Biomechanical Engineering Analysis of Pulmonary Valve Leaflet Hemodynamics and Kinematics in the Ross Procedure

The Ross procedure uses a pulmonary autograft to replace the aortic valve and aortic root for young patients with dysfunctional aortic valves [1]. However, dilation of the pulmonary autograft is one of the main causes of graft failure, which can result in valve regurgitation due to inadequate leaflet coaptation [2–6]. The inclusion technique has been described to help prevent pulmonary autograft late dilation by using Dacron graft reinforcement [4,5,7]. This is performed by including the full autograft root inside of a cylinder Dacron graft. A previous ex vivo study demonstrated that the use of the inclusion technique was associated with significantly less autograft regurgitation compared to without autograft reinforcement due to improved cusp coaptation [8]. To further improve the biomechanical performance of the pulmonary autograft in a Ross procedure, the anticommissural plication (ACP) technique has been utilized [8,9]. The ACP technique is used to generate neo-sinuses similar to native aortic roots in valve reimplantation procedures [10]. It has been shown that the use of ACP in the Ross procedure is associated with improved leaflet rapid opening and closing velocity with a trend of improved relative leaflet opening force [8]. However, the previous model did not include coronary flow from the pulmonary autograft. Leaflet kinematics were therefore evaluated without distinguishing individual cusp differences. Previous studies have demonstrated that the presence of coronary flow can have a significant impact on leaflet biomechanics and sinus hemodynamics which could in turn impact leaflet durability [11,12]. Additionally, another important yet understudied phenomenon that can affect leaflet durability is leaflet fluttering [13,14]. Leaflet fluttering has been shown to be associated with accelerated fatigue and premature failure in thin flexible structures, such as bioprosthetic leaflets, as well as structural valve deterioration in bioprosthetic valves [15–18]. Furthermore, leaflet fluttering is often connected to turbulent blood flow, a phenomenon often associated with pathologic platelet activation triggering thrombus formation and elevated wall shear stresses in the ascending aorta, which can potentially lead to endothelial lesions [19]. This is a significant and detrimental factor for poor valve hemodynamic performance [19]. Therefore, the objectives of this study were to elucidate individual pulmonary cusp's biomechanics and leaflet fluttering by including coronary flow in the Ross procedure using an ex vivo three-dimensional (3D)-printed heart simulator.

Porcine pulmonary autografts and their corresponding coronary arteries (n = 10) were harvested from a meat abattoir. Human pulmonary autografts (n = 5) were also harvested from heart transplant recipients (n = 4) whose hearts were explanted and donors (n = 1) whose hearts were not used for transplantation. All human patients did not have pulmonary valve pathologies at the time of sample explant. The human autografts were used to validate the results obtained from porcine autografts. Five porcine autografts were randomly selected to be used as nonreinforced pulmonary autograft without the Dacron grafts as controls. The other five were reinforced with straight Dacron grafts using the inclusion technique. All five human pulmonary autografts were prepared using the inclusion technique. The pulmonary autograft harvest and preparation without the coronary artery buttons have been previously described [8]. Briefly, right ventricular tissue 2 mm below the nadir of each pulmonary leaflet attachment sites was carefully dissected and preserved to not injure the coronary arteries (Fig. 1(a)). Pulmonary artery tissue up to 5 mm distal to the commissures was preserved, and the remaining pulmonary artery tissues were discarded. After the adipose tissue on the pulmonary artery was trimmed away [20], the pulmonary autografts were sized using an aortic valve sizer at the level of the commissures. Additionally, for each porcine heart, the left and right coronary arteries were dissected. Generous coronary buttons were created and coronary arteries of at least 2 cm were explanted. For human autografts' coronary arteries implantation, five additional sets of porcine coronary arteries were harvested from five other porcine hearts. The prepared pulmonary autografts and coronary arteries were then stored in vacuum sealed bags filled with normal saline at –20 °C until further use.

When ready for experiments, the pulmonary autografts were thawed at room temperature. For the control autografts, the pulmonary autograft was first sewn to an elastomeric sewing ring on a 3D-printed conduit mount using a running 4-0 polypropylene suture. The distal ends of the left and right coronary arteries were fixed onto the coronary cannulas using 6-0 polypropylene sutures. Using a 5 mm aortic punch, two coronary anastomosis sites were created above the left and right cusps. The left and right coronary buttons were trimmed and anastomosed to the corresponding sinuses onto the pulmonary autografts above the right and left leaflets, respectively, using running 5-0 polypropylene sutures. The distal pulmonary artery was connected to the outflow mount using a zip tie (Fig. 1(b)).

