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Hollingworth, Mathew (2019) Comparison of primary and stem cell-derived osteoblasts and the influence of nanoscale mechanotransduction on cell behaviour. PhD thesis, University of Nottingham.

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Abstract

The use of stem cells in regenerative bone strategies holds great promise, however it is becoming increasingly clear that stem cell-derived osteoblasts differ in behaviour and function to native osteoblasts. Pluripotent embryonic stem cells (ESCs) present a useful model to study developmental biology and in particular, osteogenesis in vitro; information which can be used to further inform and develop regenerative strategies for bone repair. Here, the differentiation of ESCs is compared to primary calvarial osteoblasts. Whilst both cell types exhibit mineralisation, expression of bone specific proteins and increased ALP activity, marked differences are observed in these characteristics over 35 days, particularly in mineral formation.

The range of factors influencing osteogenesis is well known, but recently the effects of nanoscale mechanotransduction have been observed; allowing osteogenesis of mesenchymal stem cells in the absence of conventional osteogenic medium supplements. Here we study for the first time the effect of nanoscale mechanotransduction on ESCs, comparing established ESC differentiation techniques to 1000Hz, 30nm vibrational stimulation, alongside similar studies of calvarial osteoblasts. Although nanovibration allows formation of mineralised osteoblasts cultures indicative of bone phenotype as assessed by immunocytochemistry and qPCR, this differentiation is delayed compared to conventional medium supplementation. However 1000Hz stimulation promoted higher ALP activity and produced mineral composition more similar to mature bone tissue, revealing its importance in osteogenesis.

Mechanisms by which nanovibration influences cell behaviour and differentiation were also explored, revealing influence over cell morphology, spreading and actin cytoskeleton organisation in both cell types. 1000Hz stimulation also promoted focal adhesion complex maturation, with focal adhesion kinase (FAK) activity implicated in this process, particularly in mESCs. This mechanosensing effect was confirmed by metabolomics analysis, suggesting FAK activity plays a significant role in ESC fate whilst not as important in calvarial osteoblasts. The influence of nanovibration over migratory behaviour of osteoblasts was also observed; an important part of bone remodelling that is less well characterised than other aspects of osteoblast behaviour. Migratory ability developed following conventional osteogenic culture, however 1000hz stimulation had an overriding influence over this; delaying migratory behaviour in a similar manner to osteogenesis.

This work reinforces differences in stem cell-derived osteoblasts and primary calvarial osteoblasts; which has significant implications on the use of stem cells for regenerative bone therapies. However we also highlight the importance of nanoscale mechanotransduction in osteogenesis; 1000Hz stimulation helping to improve osteogenic outcomes particularly when combined with conventional methods. It is hoped this combination approach will help bridge the current gap between stem cell-derived osteoblasts and their native counterparts.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Buttery, Lee Alexander, Morgan Ehrbar, Martin
Keywords:	osteogenesis, bone, nanovibration, bioreactor, mechanotransduction, stem cells
Subjects:	Q Science > QH Natural history. Biology > QH573 Cytology
Faculties/Schools:	UK Campuses > Faculty of Science > School of Pharmacy
Item ID:	56107
Depositing User:	Hollingworth, Mathew
Date Deposited:	29 Oct 2019 12:07
Last Modified:	22 Jul 2021 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/56107

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