The effect of the controlled release of basic fibroblast growth factor from ionic gelatin-based hydrogels on angiogenesis in a murine critical limb ischemic model

Abstract

The localized delivery of exogenous, angiogenic growth factors has become a promising alternative treatment of peripheral artery disease (PAD) and critical limb ischemia. In the present study, we describe the development of a novel controlled release vehicle to promote angiogenesis in a murine critical limb ischemic model. Ionic, gelatin-based hydrogels were prepared by the carbodiimide-mediated amidation reaction between the carboxyl groups of gelatin or poly-L-glutamic acid molecules and the amine groups of poly-L-lysine or gelatin molecules, respectively. The degree of swelling of the synthesized hydrogels was assessed as a function of EDC/NHS ratios and the pH of the equilibrating medium, while the release kinetic profile of basic fibroblast growth factor (FGF-2) was evaluated in human fibroblast cultures. The degree of swelling (DS) decreased from 26.5+/-1.7 to 18.5+/-2.4 as the EDC concentration varied from 0.75 to 2.5 mg/ml. Eighty percent of the FGF-2 was released at controlled rates from gelatin-polylysine (gelatin-PLL) and gelatin-polyglutamic acid (gelatin-PLG) hydrogel scaffolds over a period of 28 days. Cell adhesion studies revealed that the negatively charged surface of the gelatin-PLG hydrogels exhibited superior adhesion capabilities in comparison to gelatin-PLL and control gelatin surfaces. Laser Doppler perfusion imaging as well as CD31(+) capillary immunostaining demonstrated that the controlled release of FGF-2 from ionic gelatin-based hydrogels is superior in promoting angiogenesis in comparison to the bolus administration of the growth factor. Over 4 weeks, FGF-2 releasing gelatin-PLG hydrogels exhibited marked reperfusion with a Doppler ratio of 0.889 (+/-0.04) which was 69.3% higher than in the control groups.

Figure 1. Reaction scheme for the synthesis of ionic, gelatin-based hydrogels

Gelatin A and Gelatin B were co-crosslink with PLL (cationic gels) and PLG (anionic gels) respectively via an EDC/NHS coupling reaction.

Figure 2

Schematic representation for the induction of critical hindlimb ischemia in a murine model.

Figure 3. Degree of swelling (DS) measurements for crosslinked hydrogels at various pHs

The DS of gelatin-PLL (A) and gelatin-PLG (B) hydrogels was determined as a function of EDC/NHS molar ratios at pH 7.4 (n= 3 per group, [gelatin] = 0.1 g/mL, [PLL] = 0.01 g/mL, [PLG] = 0.01 g/mL). (C): DS of gelatin-PLL at pHs of 7.4 and 10.0 (pKa of lysine = 10.4). (D): DS of gelatin-PLG hydrogels at pHs of 7.4 and 4.5 (pKa of glutamic acid = 4.5). Statistical significance was determined at p < 0.05 (denoted by *; n=3 per group).

Figure 4. Adhesion and cytotoxicity studies of the gelatin-based hydrogels in human neonatal fibroblast cell cultures

(A): Percent adhesion of HNF cells on the hydrogel surfaces was assessed by counting the cells that showed whip-like projections and stained their cytoplasm (H&E staining, n=3 per time point per group, p < 0.05 denoted by *). (B): Cell viability in the presence of ionic gelatin hydrogels was determined with and without EDC and NHS as well as in the presence of the urea byproduct derivative. [EDC]=1.3 mg/mL, [NHS] = 0.44 mg/mL.

Figure 5. Release profile of FGF-2 from gelatin-based hydrogels

The overall release rate from gelatin A ( ), gelatin B ( ), gelatin-PLL ( ), gelatin-PLG ( ) hydrogels was monitored over 28 days (n=3 per time point per group). [gelatin] = 0.1 mg/mL; [PLL or PLG] = 0.01 mg/mL; FGF-2 = 250 ng; [EDC] = 1.3 mg/mL; [NHS] = 0.44 mg/mL.

Figure 6. Laser Doppler Perfusion Imaging intensity ratios of various treatments in a murine hindlimb ischemic model

Paw blood flow velocity (n = 5 mice per time point per group) was determined at specified time points for 8 weeks. Representative Doppler ratios of PBS injection (●), gelatin-PLL (◆), gelatin-PLG (■) gelatin A with FGF-2 (△), gelatin B with FGF-2 (○), FGF-2 bolus injection (□), gelatin-PLL with FGF-2 (▲), and gelatin-PLG with FGF-2 (×). Statistical significance was determined at p < 0.01 for all treatment groups (denoted by *). Concentrations of constituents: [gelatin]=0.1 g/mL; [PLL or PLG]=0.01 g/mL; [FGF-2]=250 ng; [EDC]=1.3 mg/mL; [NHS]=0.44 mg/mL.

Figure 7. Representative immunohistological staining (CD31+) images of ischemic hindlimb quadriceps muscle at 8 weeks

(A): CD31+ staining of a non-ischemic limb; (B): Gelatin-PLG treatment; (C): Bolus injection of FGF-2; (D): Gelatin-PLG releasing FGF-2. Concentrations of constituents: [gelatin]=0.1 g/mL; [PLL or PLG]=0.01 g/mL; 250 ng of FGF-2; [EDC]=1.3 mg/mL; [NHS]=0.44 mg/mL.

Figure 8. CD31+ capillary density measurements

At two, four, and eight weeks, positively stained capillaries were counted in five fields per slide in triplicates by a blinded observer and the treatment groups compared to the controls. Statistical significance was determined at p < 0.05 and denoted by *.