INMdok (Leibniz Institute for New Materials)
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Collagen density defines 3D migration of CTLs and their consequent cytotoxicity against tumor cells
Get PDFSolid tumors are often characterized by condensed extracellular matrix (ECM). The impact of dense ECM on cytotoxic T lymphocytes (CTL) function is not fully understood. Here, we report that CTL-mediated cytotoxicity is substantially impaired in dense collagen matrices. Although the intrinsic killing machinery including expression of cytotoxic proteins and degranulation was intact, CTL motility was substantially compromised in dense collagen. We found that for 3D CTL migration, persistence and velocity was regulated by collagen stiffness and the porosity, respectively. Interestingly, 3D CTL velocity is strongly correlated with their nuclear deformability/flexibility during migration, which is regulated by the microtubule network. Moreover, CTL migration was completely abolished by inhibition of actin polymerization and or myosin IIA. Remarkably, disruption of the microtubule-networks significantly improves the impaired migration, search efficiency, and cytotoxicity of CTLs in dense collagen. Our work suggests the microtubule network as a promising target to rescue impaired CTL killing capacity in solid tumor related scenarios
Development of bioactive catechol functionalized nanoparticles applicable for 3D bioprinting
Get PDFEfficient wound treatments to target specific events in the healing process of chronic wounds constitute a significant aim in regenerative medicine. In this sense, nanomedicine can offer new opportunities to improve the effectiveness of existing wound therapies. The aim of this study was to develop catechol bearing polymeric nanoparticles (NPs) and to evaluate their potential in the field of wound healing. Thus, NPs wound healing promoting activities, potential for drug encapsulation and controlled release, and further incorporation in a hydrogel bioink formulation to fabricate cell-laden 3D scaffolds are studied. NPs with 2 and 29 M % catechol contents (named NP2 and NP29) were obtained by nanoprecipitation and presented hydrodynamic diameters of 100 and 75 nm respectively. These nanocarriers encapsulated the hydrophobic compound coumarin-6 with 70% encapsulation efficiency values. In cell culture studies, the NPs had a protective effect in RAW 264.7 macrophages against oxidative stress damage induced by radical oxygen species (ROS). They also presented a regulatory effect on the inflammatory response of stimulated macrophages and promoted upregulation of the vascular endothelial growth factor (VEGF) in fibroblasts and endothelial cells. In particular, NP29 were used in a hydrogel bioink formulation using carboxymethyl chitosan and hyaluronic acid as polymeric matrices. Using a reactive mixing bioprinting approach, NP-loaded hydrogel scaffolds with good structural integrity, shape fidelity and homogeneous NPs dispersion, were obtained. The in vitro catechol NPs release profile of the printed scaffolds revealed a sustained delivery. The bioprinted scaffolds supported viability and proliferation of encapsulated L929 fibroblasts over 14 days. We envision that the catechol functionalized NPs and resulting bioactive bioink presented in this work offer promising advantages for wound healing applications, as they: 1) support controlled release of bioactive catechol NPs to the wound site; 2) can incorporate additional therapeutic functions by co-encapsulating drugs; 3) can be printed into 3D scaffolds with tailored geometries based on patient requirements
Effect of Subsurface Microstructures on Adhesion of Highly Confined Elastic Films
Get PDFPolymer adhesive films sandwiched between two rigid solids are a common bonding strategy. The mechanics and consequently the adhesion of such geometrically confined films depend mainly on their thickness, Young's modulus, and the Poisson's ratio of the material. In this work, we explore the effect of a micropatterned subsurface embedded into the adhesive layer. We compare experiments with three-dimensional numerical simulations to evaluate the impact of the microstructure on the contact stiffness and effective modulus. The results are used to extend a previously proposed size scaling argument on adhesion from incompressible to slightly compressible films to account for the silicone used in our study with a Poisson's ratio of 0.495. In addition, interfacial stress distributions between the elastic film and the glass disc are obtained from plane strain simulations to evaluate characteristic adhesion failures such as edge cracks and cavitation. Overall, the micropatterned subsurface has a large impact on the contact stiffness, the interfacial stress distribution, and the detachment behavior; however, the adhesion performance is only slightly improved in comparison to a non-patterned subsurface
Optoacoustically induced auditory brainstem responses in the mouse model enhanced through an absorbing film
