Effect of laser energy, substrate film thickness and bioink viscosity on viability of endothelial cells printed by Laser-Assisted Bioprinting

Biofabrication of three dimensional tissues by Laser-Assisted Bioprinting (LAB) implies to develop specific strategies for assembling the extracellular matrix (ECM) and cells. Possible strategies consist in (i) printing cells onto or in the depth of ECM layer and/or (ii) printing bioinks containing both cells and ECM-like printable biomaterial. The aim of this article was to evaluate combinatorial effects of laser pulse energy, ECM thickness and viscosity of the bioink on cell viability. A LAB workstation was used to print Ea.hy926 endothelial cells onto a quartz substrate covered with a film of ECM mimicking Matrigel™. Hence, effect of laser energy, Matrigel™ film thickness and bioink viscosity was addressed for different experimental conditions (8-24 μJ, 20-100 μm and 40-110 mPa s, respectively). Cell viability was assessed by live/dead assay performed 24 h post-printing. Results show that increasing the laser energy tends to augment the cell mortality while increasing the thickness of the Matrigel™ film and the viscosity of the bioink support cell viability. Hence, critical printing parameters influencing high cell viability have been related to the cell landing conditions and more specifically to the intensity of the cell impacts occurring at the air-ECM interface and at the ECM-glass interface.     

Laser scribing on HOPG for graphene stamp printing on silicon wafer

Highly oriented pyrolytic graphite (HOPG) was scribed by pulsed laser beam to produce square patterns. Patterning of HOPG surface facilitates the detachment of graphene layers during contact printing. Direct HOPG-to-substrate and glue-assisted stamp printing of a few-layers graphene was compared. Printed graphene sheets were visualized by optical and scanning electron microscopy. The number of graphene layers was measured by atomic force microscopy. Glue-assisted stamp printing allows printing relatively large graphene sheets (40×40 μm) onto a silicon wafer. The presented method is easier to implement and is more flexible than the majority of existing ways of placing graphene sheets onto a substrate.     

Laser-based techniques for living cell pattern formation

In the production of biosensors or artificial tissues a basic step is the immobilization of living cells along the required pattern. In this paper the ability of some promising laser-based methods to influence the interaction between cells and various surfaces is presented. In the first set of experiments laser-induced patterned photochemical modification of polymer foils was used to achieve guided adherence and growth of cells to the modified areas: (a) Polytetrafluoroethylene was irradiated with ArF excimer laser (λ=193 nm, FWHM=20 ns, F=9 mJ/cm²) in presence of triethylene–tetramine liquid photoreagent; (b) a thin carbon layer was produced by KrF excimer laser (λ=248 nm, FWHM=30 ns, F=35 mJ/cm²) irradiation on polyimide surface to influence the cell adherence. It was found that the incorporation of amine groups in the PTFE polymer chain instead of the fluorine atoms can both promote and prevent the adherence of living cells (depending on the applied cell types) on the treated surfaces, while the laser generated carbon layer on polyimide surface did not effectively improve adherence. Our attempts to influence the cell adherence by morphological modifications created by ArF laser irradiation onto polyethylene–terephtalate surface showed a surface–roughness dependence. This method was effective only when the Ra roughness parameter of the developed structure did not exceed the 0.1 micrometer value. Pulsed laser deposition with femtosecond KrF excimer lasers (F=2.2 J/cm²) was effectively used to deposit structured thin films from biomaterials (endothelial cell growth supplement and collagen embedded in starch matrix) to promote the adherence and growth of cells. These results present evidence that some surface can be successfully altered to induce guided cell growth.     

Relationship between adsorbed fibronectin and cell adhesion on a honeycomb-patterned film

Substratum surface morphology plays a vital roles in cellular behavior. Here, we characterized adsorption of fibronectin (Fn) as a typical cell adhesion protein onto honeycomb-patterned films made of poly(ε-caprolactone) (PCL) by using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). In order to determine how cells adhere to a honeycomb-patterned film, focal adhesion of cardiac myocytes (CMYs) and endothelial cells (ECs) on the films were studied by using fluorescence labeling of vinculin. Fn adsorbs around the pore edges to form ring-shaped structures. CMYs and ECs adhere onto the honeycomb-patterned films at focal contact points localized around pore edges distributed over the entire cellular surface. The focal contact points on the honeycomb-patterned films correspond well with the adsorption sites of Fn. We suggest that the cell response to honeycomb-patterned films is associated with the adsorption pattern of Fn on the film.     

