Low-frequency magnetic response of gold nanoparticles.

Gold nanoparticles (AuNPs) exposed to low frequency magnetic fields have shown promise in enhancing biological processes, such as cellular reprogramming. Despite the experimental evidence, a comprehensive understanding of the underlying physical principles and the corresponding theory remains elusive. The most common hypothesis is that functionalized nanoparticles transiently amplify magnetic fields, leading to improved cellular reprogramming efficiency. However, a detailed investigation on this topic is lacking. This paper bridges this knowledge gap by conducting a comprehensive investigation on the magnetic response of surface-modified AuNPs exposed to magnetic fields with frequencies up to hundreds of MHz. Starting with the inherent properties of bulk gold material, we explore a wide range of magnetic susceptibilities that might result from the redistribution of charge carriers due to bond molecules on the particle surfaces. Through analytical models and numerical electromagnetic simulations, we examine various geometric factors that can enhance the magnetic response, including the number of particles, spatial distribution, size, and shape. Our broad investigation provides researchers with analytical and numerical estimates of the magnetic response of nanoparticles, and the associated limits that can be expected. We found that a magnetic field enhancement comparable to the incident field requires very high magnetic susceptibilities, well beyond the values measured in functionalized gold nanoparticles thus far.

Laser Synthesis of Cerium-Doped Garnet Nanoparticles.

The application of a pulsed laser ablation technique for the generation of cerium-doped garnet nanoparticles in liquids is investigated. The morphological and optical properties of the obtained nanoparticles are demonstrated. Features introduced by the single crystals of Gd3Al2.4Ga2.6O12:Ce3+, Lu3Al5O12:Ce3+, and Y3Al1.25Ga3.75O12:Ce3+ from which the nanoparticles are generated, as well as the parameters of a liquid media on the garnet nanoparticle generation are experimentally studied using TEM and UV-Vis spectroscopy methods. It is shown how the size, shape, and internal structure of the nanoparticles are related to the external laser ablation conditions, as well as to the laser melting processes of NPs in the colloidal solutions. This work provides important information about the generated nanoparticles, which can be used as building blocks for specially designed structures with predetermined optical properties.

344Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity.

Generation of human neuronal networks by three-dimensional (3D) bioprinting is promising for drug testing and hopefully will allow for the understanding of cellular mechanisms in brain tissue. The application of neural cells derived from human induced-pluripotent stem cells (hiPSCs) is an obvious choice, since hiPSCs provide access to cells unlimited in number and cell types that could be generated by differentiation. The questions in this regard include which neuronal differentiation stage is optimal for printing of such networks, and to what extent the addition of other cell types, especially astrocytes, supports network formation. These aspects are the focus of the present study, in which we applied a laser-based bioprinting technique and compared hiPSC-derived neural stem cells (NSCs) with neuronal differentiated NSCs, with and without the inclusion of co-printed astrocytes. In this study, we investigated in detail the effects of cell types, printed droplet size, and duration of differentiation before and after printing on viability, as well as proliferation, stemness, differentiation potential, formation of dendritic extensions and synapses, and functionality of the generated neuronal networks. We found a significant dependence of cell viability after dissociation on differentiation stage, but no impact of the printing process. Moreover, we observed a dependence of the abundance of neuronal dendrites on droplet size, a marked difference between printed cells and normal cell culture in terms of further differentiation of the cells, especially differentiation into astrocytes, as well as neuronal network formation and activity. Notably, there was a clear effect of admixed astrocytes on NSCs but not on neurons.

Development of a Novel Valve-Controlled Drug-Elutable Microstent for Microinvasive Glaucoma Surgery: In Vitro and Preclinical In Vivo Studies.

