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Benedikt Weber,
Jacques Scherman,
Maximilian Y Emmert,
Juerg Gruenenfelder,
Renier Verbeek,
Mona Bracher,
Melanie Black,
Jeroen Kortsmit,
Thomas Franz,
Roman Schoenauer,
Laura Baumgartner,
Chad Brokopp,
Irina Agarkova,
Petra Wolint, Gregor Zund,
Volkmar Falk,
Peter Zilla,
Simon P Hoerstrup
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ABSTRACT: 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 remodelling 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.
European Heart Journal 03/2011; 32(22):2830-40. · 10.48 Impact Factor
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Dörthe Schmidt,
Petra E Dijkman,
Anita Driessen-Mol,
Rene Stenger,
Christine Mariani,
Arja Puolakka,
Marja Rissanen,
Thorsten Deichmann,
Bernhard Odermatt,
Benedikt Weber,
Maximilian Y Emmert, Gregor Zund,
Frank P T Baaijens,
Simon P Hoerstrup
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ABSTRACT: The aim of this study was to demonstrate the feasibility of combining the novel heart valve replacement technologies of: 1) tissue engineering; and 2) minimally-invasive implantation based on autologous cells and composite self-expandable biodegradable biomaterials.
Minimally-invasive valve replacement procedures are rapidly evolving as alternative treatment option for patients with valvular heart disease. However, currently used valve substitutes are bioprosthetic and as such have limited durability. To overcome this limitation, tissue engineering technologies provide living autologous valve replacements with regeneration and growth potential.
Trileaflet heart valves fabricated from biodegradable synthetic scaffolds, integrated in self-expanding stents and seeded with autologous vascular or stem cells (bone marrow and peripheral blood), were generated in vitro using dynamic bioreactors. Subsequently, the tissue engineered heart valves (TEHV) were minimally-invasively implanted as pulmonary valve replacements in sheep. In vivo functionality was assessed by echocardiography and angiography up to 8 weeks. The tissue composition of explanted TEHV and corresponding control valves was analyzed.
The transapical implantations were successful in all animals. The TEHV demonstrated in vivo functionality with mobile but thickened leaflets. Histology revealed layered neotissues with endothelialized surfaces. Quantitative extracellular matrix analysis at 8 weeks showed higher values for deoxyribonucleic acid, collagen, and glycosaminoglycans compared to native valves. Mechanical profiles demonstrated sufficient tissue strength, but less pliability independent of the cell source.
This study demonstrates the principal feasibility of merging tissue engineering and minimally-invasive valve replacement technologies. Using adult stem cells is successful, enabling minimally-invasive cell harvest. Thus, this new technology may enable a valid alternative to current bioprosthetic devices.
Journal of the American College of Cardiology 08/2010; 56(6):510-20. · 14.16 Impact Factor
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ABSTRACT: Fetal stem cells represent a promising cell source for heart valve tissue engineering. In particular, amniotic fluid-derived cells (AFDC) have been shown to lead to autologous fetal-like heart valve tissues in vitro for pediatric application. In order to expand the versatility of these cells also for adult application, cryopreserved AFDC were investigated as a potential life-long available cell source for heart valve tissue engineering.
Human AFDC were isolated using CD133 magnetic beads, and then differentiated and analyzed. After expansion of CD133- as well as CD133+ cells up to passage 7, a part of the cells was cryopreserved. After four months, the cells were re-cultured and phenotyped by flow cytometry and immunohistochemistry, including expression of CD44, CD105, CD90, CD34, CD31, CD141, eNOS and vWF, and compared to their non-cryopreserved counterparts. The stem cell potential was investigated in differentiation assays. The viability of cryopreserved AFDC for heart valve tissue engineering was assessed by creating heart valve leaflets in vitro.
After cryopreservation, amniotic fluid-derived CD133- and CD133+ cells retained their stem cell-like phenotype, expressing mainly CD44, CD90 and CD105. This staining pattern was comparable to that of their non-cryopreserved counterparts. Moreover, CD133- cells demonstrated differentiation potential into osteoblast-like and adipocyte-like cells. CD133+ cells showed characteristics of endothelial-like cells by eNOS, CD141 and beginning vWF expression. When used for the fabrication of heart valve leaflets, cryopreserved CD133- cells produced extracellular matrix elements comparable to their non-cryopreserved counterparts. Moreover, the resulting tissues showed a cellular layered tissue formation covered by functional endothelia. The mechanical properties were similar to those of tissues fabricated from non-cryopreserved cells.
