Name: an experimental and finite element protocol to investigate the transport of neutral and charged solutes across articular cartilage
Uploaded: 2017-04-23T13:00:00
Description: 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

De auteurs willen graag hun dank aan de heer Jeroen van den Berg en de heer Matthijs Wassink uit de groep ontwikkeling monteurs aan het UMC Utrecht bedanken voor hun hulp bij het verpakken van proces van de osteochondrale pluggen. Dit werk werd ondersteund door een subsidie ​​van Nederlands Arthritis Foundation.

LET OP: Het protocol hier gepresenteerde is overgenomen uit de experimentele en computationele procedures van de recente research papers 6, 8, 9. Het protocol wordt weergegeven in figuur 1. De kadavers materialen werden verzameld met toestemming van de veterinaire faculteit van de Universiteit Utrecht. 1. Monster bereiding en Bath <ol><li>...

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	<li>Arkill, K. P., Winlove, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solute+transport+in+the+deep+and+calcified+zones+of+articular+cartilage.">Solute transport in the deep and calcified zones of articular cartilage.</a> Osteoarthritis Cartilage. 16 (6), 708-714 (2008).</li><li>Chin, H. C., Moeini, M., Quinn, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solute+transport+across+the+articular+surface+of+injured+cartilage.">Solute transport across the articular surface of injured cartilage.</a> Arch Biochem Biophys. 535 (2), 241-247 (2013).</li><li>Leddy, H. A., Guilak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+effects+of+compression+on+macromolecular+diffusion+in+articular+cartilage.">Site-specific effects of compression on macromolecular diffusion in articular cartilage.</a> Biophys J. 95 (10), 4890-4895 (2008).</li><li>Leddy, H. A., Guilak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-Specific+Molecular+Diffusion+in+Articular+Cartilage+Measured+using+Fluorescence+Recovery+after+Photobleaching.">Site-Specific Molecular Diffusion in Articular Cartilage Measured using Fluorescence Recovery after Photobleaching.</a> Annals of Biomedical Engineering. 31 (7), 753-760 (2003).</li><li>Gu, W. Y., Yao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hydration+and+fixed+charge+density+on+fluid+transport+in+charged+hydrated+soft+tissues.">Effects of hydration and fixed charge density on fluid transport in charged hydrated soft tissues.</a> Ann Biomed Eng. 31 (10), 1162-1170 (2003).</li><li>Pouran, B., Arbabi, V., Zadpoor, A. A., Weinans, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+effects+of+external+bath+osmolality,+solute+concentration,+and+electrical+charge+on+solute+transport+across+articular+cartilage.">Isolated effects of external bath osmolality, solute concentration, and electrical charge on solute transport across articular cartilage.</a> Medical Engineering and Physics. 38 (12), 1399-1407 (2016).</li><li>Kulmala, K. A. 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=Diffusion+coefficients+of+articular+cartilage+for+different+CT+and+MRI+contrast+agents.">Diffusion coefficients of articular cartilage for different CT and MRI contrast agents.</a> Medical Engineering &amp; Physics. 32 (8), 878-882 (2010).</li><li>Arbabi, V., Pouran, B., Weinans, H., Zadpoor, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+Neutral+Solute+Across+Articular+Cartilage:+The+Role+of+Zonal+Diffusivities.">Transport of Neutral Solute Across Articular Cartilage: The Role of Zonal Diffusivities.</a> Journal of Biomechanical Engineering. 137 (7), 071001-071001 (2015).</li><li>Arbabi, V., Pouran, B., Weinans, H., Zadpoor, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphasic+modeling+of+charged+solute+transport+across+articular+cartilage:+Application+of+multi-zone+finite-bath+model.">Multiphasic modeling of charged solute transport across articular cartilage: Application of multi-zone finite-bath model.</a> J Biomech. 49 (9), 1510-1517 (2016).</li><li>Arbabi, V., Pouran, B., Weinans, H., Zadpoor, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+artificial+neural+networks+for+robust+estimation+of+the+diffusion+coefficients+across+cartilage.">Combined artificial neural networks for robust estimation of the diffusion coefficients across cartilage.</a> , (2016).</li><li>Arbabi, V., Pouran, B., Weinans, H., Zadpoor, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+inverse-forward+artificial+neural+networks+for+fast+and+accurate+estimation+of+the+diffusion+coefficients+of+cartilage+based+on+multi-physics+models.">Combined inverse-forward artificial neural networks for fast and accurate estimation of the diffusion coefficients of cartilage based on multi-physics models.</a> Journal of Biomechanics. , (2016).</li><li>Sophia Fox, A. J., Bedi, A., Rodeo, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Basic+Science+of+Articular+Cartilage:+Structure,+Composition,+and+Function.">The Basic Science of Articular Cartilage: Structure, Composition, and Function.</a> Sports Health. 1 (6), 461-468 (2009).</li><li>Ateshian, G., Weiss, J., Holzapfel, G. A., Kuhl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+17.">Ch. 17.</a> Computer Models in Biomechanics. , 231-249 (2013).</li><li>Ateshian, G. A., Maas, S., Weiss, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphasic+Finite+Element+Framework+for+Modeling+Hydrated+Mixtures+With+Multiple+Neutral+and+Charged+Solutes.">Multiphasic Finite Element Framework for Modeling Hydrated Mixtures With Multiple Neutral and Charged Solutes.</a> Journal of Biomechanical Engineering. 135 (11), 111001-111001 (2013).