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Evaluating donor variability of ovine adult stem cells - implications for orthopaedic animal models

Hassan, Eatelaf Muhammad

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Eatelaf Muhammad Hassan


Articular cartilage is a specialised tissue which covers the articulating surfaces at the end of the mammalian bone. This thin layer performs essential functions such as load bearing and shock absorption in joints. However, it lacks the ability to satisfactorily self-repair. This characteristic of the cartilage tissue makes this tissue of interest to tissue engineering and regenerative medicine therapies.
Within sheep populations, there is a significant individual variation in the characteristics of the ovine bone marrow-derived mesenchymal stem cells (oMSCs). These variations are demonstrated in the differentiation responses of oMSCs to multiple inducing factors such as nutrients and biomechanics. These differences may impact on the choices of treatment strategies that are used within regenerative medicine.
This study aims to investigate the variation in characteristics of oMSCs and to design in vitro growth protocols for oMSCs in 3D culture. These 3D growth conditions will be compared for static and dynamic culture. For comparison, native articular cartilage and engineered cartilage structures were also characterised through the measurement of mechanical and biochemical properties in addition to histological and immunohistological assessment.
Mechanical conditioning of the cells and 3D constructs was carried out using the Magnetic Ion Channel Activation (MICA) technology developed previously. The pioneering bio-magnetic technology offers a novel way to stimulate mechanically sensitive membrane channels using remotely controlled magnetic nanoparticles (MNPs) which lead to the differentiation of bone marrow-derived stromal stem cells in vitro.
This thesis has effectively demonstrated that there is a clear individual variation between the sheep donors in regard to oMSCs characterisation and tri-lineage differentiation potential. The effect of this variation on dynamic and static culture has been identified. The study has also demonstrated that mechanical stimulation of the transient receptor potential cation channel subfamily V member 4 (TRPV-4) enhances chondrogenic differentiation of oMSCs Mechanical stimulation of oMSCs for their differentiation towards chondrogenesis in 3D hydrogels using the MICA technology, firstly Twik-related potassium channel 1 (TREK-1) ion canal was targeted. However, no consistent response in all the ovine donors was observed. The total amounts of sGAG, total collagen and total protein produced by the cells during culture was enhanced in mechanically stimulated gels for potentially only three out of the five donors. However, mechanical testing revealed significant increase in Young’s Modulus for the dynamically cultured gels compared to static gels.
Whereas, mechanical stimulation oMSCs targeting the Transient receptor potential cation channel subfamily V member 4 (TRPV4) resulted in increased cellular responses as shown by mechanical testing, biochemical analysis, histological and immunobiological analysis compared to TREK-1 targeted samples.
In summary, this thesis sets out to systematically explore the ovine mesenchymal stem cell population derived from multiple donors. With this information, we can ultimately start to address the issues of personalised approaches to regenerative therapies.

Keywords ovine MSCs, MNPs, MICA technology, articular cartilage, chondrogenesis, cell therapy, regenerative medicine, bone marrow derived mesenchymal stem cell


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