Autologous Human Tissue-Engineered Heart Valves
Prospects for Systemic Application
Background— Tissue engineering represents a promising approach for the development of living heart valve replacements. In vivo animal studies of tissue-engineered autologous heart valves have focused on pulmonary valve replacements, leaving the challenge to tissue engineer heart valves suitable for systemic application using human cells.
Methods and Results— Tissue-engineered human heart valves were analyzed up to 4 weeks and conditioning using bioreactors was compared with static culturing. Tissue formation and mechanical properties increased with time and when using conditioning. Organization of the tissue, in terms of anisotropic properties, increased when conditioning was dynamic in nature. Exposure of the valves to physiological aortic valve flow demonstrated proper opening motion. Closure dynamics were suboptimal, most likely caused by the lower degree of anisotropy when compared with native aortic valve leaflets.
Conclusions— This study presents autologous tissue-engineered heart valves based on human saphenous vein cells and a rapid degrading synthetic scaffold. Tissue properties and mechanical behavior might allow for use as living aortic valve replacements.
Heart valve replacement represents a common surgical therapy for end-stage valvular heart diseases. Yet, currently used heart valve prostheses lack the ability to grow, repair, and remodel. Tissue engineering of living autologous heart valves, based on the recipient’s cells and a biodegradable carrier material (scaffold), has been demonstrated as a feasible alternative and might overcome the limitations of state-of-the-art therapies.
The first successful replacement of a single pulmonary valve leaflet with a tissue-engineered equivalent, based on a synthetic biodegradable scaffold, was demonstrated in lambs in 1995.1,2 As a major milestone toward clinical application, tissue-engineered trileaflet heart valves were shown to function successfully in sheep for up to 8 months.3–5 These valves remodeled in vivo and an increase in valve diameter was reported with the growth of the animal.3,4 Whether this was a result of growth or dilatation needs to be elucidated. The valves in the referenced studies did not possess sufficient neo-tissue and mechanical strength for systemic pressure application, such as the replacement of the aortic valve. Furthermore, the valves were engineered using animal cells, leaving the demanding challenge to generate valves from human cells for future clinical application.
Mechanical conditioning during tissue culture has been shown to enhance tissue formation and, thereby, improve the mechanical strength.6–8 Recently, a novel in vitro conditioning strategy, mimicking the cardiac cycle especially during diastole, in combination with a novel cell seeding procedure using fibrin gel, has been developed to further increase tissue properties and mechanical strength of tissue-engineered human heart valves.9,10 Applying this new methodology, this study presents tissue-engineered heart valves, based on vascular human cells and a synthetic biodegradable scaffold, that might have adequate tissue properties and mechanical strength to serve as human aortic valve replacements.
Heart Valve Scaffolds
Stented trileaflet heart valve scaffolds were fabricated as described previously.10 In short, the stents were prepared from Fastacryl (Vertex-dental, Zeist, the Netherlands) and the leaflets from nonwoven polyglycolic acid (PGA) (thickness 1.0 mm, specific gravity 70 mg/cm3; Cellon, Bereldange, Luxembourg), coated with poly-4-hydroxybutyrate (P4HB) (provided by Symetis Inc, Zürich, Switzerland). A mold was used to define the shape of the leaflets. The scaffolds were sterilized using 70% ethanol.
Cell Culture and Seeding of Scaffolds
Cells, harvested by plating,11 from the human vena saphena magna of a 77-year-old man were used. Immunohistochemistry was performed to characterize cell phenotype using the Ventana Benchmark automated staining system (Ventana Medical Systems, Tucson, Ariz) and the following primary antibodies: anti-vimentin, anti-desmin (clone 3B4 and D33; DakoCytomation, Glostrup, Denmark), and anti-α-smooth muscle actin (α-SMA, clone 1A4; Sigma, St. Louis, Mo). The medium for cell expansion consisted of DMEM Advanced (Gibco, New York, NY) supplemented with 10% fetal bovine serum (Biochrom, Berlin, Germany), 1% GlutaMax (Gibco), and 0.1% gentamycin (Biochrom, Berlin, Germany). The medium used for seeding and tissue culture contained additional gentamycin (0.3%) and L-ascorbic acid 2-phosphate (0.25 mg/mL; Sigma). The scaffolds were placed in medium overnight to facilitate cell attachment. Seeding was performed at a density of 4 to 5×106 cells (passage 7) per cm2 of scaffold using fibrin as a cell carrier for each leaflet separately.9,10 The seeded valves were placed either into the diastolic pulse duplicator (DPD) or culture flasks.