Purpose: Microinvasive glaucoma surgery (MIGS) has become an important treatment approach for primary open-angle glaucoma, although the safe and long-term effective lowering of intraocular pressure with currently available implants for MIGS is not yet achieved to a satisfactory extent. The study focusses on the development and in vitro and in vivo testing of a novel microstent for MIGS. Methods: A silicone elastomer-based microstent was developed. Implants were manufactured using dip coating, fs-laser cutting, and spray coating. Within the current study no antifibrotic drug was loaded into the device. Sterilized microstents were analyzed in vitro regarding pressure-flow characteristics and biocompatibility. Six New Zealand white rabbits were implanted with a microstent draining the aqueous humor from the anterior chamber into the subconjunctival space. Drainage efficacy was evaluated using oculopressure tonometry as a transient glaucoma model. Noninvasive imaging was performed. Results: Microstents were manufactured successfully and characterized in vitro. Implantation in vivo was successful for four animals with additional device fixation. Without additional fixation, dislocation of microstents was found in two animals. Safe and effective intraocular pressure reduction was observed for the four eyes with correctly implanted microstent during the 6-month trial period. Conclusions: The described microstent represents an innovative treatment approach for MIGS. The incorporation of a selectively antifibrotic drug into the microstent drug-elutable coating will be addressed in future investigations. Translational Relevance: The current preclinical study successfully provided proof of concept for our microstent for MIGS which is suitable for safe and effective intraocular pressure reduction and offers promising perspectives for the clinical management of glaucoma.

Laser-Induced µ-Rooms for Osteocytes on Implant Surface: An In Vivo Study.

Laser processing of dental implant surfaces is becoming a more widespread replacement for classical techniques due to its undeniable advantages, including control of oxide formation and structure and surface relief at the microscale. Thus, using a laser, we created several biomimetic topographies of various shapes on the surface of titanium screw-shaped implants to research their success and survival rates. A distinctive feature of the topographies is the presence of "µ-rooms", which are special spaces created by the depressions and elevations and are analogous to the µ-sized room in which the osteocyte will potentially live. We conducted the comparable in vivo study using dental implants with continuous (G-topography with µ-canals), discrete (S-topography with µ-cavities), and irregular (I-topography) laser-induced topographies. A histological analysis performed with the statistical method (with p-value less than 0.05) was conducted, which showed that G-topography had the highest BIC parameter and contained the highest number of mature osteocytes, indicating the best secondary stability and osseointegration.

Laser printing: trends and perspectives.

In this paper, I present my personal view on the possible development and applications of laser printing technologies based on laser-induced forward transfer of inorganic and biological materials. Laser printing of micro- and nanoparticles, living cells, and microorganisms are discussed.

Capillary-like Formations of Endothelial Cells in Defined Patterns Generated by Laser Bioprinting.

Bioprinting is seen as a promising technique for tissue engineering, with hopes of one day being able to produce whole organs. However, thick tissue requires a functional vascular network, which naturally contains vessels of various sizes, down to capillaries of ~10 µm in diameter, often spaced less than 200 µm apart. If such thick tissues are to be printed, the vasculature would likely need to be printed at the same time, including the capillaries. While there are many approaches in tissue engineering to produce larger vessels in a defined manner, the small capillaries usually arise only in random patterns by sprouting from the larger vessels or from randomly distributed endothelial cells. Here, we investigated whether the small capillaries could also be printed in predefined patterns. For this purpose, we used a laser-based bioprinting technique that allows for the combination of high resolution and high cell density. Our aim was to achieve the formation of closed tubular structures with lumina by laser-printed endothelial cells along the printed patterns on a surface and in bioprinted tissue. This study shows that such capillaries are directly printable; however, persistence of the printed tubular structures was achieved only in tissue with external stimulation by other cell types.

Metal Nanoparticles in Laser Bioprinting.

Laser bioprinting is a promising method for applications in biotechnology, tissue engineering, and regenerative medicine. It is based on a microdroplet transfer from a donor slide induced by laser pulse heating of a thin metal absorption film covered with a layer of hydrogel containing living cells (bioink). Due to the presence of the metal absorption layer, some debris in the form of metal nanoparticles is printed together with bioink microdroplets. In this article, experimental investigations of the amount of metal nanoparticles formed during the laser bioprinting process and transported in bioink microdroplets are performed. As metal absorption layers, Ti films with the thickness in the range of 25-400 nm, produced by magnetron spattering, were applied. Dependences of the volume of bioink microdroplets and the amount of Ti nanoparticles within them on the laser pulse fluence were obtained. It has been experimentally found that practically all nanoparticles remain in the hydrogel layer on the donor slide during bioprinting, with only a small fraction of them transferred within the microdroplet (0.5% to 2.5%). These results are very important for applications of laser bioprinting since the transferred metal nanoparticles can potentially affect living systems. The good news is that the amount of such nanoparticles is very low to produce any negative effect on the printed cells.

In Vitro Development of Human iPSC-Derived Functional Neuronal Networks on Laser-Fabricated 3D Scaffolds.

