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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
86 results
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Regulation of Abnormal Atrioventricular Valve Development
Authors: Huseyin C. Yalcin, Sarah Ajaeb and Jonathan T. ButcherAbstractHemodynamic forces play an essential role in driving the morphogenesis of the embryonic heart and its valves. We previously found that genes that regulate heart morphogenesis like BMP2 and VEGFA, have hemodynamically specific patternings. However, how mechanical signals like wall shear stress (WSS) affect these expression levels to regulate heart morphogenesis is not fully understood. A detailed understanding of the hemodynamic environment and gene expression patterns inside normally and abnormally growing embryonic hearts would help inform the clinical progression of congenital heart defects and develop treatment strategies to restore these defects. We previously developed a system to quantify local fluid forces within growing embryonic heart where we combined imaging modalities with computational fluid dynamics (CFD), and quantified the hemodynamics within the AV canal and OFT in chicks. In the current study, we analyzed how altered hemodynamics drives changes in local gene expression and downstream morphogenesis of AV valves. Perturbed blood flow was created via either left atrial ligation (LAL) at HH24 or right atrial ligation (RAL) at HH25, to constrict blood flow on respective side of the heart. Hemodynamic environment in the AV canal for operated embryos were compared to control groups via doppler ultrasound at HH31. Gene expression levels were analyzed using RT-PCR. Heart morphology was analyzed at HH31 via micro-CT and histology. RT-PCR results at HH25 showed that LAL caused a drastic decrease in gene expression levels in LV myocardium (VEGFA expression 18.2±5.6%, BMP2 expression 21.7±1.4% compared to controls) and in AV cushions (VEGFA expression 9.8±8.1%, BMP2 expression 3.5±1.9 %), whereas in RV myocardium, expression levels were not affected significantly (VEGFA expression 77.5±5.8% , BMP2 expression 66.0±10.0%). Doppler ultrasound showed that neither peak blood velocities nor time averaged velocities in right and left AV canal are different for both LAL and RAL at HH31, suggesting WSS levels are not different between experimental groups (max shear stress 287dynes/cm2) at that stage. For LAL embryos, left AV valve area was smaller (48±4% vs. 67±6%, p<0.05, valve areas normalized to total left and right AV valve areas) and right valve area was bigger (52±4% vs. 33±6%, p<0.05) compared to controls, whereas in RAL embryos, opposite was true at HH31, right AV valve are was smaller (12±5 % vs. 33±6%, p<0.05) and left AV valve area was larger (88±5% vs %67±6, p<0.05). The ratio of right ventricle to left ventricle was significantly larger in LAL embryos compared to RAL embryos (1.1 vs 0.55). Our results show that morphological abnormalities follow the alterations in gene expression levels which are caused by changes in mechanical signals due to altered hemodynamics. We found that at later stages hemodynamics is restored to keep the blood circulation normal, with an altered morphology.
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Contribution of epicardially derived cells to the leaflets of the murine AV valves
Authors: John Burch, Maurice Van den Hoff, Marie Lockhart, Laura Briggs and Andy WesselsAbstractTo determine the spatiotemporal contribution of epicardially derived cells (EPDCs) to the leaflets of the developing atrioventricular (AV) valves in the murine heart we have used a mWt1/IRES/GFP-Cre mouse and traced the fate of EPDCs from embryonic day (ED)10 until birth. Migration of EPDCs into the mesenchyme of the AV cushions starts around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets that derive from the lateral atrioventricular cushions, i.e. the mural leaflet of the left AV valve and the lateral leaflet of the right AV valve. In these developing leaflets the EPDCs eventually largely replace the endocardially-derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the respective leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in heart development.
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Matrix-controlled In-growth Selectivity as a Principle of Heart Valve Regeneration
Authors: Neil Davies, Mona Bracher, Michelle Sun, Deon Bezuidenhout and Peter ZillaAbstractSpontaneous scaffold based tissue regeneration may be a promising alternative to in vitro tissue engineered heart valve prostheses. Selective promotion of ingrowth of desired cells versus suppression of potentially harmful cell invasion would be a key principle of such an approach. We have begun to demonstrate the utility of hydrogels engineered with specific adhesive and degradation peptide sites towards achieving this long-term goal. Polyethylene glycol hydrogels that through Michaels addition chemistry allow for both polymerization via enzymatically (matrix metalloproteinase (MMP)) degradable peptides and appendage of adhesive peptides were developed. Hydrogels were crosslinked with either a relatively enzymatically promiscuous peptide (MMP-pep) or sequences specifically degradable by either MMP-14 or MMP-9 (MMP-14pep, MMP-9pep). MMPs are of interest as there is evidence of cell specific expression. For example in vascular cells under physiological conditions MMP-9 is expressed at low levels except in macrophages. Adhesive peptides utilised were RGD, YIGSR and/or PHRSN. A range of primary vascular cells (smooth muscle cells (SMC), endothelial cells (EC), valvular intestitial cells (VIC) and fibroblasts (FB)) were cultured individually or as spheroids in 2 or 3-D. Migration and invasion were then assessed by time-lapse phase and confocal microscopy. ECs were shown to migrate significantly more rapidly on a surface containing YIGSR/RGD relative to other peptide combinations whilst the migration of SMC was unaffected. Hydrogels polymerized with MMP-14pep or MMP-9pep were shown to be highly preferentially cleaved by their relative enzymes. VICs, Fb and SMC primarily utilised MMPs to invade the hydrogels as demonstrated by inhibition of sprout formation by the MMP inhibitor, GM6001. VICs and FB invaded the gels in the following order, MMP-pep>>MMP-14>MMP-9. However, SMC showed a marked preference for invasion into MMP-14pep crosslinked hydrogels above that observed in their MMP-pep and MMP-9pep counterparts. Confocal microscopy analysis also showed a significantly more branched and interconnected pattern of invasion for SMC in MMP-14pep hydrogels. Thus we have begun to show that through engineering of extracellular matrix mimics with precise enzymatic and adhesive recognition sites it is possible to selectively influence specific cell types invasive behaviour suggesting that cell-specific ingrowth scaffolds may be achievable.
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Investigating the Contribution of Pathological Levels of Cyclic Strain on Vascular and Valvular Calcification
Authors: Zannatul Ferdous, Hanjoong Jo and Robert NeremAbstractAortic stenosis and atherosclerosis tend to coexist in most patients with cardiovascular disease; however, the causes and mechanisms of calcification are still not clearly understood. To understand the contributions of physiological (10%) and pathological (5%, 15%) levels of cyclic strain in calcification, we used a model system of tissue-engineered collagen gels containing human aortic smooth muscle cells (HASMC) and human aortic valvular interstitial cells (HAVIC), both isolated from non-calcific heart transplant tissues. The tissue engineered collagen gels were cultured in standard osteogenic media for three weeks in a custom designed bioreactor and all assessments were performed at the end of the culture period. The major finding of this study was that bone morphogenic protein (BMP) -2, -4 and transforming growth factor (TGF)-β1 mRNA expression significantly changed in response to the magnitude of strain in valvular cells, while the least expression was observed for the representative 10% physiological strain. On the other hand, these mRNA expressions in vascular cells responded to strain, but did not vary due to the magnitude (5% versus 10% versus 15%) of strain. When the BMP-2 and BMP-4 protein expression was detected using immunostaining, we observed that only valvular cells showed greater BMP-2 expression for 5% and 15% strain when compared to 10% strain within the same cell type. Our results suggest that cell mediated differences exist between vascular and valvular cells in their response to different levels of cyclic strain.
