Ho, Jasmine Onn Yee; (2023) Dynamic hypoxic pre-conditioning of cells seeded in tissue-engineered scaffold to improve neovascularisation. Doctoral thesis (Ph.D), UCL (University College London).

Text Jasmine Ho_40368539_PhD Thesis.pdf - Accepted Version Access restricted to UCL open access staff until 1 August 2026. Download (19MB)

Abstract

Introduction: Tissue engineering (TE) is the potential solution to the global shortage of tissue and organs. However, the lack of adequate angiogenesis to TE scaffolds during the initial stages of implantation has hindered its success in vivo. Mesenchymal stem cells (MSC) have the most established track record for translational regenerative therapy and have been widely used in combination with TE scaffolds. Hypoxia is one of the main potentiators for upregulating angiogenic factors in MSC. However, fine-tuning their cellular function and behaviour is still not fully understood. This study aims to help increase the understanding of this process by determining the effects of in vitro hypoxic conditioning on enhancement of angiogenesis of MSC for the purpose of pre-clinical translational for TE application. Methods: The angiogenic potential of 3 different tissue sources (bone marrow, umbilical cord and adipose) MSC were initially determined for downstream pre-clinical application. We established the appropriate regime for in vitro dynamic hypoxia conditions in 2D and 3D hydrogel to enhance MSC angiogenic pathway using real-time continuous oxygen sensors and angiogenic cytokine profiling. Cell metabolism and proliferation effects were also evaluated using intravital Realtime-glo, D-luciferin (on transduced MSC) and microscopic Live-Dead stain techniques. We optimised seeding of cells on the tissue engineered dermal (INTEGRA®) for in vivo translational purpose and used targeted in vitro and ex vivo angiogenesis assays, which helped to determine aspects of the MSC conditioned media on endothelial migration, proliferation, morphogenesis and matrix degradation. Finally, the functional reproducibility of the in vitro angiogenic response was assessed using in vivo angiogenesis CAM assay and murine diabetic wound healing models. Results: Adipose derived MSC (adMSC) were found to have the most angiogenic potential in response to hypoxic conditioning. Dynamic hypoxia (DH) regime of changing oxygen levels from 21% to 1% when transitioning from T-flask subculture to multiwell plate seeding was most effective at eliciting pro-angiogenic response from adMSC for both in vitro 2D and 3D models compared to controls using static normoxia (21% oxygen) and static hypoxia (1%). Low seeding density of adMSC was found to be the most appropriate to ensure optimised cell adherence and survival post-seeding on TE dermal scaffold (INTEGRA®). It also minimised on localised hypoxic gradient induced oxidative stress by the seeded cells when compared to high seeding density techniques found on non-invasive oxygen monitoring. Conditioned media from DH seeded adMSC was shown to have enhanced angiogenic proteomic profile compared to the controls. In vitro angiogenesis assays showed better human endothelial cell migration and morphogenesis in scratch assay and tubular formation assay compared to controls. Preliminary ex vivo organ assay results using novel human umbilical arterial rings showed better endothelial out-sprouting and migration through embedded matrix compared to controls. Results from in vivo transplantation of adMSC seeded INTEGRA® scaffold showed a mixed response in the CAM assay, highlighting an unaccounted scaffold effect from INTEGRA® from the host. Histological sections showed increased vascular and host tissue infiltration into the scaffold. When evaluating the functional angiogenesis in murine wound healing models, although DH adMSC seeded scaffolds showed non-statistically significant increased rate of wound closure, there was significantly greater vessel density within the scaffold on histological evaluation in this group compared to controls. Conclusion: The results provide a better comprehension of how cells behave in 2D and 3D environments when cultured in dynamically changing oxygen environments. The study addresses important issues, such as the effects of chronic hypoxia on MSC, and how dynamic hypoxia can enhance angiogenic signalling. It also offers a crucial understanding of the in vitro oxygen culture environments for future research applications. Further insight into cell-scaffold interaction during in vivo transplantation was also established. The importance of having an appropriate in vivo model to determine if such in vitro angiogenic enhancement would translate to functionally improving neoangiogenesis and subsequent tissue regeneration in vivo was also highlighted in this study. Improving and advancing research into optimising and evaluating the in vitro environment for clinical application will undoubtedly have a huge impact on the future of cell therapy for regenerative medicine purposes.

Download activity - last month

Export as JSONXMLCSV

Download activity - last 12 months

Export as CSVXMLJSON

Downloads by country - last 12 months

Export as XMLCSVJSON

Archive Staff Only

View Item