</li><li>Arbabi, V., Pouran, B., Weinans, H., Zadpoor, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+neutral+solute+across+articular+cartilage+and+subchondral+plate.">Transport of neutral solute across articular cartilage and subchondral plate.</a> , (2016).</li><li>Pouran, B., Arbabi, V., Weinans, H., Zadpoor, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+multiphysics+models+to+efficient+design+of+experiments+of+solute+transport+across+articular+cartilage.">Application of multiphysics models to efficient design of experiments of solute transport across articular cartilage.</a> Comput Biol Med. 78, 91-96 (2016).</li><li>Hosseini, S. M., Wu, Y., Ito, K., Donkelaar, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+superficial+collagen+fibrils+for+the+function+of+articular+cartilage.">The importance of superficial collagen fibrils for the function of articular cartilage.</a> Biomechanics and Modeling in Mechanobiology. 13 (1), 41-51 (2013).</li><li>Alexopoulos, L. G., Williams, G. M., Upton, M. L., Setton, L. A., Guilak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+changes+in+the+biphasic+mechanical+properties+of+the+chondrocyte+pericellular+matrix+in+articular+cartilage.">Osteoarthritic changes in the biphasic mechanical properties of the chondrocyte pericellular matrix in articular cartilage.</a> Journal of Biomechanics. 38 (3), 509-517 (2005).</li><li>Felson, D. T., 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=Osteoarthritis:+new+insights.+Part+1:+the+disease+and+its+risk+factors.">Osteoarthritis: new insights. Part 1: the disease and its risk factors.</a> Ann Intern Med. 133 (8), 635-646 (2000).</li><li>Kokkonen, H. T., Jurvelin, J. S., Tiitu, V., Toyras, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+mechanical+injury+of+articular+cartilage+using+contrast+enhanced+computed+tomography.">Detection of mechanical injury of articular cartilage using contrast enhanced computed tomography.</a> Osteoarthritis Cartilage. 19 (3), 295-301 (2011).</li><li>Raya, J. G., 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=Diffusion-tensor+imaging+of+human+articular+cartilage+specimens+with+early+signs+of+cartilage+damage.">Diffusion-tensor imaging of human articular cartilage specimens with early signs of cartilage damage.</a> Radiology. 266 (3), 831-841 (2013).</li><li>Tavakoli Nia, 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=Aggrecan+Nanoscale+Solid-Fluid+Interactions+Are+a+Primary+Determinant+of+Cartilage+Dynamic+Mechanical+Properties.">Aggrecan Nanoscale Solid-Fluid Interactions Are a Primary Determinant of Cartilage Dynamic Mechanical Properties.</a> ACS Nano. 9 (3), 2614-2625 (2015).</li><li>Arbabi, V., Campoli, G., Weinans, H., Zadpoor, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+cartilage+properties+using+indentation+tests,+finite+element+models,+and+artificial+neural+networks.">Estimation of cartilage properties using indentation tests, finite element models, and artificial neural networks.</a> , (2014).</li><li>Arbabi, V., Pouran, B., Campoli, G., Weinans, H., Zadpoor, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+mechanical+and+physical+properties+of+cartilage+by+coupling+poroelastic-based+finite+element+models+of+indentation+with+artificial+neural+networks.">Determination of the mechanical and physical properties of cartilage by coupling poroelastic-based finite element models of indentation with artificial neural networks.</a> Journal of Biomechanics. 49 (5), 631-637 (2016).</li><li>Kokkonen, H. T., 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=Computed+tomography+detects+changes+in+contrast+agent+diffusion+after+collagen+cross-linking+typical+to+natural+aging+of+articular+cartilage.">Computed tomography detects changes in contrast agent diffusion after collagen cross-linking typical to natural aging of articular cartilage.</a> Osteoarthritis and Cartilage. 19 (10), 1190-1198 (2011).</li><li>Decker, S. G., Moeini, M., Chin, H. C., Rosenzweig, D. H., Quinn, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+and+Distribution+of+Fluorescent+Solutes+near+the+Articular+Surface+of+Mechanically+Injured+Cartilage.">Adsorption and Distribution of Fluorescent Solutes near the Articular Surface of Mechanically Injured Cartilage.</a> Biophysical Journal. 105 (10), 2427-2436 (2013).</li><li>Silvast, T. S., Jurvelin, S. J., Tiitu, V., Quinn, T. M., Töyräs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bath+Concentration+of+Anionic+Contrast+Agents+Does+Not+Affect+Their+Diffusion+and+Distribution+in+Articular+cartilage+In+Vitro.">Bath Concentration of Anionic Contrast Agents Does Not Affect Their Diffusion and Distribution in Articular cartilage In Vitro.</a> Cartilage. 4 (1), 42-51 (2013).</li><li>Silvast, T. S., Jurvelin, J. S., Lammi, M. J., T&#246;yr&#228;s, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pQCT+study+on+diffusion+and+equilibrium+distribution+of+iodinated+anionic+contrast+agent+in+human+articular+cartilage+-+associations+to+matrix+composition+and+integrity.">pQCT study on diffusion and equilibrium distribution of iodinated anionic contrast agent in human articular cartilage - associations to matrix composition and integrity.</a> Osteoarthritis and Cartilage. 17 (1), 26-32 (2009).</li><li>Pouran, B., Arbabi, V., Villamar, J., Weinans, H., Zadpoor, A. 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Stelden wij een experimenteel protocol in combinatie met een eindige elementen modellering procedure om de diffusie van neutrale en geladen opgeloste stoffen in gewrichtskraakbeen bestuderen. Volgens de recente studies zou het voorgestelde modellen nauwkeurig te beschrijven het transport van neutrale (bifasische-opgeloste stof) en negatief geladen (meerfasige) opgeloste stoffen in verschillende zones van gewrichtskraakbeen 8, 9. Het wordt algemeen aangenomen dat...

