The cell-stretcher: A novel device for the mechanical stimulation of cell populations.

Mechanical stimulation appears to be a critical modulator for many aspects of biology, both of living tissue and cells. The cell-stretcher, a novel device for the mechanical uniaxial stimulation of populations of cells, is described. The system is based on a variable stroke cam-lever-tappet mechanism which allows the delivery of cyclic stimuli with frequencies of up to 10 Hz and deformation between 1% and 20%. The kinematics is presented and a simulation of the dynamics of the system is shown, in order to compute the contact forces in the mechanism. The cells, following cultivation and preparation, are plated on an ad hoc polydimethylsiloxane membrane which is then loaded on the clamps of the cell-stretcher via force-adjustable magnetic couplings. In order to show the viability of the experimentation and biocompatibility of the cell-stretcher, a set of two in vitro tests were performed. Human epithelial carcinoma cell line A431 and Adult Mouse Ventricular Fibroblasts (AMVFs) from a dual reporter mouse were subject to 0.5 Hz, 24 h cyclic stretching at 15% strain, and to 48 h stimulation at 0.5 Hz and 15% strain, respectively. Visual analysis was performed on A431, showing definite morphological changes in the form of cellular extroflections in the direction of stimulation compared to an unstimulated control. A cytometric analysis was performed on the AMVF population. Results show a post-stimulation live-dead ratio deviance of less than 6% compared to control, which proves that the environment created by the cell-stretcher is suitable for in vitro experimentation.

Analysis of long- and short-range contribution to adhesion work in cardiac fibroblasts: an atomic force microscopy study.

Atomic force microscopy (AFM) for single-cell force spectroscopy (SCFS) and Poisson statistic were used to analyze the detachment work recorded during the removal of gold-covered microspheres from cardiac fibroblasts. The effect of Cytochalasin D, a disruptor of the actin cytoskeleton, on cell adhesion was also tested. The adhesion work was assessed using a Poisson analysis also derived from single-cell force spectroscopy retracting curves. The use of Poisson analysis to get adhesion work from AFM curves is quite a novel method, and in this case, proved to be effective to study the short-range and long-range contributions to the adhesion work. This method avoids the difficult identification of minor peaks in the AFM retracting curves by creating what can be considered an average adhesion work. Even though the effect of actin depolymerisation is well documented, its use revealed that control cardiac fibroblasts (CT) exhibit a work of adhesion at least 5 times higher than that of the Cytochalasin treated cells. However, our results indicate that in both cells short-range and long-range contributions to the adhesion work are nearly equal and the same heterogeneity index describes both cells. Therefore, we infer that the different adhesion behaviors might be explained by the presence of fewer membrane adhesion molecules available at the AFM tip-cell interface under circumstances where the actin cytoskeleton has been disrupted.

Exploring the elasticity and adhesion behavior of cardiac fibroblasts by atomic force microscopy indentation.

AFM was used to collect the whole force-deformation cell curves. They provide both the elasticity and adhesion behavior of mouse primary cardiac fibroblasts. To confirm the hypothesis that a link exists between the membrane receptors and the cytoskeletal filaments causing therefore changing in both elasticity and adhesion behavior, actin-destabilizing Cytochalsin D was administrated to the fibroblasts. From immunofluorescence observation and AFM loading/unloading curves, cytoskeletal reorganization as well as a change in the elasticity and adhesion was indeed observed. Elasticity of control fibroblasts is three times higher than that for fibroblasts treated with 0.5 µM Cytochalasin. Moreover, AFM loading-unloading curves clearly show the different mechanical behavior of the two different cells analyzed: (i) for control cells the AFM cantilever rises during the dwell time while cells with Cytochalasin fail to show such an active resistance; (ii) the maximum force to deform control cells is quite higher and as far as adhesion is concern (iii) the maximum separation force, detachment area and the detachment process time are much larger for control compared to the Cytochalasin treated cells. Therefore, alterations in the cytoskeleton suggest that a link must exist between the membrane receptors and the cytoskeletal filaments beneath the cellular surface and inhibition of actin polymerization has effects on the whole cell mechanical behavior as well as adhesion.

Atomic force microscopy of 3T3 and SW-13 cell lines: an investigation of cell elasticity changes due to fixation.

Mechanical properties of single cells are of increasing interest both from a fundamental cell biological perspective and in the context of disease diagnostics. In this respect, atomic force microscopy (AFM) has become a powerful tool for imaging and assessing mechanical properties of biological samples. However, while these tests are typically carried out on chemically fixed cells, the most important data is that on living cells. The present study applies AFM technique to assess the Young's modulus of two cell lines: mouse embryonic fibroblasts (NIH/3T3) and human epithelial cancer cells (SW-13). Both living cells and those fixed with paraformaldehyde were investigated. This analysis quantifies the difference between Young's modulus for these two conditions and provides a coefficient to relate them. Knowing the relation between Young's modulus of living and fixed cells, allows carrying out and comparing data obtained during steady-state measurements on fixed cells that are more frequently available in the clinical and research settings and simpler to maintain and probe.

