NOTE: End date is now 11/30/2014 with PostDoc change in institution, per NSBRI (Ed., 1/15/14)

NOTE: End date changed to 11/30/2013 per NSBRI (Ed., 10/24/13)

Little is known about the effects of spaceflight on articular cartilage health. Unlike bone and muscle, cartilage lacks the ability to regenerate. Once a catabolic cascade is triggered, it usually results in osteoarthritis. Some studies have shown degradation of the articular cartilage in response to unloading and prolonged bedrest. However, the underlying molecular mechanisms of articular cartilage degradation in response to unloading are still elusive. The overall goal of my research is to understand and elucidate key molecular mechanisms involved in response of cartilage to changes in gravitational forces. The majority of my focus is at the cell level, with analyses of potential approaches to translate these changes to tissue and joint level. To begin to address this problem, I investigated the effects of simulated microgravity on chondrocytes using the rotating wall vessel (RWV) bioreactor. Also, in collaboration with Dr. Jeff Willey, I studied the combined effects of radiation and simulated microgravity on articular cartilage health. I found that chondrocytes exposed to simulated microgravity undergo morphological rearrangement of the actin cytoskeleton. This change was not observed in irradiated cells; however, when cells were exposed to both radiation and simulated microgravity, they developed long stress-like fibers, resembling a more fibroblast morphology as compared to the cortical control chondrocytes. Gene expression analyses confirmed that cells exposed to both radiation and simulated microgravity express more collagen I and less collagen II and aggrecan, which is characteristic for de-differentiated chondrocytes. We also determined that irradiated chondrocytes upregulate R-spondin1. R-spondin1 is a positive regular of Wnt signaling that has been shown to protect against radiation-induced damage to the oral mucosa and intestine. R-spondin1 has also been shown to protect against inflammatory bone damage in a mouse model of arthritis. Our results are interesting given R-spondin1 is upregulated in response to radiation, but not radiation and microgravity. If chondrocytes in normal gravity respond to radiation by producing "radioprotectant" genes, simulated microgravity is inhibiting that effect, making cartilage cells more susceptible to radiation-induced damage in microgravity conditions. This data shows that microgravity may inhibit the radio-protective mechanism of chondrocytes, suggesting a combined synergistic, degradatory effect of simulated microgravity and radiation, which mimics the environment of spaceflight. The mechanisms responsible for these morphological and signal transduction changes in response to simulated microgravity and radiation are still under investigation. Another unexpected and compelling finding was that chondrocytes respond to simulated microgravity by up-regulating sclerostin, an inhibitor of Wnt signaling known to induce bone density loss in space. However, according to a recent study, sclerostin may prevent cartilage from further degradation by decreasing production of matrix degrading genes, suggesting a chondro-protective role for sclerostin in this tissue. Therapeutic treatments using an antibody against sclerostin have shown promising results to prevent bone density loss in unloading conditions. However, the effects of blocking sclerostin on neighboring tissue, such as articular cartilage have not been addressed and may have adverse effects. My goal for next year is to compare results of cells exposed to microgravity to cells incubated in hydrostatic pressure, an environment known to induce expression of anabolic genes in articular cartilage. In addition, I will investigate the role of primary cilia in microgravity, an important organelle involved in mechanotransduction that has also been associated with the Wnt signaling pathway.

NOTE: End date is now 11/30/2014 with PostDoc change in institution, per NSBRI (Ed., 1/15/14)

NOTE: End date changed to 11/30/2013 per NSBRI (Ed., 10/24/13)

