Development of an Ex Vivo Mitral Valve Evaluation Model Using a Pulsatile Flow Simulator

Summary

We present an ex vivo model for mounting lamb mitral valves into a pulsatile simulator to assess valve function under physiologic conditions. This setup enables quantitative evaluation of mitral valve dynamics using biological tissue in a reproducible, anatomically accurate configuration.

Abstract

Surgical mitral valve repair remains a challenging procedure. Although several repair techniques have been defined, data comparing their hemodynamic effects are lacking. The commercially available pulse duplicators are commonly used to simulate blood circulation through mechanical or 3D printed cardiac valves. However, due to the specific structure and working mechanism of the mitral valve, the experiments on surgical techniques require the use of biological tissues. Ex vivo lamb mitral valves are suitable for such experiments, but the methods for mounting these valves to the pulse duplicator system (PDS) are not well defined. To address this, we modified the system by 3D printing and silicone molding as a mitral valve holder. We excised the mitral valve from a lamb heart for each experiment, including its annulus and subvalvar apparatus. We implanted this into the atrioventricular (mitral) valve area of the test machine using the silicone holder. Papillary muscle tension was simulated by tying sutures around the chordae-papillary junctions and passing these sutures through the release hole at the bottom of the ventricular chamber. Initial testing of the valve competence was conducted at a heart rate of 120 beats per minute and a cardiac output of 2 L/min. Valve regurgitation and the pressure gradient between the atrial and ventricular chambers were measured using pulse duplicator electromagnetic flowmeters and validated with echocardiography. Baseline hemodynamic testing demonstrated consistent valve function across five experiments, with a mean regurgitation fraction of 21.1% and echo-derived transmitral gradients ranging from 5.15 to 8.13 mmHg. Stroke volumes and peak flow rates varied among specimens, reflecting physiological variability within the pediatric model.

Introduction

Mitral valve repair has evolved significantly, with contemporary surgical techniques offering enhanced efficacy and improved long-term outcomes¹. Although many centers have standardized mitral repair strategies, a persistent gap remains in the comprehensive evaluation of the hemodynamic impact of these interventions². Optimizing repair strategies, particularly for anatomically complex or pediatric cases, requires models that allow for controlled, repeatable physiological testing.

The primary objective of this study was to develop a reproducible ex vivo model using biological mitral valves, enabling precise assessment of valve dynamics under physiological flow conditions. Pulse duplicator systems have significantly advanced the field of valve biomechanics by facilitating ex vivo analysis of valve function under near-physiological conditions^3,4,5. These systems have been applied to aortic and pulmonary valves and, more recently, to the mitral valve. However, the aim for developing this technique comes from the unique anatomical and functional complexity of the mitral valve, particularly its subvalvular apparatus, which necessitates biologically accurate models. There exists a substantial body of literature on ex vivo models of the mitral valve; however, none of these studies provide a detailed description of the harvesting technique or highlights the critical steps required to preserve the structural integrity of the mitral valve and subvalvular apparatus during extraction^6,7. Moreover, literature lacks practical guidance on how to successfully integrate the excised valve into a pulse duplicator system, which is essential for achieving physiologically relevant hemodynamic simulation.

In adult cardiac surgery, several repair techniques have been assessed using ex vivo models, such as edge-to-edge repair, chordal replacement, and papillary approximation⁵. These techniques have yielded valuable biomechanical insights. In contrast, pediatric applications remain underexplored, with only limited ex vivo studies addressing congenital anomalies like mitral clefts. One such study demonstrated the importance of complete cleft closure to restore valve competence⁶.

This method is particularly appropriate for researchers and clinicians interested in assessing repair outcomes for congenital or complex mitral valve pathologies in a controlled environment. By harvesting the mitral apparatus from lamb hearts, chosen for their anatomical similarity to pediatric valves, and mounting them in a custom silicone holder within a pulsatile flow simulator, the system replicates realistic hemodynamics and allows for serial testing. Importantly, this model avoids live animal experimentation and provides an adaptable platform for simulating regurgitation, stenosis, cleft, or subvalvular fusion⁸.

