Nanofiber polymeric scaffold for slow and steady release of VEGF and ECs to induce efficient angiogenesis and healing in a mouse model of hind limb ischemia

The primary aim of the project is the application of a biocompatible polymeric nanofiber scaffold, and the comparison with micrometric biological scaffold, both combined with endothelial cells (ECs) and vascular endothelial growth factor (VEGF) in a hindlimb ischemia mouse model in order to stimulate the angiogenis and muscle protection. Firstly, the hindlimb ischemic model (HLIM) was started to be developed, surgically ligating and transecting the left femoral artery of BALB/C mice, under operating microscope. Postoperative clinical assessment was performed according to the Tarlov score and the Modified Ischemia score. All hindlimb ischemia were assessed at day 1 and day 7. At day 7 mice were euthanized using cervical dislocation method. Cross-section of quadriceps, tibialis anterior and gastrocnemius muscle of Balb/c one week after surgery was performed in all HLIM experiments. A 2D and 3d culture of the ECs was performed. The tube formation analysis was executed counting numbers of the tubes in 3D consisting of two or more CD31 positive cells that formed a tube-like structure. Integra dermal substitute and PCL nanofiber scaffold were compared. The PCL scaffold alone showed a lower response in term of cell colonization and ischemia recovery compared to the Integra alone while comparing the results of ECs seeded on both matrix they were similar. Histologically, numerous perfusable vessels were observed within the muscle in the ESc seeded on Integra® group and overall capillary density was increased in these groups in ischemic hindlimb muscle. The migration of host cells into the scaffolds was analysed by the infiltration of host cells into scaffolds and sub-sequential migration of the ECs in the surrounding tissues. Parallel to these experiments, we started to performe also the same experiments using the Integra and the PCL nanoscaffold seeding Adipose Cells. The results were more encouraging than with the ECs as the regenerative potential found in the SVF appeared to be even more promising in the promoting the microvascularization