the negative aspects of extended unloading on bone healing. This demonstrates that small amounts of artificial loading at the fracture site during prolonged unloading may be beneficial to fracture healing.

4. Research for the upcoming year will be aimed at biomarker discovery for molecular targets regulating musculoskeletal mechanosensation, and/or regeneration, using our newly developed fracture healing model of bone unloading. Biomarkers will be assayed via high throughput functional gene expression modalities and correlated to fracture healing progression. Special attention to biological processes implicated in bone pathologies associated with disuse and aging, such as angiogenesis, oxidative stress, senescence, autophagy, wingless/integrated (WNT) signaling, and inflammation, will be assessed via callus gene expression during mechanical unloading and reambulation during the early stages of fracture repair. This will increase our knowledge of putative molecular targets for augmenting bone mass and fracture healing during prolonged unloading found during spaceflight. Furthermore, the role of Cx43 in mature osteoblasts and osteocytes using a transgenic mouse model (DMP1-Cre CX43 fl/fl) will be utilized to assess the role of bone targeted Cx43, as a potential countermeasure, in alleviating disuse and fracture healing associated with bone formation impairments during spaceflight.

Astronauts exposed to long periods of unloading due to extended spaceflight experience on average a decrease in bone strength at 2.0 – 2.5% per month. This sharp decline in bone strength can predispose astronauts to fragility fractures, especially when re-entering a gravity-based loading environment due to extra-vehicular activities, extraterrestrial exploration, off-nominal spacecraft landings, and or upon return to Earth. While emerging evidence suggests that unloading, as would occur in microgravity during spaceflight, impairs fracture healing, the cellular and molecular mechanisms by which this occurs largely remains unknown due to a lack of ground based rodent analog models mimicking spaceflight conditions. Understanding the mechanisms underlying the microgravity induced impairment in bone regeneration following fracture will lead to the development of new countermeasure targets. One potential countermeasure target is Connexin 43 (Cx43), the primary gap junction protein in bone. Gap junctions facilitate intercellular communication between neighboring bone cells such as osteoblasts and osteocytes and have been strongly implicated in bone fracture healing and bone adaptation to the mechanical environment.

In order to study how the duration of microgravity and Cx43 affect fracture healing outcomes, a novel murine healing model undergoing different periods of unloading before and during fracture healing will be developed and characterized. This model will be created by combining the ground-based microgravity analog, hindlimb unloading, in conjunction with an established mouse endochondral bone healing model, the stabilized open surgical femoral fracture model. Bone healing outcomes via molecular, histological, mechanical, and cellular techniques will be evaluated in wildtype and Cx43 transgenic mice. Furthermore, biomarker characterization of healing progression will be evaluated. The outcomes of this research will provide better mechanistic insight into how microgravity and gravitational reloading such as that found during spaceflight and terrestrial exploration, respectively, affects bone healing. Furthermore, this proposal will identify whether possible treatment strategies targeting Cx43 and or other biological targets is an efficacious approach to augment bone healing during microgravity.