We have conclusively established methodology to successfully induce disuse in vitro, using the Synthecon slow turning lateral vessel (rotating wall vessel) bioreactor system. Building on Year 2 experiments, we now show that three days of culture in the bioreactor consistently up-regulates disuse-associated genes like sclerostin in these osteocyte populations. Moreover, we have confirmed across independent biological replicate experiments that exposure to disuse does sensitize osteocytes to the formation of PMD during re-loading. We are now working to define the mechanism by which disuse increases susceptibility to PMD.

We have established a model of defective PMD repair in osteocytes by knockout of a protein called Prkd1, which is involved in membrane repair. This model demonstrated impaired adaptation to loading, consistent with a critical role for PMD-mediated mechanisms in bone mechanobiology. In the third year of the grant, we have conclusively established that enhancing membrane stability / repair can have therapeutic implications in terms of modifying the skeleton’s response to changes in mechanical loading. Specifically, treating mice (in vivo) or immortalized or primary osteocytes (in vitro) with a Food and Drug Administration (FDA) approved drug agent that enhances membrane stability appears to fully rescue the defects caused by impaired PMD repair from Prkd1-deficiency. In particular, we have found that treatment of cells with Poloxamer 188 rescues PMD repair rates in Prkd1-inhibited or Prkd1-knockout osteocytes to control levels, and improves postwounding survival in these cells, even though it does not appear to further enhance repair behavior in wildtype (control) cells.

We continue to support the professional development of students, having now supported a total of four PhD students, five medical students, four undergraduate students, and a high school student in completion of our funded experiments over the past two years. One PhD student has scheduled his PhD defense for January 2024. All of the students involved have received authorship on either journal manuscripts or conference abstracts stemming from their contributions. Therefore, this grant continues to support the career development of the next generation of scientists.

PLEASE NOTE: we are requesting a 1 year No Cost Extension (NCE) to conclude in vivo disuse experiments, in vivo experiments with Prkd1 and Sptbn1 CKO mouse models, and in vivo experiments with Poloxamer 188 treatment as described in our Annual Progress Report.

We have developed and validated a method to successfully induce disuse in vitro, using the Synthecon slow turning lateral vessel (rotating wall vessel) bioreactor system. In pilot experiments earlier this year, we developed cell culture protocols to seed osteocytes onto scaffold materials, expose the osteocytes to several days of disuse, and then subject them to varying levels of mechanical shear stress to simulate re-loading after disuse. We have validated this disuse model, showing that three days of culture in the bioreactor consistently up-regulates disuse-associated genes like sclerostin in these osteocyte populations. Moreover, we have developed a successful protocol to expose these cells to re-loading via fluid shear. We had initially proposed to re-seed the cells from the disuse-exposed scaffolds into flow chambers to determine effects of re-loading, but instead employed our more recently developed turbulent fluid shear stress model, finding that we are able to effectively wound the cells in situ on the scaffolds. We are excited to report that our initial hypotheses were confirmed – i.e., that exposure to disuse does appear to sensitize osteocytes to the formation of PMD during re-loading. These results are consistent with our earlier in vivo studies, and will serve as a launching point for our upcoming experiments aimed at helping osteocytes better survive and respond to the stress of re-loading after exposure to prolonged disuse.

We are also interested in understanding what signals are specifically produced in mechanically loaded bone cells (osteocytes) that develop PMD (PMD+) as compared to cells that are loaded but do not develop PMD (PMD-), and understanding how disuse impacts these signaling pathways. This will help us test and establish the importance of PMD in bone’s sensation of load, helping us to understand if this mechanism represents a viable target for modifying bone’s adaptation to changes in loading. Over the last year, we have continued experiments that mechanically load the osteocytes, sort them based on whether they developed a PMD during loading, and then analyze the molecular signature (gene expression trends) in the PMD+ as compared to PMD- cells. These studies are still ongoing, but preliminary results suggest that the PMD+ cells are critical for initiating the earliest responses to application of a mechanical load.

