04 Fakultät Energie-, Verfahrens- und Biotechnik

Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/5

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Author: search.filters.author.Tovar, Günter E. M.Start date: 2000

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    ItemOpen Access
    Precision 3D‐printed cell scaffolds mimicking native tissue composition and mechanics
    (2020) Erben, Amelie; Hörning, Marcel; Hartmann, Bastian; Becke, Tanja; Eisler, Stephan A.; Southan, Alexander; Cranz, Séverine; Hayden, Oliver; Kneidinger, Nikolaus; Königshoff, Melanie; Lindner, Michael; Tovar, Günter E. M.; Burgstaller, Gerald; Clausen‐Schaumann, Hauke; Sudhop, Stefanie; Heymann, Michael
    Cellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell‐ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell‐ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two‐photon stereolithography is adopted to print up to mm‐sized high‐precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein‐based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two‐pass printing or post‐print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7‐300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D‐lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single‐cell and tissue dynamics in response to defined mechanical and bio‐molecular cues and is ultimately scalable to full organs.
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    Nano-in-micro-particles consisting of PLGA nanoparticles embedded in chitosan microparticles via spray-drying enhances their uptake in the olfactory mucosa
    (2021) Spindler, Lena Marie; Feuerhake, Andreas; Ladel, Simone; Günday, Cemre; Flamm, Johannes; Günday-Türeli, Nazende; Türeli, Emre; Tovar, Günter E. M.; Schindowski, Katharina; Gruber-Traub, Carmen
    Intranasal delivery has gained prominence since 1990, when the olfactory mucosa was recognized as the window to the brain and the central nervous system (CNS); this has enabled the direct site specific targeting of neurological diseases for the first time. Intranasal delivery is a promising route because general limitations, such as the blood-brain barrier (BBB) are circumvented. In the treatment of multiple sclerosis (MS) or Alzheimer’s disease, for example, future treatment prospects include specialized particles as delivery vehicles. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are well known as promising delivery systems, especially in the area of nose-to-brain (N2B) delivery. Chitosan is also broadly known as a functional additive due to its ability to open tight junctions. In this study, we produced PLGA nanoparticles of different sizes and revealed for the first time their size-time-dependent uptake mechanism into the lamina propria of porcine olfactory mucosa. The intracellular uptake was observed for 80 and 175 nm within only 5 min after application to the epithelium. After 15 min, even 520 nm particles were detected, associated with nuclei. Especially the presence of only 520 nm particles in neuronal fibers is remarkable, implying transcellular and intracellular transport via the olfactory or the trigeminal nerve to the brain and the CNS. Additionally, we developed successfully specialized Nano-in-Micro particles (NiMPs) for the first time via spray drying, consisting of PLGA nanoparticles embedded into chitosan microparticles, characterized by high encapsulation efficiencies up to 51%, reproducible and uniform size distribution, as well as smooth surface. Application of NiMPs accelerated the uptake compared to purely applied PLGA nanoparticles. NiMPs were spread over the whole transverse section of the olfactory mucosa within 15 min. Faster uptake is attributed to additional paracellular transport, which was examined via tight-junction-opening. Furthermore, a separate chitosan penetration gradient of ∼150 µm caused by dissociation from PLGA nanoparticles was observed within 15 min in the lamina propria, which was demonstrated to be proportional to an immunoreactivity gradient of CD14. Due to the beneficial properties of the utilized chitosan-derivative, regarding molecular weight (150-300 kDa), degree of deacetylation (80%), and particle size (0.1-10 µm) we concluded that M2-macrophages herein initiated an anti-inflammatory reaction, which seems to already take place within 15 min following chitosan particle application. In conclusion, we demonstrated the possibility for PLGA nanoparticles, as well as for chitosan NiMPs, to take all three prominent intranasal delivery pathways to the brain and the CNS; namely transcellular, intracellular via neuronal cells, and paracellular transport.
