bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

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.
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

AbstractThe 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

AbstractAortic 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

AbstractAortic 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

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.
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

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.
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

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.
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

AbstractSpontaneous 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

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.