Pokročilé biomateriály

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In vitro culture of leukemic cells in collagen scaffolds and carboxymethyl cellulose-polyethylene glycol gel
(PeerJ, 2024-12-06) Svozilova, Hana; Vojtová, Lucy; Matulová, Jana; Bruknerova, Jana; Poláková, Veronika; Radová, Lenka; Doubek, Michael; Plevová, Karla; Pospíšilová, Šárka
Background: Chronic lymphocytic leukemia (CLL) is a common adult leukemia characterized by the accumulation of neoplastic mature B cells in blood, bone marrow, lymph nodes, and spleen. The disease biology remains unresolved in many aspects, including the processes underlying the disease progression and relapses. However, studying CLL in vitro poses a considerable challenge due to its complexity and dependency on the microenvironment. Several approaches are utilized to overcome this issue, such as co-culture of CLL cells with other cell types, supplementing culture media with growth factors, or setting up a three-dimensional (3D) culture. Previous studies have shown that 3D cultures, compared to conventional ones, can lead to enhanced cell survival and altered gene expression. 3D cultures can also give valuable information while testing treatment response in vitro since they mimic the cell spatial organization more accurately than conventional culture. Methods: In our study, we investigated the behavior of CLL cells in two types of material: (i) solid porous collagen scaffolds and (ii) gel composed of carboxymethyl cellulose and polyethylene glycol (CMC-PEG). We studied CLL cells' distribution, morphology, and viability in these materials by a transmitted-light and confocal microscopy. We also measured the metabolic activity of cultured cells. Additionally, the expression levels of MYC, VCAM1, MCL1, CXCR4, and CCL4 genes in CLL cells were studied by qPCR to observe whether our novel culture approaches lead to increased adhesion, lower apoptotic rates, or activation of cell signaling in relation to the enhanced contact with co-cultured cells. Results: Both materials were biocompatible, translucent, and permeable, as assessed by metabolic assays, cell staining, and microscopy. While collagen scaffolds featured easy manipulation, washability, transferability, and biodegradability, CMC-PEG was advantageous for its easy preparation process and low variability in the number of accommodated cells. Both materials promoted cell-to-cell and cell-to-matrix interactions due to the scaffold structure and generation of cell aggregates. The metabolic activity of CLL cells cultured in CMC-PEG gel was similar to or higher than in conventional culture. Compared to the conventional culture, there was (i) a lower expression of VCAM1 in both materials, (ii) a higher expression of CCL4 in collagen scaffolds, and (iii) a lower expression of CXCR4 and MCL1 (transcript variant 2) in collagen scaffolds, while it was higher in a CMC-PEG gel. Hence, culture in the material can suppress the expression of a pro-apoptotic gene ( MCL1 in collagen scaffolds) or replicate certain gene expression patterns attributed to CLL cells in lymphoid organs (low CXCR4, high CCL4 in collagen scaffolds) or blood (high CXCR4 in CMC-PEG).
Porcine model of a complicated skin and soft tissue infection caused by Pseudomonas aeruginosa
(CZECH ACADEMY AGRICULTURAL SCIENCES, 2024-09-10) Lipový, Břetislav; Vacek, Lukáš; Polaštík Kleknerová, Dominika; Jeklová, Edita; Lišková, Lenka; Holoubek, Jakub; Matýsková, Dominika; Růžička, Filip
Pseudomonas aeruginosa poses a significant threat to both immunocompetent and immunocompromised individuals, often resulting in life -threatening infections. With increasing antimicrobial resistance, novel therapeutic strategies are urgently needed. Although animal models are crucial for preclinical studies, limited data are available for porcine models, more specifically for P. aeruginosa complicated skin and soft tissue infections (cSSTIs). This study presents a novel porcine model inducing and sustaining cSSTI for 14 days. Six pigs (120 wounds) were used for the development of infections, and within this group, two pigs (40 wounds) were used to evaluate the progression of the cSSTI infection. The model demonstrated bacterial loads of more than 10 7 CFU/ gram of tissue or higher. The cSSTI fully developed within three days and remained well above these levels until day 14 post -infection. Due to the immunocompetence of this model, all the immunological processes associated with the response to the presence of infection and the wound healing process are preserved.