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The Role of Integrins on the Regulation of Contractility in Mitral Valve
Authors: Marzieh Zamani, Padmini Sarathchandra, Najma Latif, Magdi H. Yacoub and Adrian H. ChesterAbstractThe mechanical properties of heart valves are modulated by the contractile response of valve interstitial cells (VICs). It is unknown how the contractile responses of VICs are translated to the extra cellular matrix to alter the stiffness of the valve tissue. We have studied the signalling mechanisms of integrin subunits and specifically α2β1 integrin, a predominant collagen binding molecule, in mediating the contractility of porcine mitral valve tissue and cells. The expression of integrins was analysed by immunostaining of cells. The contractility of porcine mitral VICs in the presence and absence of a α2β1 integrin blocking antibody and the effect of different signalling inhibitors was analysed in a collagen gel. The effect of modulators of contractility was determined on mitral valve tissue incubated in organ baths which challenged with endothelin-1 (10-10-10-7M) following 24 hours incubation with α2β1 integrin blocking antibody. Immunohistochemistry confirmed positive staining for α2β1 integrin and the expression of α1, α2, α3 and β1 in the VICs. The α2β1 blocking antibody was able to significantly reduce the contraction of collagen gels from 63.6±7.8 to 23.4±4.3 % (p<0.001) of control (no cells in gel). PF573228, the inhibitor of focal adhesion kinase, caused a reduction of gel contraction by 30% of the control. The maximum contractile response of cusp tissue to ET-1 maintained under isometric conditions in tissue baths was significantly reduced from 9.5±5.0 to 3.0±2.5mN/g (p<0.001) wet weight by the presence of the α2β1 integrin blocking antibody. These results demonstrate a role for α2β1 integrin in linking the contractile response of valve cells to the extra cellular matrix and highlight further the complex interactions between the cells and the extra cellular matrix of the valve.
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Modeling Flow-Responsive Gene Regulation In Native Valve Tissue
Authors: Arpi Siyahian, Fariba Chalajour, Xiaoyuan Ma, Frank L. Hanley and R. Kirk RiemerAbstractA variety of approaches to heart valve tissue engineering are underway by many groups of investigators. We believe that a thorough understanding of the biophysical and biomolecular interactions that mediate normal leaflet homeostasis will inform improved approaches to the engineering of truly regenerative heart valve grafts. However, our understanding of the fundamental biology of normal valve tissue is still quite limited. This informational gap has led us to develop an ex vivo system for long-term valve culture in order to study the mechanobiology of native tissue. Using this system, we have cultured trileaflet rat heart valves with and without flow-induced valve cycling. We report here the preliminary findings about the effects of both ex vivo culture and of valve cycling on pulmonary leaflet cell gene expression following 7 days of flow culture. Expression of leaflet mRNA was queried using Affymetrix whole genome microarrays and the results analyzed using GeneSpring, EXPLAIN and MetaCore (GeneGO) software. Our analysis revealed 2147 genes whose expression was altered under flow conditions, and 1918 genes under static conditions. 1501 genes were common to both conditions. The effects of culture on gene expression affected multiple pathways, but the most active gene groups involved pathways for cytoskeletal remodeling, development, and cell adhesion. The genes involved in these pathways were altered in both culture conditions. Changes in cytoskeletal and ECM remodeling genes were more prominent in the static condition, while those involved in VEGF signaling were seen under flow condition. Our previously reported histological analyses of cultured rat pulmonary valve leaflet tissue revealed that flow promotes the maintenance of tissue integrity while the lack of flow leads to extracellular matrix remodeling and fibrinoid tissue formation. These gene expression analyses confirm the expected role of flow in maintaining leaflet tissue integrity as evidenced by its induction of VEGF signaling genes, while the lack of flow induces changes in genes involved in extracellular matrix and cytoskeletal remodeling.
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Inflammation Drives Endothelial-to-Mesenchymal Transition and Interstitial Calcification in 3D in Vitro Culture
Authors: Jennifer Richards, Emily Farrar, Gretchen Mahler and Jonathan ButcherAbstractCalcific aortic stenosis is a serious pathology that accounts for 43% of those suffering from heart valve disease. Valve disease initially presents with inflammation and endothelial dysfunction, which can lead to an endothelial-to-mesenchymal (EndMT) shift in the endothelial layer of the valve. It is not yet well understood how valve endothelial cells (VEC) regulate valve interstitial cells (VIC) in inflammatory or osteogenic environments. We here present a 3D culture system that models cellular responses and interactions when exposed to different environmental conditions. Porcine aortic VEC and/or VIC were cultured in mechanically constrained 3D type I collagen hydrogels for up to 14 days. Early disease studies introduced different dosages of TNFα to VEC on gel surfaces for up to 48 hours. EndMT was assessed via real time PCR and cell invasion within the matrix. Apoptosis and proliferation in VEC via TUNEL and anti-BrdU IHC staining were also evaluated in early inflammatory conditions. Later early-stage disease studies introduced osteogenic and inflammatory environments to 3D VIC/VEC gels through the addition of osteogenic differentiation factors into the culture media (OGM), or 30 ng/ml TNFα. Calcium deposition within the matrix was measured via Alizarin Red staining. Real time PCR was used to evaluate the expression of calcification-related genes such as osteocalcin and runx-2. Early inflammatory conditions as induced by addition of TNFα for 48 hours stimulated EndMT-like VEC activation, including matrix invasion and upregulation of αSMA and snail. Addition of TNFα increases VEC proliferation in a dose dependent manner. TNFα does not significantly increase apoptosis, even at the highest dosage levels. VIC gels cultured in OGM for 14 days significantly calcified and expressed osteocalcin and runx-2. Co-culture with VEC inhibited both matrix calcium deposition and osteoblastic differentiation. When 30 ng/ml TNFα was added to VIC 3D cultures, TNFa induced increased matrix calcium deposition in 14 days, which was then mitigated by co-culture with VEC. The inhibitory effect of VEC on VIC calcification in OGM was blocked when TNFα was introduced to the culture. These results demonstrate that inflammatory microenvironments induce calcification in valve interstitial cells. There is a protective role for the valve endothelium in VIC calcification, which could be inhibited by EndMT induced by the inflammatory conditions found in diseased valves. We found that apoptosis is not a driving factor in inflammatory activation of VEC or osteogenic differentiation of VIC. Targeting TNFα and EndMT signaling could be important therapeutic strategies. More generally, targeting VEC is a promising approach for early treatment of valve disease. This co-culture 3D system is a powerful tool to elucidate mechanisms of valve disease.
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Atypical expression and regulation of smooth muscle markers in human calcified valves
Authors: Najma Latif, Padmini Sarathchandra, Magdi H. Yacoub and Adrian H. ChesterAbstractCalcific aortic stenosis and atherosclerosis share many similar characteristics suggesting that similar mediators and pathogenic pathways are involved. In both conditions, the native cells are quiescent. However upon damage, both valve interstitial cells (VICs) and smooth muscle cells (SMCs) in the valve and the vascular wall respectively undergo remodelling and transformation. We hypothesise that the smooth muscle cell phenotype plays a role during the calcification process. 12 normal and 16 calcified valves were analysed for smooth muscle markers including smooth muscle α-actin, smooth muscle myosin, calponin, caldesmon, desmin, SM1 and SM2 by immunocytochemistry. The expression of myocardin-related transcription factors, (MRTFA/B) as key regulators of smooth muscle gene expression, was also evaluated. Normal human valves demonstrated the expression of smooth muscle markers localised to the base of the valve. Occasionally smooth muscle markers were seen in small groups of cells in the region from the base to the central region of the valve but never in the region from the central to the co-apting edge. Smooth muscle markers in all the calcified valves demonstrated an increased and aberrant expression. The expression of smooth muscle myosin, SM1, SM2, calponin and smooth muscle α-actin was found to be abundantly expressed around calcified nodules and distal to the nodules. The pattern of staining was frequently localised along the majority of the valve tissue as well as in isolated cells. Additionally, regions of the endothelium and endothelial cells lining the vessels were found to be positive for smooth muscle markers in 9/16 calcified valves. Normal valves did not demonstrate any expression of MRTFs. However, MRTF-A and MRTF-B were induced in calcified valves in a similar pattern to the expression of smooth muscle markers. They were also induced in the endothelium and the endothelial cells lining some vessels. Importantly, the expression of MRTFs was nuclear in both VICs and endothelial cells indicative of activation. TGFβ1 (10ng/ml) was able to induce and translocate the expression of MRTF-A to the nucleus in VICs within 4 hours in some isolates. Calcified valves harbour a greatly increased number of smooth muscle marker-positive VICs and endothelial cells. This aberrant expression in endothelial cells may emulate endothelial to mesenchymal transformation (EMT) during embyonic valvulogenesis. Concomittantly, calcified valves expressed MRTFs, key regulators of smooth muscle gene expression. The presence of smooth muscle cell markers and MRTFs in calcified valves suggest a role for this cell phenotype in the development of the calcification process.
