Tubular silk scaffolds for small diameter vascular grafts

Vascular surgeries such as coronary artery bypass require small diameter vascular grafts with properties that are not available at this time. Approaches using synthetic biomaterials have been not completely successful in producing non-thrombogenic grafts with inner diameters less than 6 mm, and there is a need for new biomaterials and graft designs. We propose silk fibroin as a microvascular graft material and describe tubular silk scaffolds that demonstrate improved properties over existing vascular graft materials. Silk tubes produced using an aqueous gel spinning technique were first assessed in vitro in terms of thrombogenicity (thrombin and fibrinogen adsorption, platelet adhesion) and vascular cell responses (endothelial and smooth muscle cell attachment and proliferation) in comparison with polytetrafluoroethylene (PTFE), a synthetic material most frequently used for vascular grafts. Silk tubes were then implanted into the abdominal aortas of Sprague-Dawley rats. At time points of 2 weeks and 4 weeks post implantation, tissue outcomes were assessed through gross observation (acute thrombosis, patency) and histological staining (H&E, Factor VIII, smooth muscle actin). Over the 4-week time period, we observed graft patency and endothelial cell lining of the lumen surfaces. These results demonstrate the feasibility of using silk fibroin as a vascular graft material and some advantages of silk tubes over the currently used synthetic grafts.     

The in vivo performance of small-caliber nanofibrous polyurethane vascular grafts

We evaluated the association between linear standard Heart Rate Variability (HRV) measures and vascular, renal and cardiac target organ damage (TOD). A retrospective analysis was performed including 200 patients registered in the Regione Campania network (aged 62.4 ± 12, male 64%). HRV analysis was performed by 24-h holter ECG. Renal damage was assessed by estimated glomerular filtration rate (eGFR), vascular damage by carotid intima-media thickness (IMT), and cardiac damage by left ventricular mass index. Significantly lower values of the ratio of low to high frequency power (LF/HF) were found in the patients with moderate or severe eGFR (p-value < 0.001). Similarly, depressed values of indexes of the overall autonomic modulation on heart were found in patients with plaque compared to those with a normal IMT (p-value <0.05). These associations remained significant after adjustment for other factors known to contribute to the development of target organ damage, such as age. Moreover, depressed LF/HF was found also in patients with left ventricular hypertrophy but this association was not significant after adjustment for other factors. Depressed HRV appeared to be associated with vascular and renal TOD, suggesting the involvement of autonomic imbalance in the TOD. However, as the mechanisms by which abnormal autonomic balance may lead to TOD, and, particularly, to renal organ damage are not clearly known, further prospective studies with longitudinal design are needed to determine the association between HRV and the development of TOD.     

Predicting the compliance of small diameter vascular grafts from uniaxial tensile tests

The compliance hypothesis states that the compliance of vascular grafts should match that of the host artery for optimal patency. Although this has not been proven, the literature shows that much effort has gone into measuring compliance. Uniaxial circumferential tensile tests are simpler than compliance tests, but do not give the compliance (a multiaxial property) directly. Therefore, we have used mechanical models to correlate the two. Simple models suffer from inappropriate simplifying assumptions. In a clinically useful range, a Laplace law model and an incremental elasticity model do not predict the compliance from the rigidity as well as does a model derived from finite elasticity. This latter model has helped locate sources of errors. Variations in graft thickness, diameter, and anisotropy may be responsible for scatter in the experimental correlations between compliance and uniaxial rigidity.     

Effect of shearing on formation of silk fibers from regenerated Bombyx mori silk fibroin aqueous solution

In this paper, the spinnable regenerated silk fibroin aqueous solution with high concentration was prepared and the regenerated silk fibers were obtained from the aqueous solution by two different spinning processes at ambient temperature. The orientation of these fibers was characterized by polarizing microscope. Their secondary structure was investigated by Raman spectroscopy and related mechanical properties were also measured. These data showed that shearing is an important step for increasing orientation and silk II (β-sheet) structure, and the mechanical properties of the regenerated silk fibers can also be improved by shearing.     

An ESR study of spin-labeled silk fibroin membranes and spin-labeled glucose oxidase immobilized in silk fibroin membranes

This article describes the characteristics of silk fibroin membranes and glucose oxidase, immobilized in membranes as determined by a variety of physical methods, mainly the spin-label electron spin resonance (ESR) method. The properties of membranes insolubilized by different methods, i. e., immersion in 80% methanol aqueous solution, uniaxially drawing by placing on a stretcher, and hydration by placing in a desiccator of 96% relative humidity (RH) for 17 h, are compared. The results are also analyzed in relation to ESR spectra of spin-labeled immobilized glucose oxidase and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy as a model of the substrate. It is concluded that the heterogeneous structures of the swollen membranes in water differ locally among membranes insolubilized by different methods, but the immobilized state of the enzyme in such membranes is mostly similar. This is correlated to the fact that the thermal or pH stabilities are essentially same among glucose-oxidase-immobilized silk fibroin membranes insolubilized by different methods.     

Flexibility regeneration of silk fibroin in vitro

Although natural silk fibers have excellent strength and flexibility, the regenerated silk materials generally become brittle in the dry state. How to reconstruct the flexibility for silk fibroin has bewildered scientists for many years. In the present study, the flexible regenerated silk fibroin films were achieved by simulating the natural forming and spinning process. Silk fibroin films composed of silk I structure were first prepared by slow drying process. Then, the silk fibroin films were stretched in the wet state, following the structural transition from silk I to silk II. The difference between the flexible film and different brittle regenerated films was investigated to reveal the critical factors in regulating the flexibility of regenerated silk materials. Compared with the methanol-treated silk films, although having similar silk II structure and water content, the flexible silk films contained more bound water rather than free water, implying the great influence of bound water on the flexibility. Then, further studies revealed that the distribution of bound water was also a critical factor in improving silk flexibility in the dry state, which could be regulated by the nanoassembly of silk fibroin. Importantly, the results further elucidate the relation between mechanical properties and silk fibroin structures, pointing to a new mode of generating new types of silk materials with enhanced mechanical properties in the dry state, which would facilitate the fabrication and application of regenerated silk fibroin materials in different fields.     

