Pre-Vascularized Modular Tissue Engineering System

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SEM images of microtubes of silk fibroin and silk fibroin/PEO blends: (A) silk fibroin, (B) 99/1 wt %, (C) 98/2 wt %, (D) 90/10 wt %, (E) 80/20 wt % (silk fibroin/PEO).Pore sizes (μm) of silk fibroin/PEO blend microtubes as a function of microtube treatment. Surface pore sizes of the silk microtubes of different silk fibroin/PEO blends were measured and quantified both along the tube surface and cross-section using SEM images and ImageJ software. Microtube treatments are given either as 100% silk fibroin (SILK) or according to wt % of PEO in a silk fibroin/PEO blend (e.g. PEO01 represents a 99/1 wt % silk fibroin/PEO blend). * indicates P<0.01 with PEO10, PEO20; ** indicates P<0.01 with PEO02, PEO10, PEO20; *** indicates P<0.01 with PEO01, PEO02, PEO10, PEO20 (two-sample t-test).Burst pressure of silk microtubes as a function of microtube treatment. Internal pressure of the microtubes was increased by flowing water through the microtube, keeping one end of the flow loop closed. Pressure at point of failure was recorded with a digital manometer (n=5-6). * indicates P<0.05 with PEO20; ** indicates P<0.01 with PEO10, PEO20 (two-sample t-test). Permeability of silk microtubes as a function of tube porosity. Silk microtubes embedded in a collagen gel were perfused with labeled BSA, and fluorescent confocal microscopy images are shown after 10 minutes of perfusion. Edges of microtubes are shown at the right of each image with labeled BSA diffusing out to the left of the microtube (edge of microtube given by dashed line). Labeled BSA can be seen perfusing through the middle of the 100% silk fibroin tube and is faintly visible in the PEO01 tube. Labeled protein is not visible in the middle of silk microtubes with greater percentages of PEO due to the opaqueness of the tubes. Scale bars=300 microns.Perfusion of GFP-transduced human umbilical vein endothelial cells (HUVECs). Silk microtubes embedded in a collagen gel were perfused with green fluorescent protein (GFP) transduced HUVECs. GFP-HUVECs were perfused back and forth at a rate of 4 μL/min through the silk microtubes using a syringe pump, and fluorescent confocal microscopy images are shown after 3 days of perfusion. Sections of the microtubes are shown at the right of each image with GFP-HUVECs present in the channel of the microtube (edge of microtube given by dashed line). These cells are visible in the 100% silk fibroin and PEO01 tubes, but their presence in the microtubes with greater percentages of PEO is obscured due to the opaqueness of the tubes. The silk microtubes were a significant barrier to cell migration. At low porosity, GFP-HUVEC migration was completely blocked; at higher porosities (PEO10 and PEO20) cell migration was limited to a few cells (outlined by dashed ovals) over the entire microtube. Scale bars=300 microns.
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Inventors
Michael Lovett
Department of Biomedical Engineering, Tufts University
Xianyan Wang
Department of Biomedical Engineering, Tufts University
Christopher Cannizzaro
Harvard-M.I.T. Division for Health Sciences and Technology
Professor Gordana Vunjak-Novakovic
Department of Biomedical Engineering, Columbia University
External Link (gvn.hostedplace.com)
Professor David Kaplan
Department of Biomedical Engineering, Tufts University
External Link (engineering.tufts.edu)
Managed By
Jon Gilbert
MIT Technology Licensing Officer
Patent Protection

Tubular Silk Compositions and Methods of Use Thereof

US Patent Pending 2012-0123519
Publications
Silk Fibroin Microtubes for Blood Vessel Engineering
Biomaterials, Dec 28, 2007, p. 5271-9
Gel Spinning of Silk Tubes for Tissue Engineering
Biomaterials, Dec 29, 2008, p. 4650-7
Tubular Silk Scaffolds for Small Diameter Vascular Grafts
Organogenesis, Oct 1, 2010, p. 217-224

Applications

Silk fibroin microtubules with varying sizes of porosity and diameters between 100 um and 6 mm may be used in vitro as a tool for diffusion of oxygen and nutrients into microtubule-embedded hydrogel, a model for human microvasculature, and a scaffold for tissue engineering. They can be used in vivo as synthetic microvascular grafts in cases of peripheral artery disease. 

Problem Addressed

While attempts to engineer artificial macrovessels have been met with moderate success, methods to create high quality artificial microvessels remain elusive. Macrovascular grafts (6-7 mm inner diameter) made from materials such as polytetrafluoroethylene (ePTFE, Teflon™) and polyethylene terephthalate (PET, Dacron™) perform at a "gold standard" of 75% for 5-year patency. However, microvascular grafts (less than a 1 mm inner diameter) made from the same materials fall below this standard. In fact, no microvascular grafts, natural or synthetic, has been fully accepted into routine clinical practice. As a result, there is a medical need to develop new methods to produce longer-lasting microvascular grafts. 

Technology

This invention discloses a method to produce silk fibroin microtubules that can act as small-caliber (<6 mm inner diameter) vessel surrogates. Silk fibroin, derived from Bombyx mori silkworm cocoons, is biocompatible, slow to degrade, nontoxic, and mechanically robust. It also experiences low thrombicity and immunogenicity. These properties make it a perfect candidate for a variety of biomedical applications, including microvascular grafting. Silk fibroin microtubules of different sizes and porosities were produced by coating various sizes of steel wires with layers of silk fibroin/ polyethylene oxide (PEO) aqueous solutions to create tubes of beta-sheet structure. Microtubule strength, measured via a digital manometer by flowing water through tubes with one obstructed end until the tubes burst, was shown to be very high for lower porosity microtubules (as high as 2780 mmHg), while higher porosity microtubules showed lower strengths (680 mmHg). These values can be compared to human saphenous veins, which on average have a burst strength of 1680 mmHg. Protein permeability, as measured by perfusing the tubes with fluorescently labeled bovine albumin serum, was determined to be as low as 1.1x10-5  tcm/s for low porosity microtubules, to 9.4x10-4 cm/s for high porosity microtubules. Like the burst strengths, these values also span the range of protein permeability in human vessels. Finally, cell diffusion, measured by perfusing the tubes with human umbilical vein endothelial cells, showed that low porosity microtubules were impermeable to cells, while high porosity microtubules allowed for the diffusion of only a few cells per centimeter of microtubule over a 3-day perfusion period. The low level of cell diffusion suggests that these microtubules may be pre-endothelialized before being implanted as a graft to prevent thrombosis. Altogether, these properties show a wide variety of range depending on tube size and porosity, which suggests that these silk fibroin microtubules may act as a good surrogate to human vessels in artificial microvascular grafts. They are generally capable of withstanding physiological pressures while allowing for protein diffusion and endothelialization, and are likely to perform at the "golden standard" of vascular grafts. They are also relatively easy to manufacture, making them an attractive candidate for further development. 

Advantages

  • Slow degradation and high biocompatibility
  • Tailorable to different pore sizes, burst strengths, and protein and cell permeability depending on application
  • Pre-endothelialization capability
  • Ease of manufacture