Kosuke Ohgo

Preparation of non-woven nanofibers of Bombyx mori silk, Samia cynthia ricini silk and recombinant hybrid silk with electrospinning method

Abstract Electrospinning is a good method to obtain nanoscale fibers from polymer solutions. In this paper, we successfully prepared non-woven nanofibers of Bombyx mori and Samia cynthia ricini silk fibroins, and of the recombinant hybrid fiber involving the crystalline domain of B. mori silk and non-crystalline domain of S. c. ricini silk from hexafluoroacetone (HFA) solution using electrospinning method. 13 C cross polarization/magic angle spinning NMR spectroscopy was used to monitor the structural change of silk fibroins together with the detection of the residual HFA during the process of the fiber formation. Scanning electronic microscope was used to determine the diameters and their distributions of the fibers.

Comparative study of silk fibroin porous scaffolds derived from salt/water and sucrose/hexafluoroisopropanol in cartilage formation.

The purpose of this study is to create a new silk fibroin scaffold with sufficient three-dimensional morphology and porous structure for cartilage formation. We have applied sucrose particles sized around 300 to 500 microm as porogens compared to equal-sized salt particles. After the porogen was leached out with water, scaffolds were prepared with fibroin derived from sucrose/hexafluoroisopropanol (Su/H) or salt/water (Sa/W) based composites. A compression test indicated that the Sa/W fibroin was much harder than the Su/H fibroin, but a protease enzyme digested the Sa/W fibroin more quickly than Su/H fibroin. Rabbit ear chondrocytes were seeded onto the scaffolds for 4-8 week in vitro culture and histological analyses were performed. The distribution of cartilage formation in Safranin O staining was more homogenous in Su/H fibroin than that of Sa/W fibroin. The overall amount of cartilage was significantly better in the Su/H fibroin than that in the Sa/W fibroin. However, the inner structure of pore wall in the Sa/W fibroin was rough and microporous with cartilage matrix deposition, while that in the Su/H fibroin was thin and homogenous. Since mature cartilage gradually regenerates to fill the porous space, slowly degradable Su/H fibroin should be a better candidate for cartilage formation.

Investigation of structural transition of regenerated silk fibroin aqueous solution by Rheo-NMR spectroscopy.

In this study we applied Rheo-NMR to investigate the structural change of Bombyx mori silk fibroin in aqueous solution under shear. Monitoring the time dependence of 1H solution NMR spectra of silk fibroin subjected to constant shear strain, signal intensities of random coil decreased suddenly during shear while peaks from beta-sheet structure did not arise in the solution spectra. After these experiments, an aggregate of silk was found in the Couette flow cell and its secondary structure was determined as beta-sheet by 13C solid-state NMR. In conclusion the moderate shear applied here triggered the change in the secondary structure.

Determination of the torsion angles of alanine and glycine residues of model compounds of spider silk (AGG)(10) using solid-state NMR methods.

Spiders synthesize several kinds of silk fibers. In the primary structure of spider silk, one of the major ampullate (dragline, frame) silks, spidroin 1, and flagelliform silk (core fibers of adhesive spiral), there are common repeated X-Gly-Gly (X = Ala, Leu, Pro, Tyr, Glu, and Arg) sequences, which are considered to be related to the elastic character of these fibers. In this paper, two dimensional spin diffusion solid-state NMR under off magic angle spinning (OMAS), 13C chemical shift contour plots, and Rotational Echo DOuble Resonance (REDOR) were applied to determine the torsion angles of one Ala and two kinds of Gly residues in the Ala-Gly-Gly sequence of 13C=O isotope-labeled (Ala-Gly-Gly)10. The torsion angles were determined to be (φ, ψ) = (−90°, 150° ) within an experimental error of ±10° for each residue. This conformation is characterized as 31 helix which is in agreement with the structure proposed from the X-ray powder diffraction pattern of poly(Ala-Gly-Gly). The 31 helix of (Ala-Gly-Gly)10 does not change by formic acid treatment although (Ala-Gly)15 easily changes from the silk I conformation (the structure of Bombyx mori silk fibroin before spinning in the solid state) to silk II conformation (the structure of the silk fiber after spinning) by such treatment. Thus, the 31 helix conformation of (Ala-Gly-Gly)10 is considered very stable. Furthermore, the torsion angles of the 16th Leu residue of (Leu-Gly-Gly)10 were also determined as (φ, ψ) = (−90°, 150° ) and this peptide is also considered to take 31 helix conformation.

