Axonal Guidance Using Biofunctionalized Straining Flow Spinning Regenerated Silk Fibroin Fibers as Scaffold.

After an injury, the limited regenerative capacity of the central nervous system makes the reconnection and functional recovery of the affected nervous tissue almost impossible. To address this problem, biomaterials appear as a promising option for the design of scaffolds that promote and guide this regenerative process. Based on previous seminal works on the ability of regenerated silk fibroin fibers spun through the straining flow spinning (SFS) technique, this study is intended to show that the usage of functionalized SFS fibers allows an enhancement of the guidance ability of the material when compared with the control (nonfunctionalized) fibers. It is shown that the axons of the neurons not only tend to follow the path marked by the fibers, in contrast to the isotropic growth observed on conventional culture plates, but also that this guidance can be further modulated through the biofunctionalization of the material with adhesion peptides. Establishing the guidance ability of these fibers opens the possibility of their use as implants for spinal cord injuries, so that they may represent the core of a therapy that would allow the reconnection of the injured ends of the spinal cord.

The Spider Silk Standardization Initiative (S3I): A powerful tool to harness biological variability and to systematize the characterization of major ampullate silk fibers spun by spiders from suburban Sydney, Australia.

The true stress-true strain curves of 11 Australian spider species from the Entelegynae lineage were tensile tested and classified based on the values of the alignment parameter, α*, in the framework of the Spider Silk Standardization Initiative (S3I). The application of the S3I methodology allowed the determination of the alignment parameter in all cases, and were found to range between α* = 0.03 and α* = 0.65. These data, in combination with previous results on other species included in the Initiative, were exploited to illustrate the potential of this approach by testing two simple hypotheses on the distribution of the alignment parameter throughout the lineage: (1) whether a uniform distribution may be compatible with the values obtained from the studied species, and (2) whether any trend may be established between the distribution of the α* parameter and phylogeny. In this regard, the lowest values of the α* parameter are found in some representatives of the Araneidae group, and larger values seem to be found as the evolutionary distance from this group increases. However, a significant number of outliers to this apparent general trend in terms of the values of the α* parameter are described.

Strategies for the Biofunctionalization of Straining Flow Spinning Regenerated Bombyx mori Fibers.

High-performance regenerated silkworm (Bombyx mori) silk fibers can be produced efficiently through the straining flow spinning (SFS) technique. In addition to an enhanced biocompatibility that results from the removal of contaminants during the processing of the material, regenerated silk fibers may be functionalized conveniently by using a range of different strategies. In this work, the possibility of implementing various functionalization techniques is explored, including the production of fluorescent fibers that may be tracked when implanted, the combination of the fibers with enzymes to yield fibers with catalytic properties, and the functionalization of the fibers with cell-adhesion motifs to modulate the adherence of different cell lineages to the material. When considered globally, all these techniques are a strong indication not only of the high versatility offered by the functionalization of regenerated fibers in terms of the different chemistries that can be employed, but also on the wide range of applications that can be covered with these functionalized fibers.

Unexpected high toughness of Samia cynthia ricini silk gut.

Silk gut fibers were produced from the silkworm Samia cynthia ricini silk glands by the usual procedure of immersion in a mildly acidic solution and subsequent stretching. The morphology of the silk guts was assessed by scanning electron microscopy, and their microstructure was assessed by infrared spectroscopy and X-ray diffraction. It was found that both naturally spun and Samia silk guts share a common semicrystalline microstructure. The mechanical characterization of the silk guts revealed that these fibers show an elastomeric behavior when tested in water, and exhibit a genuine ground state to which the fiber may revert independently of its previous loading history. In spite of its large cross-sectional area compared with naturally spun silk fibers, Samia silk guts show values of work to fracture up to 160 MJ m-3, much larger than those of most of their natural counterparts, and establish a new record value for this parameter in silk guts.

Conversion of the OmpF Porin into a Device to Gather Amyloids on the E. coli Outer Membrane.

Protein amyloids are ubiquitous in natural environments. They typically originate from microbial secretions or spillages from mammals infected by prions, currently raising concerns about their infectivity and toxicity in contexts such as gut microbiota or soils. Exploiting the self-assembly potential of amyloids for their scavenging, here, we report the insertion of an amyloidogenic sequence stretch from a bacterial prion-like protein (RepA-WH1) in one of the extracellular loops (L5) of the abundant Escherichia coli outer membrane porin OmpF. The expression of this grafted porin enables bacterial cells to trap on their envelopes the same amyloidogenic sequence when provided as an extracellular free peptide. Conversely, when immobilized on a surface as bait, the full-length prion-like protein including the amyloidogenic peptide can catch bacteria displaying the L5-grafted OmpF. Polyphenolic molecules known to inhibit amyloid assembly interfere with peptide recognition by the engineered OmpF, indicating that this is compatible with the kind of homotypic interactions expected for amyloid assembly. Our study suggests that synthetic porins may provide suitable scaffolds for engineering biosensor and clearance devices to tackle the threat posed by pathogenic amyloids.

