Isoflavonoid-Antibiotic Thin Films Fabricated by MAPLE with Improved Resistance to Microbial Colonization.

Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) bacteria represent major infectious threats in the hospital environment due to their wide distribution, opportunistic behavior, and increasing antibiotic resistance. This study reports on the deposition of polyvinylpyrrolidone/antibiotic/isoflavonoid thin films by the matrix-assisted pulsed laser evaporation (MAPLE) method as anti-adhesion barrier coatings, on biomedical surfaces for improved resistance to microbial colonization. The thin films were characterized by Fourier transform infrared spectroscopy, infrared microscopy, and scanning electron microscopy. In vitro biological assay tests were performed to evaluate the influence of the thin films on the development of biofilms formed by Gram-positive and Gram-negative bacterial strains. In vitro biocompatibility tests were assessed on human endothelial cells examined for up to five days of incubation, via qualitative and quantitative methods. The results of this study revealed that the laser-fabricated coatings are biocompatible and resistant to microbial colonization and biofilm formation, making them successful candidates for biomedical devices and contact surfaces that would otherwise be amenable to contact transmission.

Laser Direct-Write Onto Live Tissues: A Novel Model for Studying Cancer Cell Migration.

Investigation into the mechanisms driving cancer cell behavior and the subsequent development of novel targeted therapeutics requires comprehensive experimental models that mimic the complexity of the tumor microenvironment. Recently, our laboratories have combined a novel tissue culture model and laser direct-write, a form of bioprinting, to spatially position single or clustered cancer cells onto ex vivo microvascular networks containing blood vessels, lymphatic vessels, and interstitial cell populations. Herein, we highlight this new model as a tool for quantifying cancer cell motility and effects on angiogenesis and lymphangiogenesis in an intact network that matches the complexity of a real tissue. Application of our proposed methodology offers an innovative ex vivo tissue perspective for evaluating the effects of gene expression and targeted molecular therapies on cancer cell migration and invasion. J. Cell. Physiol. 231: 2333-2338, 2016. © 2016 Wiley Periodicals, Inc.

Time-Resolved Imaging Study of Jetting Dynamics during Laser Printing of Viscoelastic Alginate Solutions.

Matrix-assisted pulsed-laser evaporation direct-write (MAPLE DW) has been successfully implemented as a promising laser printing technology for various fabrication applications, in particular, three-dimensional bioprinting. Since most bioinks used in bioprinting are viscoelastic, it is of importance to understand the jetting dynamics during the laser printing of viscoelastic fluids in order to control and optimize the laser printing performance. In this study, MAPLE DW was implemented to study the jetting dynamics during the laser printing of representative viscoelastic alginate bioinks and evaluate the effects of operating conditions (e.g., laser fluence) and material properties (e.g., alginate concentration) on the jet formation performance. Through a time-resolved imaging approach, it is found that when the laser fluence increases or the alginate concentration decreases, the jetting behavior changes from no material transferring to well-defined jetting to well-defined jetting with an initial bulgy shape to jetting with a bulgy shape to pluming/splashing. For the desirable well-defined jetting regimes, as the laser fluence increases, the jet velocity and breakup length increase while the breakup time and primary droplet size decrease. As the alginate concentration increases, the jet velocity and breakup length decrease while the breakup time and primary droplet size increase. In addition, Ohnesorge, elasto-capillary, and Weber number based phase diagrams are presented to better appreciate the dependence of jetting regimes on the laser fluence and alginate concentration.

In Vitro/In Vivo Toxicity Evaluation and Quantification of Iron Oxide Nanoparticles.

Increasing biomedical applications of iron oxide nanoparticles (IONPs) in academic and commercial settings have alarmed the scientific community about the safety and assessment of toxicity profiles of IONPs. The great amount of diversity found in the cytotoxic measurements of IONPs points toward the necessity of careful characterization and quantification of IONPs. The present document discusses the major developments related to in vitro and in vivo toxicity assessment of IONPs and its relationship with the physicochemical parameters of IONPs. Major discussion is included on the current spectrophotometric and imaging based techniques used for quantifying, and studying the clearance and biodistribution of IONPs. Several invasive and non-invasive quantification techniques along with the pitfalls are discussed in detail. Finally, critical guidelines are provided to optimize the design of IONPs to minimize the toxicity.

