The role of the biophysical cues on the initiation, progression and maintenance of a chondrogenic phenotype in human stem cells
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2022Author:
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2024-12-31Citation:
Chariyev-Prinz, Farhad, The role of the biophysical cues on the initiation, progression and maintenance of a chondrogenic phenotype in human stem cells, Trinity College Dublin.School of Engineering, 2022Download Item:
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Articular Cartilage Tissue Engineering (AC TE) is a promising strategy for the treatment of small cartilage defects, however complete mimicry of the native tissue remains a significant challenge. Mechanical cues like dynamic compression (DC) and hydrostatic pressure (HP) are commonly used to enhance the chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs) when seeking to engineer functional cartilage grafts. Despite growing evidence that biophysical cues regulate the chondrogenic differentiation of MSCs and the development of a stable chondrocyte-like phenotype, there is a limited understanding for how in vitro culture conditions will influence the response of chondrogenically primed MSCs to mechanical stimulation. Therefore, the overarching goal of this thesis was to assess how DC and HP regulate the initiation, progression and maintenance of a chondrogenic phenotype in human MSCs (hMSCs) maintained in altered culture conditions.
To achieve this goal this dissertation first sought to examine how cell seeding density and biomaterial concentration/stiffness would regulate the development of engineered cartilage tissue. A higher cell density, as well as lower concentrations (2.5%) of fibrin, were associated with superior chondrogenic differentiation of hMSCs in static conditions. When these cells were subjected to HP, it was found that the application of HP did not further enhance chondrogenic differentiation. However, when hMSCs were subjected to HP once exogenous TGF-β3 was removed from the culture environment after a 3-week priming period, they responded positively to the application of such a mechanical stimulus. Due to the observed importance of chondrogenic priming, the thesis next explored the role of cartilage graft maturity on the cellular response to HP. To this end fibrin encapsulated hMSCs were chondrogenically primed for different periods (0, 1, 3 and 5 weeks) before being subjected to HP in the absence of exogenous TGF-β3. The application of HP in the absence of growth factor priming supported a significant upregulation of SOX9, however the expression of downstream chondrogenic markers was not increased. The application of HP during the latter stages of maturation, specifically after 5 weeks of chondrogenic priming, resulted in the most anabolic response to mechanical loading.
Having identified conditions where chondrogenically primed hMSCs respond positively to the application of HP, this thesis next sought to examine how they would respond to the application of DC. As part of addressing this question, this thesis first explored if the application of DC alone in the absence of exogenous growth factors could initiate chondrogenesis of hMSCs. While a biomaterial stiffness and DC strain magnitude dependant upregulation of the chondrogenic transcription factor SOX9 could be observed, no upregulation of other chondrogenic markers was observed. When applied in the presence of TGF-β3, DC was only found to benefit the chondrogenic differentiation of hMSCs in suboptimal culture conditions. The response of chondrogenically primed hMSCs to DC was also assessed after exogenous growth factors were removed from the media. After a 3-week priming period in the presence of TGF-β3, the engineered grafts responded negatively to the application of DC, particularly less mature tissues engineered using lower initial seeding densities. The results of both the HP and DC studies point to the importance of allowing engineered cartilage tissues to reach a certain level of maturity before subjecting them to challenging loading conditions.
In summary, this thesis provides compelling evidence that mechanical stimulation per se is not always beneficial in regard to the chondrogenic differentiation of hMSCs and subsequent levels of ECM deposition. Experimental parameters like cell density, graft maturity and the specifics of the loading regimes are likely to act interdependently and thus affect the final phenotype of the cartilage graft.
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European Union?s Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 721432
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https://tcdlocalportal.tcd.ie/pls/EnterApex/f?p=800:71:0::::P71_USERNAME:FCHARIYEDescription:
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Author: Chariyev-Prinz, Farhad
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Kelly, DanielPublisher:
Trinity College Dublin. School of Engineering. Discipline of Mechanical & Manuf. EngType of material:
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