Structural insights into the elastin mimetic (LGGVG)6 using solid-state 13C NMR experiments and statistical analysis of the PDB.

Elastin is a crosslinked hydrophobic protein found in abundance in vertebrate tissue and is the source of elasticity in connective tissues and blood vessels. The repeating polypeptide sequences found in the hydrophobic domains of elastin have been the focus of many studies that attempt to understand the function of the native protein on a molecular scale. In this study, the central residues of the (LGGVG)(6) elastin mimetic are targeted. Using a combination of a statistical analysis based on structures in the Brookhaven Protein Data Bank (PDB), 1D cross-polarization magic-angle-spinning (CPMAS) NMR spectroscopy, and 2D off-magic-angle-spinning (OMAS) spin-diffusion experiments, it is determined that none of the residues are found in a singular regular, highly ordered structure. Instead, like the poly(VPGVG) elastin mimetics, there are multiple conformations and significant disorder. Furthermore, the conformational ensembles are not reflective of proteins generally, as in the PDB, suggesting that the structure distributions in elastin mimetics are unique to these peptides and are a salient feature of the functional model of the native protein.

Resolving Nitrogen-15 and Proton Chemical Shifts for Mobile Segments of Elastin with Two-dimensional NMR Spectroscopy

Background: Little is known about the structure of elastin, the abundant elastomeric protein in vertebrate tissue. Results: Observed chemical shifts do not match predictions for random coil, helix, or sheet. Conclusion: The structural makeup of elastin is heterogeneous and possibly unique in nature. Significance: The chemical shift index needs refinement for structural studies of Gly-rich proteins. In this study, one- and two-dimensional NMR experiments are applied to uniformly 15N-enriched synthetic elastin, a recombinant human tropoelastin that has been cross-linked to form an elastic hydrogel. Hydrated elastin is characterized by large segments that undergo “liquid-like” motions that limit the efficiency of cross-polarization. The refocused insensitive nuclei enhanced by polarization transfer experiment is used to target these extensive, mobile regions of this protein. Numerous peaks are detected in the backbone amide region of the protein, and their chemical shifts indicate the completely unstructured, “random coil” model for elastin is unlikely. Instead, more evidence is gathered that supports a characteristic ensemble of conformations in this rubber-like protein.

Backbone motion in elastin's hydrophobic domains as detected by 2H NMR spectroscopy†

The elasticity of vertebrate tissue originates from the insoluble, cross-linked protein elastin. Here, the results of variable-temperature (2) H NMR spectra are reported for hydrated elastin that has been enriched at the Hα position in its abundant glycines. Typical powder patterns reflecting averaged quadrupolar parameters are observed for the frozen protein, as opposed to the two, inequivalent deuterons that are detected in a powder sample of enriched glycine. The spectra of the hydrated elastin at warmer temperatures are dominated by a strong central peak with features close to the baseline, reflective of both isotropic and very weakly anisotropic motions.

Tropoelastin Implants That Accelerate Wound Repair

A novel, pure, synthetic material is presented that promotes the repair of full-thickness skin wounds. The active component is tropoelastin and leverages its ability to promote new blood vessel formation and its cell recruiting properties to accelerate wound repair. Key to the technology is the use of a novel heat-based, stabilized form of human tropoelastin which allows for tunable resorption. This implantable material contributes a tailored insert that can be shaped to the wound bed, where it hydrates to form a conformable protein hydrogel. Significant benefits in the extent of wound healing, dermal repair, and regeneration of mature epithelium in healthy pigs are demonstrated. The implant is compatible with initial co-treatment with full- and split-thickness skin grafts. The implants superiority to sterile bandaging, commercial hydrogel and dermal regeneration template products is shown. On this basis, a new concept for a prefabricated tissue repair material for point-of-care treatment of open wounds is provided.