Tissue-engineered constructs

Abstract

The present invention provides constructs including a tubular biodegradable polyglycolic acid scaffold, wherein the scaffold may be coated with extracellular matrix proteins and substantially acellular. The constructs can be utilized as an arteriovenous graft, a coronary graft, a peripheral artery bypass conduit, or a urinary conduit. The present invention also provides methods of producing such constructs.

We claim: 
     
         1 . A construct comprising a tubular biodegradable non-woven polyglycolic acid scaffold, wherein the density of the polyglycolic acid is about 45 mg/cc to about 75 mg/cc and said density is uniform across the entire tubular scaffold, wherein the thickness of the polyglycolic acid is about 0.8 to about 1.2 mm and wherein the thickness of the fibers within the polyglycolic acid is about 5 to about 20 μm. 
     
     
         2 . The construct of  claim 1 , wherein the length of the tubular biodegradable polyglycolic acid scaffold is about 1 cm to about 100 cm. 
     
     
         3 . The construct of  claim 1 , wherein the inner diameter of the tubular biodegradable polyglycolic acid scaffold is about 3 mm to about 6 mm. 
     
     
         4 . The construct of  claim 1 , further comprising non-biodegradable polyethylene terephthalate supports at each end of the tubular biodegradable polyglycolic acid scaffold, wherein, wherein the porosity of the polyethylene terephthalate is 2≧200 cc/min/cm 2  and the supports permit the attachment and growth of cells. 
     
     
         5 . The construct of  claim 1 , wherein the construct is substantially free of heavy metal contaminants. 
     
     
         6 . The construct of  claim 5 , wherein the construct comprises trace amounts of heavy metal contaminants selected from the group consisting of: aluminum, barium, calcium, iodine, lanthanum, magnesium, nickel, potassium and zinc. 
     
     
         7 . The construct of  claim 1 , wherein the construct further comprises extracellular matrix proteins. 
     
     
         8 . The construct of  claim 7 , wherein the thickness of the extracellular matrix proteins is greater than about 200 micrometers at the thinnest portion of the matrix. 
     
     
         9 . A method of producing a tubular polyglycolic acid construct comprising:
 (a) providing a biodegradable polyglycolic acid sheet, wherein the density of the polyglycolic acid is about 45 mg/cc to about 75 mg/cc and the thickness of the polyglycolic acid sheet is about 0.8 to about 1.2 mm,   (b) wrapping the polyglycolic acid sheet around a mandrel such that opposite edges of the polyglycolic acid sheet meet at an interface;   (c) pulling polyglycolic acid fibers from each opposing edge of the sheet across the interface, and   (d) forming a seam by entangling said pulled polyglycolic acid fibers from one side of the interface with the polyglycolic acid fibers on the opposite side of the interface, wherein the density of the polyglycolic acid at the seam is about 45 mg/cc to about 75 mg/cc and the thickness of the polyglycolic acid at the seam is about 0.8 to about 1.5 mm,   thereby producing a tubular biodegradable polyglycolic acid construct with a uniform polyglycolic acid density.   
     
     
         10 . The method of  claim 9 , further comprising treating the tubular construct to remove heavy metal contaminants. 
     
     
         11 . The method of  claim 10 , wherein the seam remains intact following said treatment. 
     
     
         12 . The method of  claim 9 , further comprising treating the tubular construct to increase the rate of polyglycolic acid degradation. 
     
     
         13 . The method of  claim 12 , wherein the seam remains intact following said treatment. 
     
     
         14 . A tubular construct comprising extracellular matrix proteins and polyglycolic acid having thickness greater than about 200 μm at the thinnest portion of the construct and having an internal diameter of ≧3 mm, wherein the construct is intimal hyperplasia and calcification resistant, wherein the polyglycolic acid comprises less than 33% of the cross-sectional area of said construct and wherein the construct is substantially acellular comprising less than 5% intact cells. 
     
     
         15 . The tubular construct of  claim 14 , wherein the construct is substantially acellular comprising less than 1% intact cells. 
     
     
         16 . The tubular construct of  claim 14 , wherein the inner diameter of the tubular construct is about 3 mm to about 6 mm. 
     
     
         17 . The tubular construct of  claim 14 , wherein the length of the construct is about 1 cm to about 100 cm. 
     
     
         18 . The tubular construct of  claim 14 , wherein the construct is impermeable to fluid leakage up to at least 200 mm Hg. 
     
     
         19 . The tubular construct of  claim 14 , wherein the construct is selected from the group consisting of an arteriovenous graft, a coronary graft, diseased peripheral artery bypass conduit, fallopian tube replacement and a urinary conduit. 
     
