Shear-stress preconditioning and tissue-engineering-based paradigms for generating arterial substitutes

Abstract

In situ tissue engineering using shear-stress preconditioning and adhesive biomolecules is a new approach to autologous tissue engineering. In the present study, novel tissue-engineering grafts (TEGs) were preconditioned within an in vitro pulsatile flow circuit, with and without the addition of fibronectin (FN), to establish whether low-shear-stress conditions promoted endothelial cell (EC) retention and differentiation. TEGs ( n =24) were generated by the contraction and compaction of collagen(I) by porcine aortic smooth-muscle cells (SMCs) on to a compliant polyester graft scaffold. ECs were radiolabelled with [(111)In]indium tropolonate and seeded on to the luminal surface of the TEGs. Following organ culture in a bioreactor (7 days), TEGs were split into four groups ( n =six TEGs per group): Group A acted as controls with TEGs unmodified and seeded with radiolabelled ECs; Group B underwent luminal pre-coating with FN (75 microg/ml) prior to EC seeding; Group C underwent preconditioning within a pulsatile flow circuit at 10-20 microN (1-2 dyn)/cm(2) for 7 days prior to EC seeding, and Group D TEGs were preconditioned for 7 days at 1-2 dyn/cm(2), followed by luminal pre-coating with FN prior to EC seeding. The resistance to physiological shear stress of the seeded ECs was assessed using a gamma-radiation counter within a physiological flow circuit producing an arterial waveform with a mean shear stress of 93.2 microN (9.32 dyn)/cm(2). Environmental scanning electron microscopy (ESEM) was used to determine the distribution and degree of differentiation of the attached Ecs, and tissue-type-plasminogen-activator (tPA) assays provided a measure of function and viability. EC resistance to shear stress at 93.2 microN/cm(2) was significantly enhanced by a period of preconditioning (Group C) at 10-20 microN/cm(2), surface modification with FN (Group B), or both (Group D) when compared with control grafts (Group A). However, TEGs coated with FN whether preconditioned (Group D) or not (Group B) demonstrated the best results for EC retention. ESEM demonstrated near-confluent differentiated flattened ECs in both these cases. EC function was demonstrated by a steady increase in tPA production. Low-shear-stress preconditioning of TEGs enhances EC retention in vitro with an additional advantage demonstrated by pre-treatment with FN prior to endothelialization. These findings may be exploited in the development of tissue-engineered constructs to maintain a confluent endothelial lining.