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. 2020 Feb:230:119567.
doi: 10.1016/j.biomaterials.2019.119567. Epub 2019 Oct 23.

Fabricating mechanically improved silk-based vascular grafts by solution control of the gel-spinning process

Affiliations

Fabricating mechanically improved silk-based vascular grafts by solution control of the gel-spinning process

Maria Rodriguez et al. Biomaterials. 2020 Feb.

Abstract

There is a large unmet need for off-the-shelf biomaterial options to supplant venous autografts in bypass and reconstructive surgical procedures. Existing graft alternatives formed from non-degradable synthetic polymers are not capable of maintaining long-term patency and are thus not indicated for <6 mm inner diameter bypass procedures. To fill this void, degradable silk-based biomaterials have been proposed that can maintain their mechanical properties (i.e. compliance) while facilitating slow but progressive biomaterial remodeling and host integration mediated by cellular colonization. The goal of the present study was to enhance the porosity of gel-spun silk tubes, to facilitate faster degradation rates and improve cellularity, and thus improve host integration over time in vivo, while maintaining requisite mechanical functions. Silk solutions with a range of molecular weight distributions and, in turn, viscosities were used to generate tubes of varying porosities. A decrease in solution concentration correlated with an increase in mean pore size and overall porosity through a density-dependent mechanism. Tubes were mechanically analyzed, and these properties were the basis of an analytical model used to correlate tube formulations to structural compliance, which were shown to be similar to the saphenous vein. Tubes were also tested for suture retention to ensure surgical utility despite increased porosity. Tubes were implanted in the abdominal aorta of Sprague-Dawley rats via an end-to-end anastomosis model. Tubes with higher porosities showed early improvements in cell colonization that progressively increased over time; conversely, the dense architecture of less porous grafts (20MB) inhibited cell ingrowth and resulted in minimal biomaterial degradation at the 6-month time point. None of the highly porous tubes (5 MB and 10MB) remained patent at 6 months, likely due remodeling inducing bulk mechanical failure or a compromised blood-material interface.

Keywords: Mechanical properties; Porosity; Silk; Vascular grafts.

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Figures

Figure 1.
Figure 1.. Viscosity of silk solution needed for gel-spinning.
Silk solutions of increasing boiling times (5MB, 10MB, 20MB and 30MB which correlate to decreasing molecular weights) and increasing concentrations were prepared. The viscosity of these silk solutionswere characterized with a cone-plate viscometer. A trend of increase in viscosity with decreasing boiling time (increasing molecular weight) and increasing solution concentration was seen.
Figure 2.
Figure 2.. Morphological characterization of silk tubes showed control of silk solution boiling time affected tube structure.
(A) Tubes formed from 5MB, 10MB, 20MB, 30MB, (14%,16%,26%,34% w/v concentrations, respectively) characterized by SEM showed different pore structures after lyophilization. Scale bars 100 μm for cross-sectional images. Inset shows the inner lumen of each tube (inset scale bar =500 μm). (B) Mercury Intrusion Porosimetry was used to evaluate the pore size distribution (between 10 nm and ~300 μm) for tubes shown in panel (A). The dark hashed section of the histogram indicates the overlap between each MB group.
Figure 3.
Figure 3.. In vitro Enzymatic Degradation.
Sections of tubes (10 mg each) formed from 5MB, 10MB, 20MB, 30MB, (14%,16%,26%,34% w/v concentrations, respectively) were subject to Protease XIV enzyme exposure for 14 days under conditions of constant orbital shaking. A 5MB group was subject to PBS (without enzyme) as a control. Dry tubes were weighed at the onset of the study and samples were removed from enzyme solutions, rinsed in DI water, dried and re-weighed at each time point. Enzyme solution was replaced at each measurement interval. While all groups exposed to enzyme lost mass throughout the study, the 5MB group was the fastest to degrade, likely due to rapid fluid transport through the large pores.
Figure 4.
Figure 4.. Mechanical Properties of vascular grafts.
Uniaxial tensile properties were determined for a range of silk formulations to determine the difference in properties as a function of molecular weight and concentration. Concentrations of 9 % w/v for 5MB, 14 % w/v for 10MB, 17 % for 15MB and 25% w/v for 20MB silk solutions were tested. (A) From the resulting stress strain curves (representative curves shown here for low (20 MB), medium (10 MB) and high (5 MB) molecular weights) the young’s modulus was calculated from the slope of the linear region below 5% (C), and Poisson ratio was calculated as the ratio of transverse strain to axial strain (D). The suture retention strength (B) was defined as the force required to pull through the tube or cause the wall to fail. There was a linear trend with decreasing elastic moduli (indicative of greater elasticity) and decreasing suture retention strength as the molecular weight increased and the concentration decreased.
Figure 5.
Figure 5.. Experimental and model compliance of vascular grafts.
(A) Compliance was measured experimentally using a custom pressurization device, composed of a programmable syringe pump, a holding chamber, a camera and two in-line pressure transducers. Tubes made with concentrations of 8 % w/v for 5MB, 14 % w/v for 10MB, 18 % for 15MB and 25% w/v for 20MB silk solutions were tested. (B) A Finite Element Model was made using ABAQUS simulating the experimental compliance test. The model considered a cylinder fixed at both ends, the longitudinal, radial and circumferential stresses were calculated using quasi static analysis where the cylinder was pressurized from 0 to 200 mm Hg in increments of 0.005 mm Hg. The predicted compliance as a function of thickness and elastic modulus is plotted (right). The predicted compliance, experimental compliance and compliance values derived from literature are compared (left).
Figure 6.
Figure 6.. Histological cross-sections of 20 MB grafts 1, 3- and 6-months post-implantation.
Adjacent sections were stained for Verhoff’s elastic (VE), trichrome, Factor VIII (FVIII) and smooth muscle actin (SMA). For each stain images are shown in low (20×, top) and high magnification (40×, bottom) corresponding to the box in the image above. Arrows indicate areas of clear positive IHC staining. Histology images shown were taken midgraft. The scale bar (500 μm) is common to all images.
Figure 7.
Figure 7.. Histological cross-sections of 10 MB (top) and 5 MB grafts 1-month post-implantation.
Adjacent sections were stained for Verhoff’s elastic (VE), trichrome, Factor VIII (FVIII) and smooth muscle actin (SMA). For each stain images are shown in low (20x, top) and high magnification (40x, bottom) corresponding to the box in the image above. Arrows indicate areas of clear positive IHC staining. Histology images shown were taken midgraft The scale bar (500 μm) is common to all images.

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