Suppression of triple-negative breast cancer metastasis by pan-DAC inhibitor panobinostat via inhibition of ZEB family of EMT master regulators.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial-to-mesenchymal transition (EMT) is a key contributor in the metastatic process. We previously showed the pan-deacetylase inhibitor LBH589 induces CDH1 expression in TNBC cells, suggesting regulation of EMT. The purpose of this study was to examine the effects of LBH589 on the metastatic qualities of TNBC cells and the role of EMT in this process. A panel of breast cancer cell lines (MCF-7, MDA-MB-231, and BT-549), drugged with LBH589, was examined for changes in cell morphology, migration, and invasion in vitro. The effect on in vivo metastasis was examined using immunofluorescent staining of lung sections. EMT gene expression profiling was used to determine LBH589-induced changes in TNBC cells. ZEB overexpression studies were conducted to validate requirement of ZEB in LBH589-mediated proliferation and tumorigenesis. Our results indicate a reversal of EMT by LBH589 as demonstrated by altered morphology and altered gene expression in TNBC. LBH589 was shown to be a more potent inhibitor of EMT than other HDAC inhibitors, SAHA and TMP269. Additionally, we found that LBH589 inhibits metastasis of MDA-MB-231 cells in vivo. These effects of LBH589 were mediated in part by inhibition of ZEB, as overexpression of ZEB1 or ZEB2 mitigated the effects of LBH589 on MDA-MB-231 EMT-associated gene expression, migration, invasion, CDH1 expression, and tumorigenesis. These data indicate therapeutic potential of LBH589 in targeting EMT and metastasis of TNBC.

Synthesis of Silicon and Germanium Oxide Nanostructures via Photonic Curing; a Facile Approach to Scale Up Fabrication.

Silicon and Germanium oxide (SiOx and GeOx) nanostructures are promising materials for energy storage applications due to their potentially high energy density, large lithiation capacity (~10X carbon), low toxicity, low cost, and high thermal stability. This work reports a unique approach to achieving controlled synthesis of SiOx and GeOx nanostructures via photonic curing. Unlike conventional methods like rapid thermal annealing, quenching during pulsed photonic curing occurs rapidly (sub-millisecond), allowing the trapping of metastable states to form unique phases and nanostructures. We explored the possible underlying mechanism of photonic curing by incorporating laws of photophysics, photochemistry, and simulated temperature profile of thin film. The results show that photonic curing of spray coated 0.1 M molarity Si and Ge Acetyl Acetate precursor solution, at total fluence 80 J cm-2 can yield GeOx and SiOx nanostructures. The as-synthesized nanostructures are ester functionalized due to photoinitiated chemical reactions in thin film during photonic curing. Results also showed that nanoparticle size changes from ~48 nm to ~11 nm if overall fluence is increased by increasing the number of pulses. These results are an important contribution towards large-scale synthesis of the Ge and Si oxide nanostructured materials which is necessary for next-generation energy storage devices.

Generation of Ag-Ag(2)O complex nanostructures by excimer laser ablation of Ag in water.

Pulsed laser ablation in liquid (PLAL) has been well established as a facile method to produce nanoparticles from bulk materials, but it is still insufficient for fabricating anisotropic and complex nanostructures, especially without the use of surfactants. Here, we demonstrate that silver (Ag) nanosheets can be produced by pulsed excimer laser ablation of bulk Ag in water via laser re-processing of the laser-produced primary clusters. We also show that by combining PLAL and drop evaporation, rice-shaped Ag-Ag(2)O particles and their assemblies can be generated on Si substrates, because the interior flow of an evaporating colloidal drop could redistribute the laser-produced primary clusters, which results in the formation of complex nanostructures. These results show that PLAL is able to fabricate novel micro-/nanostructures while keeping its merit of "clean" fabrication.

Electric Cell-Substrate Impedance Sensing (ECIS) as a Platform for Evaluating Barrier-Function Susceptibility and Damage from Pulmonary Atelectrauma.

