Observation of nanoparticle internalization on cellular membranes by using noninterferometric widefield optical profilometry

We demonstrate the observation of gold-nanoparticle internalization in membranes of living cells by using noninterferometric widefield optical profilometry (NIWOP). The NIWOP technique can trace the height of an 80 nm gold particle on the membrane by calibrating the change of light intensity scattered from the particle along the optical axis. On the membrane, the depth resolution based on the scattering signal is similar to that based on the reflection signal, nearly 20 nm. Comparing the heights of the nanoparticle and the nearby cell membranes, we can identify the occurrence of particle internalization. Combining fluorescence microscopy with NIWOP, we also find actin aggregation around the site of the internalization process, which is an indication of endocytosis.     

Influence of nickel ions on human multipotent mesenchymal stromal cells (hMSCs)

Nickel‐Titanium‐Shape‐Memory‐Alloys (NiTi‐SMA) are of biomedical interest due to an unusual range of pure elastic deformability (superelasticity) and the shape memory effect which allows this material to return to a predictable previously memorized shape after external changes in temperature. HMSCs (human multipotent mesenchymal stromal cells) are currently the most promising cell type for regenerative medicine and tissue engineering, due to the ability to differentiate into several tissues such as bone, tendon, cartilage or muscle. For tissue engineering newly developed porous NiTi‐SMA materials are evaluated preloaded with hMSCs. For biocompatibility testing the high nickel content (50 %at) of NiTi‐SMA plays a critical role. To analyse the influence of Ni‐ions on hMSCs viability and activation, cells were cultured with or without NiCl₂ for 24h and 7days. Cells were either seeded in media containing NiCl₂ or the NiCl₂ was later added to already adherent cells. Cell metabolism, proliferation and viability were analysed by alamarBlue^TM assay or fluorescence microscopy. Cytokine (IL‐6, 8, 11) release from hMSCs was determined by ELISA . NiCl₂ concentrations below 25 μg/ ml were well tolerated by the cells. A significant decrease in cell proliferation occurred at threshold values of 200 μg/ ml (24 h) and 25 μg/ ml (7 d). There was a significant, dose dependent increase in the release of IL‐8 from hMSCs cultured in the presence of sub toxic NiCl₂ concentrations. The present study demonstrates for the first time that high but non‐toxic concentrations of Ni²⁺ are capable to activate hMSCs. Thus high Ni²⁺ concentrations apart from allergen‐ or particle‐induced inflammation, may lead to tissue inflammation in the vicinity of a NiTi‐SMA implant in vivo and subsequently to implant failure e.g. due to implant loosening.     

Aberration compensation of holographic particle images using digital holographic microscopy

Characterisation of small and large-scale vortices in turbulent flows demands a system with high spatial resolution. The measurement of high spatial resolution, three-dimensional vector displacements in fluid mechanics using holography, is usually hampered by aberration. Aberration poses some problems in particle image identification due to low fidelity of real image reconstruction. Phase mismatch between the recording and the reconstruction waves was identified as the main source of aberration in this study. This paper demonstrates how aberration compensation can be achieved by cross-correlating the complex amplitude of an aberrated reconstructed object with the phase conjugate of a known reference object in the plane of the hologram (frequency space). Results favourably show significant increase in Strehl ratio and suppression of background noise that are more pronounced for particle images of 10 and 5 microns. It is clear from the work conducted that wavefront aberration measurement and compensation of holographic microscopic objects are now possible with the use of a variant digital holographic microscope.     

Enhanced mesenchymal stromal cell recruitment via natural killer cells by incorporation of inflammatory signals in biomaterials

An exacerbated inflammatory response questions biomaterial biocompatibility, but on the other hand, inflammation has a central role in the regulation of tissue regeneration. Therefore, it may be argued that an ‘ideal’ inflammatory response is crucial to achieve efficient tissue repair/regeneration. Natural killer (NK) cells, being one of the first populations arriving at an injury site, can have an important role in regulating bone repair/regeneration, particularly through interactions with mesenchymal stem/stromal cells (MSCs). Here, we studied how biomaterials designed to incorporate inflammatory signals affected NK cell behaviour and NK cell–MSC interactions. Adsorption of the pro-inflammatory molecule fibrinogen (Fg) to chitosan films led to a 1.5-fold increase in adhesion of peripheral blood human NK cells, without an increase in cytokine secretion. Most importantly, it was found that NK cells are capable of stimulating a threefold increase in human bone marrow MSC invasion, a key event taking place in tissue repair, but did not affect the expression of the differentiation marker alkaline phosphatase (ALP). Of significant importance, this NK cell-mediated MSC recruitment was modulated by Fg adsorption. Designing novel biomaterials leading to rational modulation of the inflammatory response is proposed as an alternative to current bone regeneration strategies.     

