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Macroporous poly(vinyl alcohol) cryogels (PVACGs) are physical gels formed via cryogenic processing of polymer solutions. The properties of PVACGs depend on many factors: the characteristics and concentration of PVA, the absence or presence of foreign solutes, and the freezing-thawing conditions. These factors also affect the macroporous morphology of PVACGs, their total porosity, pore size and size distribution, etc. In this respect, there is the problem with developing a scientifically-grounded classification of the morphological features inherent in various PVACGs. In this study PVA cryogels have been prepared at different temperatures when the initial polymer solutions contained chaotropic or kosmotropic additives. After the completion of gelation, the rigidity and heat endurance of the resultant PVACGs were evaluated, and their macroporous structure was investigated using optical microscopy. The images obtained were treated mathematically, and deep neural networks were used for the classification of these images. Training and test sets were used for their classification. The results of this classification for the specific deep neural network architecture are presented, and the morphometric parameters of the macroporous structure are discussed. It was found that deep neural networks allow us to reliably classify the type of additive or its absence when using a combined dataset.
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Criogéis/química , Redes Neurais de Computação , Polímeros/química , Álcool de Polivinil/química , Módulo de Elasticidade , Congelamento , Temperatura Alta , PorosidadeRESUMO
Freeze-thaw processing of bovine serum albumin (BSA) aqueous solutions, which contain also the additives of denaturants (urea in this case) and thiol-bearing reductants [cysteine (Cys) in this case] leads to the formation of wide-pore cryogels. The properties and porous morphology of these spongy gel matrices were demonstrated to depend on the initial concentration of all precursors and on the freezing/frozen storage temperature. The optimum conditions for preparing such BSA-based cryogels were found to be as follows: [BSA] = 3-5 g dL(-1), [urea] = 0.5-2.0 mol L(-1), [Cys] = 0.01 mol L(-1), and freezing temperatures in the range of -15 to -20 °C. The size of gross pores in thus prepared cryogels is â¼50-150 µm. The spatial network of BSA-cryogels was shown to be cross-linked chemically via interchain disulfide bridges. The significant role of hydrophobic interactions in the stabilization of 3D networks of these cryogels is inferred, as well as the supposition about the relay-race sequence mechanism of the intermolecular disulfide cross-link formation is made.
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Criogéis/química , Congelamento , Desnaturação Proteica , Soroalbumina Bovina/químicaRESUMO
Cultivation and proliferation of stem cells in three-dimensional (3-D) scaffolds is a promising strategy for regenerative medicine. Mesenchymal stem cells with their potential to differentiate in various cell types, cryopreserved adhesion-based in fabricated scaffolds of biocompatible materials can serve as ready-to-use transplantation units for tissue repair, where pores allow a direct contact of graft cells and recipient tissue without further preparation. A successful cryopreservation of adherent cells depends on attachment and spreading processes that start directly after cell seeding. Here, we analyzed different cultivation times (0.5, 2, 24 h) prior to adhesion-based cryopreservation of human mesenchymal stem cells within alginate-gelatin cryogel scaffolds and its influence on cell viability, recovery and functionality at recovery times (0, 24, 48 h) in comparison to non-frozen control. Analysis with confocal laser scanning microscopy and scanning electron microscopy indicated that 2 h cultivation time enhanced cryopreservation success: cell number, visual cell contacts, membrane integrity, motility, as well as spreading were comparable to control. In contrast, cell number by short cultivation time (0.5 h) reduced dramatically after thawing and expanded cultivation time (24 h) decreased cell viability. Our results provide necessary information to enhance the production and to store ready-to-use transplantation units for application in bone, cartilage or skin regenerative therapy.
