NMR Analyses and Statistical Modeling of Biobased Polymer Microstructures-A Selected Review.

NMR analysis combined with statistical modeling offers a useful approach to investigate the microstructures of polymers. This article provides a selective review of the developments in both the NMR analysis of biobased polymers and the statistical models that can be used to characterize these materials. The information obtained from NMR and statistical models can provide insights into the microstructure and stereochemistry of appropriate biobased polymers and establish a systematic approach to their analysis. In suitable cases, the analysis can help optimize the synthetic procedures and facilitate the development of new or modified polymeric materials for various applications. Examples are given of the studies of poly(hydroxyalkanoates), poly(lactic acid), and selected polysaccharides, e.g., alginate, pectin, and chitosan. This article may serve as both a reference and a guide for future workers interested in the NMR sequence analysis of biobased materials.

Thick silk fibroin vascular graft: A promising tissue-engineered scaffold material for abdominal vein grafts in middle-sized mammals.

Abdominal vein replacement with synthetic tissue-engineered vascular grafts constructed from silk-based scaffold material has not been reported in middle-sized mammals. Fourteen canines that underwent caudal vena cava replacement with a silk fibroin (SF) vascular graft (15 mm long and 8 mm diameter) prepared with natural silk biocompatible thread were allocated to two groups, thin and thick SF groups, based on the graft wall thickness. The short-term patency rate and histologic reactions were compared. The patency rate at 2 weeks after replacement in the thin and thick SF groups was 50% and 88%, respectively (p = 0.04). CD31-positive endothelial cells covered the luminal surface of both groups at 4 weeks. The elastic modulus of the thick SF graft was significantly better than that of the thin SF graft (0.0210 and 0.0007 N/m2, p < 0.01). Roundness of thick SF groups (o = 0.8 mm) was better than thin SF (o = 2.0 mm). There was significant difference between the groups (p = 0.01). SF vascular grafts are a promising tissue-engineered scaffold material for abdominal venous system replacement in middle-sized mammals, with thick-walled grafts being superior to thin-walled grafts.

Characterization and promotion of endothelialization of Bombyx mori silk fibroin functionalized with REDV peptide.

In the development of small-diameter vascular grafts, it is crucial to achieve early-stage endothelialization to prevent thrombus formation and intimal hyperplasia. Silk fibroin (SF) from Bombyx mori is commonly used for such grafts. However, there is a need to expedite endothelialization post-implantation. In this study, we functionalized SF with Arg-Glu-Asp-Val (REDV) (SF + REDV) using cyanuric chloride to enhance endothelialization. The immobilization of REDV onto SF was confirmed and the amount of immobilized REDV could be calculated by 1H NMR. Furthermore, the conformational changes in Tyr, Ser, and Ala residues in [3-13C]Tyr- and [3-13C]Ser-SF due to REDV immobilization were monitored using 13C solid-state NMR. The REDV immobilized onto the SF film was found to be exposed on the film's surface, as confirmed by biotin-avidin system. Cell culture experiments, including adhesiveness, proliferation, and extensibility, were conducted using normal human umbilical vein endothelial cells (HUVEC) and normal human aortic smooth muscle cells (HAoSMC) on both SF and SF + REDV films to evaluate the impact of REDV on endothelialization. The results indicated a trend towards promoting HUVEC proliferation while inhibiting HAoSMC proliferation. Therefore, these findings suggest that SF + REDV may be more suitable than SF alone for coating small-diameter SF knitted tubes made of SF threads.

Bombyx mori Silk Fibroin and Model Peptides Incorporating Arg-Gly-Asp Motifs and Their Application in Wound Dressings.

Skin plays an important role in protecting the human body from the environment, dehydration, and infection. Burns, wounds, and disease cause the skin to lose its role, but tissue-engineered skin substitutes offer the opportunity to restore skin loss. Silk fibroin from Bombyx mori (SF) has proven to be an excellent wound dressing material. In this study, we aim to develop an excellent wound dressing material by introducing three-residue sequence Arg-Gly-Asp (RGD), which is the most well-known adhesion site of fibronectin, in the films of SF and the model peptide. Its usefulness as a wound dressing material was evaluated both in vitro and in vivo. First, we showed that the flexible structures of the RGD sequence are still maintained in SF with a rigid antiparallel ß-sheet structure using NMR in association with excellent wound dressings of SF containing RGD. Then, in in vitro experiments, two types of normal cells derived from human skin, normal human neonatal epidermal keratinocytes and normal human neonatal dermal fibroblasts, were used to evaluate the cell adhesion. On the other hand, in in vivo experiments, the study was conducted using a rat model of a whole skin layer defect wound. The results showed that the high-functionalized SF developed here has the potential to play a significant role in the field of wound dressings.

