Michael Ignatyuk

In this study, the neurorehabilitation potential of combined and isolated intermittent hypercapnia and hypoxia exposure was evaluated following photochemically induced cerebral thrombosis in rats. Particular attention was given to the roles of possible neuroplasticity mechanisms mediated by VEGF and BDNF, as well as the potential of hypercapnic–hypoxic interventions to synergistically amplify the therapeutic effects of pharmacological neuroprotectants during recovery. A total of 50 male Wistar rats were randomly assigned to five equal groups (n = 10 per group), each undergoing a course of respiratory interventions lasting 30 min per day for 15 sessions. The groups included (1) a normobaric hypoxia (PO₂ ≈ 90 mmHg) group, (2) a permissive hypercapnia (PCO₂ ≈ 50 mmHg) group, (3) a combined hypercapnic hypoxia (PO₂ ≈ 90 mmHg, PCO₂ ≈ 50 mmHg) group, (4) a control group, and (5) a sham-operated group. Following the rehabilitation protocol, animals exposed to hypercapnic hypoxia exhibited a two-fold reduction in stroke volume compared with controls, significant improvement in motor coordination (as assessed via the rotarod test), and marked upregulation of VEGF and BDNF expression within the ischemic brain region. Notably, only the HH group showed a decrease in serum neuron-specific enolase (NSE) levels. These findings indicate that hypercapnic hypoxia exerts a possible neurorehabilitative effect after focal ischemic injury, superior to that of isolated hypoxia or hypercapnia. Possible mechanisms underlying this outcome may involve activation of neurotrophic (BDNF) and angiogenic (VEGF) signaling pathways.

The rapid development of 3D bioprinting technology has not been critically evaluated for its potential clinical applications… The ability of 3D manufacturing to create organ-like structures obscures the fact that the formed grafts are not physiologically relevant. We hypothesize that researchers do not use techniques that allow for the evaluation of the micro-architectonics of formed implants and mainly focus on biocompatibility and commonly observed immunological responses. This study aims to investigate the morphological landscape of the basics of 3D bioprinting through a systematic review of the outcomes of the experimental implantation of bioprinted constructs. A systematic search was conducted in the PubMed database using the following query: (bioprinting OR printing OR bioprinted OR printed OR bioinks) AND (cell OR cells) AND (implantation OR implanted OR in vivo) AND (goat OR porcine OR pig OR swine OR dog OR rabbit OR sheep) NOT (human OR humans). This systematic review evaluated the preformed studies of the in vivo assessment the 3D-bioprinted constructs, and 41 articles meeting the inclusion criteria were selected. We concluded that 3D bioprinting has limited applications for forming living tissue for orthotopic implantation. Additionally, quantitative methods for evaluating the properties and morphological quality of implanted bioprinted constructs have not been developed for tissue engineering applications.

Background/Objectives: Radiobiology has shown heterogeneity in the sensitivity of cells to ionizing radiation, depending on a variety of conditions. The presence of an extracellular matrix (ECM) appears to confer a radioprotective effect on cells and can influence the cellular microenvironment by modulating the availability of oxygen and nutrients, which can affect cellular metabolism and stress responses. A three-dimensional cell culture allows the synergistic effect on cell survival to be obtained based not only on the radioprotective properties of the extracellular matrix but also on the stress-resistant endogenous properties of the cell culture. The aim of this study was to investigate the survival of chondrocytes in a 3D cell culture during high-dose ionizing irradiation. Methods: The properties of nasal chondrocytes were evaluated using a pellet culture model in which the cells were surrounded by a de novo synthesized extracellular matrix. Tissue cultures were exposed by gamma radiation at doses of 10, 100, and 1300 Gy. Cell viability was assessed after 2 days of irradiation by live/dead staining using confocal scanning laser microscopy. Results: Tissue-cultured chondrocytes survive after gamma-irradiation of low (10 Gy), medium (100 Gy), and high (1300 Gy) dosages; however, after irradiation of 1300 Gy, the percentage of surviving cells was lower. The average percentages of viable cells were evaluated as 82%, 79%, and 63% in low-, medium-, and high-dose groups, respectively. Conclusions: Under determined conditions, human cells are able to survive at doses of ionizing radiation that are significantly higher than the current limits.

