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Project Title:  Investigating the Effects of Simulated Microgravity Duration and Connexin 43 Deficiency on Bone Fracture Healing Reduce
Fiscal Year: FY 2022 
Division: Human Research 
Research Discipline/Element:
TRISH--TRISH 
Start Date: 09/01/2020  
End Date: 08/31/2023  
Task Last Updated: 01/07/2024 
Download report in PDF pdf
Principal Investigator/Affiliation:   Buettmann, Evan  Ph.D. / Virginia Commonwealth University 
Address:  Department of Biomedical Engineering 
 
Richmond , VA 23284-9097 
Email: egbtg6@gmail.com 
Phone: 636-236-5676  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: Virginia Commonwealth University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Donahue, Henry  Ph.D. MENTOR: Virginia Commonwealth University 
Project Information: Grant/Contract No. NNX16AO69A-P0501 
Responsible Center: TRISH 
Grant Monitor:  
Center Contact:   
Unique ID: 14107 
Solicitation / Funding Source: 2020 TRISH-RFA-2001-PD: Translational Research Institute for Space Health (TRISH) Postdoctoral Fellowships 
Grant/Contract No.: NNX16AO69A-P0501 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:  
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Human Research Program Elements: None
Human Research Program Risks: None
Human Research Program Gaps: None
Flight Assignment/Project Notes: NOTE: End date changed to 08/31/2023 per TRISH (Ed., 8/4/22).

Task Description: POSTDOCTORAL FELLOWSHIP

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.

Research Impact/Earth Benefits: This project has the capability to address the major NASA Human Research Program (HRP) Gaps in Osteo 1 (Risk of Bone Fracture due to Spaceflight-induced Changes to Bone) and Fracture 1 (We Don’t Understand how Spaceflight Affects Fracture Healing).

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).

Task Progress & Bibliography Information FY2022 
Task Progress: 1. 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 by tail suspension (HLU), 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 (Aim 1) and Cx43 transgenic mice (Aim 2). 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.

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.

Bibliography: Description: (Last Updated: 01/11/2023) 

Show Cumulative Bibliography
 
Awards Buettmann EG. "American Society for Bone and Mineral Research (ASBMR) 2021 Meeting Research Travel Award, San Diego, October 1-4, 2021." Oct-2021
Awards Buettmann EG. "ISFR Biennial Meeting 2022 Travel Award, Edinburgh, Scotland, September 5 – 7, 2022." Sep-2022
Awards Buettmann EG. "ISFR Biennial Meeting 2022 Poster Award, Edinburgh, Scotland, September 5 – 7, 2022." Sep-2022
Awards Buettmann EG. "Virginia Commonwealth University (VCU) Postdoctoral Travel Award, May 2022." May-2022
Books/Book Chapters DeNapoli RC, Buettmann EG, Donahue HJ. "Cellular and Molecular Biology in Bone Remodeling." in "Osteoporotic Fracture and Systemic Skeletal Disorders." Ed. Takahashi, H.E., Burr, D.B., Yamamoto, N. (eds). Springer, Singapore. https://doi.org/10.1007/978-981-16-5613-2_1 , Sep-2021
Project Title:  Investigating the Effects of Simulated Microgravity Duration and Connexin 43 Deficiency on Bone Fracture Healing Reduce
Fiscal Year: FY 2021 
Division: Human Research 
Research Discipline/Element:
TRISH--TRISH 
Start Date: 09/01/2020  
End Date: 08/31/2023  
Task Last Updated: 09/15/2022 
Download report in PDF pdf
Principal Investigator/Affiliation:   Buettmann, Evan  Ph.D. / Virginia Commonwealth University 
Address:  Department of Biomedical Engineering 
 
Richmond , VA 23284-9097 
Email: egbtg6@gmail.com 
Phone: 636-236-5676  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: Virginia Commonwealth University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Donahue, Henry  Ph.D. MENTOR: Virginia Commonwealth University 
Project Information: Grant/Contract No. NNX16AO69A-P0501 
Responsible Center: TRISH 
Grant Monitor:  
Center Contact:   
Unique ID: 14107 
Solicitation / Funding Source: 2020 TRISH-RFA-2001-PD: Translational Research Institute for Space Health (TRISH) Postdoctoral Fellowships 
Grant/Contract No.: NNX16AO69A-P0501 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:  
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Human Research Program Elements: None
Human Research Program Risks: None
Human Research Program Gaps: None
Flight Assignment/Project Notes: NOTE: End date changed to 08/31/2023 per TRISH (Ed., 8/4/22).

