Name: an in vitro model of a parallel-plate perfusion system to study bacterial adherence to graft tissues
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues at JoVE.com

This study was sponsored by a grant of the Research Fund KU Leuven (OT/14/097) given to RH. TRV was Postdoctoral Fellow of the FWO Research Foundation - Flanders (Belgium; Grant Number - 12K0916N) and RH is supported by the Clinical Research Fund of UZ Leuven.

1. Preparing Graft Tissues for In Vitro Studies
Note: Three types of tissues were used: Bovine Pericardium patch (BP), Cryopreserved Homograft (CH) and Bovine Jugular Vein grafts (BJV). In case of BJV conduit and CH (tissue processed by the European Homograft Bank (EHB) and stored in liquid nitrogen prior to use), both the wall and valvular leaflets were used. BP patch and BJV conduit were purchased from the manufacturers. Prior to use, thaw the CH following the EHB instructions<sup cla...

<ol>
	<li>Que, Y. A., Moreillon, P. <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.">Infective endocarditis.</a> Nature Reviews Cardiology. 8 (6), 322-336 (2011).</li><li>Werdan, K., 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=Mechanisms+of+infective+endocarditis:+pathogen-host+interaction+and+risk+states.">Mechanisms of infective endocarditis: pathogen-host interaction and risk states.</a> Nature Reviews Cardiology. 11 (1), 35-50 (2014).</li><li>Moreillon, P., Que, Y. A. <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.">Infective endocarditis.</a> The Lancet. 363 (9403), 139-149 (2004).</li><li>Jalal, Z., 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=Selective+propensity+of+bovine+jugular+vein+material+to+bacterial+adhesions:+An+in+vitro+study.">Selective propensity of bovine jugular vein material to bacterial adhesions: An in vitro study.</a> International Journal of Cardiology. 198, 201-205 (2015).</li><li>Sharma, A., Cote, A. T., Hosking, M. C. K., Harris, K. 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+Systematic+Review+of+Infective+Endocarditis+in+Patients+With+Bovine+Jugular+Vein+Valves+Compared+With+Other+Valve+Types.">A Systematic Review of Infective Endocarditis in Patients With Bovine Jugular Vein Valves Compared With Other Valve Types.</a> JACC Cardiovascular Interventions. 10 (14), 1449-1458 (2017).</li><li>Malekzadeh-Milani, 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=Incidence+and+predictors+of+Melody(R)+valve+endocarditis:+a+prospective+study.">Incidence and predictors of Melody(R) valve endocarditis: a prospective study.</a> Archives of Cardiovascular Diseases. 108 (2), 97-106 (2015).</li><li>Hill, E. 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=Management+of+prosthetic+valve+infective+endocarditis.">Management of prosthetic valve infective endocarditis.</a> American Journal of Cardiology. 101 (8), 1174-1178 (2008).</li><li>Claes, 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=Clumping+factor+A,+von+Willebrand+factor-binding+protein+and+von+Willebrand+factor+anchor+Staphylococcus+aureus+to+the+vessel+wall.">Clumping factor A, von Willebrand factor-binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall.</a> Journal of Thrombosis and Haemostasis. 15 (5), 1009-1019 (2017).</li><li>Fowler, 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=Cellular+invasion+by+Staphylococcus+aureus+involves+a+fibronectin+bridge+between+the+bacterial+fibronectin-binding+MSCRAMMs+and+host+cell+beta1+integrins.">Cellular invasion by Staphylococcus aureus involves a fibronectin bridge between the bacterial fibronectin-binding MSCRAMMs and host cell beta1 integrins.</a> European Journal of Cell Biology. 79 (10), 672-679 (2000).</li><li>Patti, J. M., Hook, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+adhesins+recognizing+extracellular+matrix+macromolecules.">Microbial adhesins recognizing extracellular matrix macromolecules.</a> Current Opinion in Cell Biology. 6 (5), 752-758 (1994).</li><li>Massey, R. C., 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=Fibronectin-binding+protein+A+of+Staphylococcus+aureus+has+multiple,+substituting,+binding+regions+that+mediate+adherence+to+fibronectin+and+invasion+of+endothelial+cells.">Fibronectin-binding protein A of Staphylococcus aureus has multiple, substituting, binding regions that mediate adherence to fibronectin and invasion of endothelial cells.</a> Cellular Microbiology. 3 (12), 839-851 (2001).</li><li>Jashari, 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=Belgian+and+European+experience+with+the+European+Homograft+Bank+(EHB)+cryopreserved+allograft+valves--assessment+of+a+20+year+activity.">Belgian and European experience with the European Homograft Bank (EHB) cryopreserved allograft valves--assessment of a 20 year activity.</a> Acta Chirurgica Belgica. 110 (3), 280-290 (2010).</li><li>Cheatham, J. 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=Clinical+and+hemodynamic+outcomes+up+to+7+years+after+transcatheter+pulmonary+valve+replacement+in+the+US+melody+valve+investigational+device+exemption+trial.">Clinical and hemodynamic outcomes up to 7 years after transcatheter pulmonary valve replacement in the US melody valve investigational device exemption trial.</a> Circulation. 131 (22), 1960-1970 (2015).</li><li>Que, Y. 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=Fibrinogen+and+fibronectin+binding+cooperate+for+valve+infection+and+invasion+in+Staphylococcus+aureus+experimental+endocarditis.">Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis.</a> The Journal of Experimental Medicine. 201 (10), 1627-1635 (2005).</li><li>Veloso, T. 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=Bacterial+adherence+to+graft+tissues+in+static+and+flow+conditions.">Bacterial adherence to graft tissues in static and flow conditions.</a> The Journal of Thoracic and Cardiovascular Surgery. 155 (1), 325-332 (2018).</li><li>Liesenborghs, L., Verhamme, P., Vanassche, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus,+master+manipulator+of+the+human+hemostatic+system.">Staphylococcus aureus, master manipulator of the human hemostatic system.</a> Journal of Thrombosis and Haemostasis. 16 (3), 441-454 (2018).</li><li>Chiu, J. 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=Shear+stress+increases+ICAM-1+and+decreases+VCAM-1+and+E-selectin+expressions+induced+by+tumor+necrosis+factor-[alpha]+in+endothelial+cells.">Shear stress increases ICAM-1 and decreases VCAM-1 and E-selectin expressions induced by tumor necrosis factor-[alpha] in endothelial cells.</a> Artheriosclerosis, Thrombosis, and Vascular Biology. 24 (1), 73-79 (2004).</li><li>Jockenhoevel, S., Zund, G., Hoerstrup, S. P., Schnell, A., Turina, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+tissue+engineering:+a+new+laminar+flow+chamber+for+in+vitro+improvement+of+mechanical+tissue+properties.">Cardiovascular tissue engineering: a new laminar flow chamber for in vitro improvement of mechanical tissue properties.</a> ASAIO Journal. 48 (1), 8-11 (2002).</li><li>Veltrop, M. H. 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=Bacterial+Species-+and+Strain-Dependent+Induction+of+Tissue+Factor+in+Human+Vascular+Endothelial+Cells.">Bacterial Species- and Strain-Dependent Induction of Tissue Factor in Human Vascular Endothelial Cells.</a> Infection and Immunity. 67 (11), 6130-6138 (1999).</li></ol>

