The influence of tool quality on the machining of additive manufactured and powder metallurgy titanium alloys.

This study was carried out to investigate the impact the quality of the drill bits has on the machining behavior of additive manufacturing (AM) and powder metallurgy (PM) titanium alloys. Therefore, commercially available drill bits which typically reflect two extremes of drill bit quality were selected. The performance of coated carbide twist drills, typically recommended for the drilling of wrought titanium alloys was compared with that of high-speed steel (HSS) drills. The average torque value, specific cutting energy (SCE), and tool wear were used to evaluate the drilling performance of AM and PM titanium alloys. The results of drilling tests revealed the application of the coated carbide drill resulted in lower torque and SCE values, less flank wear, and lower build-up-edge (BUE) compared with the uncoated HSS drill bits for AM fabricated titanium alloys. However, the carbide drill appeared to offer negligible improvement over the uncoated HSS drill when employed with the PM fabricated titanium alloy. In spite of the improvement in the drilling performance offered by the carbide drills for the AM titanium alloy, TiB intermetallic particles (part of the AM titanium microstructure) contributed to the damage of the coated carbide drill which would limit the drill lifetime.

Multisensor-integrated organs-on-chips platform for automated and continual in situ monitoring of organoid behaviors.

Organ-on-a-chip systems are miniaturized microfluidic 3D human tissue and organ models designed to recapitulate the important biological and physiological parameters of their in vivo counterparts. They have recently emerged as a viable platform for personalized medicine and drug screening. These in vitro models, featuring biomimetic compositions, architectures, and functions, are expected to replace the conventional planar, static cell cultures and bridge the gap between the currently used preclinical animal models and the human body. Multiple organoid models may be further connected together through the microfluidics in a similar manner in which they are arranged in vivo, providing the capability to analyze multiorgan interactions. Although a wide variety of human organ-on-a-chip models have been created, there are limited efforts on the integration of multisensor systems. However, in situ continual measuring is critical in precise assessment of the microenvironment parameters and the dynamic responses of the organs to pharmaceutical compounds over extended periods of time. In addition, automated and noninvasive capability is strongly desired for long-term monitoring. Here, we report a fully integrated modular physical, biochemical, and optical sensing platform through a fluidics-routing breadboard, which operates organ-on-a-chip units in a continual, dynamic, and automated manner. We believe that this platform technology has paved a potential avenue to promote the performance of current organ-on-a-chip models in drug screening by integrating a multitude of real-time sensors to achieve automated in situ monitoring of biophysical and biochemical parameters.

Detection of mRNA in living cells by double-stranded locked nucleic acid probes.

Double-stranded probes are homogeneous biosensors for rapid detection of specific nucleotide sequences. These double-stranded probes have been applied in various molecular sensing applications, such as real-time polymerase chain reaction and detection of bacterial 16S rRNA. In this study, we present the design and optimization of double-stranded probes for single-cell gene expression analysis in living cells. With alternating DNA/LNA monomers for optimizing the stability and specificity, we show that the probe is stable in living cells for over 72 hours post-transfection and is capable of detecting changes in gene expression induced by external stimuli. The probes can be delivered to a large number of cells simultaneously by cationic liposomal transfection or to individual cells selectively by photothermal delivery. We also demonstrate that the probe quantifies intracellular mRNA in living cells through the use of an equilibrium analysis. With its effectiveness and performance, the double-stranded probe represents a broadly applicable approach for large-scale single-cell gene expression analysis toward numerous biomedical applications, such as systems biology, cancer, and drug screening.

Role of graphene concentration on electrochemical and tribological properties of graphene-poly(methyl methacrylate) composite coatings.

This study aims to investigate the influence of graphene nanoplatelet (GNP) concentration on the electrochemical and tribological properties of GNP-poly(methyl methacrylate) (PMMA) composite coatings. GNP-PMMA coatings were prepared with varying GNP concentrations (0.5, 1.0, 3.0, and 5.0 wt %) using the drop-casting method onto AA6061 aluminum alloy substrates. Results showed that the addition of 1.0 wt % GNP increased the tensile strength of PMMA but further increase reduced the tensile strength and fracture strain of the composites. Permeability studies indicated that 1.0GNP-PMMA had the lowest water vapour transition rate. All GNP-PMMA coatings showed a higher coating resistance and impedance modulus at the lowest frequency compared to neat PMMA with 1.0GNP-PMMA having the highest |Z|0.01 Hz value in comparison to the composites with higher GNP concentrations. According to Raman mapping, an increase in the concentration of GNP in the composite resulted in the agglomeration of graphene, which caused the debonding of the graphene-PMMA interfaces and also resulted in a higher number of shear fronts and other defects on the fracture surface that reduced barrier properties of graphene. The specific wear rate of 1.0GNP-PMMA was lower than that of neat PMMA, indicating improved wear resistance. The coefficient of friction was lowest for 5.0GNP-PMMA, although this was due to a higher amount of material being transferred to the counterface. Accordingly, optimizing the GNP concentration enables the development of high-performance PMMA coatings with enhanced strength, improved barrier properties, and reduced wear rates, making them well-suited for applications such as corrosion protection and tribological coatings.