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Characterization of Early Porcine Aortic Valve Disease
Authors: Krista L. Sider, Andrea V. Kwong, Bowen Wei and Craig A. SimmonsAbstractIn calcific aortic valve disease (CAVD), pathological differentiation of valve interstitial cells (VICs) and lesion formation occur focally in the fibrosa layer. We have shown that VIC pathological differentiation in vitro is sensitive to matrix stiffness. We therefore hypothesized that focal changes in the stiffness of the fibrosa with CAVD progression correlate with alterations in the extracellular matrix (ECM) and pathological cell phenotypes in vivo. Twenty-four male Yorkshire pigs were fed either a control or experimental diet supplemented with 12% lard and 1.5% cholesterol for 2 or 5 months (n = 6 per group). Effective moduli were measured in focal regions of the fibrosa (n = 295) and ventricularis (n = 142) layers of intact right coronary aortic valve leaflets by micropipette aspiration. Histology was conducted within radial center sections and selected focal test locations within the fibrosa from each leaflet to characterize local ECM and cell phenotypes. Early stage disease was induced by the experimental diet, as evidenced by more significant proteoglycan-rich lesions (~10 - 200 µm thick) onlayed on the fibrosa (p < 0.05, controlling for genetic variability). Interestingly, lipid deposition was seen in only 28% of regions with early lesions, and 15% of regions without early lesions. No osteoblastic or macrophage cells were present in control or experimental valves, with only slight myofibroblast presence at the base of some 5 month lesions. Chondrogenic Sox9-positive cells were observed extensively and were positively correlated with proteoglycan content (p < 0.0001). Regions within the fibrosa were significantly stiffer than those in the ventricularis in both control and experimental valves (p <0.001). Although there were no significant differences between the control and experimental overall tissue moduli at these early time points, modulus heterogeneity did increase with disease. Furthermore, lesions in the fibrosa had lower moduli than non-lesion fibrosa locations (p < 0.06). These soft lesions contained significantly more proteoglycan (p <0.001) and Sox9 expression (p = 0.014) than normal fibrosa tissue. In conclusion, ECM remodelling can occur in a porcine model of early CAVD in the absence of lipid deposition, inflammatory cells, osteoblasts, or myofibroblasts, but with significant proteoglycan-rich lesion and chondrogenic cell presence. Mechanically soft proteoglycan-rich lesions may support chondrogenic VIC differentiation, providing new insights into early CAVD pathogenesis.
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The role of Insulin-like Growth Factor-1 in Calcification of Human Aortic Valves
AbstractInsulin-like Growth Factor-1 (IGF-1) is a multi-functional protein that plays a role in survival, growth, proliferation and differentiation of various cell types. Particularly in the bone, IGF-1 is involved in tissue formation and growth, and the lack of expression of the gene leads to striking features such as short bone phenotype and low bone mineral density. In atherosclerosis, IGF system has been shown to stimulate vascular smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) synthesis contributing to maintenance of plaque stability. Consequently, a reduction of IGF-1 in atherosclerotic plaques has been suggested to increase SMC apoptosis and reduce ECM synthesis leading to weakening of the plaque. Despite extensive studies on its role in various organs and disease models, there is very little known about the effects of IGF-1 in human aortic valve (hAV) tissue that can suffer damaging effects such as cell death, differentiation and tissue mineralization, resulting into Aortic Valve Stenosis (AVS). The objective of the present study is therefore to investigate potential function of IGF-1 in this disease model. Human aortic valve samples were collected from patients undergoing aortic valve replacement or heart transplantation. The tissue expression of IGF-1 was analysed at mRNA level by quantitative Realtime PCR in calcified hAV (n=10-14) and non-calcified hAV (n=3-6), using the comparative Ct method. In vitro, the expression of endogenous IGF-1 was investigated on human valve interstitial cells (hVICs) upon administration of calcifying medium with or without transforming growth factor-beta1 (TGF-b1), a known pro-mineralization molecule (n=3). In addition to the expression of IGF-1, alkaline phosphatase (ALPL), TGF-b1 and osteopontin (OPN) were also examined at RNA level. IGF-1 expression was significantly up-regulated in the diseased valves compared to the controls (4.10 +/- 1.20 calcified vs. 1.10 +/- 0.76 control, p=0.0127). In vitro, there was an up-regulation of IGF-1 (p=0.06) and TGF-b1 expression (p=0.02) upon TGF-b1 stimulation. OPN expression was also highly up-regulated in calcifying media treated cultures, independent of TGF-b1 treatment. ALPL expression in contrast, was higher in normally growing cells compared to TGF-b1 and/or calcifying media treated cells (p=0.05). The significant elevation of IGF-1 in calcified valves in this small patient cohort strongly suggests that IGF-1 might be a key player in AVS disease. Whether the elevated IGF-1 expression is an effect or the cause of calcification is currently under investigation. Our preliminary data on TGF-b1 treatment of hVICs suggests that IGF-1 expression might be an effect of cells to counter apoptotic death conferred by TGF-b1 treatment. Further work on the detailed molecular mechanisms involved in IGF-1 action and its downstream signalling pathways is needed.
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Reversible Effects of Polyunsaturated Fatty Acids on Activation of Interstitial Cells From Porcine Aortic Valves
Authors: Wolfgang Witt, Anett Jannasch, Klaus Matschke and Thomas WaldowAbstractValvular interstitial cells (VICs) constitute the most prominent cell type in aortic valve cusps. VICs are a heterogeneous group of cells of multiple origins with mostly fibroblast properties. Like fibroblasts in general, VICs can acquire an activated state (myofibroblasts) upon exposure to several stimuli including mediators of inflammation, growth factors, mechanical stress or changes of ECM composition. An increase of activated VICs in adult valves is considered as indicator of pathological developments. Cultivation of VICs using standard 2D cell culture procedures also induces VIC activation resulting in contraction and spontaneous nodule formation. Our previous work has shown that this phenotype switch can be reversed by exposure to polyunsaturated fatty acids (PUFAs). In order to investigate underlying cellular mechanisms of the PUFA effects, VICs from porcine aortic valves were isolated and subcultured on collagen-coated surfaces. Spontaneous nodule formation after transfer to uncoated polystyrene was completely blocked by docosahexaenoic acid (DHA) and arachidoic acid (ARA) whereas eicosapentaenoic acid and a commercial extract from fish oil (Omacor) were less active. Oleic acid and palmitic acid were without effect. Treatment with ARA or DHA reduced the expression of myofibroblast marker proteins, α-smooth muscle actin (SMA) and myosin II, of a key enzyme of collagen synthesis (prolyl 4-hydroxylase), and of the phosphorylated (inactive) form of the F-actin severing protein, cofilin. In contrast, the abundance of fibroblast marker S100A4 was increased after treatment with PUFAs. The steady state level of active RhoA was reduced in the presence of DHA and ARA, and inhibition of RhoA or ROCK elicited the same effects as PUFAs. Finally, exposure to ARA and DHA reduced the G/F-actin ratio, and stabilization of F-actin with jasplakinolide blocked the effect of PUFAs on the expression of myofibroblast markers and on nodule formation. After culturing VICs with PUFAs for 14 days and subsequently in the absence of PUFAs for 4 days, cells regained the myofibroblast phenotype, showing the PUFA-induced phenotype switch was fully reversible. In conclusion, the results suggest that the differentiation of VICs to myofibroblasts can be truly reversed by certain PUFAs via the RhoA – ROCK – G-actin pathway, whereas an alternative mechanism, the preferential outgrowth of a undifferentiated subpopulation of VICs, seems to be unlikely since the PUFA effect was fully reversible.
