Dermal fibroblasts positively contribute throughout the wounding response by secreting a profile of pro- and anti-inflammatory cytokines in the wound milieu. However, a chronically inflamed environment will, cause detrimental effects on the functional, secretory, and molecular properties of these cells. This study aims to understand how the effect of the pro-inflammatory cytokine TNF-α modulates both healthy and diabetic dermal fibroblast phenotype. To mimic a chronic inflammatory environment and assess whether fibroblasts respond similarly in different anatomical sites, donor-matched fibroblasts from face and scalp were pre-incubated for 3 days with different concentrations (2.5, 25 or 250 ng/ml) of TNF-α. All concentrations significantly impaired proliferation by day 14 in cells from both sites and stimulated (papillary) metabolic activity at day 14. However, this did not correlate with an increase in papillary cell senescence since this did not appear until passage 17, and then only at a supra pathophysiological concentration. Migration of dermal fibroblasts, assessed by the scratch assay. TNF-α significantly inhibited the cells migration, particularly in diabetic fibroblasts, suggesting they are more sensitive to TNF-α. Since TNF-α may stimulate the secretion of soluble paracrine factors by dermal fibroblasts, conditioned medium was collected to assess its effect on other dermal fibroblasts, however, this had no significant effect on migration. However, using gelatin zymography, it was found that TNF-α did stimulate the secretion of soluble paracrine factors that induce MMP activity in non-diabetic fibroblasts, mirroring previous observations that a pro-inflammatory environment can increase proteolytic activity, and indicating that diabetic fibroblasts were again more sensitive than healthy. No difference was observed with MMP-9 activity and nor did the results with dermal fibroblasts reach statistical significance, perhaps because of a relatively low n-number. The ability of TNF-α to modulate the expression of genes associated with the ECM (MMP-1, -2, -9, TIMP-1, and -2) and senescence (Sirt1 and 6) was investigated. There was no change in Sirt1 and Sirt6 expression and no evidence of paracrine effects (conditioned medium) on any of the genes. TNF-α significantly induced mRNA expression of MMP-1 in healthy non-scratched and scratched diabetic fibroblasts, and TIMP-1 in healthy non-scratched cells. There was also considerable donor variability that prevented statistical significance being achieved under the other conditions. The secretion of various cytokines associated with inflammation was compared in healthy and diabetic fibroblasts in the presence and absence of TNF-α. Seven cytokines were secreted, by healthy and diabetic male and female fibroblasts, although diabetic female fibroblasts did not secrete two of them. TNF-α stimulated secretion of cytokines in healthy and diabetic, male and female cells but the profiles of those released were different between the different groups. There was no TNF-α induced paracrine effect on cytokine secretion by healthy dermal fibroblasts. In conclusion, changes in the microenvironment and the influx of pro inflammatory cytokines may significantly alter the dermal fibroblast phenotype. Understanding these functional and molecular changes in response to inflammatory cytokines will give a better understanding of the differences between fibroblast activity in normal physiological wound healing and chronic or diabetic non-healing wounds.

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. / Plastic Surgery and Burns Research unit