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5th Biennial Conference on Heart Valve Biology and Tissue Engineering
- Conference date: 18-20 May 2012
- Location: Mykonos Island, Greece
- Volume number: 2012
- Published: 01 May 2012
61 - 80 of 86 results
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Immunological Effects of [alpha]Gal-positive and [alpha]Gal Knockout Biological Heart Valves
Authors: Christopher G. A. McGregor, Heide Kogelberg and Guerard W. ByrneAbstractAs immune responses likely contribute to degeneration of bioprosthetic heart valves (BHVs), this nonhuman primate study compares the anti-Gal antibody response to BHVs from wild type (WT) pigs (current BHVs) and those from α-galactosyltransferase knockout (GTKO) pigs. Stented glutaraldehyde fixed BHVs from WT (n=4) and GTKO (n=3) pigs were commercially manufactured and implanted in the mitral position in nonhuman primates using standard surgical techniques. Recipients were treated with Lovenox (1mg/kg BID) for 5 weeks reducing to 1mg/kg daily for one week and then discontinued. Recipients were sensitized to the αGal antigen by immunization to match IgG levels found in humans. Serum antibody responses were monitored by ELISA. WT BHVs were explanted at 3 hours, 1 year, 2 years and the fourth is currently ongoing at 3 years. One GTKO BHV was explanted at 218 days. The remaining two GTKO BHVs are currently ongoing at 3 years. After immunization, circulating anti-Gal IgG levels were comparable in both groups at the time of implantation and were equal or greater than human levels. Anti-Gal antibody levels decreased in both WT and GTKO recipients after implantation. WT recipients, however, retained elevated levels (greater than 20% preimplant values) of anti-Gal IgG for at least the first year post implant. Conversely anti-Gal IgG levels in all GTKO recipients fell within one month and remained at less than 20% preimplant values. Analysis of the area under the curve showed a significant increase of anti-Gal IgG in the WT BHV group compared to GTKO BHV recipients (p<0.01). In this nonhuman primate model, the persistently and significantly (p<0.01) elevated levels of anti-Gal IgG antibody observed in WT but not GTKO BHV recipients, indicate continuing immune stimulation to the αGal antigen. Current commercially available BHVs are similarly known to contain αGal antigen. Anti-Gal antibody has been shown to increase calcification of processed pericardium in the rat implant model. These data support the hypothesis that preformed and induced anti-Gal antibody in BHV recipients may initiate an early immune response, which promotes subsequent calcification. BHVs made from GTKO pigs would eliminate this major xenoreactive antigen and provide a prosthesis with reduced immunogenicity. Such BHVs made from GTKO pigs may have greater durability and be potentially used in younger patients with more active immune systems.
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Stress-Fiber Remodeling in 3D: ‘Contact Guidance vs Stretch Avoidance’?
Authors: Jasper Foolen and Frank BaaijensAbstractWhen engineering heart valves in vitro, matrix anisotropy is considered important for long-term in vivo functionality. However, it is not fully understood how to guide, maintain and control matrix anisotropy. Experiments suggest that collagen anisotropy is affected by actin-mediated cell traction and associated cellular orientation. Although cellular orientation in 2D can be manipulated via imposed uniaxial cyclic stretch, 3D data are lacking. We questioned how cyclic stretch influences actin and collagen orientation in 3D constructs. A novel micro-tissue platform system was designed, able to dynamically and biochemically load small-scale cell-populated fibrous tissues. Flexible membranes of Bioflex culture plates were provided with a rectangular array silicone posts. These silicone posts constrained a contracting gel mixture of human vena saphena cells (HVSC), collagen type I and matrigel. The constrained tissues were subjected to pure uniaxial cyclic stretch (10%, 0.5Hz, in the presence or absence of agents) on the Flexcell system. F-actin was taken as a measure for the cell traction direction, and the F-actin orientation was quantified throughout the complete tissue thickness (~ 300m) using fiber-tracking software, and was fitted using a bi-model distribution function. Uniaxial cyclic stretching for 3 days (preceded by 3 days of static constraint) resulted in stress-fibers that were oriented perpendicular to the stretching direction only at tissue surfaces, as generally observed in 2D. Strikingly, however, in the tissue core F-actin (and cell) and collagen orientation was biaxial. Immediate cyclic stretching, starting before polymerization of the collagen matrix, resulted in a strong stretch avoidance throughout the tissue, of both F-actin and collagen. We systematically investigated the effect of biochemical treatment, including MMP1 to perturb matrix integrity, MMP-1 + ROCK-inhibitor to counteract the possible MMP1-induced response, as well the effect of a lower and a higher initial collagen density. Experimental data suggests that F-actin stress-fibers avoid cyclic stretch in 3D, unless collagen contact guidance dictates otherwise.
