Sovremennye Tehnologii v Medicine最新文献

Patient-specific in vitro tumor models are a promising platform for studying the mechanisms of oncogenesis and personalized selection of drugs. In case of glial brain tumors, development and use of such models is particularly relevant as the effectiveness of such tumor treatment remains extremely unsatisfactory. The aim of the study was to develop a model of a 3D tumor glioblastoma spheroid based on a patient's surgical material and to study its metabolic characteristics by means of fluorescence lifetime imaging microscopy of metabolic coenzymes.

Materials and methods: The study was conducted with tumor samples from patients diagnosed with glioblastoma (Grade IV). To create spheroids, primary cultures were isolated from tumor tissue samples; the said cultures were characterized morphologically and immunocytochemically, and then planted into round-bottom ultra low-adhesion plates. The number of cells for planting was chosen empirically. The characteristics of the growth of cell cultures were compared with spheroids from glioblastomas of patients with U373 MG stable line of human glioblastoma. Visualization of autofluorescence of metabolic coenzymes of nicotinamide adenine dinucleotide (phosphate) NAD(P)H and flavin adenine dinucleotide (FAD) in spheroids was performed by means of an LSM 880 laser scanning microscope (Carl Zeiss, Germany) with a FLIM module (Becker & Hickl GmbH, Germany). The autofluorescence decay parameters were studied under normoxic and hypoxic conditions (3.5% О₂).

Results: An original protocol for 3D glioblastoma spheroids cultivation was developed. Primary glial cultures from surgical material of patients were obtained and characterized. The isolated glioblastoma cells had a spindle-shaped morphology with numerous processes and a pronounced granularity of cytoplasm. All cultures expressed glial fibrillary acidic protein (GFAP). The optimal seeding dose of 2000 cells per well was specified; its application results in formation of spheroids with a dense structure and stable growth during 7 days. The FLIM method helped to establish that spheroid cells from the patient material had a generally similar metabolism to spheroids from the stable line, however, they demonstrated more pronounced metabolic heterogeneity. Cultivation of spheroids under hypoxic conditions revealed a transition to a more glycolytic type of metabolism, which is expressed in an increase in the contribution of the free form of NAD(P)H to fluorescence decay.

Conclusion: The developed model of tumor spheroids from patients' glioblastomas in combination with the FLIM can serve as a tool to study characteristics of tumor metabolism and develop predictive tests to evaluate the effectiveness of antitumor therapy.

Current technologies of plasma electrolytic oxidation (PEO) for modifying the surfaces of dental implants made of the Grade IV titan alloy provide predictable long-term results in implant dentistry. The aim of the study is to evaluate the efficacy of PEO technology comparing two types of surface modification of dental implants made of VT1-0 medical titanium alloy.

Materials and methods: 50 IRIS dental implants (Scientific Production Company LICOSTOM, Russia), 10-mm long and 4 mm in diameter, were manufactured from the VT1-0 alloy. The implant surface was treated by two PEO methods: 1) in the aqueous solution of alkaline electrolyte without any additional modifiers (PEO-Ti); 2) in the aqueous solution of orthophosphoric acid-based electrolyte containing calcium carbonate (PEO-Ca). Implants made of VT1-0 alloy after milling and without additional treatment served as control samples. The implant surfaces were studied by electron microscopy and energy dispersive X-ray spectrometry. Some of the implants were installed in sheep, samples were obtained at 2, 4, and 8 weeks and studied by microcomputer tomography.

Results: Regardless of the electrolyte composition, a highly developed porous surface was formed in the samples with PEO-modified surfaces. The surface of the PEO-Ti samples in a simple unmodified electrolyte was characterized by a large number of open pores with a wide range of size distribution from 200 nm to 3 μm. The pore size distribution was of a monomodal character, with a maximum near 0.23 μm. The PEO samples in the Ca-containing electrolyte had pores also in a wide range from ~80 nm to ~7 μm. The pore distribution, in contrast to PEO-Ti, was bimodal in nature, with the main maximum in the region of 1.05 μm and the concomitant maximum near 2.45 μm.The obtained surfaces of both types (PEO with Ca and Ti) possessed high purity and optimal microroughness for osseointegration. Both types of PEO treatment (PEO with Ca and Ti) have demonstrated a similar osseointegrative potential, nevertheless, the surface of the PEO-Ca showed a better contact with the implant surface (49.8%) than PEO-Ti (42.4%) obviously due to the presence of calcium in its composition.

Conclusion: The PEO-formed implant surfaces demonstrate high osseointegrative properties after any variants of treatment and show the potential for application in osteoporosis.

