Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Technion Israel Institute of Technology

Large soft tissue defects involve significant tissue loss, requiring surgical reconstruction. Autologous flaps are occasionally scant, demand prolonged transfer surgery, and induce donor site morbidity. The present work set out to fabricate an engineered muscle flap bearing its own functional vascular pedicle for repair of a large soft tissue defect in mice. Full-thickness abdominal wall defect was reconstructed using this engineered vascular muscle flap. A 3D engineered tissue constructed of a porous, biodegradable polymer scaffold embedded with endothelial cells, fibroblasts, and/or myoblasts was cultured in vitro and then implanted around the femoral artery and veins before being transferred, as an axial flap, with its vascular pedicle to reconstruct a full-thickness abdominal wall defect in the same mouse. Within 1 wk of implantation, scaffolds showed extensive functional vascular density and perfusion and anastomosis with host vessels. At 1 wk posttransfer, the engineered muscle flaps were highly vascularized, were well-integrated within the surrounding tissue, and featured sufficient mechanical strength to support the abdominal viscera. Thus, the described engineered muscle flap, equipped with an autologous vascular pedicle, constitutes an effective tool for reconstruction of large defects, thereby circumventing the need for both harvesting autologous flaps and postoperative scarification.

An engineered muscle flap for reconstruction of large soft tissue defects.

Abdominal wall reconstruction following extensive tissue loss is essential and can be achieved using autologous flaps. However, their use is limited due to their inadequate availability and due to post-operative donor site scarification. This work presents a step-by-step technique for fabrication of a vascularized muscle flap, to be applied in full-thickness abdominal wall defect reconstruction. Poly L-lactic acid/poly lactic-co-glycolic acid scaffolds, prepared using a salt leaching technique, were used as the supporting matrix in vitro for simultaneously seeded endothelial cells, fibroblasts and myoblasts. The cell-embedded graft was then implanted around femoral artery and vein vessels, which provided a central blood supply. Vascularization and perfusion were achieved by capillary sprouting from the main host vessel into the graft. A thick and vascularized tissue was formed within one week, and was then transferred as an autologous flap together with its main vessels, to a full-thickness abdominal wall defect. The flap remained viable after transfer and featured sufficient mechanical strength to support the abdominal viscera. Thus, this engineered muscle flap can be used as an alternative source for autologous flaps to reconstruct full-thickness abdominal wall defects.

A method for constructing vascularized muscle flap.

Perivascularly implanted matrix embedded endothelial cells (MEECs) are potent regulators of inflammation and intimal hyperplasia following vascular injuries. Endothelial cells (ECs) in collagen scaffolds adopt a reparative phenotype with significant therapeutic potential. Although the biology of MEECs is increasingly understood, tuning of scaffold properties to control cell-substrate interactions is less well-studied. It is hypothesized that modulating scaffold degradation would change EC phenotype. Scaffolds with differential degradation are prepared by cross-linking and predegradation. Vascular injury increases degradation and the presence of MEECs retards injury-mediated degradation. MEECs respond to differential scaffold properties with altered viability in vivo, suppressed smooth muscle cell (SMC) proliferation in vitro, and altered interleukin-6 and matrix metalloproteinase-9 expression. When implanted perivascularly to a murine carotid wire injury, tuned scaffolds change MEEC effects on vascular repair and inflammation. Live animal imaging enables real-time tracking of cell viability, inflammation, and scaffold degradation, affording an unprecedented understanding of interactions between cells, substrate, and tissue. MEEC-treated injuries improve endothelialization and reduce SMC hyperplasia over 14 d. These data demonstrate the potent role material design plays in tuning MEEC efficacy in vivo, with implications for the design of clinical therapies.

Tuning of Collagen Scaffold Properties Modulates Embedded Endothelial Cell Regulatory Phenotype in Repair of Vascular Injuries In Vivo.

Biomaterials exhibit dramatically varying behavior and efficacy in different tissue microenvironments. On p. 2220, S. Unterman, N. Artzi, and co-workers describe how tuning material properties can enhance efficacy of implanted endothelial cells in the injured perivascular environment. The rational design of materials for optimal in vivo efficacy is dependent on real-time monitoring of tissue, material (red), and implanted cell (green) state using live animal fluorescent imaging.

Cell-Substrate Interactions: Tuning of Collagen Scaffold Properties Modulates Embedded Endothelial Cell Regulatory Phenotype in Repair of Vascular Injuries In Vivo (Adv. Healthcare Mater. 15/2015).

Implantable 3D engineered vascular tissue constructs can be formed by co-culturing endothelial and fibroblast cells on macroporous scaffolds. Here we show that these constructs can be used for studying the dynamics of neovascular formation in-vitro by a combination of live confocal imaging and an array of image processing and analysis tools, revealing multiple distinct stages of morphogenesis. We show that this process involves both vasculogenic and angiogenic elements, including an initial endothelial multicellular cluster formation followed by rapid extensive sprouting, ultimately resulting in a stable interconnected endothelial network morphology. This vascular morphogenesis is time-correlated with the deposition and formation of an extensive extra-cellular matrix environment. We further show that endothelial network junctions are formed by two separate morphogenic mechanisms of anastomosis and cluster thinning.

Article	Year
An engineered muscle flap for reconstruction of large soft tissue defects. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24711414	2014
A method for constructing vascularized muscle flap. Methods (San Diego, Calif.)\| PubMed ID: 25843607	2015
Tuning of Collagen Scaffold Properties Modulates Embedded Endothelial Cell Regulatory Phenotype in Repair of Vascular Injuries In Vivo. Advanced healthcare materials\| PubMed ID: 26333178	2015
Cell-Substrate Interactions: Tuning of Collagen Scaffold Properties Modulates Embedded Endothelial Cell Regulatory Phenotype in Repair of Vascular Injuries In Vivo (Adv. Healthcare Mater. 15... Advanced healthcare materials\| PubMed ID: 26503521	2015
Vasculogenic dynamics in 3D engineered tissue constructs. Scientific reports\| PubMed ID: 26648270	2015

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