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Computational Structural Biomechanical Models to Guide Tissue Engineered Heart Valve Leaflet Fabrication
AbstractA computational structural deterministic modeling strategy has been developed and experimentally validated to (1) assist tissue engineering scaffold fabrication, and as a consequence to improve in vivo scaffolds performances, and (2) provide a better understanding of cellular mechanical and metabolic response to local micro-structural deformations of the extracellular matrix (ECM). Image analysis software was developed and tested on electrospun poly (ester urethane) urea (PEUU) scaffolds, collagen gels, decellularized tissues. The algorithm analyzed SEM and multi-photon images (maximum imaging penetration depth: 160 µm) providing a full 3D characterization of engineered constructs morphology (n ≥ 6). The detected material topologies were adopted to generate statistically equivalent scaffold biomechanical models minimizing the difference between the real material and network model architectural features. The mechanical response at the macro scale was fully characterized by stress control biaxial tests (n ≥ 6). The experimental biaxial response was used to calibrate a Finite Element Model able to predict, for a given material topology, the mechanical response at both organ (cm), cells (100 µm) and fiber (1 µm) levels. Scaffold networks models were imported in Abaqus, Yeoh strain energy with incompressibility hypothesis and t2d2h elements were adopted. Stress vs. strain prediction was produced for four different scaffold groups: isotropic ES-PEUU, anisotropic ES-PEUU, Vascular Smooth Muscle Cells integrated PEUU, Polystyrene micro-spheres integrated (10 µm diameter) proving the flexibility of the modeling approach. At mesoscopic level nuclear aspect ratio vs. strain curve for the rat VSMCs embedded into the scaffold was produced and compared with previous experimental findings. At the microscopic level the single fiber initial shear modulus was quantified from the strain energy function material parameters, and compared with Atomic Force Microscopy measurements on single PEUU fibers. The developed generalist modeling approach bridges scaffold fabrication parameters, micro architecture, and organ level - cell level mechanical response.
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Experimental and Computational Studies of the Aortic Bi-leaflet Mechanical Heart Valve (BMHV) Hemodynamics in an Idealized Left Ventricle
AbstractWe aim to validate the performance of our fluid structure interaction (FSI) simulations of a bi-leaflet mechanical heart valve in the aortic position., whose motion is driven by the beating left ventricle. We compare in vitro experiments and computations performed on an idealized model of the left ventricle (LV) with a St. Jude Medical Regent heart valve in the aortic position. The idealized LV consists of a truncated tetrahedron representing a simplified LV chamber with a single deformable surface made of silicone. The silicone surface simulates the lateral wall of the LV. The deformation of the LV is controlled by pressurizing the fluid surrounding the LV chamber via a Vivitro Superpump (Vivitro Systems; British Columbia). The experimental model simulates physiological flow rates and pressures that occur in the LV. The three-dimensional motion of the deformable surface is tracked using high speed cameras and reconstructed using a direct linear transformation. The reconstruction of the motion of the deformable surface has been validated against fluid volume flux of the LV measured using two Transonic Systems Inc. flow probes (Model ME-PXN; Ithaca, NY). The leaflet motion of the SJM is tracked using 2D photogrammetry with a single high speed camera. A two-dimensional DPIV system (LaVision GmbH; Goettingen, Germany) is used to acquire fluid velocity measurements within the ventricle and in the aortic position of the flow field through the cardiac cycle. In the computation, the ventricle kinematics, measured from experiment, is treated as an immersed boundary by the Curvilinear Immersed Boundary (CURVIB) method ( Ge et al., J. Comp. Physics, 2007). The leaflet kinematics of the BMHV are computed from the fluid-structure interaction solver FSI-CURVIB (Borazjani et al., J. Comp. Physics, 2008). The computational domain is a structured mesh of 8 million grid points with a physical timestep of 1 ms. We have previously shown the capability of the FSI solver to resolve the BMHV kinematics and the resulting flow patterns in a stand-alone aorta (Borazjani et al., J. Comp. Physics, 2008). The comparison between the experimental and computational results shows good agreement for the ventricular flow pattern. The details of the validation of coupled LV-BMHV simulations will be discussed in the presentation.
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Quantification of the Role of Glycosaminoglycans on the Tensile and Viscoelastic Properties of the Aortic Valve Leaflet
Authors: Hubert Tseng, Eric J. Kim, Connell Patrick S. and K. Jane Grande-AllenAbstractThe spongiosa is the middle of three layers of the aortic valve leaflet and contains the proteoglycan (PG) versican and glycosaminoglycan (GAG) hyaluronan (HA) in large quantities. The resulting versican-HA aggregates allow the layer to retain large quantities of water, giving the layer a gel-like consistency. This layer is the least understood layer of the leaflet as it pertains to its role in valvular function and mechanics. However, the GAGs in this layer are perceived to be important to valve function, as the loss of GAGs in bioprosthetic heart valves coincides with its failure. The functions attributed to these GAGs include: (1) lubricating shear between the outer layers during flexure and tension; and (2) filling large volumes, resisting compression, and dampening shock from valve closure. The lack of understanding of GAGs role in valvular function is due to the difficulty in its isolation from the rest of the leaflet. For our study, rather than isolate the spongiosa, we enzymatically digest GAGs from the spongiosa via hyaluronidase. Previous studies have shown that the complete enzymatic removal of GAGs from heart valve leaflets greatly increases flexural rigidity. In this study, we instead varied the amounts of GAGs in the leaflets and investigated the effects on the tensile properties of the native leaflet; native leaflets in tension have a bilinear stress-strain curve, little hysteresis and minimal relaxation. GAGs were depleted using varying concentrations (0, 1, 2, 5 U/mL) and application times (8, 24 h) to yield a gradient of 4 GAG amounts ranging between 20-40 μg GAG/mg dry weight of native tissue, although with little change in water content. GAG depletion from the spongiosa was also confirmed using Alcian blue staining, which also verified no gross changes to the outer layers. Leaflets in these GAG depletion gradient conditions are in the progress of being mechanically tested to elucidate the effects of GAGs on the tensile elastic and viscoelastic properties of the native leaflet. This work is a component of a broader effort to elucidate the important role of GAGs and PGs in the function of native and diseased valves and in the design of improved valve replacements.
