A two-layer bioresorbable vascular graft (BVG) with an internal diameter of 2 mm has been developed from biopolymer bioresorbable materials: synthetic polymer polycaprolactone and high-molecular compound of natural origin—gelatin. BVG has a highly porous structure, physical and mechanical characteristics close to a natural blood vessel of a similar diameter, and water permeability of less than 10 mL/(cm2 min). The graft is biocompatible and, when implanted into the infrarenal aorta of a rat, demonstrates blood flow parameters (systolic and diastolic velocity and resistivity index) close to the corresponding parameters of the rat aorta. Under in vivo functioning conditions, the two-layer vascular graft is resistant to stratification and does not provoke an inflammatory reaction, and after 8 weeks of functioning, the formation of neointima is observed on the inner surface of the BVG against the background of the integration of the graft into the body’s surrounding tissues.

Viscoelastic hydrogels based on animal tissue extracts are considered promising biomimetics of the extracellular matrix (ECM) due to their proven effectiveness for stimulating the regeneration of the liver, pancreas and articular cartilage. Cryostructuring is an approach that makes it possible to give polymer scaffolds macroporosity and provide mechanical strength. preparation of a new macroporous cryogenically structured biomimetic of ECM based on a commercially available concentrated collagen-containing solution and evaluation of the possibilities of its application in tissue engineering. The target spongy collagen-containing material was obtained by sequential freezing of a concentrated collagen-containing solution, its subsequent lyophilization and chemical tanning by treatment with an alcohol solution of carbodiimide. The morphology of the cryostructured multicomponent collagen-containing material was studied using optical and scanning electron microscopy (SEM) using lanthanide contrast. The cytotoxicity of the scaffold was studied on the culture of human adipose-derived stem cells (hADSCs). Adhesion and proliferation of hADSCs on the scaffold surface were studied on the 7th day of cultivation. The compression modulus of elasticity of the obtained collagen–containing material in the swollen state in water was 35,3 ± 2,2 kPa, the total water-holding capacity of the material was 45.80 ± 0.46 ml/g of polymer, and the degree of swelling of the walls of macropores was 3.99 ± 0.31 ml/g. During SEM examination and histological staining with hematoxylin and eosin, a broad-pored structure was observed on the surface and cross-section of the disc. The pores in the upper part are larger (the average diameter is not less than ~ 30 µm) than the pores in the lower part of the sponge (the average diameter is not more than ~ 30 µm) due to the occurrence of a vertical temperature gradient. The matrix did not have cytotoxicity relative to the hADSCs. In the sample, active proliferation of hADSCs was observed on the surface of the scaffold. It was shown that the developed cryostructurates based on a concentrated collagen-containing solution had supermacroporosity and a compression modulus of elasticity of 35.3 ± 2.2 kPa. The absence of cytotoxicity and the ability to maintain adhesion and proliferation of hADSCs indicate the possibility of using cryogenically structured biomimetic of the extracellular matrix in tissue engineering and regenerative medicine.

