Ex Vivo Remodeling of Excised Blood Vessels for Vascular Grafts

Abstract

The present invention provides an ex vivo vascular remodeling methods and system by which an excised, small diameter blood vessel can be harvested and expanded to provide viable vascular grafts, as demonstrated at the physical and molecular levels, and as optimized in vivo. The tissue-engineered vessels generated by the present invention closely resemble native vessels in terms of structure, histologically, including endothelial coverage and intricate structural components such as the internal elastic lamina, viability (as measured with MTT assay and TUNEL analysis), and function (vasoactivity, mechanical and biomechanical properties). Thus, the resulting vascular grafts behave in a manner similar to native arteries in terms of mechanical integrity, and provide clinically relevant patency rates when implanted in vivo. Moreover, the ex vivo methods and system permit the precise control of the mechanical environment involving the excised vessel, while at the same time permitting carefully monitoring of the resulting growth/remodeling, thereby opening new avenues of research regarding the mechanical stimuli responsible for specific aspects of remodeling in vivo.

1 . A remodeled excised small blood vessel, having increased diameter, length or wall thickness, or any combination thereof as compared with its pre-excised dimensions, produced by physically remodeling and tissue engineering a small, excised blood vessel, while maintaining its viability while the blood vessel has been subjected to precisely controlled, ex vivo mechanical perfusion and extension environment, with real-time monitoring, for a time sufficient to effect the increase. 
     
     
         2 . The remodeled vessel of  claim 1 , wherein the excised vessel is a small artery or a vein. 
     
     
         3 . The remodeled vessel of  claim 1 , wherein the remodeled vessel is an arterial or a venous graft for use in patient in need of such graft. 
     
     
         4 . The remodeled vessel of  claim 1 , wherein during remodeling within the precisely controlled, ex vivo mechanical perfusion and extension environment, the excised vessel is subjected to applied pressure, shear, and strain under controlled conditions, such that transmural pressure drop regulates wall thickness, longitudinal tension regulates length, and flow-induced shear stress regulates inner diameter of the remodeled vessel. 
     
     
         5 . The remodeled vessel of  claim 1 , the process of producing the remodeled vessel further comprising:
 cannulating each end of the excised vessel to two sliding stainless steel tubes that are cannulated on each end and that are contained within an enclosed medium-filled chamber;   gradually mechanically extending the vessel without rupture by slowly extending the stainless steel tubes on which the vessel is mounted within the enclosed medium-filled chamber; while   maintaining viability of the vessel by perfusing the vessel under precisely controlled and real-time monitored flow and pressure regimes within traditional cell or organ culture conditions, maintaining temperature, pH, pO 2 , pCO 2 , and nutrients at viable levels.   
     
     
         6 . The remodeled vessel of  claim 5 , wherein the excised vessel is a small artery or a vein. 
     
     
         7 . The remodeled vessel of  claim 5 , wherein the remodeled vessel is an arterial or a venous graft for use in patient in need of such graft. 
     
     
         8 . The remodeled vessel of  claim 5 , wherein during remodeling within the precisely controlled, ex vivo mechanical perfusion and extension environment, the excised vessel is subjected to applied pressure, shear, and strain under controlled conditions, such that transmural pressure drop regulates wall thickness, longitudinal tension regulates length, and flow-induced shear stress regulates inner diameter of the remodeled vessel. 
     
     
         9 . The method of  claim 5 , wherein length of the remodeled vessel is increased at least 100% over its native length when excised, and wherein more than 50% of the increased length is retained after recoil when the remodeled vessel is removed from the stainless steel tubes. 
     
     
         10 . A method for determining the effect of precisely controlling flow and pressure regimes in a mechanical perfusion and extension process for remodeling and tissue engineering an excised blood vessel, in response to a mechanical load, the method comprising:
 detecting and quantifying spatial expression and distribution of a glycoprotein marker in cells of the remodeled vessel to determine the region(s) of a TN-C promoter responsible for mechanosensitivity; and   determining molecular regulation of the remodeled blood vessel in, and as a result of, mechanical remodeling, wherein the physical remodeling process comprises subjecting the excised blood vessel to precisely controlled, ex vivo mechanical perfusion and extension, with real-time monitoring, for a time sufficient to effect an increase in diameter, length or wall thickness, or any combination thereof of the vessel while maintaining its viability.   
     
     
         11 . The method of  claim 10 , comprising detecting and quantifying spatial expression and distribution of mRNA of the glycoprotein marker in cells of the remodeled vessel. 
     
     
         12 . The method of  claim 10 , further comprising applying the determined molecular regulation to modifying the controlled flow and pressure regimes. 
     
     
         13 . The method of  claim 10 , comprising a single excised blood vessel, or multiple excised blood vessels run in parallel, each vessel contained within its own housing, comprising corresponding chambers and needle valves. 
     
     
         14 . The method of  claim 10 , wherein during remodeling within the precisely controlled, ex vivo mechanical perfusion and extension environment, the excised vessel is subjected to applied pressure, shear, and strain under controlled conditions, such that transmural pressure drop regulates wall thickness, longitudinal tension regulates length, and flow-induced shear stress regulates inner diameter of the remodeled vessel. 
     
     
         15 . The method of  claim 10  for determining the effect of the precisely controlling flow and pressure regimes in a mechanical process for remodeling and tissue engineering the excised blood vessel, in response to a mechanical load, wherein the physical remodeling process further comprises:
 cannulating each end of the excised vessel to two sliding stainless steel tubes that are cannulated on each end and that are contained within an enclosed medium-filled chamber; 
 gradually mechanically extending the vessel without rupture by slowly extending the stainless steel tubes on which the vessel is mounted within the enclosed medium-filled chamber; while 
 maintaining viability of the vessel by perfusing the vessel under precisely controlled and real-time monitored flow and pressure regimes within traditional cell or organ culture conditions, maintaining temperature, pH, pO 2 , pCO 2 , and nutrients at viable levels. 
 
     
     
         16 . The method of  claim 15 , comprising detecting and quantifying spatial expression and distribution of mRNA of the glycoprotein marker in cells of the remodeled vessel. 
     
     
         17 . The method of  claim 15 , further comprising applying the determined molecular regulation to modifying the controlled flow and pressure regimes. 
     
     
         18 . The method of  claim 15 , comprising a single excised blood vessel or multiple excised blood vessels run in parallel, each vessel contained within its own housing, comprising corresponding chambers and needle valves. 
     
     
         19 . The method of  claim 15 , wherein during remodeling within the precisely controlled, ex vivo mechanical perfusion and extension environment, the excised vessel is subjected to applied pressure, shear, and strain under controlled conditions, such that transmural pressure drop regulates wall thickness, longitudinal tension regulates length, and flow-induced shear stress regulates inner diameter of the remodeled vessel. 
     
     
         20 . The method of  claim 15 , wherein length of the remodeled vessel is increased at least 100% over its native length when excised, and wherein more than 50% of the increased length is retained after recoil when the remodeled vessel is removed from the stainless steel tubes.