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Biomechanical Stimuli Effects on Valve Endothelial Cell Anti-thrombotic Mechanisms
AbstractBecause of the degeneration and thrombosis in artificial heart valve implants, it is important to understand the anti-thrombotic mechanisms of cardiac valve endothelial cells (VECs). These anti-thrombotic mechanisms can be integrated into poly(ethylene glycol) diacrylate (PEGDA) tissue-engineered heart valve (TEHV) design. This work will study the effects of (1) PEGDA hydrogel stiffness and (2) specific extracellular matrix (ECM) adhesive peptides on VEC phenotype and anti-thrombotic mechanisms. PEGDA 10% (w/v) hydrogels of MWs 3.4 and 20kDa were polymerized to apply different substrate rigidities in VEC culture. Thiol-ene reactions were used to covalently bind laminin- and fibronectin- derived peptides to the acrylate groups on PEGDA hydrogel surfaces. Laminin-derived peptide motif RKRLQVQLSIRT (RKR) and fibronectin adhesive peptide RGD were modified to include an additional cysteine at the end of each sequence, introducing a free thiol to undergo the thiol-ene reaction. Thiol-PEG-fluorescein (SH-PEG-FITC) served as a negative adhesive substrate control. Porcine aortic VECs were seeded onto each of the ECM-hydrogel combinations and cultured for 2, 6, and 10 days. Cell phenotype, adhesion, and proliferation were then assessed. Analysis of specific VEC anti-thrombotic protein regulation is in progress. At each time point, samples will be analyzed for maintenance of VEC phenotype and expression of thrombotic (von Willebrand Factor [VWF]) and anti-thrombotic (VWF cleaving enzyme [ADAMTS-13], eNOS, PGI2, tPA) proteins using histochemistry and qRT-PCR. Addition of histamine has been shown to stimulate rapid release of thrombogenic ultra-large VWF (ULVWF) strings by vascular endothelial cells. This method will be used to study VEC ULVWF string production and the associated cleavage activity of ADAMTS-13. Control of the hemostatic process will be quantified via western blot and ELISAs. The 3.4 and 20kDa MW PEGDA hydrogels had compressive moduli of 131±5 and 7.5±2kPa, respectively. Binding different concentrations of SH-PEG-FITC onto the gel surfaces showed that acrylate saturation was achieved for both MW compositions using ~5mM of peptide solution. After 2 days, the VECs on the stiffer 3.4kDa RKR gels appeared spread and elongated, whereas the 3.4kDa RGD seeded VECs had cobblestone morphology. VEC adhesion on the RKR and RGD 20kDa gels was observed, but with limited spreading. The cultured VECs may prefer the stiffer 3.4kDa gels over the softer 20kDa gels. After 10 days, VECs on the 3.4kDa RGD gels had minimal proliferation, while VECs on RKR grew confluent, were cobblestone shaped, and expressed VWF. Results suggest that both substrate rigidity and adhesive substrate greatly influence VEC survival, and likely affects anti-thrombotic regulation. Future studies include the use of basal lamina components collagen IV and perlecan peptides to evaluate changes in VEC behavior.
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Biomechanical Activation of Human Valvular Interstitial Cells from Early Stage of CAVD
AbstractAortic Valve (AV) leaflets are exposed to complex biomechanical stimulations, which has been linked to the progression of Calcific Aortic Valve Disease (CAVD) characterized by valvular interstitial cells (VICs) adopting an osteogenic phenotype, remodeling of the ECM and biomineralization. Initial asymptomatic phases of CAVD, named Aortic Valve Sclerosis (AVSc), include thickening of the cusps whereas advanced stages, Aortic Valve Stenosis (AVS), are associated with severe calcification. Prospective clinical studies of CAVD are hampered by the typically slow and variable progression of the disease. In addition, patients who present with AVS are already in the stage of severe calcification where damage to the AV leaflets is too severe to be reversed by drug therapy. Finally, due to its asymptomatic presentation little is known about the AVSc stage. The aim of this study is to develop tissue- and cell-based models to uncover the biomechanical forces leading to activation of VICs in early, asymptomatic AVSc patients. We investigate the impact of biological (BMP4 pathway) and mechanical (tensile stretch) forces leading to VICs osteogenic transdifferentiation, and biomineralization of the fibrosa layer. Human, surgically resected, AV tissue from AVSc patients were characterized for cellular and extracellular markers and compared to healthy controls and AVS tissues. BMP4 and tensile stretch induce osteogenic marker expression and biomineralization of the fibrosa in non-calcified AVSc-derived tissue. VICs were isolated from patients and induced to transdifferentiate in either 2D cell culture or 3D Tissue Engineering valve model based on decellularized porcine AV scaffold repopulated with human-derived cells. Our results show that the synergistic combination of biological (BMP4) and mechanical (tensile stretch) forces is required to promote SMA, RUNX2, OPN, and ON expression of AVSc-derived cells. These results provide a novel study model of early asymptomatic AVSc that combine biological and mechanical stimulation to induce activation of AVSc-derived VICs and biomineralization of AVSc tissue. This model could be used to unveil the molecular mechanisms lading to VIC activation and to test possible future small drug to control cellular activation and tissue remodeling.