Introduction: Langerhans cell histiocytosis (LCH) is a rare clonal myeloid disorder characterized by tissue infiltration with pathological antigen-presenting cells morphologically and phenotypically similar to Langerhans cells. Activating mutations in the MAPK/ERK signaling pathway play a key role in the pathogenesis of LCH. The aim of this study was to characterize the morphological and molecular features of the neoplastic process. Materials and Methods: This study presents a clinical case of a 34-year-old patient with a rare mixed form of LCH combined with Erdheim-Chester disease (ECD), involving multiple organs including kidneys, skin, bones, ENT organs, eyes, and the central nervous system. The patient also had diabetes insipidus, chronic external otitis, and presumptive hereditary forms of anemia and hemochromatosis. A comprehensive histopathological evaluation was performed, including histochemical, immunohistochemical, and immunofluorescent analyses, alongside molecular genetic testing using whole exome sequencing (WES) of blood and skin samples. The patient demonstrated a partial clinical and laboratory response to targeted therapy with trametinib and desmopressin. Results: Comparison of WES data from blood and skin revealed 71,953 shared variants consistent with likely germline changes, as well as 3,081 unique somatic variants in skin and 2,633 unique variants in blood. Low-level mosaic BRAF V600E mutation (VAF = 1%) was detected in the skin, alongside pathogenic germline variants in the STEAP3 (c.523-2A>T, NM_182915.3) and SLC40A1 (c.332T>A, p.Met111Lys, NM_014585.6) genes. Immunofluorescence analysis of the trephine biopsy revealed diverse cellular populations, including sparse tryptase-positive mast cells, which showed minimal contact with each other or other labeled cells. The populations of Vimentin⁺CD11b^–CD34⁺ and Vimentin⁺CD11b⁺CD34^– cells (excluding endothelial cells) accounted for median values of 0.68% and 7.78%, respectively, of the total Vimentin⁺ cells. Regarding CD68 and S100 expression, CD68⁺ cells predominated (median = 55.61%), S100⁺ cells constituted a median of 43.02%, whereas CD68⁺S100⁺ cells were the rarest (median = 2.174%), reflecting the transition of Langerhans cells to LCH cells, encompassing up to 4.81% of the cellular population. This hybrid microenvironment paralleled the patient’s systemic inflammatory activity (elevated acute-phase reactants), multi-organ involvement, and renal interstitial fibrosis/tubular atrophy with reduced kidney function, without serological or histological evidence of vasculitis. Conclusion: This case illustrates the histological heterogeneity and hybrid pathology of mixed histiocytosis, where canonical dendritic LCH cells coexist with macrophage-rich ECD-like stroma. An integrated approach involving a multidisciplinary clinical team, thorough molecular genetic testing, and the application of targeted therapy is essential for improving prognosis and quality of life in young patients.

Degenerative disc disease (DDD) is a major cause of chronic low back pain (LBP), yet the molecular mechanisms driving disc degeneration and pain remain poorly understood. This study analyzed intervertebral disc (IVD) tissue from 36 young patients (median age = 36.00 [31.00, 42.50] years) with herniated discs and LBP, alongside healthy controls, to investigate changes in the extracellular matrix (ECM) and neurochemical alterations. Disc degeneration was assessed using MRI (Pfirrmann grading) and histology (Sive’s criteria). Histochemical and immunohistochemical methods were used to evaluate aggrecan content, calcification, and the expression of nerve growth factor (NGF), substance P (SP), and S-100 protein. MRI findings included Pfirrmann grades V (30.55%), IV (61.11%), III (5.56%), and II (2.78%). Severe histological degeneration (10–12 points) was observed in three patients. Aggrecan depletion correlated with longer pain duration (r = 0.449, p = 0.031). NGF expression was significantly elevated in degenerated discs (p = 0.0287) and strongly correlated with SP (r = 0.785, p = 5.268 × 10⁻⁹). Free nerve endings were identified in 5 cases. ECM calcification, present in 36.1% of patients, was significantly associated with radiculopathy (r = 0.664, p = 0.005). The observed co-localization of NGF and SP suggests a synergistic role in pain development. These results indicate that in young individuals, aggrecan loss, neurochemical imbalance, and ECM calcification are key contributors to DDD and chronic LBP.