<html> Moleculair transport speelt een cruciale rol bij de homeostase van scharnierende verbindingen, afgifte van therapeutische middelen aan gewrichtskraakbeen en contrast-versterkte beeldvorming kraakbeen 1, 2, 3. Factoren zoals kraakbeen integratie en intactheid, opgeloste lading en grootte alsmede osmolaliteit en concentratie bad in contact met kraakbeen kan de transportsnelheid 4, 5, 6 te beïnvloeden. Het transport van opgeloste stoffen, hetzij neutraal of geladen, kunnen verschillend tussen gewrichtskraakbeen zones, omdat elke zone bestaat uit verschillende concentraties en oriëntaties van grote extracellulaire matrix moleculen, namelijk proteoglycanen (PG) en collageen type II 1, 7, 8, 9,lass = &quot;xref&quot;&gt; 10, 11. Wat nog belangrijker is, kan het transport van geladen opgeloste stoffen in hoge mate afhankelijk van de concentratie van proteoglycanen die negatieve vaste ladingen in de extracellulaire matrix die toeneemt in gewrichtskraakbeen 8, 9 zijn. Die parameters bijzonder vaste ladingsdichtheid (FCD), kan de oriëntatie van collageenfibrillen en het watergehalte variatie tussen kraakbeen veranderingen ondergaan osteoartritis (OA) verloopt, waarbij het belang van het bestuderen diffusie door kraakbeen betekent. In de huidige studie, een protocol gebaseerd op vooraf vastgestelde experimentele en computationele studies 6, 8, 9 voorgesteld om nauwkeurig onderzoeken diffusie onder verschillende randvoorwaarden met neutrale en geladen opgeloste stoffen in een eindig-bad model diffusie. THij voorgestelde methoden bestaan ​​uit micro-CT beeldvorming (micro-CT) van een systeem dat kraakbeen en een eindig-bad met geavanceerde bifasische opgeloste stof en meerfasige eindige elementenmodel. Deze modellen stellen verkrijgen van de diffusiecoëfficiënten van neutrale en geladen moleculen en fcds in verschillende zones van gewrichtskraakbeen. Met deze modellen kan een beter inzicht in het gedrag van de diffunderende neutraal geladen moleculen die kunnen worden gebruikt om de interacties tussen kraakbeen en overlappen eindige-bad onderzoeken krijgen.

