Biophysical Regulation of Stem Cell Osteogenic Lineage Commitment: A Role for the Cytoskeleton
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STAVENSCHI, ELENA, Biophysical Regulation of Stem Cell Osteogenic Lineage Commitment: A Role for the Cytoskeleton, Trinity College Dublin.School of Engineering, 2019Download Item:
Abstract:
Osteoporosis is a debilitating disease characterised by weakened bone architecture due to increased resorption activity of osteoclasts and diminished bone deposition by mesenchymal stem cell (MSC) derived osteoblasts. Current osteoporosis treatments target activity of osteoclasts but fail to promote bone formation. Bone formation is modulated by biophysical cues and MSCs play an important role in this process by undergoing osteogenic lineage commitment to sustain loading induced adaptation. However, if and how mechanical cues developed during locomotion in the bone marrow niche directly affect the ability of MSCs to commit towards the osteogenic lineage is unknown. Understanding how biophysical cues can modulate MSC osteogenic lineage commitment will enable the identification of novel anabolic therapeutic targets to treat bone disorders such as osteoporosis.
The overall objective of this thesis is to investigate if and how changes in the marrow mechanical environment can directly regulate the osteogenic lineage commitment of MSCs. Specifically, to investigate the effect of bone marrow associated physiologically relevant 1) oscillatory fluid flow induced shear stress and 2) pressure mechanical cues on the ability of MSCs to undergo osteogenic lineage commitment. Furthermore, the mechanisms by which MSCs responds to the pressure biophysical cues will be explored, focusing on 3) cytoskeletal elements and 4) the cytoskeletal extension, the primary cilium.
Initially, a systematic approach was employed to determine whether bone marrow physiologically relevant fluid flow induced shear stress can directly affect the ability of MSCs to undergo osteogenic lineage commitment. MSCs were exposed to oscillatory fluid flow (OFF) of 1Pa, 2Pa and 5Pa shear stress magnitudes at human locomotion associated frequencies of 0.5Hz, 1Hz and 2 Hz for 1hr, 2hrs and 4hrs of stimulation. In this study it was found that OFF elicits a positive osteogenic response in MSCs in a shear stress magnitude, frequency, and duration dependent manner that is gene specific. Based on the mRNA expression of osteogenic markers Cox2, Runx2 and Opn after short-term fluid flow stimulation, it was identified that a regime of 2Pa shear stress magnitude and 2Hz frequency induces the most robust and reliable upregulation in osteogenic gene expression. Furthermore, long-term mechanical stimulation utilising this regime, elicits a significant increase in collagen and mineral deposition demonstrating that mechanical stimuli predicted within the marrow is sufficient to directly promote osteogenesis.
In the next chapter, the effect of physiologically relevant pressure cues was explored since fluid flow within the marrow is dependent on pressurisation effects and little is known about its involvement in MSC osteogenesis. A systematic approach was employed whereby MSCs were exposed to cyclic hydrostatic pressures (CHP) of 10kPa, 100kPa and 300kPa magnitudes at frequencies of 0.5Hz, 1Hz and 2Hz for 1hr, 2hrs and 4hrs of stimulation. CHP was found to elicit a positive but variable early osteogenic response in MSCs in a magnitude and frequency dependent manner that is gene specific. COX2 expression elicited magnitude dependent effects which were not present for RUNX2 and OPN mRNA expression. However, the most robust pro-osteogenic response was found at the highest magnitude (300kPa) and frequency regimes (2Hz). Interestingly, long-term mechanical stimulation utilising 2Hz frequency elicited a magnitude dependent release of adenosine triphosphate (ATP) however, all magnitudes promoted similar levels of collagen synthesis and significant mineral deposition, demonstrating that lineage commitment is CHP magnitude independent. Therefore, this study demonstrates that physiological levels of pressures, as low as 10kPa, within the bone can drive MSC osteogenic lineage commitment.
In the last two studies, the involvement of the cytoskeleton in transduction of pressure cues into an osteogenic response were explored. Initially, it was demonstrated that CHP is a potent mediator of cytoskeletal reorganisation and increases in early osteogenic responses in MSCs. In particular, the intermediate filament (IF) network associated cytoskeletal element was shown to undergo breakdown and reorganisation with a recoiling effect towards the perinuclear region. Furthermore, this IF remodelling was found to be paramount for loading induced MSC osteogenesis, revealing a novel mechanism of MSC mechanotransduction. In addition, it was identified that chemical disruption of intermediate filaments with Withaferin A induces a similar mechanism of IF breakdown and remodelling and subsequent increase in osteogenic gene expression in MSCs, exhibiting a potential mechanotherapeutic effect to enhance MSC osteogenesis. This study, therefore, highlights a novel mechanotransduction mechanism of pressure-induced MSC osteogenesis involving the understudied cytoskeletal structure, the intermediate filament, and demonstrates a potential new therapy to enhance bone formation in bone loss diseases such as osteoporosis.
Lastly, the role of cytoskeletal extension, the primary cilium was examined in relation to MSC associated pressure mechanotransduction. CHP stimulation of MSCs elicits a rapid release of ATP into the extracellular space, a reorganization of the intermediate filament network with concentration in the perinuclear region and an inhibition of proliferation. Furthermore, CHP results in a modulation of the primary cilium length. Importantly, the integrity of the primary cilium was found to be paramount for pressure induced ATP release and inhibition of proliferation, demonstrating a novel role for the cilium in MSC pressure mechanotransduction. Moreover, primary cilium expression was determined to play a pivotal role in pressure induced remodelling of intermediate filament network, which indicates that the cilium may be upstream of this IF mechanism demonstrated previously. Overall, this study highlights a novel pressure induced mechanotransduction mechanism pertaining to the expression of the primary cilium, which mediates purinergic signalling, anti-proliferative and cytoskeletal remodelling effects in response to pro-osteogenic pressure stimuli.
To conclude, in this thesis it was demonstrated that MSCs are mechanosensitive to physiological bone marrow associated mechanical cues such as fluid shear and pressure and they can have a direct effect on MSC osteogenic lineage commitment. Furthermore, the transduction of pressure mechanical cues was demonstrated to occur via novel mechanisms pertaining to the cytoskeletal associated intermediate filaments and cytoskeletal extension, the primary cilium. Moreover, MSC osteogenesis can be modulated by pharmacologically targeting the disassembly of the intermediate filament network using Withaferin A. These studies provide a framework for bioreactor based orthopaedic strategies and mechanobiology studies to identify biological targets for bone anabolism and how alterations to such mechanisms may lead to debilitating diseases such as osteoporosis.
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European Research Council (ERC)
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https://tcdlocalportal.tcd.ie/pls/EnterApex/f?p=800:71:0::::P71_USERNAME:STAVENSEDescription:
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Author: STAVENSCHI, ELENA
Sponsor:
European Research Council (ERC)Advisor:
Hoey, DavidPublisher:
Trinity College Dublin. School of Engineering. Discipline of Mechanical & Manuf. EngType of material:
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Bone, Mechanobiology, Stem Cells, CytoskeletonMetadata
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