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.

Tissue-engineered valved conduits in the pulmonary circulation.

OBJECTIVE: Bioprosthetic and mechanical valves and valved conduits are unable to grow, repair, or remodel. In an attempt to overcome these shortcomings, we have evaluated the feasibility of creating 3-leaflet, valved, pulmonary conduits from autologous ovine vascular cells and biodegradable polymers with tissue-engineering techniques. METHODS: Endothelial cells and vascular medial cells were harvested from ovine carotid arteries. Composite scaffolds of polyglycolic acid and polyhydroxyoctanoates were formed into a conduit, and 3 leaflets (polyhydroxyoctanoates) were sewn into the conduit. These constructs were seeded with autologous medial cells on 4 consecutive days and coated once with autologous endothelial cells. Thirty-one days (+/-3 days) after cell harvesting, 8 seeded and 1 unseeded control constructs were implanted to replace the pulmonary valve and main pulmonary artery on cardiopulmonary bypass. No postoperative anticoagulation was given. Valve function was assessed by means of echocardiography. The constructs were explanted after 1, 2, 4, 6, 8, 12, 16, and 24 weeks and evaluated macroscopically, histologically, and biochemically. RESULTS: Postoperative echocardiography of the seeded constructs demonstrated no thrombus formation with mild, nonprogressive, valvular regurgitation up to 24 weeks after implantation. Histologic examination showed organized and viable tissue without thrombus. Biochemical assays revealed increasing cellular and extracellular matrix contents. The unseeded construct developed thrombus formation on all 3 leaflets after 4 weeks. CONCLUSION: This experimental study showed that valved conduits constructed from autologous cells and biodegradable matrix can function in the pulmonary circulation. The progressive cellular and extracellular matrix formation indicates that the remodeling of the tissue-engineered structure continues for at least 6 months.

Prediction of safe duration of hypothermic circulatory arrest by near-infrared spectroscopy.

OBJECTIVE: Hypothermic circulatory arrest is widely used for adults with aortic arch disease as well as for children with congenital heart disease. At present, no method exists for monitoring safe duration of circulatory arrest. Near-infrared spectroscopy is a new technique for noninvasive monitoring of cerebral oxygenation and energy state. In the current study, the relationship between near-infrared spectroscopy data and neurologic outcome was evaluated in a survival piglet model with hypothermic circulatory arrest. METHODS: Thirty-six piglets (9.36 +/- 0.16 kg) underwent circulatory arrest under varying conditions with continuous monitoring by near-infrared spectroscopy (temperature 15 degrees C or 25 degrees C, hematocrit value 20% or 30%, circulatory arrest time 60, 80, or 100 minutes). Each setting included 3 animals. Neurologic recovery was evaluated daily by neurologic deficit score and overall performance category. Brain was fixed in situ on postoperative day 4 and examined by histologic score. RESULTS: Oxygenated hemoglobin signal declined to a plateau (nadir) during circulatory arrest. Time to nadir was significantly shorter with lower hematocrit value (P <.001) and higher temperature (P <.01). Duration from reaching nadir until reperfusion ("oxygenated hemoglobin signal nadir time") was significantly related to histologic score (r (s) = 0.826), neurologic deficit score (r (s) = 0.717 on postoperative day 1; 0.716 on postoperative day 4), and overall performance category (r (s) = 0.642 on postoperative day 1; 0.702 on postoperative day 4) (P <.001). All animals in which oxygenated hemoglobin signal nadir time was less than 25 minutes were free of behavioral or histologic evidence of brain injury. CONCLUSION: Oxygenated hemoglobin signal nadir time determined by near-infrared spectroscopy monitoring is a useful predictor of safe duration of circulatory arrest. Safe duration of hypothermic circulatory arrest is strongly influenced by perfusate hematocrit value and temperature during circulatory arrest.

New pulsatile bioreactor for in vitro formation of tissue engineered heart valves.

Two potential obstacles to the creation of implantable tissue engineered heart valves are inadequate mechanical properties (ability to withstand hemodynamic stresses) and adverse host-tissue reactions due to the presence of residual nondegraded polymer scaffold. In an attempt to address these problems, we developed an in vitro cell culture system that provides physiological pressure and flow of nutrient medium to the developing valve constructs. It is anticipated that in vitro physical stress will stimulate the tissue engineered heart valve construct to develop adequate strength prior to a possible implantation. Long-term in vitro development will be realized by an isolated and thereby contamination-resistant system. Longer in vitro development will potentially enable more complete biodegradation of the polymeric scaffold during in vitro cultivation. This new dynamic bioreactor allows for adjustable pulsatile flow and varying levels of pressure. The system is compact and easily fits into a standard cell incubator, representing a highly isolated dynamic cell culture setting with maximum sterility, optimal gas supply and stable temperature conditions especially suited for long-term experiments.

