The present study focused on the development of three layered small-diameter (<6 mm) extracellular matrix (ECM)-based vessels. These were engineered artificially through the freeze-drying technique. A layer of decellularized bovine aorta (DAM) was deposited on a mandrel and, after lyophilization, it was dipped into a poly-L-lactide acid (PLLA)/polyethylene glycol (PEG) 2000 dichloromethane solution then quickly wrapped with a pre-prepared thin DAM sheet. Mechanical properties of three-layered scaffolds were evaluated by means of uniaxial tensile measurement. Furthermore, human endothelial and smooth muscle cells were seeded on internal and external scaffold surfaces, respectively, and co-cultured for 7 days. Our results demonstrate that i) ECM components provide suitable stimuli for cell adhesion and proliferation, ii) the microporous intermediate PLLA/PEG2000 layer is responsible for the scaffold resistance and iii) the layered deposition technique can be considered a valuable method to obtain layered vascular scaffolds of different sizes and with a good compromise between stiffness and elasticity for optimal cell organization.

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