Real-Time Monitoring of the Structural Transition of Bombyx mori Liquid Silk under Pressure by Solid-State NMR.

Silk fibroin is stored in the silk glands of Bombyx mori silkworms as a condensed aqueous solution called liquid silk. It is converted into silk fibers at the silkworm's spinnerets under mechanical forces including shear stress and pressure. However, the detailed mechanism of the structural transition of liquid silk to silk fibers under pressure is not well understood. Magic angle spinning (MAS) in solid-state nuclear magnetic resonance (NMR) can exert pressure on liquid samples in a quantitative manner. In this study, solid-state NMR was used to quantitatively analyze the impact of pressure on the structural transition of liquid silk. A combination of 13C DD-MAS and CP-MAS NMR measurements enabled the conformation and dynamics of the crystalline region of the silk fibroin (both before (Silk Ip) and after (Silk IIp) the structural transition) to be detected in real time with atomic resolution. Spectral analyses proposed that the pressure-induced structural transition from Silk Ip to Silk IIp proceeds by a two-step autocatalytic reaction mechanism. The first reaction step is a nucleation step in which Silk Ip transforms to single lamellar Silk IIp, and the second is a growth step in which the single lamellar Silk IIp acts as a catalyst that reacts with Silk Ip molecules to further form Silk IIp molecules, resulting in stacked lamellar Silk IIp. Furthermore, the rate constant in the second step shows a significant pressure dependence, with an increase in pressure accelerating the formation of large stacked lamellar Silk IIp.

A review on the structure of Bombyx mori silk fibroin fiber studied using solid-state NMR: An antipolar lamella with an 8-residue repeat.

Silk fibroin (SF) fiber from the silkworm Bombyx mori in the Silk II form has been used as an excellent textile fiber for over 5000 years. Recently it has been developed for a range of biomedical applications. Further expansion of these uses builds on the excellent mechanical strength of SF fiber, which derives from its structure. This relationship between strength and SF structure has been studied for over 50 years, but it is still not well understood. In this review, we report the use of solid-state NMR to study stable-isotope labeled SF fiber and stable-isotope labeled peptides including (Ala-Gly)15 and (Ala-Gly-Ser-Gly-Ala-Gly)5 as models of the crystalline fraction. We show that the crystalline fraction is a lamellar structure with a repetitive folding using ß-turns every eighth amino acid, and that the sidechains adopt an antipolar arrangement rather than the more well-known polar structure described by Marsh, Corey and Pauling (that is, the Ala methyls in each layer point in opposite directions in alternate strands). The amino acids Ser, Tyr and Val are the next most common in B. mori SF after Gly and Ala, and occur in the crystalline and semi-crystalline regions, probably defining the edges of the crystalline region. Thus, we now have an understanding of the main features of Silk II but there is still a long way to go.

Recombinant Spider Silk Fiber with High Dimensional Stability in Water and Its NMR Characterization.

Spider dragline silk has unique characteristics of strength and extensibility, including supercontraction. When we use it as a biomaterial or material for textiles, it is important to suppress the effect of water on the fiber by as much as possible in order to maintain dimensional stability. In order to produce spider silk with a highly hydrophobic character, based on the sequence of ADF-3 silk, we produced recombinant silk (RSSP(VLI)) where all QQ sequences were replaced by VL, while single Q was replaced by I. The artificial RSSP(VLI) fiber was prepared using formic acid as the spinning solvent and methanol as the coagulant solvent. The dimensional stability and water absorption experiments of the fiber were performed for eight kinds of silk fiber. RSSP(VLI) fiber showed high dimensional stability, which is suitable for textiles. A remarkable decrease in the motion of the fiber in water was made evident by 13C solid-state NMR. This study using 13C solid-state NMR is the first trial to put spider silk to practical use and provide information regarding the molecular design of new recombinant spider silk materials with high dimensional stability in water, allowing recombinant spider silk proteins to be used in next-generation biomaterials and materials for textiles.