Pharmaceutical Metabolite Identification in Lettuce (Lactuca sativa) and Earthworms (Eisenia fetida) Using Liquid Chromatography Coupled to High-Resolution Mass Spectrometry and In Silico Spectral Library
(Springer-Verlag, 2024-09-10) Fučík, Jan; Fučík, Stanislav; Rexroth, Sascha; Sedlář, Marian; Zlámalová Gargošová, Helena; Mravcová, Ludmila
Pharmaceuticals released into the aquatic and soil environments can be absorbed by plants and soil organisms, potentially leading to the formation of unknown metabolites that may negatively affect these organisms or contaminate the food chain. The aim of this study was to identify pharmaceutical metabolites through a triplet approach for metabolite structure prediction (software-based predictions, literature review, and known common metabolic pathways), followed by generating in silico mass spectral libraries and applying various mass spectrometry modes for untargeted LC-qTOF analysis. Therefore, Eisenia fetida and Lactuca sativa were exposed to a pharmaceutical mixture (atenolol, enrofloxacin, erythromycin, ketoprofen, sulfametoxazole, tetracycline) under hydroponic and soil conditions at environmentally relevant concentrations. Samples collected at different time points were extracted using QuEChERS and analyzed with LC-qTOF in data-dependent (DDA) and data-independent (DIA) acquisition modes, applying both positive and negative electrospray ionization. The triplet approach for metabolite structure prediction yielded in a total of 3,762 pharmaceutical metabolites, and in silico mass spectral library was created based on these predicted metabolites. This approach resulted in the identification of 26 statistically significant metabolites (p<0.05), with DDA+ and DDA- outperforming DIA modes by successfully detecting 56/67 sample type:metabolite combinations. Lettuce roots had the highest metabolite count (26), followed by leaves (6) and earthworms (2). Despite the lower metabolite count, earthworms showed the highest peak intensities, closely followed by roots, with leaves displaying the lowest intensities. Common metabolic reactions observed included hydroxylation, decarboxylation, acetylation, and glucosidation, with ketoprofen-related metabolites being the most prevalent, totaling 12 distinct metabolites. In conclusion, we developed a high-throughput workflow combining open-source software with LC-HRMS for identifying unknown metabolites across various sample types.
Assessing Earthworm Exposure to a Multi-Pharmaceutical Mixture in Soil: Unveiling Insights through LC-MS and MALDI-MS Analyses, and Impact of Biochar on Pharmaceutical Bioavailability
(Springer-Verlag, 2024-07-19) Fučík, Jan; Jarošová, Jana; Baumeister, Andreas; Rexroth, Sascha; Navrkalová, Jitka; Sedlář, Marian; Zlámalová Gargošová, Helena; Mravcová, Ludmila
In the European circular economy, agricultural practices introduce pharmaceutical (PhAC) residues into the terrestrial environment, posing a potential risk to earthworms. This study aimed to assess earthworm bioaccumulation factors (BAFs), the ecotoxicological effects of PhACs, the impact of biochar on PhAC bioavailability to earthworms and their persistence in soil and investigate earthworm uptake mechanisms along with the spatial distribution of PhACs. Therefore, earthworms were exposed to contaminated soil for 21 days. The results revealed that BAFs ranged from 0.0216 to 0.329, with no significant ecotoxicological effects on earthworm weight or mortality (p>0.05). Biochar significantly influenced the uptake of 14 PhACs on the first day (p<0.05), with diminishing effects over time, and affected significantly the soil-degradation kinetics of 16 PhACs. Moreover, MALDI-MS analysis revealed that PhAC uptake occurs through both the dermal and oral pathways, as pharmaceuticals were distributed throughout the entire earthworm tissue without specific localization. In conclusion, this study suggests ineffective PhAC accumulation in earthworms, highlights the influence of biochar on PhAC degradation rates in soil, and suggests that uptake can occur through both earthworm skin and oral ingestion.
The role of glycerol in manufacturing freeze-dried chitosan and cellulose foams for mechanically stable scaffolds in skin tissue engineering
(Elsevier, 2024-07-02) Verčimáková, Katarína; Karbowniczek, Joanna; Sedlář, Marian; Stachewicz, Urszula; Vojtová, Lucy
Various strategies have extensively explored enhancing the physical and biological properties of chitosan and cellulose scaffolds for skin tissue engineering. This study presents a straightforward method involving the addition of glycerol into highly porous structures of two polysaccharide complexes: chitosan/carboxymethyl cellulose (Chit/CMC) and chitosan/oxidized cellulose (Chit/OC); during a one-step freeze-drying process. Adding glycerol, especially to Chit/CMC, significantly increased stability, prevented degradation, and improved mechanical strength by nearly 50%. Importantly, after 21 days of incubation in enzymatic medium Chit/CMC scaffold has almost completely decomposed, while foams reinforced with glycerol exhibited only 40% mass loss. It is possible due to differences in multivalent cations and polymer chain contraction, resulting in varied hydrogen bonding and, consequently, distinct physicochemical outcomes. Additionally, the scaffolds with glycerol improved the cellular activities resulting in over 40% higher proliferation of fibroblast after 21 days of incubation. It was achieved by imparting water resistance to the highly absorbent material and aiding in achieving a balance between hydrophilic and hydrophobic properties. This study clearly indicates the possible elimination of additional crosslinkers and multiple fabrication steps that can reduce the cost of scaffold production for skin tissue engineering applications while tailoring mechanical strength and degradation.

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