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Técnicas de Cultura Celular por Lotes/instrumentação , Criopreservação/métodos , Regeneração Tecidual Guiada/instrumentação , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Engenharia Tecidual/instrumentação , Alicerces Teciduais , Alginatos/química , Técnicas de Cultura Celular por Lotes/métodos , Adesão Celular/fisiologia , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Criogéis/química , Desenho de Equipamento , Análise de Falha de Equipamento , Gelatina/química , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Medicina Regenerativa/instrumentaçãoRESUMO
Poly(vinyl alcohol) (PVA) physical cryogels that contained the additives of o-, m-, and p-bis-phenols or phenol were prepared, and their physico-chemical characteristics and macroporous morphology and the solute release dynamics were evaluated. These phenolic additives caused changes in the viscosity of initial PVA solutions before their freeze-thaw processing and facilitated the growth in the rigidity of the resultant cryogels, while their heat endurance decreased. The magnitude of the effects depended on the interposition of phenolic hydroxyls in the molecules of the used additives and was stipulated by their H-bonding with PVA OH-groups. Subsequent rinsing of such "primary" cryogels with pure water led to the lowering of their rigidity. The average size of macropores inside these heterophase gels also depended on the additive type. It was found also that the release of phenolic substances from the additive-containing cryogels occurred via virtually a free diffusion mechanism; therefore, drug delivery systems such as PVA cryogels loaded with either pyrocatechol, resorcinol, hydroquinone, or phenol, upon the in vitro agar diffusion tests, exhibited antibacterial activity typical of these phenols. The promising biomedical potential of the studied nanocomposite gel materials is supposed.
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Immobilization of microorganisms on/in insoluble carriers is widely used to stabilize functional activity of microbial cells in industrial biotechnology. We immobilized Rhodococcus ruber, an important hydrocarbon degrader, on biosurfactant-coated sawdust. A biosurfactant produced by R. ruber in the presence of liquid hydrocarbons was found to enhance rhodococcal adhesion to solid surfaces, and thus, it was used as a hydrophobizing agent to improve bacterial attachment to a sawdust carrier. Compared to previously used hydrophobizers (drying oil and n-hexadecane) and emulsifiers (methyl- and carboxymethyl cellulose, poly(vinyl alcohol), and Tween 80), Rhodococcus biosurfactant produced more stable and homogenous coatings on wood surfaces, thus resulting in higher sawdust affinity to hydrocarbons, uniform monolayer distribution of immobilized R. ruber cells (immobilization yield 29-30 mg dry cells/g), and twofold increase in hydrocarbon biooxidation rates compared to free rhodococcal cells. Two physical methods, i.e., high-resolution profilometry and infrared thermography, were applied to examine wood surface characteristics and distribution of immobilized R. ruber cells. Sawdust-immobilized R. ruber can be used as an efficient biocatalyst for hydrocarbon transformation and degradation.
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Células Imobilizadas/metabolismo , Hidrocarbonetos/metabolismo , Rhodococcus/metabolismo , Tensoativos/metabolismo , Madeira/microbiologia , Aderência Bacteriana , Biotecnologia/métodos , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Rhodococcus/química , Rhodococcus/fisiologiaRESUMO
Physical macroporous poly(vinyl alcohol)-based cryogels formed by the freeze-thaw technique without the use of any foreign cross-linkers are of significant interests for biomedical applications. In the present study, such gel materials loaded with the antimicrobial substances were prepared and their physicochemical properties were evaluated followed by an assessment of their potential to serve as drug carriers that can be used as implants for the treatment of infected wounds. The antibiotic Ceftriaxone and the antimycotic Fluconazole were used as antimicrobial agents. It was shown that the Ceftriaxone additives caused the up-swelling effects with respect to the cryogel matrix and some decrease in its heat endurance but did not result in a substantial change in the gel strength. With that, the drug release from the cryogel vehicle occurred without any diffusion restrictions, which was demonstrated by both the spectrophotometric recording and the microbiological agar diffusion technique. In turn, the in vivo biotesting of such drug-loaded cryogels also showed that these materials were able to function as rather efficient antimicrobial implants injected in the artificially infected model wounds of laboratory rabbits. These results confirmed the promising biomedical potential of similar implants.
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Visible light irradiation of an aqueous solution of sodium alginate and organometallic complex [(C5H5)Fe(toluene)]BF4 transforms it into a rigid hydrogel due to crosslinking of the carboxylate groups by the iron ions. Irradiation of the same iron complex together with K2S2O8 initiates the polymerization of acrylamide, which provides an efficient method for light-controlled one-step preparation of alginate-polyacrylamide double network hydrogels, which are capable of gluing wet glass with 100-200 kPa shear strength.