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Gelatin and Decorin are Suitable Candidates for Application in Tissue-Engineered Matrices - an Immunological In Vitro Study
AbstractInflammatory and immune-mediated responses to extracellular matrix proteins (ECMp) or biomaterials mainly determine the acceptance of tissue-engineered scaffolds like heart valve grafts. Therefore, we focused on detailed analysis of those responses induced either by the single ECMp gelatin and the proteoglycan decorin or by electrospun scaffolds composed of both proteins. Proliferation assays were performed to analyze response of T cell subsets by flow cytometry. Therefore, human peripheral blood mononuclear cells (PBMCs) were co-cultured with bovine decorin, porcine gelatin or with electrospun scaffolds generated of poly-ε-caprolacton (PCL) with either gelatin alone or in combination with decorin under low-dose anti-CD3 treatment for 5 days. Moreover, monocyte-derived immature dendritic cells (DC) were co-cultured with decorin and gelatin to determine maturation effects by specific surface markers and flow cytometric analysis compared to a Lipopolysaccharride (LPS) stimulated control. Supernatants of both T cell and DC cultures were analyzed for the pro-inflammatory cytokines IFNγ, TNFα and IL-6 by cytometric bead arrays or ELISA. Additionally, complement activation by decorin and gelatin was screened by incubation with pooled human serum and measuring induced C5a release by standard ELISA. In general, a slight reduction of the T cell subset proliferation compared to the anti-CD3 stimulated positive control in CFSE-based assays could be demonstrated for bovine decorin and porcine gelatin. Both electrospun scaffold types did not alter the proliferation response. Additionally, neither decorin nor gelatin induced a distinct proinflammatory cytokine secretion of IFNγ and TNFα relative to the control. The overall high secretion level of IL-6 was not affected by both proteins and electrospun scaffolds. Remarkably, a trend for reduced IFNγ and TNFα release could be observed for both scaffold types. Moreover, the surface marker expression pattern of DC revealed that decorin as well as gelatin were not able to induce DC maturation due to low expression levels for CD83 and fairly low TNFα secretion when compared to the LPS control. Analyzing complement activating capacities of both ECMp as part of the first line innate immune response, decorin as well as gelatin did not induce enhanced C5a release compared to controls. The results presented here illustrate the important role of evaluating complex immune responses to single ECMp as well as to electrospun scaffolds to facilitate an efficient selection of immunological inert tissue-engineered matrices for their future application as heart valve substitutes. Thus, gelatin as well as decorin are promising candidates for tissue engineering scaffolds as seen by their low immunogeneic properties as single ECMp or as part of electrospun blends.
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Decellularized In-Vitro Tissue-Engineered Heart Valves - First In-Vivo Results
AbstractDecellularized xenogeneic and allogeneic heart valves are used as matrices for tissue regeneration. However, xenografts are associated with a risk of immunogenic reactions or disease transmission and homografts are sparse. Further, remodeling capacity of these matrices is questioned, as cell infiltration is limited. Alternatively, biodegradable synthetic materials are attractive as of their unlimited supply and freedom in valve geometry, with sufficient capacity for cell infiltration and neotissue formation and remodeling, but with cell-mediated leaflet retraction and thickening as common problem with in-vivo application. To overcome cell-mediated leaflet retraction and thickening and the limitations when using xenografts or homografts, we propose to decellularize in-vitro cultured tissue-engineered heart valves as off-the-shelf matrices for in-vivo regeneration. Tissue-engineered heart valves are grown based on ovine vascular cells seeded onto PGA/P4HB valvular shaped scaffolds and exposed to dynamic loading in bioreactors for 4 weeks. Decellularization of in-vitro cultured tissue-engineered heart valves is demonstrated feasible with efficient cell removal and preservation of the collagen architecture and tissue strength. Storage of these valves up to 18 months did not affect tissue properties. Decellularization strongly reduced leaflet retraction and, therewith, improved valvular function up to 24 hours in a valve tester. In-vivo performance of decellularized in-vitro cultured tissue-engineered heart valves after trans-apical implantation in pulmonary position in sheep was evaluated after 8 (n=1), 16 (n=1) and 24 (n=1) weeks. Complete cellular repopulation was demonstrated within 8 weeks with excellent in-vivo performance and no signs of tissue thickening. Moderate regurgitation developed after 16 weeks with leaflet prolapse after 24 weeks and a reduced leaflet area, likely due to minimal coaptation in the current valve design. Minimal coaptation makes the leaflets prone to prolapse with direct loss of coaptation when repopulating cells exert traction forces. Mechanical analyses demonstrated tissue remodeling with a trend towards the development of anisotropic tissue properties in time, demonstrating the promising nature of decellularized in-vitro cultured tissue-engineered heart valves for in-vivo regeneration. Efforts are ongoing to increase the number of samples and to optimize valve design and geometry to increase leaflet coaptation and maintain optimal valve performance. The European Union's Seventh Framework Program is acknowledged for funding this study
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Modulating the Inflammatory Response for In Situ Tissue Engineering – The Role of MCP-1
More LessAbstractInflammation is not merely a detrimental response to biomaterials. It can be considered as a natural agent of tissue remodeling, which is pivotal for the approach of in situ cardiovascular tissue engineering. Recently, a biodegradable synthetic scaffold was shown to remodel into a fully functional healthy blood vessel via an inflammation-mediated response. In fact, it was shown that initial infiltration of immune cells (i.e. monocytes) is indispensible for long-term remodeling of scaffolds and functionality of neo-tissues. This remodeling process is orchestrated by potent signaling molecules, of which monocyte chemotactic protein-1 (MCP-1) was identified as a key player. However, the exact mechanism remains unclear. We hypothesize that MCP-1 initiates a desired wound healing cascade by recruiting favorable monocyte and macrophage (M2 type) subpopulations in the implanted scaffold. To investigate the interactions between circulating cells and scaffolds, we have developed and validated a meso-fluidics setup that exposes small-scale 3D scaffolds to a circulating cell suspension in pulsatile flow with pressure and shear stress on the scaffold surface in the physiologic range for aortic valve and small diameter arteries. Electrospun polycaprolactone (PCL) scaffolds were loaded with fibrin gel with and without MCP-1 and placed into the fluidics setup. Human peripheral blood mononuclear cells (hPBMC) were isolated from healthy donors by density gradient centrifuging. The cells were resuspended in culture medium and circulated in the fluidics setup for up to 72 hours at a pulsatile flow (1Hz), with a peak pressure of approximately 100 mmHg and peak shear stress of 1.5 Pa on the scaffold surface. The cell suspensions were characterized at various time points by flow cytometry. The hPBMC suspension at the start consisted of CD45+ lymphocytes and monocytes. After 16 hours in the fluidics setup, the distinct monocyte subpopulation (8-20% of hPBMC) was no longer present in the medium, regardless of MCP-1 presence. This suggests monocyte activation by the scaffold, resulting in cell adhesion and monocyte-to-macrophage differentiation. Moreover, whole-mount immunostaining of the scaffolds demonstrated that addition of MCP-1 resulted in a significant increase in CD163 expression, which indicates the presence of a favorable monocyte subpopulation and macrophage polarization towards a wound healing M2 phenotype. Our results show that the PCL/fibrin scaffold evokes a response from circulating monocytes, resulting in rapid cell adhesion and infiltration. Moreover, this initial response can be modulated using MCP-1 to promote favorable M2 macrophage polarization, initiating the wound healing cascade that is necessary for long term remodeling of the synthetic scaffold.
