Tissue-engineered constructs
Inventors
Dahl, Shannon L. M. • Niklason, Laura E. • STRADER, Justin T. • Tente, William E. • LUNDQUIST, Joseph J.
Assignees
Publication Number
US-10934522-B2
Publication Date
2021-03-02
Expiration Date
2032-01-06
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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.
Core Innovation
The invention provides tissue-engineered constructs comprising a tubular biodegradable polyglycolic acid (PGA) scaffold with a uniform density of about 45 mg/cc to 75 mg/cc, which may be coated with extracellular matrix proteins and is substantially acellular. The tubular scaffold has an inner diameter greater than about 3 mm, lengths ranging from 1 cm to 100 cm, thickness between 0.8 mm and 1.5 mm, and high porosity of about 90% to 98%. The constructs may include non-biodegradable polyethylene terephthalate supports at each end to facilitate attachment and growth of cells.
The invention addresses the clinical need for effective, rapidly available, and reliable vascular grafts when autologous vessels are unavailable or unsuitable due to prior harvest or disease. Current alternatives such as synthetic PTFE grafts have poor patency rates and complications including infection and thrombosis, while decellularized bovine or human allograft vessels have issues with aneurysm, calcification, and thrombosis. Tissue-engineered grafts requiring extensive autologous cell culture are costly and time-consuming. Thus, there is a need for tissue-engineered constructs with good patency that resist dilatation, calcification, and intimal hyperplasia and can be produced cost-effectively and timely.
The invention includes methods for producing tubular PGA constructs by wrapping a PGA sheet around a mandrel, entangling fibers at the seam to ensure uniform density and thickness, and treating the construct to remove heavy metal contaminants and enhance degradation or wettability. Cells at passage 6 or less are seeded on the scaffold and cultured to produce extracellular matrix proteins, followed by decellularization to produce substantially acellular, immune- and calcification-resistant constructs with minimal PGA content. The constructs exhibit mechanical properties suitable for vascular graft applications, including high burst pressure, sufficient suture strength, impermeability to fluids, and resistance to immune response and calcification.
Claims Coverage
The patent includes one independent claim focused on a method of producing a tubular construct, with several dependent claims specifying further features and conditions.
Method for producing a tubular PGA construct with uniform density and heavy metal removal
The method involves providing a tubular biodegradable polyglycolic acid construct with uniform density of about 45 mg/cc to 75 mg/cc and dimensions of inner diameter about 3 mm to 6 mm and length 1 cm to 100 cm, followed by scouring to remove heavy metal contaminants while maintaining entangled PGA fibers.
Seeding and culturing of human cells on the PGA construct
Seeding human cells at passage 10 or less onto the tubular PGA construct and culturing them to secrete extracellular matrix proteins on the scaffold.
Decellularization producing substantially acellular and calcification-resistant construct
Decellularizing the cultured construct to obtain a substantially acellular product comprising less than 5% intact cells, with resistance to calcification.
Degradation of PGA to reduce polymer content
Degrading the PGA such that it comprises less than 5% of the construct cross-sectional area, producing a construct with extracellular matrix thickness greater than about 200 µm, minimal intimal hyperplasia (<1 mm at 6 months), and limited dilation (<50% beyond implant diameter).
Use of non-biodegradable supports at construct ends
Employing non-biodegradable supports at each end of the PGA tubular construct.
Decellularization without sodium dodecyl sulfate
Performing the decellularization step in the absence of SDS.
Use of endonucleases in decellularization
Incorporating an endonuclease step during decellularization to digest DNA.
Use of cells isolated from human aorta
Using cells isolated from human aorta, particularly smooth muscle cells, for seeding.
Specific culture medium composition
Culturing cells in medium comprising about 11% to about 30% human serum for the first 2-6 weeks and about 1% to 10% human serum for at least an additional 4 weeks, optionally supplemented with high glucose, insulin, bFGF, and EGF.
Seeding cell density
Seeding cells onto the PGA scaffold at about 0.5×10⁶ to 2×10⁶ cells per cm length of construct.
Extracellular matrix protein composition
Resulting extracellular matrix proteins comprising hydroxyproline at greater than 40 µg/mg up to about 60 µg/mg dry weight.
Control of heavy metal contaminants
Limiting heavy metal contaminants to trace amounts selected from aluminum, barium, calcium, iodine, lanthanum, magnesium, nickel, potassium, and zinc.
These claims collectively describe a method for producing tubular biodegradable PGA constructs with uniform fiber density, removal of contaminants, seeding and culturing of human cells to create extracellular matrix-rich, substantially acellular grafts with minimal polymer content, suitable mechanical and biological properties, and controlled composition for vascular applications.
Stated Advantages
Provides rapidly available tissue-engineered constructs with good patency and resistance to dilatation, calcification, and intimal hyperplasia.
Enables off-line production and storage of grafts, allowing immediate availability to patients with no wait time.
Allows use of allogeneic cells pooled from multiple donors to produce many grafts, improving economies of scale compared to autologous approaches.
Produces grafts that are mechanically robust with burst pressure and suture strength comparable to native vessels.
Produces substantially acellular, immune- and calcification-resistant constructs, reducing risk of immune rejection and calcific degradation.
Achieves grafts impermeable to fluid leakage up to at least 200-400 mm Hg.
Documented Applications
Arteriovenous grafts for hemodialysis access.
Coronary grafts for bypassing blocked coronary arteries.
Peripheral artery bypass conduits for treating peripheral arterial disease.
Urinary conduits for urinary diversion after cystectomy, connecting ureters to the skin through a stoma.
Fallopian tube replacements to address scarring and infertility due to blocked or damaged fallopian tubes.
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