Search Results (13)

The development of highly sensitive approaches for detecting tumor cells in biological samples remains a critical challenge in laboratory and clinical oncology. In this study, we investigated the structural and magnetic properties of iron oxide nanoparticles incorporated into cellulose microspheres of two size ranges (~100 and ~700 μm) and evaluated their potential for targeted tumor cell isolation. In the smaller microspheres, magnetite-based magnetic nanoparticles (MNPs) were synthesized in situ via co-precipitation, whereas pre-synthesized MNPs were embedded into the larger microspheres. The geometrical characteristics of the resulting magnetic cellulose microspheres (MSCMNs) were assessed by confocal microscopy. Transmission electron microscopy and X-ray diffraction analyses revealed an average magnetic core size of approximately 17 nm. Magnetic properties of the MNPs within MSCMNs were characterized using a highly sensitive nonlinear magnetic response technique, and their dynamic parameters were derived using a formalism based on the stochastic Hilbert–Landau–Lifshitz equation. To evaluate their applicability in cancer diagnostics and treatment monitoring, the MSCMNs were functionalized with a TKD peptide that selectively binds membrane-associated Hsp70 (mHsp70), yielding TKD@MSCMNs. Magnetic separation enabled the isolation of tumor cells from biological fluids. The specificity of TKD-mediated binding was confirmed using Flamma648-labeled Hsp70 and compared with control alloferone-conjugated microspheres (All@MSCMNs). The ability of TKD@MSCMNs to selectively extract mHsp70-positive tumor cells was validated using C6 glioma cells and mHsp70-negative FetMSCs controls. Following co-incubation, the extraction efficiency for C6 cells was 28 ± 14%, significantly higher than that for FetMSC (7 ± 7%, p < 0.05). These findings highlight the potential of TKD-functionalized magnetic cellulose microspheres as a sensitive platform for tumor cell detection and isolation. Full article

Background/Objectives: A comparative study of silver (Ag), titanium nitride (TiN), zirconium nitride (ZrN), and copper (Cu) coatings on titanium (Ti) disks, considering the specifications of a microporous skin- and bone-integrated titanium pylon (SBIP), was performed to assess their biocompatibility, osseointegration, and mechanical properties. Methods: To assess cytotoxicity and biocompatibility, Ti disks with various metal coatings were co-cultured with FetMSCs and MG-63 cells for 1, 3, 7, and 14 days and subsequently evaluated using a cell viability assay, as supported by SEM and confocal microscopy studies. The antimicrobial activity of the selected four materials coating the implants was tested against S. aureus by mounting Ti disks onto the surface of LB agar dishes spread with a bacterial suspension and measuring the diameter of the growth inhibition zones. Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) analysis of the relative gene expression of biomarkers that are associated with extracellular matrix components (fibronectin, vitronectin, type I collagen) and cell adhesion (α2, α5, αV integrins), as well as of osteogenic markers (osteopontin, osteonectin, TGF-β1, SMAD), was performed during the 14-day follow-up period. Additionally, the activity of matrix metalloproteinases (MMP-1, -2, -8, -9) was assessed. Results: All samples with metal coatings, except the copper coating, demonstrated a good cytotoxicity profile, as evidenced by the presence of a cellular monolayer on the sample surface on the 14th day of the follow-up period (as shown by SEM and inverted confocal microscopy). All metal coatings enhanced MMP activity, as well as cellular adhesion and osteogenic marker expression; however, TiN showed the highest values of these parameters. Significant inhibition of bacterial growth was observed only in the Ag-coated Ti disks, and it persisted for over 35 days. Conclusions: The silver-based coating, due to its high antibacterial activity, low cytotoxicity, and biointegrative capacity, can be recommended as the coating of choice for microporous titanium implants for further preclinical studies. Full article

It has been previously shown that some short peptides are involved in various cellular processes, such as transcription modulation and regulation of differentiation mechanisms. In particular, the effect of peptides on the neuronal differentiation of human periodontal ligament stem cells has been demonstrated. The goal of this study was to assess the effect of KED, EDR, and AEDG short peptides in stimulating the transdifferentiation of fetal MSCs into induced neuronal cells and prevention of their senescence. We applied a novel in vitro technique for neuronal cell generation, which combines the use of microRNAs, transcription factors, and small molecules to transdifferentiate fetal mesenchymal stem cells into induced cortical neurons. It was shown that the application of AEDG and KED short peptides at the end of the transdifferentiation process decreases the expression of the cell cycle marker p21 by 15% and beta-galactosidase activity by 1.51–2.4 times. However, short peptides did not affect the expression levels of TUj-1 and LaminB1, whose expression also changes during neuronal differentiation. The experiments indicate the potential of AEDG and KED short peptides as modulators of neurogenesis and geroprotectors and suggest that they can be used as stimulators of neuronal differentiation. Full article

