BACKGROUND: antiseptic solutions and abrasive agents are widely used in dentistry to decontaminate surfaces, which can have a cytotoxic effect on progenitor cells. At the same time, there are no comparative studies evaluating the cytocompatible properties of such substances in cultures of multipotent human mesenchymal cells. AIM: to comprehensively evaluate the immediate and delayed cytotoxic effects of antiseptics and abrasives used in dentistry on MSCs of human adipose tissue (MSC AT) in vitro. METHODS: Human MSC AT was incubated with solutions of antiseptics (Miramistin 0.01%, Chlorhexidine 0.05%, Octenicept) and abrasive powders (sodium bicarbonate, glycine, erythritol) in initial concentrations and at dilutions of 2, 4 and 10 times. Cell viability was assessed immediately after exposure and after 24 hours using an MTT test and fluorescence microscopy. RESULTS: All substances demonstrated dose-dependent cytotoxicity. Among antiseptics, Miramistin turned out to be the least toxic. Chlorhexidine at baseline significantly reduced cell survival in both immediate and delayed assessment. Octenicept caused pronounced cell death and led to cell fixation. Among the abrasives, glycine and soda had acceptable cytocompatibility. Erythritol showed high cytotoxicity. CONCLUSION: For preserving the regenerative potential of MSC AT, Miramistin in diluted form and sodium bicarbonate are the most biocompatible agents used for decontamination. The use of Octenicept and erythritol in clinically significant concentrations is associated with irreversible damage to progenitor cells.

Relevance. Studies on the development of squamous cell carcinoma of the oral mucosa are of paramount importance due to the widespread of the disease and its aggressive course. When histological examination of the development of squamous cell carcinoma of the oral mucosa is not always possible to determine the first signs of malignancy. Squamous cell carcinoma can develop from epithelial hyperplasia and epithelial dysplasia of varying severity. In addition to histological research methods, immunohistochemical method is widely used for tumor diseases, in which the Ki-67 protein is used, with which the proliferation of epithelial cells can be determined, as well as the P53 protein encoding the TP53 gene, which is a suppressor of tumor growth. It is activated only in the presence of damage to the cell genome. The aim was to evaluate the expression of Ki-67 and P53 proteins in the development of dysplasia and squamous cell carcinoma of the oral mucosa. Materials and Methods. Four groups were identified for the study: group 1-16 patients (34.7%) diagnosed with epithelial hyperplasia, group 2-8 patients (17.3%) diagnosed with low-grade epithelial dysplasia, group 3-9 (19.5%) with a diagnosis of “high-grade epithelial dysplasia”, group 4-13 (28.2%) with a diagnosis of “squamous cell carcinoma”. Mouse monoclonal antibodies to Ki-67 (clone MM1, Diagnostic Biosystems, USA) was used to determine cell proliferation. P53 expression was determined using mouse monoclonal antibodies to the P53 protein (Clone D 07, Novocastra, UK). Monoclonal rabbit antibodies P53 (Clone Y5 Epitomics, USA) were used to study only the “mutant type” of the P53 protein. Results and Discussion. The expression of Ki-67 and P53 proteins were observed in all groups. However, the minimum number of immunopositive cells in the study of P53 (Clone D 07) and P53 (Clone Y5) was observed in the epithelial hyperplasia group, and the maximum in the squamous cell carcinoma group. The increase in the number of stained cells significantly increased as the degree of epithelial dysplasia increased from epithelial hyperplasia and low grade epithelial dysplasia to high grade epithelial dysplasia, etc. Conclusion. Thus, the detection of the expression of the P53 protein (Clone Y5) in the epithelium indicates the presence of changes in the genetic apparatus and metabolism of cells, which can be used in the early diagnosis of squamous cell carcinoma of the oral mucosa.

