V. G. Bogush

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.

In vitro and in vivo biocompatibility studies of a recombinant analogue of spidroin 1 scaffolds

The goal of this study was to generate porous scaffolds from the genetically engineered protein, an analogue of Nephila clavipes spidroin 1 (rS1/9) and to assess the properties of new rS1/9 scaffolds essential for bioengineering. The salt leaching technique was used to make the rS1/9 scaffolds of interconnected macroporous structure with spontaneously formed micropores. The tensile strength of scaffolds was 18 ± 5 N/cm(2). Scaffolds were relatively stable in a phosphate buffer but degraded in oxidizing environment after 11 weeks of incubation. Applicability of the recombinant spidroin 1 as a substrate for cell culture was demonstrated by successful 3T3 cells growth on the surface of rS1/9 films (270 ± 20 cells/mm(2) vs. 97 ± 8 cells/mm(2) on the glass surface, p < 0.01). The 3T3 fibroblasts readily proliferated within the rS1/9 scaffold (from initially plated 19 ± 2 cells/mm(3) to 3800 ± 304 cells/mm(3) after 2 weeks). By this time, cells were uniformly distributed between the surface and deeper layers (27% ± 8% and 33% ± 4%, respectively; p > 0.05), whereas the initial distribution was 58% ± 7% and 11% ± 8%, respectively; p < 0.05). The rS1/9 scaffolds implanted subcutaneously into Balb/c mice were well tolerated. Over a 2-month period, the scaffolds promoted an ingrowth of de novo formed vascularized connective tissue elements and nerve fibers. Thus, scaffolds made of the novel recombinant spidroin 1 analogue are potentially applicable in tissue engineering.

3D nanostructural analysis of silk fibroin and recombinant spidroin 1 scaffolds by scanning probe nanotomography

We use serial section scanning probe nanotomography to study the 3D structure, nanoporosity and pore interconnectivity of biocompatible scaffolds made of fibroin and recombinant spidroin 1 (rS1/9). Significant nanoporosity (24%) and pore percolation is detected for rS1/9 scaffolds. It is assumed to contribute to higher in vivo tissue regeneration efficiency of rS1/9 scaffolds.

Novel 3D-microcarriers from recombinant spidroin for regenerative medicine

Microcarriers generated from recombinant spidroin 1F9 are suitable for use as an injection material. The microcarriers were a heterogeneous mixture of microgel particles ranging from 50 to 300 µm in size with the predominance of particles of 50–150 µm. The surface of these microparticles had a complex topography and ensured efficient cultivation of primary and immortalized fibroblasts. Intradermal injections of microgel suspensions into the area of full-thickness skin wounds did not lead to the development of acute inflammation in mice; instead, they accelerated the recovery of skin tissue and stimulated neurogenesis and angiogenesis.

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.

Functional analysis of the engineered cardiac tissue grown on recombinant spidroin fiber meshes.

In the present study, we examined the ability of the recombinant spidroin to serve as a substrate for the cardiac tissue engineering. For this purpose, isolated neonatal rat cardiomyocytes were seeded on the electrospun spidroin fiber matrices and cultured to form the confluent cardiac monolayers. Besides the adhesion assay and immunostaining analysis, we tested the ability of the cultured cardiomyocytes to form a functional cardiac syncytium by studying excitation propagation in the cultured tissue with the aid of optical mapping. It was demonstrated that recombinant spidroin fiber meshes are directly suitable for the adherence and growth of the cardiomyocytes without additional coating with the attachment factors, such as fibronectin.

Biodegradable matrices from regenerated silk of Bombix mori

The structure and biological properties of devices obtained from regenerated fibroin of Bombix mori silk in the form of films, threedimensional matrices, and tubes intended for regenerative medicine were studied. It is shown that regenerated fibroin forms a substrate that maintains adhesion and proliferation of eukary� otic cells by forming a structure that enables homoge� neous distribution of proliferating cells both on the cell surface and in deep matrix layers. The obtained devices are characterized by a high strength and elas� ticity. Matrices implanted into experimental animals undergo diodegradation and neovascularization with time. The properties of devices obtained from regener� ated fibroin allow them to be considered as a base in designing artificial biological analogues of tissue struc� tures. The key problem of modern medicine is the devel�

Characterization of biodegradable cell micro and macro carriers based on recombinant spidroin

Gels, microgels, and matrices were prepared based on a previously developed recombinant spidroin, 1F9, produced by Saccharomyces cerevisiae yeast strain; their physical, chemical, and biological properties were investigated. It was shown that microgels obtained from 2.5% hydrogel are sized in the range of 50–300 μm, with a predominance of particles of 50–150 μm in diameter. The microgel particles were stable in neutral, acidic, and alkaline media; the destruction of 50% (wt.) of the particles occurred in corrosive media (Fenton’s reagent) within three weeks. The microgel surface, which was studied by laser scanning confocal microscopy and scanning electron microscopy, has a complex relief in which there are both nano-(250–400 nm) and microstructures (3–7 μm). It was shown that particles of both microgel and matrices are capable of adhesion and support the proliferation of 3T3 murine fibroblasts in vitro.

Left helix of polyproline II type and genesis of β-structures in spidroins 1 and 2 and their recombinant analogs

The distribution of secondary structure elements along the polypeptide chains of spider silk proteins spidroins 1 and 2 and their recombinant analogs has been studied by statistical methods. It was found that these proteins as monomers contain only traces of β-structure, while the Ala-rich and the Gly-rich regions are predicted as α-helices and as left-handed helices of polyproline II type. Analysis of literature and our CD data shows that the major polypeptide chain conformation of spidroins 1 and 2 and their recombinant analogs in aqueous solutions is the polyproline II helix, with some α-helices and a very small share of β-structures. The transition to the state with extended conformations, which are characteristic of mature silk fibers, requires dehydration of the polypeptide backbone. Thus, the genesis of β-structure in spider web proteins is determined by the conditions of transitions between the main regular backbone conformations.

Recombinant 1F9 spidroin microgels for murine full-thickness wound repairing

The study of the stimulating effect of the microgels (MGs) based on recombinant 1F9 spidroin on the regeneration of the deep skin wound in mice was carried out. The use of spidroin MGs was shown to increase significantly the quality of healing compared to the control. The introduction of the MG in the wound edges led to recovery of all the structural elements of the skin: the epidermis, the dermis, including vascular and nervous network, in the periphery of the wound underlying muscles, and skin appendages (sebaceous and sweat glands and hair follicles) was revealed.