Yasumoto Nakazawa

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.

Structure ofBombyx mori silk fibroin before spinning in solid state studied with wide angle x-ray scattering and13C cross-polarization/magic angle spinning NMR

The structure of a crystalline form of Bombyx mori silk fibroin, commonly found before the spinning process (known as silk I), has been proposed as a repeated β-turn type II-like structure by combining data obtained from solid-state two dimensional spin-diffusion nuclear magnetic resonance and rotational-echo double-resonance (T. Asakura et al., J Mol Biol, in press). In this paper, the WAXS pattern of alanine-glycine alternating copolypeptide, (Ala-Gly)15 with silk I form which was used for a silk I model of B. mori silk fibroin was observed. The pattern calculated with the silk I model proposed by us is well reproduced the observed one, indicating the validity of the proposed silk I model. In addition, two peptides of the other repeated sequences which contain Tyr or Val residues in the silk fibroin,23 were synthesized; (Ala-Gly-Tyr-Gly-Ala-Gly)5 and (X-Gly)15 where X is Tyr for the 7th, 15th and 23th residues, and Val for the 11th residue and Ala for other residues. There are no sharp peaks in the WAXS patterns, and therefore both samples are in the non-crystalline state. This is in agreement with the 13C CP/MAS NMR result, where the conformation is mainly random coil.

Development of Small-Diameter Vascular Grafts Based on Silk Fibroin Fibers from Bombyx mori for Vascular Regeneration

In the field of surgical revascularization, the need for functional small-diameter (1.5–4.0 mm in diameter) 1vascular grafts is increasing. Several synthetic biomaterials have been tested for this purpose, but in many cases they cause thrombosis. In this study, we report the development of small-diameter vascular grafts made from silk fibroin fibers from the domestic silkworm Bombyx mori or recombinant silk fibroin fibers from a transgenic silkworm. The vascular grafts were prepared by braiding, flattening and winding the silk fibers twice onto a cylindrical polymer tube followed by coating with an aqueous silk fibroin solution. The grafts, which are 1.5 mm in inner diameter and 10 mm in length, were implanted into rat abdominal aorta. An excellent patency (ca. 85%, n= 27) at 12 months after grafting with wild-type silk fibers was obtained. Endothelial cells and smooth muscle cells migrated into the silk fibroin graft early after implantation, and became organized into an endothelium and a media-like smooth muscle layer.

Silk structure studied with nuclear magnetic resonance.

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Small‐Diameter Silk Vascular Grafts (3 mm Diameter) with a Double‐Raschel Knitted Silk Tube Coated with Silk Fibroin Sponge

see front matter 2012 Elsevier B.V. A http://dx.doi.org/10.1016/j.pnmrs.2012.08.001 Abbreviations: CP-MAS, cross polarization magic correlation spectra with dipolar assisted rotational re CRAMPS, combined rotation and multiple pulse spect natural abundance double quantum transfer experime evaluation and reconstruction; REDOR, rotational-ech INEPT, insensitive nuclei enhanced by polarization; M after spinning; MaSp1, dragline silk spidroin 1; MaSp ⇑ Corresponding author. Fax: +81 423837733. E-mail address: asakura@cc.tuat.ac.jp (T. Asakura) 2012 Elsevier B.V. All rights reserved.

The interaction of amyloid Aβ(1-40) with lipid bilayers and ganglioside as studied by 31P solid-state NMR

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.

