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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
51 - 86 of 86 results
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Fluid-Structure Interaction Simulation of an Aortic Bi-Leaflet Mechanical Heart Valve in a Patient-Specific Left Heart
Authors: Trung Bao Le and Fotis SotiropoulosAbstractA large-scale kinematic model is developed for animating the left ventricle (LV) wall to drive the fluid-structure interaction (FSI) between the ensuing blood flow and a mechanical heart valve prosthesis implanted in the aortic position of an anatomic LV/aorta configuration. The kinematic model is of lumped type and employs a cell-based, FitzHugh-Nagumo framework to simulate the motion of the LV wall in response to an electrical wavefront propagating along the heart wall. The emerging large-scale LV wall motion exhibits complex contractile mechanisms that include contraction (twist) and expansion (untwist). The kinematic model is shown to yield global LV motion parameters that are well within the physiologic range throughout the cardiac cycle. The FSI between the leaflets of the mechanical heart valve and the blood flow driven by the dynamic LV wall motion and mitral inflow is simulated using the curvilinear immersed boundary (CURVIB) method (Ge et al., J. Comp. Physics., 2007 and Borazjani et al., J. Comp. Physics, .2008) implemented in conjunction with a domain decomposition approach. The computational results show that the simulated flow patterns are in good qualitative agreement with in vivo observations. The simulations also reveal complex kinematics of the valve leaflets, thus, underscoring the need for patient-specific simulations of heart valve prosthesis and other cardiac devices.
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Functional Performance and Biomechanics of Decellularized Human Aortic Valves
Authors: Stacy M. G. Arnold, Steven Goldstein and David C. GaleAbstractAllograft decellularization based on detergents and/or enzymes to reduce antigenicity has been reported to damage tissue structure. We applied to human aortic valves a non-detergent/non-protease decellularization treatment prior to cryopreservation. To ensure that the method did not negatively impact valve structure, we submitted decellularized and conventional human aortic valves to pulsatile flow characterizations and accelerated wear testing (to 80 million cycles) under both normal and elevated aortic valve flow conditions. The biomechanical properties of decellularized aortic valve tissues were compared to those of non-decellularized aortic valves. Valve performance was assessed using six conventional and six decellularized human aortic valves (internal valve diameter of 21mm±1mm). Valves were placed in accelerated wear testers, at a cycle rate of 200beats/min as specified in ISO 5840. All valves underwent pulsatile flow characterization before testing and at 20 million cycle intervals up to 80 million loading cycles (pressure>100mmHg). Images captured during pulsatile testing at peak systole and diastole were used to evaluate proper valve function, full leaflet coaptation and wear related damage. Tissue biomechanics was evaluated pair-wise using ten bisected valves, half conventionally treated, half decellularized. For each, conduit and leaflet circumferential ultimate tensile strength (UTS), and conduit and myocardium suture retention strength were evaluated. Compared to conventional human aortic heart valves, the decellularized valves showed comparable valve performance based on effective orifice area before accelerated testing (1.57±0.3 vs 1.51±0.2 cm2, decellularized and conventional, respectively) and similar non-significant decrease after reaching an accumulated 80 million cycles (1.40±0.2 vs. 1.45±0.3 cm2). Both populations met the minimum performance requirements specified in ISO 5840 for effective orifice area and regurgitant fraction. There was no significant increase in retrograde flow due to post-wear leakage. Biomechanical testing demonstrated leaflet and conduit circumferential UTS and conduit and myocardium suture retention strength of decellularized valves were equal to or greater than the conventional valves. From these measurements we conclude that there is no quantifiable impact on valve functionality, performance or biomechanics due to the SynerGraft® (non-detergent based) decellularization process.