The creation of a cell-engineered pancreatic construct (CEPC) from islets of Langerhans and biocompatible matrix carrier (framework/scaffold), which imitates the native microenvironment of pancreatic tissue, is an approach to the treatment of type I diabetes mellitus (T1D).The objective of this work is to conduct a comparative analysis of the functional efficacy of CEPC and isolated rat islets of Langerhans after intraperitoneal administration into rats with experimental T1D.Materials and method. T1D was induced in rats by injecting low-dose (15 mg/ kg) streptozotocin (STZ) for 5 days. CEPC samples were created using viable and functional allogeneic isolated islets of Langerhans and tissue-specific scaffold obtained by decellularization of human pancreatic fragments. The rats received intraperitoneal injection of allogeneic islets of Langerhans (experimental group 1, n = 4) and CEPC (experimental group 2, n = 4). Control group rats received no treatment (n = 4). Blood glucose levels in the rats were measured, and the pancreas and kidneys of the experimental animals were examined histologically. The follow-up period for all animals continued for 10 weeks. Results. In experimental group 1, on day 7 after injection of Langerhans islets, glycemia decreased significantly from 28.2 ± 4.2 mmol/L to 13.4 ± 2.6 mmol/L. This fall persisted for 7 weeks, following which blood sugar increased to nearly their initial levels (prior to islets administration). In experimental group 2, on day 7 after CEPC administration, there was a more noticeable drop in blood sugar levels from 25.8 ± 5.1 mmol/L to 6.3 ± 2.7 mmol/L compared to experimental group 1. By the 10th week of the experiment, the average glucose level was two times lower than it was at the beginning. Blood glucose levels dropped more sharply in the CEPC group than in the islet group (by 75.6% and 52.5%, respectively).Conclusion. In T1D rats, CEPC has a more potent antidiabetic effect than islets of Langerhans. Thus, it has been shown that a tissue-specific scaffold may be used to create bioartificial pancreas in order to increase the functional efficiency of islets.

Objective: to obtain a stable mouse model of type 1 diabetes mellitus (T1DM) using streptozotocin (STZ), which has a toxic effect on pancreatic beta cells.Materials and methods. Experiments were performed on 30 white non-diabetic male mice of the SHK colony, which were injected intraperitoneally with STZ at a dose of 200 mg/ kg by two methods: 15 animals (group 1) once and 15 animals (group 2) intermittently – 5 consecutive days at 40 mg/kg per day.Results. In group 1, one mouse died after 2 days due to hypoglycemic coma, 4 mice developed hyperosmolar hyperglycemia (>33.3 mmol/l), 3 mice had spontaneous remission of diabetes, and 7 mice had stabilized hyperglycemia at levels close to 20 mmol/l. In group 2, only one mouse showed spontaneous remission of diabetes, while the remaining 14 animals showed stable diabetes with average hyperglycemia levels moderately above 20 mmol/L until the end of the 4-week follow-up. A histological study of the pancreas of these animals confirmed the destructive effect of STZ on islets in the form of mass death of insulin-producing β-cells.Conclusion. Split-dose intraperitoneal injection of STZ provides a stable experimental T1DM in 93% of laboratory mice.

Objective : to study the effect of trypsin pretreatment in the porcine articular cartilage decellularization protocol on the ability to restore the biochemical composition and functional properties of the resulting finely dispersed tissue-specific scaffold when co-cultured with human adipose-derived stem cells (hADSCs). Materials and methods . Porcine articular cartilage was micronized to a maximum size of 250 μm. The resulting porcine articular cartilage microparticles (CMps) were treated with trypsin (0.05, 0.25, 0.50%) / EDTA solution at +37 °C for 24 hours. Then, the CMps were successively incubated for 24 hours in three surfactant solutions containing 0.1% sodium dodecyl sulfate and increasing concentration of Triton X-100 (1, 2, 3%) at room temperature and in DNase I solution at +37 °C for 48 hours. The degree of change in the biochemical composition and the ability of decellularized CMps (DCMps) scaffolds within cell-engineered constructs (CECs) to support hADSC adhesion and proliferation, as well as their potential ability to exert a stimulatory regenerative effect, were then assessed. DNA, glycosaminoglycans (GAGs) and collagen content in the DCMps and CECs were examined. The morphology of the samples was examined using histological and immunohistochemistry staining. Results . Histological analysis showed that there were no cells and detritus in the DCMp samples. Pretreatment of CMps samples гыштп a solution with the lowest content of trypsin (0.05%) / EDTA in the samples retained 5.14 ± 0.87 ng/mg DNA in the samples, while GAG content decreased to 5.34 ± 0.9 μg/mg and collagen to 154 ± 34 μg/mg. By day 28 of CEC cultivation, adherent cells had produced their own extracellular matrix (ECM) containing GAGs and collagen. The amount of DNA in it was 6.30 ± 0.11 μg/CEC and that of GAGs was 19.36 ± 0.73 μg/CEC. Conclusion . Pretreatment with trypsin allows achieving uniformly complete decellularized CMps. At the same time, onset of changes in the ECM composition indicates a decrease in the ability of hADSCs to synthesize GAGs and type II collagen during co-culturing with DCMps. The increased proliferative activity of adherent hADSCs, as well as the tissue specificity of the DCMp scaffold will allow further research towards a hydrogel matrix capable of enhancing the specific and stimulating regenerative potential when co-cultured with cells of the same phenotype.

