Latif, Arsalan
(2021)
Developing stromal instructive niches to promote wound healing.
PhD thesis, University of Nottingham.
Abstract
Wound healing is a complex biological process involving various cell types across spatial and temporal phases. Dysregulation in any of these phases, such as in the case of diabetic wounds, burn injuries or implantation of medical devices may result in non-healing wounds, chronic wounds, and/or fibrosis. The accelerated formation of granulation tissue has been studied to promote and accelerate wound healing. Fibroblasts are the prevalent cell type of the granulation tissue and have been shown to play an important role in both healing processes and fibrosis, governed by the nature of biochemical stimuli, e.g., from immune cells such as macrophages, and the physicochemical characteristics of the microenvironment and/or implanted biomaterials. Biomaterial surface chemistry is known to influence phenotype and functions of various cell types including fibroblasts. Hence, the overall aim of this study was to discover new polymer chemistries that regulate/modulate fibroblast behaviour and phenotype, thereby governing the outcome of wound healing.
To study the effect of polymer chemistry on human fibroblasts, a high throughput screening of 300 homo-polymers from a meth(acrylate) and acrylamide library was performed. Polymers that supported cell attachment and spreading, while modulating proliferation and differentiation to myofibroblasts, were identified. Polymers such as poly(tetrahydro furfuryl acrylate) (pTHFuA) promoted cell proliferation while suppressing differentiation, while polymers such as poly(ethylene glycol phenyl ether acrylate) (pEGPEA) suppressed proliferation and promoted fibroblast differentiation to myofibroblasts.
Selected ‘hit’ polymers from the high-throughput screen were synthesised, characterised, and scaled up onto coupon sized surfaces for further phenotypic and functional studies. Fibroblasts cultured on these surfaces were shown to adopt distinct anti-fibrotic and pro-fibrotic profiles, by augmenting cytokine secretion and gene expression of ECM components. Moreover, polymers were observed to directly influence fibroblast migration and proliferation, as assessed in a 2D - wound healing assay; cells cultured on pTHFuA and poly(divinyl adipate) (pDVAd) were observed to have a higher rate of wound closure compared to cells cultured on standard TCP.
To elucidate the mechanisms of the observed polymer induced modulation in behaviour and phenotype of fibroblasts, the stiffness, and the thickness of adsorbed proteins from serum supplemented culture medium on scaled-up polymers was investigated. Quantitative analysis revealed that fibroblast phenotype was modulated by adsorption of selective proteins as opposed to stiffness of the polymers or thickness of adsorbed protein layer.
Lastly, the clinical applicability of identified anti- and pro-fibrotic chemistries was pursued by fabricating these chemistries into microparticles, for application as in situ scaffolds to accelerate wound healing. The particles supported fibroblast attachment and influenced proliferation. Moreover, fibroblast – microparticle systems were shown to augment fibroblast and macrophage cytokine secretion and gene expression of collagens towards anti-fibrotic and pro-fibrotic outcomes, thereby accelerating wound healing.
In summary, findings from this study demonstrate the ability of polymer surfaces to modulate fibroblast phenotype and behaviour. The observations presented and discussed in this study, provide a framework through which bio-instructive therapeutics to direct outcome of wound healing could be developed.
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