Therapeutic Angiogenesis in Critical Limb Ischemia via the Localized Delivery of bFGF from Ionic Hydrogels Hans Layman1, Toby Brooks1, Maria-Grazia Spiga3, Ph.D., Keith Webster, Ph.D., Si Pham2, MD, Fotios M. Andreopoulos1,2, Ph.D. 1Department of Biomedical Engineering, University of Miami, Miami, FL, USA. 2Department of Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA 3 Vascular Biology Institute, Miller School of Medicine, University of Miami, Miami, FL, USA Characterization of Hydrogels Methods and Results Summary Degree of Swelling Experiments Surgical Technique Schematic The development of peptide-releasing scaffolds capable of stimulating revascularization of ischemic or underperfused tissues remains an area of continuous scientific interest in therapeutic angiogenesis. Herein, we report the synthesis and characterization of ionic, gelatin-based scaffolds as potential vehicles for localized angiogenic growth factor delivery. Ionic gel scaffolds were prepared by co-polymerizing gelatin with poly-L-lysine or poly-L-glutamic acid using a water-soluble EDC/NHS coupling mixture. Basic fibroblast growth factor (bFGF) was incorporated within the gel scaffolds during polymerization, and its release kinetics was assessed in vitro as a function of gel properties. A murine, hindlimp ischemic model was utilized to assess the effectiveness of the bFGF-releasing gelatin scaffolds to induce revascularization. The bFGF-containing gel scaffolds were placed adjacent to the ligated region and revascularization of the leg was determined via laser Doppler perfusion imaging and the release kinetics of bFGF was determined in vivo using an ELISA assay. The bFGF-releasing scaffolds were superior in inducing angiogenesis in comparison to bolus injections of bFGF. Figure 2. Degree of swelling (DS) as a function of reaction conditions. The ratio of crosslinking reagent was varied and gels were crosslinked via EDC/NHS activation in an aqueous buffer at a pKa value nominal to the ionic polymer co-polymerized with the gelatin (i.e. pKa for glutamic acid is 4.5; pKa for lysine is 10.4). Figure 5. Points of ligation and region of excision along the superficial femoral artery and vein, deep femoral artery and vein, and the external iliac artery and vein for simulation of critical limb ischemia in a murine model. Laser Doppler Perfusion Images In Vitro and In Vivo Release of bFGF In Vitro Release Kinetics In Vivo Release Kinetics Figure 6. Doppler image comparisons of treatment groups over an eight-week time course. A: Control group with no surgery; B: Control group with ligation and excision of the femoral artery; C: Treatment with crosslinked gelatin B with poly-L-glutamic acid loaded with bFGF; D: Treatment with a bolus injection of bFGF to the ischemic wound site. Synthesis of Ionic, Crosslinked Scaffolds Figure 7. Comparison of Doppler ratios over eight weeks. The right femoral artery was ligated, excised between the external iliac and saphenous branches and the treatment group was implanted a the site of injury. The ischemic leg was scanned one hour post-surgery, and every seven days after surgery for four weeks. N= 3 animals per time point per group. (** - All groups excluding Gel A/FGF are statistically significant compared to controls; *** - All treatment groups are significant compared to controls). Figure 4. In vivo bFGF release over 8 weeks. bFGF (human recombinant) content in each gel scaffold was 250 ng. Quadriceps muscle tissue was harvested, washed of blood, homogenized in a tissue grinder and incubated for 30 minutes at room temperature in a lysis buffer. The homogenate was centrifuged at 3000 rpm for 25 minutes. The supernatant was then extracted and diluted at a ratio of 1:10,000 in phosphate buffered saline (PBS, pH = 7.4). The bFGF concentration of the supernatant was determined with an ELISA kit. Figure 3. bFGF release from ionic, gelatin-based gel scaffolds over 28 days. bFGF (human recombinant) content in each gel scaffold was 250 ng. Hydrogels were incubated at 37 °C and 5% CO2 atmosphere and the released bFGF was determined by assaying the surrounding medium with an ELISA kit at defined time points. A: the total release profile over 28 days; B: initial burst release of bFGF over 120 hours. Acknowledgements Figure 1. Overall reaction schematic for the synthesis of ionic, gelatin-based gel scaffolds via water-soluble carbodiimide activation. The reaction occurs under mild conditions at pH 7.4 and 4 °C for 10 minutes. This work was partially funded by an NIH grant to FMA (RO3-NIAMS AR505018) and the Departments of Biomedical Engineering and Surgery at the University of Miami.