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The Congenital Bicuspid Aortic Valve can Experience High Frequency Unsteady Shear Stresses on its Leaflet Surface
AbstractBicuspid aortic valve (BAV) is a common congenital malformation affecting 1-2% of the population and is highly correlated to early calcification of the valve leaflets. Two widely held hypothesis for this correlation are (1) altered shape of the BAV results in altered fluid mechanical environment, leading to pro-calcification biology responses, and (2) inherent genetic defects results in pre-disposition of the tissues to calcify. In the current study, we tested the first hypothesis with porcine valve models in an in vitro flow loop. One BAV model and one tricuspid aortic valve (TAV) model were constructed using healthy porcine AV leaflets and tested in a physiological pulsatile flow loop. Fluid velocities near the center of the aortic surface of the valve leaflets were measured with Laser Doppler Velocimetry at a spatial resolution of 89 microns, and ensemble average shear stresses were calculated at various time points in the cardiac cycle. Unsteadiness of flow near the valve leaflets was quantified with variance analysis and power spectral analysis. Particle Image Velocimetry was used to visualize flow fields downstream of the valves and in the sinuses. The leaflets of the BAV model experienced shear stresses on the aortic surface with magnitudes similar to that of the TAV. However, flow near the BAV leaflets had high frequency unsteadiness components, especially during mid- to late- systole, and had high cycle-to-cycle magnitude variability, indicating that shear stresses will have similar unsteadiness and magnitude variability. These are most likely due to the stenosis in the BAV and the skewed forward flow, which collided with the aorta wall. In conclusion, our study indicated that some BAVs could experience high frequency unsteadiness and cycle-to-cycle magnitude variability on the valve leaflets because of its geometry. We speculate that, together with genetic factors, such adverse mechanical force environment could play a role in causing early calcification in the BAV leaflets.
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Autologous Bone Marrow Mononuclear Cell-Based Tissue Engineered Heart Valves: First Experiences with a One-step Intervention in Primates
AbstractA living heart valve with regeneration capacity based on autologous cells and minimally invasive implantation technology would represent a substantial improvement upon contemporary heart valve prostheses. This study investigates the feasibility of injectable, marrow stromal cell-based, autologous, living tissue engineered heart valves (TEHV) generated and implanted in a one-step intervention in non-human primates. Trileaflet heart valves were fabricated from non-woven biodegradable synthetic composite scaffolds and integrated into self-expanding nitinol stents. During the same intervention autologous bone marrow-derived mononuclear cells were harvested, seeded onto the scaffold matrix, and implanted transapically as pulmonary valve replacements into non-human primates (n=6). The transapical implantations were successful in all animals and the overall procedure time from cell harvest to TEHV implantation was 118±17 min. In vivo functionality assessed by echocardiography revealed preserved valvular structures and adequate functionality up to 4 weeks post implantation. Substantial cellular remodeling and in-growth into the scaffold materials resulted in layered, endothelialized tissues as visualized by histology and immunohistochemistry. Biomechanical analysis showed non-linear stress-strain curves of the leaflets, indicating replacement of the initial biodegradable matrix by living tissue. Here we provide a novel concept demonstrating that heart valve tissue engineering based on a minimally invasive technique for both cell harvest and valve delivery as a one-step intervention is feasible in non-human primates. This innovative approach may overcome the limitations of contemporary surgical and interventional bioprosthetic heart valve prostheses.
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The role of the epigenetic factor in valvulogenesis in zebrafish in vivo
Authors: Hae Jin Kang and Morteza GharibAbstractIn this study, we focused on wall shear stress as an epigenetic factor to initiate valve formation. Fast or oscillatory flow is induced in an embryonic zebrafish vessel to mimic the flow in atrioventricular canal (AVC). By creating an environment similar to AVC in non-cardiac region in vivo, we propose to seek a better understanding of the mechanisms associated with the induction of valvulogenesis signaling pathways. Polydimethylsiloxane is injected at the vessel wall of embryonic zebrafish, creating fast or oscillatory flow, therefore modulating local wall shear stress (WSS). Particle image velocimetry (PIV) technique is used to measure blood flow velocity. Whole mount in situ hybridization is performed to detect kruppel-like factor 2a (klf2a) expression. Klf2a is used as a marker gene since it responds to shear stress and is crucial for valve formation. Using PIV, WSS is calculated. In situ hybridization showed klf2a expression in the vessel as a result of either high WSS or oscillatory WSS. Klf2a is not expressed in the vessel during normal embryonic development. With a new gel injection technique, local WSS in the zebrafish vessel has changed, inducing expression of klf2a, an important gene for the initiation of valve formation. Expression of different genes such as TGF-β family that are involved in valve formation will be studied further to establish an innovative model to explore valvulogenesis in vivo.