Get PDFSignificance: Optoacoustic stimulation offers an alternative stimulation strategy for the hearing organ. To serve as the base for a novel auditory prosthesis, the optoacoustic stimulation must be biocompatible and energy-saving. Aim: Enhancing the efficiency of optoacoustic stimulation while reducing the energy input in a suited animal model. Approach: Optoacoustically induced auditory brainstem responses (oABRs) were recorded after the pulsed laser irradiation of the tympanic membrane (TM) in mice. The results were compared with the ABRs induced through acoustic click stimulation. In addition, self-adhesive absorbing films were applied on the TM before the optoacoustic stimulation to investigate their effect on the resulting ABRs. Results: Using an absorbing film on the TM during optical stimulation led to considerably enhanced oABR wave I amplitude values compared with the stimulation of the bare TM. When using our stimulation strategy, we induced oABR waves in the 50% to 60% range of the acoustical stimulation reached with 80-dB SPL click stimuli. Conclusions: The mouse model can be used for certain developmental work for an optoacoustic auditory prosthesis. Using absorbing films on the TM during optical stimulation considerably enhances oABR wave I amplitude. Optimization of the stimulation strategy could further enhance the efficiency within biocompatibility margins
One-Pot Synthesis of Copper Iodide-Polypyrrole Nanocomposites
Get PDFA novel one-pot chemical synthesis of functional copper iodide-polypyrrole composites, CuI-PPy, has been proposed. The fabrication process allows the formation of nanodimensional metal salt/polymer hybrid structures in a fully controlled time- and concentration-dependent manner. The impact of certain experimental conditions, viz., duration of synthesis, sequence of component addition and concentrations of the intact reagents on the structure, dimensionality and yield of the end-product was evaluated in detail. More specifically, the amount of marshite CuI within the hybrid composite can be ranged from 60 to 90 wt.%, depending on synthetic conditions (type and concentration of components, process duration). In addition, the conditions allowing the synthesis of nano-sized CuI distributed inside the polypyrrole matrix were found. A high morphological stability and reproducibility of the synthesized nanodimensional metal-polymer hybrid materials were approved. Finally, the electrochemical activity of the formed composites was verified by cyclic voltammetry studies. The stability of CuI-PPy composite deposited on the electrodes was strongly affected by the applied anodic limit. The proposed one-pot synthesis of the hybrid nanodimensional copper iodide-polypyrrole composites is highly innovative, meets the requirements of Green Chemistry and is potentially useful for future biosensor development. In addition, this study is expected to generally contribute to the knowledge on the hybrid nano-based composites with tailored properties
Optoregulated force application to cellular receptors using molecular motors
Get PDFProgress in our understanding of mechanotransduction events requires noninvasive methods for the manipulation of forces at molecular scale in physiological environments. Inspired by cellular mechanisms for force application (i.e. motor proteins pulling on cytoskeletal fibers), we present a unique molecular machine that can apply forces at cell-matrix and cell-cell junctions using light as an energy source. The key actuator is a light-driven rotatory molecular motor linked to polymer chains, which is intercalated between a membrane receptor and an engineered biointerface. The light-driven actuation of the molecular motor is converted in mechanical twisting of the entangled polymer chains, which will in turn effectively “pull” on engaged cell membrane receptors (e.g., integrins, T cell receptors) within the illuminated area. Applied forces have physiologically-relevant magnitude and occur at time scales within the relevant ranges for mechanotransduction at cell-friendly exposure conditions, as demonstrated in force-dependent focal adhesion maturation and T cell activation experiments. Our results reveal the potential of nanomotors for the manipulation of living cells at the molecular scale and demonstrate a functionality which at the moment cannot be achieved by other technologies for force application
Nanoscale disintegration kinetics of mesoglobules in aqueous poly(N-isopropylacrylamide) solutions revealed by small-angle neutron scattering and pressure jumps
Get PDFIdentification and control of the disintegration mechanism of polymer nanoparticles are essential for applications in transport and release including polymer delivery systems. Structural changes during the disintegration of poly(N-isopropylacrylamide) (PNIPAM) mesoglobules in aqueous solution are studied in situ and in real time using kinetic small-angle neutron scattering with a time resolution of 50 ms. Simultaneously length scales between 1 and 100 nm are resolved. By initiating phase separation through fast pressure jumps across the coexistence line, 3 wt% PNIPAM solutions are rapidly brought into the one-phase state. Starting at the same temperature (35.1 °C) and pressure (17 MPa) the target pressure is varied over the range 25–48 MPa, allowing to systematically alter the osmotic pressure of the solvent within the mesoglobules. Initially, the mesoglobules have a radius of gyration of about 80 nm and contain a small amount of water. Two disintegration mechanisms are identified: (i) for target pressures close to the coexistence line, single polymers are released from the surface of the mesoglobules, and the mesoglobules decrease in size, which takes ∼30 s. (ii) For target pressures more distant from the coexistence line, the mesoglobules are swollen by water, and subsequently the chains become more and more loosely associated. In this case, disintegration proceeds within less than 10 s, controlled by the osmotic pressure of the solvent