To prepare a Ross autograft using the inclusion technique, straight Dacron grafts that were 6–7 mm larger in diameter compared to those of the pulmonary autografts were used. The right ventricular tissue was attached to the proximal end of the Dacron graft to generate the proximal suture line using a running 4-0 polypropylene suture. Again using 4-0 polypropylene sutures, the pulmonary valve commissures were then suspended and attached to the Dacron graft, respecting its native commissure rotational degrees. To generate the ACP autografts, the Dacron graft between the commissures at the level of the commissures was plicated by taking roughly 2 mm of the graft above each sinus with horizontal mattress sutures. The distal end of the pulmonary artery was attached to the Dacron graft using a running 4-0 polypropylene suture to complete the middle suture line (Fig. 1(c)). This reinforced pulmonary autograft was attached to the conduit and the outflow mounts in the same fashion as described above. The left and right coronary arteries were attached to the reinforced pulmonary autograft in the same fashion. When performing the anastomosis, care was taken to ensure both the pulmonary artery and the Dacron graft were included in each stitch of the coronary button anastomosis (Fig. 1(d)). Each porcine and human autograft was randomized to be prepared as an ACP autograft or NACP (NACP) autograft first. The same autografts were used twice for a paired comparison. To prepare a NACP autograft, no ACP plication suture was placed. To change an ACP autograft to a NACP autograft, or vice versa, the middle suture line was removed followed by the removal or addition of ACP sutures, respectively. The middle suture line was finally performed again. Note that different sizes of polypropylene sutures were selected to anastomose tissues with different thickness and stiffness, consistent with what is being performed clinically in the operating room. The collection and use of human autografts were approved by the Institutional Review Board at Stanford University, and patient consent was obtained.

where F1 marks the flow rate at the beginning of systolic forward flow, F2 marks the flow rate at the end of systolic forward flow, F3 marks the flow rate at when outflow valve closing and leakage begins, and F4 marks the flow rate at the end of leakage cycle.

High-speed videography with 1280 × 1024 resolution, which correlated to a pixel resolution of 20 pixels per mm, at 1057 frames per second (Chronos 1.4, Kron Technologies, Burnaby, BC, Canada) was recorded from an en face view from the aortic chamber. This allowed for leaflet kinematics analyses. Lastly, echocardiography data were obtained using a Phillips iE33 system with an S5-1 transthoracic probe (Koninklijke Philips NV, Amsterdam, The Netherlands). Continuous-wave Doppler data was gathered from the en face aortic echocardiography port. Analysis was performed using the iE33 on-board software and a Siemens Syngo Dynamics workstation (Siemens Medical Solutions USA, Inc., Ann Arbor, MI). Specifically, the mean gradient across the pulmonary valve was obtained by taking the mean of instantaneous gradient across the pulmonary valve, and the gradient was calculated by the modified Bernoulli equation as follows: $ΔP=4×flow2$ [26]. Derivation of this modified Bernoulli equation is shown in Appendix.

Leaflet motion tracking of the collected high-speed videography data was completed using loggerpro3 (Vernier, Beaverton, OR). The center point on the leading edge of all three cusps was tracked throughout a complete cardiac cycle (Fig. 2(a)). The systolic phase after the valve was fully open was isolated for flutter analysis. Data processing and analysis were performed using matlab (R2020a, MathWorks Inc., Natick, MA) by first importing the raw positional data. Specifically, in-plane displacement plots were generated from the raw two-dimensional positional data at each time point. For the rapid opening and closing leaflet analysis, velocity plots were generated by taking the slope of the displacement regression model at each time point. The rapid leaflet opening and rapid leaflet closing phases were selected [27] and fitted using a linear regression model on the displacement plots, and the mean velocities during leaflet rapid opening and rapid closing were then calculated by taking the average of instantaneous velocities derived during the rapid leaflet opening phase and the rapid leaflet closing phase for each cusp. Relative force during leaflet rapid opening and rapid closing were calculated by taking the derivative of the velocity plots to obtain the acceleration. This acceleration was then normalized to porcine control aortic valve acceleration of each cusp measured in a previous study [9]. This relative acceleration was used as an estimation of relative force compared to porcine control aortic valve. To analyze leaflet fluttering, the mid to late systole phase was used when the valve was fully open before rapid leaflet closure, as this phase was when leaflet fluttering occurred primarily. Each peak and trough on the displacement plot during the systolic flutter phase was identified (Fig. 2(b)). Leaflet flutter frequency was calculated by counting the number of peaks divided by the flutter duration across a cardiac cycle after the valve was open fully. Leaflet flutter amplitude was measured by taking the average of the distances between each pairs of peak and trough that are adjacent to each other. Results were tabulated for all three cusps separately.