The role of irregular unit, GAAS, on the secondary structure of Bombyx mori silk fibroin studied with 13C CP/MAS NMR and wide-angle X-ray scattering

Amyloid beta-peptide (Abeta) is a major component of plaques in Alzheimers disease, and formation of senile plaques has been suggested to originate from regions of neuronal membrane rich in gangliosides. We analyzed the mode of interaction of Abeta with lipid bilayers by multinuclear NMR using (31)P nuclei. We found that Abeta (1-40) strongly perturbed the bilayer structure of dimyristoylphosphatidylcholine (DMPC), to form a non-lamellar phase (most likely micellar). The ganglioside GM1 potentiated the effect of Abeta (1-40), as viewed from (31)P NMR. The difference of the isotropic peak intensity between DMPC/Abeta and DMPC/GM1/Abeta suggests a specific interaction between Abeta and GM1. We show that in the DMPC/GM1/Abeta system there are three lipid phases, namely a lamellar phase, a hexagonal phase and non-oriented lipids. The latter two phases are induced by the presence of the Abeta peptide, and facilitated by GM1.

Tightly winding structure of sequential model peptide for repeated helical region in Samia cynthia ricini silk fibroin studied with solid‐state NMR

Bombyx mori silk fibroin is a fibrous protein whose fiber is extremely strong and tough, although it is produced by the silkworm at room temperature and from an aqueous solution. The primary structure is mainly Ala‐Gly alternative copolypeptide, but Gly‐Ala‐Ala‐Ser units appear frequently and periodically. Thus, this study aims at elucidating the role of such Gly‐Ala‐Ala‐Ser units on the secondary structure. The sequential model peptides containing Gly‐Ala‐Ala‐Ser units selected from the primary structure of B. mori silk fibroin were synthesized, and their secondary structure was studied with 13C CP/MAS NMR and wide‐angle X‐ray scattering. The 13C isotope labeling of the peptides and the 13C conformation‐dependent chemical shifts were used for the purpose. The Ala‐Ala units take antiparallel β‐sheet structure locally, and the introduction of one Ala‐Ala unit in (Ala‐Gly)15 chain promotes dramatical structural changes from silk I (repeated β‐turn type II structure) to silk II (antiparallel β‐sheet structure). Thus, the presence of Ala‐Ala units in B. mori silk fibroin chain will be one of the inducing factors of the structural transition for silk fiber formation. The role of Tyr residue in the peptide chain was also studied and clarified to induce “locally nonordered structure.”

Bombyx mori silk fibroin scaffolds for bone regeneration studied by bone differentiation experiment

There are many kinds of silks from silkworms and spiders with different structures and properties, and thus, silks are suitable to study the structure‐property relationship of fibrous proteins. Silk fibroin from a wild silkworm, Samia cynthia ricini, mainly consists of the repeated similar sequences by about 100 times where there are alternative appearances of the polyalanine (Ala)12–13 region and the Gly‐rich region. In this paper, a sequential model peptide, GGAGGGYGGDGG(A)12GGAGDGYGAG, which is a typical sequence of the silk fibroin, was synthesized, and the atomic‐level conformations of Gly residues at the N‐ and C‐terminal ends of the polyalanine region were determined as well as that of the central Ala residue using 13C 2D spin diffusion solid‐state nuclear magnetic resonance (NMR) under off‐magic angle spinning. In the model peptide with α‐helical conformation, the torsion angle of the central Ala residue, the 19th Ala, was determined to be (ϕ, ψ) = (−60°, −50°), which was a typical α‐helical structure, but the torsion angles of two Gly residues, the 12th and 25th Gly residues, which are located at the N‐ and C‐terminal ends of the polyalanine region, were determined to be (ϕ,ψ) = (−70°, −30°) and (ϕ,ψ) = (−70°, −20°), respectively. Thus, it was observed that the turns at both ends of polyalanine with α‐helix conformation in the model peptide are tightly wound.

Evidence from 13C solid-state NMR spectroscopy for a lamella structure in an alanine-glycine copolypeptide: a model for the crystalline domain of Bombyx mori silk fiber.

Bombyx mori silk fibroin (SF) shows remarkably earlier calcification than bovine serum albumin, indicating advantage of the SF scaffold for bone regeneration. We provide evidence for the first time, that SF not only activate early differentiation markers of osteoblasts, but also activate expression of the late differentiation markers.