用桑蚕丝素蛋白制备邻苯二酚酶传感器

从蘑菇组织中提取邻苯二酚粗酶 ,利用丝素蛋白在甲醇作用下 ,其分子结构由可溶性randomcoil向不容性 β -sheet发生转变 ,从而将邻苯二酚粗酶固定在丝素蛋白膜中 ,制得邻苯一酚酶传感器。该传感器在pH6 0的KH2 PO4 -Na2 HPO4 工作介质中具有良好的响应特性 ,工作线性范围为 1 0× 10 - 5- 2 5× 10 - 4mol L ,检测限 5 0× 10 - 6 mol L ,响应时间 2min。酶经丝素蛋白的固定后具有较强的耐热性能 ,并能比较长时间保持酶的活性。该传感器在KH2 PO4 -Na2 HPO4 缓冲溶液的保存下 ,其使用寿命可达 2个月以上     

Structural characteristics and biological performance of silk fibroin nanofiber containing microalgae spirulina extract

Silk fibroin (SF) nanofiber scaffold containing microalgae Spirulina extract were prepared by electrospinning and the performance and functionality of the scaffold were evaluated. The viscosity and conductivity of the dope solution of Spirulina containing SF were examined for electrospinability and we found that the morphological structure of SF nanofiber is affected by the concentration of Spirulina extract added. The platelet adhesion and coagulation time test confirmed that the Spirulina containing SF nanofiber scaffold had excellent ability to prevent blood clotting or antithrombogenicity that is comparable to heparin. Low cytotoxicity and excellent cell adhesion and proliferation were also observed for Sprulina containing SF nanofiber scaffold by methylthiazolyldiphenyl‐tetrazolium bromide assay and confocal fluorescence microscope using fibroblast and human umbilical vein endothelial cells. Based on these results, we believe SF nanofiber scaffold containing Spirulina extract has the potential to be used as tissue engineering scaffold that requires high hemocompatibility. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 307–318, 2014.     

糖化酶在丝素膜上的固定化及性质研究

利用丝素作糖化酶的固定化载体,应用包埋法和共价交联法两种方法,制备了固定化糖化酶丝素膜,研究结果表明,共价交联法制备的酶膜活力较高,且回收率可达50%以上;与溶液酶相比,固定化酶的最适温度提高了10℃,热稳定性与贮存稳定性也有了很大提高。     

Studies on silk fibroin of the silkworm Bombyx mori

A procedure has been developed to obtain native fibroin in a pure state from the reservoir part of the silk gland. The purified protein has a sedimentation coefficient of 10 S as determined on sucrose density gradients and the amino acid composition is similar to that reported for fibroin from the cocoons. The effects of various solvents has been studied; lithium thiocyanate was found to be the solvent of choice. By in vivo labeling of fibroin with [³H]glycine and [¹⁴C]alanine it was demonstrated that fibroin synthesized in the posterior part of the gland and that stored in the reservoir part are identical.     

Effect of chitin/silk fibroin nanofibrous bicomponent structures on interaction with human epidermal keratinocytes

To fabricate a biomimetic nanostructured bicomponent scaffolds, two types of chitin/silk fibroin (SF) nanofibrous scaffolds (blend scaffolds and hybrid scaffolds) were prepared by electrospinning or simultaneous electrospinning of chitin/SF solutions. The chitin/SF bicomponent scaffolds were after-treated with water vapor, and their nanofibrous structures were almost maintained. From the cytocompatibility and cell behavior on the chitin/SF blend or hybrid nanofibrous scaffolds, the hybrid matrix with 25% chitin and 75% SF as well as the chitin/SF blend nanofibers could be a potential candidate for tissue engineering scaffolds.     

Localization of the genes for silk fibroin in silk gland cells of Bombyx mori.

Radioactive iodinated silk fibroin messenger RNA and ribosomal RNA have been used as probes to localize their genes in tissue sections of Bombyx mori by in situ hybridization. From filter hybridization experiments it is inferred that the majority of the grains produced by in situ hybridization with fibroin mRNA represents specific hybridization to fibroin genes. Sections of the posterior silk gland where silk is synthesized have been compared with those of the middle gland which does not synthesize fibroin. Glands have been analyzed from the second through the fifth (last) larval instar during feeding and moulting periods. During later stages when the gland cells increase their DNA content by polyploidization, serial sections were required to follow the distribution of grains through entire nuclei. At all stages, both ribosomal DNA and fibroin genes are distributed randomly throughout the nuclei without a preferred relationship to any nuclear structure.     

CD and small-angle x-ray scattering of silk fibroin in solution

We investigated the structure of silk fibroin dissolved in water and in water-organic solvent mixtures by CD and small-angle x-ray scattering (SAXS). CD spectra indicated a disordered secondary structure in water and a beta-sheet conformation in aqueous organic solvents, such as methanol, dioxane, and trifluoroethanol (in trifluoroethanol a transient form evolving toward beta-sheet conformation was seen just after dissolution). The SAXS technique indicated the presence of fibroin particles of lamellar shape. The molecular weight was 188,000 daltons in water and 302,000 daltons in aqueous methanol.