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Shear- and Side-dependent microRNAs and Messenger RNAs in Aortic Valvular Endothelium
AbstractAortic valve (AV) disease is a major cause of cardiovascular-linked deaths globally. In addition, AV disease is a strong risk factor for additional cardiovascular events; however, the mechanism by which it initiates and progresses is not well-understood. We hypothesize that low and oscillatory flow is present on the fibrosa side of the AV and stimulates ECs to differentially regulate microRNA (miRNA) and mRNAs and influence AV disease progression. This hypothesis was tested employing both in vitro and in vivo approaches, high throughput microarray and pathway analyses, as well as a variety of functional assays. First, we isolated and characterized side-dependent, human aortic valvular endothelial cells (HAVECs) isolated from transplant recipient AVs. We found that HAVECs express both endothelial cell markers (VE-Cadherin, vWF, and PECAM) as well as smooth muscle cell markers (SMA and basic calponin). Further, HAVECs align in the direction of the flow as well as uptake acetylated LDL. Using microarray analysis on sheared, side-specific HAVECs, we identified side- and shear-induced changes in miRNA and mRNA expression profiles. More specifically, we identified over 1000 shear-responsive mRNAs which showed robust validation (93% of those tested). We then used Ingenuity Pathway Analysis to identify key miRNAs, including those with many relationships to other genes (for example, thrombospondin and IκB) and those that are members of over-represented pathways and processes (for example, sulfur metabolism). Furthermore, we validated five shear-sensitive miRNAs: miR-139-3p, miR-148a, miR-187, miR-192, and miR-486-5p and one side-dependent miRNA, miR-370. To prioritize these miRNAs, we performed in silico analysis to group these key miRNAs by cellular functions related to AV disease (including tissue remodeling, inflammation, and calcification). Additional miRNAs of interest (including miR-7 and miR-506) were determined through analysis of overrepresented miRNA binding sequences in the shear-sensitive mRNA array. Next, to compare our in vitro HAVEC results in vivo, we developed a method to isolate endothelial-enriched, side-dependent total RNA and identify and validate side-dependent (fibrosa vs. ventricularis) miRNAs in porcine aortic valvular endothelium. From this analysis, we discovered and validated eight side-dependent miRNAs in porcine endothelial-enriched AV RNA, including one miRNA previously identified in vitro, miR-486-5p, as well as a shear-responsive miRNA cluster, miR-199a/214. Through microarray studies and in silico analysis, we have prioritized key miRNAs which may serve as master regulators of AV disease. Better understanding of AV biology and disease in terms of gene-regulation under different hemodynamic conditions will facilitate the design of a tissue-engineered valve and provide alternative treatment options.
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The Effect of Side-specific Valve Endothelial Cells on Extracellular Matrix Production by Valve Interstitial Cells
Authors: Napachanok Mongkoldhumrongkul, Magdi H. Yacoub and Adrian H. ChesterAbstractValve endothelial cells (VECs) play an important role in regulating the function of the underlying interstitial cells (VICs). However, the regulatory effects VECs from each side of the valve on the production of ECM proteins by VICs have not been studied. This study aims to determine the regulatory effects of VECs isolated from the aortic (aVECs) and ventricular (vVECs) surfaces of the aortic valve on the production of collagen, glycosaminoglycan (GAGs) and elastin by VICs. Side-specific VECs were co-cultured with VICs using Transwell® inserts and the effects on extracellular matrix production by VICs were investigated by quantifying the amount of secreted collagen and sulphate GAGs by VICs as well as examining gene expressions of collagen, GAGs and elastin components by VICs. Collagen production by VICs was significantly increased by co-culturing with aVEC and vVECs to 154.38 ± 13.71% (p=0.041) and 196.35 ± 16.59% (p=0.008), respectively, of the control (VIC culture without VECs). Furthermore, vVECs significantly enhanced production of sulphate GAGs by VICs to 217.18 ± 32.9% (p=0.008) above control whereas aVECs showed an increase of 150.08 ± 18.0%, which was non-significance. There was no significant difference between sulphate GAGs release in response to vVECs and aVECs. Only fibrillin 1, a component of elastin, gene expression was increased by co-culturing VICs with aVEC, 2.07 ± 0.34 (p=0.008), and vVECs, 2.13 ± 0.31 (p=0.03) fold increase above control. In contrast, media collected over a 48 hour period from aVEC or vVEC cultures and subsequently incubated with VICs (in the absence of VECs) showed no induction on the ECM production by VICs. In conclusion, aVECs and vVECs induce the ECM production and expression by releasing labile molecules which are degradable or lose their activities with time. Further experiments are necessary to identify the mediators that produce these effects and to determine how their release is modulated by the different flow patterns experienced by aVECs and vVECs. This study further highlights the complex interaction and communication between different cell types present in the valve cusps.