The study results suggest that the use of cell bank technology fetal amniotic fluid-derived stem cells might represent a life-long available autologous cell source for heart valve tissue engineering, and also for adult application.
The Journal of heart valve disease 08/2008; 17(4):446-55; discussion 455. · 0.81 Impact Factor
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ABSTRACT: Latest techniques enable positioning of devices into the coronary sinus (CS) for mitral valve (MV) annuloplasty. We evaluate the feasibility of non-invasive assessment to determine CS anatomy and its relation to MV annulus and coronary arteries by multi-slice CT (MSCT) in normal and insufficient MV.
Fifty patients (33 males, 17 females, age 67+/-11 years) were studied retrospectively by 64-MSCT scans for anatomical criteria regarding CS and its relation to MV annulus and circumflex artery (CX). We included 24 patients with severe mitral insufficiency and 26 with no MV disease. Diameter of MV, of proximal and distal ostium of CS, length and volume of CS, angle between anterior interventricular vein (AIV) and CS, caliber change of CX before, under/over and after CS were analysed. Different anatomical correlations were demonstrated: distance of MV annulus to CS, CX to CS.
Diameter of proximal CS ostium was significantly larger in insufficient MV compared to normal MV (11+/-2.8 mm vs 9.9+/-2.5 mm; p<0.024). CS was significantly longer in patients with insufficient MV (125.4+/-17 mm vs 108.9+/-18 mm; p<0.003) with also significant differences in volume of CS (p<0.039). Significant difference in annulus diameter, 46.1+/-6mm (insufficient MV) versus 39.5+/-7.5 mm, p<0.004 was observed. Angle CS-AIV was 103.5+/-29 degrees (range 52 degrees -144 degrees ) in insufficient valves versus 118.2+/-24.5 degrees (range 73 degrees -166 degrees ) in normal valves with a tendency to higher angles in normal valves (p=0.06). Distance of MV annulus to CS measured 16+/-4.1/14.2+/-3.6 mm (insufficient/normal MV) without significant difference between groups. In 15 patients CX ran under CS. Eighty-four percent of these patients (13/15) show a decrease in CS caliber in the area of intersection. In 14 patients CS ran over and in one patient the diameter of the CS at intersecting region was smaller. In 16 patients no direct point of contact was visible, in five patients CX to CS positioning was not evaluable.
There is a significant anatomic difference between normal and insufficient MV, which might be the basis for any interventional approaches through the CS. Exact measurements of all structures and its anatomic correlations are possible with MSCT, which allows pre-interventional planning.
European Journal of Cardio-Thoracic Surgery 04/2008; 33(4):583-9. · 2.55 Impact Factor
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ABSTRACT: A novel concept providing prenatally tissue engineered human autologous heart valves based on routinely obtained fetal amniotic fluid progenitors as single cell source is introduced.
Fetal human amniotic progenitors were isolated from routinely sampled amniotic fluid and sorted using CD133 magnetic beads. After expansion and differentiation, cell phenotypes of CD133- and CD133+ cells were analyzed by immunohistochemistry and flowcytometry. After characterization, CD133- derived cells were seeded onto heart valve leaflet scaffolds (n=18) fabricated from rapidly biodegradable polymers, conditioned in a pulse duplicator system, and subsequently coated with CD133+ derived cells. After in vitro maturation, opening and closing behavior of leaflets was investigated. Neo-tissues were analyzed by histology, immunohistochemistry, and scanning electron microscopy (SEM). Extracellular matrix (ECM) elements and cell numbers were quantified biochemically. Mechanical properties were assessed by tensile testing. CD133- derived cells demonstrated characteristics of mesenchymal progenitors expressing CD44 and CD105. Differentiated CD133+ cells showed features of functional endothelial cells by eNOS and CD141 expression. Engineered heart valve leaflets demonstrated endothelialized tissue formation with production of ECM elements (GAG 80%, HYP 5%, cell number 100% of native values). SEM showed intact endothelial surfaces. Opening and closing behavior was sufficient under half of systemic conditions.
The use of amniotic fluid as single cell source is a promising low-risk approach enabling the prenatal fabrication of heart valves ready to use at birth. These living replacements with the potential of growth, remodeling, and regeneration may realize the early repair of congenital malformations.
