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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
21 - 40 of 86 results
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Investigation of the Statin Paradox in Different Populations of VICs
Authors: Elyssa L. Monzack, Chloe M. McCoy, Kelsey A. Duxstad and Kristyn S. MastersAbstractWhile numerous clinical studies have examined the effect of HMG-CoA reductase inhibitors (statin drugs) on calcific aortic valve disease (CAVD), their conflicting results have yielded many questions regarding the nature of statin-valve interactions. One step toward better understanding this relationship is to examine the effects of statin treatment on heart valves on a cellular level. Previous work found statin treatment to have a "paradoxical" effect in vitro, decreasing osteoblastic markers in valvular myofibroblasts, while increasing those same markers in osteoblast precursor cells. This finding that statins may be able to selectively induce bone formation only in a cell type that is already prone to mineralization leads to the question of how statin treatment will affect valvular interstitial cells (VICs), a heterogeneous cell population which is capable of differentiating into an osteoblast-like phenotype, termed obVICs. In this study, we set out to determine whether obVICs would respond to statin treatment in the same manner as myofibroblasts, or if obVICs would increase bone marker expression in a manner similar to a bone-derived cell type. This work was also complemented by a gene expression analysis of calcified human valves from individuals who were or were not taking a statin drug. Porcine VICs were cultured in vitro, with or without 1 uM simvastatin, in either control or mineralization medium, where the control medium yields a heterogeneous population that is predominantly myofibroblasts, while the mineralization medium drives VICs toward an obVIC phenotype. Gene expression analysis included multiple myofibroblastic and osteoblastic markers and was conducted daily over an 8-day time course, yielding information about not only expression levels, but also their temporal dynamics. Gene expression profiles were compared between VICs and an osteoblastic cell line (MC3T3-E1) to assess similarities. Myofibroblastic and osteoblastic genes were also analyzed in aortic valves from human patients (+/- statin) undergoing aortic valve replacement surgery. Statin treatment increased osteoblastic gene expression in VICs cultured in mineralization medium (obVICs), but the same effect was not obtained in control medium. This finding suggests that VICs are capable of responding to statin treatment in a manner similar to bone cells, but only when VIC cultures are driven toward an osteoblastic phenotype. The MC3T3-E1 cells also increased osteoblastic gene expression upon statin treatment, although their basal level of osteogenic activity was substantially greater than that found in any of the obVIC cultures. Analysis of human valve data is ongoing. Overall, this study suggests that different subpopulations of VICs exhibit different and temporally dynamic responses to statin treatment, further complicating the ability to predict a clinical effect of statin drugs on CAVD.
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Mitral Valve Interstitial Cells Behaviour Under Hypoxia
AbstractMitral Valve Interstitial Cells (MVICs) are distributed throughout the valve leaflets. It is predicted that some cells exist under hypoxic conditions. Hypoxia is an important stimulus for signalling pathways that affect cell growth differentiation and function. This study examines the effect of various degrees of hypoxia on MVICs growth, survival, morphological and phenotypic behaviour. Porcine MVICs were primarily isolated and incubated under atmospheric control (20% O2), mild hypoxia (5% O2), moderate (2% O2) and severe (0.5% O2) for 1 and 3 days. Cell proliferation and cell death were assessed using biochemical assays. Cell morphology was assessed by immunofluorescence staining. Cells were also stained for phenotypic expression of endothelial, myofibroblastic and smooth muscle markers. After 24 hours incubation at the different O2 concentrations there was no significant difference in cell growth or death. After 3 days incubation cells under atmospheric O2 (150±8%*) and 5% (124±5%), 2% (146±8%*) and 0.5% (161±8%*) all showed increase in cell number compared to start of the experiment (*=P<0.05).However, there was no significant difference between each of the groups. Cell death was significantly reduced under hypoxia (atmospheric O2 (10.65%±1.72) and 5% (8.5%±1.0%), 2% (6.25%±0.24%*) and 0.5% (4.02±0.45%*) O2 (*=P<0.05). Cells were significantly bigger at 3 days under hypoxic conditions but retained the same shape. MVICs continued to express similar levels of myofibroblastic markers αSMA and Vimentin under hypoxic conditions after 3 days but showed weak expression of smooth muscle cell markers. This study serves to define the role of hypoxia in VICs in terms of cell growth, death, morphology and phenotype. These properties further highlight the specialised function of cells that reside in heart valves and have important relevance to heart valve tissue engineering.
