The Long-Term Angiogenic Benefits of Precision Micropuncture
Alexander T. Liu, MD1, Sameer Massand, MD1, Patrick C. Hancock, MD1,2, Jessica M. Collins, DO1, Dana Goldenberg BA1, Mingjie Sun, MD, PhD1, Srinivas V. Koduru, PhD1, Dino J. Ravnic, DO, MPH, MSc1
1Penn State Milton S. Hershey Medical Center, Hershey, PA 2UPMC Pinnacle Harrisburg Hospital, Harrisburg, PA
Background: The lack of rapid and stable vascularization precludes clinical success of engineered tissues. Research has focused on cellular, pharmacologic, and materials-based remedies to enhance this complex process. Yet, translatable success is missing. We recently demonstrated that precision micropuncture (MP) of the recipient macrovasculature can expedite collagen scaffold vascularization. However, vascular persistence is unknown. We hypothesize that MP-induced scaffold angiogenesis will continue to endure long-term.
Methods: The rat (n=8) femoral artery and vein were transmurally micropunctured with a 60 ?m diameter needle at defined intervals just prior to implantation of a Type I bulk collagen scaffold. The contralateral hindlimb served as a non-MP internal control. The femoral pedicle and scaffold were then circumferentially wrapped with a silicone sheet to prevent vascular ingrowth from the adjacent tissue. At the four-week timepoint, animals underwent in situ fluorescence angiography. Following explantation of the scaffolds and femoral vasculature, samples were prepared for thin section and whole mount histology. Specimens were examined for cellular infiltration, angiogenic proteins and vascular metrics using ImageJ software (NIH). Statistical significance was defined as p<0.05. Results: At four weeks, MP scaffolds demonstrated a 16% increase in nucleated cell infiltration but failed to reach statistical significance (p=0.06) over non-MP controls. However, endothelial cell (CD31) accumulation within the MP scaffolds was significantly augmented (23.6% versus 15.5%; p<0.01). This was paralleled by a relevant rise in angiogenic proteins, such as VEGFR2 in the MP samples (26.6% vs. 17.4%; p<0.01). Fluorescence angiography of whole-mount preparations revealed that the MP-induced vasculature had larger mean diameters (53.8 versus 49.3 ?m; p <0.01) and a greater number of branch points (10.5 versus 7.9 per mm˛; p=.01) which resulted in an increased scaffold vascular density (29.1% vs. 15.6%; p<0.01). The MP-induced vasculature also demonstrated a more complex architecture secondary to the extensive branching patterns. Conclusions: The angiogenic effects of precision micropuncture continue to persist for at least four weeks. Our results suggest that MP has a profound impact on scaffold vascularization emanating directly from the adjacent femoral vessels. MP predictably leads to a doubling of scaffold vascularization. The resulting combination of rapid angiogenesis and vascular stability highlights the utility of this novel microsurgical platform for the perfusion of engineered scaffolds and grafts.
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