Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.549711
Title: Bone tissue engineering using adult mesenchymal stem cells and biomimetic P-15 scaffolds
Author: Mohanram, Yamuna
ISNI:       0000 0004 0131 9427
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2011
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Abstract:
Introduction: The increasing clinical demand for bone regeneration and repair in the context of our aging population poses a challenge both to healthcare providers and society more generally. Tissue engineering provides a promising strategy to meet this clinical demand by developing functional bone construct using stem/stromal cells, scaffolds, with/without growth factors. However, several issues remain to be addressed before clinical translation of this technology. The main challenge is to identify the most appropriate combination of the three elements that can be used to achieve optimum regeneration of damaged bone tissue. A second issue relates to cell death on exposure of the graft constructs to temporary hypoxic in vivo microenvironment such as that found at fracture sites. The aim of this thesis was to investigate the osteogenic potential of human dental pulp stem/stromal cells (HDPSCs) and to determine the possible use of these cells in combination with a biomimetic collagen peptide coated with anorganic bone mineral particles (ABM-P-15) for bone tissue engineering. The secondary aim of the study was to investigate the effect of hypoxia in the osteogenic differentiation potential of commercial human bone marrow stromal cells (HBMSCs) - MultiStem® cells, on ABM-P-15 scaffolds. Methodology: HDPSCs and HBMSCs were isolated from dental pulp and bone marrow respectively. In monolayer culture, proliferation, multilineage (osteogenic, adipogenic and chondrogenic) differentiation, time point based osteogenic gene expression profile (ALP, Ctll. I, Runx2 and OCN) and also ALPSA of HDPSCs was compared to HBMSCs. Then, HDPSCs or HBMSCs were cultured on 3D ABM-P- 15 and ABM scaffolds in basal media for up to 6 weeks and samples were analyzed using confocal microscopy, SEM and histological staining. For in vivo investigation, HDPSCs on ABM- P-15 and ABM scaffolds were sealed within diffusion chambers which were implanted intraperitoneally in MFl nu/nu rmce for 8 weeks and assessed for histological and immunohistochemical staining. Meanwhile, MultiStem® cells were cultured on ABM-P-15 and ABM scaffolds under in vitro hypoxic and normoxic conditions for up to 6 weeks and were analyzed for cell attachment, growth and ALP expression. Hypoxia expanded MultiStem® cells on ABM-P-15 and ABM scaffolds in diffusion chambers were also implanted intraperitoneally in MFl nu/nu mice for 8 weeks and samples were assessed for histological and immunohistochemical staining. Results: Similar to HBMSCs, HDPSCs were proliferative and also exhibited the potential to differentiation into the three lineages (osteogenic, adipogenic and chonderogenic) in monolayer culture. Under basal culture conditions, HDPSCs expressed the osteogenic markers (COLI, ALP, Runx2 and OCN) similar to HBMSCs. However, biochemical assays confirmed higher ALP protein expression in HDPSCs to that of HBMSCs. When cultured on 3D scaffolds (ABM-P-15 and ABM scaffolds alone), ABM-P-15 enhanced both HDPSCs and HBMSCs attachment and spreading at 24 hours and cell bridge formation after 14 days. Confocal microscopic images confirmed that more HDPSCs grew on both ABM-P-15 and ABM alone compared to HBMSCs on similar scaffold groups. Both ALP staining and quantitative assays showed high ALP expressions with both HDPSCs and HBMSCs on ABM-P-15 scaffolds compared to those cultured on ABM alone. Following 8 weeks in vivo implantation, HDPSCs on ABM-P-15 and ABM alone scaffolds revealed extracellular matrix deposition that was positive for COLI, OCN and OPN and the cells on ABM-P-15 scaffolds in particular showed extensive organised collagenous matrix formation, confirmed by Sirius red staining. Under hypoxic conditions, MultiStem® cells cultured on ABM-P-15 and ABM scaffolds appeared to have reduced . ALP expression. However, after in vivo implantation, hypoxic expanded MultiStem® cells produced ECM that stained positively for COLI, OCN and OPN. Those cells on ABM-P-15 scaffolds appeared to have more organised collagen matrix compared with those on ABM alone. Conclusion: The results suggest that HDPSCs together with ABM-P-15 scaffolds could be a useful combination for bone augmentation. These cell pre-expanded in hypoxic conditions may also be useful to reduce the cell death seen after clinical implantation at the fracture site which will be beneficial during clinical translational of tissue engineering.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.549711  DOI: Not available
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