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Pentraxin 3 is a Potential Diagnostic Marker for Aortic Stenosis
AbstractPentraxin 3 (ptx3) is a member of the long pentraxin family and is rapidly produced and released by many cell types, including endothelial cells, in response to primary inflammatory signals. In our preliminary work, PTX3 gene expression was significantly increased in aortic valve tissue following exposure to elevated cyclic pressure. Consequently, we hypothesized that ptx3 would be a useful biomarker for the early diagnosis of aortic valve sclerosis. Isolated aortic VICs were treated with Angiotensin II (300nM), TNF-alpha (10 ng/ml) or elevated cyclic pressure for six hours. The cell culture supernatant was collected and used to determine ptx3 protein expression using ELISA. Total RNA was isolated from the cells and PTX3 gene expression was determined using semi-quantitative RT-PCR. In addition to cell culture studies, ptx3 protein expression was determined in hypertensive New Zealand White rabbits. Rabbits underwent Goldblatt one-clip/one-kidney surgery to induce hypertension (n=5). Four sham models served as a control. Blood pressure, echocardiography data and serum samples were collected at 0, 2 and 4 months. After 4 months, rabbits were euthanized and aortic valve tissue was collected for histological and gene expression analysis. Data from the cell culture studies showed that elevated cyclic pressure caused an increase in PTX3 gene expression and ptx3 protein expression. However, no significant changes were observed in gene or protein expression from cells treated with Ang II or TNF-alpha. Data collected from the animal model showed that blood pressure increased significantly for the experimental group but not the control group. Levels of ptx3 protein were measured from the serum and showed a slight increase over the four-month course of the experiments. The data suggest that ptx3 expression is mechanosensitive in the aortic valve and is not stimulated by biochemical factors such as TNF-alpha or Ang II. The increase in ptx3 expression in hypertensive rabbits demonstrates that this could be a potential diagnostic marker for aortic stenosis.
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Effects of Finite Deformation on Extracellular Matrix Production and Mechanical Properties
AbstractWith our ability to incorporate viable cells distributed throughout the scaffold, we are provided a unique, controllable platform to develop a generalized finite deformation framework than can be used to gain an understanding of how the evolving extracellular matrix phase contributes to the construct gross mechanical behavior. The biosynthetic response of microintegrated VSMC’s was investigated low (15%), intermediate (30%), and high (50%) strain groups. These magnitudes were chosen as they correspond to a wide range of NAR deformations and physiologically relevant. A constant, quasi-static strain rate as applied sufficient to obtain a 1 Hz cycle duration. Culture durations of 7, 14, and 21 day time points were used for each strain level to quantify the ECM synthesis capacity of VSMC microintegrated in electrospun PEUU. A static group was also preformed at each time period. Results indicate that VSMC biosynthetic behavior is function of global strain with peaked soluble collagen synthesis was observed in specimens exposed to 30% strain. Our primary goal was to elucidate the mechanical behavior characteristics of the de-novo formed ECM. We determined the matrix mechanical contribution by assuming that the total mechanical response is simply the summation of the individual phases and any potential interactions that might arise between them. We thus quantified the collagen mass fraction and utilized an enzymatic technique to remove the ECM from the constructs, then retested them to obtain the degraded scaffold only responses. Results indicated that the newly formed matrix phase exhibit a highly anisotropic biaxial response, and was over 100 fold stiffer than similar ECM formed using stiff scaffolds previously studied in our lab. Moreover, the formed ECM had predicted mechanical properties similar to glutaraldehyde treated pericardium, a common heart valve biomaterial. Interestingly, peak biosynthetic activity correlates well with in vitro principle strain levels observed in PEUU tri-leaflet valves exposed to native ovine right side pressure. This suggests that physiologic hemodynamic conditions are optimal for the development of robust ECM accretion. To our knowledge, this is the first reported study to consider the effects of large deformation and the corresponding outcomes in terms of ECM mechanical integrity. Furthermore, the results reveal interesting insights into the functional role of the matrix accretion process in engineered tissues.
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Bio-inspired Anisotropic Nanofibrillar Matrices for Heart Valve Engineering
Authors: Jerome Sohier, Ivan Carubelli, Najma Latif, Padmini Sarathchandra, Adrian H. Chester and Magdi YacoubAbstractOur strategy for heart valve tissue engineering is the use of autologous cells to populate appropriate template matrices. In this context, an important goal is to devise a suitable biomimetic scaffold that supports proper cell growth and cell-matrix interactions by reproducing the specific anisotropic fibrillar structure of valves extracellular matrix (ECM). A novel type of highly porous anisotropic nanobrillar matrices was developed and evaluated with regards to structure, mechanical properties and ability to support human adipose derived stem cell (hADSC) colonization, growth and ECM production in vitro. Nanofibrillar structures were obtained by jet-spraying poly (ε-caprolactone) dissolved in chloroform on a variably rotating drum. Morphological evaluations of the structures were performed using scanning electron microscopy while porosity was calculated from polymer density, weight and volume. 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. Human adipose derived stem cells (500,000) were top and rotary seeded on nanofibrillar discs (diameter 1 cm and thickness 0.8 mm) and cultured in 10 ml of complete medium under rotation (10 rpm) for 18 days. Histology (DAPI staining), DNA quantification and immunohistochemistry were used to characterize the resulting cellularized structures. The speed of drum rotation was adjusted up to 3000 rpm to produce highly aligned fibres (600 nm of average diameter) from the sprayed polymer. In conjunction with fibres anisotropy, the scaffolds Young's modulus was simultaneously increased (from 0.3 to 0.7 Mpa) and decreased (from 0.3 to 0.01 Mpa) longitudinally and orthogonally to fibre alignment, respectively. In addition, fibre alignment further increased scaffolds porosity from 97 % (isotropic) to 99%. Anisotropic matrices allowed a more extensive cellular invasion than isotropic scaffolds, possibly linked to their higher porosity and therefore open structure. hADSC proliferated significantly (up to 6-fold DNA increase), bridged the entire scaffolds thicknesses after 10 days and produced their own ECM as evidenced by collagen I production. This study highlights the potential of a newly developed highly porous anisotropic nanofibrillar matrices as substrate for tissue engineering, and in particular for heart valve engineering.
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PCL Scaffolds and Reduced In-Vitro Cell Expansion to Improve Engineered Valvular Tissue Formation
AbstractDifferent types of synthetic scaffolds are used for tissue engineering of heart valves. Tissue-engineered heart valves (TEHV), based on a rapid degrading polyglycolic acid (PGA) scaffold coated with poly-4-hydroxybutyrate (P4HB) and seeded with vascular derived cells, have shown promising in-vivo results. However, a major drawback of these TEHV is compaction and retraction of the leaflets causing regurgitation. It is hypothesized that this is a result of traction forces exerted by the cells, combined with an imbalance of the formed tissue and loss of mechanical integrity of the scaffold due to degradation. The aim of this study is to evaluate alternative approaches to overcome the compaction and retraction of TEHV without compromising on tissue composition and properties. The alternative approaches that are studied here are 1) the use of the slow degrading poly-ε-caprolactone (PCL) scaffold for prolonged mechanical integrity and 2) the use of lower passage vascular cells for enhanced tissue formation. Compaction, tissue formation, cell phenotype and mechanical properties of tissues based on passage 3, 5 and 7 vascular cells in PCL and PGA-P4HB scaffolds are compared. TEHV aim to be designed for humans, but since the ovine model is used to show proof of principle both human and ovine cells were used. Passage 3, 5 and 7 (p3, p5 and p7) human and ovine vascular-derived cells were seeded onto both PGA-P4HB and PCL scaffold strips (n=6 per passage and scaffold material), using fibrin as a cell carrier. After 4 weeks of culture under constrained static conditions, one strip was used for histology. The remaining strips were used for mechanical testing and biochemical assays as indicators for tissue strength and tissue formation, respectively. After 4 weeks, the tissues based on PGA-P4HB showed 50-60% compaction, while PCL-based tissues showed compaction of 0-10%. Cell passage number and species did not influence compaction. Tissue formation was comparable between passage numbers and scaffold materials in ovine while human p5 showed decreased tissue formation in both scaffold materials. Collagen content was increased with decreasing passage numbers in both species and scaffold materials. No differences in cell phenotype between the scaffold materials or cell passage numbers were observed. The Young’s modulus and the ultimate tensile strength of tissues of both species were higher in the lower passage groups of both scaffold groups. This study shows that PCL scaffolds may serve as alternative scaffold material for tissue engineering heart valves with minimal compaction and without compromising on tissue composition and properties. Cells from lower passages showed to improve tissue formation. Reducing cell expansion time will result in faster generation of TEHV, providing a more rapid treatment to patients.
