Polyetheretherketone bioactivity induced by farringtonite.

Polyetheretherketone (PEEK) is considered as an excellent biomaterial for bone grafting and connective tissue replacement. The clinical potential is, however, limited by its bioinertness, poor osteoconduction, and weak antibacterial activity. These disadvantages can be overcome by introducing suitable additives to produce mineral-polymer composites or coatings. In this work, a PEEK-based bioactive composite has been obtained by blending the polymer with magnesium phosphate (Mg3(PO4)2) particles in amounts ranging from 1 to 10 wt.% using the hot press technique. The obtained composite exhibited improved mechanical and physical properties, above the lower limits set for bone engineering applications. The tested grafts were found to not induce cytotoxicity. The presence of magnesium phosphate induced the mineralisation process with no adverse effects on the expression of the marker crucial for osteoblastic differentiation. The most promising results were observed in the grafts containing 1 wt.% of magnesium phosphate embedded within the PEEK matrix. The improved bioactivity of grafts, together with suitable physical-chemical and mechanical properties, indicate this composite as a promising orthopaedic implant material.

Microfluidic device for enhancement and analysis of osteoblast differentiation in three-dimensional cell cultures.

Three-dimensional (3D) cell cultures are to date the gold standard in biomedical research fields due to their enhanced biological functions compared to conventional two-dimensional (2D) cultures. 3D cell spheroids, as well as organoids, are better suited to replicate tissue functions, which enables their use both as in vitro models for basic research and toxicology, as well as building blocks used in tissue/organ biofabrication approaches. Culturing 3D spheroids from bone-derived cells is an emerging technology for both disease modelling and drug screening applications. Bone tissue models are mainly limited by the implementation of sophisticated devices and procedures that can foster a tissue-specific 3D cell microenvironment along with a dynamic cultivation regime. In this study, we consequently developed, optimized and characterized an advanced perfused microfluidic platform to improve the reliability of 3D bone cell cultivation and to enhance aspects of bone tissue maturation in vitro. Moreover, biomechanical stimulation generated by fluid flow inside the arrayed chamber, was used to mimic a more dynamic cell environment emulating a highly vascularized bone we expected to improve the osteogenic 3D microenvironment in the developed multifunctional spheroid-array platform. The optimized 3D cell culture protocols in our murine bone-on-a-chip spheroid model exhibited increased mineralization and viability compared to static conditions. As a proof-of-concept, we successfully confirmed on the beneficial effects of a dynamic culture environment on osteogenesis and used our platform for analysis of bone-derived spheroids produced from primary human pre-osteoblasts. To conclude, the newly developed system represents a powerful tool for studying human bone patho/physiology in vitro under more relevant and dynamic culture conditions converging the advantages of microfluidic platforms with multi-spheroid array technologies.

Novel Förster Resonance Energy Transfer probe with quantum dot for a long-time imaging of active caspases inside individual cells.

With the goal to investigate biological phenomena at a single-cell level, we designed, synthesized and tested a molecular probe based on Förster resonance energy transfer (FRET) between a highly luminescent quantum dot (QD) as a donor and a fluorophore or fluorescence quencher as an acceptor linked by a specific peptide. In principle, QD luminescence, effectively dissipated in the probe, is switched on after the cleavage of the peptide by a protease and the release of the quencher. We proposed a novel synthesis strategy of a probe. A two-step synthesis consists of: (i) Conjugation of CdTe QDs functionalized by -COOH groups of succinic acid on the nanoparticle surface with the designed specific peptide (GTADVEDTSC) using a ligand-exchange approach; (ii) A fast, high-yield reaction of amine-reactive succinimidyl group on the BHQ-2 quencher with N-terminal of the peptide. This way, any crosslinking between individual nanoparticles and any nonspecific conjugation bonds are excluded. The analysis of the product after the first step proved a high reaction yield and nearly no occurrence of unreacted QDs, a prerequisite of the specificity of our luminescent probe. Its parameters evaluated as Michaelis-Menten description of enzymatic kinetics are similar to products published by other groups. Our research is focused on the fluorescence microscopy analyses of biologically active molecules, such as proteolytic active caspases, playing important roles in cell signaling regulations in normal and diseased states. Consequently, they are attractive targets for clinical diagnosis and medical therapy. The ultimate goal of our work was to synthesize a new QD luminescent probe for a long-time quantitative monitoring of active caspase-3/7 distribution in apoptotic osteoblastic MC3T3-E1 cells treated with camptothecin. As a result of comparison, our synthetized luminescent probe provides longer imaging times of caspases than commercial products. The probe proved the stability of the luminescence signal inside cells for more than 14 days.