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Calcification of Aortic Valve leaflets is Shear Dependent and Side-specific
AbstractAortic valve (AV) sclerosis is a degenerative disease and is one of the leading causes of mortality in elderly population. AV experiences various mechanical stimuli such as pressure, shear and flexure with differing magnitudes on either sides of AV (fibrosa and ventricularis) with fibrosa side facing more dynamic environment. Normal hemodynamic conditions constantly renew and remodel the valve, whereas altered mechanical loading such as hypertension and altered shear stress can cause tissue inflammation that leads to calcification, preferentially on fibrosa side. AV calcification progressively leads to sclerosis and ultimately results in valve failure. However, the molecular and cellular processes that lead to inflammation and calcification are not very well understood. To understand the role of mechanics and underlying molecular mechanisms behind this preferential calcification, aortic side and ventricularis side of fresh porcine AVs were exposed to different shear stress patterns using an ex vivo cone and plate viscometer for 72 hours. Osteogenic medium was used to accelerate the calcification process ex vivo. To investigate the effect of shear stress magnitudes on fibrosa side, sine waveforms of amplitudes 5, 10 and 25 dynes/cm2 at a frequency of 1 Hz were used. To investigate the effects of shear stress frequency on fibrosa side, sine waveforms of 1 and 2 Hz at 10dynes/cm2 were used. Following exposure to shear, calcium levels of the samples were quantified using calcium Arsenazo assay. Von Kossa stain for mineralization was also done. Fresh porcine AVs were used as controls. Results indicated that low magnitude shear stress, 5 dyne/cm2 at 1 Hz elicited significant calcium levels on fibrosa side compared to other magnitudes and frequencies. To investigate if the oscillatory nature or the low magnitude was responsible for this high calcium response, fibrosa side was exposed to steady 5 dyne/cm2 under same experimental conditions as above. However, calcium levels at steady 5dyne/cm2 were comparable to fresh AV levels and thus non-significant. This result indicated that the magnitude in combination with the oscillatory nature of the sine 5dyne/cm2 triggered significant calcification levels on the fibrosa side of the AV leaflets. To test the side-specificity of this response, ventricularis side was also exposed to 5 dyne/cm2 under same experimental conditions as above. It is interesting to note that calcium levels on ventricularis side were not significant compared to that on the fibrosa side. This result further indicated that the expression of significant calcium levels in response to the low oscillatory shear is indeed side-specific. Thus our results suggest that the calcification of the AV leaflets is shear-dependent and side-specific. Shear dependent calcification in AV also suggests mechanobiological similarities with the atherosclerosis of blood vessels.
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Proteoglycan-Rich Leaflet Thickening in Diet-Induced Early Aortic Valve Disease
AbstractAortic valve disease (AVD) is a cell-mediated pathology without effective pharmacotherapy, and the early pathogenesis of AVD is drastically understudied. Examining the early stages of disease development may identify biomarkers and novel treatment strategies for use before an untreatable burden of calcification develops. We investigated the effects of a mildly atherogenic diet on early AVD development in mice, and the involvement of proteoglycans (PG) and Sox9 in this process. Male wild-type (WT) C57Bl/6J mice were fed a control diet or BioServ F3282, a high-fat, high-carbohydrate diet (HF/HC) with 58.7% kcal from fat (cholesterol <0.1% w/w) for four months (n = 4-6 per group). Aortic valve function was examined by high-resolution ultrasound biomicroscopy (UBM). Longitudinal aortic valve sections from formalin-fixed and paraffin-embedded hearts were stained by Movat’s pentachrome (morphological changes and ECM composition) and Osteosense 680 (calcification), then immunostained for α-smooth muscle actin (αSMA) and Sox9. Mice on the HF/HC diet for four months became significantly obese (46.7 ± 4.7 vs. 32.3 ± 0.8 g, p < 0.05) but did not develop cardiac hypertrophy. They developed mild but statistically significant hypercholesterolemia (4.7 ± 1.0 vs. 3.1 ± 0.4 mmol/L total cholesterol, p < 0.05). UBM revealed moderate decreases in aortic valve opening area along with increased left ventricular ejection time in HF/HC mice, while no mice exhibited aortic regurgitation. Significant valve thickening was found in the distal third of HF/HC leaflets (84.4 ± 10.4 vs. 37.3 ± 2.7 µm, p < 0.05), while proximal and medial regions were unaltered. Valve thickening was primarily due to PG deposition in HF/HC leaflets (11435 ± 7681 vs. 5448 ± 2948 µm2, p < 0.01), not an increase in collagen content (1729 ± 815 vs. 1771 ± 663 µm2, p = 0.87). Sox9 expression was increased in thickened HF/HC leaflets, and was most highly expressed in PG-rich lesions. HF/HC leaflets did not stain positive for αSMA, implying that changes in leaflet structure and function were not the result of actively synthetic myofibroblasts. Osteosense 680 staining was negative, denoting that the HF/HC diet did not induce leaflet microcalcification.