Biophysical insults that either reduce barrier function (COVID-19, smoke inhalation, aspiration, and inflammation) or increase mechanical stress (surfactant dysfunction) make the lung more susceptible to atelectrauma. We investigate the susceptibility and time-dependent disruption of barrier function associated with pulmonary atelectrauma of epithelial cells that occurs in acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). This in vitro study was performed using Electric Cell-substrate Impedance Sensing (ECIS) as a noninvasive evaluating technique for repetitive stress stimulus/response on monolayers of the human lung epithelial cell line NCI-H441. Atelectrauma was mimicked through recruitment/derecruitment (RD) of a semi-infinite air bubble to the fluid-occluded micro-channel. We show that a confluent monolayer with a high level of barrier function is nearly impervious to atelectrauma for hundreds of RD events. Nevertheless, barrier function is eventually diminished, and after a critical number of RD insults, the monolayer disintegrates exponentially. Confluent layers with lower initial barrier function are less resilient. These results indicate that the first line of defense from atelectrauma resides with intercellular binding. After disruption, the epithelial layer community protection is diminished and atelectrauma ensues. ECIS may provide a platform for identifying damaging stimuli, ventilation scenarios, or pharmaceuticals that can reduce susceptibility or enhance barrier-function recovery.

Generation of Ag2O micro-/nanostructures by pulsed excimer laser ablation of Ag in aqueous solutions of polysorbate 80.

A new route to synthesis of Ag(2)O micro-/nanostructures, including a mixture of cubes, pyramids, triangular plates, pentagonal rods, and bars, has been developed by pulsed excimer laser ablation of bulk silver in water using polysorbate 80 as surfactant. The polysorbate 80 played an important role in the formation of the Ag(2)O structures, and similar structures could be obtained in polysorbates 20 and 40 aqueous solutions. We have proposed a mechanism to explain the formation of Ag(2)O structures. This laser ablation method provides a unique approach to discover and fabricate new Ag(2)O morphologies.

Fabrication and formation mechanism of hollow MgO particles by pulsed excimer laser ablation of Mg in liquid.

We report on the formation of hollow MgO particles by excimer laser ablation of bulk Mg in water and aqueous solutions of sodium dodecyl sulfate (SDS) and sodium citrate (SC). Lamellar nanostructures of Mg(OH)(2) also formed in water, but the formation could be avoided by the addition of SDS or SC. Laser ablation produced not only Mg species that were oxidized into MgO and Mg(OH)(2) in water, but also cavitation bubbles. The bubble interfaces trapped the MgO nanoparticles to decrease the surface free energy of the system, finally resulting in hollow particles.

Metallic foil-assisted laser cell printing.

Laser direct-write technology such as modified laser-induced forward transfer (LIFT) is emerging as a revolutionary technology for biological construct fabrication. While many modified LIFT-based cell direct writing successes have been achieved, possible process-induced cell injury and death is still a big hurdle for modified LIFT-based cell direct writing to be a viable technology. The objective of this study is to propose metallic foil-assisted LIFT using a four-layer structure to achieve better droplet size control and increase cell viability in direct writing of human colon cancer cells (HT-29). The proposed four layers include a quartz disk, a sacrificial and adhesive layer, a metallic foil, and a cell suspension layer. The bubble formation-induced stress wave is responsible for droplet formation. It is found that the proposed metallic foil-assisted LIFT approach is an effective cell direct-write technology and provides better printing resolution and high post-transfer cell viability when compared with other conventional modified LIFT technologies such as matrix-assisted pulsed-laser evaporation direct-write; at the same time, the possible contamination from the laser energy absorbing material is minimized using a metallic foil.

Excimer laser ablation of a Pt target in water: the observation of hollow particles.

Micro/nanoparticles were fabricated by pulsed-excimer-laser ablation of a Pt target in water. Three kinds of hollow Pt particles (coalesced by micrograins, assembled by nanocrystals or with smooth shells) were observed together with solid particles using different laser fluences (2.3-6.8 J cm(-2)) and after 6000 laser shots. We propose that the hollow particles were formed on laser-produced bubbles which provided thermodynamically preferred nucleation sites and diffusion sinks for the laser-fabricated Pt clusters or particles. Although the hollow particles are a small proportion, the results have extended the scope of particles that pulsed-laser ablation in liquid can fabricate, and have enriched the mechanistic scenario of laser ablation and nanostructure formation in liquid.