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    Physical interactions strengthen chemical gelatin methacryloyl gels
    (2019) Rebers, Lisa; Granse, Tobias; Tovar, Günter E. M.; Southan, Alexander; Borchers, Kirsten
    Chemically cross-linkable gelatin methacryloyl (GM) derivatives are getting increasing attention regarding biomedical applications. Thus, thorough investigations are needed to achieve full understanding and control of the physico-chemical behavior of these promising biomaterials. We previously introduced gelatin methacryloyl acetyl (GMA) derivatives, which can be used to control physical network formation (solution viscosity, sol-gel transition) independently from chemical cross-linking by variation of the methacryloyl-to-acetyl ratio. It is known that temperature dependent physical network formation significantly influences the mechanical properties of chemically cross-linked GM hydrogels. We investigated the temperature sensitivity of GM derivatives with different degrees of modification (GM2, GM10), or similar degrees of modification but different methacryloyl contents (GM10, GM2A8). Rheological analysis showed that the low modified GM2 forms strong physical gels upon cooling while GM10 and GM2A8 form soft or no gels. Yet, compression testing revealed that all photo cross-linked GM(A) hydrogels were stronger if cooling was applied during hydrogel preparation. We suggest that the hydrophobic methacryloyl and acetyl residues disturb triple helix formation with increasing degree of modification, but additionally form hydrophobic structures, which facilitate chemical cross-linking.
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    Acid catalyzed cross‐linking of polyvinyl alcohol for humidifier membranes
    (2021) Michele, Andre; Paschkowski, Patrick; Hänel, Christopher; Tovar, Günter E. M.; Schiestel, Thomas; Southan, Alexander
    Polyvinyl alcohol (PVA) is a hydrophilic polymer well known for good film forming properties, high water vapor permeance JW, and low nitrogen permeance. However, depending on molar mass and temperature, PVA swells strongly in water until complete dissolution. This behavior affects the usability of PVA in aqueous environments and makes cross‐linking necessary if higher structural integrity is envisaged. In this work, PVA networks are formed by thermal cross‐linking in the presence of p‐toluenesulfonic acid (TSA) and investigated in a design of experiments approach. Experimental parameters are the cross‐linking period tc, temperature ϑ and the TSA mass fraction wTSA. Cross‐linking is found to proceed via ether bond formation at all reaction conditions. Degradation is promoted especially by a combination of high wTSA, tc and ϑ. Thermal stability of the networks after preparation is strongly improved by neutralizing residual TSA. Humidification membranes with a JW of 6423 ± 63.0 gas permeation units (GPU) are fabricated by coating PVA on polyvinyliden fluoride hollow fibers and cross‐linking with TSA. Summarizing, the present study contributes to a clearer insight into the cross‐linking of PVA in presence of TSA, the thermal stability of the resulting networks and the applicability as selective membrane layers for water vapor transfer.
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    Unravelling parameter interactions in calcium alginate/polyacrylamide double network hydrogels using a design of experiments approach for the optimization of mechanical properties
    (2024) Gorke, Oliver; Stuhlmüller, Marc; Tovar, Günter E. M.; Southan, Alexander
    Calcium alginate/polyacrylamide double network hydrogels were reported to be exceptionally tough. However, literature reports so far varied the sample compositions mainly by one parameter at a time approaches, thus only drawing an incomplete picture of achievable material properties. In this contribution, sample compositions are varied according to a face-centered central composite experimental design taking into account the four parameters of alginate concentration cAlg, high/low molar mass alginate mixing ratio RP, acrylamide concentration cAAm, and N,N′-methylenebisacrylamide concentration cMBA. Each sample composition is investigated in triplicate. Thus, 75 samples were investigated by tensile testing, and a detailed analysis of the significant parameters and parameter interactions influencing the mechanical properties is conducted. The data shows that two parameter interactions, involving all four tested parameters, have a large effect on the Young's modulus, the strength, the toughness and the strain at material failure. As a consequence, it becomes evident that the experimental procedure from previous studies did not always result in optimum sample compositions. The results allow optimization of the mechanical properties within the studied parameter space, and a new maximum value of the strength of 710 kPa is reported. The data also give rise to the assumption that other parameters and parameter interactions ignored also in this study may allow further tailoring of mechanical properties.
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    Influence of light conditions on the production of chrysolaminarin using Phaeodactylum tricornutum in artificially illuminated photobioreactors
    (2023) Frick, Konstantin; Ebbing, Tobias; Yeh, Yen‐Cheng; Schmid‐Staiger, Ulrike; Tovar, Günter E. M.
