One of the key and actively developing areas of regenerative medicine is tissue-engineering. There is no doubt that the use of tissue-engineering products can have a significant impact on the efficiency of repair of damaged tissues and organs. However, before being used in clinical practice, tissue-engineering products require thorough preclinical studies to confirm their safety and efficacy, both with in vitro models and in experimental animals. This paper presents preclinical studies of a tissue-engineered construct, based on a hydrogel biopolymer scaffold carrier (consisting of blood plasma cryoprecipitate and collagen) with encapsulated mesenchymal stem cells, to evaluate its biocompatibility in vivo. The results were analyzed using histomorphology and transmission electron microscopy. It was shown that when implanted into animal (rat) tissues, the implants were completely replaced by connective tissue components. We also confirmed that no acute inflammation occurred in response to the scaffold implantation. The observed processes of cell recruitment to the scaffold from the surrounding tissues, the active formation of collagen fibers and the absence of acute inflammation testified that the regeneration process was ongoing in the implantation area. Thus, the presented tissue-engineered construct shows promise for becoming an effective tool for regenerative medicine in the future and may be used, in particular, to repair soft tissues.

The aim of the investigation is to study structural alterations of autologous omental adipose tissue in a silicon conduit and to evaluate its possible use for regeneration of the sciatic nerve in diastasis.Materials and Methods. Mature outbred male Wistar rats have been used in the study. The animals were divided into 7 experimental groups with complete transection of the sciatic nerve on the right side at the mid-third level of the thigh. The ends of the transected nerve were pulled apart, inserted into a silicon conduit, and secured to the epineurium. The conduit of group 1 (control) was filled with a saline solution; in group 2, it was filled with an autologous omental adipose tissue with saline solution. Intravital labeling of the omental adipose tissue with the lipophilic PKH 26 dye (in group 3) was used for the first time to find out whether the omental cells were involved in formation of the regenerating nerve. Diastasis in groups 1-3 was 5 mm, the postoperative period was 14 weeks. The dynamics of the omental adipose tissue changes in groups 4-7 was assessed by placing the omental tissues into the conduit covering 2 mm of diastasis. The postoperative period was 4, 14, 21, and 42 weeks.Results. In group 2 (omental adipose tissue + saline), the clinical condition of the damaged limb after 14 weeks may be evaluated as satisfactory and approximating to the intact parameters as compared to group 1 where the conduit was filled with a saline solution only. The sum of large and medium-sized nerve fibers in group 2 was 2.7 times greater than that in group 2. The milled omental adipose tissue inside the conduit changed its volume and structure in nerve diastasis and was constantly utilized up to complete elimination over time. The omental cells integrated into the newly formed nerve in the graft area.Conclusion. As a graft, the adipose tissue of the autologous omentum produces a stimulating effect on the post-traumatic regeneration of the sciatic nerve.

AIM OF STUDY Was to compare the dynamics of engraftment of skin micrographs in a burn wound when using protectors from an allodermal graft and from a hydrogel coating.MATERIAL AND METHODS The experimental study was conducted on 18 rats with a scab formed 3 days after modeling a deep burn with an area of 20% of the body surface. Partial fascial necrectomy was performed: two rounded sections of the sling with a diameter of 25 mm were excised. 6 automicrographs of skin 4x4 mm, 0.3 mm thick, were applied to each surface freed from the scab. In each animal, micrographs on one of the wounds were covered with a hydrogel protector, on the other with an allodermotransplant from another animal of the group. A secondary aseptic dressing was applied to the protectors. On the 5th and 20th days after the operation, the state of micrographs was studied: blood circulation — according to laser Doppler flowmetry, microstructure in vivo — using optical coherence tomography, microstructure ex vivo — according to histological examination of biopsies.ReSUlTS Differences in the rate of restoration of blood circulation of micrographs in the early stages of the postoperative period were found. In the first 5 days, the perfusion of micrographs under an allodermal protector exceeded the indicator in micrographs under a hydrogel coating by 44 [21; 51] % (p=0.031) due to the contribution of endothelial and neurogenic mechanisms of blood flow modulation. Starting from day 10, the differences in perfusion were levelled, but there were signs of more active endothelial regulation of blood flow under the skin (p=0.028). Histologically, the appearance of full-blooded capillaries was revealed earlier in micrographs under the alloderm than when using a hydrogel protector. By 20 days, under the condition of regular change of hydrogel coatings, the area of wound healing under the studied coatings did not significantly differ. However, the structure of the integumentary tissue under the alloderm according to the optical coherence tomography data was closer to normal skin than when using a hydrogel protector.CONClUSIONS From the point of view of the physiology of the wound process, alloderm is the preferred option of an autograft protector in comparison with a hydrogel coating, which is probably due to the paracrine biological activity of the alloderm. However, hydrogel coatings can provide a comparable level of efficiency, provided they are regularly changed and, potentially, given the properties of cytokine activity.