Electrokinetic focusing and separation of mammalian cells in conductive biological fluids.

Active manipulation of cells, such as trapping, focusing, and isolation, is essential for various bioanalytical applications. Herein, we report a hybrid electrokinetic technique for manipulating mammalian cells in physiological fluids. This technique applies a combination of negative dielectrophoretic force and hydrodynamic drag force induced by electrohydrodynamics, which is effective in conductive biological fluids. With a three-electrode configuration, the stable equilibrium positions of cells can be adjusted for separation and focusing applications. Cancer cells and white blood cells can be positioned and isolated into specific locations in the microchannel under both static and dynamic flow conditions. To investigate the sensitivity of the hybrid electrokinetic process, AC voltage, frequency, and bias dependences of the cell velocity were studied systematically. The applicability of the hybrid electrokinetic technique for manipulating cells in physiological samples is demonstrated by continuous focusing of human breast adenocarcinoma spiked in urine, buffy coats, and processed blood samples with 98% capture efficiency.

Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes.

Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.

Hybridization kinetics of double-stranded DNA probes for rapid molecular analysis.

This study reports the hybridization kinetics of double-stranded DNA probes for rapid molecular analysis. Molecular binding schemes based on double-stranded DNA probes have been developed for quantitative detection of various biomolecules, such as nucleic acids and DNA binding proteins recently. The thermodynamic competition between the target and the competitor in binding to the probe provides a highly specific mechanism for molecular detection. The kinetics of the double-stranded DNA probe, on the other hand, represent another key aspect toward its general applicability for a wide set of biomedical applications. Herein we report a systematic investigation of the kinetics of double-stranded DNA probes. The signal-to-background ratio and assay time of the double-stranded DNA probes are optimized at a high ionic strength (over 100 mM NaCl). Both the donor probe and the quencher probe sequences are shown to be important in the hybridization kinetics. A long sticky end of the probe is able to dramatically accelerate the kinetics of the assay. To provide a quantitative description of the kinetics, a two-stage binding model is developed to describe the major features of the kinetics of the assay. The sensitivity of the kinetic model and the dominant affinity constants are studied. The study provides a general guideline for the design of the probes for reducing the total assay time. With an appropriate design of the probes, the assay can be finished within minutes at room temperature.

Collective Cell Migration in 3D Epithelial Wound Healing.

Collective cell migration plays a pivotal role in development, wound healing, and metastasis, but little is known about the mechanisms and coordination of cell migration in 3D microenvironments. Here, we demonstrate a 3D wound healing assay by photothermal ablation for investigating collective cell migration in epithelial tissue structures. The nanoparticle-mediated photothermal technique creates local hyperthermia for selective cell ablation and induces collective cell migration of 3D tissue structures. By incorporating dynamic single cell gene expression analysis, live cell actin staining, and particle image velocimetry, we show that the wound healing response consists of 3D vortex motion moving toward the wound followed by the formation of multicellular actin bundles and leader cells with active actin-based protrusions. Inhibition of ROCK signaling disrupts the multicellular actin bundle and enhances the formation of leader cells at the leading edge. Furthermore, single cell gene expression analysis, pharmacological perturbation, and RNA interference reveal that Notch1-Dll4 signaling negatively regulates the formation of multicellular actin bundles and leader cells. Taken together, our study demonstrates a platform for investigating 3D collective cell migration and underscores the essential roles of ROCK and Notch1-Dll4 signaling in regulating 3D epithelial wound healing.

Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement.

When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

Automated microfluidic platform of bead-based electrochemical immunosensor integrated with bioreactor for continual monitoring of cell secreted biomarkers.

