Stephen Brigido, Nicole Protzman

ABSTRACT

Effective wound healing using biomaterials depends on the presence of extracellular matrix (ECM) components that support and guide the body’s natural regenerative processes. Macrophages, key immune cells derived from monocytes, play a pivotal role in both the initiation and resolution phases of tissue repair. During early wound healing, macrophages drive the inflammatory response, whereas in later stages, they contribute to tissue remodeling and resolution. Chronic wounds often result from dysregulated macrophage activation, which impairs normal healing. This study aimed to evaluate the influence of a decellularized, dehydrated human amniotic membrane (DDHAM) on monocyte-to-macrophage differentiation and activation in vitro. Human monocytes were isolated from peripheral blood of healthy donors and cultured on standard tissue culture plates (CB), collagen type I-coated plates (COL), and DDHAM-coated plates. Proinflammatory (M1) macrophage differentiation was induced using granulocyte-macrophage colony stimulating factor (GM-CSF) followed by activation with a proinflammatory stimulus comprising lipopolysaccharide (LPS) and interferon-gamma (IFN-?). The results demonstrated that DDHAM significantly enhanced monocyte differentiation compared to CB and COL controls, as evidenced by increased cell size, viability, expression of macrophage-specific genes, and secretion of soluble factors. Notably, macrophages differentiated on DDHAM and activated with inflammatory signals exhibited a marked reduction in expression of several LPS inducible NF-?B target genes, with IL12?, encoding the IL12p40 subunit, showing the most pronounced downregulation (p < 0.001). Mechanistic studies revealed that DDHAM-mediated effects on differentiation were dependent on ?2 integrins.