Hyaluronate lyase from streptomyces hyalurolyticus

Research grade hyaluronic acid (average molecular weight 74 kDa) was obtained from Lifecore Biomedical (Chaska, MN, USA). The cross-linkers for hydrogel formation were synthesized adapted from a protocol of Vibert et al. 2009 [14 (link)]. The compounds glucosamine hydrochloride and ammonium chloride, hyaluronidase type IV from bovine testes and hyaluronate lyase from Streptomyces hyalurolyticus were obtained from Sigma-Aldrich (Darmstadt, Germany). Linear RGD was purchased from PSL (Heidelberg, Germany). The Thermo Fisher Scientific Live/Dead Double Staining Kit was purchased from Merck (Darmstadt, Germany).
Normal human dermal fibroblasts (NHDF) with fibroblast basal medium (FGM2) and supplements (FGM2 Supplements) were purchased from Promocell (Heidelberg, Germany).

HUVEC, human umbilical vein endothelial cells (Gibco, Waltham, MA, USA); M200 culture medium (Gibco, Waltham, MA, USA); DMEM High Glucose w/o Sodium Pyruvate w/ L-Glutamine (Euro Clone, Pero, Italy); TNF-α (Sino Biological Inc., Wayne, NJ, USA); Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA); Hyaluronate Lyase from Streptomyces hyalurolyticus (Sigma-Aldrich, St. Louis, MO, USA); Transwell system with filter of 0.4 µm pore size, 6.5 mm diameter (Corning, New York, NY, USA); FITC labelled dextran (Mw~250,000, Sigma-Aldrich, St. Louis, MO, USA); heparinases I–II–III (form F. heparinum, Seikagaku, Tokyo, Japan); 2-Aminoacridone, AMAC-(Sigma-Aldrich, St. Louis, MO, USA); Chondroitinase ABC (from Proteus vulgaris, Seikagaku, Tokyo, Japan); 3-hydroxybiphenol (Fluka, Buchs, Switzerland); D-Glucuronic acid (Sigma-Aldrich, St. Louis, MO, USA); prostaglandin E1 and indomethacin (Sigma); all chemicals were purchased by Sigma-Aldrich (St. Louis, MO, USA).