There is an increasing interest in developing microfluidic bioreactors and organs-on-a-chip platforms combined with sensing capabilities for continual monitoring of cell-secreted biomarkers. Conventional approaches such as ELISA and mass spectroscopy cannot satisfy the needs of continual monitoring as they are labor-intensive and not easily integrable with low-volume bioreactors. This paper reports on the development of an automated microfluidic bead-based electrochemical immunosensor for in-line measurement of cell-secreted biomarkers. For the operation of the multi-use immunosensor, disposable magnetic microbeads were used to immobilize biomarker-recognition molecules. Microvalves were further integrated in the microfluidic immunosensor chip to achieve programmable operations of the immunoassay including bead loading and unloading, binding, washing, and electrochemical sensing. The platform allowed convenient integration of the immunosensor with liver-on-chips to carry out continual quantification of biomarkers secreted from hepatocytes. Transferrin and albumin productions were monitored during a 5-day hepatotoxicity assessment in which human primary hepatocytes cultured in the bioreactor were treated with acetaminophen. Taken together, our unique microfluidic immunosensor provides a new platform for in-line detection of biomarkers in low volumes and long-term in vitro assessments of cellular functions in microfluidic bioreactors and organs-on-chips.

Spatiotemporal dynamics of microRNA during epithelial collective cell migration.

MicroRNAs (miRNAs) are small, noncoding RNAs variably involved in a wide variety of developmental and regenerative programs. Techniques for monitoring the spatiotemporal expression of miRNA in living cells are essential to elucidate the roles of miRNA during these complex regulatory processes. The small size, low abundance, sequence similarity, and degradation susceptibility of miRNAs, however, make their detection challenging. In this study, we detail a double-stranded locked nucleic acid (dsLNA) probe for detecting intracellular miRNAs during epithelial collective migration. The dsLNA probe is capable of detecting the dynamic regulation and dose-dependent modulation of miRNAs. The probe is applied to monitor the spatial distribution of miRNA expression of a migrating epithelium. Our results reveal a gradient of miRNA over the first one hundred microns from the leading edge and show the involvement of miR-21 in the complex regulation of transforming growth factor beta modulated epithelial migration. With its ease of use and capacity for real-time monitoring of miRNAs in living cells, the dsLNA probe carries the potential for studying the function and regulation of miRNA in a wide spectrum of complex biological processes.

Microfluidics for Advanced Drug Delivery Systems.

Considerable efforts have been devoted towards developing effective drug delivery methods. Microfluidic systems, with their capability for precise handling and transport of small liquid quantities, have emerged as a promising platform for designing advanced drug delivery systems. Thus, microfluidic systems have been increasingly used for fabrication of drug carriers or direct drug delivery to a targeted tissue. In this review, the recent advances in these areas are critically reviewed and the shortcomings and opportunities are discussed. In addition, we highlight the efforts towards developing smart drug delivery platforms with integrated sensing and drug delivery components.

Single cell nanobiosensors for dynamic gene expression profiling in native tissue microenvironments.

A gold nanorod-locked nucleic acid nano-biosensor for dynamic single-cell gene expression analysis in living cells and tissues is developed. The nanoparticle facilitates endocytic delivery and dynamic monitoring of the gene expression in human umbilical cord endothelial cells, mouse skin tissues, mouse retina tissues, and mouse cornea tissues at the single-cell level.

Notch1-Dll4 signalling and mechanical force regulate leader cell formation during collective cell migration.

At the onset of collective cell migration, a subset of cells within an initially homogenous population acquires a distinct 'leader' phenotype with characteristic morphology and motility. However, the factors driving the leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined. Here we use single-cell gene expression analysis and computational modelling to show that the leader cell identity is dynamically regulated by Dll4 signalling through both Notch1 and cellular stress in a migrating epithelium. Time-lapse microscopy reveals that Dll4 is induced in leader cells after the creation of the cell-free region and leader cells are regulated via Notch1-Dll4 lateral inhibition. Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer. Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signalling to dynamically regulate the density of leader cells during collective cell migration.

A novel microchannel-based device to capture and analyze circulating tumor cells (CTCs) of breast cancer.

Circulating tumor cells (CTCs) have been shown in many studies as a possible biomarker for metastasis and may be instrumental for the spread of the disease. Despite advances in CTC capturing technologies, the low frequency of CTCs in cancer patients and the heterogeneity of the CTCs have limited the wide application of the technology in clinic. In this study, we investigated a novel microfluidic technology that uses a size- and deformability-based capture system to characterize CTCs. This unique platform not only allows flexibility in the selection of antibody markers but also segregates the CTCs in their own chambers, thus, enabling morphological, immunological and genetic characterization of each CTC at the single cell level. In this study, different breast cancer cell lines including MCF7, MDA-MB-231 and SKBR3, as well as a panel of breast cancer biomarkers were used to test the device. The technology can capture a wide range of cells with high reproducibility. The capturing efficiency of the cells is greater than 80%. In addition, the background of leukocytes is minimized because individual cells are segregated in their own chambers. The device captured both epithelial cancer cells such as MCF7 and SKBR3 and mesenchymal cells such as MDA-MB-231. Immunostaining of the captured cells on the microchannel device suggests that a panel of breast cancer biomarkers can be used to further characterize differential expression of the captured cells.