<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

<html> Osteoartritis (OA) is een slopende ziekte die is geassocieerd met degeneratie van gewrichtskraakbeen en subchondrale bot. Degeneratie van gewrichtskraakbeen verslechtert de dragende functie vrijwel zoals ervaart enorme chemische afbraak, namelijk proteoglycanverlies en collageenfibril verstoring. Een veelbelovende manier om chemische schade mechanismen te onderzoeken tijdens de OA is aan het kraakbeen monsters bloot te stellen aan een externe opgeloste stof en bewaken van de diffusie van de moleculen. De mate van kraakbeenschade (dwz concentratie en configuratie van essentiële macromoleculen) is toegevoegd botsingsstralingsmodellen energieverlies externe opgeloste stoffen tijdens het bewegen over gewrichtskraakbeen creëert verschillende diffusie-eigenschappen in vergelijking met gezonde kraakbeen. In deze studie introduceren we een protocol, dat bestaat uit meerdere stappen en is gebaseerd op eerder ontwikkelde experimentele micro-C14px; &quot;&gt; omputed T omography (micro-CT) en eindige elementen modellering Het transport van geladen en ongeladen gejodeerde moleculen eerst geregistreerd met behulp van micro-CT, gevolgd door het aanbrengen bifasische opgeloste stof en meerfasige eindige elementenmodel diffusie coëfficiënten te verkrijgen. en vaste ladingsdichtheden in kraakbeen zones.

De representatieve resultaten die hier worden overgenomen uit eerder onderzoek papers 6, 8, 9, 16. OA, kraakbeen ondergaat aanzienlijke veranderingen belangrijker GAG verlies en collageenfibrillen beschadigingen 17, 18, <sup class=...

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

Wij stellen een protocol voor het transport van geladen en ongeladen moleculen over gewrichtskraakbeen met behulp van recent ontwikkelde experimentele en numerieke methoden onderzocht.

Een experimentele en Finite Element Protocol bij het vervoer van neutrale en geladen opgeloste stoffen over gewrichtskraakbeen Onderzoek

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

The overall goal of this procedure it to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of experimental and numerical methods. This method can help answer key questions related to physical properties of articular cartilage, such as diffusion coefficient and fixed charge density in different zones. The main advantage of this technique is that it adopts a finite path, experimental model based on microcomputer tomography of transport of radiopaque molecules.To begin sample preparation, drill out an 8.5 millimeter diameter cylindrical osteochondral plug while spraying room temperature phosphate buffered saline, PBS, onto the drilling area. Insert the plug into an approximately 2.5 centimeter length of heat shrink tubing so that the bone layer is flush with the end, and then cover the cartilage surface with wet cotton. Then use a hot air gun to shrink the tubing around the sides of the plug.Load a finite volume of a 420 millimolar ioxaglate or iodixanol solution onto the cartilage surface inside the tubing to prepare a negatively charged or neutral solute bath respectively. Seal the open end of the tube with a cork. Place the sample with the corked end up on a sample holder mounted on the motorized stage of a micro-CT.Scan over a field of view containing the cartilage, subchondral plate, and finite bath. Repeat the scan periodically over the next 48 hours or until the contrast agent concentration in the cartilage does not change over time. In the instrument software, register the 3-D images at different time points relative to the initial image and convert the reconstructed images to a 2-D TIFF file stack.Import the images into an image processing program and duplicate them to segment bath and subchondral bone separately. Next, holes in the subchondral bone are filled and redundant pixels in the bath are removed manually. The resulting segmented bath and subchondral bone images are then added mathematically.And using the wand tool, the outline of the cartilage mask is defined and added to the Region of Interest Manager. Measure the average gray value of the cartilage. Based on the initial concentrations in the bath and cartilage, convert the average gray values in the bath and cartilage to solute concentration using a linear calibration curve.Plot the solute concentrations over time. To begin the computational modeling of diffusion using FEBio software, build finite bath-based cartilage multi-zone models where the superficial zone comprises 20%of the cartilage thickness, the middle zone represents 50%and the deep zone represents 30%A table containing the information of a neutral solute is created for a Biphasic Solute model. And a table containing the information of charged solutes is created for a Multiphasic model.To model the diffusion of charged solutes with the Multiphasic model, two monovalent counter-ions to both the bath and the cartilage have to be added to avoid the effects of electric fluctuation between the bath and the tissue. Once the cartilage and finite bath zones have been modeled, assign the corresponding mechanical and physical properties, namely, Young's modulus, permeability, free diffusivity, and diffusivity to each zone for the Biphasic Solute model. Assign mechanical and physical properties, namely, Young's modulus, permeability, free diffusivity, and diffusivity, as well as fixed charge density to each zone for the Multiphasic model.Next, generate an eight node trilinear hexahedral element mesh and refine it near the boundaries. To model the diffusion of neutral solutes with the Biphasic Solute model, apply the initial solute concentration and effective pressure to the Finite Bath model. Run the Biphasic Solute model in transient mode to produce solute concentration versus time curves based on the diffusion coefficients for each cartilage zone.For the Multiphasic model in a steady state step, set the same effective fluid pressures and solute concentrations for the cartilage and finite bath while increasing fixed charged density to its desired value. In a transient step, create a well stirred finite bath by keeping the diffusion coefficient in the bath sufficiently high. Inject the solute from the bath air interface into the bath to reach the concentration value.Then remove the prescribed solute concentration boundary condition from the previous step and revert the diffusion coefficient of the finite bath to its actual diffusion coefficient. Run the Multiphasic model using an FEBio MATLAB interface to obtain solute concentration versus time curves based on applied fixed charge densities and the diffusion coefficients for each cartilage zone. Using this method, the diffusion processes of neutral an anionic contrast agents through articular cartilage were modeled with biphasic solute and Multiphasic models respectively.Both models fit well with the experimental data. The diffusion coefficients of the neutral contrast agent, iodixanol, in different cartilage zones were determined from the Biphasic Solute model, and the diffusion coefficients of the anionic contrast agent, ioxaglate, in different zones were determined from the Multiphasic model. After its development, this technique paved the way for the researchers in the field of tissue biomechanics to explore the diffusive properties of soft tissues using a combination of experimental and computational approaches.After watching this video, you should have a good understanding of how to design experimental and computational platforms regarding diffusion across articular cartilage.