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Lysophosphatidylcholine Alters Valvular Interstitial Cell Mineralization
Authors: Dena C. Wiltz, Joel D. Morrisett and K. Jane Grande-AllenAbstractCalcific aortic valve disease (CAVD) is a condition of the heart characterized by thickening and calcification of the aortic valve and can lead to aortic stenosis, narrowing of the aortic valve that can obstruct left ventricular outflow. CAVD is thought to have similarities with atherosclerosis, in which the aortic wall demonstrates thickening due to plaque buildup. A notable similarity seen between CAVD and atherosclerosis is the accumulation of lipids in the tissues. One important chemical component involved in atherosclerosis is lysophosphatidylcholine (LPC), a phospholipid derived from phosphatidylcholine. LPC concentrations have been shown to increase in atherosclerotic conditions, and induce expression of osteogenic factors by vascular smooth muscle cells. The potential for LPC to affect valve cell calcification, however, has not been previously investigated. In addition, calcification of cells from different valves warrants investigation because the aortic valve becomes more bone-like and experiences onset of calcification sooner than the mitral valve during the calcification process. This study investigated the effect of LPC on the propensity for calcification by porcine valve interstitial cells (VICs) from aortic and mitral valves. On day 0 VICs were seeded at a density of 50 000 cells/cm2 in low serum media. On day 1, the media is changed to media containing LPC in concentrations ranging from 0 to 100 µM. The cells are cultured for 8 days and then assessed for mineralization using histological stains (Alizarin Red S for calcium deposition and Von Kossa for phosphate deposition) and biochemical assays (Alkaline phosphatase activity). Significance (p <0.05) was determined using Analysis of Variance followed by Tukey post-hoc testing. Interestingly, mineralization in the VIC cultures was decreased as LPC concentration increased from 0 to 1 µM. At 10 µM, however, an increase in mineralization was observed compared to the 1 µM cultures. VICs in 100 µM LPC media began to detach within 24 hours of LPC media application. Also, VICs from different valves displayed different levels of calcification at each condition. LPC alters mineralization in VIC cultures from both aortic and mitral valves, in a concentration dependent manner. Extremely high concentrations of LPC (at and above 100 µM) can be toxic to VICs. There is a unique behavior of VICs with addition of varying concentrations of LPC, most notably that low concentrations (below 10 µM) actually reduced mineralization. Other factors, such as effects of LPC on VIC proliferation and apoptosis, will be important to investigate in future work. This study demonstrates that LPC affects the mineralization potential of valvular cells in a way that is distinct from vascular cell types.
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Developing a Rat Model of Cardiovascular Calcification to Evaluate Tissue-Engineered Heart Valve Prostheses
AbstractA small animal model to evaluate the in vivo degeneration and calcification of biological versus tissue-engineered cardiovascular prostheses in short time periods is warranted. Our study aims at developing a standardized rat model of accelerated cardiovascular calcification and lipid metabolism disorder. Male Wistar rats (n = 60; 200 – 250g) were fed ad libitum with 5 different regimens of procalcific diet (group 1: +300,000 units/kg vitamin D (VD) +2% cholesterol (CHOL) +1.5% calciumphosphate (PO4); group 2: +150,000 units/kg VD +1% CHOL +0.75% PO4; group 3: +300,000 units/kg VD +2% CHOL; group 4: +300,000 units/kg VD +1.5% PO4; group 5: +2% CHOL + 1.5% PO4; group 6: normal food). After 4, 8 and 12 weeks, animals were euthanized, organs explanted (left ventricular myocardium, aortic valve, ascending aorta, abdominal aorta, kidney and liver) and histology as well as immunohistochemistry conducted. During the study, body weight and chow intake were monitored. Heart function was examined by echocardiography, and blood serum level analyses were conducted at explantation. Unimpaired survival was 100% in all groups. Histology revealed calcification of the aortic valves after 4 weeks, while relevant calcium deposition in the aortic wall was observed only after week 8 (vonKossa staining). Aortic valves of rats with high doses of VD (groups 1, 3 and 4) were significantly more calcified than those of animals with a reduced dose of VD (group 2; p < 0.01) or no VD supplementation (group 5; p < 0.001). In all rats, early calcium deposition was located at the commissures, whereas the aortic sinus walls and especially the valve leaflets were diseased at later time points. Massive calcification was accompanied by chondroid cells and lipid-containing cells (oil red staining). However, animals on a diet with reduced VD or no VD presented a significantly higher amount of chow intake (each with p < 0.01 versus groups 1, 3, 4 and 6), paralleled by significantly larger increase in body as well as heart weight (each with p < 0.001 versus groups 1, 3, 4 and 6). All supplementation regimens resulted in early aortic valve and later aortic wall calcification. High doses of VD intake accelerated the calcium deposition, however, the somatic growth of these rats was impaired. A procalcific diet with moderate doses of VD + CHOL + PO4 seems to be most suitable for a comparative evaluation of calcifying degeneration in native and prosthetic cardiovascular tissues.
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Decellularization Diminishes the Calcifying Degeneration of Aortic Conduit Allografts
AbstractThe present study aimed at developing a small animal transplantation model of accelerated calcifying degeneration, in order to evaluate degenerative in vivo processes in biological heart valves and vascular implants. Male Wistar rats (recipients) with an interventionally induced aortic insufficiency grade II – III (AI; day -14) were fed with a diet containing high-dose vitamin D, cholesterol and calciumphosphate. Aortic conduits of Sprague-Dawley rats (donors) were decellularized according to a detergent-based protocol and infrarenally implanted (day 0) in an end-to-side manner in the recipients (group A; n = 6). Cryopreserved implants served as controls (group B; n = 6). Doppler sonography was conducted at days -14, 0, 28 and 84. Graft explantation, histological and immunohistochemical analyses were performed at days 28 and 84. In all recipients AI grade II – III with subsequent reversed diastolic flow in the abdominal aorta was confirmed. Sonographic competence of the conduit perfusion and overall survival were 100%. After 12 weeks severe calcification of the native aortic media as well as of the aortic conduit implants was observed (vKossa staining), however, in group A diet-induced calcification was significantly lower as compared to group B (p <0.01). Histological evaluation of the conduit implants revealed an intimal hyperplasia, involving α-smooth muscle actin expressing cells, with an increased intima-to-media ratio (p < 0.001) and inflammatory activity (CD3+) in group B versus group A. During the later follow-up, intimal hyperplasia and severe calcification aggravated. After 12 weeks, in opposite to group A explants, all grafts of group B contained Syndecan-3-expressing cells with a chondroid phenotype. Our rapidly calcifying rat transplantation model enables detailed evaluation of native and tissue-engineered aortic conduits, especially in terms of degenerative processes. Compared to cryopreserved grafts, decellularization significantly diminished the calcifying degeneration and intimal hyperplasia of aortic conduit implants. Further work will focus on the characterization of the de novo interstitial repopulation, particularly on the nature of the chondroid cells and their role in graft degeneration.