The main problem in the field of tumor immunotherapy is the lack of reliable biomarkers that allow pre-determining the susceptibility of individual patients to treatment, as well as insufficient knowledge about the resistance mechanisms. Biomarkers based on the autofluorescence of metabolic coenzymes in immune cells can become a powerful new predictor of early tumor response to treatment, whereas the optical FLIM method can be a tool to predict the effectiveness of immunotherapy, which allows preserving the spatial structure of the sample and obtaining results on the metabolic status of immune cells in real time. The aim of the study is to conduct a metabolic autofluorescence imaging study of the NAD(P)H metabolic coenzyme in immune cells of freshly isolated lymph nodes as a potential marker for assessing the effectiveness of an early response to immunotherapy.

Materials and methods: The study was carried out on C57Bl/6 FoxP3-EGFP mice with B16F0 melanoma implanted near the inguinal lymph node. The mice were injected with antibodies to CTLA-4 (Bio X Cell, USA) (250 μg per mouse, intraperitoneally on days 7, 8, 11, and 12 of the tumor growth). FLIM images in the nicotinamide adenine dinucleotide (phosphate) coenzyme (NAD(P)H) channel (excitation - 375 nm, reception - 435-485 nm) were received using an LSM 880 fluorescent confocal laser scanning microscope (Carl Zeiss, Germany) equipped with a FLIM Simple-Tau module 152 TCSPC (Becker & Hickl GmbH, Germany). Flow cytometry was conducted using a BD FACSAria III cell sorter (BD Biosciences, USA).

Results: Immunotherapy with checkpoint inhibitors resulted in marked metabolic rearrangements in T cells of freshly isolated lymph nodes in responder mice, with inhibition of the tumor growth. Fluorescence lifetime imaging data on NAD(P)H indicated an increase in the free fraction of NADH α₁, a form associated with glycolysis to meet high demands of the activated T cells and pro-inflammatory cytokine synthesis. In contrast, non-responder mice with advanced tumors showed low values of the ratio of free fraction to bound α₁/α₂, which may be related to mechanisms of resistance to therapy.The response to immunotherapy was verified by data on the expression of activation and proliferation markers by means of flow cytometry. The authors observed an increase in the production of the pro-inflammatory cytokine IFN-γ in effector T cells in responder mice compared to untreated controls and non-responders. In addition, an increase in the expression of the surface activation markers CD25 and CD69 was registered compared to untreated controls.

Conclusion: Use of the FLIM method allowed to demonstrate that autofluorescence of the NAD(P)H coenzyme is sensitive to the response to checkpoint immunotherapy and can be used as a reliable marker of the effectiveness of response to treatment.

The aim of the study is to evaluate the efficacy of various types of hybrid technology in compare to the classical repair of the aortic arch of type I aortic dissection treatment in the in-hospital period.

Materials and methods: A retrospective observational study has been conducted, the results of surgical treatment of 213 patients with DeBakey type I aortic dissection operated on within the period from 2001 to 2017 were compared. Patients were divided into three groups: in group 1, patients undergone a hemiarch type of aortic repair or the total arch replacement (n=121); in group 2, a hemiarch aortic reconstruction and implantation of bare metal stent was performed (n=55); in group 3, a frozen elephant trunk technique was used (n=37). Taking into consideration the retrospective character of the investigation and nonequivalence of the groups by separate characteristics, they were equalized to improve the reliability of the results using the PSM (propensity score matching) pseudorandomization method. As a result, three groups of comparison were formed which were equalized by the PSM method and called PSM 1, 2, and 3. The mortality and complication rate in the in-hospital period, as well as the frequency of false lumen thrombosis development depending on the treatment method, have been analyzed.

Results: The mortality rate in the PSM 1 group was 15 patients: group 1 (standard technique) - 10 patients (9%), group 2 (uncoated stents) - 5 patients (11%). A significant difference was found in the number of major bleedings (group 1 - 8%, group 2 - 21%, p=0.031) and cases of bowel ischemia (group 1 - 1%, group 2 - 9%, p=0.028). Complete false lumen thrombosis of the thoracic aorta was observed significantly more often in group 1 than in group 2 (22% vs 5%, p=0.015).In the examined group PSM 2, hospital mortality rate was 4 patients: group 1 - 3 patients (12%), group 3 - 1 patient (3%). No differences between the groups were found in the number of complications. In group 3, complete false lumen thrombosis of the thoracic aorta was observed in 59% of cases, whereas in group 1 it was found only in 4% of patients (p<0.001).In comparison group PSM 3, the mortality was 8 patients: group 2 - 5 patients (11%), group 3 - 3 patients (9%). The number of neurological complications differed significantly: in group 2 - 27%, in group 3 - 6% (p=0.019). Besides, 3% of cases of complete false lumen thrombosis were found in group 2, while there appeared 55% (p<0.001) of such patients in group 3.

