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Vol 20, No 4 (2025)
Reviews
Cellular senescence in cartilage and bone tissue aging: role in disease development and therapeutic potential
Abstract
Cellular senescence induced by various types of stress leads to irreversible cell cycle arrest and alterations in gene expression, metabolism, chromatin organization, and activation of anti-apoptotic signaling pathways. This process results in the formation of a pro-inflammatory secretome, known as the senescence-associated secretory phenotype, which triggers chronic sterile inflammation (inflammaging).
This review summarizes current evidence on the role of cellular senescence in age-related changes and diseases of the joints and skeletal system, as well as potential therapeutic strategies.
Based on an articles search in the Google Scholar, PubMed (MEDLINE), Scopus, and Web of Science databases using specific keywords and their combinations and the AMSTAR 2 methodology, this review presents the role of cellular senescence in age-related changes and diseases of the joints and skeletal system. Data are presented demonstrating the presence of senescent cells with a senescence-associated secretory phenotype expressing the p16Ink4a protein in the microenvironment of cartilage and bone tissue. It was demonstrated that with advancing age, stem and progenitor cells, osteoblasts, osteocytes, and chondrocytes express senescence markers and acquire characteristic aging-associated traits such as genomic instability, telomere shortening, epigenetic modifications, proteostasis loss, and mitochondrial dysfunction. It has been demonstrated that cellular senescence and the development of the secretory phenotype are causally linked to chronic inflammation and contribute to the development of osteoporosis and osteoarthritis.
A new strategy for the treatment of osteoporosis and osteoarthritis—senotherapy—is presented, in which the therapeutic targets are senescent cells (senolytics) or various mechanisms responsible for their survival and transformation into the senescence-associated secretory phenotype (senomorphics).
Evidence shows that advances in senescence research over the past decade have laid the groundwork for novel therapeutic approaches targeting joint and bone diseases. Further investigation of the endocrine activity of senescence-associated secretory phenotype cartilage and bone cells is needed to provide the basis for understanding interactions among different systems of the aging organism and for developing strategies for the simultaneous treatment of age-related disorders.
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Fluorescence lifetime imaging microscopy in immuno-oncology: tracking tumor heterogeneity, cell death, and immune response dynamics
Abstract
Fluorescence lifetime imaging microscopy (FLIM) has developed significantly over the past two decades and is now a powerful tool in biomedical research. Recent advances in fluorescent probes have greatly expanded the range of its potential applications. As fluorescence lifetime is highly sensitive to microenvironmental and molecular changes, FLIM is a promising technique for detecting pathological conditions, including cancer, and monitoring the efficacy of antineoplastic therapies. This technology allows for the observation of tumor structure and the real-time monitoring of dynamic processes, enabling researchers to probe living cancer cells and their microenvironment with remarkable precision. FLIM is especially valuable for developing and evaluating immunotherapeutic strategies. In solid tumor therapy; in particular, it is crucial to assess how treatment affects tumor metabolism and heterogeneity, cell death mechanisms, and immune response dynamics.
This review provides a comprehensive analysis of current research supporting the feasibility of FLIM as a key research technique to advance cancer immunotherapy.
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Original Study Articles
Properties of osteoplastic matrices based on polylactide microparticles and platelet-rich plasma impregnated with adenoviral constructs carrying BMP2 gene
Abstract
BACKGROUND: The number of patients requiring bone graft procedures is rising every year. Gene-activated osteoplastic matrices represent a promising alternative to traditional bone grafting methods, as they enable sustained and targeted expression of osteoinductive genes directly within the defect area.
AIM: The work aimed to evaluate the properties of gene-activated matrices based on polylactide microparticles and platelet-rich plasma impregnated with adenoviral constructs carrying the BMP2 gene.
METHODS: Light and fluorescence microscopy, flow cytometry, spectrophotometry, real-time polymerase chain reaction, histological staining, histomorphometric analysis, MTT assay, and biochemical assays were performed.
RESULTS: The optimal concentration of adenoviral vectors carrying the BMP2 gene for impregnation into matrices based on polylactide microparticles and platelet-rich plasma was determined using the MTT assay and flow cytometry. The resulting gene-activated matrices were shown to be non-cytotoxic and to stimulate the active proliferation of multipotent mesenchymal stromal cells. As assessed by spectrophotometry, the matrices released genetic constructs in a sustained manner over 15 days. Fluorescence microscopy and real-time polymerase chain reaction confirmed effective and gradual transduction of cell cultures. Histological analysis of tissue sections obtained 28 days after intramuscular implantation in rats demonstrated in vivo biocompatibility of the matrices. The gene-activated matrices induced osteogenic differentiation of adipose tissue–derived multipotent mesenchymal stromal cells, confirmed by increased expression of osteogenic differentiation markers, elevated alkaline phosphatase activity, and extracellular matrix mineralization.
CONCLUSION: The developed gene-activated matrices composed of polylactide microparticles and platelet-rich plasma and incorporating adenoviral vectors carrying BMP2 gene demonstrated effectiveness in in vitro experiments and may be used for repair of bone tissue defects.