Stretching-Induced Conformational Transition of [3-13C]Ser- and [3-13C]Tyr-Antheraea yamamai Silk Fibroin before Spinning Investigated with 13C Solid-State NMR Spectroscopy.

The conformational transition of [3-13C]Ser- and [3-13C]Tyr-Antheraea yamamai silk fibroin before spinning induced by stretching was investigated with 13C CP/MAS NMR spectroscopy. The α-helix content of the silk fibroin before stretching was found to be 31.6% based on the Ala and Ser peaks. With increasing stretching ratio, the α-helix and the random coil Ala Cß peaks decreased gradually, while the ß-sheet peak was observed at a stretching ratio of ×5 and increased rapidly upon further stretching. For Ser residue, the α-helix peak decreased monotonically with increasing stretching ratio, but the random coil peak increased slightly till the stretching ratio of ×5 and then decreased. A small ß-sheet peak was observed before stretching and then increased rapidly starting from the stretching ratio of ×7. In contrast, a gradual decrease of random coil peak and an increase of ß-sheet peak were observed for the Tyr residue. The results of this investigation may be helpful for further studies of fiber formation mechanism in A. yamamai and in the future design of artificial silk materials.

Structure of silk I (Bombyx mori silk fibroin before spinning) in the dry and hydrated states studied using 13C solid-state NMR spectroscopy.

Nowadays, much attention has been paid to Bombyx mori silk fibroin (SF) by many researchers because of excellent physical properties and biocompatibility. These superior properties originate from the structure of SF and therefore, the structural analysis is a key to clarify the superiority. Here we concentrated on silk I structure (SF structure before spinning). We showed that silk I* (the structure of (GAGAGS)n which is a main part of SF) is a repeated type II ß-turn, neither α-helix nor random coil, from the conformation-dependent 13C NMR chemical shift data. This conclusion is different from that obtained using IR by many researchers. Next, the formation of silk I* structure was investigated at molecular level using 13C solid-state NMR spectroscopy. Three kinds of 13C INEPT, CP/MAS and DD/MAS NMR spectra were observed for SF, [3-13C]Ser- and [3-13C]Tyr-SF, the crystalline fraction obtained by chymotrypsin treatment of SF and their model peptide with silk I structures in the dry and hydrated states. Especially, the presence of the sequences containing Tyr, (((GX)m1GY)m2 where X = A or V) with random coil conformations adjacent to (GAGAGS)n is an essence to get water-soluble SF and the formation of silk I* structure of (GAGAGS)n.

Formylation of Recombinant Spider Silk in Formic Acid and Wet Spinning Studied Using Nuclear Magnetic Resonance and Infrared Spectroscopies.

We reported wet spinning of recombinant spider silk protein (RSSP) and formylation of RSSP in formic acid (FA). First, FA was selected as the spinning solvent and the detailed spinning condition was determined. Next, the mechanical property was compared between the RSSP fiber spun after allowing the spinning solution dissolved in FA to stand for 2 days and the fiber spun immediately after being dissolved in FA for 4 h. The tensile strength of the former fiber was lower than the strength of the latter fiber. This difference can be explained by the difference in the degree of formylation as follows. FA is a known formylating agent, although most researchers who prepared silk fiber by wet spinning with FA have not pointed out about formylation. The formylation of the Ser OH group was confirmed by 13C solution nuclear magnetic resonance (NMR), and the time course of formylation of the RSSP film prepared from the FA solution was tracked by Fourier transform infrared spectroscopy. The 13C solid-state NMR spectra were also compared between two kinds of the formylated RSSP fibers and indicated that the packing state was tighter for the latter fiber than the former one, which could explain higher tensile strength of the latter fiber in the dry state. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the RSSP sample decomposed gradually with storage time in FA and the decomposition has begun partly even at 2 h after dissolution in FA. The decomposition by formylation seems to have no significant effect on the backbone structure of the RSSP fiber, although the packing of the fiber becomes loose as a whole. Finally, preliminary trial of deformylation of the formylated RSSP fiber was performed.

