Minimally-Invasive Implantation of Living Tissue Engineered Heart Valves

A Comprehensive Approach From Autologous Vascular Cells to Stem Cells

Cell Harvest

Autologous myofibroblasts were harvested from ovine jugular veins as previously described (13). Endothelial cells were harvested from ovine carotid arteries. The myofibroblasts were expanded in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% lamb serum (Gibco, Invitrogen, Carlsbad, California), 1% GlutaMax (Gibco), and 0.1% gentamycin (Biochrom, Berlin, Germany). The endothelial cells were cultured in endothelial basal medium (EBM-2, complemented with 0.1% vascular endothelial growth factor, 0.4% human fibroblasts growth factor, 0.1% human recombinant long–insulin-like growth factor-1, 0.1% human epidermal growth factor, 0.1% gentamycin and amphotericin, 0.04% hydrocortisone, 0.1% heparin, 0.1% ascorbic-acid [Cambrex, Verviers, Belgium]) supplemented with 10% lamb serum. All animals received humane care, and the study was approved by the ethics committee (IMM Recherche, Paris, France) and was in compliance with the “Guide for the Care and Use of Laboratory Animals,” published by the National Institutes of Health (NIH publication no. 85-23, revised 1985).

Heart Valve Scaffold Fabrication

Trileaflet heart valve scaffolds (n = 16), were fabricated from nonwoven polyglycolic acid meshes (thickness 1.0 mm; specific gravity 70 mg/cm³; Cellon, Bereldange, Luxembourg), coated with 1.75% poly-4-hydroxybutyrate (MW: 1 × 10⁶; Tepha, Lexington, Massachusetts) by dipping into a tetrahydrofuran solution (Fluka, Steinheim, Germany). After solvent evaporation, physical bonding of adjacent fibers and continuous coating was achieved. Poly-4-hydroxybutyrate is a biologically derived rapidly degradable biopolymer, which besides being strong and pliable, is thermoplastic (61°C), and can be molded into 3-dimensional shapes. From the polyglycolic acid/poly-4-hydroxybutyrate composite scaffold material, the heart valve scaffolds were fabricated by using a heat-application welding technique. Thereafter, the scaffolds were integrated into radially self-expandable nitinol stents (length = 38 mm, outer diameter [OD] = 30 mm when fully expanded at 37°C; pfm AG, Köln, Germany) by attaching the scaffold matrix to the inner surface of the nitinol stent wires using single interrupted sutures (5-0 Prolene, Ethicon, Somerville, New Jersey). After vacuum drying overnight, the scaffolds were placed in 70% ethanol (Sigma, St. Louis, Missouri) for 4 h to obtain sterility. The ethanol was allowed to evaporate overnight, the scaffolds were washed in PBS (Sigma) and incubated overnight in medium DMEM Advanced, supplemented with 2.5% lamb serum, 1% GlutaMax, 0.3% gentamycin, and additional L-ascorbic acid 2-phosphate (0.25 mg/ml, Sigma), referred to as TE-medium, to facilitate cell attachment by deposition of proteins.

Cell Seeding and In Vitro Culturing of TEHV

Seeding of the myofibroblasts onto the stented heart valve scaffolds (1.5 ± 0.04 × 10⁶cells/cm²) was performed using fibrin as cell carrier (14). After seeding, the scaffolds were placed into pulse duplicator systems, exposing the leaflets to increasing dynamic strains, as previously described (15). Medium was replaced every 2 to 3 days. After 11 to 12 days, the TEHV were seeded with endothelial cells (1.6 ± 0.2 × 10⁵cells/cm²) and cultured for 2 to 3 additional days (n = 12). For optimized surface coverage of the free edges, a subgroup of TEHV (n = 4) was cultured for 6 additional days under the same dynamic straining and in addition flow conditioning (nonpulsatile, low-pressure flow).

TEHV Delivery Procedures

After the in vitro culture period, the OD was decreased from 30 mm to 10 mm (Fig. 2A to 2D)using a custom-built tubular-shaped crimping and delivery system (Acrostak, Geneva, Switzerland). The TEHV were left in the device for 30 min, representing a clinically relevant time frame. Thereafter, the TEHV were either implanted (n = 6) or directly analyzed (n = 5; 1 sample was excluded because of technical preservation problems).