Circulation 10/2007; 116(11 Suppl):I64-70. · 14.74 Impact Factor
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Isabella Sudano,
Andreas J Flammer,
Frank Hermann,
Thomas Syburra,
Priska Kaiser,
Astrid Hirt,
Matthias Hermann,
Roberto Corti,
Frank Ruschitzka, Gregor Zund,
Georg Noll
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ABSTRACT: Sleep apnoea syndrome is frequent in patients with heart failure and associated with a worse prognosis. We evaluated a new device (Auricall) for non-invasive, continuous recording of oxygen saturation (SpO(2)) and heart rate (HR) in patients with heart failure. We studied 20 patients (mean age 48.43+/-14.4 years, NYHA class II-III). All patients were requested to carry the device for at least 36 h and to write a diary during the recording time. Satisfactory recording of SpO(2) and HR was possible to obtain in 18 of 20 patients. Indeed 9 out of 18 patients showed significant periodic changes in SpO(2) during sleep. Therefore, Auricall is a useful tool to non-invasively monitor SpO(2) and HR in patients with heart failure and to detect breathing disorders in these patients.
International journal of cardiology 09/2007; 129(1):141-3. · 7.08 Impact Factor
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ABSTRACT: This study demonstrates the engineering of biologically active heart valve leaflets using prenatally available human umbilical cord-derived progenitor cells as the only cell source. Wharton's Jelly-derived cells and umbilical cord blood-derived endothelial progenitor cells were subsequently seeded on biodegradable scaffolds and cultured in a biomimetic system under biochemical or mechanical stimulation or both. Depending on the stimulation, leaflets showed mature layered tissue formation with functional endothelia and extracellular matrix production comparable with that of native tissues. This demonstrates the feasibility of heart valve leaflet fabrication from prenatal umbilical cord-derived progenitor cells as a further step in overcoming the lack of living autologous replacements with growth and regeneration potential for the repair of congenital malformation.
Tissue Engineering 12/2006; 12(11):3223-32. · 4.02 Impact Factor
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ABSTRACT: Tissue-engineered living blood vessels (TEBV) with growth capacity represent a promising new option for the repair of congenital malformations. We investigate the functionality of TEBV with endothelia generated from human umbilical cord blood-derived endothelial progenitor cells.
Tissue-engineered living blood vessels were generated from human umbilical cord-derived myofibroblasts seeded on biodegradable vascular scaffolds, followed by endothelialization with differentiated cord blood-derived endothelial progenitor cells. During in vitro maturation the TEBV were exposed to physiologic conditioning in a flow bioreactor. For functional assessment, a subgroup of TEBV was stimulated with tumor necrosis factor-alpha. Control vessels endothelialized with standard vascular endothelial cells were treated in parallel. Analysis of the TEBV included histology, immunohistochemistry, biochemistry (extracellular matrix analysis, DNA), and biomechanical testing. Endothelia were analyzed by flow cytometry and immunohistochemistry (CD31, von Willebrand factor, thrombomodulin, tissue factor, endothelial nitric oxide synthase).
Histologically, a three-layered tissue organization of the TEBV analogous to native vessels was observed, and biochemistry revealed the major matrix constituents (collagen, proteoglycans) of blood vessels. Biomechanical properties (Young's modulus, 2.03 +/- 0.65 MPa) showed profiles resembling those of native tissue. Endothelial progenitor cells expressed typical endothelial cell markers CD31, von Willebrand factor, and endothelial nitric oxide synthase comparable to standard vascular endothelial cells. Stimulation with tumor necrosis factor-alpha resulted in physiologic upregulation of tissue factor and downregulation of thrombomodulin expression.
These results indicate that TEBV with tissue architecture and functional endothelia similar to native blood vessels can be successfully generated from human umbilical cord progenitor cells. Thus, blood-derived progenitor cells obtained before or at birth may enable the clinical realization of tissue engineering constructs for pediatric applications.
