Effects of hydroxyapatite on endothelial network formation in collagen/fibrin composite hydrogels in vitro and in vivo

Acta Biomater. 2014 Jul;10(7):3091-7. doi: 10.1016/j.actbio.2014.03.010. Epub 2014 Mar 18.


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.

Keywords: Collagen; Fibrin; Hydroxyapatite; In vivo; Vasculogenesis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Coculture Techniques
  • Collagen*
  • Durapatite*
  • Fibrin*
  • Humans
  • Hydrogels*
  • In Vitro Techniques
  • Male
  • Mice
  • Mice, SCID


  • Hydrogels
  • Fibrin
  • Collagen
  • Durapatite