(Circulation. 2000;102:III-44.)
© 2000 American Heart Association, Inc.
Surgery for Valvular Heart Disease |
Functional Living Trileaflet Heart Valves Grown In Vitro
From the Departments of Cardiovascular Surgery (S.P.H., R.S., S.D., J.W., E.A.B., K.J.G., J.S.S., S.K., J.E.M.) and Cardiology (A.M.M.), Children’s Hospital Boston, the Department of Pathology (F.J.S.), Brigham and Women’s Hospital, and the Department of Surgery (J.P.V.), Massachusetts Hospital Boston, Harvard Medical School, Boston, Mass; and Tepha Inc (D.P.M.), Cambridge, Mass.
Correspondence to Simon Philipp Hoerstrup, MD, Tissue Engineering Research, Department of Cardiovascular Research, Clinic for Cardiovascular Surgery, University Hospital, Raemistrasse 100, CH-8091 Zurich, Switzerland. E-mail simon_philipp.hoerstrup{at}chir.usz.ch
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Abstract |
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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.
Key Words: tissue • valves • cells • prosthesis
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Introduction |
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Valve replacement represents the most common surgical therapy for end-stage valvular heart disease, with >60 000 implantations in the United States and 170 000 worldwide.1 Valve replacement surgery is efficacious, and it substantially changes the natural history of valvular disease.2 However, mechanical valves are associated with a substantial risk of thromboembolism, and tissue valves suffer from structural dysfunction due to progressive tissue deterioration.1 3 4 Because all clinically used tissue valve substitutes are nonviable, they have no potential to grow, to repair, or to remodel. Therefore, their durability is limited, especially in growing children.5
In an attempt to address the shortcomings of current valve options, we previously reported the feasibility of replacing a single pulmonary valve leaflet by a tissue-engineered (TE) autologous leaflet.6 In subsequent studies, we focused on the in vitro generation of a complete trileaflet heart valve.7 A substantial limitation was structural and mechanical "immaturity" of the constructs, which had insufficient mechanical properties and functional performance after implantation. Subsequently, more durable scaffold materials that provided better mechanical function were tested. However, because of their prolonged degradation time, they persisted in vivo and were not sufficiently replaced by autologous tissue.8 The ideal concept of a TE heart valve includes formation of functional valve constructs on the basis of a rapidly absorbable scaffold. The scaffold provides a temporary biomechanical profile until the cells produce their own matrix proteins. The structural integrity and biomechanical profile of the TE heart valves ultimately depend on this matrix formation.
We hypothesized that in vitro exposure of the developing tissue to physical signals similar to those encountered in vivo may result in more mature TE heart valves with more favorable functional performance. Accordingly, we developed a new TE approach that made use of an in vitro pulse duplicator system and a novel rapidly bioabsorbable composite scaffold material. The present study design included 2 experimental steps: the first set of experiments was undertaken to investigate whether a biomimetic culture environment guides tissue development to more mature TE heart valves in vitro, and the in vivo study that followed was performed to assess the practical utility and performance of these valve constructs.
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Methods |
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Bioabsorbable Trileaflet Valve Scaffold
Nonwoven polyglycolic-acid mesh (PGA, thickness 1.0 mm, specific gravity 69 mg · cm3, Albany Int) was coated with a thin layer of poly-4-hydroxybutyrate (P4HB, molecular weight 1x106, PHA 4400, Tepha Inc) by dipping into a tetrahydrofuran solution (1% [wt/vol] P4HB). After solvent evaporation, a continuous coating and physical bonding of adjacent fibers was achieved. P4HB is a biologically derived rapidly absorbable biopolymer that is not only strong and pliable but also thermoplastic (61°C) so that it can be molded into almost any shape. From the PGA/P4HB composite scaffold material, trileaflet valve scaffolds were fabricated by using a heat-application welding technique. The constructs were then cold gas–sterilized with ethylene oxide.
Cell Isolation and Culture
The general approach to cell isolation, culture, and seeding has
been previously described in detail.9 Briefly, 2- to 3-cm
segments of carotid artery were harvested from lambs (13±2.4 kg).