The use of 3D-printed gene-activated bone grafts represents a highly promising approach in the fields of dentistry and orthopedics. Bioresorbable poly-lactic-co-glycolic acid (PLGA) scaffolds, infused with adenoviral constructs that carry osteoinductive factor genes, may provide an effective alternative to existing bone grafts for the reconstruction of extensive bone defects. This study aims to develop and investigate the properties of 3D scaffolds composed of PLGA and adenoviral constructs carrying the BMP2 gene (Ad-BMP2), both in vitro and in vivo. The elastic modulus of the disk-shaped PLGA scaffolds created using a specialized 3D printer was determined by compressive testing in both axial and radial directions. In vitro cytocompatibility was assessed using adipose-derived stem cells (ADSCs). The ability of Ad-BMP2 to transduce cells was evaluated. The osteoinductive and biocompatible properties of the scaffolds were also assessed in vivo. The Young’s modulus of the 3D-printed PLGA scaffolds exhibited comparable values in both axial and radial compression directions, measuring 3.4 ± 0.7 MPa for axial and 3.17 ± 1.4 MPa for radial compression. The scaffolds promoted cell adhesion and had no cytotoxic effect on ADSCs. Ad-BMP2 successfully transduced the cells and induced osteogenic differentiation in vitro. In vivo studies demonstrated that the 3D-printed PLGA scaffolds had osteoinductive properties, promoting bone formation within the scaffold filaments as well as at the center of a critical calvarial bone defect.

Introduction. When examining the oral mucosa (OM) in the presence of an inflammatory process, the initial signs of malignancy are not always visible. Amid an inflammatory infiltrate, benign changes such as epithelial hyperplasia (EH), epithelial dysplasia (ED) of varying severity, and squamous cell carcinoma (SCC) can be hidden. The aim of the paper was to study the proliferative activity of epithelial cells of the OM, TERT expression, and amplification of telomerase TERC gene in the development of epithelial hyperplasia, dysplasia, and SCC. Materials and methods. We divided 60 patients into four groups: Group 1 with EH (n=16, 26.6%); Group 2 with low-grade epithelial dysplasia (LGED) [n=16, 26.6%]; Group 3 with high-grade epithelial dysplasia (HGED) [n=13, 21.6%]; and Group 4 with SCC (n=15, 25%). IHC and histological studies were carried out using standard methods; expression to Ki-67 protein and telomerase activity were evaluated. The FISH method was used with LSP TERC and CCP3 DNA probes. Results. High-cell proliferation and telomerase activity were revealed by IHC study in all groups. Amplification of TERC RNA site in chromosome 3 by FISH in Group 1 was almost absent and showed a 2:2 signal ratio in 1.3% of cases; in other groups, the amplification of TERC gene was positive: in groups 2, 3, and 4 it was in 83.9%, 97.3%, and 89.8% of cases, respectively. Conclusion. Amplification of TERC site signals in chromosome 3 is detected during malignization which may be the main criterion for early diagnosis of malignization of OM epithelium. Keywords: squamous cell carcinoma, epithelial dysplasia, TERC amplification, telomerase

Multi-organ failure is one of the common causes of fatal outcome in COVID-19 patients. However, the pathogenetic association of the SARS-CoV-2 viral load (VL) level with fatal dysfunctions of the lungs, liver, kidneys, heart, spleen and brain, as well as with the risk of death in COVID-19 patients remains poorly understood. SARS-CoV-2 VL in the lungs, heart, liver, kidneys, brain, spleen and lymph nodes have been measured by RT qPCR using the following formula: NSARS-CoV−2/NABL1 × 100. Dissemination of SARS-CoV-2 in 30.5% of cases was mono-organ, and in 63.9% of cases, it was multi-organ. The average SARS-CoV-2 VL in the exudative phase of diffuse alveolar damage (DAD) was 60 times higher than in the proliferative phase. The SARS-CoV-2 VL in the lungs ranged from 0 to 250,281 copies. The “pulmonary factors” of SARS-CoV-2 multi-organ dissemination are the high level of SARS-CoV-2 VL (≥4909) and the exudative phase of DAD. The frequency of SARS-CoV-2 dissemination to lymph nodes was 86.9%, heart–56.5%, spleen–52.2%, liver–47.8%, kidney–26%, and brain–13%. We found no link between the SARS-CoV-2 VL level in the liver, kidneys, and heart and the serum level of CPK, LDH, ALP, ALT, AST and Cr of COVID-19 patients. Isolated detection of SARS-CoV-2 RNA in the myocardium of COVID-19 patients who died from heart failure is possible. The pathogenesis of COVID-19-associated multi-organ failure requires further research in a larger cohort of patients.