These studies reveal that a high-fat Western diet lacking cholesterol can induce changes in the ECM and functional properties of a WT mouse aortic valve. This early AVD is characterized by thickened leaflets with lesions that are PG-rich and have increased Sox9 expression. Although AVD is considered to be driven by pathological myofibroblast differentiation leading to fibrosis and calcification, the early changes observed herein occur independently of myofibroblast activation, and provide new insight into the initiation and pathogenesis of AVD.
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Angiotensin Receptor Blocker Has no Effect on Atherosclerotic Factors in AVS
AbstractAortic valve sclerosis (AVS) is a chronic progressive disease affecting 25% of the population over the age of 65. Despite this high prevalence, there are currently no preventative therapies which inhibit the progression of AVS. This study sought to determine the effects of an angiotensin II type 1 receptor blocker (ARB), alone or in combination with a statin, on AVS. Male New Zealand White rabbits were fed either regular chow (Control, n=5) or an atherogenic diet for a period of 18 months to induce AVS. Recognizing the clinical reality, therapy was introduced after disease onset. After 12 months, rabbits were block randomly assigned to four groups receiving either no treatment (Cholesterol, n=6), olmesartan medoxomil (Olmesartan, n=7), atorvastatin calcium (Atorvastatin, n=7), or a combination of both drugs (Combination, n=7) for the final 6 months. Magnetic resonance imaging (MRI) was used to monitor disease progress throughout the treatment period. After sacrifice, valve lesions were analyzed using histology and immunohistochemistry. In vivo disease monitoring yielded no discernible treatment effect. While Cholesterol cusps were significantly thicker than Control throughout the treatment period (0.465 ± 0.030 vs 0.388 ± 0.023mm for Cholesterol and Control, respectively, at 18 months), the various treatments had no positive effect on cusp thickness, and were all identical to Cholesterol at 18 months. Aortic valve area provided similar results; while significant disease was established (0.379 ± 0.033 vs 0.623 ± 0.074cm2 for Cholesterol and Control, respectively, at 18 months), there were no significant differences between treatment groups. Histological analysis of Cholesterol, Atorvastatin, Olmesartan, and Combination cusps revealed fibrosal thickening, lipid deposition, macrophage infiltration, and minor calcification. However, morphological analysis did not reveal significant differences in lesion composition among the treatment groups. Treatment efficacy was confirmed by analysis of aortic lesion area which revealed a significant reduction of atherosclerosis in Olmesartan-treated animals. Neither olmesartan medoxomil nor atorvastatin calcium, alone or in combination, provide demonstrable benefit in the treatment of established AVS despite success in the treatment of atherosclerosis.