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Decellularized Heart Valve Prostheses and the Anticalcific Potential of Simvastatin
AbstractDecellularization is a proven approach to decelerate the degenerative processes which lead to the failure of heart valve implants. In order to further delay the calcifying in vivo degeneration of tissue-engineered grafts, antiarteriosclerotic and antiinflammatory substances may be advantageous, and some in vitro reports on HMGCoA reductase inhibitors have shown encouraging results, whereas clinical trials have failed to prove a positive in vivo result. Male Wistar rats (recipients) underwent an interventional generation of aortic insufficiency grade II – III (day -14) and were fed with a procalcific diet of high-dose vitamin D, cholesterol and calciumphosphate, additionally supplemented with simvastatin (group S; n = 6). Identically treated animals fed with the same diet, not supplemented with simvastatin, served as controls (group C; n = 6). Aortic conduits of Sprague-Dawley rats (donors) were decellularized according to a detergent-based protocol and infrarenally implanted (day 0) in an end-to-side manner in the recipients. Echocardiography, doppler sonography of the implant and blood serum analyses were conducted at days -14, 0 and 28. Graft explantation, histological and immunohistochemical analyses as well as quantitative real time PCR were performed after 4 weeks. Acute AI grade II – III with echocardiographically confirmed reversed diastolic flow in the whole aorta caused significant left ventricular (LV) dilatation as well as decrease of LV ejection fraction (p <0.001 at day 28) and resulted in a mortality of 8%. Sonographic competence of the conduit perfusion and overall survival of the transplanted rats were 100%. After 4 weeks of simvastatin treatment, calcification of the implants was significantly lower in group C (p < 0.01), whereas especially the aortic valve and the ascending aorta were strained by a decreased calcium burden (vonKossa staining). Histological evaluation of the conduit implants revealed an intimal hyperplasia, involving α-smooth muscle actin expressing cells, with an increased intima-to-media ratio (p < 0.05) in the aortic walls of group S and in the aortic valves of group C. RNA analysis by quantitative PCR was performed to confirm these results. The present study in a standardized rat transplantation model failed to show an early benefit of the HMGCoA reductase inhibitor simvastatin to diminish the calcification of decellularized aortic conduit implants. Furthermore, existing literature in this field is contradictory, and therefore, further experiments with more detailed analyses and long-term observations are warranted.
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Contribution of Specific Glycosaminoglycans to the Relaxation Properties of the Aortic Valve
Authors: Borghi Borghi, Carubelli Ivan, Adrian H. Chester and Magdi H. YacoubAbstractThe aortic valve (AV) is characterized by a complex mechanical behavior which is closely linked to its structural components. The central layer of the AV is rich in glycosaminoglycans (GAGs) which play an important role in the biomechanics of the AV. In this study the effect of selective GAGs depletion on time dependent mechanical behavior of porcine AV was analyzed. Fresh strips of porcine AV cusps were cut in either in the radial or in the circumferential direction and mounted on a tensile testing system (Bose Electroforce) for mechanical testing. Three groups of valves were treated enzymatically, in order to remover either all the GAGs (group 1), the sulphated GAGs only (group 2) or the non-sulphated GAGs only (group 3). Each group had a control group. Mechanical tests were performed on each strip and stress relaxation kinematics as well as relaxation percentage were compared between treated and untreated specimens. Tensile and stress relaxation tests were performed on the strips under physiological load levels. The reduced stress relaxation function was fitted to the experimental data using a two phase (τ1 and τ2) exponential decay model. Relaxation percentage was significantly lower in group 1 for both circumferential (group 1: 20.58% vs control 28.34%, p = 0.004) and radial strips (group 1: 18.89% vs control 28.85%, p = 0.006). In this group, the early relaxation value (τ1) markedly decreased in the radial direction (group 1: 7.86s vs control: 10.35s, p = 0.0041) while no statistical difference was achieved in the circumferential direction. When looking at selective GAGs depletion, an effect of the hyaluronic acid depletion (group 3) was seen on the excursion of circumferentially oriented strips (group 2: 23.15% vs control: 27.76%, p = 0.049). No major effect was seen comparing the results of group 2 with its control. No effect on t2 was found. Histology confirmed the successful GAGs depletion. The presence of GAGs influences the biomechanics of the AV in terms of time dependent mechanical properties. The presence of hyaluronic acid has a distinctive effect on the relaxation excursion of the cusps while no effect was apparent for sulphated GAGs. These results provide further insight into the relationship between structure and fuction in the AV.
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Characterization of Sex-Related Differences in Valvular Interstitial Cells
Authors: Chloe McCoy, Dylan Q. Nicholas and Kristyn S. MastersAbstractAlthough the pathogenesis of calcific aortic valve disease (CAVD) is not well understood, males have been identified as having a two-fold increased risk for the disease when compared to females. In this study, we examined gene expression profiles in healthy pigs and measured markers of disease in vitro to determine whether the differences in clinical risk between males and females translate into measurable intrinsic differences on the cellular scale. In addition, we also investigated potential sex-related differences in cellular response to TGF-β1, an inflammatory stimulus known to be elevated in calcified human aortic valve explants. mRNA was isolated from three male and three female porcine aortic valves (denuded of endothelial cells) and hybridized to Affymetrix® GeneChip Porcine Genome microarrays. Mean expression values of each probe set in the male samples were compared with those in the female samples and biological processes were analyzed from the dataset for overrepresentation using Gene Ontology term enrichment analysis. From the microarrays there were 183 genes identified as being significantly (fold change>2; P<0.05) different in healthy male versus female aortic valve leaflets. Within this significant gene list there were 298 overrepresented biological processes, several of which are relevant to pathways identified in CAVD pathogenesis. In particular, pathway analysis indicated that cellular proliferation, apoptosis, cell migration, ossification, and extracellular matrix reorganization were all significantly represented in the data set. In vitro culture of male and female porcine valve cells also revealed intrinsic differences between sexes, with male cells exhibiting higher proliferation, apoptosis, and expression of αSMA after five days of culture. When exposed to TGF-β1, male cells grown in serum-free culture were found to be more sensitive to the inflammatory cytokine, with dramatically decreased apoptosis and proliferation compared to a marginal decrease in apoptosis and no change in proliferation in female cells. These data suggest that some sex-related propensity for CAVD may be present on the cellular level in healthy subjects, possibly resulting in a differential response to systemic factors that promote disease onset and progression. These results also offer motivation for further sex-related studies with valve cells to better determine possible genetic contributors and to explore sex-related susceptibilities for valve calcification.
