Controlled cellular orientation on PLGA microfibers with defined diameters

In this study, we investigated the effects of the diameter of microfibers on the orientation (angle between cells’ major axis and the substrate fiber long axis) of adhered cells. For this purpose, mouse fibroblast L929 cells were cultured on the surface of PLGA fibers of defined diameters ranging from 10 to 242 μm, and their adhesion and alignment was quantitatively analyzed. It was found that the mean orientation of cells and the spatial variation of cell alignment angle directly related to the microfiber diameter. Cells that were cultured on microfibrous scaffolds oriented along the long axis of the microfiber and the orientation increased as the fiber diameter decreased. For the fiber diameter of 10 μm, the mean orientation was 3.0 ± 0.2° (mean ± SE), whereas for a diameter of 242 μm, it decreased to 37.7 ± 2.1°. Using these studies we demonstrate that fibroblasts have a characteristic alignment on microscale fibers and that the microscale fiber diameter plays a critical role in cellular orientation. The ability to control cellular alignment on engineered tissue scaffold can be a potentially powerful approach to recreate the microscale architecture of engineered tissues. This may be important for engineering a variety of human tissues such as tendon, muscle and nerves as well as applications in 3D tissue culture and drug screening.     

Microfluidic alignment of collagen fibers for in vitro cell culture

Three dimensional gels of aligned collagen fibers were patterned in vitro using microfluidic channels. Collagen fiber orientation plays an important role in cell signaling for many tissues in vivo, but alignment has been difficult to realize in vitro. For microfluidic collagen fiber alignment, collagen solution was allowed to polymerize inside polydimethyl siloxane (PDMS) channels ranging from 10–400 μm in width. Collagen fiber orientation increased with smaller channel width, averaging 12 ± 6 degrees from parallel for channels between 10 and 100 μm in width. In these channels 20–40% of the fibers were within 5 degrees of the channel axis. Bovine aortic endothelial cells expressing GFP-tubulin were cultured on aligned collagen substrate and found to stretch in the direction of the fibers. The use of artificially aligned collagen gels could be applied to the study of cell movement, signaling, growth, and differentiation.     

Assessment of spatial resolution of pace mapping when using body surface potentials

Using computer simulations and statistical methods, the resolution of pace mapping when used in combination with body surface potentials was systematically investigated. In an anatomical model of the human ventricular myocardium, pre-excitation sequences were initiated at 69 sites positioned along the atrioventricular (AV) ring and corresponding body surface potential maps (BSPMs) were calculated at 32 leads placed on the anterior torso. For each time after the onset of pre-excitation (every 4ms to 40ms) and each root-mean-square (RMS) noise level (5, 10, 20 and 50μV), BSPMs were cross-correlated and the spatial resolution defined as the largest pacing site separation at which the differences in correlation coefficients were not statistically significant (level p≥0.05). The findings indicate that when random RMS noise of 5μV was added to the simulated BSPMs, average spatial resolution over all 69 sites was at 20ms after the onset of pre-excitation within 3.5±0.9mm. The results provide theoretical evidence that statistical analysis of BSPMs obtained during pace mapping can offer improved means for subcentimetre identification of accessory pathways located along the AV ring.     

Cyclic strain anisotropy regulates valvular interstitial cell phenotype and tissue remodeling in three-dimensional culture

Many planar connective tissues exhibit complex anisotropic matrix fiber arrangements that are critical to their biomechanical function. This organized structure is created and modified by resident fibroblasts in response to mechanical forces in their environment. The directionality of applied strain fields changes dramatically during development, aging, and disease, but the specific effect of strain direction on matrix remodeling is less clear. Current mechanobiological inquiry of planar tissues is limited to equibiaxial or uniaxial stretch, which inadequately simulates many in vivo environments. In this study, we implement a novel bioreactor system to demonstrate the unique effect of controlled anisotropic strain on fibroblast behavior in three-dimensional (3-D) engineered tissue environments, using aortic valve interstitial fibroblast cells as a model system. Cell seeded 3-D collagen hydrogels were subjected to cyclic anisotropic strain profiles maintained at constant areal strain magnitude for up to 96 h at 1 Hz. Increasing anisotropy of biaxial strain resulted in increased cellular orientation and collagen fiber alignment along the principal directions of strain and cell orientation was found to precede fiber reorganization. Cellular proliferation and apoptosis were both significantly enhanced under increasing biaxial strain anisotropy (P<0.05). While cyclic strain reduced both vimentin and alpha-smooth muscle actin compared to unstrained controls, vimentin and alpha-smooth muscle actin expression increased with strain anisotropy and correlated with direction (P<0.05). Collectively, these results suggest that strain field anisotropy is an independent regulator of fibroblast cell phenotype, turnover, and matrix reorganization, which may inform normal and pathological remodeling in soft tissues.     

