Enhanced angiogenesis by multiple release of platelet-rich plasma contents and basic fibroblast growth factor from...

The objective of this study is to evaluate the angiogenic effects induced by biodegradable gelatin hydrogel granules incorporating mixed platelet-rich plasma (PRP) growth factor mixture (PGFM) and bioactive basic fibroblast growth factor (bFGF). The PRP was prepared by a double-spinning technique for isolating animal bloods, followed by treatment with different concentrations of calcium chloride (CaCl(2)) solution. The CaCl(2) solution treatment activated the platelets of PRP, allowing the release of various growth factors, such as platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β(1), and epithelial growth factor (EGF). In the PRP treated with different CaCl(2) solutions, high amounts of representative platelet growth factor, PDGF-BB, VEGF, EGF, and TGF-β(1) were detected in the CaCl(2) concentrations of 1, 2, and 4 wt.% compared with higher or lower ones. The PRP treated was impregnated into gelatin hydrogel granules freeze-dried at 37°C for 1h, and then the percentage of PGFM desorbed from the gelatin hydrogel granules was evaluated. The percentages of PDGF-BB, VEGF, EGF, and TGF-β(1) desorbed tended to decrease with decreasing CaCl(2) concentration. Taken together, the CaCl(2) concentration to activate PRP for PGFM release was fixed at 2 wt.%. In vitro release tests demonstrated that the PGFM was released from the gelatin hydrogel granules with time. For the gelatin hydrogels incorporating PGFM and bFGF, the time profile of PDGF-BB or bFGF release was in good correspondence with that of gelatin hydrogel degradation. The gelatin hydrogel granules incorporating mixed PGFM and bFGF were prepared and intramuscularly injected to a mouse leg ischemia model to evaluate the angiogenic effects in terms of histological and laser Doppler perfusion imaging examinations. As controls, hydrogel granules incorporating bFGF, PGFM, and platelet-poor plasma were used for the angiogenic evaluation. The number of blood vessels newly formed and the percentage of anti-α-smooth muscle actin antibody-positive cells increased around ischemic sites injected with the gelatin hydrogel granules incorporating mixed PGFM and bFGF, in marked contrast to other control groups. The blood reperfusion level of ischemic tissues was enhanced by the hydrogel granules incorporating mixed PGFM and bFGF, whereas no enhancement was observed for other groups. It is concluded that the dual-release system of PGFM and bFGF from gelatin hydrogel granules shows promise as a method to enhance angiogenic effects.