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Influence of Scaffold Structure on Cell Behaviour for Valve Tissue Engineering
AbstractThe success of a tissue engineered heart valve is dependent on developing the right structure, the right interactions between the cells and the right matrix and mechanical force. Different materials have been used as scaffold, however the best processing method have not been established. We have used 2 different type of scaffold: collagen based scaffold and a nanofibrillar scaffold made from poly(ε-caprolactone) (PCL). Different seeding methods have been tested for cell compatibility of these scaffolds with human adipose derived mesenchymal stem cells (hADSCs) and with human telomerase immortalized bone marrow derived stem cells (hTERT). Nanofibrillar scaffolds have been produced by jet-spraying the polymer on a metal grid. Collagen scaffolds were made by freeze drying a 1% bovine collagen solution chemically crosslinked with EDC-NHS. Morphological evaluations of the structures were performed using scanning electron microscopy. Elastic modulus of dry scaffolds (10x6x1 mm, n=5) was measured with a planar biaxial test bench with displacement rate of 0.05 mm/s. Two different type of cells (600000/scaffolds) were seeded using different seeding methods to find the best condition: top seeding for 2 hours and then dynamically cultured using a rotary mixer, directly dynamically seeded for different time period and with different volume (5, 10 and 25 ml). DNA quantification using Hoescht 3258 and DAPI staining were used to evaluate proliferation and penetration inside the scaffold. Immunohistochemistry was used to check collagen production. PCL scaffolds were composed of non woven nanofibers (600 nm average diameter) assembled in a highly open structure. Collagen scaffold showed an interconnected porous structure with average pore size of 100 um. Nanofibrillar scaffolds showed higher elastic modulus compared to collagen (200 KPa compared to 150 KPa). The different cell seeding approaches had an effect on cellular distribution and cell number. With 10 ml of volume cells attached more after 24 hours compared to 5 ml with no further difference compared to 25 ml. Top-seeded matrices resulted in a high cell concentration on the seeded surface while rotary seeding allowed cells to attach on both scaffold sides but in fewer numbers. Regardless of seeding method, cells proliferated extensively (up to 10 and 2-fold DNA increase for hTERT and hADSC respectively ) on both scaffold, but proliferation was up to twice higher within nanofibrillar structures compared to collagen scaffolds. Both cell types were able to populate the entire area of both scaffolds over 10 and 14 culture days for hADSC and hTERT respectively. Both cell type produced their own ECM within the scaffolds as indicated by collagen I positive staining. Jet-sprayed PCL nanofibrillar scaffolds are a promising alternative to collagen scaffolds for cellular infiltration and proliferation.
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Further Refinements in Collagen Mimetic Peptide Scaffolds for Tissue Engineering Heart Valves
AbstractCollagen is the essential protein in the extracellular matrix, which maintains the structural and mechanical integrity of tissues while providing key signals to regulate cell functions. Although animal-based collagens can be used as biomaterial for tissue engineering heart valves, they cause infections and lack flexibility. These limitations have stimulated the exploration of collagen mimetic peptides (CMPs) through a bottom-up approach using computational modeling followed by experiments to enzymatically cross-link the CMPs and produce hydrogels. The X-ray structure of triple-helices of CMP was used in software FIRST and in mutational code to identify its structural stability and hotspots. These data assisted to introduce charged residues by mutations to cross-link and to add binding motif (GFOGER) for integrin in the structure. The helical stability and self-association of the mutated CMP has been validated using molecular dynamics (MD) simulation. Experimentally, the peptide was synthesized by solid phase Fmoc chemistry and characterized by HPLC and mass spectrometry. Enzymatic cross-linking on primary amine and gel formation were obtained by incubating peptide and plasma amine oxydase (PAO) solutions in PBS at 37 and 58 °C. Peptide assembly and aggregation was monitored by turbidity (optical density at 314 nm) and morphology was analysed by transmission electron microscopy (TEM). The modelling analyses indicated the CMP to have the desired structural properties for self-assembly and high affinity towards integrin binding. The modification of the key positions with charged residues increased the possibilities for helical cross-link (gelation). In addition to cell signalling, the charged residues at the cell binding motif could further enhance the inter-helical association of the CMPs. The structural properties of the modelled CMP were reproduced in experimental conditions. Addition of PAO significantly improved turbidity of peptide solutions and lead to hydrogel formation. The peptides assembled in branched fibrillar structures around 25 nm in diameter as it was confirmed by TEM analysis. The proposed peptide promises to show some inherent structural properties of native collagen in silico and in vitro. These properties are required to produce functional scaffolds for tissue engineering heart valves.