A-Magnetic Optic-Mechanical Device to Quantify Finger Kinematics for fMRI Studies of Bimanual Coordination

Several fMRI studies have been performed to detect the neural correlates of stable bimanual coordination patterns in humans. Only few of those studies were accompanied by the on-line recording of the relative phase of fingers or hands, but none with high space and time resolutions. Conversely, the high-resolution recording of fingers’ kinematics during fMRI would permit the quantification of the instantaneous fingers’ positions, from which the instant at which transitions between different bimanual coordination patterns occur might be detected. This information could then be used to analyze fMRI data and detect the neural correlates of pattern transitions. We describe an a-magnetic optic-mechanical device (AMOMeD) able to monitor the fingers’ positions during fMRI studies on bimanual coordination with 2 mm space resolution and 1 ms time resolution. From the instantaneous fingers’ positions (recorded with an optical fiber system and a dedicated acquisition system), the oscillation amplitude, frequency, velocity and relative phase of fingers’ are calculated. The signal from the fMRI trigger can be acquired simultaneously to synchronize the behavioral outcomes with the fMRI analysis. The results of our study show that this device does not affect fMRI signals, and that fMRI data can be processed using the simultaneous behavioral information to detect the brain areas activated during the transitions between different bimanual coordination patterns.     

Molecular orientation of collagen in intact planar connective tissues under biaxial stretch

Understanding of the mechanical behavior of collagenous tissues at different size scales is necessary to understand their physiological function as well as to guide their use as heterograft biomaterials. We conducted a first investigation of the kinematics of collagen at the molecular and fiber levels under biaxial stretch in an intact planar collagenous tissue. A synchrotron small angle X-ray scattering (SAXS) technique combined with a custom biaxial stretching apparatus was used. Collagen fiber behavior under biaxial stretch was then studied with the same specimens using small angle light scattering (SALS) under identical biaxial stretch states. Both native and glutaraldehyde modified bovine pericardium were investigated to explore the effects of chemical modification to collagen. Results indicated that collagen fiber and molecular orientation did not change under equibiaxial strain, but were observed to profoundly change under uniaxial stretch. Interestingly, collagen molecular strain initiated only after approximately 15% global tissue strain, potentially due to fiber-level reorganization occurring prior to collagen molecule loading. Glutaraldehyde treatment also did not affect collagen molecular strain behavior, indicating that chemical fixation does not alter intrinsic collagen molecular stiffness. No detectable changes in the angular distribution and D-period strain were found after 80 min of stress relaxation. It can be speculated that other mechanisms may be responsible for the reduction in stress with time under biaxial stretch. The results of this first study suggest that collagen fiber/molecular kinematics under biaxial stretch are more complex than under uniaxial deformation, and warrant future studies.     

REST-PEEP: reduced scan time phase-encoded echo planar imaging

Echo planar methodology can be used for rapid acquisition in three dimensions of k-space. There are two groups of sequences which have thus far been implemented. The first group is characterized by three-dimensional acquisition from a single RF excitation. Echo planar shift mapping and echo volume imaging are single-shot chemical shift imaging and three-dimensional spatial imaging techniques, respectively. Even though these methods are extremely fast, their spatial and spectral resolutions are poor. In the second group of sequences, echo planar imaging acquisition is repeated for every phase-encode gradient step to improve upon the spatial resolution and signal-to-noise ratio at the expense of acquisition time. Here we report a novel combination of these two groups of sequences aimed at providing additional flexibility in trade-offs between the spectral bandwidth, signal-to-noise ratio, spatial resolution and imaging time. Preliminary results for both chemical shift imaging and three-dimensional spatial imaging are presented. The chemical shift imaging was optimized for excitation of the N-acetyl aspartate (NAA). An interesting consequence of our approach is that the Nyquist ghost is transferred from the spectrum to the metabolite image.     