    The light conditions are of utmost importance in any microalgae production process especially involving artificial illumination. This also applies to a chrysolaminarin (soluble 1,3-β-glucan) production process using the diatom Phaeodactylum tricornutum. Here we examine the influence of the amount of light per gram biomass (specific light availability) and the influence of two different biomass densities (at the same amount of light per gram biomass) on the accumulation of the storage product chrysolaminarin during nitrogen depletion in artificially illuminated flat-panel airlift photobioreactors. Besides chrysolaminarin, other compounds (fucoxanthin, fatty acids used for energy storage [C16 fatty acids], and eicosapentaenoic acid) are regarded as well. Our results show that the time course of C-allocation between chrysolaminarin and fatty acids, serving as storage compounds, is influenced by specific light availability and cell concentration. Furthermore, our findings demonstrate that with increasing specific light availability, the maximal chrysolaminarin content increases. However, this effect is limited. Beyond a certain specific light availability (here: 5 µmolphotons gDW-1 s-1) the maximal chrysolaminarin content no longer increases, but the rate of increase becomes faster. Furthermore, the conversion of light to chrysolaminarin is best at the beginning of nitrogen depletion. Additionally, our results show that a high biomass concentration has a negative effect on the maximal chrysolaminarin content, most likely due to the occurring self-shading effects.
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    Coumarin-4-ylmethyl- and p-hydroxyphenacyl-based photoacid generators with high solubility in aqueous media: synthesis, stability and photolysis
    (2020) Adatia, Karishma K.; Halbritter, Thomas; Reinfelds, Matiss; Michele, Andre; Tran, Michael; Laschat, Sabine; Heckel, Alexander; Tovar, Günter E. M.; Southan, Alexander
    (Coumarin‐4‐yl)methyl (c4m) and p‐hydroxyphenacyl (pHP)‐based compounds are well known for their highly efficient photoreactions, but often show limited solubility in aqueous media. To circumvent this, we synthesized and characterized the two new c4m and pHP‐based photoacid generators (PAGs), 7‐[bis(carboxymethyl)amino]‐4‐(acetoxymethyl)coumarin (c4m‐ac) and p‐hydroxyphenacyl‐2,5,8,11‐tetraoxatridecan‐13‐oate (pHP‐t), and determined their solubilities, stabilities and photolysis in aqueous media. The two compounds showed high solubilities in water of 2.77 mmol L−1±0.07 mmol L−1 (c4m‐ac) and 124.66 mmol L−1±2.1 mmol L−1 (pHP‐t). In basic conditions at pH 9, solubility increased for c4m‐ac to 646.46 mmol L−1±0.63 mmol L−1, for pHP‐t it decreased to 34.68 mmol L−1±0.62 mmol L−1. Photochemical properties of the two PAGs, such as the absorption maxima, the maximum molar absorption coefficients and the quantum yields, were found to be strongly pH‐dependent. Both PAGs showed high stabilities s24h ≥95 % in water for 24 h, but decreasing stability with increasing pH value due to hydrolysis. The present study contributes to a clearer insight into the synthesis, solubilities, stabilities, and photolysis of c4m and pHP‐based PAGs for further photochemical applications when high PAG concentrations are required, such as in polymeric foaming.
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    Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods
    (2020) Keller, Silke; Liedek, Anke; Shendi, Dalia; Bach, Monika; Tovar, Günter E. M.; Kluger, Petra J.; Southan, Alexander
    Azide-bearing cell-derived extracellular matrices (“clickECMs”) have emerged as a highly exciting new class of biomaterials. They conserve substantial characteristics of the natural extracellular matrix (ECM) and offer simultaneously small abiotic functional groups that enable bioorthogonal bioconjugation reactions. Despite their attractiveness, investigation of their biomolecular composition is very challenging due to the insoluble and highly complex nature of cell-derived matrices (CDMs). Yet, thorough qualitative and quantitative analysis of the overall material composition, organisation, localisation, and distribution of typical ECM-specific biomolecules is essential for consistent advancement of CDMs and the understanding of the prospective functions of the developed biomaterial. In this study, we evaluated frequently used methods for the analysis of complex CDMs. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and (immune)histochemical staining methods in combination with several microscopic techniques were found to be highly eligible. Commercially available colorimetric protein assays turned out to deliver inaccurate information on CDMs. In contrast, we determined the nitrogen content of CDMs by elementary analysis and converted it into total protein content using conversion factors which were calculated from matching amino acid compositions. The amount of insoluble collagens was assessed based on the hydroxyproline content. The Sircol™ assay was identified as a suitable method to quantify soluble collagens while the Blyscan™ assay was found to be well-suited for the quantification of sulphated glycosaminoglycans (sGAGs). Eventually, we propose a series of suitable methods to reliably characterise the biomolecular composition of fibroblast-derived clickECM.
