Effect of Recombinant Spidroins Self-Assembly on Rheological Behavior of Their Dispersions and Structure of Electrospun Nanofibrous Materials.

The effect of primary amino acid sequence in recombinant spidroins on their spatial organization is crucial for the fabrication of artificial fibers and fibrous materials. This study focuses on the rheological properties of aqueous and alcoholic solutions of recombinant analogs of natural spidroins (rS1/9 and rS2/12), as well as the structure of their films and nanofibrous materials. Non-Newtonian flow behavior of aqueous solutions of these proteins was observed at certain concentrations in contrast to their solutions in hexafluoroisopropanol. The secondary structure of recombinant spidroins was addressed by IR spectroscopy, whereas their self-organization in various solvents was studied by AFM and cryo-TEM. The influence of the solvent on the structure and properties of the films and nanofibrous materials produced by electrospinning has been established.

Composite Coatings Based on Recombinant Spidroins and Peptides with Motifs of the Extracellular Matrix Proteins Enhance Neuronal Differentiation of Neural Precursor Cells Derived from Human Induced Pluripotent Stem Cells.

The production and transplantation of functionally active human neurons is a promising approach to cell therapy. Biocompatible and biodegradable matrices that effectively promote the growth and directed differentiation of neural precursor cells (NPCs) into the desired neuronal types are very important. The aim of this study was to evaluate the suitability of novel composite coatings (CCs) containing recombinant spidroins (RSs) rS1/9 and rS2/12 in combination with recombinant fused proteins (FP) carrying bioactive motifs (BAP) of the extracellular matrix (ECM) proteins for the growth of NPCs derived from human induced pluripotent stem cells (iPSC) and their differentiation into neurons. NPCs were produced by the directed differentiation of human iPSCs. The growth and differentiation of NPCs cultured on different CC variants were compared with a Matrigel (MG) coating using qPCR analysis, immunocytochemical staining, and ELISA. An investigation revealed that the use of CCs consisting of a mixture of two RSs and FPs with different peptide motifs of ECMs increased the efficiency of obtaining neurons differentiated from iPSCs compared to Matrigel. CC consisting of two RSs and FPs with Arg-Gly-Asp-Ser (RGDS) and heparin binding peptide (HBP) is the most effective for the support of NPCs and their neuronal differentiation.

Nonwoven spidroin materials as scaffolds for ex vivo cultivation of aortic fragments and dorsal root ganglia.

Recombinant spidroins (RS; the analogues of silk proteins of spider's web) have multiple properties beneficial for bioengineering, including their suitability for electrospinning and thus, for production of materials with oriented fibers. This makes RS-based matrices potentially effective in stimulating regeneration of peripheral nerves. The restoration of injured nerves also depends on prompt regrowth of blood vessels. Therefore, prospective scaffold materials for neuro-regenerative therapy should positively affect both the nerves and the blood vessels. Currently, the experimental models suitable for culturing and quantitative assessment of the vascular and neuronal cells on the same material are lacking. Here, we assessed the suitability of electrospun RS-based matrices for cultivation of the mouse aorta and dorsal root ganglia (DRG) explants. We also quantified the effects of matrix topography upon both types of tissues. The RS-based materials have effectively supported aortic explants survival and sprouting. The cumulative length of endothelial sprouts on rS1/9-coated inserts was significantly higher as compared to type I collagen coatings, suggesting stimulatory effects on angiogenesis in vitro. In contrast to matrices with random fibers, on matrices with parallel fibers the migration of both smooth muscle and endothelial cells was highly oriented. Furthermore, alignment of RS fibers effectively directs the growth of axons and the migration of Schwann cells from DRGs. Thus, the electrospun RS matrices are highly suitable to culture both, the DRGs and aortic explants and to study the effects of matrix topography on cell migration. This model has a high potential for further endeavor into interactions of nerve and vascular cells and tissues.

Spidroin Silk Fibers with Bioactive Motifs of Extracellular Proteins for Neural Tissue Engineering.