Three-Dimensional Quantitative Micromorphology of Pre- and Post-Implanted Engineered Heart Valve Tissues

There is a significant gap in our knowledge of engineered heart valve tissue (EHVT) development regarding detailed three-dimensional (3D) tissue formation and remodeling from the point of in vitro culturing to full in vivo function. As a step toward understanding the complexities of EHVT formation and remodeling, a novel serial confocal microscopy technique was employed to obtain 3D microstructural information of pre-implant (PRI) and post-implant for 12 weeks (POI) EHVT fabricated from PGA:PLLA scaffolds and seeded with ovine bone-marrow-derived mesenchymal stem cells. Custom scaffold fiber tracking software was developed to quantify scaffold fiber architectural features such as length, tortuosity, and minimum scaffold fiber–fiber separation distance and scaffold fiber orientation was quantified utilizing a 3D fabric tensor. In addition, collagen and cellular density of ovine pulmonary valve leaflet tissue were also analyzed for baseline comparisons. Results indicated that in the unseeded state, scaffold fibers formed a continuous, oriented network. In the PRI state, the scaffold showed some fragmentation with a scaffold volume fraction of 7.79%. In the POI specimen, the scaffold became highly fragmented, forming a randomly distributed short fibrous network (volume fraction of 2.03%) within a contiguous, dense collagenous matrix. Both PGA and PLLA scaffold fibers were observed in the PRI and POI specimens. Collagen density remained similar in both PRI and POI specimens (74.2 and 71.5%, respectively), though the distributions in the transmural direction appeared slightly more homogenous in the POI specimen. Finally, to guide future 2D histological studies for large-scale studies (since acquisition of high-resolution volumetric data is not practical for all specimens), we investigated changes in relevant collagen and scaffold metrics (collagen density and scaffold fiber orientation) with varying section spacing. It was found that a sectioning spacing up to 25 μm (for scaffold morphology) and 50 μm (for collagen density) in both PRI and POI tissues did not result in loss of information fidelity, and that sectioning in the circumferential or radial direction provides the greatest preservation of information. This is the first known work to investigate EHVT microstructure over a large volume with high resolution and to investigate time evolving in vivo EHVT morphology. The important scaffold fiber structural changes observed provide morphological information crucial for guiding future structurally based constitutive modeling efforts focused on better understanding EHVT tissue formation and remodeling.     

Quantification of vertical-fiber defect in cattle hide by small-angle light scattering.

Vertical-fiber defect (VFD), an abnormal arrangement of collagen fibers in hides of certain cattle breeds, is still not fully understood. Prior work has been limited to subjective histological examinations from hide biopsies. A device using small angle light scattering (SALS) was used to quantify the collagen fiber orientation of sections taken from hide biopsies. Sections were chosen from the Hereford cattle breed and classified by conventional observation as belonging to either the normal, intermediate, or vertical phenotypes. The vertical fibers occur only in the upper reticular dermis, with the fibers in the lower reticular dermis lying parallel to the plane of the hide in all phenotypes. By SALS the vertical phenotype was found to be significantly different from the normal phenotype, whilst the intermediate phenotype was found to be structurally indistinguishable from the vertical one. No evidence was found for the existence of other phenotypes.     

Characteristics of a charged-coupled-device-based optical mapping system for the study of cardiac arrhythmias

We develop an optical fluorescent mapping system that is able to record the action potential wavefront propagation within cardiac tissue samples with high spatial and temporal resolutions. The system's main component, the fluorescence acquisition device (customized CCD camera), offers a high spatial resolution of 128 x 128 pixels, with 12-bit digitization and a frame rate of 490 frames/s. The system is designed and implemented to image an area of approximately 20 x 20 mm at its minimum object distance of 140 mm, corresponding to a spatial resolution of approximately 3 line pairs/mm. Experiments using this system with di-4-ANEPPS-stained canine cardiac tissues with stimulated action potentials through external electrodes result in successful mappings of the distribution and propagation of the action potential wavefronts, showing the system's sensitivity to the change in fluorescence intensity in regions of action potentials. These data demonstrate this optical mapping system as a powerful device in the study of cardiac arrhythmia mechanisms.     