Caspase-8 Deficient Osteoblastic Cells Display Alterations in Non-Apoptotic Pathways.

Caspase-8 is the key component of the receptor-mediated (extrinsic) apoptotic pathway. Immunological localization of active caspase-8 showed its presence in osteoblasts, including non-apoptotic ones. Further in vivo exploration of caspase-8 functions in the bone is hindered by the fact that the caspase-8 knock-out is lethal prenatally. Examinations were thus performed using individual cell populations in vitro. In this study, caspase-8 was eliminated by the CRISPR/cas9 technology in MC3T3-E1 cells, the most common in vitro model of osteoblastic populations. The aim of the work was to specify the consequences of caspase-8 deficiency on non-apoptotic pathways. The impact on the osteogenic gene expression of the osteoblastic cells along with alterations in proliferation, caspase cascades and rapamycin induced autophagy response were evaluated. Osteogenic differentiation of caspase-8 deficient cells was inhibited as these cells displayed a decreased level of mineralization and lower activity of alkaline phosphatase. Among affected osteogenic genes, based on the PCR Array, major changes were observed for Ctsk, as down-regulated, and Gdf10, as up-regulated. Other significantly down-regulated genes included those coding osteocalcin, bone morphogenetic proteins (-3, -4 and -7), collagens (-1a1, -14a1) or Phex. The formation of autophagosomes was not altered in rapamycin-treated caspase-8 deficient cells, but expression of some autophagy-related genes, including Tnfsf10, Cxcr4, Dapk1 and Igf1, was significantly downregulated. These data provide new insight into the effects of caspase-8 on non-apoptotic osteogenic pathways.

Miniaturized bioluminescence technology for single-cell quantification of caspase-3/7.

Correct determination of the instantaneous level and changes of relevant proteins inside individual cells is essential for correct interpretation and understanding of physiological, diagnostic, and therapeutic events. Thus, single-cell analyses are important for quantification of natural cellular heterogeneity, which cannot be evaluated from averaged data of a cell population measurements. Here, we developed an original highly sensitive and selective instrumentation and methodology based on homogeneous single-step bioluminescence assay to quantify caspases and evaluate their heterogeneity in individual cells. Individual suspended cells are selected under microscope and reliably transferred into the 7 µl detection vials by a micromanipulator. The sensitivity of the method is given by implementation of photomultiplying tube with a cooled photocathode working in the photon counting mode. By optimization of our device and methodology, the limits of detection and quantitation were decreased down to 2.1 and 7.0 fg of recombinant caspase-3, respectively. These masses are lower than average amounts of caspase-3/7 in individual apoptotic and even non-apoptotic cells. As a proof of concept, the content of caspase-3/7 in single treated and untreated HeLa cells was determined to be 154 and 25 fg, respectively. Based on these results, we aim to use the technology for investigations of non-apoptotic functions of caspases.

A single-cell analytical approach to quantify activated caspase-3/7 during osteoblast proliferation, differentiation, and apoptosis.

The protein heterogeneity at the single-cell level has been recognized to be vital for an understanding of various life processes during animal development. In addition, the knowledge of accurate quantity of relevant proteins at cellular level is essential for appropriate interpretation of diagnostic and therapeutic results. Some low-copy-number proteins are known to play a crucial role during cell proliferation, differentiation, and also in apoptosis. The fate decision is often based on the concentration of these proteins in the individual cells. This is likely to apply also for caspases, cysteine proteases traditionally associated with cell death via apoptosis but recently being discovered also as important factors in cell proliferation and differentiation. The hypothesis was tested in bone-related cells, where modulation of fate from apoptosis to proliferation/differentiation and vice versa is particularly challenging, e.g., towards anti-osteoporotic treatments and anti-cancer strategies. An ultrasensitive and highly selective method based on bioluminescence photon counting was used to quantify activated caspase-3/7 in order to demonstrate protein-level heterogeneity in individual cells within one population and to associate quantitative measurements with different cell fates (proliferation, differentiation, apoptosis). The results indicate a gradual increase of caspase-3/7 activation from the proliferative status to differentiation (more than three times) and towards apoptosis (more than six times). The findings clearly support one of the putative key mechanisms of non-apoptotic functions of pro-apoptotic caspases based on fine-tuning of their activation levels.

Osteogenic impact of pro-apoptotic caspase inhibitors in MC3T3-E1 cells.