The interaction of neural progenitor cells (NPCs) with the extracellular matrix (ECM) plays an important role in neural tissue regeneration. Understanding which motifs of the ECM proteins are crucial for normal NPC adhesion, proliferation, and differentiation is important in order to create more adequate tissue engineered models of neural tissue and to efficiently study the central nervous system regeneration mechanisms. We have shown earlier that anisotropic matrices prepared from a mixture of recombinant dragline silk proteins, such as spidroin 1 and spidroin 2, by electrospinning are biocompatible with NPCs and provide good proliferation and oriented growth of neurites. This study objective was to find the effects of spidroin-based electrospun materials, modified with peptide motifs of the extracellular matrix proteins (RGD, IKVAV, and VAEIDGIEL) on adhesion, proliferation, and differentiation of directly reprogrammed neural precursor cells (drNPCs). The structural and biomechanical studies have shown that spidroin-based electrospun mats (SBEM), modified with ECM peptides, are characterized by a uniaxial orientation and elastic moduli in the swollen state, comparable to those of the dura mater. It has been found for the first time that drNPCs on SBEM mostly preserve their stemness in the growth medium and even in the differentiation medium with brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, while addition of the mentioned ECM-peptide motifs may shift the balance toward neuroglial differentiation. We have demonstrated that the RGD motif promotes formation of a lower number of neurons with longer neurites, while the IKVAV motif is characterized by formation of a greater number of NF200-positive neurons with shorter neurites. At the same time, all the studied matrices preserve up to 30% of neuroglial progenitor cells, phenotypically similar to radial glia derived from the subventricular zone. We believe that, by using this approach and modifying spidroin by various ECM-motifs or other substances, one may create an in vitro model for the neuroglial stem cell niche with the potential control of their differentiation.

Effects of Recombinant Spidroin rS1/9 on Brain Neural Progenitors After Photothrombosis-Induced Ischemia.

The existence of niches of stem cells residence in the ventricular-subventricular zone and the subgranular zone in the adult brain is well-known. These zones are the sites of restoration of brain function after injury. Bioengineered scaffolds introduced in the damaged loci were shown to support neurogenesis to the injury area, thus representing a strategy to treat acute neurodegeneration. In this study, we explored the neuroprotective activity of the recombinant analog of Nephila clavipes spidroin 1 rS1/9 after its introduction into the ischemia-damaged brain. We used nestin-green fluorescent protein (GFP) transgenic reporter mouse line, in which neural stem/progenitor cells are easily visualized and quantified by the expression of GFP, to determine the alterations in the dentate gyrus (DG) after focal ischemia in the prefrontal cortex. Changes in the proliferation of neural stem/progenitor cells during the first weeks following photothrombosis-induced brain ischemia and in vitro effects of spidroin rS1/9 in rat primary neuronal cultures were the subject of the study. The introduction of microparticles of the recombinant protein rS1/9 into the area of ischemic damage to the prefrontal cortex leads to a higher proliferation rate and increased survival of progenitor cells in the DG of the hippocampus which functions as a niche of brain stem cells located at a distance from the injury zone. rS1/9 also increased the levels of a mitochondrial probe in DG cells, which may report on either an increased number of mitochondria and/or of the mitochondrial membrane potential in progenitor cells. Apparently, the stimulation of progenitor cells was caused by formed biologically active products stemming from rS1/9 biodegradation which can also have an effect upon the growth of primary cortical neurons, their adhesion, neurite growth, and the formation of a neuronal network. The high biological activity of rS1/9 suggests it as an excellent material for therapeutic usage aimed at enhancing brain plasticity by interacting with stem cell niches. Substances formed from rS1/9 can also be used to enhance primary neuroprotection resulting in reduced cell death in the injury area.

Akt and Src mediate the photocrosslinked fibroin-induced neural differentiation.

Neural transplantation is a promising modality for treatment of neurodegenerative diseases, traumatic brain injury and stroke. Biocompatible scaffolds with optimized properties improve the survival of transplanted neural cells and differentiation of progenitor cells into the desired types of neurons. Silk fibroin is a biocompatible material for tissue engineering. Here, we describe thin-film scaffolds based on photocrosslinked methacrylated silk fibroin (FBMA). These scaffolds exhibit an increased mechanical stiffness and improved water stability. Photocrosslinking of fibroin increased its rigidity from 25 to 480 kPa and the contact angle from 59.7 to 70.8, the properties important for differentiation of neural cells. Differentiation of SH-SY5Y neuroblastoma cells on FBMA increased the length of neurites as well as the levels of neural differentiation markers MAP2 and ßIII-tubulin. Growth of SH-SY5Y cells on the unmodified fibroin and FBMA substrates led to a spontaneous phosphorylation of Src and Akt protein kinases critical for neuronal differentiation; this effect was paralleled by neural cell adhesion molecule elevation. Thus, FBMA is an easily manufactured, cytocompatible material with improved and sustainable properties applicable for neural tissue engineering.

