The obtaining of microcarriers for the cell culture and delivery is an urgent task of tissue engineering and regenerative medicine. The novel method of surface modification of alginate microcarriers in the form of microspheres with a diameter of 200–300 μm was developed. The described method consists in covalent crosslinking between collagen and surface of alginate microcarriers. It was shown that the method makes it possible to completely modify the surface of the alginate microcarrier, which can be used to improve the biological properties of the microcarrier. Such microcarriers with improved biological properties can be considered as effective systems for cell delivery and culture.

Creation of new effective bio-artificial structures for tissue engineering and regenerative medicine requires development and implementation of new technological approaches for analysis of micro- and nanostructural features of constructs based on biomaterials and their interaction with cells. A new method of three-dimensional multiparametric analysis of nanostructure, scanning optical probe nanotomography, is presented in this paper, applied to the analysis of cells and biomaterials. Correlative reconstruction of fluorescent marker distributions and nanostructure features allows quantitative evaluation of a number of parameters of three-dimensional nanomorphology of fibroblasts and human hepatocarcinoma cells Hep-G2, adhered to biodegradable scaffolds based on silk fibroin. The developed technology with use of scanning optical probe nanotomography is applicable to investigation of three-dimensional micro- and nanostructure features of biomaterials and cells of different types.

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies. The aim of the work was to study the effect of biodegradable silk fibroin-based scaffolds containing GDNF on the corneal regeneration process. During cultivate the highest keratocytes proliferative activity was registered with scaffolds containing 250 ng/ml and 500 ng/ml GDNF. In mice with an experimental model of epithelial-stromal damage to the cornea, silk fibroin-based scaffolds containing GDNF in various concentrations were used (in groups 1, 2 and 3 silk fibroin-based scaffolds containing GDNF in a concentration of 50 ng/ml, 250 ng/ml and 500 ng/ml, respectively; in group 4 - silk fibroin-based scaffolds without GDNF; in group 5 - a solution of GDNF with concentration of 500 ng/ml; group 6- control). The area of the corneal epithelial defect in groups 2, 3, and 5 was less than in the other groups. The most pronounced positive immunohistochemical reaction with antibodies to Bcl2, Bax, phosphoERK1/2 and phospho-JNK1/2, Ki67, Gap43 was observed in groups 2 and 3. Thus, silk fibroin-based scaffolds with GDNF stimulate the epithelialization process, proliferative activity of epithelial cells and keratocytes, accelerate the formation of the stromal nerve plexus and exhibit anti-apoptotic activity.

Objective: to analyze the 3D micro- and nanostructure and quantitative morphological parameters of rat lung tissue. Materials and methods. Wistar rat lung tissue samples were obtained for the study. The 3D structure of the lung tissue was studied via scanning probe nanotomography using an experimental setup combining an ultramicrotome and a scanning probe microscope. Results. Nanoscale images and 3D nanotomographic reconstructions of the interalveolar septal sections of the rat lung were obtained. Morphological parameters (average roughness and specific effective area) of the interalveolar septal surface were determined. It was found that the average roughness of the reconstructed septal surface was 345.4 ± 24.5 nm, and the specific effective area was 2.7 ± 0.2 units. Conclusions. Results obtained demonstrate that scanning probe nanotomography allows to quantify lung morphology. The use of scanning probe nanotomography for 3D analysis of the structure and characteristics of lung tissue will increase the efficiency of future developments on creation of new criteria for diagnosing pathological conditions.

Objective : to develop a method for obtaining scaffolds based on natural silk fabric and to study their biocompatibility in vitro. Materials and methods . To obtain biodegradable scaffolds based on natural silk fabric, we propose treating natural silk fabric with a water-ethanol solution of calcium chloride. Differences in the structure of the resulting scaffolds were identified via scanning electron microscopy. Conclusion . The resulting scaffolds are non-toxic to cells and support cell adhesion and proliferation. Our studies make it possible to consider the resulting biodegradable scaffolds as promising constructs for tissue engineering and regenerative medicine.

The development of effective and versatile microcarriers is a pressing issue in tissue engineering and regenerative medicine. The objective of this wor k is to create biocompatible fiber microparticles from the cocoons of the Bombyx mori silkworm, and to study their structure and biological properties. Materials and methods . In obtaining microparticles, the Bombyx mori cocoons washed from sericin were cryo-milled in liquid nitrogen. The structure of the resulting microparticles was analyzed via scanning electron microscopy. The cytotoxicity of the obtained fibers was assessed using MTT-cell culture assay of 3T3 mouse fibroblasts. Cell adhesion analysis was performed using the Hep-G 2 human hepatocarcinoma cell line. Cell visualization was performed by staining the nuclei with DAPI fluorescent dye. Results . Natural silk microparticles were obtained in the form of cylindrical fibers with 200–400 μm average length and 15 μm diameter. It was shown that the surface of the resulting microparticles has a rough relief; no pores were found. The microparticles are non-toxic for 3T3 mouse fibroblasts, they maintain a high level of adhesion by human hepatocellular carcinoma HepG 2 cells . Conclusion. The method developed by us for fabrication of biocompatible silk fibroin microparticles in the form of fibers without using toxic reagents and significant time costs is promising for cell cultivation and delivery to the damaged area for tissue and organ regeneration.