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An Analysis of Tissue-Engineered Pulmonary Valves Implanted in the Elderly Ovine Model
AbstractBioprosthetic heart valve replacement, recommended for patients older than 65 years of age, increases the quality of life, however grafts exhibit limited functionality due to degradation and calcification. Decellularized and tissue-engineered pulmonary valves (PV) get repopulated by autologous interstitial cells when implanted into juvenile sheep. Thus, in vivo matrix guided regeneration results in restored valve function. In this study, we investigated the regeneration capacity in elderly sheep. Sheep (Ø 7 yrs old, Ø weight of 88.5 kg) received pulmonary valve replacement. Decellularized PV (n=6), decellularized PV coated with proangiogenic CCN1 (n=6), and decellularized, CCN1 coated and reendothelized PV (n=6) were implanted in orthotopic position. For endothelialization PV were seeded with autologous endothelial cells (EC) differentiated from EPC from peripheral blood. Cells were flow adapted in a pulsatile bioreactor system prior implantation. PV functional analysis in vivo was realized by echocardiography, directly after implantation and prior explantation. Allografts were explanted after six and twelve months in vivo and investigated in respect to endothelium coverage, to the integrity of the extracellular matrix, and to the degree and cellular identity of invaded cells by histological and immunochemical stains. All allografts showed good to adequate function, no stenosis, low gradients and only occasional insufficiency without clinical symptoms. No signs of degradation of the extracellular matrix and minimal calcification restricted at the anastomosis of the grafts were found. All grafts were repopulated with cells but to various degree. Only one, explanted after one year, showed complete repopulation including the leaflets. In general, cell-density in the pulmonary artery was higher on the adventitial than on the luminal side and leaflets were better repopulated at the ventricular than at the arterial side. The majority of cells expressed sm-alpha-actin. Endothelial cells located on the luminal side appeared in some grafts as intact complete monolayer. Beside a slight tendency of better repopulation observed in the reendothelialized PV group, no significant difference of cell densities was found among the groups. Autologous repopulation of decellularized heart valve matrices implanted in the orthotopic position in the elderly sheep demonstrates retained regenerative capacity even in the older organism. This observation combined with the fact that no functional loss must have taken into account, implantation of decellularized heart valves matrices in patients over age 65 remains a therapy option to overcome the drawbacks of current used bioprosthesis.
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Design of a Novel Ex Vivo Bioreactor to Investigate the Effect of Pressure Induced Stretch on Aortic Valve Biology
More LessAbstractA variety of ex vivo bioreactors have been developed to study the mechanobiology of valves and valve substitutes. Especially, bioreactors to mimic the individual mechanical stimuli (stretch, pressure and shear), have been well established. Going forward, it is essential to characterize the structural and biological changes in the aortic valve when subjected to a combination of these stimuli. Especially, since it is expected that a synchronized exposure to combined mechanical stimuli has a significantly different effect than the effect caused by individual stimulus. To this end, a novel bioreactor was designed, fabricated and validated to study the effect of stretch induced by the physiological transvalvular pressure gradient on fresh porcine aortic valve. The ex vivo bioreactor developed in the current study can maintain consistent physiological loading conditions with independent control of the flow rate, pressure and frequency to reproduce clinically relevant mechanical conditions. The design consists of a linear actuator system driving the culture media through the valve at a prescribed flow rate. The pressure levels on either side of the valve can be controlled by using a compliance chamber on the aortic side which is connected to a compressed air circuit. The linear drive system has a piston arrangement at its end, and drives the media in a rigid cylindrical ventricle section. The media returns to the ventricular side though a compliant channel during the reverse motion of the linear actuator. The entire setup is placed inside an incubator maintained at 370 C and 5% CO2. The flow and pressure variations were observed to be physiologically accurate and consistent over the time period of the experiment. Other salient features include low volume of the entire setup, and replaceable valve mounting mechanism to accommodate different sized valves. To ensure that the valve cells are healthy and native phenotype is maintained in the bioreactor, porcine aortic valves were mounted to the stents and subjected to normal physiological conditions of 80/120mmHg pressure, 5 LPM flow rate of Dulbecco’s modified eagle media(DMEM). The entire setup was placed inside an incubator. After 48 hours of culture, Movat Pentachrome stain was done to observe the tissue morphology, and DAPI stain was done to assess the cell viability. Fresh porcine AV leaflets were used as controls. The results were comparable to fresh controls, and indicated that the bioreactor is capable of preserving leaflet morphology and cell viability when cultured under normal physiological conditions for 48 hours.
