140. The Early Angiogenic Effect of Arterial and Venous Micropuncture Using In Vivo And In Vitro Models

PURPOSE: Attempts at tissue engineered replacements are limited by slow vascularization upon implantation. Micropuncture (MP) is a novel surgical technique that can be used to rapidly induce recipient site angiogenesis and adjacent biomaterial vascularization. Our initial studies demonstrated MP utility when concurrently delivered to both the femoral artery and vein. However, the individual vascular contribution is unknown. Here we hypothesize that early MP-induced angiogenesis is dependent on concurrent arterial and venous targeting. METHODS: 60um diameter MP was performed at defined intervals on the rat femoral artery (MPA; n=10), vein (MPV, n=10), or both (MPAV, n=10) just prior to implantation of a collagen scaffold. Baseline in vivo controls (n=10) did not undergo MP. Angiogenesis into the scaffold was assessed at Day 3 using in situ fluorescence angiography and endothelial cell (EC; CD31) quantification. Additionally, ECs were extracted from the rat femoral artery (ECA) and vein (ECV) for complementary in vitro studies. After culture expansion, 0.5x106 ECA, ECV, or their coculture in a 1:1 ratio (ECAV) were seeded onto a collagen scaffold and subjected to 60um MP (80 MP/well). Control ECs did not undergo MP. Conditioned media was evaluated at 24 hours (ECA or ECV alone vs. non-MP controls). Tube formation was evaluated at 72 hours for ECs that had undergone MP, with at least ten images (10x) per well. All in vitro studies were conducted in duplicate. Statistical significance was defined as p<0.05. RESULTS: In vivo, MP significantly increased scaffold vascularization, with the most profound effects observed in MPAV (p<0.05). Interestingly, MPV showed greater vascular density compared to MPA (p<0.05). CD31 staining corroborated the trends observed with perfusion angiography. When extracted rat femoral ECs were examined in vitro 24 hours after MP they showed substantial alterations in MP-induced angiogenic protein expression based on semi-quantitative ELISA when compared to non-MP controls. Using only MP-EC tube formation studies, the mean tube formation was highest in ECAV (7364 µm) compared to ECA (5428 µm) and ECV (3082 µm), although the difference was not statistically significant. CONCLUSIONS: MP appears to stimulate rapid capillary formation both in vivo and in vitro through differential protein expression in arterial and venous EC. While arterial and venous MP alone can trigger microvascular formation, the angiogenic effects appear most noteworthy when both cell types are targeted concurrently. As our understanding of MP deepens, refined microsurgical therapies to induce angiogenesis could play an important role in advancing tissue engineering.