Objective: to study the effect of a conditioned medium of mesenchymal stromal cells (MSCs) from different sources on human chondrocyte proliferation.Materials and methods. To confirm functional activity, chondrocytes were cultured in a cartilage cell-engineered construct (CEC), including 5 × 105 cells and 5 mg of tissue-specific matrix from decellularized cartilage. The conditioned medium was obtained after culturing MSCs derived from human adipose tissue (AT), MSCs derived from the pulp of primary teeth and MSCs isolated from umbilical cord-derived Wharton’s jelly in a complete cell growth medium (CCGM). To evaluate the effect of MSC-derived secretome on chondrocyte proliferation, the conditioned medium, diluted 1 : 1 with CCGM, was added to wells containing chondrocytes. The effect of MSCs on human chondrocyte proliferation was studied by indirectly coculturing cells in CCGM using Transwell inserts. 5 × 104 MSCs were applied to the bottom of the lower chamber, and 5 × 104 human chondrocytes and 5 mg of matrix were placed in the upper chamber. Chondrocyte proliferation was assessed at days 7 and 14 by DNA quantification. Interleukin-6 content was determined as a marker of secretory activity of MSCs in the conditioned medium. The morphology of the samples was studied using histological staining methods.Results. The ability of chondrocytes to produce cartilage-specific extracellular matrix was confirmed when forming cartilage CEC with tissue-specific matrix in a chondrogenic differentiation medium. When comparing the effect of the conditioned medium of MSCs obtained from different sources on the growth of human chondrocytes in vitro, increased proliferation was observed in all samples compared to controls. Indirect co-culture of MSCs with chondrocytes as part of CEC showed increased DNA amount in all samples at day 14, with the amount of DNA in the sample with MSC conditioned medium significantly higher than the control.Conclusion. Studies on the effect of MSC conditioned medium on chondrocyte proliferation in 2D culture indicate a possible regenerative potential of MSCs for cartilage tissue repair. Within the scope of this work, we did not identify significant differences in the effect of secretome derived from MSCs that were obtained from different sources on chondrocyte proliferation. However, additional in vivo studies are warranted in the future.

Creation of a bioartificial pancreas, including a cell-engineered construct (CEC) formed from pancreatic islets (islets of Langerhans) and a biocompatible matrix mimicking the native microenvironment of pancreatic tissue, is one of the approaches to the treatment of type 1 diabetes mellitus (T1D). Objective: to conduct preliminary in vivo studies of the functional efficacy of intraperitoneal injection of a cell-engineered pancreatic endocrine construct and a suspension of rat pancreatic islets in an experimental T1D model. Materials and methods. Tissue-specific scaffold was obtained by decellularization of human pancreatic fragments. The viability and functional activity of rat islets isolated with collagenase were determined. Experimental T1D was modeled by intraperitoneal injection of low-dose streptozotocin and incomplete Freund's adjuvant into rats. The rats were intraperitoneally injected twice with pancreatic CEC (n = 2) or islet suspension (n = 1). Glucose levels in the blood and urine of the rats were assessed. Histological examination of organs (pancreas and kidneys) of the experimental animals was carried out. Results. After the first injection, blood glucose levels gradually decreased in all animals by more than 47% of the initial values; by follow-up day 24, the glucose level rose to the initial hyperglycemic values. After repeated administration, a 63.4% decrease in glycemic level was observed in the rats with pancreatic CEC and a 47.5% decrease in the one with islet suspension. At week 5 of the experiment, blood glucose levels gradually increased in all animals. At the same time, the glycemic index of the rat with injected pancreatic CEC was 62% lower than the glycemic index of the rat with injected islets. Conclusion. Allogeneic pancreatic islets in pancreatic CEC increase the duration of stable glycemic level in T1D rats.