We present a 3D study of nanostructural features of a bioprinted tissue spheroid interacting with polyurethane dual-scale biocompatible scaffold manufactured by three-dimensional printing and electrospinning. Three-dimensional analysis of fibroblasts interacting with electrospun polyurethane fibers was conducted using scanning probe nanotomography with an experimental setup combining ultramicrotome and a scanning probe microscope. Three-dimensional reconstruction demonstrates direct visualization of cell membrane protrusions and coherent cell-fiber interfaces, the formation of which is a prerequisite for an efficient tissue engineered implant. Analysis of obtained 3D data allows for quantitative calculation of the important morphological parameters of adhered cells, scaffolds, and cell-scaffold interfaces. The proposed method may be successfully applied to investigate 3D cell-scaffold constructs at nanoscale.

Aim. To develop the protocols for liver and kidney tissue decellularization, and to develop an analysis of the proliferative activity of human Hep-G2 hepatocarcinoma cells on various carriers.Materials and methods. Decellularization of the liver and kidneys was performed by perfusion of detergent solutions with gradually increasing concentrations of Triton X-100 (1, 2 and 3%). A histological analysis of the obtained samples was performed, and the method of optical and scanning electron microscopy was used to study the obtained samples. The proliferative activity of human Hep-G2 hepatocarcinoma cells was studied on the obtained samples of decellularized liver and kidney tissue.Results. Decellularization of the organ does not lead to changes in the specific structure of the tissue matrix. Microparticles with an average size of 200 μm were made from their decellularized matrix of liver and kidney tissues. The level of proliferative activity of human Hep-G2 hepatocarcinoma cells cultured on microparticles from a decellularized liver was significantly higher than on microparticles from a decellularized kidney.Conclusion. The decellularized matrix retains the native three-dimensional structure of the tissue. The level of cell proliferative activity is significantly higher on microparticles from the decellularized liver, which confirms the preservation of the specificity of the extracellular matrix of the tissue after the process of decellularization.

Traumatic brain injury is one of the leading causes of disability among the working-age population worldwide. Despite attempts to develop neuroprotective therapeutic approaches, including pharmacological or cellular technologies, significant advances in brain regeneration have not yet been achieved. Development of silk fibroin-based biomaterials represents a new frontier in neuroregenerative therapies after brain injury. In this study, we estimated the short and long-term effects of silk fibroin scaffold transplantation on traumatic brain injury and biocompatibility of this biomaterial within rat neuro-vascular cells. Silk fibroin microparticles were injected into a brain damage area 1 day after the injury. Silk fibroin affords neuroprotection as judged by diminished brain damage and recovery of long-term neurological functions. We did not detect considerable toxicity to neuro-vascular cells cultured on fibroin/fibroin-gelatin microparticles in vitro. Cultivation of primary cell cultures of neurons and astrocytes on silk fibroin matrices demonstrated their higher viability under oxygen-glucose deprivation compared to 2D conditions on plastic plates. Thus, we conclude that scaffolds based on silk fibroin can become the basis for the creation of constructs aimed to treat brain regeneration after injury.

The aim of the investigation was to research the effect of preparation method and composition of silk fibroin and gelatin scaffolds on biological properties.Materials and Methods. Silk fibroin, gelatin and their blend with different mass ratio scaffolds were prepared by electrospinning. To research scaffold's structure light microscopy, scanning electron microscopy and confocal laser scanning microscopy were applied. Adhesion and proliferation of mice fibroblast 3T3 cell line were investigated to test biocompatibility of constructed scaffolds.Results. Optimal parameters of device and fiber obtaining parameters were selected. Fibrous porous three-dimensional structure of investigated scaffolds was revealed. It was established that cell proliferative activity on electrospun scaffolds was significantly higher than on casting films. Addition of gelatin to scaffold composition increases cell proliferation.Conclusions. Electrospun silk fibroin/gelatin scaffolds contain such polymers with mass ratio equal to 1: 3 have significant greater ability to maintain cell and proliferation than fibroin and gelatin scaffolds.