The current treatment of radiation-induced skin wounds utilizes mainly conventional therapies, including topical steroids, creams, ointments, and hydrogel dressings, which do not take into account the immunologic changes that occur in the skin after radiation exposure. Therefore, it is relevant to consider alternative therapies and their impact on changes in the immune landscape of the skin. The aim of this study was to investigate the effect of allogeneic minimally manipulated keratinocytes and fibroblasts on rat skin repair and the development of immune responses. We found that the use of cell therapy compared to treatment with syntazone ointment and no treatment resulted in faster healing and a reduction in the size of radiation-induced skin wounds, area of inflammation, and edema. Additionally, in the group receiving the cell therapy application, there was an observed increase in the number of mast cells (MCs), activation of MC interaction with M2 macrophages, a reduction in the direct contact of MCs with the vascular bed, an increase in the content of collagen fibers due to the intensification of collagen fibrillogenesis, and a restoration of their histotopographic organization. Thus, the positive effect of cell therapy based on allogeneic minimally manipulated keratinocytes and fibroblasts on skin regeneration indicated that it can be used in clinical practice to improve the effectiveness of rehabilitation after radiation therapy.

Chimeric antigen receptor (CAR)-T cell therapy yields good results in the treatment of various hematologic malignancies. However, the efficacy of CAR-T cell therapy against solid tumors has proven to be limited, primarily because the tumor-associated extracellular matrix (ECM) creates an intractable barrier for the cytotoxic CAR-T cells that are supposed to kill cancer cells. This review unravels the multifaceted role of the tumor-associated ECM in impeding CAR-T cell infiltration, survival, and functions within solid tumors. We analyze the situations when intratumoral ECM limits the efficacy of CAR-T cell therapy by being a purely physical barrier that complicates lymphocyte penetration/migration and also acts as an immunosuppressive factor that impairs the antitumor activities of CAR-T cells. In addition, we highlight promising approaches such as engineering CAR-T cells with improved capabilities to penetrate and migrate into/through the intratumoral ECM, combination therapies aimed at attenuating the high density and immunosuppressive potential of the intratumoral ECM, and others that enable overcoming ECM-related obstacles. A detailed overview of the data of relevant studies not only helps to better understand the interactions between CAR-T cells and the intratumoral ECM but also outlines potential ways to more effectively use CAR-T cell therapy against solid tumors.