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3D Engineered Micro-Tissue Models to Study Cardiovascular (Patho)biology and Regeneration
AbstractEngineered tissue models find their application in studying normal and pathological tissue development and the associated testing of potential therapies. In addition, they provide powerful tools for technology development for regenerative medicine and optimization of regenerative therapies. We have developed a range of ‘humanized’ engineered cardiovascular model systems – consisting of engineered tissue, (hemo)dynamic loading platforms, and readouts of tissue development and mechanical function – for the optimization of in-vitro and in-situ tissue engineering strategies of heart valves and vessels. The model systems can be adapted to simulate either healthy or diseased tissue development or healthy and diseased loading environments (e.g. high blood pressure). A first range of systems consists of cardiovascular tissues (strips or cross-shaped morphology, mm range), engineered from human myofibroblasts seeded on natural (fibrin) or synthetic (PGA) degradable polymer scaffolds, and loaded in series on an adapted Flexcell device to study the mechanobiology of collagen remodeling of engineered tissue. Vital collagen imaging (CNA35) and on-line assessment of structure-function properties indicated that stochastic rather than cyclic loading of the strips resulted in increased collagen formation, organization and tissue strength. A change of loading direction resulted in complete tissue remodeling with collagen re-orientation within 48 hours. Currently, we are using the systems to investigate the pathomorphogenesis of radiation-induced fibrosis of heart valve tissue. A second system consists of a microfluidics-based setup to study circulating cell recruitment, migration and differentiation in small 3D electrospun PCL scaffolds under physiological hemodynamic loading conditions as a model of in-situ regeneration. The system is mounted onto the stage of an inverted confocal microscope to follow cell fate and tissue development in real-time. By changing the architecture, bioactivity and mechanical properties of the scaffold, the effects of these parameters on in-situ tissue formation can be assessed. Circulating cell suspensions of changing composition/activation as well as changing hemodynamic loading conditions will be used to mimic healthy/diseased conditions and to investigate their effects on in-situ tissue regeneration. These studies demonstrate the use of versatile experimental model approaches to provide detailed insight into tissue (patho)morphogenesis, adaptation and regeneration in a real-time and high-throughput fashion.
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Ice-Free-Cryopreservation Attenuates Calcification in Allograft Heart Valves
AbstractThe objective of the study was to attenuate calcification in allogeneic ovine pulmonary heart valves. Six valves of white face sheep were ice-free-cryopreserved (STUDY) in 12.6 mol/L cryoprotectant (4.65, 4.65, and 3.31 mol/L of DMSO, formamide and 1,2-propanediol) and stored at -80°C. 6 control valves were cryopreserved by controlled slow rate freezing in 1.4 mol/L DMSO and stored in vapor-phase nitrogen (CONTROL). After 7 months in vivo explanted valves were processed for histopathology. Gross morphology showed significantly thickened leaflets in the CONTROL group. Histopathology revealed a marked calcification in the leaflet stroma and conduit wall. STUDY valves in contrast demonstrated well preserved ECM structures, no leaflet thickening, inflammation and only neglectable calcification in the conduit wall. Only discrete panus formation was noted migrating from the ventricularis onto the leaflet. Ice-free-cryopreservation enables attenuation of calcification in ovine allografts valves. The observed decreased calcification warrant improved long-term function.
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Sufficient Tissue Engineered Heart Valves: A Question of Cell Source?
Authors: Miriam Weber, Julia Frese, Nima Hatam, Joerg Sachweh, Thomas Schmitz-Rode, Stefanl Jockenhoevel and Petra MelaAbstractA promising approach to solve the problems of currently used heart valve prostheses, e.g. degeneration, need for anticoagulation and risk of endocarditis, is the tissue engineering of heart valves. By using patient derived cells and fibrin as a scaffold for these valves we aim at completely autologous heart valves which have the potential to grow and hence are especially interesting for valve replacement in paediatric surgery. However, a major obstacle on the way to clinical application of tissue engineered heart valves (TEHV) is the cell-mediated tissue contraction which leads to the shrinkage of the valve’s leaflets and thus to its insufficiency. Several groups tested TEHV in the pulmonary position in the sheep model and reported mild to moderate regurgitation already at a short postimplantation time. Our goal was to analyse the influence of the cell source on the sufficiency of TEHV. Different cell sources, among which ovine carotid artery (OCA) and umbilical artery (OUA), were compared on their contractility and contraction of fibrin gels in which the cells were embedded. Cell phenotype was characterized by immunostaining of α-smooth muscle actin (α-SMA) and myosin light-chain kinase (MLCK) as markers for cell contractile activity. For the gel contraction assay, fibrin gels with a cell concentration of 5 × 10^6/ml were moulded in a 24-well plate (n ≥ 3) and their retraction was evaluated over 15 days by measuring the gels' area in relation to their original area. Hydroxyproline content, cell proliferation and burst strength were also determined. Ovine carotid artery cells exhibited a highly contractile myofibroblast phenotype (high α-SMA and MLCK expression), while OUA cells were mostly non-contractile fibroblasts with only few cells expressing α-SMA and MLCK. After 15 days, OCA embedded gels were contracted to 30.6 ± 2.0% of the original size while OUA gels maintained a size of 83.2 ± 3.7% of the initial area. To directly correlate cell contractility and valve sufficiency we moulded fibrin based heart valves using OUA and OCA cells. All valves were conditioned statically for 14 days in the closed-leaflet configuration and successively dynamically in the open-leaflet configuration in bioreactors for 30 days. While the leaflets of TEHV with OCA cells contracted and led to insufficient valves at the end of the conditioning protocol, we were able to produce completely sufficient heart valves after in vitro conditioning using non-contractile OUA cells. In an on-going animal study OUA embedded TEHV, after being seeded with endothelial cells, are implanted in the pulmonary artery in lambs to analyse their growth potential and their hemodynamic performance in vivo. Transesophageal echocardiography showed both in colour Doppler and in cw Doppler mode that after fourteen weeks a first implanted valve was still completely sufficient and showed no sign of cell-mediated tissue contraction.