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Highly porous composite poly(vinyl alcohol) (PVA) cryogels loaded with the poly(3-hydroxybutyrate) (PHB) microbeads containing the drug, simvastatin (SVN), were prepared via cryogenic processing (freezing-storing frozen-defrosting) of the beads' suspensions in aqueous PVA solution. The rigidity of the resultant composite cryogels increased with increasing the filler content. Optical microscopy of the thin section of such gel matrices revealed macro-porous morphology of both continuous (PVA cryogels) and discrete (PHB-microbeads) phases. Kinetic studies of the SVN release from the drug-loaded microbeads, the non-filled PVA cryogel and the composite material showed that the cryogel-based composite system could potentially serve as a candidate for the long-term therapeutic system for controlled drug delivery. Such PHB-microbeads-containing PVA-cryogel-based composite drug delivery carriers were unknown earlier; their preparation and studies have been performed for the first time.
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Bone tissue engineering strategy involves the 3D scaffolds and appropriate cell types promoting the replacement of the damaged area. In this work, we aimed to develop a fast and reliable clinically relevant protocol for engineering viable bone grafts, using cryopreserved adipose tissue-derived mesenchymal stromal cells (MSCs) and composite 3D collagen-nano-hydroxyapatite (nanoHA) scaffolds. Xeno- and DMSO-free cryopreserved MSCs were perfusion-seeded into the biomimetic collagen/nanoHA scaffolds manufactured by cryotropic gelation and their osteoregenerative potential was assessed in vitro and in vivo. Cryopreserved MSCs retained the ability to homogenously repopulate the whole volume of the scaffolds during 7 days of post-thaw culture. Moreover, the scaffold provided a suitable microenvironment for induced osteogenic differentiation of cells, confirmed by alkaline phosphatase activity and mineralization. Implantation of collagen-nanoHA cryogels with cryopreserved MSCs accelerated woven bone tissue formation, maturation of bone trabeculae, and vascularization of femur defects in immunosuppressed rats compared to cell-free collagen-nanoHA scaffolds. The established combination of xeno-free cell culture and cryopreservation techniques together with an appropriate scaffold design and cell repopulation approach accelerated the generation of viable bone grafts.
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Criogéis , Células-Tronco Mesenquimais , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colágeno/farmacologia , Criopreservação , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Ratos , Engenharia Tecidual/métodos , Alicerces TeciduaisRESUMO
Various gelatin-containing gel materials are used as scaffolds for animal and human cell culturing within the fields of cell technologies and tissue engineering. Cryostructuring is a promising technique for the preparation of efficient macroporous scaffolds in biomedical applications. In the current study, two new gelatin-based cryostructurates were synthesized, their physicochemical properties and microstructure were evaluated, and their ability to serve as biocompatible scaffolds for mammalian cells culturing was tested. The preparation procedure included the dissolution of Type A gelatin in water, the addition of urea to inhibit self-gelation, the freezing of such a solution, ice sublimation in vacuo, and urea extraction with ethanol from the freeze-dried matter followed by its cross-linking in an ethanol medium with either carbodiimide or glyoxal. It was shown that in the former case, a denser cross-linked polymer phase was formed, while in the latter case, the macropores in the resultant biopolymer material were wider. The subsequent biotesting of these scaffolds demonstrated their biocompatibility for human mesenchymal stromal cells and HepG2 cells during subcutaneous implantation in rats. Albumin secretion and urea synthesis by HepG2 cells confirmed the possibility of using gelatin cryostructurates for liver tissue engineering.
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Targeted drug release is a significant research focus in the development of drug delivery systems and involves a biocompatible polymeric carrier and certain medicines. Cryostructuring is a suitable approach for the preparation of efficient macroporous carriers for such drug delivery systems. In the current study, the cryogenically structured carriers based on alginate/chondroitin sulfate mixtures were prepared and their physicochemical properties and their ability to absorb/release the bactericides were evaluated. The swelling parameters of the polysaccharide matrix, the amount of the tightly bound water in the polymer and the sulfur content were measured. In addition, FTIR and UV spectroscopy, optical and scanning microscopy, as well as a standard disk diffusion method for determining antibacterial activity were used. It was shown that alginate/chondroitin sulfate concentration and their ratios were significant factors influencing the swelling properties and the porosity of the resultant cryostructurates. It was demonstrated that the presence of chondroitin sulfate in the composition of a polymeric matrix slowed down the release of the aminoglycoside antibiotic gentamicin. In the case of the NH2-free bactericide, dioxidine, the release was almost independent of the presence of chondroitin sulfate. This trend was also registered for the antibacterial activity tests against the Escherichia coli bacteria, when examining the drug-loaded biopolymeric carriers.