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Figure 2

Crimping of TEHV for Transapical Delivery

The diameter was gradually reduced by a factor of 2.5 (A to D)before the tissue engineered heart valve (TEHV) was introduced into the implantation device.

In Vitro Valve Testing

The in vitro valve performance of a TEHV after simulated delivery procedure was visualized in a custom-built valve tester (16), using a high-speed video camera (Phantom version 9.0, Vision Research, Wayne, New Jersey). The TEHV was exposed to physiological systemic flows for 20 min, and the resulting pressure and flow profiles were measured using flow (Transonic Systems, Ithaca, New York) and pressure sensors (Becton Dickinson, Erembodegem, Belgium). The amount of regurgitation, the effective orifice area, and the mean and maximal systolic pressure gradients were calculated and averaged over 5 cycles.

TEHV Implantation and In Vivo Functionality

For evaluation of in vivo functionality TEHV were minimally-invasively delivered into the pulmonary valve position using an anterolateral-thoracic access and antegrade approach. The valves were crimped and loaded onto a custom-made inducing system (OD = 12 mm) consisting of a rigid tube and pusher. After minithoracotomy and after pericardiotomy, the right ventricle was punctured using needle through pursestring sutures. Subsequently, the inducing system was inserted, and the valve delivered into the pulmonary artery under visualization by fluoroscopy. After the optimal position for the pulmonary heart valve replacement was defined by angiography, the valve was pushed out of the delivery system and allowed to open (Figs. 3A and 3B). Thereby, the radial forces of the nitinol stent pressed the native heart valve leaflets to the artery wall and kept them fixed. Afterward, the appropriate position and functionality of the implanted valve was visualized by angiography. Moreover, the in vivo functionality was monitored using transthoracic echocardiography during the procedure, immediately after implantation, and then weekly up to 8 weeks. Anticoagulation therapy was maintained as in conventional bioprosthetic heart valves for 7 days. The animals were sacrificed, and the TEHV were explanted after 4 weeks (n = 3) and 7 to 8 weeks (n = 3).

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Figure 3

Delivery of the TEHV

Schematic view of transapical valve delivery (A)and positioning (B)in the pulmonary position. The delivery system loaded with the crimped tissue engineered heart valve (TEHV) is inserted transapically into the right ventricle (a). The delivery system consists of an inner pusher (c)and a sheath (d). The crimped TEHV integrated into the nitinol stent (b)is carefully deployed into the pulmonary position by slowly advancing the inner pusher (c)into the sheath system (d).

Qualitative Tissue Analyses

The TEHV (n = 16), either directly or after having functioned as implant, were evaluated macroscopically, and tissue composition was analyzed qualitatively by immunohistology and compared with native valve leaflets. The tissue sections were studied by hematoxylin and eosin staining for general tissue morphology, Masson-Trichrome staining for deposition of collagen, and Elastin van Gieson staining for detection of collagen and elastic fibers. Additionally, immunohistology was performed using the Ventana Benchmark automated staining system (Ventana Medical Systems, Oro Valley, Arizona) and antibodies for α-smooth muscle actin (α-SMA [clone 1A4, Sigma]) and von Willebrand factor (affinity purified rabbit antibodies, DakoCytomation, Copenhagen, Denmark). Primary antibodies were detected with the Ventana iVIEW DAB detection kit, yielding a brown reaction product. Furthermore, tissue samples were fixed in 2% glutaraldehyde (Sigma) and sputtered with platin to examine the surface structure by scanning electron microscopy.