Polymer pixel enhancement by laser-induced forward transfer for sensor applications

This paper presents polymer pixel printing for applications in chemoselective sensors where nanosecond laser direct transfer methods, with a triazene polymer (TP) acting as a Dynamic Release Layer (DRL), are used. A systematic study of laser fluence, donor film morphology and both single- and multiple-pixel deposition were optimized with the final goal to obtain continuous pixels of sensitive polymers, polyethylenimine (PEI) and polyisobutylene (PIB), on SAW surfaces. Morphology characterization after the laser transfer has been performed by Optical Microscopy and Scanning Electron Microscopy (SEM). The responses of the coated transducers were measured after deposition with different laser fluences and it was found that a fluence under 625 mJ/cm² was required in order to prevent damage of the interdigital transducers (IDT) of the sensor devices. The sensitivity of the polymer coated devices to acetone concentrations gives an indication that LIFT can be used for printing sensitive polymer pixels onto transducer devices.     

Osteoblast cell response to a CO2 laser modified polymeric material

Lasers are an efficient technology, which can be applied for the surface treatment of polymeric biomaterials to enhance insufficient surface properties. That is, the surface chemistry and topography of biomaterials can be modulated to increase the biofunctionality of that material. By employing CO₂ laser patterning and whole area processing of nylon 6,6 this paper details how the surface properties were significantly modified. Samples, which had undergone whole area processing, followed the current theory in which the advancing contact angle, θ, with water decreased and the polar component, γ^p, increased upon an increase in surface roughness. For the patterned samples it was observed that θ increased and γ^P decreased. This did not follow the current theory and can be explained by a mixed-state wetting regime. By seeding osteoblast cells onto the samples for 24 h and 4 days the laser surface treatment gave rise to modulated cell response. For the laser whole area processing, θ and γ^P correlated with the observed cell count and cover density. Owed to the wetting regime, the patterned samples did not give rise to any correlative trend. As a result, CO₂ laser whole area processing is more likely to allow one to predict biofunctionality prior to cell seeding. Moreover, for all samples, cell differentiation was evidenced. On account of this and the modulation in cell response, it has been shown that laser surface treatment lends itself to changing the biofunctional properties of nylon 6,6.     

The comparison of laser surface designing and pigment printing methods for the product quality

Developing new designs by using the computer and transferring the designs that are obtained to textile surfaces will not only increase and facilitate the production in a more practical manner, but also help you create identical designs. This means serial manufacturing of the products at standard quality and increasing their added values. Moreover, creating textile designs using the laser will also contribute to the value of the product as far as the consumer is concerned because it will not cause any wearing off and deformation in the texture of the fabric unlike the other methods.In the system that has been designed, the laser beam at selected wavelength and intensity was directed onto a selected textile surface and a computer-controlled laser beam source was used to change the colour substances on the textile surface.Pigment printing is also used for designing in textile and apparel sector. In this method, designs are transferred to the fabric manually by using dyestuff.In this study, the denim fabric used for the surfacing trial was 100% cotton, with a weft count per centimeter of 20 and a warp count per centimeter of 27, with fabric weight of 458 g/m².The first step was to prepare 40 pieces of denim samples, half of which were prepared manually pigment printing and the other half by using the laser beam. After this, some test applications were done. The tensile strength, tensile extension and some fastness values of designed pieces with two methods were compared according to the international standards.     

Effects of the Thermal Environment on Articular Chondrocyte Metabolism: A Fundamental Study to Facilitate Establishment of an Effective Thermotherapy for Osteoarthritis

Aim: To facilitate establishment of an effective thermotherapy for osteoarthritis (OA), we investigated the effects of the thermal environment on articular chondrocyte metabolism in vitro. Methods: Chondrocytes were isolated from porcine knee joints, and cultured at 32°C, 37°C and 41°C. Cell proliferation and viability were assessed at Days 2, 4 and 8. In addition, TdT-mediated dUTP nick end labeling (TUNEL) assay was performed at Day 3 to determine the proportion of apoptotic chondrocytes. Analysis of genes specific for factors related to the cartilage extracellular matrix (ECM), cartilage destruction, and cartilage protection was performed at Day 2. Furthermore, evaluation of heat stress tolerance, and heat shock protein 70 (HSP70) mRNA expression and protein synthesis was performed at Day 2 and 3, respectively. Results: Cell proliferation was more at 37°C than at 32°C and 41°C. Cell viability and the number of TUNEL-positive cells were not affected until Day 8 and 3, respectively. The expression of the ECM-related genes was up-regulated at higher temperature. The expression of MMP13, a type II collagen destructive enzyme, and that of TIMP1 and TIMP2, which are MMP inhibitors, were up-regulated at higher temperatures. Finally, the chondrocytes cultured at 41°C may acquire heat stress tolerance, in part, due to the up-regulation of HSP70, and may inhibit apoptosis induced by various stresses, which is observed in OA. Conclusions: The thermal environment affects articular chondrocyte metabolism, and a heat stimulus of approximately 41°C could enhance chondrocyte anabolism and induce heat stress tolerance.     