Design of a novel MEMS platform for the biaxial stimulation of living cells.

Micromechanical systems are increasingly being used as tools in biological applications, since their characteristic dimensions permit to operate at the same length scale of the structures under investigation. Here, we present a methodology for the design, fabrication and operation of a tool for the assessment of mechanical properties of single cells. In particular, we describe a microsystems platform to study bio-mechanical response of single living cells to in-plane biaxial stretching. The proposed device employs a new linkage design in order to obtain the displacement of the quadrants of a sliced circular plate in mutually-orthogonal directions using just one linear actuator. With this linkage geometry, the whole device has only one degree of freedom. This results in a very predictable and reliable mechanical behaviour, thereby allowing use a simple and easily available control electronics. Results of this study have relevance for the design of a powerful yet simple BioMEMS platform for the characterization of living cells as in-plane bi-axial loading simulated the conditions experienced by cells in vivo more realistically than a uniaxial stretching.

Modeling of a microfluidic channel in the presence of an electrostatic induced cross-flow.

Amongst the processes that have been implemented in microfluidic devices, electrophoretic transport of charged molecules, along microfluidic channels, is one of the most commonly found. However, less work has been done about continuous, pressure gradient driven flow systems where an electric field is applied orthogonally with respect to the microchannel walls. The perspective applications of this technique, include continuous flow separation and concentration of analyte molecules, and the kinetic control of surface reactions. In order to dimensioning and optimizing such a device, a mathematical model has been formulated and analyzed both with numeric and analytic methods. The given solutions let the designer of microfluidic devices able to estimate the concentration profiles along the microchannel length, as a function of the main system parameters. As a practical example of application which could be of great interest in biotechnology applications, the results relative to the simulation of the electrostatic induced cross flow of single strand DNA oligonucleotides of about 20 bases has been reported.

Effectiveness of 1 mol L-1 citric acid and 15% EDTA irrigation on smear layer removal.

AIM: The aim of this study was to evaluate in vitro the cleansing and smear layer removal capability of alternate canal irrigation with citric acid and NaOCl. METHODOLOGY: Eighty-one teeth were divided into three groups on the basis of the type of instrumentation, namely, manual stainless steel, Ni-Ti mechanized ProFile .04 taper or MACXim. The groups were further divided on the basis of irrigation protocol: 5% NaOCl alone, NaOCl alternated with 1 mol L-1 citric acid solution or a combination of 15% EDTA and Cetrimide solution. After longitudinal sectioning, dentinal walls were microphotographed with scanning electron microscopy at x300 and x1000 magnifications. Qualitative and quantitative cleansing level evaluations were performed using computerized image analysis software. Data were statistically evaluated using Kruskal-Wallis analysis and t-test. RESULTS: Qualitative evaluation at x300 and x1000 showed no statistically significant differences in cleansing ability between citric acid, EDTA and NaOCl groups. Quantitative evaluation of smear layer removal, measured as open tubules/total dentinal surface ratio, showed that 1 mol L-1 citric acid solution was comparable to EDTA (11.97% vs. 10.36%) (NS); in samples treated with ProFile .04 taper instruments citric acid was most effective (16.17%), whilst in the group treated with manual instrumentation EDTA and Cetrimide were the most effective (11.94%). Specimens irrigated with 5% NaOCl demonstrated significantly more cleansing than those obtained in the other two groups (P < 0.001). CONCLUSIONS: 1 mol L-1 citric acid solution was as effective in removing smear layer as EDTA, but was superior in specimens treated with ProFile .04 taper instruments.

Mechanical and chemical consequences of the residual stresses in plasma sprayed hydroxyapatite coatings.

The residual stresses in thick hydroxyapatite coatings, deposited by plasma spraying, have been determined experimentally using Raman piezo-spectroscopy. The stress dependence of the centre position of the 980 cm 1 Raman band, owing to the symmetric stretching of the phosphate ion, PO3(4), has been established and found to be 2.47 cm 3 GPa 1. Using this calibration, the residual stresses in hydroxyapatite coatings deposited onto Ti-6A1-4V substrates in air have been found to be 100 MPa (tensile), whereas those deposited in a vacuum have been found to be 60 MPa (compressive). Although desirable from a mechanical point of view, it is shown that coating under residual compression are thermodynamically more stable and, hence, the dissolution of the ionic species, necessary in the exchange between bone and hydroxyapatite coating, can be impeded. It is calculated that for the coating under examination the stresses have an effect comparable with almost an order of magnitude change of the [OH] concentration. The analysis explains the dissolution behaviour of hydroxyapatite coatings subject to cyclic stress reported previously.