The Vivitro Pulse Duplicator System was employed in this study to assess the efficacy of a mitral valve and simulate cardiac function. The procedure commenced with the activation of the system software, which was followed by the selection of the appropriate waveform parameters, such as the systolic-to-diastolic ratio, heart rate (beats per minute), and wavelength. The mitral valve was subsequently inserted into the testing chamber of the duplicator, and all requisite sensors were connected. The calibration file was opened, and the system was simultaneously initiated to ensure that all air was removed from the sensors and tubing after the sensor was installed. In order to guarantee precise measurements, it was imperative that the entire fluid circuit be devoid of oxygen and entirely filled with saline solution. When the system was in steady-state operation, the stroke volume was changed to 25 mL. Data acquisition started by recording 10 cycles when the system was found to have no leaks or errors. The data generated was then exported in spreadsheet format for further analysis. A quantitative assessment of the study was made easier by the system's ability to compute most of the needed parameters automatically, including regurgitant volume and several pressure measurements (Figure 1A).

In summary, this ex vivo model offers a standardized, biologically relevant approach to studying mitral valve repair, bridging the gap between in silico modeling and in vivo testing, and expanding the translational value of surgical innovation.

Protocol

This study was conducted using lamb hearts obtained post-mortem from a licensed abattoir. According to the decision of the Istanbul University-Cerrahpaşa Local Ethics Committee for Animal Experiments (2025/11), this work falls under the exemption outlined in Article 8 (8-k1) of the HADYEK directive and therefore did not require ethical approval .

1. Experiment setup

1. Duplicate the mitral inflow component of the pulse duplicator system using 3D modeling software. Create a digital copy of the existing geometry to ensure compatibility. Print the design using PLA filament on a 3D printer. Use the printed mold to cast the silicone inflow chamber. After curing, integrate the silicone part into the system, making sure it fits securely and maintains a proper seal (Figure 1B,C).
2. Procure lamb hearts from a nearby abattoir, with immediate attention given to ensuring that the hearts were obtained post-mortem to preserve their physiological state. Following collection, store the hearts separately at -20 °C in a freezer to maintain their structural integrity, or promptly utilize them in experimental procedures.
   NOTE: All experiments were conducted under appropriate safety precautions. Gloves and hoods were used throughout, and all surgical instruments were sterilized with alcohol after each use.

2. Ha​rvesting the mitral valve

1. Ventriculotomy and resection of the right ventricle and atrium
   1. Make a transverse incision at the apex of the heart using surgical scissors. Extend the incision superiorly toward the right ventricle along the interventricular groove. Take care to avoid injuring the papillary muscles during this dissection. Refer to Figure 2A - B for the incision trajectory and anatomical landmarks.
   2. Resect the free wall of the right ventricle using surgical scissors to extend the exposure. Continue by excising the right atrium en bloc to fully expose the interventricular septum. This exposure is critical for the subsequent dissection of the subvalvular structures (Figure 3A,B).
2. Incision of the interventricular septum (IVS)
   1. Visualize the mitral valve from the aortic root into the left ventricular cavity. Identify the anterior leaflet based on its orientation beneath the aortic valve plane. Ensure full exposure of the leaflet prior to septal incision (Figure 4A).
   2. Using fine surgical scissors, incise the IVS, beginning from the aortic root at the contralateral side of the anterior mitral leaflet. Extend the incision downward along the septum toward the previously made apical cut. Open the septum to expose the left ventricular cavity without damaging subvalvular structures (Figure 4B).
3. Papillary muscle dissection
   1. Expose the left ventricle and excise the papillary muscles using fine scissors. Preserve the chordae tendineae to maintain mitral valve functionality (Figure 4C).
4. Left atrium excision
   1. Excise the left atrial appendage circumferentially to improve visualization of the mitral annulus from above (Figure 5A,B).
   2. Translocate the papillary muscles upward through the left atrium to invert the mitral valve and safely expose them from the ventricular surface for excision (Figure 6A).
   3. Trim excess tissue surrounding the mitral annulus, ensuring at least 10 mm of tissue is preserved circumferentially to maintain anatomical integrity and function (Figure 6B).
5. Pulmonary and aortic tissue removal
   1. Remove any remaining tissue from the aorta and pulmonary artery to prevent interference with the placement of the mitral apparatus into the silicone mold.