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The processes of cryostructuring in polymeric systems, the techniques of the preparation of diverse cryogels and cryostructurates, the physico-chemical mechanisms of their formation, and the applied potential of these advanced polymer materials are all of high scientific and practical interest in many countries. This review article describes and discusses the results of more than 40 years of studies in this field performed by the researchers from the A.N.Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences-one of the key centers, where such investigations are carried out. The review includes brief historical information, the description of the main effects and trends characteristic of the cryostructuring processes, the data on the morphological specifics inherent in the polymeric cryogels and cryostructurates, and examples of their implementation for solving certain applied tasks.
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This study describes the preparation of nanoparticles derived from bovine serum albumin (BSA) in comparison with the formation of nanoparticles composed of human serum albumin (HSA), when the same preparation procedure was used in both cases. To obtain protein nanoparticles, the method of desolvation with ethanol was employed, followed by the stabilization with urea and cysteine. It was shown that, upon transition from HSA to BSA, the particles with smaller sizes and with a narrower polydispersity were formed. The possibility of the immobilization of the antitumor drug hydroxyurea in such protein nanoparticles by adsorption and inclusion methods has been shown. The drug release profile from the polymer matrix was established.
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Wide-pore proteinaceous freeze-thaw spongy gels were synthesized via the cryotropic gelation technique using the bovine blood serum or its diluted solutions as the protein-containing precursors. The feed systems also included the denaturant (urea) and the thiol-reductant (cysteine). The gel-fraction yield decreased and the swelling degree of the walls of macropores in such heterophase matrices increased with decreasing the initial protein concentration. The optimum freezing temperature was found to be within a rather narrow range from -15 to -20 °C. In this case, the average size of the macropores in the resultant cryogels was 90-110 µm. The suitability of such soft wide-pore gel materials for the application as the carriers of peptide bioregulators was demonstrated in the in vitro experiments, when the posterior segments of the Pleurodeles waltl adult newts' eyes were used as a model biological target. It was shown that a statistically reliable protective effect on the state of the sclera, vascular membrane and retinal pigment epithelium, as well as on the viability of fibroblasts, was inherent in the proteinaceous cryogels loaded with the peptide bioregulator (Viophtan-5™) isolated from the bovine eye sclera.
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Recent studies undoubtedly show that the mammalian target of rapamycin (mTOR) and the Hippo-Yes-associated protein 1 (YAP) pathways are important mediators of mechanical cues. The crosstalk between these pathways as well as de-regulation of their signaling has been implicated in multiple tumor types, including liver tumors. Additionally, physical cues from 3D microenvironments have been identified to alter gene expression and differentiation of different cell lineages. However, it remains incompletely understood how physical constraints originated in 3D cultures affect cell plasticity and what the key mediators are of such process. In this work, we use collagen scaffolds as a model of a soft 3D microenvironment to alter cellular size and study the mechanotransduction that regulates that process. We show that the YAP-mTOR axis is a downstream effector of 3D cellular culture-driven mechanotransduction. Indeed, we found that cell mechanics, dictated by the physical constraints of 3D collagen scaffolds, profoundly affect cellular proliferation in a YAP-mTOR-mediated manner. Functionally, the YAP-mTOR connection is key to mediate cell plasticity in hepatic tumor cell lines. These findings expand the role of YAP-mTOR-driven mechanotransduction to the control hepatic tumor cellular responses under physical constraints in 3D cultures. We suggest a tentative mechanism, which coordinates signaling rewiring with cytoplasmic restructuring during cell growth in 3D microenvironments.
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The goal of this study was to reveal how the chemical modification, succinylation in this case, of the wide-pore serum-albumin-based cryogels affects on their osmotic characteristics (swelling extent), biodegradability and ability to be loaded with the bactericide substance - dioxidine, as well as on its release. The cryogels were prepared via the cryogenic processing (freezing - frozen storage - thawing) of aqueous solutions containing bovine serum albumin (50 g/L), denaturant (urea or guanidine hydrochloride, 1.0 mol/L) and reductant (cysteine, 0.01 mol/L). Freezing/frozen storage temperatures were either -15, or -20, or -25 °C. After defrosting, spongy cryogels were obtained that possessed the system of interconnected gross pores, whose shape and dimensions were dependent on the freezing temperature and on the type of denaturant introduced in the feed solution. Subsequent succinylation of the resultant cryogels caused the growth of the swelling degree of the pore walls of these spongy materials, resulted in strengthening of their resistance against of trypsinolysis and gave rise to an increase in their loading capacity with respect to dioxidine. With that, the microbiological tests showed a higher bactericidal activity of the dioxidine-loaded sponges based on the succinylated albumin cryogels as compared to that of the drug-carriers based on the non-modified protein sponges.