Reconstruction of Cardiac Ventricular Geometry and Fiber Orientation Using Magnetic Resonance Imaging

An imaging method for the rapid reconstruction of fiber orientation throughout the cardiac ventricles is described. In this method, gradient-recalled acquisition in the steady-state (GRASS) imaging is used to measure ventricular geometry in formaldehyde-fixed hearts at high spatial resolution. Diffusion-tensor magnetic resonance imaging (DTMRI) is then used to estimate fiber orientation as the principle eigenvector of the diffusion tensor measured at each image voxel in these same hearts. DTMRI-based estimates of fiber orientation in formaldehyde-fixed tissue are shown to agree closely with those measured using histological techniques, and evidence is presented suggesting that diffusion tensor tertiary eigenvectors may specify the orientation of ventricular laminar sheets. Using a semiautomated software tool called HEARTWORKS, a set of smooth contours approximating the epicardial and endocardial boundaries in each GRASS short-axis section are estimated. These contours are then interconnected to form a volumetric model of the cardiac ventricles. DTMRI-based estimates of fiber orientation are interpolated into these volumetric models, yielding reconstructions of cardiac ventricular fiber orientation based on at least an order of magnitude more sampling points than can be obtained using manual reconstruction methods. © 2000 Biomedical Engineering Society. PAC00: 8761-c, 8757Gg     

A combined solution exchange/plunge-freezing device for skinned muscle fibers

For many contractility studies, defined functional states of skinned muscle fiber preparations can be introduced by application of standardized perfusion protocols with large varieties of experimental solutions. Functionally important subcellular element distributions in the myoplasm and in the sarcoplasmic reticulum can be measured with high spatial resolution by electron microscopic microanalysis. Capturing these subcellular ion distributions requires their rapid immobilization by quick-freezing. We therefore combined a plunge-freezing device with a semiautomatic solution exchanger to reproducibly perfuse skinned muscle fiber bundles with multiple solutions. The isometric tension produced is simultaneously recorded as an indicator for the functional state. The samples can be quick-frozen at any chosen time of the tension transient. A cryoglueing technique finally delivers specimens suitable for cryoultramicrotomy.     

Local elasticity imaging of vascular tissues using a tactile mapping system

This study aimed to map the elasticity of a natural artery at the micron level by using a tactile mapping system (TMS) that was recently developed for characterization of the stiffness of tissue slices. The sample used was a circumferential section (thickness, approximately 1 mm) of a small-caliber porcine artery (diameter, approximately 3 mm). Elasticity was measured with a probe of diameter 1 μm and a spatial resolution of 2 μm at a rate of 0.3 s per point, without significant sample invasion. Topographical measurements were also performed simultaneously. Wavy regions of high elasticity, layered in the circumferential direction, were measured at the tunica media, which was identified as an elastin-rich region. The Young’s modulus of the elastin-rich region in the media was 50.8 ± 13.8 kPa, and that of the elastin-rich region of the lamina elastica interna was 69.0 ± 12.8 kPa. Both these values were higher than the Young’s modulus of the other regions in the media, including smooth muscle cells and collagen fibrils (17.0 ± 9.0 kPa). TMS is simple and inexpensive to perform and allows observation of the distribution of the surface elastic modulus at the extracellular matrix level in vascular tissue. TMS is expected to be a powerful tool in evaluation of the maturation and degree of reconstruction in the development of tissue-engineered or artificial tissues and organs.     

Novel multi-sided,microelectrode arrays for implantable neural applications

A new parylene-based microfabrication process is presented for neural recording and drug delivery applications. We introduce a large design space for electrode placement and structural flexibility with a six mask process. By using chemical mechanical polishing, electrode sites may be created top-side, back-side, or on the edge of the device having three exposed sides. Added surface area was achieved on the exposed edge through electroplating. Poly(3,4-ethylenedioxythiophene) (PEDOT) modified edge electrodes having an 85-μm² footprint resulted in an impedance of 200 kΩ at 1 kHz. Edge electrodes were able to successfully record single unit activity in acute animal studies. A finite element model of planar and edge electrodes relative to neuron position reveals that edge electrodes should be beneficial for increasing the volume of tissue being sampled in recording applications.     

