PURPOSE: to characterize the in vivo effect of hypoxic EPC-CM-loaded NPs, comparing it to hypoxic EPC-CM. METHODS: EPCs were obtained from peripheral blood of healthy donors and cultured for 24 h at 1%O2 in growth factor- and serum-free medium to obtain CM. 36 rats were divided into 3 groups: control (vehicle); EPC-CM; EPC-CM NPs. For each animal, 500 μL injections were performed at 3 sites into the ischemic hindlimb, immediately after ischemia and after 1 week. The effect of ischemia was evaluated both using Laser Doppler Blood Flow imaging (ratio between ischemic and contralateral limb), histology (hematoxylin/eosin, H/E, staining) to evaluate the inflammatory reaction and immunohistochemistry to evaluate capillary (CD31+) and artery (alpha-smooth muscle actin, alpha-SMA+) number per mm2. RESULTS: A significant increase of perfusion was observed at 2 weeks in CM-NP vs. both CM and control (p<0.05). In control, tissues showed a normal morphology, while in both CM and CM-NP, at 1 week, we observed myocyte coagulative necrosis with enlarged interstitium mainly populated by inflammatory infiltrates and thin-walled new vessels, more extensively in CM-NP vs. CM. At 2 weeks, fibrosis replacement of necrotic myocytes was found together with new vessels. Immunohistochemistry evidenced a significant increase of capillaries in rats treated with both CM and CM-NPs. Treatment with CM-NP significantly increased capillary number at both 1 (p<0.05 vs. control) and 2 weeks (p<0.0005 vs. control and p<0.005 vs. CM), while CM treatment had a significantly higher effect than control only at 2 weeks (p<0.005). No significant difference in the numbers of arteries among different groups was observed, suggesting a more pronounced effect on angiogenesis rather than arteriogenesis. CONCLUSIONS: Novel therapeutic strategies based on EPC paracrine factors may replace cell transplantation, as "cell-free" therapy could overcome the risk of adverse immunological reactions and the problem of heterologous rejection. Release of EPC-CM from loaded NPs was effective for blood flow and capillary enhancement in an in vivo model of hindlimb ischemia, underlining the advantages of using controlled release in regenerative medicine.
Endothelial progenitor cell-conditioned medium delivery by polymer nanoparticles in an ischemic hindlimb model
Felice, F.;PIRAS, ANNA MARIA;CHIELLINI, FEDERICA;SOLARO, ROBERTO;DI STEFANO, ROSSELLA
2015-01-01
Abstract
PURPOSE: to characterize the in vivo effect of hypoxic EPC-CM-loaded NPs, comparing it to hypoxic EPC-CM. METHODS: EPCs were obtained from peripheral blood of healthy donors and cultured for 24 h at 1%O2 in growth factor- and serum-free medium to obtain CM. 36 rats were divided into 3 groups: control (vehicle); EPC-CM; EPC-CM NPs. For each animal, 500 μL injections were performed at 3 sites into the ischemic hindlimb, immediately after ischemia and after 1 week. The effect of ischemia was evaluated both using Laser Doppler Blood Flow imaging (ratio between ischemic and contralateral limb), histology (hematoxylin/eosin, H/E, staining) to evaluate the inflammatory reaction and immunohistochemistry to evaluate capillary (CD31+) and artery (alpha-smooth muscle actin, alpha-SMA+) number per mm2. RESULTS: A significant increase of perfusion was observed at 2 weeks in CM-NP vs. both CM and control (p<0.05). In control, tissues showed a normal morphology, while in both CM and CM-NP, at 1 week, we observed myocyte coagulative necrosis with enlarged interstitium mainly populated by inflammatory infiltrates and thin-walled new vessels, more extensively in CM-NP vs. CM. At 2 weeks, fibrosis replacement of necrotic myocytes was found together with new vessels. Immunohistochemistry evidenced a significant increase of capillaries in rats treated with both CM and CM-NPs. Treatment with CM-NP significantly increased capillary number at both 1 (p<0.05 vs. control) and 2 weeks (p<0.0005 vs. control and p<0.005 vs. CM), while CM treatment had a significantly higher effect than control only at 2 weeks (p<0.005). No significant difference in the numbers of arteries among different groups was observed, suggesting a more pronounced effect on angiogenesis rather than arteriogenesis. CONCLUSIONS: Novel therapeutic strategies based on EPC paracrine factors may replace cell transplantation, as "cell-free" therapy could overcome the risk of adverse immunological reactions and the problem of heterologous rejection. Release of EPC-CM from loaded NPs was effective for blood flow and capillary enhancement in an in vivo model of hindlimb ischemia, underlining the advantages of using controlled release in regenerative medicine.File | Dimensione | Formato | |
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