Du, Qiran
(2025)
Combinatorial screening of glycosaminoglycans for immune-instructive properties.
PhD thesis, University of Nottingham.
Abstract
Biomaterials are extensively utilised in implantable medical devices (IMD), but their use can trigger varying degrees of immune responses in the human body. Macrophages play a critical role in mediating the immune response and maintaining tissue homeostasis. Upon activation by cytokines, endotoxins, or other danger signals, macrophages can adopt a spectrum of functional phenotypes, with pro-inflammatory M1 (classically activated) and anti-inflammatory M2 (alternatively activated) representing the extremes of this spectrum, which secrete various soluble molecules, including cytokines and chemokines, regulating inflammation and promoting tissue healing. Understanding how macrophages respond to biomaterials is essential for modulating specific biological outcomes. Consequently, the development of immune-instructive biomaterials that focus on macrophage phenotype modulation is of considerable interest.
Glycosaminoglycans (GAGs), key constituents of the extracellular matrix (ECM), are linear polysaccharides composed of repeating disaccharides that can be divided into four main types: hyaluronic acid (HA), chondroitin sulphate/dermatan sulphate (CS/DS), heparin/heparan sulphate (HP/HS) and keratan sulphate (KS). They have been recognised for their role in regulating macrophage activity and immune responses, positioning them as promising candidates for therapeutic applications in tissue regeneration and wound healing. Despite their potential, the optimal composition and combination of GAGs for these purposes remains undefined. The overall goal of this study was to investigate the effect of different combinations of GAGs on the macrophage phenotype using an unbiased screening strategy to identify ‘hit’ GAGs with immunomodulatory properties capable of modulating immune responses during wound healing.
This thesis employs a systematic and combinatorial screening strategy to explore the immune-modulatory properties of GAGs, using a library of 82 mixtures comprising 10 individual GAGs and 72 combinations, varying in type, molecular weight, and degree of sulphation. Peripheral blood monocytes from healthy human donors were cultured with GAGs and allowed to differentiate into macrophages over a 6-day period, after which their phenotype was characterised. Here, the phenotype of the macrophages was determined by quantifying pro-inflammatory and anti-inflammatory cytokines and chemokines, as well as through immunostaining of specific cell membrane protein markers.
Optimal conditions for GAG activity were identified, with CS (a representative GAG) being shown to promote macrophage polarisation in a concentration-dependent manner. ‘Hit’ GAGs were selected through multi-dimensional comparative analyses, including hierarchical cluster analysis, principal component analysis (PCA), and GAG fraction comparison, based on the cytokine profiles produced by macrophages cultured on GAG-coated surfaces for 6 days. The screening process identified key GAG formulations, including GAG-comb 33 (DSH001/2) and GAG-comb 63 (70% HEP-POLY4 and 30% CS-EI), which effectively modulated macrophage polarisation, showing immune-instructive properties. These formulations were subsequently tested in wound healing models. In vitro studies demonstrated that these GAGs promoted human foreskin fibroblast-mediated wound closure. 3D OrbiSIMS analysis revealed significant changes in the metabolic profiles of macrophages following treatment with the GAGs, which may explain the observed acceleration of wound closure, facilitated by their conditioned media. In vivo studies further confirmed the therapeutic efficacy of GAG-comb 63, which facilitated immune modulation and tissue regeneration, significantly improving wound healing outcomes in a diabetic model after 7 days of treatment.
This thesis provides a comprehensive evaluation of the immunomodulatory properties of GAGs, identifying key combinations that promote macrophage polarisation and support tissue regeneration and wound healing. The findings offer valuable insights into the therapeutic potential of GAGs, particularly for diabetic wound healing, and establish a solid foundation for their broader application in immune modulation and regenerative medicine.
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