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    Differentiation of physical and chemical cross-linking in gelatin methacryloyl hydrogels
    (2021) Rebers, Lisa; Reichsöllner, Raffael; Regett, Sophia; Tovar, Günter E. M.; Borchers, Kirsten; Baudis, Stefan; Southan, Alexander
    Gelatin methacryloyl (GM) hydrogels have been investigated for almost 20 years, especially for biomedical applications. Recently, strengthening effects of a sequential cross-linking procedure, whereby GM hydrogel precursor solutions are cooled before chemical cross-linking, were reported. It was hypothesized that physical and enhanced chemical cross-linking of the GM hydrogels contribute to the observed strengthening effects. However, a detailed investigation is missing so far. In this contribution, we aimed to reveal the impact of physical and chemical cross-linking on strengthening of sequentially cross-linked GM and gelatin methacryloyl acetyl (GMA) hydrogels. We investigated physical and chemical cross-linking of three different GM(A) derivatives (GM10, GM2A8 and GM2), which provided systematically varied ratios of side-group modifications. GM10 contained the highest methacryloylation degree (DM), reducing its ability to cross-link physically. GM2 had the lowest DM and showed physical cross-linking. The total modification degree, determining the physical cross-linking ability, of GM2A8 was comparable to that of GM10, but the chemical cross-linking ability was comparable to GM2. At first, we measured the double bond conversion (DBC) kinetics during chemical GM(A) cross-linking quantitatively in real-time via near infrared spectroscopy-photorheology and showed that the DBC decreased due to sequential cross-linking. Furthermore, results of circular dichroism spectroscopy and differential scanning calorimetry indicated gelation and conformation changes, which increased storage moduli of all GM(A) hydrogels due to sequential cross-linking. The data suggested that the total cross-link density determines hydrogel stiffness, regardless of the physical or chemical nature of the cross-links.
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    Covalent incorporation of tobacco mosaic virus increases the stiffness of poly(ethylene glycol) diacrylate hydrogels
    (2018) Southan, Alexander; Lang, Tina; Schweikert, Michael; Tovar, Günter E. M.; Wege, Christina; Eiben, Sabine
    Hydrogels are versatile materials, finding applications as adsorbers, supports for biosensors and biocatalysts or as scaffolds for tissue engineering. A frequently used building block for chemically cross-linked hydrogels is poly(ethylene glycol) diacrylate (PEG-DA). However, after curing, PEG-DA hydrogels cannot be functionalized easily. In this contribution, the stiff, rod-like tobacco mosaic virus (TMV) is investigated as a functional additive to PEG-DA hydrogels. TMV consists of more than 2000 identical coat proteins and can therefore present more than 2000 functional sites per TMV available for coupling, and thus has been used as a template or building block for nano-scaled hybrid materials for many years. Here, PEG-DA (Mn = 700 g/mol) hydrogels are combined with a thiol-group presenting TMV mutant (TMVCys). By covalent coupling of TMVCys into the hydrogel matrix via the thiol-Michael reaction, the storage modulus of the hydrogels is increased compared to pure PEG-DA hydrogels and to hydrogels containing wildtype TMV (wt-TMV) which is not coupled covalently into the hydrogel matrix. In contrast, the swelling behaviour of the hydrogels is not altered by TMVCys or wt-TMV. Transmission electron microscopy reveals that the TMV particles are well dispersed in the hydrogels without any large aggregates. These findings give rise to the conclusion that well-defined hydrogels were obtained which offer the possibility to use the incorporated TMV as multivalent carrier templates e.g. for enzymes in future studies.