The Annals of thoracic surgery 11/2006; 82(4):1465-71; discussion 1471. · 3.74 Impact Factor
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Simon P Hoerstrup,
Ian Cummings Mrcs,
Mario Lachat,
Frederick J Schoen,
Rolf Jenni,
Sebastian Leschka,
Stefan Neuenschwander,
Dörthe Schmidt,
Anita Mol,
Christina Günter,
Mathias Gössi,
Michele Genoni, Gregor Zund
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ABSTRACT: Living autologous vascular grafts with the capacity for regeneration and growth may overcome the limitations of contemporary artificial prostheses. Particularly in congenital cardiovascular surgery, there is an unmet medical need for growing replacement materials. Here we investigate growth capacity of tissue-engineered living pulmonary arteries in a growing lamb model.
Vascular grafts fabricated from biodegradable scaffolds (ID 18+/-l mm) were sequentially seeded with vascular cells. The seeded constructs were grown in vitro for 21 days using biomimetic conditions. Thereafter, these tissue-engineered vascular grafts (TEVGs) were surgically implanted as main pulmonary artery replacements in 14 lambs using cardiopulmonary bypass and followed up for < or = 100 weeks. The animals more than doubled their body weight during the 2-year period. The TEVG showed good functional performance demonstrated by regular echocardiography at 20, 50, 80, and 100 weeks and computed tomography-angiography. In particular, there was no evidence of thrombus, calcification, stenosis, suture dehiscence, or aneurysm. There was a significant increase in diameter by 30% and length by 45%. Histology showed tissue formation reminiscent of native artery. Biochemical analysis revealed cellularity and proteoglycans and increased collagen contents in all of the groups, analogous to those of native vessels. The mechanical profiles of the TEVG showed stronger but less elastic tissue properties than native pulmonary arteries.
This study provides evidence of growth in living, functional pulmonary arteries engineered from vascular cells in a full growth animal model.
Circulation 08/2006; 114(1 Suppl):I159-66. · 14.74 Impact Factor
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Dörthe Schmidt,
Anita Mol,
Christian Breymann,
Josef Achermann,
Bernhard Odermatt,
Matthias Gössi,
Stefan Neuenschwander,
René Prêtre,
Michele Genoni, Gregor Zund,
Simon P Hoerstrup
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ABSTRACT: Heart valve tissue engineering is a promising strategy to overcome the lack of autologous growing replacements, particularly for the repair of congenital malformations. Here, we present a novel concept using human prenatal progenitor cells as new and exclusive cell source to generate autologous implants ready for use at birth.
Human fetal mesenchymal progenitors were isolated from routinely sampled prenatal chorionic villus specimens and expanded in vitro. A portion was cryopreserved. After phenotyping and genotyping, cells were seeded onto synthetic biodegradable leaflet scaffolds (n=12) and conditioned in a bioreactor. After 21 days, leaflets were endothelialized with umbilical cord blood-derived endothelial progenitor cells and conditioned for additional 7 days. Resulting tissues were analyzed by histology, immunohistochemistry, biochemistry (amounts of extracellular matrix, DNA), mechanical testing, and scanning electron microscopy (SEM) and were compared with native neonatal heart valve leaflets. Fresh and cryopreserved cells showed comparable myofibroblast-like phenotypes. Genotyping confirmed their fetal origin. Neo-tissues exhibited organization, cell phenotypes, extracellular matrix production, and DNA content comparable to their native counterparts. Leaflet surfaces were covered with functional endothelia. SEM showed cellular distribution throughout the polymer and smooth surfaces. Mechanical profiles approximated those of native heart valves.
Prenatal fetal progenitors obtained from routine chorionic villus sampling were successfully used as an exclusive, new cell source for the engineering of living heart valve leaflets. This concept may enable autologous replacements with growth potential ready for use at birth. Combined with the use of cell banking technology, this approach may be applied also for postnatal applications.
Circulation 08/2006; 114(1 Suppl):I125-31. · 14.74 Impact Factor
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ABSTRACT: Invasive coronary angiography (ICA) is the gold standard for the diagnosis of coronary artery disease and also for imaging procedures for preoperative planning of coronary artery bypass grafting (CABG). Sixteen-multidetector row computed tomography (MDCT) represents an alternative depiction of coronary vessels.
Preoperative exams included ICA and MDCT in 50 patients. Two blinded surgical readers independently investigated both diagnostic modalities regarding location, severity, and morphology of the stenoses. The right coronary artery, left anterior descending branch, and circumflex branch--each divided in 3 sections--and the left main artery with a diameter (3) 1.5 mm were rated in both procedures, and the percentage of complete evaluations by MDCT was assessed.