Introduction. The use of immunomodulators to regulate reparative processes in affected organs and tissues remains a pressing issue. Of greatest interest is liver regeneration after extended hepatic resection (EHR) in donors in right lobe living related donor liver transplantation. We propose a transdermal therapeutic system (TTS) with an immunomodulator to enhance the natural process of liver tissue regeneration. Objective: to study the effect of transdermal administration of immunomodulator sodium aminodihydrophthalazinedione on early recovery processes in the liver after EHR in in vivo experiments. Materials and methods. Sodium aminodihydrophthalazinedione was used as an active substance in TTS in the form of powder for preparation of intramuscular injection solution (Galavit®, SELVIM LLC). An experimental EHR model was performed on 22 male Wistar rats weighing 350–380 g. After HER, all animals were divided into two groups. Group 1 (n = 10) consisted of untreated animals. In group 2 (n = 12), TTS was applied immediately after liver resection. The experiment lasted for 48 hours; the TTS was changed once after 24 hours from the beginning of application. Results. In either group, there was no significant difference in the weight of liver remnant gain and in biochemical blood parameters at 48 hours after EHR. Assessment of the mitotic index (MI) of hepatocytes 48 hours after EHR revealed a significant increase in MI in both groups in comparison with the baseline (before liver resection) equal to 0.14 ± 0.07‰. The MI in group 1 and group 2 animals was 12.70 ± 4.9‰ and 17.43 ± 4.90‰, respectively (p ≤ 0.05). Conclusion. Studies on the regenerative activity of sodium aminodihydrophthalazinedione TTS on an experimental EHR model in rats showed that this drug form had a pronounced stimulating effect on the mitotic activity of liver cells.

Introduction. Developing a tissue-engineered pancreatic construct (TEPC) involves a search for matrices/scaffolds capable of mimicking the structure and composition of the natural extracellular matrix (ECM), which is an important component of the tissue microenvironment. A cell-free, tissue-specific matrix obtained from pancreas decellularization seems to be the most suitable for creation of a TEPC. The choice of pancreatic tissue decellularization protocol should take into account the morphological characteristics of the original pancreas. Preservation of the architectonics and composition of the native tissue in the decellularized pancreas matrix (DPM), and the presence of native ECM components allow for creation of conditions for prolonged vital activity of functionally active islet (insulin-producing) cells when creating TEPC. Objective : to determine the optimal parameters for decellularization of deceased donor pancreas with fibrosis, lipomatosis, and without pronounced signs of fibrosis and lipomatosis. Materials and methods . We used the caudal part of the pancreas obtained after multiorgan procurement from deceased donors, which was unsuitable for transplantation. Tissue-specific matrix was obtained by a combination of physical and chemical methods of pancreatic decellularization. A freeze-thaw cycle protocol and two protocols using osmotic shock were used. Samples of initial pancreatic tissue and decellularized fragments were subjected to histological analysis. Result s. It was shown that a physico-chemical method with freeze-thaw cycles is suitable for effective pancreatic decellularization in severe lipomatosis; a physico-chemical method using osmotic shock, but different protocol variants, is suitable for pancreas with diffuse fibrosis and for pancreas without pronounced signs of fibrosis and lipomatosis. Conclusion . For complete human pancreatic decellularization, the protocol should be correlated with histological features of the original tissue.