Tissue Culture and Conditioning
The DPD has been described in detail before.10 In short, the DPD consisted of a bioreactor, in which the valve was placed, and a medium reservoir. The valves were perfused (4 mL/min) with medium from the reservoir to provide fresh nutrients continuously. By applying a dynamic pressure difference over the valve (1 Hz) in the bioreactor, the diastolic phase of the cardiac cycle was mimicked.
Each engineered valve consisted of three independently seeded leaflets, which are each considered as n=1. The leaflets were divided into 3 groups: (1) dynamically conditioned leaflets (n=12) cultured in the DPD using pressure differences of 5 to 30 mm Hg; (2) statically conditioned leaflets (n=12) cultured in the DPD under continuous perfusion only; and (3) statically cultured leaflets (n=6) in culture flasks. The groups were referred to as dynamic conditioning, static conditioning, and control, respectively. The dynamically conditioned leaflets were analyzed after 2 (n=3), 3 (n=3), and 4 (n=6) weeks, whereas the leaflets of the other 2 groups were analyzed after 4 weeks only.
Qualitative Tissue Formation Analyses
Tissue composition was studied by histology. Samples were fixed in phosphate-buffered formalin and embedded in paraffin; 5- to 10-μm sections were studied by hematoxylin and eosin staining for general tissue morphology and Trichrome Masson staining for collagen formation.
Quantitative Tissue Formation Analyses
Tissue formation was quantitatively determined from DNA amount, as an indicator of cell number, glycosaminoglycans (GAGs), and hydroxyproline, as an indicator of collagen content, per mg dry weight of tissue. For DNA and GAG analyses, lyophilized tissue samples were digested in papain solution (100 mmol/L phosphate buffer, 5 mmol/L L-cysteine, 5 mmol/L ethylenediaminetetraacetic acid [EDTA], and 125 to 140 μg papain/mL) at 60°C for 16 hours. The DNA content was determined using the Hoechst dye method12 and a standard curve from calf thymus DNA (Sigma). The GAG content was determined using a modification of the assay described by Farndale et al13 and a standard curve from chondroitin sulfate from shark cartilage (Sigma). The hydroxyproline content was determined using a modification of the assay described by Huszar et al14 and a standard curve from trans-4-hydroxyproline (Sigma). A 1-to-8.8 ratio of hydroxyproline to collagen was assumed. By normalizing the collagen and GAG content for the amount of DNA, a measure for the amount of these matrix components produced per cell was obtained.
Evaluation of Mechanical Properties
The mechanical properties of the leaflets and the unseeded scaffold material were measured in radial and circumferential direction using a uniaxial tensile tester (custom-built, equipped with a 20-N load cell). The dimensions of the tissue samples were 8 to 12×2 to 3 mm. The forces acting on the tissues as a response to elongation were represented in stress-strain curves. The ultimate tensile strength, indicative for tissue strength, and elongation at break, indicative for tissue extensibility, were obtained from the stress-strain curves. The modulus, indicative of tissue stiffness, was calculated as the slope of the linear part of the stress-strain curve. Anisotropic properties were defined as differences between the moduli in the 2 directions.
