A seed grant from the University of Florida - College of Medicine is gratefully acknowledged. Graduate studies (Manuel Salinas) were supported through a minority opportunities in biomedical research programs - research initiative for scientific enhancement (MBRS-RISE) fellowship: NIH/NIGMS R25 GM061347. Financial support from the Wallace H. Coulter Foundation through Florida International University's, Biomedical Engineering Department is also gratefully acknowledged. Finally, the authors thank the following students for their assistance during various stages of the experimental process: Kamau Pier, Malachi Suttle, Kendall Armstrong and Abraham Alfonso.

1. Preparation
<ol>
	<li>Design and fabricate an assembly to accommodate a tri-leaflet valve geometry. This will at minimum include a valve holder to suture-in the valve leaflets&#160;and a tube to house the valve holder and surrounding accessories to secure the assembly onto the pulse duplicator system. In our case, we utilized a commercially available pulse duplicator system available from ViVitro Labs Inc. (Victoria, BC). Valve holder design as well as pre and post assembly configurations are depicted in Fig...

<ol>
	<li>Rajamannan, N. 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=Calcific+aortic+valve+disease:+not+simply+a+degenerative+process:+A+review+and+agenda+for+research+from+the+National+Heart+and+Lung+and+Blood+Institute+Aortic+Stenosis+Working+Group.+Executive+summary:+Calcific+aortic+valve+disease-2011+update.">Calcific aortic valve disease: not simply a degenerative process: A review and agenda for research from the National Heart and Lung and Blood Institute Aortic Stenosis Working Group. Executive summary: Calcific aortic valve disease-2011 update.</a> Circulation. 124, 1783-1791 (2011).</li><li>Marijon, E., Mirabel, M., Celermajer, D. S., Jouven, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheumatic+heart+disease.">Rheumatic heart disease.</a> Lancet. 379, 953-964 (2012).</li><li>Karaci, A. 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=Surgical+treatment+of+infective+valve+endocarditis+in+children+with+congenital+heart+disease.">Surgical treatment of infective valve endocarditis in children with congenital heart disease.</a> J. Card. Surg. 27, 93-98 (2012).</li><li>Knirsch, W., Nadal, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infective+endocarditis+in+congenital+heart+disease.">Infective endocarditis in congenital heart disease.</a> Eur. J. Pediatr. 170, 1111-1127 (2011).</li><li>Korossis, S. A., Fisher, J., Ingham, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+valve+replacement:+a+bioengineering+approach.">Cardiac valve replacement: a bioengineering approach.</a> Biomed. Mater. Eng. 10, 83-124 (2000).</li><li>Ghanbari, 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=Polymeric+heart+valves:+new+materials,+emerging+hopes.">Polymeric heart valves: new materials, emerging hopes.</a> Trends Biotechnol. 27, 359-367 (2009).</li><li>Mol, A., Smits, A. I., Bouten, C. V., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+heart+valves:+advances+and+current+challenges.">Tissue engineering of heart valves: advances and current challenges.</a> Expert Rev. Med. Devices. 6, 259-275 (2009).</li><li>Ramaswamy, 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=The+role+of+organ+level+conditioning+on+the+promotion+of+engineered+heart+valve+tissue+development+in+using+mesenchymal+stem+cells.">The role of organ level conditioning on the promotion of engineered heart valve tissue development in using mesenchymal stem cells.</a> Biomaterials. 31, 1114-1125 (2010).</li><li>Sacks, M. S., Schoen, F. J., Mayer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineering+challenges+for+heart+valve+tissue+engineering.">Bioengineering challenges for heart valve tissue engineering.</a> Annu. Rev. Biomed. Eng. 11, 289-313 (2009).</li><li>Zamorano, J. 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=EAE/ASE+recommendations+for+the+use+of+echocardiography+in+new+transcatheter+interventions+for+valvular+heart+disease.">EAE/ASE recommendations for the use of echocardiography in new transcatheter interventions for valvular heart disease.</a> J. Am. Soc. Echocardiogr. 24, 937-965 (2011).</li><li>ANSI/AAMI/ISO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+Implants+-+Cardiac+Valve+Prostheses.">Cardiovascular Implants - Cardiac Valve Prostheses.</a> Assoc. Adv. Med. Instrum. 