bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating the dynamics of bulk snow density in dry and moist conditions using a one-dimensional model
Authors: De Michele C. , Avanzi F. , Ghezzi A. , Jommi C.
Published: July 2012

Abstract. Snowpack is a complicated multiphase mixture with mechanical, hydraulic, and thermal properties, highly variable within the year in response to climatic forcings. Bulk density is a macroscopic property of the snowpack used, together with snow depth, to quantify the water stored. In seasonal snowpacks, the bulk density is characterized by a strong non-linear behaviour due to the occurrence of both dry and wet conditions. In literature, bulk snow density estimates are obtained principally with multiple regressions, and snowpack models have put the attention principally on the snow depth and snow water equivalent. Here a one-dimensional model for the temporal dynamics of the bulk snow density has been proposed, accounting for both dry and moist conditions. The model assimilates the snowpack to a two-constituent mixture: a dry part including ice structure, and air, and a wet part constituted by liquid water. It describes the dynamics of three variables: the depth and density of the dry part and the depth of liquid water. The model has been calibrated and validated against hourly data registered in two SNOTEL stations, Western US, with mean values of the Nash-Sutcliffe coefficient ≈0.90–0.92.
Focus:
Authors: Darling Elizabeth
Published: July 2012

This article explores the architectural little magazine Focus, which was produced by students at London's Architectural Association between 1938 and 1939. It considers the role this magazine played in the development of architectural modernism in the UK at the end of the 1930s through an analysis of its form, content, and distribution networks. More generally, it considers how Focus typifies the significance of the written word to the practice of architecture.
Picturing Smartness:
Authors: Newton Leslie
Published: July 2012

In this article, I examine the literary single-panel cartoons and caricatures in smart magazines the New Yorker, Vanity Fair, and Esquire, which reflect each magazine's bibliographic code, emphasizing a burgeoning “smart” audience, and efforts to promote popular cultural tastes and celebrities. While Bourdieu discusses the circulation of symbolic capital of the avant-garde, the New Yorker, Vanity Fair, and Esquire market to a wider audience, conflating economic, social, and cultural capital by their published contents and creating an omnivorous readership that recognizes the consecrated modern cultural celebrities while eschewing—and laughing at—the exclusive elitism of other publications.
Regulation of Abnormal Atrioventricular Valve Development
Authors: Yalcin Huseyin C. , Ajaeb Sarah , Butcher Jonathan T.
Published: July 2012

Hemodynamic 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.
Effects of Finite Deformation on Extracellular Matrix Production and Mechanical Properties
Authors: Stella John A. , Amoroso Nicholas J. , D’Amore Antonio , Mayer, Jr. John E. , Wagner William R. , Sacks Michael S.
Published: July 2012

With 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.
Fibrin Based Tissue Engineered Heart Valves Featuring the Sinuses of Valsalva
Authors: Mela Petra , Schermer Thomas , Frese Julia , Schmitz-Rode Thomas , Jockenhoevel Stefan
Published: July 2012

The 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.
Human Valve Interstitial Cells Demonstrate Transdifferentiation Potential
Authors: Latif Najma , Sarathchandra Padmini , Chester Adrian H , Yacoub Magdi H,
Published: July 2012

Aortic 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.
The Role Of BMP/Wnt Signalling In Human Heart Valve Calcification
Authors: Vis Paul Riem , Chester Adrian , Latif Najma , Sarathchandra Padmini , Kluin Jolanda , Yacoub Magdi
Published: July 2012

Aortic 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.
Mitral Valve Interstitial Cells Behaviour Under Hypoxia
Authors: Salhiyyah Kareem , Marzieh Zamani , Sarathchandra Padmini , Latif Najma , Yacoub Magdi , Chester Adrian H.
Published: July 2012

Mitral 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.
Developing a Rat Model of Cardiovascular Calcification to Evaluate Tissue-Engineered Heart Valve Prostheses
Authors: Assmann Alexander , Zwirnmann Kai , Munakata Hiroshi , Delfs Christofer , Horstkötter Kim , Poudel-Bochmann Bhawana , Kamiya Hiroyuk , Lichtenberg Artur , Akhyari Payam
Published: July 2012

A 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.
Decellularization Diminishes the Calcifying Degeneration of Aortic Conduit Allografts
Authors: Assmann Alexander , Horstkötter Kim , Munakata Hiroshi , Delfs Christofer , Bhawana Poudel-Bochmann Bhawana Poudel-Bochmann , Boeken Udo , Akhyari Payam , Lichtenberg Artur
Published: July 2012

The 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.
Matrix-controlled In-growth Selectivity as a Principle of Heart Valve Regeneration
Authors: Davies Neil , Bracher Mona , Sun Michelle , Bezuidenhout Deon , Zilla Peter
Published: July 2012

Spontaneous 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.
3D Engineered Micro-Tissue Models to Study Cardiovascular (Patho)biology and Regeneration
Authors: Bouten Carlijn V. , van Geemen Daphne , de Jonge Nicky , Smits Anthal I. , Driessen-Mol Anita , Baaijens Frank P.
Published: July 2012

Engineered 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.
Ice-Free-Cryopreservation Attenuates Calcification in Allograft Heart Valves
Authors: Lisy Milan , Brockbank Kelvin G.M. , Schenke-Layland Katja , Fend Falko , Moritz Anton , Stock Ulrich A.
Published: July 2012

The 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.
Investigation of the Suitability of Decellularised Porcine Pericardium for Mitral Valve Reconstruction
Authors: Morticelli Lucrezia , Thomas Daniel , Ingham Eileen , Korossis Sotiris
Published: July 2012

The 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.
Integration of Microstructural Architecture of the Mitral Valve into an Anatomically Accurate Finite Element Mesh
Authors: Amini Rouzbeh , van Loosdregt Inge , Koomalsingh Kevin , Gorman Robert C. , Gorman Joseph H. , Sacks Michael S.
Published: July 2012

Although 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.
Valve Calcification on Computed Tomography Can Estimate Aortic Stenosis Severity
Authors: Chitsaz Sam , Blackshear Charlie , Tegegn Nebiyu , Wisneski Andrew , Azadani Ali N. , Ge Liang , Hope Michael D. , Tseng Elaine E.
Published: July 2012

Use 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.
Functional Mitral Regurgitation Is a Main Determinant Of Adverse Outcome In Patients With Heart Failure Due To Non-Ischemic Dilated Cardiomyopathy.
Authors: Mauro Toniolo , Andrea Rossi , Mariantonietta Cicoira , Corinna Bergamini , Corrado Vassanelli
Published: July 2012

Ischemic 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.
Fluid-Structure Interaction Simulation of an Aortic Bi-Leaflet Mechanical Heart Valve in a Patient-Specific Left Heart
Authors: Bao Le Trung , Sotiropoulos Fotis
Published: July 2012

A 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.
Functional Performance and Biomechanics of Decellularized Human Aortic Valves
Authors: Arnold Stacy M. G. , Goldstein Steven , Gale David C.
Published: July 2012

Allograft 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.
Autologous Bone Marrow Mononuclear Cell-Based Tissue Engineered Heart Valves: First Experiences with a One-step Intervention in Primates
Authors: Weber Benedikt , Emmert Maximilian Y. , Scherman Jacques , Gruenenfelder Juerg , Frese Laura , Brokopp Chad , Falk Volkmar , Zilla Peter , Hoerstrup Simon P.
Published: July 2012

A 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.