In the first year of the grant, we developed several genetic mouse models, one of which slowed the rate of PMD repair in osteocytes by knocking out a protein called Prkd1 which is involved in membrane repair. This model demonstrated impaired adaptation to loading, consistent with a critical role for PMD-mediated mechanisms in bone mechanobiology. In the second year of the grant, we have tested whether enhancing membrane stability / repair can have therapeutic implications in terms of modifying the skeleton’s response to changes in mechanical loading. We have been treating mice and isolated osteocytes with an FDA-approved drug agent that enhances membrane stability, and have completed experiments demonstrating that this drug fully rescues the defects caused by impaired PMD repair identified in our genetic mouse models. In particular, we have found that treatment of cells with Poloxamer 188 rescues PMD repair rates in Prkd1-inhibitired or Prkd1-knockout osteocytes to control levels, even though it does not appear to further enhance repair behavior in wildtype (control) cells. We are now in the middle of testing the in vivo effects of Poloxamer 188, administering it to Prkd1-deficient mice to determine if this treatment rescues bone adaptation in these mice. We are also investigating the effects of disuse on membrane repair rate and stability, using the bioreactor system mentioned at the beginning of this report.

We continue to support the professional development of students, having now supported a total of four PhD students, five medical students, three undergraduate students, and a high school student in completion of our funded experiments over the past two years. One PhD student has successfully advanced to candidacy and intends to defend his PhD within the next year. All of the students involved have received authorship on either journal manuscripts or conference abstracts stemming from their contributions. Therefore, this grant continues to support the career development of the next generation of scientists.

We initially proposed a two week single hindlimb immobilization model to stimulate disuse-induced loss of bone and muscle, as we had collected pilot data showing that mice would lose muscle and bone mass in this timeframe, and also show decreased evidence of osteocyte PMD in this window. This immobilization model has the advantage that one limb of the mouse is subjected to disuse, but the other remains mechanically loaded – allowing us to compare the effects of unloading within each animal, minimizing the number of animals needed for study and controlling for variability between mice. However, before embarking on our full studies, we first examined whether two weeks of immobilization was sufficient to properly cause bone loss. We were encouraged to find that an additional week of immobilization, three weeks in total, led to a more robust and reproducible loss of both muscle and bone mass than our originally proposed approach. Specifically, three weeks of immobilization significantly decreased muscle mass, cortical bone thickness, cortical bone area, and measurements of bone strength in the immobilized as compared to loaded limbs, whereas these trends were considerably weaker in the mice subjected to only two weeks of disuse. Therefore, we revised our experimental plans to focus on a 3 week immobilization model for all experiments moving forward, to ensure rigor and repeatability in our experiments.

We are also interested in understanding what signals are specifically produced in mechanically loaded bone cells (osteocytes) that develop PMD (PMD+) as compared to cells that are loaded but do not develop PMD (PMD-). This will help us test and establish the importance of PMD in bone’s sensation of mechanical loading, helping us to understand if this mechanism represents a viable target for modifying bone’s adaptation to changes in loading. Over the last year, we have developed methods to mechanically load the osteocytes, sort them based on whether they developed a PMD during loading, and then analyze the molecular signature (gene expression trends) in the PMD+ as compared to PMD- cells. These studies are still ongoing, but preliminary results suggest that the PMD+ cells are critical for initiating the earliest responses to application of a mechanical load.

In this first year of the grant, we have completed development of a genetic mouse model where we have made the osteocytes more susceptible to the development of PMD with loading (by knocking out a protein called Sptbn1,which is involved in membrane stability), and a model where we have slowed the rate of PMD repair in osteocytes (by knockout of a protein called Prkd1, which is involved in membrane repair). These models allow us to test the contribution of PMD-mediated events to bone adaptation to changes in mechanical loading. We have validated these models, showing enhanced fragility and impaired repair, respectively. Both of these models demonstrate impaired adaptation to loading, consistent with demonstrating an important role for PMD in bone mechanobiology. We intend to next subject these mice to disuse conditions, to test the effects of these genetic modifications on the response of bone to reduced mechanical loading and subsequent reloading during remobilization.

Excitingly, we are also keenly interested in testing whether enhancing membrane stability or repair can have therapeutic implications in terms of modifying the skeleton’s response to changes in mechanical loading. We have been treating mice and isolated osteocytes with an FDA (Food & Drug Administration) approved drug agent that enhances membrane stability; while this does not necessarily enhance the response of healthy mice or cells to mechanical loading, preliminary results suggest that this strategy can rescue the defects caused by impaired PMD repair identified in our genetic mouse models.

While we have encountered some difficulty in personnel/staff recruitment for this project due to the ongoing pandemic, we are happy to report that we have involved four PhD students, four medical students, two undergraduate students, and a high school student in completion of our funded experiments over the past year. One PhD student successfully defended her PhD and graduated earlier this year, our two undergraduate students received their Bachelor's Degrees, and all of the students involved have received authorship on either journal manuscripts or conference abstracts stemming from their contributions. Therefore, this grant is supporting the career development of the next generation of scientists.