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Decellularized Tissue Engineered Heart Valves: Infiltration, Inflammation and Regeneration?
AbstractDecellularized native biological tissue is currently investigated for its use as heart valve tissue engineering scaffold. Important features are the intact native matrix structure and retained mechanical properties. Unfortunately, these materials lack sufficient cell infiltration, reducing its remodeling potential. As shown in our recent ovine study (see abstract A. Driessen-Mol), in vitro cultured decellularized tissue-engineered heart valves facilitate complete cell infiltration, even after only 5 hours in vivo. This rapid cell infiltration is essential for remodeling. In general, all implanted biological materials evoke an inflammatory response, resulting in undesired chronic inflammation and/or fibrosis or in desired regeneration. Macrophage phenotype (M1/M2) is demonstrated to play an essential role in regulating the delicate balance between inflammation and regeneration. More in-depth insights underlying the process of cell infiltration and subsequent host inflammation response is crucial to achieve regeneration and is studied here using an in vitro model system. For this in vitro study, we used an adapted setup of the IBIDI flow chamber system and simulated native pulmonary conditions. A mix of mono- and polynuclear cells was isolated from fresh ovine blood and added to the IBIDI system to circulate along inserted pieces of tissues. Ovine tissue-engineered patches were cultured according to our protocols for heart valve tissue engineering. In short, ovine vascular cells were isolated and cultured for 4 weeks on a PGA-P4HB scaffold. The patches were decellularized afterwards. Cell infiltration into both decellularized ovine tissue-engineered patches and decellularized native ovine pulmonary leaflets was studied up to 5 hours. Thereafter, the tissues were fixed, stained with DAPI and visualized by confocal microscopy. Tremendous cell infiltration was observed at the edges in the decellularized tissue-engineered patches, whereas no clear cell infiltration was observed for the decellularized native leaflets. These first preliminary in vitro results are indicative of the host body’s capacity to rapidly infiltrate decellularized tissue-engineered matrices with inflammation-associated blood cells. Further research for cell type identification and tissue regenerative capacity is ongoing. The European Union’s Seventh Framework Program is acknowledged for funding this study.
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Marrow Stromal Cell based Stem Cell Based Transcatheter Aortic Valve Implantation: First Experiences in a Preclinical Model
AbstractWe investigate the combination of transcatheter aortic-valve implantation (TAVI) and a novel concept of stem cell-based, tissue-engineered heart-valves (TEHV) comprising minimally-invasive techniques for both, cell-harvest and valve-delivery. TAVI represents an emerging technology for the treatment of aortic-valve disease. The utilized bioprostheses are inherently prone to calcific-degeneration and recent evidence suggests even accelerated degeneration resulting from structural-damage due to the crimping-procedures. Autologous, living heart-valve prosthesis with regeneration and repair capacities would overcome such limitations. Methods: Within a one-step intervention, tri-leaflet TEHV, generated from biodegradable synthetic-scaffolds, were integrated into self-expanding nitinol-stents, seeded with autologous bone-marrow mononuclear cells, crimped and transapically delivered into adult sheep (n=12). The animals were followed up for up to 2 weeks. TEHV-functionality was assessed by fluoroscopy, echocardiography and computed-tomography. Post-mortem analysis was performed using histology, extracellular-matrix analysis and electron-microscopy. Transapical aortic implantation of TEHV was successful in all animals (n=12) and the entire procedure-time from cell-harvest to TEHV-delivery was 109±14min. Fluoroscopy and echocardiography displayed TEHV-functionality demonstrating an adequate leaflet-mobility and co-aptation. Explanted TEHV showed intact leaflet-structures with well defined cusps without signs of thrombus-formation or structural-damage. Histology and ECM analysis displayed a high cellularity indicative for an early cellular-remodelling and in-growth after 2weeks. For the first time, we demonstrate the principal feasibility of a transcatheter, stem cell-based TEHV implantation into the aortic-valve position within a one-step intervention. Its long term functionality proven, a stem cell-based TEHV approach may represent a next generation heart-valve concept extending the clinical indication of transcatheter valves beyond elderly high-risk patients.