BACKGROUND:Mineralized lesions of the jaws are often found incidentally on radiographs and computed tomography. Most of them are benign, and only a few rare cases are associated with malignant transformation. However, there is little clinical data on successful rehabilitation with implants in patients with mineralized lesions. This narrative review aimed to study the efficiency and safety of dental implantation in the area of hyperdense lesions.MATERIALS AND METHODS:A PubMed, Google Scholar, and Science Direct database search was carried out with keywords and manually.RESULTS:The literature exploration identified 323 articles; only 19 of them matched the search criteria and reported cases about dental implantation in the lesion area. It has been shown that in 84.2% of described cases, dental implantation was successful: in the osteoid osteoma, odontoma, cementoblastoma, idiopathic osteosclerosis, and condensing osteitis areas dental implantation was performed without any complications. The possibility of lesion recurrence and implant failure limited the use of dental implants in the area of osteoblastoma and cemento-osseous dysplasia. Although most cases of dental implantation in hyperdense jaw lesions were successful and were not accompanied by complications, further research is needed.

In dentistry, maxillofacial surgery, traumatology, and orthopedics, there is a need to use osteoplastic materials that have not only osteoinductive and osteoconductive properties but are also convenient for use. In the study, compositions based on collagen hydrogel were developed. Polylactide granules (PLA) or a traditional bone graft, a mixture of hydroxyapatite and β-tricalcium phosphate (HAP/β-TCP), were used for gel filling to improve mechanical osteoconductive properties of compositions. The mechanical tests showed that collagen hydrogels filled with 12 wt% highly porous PLA granules (elastic modulus 373 ± 55 kPa) or 35 wt% HAP/β-TCP granules (elastic modulus 451 ± 32 kPa) had optimal manipulative properties. All composite components were cytocompatible. The cell's viability was above 90%, and the components' structure facilitated the cell's surface adhesion. The bone morphogenetic protein-2 (BMP-2) provided osteoinductive composition properties. It was impregnated directly into the collagen hydrogel with the addition of fibronectin or inside porous PLA granules. The implantation of a collagen hydrogel with BMP-2 and PLA granules into a critical-size calvarial defect in rats led to the formation of the most significant volume of bone tissue: 61 ± 15%. It was almost 2.5 times more than in the groups where a collagen-fibronectin hydrogel with a mixture of HAP/β-TCP (25 ± 7%) or a fibronectin-free composition with porous PLA granules impregnated with BMP-2 (23 ± 8%) were used. Subcutaneous implantation of the compositions also showed their high biocompatibility and osteogenic potential in the absence of a bone environment. Thus, the collagen-fibronectin hydrogel with BMP-2 and PLA granules has optimal biocompatibility, osteogenic, and manipulative properties.

Lymphopenia is a frequent hematological manifestation, associated with a severe course of COVID-19, with an insufficiently understood pathogenesis. We present molecular genetic immunohistochemical, and electron microscopic data on SARS-CoV-2 dissemination and viral load (VL) in lungs, mediastinum lymph nodes, and the spleen of 36 patients who died from COVID-19. Lymphopenia <1 × 109/L was observed in 23 of 36 (63.8%) patients. In 12 of 36 cases (33%) SARS-CoV-2 was found in lung tissues only with a median VL of 239 copies (range 18–1952) SARS-CoV-2 cDNA per 100 copies of ABL1. Histomorphological changes corresponding to bronchopneumonia and the proliferative phase of DAD were observed in these cases. SARS-CoV-2 dissemination into the lungs, lymph nodes, and spleen was detected in 23 of 36 patients (58.4%) and was associated with the exudative phase of DAD in most of these cases. The median VL in the lungs was 12,116 copies (range 810–250281), lymph nodes—832 copies (range 96–11586), and spleen—71.5 copies (range 0–2899). SARS-CoV-2 in all cases belonged to the 19A strain. A immunohistochemical study revealed SARS-CoV-2 proteins in pneumocytes, alveolar macrophages, and bronchiolar epithelial cells in lung tissue, sinus histiocytes of lymph nodes, as well as cells of the Billroth pulp cords and spleen capsule. SARS-CoV-2 particles were detected by transmission electron microscopy in the cytoplasm of the endothelial cell, macrophages, and lymphocytes. The infection of lymphocytes with SARS-CoV-2 that we discovered for the first time may indicate a possible link between lymphopenia and SARS-CoV-2-mediated cytotoxic effect.

