Tissue-engineered constructs
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.
1 . A method of producing a tubular construct comprising:
(a) providing a tubular biodegradable polyglycolic acid (PGA) construct having an inner diameter of about 3 mm to about 6 mm and a length of 1 cm to 100 cm, wherein the tubular PGA construct comprises entangled PGA fibers, wherein the density of the PGA is about 45 mg/cc to about 75 mg/cc, and wherein said density is uniform across the entire tubular PGA construct,
(b) seeding human cells at passage 10 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, wherein the cells are cultured in medium comprising human serum, high glucose, insulin, bFGF, and EGF,
(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 calcification resistant, and
(e) degrading the polyglycolic acid construct in step (c) such that the polyglycolic acid comprises less than 5% of the cross-sectional area of said construct,
wherein the construct has an extracellular matrix protein thickness greater than about 200 μm at the thinnest portion of the construct,
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, and
wherein the construct does not dilate greater than 50% beyond its implant diameter after implantation, thereby producing a decellularized tubular construct.
2 . The method of claim 1 , wherein the (PGA) construct comprises non-biodegradable supports at each end of the construct.
3 . The method of claim 1 , wherein the decellularizing step occurs in the absence of sodium dodecyl sulfate (SDS).
4 . The method of claim 1 , wherein the decellularizing step employs an endonuclease.
5 . The method of claim 1 , wherein the construct is substantially acellular comprising less than 1% intact cells.
6 . The method of claim 1 , wherein the cells are isolated from human aorta.
7 . The method of claim 1 , wherein the cells comprise smooth muscle cells.
8 . The method of claim 1 , 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.
9 . The method of claim 1 , wherein the seeded cell and the seeded cells' progeny, collectively produce greater than 1 ng of hydroxyproline per cell over 9 weeks in culture.
10 . The method of claim 1 , wherein step (a) further comprises scouring the tubular PGA construct to remove heavy metal contaminants.
11 . The method of claim 10 , wherein the heavy metal contaminant is selected from the group consisting of aluminum, barium, calcium, iodine, lanthanum, magnesium, nickel, potassium and zinc.