Paired student t-test was used to compare the ACP and NACP groups for human autografts. To compare the porcine autografts in a pairwise fashion, analysis of variance with posthoc Tukey correction was performed. The variance was assessed using the F-test. All data analysis was performed in a blinded fashion. Continuous variables are reported as mean±standard deviation unless otherwise specified. Statistical significance was defined at p < 0.05 for all tests. Reported P values are Tukey honest significant difference–adjusted for pairwise porcine autograft comparisons and from paired t-test for human autograft comparisons, unless otherwise noted. Please note that left, right, and noncoronary cusps were not compared to each other. Rather, the cusps from each group were compared to the same cusps in another group (i.e., the left coronary cusps of human ACP autografts were compared to the left coronary cusps of human NACP autografts). Statistical significances explored in this study did not denote the differences in variance.

Representative examples of the pulmonary autografts in diastole captured by high-speed videography are shown in Fig. 3. Porcine pulmonary autograft controls without reinforcement did not have adequate leaflet coaptation, leading to a central regurgitant area most noticeable during diastole (Fig. 3(a)). ACP and NACP autografts both demonstrated improved leaflet coaptation compared to controls, as evidenced by the lack of central regurgitant area (Figs. 3(b) and 3(c)).

Figure 4 demonstrated the mean transaortic flow, and the aortic and left ventricular pressure tracings of the porcine and human autografts. The ACP and NACP autografts did not show significant autograft regurgitation. However, in porcine autografts, the regurgitant fractions were significantly different among control, ACP, and NACP autografts (p = 0.004) with porcine controls revealing higher regurgitant fractions (17.5 ± 5.9%) compared to porcine ACP (5.9 ± 3.8%, p = 0.007) and NACP autografts (6.2 ± 4.5%, p = 0.008). Autograft regurgitant fractions measured from human ACP and NACP autografts were 11.3 ± 6.5% and 14.6 ± 11.1% (p = 0.35), respectively. No other hemodynamic differences were observed between the ACP and NACP autografts in either porcine or human specimens (Tables 1 and 2).

There was no statistically significant difference in transvalvular hemodynamics between both porcine and human ACP or NACP autografts measured using two-dimensional echocardiography. For porcine autografts, the mean gradients were 10.8 ± 6.7 mmHg for ACP autografts and 10.6 ± 4.5 mmHg for NACP autografts. Compared to porcine control autografts' mean gradient of 5.2 ± 1.5 mmHg, there was no difference among the three groups (p = 0.15). In human specimens, the mean gradients from human ACP and NACP autografts were 6.6 ± 3.9 mmHg versus 6.6 ± 5.1 mmHg (p = 1), respectively.

Exemplary high-speed videography footage acquired from porcine controls and human ACP and NACP autografts are shown in Videos S1–S3 available in the Supplemental Materials on the ASME Digital Collection. Using the high-speed videometric data, leaflet motion tracking analysis (Fig. 5) demonstrated that porcine autografts, regardless of autograft preparation methodologies, demonstrated similar leaflet rapid opening mean velocity in the right (p = 0.07), left (p = 0.07), and noncoronary cusps (p = 0.50). However, human autografts showed higher leaflet rapid opening mean velocity in NACP autografts compared to ACP autografts, in the right (p = 0.02), left (p = 0.003), but not noncoronary cusp (p = 0.66). The leaflet rapid closing mean velocities were similar in all three porcine groups in the right (p = 0.89), left (p = 0.49), and noncoronary cusps (p = 0.77). In human autografts, the leaflet rapid closing mean velocities were also similar for ACP and NACP autografts in the right (p = 0.55), left (p = 0.09), and noncoronary cusp (p = 0.65).

Porcine autografts showed similar leaflet relative rapid opening force in right (p = 0.16), left (p = 0.17), and noncoronary cusp (p = 0.13) in all three groups. The porcine leaflet relative rapid closing forces were similar for all three groups in the right (p = 0.89), left (p = 0.14), and noncoronary cusp (p = 0.64). In human ACP compared to NACP autografts, there was no difference in the leaflet relative rapid opening or closing force in the right (p = 0.44 and 0.59), left (p = 0.12 and 0.58), and noncoronary cusp (p = 0.93 and 0.15).