Little is known about the effects of spaceflight on articular cartilage health, and the synovial joint in general. Unlike bone and muscle, cartilage lacks the ability to regenerate and once a catabolic cascade is triggered, it usually results in osteoarthritis. Some studies have shown degradation of the articular cartilage in response to unloading (1) and prolonged bedrest (2). However, the underlying molecular mechanisms of articular cartilage degradation in response to unloading are still elusive. My study aims to investigate the effects of simulated microgravity on chondrocytes using the RWV bioreactor. Further, in collaboration with Dr. Jeff Willey, we are studying the combined effects of radiation and simulated microgravity in articular cartilage health. My goal is to understand the molecular mechanisms involved in response to changes in gravitational forces at the cell level, and translate these changes to tissue and whole joint level. One interesting finding is that chondrocytes exposed to simulated microgravity undergo morphological rearrangement of the actin cytoskeleton. This change was not observed in irradiated cells; however, when cells were exposed to both radiation and simulated microgravity, they developed long stress-like fibers, resembling a more fibroblast morphology compared to the cortical control chondrocytes. Gene expression analyses confirmed that cells exposed to both radiation and simulated microgravity express more collagen I and less collagen II and aggrecan, which is characteristic for de-differentiated chondrocytes. The mechanotransduction mechanisms responsible for these morphological changes in response to simulated microgravity are still under investigation. Another unexpected and compelling finding is that chondrocytes respond to simulated microgravity by up-regulating sclerostin, an inhibitor of Wnt signaling known to induce bone density loss in space. However, according to a recent study, sclerostin may prevent cartilage from further degradation by decreasing production of matrix degrading genes, suggesting a chondro-protective mechanism in this tissue (3). Therapeutic treatments using an antibody against sclerostin have shown promising results to prevent bone density loss in unloading conditions; however, the effects of blocking sclerostin on neighboring tissue, such as articular cartilage, have not been addressed and may have adverse effects. In addition, irradiated chondrocytes upregulate R-spondin1, a positive regulator of Wnt signaling. R-spondin1 has been shown to protect against radiation-induced damage to the oral mucosa and intestine. Moreover, R-spondin1 has been shown to protect against inflammatory bone damage in a mouse model of arthritis (4). Our results are interesting given R-spondin1 is upregulated in response to radiation but not radiation and microgravity. If chondrocytes in normal gravity respond to radiation by producing "radioprotectant" genes, simulated microgravity is inhibiting that effect, making cartilage cells more susceptible to radiation-induced damage in microgravity conditions. This data shows that microgravity may inhibit the radio-protective mechanism of chondrocytes, suggesting a combined synergistic, degrading effect of simulated microgravity and radiation, which mimics the environment of spaceflight.

Our goal for next year is to compare our results to cells incubated in hydrostatic pressure, an environment known to induce expression of anabolic genes in articular cartilage. In addition, we will study the role of primary cilia in microgravity, an important organelle involved in mechanotransduction that has also been associated with Wnt signaling pathway.

1. Niu, H.J. et al. (2012) Acta Mechanica Sinica 28, 1488-1493.

2. Liphardt, A.M. et al. (2009) Osteoarthritis Cartilage 17, 1598-1603.

3. Chan, B.Y., et al. (2011) Osteoarthritis Cartilage 19, 874-885.

4. Zhao, J. et al. (2009) Trends Biotechnol 27, 131-136.

Aim 2: Examine changes in cell-matrix interactions in response to simulated microgravity. Preliminary data looking at CD44 expression in chondrocytes, a receptor important in cartilage homeostasis that mediates binding to hyaluronan, did not show any changes after 48 hr exposure to simulated microgravity. A time-course study will evaluate different time points, and other important cell surface receptors, such as integrins, will be evaluated.

Aim 3: Examine the effects of simulated microgravity on the cytoskeletal morphology of chondrocytes. Chondrocytes in simulated microgravity respond by re-arranging their cytoskeletal morphology. This change is more pronounced in cells exposed to radiation and simulated microgravity. The mechanotransduction mechanisms involved with these morphological changes are still under investigation.

Our goal is to investigate the effects of simulated microgravity on cartilage homeostasis, by exposing two chondrocyte cell lines to a modeled microgravity environment using a rotating wall vessel bioreactor. Cells exposed to microgravity will be compared to cells incubated under normal gravitational conditions as a control, as well as an arthritic-like cell model induced by treating chondrocytes with pro-inflammatory cytokines such as IL-1B and oncostatin M (OSM). Disruptions in cell-matrix interactions, changes in cytoskeletal morphology and gene up-regulation will be evaluated to determine changes in chondrocyte metabolism. We hypothesize that similar to bone, cartilage homeostasis can be compromised during exposure to microgravity, resulting in osteoarthritic-like conditions in astronauts after space missions. This study will give us a better insight into whether exposure to microgravity can increase the risk of developing early stages of osteoarthritis in astronauts.

Due to the limited capacity of regeneration in articular cartilage, early detection and treatment are key components to prevent the advanced stages of cartilage degradation, which is the leading cause of disability in the U.S., limiting the activities of nearly 21 million adults. Cells incubated under simulated microgravity undergo morphological changes in the actin cytoskeleton. These changes were detected after only two days in simulated microgravity and retained for seven days. Cells under normal gravitation forces maintain the characteristic cortical morphology of healthy chondrocytes, while cells under simulated microgravity developed elongated fibers and adopted a more fibroblast-like morphology. Similar changes have been detected before in osteoarthritic chondrocytes, cells exposed to mechanical loading and cells undergoing de-differentiation. In addition, we detected several changes at the RNA level of genes associated with Wnt signaling pathway. Sclerostin, an inhibitor of Wnt signaling associated with bone density loss in space, was up-regulated in chondrocytes under simulated microgravity. There is not much known about the effects of sclerostin in articular cartilage, but a recent report suggested that sclerostin may protect cartilage from degradation because this up-regulation was also associated with decreased expression of several cartilage degrading genes. However, in addition to sclerostin up-regulation we also detected up-regulation of MMP-9 and down-regulation of aggrecan, both previously associated with arthritic conditions.