Bilio-biliary anastomosis (BBA) is a critical surgical procedure that is performed with the objective of restoring bile duct continuity. This procedure is often required in cases where there has been an injury to the extrahepatic bile ducts or during liver transplantation. Despite advances in surgical techniques, the healing of BBA remains a significant challenge, with complications such as stricture formation and leakage affecting patient outcomes. The stromal vascular fraction (SVF), a heterogeneous cell population derived from adipose tissue, has demonstrated promise in regenerative medicine due to its rich content of stem cells, endothelial progenitor cells, and growth factors. The objective of this study was to evaluate the potential of locally administered autologous SVF to enhance the healing of BBAs. Bilio-biliary anastomosis was performed on a swine model (female Landrace pigs). Six swine were divided into two groups: the treatment group (n = 3) received a local application of autologous SVF around the anastomosis site immediately following BBA formation, while the control group (n = 3) received saline. The primary outcomes were assessed over an eight-week period post-surgery, and included anastomosis healing, stricture formation, and bile leakage. Histological analysis was performed to evaluate fibrosis, angiogenesis, and inflammation. Immunohistochemistry was conducted to assess healing-related markers (CD34, α-SMA) and the immunological microenvironment (CD3, CD10, tryptase). The SVF-treated group exhibited significantly enhanced healing of the BBA. Histological examination revealed increased angiogenesis and reduced fibrosis in the SVF group. Immunohistochemical staining demonstrated higher vascular density in the anastomosed area of the SVF-treated group (390 vs. 210 vessels per 1 mm², p = 0.0027), as well as a decrease in wall thickness (1.9 vs. 1.0 mm, p = 0.0014). There were no statistically significant differences in mast cell presence (p = 0.40). Immunohistochemical staining confirmed the overexpression of markers associated with tissue repair. Local injections of autologous SVF at the site of BBA have been demonstrated to significantly enhance healing and promote tissue regeneration. These findings suggest that SVF could be a valuable adjunctive therapy in BBA surgery, potentially improving surgical outcomes. However, further investigation is needed to explore the clinical applicability and long-term benefits of this novel approach in clinical practice as a minimally manipulated cell application.

Molecular hydrogen (H₂) has antioxidant, anti-inflammatory, and anti-fibrotic effects. In a rat model simulating pulmonary fibrotic changes induced by monocrotaline-induced pulmonary hypertension (MPH), we had previously explored the impact of inhaled H₂ on lung inflammation and blood pressure. In this study, we further focused the biological effects of H₂ on mast cells (MCs) and the parameters of the fibrotic phenotype of the local tissue microenvironment. MPH resulted in a significantly increased number of MCs in both the pneumatic and respiratory parts of the lungs, an increased number of tryptase-positive MCs with increased expression of TGF-β, activated interaction with immunocompetent cells (macrophages and plasma cells) and fibroblasts, and increased MC colocalization with a fibrous component of the extracellular matrix of connective tissue. The alteration in the properties of the MC population occurred together with intensified collagen fibrillogenesis and an increase in the integral volume of collagen and elastic fibers of the extracellular matrix of the pulmonary connective tissue. The exposure of H₂ together with monocrotaline (MCT), despite individual differences between animals, tended to decrease the intrapulmonary MC population and the severity of the fibrotic phenotype of the local tissue microenvironment compared to changes in animals exposed to the MCT effect alone. In addition, the activity of collagen fibrillogenesis associated with MCs and the expression of TGF-β and tryptase in MCs decreased, accompanied by a reduction in the absolute and relative content of reticular and elastic fibers in the lung stroma. Thus, with MCT exposure, inhaled H₂ has antifibrotic effects involving MCs in the lungs of rats. This reveals the unknown development mechanisms of the biological effects of H₂ on the remodeling features of the extracellular matrix under inflammatory background conditions of the tissue microenvironment.

The extracellular matrix is an organized three-dimensional network of protein-based molecules and other macromolecules that provide structural and biochemical support to tissues. Depending on its biochemical and structural properties, the extracellular matrix influences cell adhesion and signal transduction and, in general, can influence cell differentiation and proliferation through specific mechanisms of chemical and mechanical sensing. The development of body tissues during ontogenesis is accompanied by changes not only in cells but also in the composition and properties of the extracellular matrix. Similarly, tumor development in carcinogenesis is accompanied by a continuous change in the properties of the extracellular matrix of tumor cells, called ‘oncomatrix’, as the tumor matures, from the development of the primary focus to the stage of metastasis. In this paper, the characteristics of the composition and properties of the extracellular matrix of tumor tissues are considered, as well as changes to the composition and properties of the matrix during the evolution of the tumor and metastasis. The extracellular matrix patterns of tumor tissues can be used as biomarkers of oncological diseases as well as potential targets for promising anti-tumor therapies.