Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix.

Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.

Artificial extracellular matrices support cell growth and matrix synthesis of human dermal fibroblasts in macroporous 3D scaffolds.

Surface modification of materials designed for regenerative medicine may improve biocompatibility and functionality. The application of glycosaminoglycans (GAGs) and chemically sulphated GAG derivatives is a promising approach for designing functional biomaterials, since GAGs interact with cell-derived growth factors and have been shown to support fibroblast growth in two-dimensional (2D) cultures. Here, coatings with artificial extracellular matrix (aECM), consisting of the structural protein collagen I and the GAG hyaluronan (HA) or sulphated HA derivatives, were investigated for their applicability in a three-dimensional (3D) system. As a model, macroporous poly(lactic-co-glycolic acid) (PLGA) scaffolds were homogeneously coated with aECM. The resulting scaffolds were characterized by compressive moduli of 0.9-1.2 MPa and pore sizes of 40-420 µm. Human dermal fibroblasts (dFbs) colonized these aECM-coated PLGA scaffolds to a depth of 400 µm within 14 days. In aECM-coated scaffolds, collagen I(α1) and collagen III(α1) mRNA expression was reduced, while matrix metalloproteinase-1 (MMP-1) mRNA expression was increased within 7 days, suggesting matrix-degradation processes. Stimulation with TGFß1 generally increased cell density and collagen synthesis, demonstrating the efficiency of bioactive molecules in this 3D model. Thus, aECM with sulphated HA may modulate the effectivity of TGFß1-induced collagen I(α1) expression, as demonstrated previously in 2D systems. Overall, the tested aECM with modified HA is also a suitable material for fibroblast growth under 3D conditions. Copyright © 2015 John Wiley & Sons, Ltd.

Artificial extracellular matrix composed of collagen I and highly sulfated hyaluronan interferes with TGFß(1) signaling and prevents TGFß(1)-induced myofibroblast differentiation.

Sulfated glycosaminoglycans are promising components for functional biomaterials since sulfate groups modulate the binding of growth factors and thereby influence wound healing. Here, we have investigated the influence of an artificial extracellular matrix (aECM) consisting of collagen I (coll) and hyaluronan (HA) or highly sulfated HA (hsHA) on dermal fibroblasts (dFb) with respect to their differentiation into myofibroblasts (MFb). Fibroblasts were cultured on aECM in the presence of aECM-adsorbed or soluble transforming growth factor ß1 (TGFß1). The synthesis of α-smooth muscle actin (αSMA), collagen and the ED-A splice variant of fibronectin (ED-A FN) were analyzed at the mRNA and protein levels. Furthermore, we investigated the bioactivity and signal transduction of TGFß1 in the presence of aECM and finally made interaction studies of soluble HA or hsHA with TGFß1. Artificial ECM composed of coll and hsHA prevents TGFß1-stimulated αSMA, collagen and ED-A FN expression. Our data suggest an impaired TGFß1 bioactivity and downstream signaling in the presence of aECM containing hsHA, shown by massively reduced Smad2/3 translocation to the nucleus. These data are explained by in silico docking experiments demonstrating the occupation of the TGFß-receptor I binding site by hsHA. Possibly, HA sulfation has a strong impact on TGFß1-driven differentiation of dFb and thus could be used to modulate the properties of biomaterials.

Growth promoting substrates for human dermal fibroblasts provided by artificial extracellular matrices composed of collagen I and sulfated glycosaminoglycans.

The application of native extracellular matrix (ECM) components is a promising approach for biomaterial design. Here, we investigated artificial ECM (aECM) consisting of collagen I (coll) and the glycosaminoglycans (GAGs) hyaluronan (HA) or chondroitin sulfate (CS). Additionally, GAGs were chemically modified by the introduction of sulfate groups to obtain low-sulfated and high-sulfated GAG derivatives. Sulfate groups are expected to bind and concentrate growth factors and improve their bioactivity. In this study we analyzed the effect of aECM on initial adhesion, proliferation, ECM synthesis and differentiation of human dermal fibroblasts (dFb) within 8-48 h. We show that initial adhesion and cell proliferation of dFb progressively increased in a sulfate dependent manner. In contrast, synthesis of ECM components coll and HA was decreased on high-sulfated aECM coll/HA3.0 and coll/CS3.1. Furthermore, the matrix metallo-proteinase-1 (MMP-1) was down-regulated on coll/HA3.0 and coll/CS3.1 on mRNA and protein level. The fibroblast differentiation marker α-smooth muscle actin (αSMA) is not affected by aECM on mRNA level. Artificial ECM consisting of coll and high-sulfated GAGs proves to be a suitable biomaterial for dFb adhesion and proliferation that induces a "proliferative phenotype" of dFb found in the early stages of cutaneous wound healing.