Controlled Microsurgical Puncture of Vessels to Augment Angiogenesis
Patrick C. Hancock, MD, Srinivas V. Koduru, PhD, Mingjie Sun, MD, Dino J. Ravnic, DO, MPH, MSc.
Penn State College of Medicine, Hershey, PA, USA.
BACKGROUND: The formation of new blood vessels from existing vasculature, or angiogenesis, is an essential process in the integration of bioengineered tissue grafts. When angiogenesis is too slow or inadequate it can lead to tissue compromise or graft integration failure. Previous attempts to stimulate angiogenesis therapeutically using methods such as pharmacologic stimulation, cytokine supplementation, stem cells, and in situ cell homing have been limited by their suboptimal results or significant side effects. Here we describe an alternative novel microsurgical technique to safely induce rapid angiogenesis.
METHODS: Rat hindlimb macrovasculature (N=15) was micropunctured using a 60 μm diameter needle at defined intervals before implantation of a prepared Type I collagen scaffold over the punctured segment. The contralateral hindlimb served as a normal (non-micropunctured) intrinsic control. Collagen grafts and the underlying vascular segment were explanted en bloc after 24, 48, and 72 hours. Trichrome stained single cell-lined lumens were characterized by both number and diameter; from which the overall perfusable area was calculated. Endothelial cell integration was confirmed with immunofluorescence staining and western blot protein quantification of CD31/PECAM-1, von Willebrand factor (vWF), VEGFR2, Notch 1, DLL4, Tie 2, and HIF 1α. At 96 hours, fluorescence vessel painting was used for vessel analysis using ImageJ. Statistical significance was defined as p < 0.05.
RESULTS: Collagen overlying micropunctured vessels exhibited a statistically significant, approximately two-fold increase, of perfusable area at all time points. Immunofluorescence labeling revealed increased numbers of cells positive for all the aforementioned angiogenic markers in the micropunctured group compared to the control. The increased presence of endothelial cells in the experimental group was further supported by the western blot protein quantification which showed a 2.35-, 2.66-, and 7.19-fold increase of CD31 at 24, 48, and 72 hours respectively. Similar increases were seen in the quantities of vWF, VEGFR2, and Tie 2 suggesting greater endothelial cell proliferation. At 96 hours there was a significant increase in vascular density within micropunctured samples (22.04% vs. 9.06%, p<0.001).
CONCLUSIONS: Microsurgical induction of angiogenesis was first described with the creation of arteriovenous-loops. However, the time to new capillary formation is slow, limiting clinical translation. Here we describe an innovative alternative technique that leads to rapid capillary formation and perfusion. Because our application addresses the paradigm of therapeutic angiogenesis from a completely novel perspective it has enormous potential to mitigate ischemia in both conventional surgery and bioengineered applications.
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