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Subcutaneous Testing of E-spun PCL Patches Suitable for in Situ Heart Valve Tissue Engineering
AbstractIn situ tissue engineered heart valves yields a new generation of cardiovascular substitutes. The body is used as a bioreactor where it relies on the natural regenerative potential of the body. The shift from the classical way of tissue engineering to in-situ tissue engineering emphasizes the role of the scaffold. The scaffold should be able to capture and preserve cells for tissue formation and it has to maintain valve functionality while tissue is developing. The use of synthetic biomaterials is very attractive. Poly(ε-caprolactone) (PCL) is an important polymer due to its mechanical properties and miscibility with a large range of other polymers. Electrospinning attracted great interest as a production method of biomaterials for in situ tissue engineering. The electrospinning process of PCL offers a nice technique for thin fiber formation to eventually create three dimensional scaffolds with the characteristic three layers, typical for heart valves. The fibers produced with electrospinning provide a similar physical structure as the extra cellular matrix. The space between fibers needs to be large enough for cells to adhere and migrate into the scaffold. Sufficient cellular in growth is needed for tissue formation. In this study cellular in growth was measured in subcutaneously implanted electrospun PCL patches. Furthermore tissue formation and degradation of the polymer is investigated. Thirty healthy male F344 Rats are used. The implants with surrounding tissue were explanted after 2, 5, 10, 21 or 84 days and embedded in paraffin. The explanted tissues were examined using immunohistochemistry (HE,MPO, ED1, α-SMA, and PSR). The electrospun fibers had a diameter of 10 μm. SEM pictures showed controlled void spaces. In the electrospun PCL samples we found a very high cellular infiltration rate after 84 days, mean of 396.819 cells per high power field. First infiltration of mainly neutrophil granulocytes was seen, followed by macrophages. Cells infiltrate throughout the whole sample. After 84 days fibroblast were seen, which were able to produce collagen. Furthermore after 84 days, macrophage giant cells and neo-vessel formation was observed. PCL was degraded in the cytoplasm of the macrophages. Electrospun PCL scaffolds with a fiber diameter of 10 μm, are suitable for cellular in growth and tissue formation. This research forms part of the Project P1.01 iValve of the research program of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation. The financial contribution of the Nederlandse Hartstichting is gratefully acknowledged.
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Non-Cytotoxic Cross-Linking of Bioactive Porcine Matrices
Authors: Pamela Somers, Filip De Somer, Ria Cornelissen, Hubert Thierens and Guido Van NootenAbstractIncubating a porcine aortic valve matrix with a platelet gel (PG) concentrate creates a bioactive matrix which is loaded with growth factors. These matrices can be repopulated with mesenchymal stem cells. However, these recellularized matrices still elicit a host immune response. Therefore, the aim of this study was to evaluate the cytotoxicity and cross-linking effect of naturally organic compounds such as quercetin, tannic acid, caffeic acid and catechin on these matrices and to investigate the effect of these cross-linkers on the in vitro growth factor release rate. Porcine aortic heart valves were decellularized using a detergent/enzymatic treatment. Cytotoxicty of the cross-linkers was evaluated by cell culture media supplementation of 10, 100, 1000, 5000, 10000 and 20000µg/mL. These concentrations were also used to cross-link the acellular matrices. Mechanical strength of the leaflets was investigated. Also the effect of these cross-linkers on the growth factor release from the PG loaded scaffolds was evaluated by ELISA assays. Results showed that proliferation of porcine mesenchymal stem cells increased significantly with increasing concentrations of quercetin, tannic acid, caffeic acid and catechin. All compounds, except tannic acid, significantly increased mechanical strength of the matrices. Moreover, tensile strength of quercetin cross-linked matrices was comparable to the commercially available 0.625% glutaradehyde fixed valves. Furthermore, cross-linking of the matrices resulted in a decreased burst release of growth factors during the first 4 hours but prolonged the release after 24 hours when compared to non-cross-linked matrices. Natural compounds such as quercetin, caffeic acid and catechin can be used to cross-link porcine aortic valve matrices. Moreover, the in vitro release of growth factors can be prolonged which can be very advantageous in the recellularization of these scaffolds.