Compliant Substrates Enhance Macrophage Cytokine Release and NLRP3 Inflammasome Formation During Their Pro-Inflammatory Response
Get PDFImmune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1β and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1β and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses
Nanotribology of metallic glasses in corrosive environments
Get PDFMetallic glasses (MGs) are promising materials for micromechanical systems, where miniaturized components involving mechanical contact require control of friction. Nanotribological experiments on MGs in corrosive aqueous solutions are carried out using atomic force microscopy (AFM), focusing on the role of surface oxide films formed during corrosion. A new method is developed to study in situ the structure of surface oxide films. The surface oxide film has a bilayer structure as revealed by repeated scanning with the AFM tip. The dependence of friction on electrochemical potential reveals the growth mechanism of the oxide film. Friction and adhesion after different immersion times in different solutions allow to compare the physicochemical processes of surface dissolution at the interfaces of the two layers of surface films and elucidate their influence on friction. An irregular atomic-scale stick-slip friction is observed and attributed to the amorphous nature of corroded surfaces. Finally, we show three different friction processes occurring at increasing normal loads: removal of the dissolution layer at low-load regime; stress-assisted tribo-oxidation in intermediate-load regime; and tribochemical wear in high-load regime. The chemical sensitivity of nanotribology studies demonstrates a novel route to explore fundamental mechanisms of corrosion at the microscopic scale.Metallische Gläser (MG) sind vielversprechende Materialien für mikromechanische Systeme, in denen der mechanische Kontakt eine Kontrolle über Reibung erfordert. Mit Hilfe der Rasterkraftmikroskopie (AFM) wurden nanotribologische Experimente auf MG in korrosiven wässrigen Lösungen durchgeführt, wobei die Rolle von Oxidfilmen im Fokus stand. Eine neuartige Methode für die in situ-Untersuchung der Struktur der Oberflächenoxidfilme wurde entwickelt. Der Oberflächenoxidfilm weist eine zweilagige Struktur auf, die durch wiederholtes Rastern mit der AFM-Spitze nachgewiesen wurde. Die Abhängigkeit der Reibung vom elektrochemischen Potential zeigt die Wachstumsmechanismen der Oxidfilme an. Reibung und Adhäsion nach verschieden langer Immersion erlauben den Vergleich der physikochemischen Prozesse der Oberflächenauflösung an der Grenzfläche der beiden Lagen. Es wurde eine unregelmäßige stick-slip Reibung auf atomarer Skala beobachtet und auf die amorphe Natur der korrodierten Oberflächen zurückgeführt. Schließlich beschreiben wir drei verschiedene Reibungsprozesse, die mit zunehmender Normalkraft auftreten: die Abtragung der abgeschiedenen Lage bei niedrigen Auflagekräften, eine durch mechanische Spannung unterstützte Tribo-Oxidation bei mittleren Kräften sowie tribochemischen Verschleiß bei hohen Kräften. Die chemische Empfindlichkeit der nanotribologischen Studien zeigt eine neue Möglichkeit auf, grundsätzliche Mechanismen der Korrosion auf der mikroskopischen Skala zu erforschen
Profiling of cytokines, chemokines and growth factors in saliva and gingival crevicular fluid
Get PDFIn saliva and gingival crevicular fluid (GCF) soluble factors such as cytokines, chemokines and growth factors have shown a great potential serving as biomarkers for early detection and/or diagnosis of oral and systemic diseases. However, GCF and saliva, which one is a better source is still under debate. This study aimed to gain an overview of cytokines, chemokines and growth factors in saliva and GCF to pave the way for selecting suitable oral fluids for oral and systemic diseases. Multiplex cytokine assay was conducted to determine concentrations of cytokines, chemokines and growth factors in saliva and GCF samples from healthy subjects. The protocol for sample collection was carefully optimized. Stabilization, repeatability, and donor variation of the profiles were analyzed. We found that for different donors, cytokine and chemokine profiles showed unique patterns in saliva but similar patterns in GCF. In terms of growth factors, the profiles were individualized in saliva and GCF. All profiles stayed stable for the same healthy individual. In saliva, profiles of cytokines, chemokines and growth factors are individualized for different donors. In GCF, profiles of cytokines and chemokines are similar. Other factors, such as growth factors and T helper-related cytokines, are highly variable in donors. Profiles of soluble factors are not correlated in saliva and GCF. The comprehensive cytokine profiles in saliva and GCF reported in this work would serve as a good base for choosing promising cytokines for developing biomarkers in oral fluids.Competing Interest StatementThe authors have declared no competing interest