Method of manufacturing a tissue-engineered prosthesis

Abstract

Developing heart valves are exposed to dynamic strains by applying a dynamic pressure difference over the leaflets. The flow is kept to a minimum, serving only as a perfusion system, supplying the developing tissue with fresh nutrients. Standard heart valves were engineered based on B trileaflet scaffolds seeded with cells isolated from the human saphenous vein. Tissue compaction is constrained by the stent, inducing increasing pre-strain in the tissue. The dynamic strains the tissues are exposed to via the dynamic pressure difference, are estimated using finite element methods based on the mechanical properties of the neo-tissue, in order to get inside into the strain distribution over the leaflet.

1 .- 16 . (canceled) 
     
     
         17 . A bioreactor for manufacturing a tissue-engineered prosthesis having at least in an open condition a flow passage, especially human heart valves, comprising:
 a bioreactor chamber for inserting therein a seeded 3D scaffold,   at least one perfusion flow means to provide at least one perfusion flow of a nutrient medium in the bioreactor chamber, in addition pressuriser means to apply a dynamic pressure difference over the seeded 3D scaffold and/or thereon developing tissue to create strain in the 3D scaffold and/or thereon developing tissue, the pressuriser means having compressible and decompressive tubing in flow connection with the bioreactor chamber.   
     
     
         18 . The bioreactor of  claim 17 , wherein at least a portion of the compressible and decompressible tubing is placed in a cylinder surrounding the outer surface of said portion of the tubing and having a port for flow of a compressed fluid, preferably air, into the cylinder. 
     
     
         19 . The bioreactor of  claim 18 , wherein a magnet valve is provided being in flow communication with the port of the cylinder to control the flow of the compressed fluid into the cylinder. 
     
     
         20 . The bioreactor of  claim 17 , wherein a compliance chamber is provided in flow communication with the bioreactor chamber to compensate for the displacement of the nutrient medium due to the pressure difference. 
     
     
         21 . The bioreactor of  claim 17 , wherein the bioreactor chamber has a first and a second portion each provided with a pressure sensor and holding means for holding a 3D scaffold and/or the thereon developing tissue is positioned between the first and second portion. 
     
     
         22 . A method of manufacturing a tissue-engineered prosthesis having at least in an open condition a flow passage, especially human heart valves, comprising the steps of:
 placing a seeded 3D scaffold in a bioreactor chamber,   providing at least one perfusion flow of a nutrient medium in the bioreactor chamber to supply said seeded 3D scaffold and/or a thereon developing tissue with nutrients whereby a flow passage of the seeded 3D scaffold and/or the thereon developing tissue in relation to the flow passage of the finished prosthesis is restricted or zero,   applying in addition a dynamic pressure difference over the 3D scaffold and/or thereon developing tissue depending on the condition and/or stage of tissue development to create strain in the 3D scaffold and/or thereon developing tissue, and   opening the restricted or zero flow passage to its finished size.   
     
     
         23 . The method of  claim 22 , wherein the prosthesis is a heart valve having leaflets and the restriction is achieved by coaptation of the leaflets. 
     
     
         24 . The method of  claim 22 , wherein the restricted or zero flow passage is at least 20%, preferably 50%, smaller than the flow passage of the finished prosthesis. 
     
     
         25 . The method of  claim 22 , wherein the at least one perfusion flow is less than 50 ml/min, preferably less than 5 ml/min. 
     
     
         26 . The method of  claim 22 , wherein the at least one perfusion flow has substantially no pulsation. 
     
     
         27 . The method of  claim 22 , wherein the pressure difference is substantially zero at the beginning, subsequently increasing up to a mean peak pressure difference and thereafter decreasing to the end of the duration of stay of the 3D scaffold and/or thereon developing tissue in the bioreactor chamber. 
     
     
         28 . The method of  claim 22 , wherein a mean peak pressure difference averaged for 24 hours, is above 25 mmHg, preferably above 45 mmHg. 
     
     
         29 . The method of  claim 22 , wherein the mean pressure difference over a time period, which is 30-70%, preferably 45-55%, of the duration of stay in the bioreactor, is substantially zero. 
     
     
         30 . The method of  claim 29 , wherein the mean peak pressure difference averaged for 24 hours is reached after 60%, preferably after 70%, of the duration of stay in the bioreactor. 
     
     
         31 . The method of  claim 30 , wherein the modulated pressure differences per time period, preferably 24 hours, are used to provide respective strain distribution in that time period. 
     
     
         32 . method of  claim 22 , wherein the dynamic pressure difference has a frequency of 0.1 to 10 Hz, preferably 1 Hz.