The authors would like to express their gratitude to Mr. Jeroen van den Berg and Mr. Matthijs Wassink from the development mechanics group at UMC Utrecht for their help in wrapping process of the osteochondral plugs. This work was supported by a Grant from Dutch Arthritis Foundation.

NOTE: The protocol presented here is adopted from the experimental and computational procedures of recent research papers 6,8,9. The protocol is illustrated in Figure 1.
The cadaveric materials were collected with permission from veterinary faculty of Utrecht University.
1. Sample and Bath Preparation
<ol>
	<li>Drill out cylindrical osteochondral plugs (diameter of 8.5 mm) from cadaveric equine femoral condyles using custom-made drill bit (Figure 1) while spraying cool Phosphate-buffered Saline&#160;(PBS) to prevent overheating and subsequent cartilage damage.</li>
	<li>Heat-shrink the osteochondral plugs with a plastic shrinking sleeve to minimize the lateral diffusion of the overlaying bath.
	<ol>
		<li>Mount the osteochondral plug with cartilage at the top initially inside the plastic shrinking sleeve and follow by blowing hot air to it. Add wet cotton pieces on the surface of cartilage to prevent heat-related damage.</li>
	</ol>
	</li>
	<li>Prepare finite-baths of charged (e.g.,&#160;650 &#181;L, 420 mM, ioxaglate, Molecular Weight (MW) = 1,269 Da, charge = -1) and neutral (650 &#181;L, 420 mM, iodixanol, MW = 1,550 Da) solutes separately.</li>
	<li>Load the prepared finite-baths on the surface of cartilage using a syringe and place a cork plug on the wrapped sample to prevent evaporation during experiments at RT (Figure 2A). To study the neutral solute transport, place the iodixanol bath and to study the negatively charged solute transport place ioxaglate bath onto the cartilage surface.</li>
</ol>
2. Imaging and Image Processing
<ol>
	<li>Place the wrapped samples isolated with cork plug on a custom-made holder attached to the motorized stage of a micro-CT. Place the sample so that cartilage surface covered with contrast agent solution is facing upward.</li>
	<li>Scan using the micro-CT (voxel size of 40 &#215; 40 &#215; 40 &#181;m3, scan time of 2 min , tube voltage of 90 kV and tube current of 180 &#181;A) a field of view consisting of cartilage, subchondral plate, and finite-bath in several time points until equilibrium state (48 h) is reached (Figure 2A). The equilibrium state is achieved when concentration values do not change over time.</li>
	<li>Register the 3D images in different time points based on the initial image to facilitate positioning of the Region of Interest (ROI) using manufacturer&#39;s software (e.g., Analyze).</li>
	<li>Convert the 3D reconstructed micro-CT images into 2D Tagged Image File Format (TIFF) stack before processing them using manufacturer&#39;s software.</li>
	<li>Globally segment (ImageJ&#62;Adjust&#62;Threshold) the cartilage from the subchondral bone and overlying bath in the software.</li>
	<li>Obtain the average grey value of cartilage at different time points (ImageJ&#62;Analyze&#62;Measure) using the generated cartilage mask in the previous step.</li>
	<li>Given the initial bath concentration and initial contrast agent concentration in the cartilage, use a linear calibration curve to convert the average grey values to the actual concentration of solutes. Previous data support the fact that grey values maintain a linear relationship with the concentration of contrast agents.</li>
	<li>Plot the solute concentrations versus experimental time points.</li>
</ol>
3. Computational Modeling
NOTE: The diffusion in this problem is assumed to take place in 1D (along the z-axis), which complies with the experimental boundary condition. Therefore, the geometry could be arbitrarily created.
<ol>
	<li>Build finite-bath based cartilage multi-zone models: 1) cartilage consisting of superficial zone (20% of the total cartilage thickness), middle zone (50% of the total cartilage thickness) and deep zone (30% of the total cartilage thickness) 12 and 2) finite-bath in FEBio 13,14 (Figure 2B).</li>
	<li>Assign the mechanical and physical properties of different zones of cartilage and bath in FEBio. Young&#39;s modulus (10 MPa) was assumed to be high enough to resist the osmotic pressure exerted by the overlying bath and therefore protect the cartilage from excessive deformations.
	<ol>
		<li>Use a hydraulic permeability of 10-3 mm4/Ns and Poisson&#39;s ratio of 0. Use actual solute diffusion coefficient of the bath in the simulations 8,9.</li>
	</ol>
	</li>
	<li>Generate mesh (8-node trilinear hexahedral elements) and refine it near the boundaries (Figure 2B) 8,9.</li>
	<li>Biphasic-solute model
	<ol>
		<li>Apply initial solute concentration in the bath and effective pressure corresponding to it. Look at the description of effective pressure in 9,15.</li>
		<li>Run the model in transient mode to obtain solute concentration versus time curves according to the prescribed diffusion coefficients in different cartilage zones.</li>
	</ol>
	</li>
	<li>Multiphasic model 
	NOTE: The electric fluctuation between bath and tissue can be circumvented by adding two monovalent counter-ions to both the bath and the tissue.
	<ol>
		<li>For steady-state models: use the same effective fluid pressures and concentrations in cartilage and overlying bath while increasing FCD to its desired value.</li>
		<li>For transient models: create a well-stirred finite-bath by keeping the solute diffusion coefficient in the bath sufficiently high. Inject the solute from the bath-air interface into the bath to reach its desired concentration value.</li>
		<li>Transient: remove the prescribed solute concentration boundary condition in the previous step and revert the diffusion coefficient of the finite-bath to its actual diffusion coefficient.</li>
		<li>Run the model to obtain solute concentration-time curves based on applied FCDs and diffusion coefficients in different cartilage zones.</li>
	</ol>
	</li>
	<li>FEBio-MATLAB interface
	<ol>
		<li>Develop a MATLAB code to automatically perform simulation in FEBio and plot concentration-time curves (FEBio-MATLAB interface) 8,9.</li>
		<li>Change diffusion coefficients and FCDs in cartilage zones using FEBio-MATLAB interface. Run models in FEBio and extract solute concentration-time curves 8,9.</li>
		<li>Compare the obtained solute concentration-time curves with the experimental data and obtain sets of diffusion coefficients and FCDs in different cartilage zones based on minimum root mean square error (RMSE) 8,9.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54984/54984fig1.jpg" /> 
Figure 1: Experimental Setup. A) Sample extraction procedure using a custom-made drill bit. B) Micro-CT imaging procedure to monitor diffusion process. <a href="http://cloudfront.jove.com/files/ftp_upload/54984/54984fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54984/54984fig2.jpg" /> 
Figure 2: Schematic. A) Experimental design. B) Multi-zone computational model consisting of the finite bath, superficial, middle and deep zones of cartilage and associated mesh. <a href="http://cloudfront.jove.com/files/ftp_upload/54984/54984fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose.

