Effects of RGD-fused silk fibroin in a solution format on fibroblast proliferation and collagen production.

BACKGROUND: Collagen production in fibroblasts is important for skin tissue repair. Cell-adhesive Arg-Gly-Asp (RGD) peptides immobilized on scaffolds stimulate fibroblast collagen production, but RGD peptides in solution exhibit opposite effects. Transgenic silkworm technology enables the design of fusion positions for RGD peptides in silk fibroin molecules. The effect of RGD-fused silk fibroin in solution on fibroblast cell activity remains unclear. OBJECTIVE: To clarify the effects of RGD peptides fused to silk fibroin heavy (H)-chain or light (L)-chain on fibroblast proliferation and collagen production when RGD-fused silk fibroin proteins were added to the culture medium. METHODS: Silk fibers with RGD-fused H-chains (H-RGD) or L-chains (L-RGD) were degummed, dissolved, and dialyzed to prepare H-RGD or L-RGD aqueous solutions, respectively. These solutions were added to the fibroblast medium, and their proliferation and collagen production were quantified. RESULTS: Both L- and H-RGD stimulated fibroblast proliferation at a similar level, even in a solution format, but L-RGD promoted fibroblast collagen production significantly, indicating the synergistic effect of the native H-chain and RGD-fused L-chain. CONCLUSION: RGD-fused silk fibroin in solution stimulated fibroblast proliferation and collagen production, depending on the fusion position of the peptides.

Silk Fibroin Conjugated with Heparin Promotes Epithelialization and Wound Healing.

Silk fibroin (SF) has attracted attention as a base biomaterial that could be suitable in many applications because of its shape and structure. Highly functional SF has been developed to promote tissue regeneration with heparin conjugation. However, the hydrophobic three-dimensional structure of SF makes it difficult to bind to high-molecular-weight and hydrophilic compounds such as heparin. In this study, sufficient heparin modification was achieved using tyrosine residues as reaction points to improve cellular response. As it was considered that there was a trade-off between the improvement of water wettability and cell responsiveness induced by heparin modification, influences on the structure, and mechanical properties, the structure and physical properties of the SF conjugated with heparin were extensively evaluated. Results showed that increased amounts of heparin modification raised heparin content and water wettability on film surfaces even though SF formation was not inhibited. In addition, the proliferation of endothelial cells and fibroblasts were enhanced when a surface with sufficient heparin assumed its potential in assisting wound healing. This research emphasizes the importance of material design focusing on the crystal structure inherent in SF in the development of functionalized SF materials.

Compatibility Evaluation of Non-Woven Sheet Composite of Silk Fibroin and Polyurethane in the Wet State.

SF/polyurethane composite non-woven sheet was fabricated to evaluate the cardiovascular tissue engineering materials in the wet state. The compatibility and microstructure analyses were carried out on the fabricated SF/polyurethane composite non-woven sheet by thermal analysis and solid-state NMR analysis in the wet state. To evaluate the modulus of elasticity, a tensile test was performed and supported with dynamic viscoelasticity and mechanical analysis. Results showed that SF/polyurethane composites form domains within the non-woven sheet and are in a finely dispersed state while maintaining their structures at a scale of several tens of nm. Moreover, an increase of the loss tangent with low elastic modulus proved that a micromolecular interaction occurs between silk fibroin (SF) and polyurethane molecules.

Evaluation of endothelialization in the center part of graft using 3 cm vascular grafts implanted in the abdominal aortae of the rat.

In order to develop small-diameter vascular grafts, it is necessary to evaluate endothelialization, especially, in the center part at early stage. For implantation of vascular grafts of 1 cm in length to abdominal aortae of rat, endothelial cells can be formed easily by stretching anastomosis. We evaluated the endothelialization in the center part of vascular grafts by implanting vascular grafts using transgenic (TG) silk fibroin (SF) of 3 cm in length. Vascular grafts were prepared 1.5 mm in diameter and 1 and 3 cm in length using wild type (WT) SF and TG SF by braiding structure, respectively. The grafts were removed after 2 weeks or 3 months and evaluated pathologically. Endothelialization was not confirmed totally after 3 months of implantation. However, endothelialization in the center part of grafts was significantly higher in TG SF than in WT SF. No significant difference was found regarding tissue infiltration and internal diameter. The TG SF revealed migration of the endothelial cells into the center part of the vessels at the early stage. Also, tissue infiltration and remodeling is expected using SF. The 3 cm length vascular grafts can be evaluated as a new experimental system.

Effect of fibroin sponge coating on in vivo performance of knitted silk small diameter vascular grafts.

