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Overcoming wound healing complications following radiotherapy in human breast dermal fibroblasts, through the influence of preadipocytes from the stromal vascular fraction
Trevor, Lucy V.
Trevor, Lucy V.
Publication Date
2021
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The University of Bradford theses are licenced under a Creative Commons Licence.
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Accepted for publication
Institution
University of Bradford
Department
Plastic Surgery and Burns Research Unit, Centre for Skin Sciences
Awarded
2021
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Abstract
Radiotherapy has major therapeutic benefits for cancer patients, but ionizing
radiation causes damage of surrounding healthy tissues with poor wound
healing a common side effect. Therefore, further oncoplastic, reconstructive
surgery is challenging and often problematic. Current research models use
normal human dermal fibroblasts irradiated in vitro to mimic radiation
damage, but this is not comparable to ionising radiation and only measures
acute changes. Since radiotherapy may induce epigenetic changes leading
to alterations in dermal fibroblast phenotype, the first aim of this study was to
compare fibroblasts cultured from irradiated skin with non-irradiated skin. As
mesenchymal stem cells isolated from adipose tissue may offer beneficial
effects in the regenerative capacity of irradiated tissue, the second part of
this study was to compare those cultured from non-irradiated and irradiated
breast tissue.
Histological changes in the structural organisation of breast tissue in situ
from donors exposed to radiotherapy was compared to untreated breast.
Primary cultures of dermal fibroblasts from irradiated and non-irradiated
breast skin were established and comparisons quantitated in proliferation
(CyQuant), metabolism (Alamar Blue), migration (scratch-wound assay),
collagen production (Sircol), levels of proteases and protease inhibitors
(human protease/protease inhibitor array) and gene expression of COL1A1,
COL3A1, MMP1, MMP2, TIMP1 and PPAR-γ mRNA (qPCR). Cells from the
stromal vascular fraction (SVF) were cultured and characterised by
immunocytochemistry and compared to human preadipocytes sourced
commercially. The secretion of FGF, adiponectin and VEGF by the preadipocyte and the SVF mesenchymal cells was compared and the ability
of their secretome to modulate dermal fibroblast proliferation, metabolism
and migration was evaluated.
Radiotherapy caused extensive disorganisation of the reticular dermis and
flattening of the epidermal-dermal junction. Dermal fibroblasts cultured from
irradiated skin had a pronounced spindle shaped morphology with longer
thinner projections and took approximately twice as long to explant and grow.
They had a lower proliferative and higher basal metabolic rate and did not
respond to FGF-2. While they secreted similar amounts of total collagen they
demonstrated distinct differences in proteolytic enzyme and protease inhibitor
expression. This is the first report to culture cells from the SVF of irradiated
breast tissue. The cells expressed the preadipocyte markers CD10, CD73 and
CD105 and no CD45 (negative marker). SVF cells cultured displayed a typical
ASC fibroblastoid morphology. Analysis of the secretome identified the
presence of FGF, adiponectin and VEGF, while functional analysis
demonstrated a stimulatory effect on normal dermal fibroblast migration,
although irradiated dermal fibroblasts were unresponsive.
Radiotherapy induces long term, detrimental changes in breast skin. This is
the first quantitative characterisation of dermal fibroblasts and mesenchymal
cells from the SVF, subjected to ionising radiation in situ. Changes in their
phenotype that alter their function will impact on wound healing. Further
characterisation of these cells may explain their dysfunctional behaviour, and
lead to therapies to reverse or reduce this deleterious side-effect and
significantly improve treatments facilitating wound healing following radiation
injury.
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Type
Thesis
Qualification name
PhD