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Fibrin Based Tissue Engineered Heart Valves Featuring the Sinuses of Valsalva
Authors: Petra Mela, Thomas Schermer, Julia Frese, Thomas Schmitz-Rode and Stefan JockenhoevelAbstractThe implementation of the sinuses of Valsalva in both the aortic and the pulmonary position is a crucial step towards the development of functional tissue engineered heart valves with optimal hemodynamic performance and reduced risk of thrombi formation. However, the implementation of these features is not standard in tissue engineered heart valves. In our laboratory we aim at the realization of autologous heart valves starting from materials isolated from the patient (fibrinogen and cells) and shaped into 3D geometries by moulding techniques. We present two new fabrication methods that result in the realization of heart valve scaffolds reproducing the complex geometry of semilunar valves. The first concept consists of a mould in two parts: a ventricular part and a vascular part which contains three removable bulbs representing the sinuses of Valsalva; in the second concept the vascular part features three flexible protrusions shaped as the sinuses of Valsalva which can be collapsed when a vacuum is applied. The moulds were designed with the 3D CAD software Pro/Engineer (PTC, Needham, MA, USA) and manufactured by rapid prototyping. The cell embedded fibrin gel valves were produced by polymerizing a fibrinogen solution in TBS (10 mg/ml) with CaCl2, thrombin and ovine umbilical cord derived fibroblasts (10x106 /ml) suspended in TBS. Afterwards, the obtained construct was placed in a static bioreactor to be cultured on the mould for 14 days in order to avoid cell-mediated tissue contraction before transferring it to a bioreactor for dynamic cultivation of a duration of 2 weeks. To release the fibrin scaffold from the mould after static cultivation the vascular part and subsequently the three removable sinuses of Valsalva were removed (first approach), or the vascular part was collapsed and taken out as one part (second approach). The constructs were successfully released without any tearing with both approaches despite the poor mechanical properties of the fibrin gel. Valved conduits including the sinuses of Valsalva were obtained without the need for suturing any of the parts together. After static and dynamic cultivation the conduits demonstrated good compliance. The tissue development was evaluated by histology (hematoxylin, eosin and immunohistochemistry) hydroxyproline assay and DNA assay. The presence of the sinuses of Valsalva in the aortic and the pulmonary root is fundamental for the correct functioning of semilunar heart valves. The implementation of the sinuses in tissue engineered valves will lead to an improvement of the valve function and an increased durability. Ongoing research focuses on the optimization of the cell source and of the conditioning protocol as to achieve optimal extracellular matrix synthesis and mechanical properties and minimize cell mediated tissue contraction.
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New Biomaterials in Heart Valve Tissue Engineering
AbstractIn situ tissue engineering may lead to a clinically and economically attractive new generation of heart valve replacement therapies, overcoming the limitations of currently used prostheses. Development of novel scaffolds is required for the proposed in situ tissue engineering. These scaffolds must be able to carry a full mechanical load immediately after implantation, and should subsequently provide all biological cues for the recruitment and attachment of specific circulating cells, and should also induce the growth of new tissue. The properties of the scaffold materials should be optimized with respect to mechanical properties and biodegradability characteristics. Synthetic biomaterials are tunable, which enables the creation of tailor made scaffolds suitable for in situ tissue engineering of heart valves. Two different approaches towards scaffold materials have been investigated, based on a biodegradable polyester (PCL or PLLCL), and either a quadruple hydrogen-bonding ureidopyrimidinone (UPy) unit or a bis-urea (BU) moiety. Both were investigated in vivo regarding biocompatibility and degradation kinetics. Thirty rats received the UPy and BU polyester based, disk-shaped implants subcutaneously. To investigate the effects of the implants over time, samples were explanted on day 2, 5, 10, 21 and 84. Disks with surrounding tissue were fixed in 10% neutral buffered formalin. After fixation, explants were dehydrated in graded alcohols and longitudinal sections were embedded in paraffin and stained. From each explant the foreign body response was quantified per high power field. Both the UPy and the BU-based materials proved biocompatible. In the acute phase all investigated biomaterials showed an infiltration of neutrophil granulocytes and mononuclear cells. In the chronic phase encapsulation by fibroblasts took place in all cases. Degradation rates were investigated by gel permeation chromatography (GPC) after 21 and 84 days. Little degradation was observed for the PCL-based polymers over the course of the experiment. The PLLCL-based polymers with a BU moiety, however, showed little at 21 days, but marked degradation at 84 days. Which of the explored materials is best suitable for in situ tissue engineering of heart valves will depend on further experiments, investigating how much time is needed for cell recruitment, adhesion, differentiation and tissue growth.
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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Human Valve Interstitial Cells Demonstrate Transdifferentiation Potential
Authors: Najma Latif, Padmini Sarathchandra, Adrian H Chester and Magdi H, YacoubAbstractAortic stenosis and valvular degeneration is characterized by lipid accumulation, presence of cartilage and calcification. The identity and cellular origins of the cells mediating these effects are unknown. We have investigated the potential of human valve interstitial cells (VICs) to transdifferentiate into osteogenic, adipogenic and chondrogenic-like lineages and examined the presence of resident stem cells. Transdifferentiation potential of VICs was assessed after removal of stem cells. Human aortic heart valves (n=8; mean age 64.7 ± 7.5 years) from patients undergoing transplantation, free from calcification and disease, and were used to isolate VICs. Differentiation was carried out by incubating VICs for 21 days with media containing ascorbate (50µg/ml), dexamethasone (10-8M) and β-glycerophosphate (10mM) for osteogenic differentiation, with ascorbate (50µg/ml), dexamethasone (10-7M) and indomethacin (50µg/ml) for adipogenic differentiation and with insulin transferrin selenium and TGFβ1 (10ng/ml) for chondrogenic differentiation. Immunocytochemistry and fluorescence-activated cell sorting (FACS) were used to assess stem cell populations and removal of sub-populations of VICs. Mesenchymal stem cells were isolated from bone marrow samples obtained from healthy human donors (n=6) and used as positive controls. We analysed the gene expression of some of the Wnt family as potential mediators of transdifferentiation. Incubation of VICs with osteogenic media induced alkaline phosphatase and osteocalcin expression in 34.2 ± 4.6% of VICs, with adipogenic media induced oil red O, SREBP and PPARγ expression in 13.8 ± 5.1% of VICs and with chondrogenic media changed the morphology in 41.7 ± 4.8% of VICs but did not induce collagen type II, type X or aggrecan expression. Cultured VICs expressed CD44 (94.0± 4.4%), CD73 (79.4 ± 15.4%) and CD105 (6.9 ± 2.6%) in common with mesenchymal stem cells. However very low percentages of stem cells were identified in cultured VICs, CD34 (2.8% ± 0.50), CD133 (1.84 ± 0.77%), c-kit (0.72% ± 0.21) and stro-1 (1.55 ± 0.93%). Valve leaflets demonstrated only occasional positive markers for stem cells. Cultured VICs were negative for CD31, CD14, CD45, Tie-2 and flk-1. Removal of these stem cells by FACS demonstrated that purified VICs retained their ability to transdifferentiate into osteogenic and adipogenic lineages to the same degree. Gene expression of the Wnt family showed the expression of Wnt2, Wnt2B, Wnt5B, Wnt 10B. A number of frizzled receptors were detected, FZD2-5 and FZD 6-10 as well as inhibitors DKK 1-3. The population of purified human VICs have the capacity to transdifferentiate into osteogenic, adipogenic and chondrogenic-like lineages. Human valve leaflets and cultures contain a resident stem cell population which can differentiate and thus contribute to valve IC population as well as to pathological phenotypes.