The success of the regenerative process resulting from the implantation of a scaffold or a tissue-engineered structure into damaged tissues depends on a series of factors, including, crucially, the biodegradability of the implanted materials. The selection of a scaffold with appropriate biodegradation characteristics allows for synchronization of the degradation of the construct with the processes involved in new tissue formation. Thus, it is extremely important to characterize the biodegradation properties of potential scaffold materials at the stage of in vitro studies. We have analyzed the biodegradation of hybrid fibrin–collagen scaffolds in both PBS solution and in trypsin solution and this has enabled us to describe the processes of both their passive and enzymatic degradation. It was found that the specific origin of the collagen used to form part of the hybrid scaffolds could have a significant effect on the nature of the biodegradation process. It was also established, during comparative studies of acellular scaffolds and scaffolds containing stem cells, that the cells, too, make a significant contribution to changes in the biodegradation and structural properties of such scaffolds. The study results also provided evidence indicating the dependency between the pre-cultivation period for the cellular scaffolds and the speed and extent of their subsequent biodegradation. Our discussion of results includes an attempt to explain the mechanisms of the changes found. We hope that the said results will make a significant contribution to the understanding of the processes affecting the differences in the biodegradation properties of hybrid, biopolymer, and hydrogel scaffolds.

The intensity of accumulation and excretion of atrial natriuretic peptide in myocytes of the right atrium in rat models of renovascular hypertension and salt loading was studied by immunocytochemical analysis and transmission electron microscopy. The data suggest that high BP is not the decisive factor affecting secretion of atrial natriuretic peptide in atrial cardiomyocytes. The regulatory mechanisms of the accumulation and release of the peptide from myocyte granules can vary and depend on the pathogenesis of hypertension.

The activity of stem cell processes is regulated by internal and external signals of the cell "niche". In general, the niche of stem cells can be represented as the microenvironment of the cells, providing a signal complex, determining the properties of the cells. At the same time, the "niche" concept implies feedback. Cells can modify their microenvironment, supporting homeostasis or remodeling the composition and structure of the extracellular matrix. To ensure the regenerative potential of tissue engineering products the "niche" concept should be taken into account. To investigate interactions in an experimental niche, an original hydrogel biopolymer scaffold with encapsulated mesenchymal adipose-derived stem cells (ASCs) was used in this study. The scaffold provides for cell adhesion, active cell growth, and proliferative activity. Cells cultured within a scaffold are distinguished by the presence of a developed cytoskeleton and they form a cellular network. ASCs cultured within a scaffold change their microenvironment by secreting VEGF-A and remodeling the scaffold structure. Scaffold biodegradation processes were evaluated after previous culturing of the ASCs in the scaffolds for periods of either 24 h or six days. The revealed differences confirmed that changes had occurred in the properties of scaffolds remodeled by cells during cultivation. The mechanisms of the identified changes and the possibility of considering the presented scaffold as an appropriate artificial niche for ASCs are discussed.

The aim of the study was to compare two types of conduits made of either non-resorbable Reperen or resorbable Tissucol for their effects on the regeneration of the rat sciatic nerve under conditions of stump diastasis.MATERIALS AND METHODS:The experiments were carried out using outbred white male rats of the reproductive age (n=14). The animals were divided into three groups: group 1, intact (n=5), used for studying the morphology of the sciatic nerve; group 2 (n=4) - nerve plastic surgery was performed using a conduit made of non-resorbable Reperen; group 3 (n=5) - surgery was performed using a conduit made of resorbable Tissucol. The animals were anesthetized with isoflurane. After a complete transection of the sciatic nerve in the middle third of the thigh, its stumps were inserted into a conduit of an internal diameter of 2 mm and a length of 10 mm, filled with saline. Diastasis of 5 mm in length was created by spreading the nerve ends and securing the epineurium to the tube edges with 8/0 polypropylene sutures. A total count of myelinated nerve fibers was performed in the area of repair (tubulation) and the distal part of the nerve; the formation of connective tissue sheaths was assessed 14 weeks after the operation.RESULTS:According to the morphological assessment, both types of conduits (resorbable and non-resorbable) caused the similar number of fibers to restore in the distal part of the repaired nerve; clinical characteristics of the animals in both groups were close to each other and to the norm.CONCLUSION:The results allow us to consider the conduit made of non-resorbable Reperen as a device promising for neuroplasty along with the resorbable conduit made of Tissucol.

