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Functional Liver Nodules Develop De Novo in a Cell-Infused SIEA Flap: A Model of Free Flap-Based Organ-Level Tissue Engineering
Kristine C. Rustad, MD, Peter A. Than, MD, Marcelina G. Perez, BS, Alex Y. Li, BS, Michael W. Findlay, MBBS, PhD, FRACS, FACS, Sacha M.L. Khong, PhD, Geoffrey C. Gurtner, MD, FACS.
Stanford University, Stanford, CA, USA.

Purpose:
Tissue engineering has been proposed as a solution to the limited donors available for patients with end-stage organ failure. However, strategies aimed at engineering whole organs have been met with significant challenges, arguably the greatest being the difficulties in developing a de novo vascular system. We have previously proposed that microcirculatory beds found within soft tissue free flaps could be used as an autologous source of explantable microcirculation for organ-level tissue engineering. Here we demonstrate proof of this concept with auto-assembly of functional liver nodules within an SIEA flap.
Methods:
SIEA flaps were dissected in Wistar rats. The proximal and distal femoral vessels were clamped to isolate the flap microcirculation. A mixture of 1 x 10^6 ASCs, 1 x 10^6 hepatocytes and 5 x 10^5 endothelial cells were infused into the flap and allowed to incubate for 2 hours. The flaps were harvested at days 3, 14 and 28 for RNA and protein analysis and histology. Immunohistochemistry was performed looking at liver-specific markers.
Results:
Spontaneous auto-assembly of liver nodules within the SIEA flaps were noted as early as post-operative day 3. The liver nodules consisted of cells within a sinusoidal architecture that expressed albumin, cytochrome P450 enzymes, alpha-1-antitrypsin and gamma-glutamyl-transpeptidase, indicating the presence of cholangiocytes.
Conclusion:
Individual cells perfused into an SIEA flap are capable of auto-assembly within the flap into tissue that expresses liver-specific genes and proteins. The use of soft tissue free flaps as an autologous vascularized scaffold is a promising technology for organ-level tissue engineering.


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