Mechanical and Structural Analysis of the Pulmonary Valve in Congenital Heart Defects: A Presentation of Two Case Studies

Abstract

Objective:

Congenital Heart Disease (CHD) is the leading cause of pediatric mortality, with many cases affecting the right ventricular outflow tract (RVOT) or pulmonary valve (PV). Understanding the mechanics of the disease condition can provide insight into development of durable repair techniques and bioengineered replacement devices. This work presents a mechanical and structural analysis of the pulmonary valve of two pediatric cases.

Methods:

Two PV tissues were excised as part of the operative procedure. One PV was obtained from a 9-month- old with Noonan syndrome (Patient 1) and the other from a 6-month-old with tricuspid atresia (Patient 2). The leaflets were subjected to planar biaxial tensile testing and second harmonic generation (SHG) imaging for mechanical and structural evaluation.

Results and Discussion:

Patient 1 exhibited a more anisotropic mechanical response than Patient 2, with sample stiffness on par with that of adult PV tissue. Additionally, both samples showed radial and circumferential alignment of collagen fibers on the ventricularis and fibrosa sides of the leaflets, respectively. Collagen fibers on the fibrosa side were also more crimped than on the ventricularis side.

1. Introduction

Congenital Heart Defects (CHD) are the most common birth defect, affecting one out of every 100 live births in the United States and accounting for about 40,000 children per year [1–3]. CHD encompasses a wide range of cardiac anomalies, which invariably affects other organ systems [4]. CHD is typically grouped in the following categories [5]: cyanotic and acyanotic lesions, left-sided cardiac obstructive lesions, right sided cardiac obstructive lesions, and complex mixing lesions .

It is estimated that up to 20% of CHD children have specific heart defects that involve the right ventricular outflow tract (RVOT) and pulmonary valve (PV), over half of which consist of lesions associated with decreased pulmonary blood flow [3, 6]. PV stenosis and atresia (lack of valve formation), tricuspid valve atresia, Ebstein’s anomaly, and the tetralogy of Fallot all fall under this category [1].

The etiology of CHD remains largely unknown [1, 4, 7]. As pediatric patients are typically subjected to multiple invasive procedures throughout their lifetimes in order to normalize pulmonary blood flow [8, 9], a better understanding of the disease state is necessary for the development of durable repair techniques and replacement devices, particularly as relates to tissue engineering and scaffold materials [10–12].

This undertaking has proven difficult in the past due to the limited availability of published data and the small size of the samples, which have led to substantial difficulties in mechanical testing. Despite these limitations, efforts have been devoted towards investigating the human pediatric aorta [13–15] and pulmonary artery [15–17], but no evaluation of valvular mechanics, to our knowledge, has been published.

To address these gaps in the literature, the following report presents the mechanical and structural behavior of PV leaflets from two pediatric cases with different disease etiologies. Properties were evaluated through planar biaxial tensile testing and second harmonic generation (SHG) imaging.

2. Materials and Methods

2.1. Patient Characteristics

Pulmonary leaflets from two pediatric patients with different valvar pathology were obtained from Connecticut Children’s Medical Center (Hartford, CT), as shown in Figure 1. These tissue samples were obtained as part of the surgical procedure, which would have been otherwise discarded. The study was approved by the Georgia Institute of Technology Institutional Review Board. Sample thickness was measured and averaged in three distinct locations throughout the testing region using a Mitutoyo 7301 rotating thickness gage (Aurora, IL, ± 0.01 mm resolution).

Figure 1.

PV leaflet samples for Patient 1 (A) and Patient 2 (B). Ruler tick marks indicate 1 mm.

Patient 1: 9-month-old male infant with diagnosis of Noonan syndrome, dysplastic pulmonary valve, supra-valvar pulmonary stenosis and atrial septal defect. The patient underwent two pulmonary balloon valvuloplasties.

Patient 2: 6-month-old female infant with tricuspid atresia, normally related great arteries, restrictive VSD and valvar pulmonary stenosis.

2.2. Biaxial Tensile Testing

Samples were subjected to biaxial tensile testing as per Sacks and Sun [18]. Briefly, a square section of the pulmonary valve was delimited by 8 suture hooks, 2 per side, such that the circumferential direction corresponded with the X₁ testing axis and the radial with the X₂. The sample was mounted onto a testing machine in a trampoline-like fashion, and submerged in a 0.9% saline solution maintained at 37° Celsius for the duration of the test. A stress-controlled testing protocol was administered, such that the ratio of the normal Lagrangian stress components P₁₁:P₂₂ was predefined with shear terms P₁₂ = P₂₁ = 0. Samples were subjected to a minimum of 30 equibiaxial preconditioning cycles, followed by testing protocols with varied P₁₁:P₂₂ ratio. Equibiaxial results are reported, with all biaxial plots showing Green Strain, defined as E = ½ (F^TF – I), and Second Piola-Kirchoff Stress, S. Stiffness was evaluated by means of the upper tangent modulus (Figure 2).