Heart rate was 72 +/- 8 bpm. Forty-six percent of patients received a complete MDCT evaluation, and 54% received an incomplete MDCT evaluation. In 62% of these incompletely examined patients, 1 branch was not completely analyzable, in 31% 2 branches; and in 7% all 3 branches. In total, 9% of all segments were incompletely assessed. Investigators detected coronary stenoses in complete evaluations with a sensitivity of 94% and a specificity of 95%. Positive predictive value was 87% and negative predictive value was 98%. Plaque classification in soft and hard plaques was possible.
Sixteen-MDCT is not a viable alternative diagnostic tool at present. However, although the percentage of incomplete evaluated patients is more then 50%, only 9% of all segments were incompletely assessable. If this technology can be further improved, especially its software, it will become a valid diagnostic tool for coronary artery disease.
Heart Surgery Forum 02/2006; 9(2):E572-8. · 0.63 Impact Factor
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ABSTRACT: A major shortcoming in contemporary congenital heart surgery is the lack of viable replacement materials with the capacity of growth and regeneration. Here we focused on living autologous patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells (EPCs) as a ready-to-use cell source for paediatric cardiovascular tissue engineering.
EPCs were isolated from 20 ml fresh umbilical cord blood by density gradient centrifugation and myofibroblasts were harvested from umbilical cord tissue. Cells were differentiated and expanded in vitro using nutrient media containing growth factors. Before seeding, cell-phenotypes were assessed by immuno-histochemistry. Biodegradable patches fabricated from synthetic polymers (PGA/P4HB) were seeded with myofibroblasts followed by endothelialization with EPCs. All patches were cultured in a perfusion bioreactor. A subgroup of patches was additionally stimulated by cyclic strain. Analysis of the neo-tissues comprised histology, immuno-histochemistry, extracellular matrix (ECM) analysis and biomechanical testing.
Endothelial phenotypes of EPCs before seeding were confirmed by Ac-Dil-LDL, CD 31, von-Willebrand-Factor and eNOS staining. Histology of the seeded patches demonstrated layered viable tissue formation in all samples. The cells in the newly formed tissues expressed myofibroblast markers, such as desmin and alpha-SMA. The EPCs derived neo-endothelia showed constant endothelial phenotypes (CD 31, vWF). major constituents of ECM such as collagen and proteoglycans were biochemically detected. Stress-strain properties of the patches showed features of native-analogous tissues.
Living tissue engineered patches can be successfully generated from human umbilical cord derived myofibroblasts and EPCs. This new cell source may enable the tissue engineering of versatile, living, autologous replacement materials for congenital cardiac interventions.
European Journal of Cardio-Thoracic Surgery 06/2005; 27(5):795-800. · 2.55 Impact Factor
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ABSTRACT: A substantial limitation regarding present pediatric cardiac surgery is the lack of appropriate materials for the repair of congenital defects. To address this shortcoming, tissue engineering is a scientific field that aims at in vitro fabrication of living autologous grafts with the capacity of growth, repair, and regeneration. Here we focused on tissue engineered vascular grafts using human umbilical cord blood derived endothelial progenitor cells (EPCs), as a noninvasive cell source for pediatric applications.
EPCs were isolated from 20 ml fresh human umbilical cord blood by Ficoll gradient centrifugation and cultured in endothelial basal medium containing growth factors. After proliferation and differentiation cells were analyzed by immunohistochemistry and seeded onto three-dimensional (3D) biodegradable vascular scaffolds (porosity > 95%, n = 22). Twenty-four hours after seeding the vascular grafts were positioned into a pulse-duplicator-in vitro system and grown for 48 hours under biomimetic conditions. A second group was grown 6 days statically and an additional 6 days biomimetically. Controls were cultured statically. Analysis of the grafts included immunohistochemistry, histology, and scanning electron microscopy.
Preseeding differentiated EPCs indicated constant endothelial phenotypes including acetylated low-density lipoprotein, cluster of differentiation 31, von Willebrand factor, and endothelial nitric oxide synthetase. Seeded EPCs established favorable cell-to-polymer attachment and proliferation into the 3D tubular scaffolds. Both conditioned and static cellular constructs demonstrated positive staining for cluster of differentiation 31, von Willebrand factor, and expression of endothelial nitric oxide synthase.