We presented an experimental protocol combined with a finite element modeling procedure to study the diffusion of neutral and charged solutes across articular cartilage. According to our recent studies, the proposed models could accurately describe the transport of both neutral (biphasic-solute) and negatively charged (multiphasic) solutes across different zones of articular cartilage 8,9. It is widely believed that articular cartilage becomes functionally limite...

Molecular transport plays a vital role in the homeostasis of articulating joints, delivery of therapeutics to articular cartilage and contrast-enhanced cartilage imaging 1,2,3. Factors such as cartilage integration and intactness, solute charge and size as well as osmolality and concentration of bath in contact with cartilage may influence the transport rate 4,5,6. The transport of solutes, either neutral or charged, can be different between articular cartilage zones, because each zone consists of different concentrations and orientations of major extracellular matrix molecules, namely proteoglycans (PGs) and collagen type II 1,7,8,9,10,11. More importantly, the transport of charged solutes can be highly dependent on the concentration of proteoglycans comprising negative fixed charges within the extracellular matrix which increases across articular cartilage 8,9. Those parameters particularly fixed charge density (FCD), orientation of collagen fibrils and water content variation across cartilage may undergo alterations as osteoarthritis (OA) progresses, thereby signifying the importance of studying diffusion across cartilage.
In the current study, a protocol based on previously established experimental and computational studies 6,8,9 is proposed to accurately investigate diffusion under various boundary conditions using neutral and charged solutes in a finite-bath model of diffusion. The proposed methods are composed of micro-Computed Tomography imaging (micro-CT) of a system including cartilage and a finite-bath supported by advanced biphasic-solute and multiphasic finite element models. These models enable obtaining the diffusion coefficients of neutral and charged molecules as well as FCDs across various zones of articular cartilage. Using these models, one can gain better understanding of the behavior of the diffusing neutral and charged molecules that could be used to investigate the interactions between cartilage and overlaying finite-bath.