Recent clinical observations give special awareness to IE as a complication in patients having undergone valve replacement of the RVOT6,13. Dysfunction of the implanted valve in IE is the result of bacterial interaction with the endovascular graft leading to extensive inflammatory and procoagulant reactions1,14. The presented novel in vitro model allowed us to investigate if differences in tissue...

Staphylococcus aureus (S. aureus) employs a variety of virulence strategies to circumvent the host immune defense system colonizing biological or non-biological surfaces implanted in the human circulation, which leads to severe intravascular infections such as sepsis and IE1,2,3,4,5. IE remains an important treatment associated complication in patients after implantation of prosthetic heart valves while individual factors contributing to the onset of IEare not yet fully understood6,7. Under flow conditions, bacteria encounter shear forces, which they need to overcome in order to adhere to the vessel wall8. Models, which allow studying the interplay between bacteria and prosthetic valve tissue or endothelium under flow, are of interest as they reflect the in vivo situation more.
Several specific mechanisms facilitate bacterial adherence to endothelial cells (ECs) and to the exposed subendothelial matrix (ECM) leading to tissue colonization and maturation of vegetations, being essential early steps in IE9. Various staphylococcal surface proteins or MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) have been described as mediators of adhesion to host cells and to ECM proteins by interacting with molecules such as fibronectin, fibrinogen, collagen and von Willebrand factor (VWF)8,10,11. However, in view of intra-molecular folding of some virulence factors, mostly studied in static conditions, many of these interactions may have different relevance in endovascular infections in circulating blood.
Therefore, we present an in-house designed in vitro parallel-plate flow chamber model, which allows the assessment of bacterial adherence to different components of ECM and ECs in the context of tissue grafts implanted in the RVOT position. The overall purpose of the method described in this work is to study mechanisms of interaction between bacteria and underlying endovascular tissues in flow conditions, which are closely related to the in vivo environment of bloodstream pathogens such as S. aureus. This novel approach focuses on the susceptibility of graft tissue surfaces to bacterial adherence to identify potential risk factors for the development of IE.

