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Assessment of the bioactive potential of demineralised dentine matrix on bone marrow-derived, mesenchymal stem cells

Avery, Steven 2016. Assessment of the bioactive potential of demineralised dentine matrix on bone marrow-derived, mesenchymal stem cells. PhD Thesis, Cardiff University.
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Abstract

At present, the prime candidate for augmentation of bone defects are autologous bone grafts (ABGs), owing to their ability to emulate the physiological signalling environment to induce bone marrow-derived, mesenchymal stem cells (BMMSCs) to differentiate into functional osteoblasts. However, the uses of ABGs are negated due to patient complications. Dentine matrix has recently become increasingly recognised as possessing bioactive nature and has demonstrated the ability to augment bone repair in vivo. However, the constituents of dentine that confer efficacy for bone formation are poorly understood. The aim of this study was to determine the potential for demineralised dentine matrix (DDM) to induce biological responses of BMMSCs and elucidate the key mediators required for bioactivity. Isolation of a sub-population of BMMSCs yielded a population of cells with enhanced expansive capacity in vitro, with maintained mesenchymal stem cell marker expression. Application of DDM to BMMSCs significantly reduced cell expansion (0.1-10μg/mL), reduced apoptotic activity (10μg/mL) and enhanced migration (0.1μg/mL). Importantly, DDM at 10μg/mL directed osteogenesis of BMMSC by enhancing RunX2 gene expression after 5 days and deposition of a mineralised matrix after 28 days. Western blot and enzyme-linked immunosorbent assay (ELISA) analyses of DDM identified a plethora of growth factors associated with directing osteogenesis of BMMSCs, including; transforming growth factor-β1 (TGF-β1), bone morphogenetic protein-2 (BMP-2) and vascular epidermal growth factor (VEGF). Fractionation of DDM by heparin-affinity, to concentrate growth factors, resulted in diminished potential of DDM to enhance RunX2 expression and mineral deposition of BMMSCs. Depletion of decorin from DDM had no effect on osteogenic potential, however, depletion of biglycan attenuated RunX2 expression and mineral deposition. Substrates composed of silk-fibroin/gelatin (SF/G) at ratios of 75:25 supported attachment and expansion of BMMSCs with no discernible changes in morphology, compared to cells cultured on plastic. Addition of DDM to SF/G substrates resulted in enhanced BMMSC expansion (20μg/mL) and evidence of mineralised matrix deposition (5-20μg/mL), compared to un-loaded SF/G substrates. In conclusion, DDM possesses bioactive potential towards BMMSCs, which is attributable to a range of synergistically acting growth factors along with additional matrix constituents. The ability of DDM to stimulate osteogenesis demonstrates potential for future developments in the field of tissue engineering of bone.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Dentistry
Subjects: R Medicine > RK Dentistry
Uncontrolled Keywords: mesenchymal stem cells (MSCs); dentine; bone; bioactive; growth factors; matrix; biglycan; decorin; differentiation; osteogenic
Date of First Compliant Deposit: 9 January 2017
Last Modified: 04 Jun 2017 09:35
URI: http://orca.cf.ac.uk/id/eprint/97262

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