Neural progenitor cells generated from human induced pluripotent stem cells (hiPSCs) are the forefront of ″brain-on-chip″ investigations. Viable and functional hiPSC-derived neuronal networks are shaping powerful in vitro models for evaluating the normal and abnormal formation of cortical circuits, understanding the underlying disease mechanisms, and investigating the response to drugs. They therefore represent a desirable instrument for both the scientific community and the pharmacological industry. However, culture conditions required for the full functional maturation of individual neurons and networks are still unidentified. It has been recognized that three-dimensional (3D) culture conditions can better emulate in vivo neuronal tissue development compared to 2D cultures and thus provide a more desirable in vitro approach. In this paper, we present the design and implementation of a 3D scaffold platform that supports and promotes intricate neuronal network development. 3D scaffolds were produced through direct laser writing by two-photon polymerization (2PP), a high-resolution 3D laser microstructuring technology, using the biocompatible and nondegradable photoreactive resin Dental LT Clear (DClear). Neurons developed and interconnected on a 3D environment shaped by vertically stacked scaffold layers. The developed networks could support different cell types. Starting at the day 50 of 3D culture, neuronal progenitor cells could develop into cortical projection neurons (CNPs) of all six layers, different types of inhibitory neurons, and glia. Additionally and in contrast to 2D conditions, 3D scaffolds supported the long-term culturing of neuronal networks over the course of 120 days. Network health and functionality were probed through calcium imaging, which revealed a strong spontaneous neuronal activity that combined individual and collective events. Taken together, our results highlight advanced microstructured 3D scaffolds as a reliable platform for the 3D in vitro modeling of neuronal functions.

Culture surface protein coatings affect the barrier properties and calcium signalling of hESC-RPE.

Human pluripotent stem cell-derived retinal pigment epithelium (RPE) transplantation is currently under evaluation as treatment for macular degeneration. For therapeutic applications, cryostorage during cell production is typically needed with potential consequences to cell functionality. We have previously shown that the culture substrate affects human embryonic stem cell-derived RPE (hESC-RPE) properties in fresh cultures. Here, we aimed to further identify the role of RPE basement membrane proteins type IV collagen (Col-IV), laminin (LN), and nidogen-1 in the maturation and functionality of hESC-RPE after cryopreservation. In addition to cell attachment and morphology, transepithelial electrical resistance, expression of key RPE proteins, phagocytosis capacity and Ca2+ signalling were analysed. After cryostorage, attachment of hESC-RPE on culture surfaces coated with Col-IV alone was poor. Combining Col-IV and LN with or without nidogen-1 significantly improved cell attachment and barrier properties of the epithelium. Furthermore, functional homogeneity of the hESC-RPE monolayer was enhanced in the presence of nidogen-1. Our results suggest that the choice of coating proteins for the cell culture may have implications to the functional properties of these cells after cryostorage cell banking.

Cell Type-Specific Adhesion and Migration on Laser-Structured Opaque Surfaces.

Cytocompatibility is essential for implant approval. However, initial in vitro screenings mainly include the quantity of adherent immortalized cells and cytotoxicity. Other vital parameters, such as cell migration and an in-depth understanding of the interaction between native tissue cells and implant surfaces, are rarely considered. We investigated different laser-fabricated spike structures using primary and immortalized cell lines of fibroblasts and osteoblasts and included quantification of the cell area, aspect ratio, and focal adhesions. Furthermore, we examined the three-dimensional cell interactions with spike topographies and developed a tailored migration assay for long-term monitoring on opaque materials. While fibroblasts and osteoblasts on small spikes retained their normal morphology, cells on medium and large spikes sank into the structures, affecting the composition of the cytoskeleton and thereby changing cell shape. Up to 14 days, migration appeared stronger on small spikes, probably as a consequence of adequate focal adhesion formation and an intact cytoskeleton, whereas human primary cells revealed differences in comparison to immortalized cell lines. The use of primary cells, analysis of the cell-implant structure interaction as well as cell migration might strengthen the evaluation of cytocompatibility and thereby improve the validity regarding the putative in vivo performance of implant material.