Presence of ß-Turn Structure in Recombinant Spider Silk Dissolved in Formic Acid Revealed with NMR.

Spider dragline silk is a biopolymer with excellent mechanical properties. The development of recombinant spider silk protein (RSP)-based materials with these properties is desirable. Formic acid (FA) is a spinning solvent for regenerated Bombyx mori silk fiber with excellent mechanical properties. To use FA as a spinning solvent for RSP with the sequence of major ampullate spider silk protein from Araneus diadematus, we determined the conformation of RSP in FA using solution NMR to determine the role of FA as a spinning solvent. We assigned 1H, 13C, and 15N chemical shifts to 32-residue repetitive sequences, including polyAla and Gly-rich regions of RSP. Chemical shift evaluation revealed that RSP is in mainly random coil conformation with partially type II ß-turn structure in the Gly-Pro-Gly-X motifs of the Gly-rich region in FA, which was confirmed by the 15N NOE data. In addition, formylation at the Ser OH groups occurred in FA. Furthermore, we evaluated the conformation of the as-cast film of RSP dissolved in FA using solid-state NMR and found that ß-sheet structure was predominantly formed.

Characterization of a Water-Dispersed Biodegradable Polyurethane-Silk Composite Sponge Using 13C Solid-State Nuclear Magnetic Resonance as Coating Material for Silk Vascular Grafts with Small Diameters.

Recently, Bombyx mori silk fibroin (SF) has been shown to be a suitable material for vascular prostheses for small arteries. In this study, we developed a softer SF graft by coating water-dispersed biodegradable polyurethane (PU) based on polycaprolactone and an SF composite sponge on the knitted SF vascular graft. Three kinds of 13C solid-state nuclear magnetic resonance (NMR), namely carbon-13 (13C) cross-polarization/magic angle spinning (MAS), 13C dipolar decoupled MAS, and 13C refocused insensitive nuclei enhanced by polarization transfer (r-INEPT) NMR, were used to characterize the PU-SF coating sponge. Especially the 13C r-INEPT NMR spectrum of water-dispersed biodegradable PU showed that both main components of the non-crystalline domain of PU and amorphous domain of SF were highly mobile in the hydrated state. Then, the small-diameter SF artificial vascular grafts coated with this sponge were evaluated through implantation experiments with rats. The implanted PU-SF-coated SF grafts showed a high patency rate. It was confirmed that the inside of the SF grafts was covered with vascular endothelial cells 4 weeks after implantation. These results showed that the water-dispersed biodegradable PU-SF-coated SF graft created in this study could be a strong candidate for small-diameter artificial vascular graft.

Structure of Silk I (Bombyx mori Silk Fibroin before Spinning) -Type II ß-Turn, Not α-Helix.

Recently, considerable attention has been paid to Bombyx mori silk fibroin by a range of scientists from polymer chemists to biomaterial researchers because it has excellent physical properties, such as strength, toughness, and biocompatibility. These appealing physical properties originate from the silk fibroin structure, and therefore, structural determinations of silk fibroin before (silk I) and after (silk II) spinning are a key to make wider applications of silk. There are discrepancies about the silk I structural model, i.e., one is type II ß-turn structure determined using many solid-state and solution NMR spectroscopies together with selectively stable isotope-labeled model peptides, but another is α-helix or partially α-helix structure speculated using IR and Raman methods. In this review, firstly, the process that led to type II ß-turn structure by the authors was introduced in detail. Then the problems in speculating silk I structure by IR and Raman methods were pointed out together with the problem in the assignment of the amide I band in the spectra. It has been emphasized that the conformational analyses of proteins and peptides from IR and Raman studies are not straightforward and should be very careful when the proteins contain ß-turn structure using many experimental data by Vass et al. In conclusion, the author emphasized here that silk I structure should be type II ß-turn, not α-helix.

Bio-functionalized titanium surfaces with modified silk fibroin carrying titanium binding motif to enhance the ossific differentiation of MC3T3-E1.