Conclusion: The comparative analysis showed that the use of bare metal stents and hybrid prostheses demonstrated a comparable low level of in-hospital mortality compared to the standard surgical technique of aortic arch reconstruction. At the same time, the use of the bare metal stents is associated with a higher rate of perioperative complications (bleeding, postoperative bowel ischemia, neurologica

Apoptosis and necrosis during reperfusion after ischemia are key mechanisms at the cellular level leading to damage. The development of pathological conditions is preceded by intracellular calcium ion overload both at the stage of ischemia and at the stage of reperfusion. In this regard, one of the strategies aimed at reducing damage during ischemia/reperfusion is associated with the use of calcium channel blockers. The aim of the study was to study the effect of a peptide toxin, a calcium channel blocker ω-hexatoxin-Hv1a, on different types of epithelial cell death during in vitro reconstruction of ischemia/reperfusion conditions characteristic of organ transplantation.

Materials and methods: In this study, we used CHO-K1 epithelial cell culture. Changes in apoptosis, necrosis, cell index, and calcium ion concentration were assessed when modeling ischemia/reperfusion processes in vitro with the addition of a calcium channel blocker toxin. Ischemic and reperfusion injury was achieved by oxygen and nutrient deprivation followed by reperfusion in a complete nutrient medium. The measurements were performed using a multimodal plate reader-fluorimeter.

Results: An increase in apoptosis, necrosis, and the concentration of calcium ions was recorded when modeling ischemia/reperfusion processes. A decrease in the level of apoptosis and necrosis, as well as the concentration of calcium ions to a physiological level or a level close to physiological, was noted when the toxin was added at a concentration of 50 nM at the reperfusion stage. The cell index showed a faster restoration in the presence of the toxin.

Conclusion: The experimental data confirm the hypothesis of a beneficial effect of peptide calcium channel blockers on the state of epithelial cells during reperfusion after ischemia and can be considered for further study as a strategy for organ adaptation before reperfusion.

The aim of the study is to evaluate the efficacy of approaches to sampling during periodic quality control of the artificial intelligence (AI) results in biomedical practice.

Materials and methods: The approaches to sampling based on point statistical estimation, statistical hypothesis testing, employing ready-made statistical tables, as well as options of the approaches presented in GOST R ISO 2859-1-2007 "Statistical methods. Sampling procedures for inspection by attributes" have been analyzed. We have considered variants of sampling of different sizes for general populations from 1000 to 100,000 studies.The analysis of the approaches to sampling was carried out as part of an experiment on the use of innovative technologies in computer vision for the analysis of medical images and their further application in the healthcare system of Moscow (Russia).

Results: Ready-made tables have specific statistical input data, which does not make them a universal option for biomedical research. Point statistical estimation helps to calculate a sample based on given statistical parameters with a certain confidence interval. This approach is promising in the case when only a type I error is important for the researcher, and a type II error is not a priority. Using the approach based on statistical hypothesis testing makes it possible to take account of type I and II errors based on the given statistical parameters. The application of GOST R ISO 2859-1-2007 for sampling allows using ready-made values depending on the given statistical parameters.When evaluating the efficacy of the studied approaches, it was found that for our purposes, the optimal number of studies during AI quality control for the analysis of medical images is 80 items. This meets the requirements of representativeness, balance of the risks to the consumer and the AI service provider, as well as optimization of labor costs of employees involved in the process of quality control of the AI results.

Liver pathologies remain one of the leading causes of mortality worldwide. Despite a high prevalence of liver diseases, the possibilities of diagnosing, prognosing, and treating non-alcoholic and alcoholic liver diseases still have a number of limitations and require the development of new methods and approaches. In laboratory studies, various models are used to reconstitute the pathological conditions of the liver, including cell cultures, spheroids, organoids, microfluidic systems, tissue slices. We reviewed the most commonly used in vivo, in vitro, and ex vivo models for studying non-alcoholic fatty liver disease and alcoholic liver disease, toxic liver injury, and fibrosis, described their advantages, limitations, and prospects for use. Great emphasis was placed on the mechanisms of development of pathological conditions in each model, as well as the assessment of the possibility of reconstructing various key aspects of pathogenesis for all these pathologies. There is currently no consensus on the choice of the most adequate model for studying liver pathology. The choice of a certain effective research model is determined by the specific purpose and objectives of the experiment.

The aim of the study was to identify different degrees of dermal lesions in vulvar lichen sclerosus (VLS) using cross-polarization optical coherence tomography (CP OCT) based on attenuation coefficient to detect disease early manifestations and to monitor the effectiveness of treatment.

Materials and methods: The study included 10 patients without pathology and 39 patients with VLS diagnosed histologically. CP OCT was performed in vivo on the inner surface of the labia minora, in the main lesion area. From each scanning point, a 3.4×3.4×1.25-mm3 3D data array was obtained in 26 s. CP OCT examination results were compared with histological examination of specimens stained with Van Gieson's picrofuchsin.Quantitative analysis of OCT images was performed by measuring the attenuation coefficient in co-polarization and cross-polarization. For visual analysis, color-coded charts were developed based on OCT attenuation coefficients.