Endothelial cells were obtained by use of a
collagenase instillation technique, incubated for 20
minutes at 37°C and 5% CO2 in DMEM containing
0.2% collagenase type A (Boehringer-Mannheim) and
1% BSA (HyClone), and cultured on gelatin-precoated (1% gelatin,
Sigma Chemical Co) tissue culture flasks (Corning Inc) with the use of
medium 199 (GIBCO) supplemented with 10% FBS (HyClone), penicillin,
streptomycin (GIBCO), and 50 IU/mL heparin (Promega). To obtain
myofibroblasts, the remaining deendothelialized
vessel segments were minced and cultured on P100 dishes (Corning) in
DMEM (GIBCO) supplemented with 10% FBS (HyClone), penicillin, and
streptomycin (GIBCO). After migration of the myofibroblasts onto the
dishes (after 5 to 7 days), the cells were serially passaged and
expanded in a humidified incubator at 37°C and 5%
CO2. Sufficient cell numbers for cell seeding
were obtained in pure culture after 21 to 28 days. The
endothelial cells were characterized by the presence of
CD31 (platelet endothelial cell adhesion molecule [PECAM 1])
and von Willebrand factor (vWF); the myofibroblasts, by the
presence of smooth muscle actin (SMA) (Figure 1
).
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Cell Seeding and Conditioning in an In Vitro Pulse Duplicator
System
Myofibroblasts (4.5 to 5.5x106 per
cm2) were seeded onto the trileaflet valve
scaffolds and cultured in static nutrient medium (DMEM, GIBCO) for 4
days in a humidified incubator (37°C, 5% CO2).
Thereafter, the constructs (n=10) were seeded with
endothelial cells (1.5 to
2.0x106 per cm2),
transferred into a pulse duplicator system ("bioreactor," Figure 2), and grown under gradually increasing
nutrient medium flow and pressure conditions (125 mL/min at 30
mm Hg to 750 mL/min at 55 mm Hg) for 4, 7, 14, 21, and 28 days.
Controls (n=10) were grown in static nutrient medium accordingly. The
medium was changed every 7 days.
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Animal Implants
Cells were harvested, multiplied, and seeded onto the trileaflet
heart valve constructs as described above. After maturation in the
bioreactor for 14 days, the TE valves were functionally tested in the
system under high-pressure conditions (>150 mm Hg) for 60
minutes. Thereafter, they were implanted into the same lambs (n=6,
19±2.8 kg) from which the cells were initially harvested.
Anesthesia was induced with 2 mg/kg ketamine, 0.02
mg/kg atropine, and an intravenous bolus infusion of 2
mg/kg propofol and maintained by inhalational isoflurane. The heart was
exposed by a left anterolateral thoracotomy entering the chest through
the third intercostal space. Systemic anticoagulation was induced with
400 IU heparin/kg. By use of femoral arterial and right
atrial venous cannulation, normothermic
cardiopulmonary bypass was established. With the heart beating,
the main pulmonary artery was transected, and all 3 native
leaflets were excised. The TE heart valve constructs were implanted by
using running 5-0 monofilament sutures (Prolene, Ethicon). Heparin was
reversed with 300 IU protamine/kg after weaning from bypass, and the
chest was closed. No further anticoagulation was given.
Echocardiography (Hewlett-Packard Sonos 1500
Cardiac Imager equipped with a 7.5-MHz phased transducer), including
imaging from a long- and short-axis view, was performed after surgery
and at various time intervals for up to 20 weeks. The animals were
euthanized after 1 day and at 4, 6, 8, 16, and 20 weeks. Before
explantation, direct pressures were measured during surgery (Digital
Ultrasonic Measurement System, Sono Metrics Inc) proximal and distal to
the TE construct. All animals received humane care 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).
Microstructure
A representative portion of each trileaflet
valve construct was examined histologically by
hematoxylin and eosin stain (overall morphology) and Movat
pentachrome stain (for demonstration of matrix elements,
including collagen, elastin, and glycosaminoglycans
[GAGs]) and by immunohistochemistry for CD31, vWF, and SMA.