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Taurodeoxycholate Overcomes Limitations of Deoxycholate for Xenogenic Cell Removal
AbstractWhen replacement of heart valves is required there is almost no alternative to overcome the shortcomings of the conventional substitutes and the clinical outcomes of recently devised cell-depleted tissue engineered xenogeneic constructs are still controversial. Particularly, osmotic shock- and deoxycholate (DOC)-based acellular preparations that gained approval for use in surgical practice, are reported to have been fully or partly unsuccessful. The formers leading to patient deaths and the others resulting in either a high number of explantations or in successful outcomes at midterm follow-up according to different reports. Experimental evidence obtained in the present investigation indicated that inconsistent clinical outcomes of deoxycholate (DOC)-based heart valve preparations might have been related at least in part to incomplete or variable removal of xenogenic cell material following DOC solubility limitations. Therefore we explored alternatively the efficiency of taurodeoxycholate (TDOC), the highly soluble conjugated form of DOC, associated with Triton X 100 (TRI). Characterization of the resulting acellular scaffold, included shape, volume and mass analysis, quantification of residual xenoantigen alpha-Gal, histology, immunofluorescence, scanning and transmission electron microscopy as well as pulse duplicator testing at systemic pressures. In contrast to previous DOC and combined SDS (sodium dodecyl sulfate)-DOC procedures, adoption of TDOC resulted in complete removal of alpha-Gal xenoantigen, with apparent reduction of laminin and enhanced fibronectin detection by immunofluorescence. Besides cell removal from leaflet, sinus and aortic wall, detailed morphological investigation revealed unconventional aspects of the stromal matrix distribution in native and treated samples. In native samples GAG concentration in spongiosa resulted apparently comparable to that in fibrosa layer while collagen and elastic fibres, respectively, exhibited a peculiar interconnected distribution throughout the valve layers. After TRI-TDOC treatment total leaflet hydration was unchanged while mass, area and thickness decreased. The general hydrodynamic performance of the TRI-TDOC-scaffold well accorded with substantial maintenance of matrix architecture while increased post-treatment gradients and regurgitant volumes correlated with loss of ECM components and partial leaflet retraction. Considering the remarkable cell-removal efficiency and the solubility properties, TDOC is worth of further investigation in the perspective to replace DOC for obtaining xenogenic valve scaffolds free of cell remnants and detergent residues with the aim to restore valvular function in vivo or after dynamic cell culture in vitro.
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AlphaGal Residue in Xenograft Heart Valve Bioprostheses and Tissue Engineered Construct
Authors: Filippo Naso, Alessandro Gandaglia, Giovanni Comacchio, Michele Spina and Gino GerosaAbstractThe glutaraldehyde (GA) fixed heart valve bioprostheses (HVB) fail in the long term due to dystrophic degeneration. To avoid GA treatment, xenogenic tissues have been processed by detergent-based decellularization procedures (DBDP). However a complete immunogeneic tolerance by the host is not granted. Degenerative inflammatory process seems to be triggered by the persistent presence of reactive xenogeneic residual, specifically by the alpha-Gal antigen. Through the use of an ELISA assay we assessed and quantified the content of such xenoantigen in different commercial bioprosthetic heart valves and compare to that present in the native and decellularized tissues used for their manufacture. Four models of pericardial and two of porcine HVBs were investigated for the alpha-Gal content. Untreated porcine aortic leaflets (UPAL) were assessed before and after 3 different detergent-based decellularization procedure: TRICOL (Triton X100 and Sodium Cholate), DOC (Sodium Deoxycholate) and DOC-SDS (Sodium Deoxycholate and Sodium Dodecyl Sulfate). Moreover the total amount of alpha-Gal epitopes in native bovine pericardium (NBP) was determined. All the specimens react with the M86 primary monoclonal antibody and the exposed alpha-Gal epitopes is determined by an indirect ELISA assay. For each sample, the amount of alpha-Gal was expressed as numbers of epitopes *10e11 each 10 mg of wet tissue. The amount of alpha-Gal xenoantigen in pericardial HVB (1.5 ± 0.18 *10e11, n=15) was three and half times less with respect to NBP (5.1 ± 0.21 *10e11, n=9). In a model of porcine HVB the xenoantigen was not detected, in the second one the absolute value (1.37 ± 0.25 *10e11, n=9) was similar to that of the pericardial HVB and half of UPAL (2.5 ± 0.31 *10e11, n=9). DOC and DOC-SDS treatments leave on the tissue the 40% (1.02 ± 0.1 *10e11) of the epitopes originally present in the native cusps. TRICOL has proven to be able to eliminate all the alpha-Gal antigen. HVBs GA-treatment do not prevent the binding of resident alpha-Gal antigens with M86 antibodies. The investigated HVBs exhibited a non negligible amount of reactive epitopes accounting to 29.3% of those exposed by native pericardial tissue and 55% for the porcine one. Probably, the pericardial simpler structure allow a better action of the GA, which is able to ensure a greater, but non complete epitope masking. In one model of porcine HVB the alpha-Gal was not found. Regarding the different DBDPs, the removal of cell components is not a sufficient condition to ensure the elimination of the alpha-Gal epitopes. Up to date the TRICOL seems to be the only method capable of producing an alpha-Gal tissue free.
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The Cellular and Extracellular Matrix Structure of Human Pericardium for Heart Valve Tissue Engineering
AbstractThe objective of our study was to compare the histological structure and cellular organization of autologous human pericardium to that of the human aortic heart valve. Collagen, elastin and glycosaminoglycans are responsible for the mechanical properties of aortic heart valve leaflets. Aortic valve leaflets are composed primarily of collagen representing 50% of total extracellular matrix (ECM). The main collagen types in the aortic heart valve are collagen I (74% of total collagen) and collagen III (24% of total collagen). Elastin represents 13% and is responsible for the elastic properties of the valve leaflet. Collagen has a specific architecture that endows heart valve tissue with the ability to withstand circulatory forces over the course of a lifetime. Its fiber orientation, density and cell associations are very important for this purpose. Normal aortic heart valves were obtained during heart transplantation and compared to autologous human pericardium before and after dynamic conditioning using classical histological assessment, immunohistochemical analysis and confocal microscopy. The architecture of pericardial tissue is very similar to that of the normal aortic heart valve possessing well organized collagen fibers with embedded pericardial interstitial cells (PICs) forming a three dimensional network. Instead of the trilaminar histological organization present in the aortic heart valve, the pericardium possesses one layer whose densely packed collagen bundles closely resemble that of the lamina fibrosa of the native aortic heart valve by confocal microscopy. Elastin fibers are evenly distributed throughout the entire thickness of the pericardium in comparison to the specialized elastin containing layer in the lamina ventricularis of the aortic heart valve. PICs are also evenly distributed throughout the pericardium. In the inner pericardial layer facing the heart these cells have a more spindle-like shape similar to that of valvular interstitial cells (VICs) in the lamina fibrosa, while in the outer part of the pericardium these cells have a more spread-out cytoplasmic morphology interacting with more loosely distributed collagen bundles. Like in the aortic heart valve, PICs show cell-cell interactions in addition to cell-matrix interactions. Our study confirmed similarities in the cellular and ECM organization of human pericardium and the native aortic heart valve. It may be concluded that human autologous pericardium may be a favorable tissue for heart valve replacement. Autologous pericardial tissue may also avoid a negative immune system response that could adversely affect recipient graft uptake and downstream ECM remodeling events.