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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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A Pulmonary Valved Conduit of Porcine SIS Remodels into Native Tissue in an Ovine Model
Authors: Anna Fallon, Traci Goodchild, Christian Gilbert and Robert MathenyAbstractReconstruction of the pulmonary valve and outflow tract is frequently needed to repair congenital defects. Current substitutes lead to graft failure and reoperation due to calcification, shrinkage, progressive insufficiency or transvalvular gradients, and relative growth of the patient compared to the valve. CorMatrix extracellular matrix (ECM), derived from decellularized, non-crosslinked small intestine submucosa (SIS) is used for general cardiac repairs and regenerates into normal cardiac tissue with growth potential. Previously, we showed that an ECM pulmonary valve leaflet remodeled into a neo-leaflet histologically similar to native valve architecture. In this study we used an ECM valved conduit for pulmonary valve replacement in an ovine model to demonstrate its potential to remodel into native tissue. A trileaflet valved conduit was produced from CorMatrix ECM sutured into a tube then intussuscepted to form a tube within a tube. At three equidistant points the inner tube was sutured to the outer tube forming three leaflets to guide unidirectional flow with physiologic opening and closing mechanics. Under cardiopulmonary bypass the ovine pulmonary valve and pulmonary artery section was removed and replaced with the ECM valved conduit. Valve function was evaluated by echocardiography post-operatively and at bi-monthly intervals until euthanasia at 3, 5, 8, and 12 months. Histological evaluation included H and E, Movat pentachrome, von Kossa, anti-CD31, and anti-eNOS. Our echocardiography results show that a pulmonary valve constructed from ECM opens and closes completely without regurgitation or stenosis for 12 months. Grossly, explanted valves appeared similar to native valves and were remodeling after 3 months with further progression to native morphology after 5, 8 and 12 months. Histological examination showed diffuse cellular infiltration by 3 months. At 5 months, collagen organization was increased and glycosaminoglycans were distributed throughout the middle of the leaflet. At 3 months, SEM and eNOS staining demonstrated a confluent and functional endothelial lining on the pulmonary artery and hinge regions of the valve. At 5 months, this lining extended to the center of the leaflet with confluent areas at the leaflet tip. At 8 and 12 months, a tri-layered structure similar to native valve architecture was demonstrated histologically by a Movat stain with a confluent endothelial lining demonstrated by eNOS and CD31 staining. The von Kossa stain showed an absence of calcific deposits at all time points except occasionally at the suture. These results demonstrate the potential of a CorMatrix ECM pulmonary valve to remodel into endothelialized tissue that is indistinguishable from the host’s native valve both grossly and histologically. Such a regenerated valve would be expected to improve patient outcomes since it remodels into native tissue with growth potential.
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Finite Element Modeling of Transcatheter Aortic Valve Replacement
Authors: Ali N. Azadani, Sam Chitsaz, Andrew Wisneski, Natalie Lui, Julius M. Guccione, Liang Ge and Elaine E. TsengAbstractTranscatheter aortic valve replacement (TAVR) has revolutionized treatment for inoperable and high risk surgical patients with severe symptomatic aortic stenosis. Transcatheter aortic valves (TAVs) are deployed within the native diseased valve without sutures to secure them within the annulus. Oversizing of TAVs with respect to the annulus size is required to achieve appropriate anchoring. Optimal TAV function requires expansion of the frame to its nominal dimension. However, clinically TAVR results routinely in incomplete expansion of the stent frame. We have previously demonstrated that significant under-expansion results in suboptimal TAV function with impaired coaptation of TAV leaflets, but precise characteristics of TAV leaflets and frame after implantation have been poorly studied. The aim of this study was to determine the effect of TAV under-expansion as observed clinically on stress distribution and magnitude in the TAV stent and leaflets using finite element (FE) modeling. A computer aided design (CAD) model of a TAV was developed based on the 23mm Edwards-SAPIEN design and used to create a finite element (FE) model. The 3D model consists of a stent, three pericardial leaflets, a clamp compression unit and an expandable balloon. Large deformation FE simulations were conducted to model the TAVR procedure, including TAV crimping followed by balloon-expansion to 17, 21, and 23mm. Stress distribution on the stent and leaflets were determined. As the post-inflation diameter increased, the von Mises stress on the TAV stent decreased. The maximum von Mises stresses of stent after expansion to 17, 21, and 23mm were 365, 346, and 262 MPa, respectively. However, unlike the stent, the leaflet stress increased as the post-inflation diameter increased. The peak von Mises stresses after expansion were 1.5, 1.3, and 2.4 MPa, respectively. We present the first FE simulation which was developed to model the TAVR procedure from crimping and balloon inflation of the TAV. Stress on stent and leaflets after implantation is dependent on the internal diameter of the inflated stent. While stress on the stent decreases with increasing TAV expansion, stress on the leaflets increases. FE modeling can be further applied evaluate whether a specific TAV size and design is optimal for a specific patient.