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C-type Natriuretic Peptide Modulates TGF-β1-induced Synthesis of Proteoglycan by VICs
Authors: Mark Blaser, Jung Woo Kwon, Krista L. Sider, Andrea V. Kwong, Kuiru Wei and Craig A. SimmonsAbstractAortic valve disease (AVD) is a cell-mediated pathology marked by unchecked remodeling of the extracellular matrix (ECM). This process is thought to be promoted by increased levels of TGF-β1 in the valve leaflets and the concomitant loss of protective factors such as C-type natriuretic peptide (CNP). Among its many functions, TGF-β1 stimulates the expression of proteoglycan (PG), in part via mitogen activated protein kinase (MAPK)-mediated Smad signalling. We and others have observed the formation of PG-rich lesions in early diseased aortic valves. Here we investigated the relationship between TGF-β1, CNP, and PG in a mouse model of early AVD and in vitro. 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 < w/w) for four months (n = 4-6 per group). Longitudinal aortic valve sections from formalin-fixed and paraffin-embedded hearts were stained by Movat’s pentachrome and immunostained for CNP and TGF-β1. Valve interstitial cells (VICs) were isolated from healthy porcine aortic valves and treated in vitro with 5 ng/ml TGF-β1 and/or 1 µM CNP or 10 µM U0126 (inhibitor of MEK, upstream of the MAPK Erk1/2) for up to 6 days. Erk1/2 phosphorylation was measured by Western blot, while PG expression in media collected from these cultures was assayed by Alcian Blue guanidine-HCL solubilisation. Mice on the HF/HC diet for four months became obese, developed mild hypercholesterolemia, and had early AVD, as demonstrated by hemodynamic dysfunction and significant thickening of the leaflets (data shown in another abstract). Thickening was due to PG deposition (11435 ± 7681 vs. 5448 ± 2948 µm2, p < 0.01), not increased collagen content (1729 ± 815 vs. 1771 ± 663 µm2, p = 0.87). TGF-β1 levels were elevated in leaflets of mice on the HF/HC diet, particularly in the region of PG lesions. The PG lesions in HF/HC mice also had lower CNP expression than non-lesion regions. In VICs, TGF-β1 induced a 29% increase of PG expression (p < 0.05), which was significantly reduced by the addition of CNP (p < 0.05). Interactions between TGF-β1 and CNP in PG synthesis were mediated in part via Erk1/2 signaling, as TGF-β1 increased Erk1/2 phosphorylation, CNP abrogated TGF-β1-induced Erk1/2 activation, and blocking Erk1/2 signaling with U0126 significantly reduced PG expression (p = 0.06). These studies demonstrate that in early AVD, thickened leaflets are PG-rich with elevated expression of TGF-β1 and down-regulated CNP. Furthermore, TGF-β1 induces PG expression in VICs, a process which is inhibited by CNP – possibly through Erk1/2. Insight into the mechanisms by which TGF-β1 and CNP regulate the formation of PG lesions helps further our understanding of early disease progression and may aid in identifying novel medical strategies to arrest this disease before a substantive and possibly untreatable burden of calcification occurs.
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The Role Of BMP/Wnt Signalling In Human Heart Valve Calcification
Authors: Paul Riem Vis, Adrian Chester, Najma Latif, Padmini Sarathchandra, Jolanda Kluin and Magdi YacoubAbstractAortic valve calcification is a complex process that is characterised by the expression of bone markers in the valve leaflet. The Wnt pathway and bone morphogenetic proteins (BMPs) have been implicated to play a role in vascular and heart valve calcification. This study examines the regulation of Wnt3a by BMPs and the functional effects mediated by Wnt3a in human valve interstitial cells (VICs). Treatment of VICs by BMPs up-regulated the expression of Wnt3a, ALP, RUNX2, and β-catenin, as was shown by Western Blot analysis. Analysis of human pathological specimens by immunohistochemistry showed an increased level of expression of Wnt3a, Msx2 and β-catenin was localised to peri-calcific regions of the tissue. The concentration-dependent proliferation or differentiation of VICs in response to Wnt3a was measured by the incorporation of [3H]-thymidine and activity of alkaline phosphatase (ALP) respectively. Wnt3a-treated VICs proliferated in a concentration-dependent manner. At higher concentrations, Wnt3a caused a significant increase in ALP activity and expression of RUNX2. Lastly, Western blot experiments showed that BMP2 induced RUNX2 up-regulation was mediated through β-catenin/Wnt-signalling and not Smad-signalling. This study demonstrates the important role of Wnt-signalling in valve calcification in response to BMPs. Wnt3a may induce valve calcification by regulating the proliferation and osteogenic differentiation of cells within the valve. These findings may help to identify new therapeutic.
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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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Pressure and Angiotensin II Influence the Mechanical Properties of Aortic Valves
Authors: Valtresa Myles, Jun Liao and James N. WarnockAbstractElevated cyclic pressure and angiotensin II (Ang II) both promote aortic valve collagen synthesis, an early hallmark of aortic sclerosis. In the current study, it was hypothesized that the increased collagen production induced by either elevated pressure or Ang II would increase tissue stiffness. Porcine aortic valve leaflets were randomly assigned to four groups; leaflets in group 1 were treated with 10-6M Ang II, leaflets in group 2 were exposed to 80 or 120 mmHg cyclic pressure, leaflets in group 3 were treated with Ang II and exposed to cyclic pressure and leaflets in group 4 were the control. Biaxial testing was performed after 24 and 48 hours on 10mm x 10mm samples of tissue dissected from the central region of the leaflets. Four fiducial markers arranged in an approximately 4 mm x 4 mm square were placed in the center of the extracted portion of the leaflets to track tissue strain. A membrane tension (force/unit length) was applied along each axis and increased slowly from a pre-stress tension of ~0.5 N/m to a peak tension of 60 N/m. The samples were preconditioned for ten contiguous cycles, following an equibiaxial protocol of TCC:TRR = 60:60 N/m, where TCC and TRR are the tensions applied in the circumferential and radial directions, respectively. Tissue extensibility was characterized by maximum stretch along the circumferential direction (λcc) and maximum stretch along the radial direction (λrr), at an equibiaxial tension of 60 N/m. Leaflet stiffness was greater in the circumferential direction than in the radial direction, which is consistent with previous studies. The peak stretches of native valves were calculated as 1.05±0.02 and 1.40±0.02 in the circumferential and radial directions, respectively. There was no significant difference in the stiffness of native valves compared to those exposed to 0mmHg(-Ang II) or 80mmHg(-Ang II) for 24 or 48 hours. Elevated pressure increased stiffness in both directions after 24 and 48 hours. After 24 and 48 hours Ang II significantly increased stiffness in the radial direction. The combination of Ang II and elevated pressure increased stiffness in radial direction after 24 and 48 hours. Increased stiffness may be due to remodeling of the ECM. Excessive collagen production is known to hinder valve function, eventually resulting in aortic stenosis. In conclusion, the results of the present study demonstrated that both elevated pressure and Ang II play a role in the increased stiffness of aortic valve leaflets.
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Adaptation of the Mitral Valve to Chronic Ischemic Left Ventricular Failure in Swine
Authors: Bryant V. McIver, Samiya Hussain, Vinod Thourani and Muralidhar PadalaAbstractMitral valve leaflets in patients with chronic heart failure are severely stiffened, larger and fibrotic; though the mechanisms underlying such biological remodeling are currently unknown. Chronic tethering of the valve leaflets by the enlarged left ventricle, and the presence of mitral regurgitation (MR), is hypothesized to trigger fibrotic pathways that induce such remodeling. In this study, we sought to develop a large animal model of chronic ischemic left ventricular failure in which moderate leaflet tethering and MR can be repeatedly induced, and the remodeling of the mitral valve can be assessed in vivo using echocardiography. Inferior wall myocardial infarction (IMI) was induced in 16 farm swine (30-35kg) using a percutaneous transfemoral approach. Coronary angiography was performed to selectively identify the marginal branches of the left circumflex artery perfusing the inferior left ventricular (LV) wall and the posterior papillary muscle (PPM), and then these regions were infarcted using a 2 mm balloon catheter and injection of 100% ethyl alcohol into the corresponding branch. Transthoracic 2D echocardiography (TTE) of the left heart was performed pre and post-IMI in all animals, at 4 weeks and at 8 weeks. MR percentage (MR jet/left atrial area), mitral leaflet length and thickness, and mitral annular geometry were measured. Leaflet length and thickness were measured at end diastole to assure measurements were taken in the maximal unloaded state. All animals survived the procedure without complications (0% mortality). At baseline, the swine had mild MR (10.7±8%) which significantly increased to 26.6±9% immediately post-op (p<0.05), declined slightly to 23.8±7.8% at 4 weeks, and increased to 32.2±13.9% at 8 weeks. Mitral annular diameter increased from 2.7±0.5 cm pre-op, to 3.1±0.5 cm post-op (p<0.05), then to 3.8±0.4 cm (p<0.005) and 3.9±0.6 cm (p<0.005), at 4 and 8 weeks, respectively. Posterior leaflet length was 1.8±0.2 cm pre-op, and then stabilized at 1.9±0.2 cm post-op, at 4 weeks, and at 8 weeks. Anterior leaflet length was 2.3±0.3 cm at baseline and increased with the increase in MR, to 2.5±0.4 cm post-op, 2.8±0.5 cm at 4 weeks (p<0.05), and 3.2±0.2 cm at 8 weeks (p<0.001). Posterior leaflet thickness increased from 0.36±0.05 cm at baseline, to 0.37±0.07 cm post-op, and then to 0.4±0.05 cm and 0.41±0.03 cm at 4 weeks and 8 weeks, respectively. Anterior leaflet thickness was 0.4±0.05 cm at baseline and post-operatively, and 0.47±0.03 cm (p<0.01) and 0.45±0.07 cm at 4 weeks and 8 weeks, respectively. Changes in mitral leaflet geometry correspond with changes in the mitral valvular apparatus after IMI in a chronic swine model. As the mitral annulus increases in diameter, significant lengthening is seen from the anterior leaflet, while the posterior leaflet remains relatively static and tethered. The anterior leaflet thickens by 4 weeks, while the posterior leaflet remains stable.
