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

Summary

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

Abstract

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.

Introduction

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 IE^1,2,3,4,5. 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 understood

Protocol

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 instructions¹².

1. Rinse all tissues with 0.9% NaCl prior to use.
2. Prepare tissue biopsies using a disposable skin biopsy punc....

Results

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.......

Discussion

Recent clinical observations give special awareness to IE as a complication in patients having undergone valve replacement of the RVOT^6,13. Dysfunction of the implanted valve in IE is the result of bacterial interaction with the endovascular graft leading to extensive inflammatory and procoagulant reactions^1,14. The presented novel in vitro model allowed us to investigate if differences in tissue.......

Materials

List of materials used in this article

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