Objective: to compare the efficiency of regenerative processes in the liver using apoptotic bone marrow-derived mononuclear cells (BMMCs) and intact BMMCs from healthy animals on an extended liver resection (ELR) model. Materials and methods. Male Wistar rats (n = 77) with an ELR model (70–75%) were divided into 3 groups: group 1 (control with a single intraperitoneal injection of saline), group 2 (single intraperitoneal injection of unsorted intact BMMCs at a dose of 30–35 × 106, and group 3 (single intraperitoneal injection of apoptotic BMMCs at the same dose). Restoration of biochemical parameters of liver function and mass, as well as the emerging microstructural changes in hepatocytes in histological preparations, were monitored by assessing hepatocyte mitotic activity (MA) during the first 7–10 days after ELR. Results. It was found that in groups 2 and 3, as compared with group 1, there was no death after ELR modeling, and that the biochemical parameters of liver function normalized more rapidly (at days 10–14). Hepatocyte MA in group 3 sharply increased as early as on day 1, and mitotic index (MI) averaged 14‰, reaching 20.9‰ in some experiments; MI in the control group remained at the baseline by this time, while in group 2, MI was only 3.2‰. In group 3, liver mass recovered more rapidly after ELR to baseline values already at days 8–10, whereas the recovery was at day 12–14 and day 17–20 in group 2 and group 1, respectively. It was suggested that the more pronounced increase in the efficiency of regenerative processes in the liver after ELR in group 3 after using apoptotic BMMCs was due to the release from these cells of a large spectrum of formed paracrine factors, including various classes of RNA molecules involved in the regeneration process. Conclusion. Apoptotic BMMNCs have a more effective adaptive and regulatory potential than intact BMMCs because reorganizations are rapidly formed in the damaged liver cells, providing an early and more powerful activation of the targeted regenerative program.

Cell death represents the most critical pathologic entity in liver disease, which dictates pathologic consequences such as inflammation, fibrosis, and cell transformation. We analyzed the conclusions of studies on the involvement of different types of programmed cell death (PCD) in the pathogenesis of liver diseases. Three main forms of PCD (autophagy, apoptosis, necrosis) and five additional, still insufficiently studied PCD – necroptosis, ferroptosis, pyroptosis, partanatosis and entosis – observed in the liver in various acute and chronic diseases are considered. The involvement of several PCD at once in the development of any one pathology and one type of PCD in different pathologies was established. This indicates the existence of cross-regulation of metabolism in the liver cells with different levels of damage in the formation of the main dominant type of PCD. Available results indicate the possibility of attenuation (correction) of functional and morphological manifestations of PCD in the organ by controlled blocking of effector-mediated PCD pathways, as well as targeted induction of autophagy, anti-apoptotic and anti-necrotic mechanisms in liver cells.

This paper presents results obtained from a modified perfusion bioreactor designed to form tissue equivalents in conditions of long-term cultivation with the capacity to oxygenate the medium. The design of the device permits the morphology of cellular engineering structures to be studies at four cultivation time points under flow conditions and also provides for constant monitoring of culture medium pH, O-2 and CO2 contents, and major metabolite contents throughout the experiment. The functional effectiveness of the bioreactor is confirmed by the example of "growing" the tissue equivalent of cartilage for 25 days with maintenance of cell culture viability, with constant values for the main culture medium parameters and sterility.