Leaflet flutter analysis (Fig. 6) showed no statistical differences in the leaflet flutter frequency on the left (p = 0.30), nor noncoronary cusp (p = 0.40) in porcine autografts regardless of the autograft preparation technique but the flutter frequencies were different in the right coronary cusp among the three groups (p = 0.05). Specifically, porcine NACP autografts compared to controls were associated with significantly higher leaflet flutter frequency in the right coronary cusp (p = 0.05). The human autografts showed similar leaflet flutter frequency in the right (p = 0.16) and left coronary cusp (p = 0.35), but the noncoronary cusp flutter frequency was significantly higher in NACP autografts compared to ACP autografts (p = 0.02). Leaflet flutter amplitudes were similar in right (p = 0.89), left (p = 0.72), and noncoronary cusps (p = 0.70) in the three porcine autograft groups. In human autografts, the leaflet flutter amplitudes were also similar in right (p = 0.28), left (p = 0.15), and noncoronary cusps (p = 0.36) comparing ACP versus NACP.

In this study, we improved upon a previously established ex vivo Ross model to further investigate individual pulmonary cusp's hemodynamics and kinematics by including coronary flow in an ex vivo 3D-printed heart simulator. The inclusion of coronary flow is a critical component of the Ross procedure, and therefore, this ex vivo Ross model is highly clinically relevant and accurate, as this model was created the same way that is being performed surgically in the operating room. We showed that the use of the inclusion technique, with or without ACP, was associated with improved regurgitation without significant stenosis compared to pulmonary autografts prepared without reinforcement. The addition of the ACP technique further improved leaflet kinematics by decreasing leaflet rapid opening mean velocity and flutter frequency to levels similar to those measured from the control autografts.

The results from this study were largely consistent with what has been reported previously [8]. The differences in results between this project and our previous model relied upon leaflet kinematics findings where only leaflet opening and closing velocity and relative force averaged across the three cusps were reported previously, whereas, in this study, individual cusp kinematics were studied separately with additional leaflet fluttering analysis performed. Our previous model was simplified and did not include coronary buttons. In this study, we generated a more accurate and realistic Ross model and again confirmed the advantage of using the inclusion technique over the nonreinforced pulmonary autografts in terms of valve hemodynamics. Specifically, the use of the inclusion technique was associated with improved leaflet coaptation without regurgitation or stenosis. Interestingly, but also consistent with our previous finding, the porcine NACP autografts demonstrated mildly increased mean transvalvular gradient compared to controls, but the use of the ACP technique in porcine autografts was not associated with statistically significant differences in the mean transvalvular gradient. The creation of neo-sinuses by using the ACP technique likely provided additional benefits to valvular hemodynamics compared to the NACP autografts [28,29].

Studies have shown that aortic leaflet calcification more frequently affects bicuspid aortic valves , and it has been hypothesized that coronary flow originating from the ostia can greatly influence aortic leaflet mechanics and sinus hemodynamics [30,31]. Given the differences in flow between the left and right coronary arteries [11,32], we individually analyzed leaflet kinematics of each cusp in this study. In human autografts, we showed that leaflet rapid opening mean velocities were lower using the ACP technique compared to the NACP autografts in the right and left coronary cusps. However, no differences were observed in the noncoronary cusps in both porcine and human autografts with versus without ACP. We suspect that the effect of the ACP technique on leaflet kinematics was augmented due to the presence of coronary flow, and the ex vivo model presented in this article is a close imitation of the physiologic state. The favorable kinematics of the Ross autograft prepared using the ACP technique could potentially improve valve durability and delay degeneration, a common cause of Ross procedure failure [3,33]. Note that porcine controls were treated as the group with the most favorable leaflet kinematics due to their similar leaflet opening and closing velocities and relative force compared to healthy, normal aortic valves from our previous study [9]. However, future study should still be carried out to delineate any potential differences, if any, in leaflet kinematics in porcine control pulmonary valves compared to porcine control aortic valves.

