Сell clusters isolation in glioblastomas and their functional and molecular characterization using new morphometric approaches.

BACKGROUND: Digital pathology has come a long way in terms of creating tools to improve existing diagnostic approaches. However, several pathology fields, such as neuropathology, are still characterized by low coverage from machine learning tools and neural network analysis, which may be due to the complexity of the internal cellular and molecular structure of the corresponding neoplasms, including glioblastomas. METHOD: In the framework of this study, using advanced proprietary tools for obtaining images of histological slides and their deep morphometric analysis, we studied samples of 198 patients with glioblastoma with the selection of morphometric cell clusters. Also, cells of each cluster were isolated, and their proliferative, migratory, invasive activity, survival ability, aerobic glycolysis activity, and chemo- and radioresistance were studied. RESULTS: Four morphometric clusters were identified, including small-cell cluster, paracirculonuclear cluster, hypochromic cluster, and macronuclear cluster, which significantly differed in morphometric parameters and functional parameters. Hypochromic cluster cells demonstrated the highest proliferation activity; macronuclear cluster was the most active glucose consumer; paracirculonuclear cluster had the most prominent migratory and invasive activity and hypoxia resistance; small-cell cluster demonstrated predominantly average values of all parameters. Moreover, additional analysis revealed the presence of a separate subcluster of stem cell elements that correspond in their molecular properties to glioma stem cells and are present in all four clusters. It also turned out that several key molecular parameters of glioblastoma, such as mutational modifications in the EGFR, PDGFRA, and NF1 genes, along with the molecular GBM subtype, are significantly correlated with the identified cell clusters. CONCLUSIONS: Thus, the results represent an up-and-coming innovation in the practical field of digital pathology and fundamental questions of glioma carcinogenesis.

[NMDA receptors expression activity in anaplastic astrocytomas]. / Aktivnost' ekspressii NMDA-retseptorov glutamata v anaplasticheskikh astrotsitomakh.

OBJECTIVE: To study the relationship of NMDA receptors expression activity with proliferative activity and genetic properties of anaplastic astrocytomas, as well as the survival of patients with this disease. MATERIAL AND METHODS: To solve this problem, we compared the expression activity of the least studied NMDA receptors in the context under consideration, detected using immunofluorescent studies and polymerase chain reaction, with the results of histological and molecular studies, the proliferative activity of neoplasms, and the survival of patients. RESULTS: The expression activity of NMDA receptors is higher in astrocytomas, grade 3, which do not carry mutations in IDH1 and IDH2 genes. In addition, the activity of NMDA receptors expression directly correlates with proliferative activity in the tumors. The activity of NMDA receptor expression has a significant impact on the prognosis of disease-free survival. CONCLUSION: We have shown for the first time the significant role of NMDA receptors in the progression of diffuse astrocytomas, which can become the basis for creating new therapeutic and diagnostic tools.

[Experimental studies of the biomechanical properties of the cornea]. / Eksperimental'nye issledovaniya biomekhanicheskikh svoistv rogovitsy.

The review presents the results of experimental studies of the biomechanical properties of the cornea. Selective evaluation of the individual corneal structures (for example, limiting membranes) using classical mechanical tests is to a certain extent limited due to the rather small thickness of these structures and the related difficulties in sample fixation. In real practice, the use of a method better adapted for conducting such studies - atomic force microscopy (AFM) - remains promising, since on the one hand it eliminates the need for mechanical capture and retention of the sample, and on the other - provides the capability for studying its segments separately.

Detection of HOCl-driven degradation of the pericardium scaffolds by label-free multiphoton fluorescence lifetime imaging.