Recombinant Spidroins as the Basis for New Materials.

Spider web proteins are unique materials created by nature that, considering the combination of their properties, do not have analogues among natural or human-created materials. Obtaining significant amounts of these proteins from natural sources is not feasible. Biotechnological manufacturing in heterological systems is complicated by the very high molecular weight of spidroins and their specific amino acid composition. Obtaining recombinant analogues of spidroins in heterological systems, mainly in bacteria and yeast, has become a compromise solution. Because they can self-assemble, these proteins can form various materials, such as fibers, films, 3D-foams, hydrogels, tubes, and microcapsules. The effectiveness of spidroin hydrogels in deep wound healing, as 3D scaffolds for bone tissue regeneration and as oriented fibers for axon growth and nerve tissue regeneration, was demonstrated in animal models. The possibility to use spidroin micro- and nanoparticles for drug delivery was demonstrated, including the use of modified spidroins for virus-free DNA delivery into animal cell nuclei. In the past few years, significant interest has arisen concerning the use of these materials as biocompatible and biodegradable soft optics to construct photonic crystal super lenses and fiber optics and as soft electronics to use in triboelectric nanogenerators. This review summarizes the latest achievements in the field of spidroin production, the creation of materials based on them, the study of these materials as a scaffold for the growth, proliferation, and differentiation of various types of cells, and the prospects for using these materials for medical applications (e.g., tissue engineering, drug delivery, coating medical devices), soft optics, and electronics. Accumulated data suggest the use of recombinant spidroins in medical practice in the near future.

Fabrication of hydrogel scaffolds via photocrosslinking of methacrylated silk fibroin.

Silk fibroin is a promising biomaterial for tissue engineering due to its valuable mechanical and biological properties. However, being a natural product and a protein, it lacks the processability and uniform quality of an advanced synthetic material. Here we propose a way to overcome this contradiction using novel fibroin photocrosslinkable derivative (FBMA). FBMA was synthesized by methacrylation of native fibroin nucleophilic side groups. It was dissolved in either formic acid (FA) or hexafluoroisopropanol (HFIP), and the obtained solutions were photocrosslinked into hydrogel scaffolds of various structural forms including films, micropatterns, pads and macroporous sponges. UV-exposition of dry FBMA films through a photomask created complex microscaled patterns of the polymer. The nature of the solvent affected the properties of resulting hydrogels. When HFIP was used as the solvent, the resulting hydrogels had a storage modulus ∼4 times higher than that of hydrogels fabricated using FA and ∼20 times higher compared to the reference hydrogel obtained from pristine fibroin. Both FBMA-based hydrogels were biocompatible and supported fibroblast adhesion and growth in vitro. Cells cultivated on FBMA scaffolds produced with HFIP exhibited more spread phenotype at 4 and 24 h of cultivation, consistent with increased stiffness of the hydrogel. Hence, FBMA is an attractive material for fabrication of micropatterned scaffolds of centimeter-scale size with minutely tunable physico-chemical properties via convenient and reproducible technological processes, applicable for rapid prototyping.

Novel Biodegradable Polymeric Microparticles Facilitate Scarless Wound Healing by Promoting Re-epithelialization and Inhibiting Fibrosis.

Despite decades of research, the goal of achieving scarless wound healing remains elusive. One of the approaches, treatment with polymeric microcarriers, was shown to promote tissue regeneration in various in vitro models of wound healing. The in vivo effects of such an approach are attributed to transferred cells with polymeric microparticles functioning merely as inert scaffolds. We aimed to establish a bioactive biopolymer carrier that would promote would healing and inhibit scar formation in the murine model of deep skin wounds. Here we characterize two candidate types of microparticles based on fibroin/gelatin or spidroin and show that both types increase re-epithelialization rate and inhibit scar formation during skin wound healing. Interestingly, the effects of these microparticles on inflammatory gene expression and cytokine production by macrophages, fibroblasts, and keratinocytes are distinct. Both types of microparticles, as well as their soluble derivatives, fibroin and spidroin, significantly reduced the expression of profibrotic factors Fgf2 and Ctgf in mouse embryonic fibroblasts. However, only fibroin/gelatin microparticles induced transient inflammatory gene expression and cytokine production leading to an influx of inflammatory Ly6C+ myeloid cells to the injection site. The ability of microparticle carriers of equal proregenerative potential to induce inflammatory response may allow their subsequent adaptation to treatment of wounds with different bioburden and fibrotic content.