Effects of calcium-phosphate topography on osteoblast mechanobiology determined using a cytodetacher

The Human fetal osteoblast (hFOB) cell morphology, adhesion force, and proliferation on a calcium-phosphate (Ca-P) micropattern surface were investigated and the mechanobiology was investigated by a cytodetachment test. Ca-P-coated groove patterns with 3.0-μm-deep grooves (C3), 4.5-μm-deep grooves (C4), and 5.5-μm-deep grooves (C5) were produced on silicon wafers using photolithography and wet etching techniques. The grooved substrates were coated with a 200-nm-thick layer of titanium (bond coat) and a 200-nm-thick layer of calcium phosphate (top coat) using a sputtering system. Smooth Ca-P-coated Si wafers were used as control surfaces. Analysis of the scanning electron microscopy observations shows that cells on the Ca-P micropattern showed spreading and elongation. The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay indicated that C3 and C4 specimens had a significantly higher number of cells than did the control group after 5- and 15-day cultures. The cyto-compatibility of specimens was quantitatively evaluated using a cytodetacher, which directly measures the detachment shear force of an individual cell to the substrate. After 30-min culture, the cell adhesion forces were 38.4 nN for the smooth specimen, 140.8 nN for C3, 124.2 nN for C4, and 67.1 nN for C5. The results indicate that the cell adhesion force is influenced by cell shape and the Ca-P grooved patterns affect the cell shape and cytoskeletal structure, thus influence cell proliferation and cell adhesion force. The cytodetachment test with nanonewton resolution is a sensitive method for studying cell-biomaterial interaction.     

Effects of frequencies of AC modulation voltage on piezoelectric-induced images using atomic force microscopy

Lead zirconate titanate (PZT) thin film is prepared by sol-gel method on Pt/Ti electrode/SiO₂/Si wafer. Local poling is performed on the PZT film using an atomic force microscope (AFM). The topography and piezoelectric-induced (PEI) images on the polarized PZT film are recorded using AFM at piezo-responsive mode, operated with an AC voltage at varying frequencies. The best PEI image was obtained at the frequency around 300 kHz. It is explained that the change of piezoelectric vibrations and input noise signals with the frequency of AC modulation voltage affects the intensity of PEI images.     

Semi-quantitative analysis of gentian violet by surface-enhanced Raman spectroscopy using silver colloids

The viability of the application of surface-enhanced Raman spectroscopy (SERS) to the semi-quantitative analysis of the triphenylmethane dye gentian violet was examined by using activated borohydride-reduced silver colloids. Raman and SERS spectra of aqueous solutions of gentian violet at different pH values were acquired for the first time and equally intense SERS signals were obtained at both acidic and alkaline pH values. Two maxima intensities observed in the pH profile revealed the presence of different ionization states of the dye. The pH conditions for SERS were optimized over the pH range 1 to 12 and the biggest enhancement for SERS of this charged dye was found to be at pH 2.0; thus, this condition was used for semi-quantitative analysis. A good linear correlation was observed for the dependence of the signal intensities of the SERS bands at 1620 cm(-1) (R = 0.999) and 1370 cm(-1) (R = 0.952) on dye concentration over the range 10(-6) to 10(-4) mol/L, using laser excitation at 514.5 nm. At concentrations of dye above 10(-2) mol/L, the concentration dependence of the SERS signals is nonlinear. This is explained as due to the precipitation of metallic silver as well as due to saturation caused by complete coverage of the SERS substrate. A series of intensities of the band at 1620 cm(-1) measured from dye molecules proved that the single-molecule limit of gentian violet is attained at the concentration of 10(-9) mol/L.     

Bone regeneration using coculture of mesenchymal stem cells and angiogenic cells

Cellular strategies remain a crucial component in bone tissue engineering （BTE）. So far, the outcome of cell-based strategies from initial clinical trials is far behind compared to animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the Ussue-engineered constructs. Cocultures, by introducing angiogenic cells into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell-based constructs. So far, pre-clinical studies demonstrated that cocultures enhance vascularization and bone formation compared to monocultures. However, there has been no report on the application of cocultures in clinics. Therefore, this mini-review aims to provide an overview regarding （i） critical parameters in cocultures and the outcomes of cocultures compared to monocultures in the currently available pre-clinical studies using human mesenchymal stem cells implanted in orthotopic animal models; and （ii） the usage of monocultures in clinical application in BTE.     