Artificial biomaterials can significantly increase the rate of tissue regeneration. However, implantation of scaffolds leads not only to accelerated tissue healing but also to an immune response of the organism, which results in the degradation of the biomaterial. The synergy of the immune response and scaffold degradation processes largely determines the efficiency of tissue regeneration. Still, methods suitable for fast, accurate and non-invasive characterization of the degradation degree of biomaterial are highly demandable. Here we show the possibility of monitoring the degradation of decellularized bovine pericardium scaffolds under conditions mimicking the immune response and oxidation processes using multiphoton tomography combined with fluorescence lifetime imaging (MPT-FLIM). We found that the fluorescence lifetimes of genipin-induced cross-links in collagen and oxidation products of collagen are prominent markers of oxidative degradation of scaffolds. This was verified in model experiments, where the oxidation was induced with hypochlorous acid or by exposure to activated neutrophils. The fluorescence decay parameters also correlated with the changes of micromechanical properties of the scaffolds as assessed using atomic force microscopy (AFM). Our results suggest that FLIM can be used for quantitative assessments of the properties and degradation of the scaffolds essential for the wound healing processes in vivo.

Role of actin-binding proteins in the regulation of cellular mechanics.

The viscoelastic parameters of the cell can report on the cell state, cellular processes and diseases. Cell mechanics strongly rely on the properties of the cytoskeleton, an important system of subcellular filaments, especially on the high-level structures that actin forms together with actin-binding proteins (ABPs). In normal cells, components of the cytoskeleton are highly integrated, and their functions are well orchestrated. In contrast, impaired expression and functioning of ABPs lead to the increasing ability of cancer cells to resist chemotherapy and metastasize. ABP-mediated changes in the cytoskeleton architecture can lead to changes in the mechanical properties of the actin network, both locally and at the level of the whole cell. Until now, in cancer-related studies, mechanical data have been used less frequently, compared to biochemical tests or cell migration assays. Here, we will review current methods for analyzing the mechanical properties of cells and provide the available data on the contribution of ABPs in determining cell mechanical properties important for the investigation of cellular functions, particularly in cancers.

Biodegradable iron-silicon implants produced by additive manufacturing.

Due to many negative and undesirable side effects from the use of permanent implants, the development of temporary implants based on biocompatible and biodegradable materials is a promising area of modern medicine. In the presented study, we have investigated complex-shaped iron-silicon (Fe-Si) scaffolds that can be used as potential biodegradable framework structures for solid implants for bone grafting. Since iron and silicon are biocompatible materials, and their alloy should also have biocompatibility. It has been demonstrated that cells, mesenchymal stromal cells derived from the human umbilical cord (UC-MSC) and 3T3, were attached to, spread, and proliferated on the Fe-Si scaffolds' surface. Most of UC-MSC and 3T3 remained viable, only single dead cells were observed. According to the results of biological testing, the scaffolds have shown that deposition of calcium phosphate particles occurs on day one in the scaffold at the defect site that can be used as a primary marker of osteodifferentiation. These results demonstrate that the 3D-printed porous iron-silicon (Fe-Si) alloy scaffolds are promising structures for bone grafting and regeneration.

Moisture adsorption by decellularized bovine pericardium collagen matrices studied by terahertz pulsed spectroscopy and solid immersion microscopy.

In this paper, terahertz (THz) pulsed spectroscopy and solid immersion microscopy were applied to study interactions between water vapor and tissue scaffolds-the decellularized bovine pericardium (DBP) collagen matrices, in intact form, cross-linked with the glutaraldehyde or treated by plasma. The water-absorbing properties of biomaterials are prognostic for future cell-mediated reactions of the recipient tissue with the scaffold. Complex dielectric permittivity of DBPs was measured in the 0.4-2.0 THz frequency range, while the samples were first dehydrated and then exposed to water vapor atmosphere with 80.0 ± 5.0% relative humidity. These THz dielectric measurements of DBPs and the results of their weighting allowed to estimate the adsorption time constants, an increase of tissue mass, as well as dispersion of these parameters. During the adsorption process, changes in the DBPs' dielectric permittivity feature an exponential character, with the typical time constant of =8-10 min, the transient process saturation at =30 min, and the tissue mass improvement by =1-3%. No statistically-relevant differences between the measured properties of the intact and treated DBPs were observed. Then, contact angles of wettability were measured for the considered DBPs using a recumbent drop method, while the observed results showed that treatments of DBP somewhat affects their surface energies, polarity, and hydrophilicity. Thus, our studies revealed that glutaraldehyde and plasma treatment overall impact the DBP-water interactions, but the resultant effects appear to be quite complex and comparable to the natural variability of the tissue properties. Such a variability was attributed to the natural heterogeneity of tissues, which was confirmed by the THz microscopy data. Our findings are important for further optimization of the scaffolds' preparation and treatment technologies. They pave the way for THz technology use as a non-invasive diagnosis tool in tissue engineering and regenerative medicine.