Aim of this study is to research an effect of silk fi broin fi lms fabricated by casting method upon Wistar rat’s full-thickness skin wound regeneration. Materials and methods. 4 different kinds of fi lms with protein concentration equal to 20 mg/ml were fabricated: fi lms from silk fi broin aqueous solution, fi lms from silk fi broin formic acid solution, fi lms from silk fi broin aqueous solution containing 30% collagen by weight, fi lms from silk fi broin formic acid solution containing 30% collagen by weight. All kinds of fi lms were fabricated by casting method on polished Tefl on surface. Scanning electron microscopy was applied to research fi lms’ surface structure. Cytotoxicity test of the fi lms was realized on mouse 3T3 fi broblasts model by MTT assay. Manufactured fi lms were utilized to regenerate full-thickness skin wounds in Wistar rats. Results. It was shown that fi lms’ surface was characterized by micro- and nanorelief in the form of roughness. The proliferative activity of mouse 3T3 fi broblasts increased during 7 days of cytotoxicity test. Fabricated fi lms enlarge the regeneration rate of full-thickness Wistar rat skin wounds an average of 25%. Histological analysis indicated structural skin restoration without any infl ammatory tissue. Conclusion. All fabricated fi lms are non-cytotoxic and characterized by appropriate structure for the adhesion and proliferation of fi broblasts. The application of fi lms for full-thickness skin wound regeneration increases its restoration rate which is confi rmed by histological examination.

This paper presents a study of nanostructural features of contacts of bioprinted tissue spheroids with polyurethane dual scale biocompatible scaffold made by three-dimensional printing and electrospinning. Analysis of nanostructural features of cell contacts was carryed out by scanning probe microscopy with use of experimental setup combining ultramicrotome and scanning probe microscope. Measured mean cell volume is 460 ± 104 μm3, mean contact area of cells with scaffold fibers–104.8 μm2 per cell (16.7% of total cell area). Maximum distance of migrating cells from spheroid border at 48 h. is ~200 μm, what corresponds to mean velocity of cell migration more than 4 μm/h. Obtained quantitative characteristics of micro- and nanostructure of human fibroblast cell contacts with elecrospun polyurethane scaffold secure high efficacy of tissue regeneration with its usage for implanted bioprinted dual scale tissue-engineered scaffolds.

We have developed microcarriers made from silk fibroins. Microcarriers can be used as a substrate for cell cultivation and cell delivery during cell-based therapy, and for the construction of bioengineered tissue. Fibroin microcarriers were mineralized, which led to the appearance of calcium phosphate crystals on their surface. The ability of mineralized and non-mineralized microcarriers to support osteogenic differentiation of the osteoblast-like cell line MG-63 was estimated by alkaline phosphatase activity, an early marker of bone formation. The experiment showed cells actively proliferating on the surface of both mineralized and non-modified microcarriers. Culturing MG-63 on the surface of fibroin microcarriers resulted in an increase of alkaline phosphatase activity indicative of osteogenic differentiation of MG-63 cells in the absence of inductors. The level of alkaline phosphatase was higher when mineralized microcarriers were used. Alkaline phosphatase activity of MG-63 cells cultivated using traditional two-dimensional approaches were close to zero. As opposed to conventional monolayer culturing, microcarriers culture cells in a three-dimensional environment that is closer to physiological conditions. This can have a significant impact on their morphology and functional properties. During this study we also characterized mechanical properties of porous scaffolds used for microcarriers.

We have developed microcarriers made from silk fibroin. Microcarriers can be used as a substrate for cell cultivation and cell delivery during cell-based therapy and for the construction of bioengineered tissue. Fibroin microcarriers were mineralized, which led to the appearance of calcium phosphate crystals on their surface. The ability of mineralized and nonmineralized microcarriers to support osteogenic differentiation of the osteoblast-like cell line MG-63 was estimated by alkaline phosphatase activity, an early marker of bone formation. The experiment showed cells actively proliferating on the surface of both mineralized and nonmodified microcarriers. Culturing MG-63 on the surface of fibroin microcarriers resulted in an increase of alkaline phosphatase activity indicative of osteogenic differentiation of MG-63 cells in the absence of inductors. The level of alkaline phosphatase was higher when mineralized microcarriers were used. Alkaline phosphatase activity of MG-63 cells cultivated using traditional two-dimensional approaches were close to zero. As opposed to conventional monolayer culturing, microcarrier culture cells are in a three-dimensional environment that is closer to physiological conditions. This can have a significant impact on their morphology and functional properties. During this study, we also characterized mechanical properties of porous scaffolds used for microcarriers.

The aim of the investigation was to study the biological and mechanical properties of silk fibroin films and composite silk fibroin films containing 30% collagen by weight.Materials and Methods. All films were prepared by casting method using water and formic acid as solvents. Scanning electron microscopy and atomic force microscopy were applied. Human hepatoblastoma cell line Hep-G(2) was used to test film compatibility.Results. We studied surface roughness degree of the obtained films. Water-based films were found to have permeability for low molecular weight substances. Tensile strength and elasticity indices were measured for all types of films. Collagen added to film composition was revealed to have no significant effect on tensile strength, though it increased film elasticity. We studied the degradation of films. Collagen was shown to have no significant impact on film degradation rate both in phosphate-buffer saline and oxidizing media. We demonstrated with an example of cell line Hep-G(2) that water-based films exhibit higher proliferative activity.Conclusion. Silk fibroin water solution has the best properties to develop films for tissue engineering.