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Temporally controlled, low-level release of encapsulated FGF2 from PLGA microspheres promotes survival and angiogenic differentiation of human umbilical vein endothelial cells, inducing neo-vessel formation in nude mice
Jennifer McGrath, BS, Patrick Gerety, MD, Jesse A. Taylor, MD, Hyun-Duck Nah, DMD PhD.
Children's Hospital of Philadelphia, Philadelphia, PA, USA.

PURPOSE: Tissue engineering offers a regenerative solution to reconstruction of large defects throughout the body. However, the major obstacle for therapeutic applications of engineered materials is the lack of rapid vascular ingress into the construct. The maximum distance that cells can survive based on diffusion of nutrients is 150 to 200 µm, necessitating early scaffold vascularization. FGF2 is a potent growth factor for angiogenesis. Therefore, controlled delivery of FGF2 and angiogenic cells to repair sites may prove to be a new and significant therapeutic solution for vascularization of a large defect. The goal of this study was to prepare PLGA microspheres (MS) that can deliver FGF2 in a temporally controlled manner and test the efficacy of the FGF2 PLGA-MS in neo-vessel formation in mice.
METHODS:FGF2 was encapsulated in PLGA microspheres using a double emulsion solvent extraction technique. Optimal release kinetics for early controlled delivery of FGF2 was determined by manipulating polymer molecular weight and concentration during microsphere fabrication. The bioactivity of encapsulated FGF2 was assessed in vitro by the MTT assay using human umbilical cord-derived endothelial cells (HUVECs). Athymic nude mice were injected with matrigel containing no MS, low dose, or high dose FGF2 PLGA-MS, with and without HUVECs. Cell survival and microvascular density were assessed by histology at 10 and 20 days.
RESULTS: PLGA microspheres encapsulating bovine serum albumin as a model protein were fabricated to optimize release kinetics. Varying polymer molecular weight and concentration allowed for manipulation of protein release kinetics. FGF2 was then successfully and efficiently encapsulated in PLGA microspheres to achieve 40% burst with 80% total release of FGF2 by 7 days. In vitro study showed that FGF2 MS promoted cell survival in a dose dependent manner when compared to cells without growth factor (p<0.05).

When FGF2 MS were implanted with matrigel and cells, HUVECs survived and formed neo-vessels within the matrigel matrix at 20 days; however, injection of matrigel alone or matrigel with HUVECs showed no evidence of vascularization. FGF2 MS alone resulted in ingrowth of host cells into the injectable matrix but did not show vessel formation.

Matrigel and HUVECs showed greater vascularity when implanted with high-dose (5ng/droplet) FGF2 MS when compared to low-dose (1ng/droplet) FGF2 MS (24 vs 8 lumens/HPF, p<0.01; MVD 1.0% vs 0.44%, p=0.04).
CONCLUSION: Achieving controlled, sustained delivery of bioactive growth factors is a critical challenge in the clinical application of tissue engineering. We have achieved neo-vessel formation during the first 20 days in vivo by delivering a low-dose of bioactive growth factor using encapsulation in biodegradable microspheres. Encapsulation of FGF2 is a promising utility for growth factor delivery by promoting survival of implanted cells as well as recruiting host cells and inducing vascularization within an injectable matrix.

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