[Assessment of age-related changes in lens capsule biomechanics using atomic force microscopy]. / Otsenka vozrastnykh izmenenii «biomekhaniki¼ kapsuly khrustalika na osnove atomno-silovoi mikroskopii.

Studies devoted to the assessment of lens capsule biomechanics can be divided into fundamental and applied. The former are oriented towards analysis of various indicators characterizing elasticity of the capsule as a basal membrane that maintains and changes the shape of the lens, and the latter deal with widespread introduction of modern microinvasive methods of phaco surgery into clinical practice. PURPOSE: To assess age-related changes in lens capsule biomechanics based on atomic force microscopy (AFM). MATERIAL AND METHODS: The study included 50 central fragments of the anterior capsule of the human lens obtained intraoperatively during ultrasonic phacoemulsification by continuous circular capsulorhexis. The measurements were carried out in the Fast Force Volume (FFV) mode. The force curves were processed in the Nanoscope Analysis software (Bruker, USA) using the Hertz model that allows calculating the Young's modulus of the capsule sample based on the dependence of the force on the puncture depth. RESULTS: There was no statistically significant difference in the «stiffness¼ of the inner and outer surfaces before and after removal of the subcapsular epithelium (p=0.25). In all cases, the inner surface of the capsule turned out to be «harder¼ than the outer one. In this case, the ratio of Young's modulus of the inner and outer surfaces has a significant dependence on age (p<0.001). With an increase in age from 50 to 90 years, this ratio decreased from ~7 to ~1.5. This was due to a simultaneous change in Young's modulus of the opposite nature: an increase in the stiffness of the outer surface and its decrease in the inner one. CONCLUSION: It is possible to assess lens capsule biomechanics using AFM if the subcapsular epithelium is present. In this case, the objects of study are the areas of the capsule free of epithelium, and the epithelial cells themselves can be used to identify the inner surface of the capsule. Regardless of age, the stiffness of the inner surface of the anterior lens capsule significantly exceeds that of the outer surface.

Viscoelasticity in simple indentation-cycle experiments: a computational study.

Instrumented indentation has become an indispensable tool for quantitative analysis of the mechanical properties of soft polymers and biological samples at different length scales. These types of samples are known for their prominent viscoelastic behavior, and attempts to calculate such properties from the indentation data are constantly made. The simplest indentation experiment presents a cycle of approach (deepening into the sample) and retraction of the indenter, with the output of the force and indentation depth as functions of time and a force versus indentation dependency (force curve). The linear viscoelastic theory based on the elastic-viscoelastic correspondence principle might predict the shape of force curves based on the experimental conditions and underlying relaxation function of the sample. Here, we conducted a computational analysis based on this theory and studied how the force curves were affected by the indenter geometry, type of indentation (triangular or sinusoidal ramp), and the relaxation functions. The relaxation functions of both traditional and fractional viscoelastic models were considered. The curves obtained from the analytical solutions, numerical algorithm and finite element simulations matched each other well. Common trends for the curve-related parameters (apparent Young's modulus, normalized hysteresis area, and curve exponent) were revealed. Importantly, the apparent Young's modulus, obtained by fitting the approach curve to the elastic model, demonstrated a direct relation to the relaxation function for all the tested cases. The study will help researchers to verify which model is more appropriate for the sample description without extensive calculations from the basic curve parameters and their dependency on the indentation rate.

Broad-spectrum antibacterial and pro-regenerative effects of photoactivated Photodithazine-Pluronic F127-Chitosan polymer system: In vivo study.

