Name: three-dimensional echocardiographic method for the visualization and assessment of specific parameters of the pulmonary veins
Uploaded: 2020-10-28T14:00:00
Description: Watch this Scientific Journal Video about Three-Dimensional Echocardiographic Method for the Visualization and Assessment of Specific Parameters of the Pulmonary Veins at JoVE.com

This work was funded by the Hungarian Government Research Fund [GINOP-2.3.2-15-2016-00043, Sz&#237;v- &#233;s &#233;rkutat&#225;si kiv&#225;l&#243;s&#225;gk&#246;zpont (IRONHEART)].

All the patients signed informed consent before examination according to approval of the local ethical committee (OGY&#201;I/12743/2018).
1. Preparation
<ol>
	<li>Start the examination with patient preparation: ensuring at least a 4-hour fasting status, questionnaire about problems with swallowing and known upper gastrointestinal diseases.</li>
	<li>Ensure that written informed consent is read and signed.</li>
	<li>Prepare an intravenous line before the examination.</li>
	<li>Position the pa...

<ol>
	<li>Altinkaynak, D., Koktener, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+pulmonary+venous+variations+in+a+large+cohort:+Multidetector+computed+tomography+study+with+new+variations.">Evaluation of pulmonary venous variations in a large cohort: Multidetector computed tomography study with new variations.</a> Wiener klinische Wochenschrift. 131 (19-20), 475-484 (2019).</li><li>Haissaguerre, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+initiation+of+atrial+fibrillation+by+ectopic+beats+originating+in+the+pulmonary+veins.">Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins.</a> New England Journal of Medicine. 339 (10), 659-666 (1998).</li><li>Nault, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+vs.+ablation+for+the+treatment+of+atrial+fibrillation:+the+evidence+supporting+catheter+ablation.">Drugs vs. ablation for the treatment of atrial fibrillation: the evidence supporting catheter ablation.</a> European Heart Journal. 31 (9), 1046-1054 (2010).</li><li>Calkins, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HRS/EHRA/ECAS+expert+consensus+statement+on+catheter+and+surgical+ablation+of+atrial+fibrillation:+recommendations+for+patient+selection,+procedural+techniques,+patient+management+and+follow-up,+definitions,+endpoints,+and+research+trial+design:+a+report+of+the+Heart+Rhythm+Society+(HRS)+Task+Force+on+Catheter+and+Surgical+Ablation+of+Atrial+Fibrillation.">HRS/EHRA/ECAS expert consensus statement on catheter and surgical ablation of atrial fibrillation: recommendations for patient selection, procedural techniques, patient management and follow-up, definitions, endpoints, and research trial design: a report of the Heart Rhythm Society (HRS) Task Force on Catheter and Surgical Ablation of Atrial Fibrillation.</a> Heart Rhythm. 9 (4), 632-696 (2012).</li><li>Kojodjojo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+venous+isolation+by+antral+ablation+with+a+large+cryoballoon+for+treatment+of+paroxysmal+and+persistent+atrial+fibrillation:+medium-term+outcomes+and+non-randomised+comparison+with+pulmonary+venous+isolation+by+radiofrequency+ablation.">Pulmonary venous isolation by antral ablation with a large cryoballoon for treatment of paroxysmal and persistent atrial fibrillation: medium-term outcomes and non-randomised comparison with pulmonary venous isolation by radiofrequency ablation.</a> Heart. 96 (17), 1379-1384 (2010).</li><li>Packer, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoballoon+ablation+of+pulmonary+veins+for+paroxysmal+atrial+fibrillation:+first+results+of+the+North+American+Arctic+Front+(STOP+AF)+pivotal+trial.">Cryoballoon ablation of pulmonary veins for paroxysmal atrial fibrillation: first results of the North American Arctic Front (STOP AF) pivotal trial.</a> Journal of the American College of Cardiology. 61 (16), 1713-1723 (2013).</li><li>Kuck, K., Brugada, J., Albenque, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoballoon+or+Radiofrequency+Ablation+for+Atrial+Fibrillation.">Cryoballoon or Radiofrequency Ablation for Atrial Fibrillation.</a> New England Journal of Medicine. 375 (11), 1100-1101 (2016).</li><li>Knecht, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+predictors+for+acute+and+mid-term+success+of+cryoballoon+ablation+of+atrial+fibrillation+using+the+28+mm+balloon.">Anatomical predictors for acute and mid-term success of cryoballoon ablation of atrial fibrillation using the 28 mm balloon.</a> Journal of Cardiovascular Electrophysiology. 24 (2), 132-138 (2013).