311-324
Analysis of the neurogenic potential of a three-dimensional culture of mouse dorsal root ganglia explants
Abstract
BACKGROUND: Cultivation of dorsal root ganglia (DRGs) is a widely used approach for modeling physiological and pathological conditions of the peripheral nervous system in vitro. Existing scientific data indicates that postnatal DRGs contain a pool of glial stem cells capable of differentiating into neurons after injury. Therefore, isolated DRGs containing a stem cell pool may retain neurogenic potential ex vivo. However, no studies in the available scientific data have investigated the preservation of the regenerative potential of DRGs ex vivo.
AIM: This study aimed to evaluate the neurogenic potential of isolated DRGs and establish a three-dimensional organotypic culture from these explants.
METHODS: DRGs were isolated from the cervical, thoracic, and lumbar regions of the spine and cultured short-term and long-term as three-dimensional explants in Geltrex gel. The morphology, ultrastructure, and gene expression profile of the cultured DRGs were analyzed using light and electron microscopy, immunofluorescent staining, and quantitative reverse transcription polymerase chain reaction.
RESULTS: DRGs cultured in Geltrex for 14 days consistently generated a three-dimensional periganglionic network composed of radially branching elongated cells. Immunofluorescent staining with Tuj1 antibodies (neuron-specific class III β-tubulin) demonstrated that the observed cells were neuronal. Immunostaining with O4 antibodies confirmed that Tuj1-positive cells were not migrating Schwann cells. Quantitative reverse transcription polymerase chain reaction analysis of total RNA extracted from the DRG explant cultures revealed a significant increase in mRNA levels of key neurogenesis-related genes, such as Neurog1, Neurog2, and Sox2, indicating activation of a neurogenic program in the cultured explants. Light and electron microscopy showed that adult sensory neurons degenerated within the DRG explants and did not contribute to the periganglionic network. Using available confocal capabilities, we evaluated three-dimensional parameters of the periganglionic network from two-dimensional microscopic images to demonstrate the topography of Tuj1-expressing neuronal cells in a DRG explant culture.
CONCLUSION: We characterized a three-dimensional culture of DRGs. Isolated explants demonstrate regenerative capacity under in vitro conditions. They may represent a novel organotypic model with neurogenic potential. The obtained model may be relevant for studying regenerative processes in DRGs, expanding research opportunities beyond traditional stem cell–derived models.
325-336
Quantitative method for assessing contractile activity of myotubes
Abstract
BACKGROUND: Myotubes are multinucleated terminally differentiated cells widely used to study changes induced by muscle contractile activity, metabolic disorders, and myopathies. The ability to contract is a key indicator of muscle cell maturity; therefore, quantitative assessment of this property is essential for evaluating the degree of myotube differentiation. Existing methods for assessing evoked contractile activity of myotubes have several limitations, including short analyzed recording intervals and the inability to distinguish true cellular activity from artifacts.
AIM: This study aimed to develop a method for quantitative evaluation of evoked myotube contractile activity that accounts for contraction–relaxation cycles and artifact effects and enables analysis of dozens of contraction–relaxation events.
METHODS: C2C12 myotubes on days 9–11 of differentiation were electrically stimulated for 1 hour using sequential bipolar rectangular pulses with 2 ms duration (45 Hz, 1.7 mA per well) for 300 ms, followed by 700 ms of rest. Contractile activity was recorded on video (40 s at the beginning and end of each session) and quantified using existing methods based on the assessment of displacement within an image region or pixel intensity variation, as well as a newly developed method based on calculating the mean standard deviation of pixel intensity within a moving window.
RESULTS: The proposed method reduced artifact influence (focus drift and particle movement in the medium) on the myotube contractile activity index by an order of magnitude compared with existing approaches, and decreased the coefficient of variation between technical replicates twofold. Extending the analyzed recording duration (to 40 seconds) further reduced variability (by 1.4–2.3 times) compared with shorter video recordings. The Python implementation of the method is available in open access (https://github.com/maxpauel/movindex).
CONCLUSION: This study proposes a quantitative method for evaluating evoked myotube contractile activity, which enables effective elimination of artifacts associated with particle motion in the culture medium and focus instability, as well as assessment of the mean contractile activity within the imaging frame (field of view) over an extended period of time.
337-346
Epidermal growth factor receptor as a target of antitumor activity of binase mutants
Abstract
BACKGROUND: The epidermal growth factor receptor (EGFR) is one of the key proteins in cell signaling that regulates mitogen-activated protein kinase (MAPK) pathways. EGFR dysregulation is associated with various neoplasms, highlighting the importance of developing targeted EGFR inhibitors. Binase, a ribonuclease (RNase) from Bacillus pumilus, is a promising antitumor agent capable of interacting with EGFR. Its cytotoxic potential is primarily determined by its activity against intracellular RNA. Binase mutants with reduced catalytic activity, K26A and H101E, also demonstrate antitumor properties.
AIM: This study aimed to assess the effect of interactions between binase mutants and EGFR on their cytotoxic potential.