71, (2005).</li><li>Gallocher, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Durability Assessment of Polymer Trileaflet Heart Valves PhD thesis. , 313 (2007).</li><li>Carroll, R., Boggs, T., Yamaguchi, H., Al-Mously, F., DeGroff, C., Tran-Son-Tay, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+Cell+Adhesion+on+Polymeric+Heart+Valves.">Blood Cell Adhesion on Polymeric Heart Valves.</a> , (2012).</li><li>Pierre, K. K., Salinas, M., Carroll, R., Landaburo, K., Yamaguchi, H., DeGroff, C., Al-Mousily, F., Bleiweis, M., Ramaswamy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Evaluation+of+a+Novel+Tri-Leaflet+Silicone+Heart+Valve+Prosthesis.">Hydrodynamic Evaluation of a Novel Tri-Leaflet Silicone Heart Valve Prosthesis.</a> , (2012).</li><li>Cacciola, G., Peters, G. W., Schreurs, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+mechanical+analysis+of+a+stentless+fibre-reinforced+aortic+valve+prosthesis.">A three-dimensional mechanical analysis of a stentless fibre-reinforced aortic valve prosthesis.</a> J. Biomech. 33, 521-530 (2000).</li><li>De Hart, J., Cacciola, G., Schreurs, P. J., Peters, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+analysis+of+a+fibre-reinforced+aortic+valve+prosthesis.">A three-dimensional analysis of a fibre-reinforced aortic valve prosthesis.</a> J. Biomech. 31, 629-638 (1998).</li><li>Lim, W. L., Chew, Y. T., Chew, T. C., Low, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsatile+flow+studies+of+a+porcine+bioprosthetic+aortic+valve+in+vitro:+PIV+measurements+and+shear-induced+blood+damage.">Pulsatile flow studies of a porcine bioprosthetic aortic valve in vitro: PIV measurements and shear-induced blood damage.</a> J. Biomech. 34, 1417-1427 (2001).</li><li>Gutierrez, C., Blanchard, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diastolic+heart+failure:+challenges+of+diagnosis+and+treatment.">Diastolic heart failure: challenges of diagnosis and treatment.</a> Am. Fam. Physician. 69, 2609-2616 (2004).</li><li>Shi, Y., Yeo, T. J., Zhao, Y., Hwang, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+image+velocimetry+study+of+pulsatile+flow+in+bi-leaflet+mechanical+heart+valves+with+image+compensation+method.">Particle image velocimetry study of pulsatile flow in bi-leaflet mechanical heart valves with image compensation method.</a> J. Biol. Phys. 32, 531-551 (2006).</li><li>Chandran, K. B., Yoganathan, A. P., Rittgers, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biofluid Mechanics: The Human Circulation. , 277-314 (2007).</li><li>Akins, C. W., Travis, B., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+loss+for+evaluating+heart+valve+performance.">Energy loss for evaluating heart valve performance.</a> J. Thorac. Cardiovasc. Surg. 136, 820-833 (2008).</li><li>Fung, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomechanics: Circulation. , (1997).</li><li>Keener, J., Sneyd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mathematical Physiology, II: Systems Physiology. , (1998).</li><li>Quick, C. M., Berger, D. S., Noordergraaf, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparent+arterial+compliance.">Apparent arterial compliance.</a> Am. J. Physiol. 274, H1393-H1403 (1998).</li><li>Wang, Q., Jaramillo, F., Kato, Y., Pinchuk, L., Schoephoerster, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Evaluation+of+a+Minimally+Invasive+Heart+Valve+in+an+Isolated+Aortic+Root+Using+a+Modified+In+Vitro+Model.">Hydrodynamic Evaluation of a Minimally Invasive Heart Valve in an Isolated Aortic Root Using a Modified In Vitro Model.</a> J. Med. Devices. 3, 011002.1-011002.6 (2009).</li><li>Baldwin, J. T., Campbell, A., Luck, C., Ogilvie, W., Sauter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+dynamics+of+the+CarboMedics+kinetic+bileaflet+prosthetic+heart+valve.">Fluid dynamics of the CarboMedics kinetic bileaflet prosthetic heart valve.</a> Eur. J. Cardiothorac. Surg. 11, 287-292 (1997).</li></ol>

In this study, we have demonstrated the utility of modifying a commercially available pulsatile duplicator unit to accommodate tri-leaflet valve geometries so that hydrodynamic testing of polymer and native porcine valves can be performed. Specifically in our case, the system modified was a ViVitro left heart and systemic simulator system (Figure 1a) controlled via the ViViTest data acquisition system (ViVitro Systems, Inc, Victoria, BC, Canada). However, the system is not unlike several in vitro</em...