Advanced Single-Cell Mapping Reveals that in hESC Cardiomyocytes Contraction Kinetics and Action Potential Are Independent of Myosin Isoform.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) represent an attractive model to investigate CM function and disease mechanisms. One characteristic marker of ventricular specificity of human CMs is expression of the ventricular, slow ß-myosin heavy chain (MyHC), as opposed to the atrial, fast α-MyHC. The main aim of this study was to investigate at the single-cell level whether contraction kinetics and electrical activity of hESC-CMs are influenced by the relative expression of α-MyHC versus ß-MyHC. For effective assignment of functional parameters to the expression of both MyHC isoforms at protein and mRNA levels in the very same hESC-CMs, we developed a single-cell mapping technique. Surprisingly, α- versus ß-MyHC was not related to specific contractile or electrophysiological properties of the same cells. The multiparametric cell-by-cell analysis suggests that in hESC-CMs the expression of genes associated with electrical activity, contraction, calcium handling, and MyHCs is independently regulated.

Tepidiforma bonchosmolovskayae gen. nov., sp. nov., a moderately thermophilic Chloroflexi bacterium from a Chukotka hot spring (Arctic, Russia), representing a novel class, Tepidiformia, which includes the previously uncultivated lineage OLB14.

A novel aerobic moderately thermophilic bacterium, strain 3753OT, was isolated from a Chukotka hot spring (Arctic, Russia) using the newly developed technology of laser engineering of microbial systems. Сells were regular short rods, 0.4×0.8-2.0 µm in size, with a monoderm-type envelope and a single flagellum. The temperature and pH ranges for growth were 42-60 °C and pH 6.5-8.5, the optima being 50-54 °C and pH 7.3. Strain 3753OT grew chemoorganoheterotrophically on a number of carbohydrates or peptidic substrates and volatile fatty acids, and chemolithoautotrophically with siderite (FeCO3) as the electron donor. The major cellular fatty acid was branched C19 : 0. Phosphatidylethanolamine, phosphatidylglycerol and two unidentified phospholipids as well as two yellow carotenoid-type pigments were detected in the polar lipid extract. Strain 3753OT was inhibited by chloramphenicol, polymyxin B, vancomycin, streptomycin, neomycin and kanamycin, but resistant to the action of novobiocin and ampicillin. The DNA G+C content was 69.9 mol%. The 16S rRNA gene as well as 51 conservative protein sequence-based phylogenetic analyses placed strain 3753OT within the previously uncultivated lineage OLB14 in the phylum Chloroflexi. Taking into account the phylogenetic position as well as phenotypic properties of the novel isolate, the novel genus and species Tepidiforma bonchosmolovskayae gen. nov., sp. nov., within the Tepidiformaceae fam. nov., the Tepidiformales ord. nov. and the Tepidiformia classis nov. are proposed. The type strain of Tepidiforma bonchosmolovskayae is 3753OT (=VKM B-3389T=KTCT 72284T).

High-speed two-photon polymerization 3D printing with a microchip laser at its fundamental wavelength.

High-resolution, high-speed 3D printing by two-photon polymerization (2PP) with a Nd:YVO4 Q-switched microchip laser at its fundamental wavelength of 1064 nm is demonstrated. Polymerization scan speeds of up to 20 mm/s and feature sizes of 250 nm are achieved using a high repetition rate Q-switched microchip laser with a semiconductor saturable absorber mirror (SESAM) and photoresist with a new photo-initiator bearing 6-dialkylaminobenzufuran as electron donor and indene-1,3-dione moiety as electron acceptor. The obtained results demonstrate the high potential of Q-switched microchip lasers for applications in 2PP 3D printing.

Heparanase-2 protects from LPS-mediated endothelial injury by inhibiting TLR4 signalling.

The endothelial glycocalyx and its regulated shedding are important to vascular health. Endo-ß-D-glucuronidase heparanase-1 (HPSE1) is the only enzyme that can shed heparan sulfate. However, the mechanisms are not well understood. We show that HPSE1 activity aggravated Toll-like receptor 4 (TLR4)-mediated response of endothelial cells to LPS. On the contrary, overexpression of its endogenous inhibitor, heparanase-2 (HPSE2) was protective. The microfluidic chip flow model confirmed that HPSE2 prevented heparan sulfate shedding by HPSE1. Furthermore, heparan sulfate did not interfere with cluster of differentiation-14 (CD14)-dependent LPS binding, but instead reduced the presentation of the LPS to TLR4. HPSE2 reduced LPS-mediated TLR4 activation, subsequent cell signalling, and cytokine expression. HPSE2-overexpressing endothelial cells remained protected against LPS-mediated loss of cell-cell contacts. In vivo, expression of HPSE2 in plasma and kidney medullary capillaries was decreased in mouse sepsis model. We next applied purified HPSE2 in mice and observed decreases in TNFα and IL-6 plasma concentrations after intravenous LPS injections. Our data demonstrate the important role of heparan sulfate and the glycocalyx in endothelial cell activation and suggest a protective role of HPSE2 in microvascular inflammation. HPSE2 offers new options for protection against HPSE1-mediated endothelial damage and preventing microvascular disease.