Stem cells therapies offer a promising approach in translational oncology, as well as in regenerative medicine due to the tropism of these cells to the damage site. To track the distribution of stem cells, the latter could be labeled by MRI-sensitive superparamagnetic (SPM) iron oxide nanoparticles. In the current study, magnetic properties of the magnetic nanoparticles (MNPs) incorporated into the bone marrow-derived fetal mesenchymal stem cells (FetMSCs) were evaluated employing nonlinear magnetic response measurements. Synthesized dextran-coated iron oxide nanoparticles were additionally characterized by X-ray diffraction, transmission electron microscopy, and dynamic light scattering. The MNP uptake by the FetMSCs 24 h following coincubation was studied by longitudinal nonlinear response to weak alternating magnetic field with registration of the second harmonic of magnetization. Subsequent data processing using a formalism based on the numerical solution of the Fokker–Planck kinetic equation allowed us to determine magnetic and dynamic parameters and the state of MNPs in the cells, as well as in the culture medium. It was found that MNPs formed aggregates in the culture medium; they were absorbed by the cells during coincubation. The aggregates exhibited SPM regime in the medium, and the parameters of the MNP aggregates remained virtually unchanged in the cells, indicating the preservation of the aggregation state of MNPs inside the cells. This implies also the preservation of the organic shell of the nanoparticles inside FetMSCs. The accumulation of MNPs by mesenchymal stem cells gradually increased with the concentration of MNPs. Thus, the study confirmed that the labeling of MSCs with MNPs is an effective method for subsequent cell tracking as incorporated nanoparticles retain their magnetic properties. Full article

In order to scientifically search for new sources of secondary metabolites with valuable qualities for phytopharmacognosy, tasks requiring a step-by-step solution were set. The primary task is the development of technologies for obtaining in vitro highly productive biomass of cells of relict gymnosperms of the genus Sequoia, capable of accumulating various classes of secondary metabolites. The study of the accumulation and localization of secondary metabolites allowed us to evaluate the biological activity and cytotoxicity of in vitro Sequoia cultures. In our study, histochemical methods were used to determine the localization of secondary compounds (phenolic and terpenoid in nature) in plant tissues. Secondary metabolites—polyphenols, catechins, and terpenoids—are mainly localized in the epidermal, parenchymal, and conductive tissues of Sequoia leaves and stems. In callus and suspension cultures of Sequoia, secondary metabolites were localized in cell walls and vacuoles. The mineral composition of the nutrient medium (MS and WPM), the light source (photoperiod), and the endogenous content of polyphenols in the primary explant influenced the initiation and growth characteristics of the in vitro culture of Sequoia plants. Inhibition of growth in suspension cultures on the WPM nutrient medium was noted. The cultivation of Sequoia cell lines at a 16 h photoperiod stimulated the formation of polyphenols but had a negative effect on the growth of callus cultures. Extractive substances obtained from intact and callus tissues of evergreen Sequoia demonstrate high biological (fungicidal) activity and cytotoxicity. The inhibitory effect on Fusarium oxisporum was noted when 200 mg/L of Sequoia extract was added to the nutrient medium. Extracts of redwood callus cultures were low in toxicity to normal FetMSC cells but inhibited the growth of lines of “immortal” cervical HeLa cancer cells and human glioblastoma A172. Intact tissues of Sequoia plants and cell cultures initiated from them in vitro are producers of secondary metabolites with high biological activity. Full article

The terpolymers of N-vinylpyrrolidone (VP) with acrylic acid and triethylene glycol methacrylate were synthesized with more than 90% yield by radical copolymerization in ethanol from monomeric mixtures of different molar composition (98:2:2, 95:5: 2 and 98:2:5) and their monomer composition, absolute molecular masses and hydrodynamic radii in aqueous media were determined. Using the MTT test, these terpolymers were established to be low toxic for non-tumor Vero cells and HeLa tumor cells. Polymer compositions of hydrophobic dye methyl pheophorbide a (MPP) based on studied terpolymers and linear polyvinylpyrrolidone (PVP) were obtained and characterized in water solution. Quantum-chemical modeling of the MPP-copolymer structures was conducted, and the possibility of hydrogen bond formation between terpolymer units and the MPP molecule was shown. Using fluorescence microscopy, the accumulation and distribution of polymer particles in non-tumor (FetMSC) and tumor (HeLa) cells was studied, and an increase in the accumulation of MPP with both types of particles was found. Full article

