No abstract

Forming functional blood vessel networks in engineered or ischemic tissues is a significant scientific and clinical hurdle. We adapted poly(ethylene glycol) (PEG)-based hydrogels to investigate the role of mechanical properties and proteolytic susceptibility on vascularization. Four arm PEG vinyl sulfone was polymerized by Michael-type addition with cysteine groups on a slowly degraded matrix metalloprotease (MMP) susceptible peptide, GPQG↓IWGQ, or a more rapidly cleaved peptide, VPMS↓MRGG. Co-encapsulation of endothelial cells and supportive fibroblasts within the gels led to vascular morphogenesis in vitro that was robust to changes in crosslinking peptide identity, but was significantly attenuated by increased crosslinking and MMP inhibition. Perfused vasculature formed from transplanted cells in vivo in all gel types; however, in contrast to the in vitro results, vascularization in vivo was not decreased in the more crosslinked gels. Collectively, these findings demonstrate the utility of this platform to support vascularization both in vitro and in vivo.

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Co-culture of endothelial cells (EC) and mesenchymal stem cells (MSC) results in robust vascular network formation in constrained 3D 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), 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 HA. In unconstrained matrices, network formation was abolished in pure COL/FIB constructs, but was rescued by 1.25 or 2.5 mg/mL 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 20 mg/mL 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 orthopaedic tissue engineering, where both vasculogenesis and formation of a mineral phase are required for regeneration.

Minimally invasive, injectable bone tissue engineering therapies offer the potential to facilitate orthopedic repair procedures, including in indications where enhanced bone regeneration is needed for complete healing. In this study, we developed a dual-phase tissue construct consisting of osteogenic (Osteo) and vasculogenic (Vasculo) components. A modular tissue engineering approach was used to create collagen/fibrin/hydroxyapatite (COL/FIB/HA) hydrogel microbeads containing embedded human bone marrow-derived mesenchymal stem cells (bmMSC). These microbeads were predifferentiated toward the osteogenic lineage in vitro for 14 days, and they were then embedded within a COL/FIB vasculogenic phase containing a coculture of undifferentiated bmMSC and human umbilical vein endothelial cells (HUVEC). In vitro studies demonstrated homogenous dispersion of microbeads within the outer phase, with endothelial network formation around the microbeads over 14 days in the coculture conditions. Subcutaneous injection into immunodeficient mice was used to investigate the ability of dual-phase (Osteo+Vasculo) and control (Osteo, Vasculo, Blank) constructs to form neovasculature and ectopic bone. Laser Doppler imaging demonstrated blood perfusion through all constructs at 1, 4, and 8 weeks postimplantation. Histological quantification of total vessel density showed no significant differences between the conditions. Microcomputed tomography indicated significantly higher ectopic bone volume (BV) in the Osteo condition at 4 weeks. At 8 weeks both the Osteo and Blank groups exhibited higher BV compared to the Vasculo and dual Osteo+Vasculo groups. These data not only show that osteogenic microbeads can be used to induce ectopic bone formation, but also suggest an inhibitory effect on BV when undifferentiated bmMSC and HUVEC were included in dual-phase constructs. This work may lead to improved methods for engineering vascularized bone tissue, and to injectable therapies for the treatment of orthopedic pathologies in which bone regeneration is delayed or prevented.