     
         20 . The tubular construct of  claim 14 , wherein the extracellular matrix proteins comprise hydroxyproline, vitronectin, fibronectin and collagen type I, collagen type III, collagen type IV, collagen VI, collagen XI, collagen XII, fibrillin I, tenascin, decorin, byglycan, versican or asporin. 
     
     
         21 . The tubular construct of  claim 20 , wherein the extracellular matrix proteins comprise hydroxyproline at >40 μg/mg dry weight. 
     
     
         22 . The tubular construct of  claim 14 , wherein the extracellular matrix proteins are oriented circumferentially around the tubular construct. 
     
     
         23 . The tubular construct of  claim 14 , wherein the construct comprises less than 300 ng/cm of beta-actin. 
     
     
         24 . The tubular construct of  claim 14 , wherein the construct comprises less than 3% dry weight of lipids. 
     
     
         25 . The tubular construct of  claim 14 , wherein the construct comprises trace amounts of double stranded genomic DNA. 
     
     
         26 . The tubular construct of  claim 14 , wherein the construct induces than 1% calcification within 6 months of implantation. 
     
     
         27 . The tubular construct of  claim 14 , wherein the construct induces less than 1% calcification within 12 months of implantation. 
     
     
         28 . The tubular construct of  claim 14 , wherein the construct induces less than 1 mm of intimal hyperplasia thickening in native vasculature at anastomoses with the construct at 6 months of implantation. 
     
     
         29 . The tubular construct of  claim 14 , wherein the construct induces less than 0.25 mm of intimal hyperplasia thickening in native vasculature at anastomoses with the construct at 6 months of implantation. 
     
     
         30 . The tubular construct of  claim 14 , wherein the construct does not dilate greater than 50% beyond its implant diameter after implantation. 
     
     
         31 . The tubular construct of  claim 19 , wherein when the construct is a urinary conduit, the urinary conduit tolerates exposure to urine for at least 4 weeks. 
     
     
         32 . The tubular construct of  claim 19 , wherein when the construct is a urinary conduit, the urinary conduit is crystallization resistant. 
     
     
         33 . The tubular construct of  claim 14 , wherein the construct may be stored at about 2° to about 30° C. for at least 12 months. 
     
     
         34 . A method of producing a tubular construct comprising:
 (a) providing a tubular biodegradable polyglycolic acid construct having an inner diameter of about 3 mm to about 6 mm,   (b) seeding human cells at passage 6 or less on the tubular biodegradable polyglycolic acid construct,   (c) culturing the cells under conditions such that the cells secrete extracellular matrix proteins on the tubular biodegradable polyglycolic acid construct,   (d) decellularizing the construct in step (c) such that the construct is substantially acellular comprising less than 5% intact cells and wherein the construct is intimal hyperplasia and calcification resistant, and   (e) degrading the polyglycolic acid construct in step (c) such that the polyglycolic acid comprises less than 33% of the cross-sectional area of said construct, and wherein the construct has a extracellular matrix protein thickness greater than about 200 μm at the thinnest portion of the construct,   thereby producing a decellularized tubular construct.   
     
     
         35 . The method of  claim 34 , wherein the decellularizing step occurs in the absence of sodium dodecyl sulfate (SDS). 
     
     
         36 . The method of  claim 34 , wherein the decellularizing step further comprises benzonase. 
     
     
         37 . The method of  claim 34 , wherein the construct is substantially acellular comprising less than 1% intact cells. 
     
     
         38 . The method of  claim 34 , wherein the cells are obtained from a single donor or obtained a cell bank, wherein the cells in the cell bank are pooled from a plurality of donors. 
     
     
         39 . The method of  claim 34 , wherein the cells are isolated from human aorta. 
     
     
         40 . The method of  claim 34 , wherein the cells comprise smooth muscle cells. 
     
     
         41 . The method of  claim 34 , wherein the cells are at passage 3 or less. 
     
     
         42 . The method of  claim 34 , wherein the cells are at cultured in medium comprising about 11% to about 30% human serum for the first 2-6 weeks of culture and in medium comprising about 1% to about 10% human serum for at least an additional 4 weeks. 
     
     
         43 . The method of  claim 42 , wherein the medium further comprises high glucose, insulin, bFGF and EGF. 
     
     
         44 . The method of  claim 34 , wherein the cells are at seeded onto the tubular biodegradable polyglycolic acid construct at about 0.5×10 6  cells per cm length of construct to about 2×10 6  cells per cm length of construct. 
     
     
         45 . The method of  claim 34 , wherein each cell of the seeded cells, or each cell's collective progeny, produces greater than 1 ng of hydroxyproline over 9 weeks in culture.