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Valve Calcification on Computed Tomography Can Estimate Aortic Stenosis Severity
AbstractUse of coronary CT angiography for screening of coronary artery disease is being advocated for the general population. Meanwhile, CT is increasingly used in preoperative planning for transcatheter aortic valve replacement (TAVR). Although aortic stenosis (AS) severity can be evaluated by measurement of incidentally found aortic valve calcification on CT, it has not been validated by pathologic specimens and can be confounded by calcification of adjacent structures as well as motion artefact. Micro-computed tomography (microCT) provides ultra-high resolution imaging of small structures to yield excellent estimation of tissue calcification. In this study, excised aortic valves from patients with confirmed AS were used to determine if the amount of calcium on microCT correlated with severity of aortic stenosis. Thirty-five aortic valves excised during surgical valve replacement underwent micro-CT imaging with resolution of 76μm in the axial direction. Amount of calcium was determined by absolute and proportional values of calcium volume. Correlation of calcium volume and preoperative mean aortic valve gradient (MAVG), peak transaortic velocity (Vmax), and aortic valve area (AVA) on echocardiography was evaluated. For the patients who had a preoperative CT scan with acceptable image quality, the amount of valvular calcification was also measured by a well-experienced radiologist using modified Agatston algorithm. Mean amount of calcium across all valves was 603.2±398.5mm3, while mean ratio of calcium volume to total valve volume was 0.36±0.16. Mean aortic valve gradient correlated positively with both calcium volume and ratio (r=0.72, p<0.001). Vmax also positively correlated with calcium volume and ratio (r=0.69 and 0.76 respectively, p<0.001). A logarithmic curvilinear model was best fit to the correlation. Calcium volume of 480mm3 showed sensitivity and specificity of 0.76 and 0.83, respectively for severe AS diagnosis, while calcium ratio of 0.37 yielded sensitivity and specificity of 0.82 and 0.94, respectively. Calcium volume and its proportion to total valve volume were found to be good predictive parameters for severe AS when estimated radiologically. Calcium volume quantification may be a complimentary measure for AS severity evaluation in situations where aortic valve calcification is found incidentally on CT as well as in preoperative assessment of aortic valves for TAVR.
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Mitral Valve Mechanics Following Posterior Leaflet Patch Augmentation
AbstractAttention towards optimization of mitral valve repair methods is increasing. Patch augmentation strategy is used to treat functional ischemic mitral regurgitation (FIMR) and hypertrophic cardiomyopathy. When used to extend the anterior leaflet, the procedure decreases the forces exerted on the secondary chordae. The purpose of this study was therefore to investigate the force balance changes following patch augmentation of the posterior leaflet, with particular attention to the secondary chordae tendineae emanating from the posterior papillary muscle (PPM) in an FIMR simulated valve. Twelve mitral valves were obtained from 80kg pigs. An in vitro test setup simulating the left ventricle was used to hold the valves with a papillary muscle positioning system. Water pressure within the ventricular chamber was regulated manually in order to simulate different static pressures during valve closure. An oval shaped porcine pericardial patch measuring 17x29mm was introduced into the posterior leaflet approximately 2mm from the annulus and extending circumferentially from the middle of P2 to the end of the P3 scallop. In order to simulate a healthy valve, retraction of the patch was performed using sutures, which were then released to simulate patch repair. Data were acquired with and without PPM displacement to simulate the effect from one of the main contributors of FIMR, before and after patch augmentation, giving four simulation scenarios. The PPM was displaced 12mm posteriorly and 5mm apically. Dedicated miniature transducers were used to record the forces exerted on the secondary chordae tendineae. Three-way ANOVA was used to analyze the measurements. The effect of displacing the posterior papillary muscle (p < .010) and implementing patch augmentation (p<.004) are significant and independent of each other. The overall effect of displacing the PPM induced tethering on the secondary chordae tendineae from the PPM to the posterior leaflet resulting in a force increase of 28.2 %. The overall effect of implementing the patch augmentation into the posterior leaflet induced a decrease in force of 24.8 % for the healthy and PPM displaced simulations together. The repairing effect of the patch augmentation is found by comparing the specific scenarios. A 40 % increase is induced by displacing the PPM and a 31 % decrease is found by implementing the patch augmentation, leaving the repaired tethering force a mere 9 % higher than that of the healthy measurements. Posterior leaflet patch augmentation significantly reduced the forces exerted onto the secondary chordae tendineae from the PPM in both healthy and PPM displaced valves. As changes in chordal tension leads to redistribution of the total stress exerted on the valve, patch augmentation may have adverse long term influence on mitral.
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Distensibility of Decellularized and Glutaraldehyde-Preserved Aortic Full Roots as Evaluated by Computed Tomography
AbstractThe aortic root is a dynamic structure and its distensibility is considered an important factor in native and prosthetic heart valve function. Glutaraldehyde-preserved (GLUT) valves have limited durability possibly due to increased tissue stiffening. Decellularized deoxycholic acid-treated (DOA) valves exhibit excellent long-term performance in the pulmonary position in humans possibly due to preservation of valve tissue distensibility. We investigated aortic distensibility in DOA (n=8) and GLUT (n=3) aortic root prostheses in 60 kg pigs 2 weeks after orthotopic implantation. Five pigs served as controls. Using a dual source computed tomography scanner (Somatom Flash, Siemens Medical Solution, Forcheim, Germany) the cross sectional area at the level of Sinus of Valsalva (SoV), sino-tubular junction (STJ), and ascending aorta (AA), respectively, was measured in both diastole and systole. Distensibility was defined as the change in area from diastole (RR 95%) to systole (RR 15%). For assessment of the prosthetic- independent aortic distensibility we performed the same measurements in the descending aorta (AD) in all animals. Data were analyzed using students t-test and reported as a mean±SD. Native aortic distensibility was significant larger at the level of SoV (15.8%±4.9), STJ (46.7%±10.3), and AA (40.6%±9.0), compared with both DOA and GLUT aortic roots (p<0.05). No difference in distensibility between the DOA and GLUT aortic roots were observed: 8.8%±2.4 vs 6.2%±5.5 (SoV), 11.7%±3.0 vs 10.0%±7.1 (STJ), 14.9%±6.1 vs 11.8±1.7 (AA), and 15.6±1.5 vs 15.5±1.8 (DA) , respectively (p>0.05). There was no difference in mean distensibility between the three groups in the decending aorta. This is the first study to evaluate in-vivo distensibility of aortic root prostheses in vivo. Aortic root distensibility is reduced following implantation of DOA or GLUT prosthetic aortic valves. No difference was observed between DOA and GLUT valves. Long-term follow-up is needed in order to verify any changes in bioprosthetic aortic root distensibility over time.