Emerging global danger of multidrug resistant microbes makes it essential to explore new approaches to treat infections. We studied antibacterial and pro-regenerative effects of photodynamic therapy (PDT) performed with water solutions of photodithazine and its complexes with Pluronic F127 and chitosan in rat model of full thickness wound (n = 24) infected by an associated Gram-negative and Gram-positive bacteria culture. Laboratory rats were exposed to PDT 24 and 72 h after the injury. Exudate samples were collected before and after PDT for a microbiological study. Autopsy tissues were excised and fixed in formalin on day 4 of the experiment. Fixed tissues were processed and poured into paraffin. Paraffin sections were stained with hematoxylin and eosin and studied by an experienced pathologist. Microbiological analysis revealed that the photoactivation of photodithazine and its complexes suppressed the associated microflora in vivo and inhibited suppurative inflammation in the wounds. The triple Photodithazine-Pluronic F127-Chitosan system possessed the highest antibacterial activity. The morphological study revealed that PDT with photodithazine polymer complexes accelerated wound healing, promoted restoration of microcirculation, facilitated proliferation of fibroblast and vessels and stimulated collagen synthesis. The Photodithazine-Pluronic F127-Chitosan complex may be successfully applied for PDT to prevent and treat suppurative inflammatory diseases of the skin and soft tissues.

Biomechanical properties of the lens capsule: A review.

The lens capsule, a thin specialized basement membrane that encloses the crystalline lens, is essential for both the structural and biomechanical integrity of the lens. Knowing the mechanical properties of the lens capsule is important for understanding its physiological functioning, role in accommodation, age-related changes, and for providing a better treatment of a cataract. In this review, we have described the techniques used for the lens capsule biomechanical testing on the macro- and microscale and summarized the current knowledge about its mechanical properties.

Influence of acetic acid on the photocatalytic activity of photosensitiser-amphiphilic polymer complexes in the oxidation reaction of tryptophan.

The effect of acetic acid on the photosensitizing activity of dimegin (DMG), chlorin e6 (Ce6), and their complexes with amphiphilic polymers-Pluronic F-127 and poly-N-vinylpyrrolidone (PVP)-in the model reaction of tryptophan photo-oxidation has been established. It was shown that the photocatalytic activity of photosensitizers (PS), which is characterized by the effective constant of the tryptophan photo-oxidation rate (keff), increases by 1.4 times for DMG, while for Ce6, it decreases by 1.2 times in a weak acidic medium (pH ∼ 4). The influence of acetic acid on the effective constant keff of the tryptophan photo-oxidation rate in the presence of PS-amphiphilic polymer complexes is determined by the nature of the polymer. Thus, the photocatalytic activity of the PS-F127 system decreases at pH ∼ 4.0, and the photocatalytic activity of PS-PVP complexes is insensitive to the presence of acetic acid in the medium. It has been suggested that the observed effects in the PS-F127 system in a weak acidic medium (pH ∼ 4) are associated with the influence of acetic acid on the supramolecular structure of Pluronic F127.

[Experimental evidence for regenerative treatment of chronic tympanic membrane perforation]. / Eksperimental'noe obosnovanie metoda tkanevoi inzhenerii dlya zakrytiya stoikikh perforatsii barabannoi pereponki.

OBJECTIVE: The aim of our study was an experimental evaluation of tissue engineering approach to chronic tympanic membrane perforation closure. MATERIALS AND METHODS: Chronic tympanic membrane perforation models were created both sides in 12 chinchillas. Right sided perforations were divided into two equal groups (A and B) according to treatment; left sided perforations were used as a control group. Group A perforations were treated with collagen scaffold and fibroblast growth factor, group B perforations were treated with collagen scaffold only. During follow-up, we provided otovideoendoscopy for closure rates assessement every 2 weeks. In case of perforation closure, a morphological investigation of the regenerate was performed. RESULTS: Group A perforations were totally closed 2 weeks after treatment in all animals. In group B, complete closure of perforation was achieved after the third treatment procedure in one case. There were no spontaneous perforation closure in the control group. According to morphological investigation, the restoration of trilaminar structure was observed only in tissue engineering group tympanic membranes.

Digging deeper: structural background of PEGylated fibrin gels in cell migration and lumenogenesis.