Human umbilical cord derived EPCs indicated exceptional growth characteristics used for tissue engineering of vascular grafts. These cells demonstrated a constant endothelial phenotype and related functional features. Based on these results EPCs seem to be a promising autologous cell source with regard to cardiovascular tissue engineering, particularly for the repair of congenital defects.
The Annals of thoracic surgery 12/2004; 78(6):2094-8. · 3.74 Impact Factor
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ABSTRACT: To assess the feasibility of computationally simulating intracoronary blood flow based on real coronary artery geometry and to graphically depict various mechanical characteristics of this flow.
Explanted fresh pig hearts were fixed using a continuous perfusion of 4% formaldehyde at physiological pressures. Omnipaque dye added to lead rubber solution was titrated to an optimum proportion of 1:25, to cast the coronary arterial tree. The heart was stabilized in a phantom model so as to suspend the base and the apex without causing external deformation. High resolution computerized tomography scans of this model were utilized to reconstruct the three-dimensional coronary artery geometry, which in turn was used to generate several volumetric tetrahedral meshes of sufficient density needed for numerical accuracy. The transient equations of momentum and mass conservation were numerically solved by employing methods of computational fluid dynamics under realistic pulsatile inflow boundary conditions.
The simulations have yielded graphic distributions of intracoronary flow stream lines, static pressure drop, wall shear stress, bifurcation mass flow ratios and velocity profiles. The variability of these quantities within the cardiac cycle has been investigated at a temporal resolution of 1/100th of a second and a spatial resolution of about 10 microm. The areas of amplified variations in wall shear stress, mostly evident in the neighborhoods of arterial branching, seem to correlate well with clinically observed increased atherogenesis. The intracoronary flow lines showed stasis and extreme vorticity during the phase of minimum coronary flow in contrast to streamlined undisturbed flow during the phase of maximum flow.
Computational tools of this kind along with a state-of-the-art multislice computerized tomography or magnetic resonance-based non-invasive coronary imaging, could enable realistic, repetitive, non-invasive and multidimensional quantifications of the effects of stenosis on distal hemodynamics, and thus help in precise surgical/interventional planning. It could also add insights into coronary and bypass graft atherogenesis.
European Journal of Cardio-Thoracic Surgery 09/2004; 26(2):248-56. · 2.55 Impact Factor
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ABSTRACT: Tissue engineering of viable, autologous cardiovascular replacements with the potential to grow, repair and remodel represents an attractive approach to overcome the shortcomings of available replacements for the repair of congenital cardiac defects. Currently, vascular myofibroblast cells represent an established cell source for cardiovascular tissue engineering. Cell isolation requires the invasive harvesting of venous or arterial vessel segments prior to scaffold seeding, a technique which may not be preferable, especially in pediatric patients. This study evaluates cells isolated from human umbilical cord artery, umbilical cord vein and whole cord as alternative autologous cell sources for cardiovascular tissue engineering.
Cells were isolated from human umbilical cord artery (UCA), umbilical cord vein (UCV), whole umbilical cord (UCC) and saphenous vein segments (VC), and were expanded in culture. All three expanded cell groups were seeded on bioabsorbable copolymer strips and grown in vitro for 28 days. Isolated cells were characterized by flow cytometry, histology, immunohistochemistry, proliferation assays and compared to VC. Morphological analysis of the seeded polymer strips included histology, immunohistochemistry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and uniaxial stress testing.
UCA, UCV and UCC demonstrated excellent cell growth properties comparable to VC. Following isolation, all three cell groups showed myofibroblast-like morphology and characteristics by staining positive for alpha-smooth muscle actin (ASMA) and vimentin. Histology and immunohistochemistry of seeded polymers showed good tissue and extracellular matrix formation containing collagen I, III and elastin. TEM showed viable myofibroblasts and the deposition of collagen fibrils and progressive growing tissue formation, with a confluent surface, was observed in SEM. No difference was found among the mechanical properties of UCA, UCV, UCC and VC tissue engineered constructs.
Tissue engineering of cardiovascular constructs by using UCA, UCV and UCC is feasible in an in vitro environment. Cell growth, morphology, characteristics and tissue formation were comparable between UCA, UCV, UCC and VC. UCC represent an attractive, readily available autologous cell source for cardiovascular tissue engineering offering the additional benefits of utilizing juvenile cells and avoiding the invasive harvesting of intact vascular structures.
