The use of efficient and inexpensive substrates (2D matrices) for cultivation and differentiation of nerve cells in vitro is important for the creation of tissue engineering constructs intended for the treatment of nervous system pathologies. Recombinant analogues of the orb-weaver spider dragline-silk proteins spidroins 1 and 2 appear promising in addressing this task. The aim of the study was to evaluate the effect of cell substrates derived from mixtures of recombinant spidroins (RS) rS1/9 and rS2/12 with hybrid proteins (HP) containing rS1/9 monomer fused with biologically active peptides on gene expression levels of key synapse-specific proteins and viability of the human neuroblastoma SH-SY5Y cell line during directed cholinergic differentiation. A two-stage scheme of directed cholinergic differentiation of SH-SY5Y cells using retinoic acid and brain-derived neurotrophic factor (BDNF) was implemented. Cell viability was assessed via MTT assay and crystal violet staining. The mRNA levels of the studied genes were assessed by real-time PCR. Directed differentiation of the SH-SY5Y cells was marked by a significant increase in the gene expression levels of synaptophysin, synapsins I and II, and the postsynaptic protein PSD-95. The highest cell viability and increased PSD-95 expression levels were observed during differentiation on a matrix consisting of RS rS1/9 and rS2/12 mixed with the RGDS peptide (present in extracellular matrix proteins) and heparin-binding peptide (HBP, laminin fragment) containing HPs. The highest efficiency during the differentiation of the SH-SY5Y cells was demonstrated by a matrix consisting of the mixture of RS rS1/9 and rS2/12 and a HP made up by RS rS1/9 monomer fused with RGDS (the ligand of integrins) and HBP (the ligand of growth factors and syndecans). Matrices consisting of RS rS2/12 alone or the mixture of rS2/12 with HP(RGDS) showed lower efficiency, although the use of the GRGGL peptide (which interacts with the neural cell adhesion molecules and is a component of RS rS1/9) led to an increase in efficiency.

The effect of recombinant spidroin (RS) hydrogel (HG) on anterior epithelial cells and keratocytes of the human cornea was studied in vitro. Corneal injuries are highly prevalent in developing countries according to the World Health Organization. Various technologies have recently been proposed to restore the damaged surface of the cornea. Use of biodegradable silk-based materials, including recombinant analogs of the spider silk protein spidroin, is an important avenue of research in the field of wound healing and corneal regeneration. Spidroins are well known for their optimal balance of strength and elasticity. Given their biological compatibility, lack of immunogenicity, and biodegradability, spidroins provide a biomaterial for tissue engineering and regenerative medicine. HGs based on RS rS2/12-RGDS were therefore tested for cytotoxicity toward isolated corneal epithelial cells and keratocytes with regard to possible changes in cell phenotype and migratory activity. A promising outlook and therapeutic potential were demonstrated for RS-based HGs.

To follow the structural reorganization of spidroin during its spinning, the rheological behavior of low-concentration solution of recombinant spidroins rS1/9 and rS2/12 is studied on different stages of separation, purification, and lyophilization. It is shown that spidroin solutions with concentration of 1 mg mL(-1) appear to be structured liquids with yield stress around value 0.2 Pa. Distribution of hydrodynamic radii of spidroins reveals two peaks due to presence of single particles and aggregates both. Their ability for spidroin transformation from micellar to fibrillar structure under shear stress is demonstrated. Studying rheological behavior of recombinant spidroin solutions and their supramolecular organization is necessary to select the optimal spinning parameters to produce fibrous materials with required physicochemical properties.

It has been shown that recombinant spidroins rS1/9 and rS2/12 that we previously developed exhibit adhesive properties with respect to both inorganic and organic substrates. It is well known that the adhesive properties of mussel foot proteins are associated with the level of DOPA, which is formed as a result of the post-translational modification of tyrosine residues by the tyrosinase enzyme. Therefore, we used recombinant tyrosinase for in vitro modification of tyrosine residues in the recombinant rS1/9 and rS2/12 spidroins to increase their adhesion capacity. As expected, the conversion of tyrosine residues into DOPA led to an increase in the adhesion properties of these proteins, which was demonstrated in experiments on gluing plates of polyvinyl chloride, aluminum, polylactic acid, and tubular pork bone. The molecules of recombinant spidroins retained their inherent properties to form supramolecular structures, hydrogels (microgels), transparent films, and 3D matrices. Interestingly, tyrosinase-modified proteins exhibited increased cohesion in experiments on bonding different materials in the presence of water.

