Development of a new surgical sheet containing both silk fibroin and thermoplastic polyurethane for cardiovascular surgery.

PURPOSE: The surgical sheets that are currently used for congenital cardiovascular surgery have several drawbacks, including material deterioration, calcification, and pseudo-intimal proliferation resulting in hemodynamic disturbance. The aim of this study was to evaluate a newly developed sheet made from a combination of silk fibroin (SF) and a synthetic polymer, thermoplastic polyurethane (TPU), for surgical use. METHODS: The hybrid SF/TPU sheet was a non-woven fabric with nanofibers that was made using the electrospinning method. The mechanical properties of the SF/TPU sheet were characterized. To determine its biocompatibility, part of the wall of the canine descending aorta was replaced with a SF/TPU sheet as a patch. The patches were removed after 3 months and a histological examination was performed. RESULTS: The flexibility, water permeability, and suture retention strength of the SF/TPU sheet were excellent and equivalent to those of existing sheets. The SF/TPU sheet had excellent handling properties and fit well into the vascular wall without needle hole bleeding. The histological examination revealed that the intimal tissue was restored well over the intraluminal surface of the explanted SF/TPU sheet, the absence of calcium deposition, and minimal inflammatory reaction, without signs of degradation. CONCLUSION: The SF/TPU sheet had excellent mechanical properties and tissue biocompatibility. These favorable features and possible biodegradability of the SF portion warrant a long-term follow-up study.

Silk fibroin-Pellethane® cardiovascular patches: Effect of silk fibroin concentration on vascular remodeling in rat model.

Life-threatening cardiovascular anomalies require surgery for structural repair with cardiovascular patches. The biomaterial patch, derived from Bombyx mori silk fibroin (SF), is used as an alternative material due to its excellent tissue affinity and biocompatibility. However, SF lacks the elastomeric characteristics required for a cardiovascular patch. In order to overcome this shortcoming, we combined the thermoplastic polyurethane, Pellethane® (PU) with SF to develop an elastic biocompatible patch. Therefore, the purpose of this study was to investigate the feasibility of the blended SF/PU patch in a vascular model. Additionally, we focused on the effects of different SF concentrations in the SF/PU patch on its biological and physical properties. Three patches of different compositions (SF, SF7PU3 and SF4PU6) were created using an electrospinning method. Each patch type (n = 18) was implanted into rat abdominal aorta and histopathology was assessed at 1, 3, and 6 months post-implantation. The results showed that with increasing SF content the tensile strength and elasticity decreased. Histological evaluation revealed that inflammation gradually decreased in the SF7PU3 and SF patches throughout the study period. At 6 months post-implantation, the SF7PU3 patch demonstrated progressive remodeling, including significantly higher tissue infiltration, elastogenesis and endothelialization compared with SF4PU6. In conclusion, an increase of SF concentration in the SF/PU patch had effects on vascular remodeling and physical properties. Moreover, our blended patch might be an attractive alternative material that could induce the growth of a neo-artery composed of tissue present in native artery.

A novel gradient and multilayered sheet with a silk fibroin/polyvinyl alcohol core-shell structure for bioabsorbable arterial grafts.

Bioabsorbable arterial grafts can potentially improve patency and neovessel formation; however, their application in clinical settings has not been realized. In this study, we developed bioabsorbable gradient sheets based on silk fibroin (SF) and polyvinyl alcohol (PVA) with a core-shell nanofibrous structure. This gradient sheet was expected to promote vascular remodeling while we maintained its physical properties and a gradual degrading process from the luminal surface. ESP was conducted at various flow rates for SF and PVA to achieve the multilayer gradient structure. Furthermore, the elasticity of the gradient sheet could be increased by increasing the PVA flow rate; however, this reduced the tensile strength of the core-shell fibers. Notably, the physical properties of the gradient sheet did not degrade even after 7 days of immersion in a phosphate buffer saline solution, which indicates that the structure could maintain its structural integrity while resisting arterial pressure. In vitro experiments revealed that the number of endothelial cells attached to the SF/PVA sheet was notably higher than that on the cell-culture dish. The gradient sheets were implanted in rat abdominal aortas and explanted after 14 days to confirm acute-phase patency and vascular remodeling. The gradient sheets constructed with SF composed of polyurethane and PVA improved the ease of handling of the material, and these sheets resulted in a favorable vascular remodeling outcome. Our results strongly suggest that the SF/PVA-based gradient sheets described in this study can serve as a novel design for bioabsorbable arterial grafts upon further modifications.

Silk fibroin-based vascular repairing sheet with angiogenic-promoting activity of SVVYGLR peptide regenerated the damaged vascular in rats.