Effect of an absorbent overlay on the residual stress field induced by laser shock processing on aluminum samples

Laser shock processing (LSP) or laser shock peening is a new technique for strengthening metals. This process induces a compressive residual stress field, which increases fatigue crack initiation life and reduces fatigue crack growth rate. Specimens of 6061-T6 aluminum alloy are used in this investigation. A convergent lens is used to deliver 2.5 J, 8 ns laser pulses by a Q-switch Nd:YAG laser, operating at 10 Hz. The pulses are focused to a diameter of 1.5 mm onto aluminum samples. Density of 2500 pulses/cm² with infrared (1064 nm) radiation was used. The effect of an absorbent overlay on the residual stress field using this LSP setup and this energy level is evaluated. Residual stress distribution as a function of depth is assessed by the hole drilling method. It is observed that the overlay makes the compressive residual stress profile move to the surface. This effect is explained on the basis of the vaporization of the coat layer suppressing thermal effects on the metallic substrate. The effect of coating the specimen surface before LSP treatment may have advantages on improving wear and contact fatigue properties of this aluminum alloy.     

Protein fragment imaging using ink jet printing digestion technique

We have developed a capable tool for visualizing protein distribution at the sub-cell level with TOF-SIMS. This new method of visualizing a protein includes: (1) printing a digestive enzyme with ink jet (IJ) printing technology onto a tissue section, (2) measuring the two-dimensional distributions of decomposed peptide ions derived from digested protein on the tissue section by TOF-SIMS with a bismuth cluster ion (Bi₃⁺) source and using the aqueous ion enhancement method, and (3) analyzing the two-dimensional distributions of proteins using the results of proteome analysis. We have not yet tried this method onto a real tissue section. To show the possibility of this method, TOF-SIMS results of visualizing dot-patterned digested fragment peptide ions using IJ printing digestion technique onto a protein film are presented.     

Laser-assisted modification of polystyrene surfaces for cell culture applications

Laser-assisted patterning and modification of polystyrene (PS) was investigated with respect to applications in micro-fluidics and cell culture. For this purpose the wettability, the adsorption of proteins and the adhesion of animal cells were investigated as function of laser- and processing parameters. The change of surface chemistry was characterized by X-ray photoelectron spectroscopy. The local formation of chemical structures suitable for improved cell adhesion was realized on PS surfaces by UV laser irradiation. Above and below the laser ablation threshold two different mechanisms affecting cell adhesion were detected. In the first case the debris deposited on and along laser irradiated areas was responsible for improved cell adhesion, while in the second case a photolytic activation of the polymer surface including a subsequent oxidization in oxygen or ambient air is leading to a highly localized alteration of protein adsorption from cell culture media and finally to increased cell adhesion. Laser modifications of PS using suitable exposure doses and an appropriate choice of the processing gas (helium or oxygen) enabled a highly localized control of wetting. The dynamic advancing contact angle could be adjusted between 2° and 150°. The hydrophilic and hydrophobic behaviour are caused by chemical and topographical surface changes.     

Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes

Background

As the eradication of tumor cells in vivo is most efficiently performed by cytolytic T lymphocytes (CTL), various methods for priming tumor-reactive lymphocytes have been developed. In this study, a method of priming CTLs with ultraviolet (UV)-irradiated tumor cells, which results in termination of tumor cell proliferation, apoptosis, as well as upregulation of heat shock proteins (HSP) expression is described.

Methods

Peripheral blood mononuclear cells (PBMC) were primed weekly with UV-irradiated or mitomycin-treated RPMI 8226 multiple myeloma cells. Following three rounds of stimulation over 21 days, the lymphocytes from the mixed culture conditions were analyzed for anti-MM cell reactivity.