Additional samples were fixed in cacodylic acid (Sigma) for
environmental scanning electron microscopy (ESEM).
Tissue Analysis
Biochemical assays were performed for analysis of
cellular and extracellular components of the new tissue. Total DNA was
isolated and purified by sequential organic extractions with phenol and
phenol/chloroform/isoamyl alcohol and quantified by
spectrophotometry.10 For determination of total collagen
content, tissue was completely acid-digested, and total
4-hydroxyproline was measured.11 Total proteoglycan/GAG
and elastin content were quantified with a BLYSCAN and
FASTIN assay (Biocolor) after tissue extraction.
Mechanical Properties
Mechanical properties of the TE valve constructs and native
valves were evaluated by use of a mechanical tester (model Mini 55,
Instron Corp). Longitudinal matrix strips were used for the test. A
75-lb/inch2 maximum load cell was used, and the
cross-head speed was 0.5 in/min. Young’s modulus was obtained from the
slope of the initial linear section of the stress-strain curve.
Moreover, suture retention strength was measured.
Polymer Degradation Analysis
Percent residual polymer (PGA and PHA4400) in the dried tissue
was determined by gas chromatography. Lyophilized
tissue samples (
50 mg) were digested in a butanolysis reagent
(n-butanol/concentrated HCl 9:1, containing 2 mg/mL benzoic
acid as internal standard) for 2 hours at 110°C. The organic fraction
of the digests was analyzed by gas
chromatography (HP 5890, SPB1 column, Supelco).
Standard curves were generated by using glycolide and
-butyrolactone as standards.
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Results |
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In Vitro
In all valves, synchronous opening and closing of the leaflets was observed in the bioreactor, under both low-pressure (35 mm Hg) and high-pressure (>150 mm Hg) conditions. Gross appearance showed the most advanced tissue formation after 14 days (Figure 3
), without apparent differences at 21
and 28 days. All leaflets were intact, mobile, and pliable, and the
valve constructs were competent during valve closure. The controls
grown in static culture were fragile and began to lose structural
integrity after 14 days of static culture.
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Tissue Microstructure
Histology of the TE leaflets revealed cellular tissue organized in
a layered fashion with a dense outer layer and lesser cellularity in
the deeper portions after 14 days in the pulse duplicator (Figure 4). Formation of extracellular matrix was
demonstrated as predominantly GAGs and some collagen. SMA-positive
smooth muscle cells were detectable throughout the tissue. Tissue was
maximally organized after 14 days with no further increase after longer
culture duration in the pulse duplicator. The static controls showed
less tissue formation and organization at all time points. ESEM
demonstrated dense tissue and a confluent smooth surface with cell
orientation in the direction of the flow after 7 days, whereas the
controls showed a rough surface at all time points.
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Tissue Analysis
Collagen content was 129% that of native valve tissue at 14 days
and leveled off to 86% and 85% at 21 and 28 days, respectively. DNA
content of the constructs reached 80% that of native tissue at 7 days
and leveled off to 60% at 21 and 28 days. GAG content was 60% that of
native valve tissue at 14 days, with no further increase at 21 and 28
days. Elastin was not detectable in any TE leaflet up to 28 days. DNA
and collagen content were significantly lower in all specimens at all
time points in the static controls (Figure 5).
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Mechanical Testing
Suture retention strength was >50 g after 14 days (68, 65, and
66 g at 14, 21, and 28 days, respectively) versus a maximum of
12 g at 7 days in the static controls.
In Vivo
All animals survived the valve replacement procedure and had
uneventful postoperative courses. Echocardiography
performed after surgery and at 1, 2, 4, 8, 16, and 20 weeks
demonstrated mobile functioning leaflets without evidence of thrombus,
stenosis, or aneurysm formation up to 20 weeks after
implantation (Figure 6). At 16 and 20
weeks, central pulmonary regurgitation (mild to
moderate) was detected. The maximum transvalvular peak-to-peak
gradient was <10 mm Hg by direct intraoperative pressure
measurements in all TE valves at the time of explantation.
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, main pulmonary
artery). C and D, Short-axis view of the TE valve in closed (C) and
opened (D) position.