Compositions based on chitosan/β-glycerophosphate hydrogels with highly porous polylactide granules can be used to obtain moldable bone graft materials that have osteoinductive and osteoconductive properties. To eliminate the influence of such characteristics as chain length, degree of purification, and molecular weight on a designed material, the one-stock chitosan sample was reacetylated to degrees of deacetylation (DD%) of 19.5, 39, 49, 55, and 56. A study of the chitosan/β-glycerophosphate hydrogel with chitosan of a reduced DD% showed that a low degree of deacetylation increased the MSCs (multipotent stromal cells) viability rate in vitro and reduced the leukocyte infiltration in subcutaneous implantation to Wistar rats in vivo. The addition of 12 wt% polylactide granules resulted in optimal composite mechanical and moldable properties, and increased the modulus of elasticity of the hydrogel-based material by approximately 100 times. Excessive filling of the material with PLA (polylactide) granules (more than 20%) led to material destruction at a ~10% strain. Osteoinductive and osteoconductive properties of the chitosan hydrogel-based material with reacetylated chitosan (39 DD%) and highly porous polylactide granules impregnated with BMP-2 (bone morphogenetic protein-2) have been demonstrated in models of orthotopic and ectopic bone formation. When implanted into a critical-size calvarial defect in rats, the optimal concentration of BMP-2 was 10 μg/mL: bone tissue areas filled the entire material’s thickness. Implantation of the material with 50 μg/mL BMP-2 was accompanied with excessive growth of bone tissue and material displacement beyond the defect. Significant osteoinductive and osteoconductive properties of the material with 10 μg/mL of BMP-2 were also shown in subcutaneous implantation.

In 2020 the world faced the pandemic of COVID-19 severe acute respiratory syndrome caused by a new type of coronavirus named SARS-CoV-2. To stop the spread of the disease, it is crucial to create molecular tools allowing the investigation, diagnoses and treatment of COVID-19. One of such tools are monoclonal antibodies (mAbs). In this study we describe the development of hybridoma cells that can produce mouse mAbs against receptor binding domain of SARS-CoV-2 spike (S) protein. These mAbs are able to specifically detect native and denatured S proteins in all tested applications, including immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence staining of cells and immunohistochemical staining of paraffin embedded patients’ tissue samples. In addition, we showed that the obtained mAbs can efficiently block SARS-CoV-2 infection in in vitro experiments. Finally, we determined the amino acid sequence of light and heavy chains of the mAbs. This information will allow the use of corresponding peptides to establish genetically engineered therapeutic antibodies. To date multiple mAbs against SARS-CoV-2 proteins have been established, however, bigger sets of various antibodies will allow the detection and neutralization of SARS-CoV-2, even if the virus acquires novel mutations.

Magnetic levitational bioassembly of three-dimensional (3D) tissue constructs represents a rapidly emerging scaffold- and label-free approach and alternative conceptual advance in tissue engineering. The magnetic bioassembler has been designed, developed, and certified for life space research. To the best of our knowledge, 3D tissue constructs have been biofabricated for the first time in space under microgravity from tissue spheroids consisting of human chondrocytes. Bioassembly and sequential tissue spheroid fusion presented a good agreement with developed predictive mathematical models and computer simulations. Tissue constructs demonstrated good viability and advanced stages of tissue spheroid fusion process. Thus, our data strongly suggest that scaffold-free formative biofabrication using magnetic fields is a feasible alternative to traditional scaffold-based approaches, hinting a new perspective avenue of research that could significantly advance tissue engineering. Magnetic levitational bioassembly in space can also advance space life science and space regenerative medicine.