METHODS: The antiproliferative activity of binase and its mutants K26A and H101E, with residual catalytic activity of 11.0% and 0.02%, respectively, was evaluated using the MTT assay in A431 epidermoid carcinoma cells, which overexpress wild-type EGFR. Immunofluorescence analysis was used to examine the interactions of RNases with EGFR and their ability to modulate the MAPK/ERK pathway. Hypothetical protein–protein interaction models were generated using computational modeling. The antimigratory activity of RNases was assessed using the standard scratch assay.
RESULTS: Binase and its mutants reduced the proliferative activity of A431 tumor cells by 40%. Pretreatment of cells with the monoclonal anti-EGFR antibody cetuximab attenuated the cytotoxic potential of binase mutants. Computational modeling indicated that the tested RNases may interact with EGFR, with binase having a higher affinity for the ATP-binding site of the tyrosine kinase domain and the mutant derivatives binding preferentially to regions involved in receptor endocytosis. This may underlie the observed differences in EGFR internalization rates. Unlike binase and the K26A mutant, the catalytically inactive H101E mutant lacks antimigratory activity, indicating the importance of maintaining a certain level of enzymatic activity.
CONCLUSION: EGFR is a target of the cytotoxic activity of binase mutants, and their interaction inhibits the MAPK/ERK signaling pathway and causes tumor cell death.
347-358
Spatial characterization of macrophage-enriched tumor regions in triple-negative breast cancer
Abstract
BACKGROUND: The spatial organization of immune cells within tumors is an important area of investigation in studies of interactions between tumor cells and the tumor microenvironment. This is particularly relevant because some immune cells function through direct contact with their target cells, whereas others communicate over a distance via paracrine signaling involving cytokines. Thus, the topography of tumor cells and microenvironmental cells may determine the possibility and nature of intercellular interactions and thereby influence the functional effects of immune cells.
AIM: This study aimed to compare the spatial transcriptomic profiles of tumor and stromal regions enriched in macrophages in triple-negative breast cancer.
METHODS: Eight patients with triple-negative breast cancer were included. Spatial transcriptomic analysis was performed on formalin-fixed, paraffin-embedded tissue sections using high-throughput RNA sequencing with the 10X Visium platform. The annotated spots enriched in intraepithelial and stromal macrophages were used for downstream bioinformatic analysis.
RESULTS: A total of 437 differentially expressed genes were identified between the two groups of spots containing macrophages with distinct spatial localization. Spots with intraepithelial macrophages were characterized by activation of processes related to cytokine and chemokine signaling, regulation of regulatory T-cell differentiation, organization of cell–cell contacts, wound healing, and inhibition of viral activity. Spots enriched in stromal macrophages demonstrated activation of biological processes associated with the regulation of angiogenesis, cell migration and recruitment, cell adhesion, and stromal remodeling.
CONCLUSION: Macrophage topography within primary tumors of triple-negative breast cancer is associated with their functional characteristics. These fundamental findings may be useful for developing prognostic criteria and therapeutic approaches aimed at modulating the tumor microenvironment to improve long-term outcomes in patients with triple-negative breast cancer.
359-370
Culturing keratinocytes from patients with burn injury
Abstract
BACKGROUND: A burn injury, particularly when extensive and deep, leads not only to local tissue damage but also to systemic alterations that impair the skin’s regenerative capacity. Modern therapeutic approaches, including cell-based technologies, aim to accelerate skin repair in conditions where autologous tissue is insufficient. Despite using cells derived from unaffected skin regions, their viability and ability to form epithelial structures may be substantially reduced. This study investigates changes occurring in the epidermis of uninjured skin in patients with extensive burns and explores effective strategies to enhance the viability of cultured keratinocytes.
AIM: This study aimed to examine the systemic impact of burn injury on the viability of epidermal keratinocytes from uninjured skin and to develop approaches to enhance their viability during in vitro culture.
METHODS: Immunofluorescence analysis of specialized markers was performed on cryosections of skin obtained from healthy donors and patients with burn injuries. The proportions of viable cells and epidermal stem cells in keratinocyte cultures were assessed using flow cytometry. The adhesive capacity, colony-forming ability, and formation of confluent cell layers were evaluated in vitro for keratinocytes isolated from healthy donors and donors with burns.
RESULTS: Keratinocytes isolated from patients with burns exhibited reduced adhesion and viability during the first days of culture. Immunofluorescence analysis revealed abnormal expression of keratins 6 and 17 in the epidermis of uninjured skin from patients with burns, an increased proportion of cells with nuclear localization of YAP1, and decreased integrin expression, indicating the development of a pro-inflammatory phenotype. Flow cytometry demonstrated a reduction in epidermal stem cell fractions (ITGα6high/CD71low) in patients with burns. To enhance cell adhesion and survival, culture methods involving collagen coating of the culture surface and the addition of a Rho-associated kinase inhibitor to the culture medium were proposed, both of which promoted cell growth and rapid formation of a confluent layer.
CONCLUSION: A modified culture protocol incorporating type I collagen and a Rho-associated kinase inhibitor improves the efficiency of keratinocyte expansion from patients with burns. These findings may optimize the production of cell cultures for burn treatment, enhance the survival of transplanted cells, and ultimately improve the effectiveness of tissue engineering approaches in combustiology.
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