Gross appearance of all explanted trileaflet TE valves showed intact
mobile leaflets with a smooth ventricular and
arterial surface and no thrombus or stenosis. The
TE leaflets at 4, 6, and 8 weeks appeared thicker and less pliable than
the valves at 16 and 20 weeks (Figure 7).
There was an increase of the inner diameter of the valve constructs at
the level of leaflet attachments from an initial measurement of 19 to
23 mm at 20 weeks in accordance with the observed growth of the
native pulmonary artery.
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Tissue Microstructure
Histology (hematoxylin and eosin staining) showed a uniform
laminated structure with progressive thinning and organization of the
cuspal structure. At 16 and 20 weeks, the leaflets were layered with a
loose spongy layer on the ventricular (inflow) side and
fibrous layer on the arterial (outflow) side. Special
stains at 20 weeks revealed collagen in the fibrous layer and GAGs in
the central loose layer, whereas elastin could be detected near the
inflow surface (Figure 8). The structure
was uniform from base to edge. Coverage of the leaflet surface with
CD31- and vWF-positive cells was partial, principally from the proximal
attachments at 16 and 20 weeks. There was no evidence of inflammation
or residual polymer at 16 to 20 weeks. ESEM demonstrated a smooth
surface of the TE leaflets at both the inflow and outflow side as a
smooth rounded free edge of the leaflets.
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Tissue Analysis
Collagen content of the TE leaflets was 140% that of native
tissue at 4 weeks and increased to a plateau level of 180% after 8
weeks. DNA content of the constructs was 65% that of native tissue at
4 weeks and 6 weeks and increased to 77%, 100%, and 150% at 8, 16,
and 20 weeks, respectively, indicating a constant cell proliferation on
the TE leaflets. GAG content increased from 90% that of native valve
tissue at 4 weeks to 300% at week 16 and decreased to comparable to
native values (140%) at 20 weeks. Elastin was detectable in the TE
leaflets by 6 weeks.
Mechanical Testing
The tensile strength of all implanted TE valves leaflets was
initially higher than that of native tissue and decreased over the
follow-up period to be comparable to native values (130% that of
native tissue at 20 weeks). In parallel, the constructs became more
pliable, which was evaluated by a decrease of Young’s modulus and an
increase of elongation as a percentage at maximum stress. The
stress/strain curve at 20 weeks demonstrated that the mechanical
properties of the new tissue strongly resembled that of native
pulmonary valve tissue (Figure 9).
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Scaffold Material Bioabsorption Analysis
Scaffold material bioabsorption analysis of the valve
tissue demonstrated complete biodegradation of the PGA by 4 weeks and
of the P4HB by 8 weeks.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Valve replacement surgery is efficacious and substantially changes the natural history of valvular heart disease.2 However, although the overall performance of these devices is excellent, prosthesis-associated problems occur within 10 years after surgery in 30% to 35% of patients.12 Mechanical valves require lifelong anticoagulation therapy.3 Bioprosthetic valves have limited durability and may calcify prematurely, particularly in young patients.4 More important, both mechanical and bioprosthetic valves are nonviable structures and do not have the ability to grow, repair, or remodel, which is a specific problem in the pediatric patient population.5
TE applies the principles and methods of engineering to biological sciences in an attempt to create viable structures for replacement of deficient natural structures.13 The option of creating heart valves from autologous cells offers many potential advantages. These include elimination of unfavorable side effects of anticoagulation therapy, elimination of immune rejection, and the potential of growth, repair, and remodeling.
