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Title: Chondrogenic differentiation of human amniotic fluid-derived stem cells cultured in alginate and agarose models with CNP and TGFb
Author: Taylor, James
Awarding Body: Queen Mary University of London
Current Institution: Queen Mary, University of London
Date of Award: 2019
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The use of stem cells for cartilage repair and treatment of osteoarthritis has been a subject of much debate. To date, there is no consensus on the appropriate cell source that has chondrogenic potential for cartilage repair. The present study examined whether differentiation of stem cells derived from human amniotic fluid (AFSCs) are proliferative and increase gene expression and protein production of extracellular matrix (ECM) components in agarose culture after treatment with growth factors (TGFb1 and TGFb3) and/or agents which promote natriuretic peptide signalling (CNP, Dex). Following short (up to 7 days) and long-term culture (28 days) of AFSC/agarose constructs, markers for ECM synthesis (GAG, collagen, DNA) were quantified by biochemical assay. Proteoglycan and collagen distribution profiles of AFSC/agarose constructs were examined with Alcian blue staining of histological specimens and by second harmonic generation (SHG) confocal imaging. The gene expression of SOX-9, aggrecan and Type II collagen was examined by RT-qPCR. In addition, the development of sophisticated scaffold materials which release biologically active agents to incorporated cells is an increasing area of scientific research for tissue engineering applications. In separate experiments, I investigated whether the application of CNP containing microcapsules to bovine chondrocyte/agarose constructs over a 48-hour time period constituted any effect to the GAG synthesis of encapsulated cells compared to exogenous application of CNP. CNP microcapsule structures were characterised via SEM and confocal microscopy and markers for ECM synthesis (GAG, DNA) were quantified by biochemical assay. GAG synthesis was not stimulated in TGFb treated AFSCs embedded within alginate bead structures. Alginate beads also suffered from significant structural weakness after prolonged culture periods suggesting they were unsuitable for the chondrogenic differentiation of AFSCs. Co-treatment of AFSC/agarose constructs with either CNP + TGFb1, CNP + TGFb3 or Dex + TGFb1 enhanced GAG synthesis (p < 0.01) but not collagen synthesis compared to growth factor only conditions after 28 days (p > 0.05). Weak positive homogenous histological staining was detected in co-treated samples at both core and edge sections of constructs while SHG imaging confirmed some collagen deposition in co-treated samples at the core of constructs investigated. SOX-9, aggrecan and type II collagen gene expression were unaffected by all treatments investigated after 28 days relative to day 0 (all p > 0.05). Treatment of bovine chondrocyte/agarose iii constructs with CNP microcapsules enhanced GAG synthesis relative to exogenous application after 48 hours (p < 0.01). In summary, I demonstrate significant upregulation of GAG synthesis but not collagen in AFSC/agarose constructs stimulated with TGFb1/3 and Dex or CNP for 28 days but not 7 days. In addition, I also show that localised release of CNP is conducive to enhanced GAG synthesis in bovine chondrocyte/agarose constructs after 48 hours relative to exogenous application. The lack of a substantial increases in GAG synthesis upon chondrogenic growth factor treatment in conjunction with CNP or dexamethasone suggest that AFSCs may not be suitable for a cartilage tissue engineering in comparison to other stem cells types. This would therefore have an obvious impact on the use of AFSCs for cartilage engineering field but may have ramifications for their use in other tissue engineering fields. Future investigations should focus build upon the investigations carried out here with a focus on the effect of CNP on hypertrophic/osteogenic gene expression in AFSCs and the effect of CNP microcapsules on the process of AFSC chondrogenic differentiation.
Supervisor: Not available Sponsor: Rosetrees Trust ; Institute of Bioengineering
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available