Another important finding of this study is the leaflet fluttering differences observed between the ACP and NACP autografts. We hypothesize that the fluttering phenomenon observed in this study was associated with flow dynamic changes and turbulent flow as well as flow instability across the valve. Though no differences in leaflet flutter frequency were observed in porcine autografts in the left and noncoronary cusps, the flutter frequency of porcine NACP autografts was significantly higher compared to controls in the right coronary cusp. Leaflet flutter, unfortunately, is an understudied phenomenon. Studies have shown that leaflet thickness, diameter, asymmetry, and rigidity can all contribute to fluttering [13,14,34]. More importantly, increased leaflet fluttering may contribute to additional mechanical load cycles on the leaflets during systole due to the repeated bending of the leaflet, in addition to the forces experienced during valve opening and closure [35]. Clinically, leaflet flutter is known to be associated with early valve failure, such as reduced life span of bioprosthetic valves and valve calcification [13,14,34,35]. We hypothesize that by replicating a neo-sinus, the ACP technique may contribute to more physiologic fluid dynamics in the root, manifested by similar leaflet flutter frequency compared to the control autografts. The reduced leaflet fluttering associated with ACP might have a significant impact on autograft durability, as increased leaflet fluttering is associated with accelerated fatigue [15–18].

To further validate our findings, we used human pulmonary autografts to confirm our findings using the porcine autografts. In the human autografts, we observed similar leaflet flutter frequency in the right and left coronary cusps between the ACP and NACP group, but higher flutter frequency in the noncoronary cusp in the ACP autografts compared to the NACP autografts. This difference in finding compared to that in porcine autografts likely reflects the innate differences in tissue mechanical properties and cusp thickness between human and porcine pulmonary valves [36,37], even though porcine hearts have been frequently used as a human analog due to the similarities in size and anatomy [38]. Interestingly, previous studies have shown that valve degeneration and calcification were more likely to happen to the noncoronary cusp . It is possible that the beneficial increase in washout and decrease in flow-induced instability near the right and left coronary cusps plays a larger role than the neo-root geometry in valve kinematics [34]. However, the recreation of a neo-sinus may have an important impact on the noncoronary cusp's kinematics by preserving a more physiologic flow [39]. Future clinical studies should be pursued to validate the findings in this study.

This ex vivo simulation contains a few limitations. Due to the ex vivo nature of this experiment, it is difficult to fully replicate the physiologic motion and flow dynamics in the left ventricle and in the aortic root. In addition, saline was used as a working fluid in the ex vivo experiments, and it is used widely in testing explanted cardiac valves in compliance with the ISO 5840 standards. However, viscosity differences exist between saline and blood. This may lead to small-scale turbulent flow difference, although large-scale flow structures and leaflet kinematics likely remained similar between the two working fluids [40]. In this study, a linear regression model was used to fit the displacement plots, and thus there may be a degree of error associated with the velocity and acceleration data that were derived from the linear regressions. Other models should be explored in the future to mitigate this error by using a quadratic regression model, for example. Furthermore, given that some of the human autografts were obtained from heart transplantation recipients with end-stage cardiomyopathy, potential biomechanical differences may exist in these samples compared to healthy, normal tissues. Additional differences may also exist between the autografts used in this study and tissues in patients with aortic valve diseases. This study also noted improved leaflet coaptation after the use of the inclusion technique with or without ACP compared to controls via qualitative visual assessment. In future studies, quantitative measurement of leaflet coaptation height or surface area should be obtained to validate this observation. Finally, the findings obtained from this study illustrate the short-term outcomes using different techniques for the Ross procedure with indirect implications on long-term valve durability. Future clinical evaluations and long-term outcomes studies are warranted to further validate the findings reported in this study to further improve clinical practice. Additionally, the impact of coronary occlusion, partial or complete, as observed in patients with coronary artery diseases, on cusp biomechanics may also be evaluated as another potential future direction.

In conclusion, the Ross procedure using the inclusion technique with ACP is associated with excellent hemodynamics and more favorable leaflet kinematics in terms of leaflet rapid opening mean velocity, relative rapid opening force, and flutter frequency compared to NACP autografts. These results may translate to improved long-term durability of the pulmonary autografts.

We would like to thank the generous donation by Mr. M. Ian Ritchie to support this research effort.

• National Heart, Lung, and Blood Institute (NIH F32 HL158151-01, YZ; Funder ID: 10.13039/100000050).

• National Institutes of Health (Grant No. NIH R01 HL152155, YJW; Funder ID: 10.13039/100000002).

• Thoracic Surgery Foundation Resident Research Fellowship (YZ) (Funder ID: 10.13039/100005631).

• National Science Foundation Graduate Research Fellowship Program (Grant No. DGE-1656518, AMI; Funder ID: 10.13039/100000001).

None.

Abbreviations

ACP =

anticommissural plication

LCC =

left coronary cusp

NACP =

nonanticommissural plication

NCC =

noncoronary cusp

RCC =

right coronary cusp

Modified Bernoulli equation derivation used for echocardiography measurements:

Let K₁ and P₁ be the kinetic energy and pressure energy proximal to the valve.