Fabrication of a trileaflet heart valve scaffold from a polyhydroxyalkanoate biopolyester for use in tissue engineering.

Previously, we reported the implantation of a single tissue engineered leaflet in the posterior position of the pulmonary valve in a lamb model. The major problems with this leaflet replacement were the scaffold's inherent stiffness, thickness, and nonpliability. We have now created a scaffold for a trileaflet heart valve using a thermoplastic polyester. In this experiment, we show the suitability of this material in the production of a biodegradable, biocompatible scaffold for tissue engineered heart valves. A heart valve scaffold was constructed from a thermoplastic elastomer. The elastomer belongs to a class of biodegradable, biocompatible polyesters known as polyhydroxyalkanoates (PHAs) and is produced by fermentation (Metabolix Inc., Cambridge, MA). It was modified by a salt leaching technique to create a porous, three-dimensional structure, suitable for tissue engineering. The trileaflet heart valve scaffold consisted of a cylindrical stent (1 mm X 15 mm X 20 mm I.D.) containing three valve leaflets. The leaflets were formed from a single piece of PHA (0.3 mm thick), and were attached to the outside of the stent by thermal processing techniques, which required no suturing. After fabrication, the heart valve construct was allowed to crystallize (4 degrees C for 24 h), and salt particles were leached into doubly distilled water over a period of 5 days to yield pore sizes ranging from 80 to 200 microns. Ten heart valve scaffolds were fabricated and seeded with vascular cells from an ovine carotid artery. After 4 days of incubation, the constructs were examined by scanning electron microscopy. The heart valve scaffold was tested in a pulsatile flow bioreactor and it was noted that the leaflets opened and closed. Cells attached to the polymer and formed a confluent layer after incubation. One advantage of this material is the ability to mold a complete trileaflet heart valve scaffold without the need for suturing leaflets to the conduit. Second advantage is the use of only one polymer material (PHA) as opposed to hybridized polymer scaffolds. Furthermore, the mechanical properties of PHA, such as elasticity and mechanical strength, exceed those of the previously utilized material. This experiment shows that PHAs can be used to fabricate a three-dimensional, biodegradable heart valve scaffold.

Tissue engineering of heart valves: in vitro experiences.

BACKGROUND: Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. METHODS: We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. RESULTS: It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. CONCLUSIONS: Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.

Experience with spiral computed tomography as the sole diagnostic method for traumatic aortic rupture.

BACKGROUND: Spiral computed tomographic (CT) scan is an excellent screen for aortic trauma. Traditionally, aortography is performed when injury is suspected to confirm the diagnosis. We hypothesized that it is safe and expeditious to forgo aortography when the spiral CT demonstrates aortic injury. METHODS: Retrospective review of 54 patients undergoing aortic repair from July 1994 to December 1999. Spiral CT was the initial diagnostic study in 52 patients. Pseudoaneurysm or aortic wall defect in the presence of mediastinal hematoma was considered diagnostic. Angiography, initially routine, was later performed only when requested by the surgeon, and for all "nonnegative" studies (periaortic hematoma without detectable aortic injury). RESULTS: Twenty-six patients underwent angiography before operation (group 1). Nineteen group 1 spiral CTs were unequivocally diagnostic; 7 were nonnegative and angiography was required. Twenty-eight other patients underwent repair based on spiral CT alone (group 2). There was one false-positive result in both groups. There were no unexpected operative findings. Mean time from admission to diagnosis was 5.7+/-3.4 hours for group 1 and 1.7+/-1.7 hours for group 2 (p < 0.01). CONCLUSIONS: Operating on the basis of a diagnostic spiral CT is safe and expeditious. Aortography may be reserved for those with equivocal studies.

Surgically confirmed incorporation of a chronically retained neurointerventional microcatheter in the carotid artery.

A woman reported painful thrombosis of the superficial femoral artery 16 months after a transfemoral microcatheter was glued into a cerebral arteriovenous malformation and transected at the groin. When the catheter was removed, a portion was found to be incorporated into the wall of the carotid artery. This case demonstrates that portions of a retained microcatheter may be incorporated into the arterial wall while other portions may remain mobile and cause late peripheral arterial symptoms.

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.