In terms of method of production, collagen carriers are subdivided into materials obtained on the basis of extracellular matrix (ECM) components, particularly collagen-containing hydrogels and decellularized tissue. Objective : to compare in vitro the ability of biopolymer microheterogeneous collagen-containing hydrogel (BMCH) and tissue-specific matrix from decellularized porcine articular cartilage (DPAC) to support adhesion, proliferation and chondrogenic differentiation of human adipose-derived mesenchymal stem cells (hAMSCs). Materials and methods . For cartilage decellularization, we carried out treatment with surfactants (sodium dodecyl sulfate, Triton X-100) followed by exposure in DNAase. The metabolic activity of hAMSCs was assessed by PrestoBlue™ (Invitrogen, USA) staining. The morphological study of cell-engineered constructs (CECs) formed by culturing hAMSCs in the presence of matrices was performed using histological staining and scanning electron microscopy (SEM) with lanthanide contrasting. Results . The number of cells on the surface of both BMCH and DPAC increased within 14 days. Mitochondrial activity of the cells was 1.7, 1.7, and 1.3 times higher on days 3, 10, and 14 when cultured on DPAC compared to BMCH, respectively. On day 14 of cultivation in the chondrogenic culture medium, hAMSCs formed cell layers on the DPAC surface and on the BMCH surface. Cytoplasm of the cells included numerous granules, which, when stained, resembled the matrix itself. On the DPAC matrix surface, cells were more evenly distributed, whereas in the case of BMCH, cell adhesion and proliferation were observed only in certain areas. The ECM produced by the cells contained collagen and glycosaminoglycans (GAGs). Conclusion . The ability of DPAC obtained according to the developed protocol to form CECs with hAMSCs with uniform distribution of cells and their production of specific collagen- and GAG-containing ECM suggests that DPAC is effective in regeneration of damaged cartilage. Chondrogenic differentiation of hAMSCs was observed both when cultured with BMCH and with DPAC. When creating a tissue equivalent of cartilage in vitro, the advantage of using tissue-specific matrix over BMCH should be considered.

We performed a comparative study of the proliferative potential of human mesenchymal stromal cells (MSC) from three sources (tooth pulp, adipose tissue, and Wharton’s jelly) in spheroid culture; human chondroblasts served as the positive control. Histological examination revealed signs of chondrogenic differentiation in all studied cell cultures and the differences in the volume and composition of the extracellular matrix. Spheroids formed by MSC from the tooth pulp and Wharton’s jelly were characterized by low content of extracellular matrix and glycosaminoglycans. Spheroids from adipose tissue MSC contained maximum amount of the extracellular matrix and high content of glycosaminoglycans. Chondrocytes produced glycosaminoglycan-enriched matrix. Type II collagen was produced by chondrocytes (to a greater extent) and adipose tissue MSC (to a lesser extent). The results of our study demonstrate that MSC from the adipose tissue under conditions of spheroid culturing exhibited maximum chondrogenic potential.

Objective: to obtain long-lived proliferating cells with progenitor features by dedifferentiation of mature rat hepatocytes using combinations of small molecules.Materials and Methods. Hepatocytes isolated from rat liver by perfusion were cultured in the presence of a cocktail of three small molecules – Wnt signaling pathway activator (CHIR99021), TGF-β inhibitors (A83-01) and ROCK kinase (Y27632). The morphological characteristics and growth features of the culture were assessed using fluorescence and phase-contrast microscopy during cell culture. Cell proliferative activity was analyzed using real-time time-lapse imaging. The expression of surface and intracellular markers was analyzed using flow cytometry and high-resolution fluorescence microscopy.Results. Using a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, long-lived proliferating cells that express progenitor cell markers, such as α-fetoprotein and HNF4α, were obtained from mature rat hepatocytes. The cells had hepatocyte-like morphology and formed discrete clusters of proliferating cells, forming a single cell layer during culturing. Removal of the small molecules from the medium led to expansion of fibroblast-like cells and elimination of potentially progenitor hepatocyte-like cells.Conclusion. Proliferating progenitor cells can be obtained by dedifferentiation of mature hepatocytes.