Results

By day 10 of cultures, PBMCs primed using UV-irradiated tumor cells demonstrated a higher percentage of activated CD8+/CD4- T lymphocytes than non-primed PBMCs or PBMCs primed using mitomycin-treated MM cells. Cytotoxicity assays revealed that primed PBMCs were markedly more effective (p < 0.01) than non-primed PBMCs in killing RPMI 8226 MM cells. Surface expression of glucose regulated protein 94 (Grp94/Gp96) and Grp78 were both found to be induced in UV-treated MM cells.

Conclusion

Since, HSP-associated peptides are known to mediate tumor rejection; these data suggest that immune-mediated eradication of MM cells could be elicited via a UV-induced HSP process. The finding that the addition of 17-allylamide-17-demethoxygeldanamycin (17AAG, an inhibitor of HSP 90-peptide interactions) resulted in decreased CTL-induced cytotoxicity supported this hypothesis. Our study, therefore, provides the framework for the development of anti-tumor CTL cellular vaccines for treating MM using UV-irradiated tumor cells as immunogens.     

The pertinence of expression of heat shock proteins (HSPs) to the efficacy of cryopreservation in HELAs

HELAs (Hela cells, passed cells of human cervical carcinoma) were heat or cold treated (named heat or cold shock) and then resumed normal culture for 2, 4 or 8 hours respectively. The expressions of heat shock protein 70 (HSP70) and 90 (HSP90) of the HELAs were measured by Northern and Western blotting. HELAs after 4-hour culture were exposed to or cryopreserved with different concentration of dimethyl sulfoxide (Me2SO, 2.5%, 5%, 10%, 15% and 20% respectively, V/V). Meanwhile, the HELAs after different culture time (2, 4 and 6 hours of culture) were cryopreserved with 5% Me2SO. After exposure or cryopreservation, the number of live HELAs was counted and the survival rate was calculated. The results showed that heat shock increased the expression of HSP70 and HSP90 of HELAs, while cold shock decreased the expression of the two proteins. When the concentrations of Me2SO were 10%, 15% and 20%, the survival rates of HELAs after exposure to Me2SO or cryopreservation were much lower than those when the concentrations were small. The survival rates of the heat shocked HELAs were significantly higher than those of the cold shocked and control HELAs. After cryopreservation with 5% Me2SO, the survival rate of heat shocked HELAs group with 2 hours culture time was the lowest among all the groups of HELAs with different cultural time. From the results of this study, we conclude that the expressions of HSP70 and HSP90 in HELAs increased significantly after heat shock, while cold shock decreased the expressions of these two proteins. The over-expressions of HSPs in the heat shocked HELAs could protect the cells from both injury caused by potential toxicity of high concentrations of Me2SO and cryoinjury caused by the freeze-thawing/cryopreservation procedure.     

Application of magnetic poly(styrene-glycidyl methacrylate) microspheres for immunomagnetic separation of bone marrow cells

Surface-functionalized magnetic poly(styrene-glycidyl methacrylate) (PS-GMA) microspheres were prepared and coupled with Sca-1 antibody for cell selection from murine bone marrow mononuclear cells (MNCs). Biotinylated Sca-1 antibody could be directly coupled to avidin-bound magnetic microspheres. Alternatively, oxidized goat anti-mouse antibody was covalently bound onto the amino group-containing magnetic microspheres in a site-directed manner, and the resultant conjugate was coupled with non-modified Sca-1 antibody. Using the indirect antibody-bound magnetic microspheres, the purity of isolated Sca-1⁺ cells increased with bead-to-cell ratio. Using a bead-to-cell ratio of 10 beads/cell, a purity of 85% Sca-1⁺ cells corresponding to a 17-fold enrichment was achieved.     

Protective effect of transgenic expression of porcine heat shock protein 70 on hypothalamic ischemic and oxidative damage in a mouse model of heatstroke

Background  

Transgenic mice have been used to examine the role of heat shock protein (HSP)72 in experimental heatstroke. Transgenic mice that were heterozygous for a porcine HSP70β gene ([+] HSP72) and transgene-negative littermate controls ([-] HSP72), under pentobarbital sodium anesthesia, were subjected to heat stress to induce heatstroke. It was found that the overexpression of HSP72 in multiple organs improved survival during heatstroke by reducing hypotension and cerebral ischemia and damage in mice. Herein we attempted to further assess the effect of heat exposure on thermoregulatory function, hypothalamic integration, and survival in unrestrained, unanesthetized [+]HSP72 and compare with those of [-]HSP72. In this research with the transgenic mice, we first conducted several biochemical, physiologic and histological determinations and then investigated the beneficial effects of HSP72 overexpression on the identified hypothalamic deficits, thermoregulatory dysfunction, and mortality during heatstroke.     

