Page 126 - Tailoring Electrospinning Techniques for Regenerative Medicine - Marc Simonet

CHAPTER 6
cardiac output, e ective orifice area (AEO, obtained by measuring images taken with the high speed camera) are averaged values from measurements taken over a period of 20 seconds.
6.3.3 In-vivo valve performance
Valve preparation and implantation
Because of the smaller diameter of the pulmonary artery in sheep, the diameter of the electrospun PCL2kU4Un heart valve sca olds was adjusted to 21 mm as compared to the diameter used in the in- vitro tests. Additionally, in accordance with heart valve prosthesis used in open heart valve surgery, the nitinol stent was removed. To increase cellular infiltration, sca olds were immersed with a fibrin solution, consisting of sterile filtered (0.2 μm) bovine fibrinogen and bovine thrombin dissolved in DMEM Advanced (Gibco; 12491-015), supplemented with 10% FBS and 1% penicillin-streptomycin.
10 valves were implanted as pulmonary valve replacements. Sheep (63.8±5.3 kg) were placed on cardiopulmonary bypass (jugular vein to carotid artery) using an 18 French arterial cannula (Edwards Life science) for carotid access and a 27 French venous cannula (Biomedics) for Jugular cannulation. A le -sided anterolateral thoracotomy (7-10 cm) at the third or fourth intercostal space was performed (exact position was determined pre-operatively by echocardiography). A length incision in the pulmonary artery was made and the valve cusps of the
native pulmonary valve were excised. The prosthesis was implanted using 5-0 prolene continuous suture. With careful de-airing the pulmonary artery was closed using 5-0 prolene continuous suture. A er weaning from the extracorporal circulation, valve function and hemodynamics were assessed with epicardial echocardiography. All animal procedures were approved by the UMC Utrecht Animal Care Ethics Committee and were in agreement with the current Dutch law on animal experiments.
Valve explantation and characterization
A er a follow-up period of 2 (n=2), 3 (n=2), 4 (n=4) or 5 (n=2) weeks, valves were explanted. Prior to termination an epicardial echocardiography was made to assess valve performance. Explants were evaluated on fiber morphology, mechanical behavior and molecular weight. Where not stated otherwise, same analytical protocols were used as prior to implantation. Additionally, a er explantation histological stainings were performed to investigate cellular infiltration and neo-tissue formation.
A er gross inspection, the valve was freed from the reinforcement ring. Valve leaflets were transected longitudinally through the midline of the leaflet. From every valve one leaflet was used for (immuno)histochemistry. Sections were fixed in formalin 10% (v/v) before pre-embedding in 1% (w/v) agar (Eurogentec) and embedding in para in. Four-micrometer sections were stained with Mayer’s
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