Neovascularization of Peg-Fibrinogen Hydrogels through the Encapsulation of Equine Endothelial Cells

Neovascularization—the formation of new blood vessels—represents a significant area of research due to its array of applications in tissue engineering and regenerative medicine. In tissue engineered constructs, vascularization is essential to the survival of encapsulated cells and for establishing physiological relevance of the constructs to native tissue. In regenerative medicine, neovascularization provides an innovative approach to tissue repair and wound healing throughout the body. While previous studies investigated neovascularization for human applications, such techniques have yet to be fully developed in the field of veterinary regenerative medicine. This study assessed the degree to which equine endothelial progenitor cells (EPCs) could form vasculature using 3D tissue engineered constructs and the ability of co-encapsulated equine mesenchymal stem cells (MSCs) to influence vessel formation and stability.

Equine EPCs and MSCs were isolated from horse peripheral blood and bone marrow and cultured in appropriate culture media. Prior to encapsulation, EPCs and MSCs were fluorescently labeled with CellTracker Red and Green. Labeled cells were then encapsulated in a PEG-fibrinogen hydrogel precursor both separately and in combination. Photoinitiator Eosin Y was added to the cell‑laden precursor which was then pipetted into PDMS molds set on top of acrylated glass slides. Cell-laden hydrogel precursor was then crosslinked using a metal halide lamp. Molds were removed and the hydrogels were cultured in appropriate cell culture media which was changed daily. Viability of encapsulated cells was assessed using Invitrogen’s LIVE/DEAD viability assay. Vessel formation was tracked with fluorescent microscopy.

Encapsulated cells remained viable within the hydrogel at 24 and 96 hours after crosslinking as demonstrated by the LIVE/DEAD viability assay. In hydrogels containing only EPCs as well as hydrogels containing co-cultured EPCs and MSCs, tubules appeared as early as day 1 and increased substantially over a 4 day period. As expected, no tubules were detected in hydrogels containing only MSCs.

This study demonstrated that equine EPCs could produce vasculature when cultured both alone and with MSCs in 3D PEG-fibrinogen scaffolds. It was unclear if the MSCs had a significant effect on tubule formation. Ensuing research will concentrate on quantifying tubule formation through lumen density, branching points, and average vessel length. This quantitative data will help to determine the influence of MSCs on tubule formation and stability. Eventually this technology could advance to in vivo trials for ultimate use as therapy for wound healing in horses.