Caspases are proteases traditionally associated with inflammation and cell death. Recently, they have also been shown to modulate cell proliferation and differentiation. The aim of the current research was to search for osteogenic molecules affected by caspase inhibition and to specify the individual caspases critical for these effects with a focus on proapoptotic caspases: caspase-2, -3, -6, -7, -8 and -9. Along with osteocalcin (Ocn), general caspase inhibition significantly decreased the expression of the Phex gene in differentiated MC3T3-E1 cells. The inhibition of individual caspases indicated that caspase-8 is a major contributor to the modification of Ocn and Phex expression. Caspase-2 and-6 had effects on Ocn and caspase-6 had an effect on Phex. These data confirm and expand the current knowledge about the nonapoptotic roles of caspases and the effect of their pharmacological inhibition on the osteogenic potential of osteoblastic cells.

A device for investigation of natural cell mobility and deformability.

A microfluidic device made of polydimethylsiloxane was developed for continuous evaluation of natural migration mobility of many eukaryotic cells in relaxed and deformed state. The device was fabricated by standard photolithography and soft lithography techniques using the SU-8 3010 negative photoresist on a glass wafer as the master mold. The simple flow-free device exploits the chemotactic movement of cells through a set of mechanical barriers in the direction of concentration gradients of attractants. The barriers are formed by arrays of circular cross-section pillars with decreasing spacing 7, 5, and 3 µm. To pass through the obstacles, the cells are deformed and change their cytoskeletal architecture. The instantaneous migration velocities of cells are monitored in a time-lapse setup of the scanning confocal microscope. Thus, the cellular deformability and migratory activity can easily be evaluated. The functionality of the device was tested with model HeLa cells stably transfected with fluorescent Premo FUCCI Cell Cycle Sensor. The designed device has the potential to be implemented for testing the tendency of patients' tumors to metastasis.

Preparation and Analysis of Quantum Dots: Applications of Capillary Electrophoresis.

The implementation of quantum dots in analytical chemistry has already advanced from basic research activities to routine applications of commercially available fluorescent agents present in sophisticated assays kits. Nevertheless, a further development of new preparation and characterization methods of nanoparticles is still required to increase the sensitivity of analytical methods substantially. Thus, in many bioanalytical applications, important molecules such as DNA, proteins, and antibodies are routinely conjugated with fluorescent tags to reach even the absolute sensitivity, that is, the capability to detect a single molecule in complex matrices. Semiconductor quantum dots have already proved to be suitable components of highly luminescent tags, probes, and sensors with broad applicability in analytical chemistry. Quantum dots provide high extinction coefficients together with wide ranges of excitation wavelengths, size- and composition-tunable emissions, narrow and symmetric emission spectra, good quantum yields, relatively long size-dependent luminescence lifetime, and low photobleaching. Most of these properties are superior when compared with conventional organic fluorescent dyes. In this chapter, optimized procedures for the preparation of water-dispersed CdTe quantum dots; their coatings and conjugation reactions with antibodies, DNA, and macrocycles; and their analyses by capillary electrophoresis are described. The potential of capillary electrophoresis for fast analyses of nanoparticles, their conjugates with antibodies and immunocomplexes with targeted antigens, is demonstrated as an example.

Bi-Ligand Modification of Nanoparticles: An Effective Tool for Surface-Enhanced Raman Spectrometry in Salinated Environments.

Elimination of massive aggregation of nanoparticles in the sample of high ionic strength is a prerequisite for the sensitive analysis through a surface-enhanced Raman spectrometry (SERS). We present a system of silver colloid modification composed of two thiolated modifiers (3-mercaptopropionic acid and thiolated polyethylene glycol) both creating a strong Ag-S bond. At their optimal molar ratio, the polymer acts as a steric barrier preventing direct nanoparticle-nanoparticle interaction, while the low-molecular organic acid creates areas accessible for the analyte molecules. Thus, this approach is an excellent tool for sustaining both the colloidal stability and SERS sensitivity. The functionality of the system was demonstrated on the SERS analysis of myoglobin from a saline solution. The favorable creation of hot spots was achieved by laser-induced sintering.

Microfabricated liquid junction hybrid capillary electrophoresis-mass spectrometry interface for fully automated operation.