One of the current problems in regenerative medicine is the search for new approaches for skin restoration. A promising area is the use of biocompatible materials. Such constructs can serve as the basis for biomedical products designed to replace damaged tissue or function as wound dressings. In this work, photopolymerized films based on silk fibroin and methacrylated gelatin (F-MG) were created. An in vitro study revealed that the use of films as a substrate for the cultivation of NIH 3T3 fibroblasts and HaCaT keratinocytes leads to a change in the kinetics of cell growth. According to the MTT assay, the fibroblasts’ proliferation rate was lower on photopolymerized films, while that for keratinocytes was higher compared to culture plastic. The effect of the obtained films on skin regeneration was investigated in vivo in a model of a full-thickness wound of mouse skin. The use of F-MG films as wound dressings contributed to faster wound healing and more complete recovery of the skin structure compared to control (use of gauze). Animals of the experimental group exhibited the formation of hair follicles and the reduction of the scar area after 28 days.

The search for new approaches for skin restoration is an urgent task of modern regenerative medicine. A promising area is the use of biocompatible materials. Designs from them can serve as the basis for biomedical products designed to replace damaged tissue, or serve as wound dressings. In this work, photopolymerized films based on silk fibroin and methacrylated gelatin (F-MG) were created. An in vitro study revealed that the use of films as a substrate for the cultivation of NIH 3T3 fibroblasts and HaCaT keratinocytes leads to a change in the kinetics of cell growth. According to the MTT assay, the fibroblasts proliferation rate was lower on photopolymerized films, and keratinocytes’ one was higher compared to culture plastic. An assessment of the effect of the obtained films on skin regeneration was performed in vivo in a model of a full-layer wound of mouse skin. The use of F-MG films as wound dressings contributed to the acceleration of wound healing and a more complete restoration of the skin structure compared to control (use of gauze). In animals in the experimental group, the formation of hair follicles and the reduction in the scar area by 28 days were observed.

The aim of the investigation was to study toxic properties (chronic toxicity, local irritant and sensitizing effects) of medical products Hydrogel RS and Microgel RS, intended for use in regenerative medicine as implants for replacing defects of soft and bone tissues, treatment of deep burns and for in vitro cell culturing when testing medications.Materials and Methods. The work was carried out on outbred Wistar rats, Chinchilla rabbits, guinea pigs. There was investigated chronic toxicity, local irritant and sensitizing effects of medical products Hydrogel RS and Microgel RS (GosNIIGenetika, Russia) based on recombinant spidroin (with cutaneous and intramuscular routes of administration). While investigating chronic toxicity, there were recorded integral, hematological, biochemical indices. Local irritation was studied by pathomorphological examination of the areas of medical product introduction. Sensitization was studied using maximization method and closed epicutaneous applications.Results. Medical products Hydrogel RS and Microgel RS were found to be relatively safe when used during 90 days, they have no irritant effect when introduced epicutaneously and intramuscularly, no sensitizing properties and can be recommended for clinical testing.Conclusion. Medical products Hydrogel RS and Microgel RS can be recommended for clinical testing as implants for replacing defects of soft and bone tissues, treatment of deep burns and for in vitro cell culturing in testing medicines.

A comparative analysis of rheological, organoleptic, physical and chemical properties of hydrogels was performed and their biosafety was examined in cell cultures before and after radiation and tyndallization sterilization. It was shown that the radiation sterilization does not destabilize the protein molecules of hydrogels at exposure doses less than 15 kGy, thereby not changing their physical, chemical and biomedical properties and, hence, the operational characteristics of products based on hydrogels.

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.

The fermentation of a Saccharomyces cerevisiae strain producing recombinant spidroin IF9 was optimized. A simplified two-stage scheme of the process was developed; the effect of sucrose, glucose, fructose, and galactose on the efficiency of the process was investigated. The optimal concentration of sucrose in the medium and replenishment was determined. The influence of peptone, tryptone, and casein hydrolysate of various brands on the effectiveness of the fermentation was analyzed. The optimal concentrations of peptone and yeast extract in the medium and replenishment were determined. As a result, owing to the optimization, the process of fermentation was simplified, a new composition of a complex replenishment was designed, and sucrose-specific consumption was reduced by two times, whereas the expenses of peptone and yeast extract were decreased by about 2.5 times, the yield of biomass per unit of the culture broth volume grew by ∼ 40%, and that of the protein of interest increased by ∼ 60%.

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.