Objective : to study the peculiarities of the induction effect of total RNA (tRNA) from xenogenic bone marrow cells (BMCs) on regeneration processes in the recipient's native liver with extensive liver resection using an adoptive transfer model. Materials and methods . The study was carried out on an adoptive transfer model using male Wistar rats (n = 20) and guinea pigs (n = 17). The donors were rats (n = 10). 12 hours after extensive liver resection (70-75%), tRNA was isolated from BMCs and injected into intact (non-operated) recipients intraperitoneally at a dose of 30 μg/100 g of weight. The induction effect of the tRNA on operated rats was studied in 3 groups of recipients: Group 1 (control, n = 5) - administration of saline to guinea pigs; Group 2 (control, n = 10) - administration of tRNA from a donor rat to a recipient rat (allogeneic transfer); Group 3 (experiment, n = 12) - administration of tRNA from a donor rat to a recipient guinea pig (xenogeneic transfer). In histological preparations of recipient livers, after 48, 72 hours and 7 days, we studied the mitotic activity of hepatocytes and the features of the microscopic picture of the liver. The significance of differences in the compared groups was assessed using the parametric Student's t-test. Results. The ability of BMC tRNA to tissue-specifically activate regenerative and immune responses in the liver after extensive resection was found to depend on the donor and recipient species identity. Introduction of allogeneic donor tRNA in the recipient's liver resulted in predominant enhancement in hepatocyte mitotic activity (p < 0.05). The use of xenogeneic donor tRNA leads to enhanced activity of only immuno-inflammatory reactions in the recipient's liver, such as sinusoidal cell activation, lymphocytic infiltration into sinusoids, and portal tract infiltration by inflammatory cells. Conclusion. To induce regenerative processes in the liver, tRNA obtained from allogeneic BMCs should be used.

Objective: using an adoptive transfer model to study the cellular mechanisms involved in the formation of the initial stage of liver regeneration during intraperitoneal injection of a healthy recipient with apoptotic bone marrowderived mononuclear cells (BM-MNCs) from a donor after extended liver resection.Materials and methods. Male Wistar rats (n = 40) were used to create a model of adoptive transfer of apoptotic BM-MNCs (a-BM-MNCs) taken from the donor after extended liver resection to a healthy recipient. During the experiments, the animals were divided into five groups. Four experimental groups with intraperitoneal injection of the same doses to the recipient: freshly isolated BM-MNCs (group 1); BM-MNCs subjected to apoptosis for 48 hours by storage at t = 4–6 °C in phosphate-buffered saline (PBS) (group 2) or in a Custodiol HTK solution (group 3). In group 4, the animals were injected with PBS after storing BM-MNCs in it. The control animals were animals injected with saline (group 5). For selection of effective modes of apoptosis induction, BM-MNCs stained with 7AAD after incubation in solutions were analyzed by flow cytometry. Targeted transfer of regenerative signals to the recipient was assessed by the mitotic activity of hepatocytes in the liver and tubular epithelium in the kidneys, as well as by the intensity of microstructural changes in the liver 24, 48 and 72 hours after injection of the studied material.Results. BMC incubation in PBS and HTK for 48 hours at t = 4–6 °C provides the most effective accumulation of a-BM-MNCs in early apoptosis. It was shown that a-BM-MNCs retain the ability to target-focused transmission of regulatory signals to the liver supported by autophagy process during adoptive transfer. It was established that a-BM-MNCs (groups 2 and 3) in comparison to native BM-MNCs (group 1) at adoptive transfer increased the regenerative potential of the liver due to pronounced increase in the activity of autophagy processes and directed infiltration of immunomodulatory mononuclear cells in the liver.Conclusion. a-BM-MNCs create a stronger basis for development and implementation of a targeted and effective regeneration program by enhancing autophagy processes and immunomodulatory effect on mononuclear cells, which are regenerative signal carriers.