Fibrin is a well-known tool in tissue engineering, but the structure of its modifications created to improve its properties remains undiscussed despite its importance, e.g. in designing biomaterials that ensure cell migration and lumenogenesis. We sought to uncover the structural aspects of PEGylated fibrin hydrogels shown to contribute to angiogenesis. The analysis of the small-angle X-ray scattering (SAXS) data and ab initio modeling revealed that the PEGylation of fibrinogen led to the formation of oligomeric species, which are larger at a higher PEG : fibrinogen molar ratio. The improvement of optical properties was provided by the decrease in aggregates' sizes and also by retaining the bound water. Compared to the native fibrin, the structure of the 5 : 1 PEGylated fibrin gel consisted of homogenously distributed flexible fibrils with a smaller space between them. Moreover, as arginylglycylaspartic acid (RGD) sites may be partly bound to PEG-NHS or masked because of the oligomerization, the number of adhesion sites may be slightly reduced that may provide the better cell migration and formation of continuous capillary-like structures.

Tailoring the collagen film structural properties via direct laser crosslinking of star-shaped polylactide for robust scaffold formation.

Application of restructured collagen-based biomaterials is generally restricted by their poor mechanical properties, which ideally must be close to those of a tissue being repaired. Here, we present an approach to the formation of a robust biomaterial using laser-induced curing of a photosensitive star-shaped polylactide. The created collagen-based structures demonstrated an increase in the Young's modulus by more than an order of magnitude with introduction of reinforcing patterns (from 0.15 ±â€¯0.02 MPa for the untreated collagen to 51.2 ±â€¯5.6 MPa for the reinforced collagen). It was shown that the geometrical configuration of the created reinforcing pattern affected the scaffold's mechanical properties only in the case of a relatively high laser radiation power density, when the effect of accumulated thermomechanical stresses in the photocured regions was significant. Photo-crosslinking of polylactide did not compromise the scaffold's cytotoxicity and provided fluorescent regions in the collagen matrix, that create a potential for noninvasive monitoring of such materials' biodegradation kinetics in vivo.

Chitosan-g-oligo(L,L-lactide) copolymer hydrogel for nervous tissue regeneration in glutamate excitotoxicity: in vitro feasibility evaluation.

Over the last decade, a number of hydrogels attracted great attention in the area of brain tissue engineering. The hydrogels are composed of hydrophilic polymers forming 3D network in water. Their function is promoting structural and functional restoration of damaged brain tissues by providing mechanical support and navigating cell fate. This paper reports on the neurocompatibility of chitosan-g-oligo(L,L-lactide) copolymer hydrogel with primary rat cortical neuron culture. The hydrogel was produced by a molding technique on the base of photocurable composition consisting of chitosan-g-oligo(L,L-lactide) copolymer, poly(ethylene glycol) diacrylate and photosensitizer Irgacure 2959. The influence of the hydrogel on cell viability, phenotype and calcium homeostasis, mitochondrial potential and oxygen consumption rate in glutamate excitotoxicity was analyzed using primary neuron cultures obtained from a neonatal rat cortex. This study revealed that the hydrogel is non-cytotoxic. Dissociated neonatal rat cortical cells were actively attaching to the hydrogel surface and exhibited the phenotype, calcium homeostasis and mitochondrial function in both standard conditions and glutamate excitotoxicity (100 µM) similar to the control cells cultured without the hydrogel. To conclude, in this study we assessed the feasibility of the application of chitosan-g-oligo(L,L-lactide) copolymer hydrogel for tissue engineering therapy of brain injury in an in vitro model. The results support that the hydrogel is able to sustain realization of the functional metabolic activity of neonatal rat cortical cells in response to glutamate excitotoxicity.

Effect of Non-Thermal Plasma on Proliferative Activity and Adhesion of Multipotent Stromal Cells to Scaffolds Developed for Tissue-Engineered Constructs.