Single cell gene expression analysis in injury-induced collective cell migration.

Collective cell behavior in response to mechanical injury is central to various regenerative and pathological processes. Using a double-stranded locked nucleic acid probe for monitoring real-time intracellular gene expression, we examined the spatiotemporal response of epithelial cells during injury-induced collective migration and compared to the blocker assay with minimal injury as control. We showed that cells ∼150 µm from the wound edge exhibit a gradient in response to mechanical injury, expressing different genes depending on the wounding process. While release of contact inhibition is sufficient to trigger the migratory behavior, cell injury additionally induces reactive oxygen species, Nrf2 protein, and stress response genes, including heat shock protein 70 and heme oxygenase-1, in a spatiotemporal manner. Furthermore, we show that Nrf2 has an inhibitory role in injury-induced epithelial-mesenchymal transition, suggesting a potential autoregulatory mechanism in injury-induced response. Taken together, our single-cell gene expression analyses reveal modular cell responses to mechanical injury, manipulation of which may afford novel strategies for tissue repair and prevention of tumor invasion in the future.

Mapping photothermally induced gene expression in living cells and tissues by nanorod-locked nucleic acid complexes.

The photothermal effect of plasmonic nanostructures has numerous applications, such as cancer therapy, photonic gene circuit, large cargo delivery, and nanostructure-enhanced laser tweezers. The photothermal operation can also induce unwanted physical and biochemical effects, which potentially alter the cell behaviors. However, there is a lack of techniques for characterizing the dynamic cell responses near the site of photothermal operation with high spatiotemporal resolution. In this work, we show that the incorporation of locked nucleic acid probes with gold nanorods allows photothermal manipulation and real-time monitoring of gene expression near the area of irradiation in living cells and animal tissues. The multimodal gold nanorod serves as an endocytic delivery reagent to transport the probes into the cells, a fluorescence quencher and a binding competitor to detect intracellular mRNA, and a plasmonic photothermal transducer to induce cell ablation. We demonstrate the ability of the gold nanorod-locked nucleic acid complex for detecting the spatiotemporal gene expression in viable cells and tissues and inducing photothermal ablation of single cells. Using the gold nanorod-locked nucleic acid complex, we systematically characterize the dynamic cellular heat shock responses near the site of photothermal operation. The gold nanorod-locked nucleic acid complex enables mapping of intracellular gene expressions and analyzes the photothermal effects of nanostructures toward various biomedical applications.

Advances in wound-healing assays for probing collective cell migration.

Collective cell migration plays essential roles in a wide spectrum of biological processes, such as embryogenesis, tissue regeneration, and cancer metastasis. Numerous wound-healing assays based on mechanical, chemical, optical, and electrical approaches have been developed to create model "wounds" in cell monolayers to study the collective cell migration processes. These approaches can result in different microenvironments for cells to migrate and possess diverse assay characteristics in terms of simplicity, throughput, reproducibility, and multiplexability. In this review, we provide an overview of advances in wound-healing assays and discuss their advantages and limitations in studying collective cell migration.

Photodynamic inactivation of Enterococcus faecalis in dental root canals in vitro.

BACKGROUND AND OBJECTIVES: We previously reported the use of a flexible fiber optic that uniformly distributed light in the root canal space for targeting bacteria after their sensitization with methylene blue (MB). In the present study, we investigated the photodynamic effects of MB on Enterococcus faecalis species in experimentally infected root canals of extracted teeth after their sensitization with a concentration of MB that exhibits reduced dark toxicity. STUDY DESIGN/MATERIALS AND METHODS: In a model of root canal infection, 64 root canal specimens were prepared from extracted, single-rooted teeth and inoculated with E. faecalis (ATCC 29212). Three days later root canal infection was confirmed by scanning electron microscopy. The root canal systems were then incubated with 6.25 microg/ml MB for 5 minutes followed by exposure to light at 665 nm (60 J/cm(2)) that was delivered from a diode laser via a fiber optic with a diameter of 500 microm. Following photodynamic therapy (PDT) the canal content was sampled by flushing the root canals, serially diluted and cultured on blood agar. Survival fractions were calculated by counting colony-forming units. High-performance liquid chromatography (HPLC) was employed to determine the porphyrins content of E. faecalis. RESULTS: Scanning electron microscopy confirmed the presence of bacteria in the root canal system. PDT achieved 77.5% reduction of E. faecalis viability. MB alone and light alone reduced bacterial viability by 19.5% and 40.5%, respectively. HPLC did not reveal any porphyrin patterns expressed by E. faecalis. CONCLUSION: The results of this study support the need to determine the optimum MB concentration and light parameters to maximize bacterial killing in root canals.