Cultivation of human fibroblasts and multipotent mesenchymal stromal cells on mesoporous silica and mixed metal oxide films

The application of mesoporous silica and silica–titania-mixed metal oxide films prepared via sol–gel processing as substrates for cell growth was investigated. A deliberate tailoring of the chemical composition of the porous substrates with different Si:Ti ratios was achieved by using a single-source precursor based on a titanium-coordinated alkoxysilane, resulting in mesoporous silica–titania films with hydrophilic surfaces. The different coatings were investigated with respect to their applicability in the cultivation of human cells such as human fibroblasts and multipotent mesenchymal stromal cells. It was found that they promoted cell adhesion and proliferation of human fibroblasts up to a period of 14 days. After 2 weeks only single apoptotic cells could be detected on silica–titania mixed oxide films in contrast to a somewhat higher amount on silica coatings. Furthermore, none of the films inhibited osteogenic differentiation of multipotent mesenchymal stromal cells.     

Chemometric labeling of cereal tissues in multichannel fluorescence microscopy images using discriminant analysis

This paper presents a novel, semiautomatic method for microscopic identification of multicomponent samples, which allows the identification, location, and percentage quantity of each component to be determined. The method involves applying discriminant analysis to a sequence of multichannel fluorescence microscopy images via a supervised learning approach; by selecting groups of pixels that are representative for each component type in a "known" sample, a computer is "taught" how to recognize the behavior (i.e., fluorescence emission) of the various components when illuminated under different spectral conditions. The identity, quantity, and location of these components in "unknown" samples (i.e., samples with the same component types but in different ratios or distributions) can then be investigated. The technique therefore enables semiautomatic quantitative fluorescence microscopy and has potential as a quality control tool. This work demonstrates the application of the technique to artificial and natural samples and critically discusses its quality, potential, and limitations.     

From design of bio-based biocomposite electrospun scaffolds to osteogenic differentiation of human mesenchymal stromal cells

Electrospinning coupled with electrospraying provides a straightforward and robust route toward promising electrospun biocomposite scaffolds for bone tissue engineering. In this comparative investigation, four types of poly(3-hydroxybutyrate) (PHB)-based nanofibrous scaffolds were produced by electrospinning a PHB solution, a PHB/gelatin (GEL) mixture or a PHB/GEL/nHAs (hydroxyapatite nanoparticles) mixed solution, and by electrospinning a PHB/GEL solution and electrospraying a nHA dispersion simultaneously. SEM and TEM analyses demonstrated that the electrospun nHA-blended framework contained a majority of nHAs trapped within the constitutive fibers, whereas the electrospinning-electrospraying combination afforded fibers with a rough surface largely covered by the bioceramic. Structural and morphological characterizations were completed by FTIR, mercury intrusion porosimetry, and contact angle measurements. Furthermore, an in vitro investigation of human mesenchymal stromal cell (hMSC) adhesion and proliferation properties showed a faster cell development on gelatin-containing scaffolds. More interestingly, a long-term investigation of hMSC osteoblastic differentiation over 21 days indicate that hMSCs seeded onto the nHA-sprayed scaffold developed a significantly higher level of alkaline phosphatase activity, as well as a higher matrix biomineralization rate through the staining of the generated calcium deposits: the fiber surface deposition of nHAs by electrospraying enabled their direct exposure to hMSCs for an efficient transmission of the bioceramic osteoinductive and osteoconductive properties, producing a suitable biocomposite scaffold for bone tissue regeneration.     

Determination of the elastic modulus of adherent cells using spherical atomic force microscope probe

When the conventional Hertz formula is used to extract the elastic modulus, E, of cells based on the compression test using atomic force microscope spherical probe, the inconsistency between the actual situation and the assumption of the formula will lead to a large error. Using the ABAQUS for finite element modeling and analysis, here, a modified Hertz formula was developed to reduce the effects of cell radius, cell thickness, probe radius and compression depth on the extracted E of cells. Experimentally, the insensitivity of the extracted E to the compression region of cell and probe radius reflects the validity of the modified formula. Owing to the poor resolution of spherical probes, it's unlikely to know the actual thickness of cell at the measured point, which can lead to a huge error. Based on the modified formula, we further proposed an approach to control the effect of the uncertainty of cell thickness and ensured that a 10% difference in cell thickness does not incur over 10% variation in the obtained elastic modulus.

     

Simple monitoring of cancer cells using nanoparticles

Here we present a new strategy for a simple and fast detection of cancer circulating cells (CTCs) using nanoparticles. The human colon adenocarcinoma cell line (Caco2) was chosen as a model CTC. Similarly to other adenocarcinomas, colon adenocarcinoma cells have a strong expression of EpCAM, and for this reason this glycoprotein was used as the capture target. We combine the capturing capability of anti-EpCAM functionalized magnetic beads (MBs) and the specific labeling through antibody-modified gold nanoparticles (AuNPs), with the sensitivity of the AuNPs-electrocatalyzed hydrogen evolution reaction (HER) detection technique. The fully optimized process was used for the electrochemical detection of Caco2 cells in the presence of monocytes (THP-1), other circulating cells that could interfere in real blood samples. Therefore we obtained a novel and simple in situ-like sensing format that we applied for the rapid quantification of AuNPs-labeled CTCs in the presence of other human cells.     