Medical sheets are useful in surgically repair vascular disease. To avoid long-term side effects, they are to be replaced with regenerated tissue after implantation. Silk fibroin is a fibrous protein secreted by silkworm. The advantage of silk fibroin is its biocompatibility and has been used as regenerative artificial materials. The problem of its biodegradability is that the effect is time consuming. In this study, SVVYGLR peptide was used to expect promoting cell migration and accelerating the biodegradation of silk fibroin. Silk fibroin and polyurethane-based medical sheets with or without SVVYGLR peptide were implanted in rat abdominal aorta (silk fibroin/polyurethane/SVVYGLR peptide versus silk fibroin/polyurethane). The result of histological evaluation indicated that the new cell layer created under both sheets was composed of endothelial cells, smooth muscle, and fibroin in both sheets and similar to a native vessel. Both sheets did not show any excessive inflammation or calcification, and moderate biodegradability was observed. The decrease of silk fibroin indicated the biodegradability of all sheets. Silk fibroin/polyurethane/SVVYGLR peptide had many small vessels in the regenerated tissue than silk fibroin/polyurethane. This appearance indicated that SVVYGLR peptide promoted the angiogenesis in the regenerative tissue. This study suggested that SVVYGLR peptide could give the angiogenic-promoting activity to silk fibroin-based vascular repairing sheet.

Silk fibroin/polyurethane patch implantation in hyperglycemic rat model.

PURPOSE: To understand the complication and histopathological characteristics between the Silk Fibroin/Polyurethanes (SF/PU) and the host response, and to unveil the compatibility of the patch in diabetes individuals. METHODS: Rats were divided into DM and control (CT) groups, and the DM group was induced with streptozotocin. All groups underwent the SF/PU patch implantation in the abdominal aorta, and the implanted patches were evaluated at one, two, three, and four weeks after implantation. RESULTS: DM group had more fibrosis formation and a delayed endothelialization compared to the CT group. There was no evidence of chronic inflammation in both DM and CT groups. CONCLUSIONS: Fibrosis in hyperglycemic individuals could promote the formation of new vascular structures in the implanted patch such as endothelial and vascular smooth muscle cells. In summary, the SF/PU patch was no serious complications when implanted under hyperglycemia, and the patch was suitable to implant in diabetes mellitus.

Long-term patency of small-diameter vascular graft made from fibroin, a silk-based biodegradable material.

OBJECTIVE: There is an increasing need for vascular grafts in the field of surgical revascularization. However, smaller vascular grafts made from synthetic biomaterials, particularly those <5 mm in diameter, are associated with a high incidence of thrombosis. Fibroin is a biodegradable protein derived from silk. Silk fibroin from Bombyx mori provides an antithrombotic surface and serves as a scaffold for various cell types in tissue engineering. We evaluated the potential of fibroin to generate a vascular prosthesis for small arteries. METHODS: A small vessel with three layers was woven from silk fibroin thread. These fibroin-based grafts (1.5 mm diameter, 10 mm length) were implanted into the abdominal aorta of 10- to 14-week-old male Sprague-Dawley rats by end-to-end anastomosis. Polytetrafluoroethylene (PTFE)-based grafts were used as the control. To investigate the origin of the cells in the neointima and media, bone marrow transplantation was performed from green fluorescent protein (GFP) rats to wild-type rats. RESULTS: The patency of fibroin grafts at 1 year after implantation was significantly higher than that of PTFE grafts (85.1% vs 30%, P < .01). Endothelial cells and smooth muscle cells (SMCs) migrated into the fibroin graft early after implantation and became organized into endothelial and medial layers, as determined by anti-CD31 and anti-alpha-smooth muscle actin immunostaining. The total number of SMCs increased 1.6-fold from 1 month to 3 months. Vasa vasorum also formed in the adventitia. Sirius red staining of the fibroin grafts revealed that the content of collagen significantly increased at 1 year after implantation, with a decrease in fibroin content. GFP-positive cells contributed to organization of a smooth muscle layer. CONCLUSIONS: Small-diameter fibroin-based vascular grafts have excellent long-term patency. Bone marrow-derived cells contribute to vascular remodeling after graft implantation. Fibroin might be a promising material to engineer vascular prostheses for small arteries.

The effect of a silk Fibroin/Polyurethane blend patch on rat Vessels.

Patch grafts are widely used in various kind of vascular surgeries such as detect repair or dilation of vascular stenosis. Expanded polytetrafluoroethylene (ePTFE) patches are flexible and handle well, but have shown problems with calcification as they are non-bioabsorbable and therefore permanently remain in the body. It is important to develop an alternative biocompatible patch. Silk fibroin (SF) was developed as a biocompatible material, but it lacks of the elasticity required for surgery as a patch. Polyurethane (PU) is also a well-known elastomer so this study focused on the SF and the PU blend materials with a weight ratio of 5:5 (SF/PU). To evaluate the SF/PU patch, the patches were implanted into the abdominal aortas of rats, using the ePTFE patch in the control group. Because it was more flexible the SF/PU patch was easier to implant than the ePTFE patch. At 1 week after implantation, the SF/PU patch had been infiltrated with cells and collagen fiber. The ePTFE control patch did not accumulate collagen fiber until 3 months and calcification occurred at 4 weeks. The SF/PU patch did not present any signs of calcification for 3 months. This study addressed the problems associated with using SF in isolation and showed that the SF/PU patch can be considered as a useful alternative to the ePTFE to overcome the problem of calcification.