Detection of sperm within Gyrodactylus (Platyhelminthes, Monogenea) tissues using fluorescence and transmission electron microscopy

A rapid fluorescent staining method demonstrating spermatozoa within gyrodactylid monogeneans is described. Gyrodactylids fixed and stained in 2% acetic acid containing 1 μg ml⁻¹ bisBenzimide (Hoechst 33258) were viewed using epifluorescence microscopy. In addition to staining sperm in the testis, seminal vesicle and seminal receptacle, the technique highlighted sperm in the gut and cytoplasmic lining of the uterus, locations from which they had not previously been recorded. The technique was more rapid than transmission electron microscopy (TEM), which was used to confirm the observations. Fluorescence microscopy provided an overview of sperm orientation and distribution that could otherwise be obtained only from serial ultrathin sections. Using the fluorescence technique and/or TEM we have located migrating sperm in the tissues of Gyrodactylus turnbulli, G. bullatarudis and G. gasterostei, although these sperm do not appear to enter the tissues of embryos in utero. The technique can be used to study insemination patterns in gyrodactylid populations and in experimental studies of gyrodactylid reproduction. Received: 20 September 1996 / Accepted: 15 January 1997     

Surface elasticity imaging of vascular tissues in a liquid environment by a scanning haptic microscope

The objective of this study was to make an elasticity distribution image of natural arteries in a liquid environment at high resolution at the micrometer level and at a wide area at the sub-square millimeter level by improving the scanning haptic microscope (SHM), developed previously for characterization of the stiffness of natural tissues. The circumferential sections (thickness, 1.0 mm) of small-caliber porcine arteries (approximately 3-mm diameter) were used as a sample. Measurement was performed by soaking a probe (diameter, 5 μm; spatial resolution, less than 2 μm) in saline solution at an appropriate depth. The vascular tissues were segregated by multi-layering a high elasticity region with mainly elastin (50.8 ± 13.8 kPa) and a low one with mainly collagen and smooth muscle cells (17.0 ± 9.0 kPa), as observed previously in high humidity conditions. The elasticity was measured repeatedly with little change for over 4 h in a liquid environment, which enabled observation with maintenance of high precision of a large area of at least 1,200 × 100 μm, whereas the elasticity was increased with time by the dehydration of samples with shrinkage in the air, in which an averaged elasticity in the overall area was approximately doubled within 2 h. This simple, inexpensive system allows observation of the distribution of the surface elasticity at the extracellular matrix level of vascular tissues in a liquid environment close to the natural one.     

Video-rate two-photon imaging of mouse footpad – a promising model for studying leukocyte recruitment dynamics during inflammation

Leukocyte recruitment is a key host defense mechanism to infection and a salient feature of autoimmune diseases such as arthritis. The cell dynamics of these processes are difficult to study due to the challenge of tracking cells flowing in the circulation and migrating through light scattering tissues. Here, we describe a noninvasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. In the absence of inflammation, cells moved > several hundred μm/s in vessels and only rarely adhered to endothelium or entered the tissue parenchyma. In response to bacterial infection, neutrophils moved in small capillaries at reduced speeds of (14–45μm/min) and rolled in larger vessels at 5–60 μm/min. Within minutes of adoptive transfer, neutrophils entered the connective tissue and crawled with a median velocity of 7.3 μm/min. 2P imaging has excellent spatiotemporal resolution and is a promising in vivo approach to study the cellular basis of inflammation. Electronic supplementary material The online version of this article (DOI: ) contains supplementary material, which is available to authorized users. Received 23 October 2007; accepted by M. Parnham 25 October 2007     

Dielectrophoresis assisted concentration of micro-particles and their rapid quantitation based on optical means