European Journal of Cardio-Thoracic Surgery 05/2004; 25(4):635-41. · 2.55 Impact Factor
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ABSTRACT: Tissue engineering of viable, autologous cardiovascular constructs with the potential to grow, repair, and remodel represents a promising new concept for cardiac surgery, especially for pediatric patients. Currently, vascular myofibroblast cells (VC) represent an established cell source for cardiovascular tissue engineering. Cell isolation requires the invasive harvesting of venous or arterial vessel segments before scaffold seeding, a technique that may not be preferable, particularly in pediatric patients. In this study, we investigated the feasibility of using umbilical cord cells (UCC) as an alternative autologous cell source for cardiovascular tissue engineering.
Human UCC were isolated from umbilical cord segments and expanded in culture. The cells were sequentially seeded on bioabsorbable copolymer patches (n = 5) and grown in vitro in laminar flow for 14 days. The UCC were characterized by flow cytometry (FACS), histology, immunohistochemistry, and proliferation assays and were compared to saphenous vein-derived VC. Morphologic analysis of the UCC-seeded copolymer patches included histology and both transmission and scanning electron microscopy. Characterization of the extracellular matrix was performed by immunohistochemistry and quantitative extracellular matrix protein assays. The tissue-engineered UCC patches were biomechanically evaluated using uniaxial stress testing and were compared to native tissue.
We found that isolated UCC show a fibroblast-like morphology and superior cell growth compared to VC. Phenotype analysis revealed positive signals for alpha-smooth muscle actin (ASMA), desmin, and vimentin. Histology and immunohistochemistry of seeded polymers showed layered tissue formation containing collagen I, III, and glycoaminoglycans. Transmission electron microscopy showed viable myofibroblasts and the deposition of collagen fibrils. A confluent tissue surface was observed during scanning electron microscopy. Glycoaminoglycan content did not reach values of native tissue, whereas cell content was increased. The biomechanical properties of the tissue-engineered constructs approached native tissue values.
Tissue engineering of cardiovascular constructs using UCC is feasible in an in vitro environment. The UCC demonstrated excellent growth properties and tissue formation with mechanical properties approaching native tissue. It appears that UCC represent a promising alternative autologous cell source for cardiovascular tissue engineering, offering the additional benefits of using juvenile cells and avoiding the invasive harvesting of intact vascular structures.
The Annals of Thoracic Surgery 11/2002; 74(4):S1422-8. · 3.74 Impact Factor
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Simon P Hoerstrup,
Alexander Kadner,
Serguei Melnitchouk,
Andreas Trojan,
Karim Eid,
Jay Tracy,
Ralf Sodian,
Jeroen F Visjager,
Stefan A Kolb,
Jurg Grunenfelder, Gregor Zund,
Marko I Turina
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ABSTRACT: We previously demonstrated the successful tissue engineering and implantation of functioning autologous heart valves based on vascular-derived cells. Human marrow stromal cells (MSC) exhibit the potential to differentiate into multiple cell-lineages and can be easily obtained clinically. The feasibility of creating tissue engineered heart valves (TEHV) from MSC as an alternative cell source, and the impact of a biomimetic in vitro environment on tissue differentiation was investigated.
Human MSC were isolated, expanded in culture, and characterized by flow-cytometry and immunohistochemistry. Trileaflet heart valves fabricated from rapidly bioabsorbable polymers were seeded with MSC and grown in vitro in a pulsatile-flow-bioreactor. Morphological characterization included histology and electron microscopy (EM). Extracellular matrix (ECM)-formation was analyzed by immunohistochemistry, ECM protein content (collagen, glycosaminoglycan) and cell proliferation (DNA) were biochemically quantified. Biomechanical evaluation was performed using Instron(TM). In all valves synchronous opening and closing was observed in the bioreactor. Flow-cytometry of MSC pre-seeding was positive for ASMA, vimentin, negative for CD 31, LDL, CD 14. Histology of the TEHV-leaflets demonstrated viable tissue and ECM formation. EM demonstrated cell elements typical of viable, secretionally active myofibroblasts (actin/myosin filaments, collagen fibrils, elastin) and confluent, homogenous tissue surfaces. Collagen types I, III, ASMA, and vimentin were detected in the TEHV-leaflets. Mechanical properties of the TEHV-leaflets were comparable to native tissue.