At present there is a growing need for tissue engineering products, including the products of scaffold-technologies. Biopolymer hydrogel scaffolds have a number of advantages and are increasingly being used to provide means of cell transfer for therapeutic treatments and for inducing tissue regeneration. This work presents original hydrogel biopolymer scaffolds based on a blood plasma cryoprecipitate and collagen and formed under conditions of enzymatic hydrolysis. Two differently originated collagens were used for the scaffold formation. During this work the structural and mechanical characteristics of the scaffold were studied. It was found that, depending on the origin of collagen, scaffolds possess differences in their structural and mechanical characteristics. Both types of hydrogel scaffolds have good biocompatibility and provide conditions that maintain the three-dimensional growth of adipose tissue stem cells. Hence, scaffolds based on such a blood plasma cryoprecipitate and collagen have good prospects as cell carriers and can be widely used in regenerative medicine.

Immunocytochemistry and transmission electron microscopy were employed to examine the ultrastructural morphometric parameters of the left ventricular cardiomyocytes and right atrial secretory myocytes in rats during early and delayed postreperfusion periods. The revealed alterations in these cells are stereotypical, but differed by their severity, probably due to specific morphofunctional peculiarities of these heart structures.

We investigated the effect of mexicor on functional indices of erythrocytes and the structure of myocardial microcirculation in rats suffering from traumatic brain injury (TBI). At 3, 7, and 12 days after TBI, we measured the concentration of 2,3-diphosphoglycerate (2,3-DPG) and the degree of erythrocyte aggregation and their electrophoretic mobility (EPME) in the blood of rats, as well as analyzing sections of the left ventricular myocardium. The first day after the TBI, we observed a decrease in EPME, an increase of erythrocyte aggregation, and an increase of 2,3-DFG concentration in erythrocytes as compared with intact animals. Intraperitoneal injection of mexicor led to an increase of EPME and 2,3-DPG level and reduced the aggregation of erythrocytes, which was most pronounced during the 3–7 days of the post-traumatic period. Improved functional parameters of erythrocytes were accompanied by the dynamics of regenerative processes in the heart. Intraperitoneal injection of mexicor restrained architectonic damage of microvasculature and cardiomyocytes ultrastructure of the left ventricular myocardium of the heart.

The aim of the investigation is to assess the influence of salt load on atrial (ANP) and brain (BNP) natriuretic peptide production in granules of secretory cardiomyocytes in rats.Materials and Methods. The experiments were carried out on 14 white out-bred male Wistar rats weighing 280-300 g. During the experiment all the animals were treated with standard-feed diet and had unlimited access to food and water. NaCl solution was introduced per os in the dose of 1 g per 1 kg of body mass during 14 days. Arterial pressure (AP) was measured noninvasively using a tail-cuff method. ANP and BNP production of atrial cardiomyocytes was studied by means of immunohistochemistry, transmission electron microscopy, immunocytochemistry. There was performed a morphometric analysis of granules containing peptides (A-type - "mature, storing" and B-type - "dissolving").Results. Increase in the number of granules with ANP and decrease in those with BNP accompanied by elevated AP was revealed 14 days after NaCl intake as compared to intact animals.Conclusion. Natriuretic peptides metabolism is regulated by various mechanisms. Early BNP release does not promote AP reduction due to compensatory mechanism disturbance in salt-induced arterial hypertension. Increase in ANP production occurs under the influence of renin-angiotensin-aldosterone system and elevated AP. The present data can indicate adaptive reaction in response to salt loading.

Accumulation and release of atrial and brain natriuretic peptides (ANP and BNP) in right atrial cardiac muscle cells has been investigated in rats after 60 minutes and 60 days after the reperfusion start. The total ischemia was simulated by the method of V. G. Korpachev. Immunocytochemical localization of peptides in cardiomyocytes was performed in ultrathin sections using polyclonal antibodies. The intensity of accumulation/excretion of ANP and BNP were analyzed by the method of counting the number of granules (A- and B-types) with immunoreactive labels in 38 x 38 mkm2 visual fields in transmission electron microscope Morgagni 268D (FEI). The results were assessed using Mann-Whitney U-test (p < 0.05). After 60 minutes and 60 days post-reperfusion period, we detected an increase in the synthesis and release of ANP and BNP. The reaction of BNP was more pronounced than ANP. This is due to the fact that ANP is the main hormone of the natriuretic peptide system involved in the regulation of blood pressure in normal conditions, while BNP is the principal regulator of pressure in cardiovascular pathology.