One of the challenging instrumental aspects in coupling an automated CE instrument with ESI mass spectrometry (CE-MS) is finding the balance between the stability, reproducibility and sensitivity of the analysis and compatibility with the standard CE instrumentation. Here, we present a development of a new liquid junction based electrospray interface for automated CE-MS, with a focus on the technical design followed by computer modeling of transport conditions as well as characterization of basic performance of the interface. This hybrid arrangement designed as a microfabricated unit attachable to the automated CE instrument allows using of a wide range of separation capillaries with respect to their diameter, length or internal coating (e.g., for suppressed electroosmotic flow). Different compositions of the ESI liquid and background electrolyte solutions can be used if needed. The microfabricated part, prepared by laser machining from polyimide, includes a self-aligning liquid junction, a short transport channel, and a pointed sprayer for the electrospray ionization. This microfabricated part is positioned in a plastic connection block securing the separation capillary and flushing ports. Transport conditions were modelled using computer simulation and the real life performance of the interface was compared to that of a commercial sheath liquid interface. The basic performance of the interface was demonstrated by separations of peptides, proteins, and oligosaccharides.

Combination of liquid-based column separations with surface-enhanced Raman spectroscopy.

Surface-enhanced Raman spectroscopy is a constantly developing analytical method providing not only high-sensitive quantitative but also qualitative information on an analyte. Thus, it is reasonable that it has been tested as a promising detection method in column separations. Although its implementation in analytical separations is not widespread, some surprising results, like enormous signal enhancement and demonstrations of single-molecule identifications, proved in only a few special examples, indicate the potential of the method. The high detection sensitivity and selectivity would be of paramount importance in trace analyses of biologically relevant molecules in complex matrices. However, the combination of surface-enhanced Raman spectroscopy with column separation methods brings two principal issues. Interactions of analytes with metal substrates can cause deteriorations of separations and the detection can be affected by background electrolytes or elution agents. Thus, in principle, this review is on the experimental and methodological solutions to these problems. First, theoretical and practical aspects of Raman scattering, and excitation of surface plasmon in colloid suspensions of nanoparticles and on planar nanostructured substrates are briefly explained. Advances in experimental arrangements of on-line and at-line couplings with column liquid phase separation methods, including microfluidic devices, are described together with chosen analytical applications.

Time-Dependent Growth of Silica Shells on CdTe Quantum Dots.

The purpose of this study is to investigate the time dependent growth of silica shells on CdTe quantum dots to get their optimum thicknesses for practical applications. The core/shell structured silica-coated CdTe quantum dots (CdTe/SiO2 QDs) were synthesized by the Ströber process, which used CdTe QDs co-stabilized by mercaptopropionic acid. The coating procedure used silane primer (3-mercaptopropyltrimethoxysilane) in order to make the quantum dots (QDs) surface vitreophilic. The total size of QDs was dependent on both the time of silica shell growth in the presence of sodium silicate, and on the presence of ethanol during this growth. The size of particles was monitored during the first 72 h using two principally different methods: Dynamic Light Scattering (DLS), and Scanning Electron Microscopy (SEM). The data obtained by both methods were compared and reasons for differences discussed. Without ethanol precipitation, the silica shell thickness grew slowly and increased the nanoparticle total size from approximately 23 nm up to almost 30 nm (DLS data), and up to almost 60 nm (SEM data) in three days. During the same time period but in the presence of ethanol, the size of CdTe/SiO2 QDs increased more significantly: up to 115 nm (DLS data) and up to 83 nm (SEM data). The variances occurring between silica shell thicknesses caused by different methods of silica growth, as well as by different evaluation methods, were discussed.

Capillary electrophoresis, a method for the determination of nucleic acid ligands covalently attached to quantum dots representing a donor of Förster resonance energy transfer.

The synthesis and determination of the structure of a Förster resonance energy transfer probe intended for the detection of specific nucleic acid sequences are described here. The probe is based on the hybridization of oligonucleotide modified quantum dots with a fluorescently labeled nucleic acid sample resulting in changes of the fluorescence emission due to the energy transfer effect. The stoichiometry distribution of oligonucleotides conjugated to quantum dots was determined by capillary electrophoresis separation. The results indicate that one to four molecules of oligonucleotide are conjugated to the surface of a single nanoparticle. This conclusion is confirmed by the course of the dependence of Förster resonance energy transfer efficiency on the concentration of fluorescently labeled complementary single-stranded nucleic acid, showing saturation. While the energy transfer efficiency of the probe hybridized with complementary nucleic acid strands was 30%, negligible efficiency was observed with a noncomplementary strand.

Recent advances in CE-MS coupling: Instrumentation, methodology, and applications.