Figure 2.

Evaluation of stiffness and extensibility.

2.3. Structural Imaging

Tissues were imaged on a Zeiss 710 NLO inverted confocal microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA) equipped with a mode-locked Ti:Sapphire Chameleon Ultra laser (Coherent Inc., Santa Clara, CA) in combination with non-descanned detection (NDD) to look at the collagen fibers. The laser was set to 800 nm and emission was filtered from 380–430 nm. Samples were kept hydrated with saline solution during imaging to prevent drying artifacts and SHG was collected from the fibrosa and ventricularis sides of the tissue on the belly of the leaflet using a Plan-Apochromat 40x oil immersion objective. Zeiss ZEN software was used to visualize and export image stacks for analysis.

3. Results and Discussion

As shown in Figures 3 and 4, the two samples presented markedly different mechanical and structural properties, despite being from patients of similar ages (9 and 6 months for Patients 1 and 2, respectively). Similar to healthy adult and animal PV leaflets [20, 21], anisotropy was seen in both pediatric samples, with the circumferential direction being stiffer than the radial direction. Additionally, Patient 2 exhibited a less extensible mechanical response along the radial direction (maximum strains of 0.4 vs. 0.2 for Patients 1 and 2, respectively), but was more extensible along the circumferential direction. Overall, Patient 2 exhibited a more isotropic response than Patient 1.

Figure 3.

Equibiaxial response curve for (A) aged human pulmonary leaflet, (B) Patient 1, (C) Patient 2. Panel A is adopted from Pham et al. [19].

Figure 4.

Representative SHG images from the respective ventricularis and fibrosa sides of the PV leaflet for Patient 1 (A, B) and Patient 2 (C, D).

Previously published adult data from our lab shows PV stiffness at 5647 and 2179 kPa along the circumferential and radial directions, respectively [21]. It is interesting to note that the leaflet stiffness from the stenotic tissue of Patient 1 (10052 kPa and 3807 kPa in the post-transitional regions along the circumferential and radial directions respectively) was stiffer than that of adult cardiac tissue, while that of Patient 2 was more compliant along the circumferential direction (2779 kPa), but on par with that of much older adults in the radial direction (764 kPa). As tissue has been known to stiffen with age, the comparable stiffness of these tissues between pediatric tissue and that of older adults points to the extent of the mechanical effects of their diseased states.

Structurally, the leaflets exhibited similar collagen structure on each side, with straighter collagen fibers on the ventricularis side compared to the more tightly crimped collagen structure of the fibrosa side for both patients. Moreover, the collagen fibers of the ventricularis side were predominantly aligned in the radial direction for both patients, while on the fibrosa side they were more aligned along the circumferential direction. These results are consistent with previously published human and animal studies investigating collagen fiber alignment [22–24]. Patient 1 showed a higher variation of collagen fiber orientation in the fibrosa side (average fiber orientation: 47.8 ± 18.1 ° and −14.4 ± 34.14° for the ventricularis and fibrosa sides, respectively), while Patient 2 had higher variations in the ventricularis side (average fiber orientation: 86.4 ± 38.1 ° and 2.6 ± 1° for the ventricularis and fibrosa sides, respectively). The less organized distribution of collagen fibers in Patient 2 suggests remodeling on the ventricularis side [25], a point of interest for future studies investigating cardiac remodeling as a result of tricuspid atresia.

4. Conclusions

This study investigated the mechanical and structural behavior of PV leaflets from two pediatric CHD cases through planar biaxial tensile testing and SHG imaging. The two leaflets showed relatively similar collagenous structure and distribution in the two leaflets, but differing in their mechanical responses. Patient 2, suffering from tricuspid atresia, had more isotropic leaflet behavior than Patient 1, who suffered from a dysplastic PV. Interestingly, tissue stiffness was comparable to or stiffer than that of adult human PV leaflets. Continued investigation of the mechanics and structure of CHD patients can lead to a deeper understanding of this complex mechanical and structural disease environment and to future development of advanced repair and replacement devices and techniques.