Valve behavior was visualized in the “valve exerciser”15 up to 4 hours. The aortic flow was generated via a computer controlled pump. The flow profile and testing frequency were scaled to correct for the differences in viscosities between blood and phosphate-buffered saline, used for testing, to generate physiological aortic flow patterns through the valve. Images were obtained using a high-speed video camera (Phantom v9.0; Vision Research Inc, New Jersey). The flow, aortic pressure, and left ventricular pressure were monitored using flow (Transonic, New York, NY) and pressure sensors (Becton Dickinson, Erembodegem, Belgium). Mean systolic pressure gradients, regurgitation, and effective orifice area16 were calculated and averaged over 5 heart cycles at 4 stroke volumes from 39 to 72 mL. For calculation of the mean systolic pressure gradients, the scaling factor used during testing was corrected for.
Quantitative data were averaged per leaflet, subsequently averaged per group, and represented as average±standard deviation. Comparisons between groups were performed by 1-way ANOVA using Bonferroni post-hoc tests to determine significant differences (P<0.05). Student t tests were used for comparison of the radial and circumferential moduli within groups.
Statement of Responsibility
The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.
Cell Phenotype Characterization
The cells used for seeding all showed expression of vimentin (Figure 1a). None showed expression of desmin and a subpopulation showed expression of α-smooth muscle actin (Figure 1b), characterizing them as a mixture of V and VA type myofibroblasts.17
Evolution of Tissue Properties Over Time
Fabrication of PGA/P4HB trileaflet stented heart valve scaffolds was feasible (Figure 2a and 2b). All leaflets were intact after culturing (Figure 2c and 2d). The collagen and GAG content increased up to 3 weeks and was stable thereafter (Table 1). Cell number, as well as the amount of collagen and GAGs produced per cell, did not change over time.
Strength of the tissue increased with time in both circumferential and radial direction (Table 1). The modulus in circumferential direction increased with time, whereas the radial modulus and the elongation at break did not change. Anisotropy was elucidated after 4 weeks. The initial properties of the bare scaffold were lower compared with those of the tissue-engineered leaflets.
The Effect of Conditioning on Tissue Properties
Dynamic and static conditioning resulted in more homogeneous tissues with abundant amounts of collagen when compared with the control group (Figure 3). More collagen and GAG was present in the dynamic and static conditioning group when compared with the controls, whereas the number of cells was lower, resulting in higher amounts of matrix produced per cell (Table 2). The use of dynamic or static conditioning did not influence the cell number or collagen content, but the GAG content increased with dynamic conditioning because of increased GAG production per cell.
Circumferential strength was highest when using dynamic conditioning, followed by static conditioning, and lowest for the controls (Table 2). Radial strength was only increased in the dynamic conditioning group, as well as the circumferential modulus. The radial modulus was similar for the dynamic and static conditioning group, but higher when compared with the controls. Tissue extensibility did not differ between the 3 groups. Anisotropic properties were only elucidated in the dynamic conditioning group.
Comparison of Tissue-Engineered Valves to Native Human Aortic Valves
The mechanical behavior of the dynamically conditioned tissue-engineered valve leaflets, in terms of the shape of the stress-strain curve, matched those of their native counterparts in radial direction, whereas in circumferential direction the engineered leaflet properties were lower (Figure 4, native valve data kindly provided by P. Stradins18).
The dynamically conditioned leaflets showed proper opening motion when exposed to aortic valve flow conditions (Figure 5a). The effective orifice area and mean systolic pressure gradient increased from 1.01±0.12 to 1.52±0.21 cm2 and 7.0±4.8 to 11.5±3.1 mm Hg at increasing stroke volumes from 39 to 72 mL, respectively. Closure dynamics were suboptimal as coaptation was incomplete, resulting in regurgitation of 12.6±4.9% at a stroke volume of 39 mL and 18.2±4.2% at a stroke volume of 72 mL. Suboptimal closure was also visible in the pressure profile (Figure 5b), being lower than expected for aortic valves.