<table border="0" cellpadding="0" cellspacing="0" width="688">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hexabrix</td>
			<td style="width:149px;">Guerbet</td>
			<td style="width:123px;">15HX005D</td>
			<td style="width:201px;">Negatively charged contrast agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Visipaque</td>
			<td style="width:149px;">GE healthcare</td>
			<td align="right" style="width:123px;">12570511</td>
			<td>Nuetral contrast agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:149px;">Life technologies</td>
			<td align="right" style="width:123px;">10010023</td>
			<td>Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">micro-CT</td>
			<td style="width:149px;">Perkin Elmer</td>
			<td style="width:123px;"></td>
			<td>Monitoring diffusion</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Freezing-point osmometer</td>
			<td style="width:149px;">Advanced instruments</td>
			<td style="width:123px;"></td>
			<td>Measuring solution osmolality</td>
		</tr>
	</tbody>
</table>

an experimental and finite element protocol to investigate the transport of neutral and charged solutes across articular cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

The representative results provided here are adopted from previous research papers 6,8,9,16.
In OA, articular cartilage undergoes significant changes most importantly GAG loss, and collagen fibril damage 17,18,19</s...

Watch this Scientific Journal Video about An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage at JoVE.com

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

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6.2K Views.  Delft University of Technology (TU Delft). We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods. 

Video: An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points

Light is a versatile and precise means to control neuronal excitability. The recent introduction of light sensitive effectors such as channel-rhodopsin and caged neurotransmitters have led to interests in developing better means to control patterns of light in space and time that are useful for experimental neuroscience. One conventional strategy, employed in confocal and 2-photon microscopy, is to focus light to a diffraction limited spot and then scan that single spot sequentially over the region of interest. This approach becomes problematic if large areas have to be stimulated within a brief time window, a problem more applicable to photostimulation than for imaging. An alternate strategy is to project the complete spatial pattern on the target with the aid of a digital micromirror device (DMD). The DMD approach is appealing because the hardware components are relatively inexpensive and is supported by commercial interests. Because such a system is not available for upright microscopes, we will discuss the critical issues in the construction and operations of such a DMD system. Even though we will be primarily describing the construction of the system for UV photolysis, the modifications for building the much simpler visible light system for optogenetic experiments will also be provided. The UV photolysis system was used to carryout experiments to study a fundamental question in neuroscience, how are spatially distributed inputs integrated across distal dendritic branch points. The results suggest that integration can be non-linear across branch points and the supralinearity is largely mediated by NMDA receptors.

Digital micromirror devices (DMD) can generate complex patterns in time and space with which to control neuronal excitability. Issues relevant to the design, construction, and operation of DMD systems are discussed. Such a system enabled the demonstration of non-linear integration across distal dendritic branch points.

Light is a versatile and precise means to control neuronal excitability. The recent introduction of light sensitive effectors such as channel-rhodopsin ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

A Coupled Experiment-finite Element Modeling Methodology for Assessing High Strain Rate Mechanical Response of Soft Biomaterials

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. brain, liver, tendon, fat, etc.) when exposed to high strain rates. This study utilized a Split-Hopkinson Pressure Bar (SHPB) to generate strain rates of 100-1,500 sec-1. The SHPB employed a striker bar consisting of a viscoelastic material (polycarbonate). A sample of the biomaterial was obtained shortly postmortem and prepared for SHPB testing. The specimen was interposed between the incident and transmitted bars, and the pneumatic components of the SHPB were activated to drive the striker bar toward the incident bar. The resulting impact generated a compressive stress wave (i.e. incident wave) that traveled through the incident bar. When the compressive stress wave reached the end of the incident bar, a portion continued forward through the sample and transmitted bar (i.e. transmitted wave) while another portion reversed through the incident bar as a tensile wave (i.e. reflected wave). These waves were measured using strain gages mounted on the incident and transmitted bars. The true stress-strain behavior of the sample was determined from equations based on wave propagation and dynamic force equilibrium. The experimental stress-strain response was three dimensional in nature because the specimen bulged. As such, the hydrostatic stress (first invariant) was used to generate the stress-strain response. In order to extract the uniaxial (one-dimensional) mechanical response of the tissue, an iterative coupled optimization was performed using experimental results and Finite Element Analysis (FEA), which contained an Internal State Variable (ISV) material model used for the tissue. The ISV material model used in the FE simulations of the experimental setup was iteratively calibrated (i.e. optimized) to the experimental data such that the experiment and FEA strain gage values and first invariant of stresses were in good agreement.

The current study prescribes a coupled experiment-finite element simulation methodology to obtain the uniaxial dynamic mechanical response of soft biomaterials (brain, liver, tendon, fat, etc.). The multiaxial experimental results that arose because of specimen bulging obtained from Split-Hopkinson Pressure Bar testing were rendered to a uniaxial true stress-strain behavior when simulated through iterative optimization of the finite element analysis of the biomaterial.

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application.
The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging.
The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner.
The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for&#160;the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for&#160;investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...