Simulation studies of ultrasonic backscattering and B-mode images of liver using acoustic microscopy data

Data obtained from a scanning laser acoustic microscope (SLAM) were used to examine several aspects of ultrasonic backscattering from the liver. Phase interferograms from normal and abnormal human-liver specimens were digitized, and a series of algorithms was used to compute images of propagation velocity within the specimens. The propagation velocity images were then employed to simulate A- and B-mode results. These initial simulations were used to investigate how ultrasonic echo signals are related to tissue microstructure. Among the topics examined were B-mode speckling, frequency and beamwidth effects, and angulation dependencies.     

Detection of oxygen consumption of cultured adherent cells by bead injection spectroscopy

This paper describes a method for detecting oxygen consumption of adherent cell cultures. The sensing is based on oxygen-dependent quenching of the phosphorescence of a Pt-porphyrin complex immobilized on microcarrier beads, which are used as the cell culture substrate. Bead injection, a recent variant of the flow injection technique, is used to pack an aliquot of the beads into a small sensing layer that can be easily and rapidly renewed. The technique is tested on a model system of Chinese Hamster Ovary M1 cells grown on Cytodex-3 microcarrier beads. Cellular respiration is monitored through O2 consumption measured across a period of 3 min. The method is validated by detecting the impairment of aerobic metabolism caused by 1.5 mM amobarbital. Further, it is shown to have enough precision to distinguish even more subtle changes, such as the increase in oxygen consumption caused by stimulation of the muscarinic m1 receptor with 100 microM carbachol.     

In situ tracking of enzymatic breakdown of starch granules by synchrotron UV fluorescence microscopy

Synchrotron UV fluorescence microscopy was used for the first time to visualize the adsorption and diffusion of an enzyme while degrading a solid substrate. The degradation pathway of single starch granules by two amylases, optimized for biofuel production and industrial starch hydrolysis, was followed by tryptophan fluorescence (excitation at 280 nm, emission filter at 300-400 nm) and visible light imaging. Thus, both the adsorption of enzyme onto starch granules at 283 nm resolution and the resulting morphological changes were recorded at different stages of hydrolysis. It is the first time that amylases were localized on starch without staining or adding a fluorescent probe at such high resolution. This technique presents a very high potential for imaging proteins in complex systems. Its sensitivity was demonstrated by the detection of GBSS (the granular bound starch synthase) at high recording times, GBSS being present at very low levels in maize starch granules.     

Monitoring motility, spreading, and mortality of adherent insect cells using an impedance sensor

An emerging sensor technology referred to as electric cell-substrate impedance sensing (ECIS) has been extended for monitoring the behavior of insect cells including attachment, motility, and mortality. In ECIS, adherent cells were cultured on an array of eight small gold electrodes deposited on the bottom of tissue culture wells and immersed in a culture medium. Upon the attachment and spreading of cells on the gold electrode, the impedance increased because the cells acted as insulating particles to restrict the current flow. Experimental data revealed that insect cells interacted differently with various proteins used to precoat the gold electrode with concanavalin A as the best promoter to accelerate the rate of cell attachment. After the cells were fully spread, the measured impedance continued to fluctuate to reflect the constant motion and metabolic activity of the cells. As the cell behavior was sensitive to external chemicals, the applicability of ECIS for inhibition assays was demonstrated with HgCl2, trinitrotoluene, trinitrobenzene (TNB), and 2-amino-4,6-dinitrotoluene as model systems. Unlike conventional assays, the quantitative data obtained in this study are taken in real time and in a continuous fashion to depict cell motility and mortality.     

Measurement of gene expression from single adherent cells and spheroids collected using fast electrical lysis

The cytosol of a single adherent cell was collected by the electrical cell lysis method with a Pt-ring capillary probe, and the cellular messenger RNA (mRNA) was analyzed at a single-cell level. The ring electrode probe was positioned 20 microm above the cultured cells that formed a monolayer on an indium-tin oxide (ITO) electrode, and an electric pulse with a magnitude of 40 V was applied for 10 micros between the probe and the ITO electrodes in an isotonic sucrose solution. Immediately after the electric pulse, less than 1 microL of the lysed solution was collected using a micro-injector followed by RNA purification and first strand cDNA synthesis. Real-time PCR was performed to quantify the copy numbers of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression inside the single cell. The average copy numbers of GAPDH mRNA collected by the electrical cell lysis method were found to be comparable to those obtained by a simple capillary suction method. Although single-cell analysis has already been demonstrated, we have shown for the first time that the fast electrical cell lysis can be used for quantitative mRNA analysis at the single-cell level. This electrical cell lysis method was further applied for the analysis of mRNA obtained from single spheroids-the aggregated cellular masses formed during the three-dimensional culture -- as a model system to isolate small cellular clusters from tissues and organs.