Cellulose is one of the main renewable polymers whose properties are very attractive in many fields, including biomedical applications. The modification of nanocrystalline cellulose (NCC) opens up the possibility of creating nanomaterials with properties of interest as well as combining them with other biomedical polymers. In this work, we proposed the covalent modification of NCC with amphiphilic polyanions such as modified heparin (Hep) and poly(αL-glutamic acid) (PGlu). The modification of NCC should overcome two drawbacks in the production of composite materials based on poly(ε-caprolactone) (PCL), namely, (1) to improve the distribution of modified NCC in the PCL matrix, and (2) to provide the composite material with osteoconductive properties. The obtained specimens of modified NCC were characterized by Fourier-transform infrared spectroscopy and solid-state ¹³C nuclear magnetic resonance spectroscopy, dynamic and electrophoretic light scattering, as well as thermogravimetric analysis. The morphology of PCL-based composites containing neat or modified NCC as filler was studied by optical and scanning electron microscopy. The mechanical properties of the obtained composites were examined in tensile tests. The homogeneity of filler distribution as well as the mechanical properties of the composites depended on the method of NCC modification and the amount of attached polyanion. In vitro biological evaluation showed improved adhesion of human fetal mesenchymal stem cells (FetMSCs) and human osteoblast-like cells (MG-63 osteosarcoma cell line) to PCL-based composites filled with NCC bearing Hep or PGlu derivatives compared to pure PCL. Furthermore, these composites demonstrated the osteoconductive properties in the experiment on the osteogenic differentiation of FetMSCs. Full article

A new method for the synthesis of azido-propargyloxy derivatives of 1,3,5-triazine has been developed utilizing the nitrosation of hydrazyno-1,3,5-triazines. New hydrazines (2-hydrazino-4,6-bis(propargyloxy)-1,3,5-triazine and 2,4-dihydrazino-6-propargyloxy-1,3,5-triazine) were synthesized and characterized via FTIR, NMR spectroscopy and elemental analysis. The hyperbranched polymers with azide (diazide monomer) and propargyloxy terminal groups were obtained via the azide-alkyne polycycloaddition reaction of diazide and monoazide AB₂-type monomers. The antibacterial activity against Escherichia coli bacteria of 2,4,6-trispropargyloxy-1,3,5-triazine, 2-azido-4,6-bispropargyloxy-1,3,5-triazine, and 2,4-diazido-6-propargyloxy-1,3,5-triazine and their hyperbranched polymers was studied. Only 2,4-diazido-6-propargyloxy-1,3,5-triazine has weak antibacterial activity in comparison with ampicillin. The cytotoxicity of these compounds against M-HeLa, FetMSC, and Vero cell lines was also studied. 2,4,6-trispropargyloxy-1,3,5-triazine does not show any cytotoxic effect (IC₅₀ ≥ 280 µM). It was shown that the presence of an azide group in the compound directly affects the cytotoxic effect. Hyperbranched polymers have a less cytotoxic effect against M-HeLa (IC₅₀ > 100) in comparison with monomers (IC₅₀ = 90–99 µM). This makes it possible to use these polymers as the basis for biocompatible materials in biomedical applications. Full article

Using a novel method of N-substituted succinimide ring opening, new N-hydroxybutanamide derivatives were synthesized. These compounds were evaluated for their ability to inhibit matrix metalloproteinases (MMPs) and their cytotoxicity. The iodoaniline derivative of N¹-hydroxy-N⁴-phenylbutanediamide showed the inhibition of MMP-2, MMP-9, and MMP-14 with an IC₅₀ of 1–1.5 μM. All the compounds exhibited low toxicity towards carcinoma cell lines HeLa and HepG2. The iodoaniline derivative was also slightly toxic to glioma cell lines A-172 and U-251 MG. Non-cancerous FetMSC and Vero cells were found to be the least sensitive to all the compounds. In vivo studies demonstrated that the iodoaniline derivative of N¹-hydroxy-N⁴-phenylbutanediamide had low acute toxicity. In a mouse model of B16 melanoma, this compound showed both antitumor and antimetastatic effects, with a 61.5% inhibition of tumor growth and an 88.6% inhibition of metastasis. Our findings suggest that the iodoaniline derivative of N¹-hydroxy-N⁴-phenylbutanediamide has potential as a lead structure for the development of new MMP inhibitors. Our new synthetic approach can be a cost-effective method for the synthesis of inhibitors of metalloenzymes with promising antitumor potential. Full article