This review focuses on the latest development of microseparation electromigration methods in capillaries and microfluidic devices coupled with MS for detection and identification of important analytes. It is a continuation of the review article on the same topic by Kleparnik (Electrophoresis 2015, 36, 159-178). A wide selection of 161 relevant articles covers the literature published from June 2014 till May 2016. New improvements in the instrumentation and methodology of MS interfaced with capillary or microfluidic versions of zone electrophoresis, isotachophoresis, and isoelectric focusing are described in detail. The most frequently implemented MS ionization methods include electrospray ionization, matrix-assisted desorption/ionization and inductively coupled plasma ionization. Although the main attention is paid to the development of instrumentation and methodology, representative examples illustrate also applications in the proteomics, glycomics, metabolomics, biomarker research, forensics, pharmacology, food analysis, and single-cell analysis. The combinations of MS with capillary versions of electrochromatography, and micellar electrokinetic chromatography are not included.

Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells.

Analysing the chemical content of individual cells has already been proven to reveal unique information on various biological processes. Single-cell analysis provides more accurate and reliable results for biology and medicine than analyses of extracts from cell populations, where a natural heterogeneity is averaged. To meet the requirements in the research of important biologically active molecules, such as caspases, we have developed a miniaturized device for simultaneous analyses of individual cells. A stainless steel body with a carousel holder enables high-sensitivity parallel detections in eight microvials. The holder is mounted in front of a photomultiplier tube with cooled photocathode working in photon counting mode. The detection of active caspase-3/7, central effector caspases in apoptosis, in single cells is based on the bioluminescence chemistry commercially available as Caspase-Glo® 3/7 reagent developed by Promega. Individual cells were captured from a culture medium under microscope and transferred by micromanipulator into detection microvial filled with the reagent. As a result of testing, the limits of detection and quantification were determined to be 0.27/0.86 of active caspase-3/7 content in an average apoptotic cell and 0.46/2.92 for non-apoptotic cells. Application potential of this technology in laboratory diagnostics and related medical research is discussed. Graphical abstract Miniaturized device for simultaneous analyses of individual cells.

Design and validation of an STR hexaplex assay for DNA profiling of grapevine cultivars.

Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser-induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high-throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling.

Caspases and osteogenic markers--in vitro screening of inhibition impact.

Caspases, well-known players in apoptosis or inflammation, appear to have roles also in other processes such as cell differentiation. Caspase-3, in particular, was recently demonstrated to have non-apoptotic functions in osteogenesis. However, the molecular pathways involved are not yet known. Therefore, we used osteogenic PCR arrays to provide a comprehensive screening of possible interactions of caspases in general and specifically of caspase-3 in osteogenic networks. Embryonic micromass cultures derived from mouse forelimbs were established and pharmacological fluoromethylketone (FMK) inhibitors applied. Alterations were observed in expression of several genes after caspase inhibition (Bmp1, Bmp5, Bmp6, Col10a1, Col2a1, Comp, Egf, Fgfr2, Gli1, Igf1, Nog, Phex, Sox9, Spp1). The list suggests molecular interactions of caspases and osteogenic molecules and creates a background for further temporospatial and functional studies.

Self-aligning subatmospheric hybrid liquid junction electrospray interface for capillary electrophoresis.

We report a construction of a self-aligning subatmospheric hybrid liquid junction electrospray interface for CE eliminating the need for manual adjustment by guiding the capillaries in a microfabricated liquid junction glass chip at a defined angle. Both the ESI and separation capillaries are inserted into the microfabricated part until their ends touch. The distance between the capillary openings is defined by the angle between the capillaries. The microfabricated part contains channels for placement of the capillaries and connection of the external electrode reservoirs. It was fabricated using standard photolithographic/wet chemical etching techniques followed by thermal bonding. The liquid junction is connected to a subatmospheric electrospray chamber inducing the flow inside the ESI needle and helping the ion transport via aerodynamic focusing.

Recent advances in combination of capillary electrophoresis with mass spectrometry: methodology and theory.

This review focuses on the latest development of microseparation electromigration methods in capillaries and microfluidic devices with MS detection and identification. A wide selection of 183 relevant articles covers the literature published from June 2012 till May 2014 as a continuation of the review article on the same topic by Kleparnik [Electrophoresis 2013, 34, 70-86]. Special attention is paid to the new improvements in the theory of instrumentation and methodology of MS interfacing with capillary versions of zone electrophoresis, ITP, and IEF. Ionization methods in MS include ESI, MALDI, and ICP. Although the main attention is paid to the development of instrumentation and methodology, representative examples illustrate also applications in the proteomics, glycomics, metabolomics, biomarker research, forensics, pharmacology, food analysis, and single-cell analysis. The combinations of MS with capillary versions of electrochromatography and micellar electrokinetic chromatography are not included.