Novel Antimicrobial Surfaces to Defeat COVID-19 Transmission.

Antimicrobial surface coatings function as a contact biocide and are extensively used to prevent the growth and transmission of pathogens on environmental surfaces. Currently, scientists and researchers are intensively working to develop antimicrobial, antiviral coating solutions that would efficiently impede/stop the contagion of COVID-19 via surface contamination. Herein we present a flavonoid-based antimicrobial surface coating fabricated by laser processing that has the potential to eradicate COVID-19 contact transmission. Quercetin-containing coatings showed better resistance to microbial colonization than antibiotic-containing ones.

Solvent-based Extrusion 3D Printing for the Fabrication of Tissue Engineering Scaffolds.

Three-dimensional (3D) printing has been emerging as a new technology for scaffold fabrication to overcome the problems associated with the undesirable microstructure associated with the use of traditional methods. Solvent-based extrusion (SBE) 3D printing is a popular 3D printing method, which enables incorporation of cells during the scaffold printing process. The scaffold can be customized by optimizing the scaffold structure, biomaterial, and cells to mimic the properties of natural tissue. However, several technical challenges prevent SBE 3D printing from translation to clinical use, such as the properties of current biomaterials, the difficulties associated with simultaneous control of multiple biomaterials and cells, and the scaffold-to-scaffold variability of current 3D printed scaffolds. In this review paper, a summary of SBE 3D printing for tissue engineering (TE) is provided. The influences of parameters such as ink biomaterials, ink rheological behavior, cross-linking mechanisms, and printing parameters on scaffold fabrication are considered. The printed scaffold structure, mechanical properties, degradation, and biocompatibility of the scaffolds are summarized. It is believed that a better understanding of the scaffold fabrication process and assessment methods can improve the functionality of SBE-manufactured 3D printed scaffolds.

Matrix-Assisted Pulsed laser Evaporation-deposited Rapamycin Thin Films Maintain Antiproliferative Activity.

Matrix-assisted pulsed laser evaporation (MAPLE) has many benefits over conventional methods (e.g., dip-coating, spin coating, and Langmuir-Blodgett dip-coating) for manufacturing coatings containing pharmacologic agents on medical devices. In particular, the thickness of the coating that is applied to the surface of the medical device can be tightly controlled. In this study, MAPLE was used to deposit rapamycin-polyvinylpyrrolidone (rapamycin-PVP) thin films onto silicon and borosilicate optical glass substrates. Alamar Blue and PicoGreen studies were used to measure the metabolic health and DNA content of L929 mouse fibroblasts as measures of viability and proliferation, respectively. The cells on the MAPLE-deposited rapamycin-PVP surfaces exhibited 70.6% viability and 53.7% proliferation compared to a borosilicate glass control. These data indicate that the antiproliferative properties of rapamycin were maintained after MAPLE deposition.

Nanostructured manganese oxides electrode with ultra-long lifetime for electrochemical capacitors.

We describe the instantaneous fabrication of a highly porous three-dimensional (3D) nanostructured manganese oxides-reduced graphitic oxide (MnO x -rGO) electrode by using a pulse-photonic processing technique. Such nanostructures facilitate the movement of ions/electrons and offer an extremely high surface area for the electrode/electrolyte interaction. The electrochemical performance was investigated by cyclic voltammetry (CV), galvanostatic charge-discharge (GCD) and electrochemical impedance spectroscopy (EIS) with 1 M KOH as the electrolyte. The as-prepared thin film electrode exhibits excellent electrochemical performance and an ultra-long lifetime by retaining 90% of the initial capacitance even after 100 000 GCD cycles at constant areal current density of 0.4 mA cm-2. We attribute this excellent lifetime performance to the conductive reduced graphitic oxide, synergistic effects of carbon composite and the metal oxides, and the unique porous nanostructure. Such highly porous morphology also enhances the structural stability of the electrode by buffering the volume changes during the redox processes.

Chemotherapy-induced senescent cancer cells engulf other cells to enhance their survival.