New tubular conduits have been developed for the regeneration of peripheral nerves and the repair of defects that are larger than 3 cm. The conduits consist of a combination of poly(L-lactide) nanofibers and chitosan composite fibers with chitin nanofibrils. In vitro studies were conducted to assess the biocompatibility of the conduits using human embryonic bone marrow stromal cells (FetMSCs). The studies revealed good adhesion and differentiation of the cells on the conduits just one day after cultivation. Furthermore, an in vivo study was carried out to evaluate motor-coordination disorders using the sciatic nerve functional index (SFI) assessment. The presence of chitosan monofibers and chitosan composite fibers with chitin nanofibrils in the conduit design increased the regeneration rate of the sciatic nerve, with an SFI value ranging from 76 to 83. The degree of recovery of nerve conduction was measured by the amplitude of M-response, which showed a 46% improvement. The conduit design imitates the oriented architecture of the nerve, facilitates electrical communication between the damaged nerve’s ends, and promotes the direction of nerve growth, thereby increasing the regeneration rate. Full article

The combination of titania nanofilms and silver nanoparticles (NPs) is a very promising material, with antibacterial and osseointegration-induced properties for titanium implant coatings. In this work, we successfully prepared TiO₂ nanolayer/Ag NP structures on titanium disks using atomic layer deposition (ALD). The samples were studied by scanning electron microscopy (SEM), X-ray diffraction, X-ray photoelectron spectroscopy (XPS), contact angle measurements, and SEM-EDS. Antibacterial activity was tested against Staphylococcus aureus. The in vitro cytological response of MG-63 osteosarcoma and human fetal mesenchymal stem cells (FetMSCs) was examined using SEM study of their morphology, MTT test of viability and differentiation using alkaline phosphatase and osteopontin with and without medium-induced differentiation in the osteogenic direction. The samples with TiO₂ nanolayers, Ag NPs, and a TiO₂/Ag combination showed high antibacterial activity, differentiation in the osteogenic direction, and non-cytotoxicity. The medium for differentiation significantly improved osteogenic differentiation, but the ALD coatings also stimulated differentiation in the absence of the medium. The TiO₂/Ag samples showed the best antibacterial ability and differentiation in the osteogenic direction, indicating the success of the combining of TiO₂ and Ag to produce a multifunctional biocompatible and bactericidal material. Full article

Titanium oxide nanocoatings were synthesized on the surface of monocrystalline silicon and ultra-fine-grained titanium by atomic layer deposition (ALD) using titanium tetrachloride (TiCl₄) and titanium tetraisopropoxide (TTIP). The morphology of the samples was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The structure and composition were studied by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), contact angle measurements, and energy-dispersive spectroscopy (EDS). The cytological response of osteosarcoma MG-63 and human fetal mesenchymal stem cells (FetMSCs) were studied by analyzing their morphology, viability, and alkaline phosphatase activity with and without the use of medium-induced differentiation in the osteogenic direction. A significant influence of the precursor type and ALD temperature on the crystal structure, morphology, composition, and surface free energy of TiO₂ nanocoatings was found. The biocompatibility of amorphous non-stoichiometric and partially crystalline stoichiometric TiO₂ coatings was compared. Both types of cells showed faster adhesion and improved spreading on the surface for the samples from TTIP compared to those from TiCl₄ at the early stages of cultivation (2 h) due to the difference in composition and higher surface free energy. No cytotoxic effect was found on both types of coatings, nor was there a noticeable difference in cell differentiation. All ALD coatings provided excellent biocompatibility and osteoconductive properties. Full article

Secretome of multipotent mesenchymal stromal cells (MSCs) is actively used in biomedical applications such as alveolar bone regeneration, treatment of cardiovascular disease, and neurodegenerative disorders. Nevertheless, hMSCs have low proliferative potential and production of the industrial quantity of their secretome might be challenging. Human fetal multipotent mesenchymal stromal cells (FetMSCs) isolated from early human embryo bone marrow are easy to expand and might be a potential source for pharmaceutical substances production based on their secretome. However, the secretome of FetMSCs was not previously analyzed. Here, we describe the secretome of FetMSCs using LC-MALDI shotgun proteomics. We identified 236 proteins. Functional annotation of the identified proteins revealed their involvement in angiogenesis, ossification, regulation of apoptosis, and immune response processes, which made it promising for biomedical applications. The proteins identified in the FetMSCs secretome are involved in the same biological processes as proteins from previously described adult hMSCs secretomes. Nevertheless, many of the common hMSCs secretome components (such as VEGF, FGF, Wnt and TGF-β) have not been identified in the FetMSCs secretome. Full article