(84b) Gradient Biomaterials Accelerate Vascularization in Vivo

Controlled angiogenesis is essential for the clinical translation of tissue engineering. Blood vessel formation in vivo is influenced by gradients of soluble growth factors. The use of microfluidic systems has enabled the evaluation of cell behaviors in controlled, small volume applications. Studies have shown the importance of gradients of soluble factors on the directed and accelerated growth and migration of cells and vessels. The complex nature and small volume of the microfluidic systems used precludes their application in vivo. Recently, we have developed a method for generating gradients of soluble factors within our porous hydrogels which can be easily applied to increase vascular invasion depth in vivo.

Composite hydrogels of poly(ethyelene glycol) (PEG) and fibrin were generated using a particulate leaching method. These hydrogels support vascularized tissue formation but at levels insufficient for clinical application.¹Gradients of soluble factors were generated through the placement of a growth factor source within the hydrogel system distal to the tissue bed. The source used in this case was poly(lactic-co-glycolic acid) (PLGA) microspheres prepared using a double emulsion procedure. When placed in the distal region of the hydrogel system the released proteins diffuse to the tissue bed generating a gradient within the hydrogel from the microspheres to the tissue that promotes vessel invasion.

Platelet derived growth factor BB (PDGF-BB) was chosen for the following studies as it has been shown to stimulate directed growth of multiple cell types and plays an important role in vessel maturation and stabilization. Cell and tissue response depends, in part, on the magnitude of the gradient. The release of PDGF-BB was varied based on the composition of the PLGA microspheres. Using this technique, persistent gradients (greater than 50 days) were generated within the hydrogel scaffolds. The gradient, formed by the transport of PDGF-BB through the hydrogel, was modeled following Fick’s second law, which assumes that diffusion is the only mechanism of transport and that the proteins do not interact (react) with the porous structure as they diffuse. The gradient in the first 5 days is higher due to the burst release exhibited by the microspheres. From 10 days the concentration profile gradually decreases as the microspheres approach zero order (constant) release.  The model predictions were verified by measuring transport from the gel using radiolabeled proteins. 

In order to further optimize our release system prior to analysis in a murine subject, an agent-based model of angiogenesis in engineered tissues was extended to enable testing the influence of biomaterial structures and growth factor release rates on vascularization.² Parameters, including the magnitude of the concentration gradient, pore size and pore interconnectivity were varied and the depth and extent of vascularization evaluated in silico. These studies suggest that under optimized hydrogel conditions, gradients of soluble factors can increase the rate at which vessels invade the engineered tissues.

A murine subcutaneous implant model was used to examine the influence of gradients and the magnitude of gradients on vascularization. PEG/Fibrin composite hydrogels with increasing concentrations (0, 1, 10, and 100 µg/ml) of PDGF-BB were prepared and implanted. The concentration was varied to investigate the role of gradient magnitude on vascular invasion. The hydrogels were harvested at 1, 3, and 6 weeks.  Vascularization was evaluated using lectin staining and confocal imaging. Tissue structure was evaluated with histological stains.

The gradient hydrogels resulted in significantly higher vascular density within the hydrogels after 1 and 3 weeks of implantation in comparison to controls. At the highest concentration (100µg/ml), the depth of vessel invasion was 1.5X greater than any other conditions we have studied in the same model, including multiple growth factor systems described in other studies. These results show that we have developed a novel method for generating persistent gradients within porous hydrogel systems that can be implanted in vivo. The gradients rapidly accelerate vascularization into the porous hydrogels. We are currently examining vascularization in gradient hydrogels at longer time points.

References:

1. Jiang B. et al., Fibrin Loaded Porous Poly(ethylene glycol) Hydrogels Induce Vascular Network Formation. Tissue Engineering Part A. 19(1-2):224-34, 2013.

2. Mehdizadeh H. et al., Three-Dimensional Modeling of Angiogenesis in Porous Biomaterial Scaffolds. Biomaterials. 34(12):2875-87, 2013.