Task Description: POSTDOCTORAL FELLOWSHIP

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.

Research Impact/Earth Benefits: The outcomes of both of these research aims 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, it can be used to inform clinical treatment strategies on Earth in fracture patients experiencing bone and muscle loss due to extended bedrest, paralysis, or tumor resection.

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.

Task Progress & Bibliography Information FY2021 
Task Progress: 1. 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 extravehicular 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 remain 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. 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.

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.

Bibliography: Description: (Last Updated: 01/11/2023) 

Show Cumulative Bibliography
 
Abstracts for Journals and Proceedings Buettmann EG, DeNapoli RC, Abraham L, Denisco JA, Lorenz MR, Donahue HJ. "Reambulation protects against hindlimb suspension induced impairments in callus and woven bone formation during murine fracture healing." American Society of Bone and Mineral Research. San Diego, California. October 1-4 2021.

Abstracts. American Society of Bone and Mineral Research. San Diego, California. October 1-4 2021. , Oct-2021

Abstracts for Journals and Proceedings Buettmann EG, DeNapoli RC, Donahue HJ. "Investigating the effects of simulated microgravity by hindlimb suspension on murine bone fracture healing." 2021 NASA Human Research Program Investigators’ Workshop, Virtual, February 1-4, 2021.

Abstracts. 2021 NASA Human Research Program Investigators’ Workshop, Virtual, February 1-4, 2021. , Feb-2021

Articles in Peer-reviewed Journals Juhl OJ 4th, Buettmann EG, Friedman MA, DeNapoli RC, Hoppock GA, Donahue HJ. "Update on the effects of microgravity on the musculoskeletal system." npj Microgravity. 2021 Jul 23;7(1):28. https://doi.org/10.1038/s41526-021-00158-4 ; PMID: 34301942; PubMed Central PMCID: PMC8302614 , Jul-2021
Articles in Peer-reviewed Journals Buettmann EG, Goldscheitter GM, Hoppock GA, Friedman MA, Suva LJ, Donahue HJ. "Similarities between disuse and age-induced bone loss." J Bone Miner Res. 2022 Jun 30;37(8):1417-34. Review. https://doi.org/10.1002/jbmr.4643 ; PubMed PMID: 35773785; PubMed Central PMCID: PMC9378610 , Jun-2022
Project Title:  Investigating the Effects of Simulated Microgravity Duration and Connexin 43 Deficiency on Bone Fracture Healing Reduce
Fiscal Year: FY 2020 
Division: Human Research 
Research Discipline/Element:
TRISH--TRISH 
Start Date: 09/01/2020  
End Date: 08/31/2022  
Task Last Updated: 10/22/2020 
Download report in PDF pdf
Principal Investigator/Affiliation:   Buettmann, Evan  Ph.D. / Virginia Commonwealth University 
Address:  Department of Biomedical Engineering 
 
Richmond , VA 23284-9097 
Email: egbtg6@gmail.com 
Phone: 636-236-5676  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: Virginia Commonwealth University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Donahue, Henry  Ph.D. MENTOR: Virginia Commonwealth University 
Project Information: Grant/Contract No. NNX16AO69A-P0501 
Responsible Center: TRISH 
Grant Monitor:  
Center Contact:   
Unique ID: 14107 
Solicitation / Funding Source: 2020 TRISH-RFA-2001-PD: Translational Research Institute for Space Health (TRISH) Postdoctoral Fellowships 
Grant/Contract No.: NNX16AO69A-P0501 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:  
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Human Research Program Elements: None
Human Research Program Risks: None
Human Research Program Gaps: None
Task Description: POSTDOCTORAL FELLOWSHIP

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.

Research Impact/Earth Benefits:

Task Progress & Bibliography Information FY2020 
Task Progress: New project FY2020.

Bibliography: Description: (Last Updated: 01/11/2023) 

Show Cumulative Bibliography
 
 None in FY 2020