Results: According to histological examination, all patients with VLS were divided into 4 groups as per dermal lesion degree: initial (8 patients); mild (7 patients); moderate (9 patients); severe (15 patients). Typical features of different degrees were interfibrillary edema up to 250 μm deep for initial degree, thickened collagen bundles without edema up to 350 μm deep for mild degree, dermis homogenization up to 700 μm deep for moderate degree, dermis homogenization and total edema up to 1200 μm deep for severe degree.Pathological processes in dermis during VLS like interfibrillary edema and collagen bundles homogenization were visualized using CP OCT method based on values of attenuation coefficient in co- and cross-polarization channels. However, CP OCT method appeared to be less sensitive to changes of collagen bundles thickness not allowing to distinguish thickened collagen bundles from normal ones with enough statistical significance. The CP OCT method was able to differentiate all degrees of dermal lesions among themselves. OCT attenuation coefficients differed from normal condition with statistical significance for all degrees of lesions, except for mild.

Conclusion: For the first time, quantitative parameters for each degrees of dermis lesion in VLS, including initial degree, were determined by CP OCT method allowing to detect the disease at an early stage and to monitor the applied clinical treatment effectiveness.

Modern methodology of PET/CT quantitative analysis in patients with glioblastomas is not strictly standardized in clinic settings and does not exclude the influence of the human factor. Methods of radiomics may facilitate unification, and improve objectivity and efficiency of the medical image analysis. The aim of the study is to evaluate the potential of radiomics in the analysis of PET/CT glioblastoma images identifying the relationship between the radiomic features and the ¹¹С-methionine tumor-to-normal brain uptake ratio (TNR) determined by an expert in routine.

Materials and methods: PET/CT data (2018-2020) from 40 patients (average age was 55±12 years; 77.5% were males) with a histologically confirmed diagnosis of "glioblastoma" were included in the analysis. TNR was calculated as a ratio of the standardized uptake value of ¹¹C-methionine measured in the tumor and intact tissue. Calculation of radiomic features for each PET was performed in the specified volumetric region of interest, capturing the tumor with the surrounding tissues. The relationship between TNR and the radiomic features was determined using the linear regression model. Predictors were included in the model following correlation analysis and LASSO regularization. The experiment with machine learning was repeated 300 times, splitting the training (70%) and test (30%) subsets randomly. The model quality metrics and predictor significance obtained in 300 tests were summarized.

Results: Of 412 PET/CT radiomic parameters significantly correlated with TNR (p<0.05), the regularization procedure left no more than 30 in each model (the median number of predictors was 9 [7; 13]). The experiment has demonstrated a non-random linear correlation (the Spearman correlation coefficient was 0.58 [0.43; 0.74]) between TNR and separate radiomic features, primarily fractal dimensions, characterizing the geometrical properties of the image.

Conclusion: Radiomics enabled an objective determination of PET/CT image texture features reflecting the biological activity of glioblastomas. Despite the existing limitations in the application, the first results provide a good perspective of these methods in neurooncology.

The aim of the study was to compare type I collagen-based and methacryloyl gelatin-based (GelMA) hydrogels by their ability to form hyaline cartilage in animals after subcutaneous implantation of scaffolds.

Materials and methods: Chondrocytes were isolated from the costal cartilage of newborn rats using 0.15% collagenase solution in DMEM. The cells was characterized by glycosaminoglycan staining with alcian blue. Chondrocyte scaffolds were obtained from 4% type I porcine atelocollagen and 10% GelMA by micromolding and then implanted subcutaneously into the withers of two groups of Wistar rats. Histological and immunohistochemical studies were performed on days 12 and 26 after implantation. Tissue samples were stained with hematoxylin and eosin, alcian blue; type I and type II collagens were identified by the corresponding antibodies.

Results: The implanted scaffolds induced a moderate inflammatory response in both groups when implanted in animals. By day 26 after implantation, both collagen and GelMA had almost completely resorbed. Cartilage tissue formation was observed in both animal groups. The newly formed tissue was stained intensively with alcian blue, and the cells were positive for both types of collagen. Cartilage tissue was formed among muscle fibers.

Conclusion: The ability of collagen type I and GelMA hydrogels to form hyaline cartilage in animals after subcutaneous implantation of scaffolds was studied. Both collagen and GelMA contributed to formation of hyaline-like cartilage tissue type in animals, but the chondrocyte phenotype is characterized as mixed. Additional detailed studies of possible mechanisms of chondrogenesis under the influence of each of the hydrogels are needed.