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A Completely Biological, Tissue Engineered Valve Leaflet Suitable for TAVI
Authors: Todd McAllister, Nathalie Dusserre, Nicolas Chronos and Nicolas L'HeureuxAbstractClinically available transcatheter aortic valve replacement (TAVR) technologies typically use chemically fixed bovine or equine tissues for the valve leaflets. While these fixed, xenogeneic materials have been used with success in devices placed by open surgical access, the tissue thickness (>500 microns) adds significantly to the overall crossing profile of the delivery device. Complications associated with device diameter are generally reported in at least 10-20% of clinical cases, making a reduced crossing profile one of the most critical targets for second generation TAVR devices. Another limitation associated with pericardium is fatigue induced delamination. Previously we have reported clinical results with a completely autologous tissue engineered vascular graft built using a process termed sheet-based tissue engineering. Using this approach, we were able to build small diameter blood vessels with supraphysiologic burst pressures, and demonstrated clinical durability with time points out to 3 years. Importantly, this tissue engineering approach requires no chemical fixation or exogenous biomaterials. More recently, we reported initial human use with an allogeneic version of the vessel. With time points out to 1 year, the allogeneic tissue engineered material demonstrated no evidence of immune reaction. This transition to an off-the shelf, allogeneic approach enables use the material in a variety of new clinical indications, including valve reconstruction. Valve leaflets built from a single sheet, demonstrated ultimate tensile strength in excess of that for bovine valve leaflets. Of note, the thickness of the sheet was less than 200 microns, roughly 30 percent that of bovine pericardium. The tissue can also be compressed, further reducing the thickness to approximately 75 microns. This thin, durable, single layered tissue can be assembled onto commercially available TAVR devices resulting in a reduction in crossing profile of approximately 2 Fr. The valve leaflets can be sutured easily, coapt normally, and can withstand arterial backpressure. Given the non-laminated structure of the tissue engineered leaflet, the lack of synthetic materials, and the durability demonstrated in other clinical indications, this approach may provide not only a reduced crossing profile, but also improved long term clinical results.
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Imbalance in Extracellular Matrix Synthesis and Degradation as a Mechanism of Leaflet Weakening in Sporadic Mitral Valve Prolapse in Humans
AbstractSporadic mitral valve prolapse (MVP) is a common valvular disorder affecting 2-3% of the humans worldwide. Unlike MVP in Marfan’s and Ehlers-Danlos syndromes, no specific genetic mutations that impact tissue homeostasis are currently identified. Yet, significant degenerative leaflet changes are observed, manifesting as leaflet weakening and billowing. In this study, we hypothesized that assessing the matrix health by investigating the activity of matrix synthetic and degradative markers would provide insights into the leaflet degeneration processes. Mitral leaflets were obtained from eight humans (N =8) undergoing surgical repair for MVP at our institution. Fresh leaflets were rinsed, stored in sterile PBS and divided into annulus, base or edge regions and used for immunohistochemistry(IHC) and western blotting(WB). Matrix synthetic activity was assessed using assays for: prolyl-4-hydroxylase(P4H–collagen synthesis enzyme), heat shock protein-47(HSP47–chaperone for collagen folding), and lysyl oxidase(LOX–collagen cross-linking). Matrix degradation was assessed using assays for matrix metalloproteases: MMP-1(collagen I degradation), MMP-3(collagen III degradation), and MMP-9(collagen V degradation). Tissue sections were imaged and quantitative analysis was performed using an image thresholding technique. Cell viability was confirmed using DAPI, and overall tissue structure assessed using H and E staining. Tissue section closest to the mitral annulus served as the control, against which the belly and edge expressions were compared. In the synthetic pathway, P4H activity decreased from 4±1.5 at the annulus to 2±1.3 at the belly (2X) and increased to 17±12 at the edge (4X). HSP47 activity was 3.2±2 in the annular section that increased to 5.1±2 in the belly (1.6X) and 4±1.3 in the edge (1.2X). LOX expression slightly increased from the annulus to the belly (1.2X), but was significantly higher in the edge (10X). In the degradative pathway, MMP-1 expression was 8.4±6.3 at the annulus, which decreased to 5.6±2.8 in the belly (1.5X), and 6±2 in the edge (1.4X). MMP-3 expression significantly increased from the annulus to the edge, with 2.3±2 at the annulus, 7.7±5 at the belly (3.3X) and 24.5±10.6 at the edge (10.4X). MMP-9 expression decreased from the annulus (1.3±0.4) to the belly (0.7±0.2; 1.7X) and increased to 3±1 in the edge(2.4X). Collagen biosynthesis is adequate in the edge as evident from increased P4H activity, however nominal HSP47 activity indicates poor fibril maturation and folding, resulting in a weak matrix in spite of good cross linking from higher LOX expression. Increased MMP-3 and 9 activities in the edge signify degradation of collagen III and V, which may further weaken the matrix. Studies to investigate the regional differences in genetic, cellular and molecular pathways in MVP and the potential role of the elevated stress in leaflet degradation are necessary.