Quantitative Tissue Analyses

The TEHV used to study the effect of the delivery procedure (n = 2 uncrimped, and n = 2 crimped), the explants (n = 6) and their corresponding control valves and native ovine valves (n = 3) were analyzed by biochemical assays for total deoxyribonucleic acid (DNA) content, as an indicator for cell number, glycosaminoglycans (GAG) content, and hydroxyproline content, as an indicator for collagen. For measuring DNA amount, the Hoechst dye method (17) was used with a standard curve prepared from calf thymus DNA (Sigma) in addition to the samples. The GAG content was determined using a modified version of the protocol described by Farndale et al. (18), and a standard curve was prepared from chondroitin sulphate from shark cartilage (Sigma) in addition to the samples. Hydroxyproline was determined with a modified version of the protocol provided by Huszar et al. (19), and a standard curve was prepared in addition to the samples.

Mechanical Properties

The mechanical properties of the leaflets were determined in circumferential and radial direction by uniaxial tensile tests for the TEHV used to study the effect of the delivery procedure (n = 2 uncrimped, and n = 2 crimped). Furthermore, in the explants, and the corresponding control valves, thickness of the samples was determined with a PLμ 2300 optical imaging profiler (Sensofar-Tech S.L., Barcelona, Spain). Stress-strain curves were obtained using a uniaxial tensile tester (Kammrath & Weiss GmbH, Dortmund, Germany; load cell of 20N) with a constant strain rate of 1.7%/s. The ultimate tensile stress (UTS) was determined from the curves. The modulus of elasticity (further referred to as modulus) was obtained from the slope of the linear section of the stress-strain curve as an indicator for tissue stiffness.

Sandwich-Structured Heart Valve Scaffolds

These scaffolds were fabricated from biodegradable P(L,DL)LA [Poly(L-lactide-co-D,L-lactide)] multifilament fibers using a 3-dimensional valve-shaped cast and thermal fixation. Thereafter, the surfaces were coated with P(L,DL)LA based nanofibers by electrospinning in order to produce a multilayer structure mimicking the architecture of native heart valves. The P(L,DL)LA was dissolved in 1,1,1,3,3,3-hexafluoro-2-isopropanol and the polymer solution was electrospun onto the surfaces of the molded structure. The resulting sandwich-structured heart valve scaffolds were allowed to dry and were then sterilized by gamma irradiation afterward. Finally, the scaffolds were integrated into self-expanding nitinol stents (OD = 30 mm), as in experimental study A.

Cell Harvest, Isolation, and Seeding

Adult stem cells were obtained from bone marrow as well as peripheral blood of adult sheep (INRA Brouëssyand; ethical approval no. Zurich-08-04, Switzerland). For the isolation of myofibroblast-like cells, 20 ml bone marrow was aspirated from the iliac crest, and mononuclear cells were isolated using density gradient centrifugation (Histopaque-1077, Sigma) (20). Cells were collected from the interphase and cultured in DMEM (as described in study A) under humidified incubator conditions (CO₂5%, 37°C). Endothelial progenitor cells were collected from 20 ml peripheral blood, using the same density gradient and centrifugation as described previously (21,22). Subsequently, cells were exposed to endothelial basal medium (as described for study A) to enable endothelial cell differentiation.

According to study A, sandwich-structured trileaflet heart valve scaffolds (n = 4) were seeded with bone marrow-derived myofibroblast-like cells using fibrin as a cell carrier (14) and cultured under static conditions. After 6 days, the heart valves were inserted into a flow-bioreactor system (23) and exposed to pulsatile flow for 3 additional days followed by endothelialization using autologous peripheral blood-derived endothelial cells. After seeding, the constructs were kept under static conditions for 3 additional days to enable endothelial cell attachment and proliferation on the valve surfaces.

TEHV Implantation, In Vivo Functionality, and Analyses

In vivo performance up to 4 weeks of TEHV (n = 2) was evaluated in pulmonary position. Next to each implanted TEHV, an additional valve was engineered for each animal to serve as a control valve (n = 2). Minimally-invasive implantation procedures, assessment of TEHV functionality and tissue analysis methodology was performed as in the experimental study A. In addition to the immunohistology of study A, tissue sections were also stained for endothelial nitric oxide synthase (eNOS) type III (affinity purified rabbit antibodies; BD Biotechnology, Santa Cruz, California) with signal enhancement using the Ventana amplification kit.