Silk fibroin (SF) from Bombyx mori has superior properties as both a textile and a biomaterial, and has been used to functionalize the surfaces of various medical inorganic materials including titanium (Ti). In this study, we endowed SF with reversible binding ability to Ti by embedding a titanium binding motif (minTBP-1 and RKLPDA). Artificial SF proteins were first created by conjugating gene cassettes for SF motif (AGSGAG) and minTBP-1 motif with different ratios, which have been shown to bind reversibly to Ti surfaces in quartz crystal microbalance analyses. Based on these results, the functionalized SF (TiBP-SF) containing the designed peptide [TS[(AGSGAG)3 AS]2 RKLPDAS]8 was prepared from the cocoon of transgenic B. mori, which accelerates the ossific differentiation of MC3T3-E1 cells when coated on titanium substrates. Thus, TiBP-SF presents an alternative for endowing the surfaces of titanium materials with osseointegration functionality, which would allow the exploration of potential applications in the medical field.

Evaluation of small-diameter silk vascular grafts implanted in dogs.

Objectives: Vascular replacement is one treatment for cardiovascular disease. However, in blood vessels with a diameter less than 6 mm, the existing artificial vascular grafts may be occluded by thrombus formation or intimal hyperplasia. Thus, new artificial vascular grafts need to be developed. We have developed a small-diameter artificial vascular graft made of silk fibroin. The implantation of such graft has been evaluated mainly in rats. However, only a few reports describe long-term implantation in large animal models. Therefore, modified silk fibroin artificial vascular grafts were implanted in the femoral arteries of dogs, and their patency and remodeling ability were investigated. Methods: Six beagles weighing 10 to 12 kg were used for the in vivo study. Grafts (4 cm length × 3.5 mm inner diameter) were implanted in the femoral artery of 6 dogs. The patency of the graft was monitored using vascular ultrasound apparatus. At 3 months', 5 months', and 1-year postimplantation, the graft was retrieved and conducted histopathologic examination. Results: No side effects, such as ischemia, paralysis, and edema of the hind legs, were observed postimplantation. Five of the 6 grafts exhibited a high patency rate, and the lumen was covered with vascular endothelial cells in the central part of the graft 3 months' postimplantation. Conclusions: Based on these results, artificial silk fibroin vascular grafts implanted in the femoral arteries of dogs exhibit high patency and remodeling ability. Silk fibroin grafts may be clinically applicable as an artificial vascular graft in small-diameter <6 mm.

Silk fibroin vascular graft: a promising tissue-engineered scaffold material for abdominal venous system replacement.

No alternative tissue-engineered vascular grafts for the abdominal venous system are reported. The present study focused on the development of new tissue-engineered vascular graft using a silk-based scaffold material for abdominal venous system replacement. A rat vein, the inferior vena cava, was replaced by a silk fibroin (SF, a biocompatible natural insoluble protein present in silk thread), tissue-engineered vascular graft (10 mm long, 3 mm diameter, n = 19, SF group). The 1 and 4 -week patency rates and histologic reactions were compared with those of expanded polytetrafluoroethylene vascular grafts (n = 10, ePTFE group). The patency rate at 1 and 4 weeks after replacement in the SF group was 100.0% and 94.7%, and that in the ePTFE group was 100.0% and 80.0%, respectively. There was no significant difference between groups (p = 0.36). Unlike the ePTFE graft, CD31-positive endothelial cells covered the whole luminal surface of the SF vascular graft at 4 weeks, indicating better endothelialization. SF vascular grafts may be a promising tissue-engineered scaffold material for abdominal venous system replacement.

Chain-folded lamellar structure and dynamics of the crystalline fraction of Bombyx mori silk fibroin and of (Ala-Gly-Ser-Gly-Ala-Gly)n model peptides.

Solid-state NMR is a powerful analytical technique to determine the composite structure of Bombyx mori silk fibroin (SF). In our previous paper, we proposed a lamellar structure for Ala-Gly copolypeptides as a model of the crystalline fraction in Silk II. In this paper, the structure and dynamics of the crystalline fraction and of a better mimic of the crystalline fraction, (Ala-Gly-Ser-Gly-Ala-Gly)n (n = 2-5, 8), and 13C selectively labeled [3-13C]Ala-(AGSGAG)5 in Silk II forms, were studied using structural and dynamical analyses of the Ala Cß peaks in 13C cross polarization/ magic angle spinning NMR and 13C solid-state spin-lattice relaxation time (T1) measurements, respectively. Like Ala-Gly copolypeptides, these materials have lamellar structures with two kinds of Ala residues in ß-sheet, A and B, plus one distorted ß-turn, t, formed by repetitive folding using ß-turns every eighth amino acid in an antipolar arrangement. However, because of the presence of Ser residues at every sixth residue in (AGSGAG)n, the T1 values and mobilities of B decreased significantly. We conclude that the Ser hydroxyls hydrogen bond to adjacent lamellar layers and fix them together in a similar way to Velcro®.