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A Finite Element Study of Human Pulmonary Autograft Wall Stress after the Ross Procedure
AbstractDilatation of the pulmonary autograft after the Ross procedure can lead to aortic insufficiency and/or aneurysmal pathology requiring reoperation. Autograft remodeling occurs as the autograft wall is exposed to systemic pressure and higher wall stresses, which have not been quantified in humans. The aim of the study was to develop a realistic Finite Element (FE) model of the human pulmonary autograft and to perform simulations at systemic pressure to quantify the increases in autograft wall stress immediately after the Ross procedure. Autograft geometry was generated from high-resolution micro-computed tomography images of an explanted human pulmonary root to create a mesh of hexahedral elements. Constitutive equations were used to describe the regional tissue material properties of the human pulmonary root obtained from bi-axial stretch testing. LS-DYNA (LSTC Inc., Livermore, CA) FE software was used to simulate cardiac cycles at pulmonary and systemic pressure. Autograft dilatation and wall stress distribution were determined. Correlation of LS-DYNA model material properties to actual tissue stress-strain data was performed to ensure model accuracy. Human autograft dilation from pulmonary to systemic pressure was minimal (32.1 to 33.4mm) due to the non-linearity of the material properties. Less compliance was demonstrated at greater wall stresses. Significant increases in autograft wall stresses were found at systemic pressures. Maximal wall stresses increased approximately 10-fold in diastole (12.4 to 122.3 kPa) and 5-fold in systole (48.1 to 234.2 kPa), relative to the wall stresses at pulmonary pressures. Pulmonary autograft wall stress increased by an order of magnitude at systemic pressure. Initial autograft dilation at systemic pressure was minimal as validated by clinical studies. Chronically elevated wall stress may lead to pathologic remodeling and aneurysmal formation over time. The correspondence of this model with future studies of post-dilated autografts will lead to an improved understanding of tissue remodeling, and offer necessary data for developing improvements to the Ross procedure.
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Prevalence of Patients With Severe Aortic Stenosis, Low Flow And Preserved Ejection Fraction: Results From a Cath-Lab Data Base.
Authors: Toniolo Mauro, Rossi Andrea and Cicoira MariantoniettaAbstractRecent echocardiographic studies described that almost 30% of patients with severe aortic valve stenosis on the basis of aortic valve area may paradoxically have a relatively low mean gradient despite a preserved left ventricular ejection fraction (EF). However the existence of this pathologic entity has been questioned mainly for the lack of invasive data. We aimed to describe the prevalence of patients with severely reduced aortic valve area and low gradient from a consecutive series of patients with aortic stenosis and normal EF undergoing cardiac catheterization. Sixty one consecutive patients with invasively measured aortic valve area < 0,6 cmq/mq (AHA/ACC definition for severe aortic stenosis) and EF> 50% formed the study population. Each patient underwent to right and left heart catheterization for a comprehensive invasive hemodynamic evaluation. Aortic valve area was measured by Gorlin formula. Cardiac output was measured by thermodilution or Fick method. Low mean gradient was defined < 30 mmHg. 16 % of patients were characterized by low GM despite severely reduced aortic valve area. Patients with low GM were characterized by significantly higher aortic valve area (0.47±0.09 vs 0.36±0.09 cm2/m2; p=0.0008) but similar left ventricular stroke volume (SV) (65±22 vs 65±17 ml; p=0.9) and cardiac output (4.8±1.1 vs 4.7±1.0; p=0.7). The prevalence of low flow (defined as SV < 35 ml/ m2) was similar between groups (50% vs 43%; p=0.3). There was no difference in term of age (78±10 vs 79±11 years; p=0.6), female gender (50% vs 48%; p=0.5), body surface area (1.79±0.4 vs 1.80±0.4; p=0.8), pulmonary artery systolic pressures (37±9 vs 35±11 mmHg; p=0.8), LV end diastolic pressure (16±4 vs 20±7; p=0.1) and mean wedge pressure (17±7 vs 15±7; p=0.2). Patients with low GM showed a higher mean AO pressure (111±14 vs 93±14; p=0.009) but similar level of aortic distensibility (0.78±0.3 vs 0.9±0.4 ml/mmHg; p=0.3). This invasive study confirms that a substantial percent of patients may have a low GM despite a severely reduced aortic valve area and normal EF. It should be acknowledge that the barely perception of this pathologic entity might have reduced the likelihood of patients to undergo catheterization leading to underestimation of the prevalence of this condition.
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