We previously reported the successful replacement of a single pulmonary valve leaflet by an autologous TE leaflet.6 These TE valve constructs were based on the rapidly bioabsorbable scaffold material PGA. A substantial limitation of the PGA-based tissue constructs is its initial stiffness and thickness, making the creation of more complex 3D TE constructs, such as a trileaflet heart valve, difficult. As an alternative scaffold material, we subsequently evaluated polyhydroxyoctanoate (PHO and PHA3836, Tepha Inc), a biocompatible, strong, and flexible polymer. Recent experiments from our laboratory with trileaflet valve constructs fabricated from porous PHO showed promising functional in vivo results. However, PHO has a prolonged bioabsorption time, which persisted in vivo, and was not sufficiently replaced by new functional tissue after 17 weeks.8 In an attempt to create a more ideal scaffold, we developed a novel composite material consisting of a PGA mesh coated with a thin layer of P4HB. P4HB is a thermoplastic, strong, and flexible material, but it has a more rapid bioabsorption time than does PHO. This composite material combines the high porosity of PGA mesh and the added favorable mechanical properties of P4HB. Because of its thermoplasticity, it was possible to fabricate trileaflet valve scaffolds by a heat-application welding technique. In our approach to creating TE structures, the bioabsorbable materials serve as a temporary structural scaffold until the seeded cells produce their own matrix proteins. Once the scaffold is degraded, the biomechanical profile of the TE heart valves will ultimately depend on this matrix formation. In previous studies, we found that the TE constructs had either insufficient mechanical strength or functional performance. We hypothesized that growing the TE constructs in a biomimetic in vitro environment would yield more "mature" heart valve tissue with a more favorable performance in vivo. Recent studies of vascular TE demonstrated a beneficial effect of pulsatile flow with regard to TE arteries.14 Therefore, we developed an in vitro pulse duplicator system in which the TE valves were grown under gradually increasing flow and pressure conditions, thereby providing physical signals to the developing tissues comparable to those encountered in vivo. After 14 days of in vitro culture, the valves grown in the bioreactor showed significantly higher formation of matrix proteins, a more organized histological structure, and more favorable mechanical properties than did constructs grown under static culture conditions. Six of these valve constructs were then implanted into the pulmonary position of sheep for in vivo evaluation. Echocardiography showed functioning valve constructs up to 20 weeks. However, there was mild to moderate valve regurgitation present at 16 and 20 weeks that was due to central noncoaptation. This may result from shrinkage of the cuspal tissue during the process of scaffold bioabsorption and/or the observed increase of the inner diameter of the valve constructs in accordance with the native pulmonary artery growth (4 mm over the 5-month time period). A possible solution to compensate this phenomenon may be an optimized scaffold design with an initially increased coaptive area of the polymer leaflets.
Histology showed increasing organization and layering of the leaflet structure with a fibrous layer rich in collagen and a loose layer rich in GAGs and elastin near the inflow surface as well as partial coverage with endothelium. Furthermore, the extracellular matrix analysis reflected a dynamic process of growth and remodeling, with matrix constituents comparable to native tissue at 20 weeks.
Therefore, the present study suggests that remodeling of the TE heart valve occurred in vivo, yielding an organized layer and structure with many architectural features and extracellular matrix elements characteristic of the native semilunar valve.15 In addition to the microstructural similarities, the TE valves attained mechanical properties that at 20 weeks were almost indistinguishable from those of native valve tissue.
In summary, the present study describes a functional, living, completely autologous TE heart valve generated and conditioned in a biomimetic in vitro environment, which functioned satisfactorily in vivo up to 5 months. More important, the engineered valve leaflets gradually evolved to resemble the native pulmonary valve leaflet, as demonstrated by their histological, biomechanical, and biochemical characteristics. However, these results are very preliminary inasmuch as the number of implanted valves is small and the longer term fate is unknown. Our next efforts are directed at optimization of the scaffold design to incorporate sinuses of Valsalva to more closely approximate the natural shape of a semilunar heart valve and root. Moreover, the optimal cell source is still undetermined. Finally, optimization of the in vitro conditions with regard to growth factors, growth inhibitors, and pressure-loading conditions are areas for future studies.
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Acknowledgments |
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This study was supported by generous grants from the Department of Cardiac Surgery, Children’s Hospital Boston, Deutsche Forschungsgemeinschaft (Ho 2109/1-1), and the National Institutes of Health (HL-97-005). We thank Peter Morley, Central Machine Shop, Massachusetts Institute of Technology, for his technical assistance. Furthermore, we thank Jun Young Lee for providing the ESEM pictures and Dr Byung-Soo Kim for his kind help in performing the mechanical testing of the TE valve leaflets.
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