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Mechanism of Cardiovascular Tissue Immunogenicity Reduction by Ice-free Cryopreservation
Authors: Kelvin G.M. Brockbank, Alexandra Bayrak, Martina Seifert and Ulrich A. StockAbstractA variety of reasons for allograft heart valve failure have been discussed in the past and most investigators have emphasized immunological issues. Standard quantitative and qualitative cellular and matrix evaluations have not helped to solve the discussion of whether remaining allogeneic cells or potentially altered extracellular matrix contributed to the observed degeneration. Preliminary data on patients treated with decellularized allografts has recently demonstrated that decellularization did not significantly improve outcome in terms of pressure gradients and structural deterioration compared to non-decellularized allografts. These early clinical results question the validity of theories suggesting that an immune reaction to the remaining donor cells in allogeneic heart valves is the sole cause of structural deterioration.Porcine and ovine pulmonary and aortic heart valves were cryopreserved using traditional cryopreservation by freezing with 10% dimethylsulfoxide or ice-free cryopreservation in an 83% cryoprotectant formulation consisting of 4.65 mol/L dimethylsulfoxide, 4.65 mol/L formamide and 3.31 mol/L 1,2-propanediol. Cell viability was assessed using a water soluble fluorometric viability oxidation-reduction (REDOX) indicator which detects metabolic activity by both fluorescing and changing color in response to chemical reduction of the growth medium. Statistical analyses were performed using a t-test or one-way analysis of variance, p values<0.05 were considered statistically significant. Viability assessment revealed that heart valve tissues were significantly less viable in ice-free cryopreserved valves compared with frozen valves, p<0.05, due to cryoprotectant cytotoxicity. Juvenile sheep studies demonstrated that ice-free cryopreserved heart valves had minimal T-cell mediated inflammation in the valve leaflet stroma compared with frozen controls. Severe valvular stenosis with right heart failure was observed in recipients of frozen valves, the echo data revealed increased velocity and pressure gradients compared to ice-free valve recipients (p=0.0403, p=0.0591). In vitro studies have demonstrated retention of hemocompatibility, biocompatibility and reduction of ice-free cryopreserved heart valve tissue immunogenicity. Based upon these observations, it is hypothesized that preservation of extracellular matrix structure due to the absence of ice and minimal cell viability due to cryoprotectant cytotoxicity combine to decrease tissue repair activity and reduced immunogenicity. Work in progress is extending ice-free cryopreservation to other cardiovascular and orthopedic tissue engineering applications including in vitro and in vivo cell repopulation.
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Scaffolds and Stem Cells for Patient-Tailored Aortic Heart Valve Tissue Engineering
Authors: Dan Simionescu, Michael Jaeggli, Jun Liao and Agneta SimionescuAbstractValve reconstruction or regeneration with living tissue is a daunting project for biomedical engineers and surgeons alike. Our translational approach to development of living valve replacements embraces three main principles: a) the accurate replication of each patient’s 3D aortic valve architecture for optimal functionality, b) layered scaffolds that mimic the aortic valve fibrosa, ventricularis and spongiosa to prevent buckling and enhance mechanical durability and c) the presence of autologous, valvular interstitial cells (VICs) to maintain matrix homeostasis. To create anatomically correct constructs, we used digital image processing of patient chest CT images and generated solid aortic valve root 3D structures using a stereo-lithography printer. Collagenous scaffolds to be used as the fibrosa and ventricularis layers were prepared from acellular porcine pericardium and collagen/GAG hydrogels to be used as the spongiosa layer. Layered scaffolds were attached to the molds, dried, stabilized with a non-toxic polyphenolic agent, rehydrated, the spongiosa seeded with human mesenchymal stem cells and valves subjected to functionality tests in a physiological heart valve bioreactor in sterile culture medium. Engineered valves exhibited excellent functional characteristics; most cells were alive, elongated significantly and stained positive for vimentin and actin, among other markers, suggestive of mechanical stimuli-induced stem cell differentiation into VICs. Ongoing studies are focused on endothelialization of the novel valve surfaces using stem cell-derived endothelial cells and rotational 3D seeding devices. In conclusion, autologous stem cell-seeded tri-layered collagenous scaffolds shaped to recapitulate the aortic heart valve shape and mechanics may provide future foundations for patient-tailored heart valve tissue engineering.
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Adipose Fat Derived Tissue-Engineered Heart Valves
AbstractA major challenge in tissue engineering of heart valves is the in vitro creation of mature tissue structures compliant with the native valve function. Concerning the remodeling capacity of the extracellular matrix (ECM), various cell types have been investigated, including prenatal cells, umbilical cord and vascular derived cells. The pluripotency and availability of human mesenchymal stem cells has made them highly attractive for tissue engineering purposes. However, for clinical use, adult bone marrow derived mesenchymal stem cells (MSC) are suboptimal due to the highly invasive donation procedure, and the decline in MSC number and differentiation potential with increasing age of the patient. Adipose derived stem cells (ADSC) represent an interesting alternative of mesodermal origin. The easy and repeatable access to subcutaneous adipose tissue and the simple isolation procedures provide a clear advantage. Here, we investigate the suitability of ADSC as a novel cell source for tissue engineered heart valves (TEHV). Tissue Engineered (TE) heart valve leaflets (n=6) were produced, based on PGA/P4HB scaffolds seeded with human ADSC isolated from fat tissue excisions from plastic surgery. To stimulate tissue formation and induce matrix alignment, the TE leaflets were cultivated in dynamic strain bioreactors for 4 weeks. We subsequently reseeded the cultivated valves with ADSC derived endothelial cells. Differentiation into endothelial-like cells was induced by cultivation of ADSC in the presence of vascular endothelial growth factor. To determine the ECM composition of the TE leaflets, biochemical analyses for glycosaminoglycans (GAG), hydroxyproline (HYP) and DNA were performed. To further evaluate the microstructural features, tissue samples of TE leaflets were analyzed by stainings as well as by scanning electron microscopy. The mechanical properties of the ADSC derived TE leaflets were analyzed using a biaxial tensile tester. TE leaflets based on ADSC showed a homogenous vital cell distribution throughout the whole leaflet structure and the formation of a confluent endothelial lining. Furthermore, a mechanically stable matrix with GAG and collagen was demonstrated. These results indicate that ADSC represent an interesting alternative autologous mesenchymal human cell source with clinical relevance due to their easy accessibility and excellent proliferation and tissue formation capacities.
