title: Endothelial Network Formation Within Human Tissue-Engineered Skeletal Muscle.
abstract: The size of in vitro engineered skeletal muscle tissue is limited due to the lack of a vascular network in vitro. In this article, we report tissue-engineered skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. We extend our bioartificial muscle (BAM) model by coculturing human muscle progenitor cells with human umbilical vein endothelial cells (HUVECs) in a fibrin extracellular matrix (ECM). First, the optimal medium conditions for coculturing myoblasts with HUVECs were determined in a fusion assay. Endothelial growth medium proved to be the best compromise for the coculture, without affecting the myoblast fusion index. Second, both cell types were cocultured in a BAM maintained under tension to stimulate myofiber alignment. We then tested different total cell numbers containing 50% HUVECs and found that BAMs with a total cell number of 2 × 10(6) resulted in well-aligned and densely packed myofibers while allowing for improved interspersed endothelial network formation. Third, we compared different myoblast-HUVEC ratios. Including higher numbers of myoblasts improved endothelial network formation at lower total cell density; however, improvement of network characteristics reached a plateau when 1 × 10(6) or more myoblasts were present. Finally, addition of Matrigel to the fibrin ECM did not enhance overall myofiber and endothelial network formation. Therefore, in our BAM model, we suggest the use of a fibrin extracellular matrix containing 2 × 10(6) cells of which 50-70% are muscle cells. Optimizing these coculture conditions allows for a physiologically more relevant muscle model and paves the way toward engineering of larger in vitro muscle constructs.
authors.affiliation: 1 Tissue Engineering Laboratory , Department of Development and Regeneration, KU Leuven, Kortrijk, Belgium .
authors.forename: Dacha
authors.initials: D
authors.lastname: Gholobova
authors.affiliation: 1 Tissue Engineering Laboratory , Department of Development and Regeneration, KU Leuven, Kortrijk, Belgium .
authors.forename: Lieselot
authors.initials: L
authors.lastname: Decroix
authors.affiliation: 1 Tissue Engineering Laboratory , Department of Development and Regeneration, KU Leuven, Kortrijk, Belgium .
authors.forename: Vicky
authors.initials: V
authors.lastname: Van Muylder
authors.affiliation: 1 Tissue Engineering Laboratory , Department of Development and Regeneration, KU Leuven, Kortrijk, Belgium .
authors.forename: Linda
authors.initials: L
authors.lastname: Desender
authors.affiliation: 1 Tissue Engineering Laboratory , Department of Development and Regeneration, KU Leuven, Kortrijk, Belgium .
authors.forename: Melanie
authors.initials: M
authors.lastname: Gerard
authors.affiliation: 2 Laboratoire de Recherche sur la Croissance Cellulaire, la Réparation et la Régénération Tissulaires (CRRET), Faculté des Sciences et Technologie, Université Paris-Est , Créteil, France .
authors.forename: Gilles
authors.initials: G
authors.lastname: Carpentier
authors.affiliation: 3 Department of Pathology and Laboratory Medicine, Brown University , Providence, Rhode Island.
authors.forename: Herman
authors.initials: H
authors.lastname: Vandenburgh
authors.affiliation: 1 Tissue Engineering Laboratory , Department of Development and Regeneration, KU Leuven, Kortrijk, Belgium .
authors.forename: Lieven
authors.initials: L
authors.lastname: Thorrez
mesh_terms: D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods*; D005109:Extracellular Matrix / Q000737:chemistry; D005337:Fibrin / Q000737:chemistry; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology; D006801:Humans; D007150:Immunohistochemistry; D018482:Muscle, Skeletal / Q000166:cytology*; D032448:Myoblasts, Skeletal / Q000166:cytology; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D005337:Fibrin