</li><li>Cabrera, J. A., Ho, S. Y., Climent, V., Sanchez-Quintana, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+architecture+of+the+left+lateral+atrial+wall:+a+particular+anatomic+region+with+implications+for+ablation+of+atrial+fibrillation.">The architecture of the left lateral atrial wall: a particular anatomic region with implications for ablation of atrial fibrillation.</a> European Heart Journal. 29 (3), 356-362 (2008).</li><li>Kubala, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+pulmonary+veins+anatomy+is+associated+with+better+AF-free+survival+after+cryoablation+as+compared+to+atypical+anatomy+with+common+left+pulmonary+vein.">Normal pulmonary veins anatomy is associated with better AF-free survival after cryoablation as compared to atypical anatomy with common left pulmonary vein.</a> Pacing and Clinical Electrophysiology. 34 (7), 837-843 (2011).</li><li>Guler, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Pulmonary+Vein+Anatomy+and+Pulmonary+Vein+Diameters+on+Outcome+of+Cryoballoon+Catheter+Ablation+for+Atrial+Fibrillation.">Effect of Pulmonary Vein Anatomy and Pulmonary Vein Diameters on Outcome of Cryoballoon Catheter Ablation for Atrial Fibrillation.</a> Pacing and Clinical Electrophysiology. 38 (8), 989-996 (2015).</li><li>Baran, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+pulmonary+vein+ostia+anatomy+on+efficacy+of+cryoballoon+ablation+for+atrial+fibrillation.">Impact of pulmonary vein ostia anatomy on efficacy of cryoballoon ablation for atrial fibrillation.</a> Heart Beat Journal. 1, 65-70 (2017).</li><li>Sorgente, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+vein+ostium+shape+and+orientation+as+possible+predictors+of+occlusion+in+patients+with+drug-refractory+paroxysmal+atrial+fibrillation+undergoing+cryoballoon+ablation.">Pulmonary vein ostium shape and orientation as possible predictors of occlusion in patients with drug-refractory paroxysmal atrial fibrillation undergoing cryoballoon ablation.</a> Europace. 13 (2), 205-212 (2011).</li><li>Chun, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'single+big+cryoballoon'+technique+for+acute+pulmonary+vein+isolation+in+patients+with+paroxysmal+atrial+fibrillation:+a+prospective+observational+single+centre+study.">The 'single big cryoballoon' technique for acute pulmonary vein isolation in patients with paroxysmal atrial fibrillation: a prospective observational single centre study.</a> European Heart Journal. 30 (6), 699-709 (2009).</li><li>Cabrera, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+evidence+of+muscular+connections+between+contiguous+pulmonary+venous+orifices:+relevance+of+the+interpulmonary+isthmus+for+catheter+ablation+in+atrial+fibrillation.">Morphological evidence of muscular connections between contiguous pulmonary venous orifices: relevance of the interpulmonary isthmus for catheter ablation in atrial fibrillation.</a> Heart Rhythm. 6 (8), 1192-1198 (2009).</li><li>McLellan, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+vein+isolation:+the+impact+of+pulmonary+venous+anatomy+on+long-term+outcome+of+catheter+ablation+for+paroxysmal+atrial+fibrillation.">Pulmonary vein isolation: the impact of pulmonary venous anatomy on long-term outcome of catheter ablation for paroxysmal atrial fibrillation.</a> Heart Rhythm. 11 (4), 549-556 (2014).</li><li>Mansour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+anatomy+of+the+left+atrium+by+magnetic+resonance+angiography:+implications+for+catheter+ablation+for+atrial+fibrillation.">Three-dimensional anatomy of the left atrium by magnetic resonance angiography: implications for catheter ablation for atrial fibrillation.</a> Journal of Cardiovascular Electrophysiology. 17 (7), 719-723 (2006).</li><li>Ottaviano, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoballoon+ablation+for+atrial+fibrillation+guided+by+real-time+three-dimensional+transoesophageal+echocardiography:+a+feasibility+study.">Cryoballoon ablation for atrial fibrillation guided by real-time three-dimensional transoesophageal echocardiography: a feasibility study.</a> Europace. 15 (7), 944-950 (2013).</li><li>Faletra, F. F., Regoli, F., Acena, M., Auricchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Value+of+real-time+transesophageal+3-dimensional+echocardiography+in+guiding+ablation+of+isthmus-dependent+atrial+flutter+and+pulmonary+vein+isolation.">Value of real-time transesophageal 3-dimensional echocardiography in guiding ablation of isthmus-dependent atrial flutter and pulmonary vein isolation.</a> Circulation Journal. 76 (1), 5-14 (2012).</li><li>Coulombe, N., Paulin, J., Su, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+in+vivo+performance+of+second-generation+cryoballoon+for+pulmonary+vein+isolation.">Improved in vivo performance of second-generation cryoballoon for pulmonary vein isolation.</a> Journal of Cardiovascular Electrophysiology. 24 (8), 919-925 (2013).</li></ol>