3. Placement of the mitral valve into the silicone mold

1. Mitral valve measurements
   1. Measure the lengths of the anterior and posterior mitral valve leaflets along the anteroposterior axis to assess leaflet dimensions (Figure 7A,B).
   2. Record the length of the mitral valve along the mediolateral axis for use in subsequent mold fitting and dimensional analysis (Figure 7C).
   3. Estimate the craniocaudal length of the zone of coaptation as approximately 1/4^th of the anteroposterior length of the posterior leaflet. Measure this distance to assess leaflet overlap and sealing capacity.
   4. Use the measured dimensions to calculate the appropriate annular retention margin. Select a 30 mm silicone mold to ensure optimal fit and stabilization of the mitral apparatus (Figure 8A). Use the equations below:
      a = (30-x)/2
      Where a = the distance that should be left from commissures; x = the measured medial-lateral axis length.
      b = (30 - z y) /2−estimated ZC length
      Where b = represents the remaining distance left in the anterior and posterior leaflet; y = the length of the posterior leaflet; z = the length of the anterior leaflet.
   5. Following the measurements, connect all points to form a continuous circular outline, representing the area to be sutured onto the silicone mold (Figure 8B).
2. Tissue mounting and chordae tendineae attachment
   1. Mount the mitral valve tissue onto a silicone mold, using 16 U sutures to secure the annulus and maintain its anatomical positioning (Figure 9A).
   2. Tie the chordae tendineae at the insertion points of the papillary muscles using 2-0 coated braided polyester sutures.
   3. Pass the sutures through the release hole at the base of the ventricular chamber and secure them to simulate physiological papillary muscle tension. Leaflet tension was adjusted visually to ensure that the leaflet edges remained aligned with the plane of the annulus and did not protrude above it. (Figure 9B).
   4. Position the sutures under maximal tension at the left ventricular hole tract of the PDS, ensuring stabilization of the mitral valve without inducing displacement (Figure 10A,B).

4. Initial testing (Baseline model)

1. Place the prepared mitral valve model into the pulsatile flow simulator for baseline hemodynamic testing. Set the system to pediatric physiologic conditions: a heart rate of 120 beats per minute and a cardiac output of 2.0 L/min.
2. Fill the pulse duplicator system with saline, ensuring that the entire circuit is free of air bubbles to maintain accurate pressure and flow measurements.
3. Zero the pulse duplicator system prior to initialization to ensure all subsequent measurements are accurate.
4. Initialize the system, and once the desired stroke volume is achieved, click Capture to record the hemodynamic measurements.
5. Evaluate mitral valve function using echocardiographic imaging from the upper and transparent atrial chamber to assess parameters such as transmitral gradient and regurgitation severity, ensuring comprehensive functional assessment of the valve model (Figure 10C).

Results

Baseline hemodynamic data were obtained from five ex vivo experiments using native mitral valves mounted in a custom silicone holder within a pulse duplicator system. The model was perfused under standardized pediatric physiological conditions (cardiac output: 2 L/min; HR: 120 bpm), and functional assessments were performed using pressure transducers and echocardiographic imaging.

The regurgitation fraction ranged from 15.9% to 26.6%, with a mean of 21.1% ± 5.3%. Stroke volume varied ...

Discussion

The objective of this study was to develop a model replicating the mitral valve under pediatric hemodynamic state, which guided our choice to use lamb hearts as the experimental basis. This selection was driven by the close resemblance in dimensions between the lamb mitral valve and that of pediatric patients. Anatomically, the lamb heart exhibits a more ventrally tilted orientation along its longitudinal axis compared to the human heart and possesses a relatively blunt apex composed entirely of the left ventricle

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-0 Coated Braided Polyester Suture	Covidien	88863230-56	Stabilization of papillary muscles during testing
3D Printer	Ultimaker	Ultimaker S3	Used for fabricating custom silicone molds
5-0 Monofilament Polypropylene Suture, Double Armed	Covidien	VPF-720-X	Suturing the valve to the silicone mold
DeBakey Atraumatic Forceps	Aesculap	MB062R	Tissue handling during dissection
Fresh Lamb Hearts	Licensed Abattoir	—	Source of native mitral valves
Mayo Dissection Scissors	Aesculap	BC252B	General cardiac dissection
Micro Needle Holder	Aesculap	FM538R	Suturing the mitral valve
Nelson-Metzenbaum Scissors	Aesculap	BC606R	Used for suture cutting
Pulse Duplicator System	ViVitro Labs	18363	Simulated physiologic flow for valve testing
Silicone Molds	—	Custom-fabricated	Used to replicate ventricular geometry
Sterile Saline (0.9%)	Elabscience	PB180353	Medium for perfusion circuit