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Bioengineering

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.

Patroon fotostimulering met Digital Micromirror Apparaten voor het Dendritische Integratie Onderzoek Across Branch Punten

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.

Bepaling van de Transport Rate van xenobiotica en nanomaterialen via de placenta met de<em&gt; Ex vivo</em&gt; Human placentaperfusie Model

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.

Modellen en methoden om vervoer van Drug Delivery Systems Across Cellular Barrières Evalueer

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.

Human kraakbeenweefsel Fabrication behulp van drie-dimensionale 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 ...

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.

Een combinatie Experiment-eindige elementen modellering Methodologie voor het beoordelen van Hoge Strain Rate mechanische respons van Soft Biomaterialen

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.

3D Hydrogel Stellingen voor Articulaire Chondrocyte Cultuur en Kraakbeen Generation

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.

Een experimenteel Protocol voor de beoordeling van de prestaties van de nieuwe Ultrasound sondes gebaseerd op CMUT technologie in toepassing op Brain Imaging

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.

Creëren van een structureel realistische eindige elementen geometrische Model van een Cardiomyocyte te bestuderen van de rol van cellulaire architectuur in Cardiomyocyte systeembiologie

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

Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively charged aggrecans and, therefore, require improved targeting methods to increase their therapeutic response. Because cartilage has a high negative fixed charge density, drugs can be modified with positively charged drug carriers to take advantage of electrostatic interactions, allowing for enhanced intra-cartilage drug transport. Studying the transport of drug carriers is, therefore, crucial towards predicting the efficacy of drugs in inducing a biological response. We show the design of three experiments which can quantify the equilibrium uptake, depth of penetration and non-equilibrium diffusion rate of cationic peptide carriers in cartilage explants. Equilibrium uptake experiments provide a measure of the solute concentration within the cartilage compared to its surrounding bath, which is useful for predicting the potential of a drug carrier in enhancing therapeutic concentration of drugs in cartilage. Depth of penetration studies using confocal microscopy allow for the visual representation of 1D solute diffusion from the superficial to deep zone of cartilage, which is important for assessing whether solutes reach their matrix and cellular target sites. Non-equilibrium diffusion rate studies using a custom-designed transport chamber enables the measurement of the strength of binding interactions with the tissue matrix by characterizing the diffusion rates of fluorescently labeled solutes across the tissue; this is beneficial for designing carriers of optimal binding strength with cartilage. Together, the results obtained from the three transport experiments provide a guideline for designing optimally charged drug carriers which take advantage of weak and reversible charge interactions for drug delivery applications. These experimental methods can also be applied to evaluate the transport of drugs and drug-drug carrier conjugates. Further, these methods can be adapted for the use in targeting other negatively charged tissues such as meniscus, cornea and the vitreous humor.

This protocol determines equilibrium uptake, depth of penetration and non-equilibrium diffusion rate for cationic peptide carriers in cartilage. Characterization of transport properties is critical for ensuring an effective biological response. These methods can be applied for designing an optimally charged drug carriers for targeting negatively charged tissues.

Karakterisering van intrakraakbeen transport eigenschappen van Cationic Peptide Carriers

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.