Regenerated Silk Fibers Obtained by Straining Flow Spinning for Guiding Axonal Elongation in Primary Cortical Neurons.

The recovery of injured nervous tissue, one of the main goals for regenerative therapeutic approaches, is often hindered by the limited axonal regeneration ability of the central nervous system (CNS). In this regard, the identification of scaffolds that support the reconstruction of functional neuronal tissues and guide the alignment of regenerating neurons is a major challenge in tissue engineering. Ideally, the usage of such scaffolds would promote and guide the axonal growth, a crucial phase for the restoration of neuronal connections and, consequently, the nerve function. Among the materials proposed as scaffolds for CNS regeneration, silk has been used to exploit its outstanding features as a biomaterial to promote axonal regeneration. In this study, we explore, for the first time, the possibility of using high-performance regenerated silk fibers obtained by straining flow spinning (SFS) to serve as scaffolds for inducing and guiding the axonal growth. It is shown that SFS fibers promote the spontaneous organization of dissociated cortical primary cells into highly interconnected cellular spheroid-like tissue formations. Neuronal projections (i.e., axons) from these cellular spheroids span hundreds of microns along the SFS fibers that act as guides and allow the connection of distant spheroids. In addition, it is also shown that SFS fibers serve as scaffolds for neuronal migration covering short and long distances. As a consequence, the usage of high-performance SFS fibers appears as a promising basis for the development of novel therapies, leading to directed axonal regeneration.

First steps for the development of silk fibroin-based 3D biohybrid retina for age-related macular degeneration (AMD).

Age-related macular degeneration is an incurable chronic neurodegenerative disease, causing progressive loss of the central vision and even blindness. Up-to-date therapeutic approaches can only slow down he progression of the disease. OBJECTIVE: Feasibility study for a multilayered, silk fibroin-based, 3D biohybrid retina. APPROACH: Fabrication of silk fibroin-based biofilms; culture of different types of cells: retinal pigment epithelium, retinal neurons, Müller and mesenchymal stem cells ; creation of a layered structure glued with silk fibroin hydrogel. MAIN RESULTS: In vitro evidence for the feasibility of layered 3D biohybrid retinas; primary culture neurons grow and develop neurites on silk fibroin biofilms, either alone or in presence of other cells cultivated on the same biomaterial; cell organization and cellular phenotypes are maintained in vitro for the seven days of the experiment. SIGNIFICANCE: 3D biohybrid retina can be built using silk silkworm fibroin films and hydrogels to be used in cell replacement therapy for AMD and similar retinal neurodegenerative diseases.

Structure-Function Relationship of Artificial Spider Silk Fibers Produced by Straining Flow Spinning.

The production of large quantities of artificial spider silk fibers that match the mechanical properties of the native material has turned out to be challenging. Recent advancements in the field make biomimetic spinning approaches an attractive way forward since they allow the spider silk proteins to assemble into the secondary, tertiary, and quaternary structures that are characteristic of the native silk fiber. Straining flow spinning (SFS) is a newly developed and versatile method that allows production under a wide range of processing conditions. Here, we use a recombinant spider silk protein that shows unprecedented water solubility and that is capable of native-like assembly, and we spin it into fibers by the SFS technique. We show that fibers may be spun using different hydrodynamical and chemical conditions and conclude that these spinning conditions affect fiber mechanics. In particular, it was found that the addition of acetonitrile and polyethylene glycol to the collection bath results in fibers with increased ß-sheet content and improved mechanical properties.

Conduits based on the combination of hyaluronic acid and silk fibroin: Characterization, in vitro studies and in vivo biocompatibility.

We address the production of structures intended as conduits made from natural biopolymers, capable of promoting the regeneration of axonal tracts. We combine hyaluronic acid (HA) and silk fibroin (SF) with the aim of improving mechanical and biological properties of HA. The results show that SF can be efficiently incorporated into the production process, obtaining conduits with tubular structure with a matrix of HA-SF blend. HA-SF has better mechanical properties than sole HA, which is a very soft hydrogel, facilitating manipulation. Culture of rat Schwann cells shows that cell adhesion and proliferation are higher than in pure HA, maybe due to the binding motifs contributed by the SF protein. This increased proliferation accelerates the formation of a tight cell layer, which covers the inner channel surface of the HA-SF tubes. Biocompatibility of the scaffolds was studied in immunocompetent mice. Both HA and HA-SF scaffolds were accepted by the host with no residual immune response at 8 weeks. New collagen extracellular matrix and new blood vessels were visible and they were present earlier when SF was present. The results show that incorporation of SF enhances the mechanical properties of the materials and results in promising biocompatible conduits for tubulization strategies.