Nanotopography as a tool for the investigation of molecular mechanisms of osteogenesis of MSCs

Yang, Jingli (2015) Nanotopography as a tool for the investigation of molecular mechanisms of osteogenesis of MSCs. PhD thesis, University of Glasgow.

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Abstract

Nanotopographical patterning of biomaterial substrates has great potential for
biofunctionalisation of devices for clinical applications, such as in orthopaedics.
Nanotopography comprising 120 nm diameter nanopits with a partially
disordered arrangement of up to +/- 50 nm offset from a square lattice with 300
nm centre to centre spacing (NSQ50, fabricated by electron beam lithography)
has been characterized as being osteogenic. Following the finding of
osteogenesis of mesenchymal stromal cells derived from human bone marrow
(MSCs) on the NSQ50 nanotopography, MSCs cultured on ε-polycaprolactone
(PCL) embossed with the NSQ50 pattern was used for this study on molecular
mechanisms underlying NSQ50 induced MSC osteogenesis: the functional coupling
of gene expression and osteogenesis, the molecular regulatory events driving
gene expression and osteogenesis, and the possible link of metabolomics with
molecular signalling of MSCs on the NSQ50 surface.
Temporal analysis of gene expression for MSCs on the NSQ50 surface revealed
that MSC fate commitment and osteogenic differentiation was transcriptionally
controlled. The cell cycle and growth regulating transcription factor C-MYC was
found to be significantly repressed, whereas the osteogenic transcription factor
RUNX2 was up-regulated at 5 days of cell culture, and this was followed upregulation
of the osteoblast specific transcription factor osterix (OSX) at days 11
and 13. Following this transcription factor activation, osteoblast specific marker
genes were induced with increased alkaline phosphatase (ALP) observed at day
16, increased osteopontin (OPN) at day 20 and increased osteocalcin (OCN) at
day 28. These data suggested that transcription factors regulated MSC
osteogenic commitment at the early stage, and induced osteogenic specific
marker gene expression at the late stages of cell culture on the NSQ50 surface,
resulting in osteogenesis of the MSCs.
Signalling pathway analysis illustrated that bone morphogenetic protein 2 (BMP2)
was the initial signalling molecule that triggered osteogenic differentiation of
MSCs by inducing RUNX2 expression via the canonical SMAD pathway. BMP2 and
its transmembrane receptor type 1A (BMPR1A) were stimulated by
3
nanotopographical cues by 3 days of cell culture on the NSQ50 surface, whereas
the induction of other transmembrane receptors, including the low density
lipoprotein-receptor related protein 5 (LRP5) and integrin subunits α3, α4, β1,
and β3 were not observed. Inhibition of BMP2 signalling by the BMP2 antagonist
noggin resulted in down-regulation of RUNX2 and ALP. Further analysis of BMP2
signalling revealed that BMP2 also modulated expression of the microRNA (miR)-
23b which targets RUNX2. The effect of BMP2 signalling on the expression of
RUNX2 was enhanced by co-localizing with integrin αvβ5 (the vitronectin (VN)
receptor) which was found to be up-regulated after 5 days cell culture.
Metabolomics data for MSCs on the NSQ50 surface during early osteogenic
differentiation was analysed. MSC cellular metabolite analysis revealed possible
changes in bioenergetic balance with shifts towards more mitochondrial
oxidative process, possibly indicating a switch in MSCs on the surface towards
lineage-specific commitment. Further analysis of the metabolomics data
illustrated PPARG ligands from the polyunsaturated fatty acid family was downregulated,
suggesting the inhibition of adipocyte differentiation in MSCs on the
surface. The down-regulation of unsaturated fatty acids could also be involved in
the regulation of Ca2+ channels which positively regulate BMP2 expression. The
metabolomics data, together with gene expression and signalling pathway
analysis demonstrated that MSCs on the NSQ50 surface initiated osteogenic
commitment after 3 days of cell culture, with BMP2 initiating osteogenic
transcription factor stimulation of mature and functional osteoblasts on the
surface.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > QH Natural history > QH301 Biology
T Technology > T Technology (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Dalby, Professor Matthew
Date of Award: 2015
Depositing User: Dr. Jingli/ J. Yang
Unique ID: glathesis:2015-6370
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 20 May 2015 07:53
Last Modified: 25 May 2015 11:02
URI: http://theses.gla.ac.uk/id/eprint/6370

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