title: Effects of hydroxyapatite on endothelial network formation in collagen/fibrin composite hydrogels in vitro and in vivo.
abstract: Co-culture of endothelial cells (EC) and mesenchymal stem cells (MSC) results in robust vascular network formation in constrained 3-D collagen/fibrin (COL/FIB) composite hydrogels. However, the ability to form endothelial networks is lost when such gels are allowed to compact via cell-mediated remodeling. In this study, we created co-cultures of human EC and human MSC in both constrained and unconstrained COL/FIB matrices and systematically added nanoparticulate hydroxyapatite (HA, 0-20 mg ml(-1)), a bone-like mineral that has been shown to have pro-vasculogenic effects. Constructs cultured for 7 days were assayed for gel compaction, vascular network formation, and mechanical properties. In vitro, robust endothelial network formation was observed in constrained COL/FIB constructs without HA, but this response was significantly inhibited by addition of 5, 10, or 20 mg ml(-1) HA. In unconstrained matrices, network formation was abolished in pure COL/FIB constructs but was rescued by 1.25 or 2.5 mg ml(-1) HA, while higher levels again inhibited vasculogenesis. HA inhibited gel compaction in a dose-dependent manner, which was not correlated to endothelial network formation. HA affected initial stiffness of the gels, but gel remodeling abrogated this effect. Subcutaneous implantation of COL/FIB with 0, 2.5 or 2 0mg ml(-1) HA in the mouse resulted in increased perfusion at the implant site, with no significant differences between materials. Histology at day 7 showed both host and human CD31-stained vasculature infiltrating the implants. These findings are relevant to the design of materials and scaffolds for orthopedic tissue engineering, where both vasculogenesis and formation of a mineral phase are required for regeneration.
authors.affiliation: Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA.
authors.forename: Rameshwar R
authors.initials: RR
authors.lastname: Rao
authors.affiliation: Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Ceccarelli
authors.affiliation: Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA.
authors.forename: Marina L
authors.initials: ML
authors.lastname: Vigen
authors.affiliation: Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA.
authors.forename: Madhu
authors.initials: M
authors.lastname: Gudur
authors.affiliation: Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA.
authors.forename: Rahul
authors.initials: R
authors.lastname: Singh
authors.affiliation: Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA.
authors.forename: Cheri X
authors.initials: CX
authors.lastname: Deng
authors.affiliation: Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Putnam
authors.affiliation: Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA. Electronic address: jpsteg@umich.edu.
authors.forename: Jan P
authors.initials: JP
authors.lastname: Stegemann
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques; D003094:Collagen; D017886:Durapatite; D005337:Fibrin; D006801:Humans; D020100:Hydrogels; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D016513:Mice, SCID
chemical_list: D020100:Hydrogels; D005337:Fibrin; D003094:Collagen; D017886:Durapatite
keywords: Collagen; Fibrin; Hydroxyapatite; In vivo; Vasculogenesis