Assuntos
Plásticos Biodegradáveis/química , Criogéis/química , Portadores de Fármacos/química , Quinoxalinas/química , Soroalbumina Bovina/química , Antibacterianos/química , Temperatura Baixa , Congelamento , Porosidade , Água/químicaRESUMO
Wide-pore cryostructurates were prepared via freezing sodium alginate aqueous solutions with subsequent ice sublimation from the frozen samples, followed by their incubation in the ethanol solutions of calcium chloride or sulfuric acid, rinsing, and final drying. Such sequence of operations resulted in the calcium alginate or alginic acid sponges, respectively. The swelling degree of the walls of macropores in such matrices decreased with increasing polymer concentration in the initial solution. The dependence of the degree of swelling on the cryogenic processing temperature had a bell-like character with a maximum for the samples formed at -20 °C. According to 1H NMR spectroscopy, the content of mobile (non-frozen) water in the frozen water-sodium alginate systems also depended on the initial polymer concentration and freezing temperature. The cryostructurates obtained did not lose their integrity in water, saline, in an acidic medium at pH 2 for at least three weeks. Under alkaline conditions at pH 12 the first signs of dissolution of the Ca-alginate sponge arose only after a week of incubation. Microbiological testing of the model depot form of the antibiotics entrapped in the Ca-alginate cryostructurate demonstrated the efficiency of this system as the antibacterial material.
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In the present work, innovative composite biomaterials possessing bactericidal properties and based on the hexahistidine-tagged organophosphorus hydrolase (His6-OPH) entrapped in the poly(vinyl alcohol) cryogel (PVA-CG)/bacterial cellulose (BC) were developed. His6-OPH possesses lactonase activity, with a number of N-acyl homoserine lactones being the inducers of Gram-negative bacterial resistance. The enzyme can also be combined with various antimicrobial agents (antibiotics and antimicrobial peptides) to improve the efficiency of their action. In this study, such an effect was shown for composite biomaterials when His6-OPH was entrapped in PVA-CG/BC together with ß-lactam antibiotic meropenem or antimicrobial peptides temporin A and indolicidin. The residual catalytic activity of immobilized His6-OPH was 60% or more in all the composite samples. In addition, the presence of BC filler in the PVA-CG composite resulted in a considerable increase in the mechanical strength and heat endurance of the polymeric carrier compared to the BC-free cryogel matrix. Such enzyme-containing composites could be interesting in the biomedical field to help overcome the problem of antibiotic resistance of pathogenic microorganisms.
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Pine sawdust treated by a series of hydrophobising agents (drying oil, organosilicon emulsion, n-hexadecane and paraffin) was examined as carrier for adsorption immobilisation of hydrocarbon-oxidizing bacterial cells Rhodococcus ruber. It was shown that hydrophobising agents based on drying oil turned out to be optimal (among the other modifiers examined) for the preparation of sawdust carriers suitable for the efficient immobilisation. The results obtained demonstrate promising possibilities in developing a wide range of available and cheap, biodegradable cellulose-containing carriers that possess varying surface hydrophobicity.
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Biotecnologia/métodos , Hidrocarbonetos/química , Oxigênio/química , Rhodococcus/metabolismo , Madeira , Alcanos/química , Biodegradação Ambiental , Catálise , Celulose/química , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Modelos Químicos , Óleos , PinusRESUMO
A variety of cryogenically-structured polymeric materials are of significant scientific and applied interest in various areas. However, in spite of considerable attention to these materials and intensive elaboration of their new examples, as well as the impressive growth in the number of the publications and patents on this topic over the past two decades, a marked variability of the used terminology and definitions is frequently met with in the papers, reviews, theses, patents, conference presentations, advertising materials and so forth. Therefore, the aim of this brief communication is to specify the basic terms and definitions in the particular field of macromolecular science.