title: Human in vitro 3D co-culture model to engineer vascularized bone-mimicking tissues combining computational tools and statistical experimental approach.
abstract: The generation of functional, vascularized tissues is a key challenge for both tissue engineering applications and the development of advanced in vitro models analyzing interactions among circulating cells, endothelium and organ-specific microenvironments. Since vascularization is a complex process guided by multiple synergic factors, it is critical to analyze the specific role that different experimental parameters play in the generation of physiological tissues. Our goals were to design a novel meso-scale model bridging the gap between microfluidic and macro-scale studies, and high-throughput screen the effects of multiple variables on the vascularization of bone-mimicking tissues. We investigated the influence of endothelial cell (EC) density (3-5 Mcells/ml), cell ratio among ECs, mesenchymal stem cells (MSCs) and osteo-differentiated MSCs (1:1:0, 10:1:0, 10:1:1), culture medium (endothelial, endothelial + angiopoietin-1, 1:1 endothelial/osteo), hydrogel type (100%fibrin, 60%fibrin+40%collagen), tissue geometry (2 × 2 × 2, 2 × 2 × 5 mm(3)). We optimized the geometry and oxygen gradient inside hydrogels through computational simulations and we analyzed microvascular network features including total network length/area and vascular branch number/length. Particularly, we employed the "Design of Experiment" statistical approach to identify key differences among experimental conditions. We combined the generation of 3D functional tissue units with the fine control over the local microenvironment (e.g. oxygen gradients), and developed an effective strategy to enable the high-throughput screening of multiple experimental parameters. Our approach allowed to identify synergic correlations among critical parameters driving microvascular network development within a bone-mimicking environment and could be translated to any vascularized tissue.
authors.affiliation: Cell and Tissue Engineering Lab, IRCCS Istituto Ortopedico Galeazzi, Milano, 20161, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Bersini
authors.affiliation: Cell and Tissue Engineering Lab, IRCCS Istituto Ortopedico Galeazzi, Milano, 20161, Italy; PhD School in Life Sciences, Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, 20126, Italy.
authors.forename: Mara
authors.initials: M
authors.lastname: Gilardi
authors.affiliation: Cell and Tissue Engineering Lab, Gruppo Ospedaliero San Donato Foundation, Milano, 20122, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Arrigoni
authors.affiliation: Cell and Tissue Engineering Lab, Gruppo Ospedaliero San Donato Foundation, Milano, 20122, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Talò
authors.affiliation: Unit of Microscopy and Dynamic Imaging, National Centre for Cardiovascular Research (CNIC) Melchor Fernandez Almagro, Madrid, 28029-ES, Spain.
authors.forename: Moreno
authors.initials: M
authors.lastname: Zamai
authors.affiliation: Hip Department, IRCCS Istituto Ortopedico Galeazzi, Milano, 20161, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Zagra
authors.affiliation: Unit of Microscopy and Dynamic Imaging, National Centre for Cardiovascular Research (CNIC) Melchor Fernandez Almagro, Madrid, 28029-ES, Spain.
authors.forename: Valeria
authors.initials: V
authors.lastname: Caiolfa
authors.affiliation: Cell and Tissue Engineering Lab, IRCCS Istituto Ortopedico Galeazzi, Milano, 20161, Italy. Electronic address: matteo.moretti@grupposandonato.it.
authors.forename: Matteo
authors.initials: M
authors.lastname: Moretti
mesh_terms: D001842:Bone and Bones / Q000098:blood supply*; D018920:Coculture Techniques; D003198:Computer Simulation; D005109:Extracellular Matrix; D006801:Humans; D066298:In Vitro Techniques; D023822:Tissue Engineering
keywords: Bone-mimicking; Computational simulation; Design of experiment; ECM remodeling; Microvascular networks; Oxygen distribution