Early in vivo experience with tissue-engineered trileaflet heart valves.

BACKGROUND: Tissue engineering is a new approach in which techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional autologous tissue. Workers at our laboratory have focused on tissue engineering of heart valves. The present study was designed to evaluate the implantation of a whole trileaflet tissue-engineered heart valve in the pulmonary position in a lamb model. METHODS AND RESULTS: We constructed a biodegradable and biocompatible trileaflet heart valve scaffold that was fabricated from a porous polyhydroxyalkanoate (pore size 180 to 240 microm; Tepha Inc). Vascular cells were harvested from ovine carotid arteries, expanded in vitro, and seeded onto our heart valve scaffold. With the use of cardiopulmonary bypass, the native pulmonary leaflets were resected, and 2-cm segments of pulmonary artery were replaced by autologous cell-seeded heart valve constructs (n=4). One animal received an acellular valved conduit. No animal received any anticoagulation therapy. Animals were killed at 1, 5, 13, and 17 weeks. Explanted valves were examined histologically with scanning electron microscopy, biochemically, and biomechanically. All animals survived the procedure. The valves showed minimal regurgitation, and valve gradients were <20 mm Hg on echocardiography. The maximum gradient was 10 mm Hg with direct pressures. Macroscopically, the tissue-engineered constructs were covered with tissue, and there was no thrombus formation on any of the specimens. Scanning electron microscopy showed smooth flow surfaces during the follow-up period. Histological examination demonstrated laminated fibrous tissue with predominant glycosaminoglycans as extracellular matrix. 4-Hydroxyproline assays demonstrated an increase in collagen content as a percentage of native pulmonary artery (1 week 45.8%, 17 weeks 116%). DNA assays showed a comparable number of cells in all explanted samples. There was no tissue formation in the acellular control. CONCLUSIONS: Tissue-engineered heart valve scaffolds fabricated from polyhydroxyalkanoates can be used for implantation in the pulmonary position with an appropriate function for 120 days in lambs.

Functional living trileaflet heart valves grown in vitro.

BACKGROUND: Previous tissue engineering approaches to create heart valves have been limited by the structural immaturity and mechanical properties of the valve constructs. This study used an in vitro pulse duplicator system to provide a biomimetic environment during tissue formation to yield more mature implantable heart valves derived from autologous tissue. METHODS AND RESULTS: Trileaflet heart valves were fabricated from novel bioabsorbable polymers and sequentially seeded with autologous ovine myofibroblasts and endothelial cells. The constructs were grown for 14 days in a pulse duplicator in vitro system under gradually increasing flow and pressure conditions. By use of cardiopulmonary bypass, the native pulmonary leaflets were resected, and the valve constructs were implanted into 6 lambs (weight 19+/-2.8 kg). All animals had uneventful postoperative courses, and the valves were explanted at 1 day and at 4, 6, 8, 16, and 20 weeks. Echocardiography demonstrated mobile functioning leaflets without stenosis, thrombus, or aneurysm up to 20 weeks. Histology (16 and 20 weeks) showed uniform layered cuspal tissue with endothelium. Environmental scanning electron microscopy revealed a confluent smooth valvular surface. Mechanical properties were comparable to those of native tissue at 20 weeks. Complete degradation of the polymers occurred by 8 weeks. Extracellular matrix content (collagen, glycosaminoglycans, and elastin) and DNA content increased to levels of native tissue and higher at 20 weeks. CONCLUSIONS: This study demonstrates in vitro generation of implantable complete living heart valves based on a biomimetic flow culture system. These autologous tissue-engineered valves functioned up to 5 months and resembled normal heart valves in microstructure, mechanical properties, and extracellular matrix formation.

Evaluation of biodegradable, three-dimensional matrices for tissue engineering of heart valves.

