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.
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.
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.
Slow degradation and high biocompatibility
Tailorable to different pore sizes, burst strengths, and protein and cell permeability depending on application
Ease of manufacture