Tissue regeneration in vivo within recombinant spidroin 1 scaffolds.

One of the major tasks of tissue engineering is to produce tissue grafts for the replacement or regeneration of damaged tissue, and natural and recombinant silk-based polymer scaffolds are promising candidates for such grafts. Here, we compared two porous scaffolds made from different silk proteins, fibroin of Bombyx mori and a recombinant analog of Nephila clavipes spidroin 1 known as rS1/9, and their biocompatibility and degradation behavior in vitro and in vivo. The vascularization and intergrowth of the connective tissue, which was penetrated with nerve fibers, at 8 weeks after subcutaneous implantation in Balb/c mice was more profound using the rS1/9 scaffolds. Implantation of both scaffolds into bone defects in Wistar rats accelerated repair compared to controls with no implanted scaffold at 4 weeks. Based on the number of macrophages and multinuclear giant cells in the subcutaneous area and the number of osteoclasts in the bone, regeneration was determined to be more effective after the rS1/9 scaffolds were implanted. Microscopic examination of the morphology of the matrices revealed differences in their internal microstructures. In contrast to fibroin-based scaffolds, the walls of the rS1/9 scaffolds were visibly thicker and contained specific micropores. We suggest that the porous inner structure of the rS1/9 scaffolds provided a better micro-environment for the regenerating tissue, which makes the matrices derived from the recombinant rS1/9 protein favorable candidates for future in vivo applications.

Interaction of recombinant analogs of spider silk proteins 1F9 and 2E12 with phospholipid membranes.

Recombinant analogs of spider dragline silk proteins 1F9 and 2E12 are characterized by numerous repeats consisting of hydrophobic poly-Ala blocks and Gly-rich sequences with a substantial number of positively charged amino acid residues which suggest a pronounced ability to interact with negatively charged phospholipid membranes. Actually both proteins displayed substantial binding affinity towards lipid vesicles formed of acidic lipids as measured by fluorescence correlation spectroscopy (FCS) using rhodamine-labeled conjugates of the proteins. Both proteins did not induce liposome leakage, fusion or breakdown, but were able to bring about liposome aggregation. 1F9 was more active in the induction of liposome aggregation compared to 2E12. Interestingly, 2E12 markedly decreased the rate of calcium-induced liposome fusion. Circular dichroism data showed that binding of the proteins to negatively charged phosphatidylserine liposomes provoked transition from the left-handed helix of polyproline II (PPII) type to beta-structures and alpha-helices. The data suggested predominantly surface location of membrane bound proteins without significant perturbation of their hydrophobic core.

A novel model system for design of biomaterials based on recombinant analogs of spider silk proteins.

Spider dragline silk possesses impressive mechanical and biochemical properties. It is synthesized by a couple of major ampullate glands in spiders and comprises of two major structural proteins--spidroins 1 and 2. The relationship between structure and mechanical properties of spider silk is not well understood. Here, we modeled the complete process of the spider silk assembly using two new recombinant analogs of spidroins 1 and 2. The artificial genes sequence of the hydrophobic core regions of spidroin 1 and 2 have been designed using computer analysis of existing databases and mathematical modeling. Both proteins were expressed in Pichia pastoris and purified using a cation exchange chromatography. Despite the absence of hydrophilic N- and C-termini, both purified proteins spontaneously formed the nanofibrils and round micelles of about 1 microm in aqueous solutions. The electron microscopy study has revealed the helical structure of a nanofibril with a repeating motif of 40 nm. Using the electrospinning, the thin films with an antiparallel beta-sheet structure were produced. In summary, we were able to obtain artificial structures with characteristics that are perspective for further biomedical applications, such as producing three-dimensional matrices for tissue engineering and drug delivery.