The main problem with decellularization of liver tissue as a tissue-specific matrix/scaffold in liver bioengineered structures is the need to maximize the preservation of the original three-dimensional structure of the tissue and the main components of its extracellular matrix (ECM) while removing cells and genetic material. The attempts to use the existing protocols for the decellularization of other tissues and organs have been unsuccessful. The aim of the work is to develop a method for creation of a tissue-specific microdispersed matrix from decellularized porcine liver (TMM DLp). The protocol for decellularization of porcine liver (Lp) fragments has been developed on the basis of the complex application of chemical (sodium dodecyl sulfate and Triton X100), biochemical (DNase I), and physical (supercritical CO2) methods for treatment of the initial tissue. As a result of the found optimal conditions for decellularization of Lp with subsequent cryomicronization of decellularized DLp, an injectable form of the microdispersed tissue-specific matrix was obtained, which represents decellularized porcine liver microparticles with the size of 100–200 μm with the residual amount of DNA no more than 10 ± 1.5 ng/mg (less than 1.0%), with the preservation of the microstructure and basic composition of the liver ECM. According to the in vitro assessment, biocompatible properties of tissue-specific matrix samples meet the criteria of biological safety for cytotoxicity and hemolytic activity.

A method for obtaining a microdispersed tissue-specific matrix from decellularized porcine articular cartilage while maintaining morphological and functional properties of the extracellular matrix with no signs of cytotoxicity was developed. The cartilage particle size distribution in suspension after cryogenic grinding was determined using laser diffraction analysis. The range of sizes of the obtained cartilage microparticles suggests the possibility of their administration by injection (<220 μm). The combination of stages, including three freeze/thaw cycles (–196/37°C) followed by treatment with solutions of surface-active substances (surfactants), sodium dodecyl sulfate and Triton X-100, and DNase resulted in the complete removal of non-decellularized microparticles. The residual DNA content was 9.11 ± 1.13 ng/mg of tissue. The effectiveness of surfactant washing was assessed by the cytotoxicity of the matrix on the culture of human adipose derived mesenchymal stromal cells (hADSCs). To assess the hemocompatibility of the obtained samples, their hemolytic activity was studied in vitro. The adhesion and proliferation of hADSCs on the matrix surface were studied on day 21 of cultivation. The matrix did not exhibit hemolytic activity and cytotoxicity with respect to hADSCs. hADSCs in the samples were actively proliferating on the matrix surface. The biocompatibility and hemocompatibility of the obtained matrix in vitro indicate its potential for application in cartilage regenerative medicine.

One of the pressing issues in tissue engineering is on how to obtain an artificial matrix that can simulate a biological microenvironment for cells. When creating a bioengineered pancreatic construct, a tissue-specific scaffold obtained from decellularized pancreatic tissue can serve as such matrix. Objective: to obtain and study the characteristic properties of a tissue-specific pancreas scaffold from decellularized human pancreatic fragments. Materials and methods. The decellularization protocol included 3 freeze/thaw cycles, followed by treatment with surfactants (sodium dodecyl sulfate and Triton X100). At each decellularization stage, samples were routinely stained with hematoxylin and eosin and for total collagen. In addition, immunohistochemical staining of decellularized human pancreas (DHP) for type I collagen and elastic fibers was performed. Cell nuclei in the original samples and the resulting matrix were visualized using DAPI fluorescent staining. DNA quantity in the native and decellularized pancreatic tissue was determined. The cytotoxicity of the tissue-specific matrix was evaluated in vitro by direct contact. The matrix properties of DHP samples were determined using mesenchymal stem cells (MSCs) of human adipose tissue. Results. A pancreatic decellularization method is proposed. This method allows to obtain a tissue-specific matrix in the form of a connective tissue scaffold completely free of detritus with preserved thin-fiber mesh-like structure, in which elastic and collagen fibers, including type I collagen, are identified. DAPI staining confirmed the absence of nuclear material in the decellularized matrix, while residual amount of DNA did not exceed 0.1%. Absence of matrix cytotoxicity and its ability to maintain adhesion and proliferation of human adipose tissue-derived MSCs was proved. Conclusion. As one of the stages in creating a bioengineered pancreatic construct, a method has been developed for producing a biocompatible (lack of cytotoxicity and immunogenicity) tissue-specific scaffold from decellularized human pancreatic tissue. In the scaffold, the morphofunctional properties of the native extracellular matrix-based scaffolds of the pancreas are preserved. Adhesion and proliferation of cell cultures are ensured.