Living heart valve replacements are engineered using either an autologous approach based on rapid degrading synthetic scaffold materials or a nonautologous approach based on slow degrading scaffold materials or decellularized xenogeneic/allogeneic matrices. Although initially providing a mechanically stable implant, the nonautologous concept is associated with a number of disadvantages because of the persistence of foreign materials in the body. These include immunogenicity19 and most importantly the lack of growth and remodeling, an issue of particular importance in pediatric patients. In contrast, mechanical integrity and the ability to withstand systemic pressures of autologous heart valves are based on the neo-tissue only. However, autologous tissue-engineered heart valves do not yet contain sufficient neo-tissue and mechanical integrity for systemic pressure application.3–5
A fundamental requirement for the long-term performance of aortic heart valve leaflets is their highly organized collagen matrix, resulting in different mechanical properties when tested in perpendicular directions. This feature is known as anisotropy and, ideally, should be realized before implantation. Based on a novel in vitro conditioning strategy, this study presents autologous tissue-engineered human heart valves to possibly serve as aortic valve replacements.
Encompassing a relatively long period of in vitro experimentation, the present study provides insight into tissue evolution, although the amount of samples at the 2- and 3-week time points was low. Although cell number did not increase, the amount of extracellular matrix increased up to 3 weeks. Interestingly, mechanical strength and stiffness increased further after 3 weeks and anisotropic properties developed after 4 weeks. Previously, a culture period of 3 weeks has been suggested to be optimal for tissue development.3 However, longer culture periods might be required to optimize tissue organization and mechanical properties toward those required for aortic valve replacements.
This study clearly demonstrated that the mode of conditioning influenced tissue formation and mechanical integrity to a large extent. In static culture, the main cell activity was proliferation, resulting in more cells compared with the conditioned leaflets. Conditioning induced a shift of cell activity toward extracellular matrix production, resulting in more extracellular matrix, similar to when using cyclic flexure alone as mechanical stimulus.8 When using solely tension this shift was not seen, although extracellular matrix increased as well.7 Also, flow was reported to be beneficial for tissue formation.6 The effect of conditioning found in this study most likely represents a combination of the effects of flexure and tension as flow was kept to a minimum. Tissue distribution was more homogeneous when using conditioning, potentially caused by enhanced nutrient supply and removal of metabolic waste products. The dynamic nature of conditioning resulted in more active GAG production, most likely a result of a protection mechanism of the cells against the repetitive compressive strains.20 Tissue strength and stiffness increased because of conditioning, particularly when using dynamic conditioning, whereas tissue extensibility was not influenced. Anisotropy developed only when using conditioning with a dynamic character, suggesting that perhaps repetitive changes in strains during culturing are a prerequisite to render leaflets with a desired organization when using nonwoven PGA/P4HB scaffolds.
Dynamically conditioned leaflets showed comparable mechanical behavior to native adult human aortic heart valve leaflets in radial direction, but not in circumferential direction. The highly organized structure found in native human aortic valve leaflets was achieved only partly in the in vitro culture period. Functionality tests of the tissue-engineered human heart valves by exposure to physiological aortic valve flow conditions demonstrated the effective orifice area and mean systolic pressure gradients to be in the range of commonly accepted bioprostheses with a diameter of 23 mm.16 Closure dynamics were, however, suboptimal. The degree of anisotropy seems to influence closure behavior to a large extent.21 As the degree of anisotropy in the engineered leaflets is lower compared with native leaflets, the incomplete coaptation, as well as the lower pressure profile as a response to the physiological aortic flows, might be explained. We hypothesize that the valve leaflets are able to remodel after implantation to adopt a higher degree of anisotropy. Additionally, coaptation of the valves might be improved by optimization of the scaffold design. The time period of the functionality tests was short as the valves could not be kept alive in the testing setup, yielding only information on acute functionality under systemic conditions. The short-term, middle-term, and long-term functionality will be elucidated in upcoming animal studies. Creep of the tissue, caused by the assumed lack of elastin in the engineered valves, should be monitored as a possible failure mechanism.
For future clinical applications, the use of animal-derived serum and fibrin has to be avoided to provide a completely autologous approach. To prevent valve thrombosis, a confluent endothelial cell layer on the surface of the implants might be necessary. In summary, this study describes autologous tissue-engineered heart valves based on human cells that might suggest in vivo success as aortic heart valve replacements.
Presented at the American Heart Association Scientific Sessions, Dallas, Tex, November 13–16, 2005.
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