Reproducible, scalable, and cost effective fabrication and versatile characterization of tissue spheroids (TS) is highly demanded by 3D bioprinting and drug discovery. Consistent geometry, defined mechanical properties, optimal viability, appropriate extracellular matrix/cell organization are required for cell aggregates aimed for application in these fields. A straightforward procedure for fabrication and systematic multiparametric characterization of TS with defined properties and uniform predictable geometry employing non-adhesive technology is suggested. Applying immortalized and primary cells, the reproducibility of spheroid generation, the strong correlation of ultimate spheroid diameter, and growth pattern with cell type and initial seeding concentration are demonstrated. Spheroids viability and mechanical properties are governed by cell derivation. In this study, a new decision procedure to apply for any cell type one starts to work with to prepare and typify TS meeting high quality standards in biofabrication and drug discovery is suggested.

Background. Chronic endometritis is one of the causes of infertility, miscarriage, failed in vitro fertilization and embryo transfer attempts. Treatment of chronic endometritis is quite complicated due to a multifactorial nature of endometrium morpho-functional disorders. There are lots of approaches to a comprehensive treatment of chronic endometritis which indicates a lack of a single algorithm for a management of such patients to date. One of the modern, perspective and high-tech methods for treatment of chronic endometritis is photodynamic therapy (PDT). Aim. To study chronic endometritis treatment efficacy in patients with infertility when using the method of PDT and low doses of 17b-estradiol. Materials and methods. 85 female patients of reproductive age with chronic endometritis were examined and treated. All patients were divided into 2 groups: group 1 (43 patients) got treated by a method of intravenous PDT with low-dose transdermal 17b-estradiol; group 2 (42 patients) received low-dose transdermal therapy with 17b-estradiol. Clinical examination and laboratory tests, pelvic ultrasound, endometrial aspiration biopsy followed by pathomorphlogical and immunohistochemical examination were carried out to all patient. Results. Ultrasound examination revealed an increase in endometrium thickness at 12th day following PDT session in group 1 and following transdermal therapy with 17b-estradiol in group 2 resulting in endometrium state improvement almost 2 times compared with baseline values before treatment. Conclusions. PDT is a minimally invasive, gentle and safe treatment method. Due to a diffuser design laser radiation is evenly distributed in the uterine cavity. PDT reliably restores receptor function to progesterone in the endometrial glands.

OBJECTIVE:Chondrospheres represent a variant of tissue spheroids biofabricated from chondrocytes. They are already being used in clinical trials for cartilage repair; however, their biomechanical properties have not been systematically investigated yet. The aim of our study was to characterize chondrospheres in long-term in vitro culture conditions for morphometric changes, biomechanical integrity, and their fusion and spreading kinetics.RESULTS:It has been demonstrated that the increase in chondrospheres secant modulus of elasticity is strongly associated with the synthesis and accumulation of extracellular matrix. Additionally, significant interplay has been found between biomechanical properties of tissue spheroids and their fusion kinetics in contrast to their spreading kinetics.CONCLUSIONS:Extracellular matrix is one of the main structural determinants of chondrospheres biomechanical properties during chondrogenic maturation in vitro. The estimation of tissue spheroids' physical behavior in vitro prior to operative treatment can be used to predict and potentially control fusogenic self-assembly process after implantation in vivo.

The possibility of using immunohistochemical study and the method of tissue PCR in the complex diagnosis of vesicular lesions of the oral mucosa has been studied. A total clinical and laboratory examination included 57 patients, including: patients with pemphigoid bullosa (32), pemphigus vulgaris (25). Results of immunohistochemical study indicate cellular mechanisms of epithelial damage, in which IgG, C3d fragments of the complement take part, receptors for cytokine-2, as well as antibodies against E-cadherin protein. Identification of microorganisms in pathologically altered tissues by the method of PCR should be regarded as one of the triggers of the antigenic factor in the vesicular lesions of the oral mucosa. The obtained data convincingly prove the necessity of application of these methods in complex diagnostics of vesicular lesions of the oral mucosa.

