Analyzing immune response to engineered hydrogels by hierarchical clustering of inflammatory cell subsets

Understanding the immune response to hydrogel implantation is critical for the design of immunomodulatory biomaterials. To study the progression of inflammation around poly(ethylene glycol) hydrogels presenting Arg-Gly-Asp (RGD) peptides and vascular endothelial growth factor, we used temporal analysis of high-dimensional flow cytometry data paired with intravital imaging, immunohistochemistry, and multiplexed proteomic profiling. RGD-presenting hydrogels created a reparative microenvironment promoting CD206⁺ cellular infiltration and revascularization in wounded dorsal skin tissue. Unbiased clustering algorithms (SPADE) revealed significant phenotypic transition shifts as a function of the cell-adhesion hydrogel properties. SPADE identified an intermediate macrophage subset functionally regulating in vivo cytokine secretion that was preferentially recruited for RGD-presenting hydrogels, whereas dendritic cell subsets were preferentially recruited to RDG-presenting hydrogels. Last, RGD-presenting hydrogels controlled macrophage functional cytokine secretion to direct polarization and vascularization. Our studies show that unbiased clustering of single-cell data provides unbiased insights into the underlying immune response to engineered materials.