The detection and counting of micro particles having sizes comparable to biological entities can provide a tremendous impetus to rapid diagnostics and clinical applications. MEMS technology has already been used in capture and detection of such micron size entities in miniscule concentrations. For this purpose a concentration step is normally added prior to the detection process. A variety of methodologies are used for quantization of such micron size particles/entities including change in permittivity, medium impedance, magnetic permeability and other means. Although optical studies have been extensively performed prior to this, it has not been used for quantization of the micro particles. We have designed, developed and characterized a MEMS counter which captures micron size fluorescent beads using delectrophoresis (DEP) and monitors their accumulation in a 12 μm × 230 μm size channel and monitors this accumulation as growth of overall fluorescence. The field is generated by a set of finely placed interdigitated microelectrodes. As we apply an alternating voltage at 10 V_pp for a range of different frequencies we are able to capture the flowing beads and concentrate them by several orders of magnitude. This is also followed by their quantification in terms of growing fluorescence signal. For quantitating the fluorescence values a CCD (charge couple device) module fitted over an inverted fluorescence microscope is used that visualizes the whole capture process and a Labview based image acquisition software simultaneously calculates the signal intensity over these frames and arranges it temporally. Our work will have tremendous utility in developing a rapid bacterial counting procedure and will be a valuable tool in microbiological laboratories.     

Intraluminal recording of cross-sectional blood velocity distribution of human ascending aorta by ultrasound Doppler technique

A pulsed Doppler ultrasound technique was used for mapping two-dimensional blood velocity profiles in the human ascending aorta during open-heart surgery. An electronic position-sensitive device was constructed and linked to an intraluminal 10 MHz Doppler ultrasound probe. From a plane perpendicular to the central direction of blood flow, velocity mapping was performed covering the entire cross-section of the ascending aorta 6–7 cm above the valve. This method is based on a sequential sampling of velocity from continuously changing locations during a stable haemodynamic period; typically velocity points are recorded from 150–300 beats. Further processing transformed data to suit a previously developed velocity distribution model for normal blood flow in the human ascending aorta, based on multiregression analyses. In this model, the time series of data from consecutive beats were computed into an average two-dimensional profile described through one cardiac cycle. This method allows high spatial resolution (1.5 mm), in addition to the high-frequency response (200 Hz) of the modified ultrasound Doppler meter. Together with the advantage of velocity directionality and minimal time interventions, this makes the method well suited for studies on normal flow conditions as well as flow velocity distribution distal to different heart valve prostheses.     

Quantitative architectural analysis: a new approach to cortical mapping

Recent progress in anatomical and functional MRI has revived the demand for a reliable, topographic map of the human cerebral cortex. Till date, interpretations of specific activations found in functional imaging studies and their topographical analysis in a spatial reference system are, often, still based on classical architectonic maps. The most commonly used reference atlas is that of Brodmann and his successors, despite its severe inherent drawbacks. One obvious weakness in traditional, architectural mapping is the subjective nature of localising borders between cortical areas, by means of a purely visual, microscopical examination of histological specimens. To overcome this limitation, more objective, quantitative mapping procedures have been established in the past years. The quantification of the neocortical, laminar pattern by defining intensity line profiles across the cortical layers, has a long tradition. During the last years, this method has been extended to enable a reliable, reproducible mapping of the cortex based on image analysis and multivariate statistics. Methodological approaches to such algorithm-based, cortical mapping were published for various architectural modalities. In our contribution, principles of algorithm-based mapping are described for cyto- and receptorarchitecture. In a cytoarchitectural parcellation of the human auditory cortex, using a sliding window procedure, the classical areal pattern of the human superior temporal gyrus was modified by a replacing of Brodmann’s areas 41, 42, 22 and parts of area 21, with a novel, more detailed map. An extension and optimisation of the sliding window procedure to the specific requirements of receptorarchitectonic mapping, is also described using the macaque central sulcus and adjacent superior parietal lobule as a second, biologically independent example. Algorithm-based mapping procedures, however, are not limited to these two architectural modalities, but can be applied to all images in which a laminar cortical pattern can be detected and quantified, e.g. myeloarchitectonic and in vivo high resolution MR imaging. Defining cortical borders, based on changes in cortical lamination in high resolution, in vivo structural MR images will result in a rapid increase of our knowledge on the structural parcellation of the human cerebral cortex.