Glycosaminoglycan-Induced Macrophage Reprogramming Orchestrates Pro-Regenerative Crosstalk with Fibroblasts.

Abstract

Macrophages and fibroblasts play central roles in coordinating inflammation and tissue repair, whilst dysregulation of macrophage-fibroblast crosstalk drives pathological outcomes such as chronic inflammation and fibrosis. Glycosaminoglycans (GAGs), key components of the extracellular matrix (ECM), interact with cytokines and growth factors that modulate macrophage polarisation and direct fibroblast activity, making them attractive candidates for developing immune-instructive biomaterials. However, the optimal GAG compositions that promote macrophage-mediated repair and fibroblast-driven ECM remodelling remain unclear. Here, we report an unbiased screening of 82 surface-adsorbed GAG combinations to identify candidates capable of modulating macrophage-fibroblast interactions. Leveraging cytokine profiling, surface marker immunostaining, gene expression, principal component analysis (PCA), and hierarchical clustering, we identified 'hit' GAG combinations that induced distinct, non-canonical macrophage activation programmes characterised by coordinated inflammatory and regulatory signalling, alongside enhanced fibroblast proliferation. Shortlisted formulations were validated using expanded biological replication, orthogonal phenotypic, transcriptional and functional analyses, assessment in macrophage-fibroblast co-culture systems, and in an in vivo diabetic wound model. These data demonstrated that 'hit' GAG formulations modulated macrophage functional activation states, promoted collagen and ECM synthesis, facilitated vascular and lymphatic vessel formation, and accelerated wound closure. Complementary metabolomic profiling using OrbiSIMS and molecular formula prediction (MFP), revealed macrophage metabolic reprogramming as a potential mechanism underlying these pro-healing effects. Collectively, this study established an unbiased methodology for discovering immune-instructive GAGs in supporting pro-healing responses for potential wound-healing applications. STATEMENT OF SIGNIFICANCE: Glycosaminoglycans (GAGs) are key extracellular matrix components with significant potential to regulate immune responses. However, their immunomodulatory effects are highly context-dependent, varying with sulfation pattern, molecular composition, and the local microenvironment, making it challenging to identify GAG combinations that consistently promote pro-regenerative immune responses and tissue repair. Here, we developed an unbiased combinatorial screening platform to systematically identify GAG combinations that reprogram macrophages toward pro-regenerative functional states. The selected GAG combinations enhanced macrophage-fibroblast crosstalk in vitro and improved diabetic wound healing in vivo by promoting collagen deposition, angiogenesis, and lymphatic remodelling. OrbiSIMS metabolomics further revealed macrophage metabolic reprogramming associated with these regenerative responses. This work establishes a generalizable discovery framework for identifying immune-regulatory GAG formulations and provides new design principles for developing extracellular matrix-inspired immunomodulatory biomaterials for regenerative medicine.

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