Lastly, in a collaborative effort with Dr. Jeff Willey from Wake Forest Baptist Medical Center, we are investigating the combined effects of radiation and microgravity in articular cartilage. Radiation alone up-regulated many genes involved with OSM/IL-6 signaling pathway, suggesting that these cells are activating a pro-inflammatory reaction. The morphological cytoskeleton of irradiated cells exposed to simulated microgravity had a more drastic change than that of microgravity alone. Our goal for the next year is to look at changes at the tissue level by isolating articular cartilage explants from bovine hooves and exposing them to simulated microgravity. In addition we will continue our collaborative efforts with Dr. Willey, as well as further investigating the effects of Wnt signaling pathway in chondrocytes and simulated microgravity.

Our study is the first to detect sclerostin up-regulation in chondrocytes exposed to simulated microgravity. The mechanisms and effects of sclerostin up-regulation and changes to the Wnt signaling pathway on cartilage homeostasis will be further investigated. If Wnt signaling is in fact associated with the onset of osteoarthritis as suggested recently by a few studies, the findings from our current study may help with the understanding of the underlying mechanism involved in osteoarthritis, and can help provide a new therapeutic target for the millions of people suffering from arthritis here on Earth.

Lastly, we detected the up-regulation of several genes involved in OSM/IL-6 signaling pathway in samples that received a 1Gy dose of radiation. Similar expression of these same genes have been detected in chondrocytes treated with OSM and IL-1B inflammatory cytokines, suggesting that radiation may trigger a similar reaction to that of cartilage exposed to inflammation. This is another novel finding that will require further investigation.

2: Examine changes in cell-matrix interactions in response to simulated microgravity. Hypothesis: Exposure to simulated microgravity will have an effect on cell-matrix interactions resulting in a signaling cascade that alters the catabolic rate of chondrocytes.

3: Examine the effects of simulated microgravity on the cytoskeletal morphology of chondrocytes. Hypothesis: Chondrocytes respond to mechanical loading by changing their cytoskeletal morphology. Cells will have a similar change in morphology in response to unloading. Cytoskeletal changes will be studied by immunofluorescent staining and confocal microscopy techniques.

The health of space crews during and after space missions has become a major concern of NASA. Several physiological changes have been associated with long-term exposure to microgravity, including skeletal muscles atrophy, immune system dysfunction, decreased nutrient intake, cardiovascular anomalies and bone density loss. One field that has yet to be explored is the possible effects of microgravity on the health of articular cartilage health, which is constantly exposed to mechanical forces under normal gravitational conditions on Earth. Disruption of cartilage homeostasis leads to pathological conditions such as arthritis, which according to the Center for Disease Control is the leading cause of disability in the United States.

Osteoarthritis (OA) is a degenerative joint disease that limits mobility of the affected joint due to the degradation of articular cartilage, and in advanced stages, can expose the highly innervated bone tissue, producing excruciating pain in the affected joint. Because of the limited regenerative capacity of articular cartilage, it is very important to detect early changes in the catabolic and anabolic rates that can lead to cartilage degradation.

This project’s goal is to investigate the effects of microgravity on cartilage homeostasis by exposing two chondrocyte cell lines to a modeled microgravity environment using a rotating wall vessel bioreactor. Cells exposed to microgravity will be compared to cells incubated under normal gravitational conditions as a control, as well as an arthritic-like cell model induced by treating chondrocytes with pro-inflammatory cytokines such as IL-1B and oncostatin M (OSM). Disruptions in cell-matrix interactions, changes in cytoskeletal morphology and gene up-regulation will be evaluated to determine changes in chondrocyte metabolism.

Hypothesis. Similar to bone, cartilage homeostasis can be compromised during exposure to microgravity, resulting in osteoarthritic-like conditions in astronauts after space missions.

This study will give a better insight to whether exposure to microgravity can make astronauts more prone to develop early osteoarthritis. In addition, since early stages of OA are hard to diagnose due to the lack of symptoms, the researchers also plan to investigate potential markers in the synovial fluid that can help detect early stages of OA.

Due to the limited capacity of regeneration in articular cartilage, early detection and treatment are key components to prevent the advanced stages of cartilage degradation, which is the leading cause of disability in the U.S., limiting the activities of nearly 21 million adults.