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The Way to a New Generation of Bio-Prostheses
Authors: Thomas Waldow, Katrin Plötze, Matthias König and Klaus MatschkeAbstractDiseases of heart valves often make a prosthetic replacement therapy necessary. Long term goal is the development of artificial leaflet material with defining properties of mechanical prostheses (i.e. durability) with complete biocompatibility without the need for any anticoagulation therapy whatsoever. In order to achieve this aim, objective efforts are being made to use a new material that is still in the process of development. It is a film-like tissue, made of pure carbon in the form of carbon-nanotubes, which are characterized by their excellent mechanical properties (tensile strength 65GPa in comparison to steel with 0.6Gpa). The woven material has also a very small mesh width, which can be influenced by the spinning method. Due to the nature of this material, which is associated with a high surface energy, there is a need for adaptions for the use in vivo according to the requirements. Plasma Enhanced Chemical Vapor Deposition PECVD has proven to be the appropriate method for such a functionalization. It is possible to deposit amorphous hydrocarbon coatings below 60°C and in a thickness range from a few nanometers up to several microns, while at the same time providing a very low Young's modulus that ensures to withstand mechanical stress. In addition, this method allows to influence regional properties e.g. by incorporating silicon into the matrix to prevent adhesion of thrombocytes and/or to add nitrogen to give the ability to endothelial cell growing. The first project step is to evaluate in high resolution and accuracy the parameter characteristic of native valves and prostheses. Therefore the elastic modulus, flexibility, extension, expansion, banding stress, banding elasticity, banding rigidity, reversed banding strength, cantilever load, surface tension, surface structure, surface tension and intensity of surface loading are under investigation. A mix of physical, chemical and visual analysis methods are used. The parameter characteristic of the nanotube material has to match these results. In cases of mismatch a technical adjustment, e.g. during weaving or coating, is possible. At least two big advantages of this new composite material exist: The possibility to produce a compound material with regional different main characteristics by using a two-phase coating process. And on the other hand the abandonment of exogenous substances during the production process and so the material has a distinct advantage over those currently used Teflon fabrics.
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Mechanical Properties of Bioprostheses Leaflets Compared to Human Aortic Valve
Authors: Martins Kalejs, Peteris Stradins, Lacis Lacis, Iveta Ozolanta and Vladimir KasyanovAbstractHeart valve bioprostheses suffer from gradual tissue deterioration, which has a causal link with valve tissue mechanical properties. Limited data on mechanical properties of commercially available bioprostheses comparing them to native human aortic valves (AV) is available. Our objective was to determine the mechanical properties of several contemporary bioprostheses and compare them with native human and porcine aortic valves. Leaflets from 5 unchanged human AV, collected from cadaveric hearts and 5 porcine AV, and from 3 of each kind of bioprostheses - Medtronic Hancock II, Sorin Soprano and Medtronic Freestyle were analysed using uniaxial tensile tests in radial and circumferential directions. Data are presented as means ± standard deviation. In both tested directions there's a shift to the stress axis of stress-strain curve for HancockII prostheses and even more for Soprano prostheses when compared to native human valves. In circumferential direction modulus of elasticity (E) of native human AV is 15.34±3.84MPa, porcine AV - 9.7±1.3MPa, Freestyle - 9.0±3.0MPa, HancockII - 22.5±2.2MPa and Soprano - 29.5±6.0MPa. In radial direction E of native human AV is 1.98±0.15MPa, porcine AV - 1.0±0.2MPa, Freestyle - 0.8±0.3MPa, HancockII - 2.5±0.2MPa and Soprano - 15.8±5.4MPa. Xeno-aortic bioprostheses have a non-linear and anisotropic response to stress in uniaxial tensile tests similar to native AV leaflets. HancockII has gained mechanical strength but lost tissue elasticity compared to native valve tissue. Leaflets of Soprano prostheses are even more rigid and lack pronounced material anisotropy. These differences in mechanical properties may accelerate deterioration of bioprostheses, causing altered stress distribution within valve leaflets. These data provide important information about what mechanical properties future valve substitutes should conform to.
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Polymer Nanofiber Materials Matching the Mechanic Properties of Native Aortic Valve
AbstractPorous electrospun nanofiber materials are very promising as matrices for heart valve tissue engineering. Not only biocompatibility is important for this material but also the mechanical features – it has to be strong enough to withhold the pressure after implantation as well as deformable enough for better distribution of shear stress along its surface. Deformability is also crucial for stimulation of fibre production by fibroblasts on these matrices. Altogether 8 differing density variants of electrospun nanofiber materials from gelatine, polyurethane (PUR), polylactic acid (PLA) and polycaprolactone (PCL) were analysed using uniaxial tensile tests. Data were compared to mechanical properties of porcine aortic valve (AV) leaflets in radial and circumferential directions. Data are presented as means ± standard deviation. In circumferential direction modulus of elasticity (E) of native porcine AV is 9.7±1.3MPa and - 1.0±0.2MPa in radial. Ultimate stress and strain is 44.8±5.9% and 2.3±0.6 MPa in circumferential and 95.6±31.4% and 0.5±0.2MPa in radial direction for native leaflets. Closest of the materials to match the mechanical properties of porcine AV in circumferential direction was PUR with density 6.2 g/sqm showing E of 3.9±0.5 MPa, ultimate stress and strain - 5.3±1.68MPa and 141.8±43.9MPa respectively. Closest to match radial direction was gelatine with density 5.7 g/sqm showing E of 0.64±0.14 MPa, ultimate stress and strain - 0.38±0.05MPa and 82.53±10.20MPa respectively. Native AV leaflets have a non-linear and anisotropic response to stress in uniaxial tensile tests. Hence to model as precisely as possible their mechanical properties we suggest to use a combined material made in a sandwich fashion with layers of gelatine on the outside and PUR in the middle with their fibbers predominantly orientated in perpendicular directions. The other tested materials PLA and PCL either lacked strength to mimic leaflets in circumferential direction or deformability required for the radial direction.
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Functional Mitral Regurgitation Is a Main Determinant Of Adverse Outcome In Patients With Heart Failure Due To Non-Ischemic Dilated Cardiomyopathy.
AbstractIschemic mitral regurgitation has been recently demonstrated to carry important prognostic information in patients with left ventricular dysfunction due to coronary artery disease. There is no information regarding the prognostic role of functional mitral regurgitation in patients with non ischemic dilated cardiomyopathy. Patients with stable heart failure due to non-ischemic dilated cardiomyopathy were prospectively enrolled. All patients underwent a comprehensive echocardiographic assessment. Left ventricular diastolic (LVD) , systolic (LVS) diameters, left atrial diameter (LAD), ejection fraction (EF) and restrictive mitral filling pattern (RMP) was measured. Mitral regurgitant volume (RV) was measured by means of proximal isovelocity surface area method. The end point of the study was death or hospitalization for worsening heart failure. 80 patients (mean age 61±9 years; 82% male) were enrolled. 10 patients reached the end point of the study. At univariate Cox analysis, the echocardiographic variables associated with outcome were: EF (HR 0.84 95% CI 0.75 0.94; p=0.002), RMP (HR 5.2 95% CI 1.4 19.7; p=0.01) and RV (HR 1.046 95% CI 1.02 1.07; p=0.0005), LVS/BSA (HR 1.2 95% CI 1.02 1.4; p=0.03). At multivariate analysis RV remained the only variables independently associated with outcome (p=0.04). Result did not change when LVS/BSA substituted EF in the model. Receiving operator characteristics analysis documented that the area under the curve for RV in identifying patients with adverse outcome was 0.84±0.06 (95% CI 0.74 0.91) and the best cut off value for RV was 28 ml (sensitivity 80% 95% CI 44 97 and specificity 87% 95% CI 77 94). Patients with RV<28 had a survival rate of 95% after 6 years from the index echocardiogram compared with 22% in those with RV> 28 (longrank 23; p<0.0001). In patients with non-ischemic dilated cardiomyopathy, RV was a main predictor of death or hospitalization for worsening heart failure.
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Prevalence of Severe Aortic Stenosis With Low Flow in Patients With and Without Left Ventricular Dysfunction. A Consecutive Echocardiographic Population
AbstractPatients with aortic stenosis (AS) may have a severely reduced aortic valve area (AVA) and a paradoxically low mean gradient (MG). Although this condition is well known in presence of left ventricular (LV) dysfunction, it has recently been observed that it can be associated with normal ejection fraction (EF) as well. Since the prevalence of this condition with respect to LV function is not well defined, we aimed to evaluate the distribution of patients with severely reduced valve area and low MG in a group of patients regardless of EF over a set period of time. We retrospectively identified consecutive patients with severe AS (defined as aortic valve area <0.6 cmq/mq) from our echo data-base. Low MG was defined as < 40 mmHg. Left ventricular systolic dysfunction was considered as EF< 50%. 167 patients with AVA <0.6 cmq/mq formed the study population. 94 (56%) patients were characterized by high MG and 73 (44%) by low MG. Among patients with low MG, 38 were characterized by normal EF and 35 by reduced EF. Differences among groups are shown in Table 1. In this echocardiographic series of consecutive patients, the prevalence of low MG despite severely reduced AVA was high. The distribution of low MG was similar in presence of reduced and preserved EF (21 and 23% of the overall population respectively).
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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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