Small-diameter silk vascular grafts (3 mm diameter) with a double-raschel knitted silk tube coated with silk fibroin sponge.

Small-diameter (less than 6 mm in diameter) vascular grafts are highly desirable due to the large demand for surgical revascularization; however, there are no available artificial grafts. Vascular grafts of 1.5 mm diameter prepared by our group with silk fibroin fiber have been proved to be excellent grafts with remarkably high patency and remodeling, based on rat implantation experiment (Enomoto et al., 2010). In this study, a silk fibroin vascular graft with 3 mm diameter which can be used for the coronary arteries or lower extremity arteries is prepared with a double-raschel knitted Bombyx mori silk fiber tube coated with B. mori silk fibroin sponge. Here the silk sponge is prepared from an aqueous solution of the silk fibroin and poly(ethylene) glycol diglycidyl ether as porogen. Sufficient strength, proper elasticity, and protection from loose ends in the implantation process are obtained for the silk fibroin graft; low water permeability and relatively large compliance are also attained. These excellent physical properties make silk fibroin grafts suitable to be implanted in a canine model.

Lamellar structure in poly(ala-gly) determined by solid-state NMR and statistical mechanical calculations.

Lamellar structure of poly(Ala-Gly) or (AG)n in the solid was examined using 13C solid-state NMR and statistical mechanical approaches. Two doubly labeled versions, [1-13C]Gly14[1-13C]Ala15- and [1-13C]Gly18[1-13C]Ala19 of (AG)15 were examined by two-dimensional (2D) 13C spin diffusion NMR in the solid state. In addition five doubly labeled [15N,13C]-versions of the same peptide, (AG) 15 and 15 versions labeled [3-13C] in each of the successive Ala residues were utilized for REDOR and 13C CP/MAS NMR measurements, respectively. The observed spin diffusion NMR spectra were consistent with a structure containing a combination of distorted beta-turns with a large distribution of the torsion angles and antiparallel beta-sheets. The relative proportion of the distorted beta-turn form was evaluated by examination of 13C CP/MAS NMR spectra of [3-13C]Ala-(AG)15. In addition, REDOR determinations showed five kinds of atomic distances between doubly labeled 13C and 15N nuclei which were also interpreted in terms of a combination of beta-sheets and beta-turns. Our statistical mechanical analysis is in excellent agreement with our Ala Cbeta 13C CP/MAS NMR data strongly suggesting that (AG)15 has a lamellar structure.

Structure of model peptides based on Nephila clavipes dragline silk spidroin (MaSp1) studied by 13C cross polarization/magic angle spinning NMR.

To obtain detailed structural information for spider dragline spidroin (MaSp1), we prepared three versions of the consensus peptide GGLGGQGAGAAAAAAGGAGQGGYGGLGSQGAGR labeled with 13C at six different sites. The 13C CP/MAS NMR spectra were observed after treating the peptides with different reagents known to alter silk protein conformations. The conformation-dependent 13C NMR chemical shifts and peak deconvolution were used to determine the local structure and the fractional compositions of the conformations, respectively. After trifluoroacetic acid (solvent)/diethyl ether (coagulant) treatment, the N-terminal region of poly-Ala (PLA) sequence, Ala8 and Ala10, adopted predominantly the alpha-helix with a substantial amount of beta-sheet. The central region, Ala15, Ala18, and Leu26, and C-terminal region, Ala31, of the peptide were dominated by either 3(1)-helix or alpha-helix. There was no indication of beta-sheet, although peak broadening indicates that the torsion angle distribution is relatively large. After 9 M LiBr/dialysis treatment, three kinds of conformation, beta-sheet, random coil, and 3(1)-helix, appeared, in almost equal amounts of beta-sheet and random coil conformations for Ala8 and Ala10 residues and distorted 3(1)-helix at the central region of the peptide. In contrast, after formic acid/methanol and 8 M urea/acetonitrile treatments, all of the local structure tends to beta-sheet, although small amounts of random coil are also observed. The peak pattern of the Ala Cbeta carbon after 8 M urea/acetonitrile treatment is similar to the corresponding patterns of silk fiber from Bombyx mori and Samia cynthia ricini. We also synthesized a longer 13C-labeled peptide containing two PLA blocks and three Gly-rich blocks. After 8 M urea/acetonitrile treatment, the conformation pattern was closely similar to that of the shorter peptide.