Generation of functional TEHV from human MSC was feasible utilizing a biomimetic in vitro environment. The neo-tissue showed morphological features and mechanical properties of human native-heart-valve tissue. The human MSC demonstrated characteristics of myofibroblast differentiation.
Circulation 10/2002; 106(12 Suppl 1):I143-50. · 14.74 Impact Factor
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ABSTRACT: Tissue engineering represents a promising approach to in vitro creation of living, autologous replacements with the potential to grow, repair, and remodel. Particularly in a congenital operation, there is a substantial need for such implantation materials. We previously demonstrated fabrication of completely autologous, functional heart valves on the basis of peripheral vascular cells. Presently the feasibility of creating pulmonary artery conduits from human umbilical cord cells was investigated.
Human umbilical cord cells were harvested and expanded in culture. Pulmonary conduits fabricated from rapidly bioabsorbable polymers were seeded with human umbilical cord cells and grown in vitro in a pulse duplicator bioreactor. Morphologic characterization of the generated neo-tissues included histology, transmission, and scanning electron microscopy. Characterization of extracellular matrix was comprised of immunohistochemistry. Extracellular matrix protein content and cell proliferation were quantified by biochemical assays. Biomechanical testing was performed using stress-strain and burst-stress tests.
Histology of the conduits revealed viable, layered tissue and extracellular matrix formation with glycosaminoglycans and collagens I and III. Cells stained positive for vimentin and alpha-smooth muscle actin. Scanning electron microscopy showed confluent, homogenous tissue surfaces. Transmission electron microscopy demonstrated elements typical of viable myofibroblasts, such as collagen, fibrils, and elastin. Extracellular matrix proteins were significantly lower compared with native tissue; the cell content was increased. The mechanical strength of the pulsed constructs was comparable with native tissue; the static controls were significantly weaker.
In vitro fabrication of tissue-engineered human pulmonary conduits was feasible utilizing human umbilical cord cells and a biomimetic culture environment. Morphologic and mechanical features approximated human pulmonary artery. Human umbilical cord cells demonstrated excellent growth properties representing a new, readily available cell source for tissue engineering without necessitating the sacrifice of intact vascular donor structures.
The Annals of Thoracic Surgery 08/2002; 74(1):46-52; discussion 52. · 3.74 Impact Factor
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ABSTRACT: Vascular-derived cells represent an established cell source for tissue engineering of cardiovascular constructs. Previously, cell isolation was performed by harvesting of vascular structures prior to scaffold seeding. Marrow stromal cells (MSC) demonstrate the ability to differentiate into multiple mesenchymal cell lineages and would offer an alternative cell source for tissue engineering involving a less invasive harvesting technique. We studied the feasibility of using MSC as an alternative cell source for cardiovascular tissue engineering.
Human MSC were isolated from bone marrow and expanded in culture. Subsequently MSC were seeded on bioabsorbable polymers and grown in vitro. Cultivated cells and seeded polymers were studied for cell characterization and tissue formation including extracellular matrix production. Applied methods comprised flow cytometry, histology, immunohistochemistry, transmission (TEM) and scanning electron microscopy (SEM), and biochemical assays.
Isolated MSC demonstrated fibroblast-like morphology. Phenotype analysis revealed positive signals for alpha-smooth muscle actin and vimentin. Histology and SEM of seeded polymers showed layered tissue formation. TEM demonstrated formation of extracellular matrix with deposition of collagen fibrils. Matrix protein analysis showed production of collagen I and III. In comparison to vascular-derived cell constructs quantitative analysis demonstrated comparable amounts of extracellular matrix proteins in the tissue engineered constructs.
Isolated MSC demonstrated myofibroblast-like characteristics. Tissue formation on bioabsorbable scaffolds was feasible with extracellular matrix production comparable to vascular-cell derived tissue engineered constructs. It appears that MSC represent a promising cell source for cardiovascular tissue engineering.
European Journal of Cardio-Thoracic Surgery 07/2002; 21(6):1055-60. · 2.55 Impact Factor
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ABSTRACT: Accessory mitral valve tissue as the single cause for left ventricular outflow tract obstruction is a very rare cardiac malformation in normally connected hearts. We report a case in which this condition was present as single cause for left ventricular outflow tract obstruction. The surgical technique is described and a review of the literature presented.
European Journal of Cardio-Thoracic Surgery 05/1999; · 2.55 Impact Factor