<table>
	<tbody>
		<tr>
			<td>Bovine Pericardium (BP) patch, Supple Peri-Guard Pericardium</td>
			<td>Synovis Surgical Innovations, USA</td>
			<td>PC-0404SN</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Jugular Vein conduits (BJV)</td>
			<td>Contegra conduit; Medtronic Inc, USA</td>
			<td>M333105D001</td>
			<td></td>
		</tr>
		<tr>
			<td>CH cryopreserved homograft</td>
			<td>European Homograft Bank (EHB)</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Acu-Punch</td>
			<td>Acuderm Inc, USA</td>
			<td>P850 (8 mm); P1050 (10 mm)</td>
			<td></td>
		</tr>
		<tr>
			<td>human Albumin</td>
			<td>Flexbumin; Baxter, Belgium</td>
			<td>BE171464 
			LOT:16G12C</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic soy broth (TSB)</td>
			<td>Fluka, Steinheim, Germany</td>
			<td>22092-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Heart infusion broth (BHI)</td>
			<td>Fluka</td>
			<td>53286-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS).</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>5(6)-Carboxyfluorescein N-hydroxysuccinimide ester (CF)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>21878-100MG-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump (MODEL ISM444B)</td>
			<td>Ismatec BVP-Z Standard; Cole Parmer, Wertheim, Germany</td>
			<td>631942-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonication bath</td>
			<td>VWR Ultrasonic Cleaner; VWR, Radnor, Pa</td>
			<td>142-6044</td>
			<td>230V/50 -60Hz 60VA; HF45kHz, 30W</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Invitrogen by ThermoFisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>InCell Analyzer 2000 (fluorescence scanner)</td>
			<td>GE Healthcare Life Sciences, Pittsburgh, Pa</td>
			<td>29027886</td>
			<td></td>
		</tr>
		<tr>
			<td>Arium Pro VF - ultrapure water - H2O MilliQ</td>
			<td>Millipore</td>
			<td>87206462</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopic slides - Tissue Culture Chambers (1-well)</td>
			<td>Sarstedt</td>
			<td>94.6140.102</td>
			<td></td>
		</tr>
		<tr>
			<td>1-well on Lumox detachable</td>
			<td>Sarstedt</td>
			<td>94.6150.101</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel - surgical Blades</td>
			<td>Swann-Morton</td>
			<td>311</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon Silicone Tubing, 1/8&#34;ID x 1/4&#34;OD</td>
			<td>Cole-Parmer</td>
			<td>EW-95702-06</td>
			<td>Temperature range: &#8211;80 to 200&#176;C 
			Sterilize: With ethylene oxide, gamma irradiation, or autoclave for 30 min, 15 psi of pressure</td>
		</tr>
		<tr>
			<td>PharMed BPT Tubing</td>
			<td>Saint-Gobain</td>
			<td>AY242012</td>
			<td>Autoclavable 30 min at 121&#176;C</td>
		</tr>
		<tr>
			<td>Tygon LMT-55 Tubing</td>
			<td>Saint Gobain Performance Plastics&#8482;</td>
			<td>15312022</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostat</td>
			<td>BMG BIOMEDIZINTECHNIK</td>
			<td>300-0042</td>
			<td>230V, 90VA, 50Hz</td>
		</tr>
	</tbody>
</table>

an in vitro model of a parallel-plate perfusion system to study bacterial adherence to graft tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

To better understand the mechanisms behind IE development, this model enables the evaluation of bacterial and tissue associated factors present in the in vivo situation of infection onset.
In detail, the novel in vitro approach allows to quantify bacterial adhesion in flow conditions to different graft tissues by perfusing fluorescently labeled bacteria over the tissues exerting the shear stresses in t...

Watch this Scientific Journal Video about An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues at JoVE.com

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

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