The presentation of TGF-β1 during the early stage of wound healing is a prerequisite for extracellular matrix (ECM) synthesis and remodeling by activated fibroblasts, called myofibroblasts. At later stages, clearance of myofibroblasts is needed to avoid overshooting ECM production. Apoptosis of myofibroblasts and the macrophage-released anti-inflammatory cytokine IL-10 are controversially discussed as regulating cues in this context. To reveal the regulating cues, defined biomaterial scaffolds are needed to conduct in-depth in vitro studies in a physiologically relevant context. In this work, we used an in vitro biomimetic wound healing model. It consists of a 3D fibrillar matrix from collagen I and fibronectin and different temporal stimuli by TGF-β1 and IL-10. Human dermal fibroblast behavior was investigated in terms of myofibroblast differentiation (αSMA expression), matrix remodeling, proliferation and migration in the permanent or sequential presence of TGF-β1 and IL-10 over 4 days. We could show that removal of TGF-β1 after initial stimulation resulted in an increase of apoptosis of myofibroblasts. In contrast, TGF-β1 stimulation followed by IL-10 treatment did not result in increased cell apoptosis but instead led to a significant increase of cell motility and reduction of myofibroblasts. The findings suggest that myofibroblasts are a transiently "activated" fibroblastic phenotype and can be de-differentiated to fibroblasts in the presence of IL-10. Overall, our 3D ECM model allows mimicking the early and late stages of wound healing and highlights the temporal sequence of TGF-β1 and IL-10 as an important cue for completion of tissue formation and maintenance of tissue homeostasis.

TGFβ1 is a key regulator for induction of tissue remodeling after dermal wounding. We present a model of paracrine delivery of TGFβ1 for differentiation of dermal fibroblasts based on a fibrillar 3D collagen matrix and embedded TGFβ1 releasing microparticles. We found differentiation into myofibroblasts was achieved in a TGFβ1 dependent manner at much lower doses than systemic delivery. This effect is accounted to the slow and sustained TGFβ1 release mimicking paracrine cell signals.

Cell fate decisions in many physiological processes, including embryogenesis, stem cell niche homeostasis and wound healing, are regulated by secretion of small signaling proteins, called cytokines, from source cells to their neighbors or into the environment. Concentration level and steepness of the resulting paracrine gradients elicit different cell responses, including proliferation, differentiation or chemotaxis. For an in-depth analysis of underlying mechanisms, in vitro models are required to mimic in vivo cytokine gradients. We set up a microparticle-based system to establish short-range cytokine gradients in a three-dimensional extracellular matrix context. To provide native binding sites for cytokines, agarose microparticles were functionalized with different glycosaminoglycans (GAG). After protein was loaded onto microparticles, its slow release was quantified by confocal microscopy and fluorescence correlation spectroscopy. Besides the model protein lysozyme, SDF-1 was used as a relevant chemokine for hematopoietic stem and progenitor cell (HSPC) chemotaxis. For both proteins we found gradients ranging up to 50μm from the microparticle surface and concentrations in the order of nM to pM in dependence on loading concentration and affinity modulation by the GAG functionalization. Directed chemotactic migration of cells from a hematopoietic cell line (FDCPmix) and primary murine HSPC (Sca-1(+) CD150(+) CD48(-)) toward the SDF-1-laden microparticles proved functional short-range gradients in a two-dimensional and three-dimensional setting over time periods of many hours. The approach has the potential to be applied to other cytokines mimicking paracrine cell-cell interactions in vitro.

Physicochemical surface modifications of soft hydrogel layers are beneficial tools for modulating biomimetic substrate properties in cell adhesion studies. Recently, a layered system of a polyacrylamide hydrogel with a monolayer coating of maleic anhydride copolymers was introduced, however, without a detailed study of the chemical coupling mechanism. Herein it is shown that a stable coupling results from covalent binding of maleic anhydride residues to the primary amides of polyacrylamide via imide bond formation. The many anhydride moieties of the copolymer chains allow for coupling with a low‐yield reaction at mild conditions. Furthermore, hydrogen bonding and polar interactions are excluded to account for a stable binding of maleic anhydride copolymers to polyacrylamide hydrogels.