Heart valve disease often results from degenerative valve calcification1, rheumatic fever2, endocarditis3,4 or congenital birth defects. When valve damage occurs, causing stenosis and/or regurgitation valve prolapse and cannot be surgically repaired, the native valve is usually replaced by a prosthetic valve. Currently available options include mechanical valves (cage-ball valves, tilting disk valves, etc.), homograft, and bioprosthetic valves (porcine and bovine valves). Mechanical valves are often recommended for younger patients based on their durability; however the patient is required to remain on anticoagulant therapy to prevent thrombotic complications5. Homograft and biological prosthetic valves have been effective choices to avoid blood thinner therapy; however, these valves have elevated risk for fibrosis, calcification, degeneration, and immunogenic complications leading to valve failure6. Tissue-engineered valves are being investigated as an emerging technology7-9, but much still remains to be uncovered. Alternative durable, biocompatible, prosthetic valves are needed to improve the quality of life of the heart valve disease patients. Again, this valve design could replace the bioprosthesis used in transcatheter valve technology, with transcatheter approaches showing the potential for transforming the treatment of selected patients with heart valve disease10.
As stated by current standards, a successful heart valve substitute should have the following performance characteristics: &#34;1) allows forward flow with acceptably small mean pressure difference drop; 2) prevents retrograde flow with acceptably small regurgitation; 3) resists embolization; 4) resists hemolysis; 5) resists thrombus formation; 6) is biocompatible; 7) is compatible with in vivo diagnostic techniques; 8) is deliverable and implantable in the target population; 9) remains fixed once placed; 10) has an acceptable noise level; 11) has reproducible function; 12) maintains its functionality for a reasonable lifetime, consistent with its generic class; 13) maintains its functionality and sterility for a reasonable shelf life prior to implantation.&#34;11. Some of the shortcomings of existing valve prostheses may potentially be overcome by a polymer valve. Biocompatible polymers have been considered top candidates based on biostability, anti-hydrolysis, anti-oxidation, and advantageous mechanical properties such as high strength and viscoelasticity. In particular, elastomeric polymers may provide material deformation resembling native valve dynamics. Elastomers can be tailored to mimic soft tissue properties, and they may be the only artificial materials available that are bio-tolerant and that can withstand the coupled, in vivo, fluid-induced, flexural and tensile stresses, yet, move in a manner resembling healthy, native valve motion. Moreover, elastomers can be mass-produced in a variety of sizes, stored with ease, are expected to be cost-effective devices and can be structurally augmented with fibrous reinforcement.
The concept of the use of polymer materials to assemble a tri-leaflet valve is not new and has been the subject of several research investigations over the last 50 years12, which were abandoned largely due to limited valve durability. However, with the advent of novel manufacturing methodologies13,14, the reinforcement of polymer materials15,16 and potentially seamless integration of polymer valve substitutes with transcatheter valve technology, there has recently been a renewed interest and activity in developing polymer valves as a potentially viable alternative to currently available commercial valves. In this light, a protocol for enabling testing of these valves to assess hydrodynamic functionality is the first step in the evaluation process; yet commercially available pulse simulator systems generally do not come equipped to accommodate tri-leaflet valve designs and contain an annular spacing to insert commercially available heart valves (e.g. tilting disc, bi-leaflet mechanical heart valves). Secondly, polymer valves are an emerging technology whose hydrodynamics can only be assessed in a relative context. Even though native heart valve pressure and flow data is available, it is important to conduct testing of native aortic porcine valves, which are biologically similar to human valves, using the same pulsatile simulator that is used to evaluate the polymer valves so as to account for measurement differences that may be system dependent. Thus, the goal of this study was to demonstrate how a commercially available pulse simulator can be fitted with an assembly to accommodate tri-leaflet valve constructs and to systematically evaluate polymer valve hydrodynamic metrics in a relative context in comparison to mechanical and native porcine heart valve counterparts. In our case, novel tri-leaflet silicone polymer valves previously developed at the University of Florida 13 comprised the polymer valve group.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Pump</td>