Here, we demonstrate a step-by-step methodology to study the PVs, their surrounding structures and anatomical characteristics with 3D echocardiography. The above described method for 3D imaging of the PVs is an easily standardizable method, which provides high quality 3D images in most patients suitable for precise measurements. Optimal quality and orientation of the acquired images are paramount for the appropriate assessment of PV anatomy. The 3D reconstructed images enhance the visualization of the PV drainage pattern...

The drainage pattern of the pulmonary veins (PV) is highly variable with 56.5% variation in the average population1. Evaluation of the PV drainage pattern is crucial when planning PV isolation (PVI), which is the most common interventional treatment of atrial fibrillation nowadays2,3,4. Although radiofrequency catheter ablation has been the standard technology for achieving PVI, the cryoballoon (CB)-based ablation technology (CA) is an alternative method requiring less procedural time. The technique is less complicated compared with radiofrequency ablation5,6, while the efficacy and safety of CA are similar to those of radiofrequency ablation7.
The rate of procedural PV occlusion by the CB and the continuous circumferential extension of tissue injury in the PV ostium determines the permanent success of PVI after CA. One of the main determinants of PV occlusion is the variation of PV anatomy. In recent, computed tomography- (CT) and cardiac MRI-based studies, several PV parameters were identified with predictive values of short and long term success rates following CA. These parameters included variations of both the PV anatomy (left common PV, supernumerary PVs8,9,10, ostial area, ovality index8,11,12,13) and its surroundings (intervenous ridge8,14,15,16, thickness of left lateral ridge8,9,17).
Although conventional 2D echocardiography is not suitable for displaying and measuring most of the above parameters, three-dimensional transesophageal echocardiography (3D TEE) seems to be an alternative tool to visualize the PVs, as demonstrated in previous literature data18,19.
Furthermore, 3D TEE prior to PVI brings additional value compared to CT or MRI, as it not only provides data on PV characteristics for procedural design, but also clarifies whether a thrombus in the left atrial appendage (LAA) is present. This investigation is especially important prior to PVI. At the same time, 3D TEE requires less time, its procedural cost is low, and it does not expose the patient and the medical staff to radiation.
In the past, several types of CBs existed with different sizes, which made it difficult to extrapolate how the various parameters of the PVs influence the success rate of CA. Today, the newly introduced second-generation CB is used for CA, which only exists in one size. Thanks to its improved cooling effect, the second-generation CB offers a much higher performance compared to the first-generation CB20, which further highlights the importance of PV anatomy and interventional planning before PVI.