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Investigation of the Suitability of Decellularised Porcine Pericardium for Mitral Valve Reconstruction
Authors: Lucrezia Morticelli, Daniel Thomas, Eileen Ingham and Sotiris KorossisAbstractThe aim of this study was to investigate the suitability of decellularised porcine pericardium for heterotopic repair of the mitral valve (MV) leaflets, and its potential to regenerate through endogenous cell repopulation in vivo, or in vitro seeding and bioreactor conditioning. Anterior and posterior MV leaflets and pericardia were excised from porcine hearts within. The pericardia were decellularised according to the in-house protocol. Anterior and posterior leaflet, and decellularised and fresh pericardial samples were subjected to histology (H and E, Masson trichrome, Sirius Red, Miller’s elastin, Alcian blue-PAS), immunohistochemistry (collagen type I, III, IV, fibronectin, laminin, and chondroitin sulfate labelling), SEM, and uniaxial tensile testing. Samples were isolated along the radial and circumferential direction (leaflets), and perpendicular and parallel to the collagen fibres (pericardium). Biochemical assays for quantification of the sulphated GAG and collagen content of the tissues were also performed. Contact and extract cytotoxicity testing, and DNA quantification was performed to assess the decellularised pericardia. Histology revealed the trilaminar structure of the pericardium and quadrilaminar structure of the leaflets. Collagen type I and III was found in the fibrosa layers of both pericardium and leaflets, whereas fibronectin and laminin were found throughout the tissues. Decellularisation produced a completely acellular pericardial scaffold, which retained the histoarchitecture of the natural tissue. The biomechanics showed the anterior leaflets being stiffer along the circumferential direction. No significant anisotropy was observed in the biomechanics of the posterior leaflets, or fresh and decellularised pericardium. The anisotropy of the anterior leaflet was attributed to the orientation of the collagen (aligned along the circumferential direction). Biochemistry showed a significant increase in sulphated GAGs between the fresh leaflets and pericardium. No difference was found between the collagen content of the fresh leaflets and the fresh or decellularised pericardium. The decellularised pericardium showed a 99% reduction in DNA and a high loss in the GAG content compared to the fresh pericardium. The study showed that the MV leaflets and pericardium share similar histoarchitectures and comparable biomechanics. The similarity was more pronounced in the case of the posterior leaflet which was more isotropic both in terms of histoarchitecture and biomechanics. Apart from a decreased GAG content, the similarity was also apparent between the leaflets and the pericardial scaffolds. The decellularised pericardium has the potential to deliver the necessary biological and biomechanical cues to seeded or migrating cells, representing a plausible scaffold option for the regeneration of the MV leaflets in vitro or in vivo.
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Integration of Microstructural Architecture of the Mitral Valve into an Anatomically Accurate Finite Element Mesh
AbstractAlthough mitral valve (MV) repair initially restores normal leaflets coaptation and stops MV regurgitation, in long term it can also dramatically change the leaflet geometry and stress distribution that may be in-part responsible for limited repair durability. As shown for other collagenous tissues, such changes in geometry and loading reorganize the fiber architecture. In addition, MV interstitial cells may also respond to the altered stress by reducing biosynthetic function, which would affect the load-bearing capabilities of MV and its long-term durability. Thus, investigating the repair-induced MV stress and the concomitant microstructural alterations is a key step in assessing the repaired valve durability. Finite element models have been widely used for stress analysis of the mitral valve. Most of these models, however, have employed only basic constitutive models and above all ignore the complex microstructure of the MV. In addition, the geometry of the valve is usually simplified. Thus, in this work we developed a method to obtain accurate geometrical model of the ovine MV and quantify its fiber structure for the purposes of developing high fidelity computational meshes of the MV. To obtain an accurate geometry of the MV, microcomputed tomography (micro-CT) was used. The entire heart was scanned via a SIEMENS Inveon CT scanner. Three-dimensional scans were segmented semi-automatically using ScanIP segmentation software. The 3D positional data of the fiducial markers were also obtained via ScanIP masks generated by using gray-scale threshold of the CT scans. The segmented geometry was then converted to finite element meshes using ScanIP mesh free mesh generator scheme. Next, the anterior leaflet was then dissected and prepared for measurements of its fiber alignment. The positional data of each point on the accurate mesh was then projected onto the 3D marker mesh. By using a computational domain, the projected point was mapped back to the 2D flattened surface. In addition to mapping, the current method can be used to estimate the changes in connective tissue structure with deformation. This is done by for each point on the valve surface using the local right Cauchy strain tensor C using an in-plane convective curvilinear coordinate system to convect the local fiber orientation to predict the current fiber alignment. To conclude, a robust technique to quantify and map the fibrous microstructure of the MV anterior leaflet to anatomically accurate 3D MV shape derived from micro-CT imaging was developed. The method provides a framework for development of anatomically and micro-structurally accurate finite element models of MV using our tissue structure-based models. It can also be used as a means to validate predicted changes in fibrous structure due to altered stress following surgical interventions.
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