			<td>ViVitro Labs</td>
			<td></td>
			<td><a href="http://vivitrolabs.com/products/superpump/" target="_blank">http://vivitrolabs.com/products/superpump/</a></td>
		</tr>
		<tr>
			<td>Flow Meter and Probe</td>
			<td>Carolina Medical</td>
			<td>Model 501D</td>
			<td><a href="http://www.carolinamedicalelectronics.com/documents/FM501.pdf" target="_blank">http://www.carolinamedicalelectronics.com/documents/FM501.pdf</a></td>
		</tr>
		<tr>
			<td>Pressure Transducer</td>
			<td>ViVitro Labs</td>
			<td>HCM018</td>
			<td></td>
		</tr>
		<tr>
			<td>ViVitro Pressure Measuring Assembly</td>
			<td>ViVitro Labs</td>
			<td>6186</td>
			<td></td>
		</tr>
		<tr>
			<td>Valve holder</td>
			<td>WB Engineering</td>
			<td></td>
			<td>Designed by Florida International University. Manufactured by WB Engineering</td>
		</tr>
		<tr>
			<td>Pulse Duplicator</td>
			<td>ViVitro Labs</td>
			<td>PD2010</td>
			<td><a href="http://vivitrolabs.com/wp-content/uploads/Pulse-Duplicator-Accessories1.pdf" target="_blank">http://vivitrolabs.com/wp-content/uploads/Pulse-Duplicator-Accessories1.pdf</a></td>
		</tr>
		<tr>
			<td>Pulse Duplicator Data Acquisition and Control System, including ViViTest Software</td>
			<td>ViVitro Labs</td>
			<td>PDA2010</td>
			<td><a href="http://vivitrolabs.com/products/software-daq" target="_blank">http://vivitrolabs.com/products/software-daq</a></td>
		</tr>
		<tr>
			<td>Porcine Hearts and Native Aortic Valves</td>
			<td>Mary&#39;s Ranch Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bi-leaflet Mechanical Valves</td>
			<td>Saint Jude Medical</td>
			<td></td>
			<td><a href="http://www.sjm.com/" target="_blank">http://www.sjm.com/</a></td>
		</tr>
		<tr>
			<td>High Vacuum Grease</td>
			<td>Dow Corning Corporation</td>
			<td></td>
			<td><a href="http://www1.dowcorning.com/DataFiles/090007b281afed0e.pdf" target="_blank">http://www1.dowcorning.com/DataFiles/090007b281afed0e.pdf</a></td>
		</tr>
		<tr>
			<td>Glycerin</td>
			<td>McMaster-Carr</td>
			<td>3190K293</td>
			<td>99% Natural 5 gal</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>MT21031CV</td>
			<td>100 ml/heart</td>
		</tr>
		<tr>
			<td>Antimycotic/Antibiotic Solution</td>
			<td>Fisher Scientific</td>
			<td>SV3007901</td>
			<td>1 ml in 100 ml of PBS/heart; 20 ml for ViVitro System</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014-500G</td>
			<td>9 g/L of deionized water</td>
		</tr>
		<tr>
			<td>Deionized Water</td>
			<td>EMD Millipore Chemicals</td>
			<td></td>
			<td>Millipore Deionized Purification System. 1.3 L for ViVitro System, 200 ml for heart valve dissection process</td>
		</tr>
	</tbody>
</table>

protocol for relative hydrodynamic assessment of tri-leaflet polymer valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

Representative flow and pressure waveforms are shown in Figures 3, 4 and 5. The plots were averaged over the sample size of valves tested for each group, which was, n = 5, 4, and 2 valves for polymer, native porcine and bi-leaflet groups, respectively. The mean hydrodynamic metrics and the standard error of the mean for these sample sizes are presented in Table 1.
<img alt="Figure 1" src="/files/ftp_upload...

Watch this Scientific Journal Video about Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves at JoVE.com

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

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13.7K vues.  Florida International University. Il ya eu un regain d'intérêt dans le développement de valves polymères. Ici, les objectifs sont de démontrer la faisabilité de la modification d'un duplicateur d'impulsion commerciale pour accueillir géométries tri-leaflet et de définir un protocole de présenter des données hydrodynamiques de soupape polymères par rapport aux données de valve native et prothétique recueillies dans des conditions quasi-identiques.