Purpose. To study prerequisites for a development of artificial cornea bioengineered design based on recombinant spidroin tissue matrix by behavior evaluation of 2D (planar) and 3D cell (threedimensional) cultures on its surface.Material and methods. We studied epithelioid and stromal primary cell cultures (MSC-L) received from the limbal zone of post-mortem donor eyes. Cells were seeded on Petri dishes and on cavities of cultural trays (Corning, USA). To get spheroid structures the cells after the second passage underwent the centrifuge and were seeded on agarous trays then were cultivated in thermostatic chamber (Cell-IQ, Chip Man Technologies, Finland) under standard conditions (37° C, 5% CO2). Control over cell growth and morphology in trays was conducted under inverted microscope CKX41 (Olympus, Japan). To count the cell quantity and their viability the automatic cell counter Countess (Invitrogen, USA) was used, to analyze the surface proteins expression the flow cytofluorimetry was applied. For matrices colonization we used the 3rd passage MSC-L and 7-day spheroids of MSC-L origin. To evaluate 2D and 3D cell cultures growth on the surface of membranous matrices of recombinant spidroin, to estimate its non-toxicity and adhesiveness the immunohistochemistry, light time-lapse microscopy (Cell-IQ, Chip Man Technologies, Finland), laser scanning confocal microscopy (FluoView FV10i, Olympus, Japan) and raster electronic microscopy (CamScan, Japan) were incorporated.Results. Few hours after cell seeding there was active cells’ attachment to the substrate. Attached cells were characterized by rounded, oval or polygonal ordonnance. 24 hours later bipolar elongated cells and islets of migrating epithelioid cells appearance were observed. In the incubation process under gravity force the spheroids were accumulated predominantly in the central zone of the matrix, 2 hours later an active migration of spheroids surface zone epithelioid cells was registered on the membrane. After 24 hours of incubation all seeded on the surface of membranous matrix cells possessed a mesenchyme-like phenotype. Spheroids had an ability to merge limitlessly, later we observed a new microtissue formation with epithelioid cells on the surface and mesenchyme-like cells in the central area. Both solitary spheroids and merger-derived microtissue contained epithelial and mesenchymal components as well as regularly organized fibrils of extracellular matrix.Conclusions. According to aforementioned data the development of artificial cornea bioengineered cell-tissue constructions based on the technology of 3D cell spheroids cultivation derived from multipotent stem cells of the limbus and spidroin matrix presents a promising prospect requiring a further profound investigation.

Purpose. To study prerequisites for a development of artificial cornea bioengineered design based on recombinant spidroin tissue matrix by behavior evaluation of 2D (planar) and 3D cell (threedimensional) cultures on its surface. Material and methods. We studied epithelioid and stromal primary cell cultures (MSC-L) received from the limbal zone of post-mortem donor eyes. Cells were seeded on Petri dishes and on cavities of cultural trays (Corning, USA). To get spheroid structures the cells after the second passage underwent the centrifuge and were seeded on agarous trays then were cultivated in thermostatic chamber (Cell-IQ, Chip Man Technologies, Finland) under standard conditions (37° C, 5% CO2). Control over cell growth and morphology in trays was conducted under inverted microscope CKX41 (Olympus, Japan). To count the cell quantity and their viability the automatic cell counter Countess (Invitrogen, USA) was used, to analyze the surface proteins expression the flow cytofluorimetry was applied. For matrices colonization we used the 3rd passage MSC-L and 7-day spheroids of MSC-L origin. To evaluate 2D and 3D cell cultures growth on the surface of membranous matrices of recombinant spidroin, to estimate its non-toxicity and adhesiveness the immunohistochemistry, light time-lapse microscopy (Cell-IQ, Chip Man Technologies, Finland), laser scanning confocal microscopy (FluoView FV10i, Olympus, Japan) and raster electronic microscopy (CamScan, Japan) were incorporated. Results. Few hours after cell seeding there was active cells’ attachment to the substrate. Attached cells were characterized by rounded, oval or polygonal ordonnance. 24 hours later bipolar elongated cells and islets of migrating epithelioid cells appearance were observed. In the incubation process under gravity force the spheroids were accumulated predominantly in the central zone of the matrix, 2 hours later an active migration of spheroids surface zone epithelioid cells was registered on the membrane. After 24 hours of incubation all seeded on the surface of membranous matrix cells possessed a mesenchyme-like phenotype. Spheroids had an ability to merge limitlessly, later we observed a new microtissue formation with epithelioid cells on the surface and mesenchyme-like cells in the central area. Both solitary spheroids and merger-derived microtissue contained epithelial and mesenchymal components as well as regularly organized fibrils of extracellular matrix. Conclusions. According to aforementioned data the development of artificial cornea bioengineered cell-tissue constructions based on the technology of 3D cell spheroids cultivation derived from multipotent stem cells of the limbus and spidroin matrix presents a promising prospect requiring a further profound investigation.