Dynamic alterations of composition and mechanics of the extracellular matrix are suggested to modulate cellular behavior including plasticity of macrophages (MPhs) during wound healing. In this study, engineered 3D fibrillar matrices based on naturally occurring biopolymers (collagen I, glycosaminoglycans (GAGs)) are used to mimic matrix stiffening as well as modification by sulfated and nonsulfated GAGs at different stages of wound healing. Human MPhs are found to sensitively respond to these microenvironmental cues in terms of polarization toward proinflammatory or wound healing phenotypes over 6 days in vitro. MPhs exhibit a wound healing phenotype in stiffer matrices as determined by protein and gene expression of relevant cytokines (IL10, IL12, and TNFα). Presence of sulfated and nonsulfated GAGs inhibits this polarization effect. Furthermore, control experiments on 2D matrices stress the relevance of using stiffness-controlled 3D matrices, as MPhs show a reciprocal polarization behavior depending on GAG presence. Hence, the results indicate a strong influence of dimensionality, stiffness, and GAG presence of the biomaterial scaffold on MPh polarization and emphasize the need for matrices closely mimicking the 3D in vivo context with a variable stiffness and GAG composition in in vitro studies.

The homeostasis of hematopoietic stem and progenitor cells (HSC) in the bone marrow is regulated by a complex interplay of exogenous signals, including extracellular matrix (ECM) molecules, cell-cell contacts, and cytokines. To investigate the influence of spatial restriction and adhesive interactions on HSC fate decisions, we prepared a set of fibronectin-coated micrometer-sized cavities. Analysis of human CD133+ HSCs isolated after culture on these surfaces revealed that proliferation and differentiation is decreased when HSCs are supported by substrates with small microcavities. Single cell analysis of adherent cells also revealed decreased DNA synthesis and higher levels of HSC marker expression inside the smaller cavities. Increasing the cytokine concentration highlighted the tight balance of adhesion related signals and soluble cues acting on HSC fate decisions. Our results suggest that confining human HSCs in ECM-coated microcavities is a possible method to maintain these cells in a quiescent and immature state, an important advantage for several HSC applications.

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Persistent inflammation and impaired repair in dermal wound healing are frequently associated with cell–cell and cell–matrix miscommunication. A direct coculture model of primary human myofibroblasts (MyoFB) and M‐CSF‐differentiated macrophages (M‐Mɸ) in fibrillar three‐dimensional Collagen I (Coll I) matrices is developed to study intercellular interactions. The coculture experiments reveal the number of M‐Mɸ regulated MyoFB dedifferentiation in a dose‐dependent manner. The amount of MyoFB decreases in dependence of the number of cocultured M‐Mɸ, even in the presence of MyoFB‐inducing transforming growth factor β1 (TGF‐β1). Gene expression analysis of matrix proteins (collagen I, collagen III, ED‐A‐fibronectin) confirms the results of an altered MyoFB phenotype. Additionally, M‐Mɸ is shown to be the main source of secreted cytokine interleukin‐10 (IL‐10), which is suggested to affect MyoFB dedifferentiation. These findings indicate a paracrine impact of IL‐10 secretion by M‐Mɸ on the MyoFB differentiation status counteracting the TGF‐β1‐driven MyoFB activation. Hence, the in vitro coculture model simulates physiological situations during wound resolution and underlines the importance of paracrine IL‐10 signals by M‐Mɸ. In sum, the 3D Coll I‐based matrices with a MyoFB–M‐Mɸ coculture form a highly relevant biomimetic model of late stages of wound healing.