<table>
	<tbody>
		<tr>
			<td>4D Cardio-view 3 software</td>
			<td>Tomtec Imaging Systems GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Epiq 7G scanner</td>
			<td>Philips</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Q-Lab Software</td>
			<td>Philips</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X5-1 transducer</td>
			<td>Philips</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional echocardiographic method for the visualization and assessment of specific parameters of the pulmonary veins

The dimensions of the pulmonary veins are important parameters when planning pulmonary vein isolation (PVI), especially with the cryoballoon ablation technique. Acknowledging the dimensions and anatomical variations of the pulmonary veins (PVs) may improve the outcome of the intervention. Conventional 2D transoesophageal echocardiography can only provide limited data about the dimensions of the PVs; however, 3D echocardiography can further evaluate relevant diameters and areas of the PVs, as well as their spatial relationship to surrounding structures. In previous literature data, parameters influencing the success rate of PVI have already been identified. These are the left lateral ridge, the intervenous ridge, the ostial area of the PVs and the ovality index of the ostium. Proper imaging of the PVs by 3D echocardiography is a technically challenging method. One crucial step is the collection of images. Three individual transducer positions are necessary to visualize the important structures; these are the left lateral ridge, the ostium of the PVs and the intervenous ridge of the left and right PVs. Next, 3D images are acquired and saved as digital loops. These datasets are cropped, which result in the en face views displaying spatial relationships. This step can also be employed to determine the anatomical variations of the PVs. Finally, multiplanar reconstructions are created to measure each individual parameter of the PVs.
Optimal quality and orientation of the acquired images are paramount for the appropriate assessment of PV anatomy. In the present work, we examined the 3D visibility of the PVs and the suitability of the above method in 80 patients. The aim was to provide a detailed outline of the essential steps and potential pitfalls of PV visualization and assessment with 3D echocardiography.

Using the above-described image acquisition protocol, the first step is to visualize the left atrial appendage (LAA) using 2D acquisition (Figure 1). The probe is in the upper (or mid) transoesophageal position at 20-45&#176;. The image shows the LAA. The left lateral ridge and the left upper PV is displayed at 60-80&#176; (Figure 2), and then the 3D dataset is acquired and confirmed by cropping the dataset in order to visualize the LAA and the left lateral ridg...

Watch this Scientific Journal Video about Three-Dimensional Echocardiographic Method for the Visualization and Assessment of Specific Parameters of the Pulmonary Veins at JoVE.com

Three-Dimensional Echocardiographic Method for the Visualization and Assessment of Specific Parameters of the Pulmonary Veins

The dimensions of the pulmonary veins (PV) are important parameters when planning pulmonary vein isolation. 2D transoesophageal echocardiography can only provide limited data about the PVs; however, 3D echocardiography can evaluate relevant diameters and areas of the PVs, as well as their spatial relationship to surrounding structures.

The dimensions of the pulmonary veins are important parameters when planning pulmonary vein isolation (PVI), especially with the cryoballoon ablation ...

Acknowledging the dimensions and anatomical variations of the pulmonary veins is important when planning pulmonary vein isolation, which contributes to improving the outcome of the intervention.3D TEE is an alternative tool to visualize the pulmonary veins before pulmonary vein isolation because it can evaluate relevant diameters in areas of the pulmonary veins, and their spatial relationship to surrounding structures.Conventional 2D echocardiography is not suitable for displaying and measuring most of the specific pulmonary veins parameters that affect the pulmonary veins isolation outcome.However, the 3D method can overtake the 2D limitations.3D TEE could replace the CT in the visualization of pulmonary veins with the advantages that it requires less time, the cost is lower and it doesn't expose the patient to radiation.Before beginning the image acquisition, position the patient in a left lateral decubitus position.Set up echocardiographic and oxygen saturation monitoring.For visualization of the left pulmonary veins, insert the probe into the esophagus at approximately 30-40 cm from the front teeth and use 2D image acquisition at 20-45

three-dimensional-echocardiographic-method-for-visualization

Research

JoVE Journal

Bioengineering

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via semiautomatic segmentation, from an in vivo computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 &deg;. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.

A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

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 ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides &#160;mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...