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Fibronectin Coating Triggers In Vivo Recellularization of Tissue-Engineered Aortic Conduits
AbstractStrategies that aim at an acceleration of in vivo neoendothelialization and medial repopulation are warranted to increase the integrity and functionality of tissue-engineered cardiovascular implants. Proactive coating of decellularized implants represents a promising approach, however, little is known about the in vivo fate of coated agents over time. Detergent-decellularized aortic rat conduits (n = 24) were coated with covalently Alexa488-labelled (green emission) fibronectin (FN; 50 µg/ml, 24 hours) and implanted in the infrarenal aorta of wildtype Wistar rats (Group A; n = 12). Uncoated implants served as controls (Group B; n = 12). Before implantation and at postoperative day 1 and week 1, 4 and 8, fluorescence-based detection of FN coating was performed. Cellular repopulation was examined by histology and immunohistochemistry. All rats survived without clinical or sonographic signs of lower body malperfusion. Confocal microscopy of the aortic conduits revealed bright green FN fluorescence before and 1 day after implantation on the luminal as well as on the adventitial surface. The signal intensity decreased after 1 week, but was still present after 4 and 8 weeks. Four weeks after the operation, the luminal surface in the perianastomotic regions of Group A was completely neoendothelialized (vWF+) and a myofibroblast hyperplasia (αSMA+) with increased ratio of intima-to-media (I/M) thickness occurred. After 8 weeks I/M was significantly increased in Group A versus Group B (p < 0.01). At the same time point, medial repopulation starting at the adventitial zone was observed in group A, while only marginal repopulation occurred in group B (p < 0.001). In both groups vonKossa staining revealed sparse medial calcification and staining against inflammatory cell markers (CD3 & CD68) was negative. In our standardized rat transplantation model, a biofunctional protein coating of cardiovascular implants in the systemic circulation proved feasible and persistent up to 8 weeks. FN surface coating of aortic conduits induced a significantly increased medial recellularization, originating from the adventitial surface. The role of intimal hyperplasia and the relevance thereof needs further investigation.
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A New Approach to Heart Valve Tissue Engineering Based on the Modification of Human Pericardial Tissue
AbstractThe main problem of currently used xenogeneic biological heart valves is the development of degenerative changes leading to valve failure. Reoperation is required in approximately 65% of patients at 15 years after implantation. The challenge of heart valve tissue engineering is to create a new type of biological prosthesis for clinical use. The aim of our study is to construct a living autologous human pericardial heart valve that will have optimal mechanical properties and a similar histological structure as the normal aortic heart valve. Three leaflet heart valve constructs made from human pericardium were attached onto a plastic holder and cultured under dynamic conditions for up to four weeks. After this time conditioned pericardial samples were compared to control unconditioned pericardial samples from the same patient and to that of the normal aortic heart valve. Histological, immunohistochemical and biomechanical assessments were performed. Pericardial interstitial cells (PICs) are able to respond to mechanical stresses by proliferating and differentiating into an active (myofibroblast-like) phenotype and are able to produce new extracellurar matrix (ECM). A threefold increase in PIC number and a twofold increase in smooth muscle actin (SMA) positive cells was observed after dynamic conditioning. These measurements were statistically significant (p<0.001). The histological structure of conditioned pericardium is very similar to the normal aortic heart valve and dynamic conditioning was shown to be important for PIC activation. Uniaxial tensile tests were performed to compare the mechanical properties of conditioned pericardium with the native aortic heart valve. Our results indicate that the secant elastic modulus of pericardium before and after conditioning (13.1 ± 8.3 Mpa) is comparable to the native aortic heart valve. Autologous human pericardium mimics the natural structure of the normal aortic heart valve and has similar mechanical properties. PICs are activated to an active VIC-like phenotype by mechanical conditioning. Our pericardial heart valve construct also possesses optimal hemodynamic properties by echocardiographic measurements similar to the healthy aortic heart valve. Acknowledgements: Supported by the Grant Agency of the Ministry of Health of the Czech Republic (project No. NT 11270.
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Development of In-Body Tissue-Engineered, Completely Autologous Valve (BIOVALVE)
AbstractUsing simple, safe, and economical in-body tissue engineering, autologous valved conduits (BIOVALVEs) with the sinus of Valsalva and without any artificial support materials were developed in animal recipients’ bodies. In this study, the feasibility of theBIOVALVE as an aortic valve was evaluated in a goat model. BIOVALVEs were prepared by 2-month embedding of the molds, assembled using 2 types of specially designed plastic rods, in the dorsal subcutaneous spaces of goats. One rod had 3 projections, resembling the protrusions of the sinus of Valsalva. Completely autologous connective tissue BIOVALVEs with 3 leaflets in the inner side of the conduit with the sinus of Valsalva were obtained after removing the molds from both terminals of the harvested implants with complete encapsulation. The BIOVALVE leaflets had appropriate strength and elastic characteristics similar to those of native aortic valves; thus, a robust conduit was formed. Tight valvular coaptation and sufficient open orifice area were observed in vitro. BIOVALVEs (n=3) were implanted in the specially designed apico-aortic bypass for 2 months as a pilot study under the systemic circulation. Postoperative echocardiogram and angiogram showed smooth movement of the leaflets with little regurgitation (2.6 ±1.1 L/min). The α-SMA–positive cells migrated in the tissue of the conduit significantly with rich angiogenesis and expanded toward the leaflet tip. At the sinus portions, marked elastic fibers were formed. The luminal surface was covered with thin pseudointima without thrombus formation. Completely autologous BIOVALVEs with robust and elastic characteristics satisfied the higher requirements of systemic circulation in goats for 2 months with the potential for valvular tissue regeneration.
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