Lamellar Structure in Alanine-Glycine Copolypeptides Studied by Solid-State NMR Spectroscopy: A Model for the Crystalline Domain of Bombyx mori Silk Fibroin in Silk II Form.

Bombyx mori silk fibroin (SF) fibers with excellent mechanical properties have attracted widespread attention as new biomaterials. However, the structural details are still not conclusive. Here, we propose a lamellar structure for the crystalline domain of the SF fiber based on structural analyses of the Ala Cß peaks in the 13C cross-polarization/magic angle spinning NMR spectra of (Ala-Gly)m (m = 9, 12, 15, and 25) and 13C selectively labeled (Ala-Gly)15 model peptides. Namely, three Ala Cß peaks with relative intensities of 1:2:1 obtained by deconvolution were assigned to two kinds of ß-sheet and a ß-turn, which are interpreted as a lamellar structure formed by repetitive folding using ß-turns every eighth amino acid, for which the basic structure is (Ala-Gly)4 in an antipolar arrangement. The dynamics and intermolecular arrangement were further studied using 13C solid-state spin-lattice relaxation time observations and the rotational echo double resonance experiments, respectively.

Structure and Dynamics of Spider Silk Studied with Solid-State Nuclear Magnetic Resonance and Molecular Dynamics Simulation.

This review will introduce very recent studies using solid-state nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulation on the structure and dynamics of spider dragline silks conducted by the author's research group. Spider dragline silks possess extraordinary mechanical properties by combining high tensile strength with outstanding elongation before breaking, and therefore continue to attract attention of researchers in biology, biochemistry, biophysics, analytical chemistry, polymer technology, textile technology, and tissue engineering. However, the inherently non-crystalline structure means that X-ray diffraction and electron diffraction methods provide only limited information because it is difficult to study the molecular structure of the amorphous region. The most detailed picture of the structure and dynamics of the silks in the solid state experimentally have come from solid-state NMR measurements coupled with stable isotope labeling of the silks and the related silk peptides. In addition, combination of solid-state NMR and MD simulation was very powerful analytical tools to understand the local conformation and dynamics of the spider dragline silk in atomic resolution. In this review, the author will emphasize how solid-state NMR and MD simulation have contributed to a better understanding of the structure and dynamics in the spider dragline silks.

Biodegradable Extremely-Small-Diameter Vascular Graft Made of Silk Fibroin can be Implanted in Mice.

AIM: Synthetic vascular grafts are widely used in surgical revascularization, mainly for medium- to large-sized vessels. However, synthetic grafts smaller than 6 mm in diameter are associated with a high incidence of thrombosis. In this study, we evaluated silk fibroin, a major protein of silk, with high biocompatibility and biodegradability, as a useful material for extremely-small-diameter vascular grafts. METHODS: A small-sized (0.9 mm inner diameter) graft was braided from a silk fibroin thread. The right carotid arteries of 8- to 14-week-old male C57BL/6 mice were cut at the midpoint, and fibroin grafts (5- to 7-mm in length) were transplanted using a cuff technique with polyimide cuffs. The grafts were harvested at different time points and analyzed histologically. RESULTS: CD31＋ endothelial cells had already started to proliferate at 2 weeks after implantation. At 4 weeks, neointima had formed with α-smooth muscle actin+ cells, and the luminal surface was covered with CD31＋endothelial cells. Mac3＋ macrophages were accumulated in the grafts. Graft patency was confirmed at up to 6 months after implantation. CONCLUSION: This mouse model of arterial graft implantation enables us to analyze the remodeling process and biocompatibility of extremely-small-diameter vascular grafts. Biodegradable silk fibroin might be applicable for further researches using genetically modified mice.