We studied the effect of non-thermal argon plasma on proliferative activity of bone marrow multipotent stromal cells in vitro. Treatment of stromal cell suspension with pure argon did not affect their proliferation. The cells treated with non-thermal argon plasma and explanted in the treatment medium demonstrated growth inhibition by 30-40% in comparison with the control. Multipotent stromal cells treated with plasma and after centrifugation explanted in normal medium within 12 min demonstrated accelerated growth. The total cell growth from the pellet and supernatant significantly exceeded the control values. We also analyzed adhesion and proliferative activity of multipotent stromal cells treated with non-thermal plasma on bioresorbable carriers. The cells adhered and proliferated on all types of studied samples. Adhesion properties of scaffolds differed. Caprolactone was found to be the most suitable material for adhesion and proliferation of multipotent stromal cells.

Viscoelastic mapping of cells based on fast force volume and PeakForce Tapping.

Development of fast force volume (FFV), PeakForce Tapping (PFT), and related AFM techniques allow fast acquisition and mapping of a sample's mechanical properties. The methods are well-suited for studying soft biological samples like living cells in a liquid environment. However, the question remains how the measured mechanical properties are related to those acquired with the classical force volume (FV) technique conducted at low indentation rates. The difference is coming mostly from the pronounced viscoelastic behavior of cells, making apparent elastic parameters depending on the probing rate. Here, the viscoelastic analysis was applied directly to the force curves acquired with force volume or PeakForce Tapping by their post-processing based on the Ting's model. Maps from classical force volume, FFV and PFT obtained using special PFT cantilevers and cantilevers modified with microspheres were compared here. With the correct viscoelastic model, which was found to be the power-law rheology model, all the techniques have provided self-consistent results. The techniques were further modified for the mapping of the viscoelastic model-independent complex Young's modulus.

Interrogating Parkinson's disease associated redox targets: Potential application of CRISPR editing.

Loss of dopaminergic neurons in the substantia nigra is one of the pathogenic hallmarks of Parkinson's disease, yet the underlying molecular mechanisms remain enigmatic. While aberrant redox metabolism strongly associated with iron dysregulation and accumulation of dysfunctional mitochondria is considered as one of the major contributors to neurodegeneration and death of dopaminergic cells, the specific anomalies in the molecular machinery and pathways leading to the PD development and progression have not been identified. The high efficiency and relative simplicity of a new genome editing tool, CRISPR/Cas9, make its applications attractive for deciphering molecular changes driving PD-related impairments of redox metabolism and lipid peroxidation in relation to mishandling of iron, aggregation and oligomerization of alpha-synuclein and mitochondrial injury as well as in mechanisms of mitophagy and programs of regulated cell death (apoptosis and ferroptosis). These insights into the mechanisms of PD pathology may be used for the identification of new targets for therapeutic interventions and innovative approaches to genome editing, including CRISPR/Cas9.

Tissue Engineered Neural Constructs Composed of Neural Precursor Cells, Recombinant Spidroin and PRP for Neural Tissue Regeneration.

We have designed a novel two-component matrix (SPRPix) for the encapsulation of directly reprogrammed human neural precursor cells (drNPC). The matrix is comprised of 1) a solid anisotropic complex scaffold prepared by electrospinning a mixture of recombinant analogues of the spider dragline silk proteins - spidroin 1 (rS1/9) and spidroin 2 (rS2/12) - and polycaprolactone (PCL) (rSS-PCL), and 2) a "liquid matrix" based on platelet-rich plasma (PRP). The combination of PRP and spidroin promoted drNPC proliferation with the formation of neural tissue organoids and dramatically activated neurogenesis. Differentiation of drNPCs generated large numbers of ßIII-tubulin and MAP2 positive neurons as well as some GFAP-positive astrocytes, which likely had a neuronal supporting function. Interestingly the SPRPix microfibrils appeared to provide strong guidance cues as the differentiating neurons oriented their processes parallel to them. Implantation of the SPRPix matrix containing human drNPC into the brain and spinal cord of two healthy Rhesus macaque monkeys showed good biocompatibility: no astroglial and microglial reaction was present around the implanted construct. Importantly, the human drNPCs survived for the 3 month study period and differentiated into MAP2 positive neurons. Tissue engineered constructs based on SPRPix exhibits important attributes that warrant further examination in spinal cord injury treatment.