千瓦级双包层光纤激光器冷却方案设计理论和实验研究

尽管双包层光纤激光器的散热性能好于传统的固体激光器的散热性能,光纤激光器中的热沉积仍然是限制提高其输出功率的重要因素.以双端抽运的400 W双包层光纤激光器为实例,定量分析了光纤内的热沉积分布.根据所建立的散热模型,为了确保千瓦级双包层光纤激光器安全稳定的运行,抽运端附近的对流换热系数应大于2.8×10^-2W·cm^-2K^-1.据此设计出高功率双包层光纤激光器抽运端冷却装置并成功应用在激光系统中,获得了千瓦级的激光输出. 关键词： 热沉积 散热 双包层光纤激光器     

Multilayer laser printing for Organic Thin Film Transistors

Functional laser printed Organic Thin Film Transistors (OTFTs) have been achieved from multilayer substrates composed with semiconductor and electrodes. The p-type copper phthalocyanine (CuPc) was used to form the active layer. Different kinds of metallic materials were used for source and drain electrodes. Multilayer donor substrates were prepared by the successive depositions of materials by either thermal evaporation under vacuum or laser printing. The materials were transferred together in a single step onto a receiver substrate by laser pulses in the picosecond regime. The latter substrate formed the gate and the dielectric of the transistor. The results are compared with the step-by-step laser printing process, where electrodes and organic layer were successively printed from two different donor substrates. The multilayer laser printing reveals an improvement of the performances of the OTFT devices.     

Nondestructive isolation of single cultured animal cells by femtosecond laser-induced shockwave

A shockwave induced by focusing an intense femtosecond laser onto a culture medium under a microscope was used to manipulate single cultured animal cells. Mouse NIH3T3 fibroblasts were cultured on a collagen matrix and placed on a microscope installation that included an intense femtosecond laser. Cells were detached individually from the matrix by the shockwave, after their filopodia were cut by focusing the laser directly on them. The appearance and locomotion of cells after detachment was monitored with a CCD camera. Filopodia of the detached cells were regenerated, and the cell adhered again to the matrix within 4 hrs after detachment. When the shockwave was induced at a pulse energy of 0.72 J/pulse, 80% of cells were successfully detached from the culture plate in a non-destructive manner. The force required to detach a cell was estimated to be a few N/m², which is larger than the photon force resulting from conventional laser trapping. PACS 42.62.Be; 47.40.Nm; 87.80.Rb     

Hyperbranched polymers – their application in printing inks

Urania^® flexographic printing ink was modified with the commercially available hyperbranched polymer Boltorn H2004. The surface tension of the printing inks was measured and the rheology behaviour of the printing inks was assessed. The flow curves (shear stress vs. shear rate) for the printing inks exhibited pseudoplastic behaviour. Modified flexographic inks were laboratory printed on three different plastic films: polyethylene, polypropylene and polyethylene terephthalate. The impact of a small amount of hyperbranched polymer on printing ink colour was examined by optical density, total colour difference ΔE ^? (CIE L ^?, a ^?, b ^?) and the gloss of the printed film. In general, the addition of the hyperbranched polymer improved the colour fastness and hardness resistance. The influence of the hyperbranched polymer on the rheological properties, optical density, CIE LAB coordinates and colour fastness is discussed.     

Pulsed laser CVD investigations of single-wall carbon nanotube growth dynamics

The nucleation and rapid growth of single-wall carbon nanotubes (SWNTs) were explored by pulsed-laser assisted chemical vapor deposition (PLA-CVD). A special high-power, Nd:YAG laser system with tunable pulse width (>0.5 ms) was implemented to rapidly heat (>3×10⁴°C/s) metal catalyst-covered substrates to different growth temperatures for very brief (sub-second) and controlled time periods as measured by in situ optical pyrometry. Utilizing growth directly on transmission electron microscopy grids, exclusively SWNTs were found to grow under rapid heating conditions, with a minimum nucleation time of >0.1 s. By measuring the length of nanotubes grown by single laser pulses, extremely fast growth rates (up to 100 microns/s) were found to result from the rapid heating and cooling induced by the laser treatment. Subsequent laser pulses were found not to incrementally continue the growth of these nanotubes, but instead activate previously inactive catalyst nanoparticles to grow new nanotubes. Localized growth of nanotubes with variable density was demonstrated through this process and was applied for the reliable direct-write synthesis of SWNTs onto pre-patterned, catalyst-covered metal electrodes for the synthesis of SWNT field-effect transistors.