A comparative analysis of the proliferative activity of inflammatory infiltrate cells and the distribution of collagen type I and III in granulomas formed in the area of contact of mesh materials made of polypropylene (PP Std Light) and titanium-coated polypropylene (TiMesh) was performed via polarized microscopy and immunohistochemical detection of Ki-67 protein. Mesh materials were implanted in the soft tissues of the lumbar region of rats. The number of proliferative cells around implants made of the standard polypropylene (PP Std Light) 7 days after the operation was detected with the use of antibodies to Ki-67 protein to be less than the number of those around titanium-coated polypropylene (TiMesh) (29.1 ± 5.7 and 33.6 ± 3.1%, respectively, p < 0.001). The similar differences were revealed 1 month after the beginning of the experiment (15.9 ± 4.3 and 26.9 ± 3.6%, respectively, p < 0.001). Various types of collagen fibers in granulomas around the implanted mesh materials were detected in polarized light with the use of sections stained with Sirius Red. The ratios between collagen fibers type I and III in granulomas around PP Std Light and TiMesh materials determined 7 days after the operation were 1.085 ± 0.022 and above 1.107 ± 0.013, respectively (p = 0.017). The ratio increased to 1.174 ± 0.036 and 1.246 ± 0.102, respectively (p = 0.045) 1 month after the operation. The obtained results suggest that the titanium coating of polypropylene stimulates the formation of collagen type I and more mature connective tissue around mesh endoprostheses.

Pigment Epithelium-Derived Factor (PEDF) is a secreted glycoprotein belonging to the family of non-inhibitory serpins. It is known, that in cases of complicated myopia, the content of PEDF in aqueous humor of the anterior chamber is significantly reduced. Here we examined a bulk of Tenon's capsule samples obtained from various groups of myopes, to examine PEDF processing in progressive myopia. We have analyzed the distribution of full length PEDF50 and its truncated form PEDF45 in the soluble and insoluble fractions extracted from Tenon's capsule of myopic and control (non-myopic) patients using SDS-polyacrylamide gel electrophoresis, as well as monitored the proteolytic degradation of PEDF ex vivo by enzyme-linked immunosorbent assay. These results were complemented by PEDF mRNA analysis in correspondent tissues by using qPCR and immunohistochemistry analysis of PEDF distribution in normal and myopic specimens. We found that in the Tenon's capsule of patients suffering from a high myopia the level of "soluble" 45 kDa PEDF reduced by 2-fold, while the content of "insoluble" 50 kDa form of PEDF was increased by 4-fold compared to controls. Excessive amount of PEDF50 in myopic specimens have been shown to correlate with the abrogated PEDF processing rather than with an increase of its expression. Moreover, immunohistochemical staining of the myopic Tenon's capsule tissue sections revealed the halo of deposited PEDF50 in the fibroblast extracellular space. These findings suggest that in myopia limited proteolysis of PEDF is altered or abrogated. Accumulation of full-length PEDF insoluble aggregates in the fibroblast intercellular space may affect cell survival and consequently causes the destructive changes in the extracellular matrix of the eye connective tissues. As a result, the abrogation of full-length PEDF normal processing can be an important mechanism leading to biomechanical destabilization of the scleral capsule and myopia progression.

An immunohistochemical study on the cells proliferative activity by Ki-67 protein and localization of the matrix metalloproteinase-9 and the inhibitor of matrix metalloproteinase-1 was carried out at the benign prostatic hyperplasia (BPH) and the adenocarcinoma (AC) of different Gleason's grades. A significant decrease of the MMP-9 and TIMP-1 level in the AC of different gradations was observed. A moderate positive correlation between the Gleason score and cell proliferation Ki-67 index (rs = 0.674) and a moderate negative correlation with the level of such a score and expression of MMP-9 (rs = -0.660) was detected. A weak negative correlation exists also between the level of proliferative activity of secretory cells and the expression of MMP-9 by tumor cells (rs = -0.369). The invasive properties of AC cells that promote a degradation of the basal membrane and connective tissue in prostate may be explained by the imbalance between the MMP-9 and TIMP-1, which expression is significantly reduced in AC, in comparison with BPH.