Liquid-Infused Structured Titanium Surfaces: Antiadhesive Mechanism to Repel Streptococcus oralis Biofilms.

To combat implant-associated infections, there is a need for novel materials which effectively inhibit bacterial biofilm formation. In the present study, the antiadhesive properties of titanium surface functionalization based on the "slippery liquid-infused porous surfaces" (SLIPS) principle were demonstrated and the underlying mechanism was analyzed. The immobilized liquid layer was stable over 13 days of continuous flow in an oral flow chamber system. With increasing flow rates, the surface exhibited a significant reduction in attached biofilm of both the oral initial colonizer  Streptococcus oralis and an oral multispecies biofilm composed of S. oralis, Actinomyces naeslundii, Veillonella dispar, and Porphyromonas gingivalis. Using single cell force spectroscopy, reduced S. oralis adhesion forces on the lubricant layer could be measured. Gene expression patterns in biofilms on SLIPS, on control surfaces, and expression patterns of planktonic cultures were also compared. For this purpose, the genome of S. oralis strain ATCC 9811 was sequenced using PacBio Sequel technology. Even though biofilm cells showed clear changes in gene expression compared to planktonic cells, no differences could be detected between bacteria on SLIPS and on control surfaces. Therefore, it can be concluded that the ability of liquid-infused titanium to repel S. oralis biofilms is mainly due to weakened bacterial adhesion to the underlying liquid interface.

Comparison of two prototypes of a magnetically adjustable glaucoma implant in rabbits.

Glaucoma drainage devices are used in surgical glaucoma therapy. Success of controlling the intraocular pressure is limited due to fibrous implant encapsulation and fibrin coating on the implant which lead to drainage obstructions. An innovative implant with a magnetically adjustable valve was developed. The valve opening of the implant should eliminate inflammatory products from the outflow area and affect fibrous tissue formation to achieve a sufficient long-term aqueous humour outflow. Lifting of this valve should disturb cell adhesion by exerting mechanical forces. Before testing this hypothesis, the flow characteristics of glaucoma drainage devices, especially the outflow resistance by regular IOP, should be considered in a pilot study, as they are important in preventing too low postoperative intraocular pressure known as ocular hypotony. Therefore, two prototypes of the innovative implant differing in their valve area design were examined regarding their flow characteristics in a limited animal experiment lasting two weeks. Ten healthy New Zealand White rabbits were divided into two groups (A & B) with different implanted prototypes. Daily, tonometry and direct ophthalmoscopy were performed to assess the intraocular pressure and the inflammatory reaction of the eye. After two weeks, the rabbits were euthanised to evaluate the initially histological inflammatory reaction to the implant. In group A, one case of hypotony emerged. When considering the entire observation period, a highly statistically significant difference between the intraocular pressure in the operated eye and that in the control eye was detected in group A (p < 0.0001) in contrast to group B (p = 0.0063). The postoperative inflammatory signs decreased within two weeks. Histologically, a typical but low level foreign body reaction with macrophages and lymphocytes as well as mild to moderate fibrosis was seen after the short experimental period. Based on our tonometric results, prototype B seems to be the system of choice for further research assessing its long-term function and biocompatibility.

Nanofabrication of High-Resolution Periodic Structures with a Gap Size Below 100 nm by Two-Photon Polymerization.

In this paper, approaches for the realization of high-resolution periodic structures with gap sizes at sub-100 nm scale by two-photon polymerization (2PP) are presented. The impact of laser intensity on the feature sizes and surface quality is investigated. The influence of different photosensitive materials on the structure formation is compared. Based on the elliptical geometry character of the voxel, the authors present an idea to realize high-resolution structures with feature sizes less than 100 nm by controlling the laser focus position with respect to the glass substrate. This investigation covers structures fabricated respectively in the plane along and perpendicular to the major axis of voxel. The authors also provide a useful approach to manage the fabrication of proposed periodic structure with a periodic distance of 200 nm and a gap size of 65 nm.