Ghaemmaghami, AmirAlromaihi, Reem2024-12-232024-03https://hdl.handle.net/20.500.14154/74403Chapter 3: Development of a Novel Screening Platform for Secretome profiling for the Discovery of Immune-Responsive Polymers This study presents an innovative screening platform based on the polymer microarray concept, designed to profile the secretome for each polymer separately. The aim is to uncover new polymer chemistries that can regulate or modulate macrophage phenotype, thereby guiding tissue integration and enhancing healing processes post-implantation. The novel microchamber (7 mm × 7 mm wells) based polymer array was developed using a contact printing fabrication technique and ProPlate® multi-well chambers. It comprises 56 polymer spots/wells, with 16 wells per slide and four slides per plate. It is designed to be compatible with the robotic printing of polymers and is well-suited for secretome profiling of both adherent and non-adherent cells. This platform was successfully validated, with its effectiveness proven through a detailed study assessing the impact of chemically diverse homopolymers on macrophage viability, attachment, and differentiation into pro inflammatory (M1) or anti-inflammatory (M2) phenotypes. Eighty-one acrylate and acrylamide polymers were printed into individual wells and cultured with primary human monocyte-derived macrophages (MDMs) for six days. The polymers that ensured high MDMs viability (>80%) were then selected for attachment and secretome analyses, which involved measuring the production of cytokines TNF-α (Tumor Necrosis Factor-alpha), IL-6 (Interleukin-6), IL-10 (Interleukin-10), TGF-β (Transforming Growth Factor-beta), and CCL-18 (Chemokine (C-C motif) Ligand 18) by cells cultured on them. These cytokines are key indicators of the macrophages’ immune responses and can signal pro inflammatory or anti-inflammatory activity. Several Polymers that supported cell attachment and modulated MDM phenotypes were identified. Polymers such as poly(carbazol-9-yl ethyl acrylate) (pCzEA) and poly(3-hydroxy-2,2- dimethylpropyl 3-hydroxy-2,2-dimethylpropionate diacrylate) (pHDMPDA) were categorised as M2-like, while poly(1,1,1-trimethylolpropane trimethacrylate) (pTMOPTMA) and poly([methylthio]ethyl methacrylate) (pMTEMA) were categorised as M1-like. The polymer poly(glycerol propoxylate triacrylate) (pGPOTA) retained macrophages in a naive state, akin to inactivated (M0) macrophages. Chapter 4: Evaluating hit Polymers: Exploring the Impact of Seven Promising Polymers on Macrophage Behaviours and in-vitro Wound Healing The seven ‘hit’ polymers with most consentient immune modulatory properties in the screening were synthesised, characterised, and scaled up to larger 13 mm surfaces. This scale-up was essential for further phenotypic and functional analyses of these 'hit’ polymers. MDMs showed distinct anti- and pro-inflammatory characteristics when cultured on these scaled up polymer surfaces. They showed marked increases/decreases in cytokine production and notable changes in both cell surface markers and gene expression levels. Furthermore, these polymers indirectly affected fibroblast migration and proliferation in a 2D wound healing assay using MDMs-conditioned media. Wounds closed faster when fibroblasts were exposed to media from macrophages cultured on polymers that favoured M2-like phenotypes like pHDMPDA and pCzEA, and intriguingly pMTEMA, an M1- polymer, outperforming the tissue culture plastic (TCP) control which is highly pro-fibroblast proliferation. This suggests that these polymers indirectly influence stromal cells during tissue healing and regeneration through paracrine signalling, demonstrating their potential in improving healing outcomes. The mechanisms behind the polymer-induced changes in MDMs phenotype were also explored, focusing on the polymers’ stiffness and the thickness of the protein layer adsorbed from the culture medium. However, unfortunately, there was no apparent correlation. Chapter 5: The Impact of Selected Polymers on Fibroblast Behaviours and in vitro Scratch Assay Having shown the paracrine effect of macrophage conditioned media in previous chapter, in this chapter I examined the direct impact of selected polymers on fibroblasts, another key cell type in wound healing, by culturing these cells on polymer surfaces to evaluate their ability to modify fibroblasts behaviour. All polymer surfaces supported fibroblast viability, adhesion and proliferation. They also induced variable secretome profiles and impacted the wound closure rate, with cells cultured on pHDMPDA and pTMOPTMA showing distinct morphological characteristics including variations in cell area and perimeter. Conclusion This study's innovative approach utilising a microchamber based polymer array platform has successfully identified novel immune-instructive polymers capable of steering macrophage activity through secretome analysis, a method surpassing previous techniques reliant on surface markers. The identified polymers, by promoting distinct immune and wound healing responses, could pave the way for the development of next-generation medical devices and regenerative therapies that are finely tuned to harness and direct the body's own healing mechanismsBackground Medical devices, from implants to gene/drug delivery systems and biosensors, often elicit immune reactions ranging from transient inflammation to chronic inflammation and fibrosis, known as the foreign body reaction (FBR). These immune responses can lead to device failure and tissue damage, highlighting the need for biomaterials that not only avoid these reactions but actively guide positive immune interactions for tissue integration and healing. The role of macrophages, with their highly plastic nature, is critical in the orchestration of these immune responses, making them a focal point for biomaterial design aimed at minimising FBR and enhancing implant integration. High-throughput screening using polymer microarrays has been increasingly utilised to accelerate the discovery of new biomaterials. Previously, this strategy has identified acrylate and acrylamide polymers that can modulate the polarisation state of macrophages based on their expression of surface markers using fluorescent labels and microscopy. Unlike the macrophage ‘secretome’, surface marker expression only provides a partial insight into the macrophage functional phenotype. However, since conventional polymer microarrays do not have defined cell culture chambers for individual polymer spots, investigating the secretome in this format is not feasible.305enmacrophage phenotypesImmune-instructivepolymersThesis