In chemotherapy-treated breast cancer, wild-type p53 preferentially induces senescence over apoptosis, resulting in a persisting cell population constituting residual disease that drives relapse and poor patient survival via the senescence-associated secretory phenotype. Understanding the properties of tumor cells that allow survival after chemotherapy treatment is paramount. Using time-lapse and confocal microscopy to observe interactions of cells in treated tumors, we show here that chemotherapy-induced senescent cells frequently engulf both neighboring senescent or nonsenescent tumor cells at a remarkable frequency. Engulfed cells are processed through the lysosome and broken down, and cells that have engulfed others obtain a survival advantage. Gene expression analysis showed a marked up-regulation of conserved macrophage-like program of engulfment in chemotherapy-induced senescent cell lines and tumors. Our data suggest compelling explanations for how senescent cells persist in dormancy, how they manage the metabolically expensive process of cytokine production that drives relapse in those tumors that respond the worst, and a function for their expanded lysosomal compartment.

Directed self-assembly software for single cell deposition.

Laser direct-write (LDW) bioprinting methods offer a diverse set of tools to design experiments, fabricate tissue constructs and to cellular microenvironments all in a CAD/CAM manner. To date, we have just scratched the surface of the system's potential and for LDW to be utilized to its fullest, there are many distinct hardware and software components that must be integrated and communicate seamlessly. In this perspective article, we detail the development of novel graphical user interface (GUI) software to improve LDW capability and functionality. The main modules in the control software correspond to cell transfer, microbead fabrication, and micromachining. The modules make the control of each of these features, and the management of printing programs that utilize one or more features, to be facile. The software also addresses problems related to construct scale-up, print speed, experimental conditions, and management of sensor data. The control software and possibilities for integrated sensor data are presented.

Study of gelatin as an effective energy absorbing layer for laser bioprinting.

Laser-induced forward transfer printing, also commonly known as laser printing, has been widely implemented for three-dimensional bioprinting due to its unique orifice-free nature during printing. However, the printing quality has the potential to be further improved for various laser bioprinting applications. The objectives of this study are to investigate the feasibility of using gelatin as an energy absorbing layer (EAL) material for laser bioprinting and its effects on the quality of printed constructs, bioink printability, and post-printing cell viability and process-induced DNA damage. The gelatin EAL is applied between the quartz support and the coating of build material, which is to be printed. Printing quality can be improved by EAL-assisted laser printing when using various alginate solutions (1%, 2%, and 4%) and cell-laden bioinks (2% alginate and 5 × 106 cells ml-1 in cell culture medium). The required laser fluence is also reduced due to a higher absorption coefficient of gelatin gel, in particular when to achieve the best printing type/quality. The post-printing cell viability is improved by ∼10% and DNA double-strand breaks are reduced by ∼50%. For all the build materials investigated, the gelatin EAL helps reduce the droplet size and average jet velocity.

Effects of living cells on the bioink printability during laser printing.

Laser-induced forward transfer has been a promising orifice-free bioprinting technique for the direct writing of three-dimensional cellular constructs from cell-laden bioinks. In order to optimize the printing performance, the effects of living cells on the bioink printability must be carefully investigated in terms of the ability to generate well-defined jets during the jet/droplet formation process as well as well-defined printed droplets on a receiving substrate during the jet/droplet deposition process. In this study, a time-resolved imaging approach has been implemented to study the jet/droplet formation and deposition processes when printing cell-free and cell-laden bioinks under different laser fluences. It is found that the jetting behavior changes from no material transferring to well-defined jetting with or without an initial bulgy shape to jetting with a bulgy shape/pluming/splashing as the laser fluence increases. Under desirable well-defined jetting, two impingement-based deposition and printing types are identified: droplet-impingement printing and jet-impingement printing with multiple breakups. Compared with cell-free bioink printing, the transfer threshold of the cell-laden bioink is higher while the jet velocity, jet breakup length, and printed droplet size are lower, shorter, and smaller, respectively. The addition of living cells transforms the printing type from jet-impingement printing with multiple breakups to droplet-impingement printing. During the printing of cell-laden bioinks, two non-ideal jetting behaviors, a non-straight jet with a non-straight trajectory and a straight jet with a non-straight trajectory, are identified mainly due to the local nonuniformity and nonhomogeneity of cell-laden bioinks.