We have studied the accumulation and excretion of atrial (ANP) and brain (BNP) natriuretic peptides in the early and late postreperfusion period (60 min and 60 days) in the myocardium of the right atrium in rats. The model of total ischemia proposed by Korpachev et al. (1982) was used. Immunocytochemical localization of peptides in cardiomyocytes was performed on ultrathin sections using the polyclonal antibodies. The intensities of accumulation (excretion) of ANP and BNP were analyzed by counting the immunolabeled granules (types A and B) with a transmission electron microscope. At 60 min and 60 days of the postreperfusion period, an increase in the synthesis and release of ANP and BNP was found. A more pronounced BNP reaction could be explained by the fact that, under normal conditions, the main hormone of the natriuretic peptide system regulating blood pressure is ANP, while BNP regulates blood pressure in cardiovascular pathology.

Brain natriuretic peptide (BNP) participates in electrolyte balance maintenance in the body playing a critical part in the pathogenesis of cardiovascular diseases, and has the prognostic value in clinical presentation. It is of interest to analyze the peculiarities of BNP interaction with medicinal drugs, e.g. Mexidol, an antihypoxic agent of metabolic type that has a cardioprotective effect and is widely used in cardiology. The effect of Mexidol on BNP in a post-reperfusion period was studied for the first time.The aim of the investigation was to estimate the effect of Mexidol on BNP accumulation and release intensity in cardiomyocyte granules in rats in a post-reperfusion period.Materials and Methods. The experiments were carried out on 25 outbred male rats weighing 220-250 g. Total ischemia (10 min) was modeled by cardiovascular bundle compression according to Korpachev. Mexidol was administered intermittently, it being injected intraperitoneally after resuscitation, every 20 min within the first hour. BNP accumulation and release intensity was assessed by a quantitative analysis of immunolabeled granules of atrial myocytes under a transmission electron microscope.Results. Mexidol administered at a dose of 25 mg/kg body mass within the first hour reperfusion after 10 min of total ischemia has a positive prolonged effect on BNP: after 60 days of a postperfusion period the processes of peptide accumulation and release in atrial myocytes of rats enhance resulting in an additional cardioprotective effect. The increase of BNP release against high synthetic and proliferative activity of fibroblasts contributes to the reduction of cardiosclerosis development in a long-term post-reperfusion period.The study of immunolabeled granules of BNP myocytes in rat right atrium enabled to discover a new mechanism of a cardioprotective effect of Mexidol in a long-term post-reperfusion period.Conclusion. Mexidol has a prolonged effect on brain natriuretic peptide and significantly enhances its accumulation and release in atrial cardiomyocytes of rats in a long-term post-reperfusion period having an additional cardioprotective effect and reducing cardiosclerosis development.

Effects of successive exposure to ionizing irradiation and low-intensity broadband red light on electrical activity of the heart and myocardium microstructure were studied in rats. Lowintensity red light corrected some ECG parameters, in particular, it normalized QT and QTc intervals and voltage of R and T waves. Changes in ECG parameters were followed by alterations in microstructure of muscle fi laments in the myocardium of treatment group animals comparing to control group.

We studied the intensities of accumulation and secretion of brain natriuretic peptide (BNP) in the early postreperfusion period, i.e., 5 min and 1 h after the start of reperfusion in the myocardium of the right atrium of rats. Total ischemia was modeled using Korpachev’s method. Immunocytochemical detection of peptide distribution in secretory cardiomyocytes was performed in ultrathin sections using polyclonal antibodies to BNP. The immunoreactive granules of A and B types were counted in optical fields of 38 × 38 µm2 using a transmission electron microscope. Five minutes after the start of reperfusion, the number of BNPcontaining secretory granules was similar to that observed in the control animals. After a 1-h postreperfusion period, we observed active formation and release of BNP because the number of granules of A and B-types increased by 134 and 210%, respectively, as compared to the intact group. These data demonstrate the stimulating effects of ischemic and reperfusion factors on the processes of synthesis and secretion of BNP in this period.