A crucial factor in tissue engineering of heart valves is the type of scaffold material. In the following study, we tested three different biodegradable scaffold materials, polyglycolic acid (PGA), polyhydroxyalkanoate (PHA), and poly-4-hydroxybutyrate (P4HB), as scaffolds for tissue engineering of heart valves. We modified PHA and P4HB by a salt leaching technique to create a porous matrix. We constructed trileaflet heart valve scaffolds from each polymer and tested them in a pulsatile flow bioreactor. In addition, we evaluated the cell attachment to our polymers by creating four tubes of each material (length equals 4 cm; inner diameter, 0.5 cm), seeding each sample with 8,000,000 ovine vascular cells, and incubating the cell-polymer construct for 8 days (37 degrees C and 5% CO2). The seeded vascular constructs were exposed to continuous flow for 1 hour. Analysis of samples included DNA assay before and after flow exposure, 4-hydroxyproline assay, and environmental scanning electron microscopy (ESEM). We fabricated trileaflet heart valve scaffolds from porous PHA and porous P4HB, which opened and closed synchronously in a pulsatile bioreactor. It was not possible to create a functional trileaflet heart valve scaffold from PGA. After seeding and incubating the PGA-, PHA-, and P4HB-tubes, there were significantly (p < 0.001) more cells on PGA compared with PHA and P4HB. There were no significant differences among the materials after flow exposure, but there was a significantly higher collagen content (p < 0.017) on the PGA samples compared with P4HB and PHA. Cell attachment and collagen content was significantly higher on PGA samples compared with PHA and P4HB. However, PHA and P4HB also demonstrate a considerable amount of cell attachment and collagen development and share the major advantage that both materials are thermoplastic, making it possible to mold them into the shape of a functional scaffold for tissue engineering of heart valves.

A low-flow adaptation phase improves shear-stress resistance of artificially seeded endothelial cells.

INTRODUCTION: The purpose of this study was to evaluate the effect of different adaptation phases on the shear-stress resistance of endothelial cells seeded artificially onto vascular prostheses and biological heart valves. MATERIAL AND METHODS: Human endothelial cells (EC), fibroblasts (FB), and smooth muscle cells (SMC) were isolated from vena saphena magna pieces and expanded in culture. Group A: 15 polyurethane vascular grafts (20 mm diameter) were seeded with FB and SMC (53 +/- 1.2 million cells), followed by EC seeding (39 +/- 0.9 million cells). Group B: eight stentless porcine valves (Freestyle, Medtronic, USA) were seeded with FB (68 +/- 1.5 million cells) and EC (42 +/- 1.1 million cells). Shear-stress testing was done under pulsatile flow (pulse rate: 80 pulses/min.). Adaptation phase: flow was set to 0.9 +/- 0.3 l/min (systolic pressure: 40 - 50 mm Hg). High flow was 3.2 +/- 0.6 l/min. (systolic pressure: 140 - 160 mm Hg) and lasted over four hours in all groups. The vascular grafts were divided into three groups (n = 5 each): group 1 (high flow immediately), group 2 (adaptation phase of 15 minutes), and group 3 (adaptation phase of 30 minutes). The valves either were given high flow immediately (n = 4) or had an adaptation phase of 30 minutes (n = 4). Specimens were obtained after cell seeding, before, and after perfusion. RESULTS: A confluent EC layer was achieved on all grafts. After perfusion without adaptation, large defects within the cell layer were found. No FB and SMC were seen at the bottom of these defects. In group B, the defects were largest on the ventricular surface of the leaflets. After an adaptation phase of 15 minutes in group A, only a few defects within the EC layer were detected with a still confluent FB and SMC. After a 30-minute adaptation phase defects within the EC layer were very rare and no interruption of the underlying FB and SMC layer was seen. Immunohistochemical staining for factor VIII and CD31 proved the EC to be viable and staining for collagen IV and laminin revealed the formation of a basement membrane. After perfusion, the specimen also stained positive for eNOS. CONCLUSION: An adaptation phase of 30 minutes proved to be sufficient to allow artificially seeded endothelial cells to adapt to shear stress. The formation of a basement membrane was of great importance for the maintenance of a confluent EC layer.