Vascular grafts under 5 mm or less in diameter are not developed due to a problem caused by early thrombus formation, neointimal hyperplasia, etc. Bombyx mori silk fibroin (SF) which has biodegradability and tissue infiltration is focused as tube and coating material of vascular grafts. Coating is an important factor to maintain the strength of the anastomotic region of vascular grafts, and to prevent the blood leak from the vascular grafts after implantation. Therefore, in this research, we focused on the SF concentration of the coating solution, and tissue infiltration and remodeling were compared among each SF concentration. Silk poly (-ethylene) glycol diglycidyl ether (PGDE) coating with concentrations of 1.0%, 2.5%, 5.0%, and 7.5% SF were applied for the double-raschel knitted small-sized vessel with 1.5 mm diameter and 1cm in length. The grafts were implanted in the rat abdominal aorta and removed after 3 weeks or 3 months. Vascular grafts patency was monitored by ultrasound, and morphological evaluation was performed by histopathological examination. SF concentration had no significant effects on the patency rate. However, tissue infiltration was significantly higher in the sample of 2.5% SF in 3 weeks, and 1.0% and 2.5% SF in 3 months. Also, in comparison of length inside of the graft, stenosis were not found in 3 weeks, however, found with 5.0% and 7.5% in 3 months. From these results, it is clear that 2.5% SF coating is the most suitable concentration, based on the characteristics of less stenosis, early tissue infiltration, and less neointimal hyperplasia.

Biological reaction to small-diameter vascular grafts made of silk fibroin implanted in the abdominal aortae of rats.

BACKGROUND: Bombyx mori silk fibroin (SF) is biocompatible and degradable and has been proposed as a new material for small-diameter vascular grafts. We compared biological reactions to vascular grafts made of SF and polyethylene terephthalate (PET) to reveal the potential ability of SF as a base and/or coating materials for vascular prostheses. METHODS: SF was combined with PET or gelatin (G) to make 4 types of vascular grafts (SF/SF, SF/G, PET/SF, and PET/G, shown as "base/coating material," respectively), which are 1.5 mm in diameter and 10 mm in length. The 4 types of grafts (n = 6, respectively) were implanted into rat abdominal aortae and explanted 2 weeks or 3 months later. RESULTS: Two weeks after implantation, there are no significant differences among the 4 kinds of grafts in biological reactions evaluated by histopathologic examination. However, a remarkable difference was observed after 3 months. The area of tissue infiltration into the inside of the graft wall was approximately 2.5 times larger in SF/SF than that in PET/G. The endothelialization was achieved almost 100% in SF/SF, despite only 50% was achieved in PET/G. CONCLUSIONS: Results show that SF has a higher potential as a base of vascular grafts than the commercially available PET/G graft. The larger tissue infiltration area in PET/SF compared with that in PET/G also indicates the potential of SF as a coating material. In the present study, SF delivered promising results as base and coating materials for small-diameter vascular prostheses.

Evaluation of changes in left ventricular myocardial function observed in canine myocardial dysfunction model using a two-dimensional tissue tracking technique.

This study was conducted to assess the ability of two-dimensional tissue tracking (2DTT) to evaluate changes in left ventricular (LV) myocardial function associated with sustained high electrical pacing. Pacemakers were implanted at the right ventricular (RV) apex of five female Beagles, and sustained high electrical pacing of 250 beats per minute (bpm) was performed for three consecutive weeks. Conventional echocardiography and 2DTT were performed at baseline, and at every week for three weeks with pacing. The baseline parameters were then compared to those of weeks 1, 2, and 3. Three weeks of pacing resulted in significant reduction of radial and circumferential global strains (p < 0.001). Regional analysis revealed reduction of segmental strains in both radial and circumferential directions, as well as increased dyssynchrony after three weeks of pacing in the radial direction (p = 0.0007). The results of this study revealed the ability of 2DTT to measure radial and circumferential strains in dogs with sustained high-electrical pacing, and allowed assessment of global and regional myocardial function and the degree of dyssynchrony.

Extreme tetralogy of Fallot in a dog.

A four-month-old female Labrador retriever was brought to the Tokyo University of Agriculture and Technology Animal Medical Center for examination of its main symptoms of cough, tachypnea and exercise intolerance. Upon examination, the dog was found to have cyanosis and inadequate growth. Echocardiography revealed tetralogy of Fallot. Cardiac catheterization confirmed that the main pulmonary artery was completely occluded and that blood flowed from the aorta to the pulmonary artery. Accordingly, the animal was diagnosed with extreme tetralogy of Fallot.

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.

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) vascular 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.