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Low correlations of cell culture data with clinical outcomes pose major medical challenges with costly consequences. While the majority of biomaterials are tested using in vitro cell monocultures, the importance of synergistic interactions between different cell types on paracrine signalling has recently been highlighted. In this proof-of-concept study, we asked whether the first contact of surfaces with whole human blood could steer the tissue healing response. This hypothesis was tested using alkali-treatment of rough titanium (Ti) surfaces since they have clinically been shown to improve early implant integration and stability, yet blood-free in vitro cell cultures poorly correlated with in vivo tissue healing. We show that alkali-treatment, compared to native Ti surfaces, increased blood clot thickness, including platelet adhesion. Strikingly, blood clots with entrapped blood cells in synergistic interactions with fibroblasts, but not fibroblasts alone, upregulated the secretion of major factors associated with fast healing. This includes matrix metalloproteinases (MMPs) to break down extracellular matrix and the growth factor VEGF, known for its angiogenic potential. Consequently, in vitro test platforms, which consider whole blood-implant interactions, might be superior in predicting wound healing in response to biomaterial properties.

More people die annually from cardiovascular diseases than from any other cause. In particular, patients who suffer from myocardial infarction may be affected by ongoing adverse remodeling processes of the heart that may ultimately lead to heart failure. The introduction of stem and progenitor cell-based applications has raised substantial hope for reversing these processes and inducing cardiac regeneration. However, current stem cell therapies using single-cell suspensions have failed to demonstrate long-lasting efficacy due to the overall low retention rate after cell delivery to the myocardium. To overcome this obstacle, the concept of 3D cell culture techniques has been proposed to enhance therapeutic efficacy and cell engraftment based on the simulation of an in vivo-like microenvironment. Of great interest is the use of so-called microtissues or spheroids, which have evolved from their traditional role as in vitro models to their novel role as therapeutic agents. This review will provide an overview of the therapeutic potential of microtissues by addressing primarily cardiovascular regeneration. It will accentuate their advantages compared to other regenerative approaches and summarize the methods for generating clinically applicable microtissues. In addition, this review will illustrate the unique properties of the microenvironment within microtissues that makes them a promising next-generation therapeutic approach.

Background: Heterotopic heart transplantation (HHT) in rodent animal models represents an important technique enabling studies on organ transplantation immunology and pharmaceutical development. Recent investigations used nonworking HHT designs, with the left ventricle (LV) bypassed in the anastomosis system. In spite of their principal success, the lack of orthogonal ventricular filling leads to myocardial atrophy. However, when focusing on the cellular and molecular mechanisms involved in the in vivo remodeling of the myocardium or cell-based cardiovascular implants, a nonworking model is suboptimal as it lacks the native-analogous hemodynamic and metabolic situation. Here we present the hemodynamic and electrical assessment of a biventricularly loaded murine HHT method without the need for a combined heart-lung transplantation approach., Methods: Heterotopic transplantations (n = 13) were performed on C57BL/6J-(H-2b) inbred mice (n = 13 donors, n = 13 recipients) by creating end-to-side anastomoses between the donors' cranial vena cava (CrVC) and the recipients' abdominal caudal vena cava (CVC), between the donors' ascending aorta and the recipients' abdominal aorta (aAo), and between the grafts' pulmonary trunk and the left atrium. After transplantation, a hemodynamic assessment using echocardiography (including 2D speckle tracking analysis) and electrocardiography was performed., Results: The loaded HHT procedure in the mice was performed with an overall success rate of 61%. In 3 of the remaining 5 cases, only atrial function was restored. The median duration of the entire surgical procedure for the recipient animal was 190 (IQR 180-250) min. The mean heart rate in the loaded HHT group was 355 ± 6 bpm in comparison to the control group with an in situ heart rate of 418 ± 61 bpm. A native-like closing and opening pattern of the aortic and mitral valves (visible on both 2D and M-mode images) was observed, confirming a native-analogous loading of the LV. Pulsed-wave Doppler provided visualization of the flow across the region of anastomoses between the pulmonary trunk and the left atrium, reaching a mean maximum velocity of 382 ± 12 mm/s. Exemplary 2D speckle tracking analysis of the LV free wall and interventricular septum revealed some differences in vector directions in one animal when compared to the orthotopic native heart, indicating an asynchronous movement of the LV., Conclusions: These results demonstrate the technical (micro)surgical feasibility of a fully loaded HHT procedure in the murine model without using a combined heart-lung transplantation approach. The acute hemodynamic performance of the HHT grafts approximated the native orthotopic situation. This model may open up new options for the investigation of cellular and molecular questions in the murine cardiovascular in vivo system in the near future., (© 2016 S. Karger AG, Basel.)

The use of valved stents for minimally invasive replacement of semilunar heart valves is expected to change the extracellular matrix and mechanical function of the native artery and may thus impair long-term functionality of the implant. Here we investigate the impact of the stent on matrix remodeling of the pulmonary artery in a sheep model, focusing on matrix composition and collagen (re)orientation of the host tissue. Ovine native pulmonary arteries were harvested 8 (n = 2), 16 (n = 4) and 24 (n = 2) weeks after transapical implantation of self-expandable stented heart valves. Second harmonic generation (SHG) microscopy was used to assess the collagen (re)orientation of fresh tissue samples. The collagen and elastin content was quantified using biochemical assays. SHG microscopy revealed regional differences in collagen organization in all explants. In the adventitial layer of the arterial wall far distal to the stent (considered as the control tissue), we observed wavy collagen fibers oriented in the circumferential direction. These circumferential fibers were more straightened in the adventitial layer located behind the stent. On the luminal side of the wall behind the stent, collagen fibers were aligned along the stent struts and randomly oriented between the struts. Immediately distal to the stent, however, fibers on both the luminal and the adventitial side of the wall were oriented in the axial direction, demonstrating the stent impact on the collagen structure of surrounding arterial tissues. Collagen orientation patterns did not change with implantation time, and biochemical analyses showed no changes in the trend of collagen and elastin content with implantation time or location of the vascular wall. We hypothesize that the collagen fibers on the adventitial side of the arterial wall and behind the stent straighten in response to the arterial stretch caused by oversizing of the stent. However, the collagen organization on the luminal side suggests that stent-induced remodeling is dominated by contact guidance., (© 2016 S. Karger AG, Basel.)

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

Amniotic fluid cells (AFCs) have been proposed as a valuable source for tissue engineering and regenerative medicine. However, before clinical implementation, rigorous evaluation of this cell source in clinically relevant animal models accepted by regulatory authorities is indispensable. Today, the ovine model represents one of the most accepted preclinical animal models, in particular for cardiovascular applications. Here, we investigate the isolation and use of autologous ovine AFCs as cell source for cardiovascular tissue engineering applications. Fetal fluids were aspirated in vivo from pregnant ewes (n = 9) and from explanted uteri post mortem at different gestational ages (n = 91). Amniotic non-allantoic fluid nature was evaluated biochemically and in vivo samples were compared with post mortem reference samples. Isolated cells revealed an immunohistochemical phenotype similar to ovine bone marrow-derived mesenchymal stem cells (MSCs) and showed expression of stem cell factors described for embryonic stem cells, such as NANOG and STAT-3. Isolated ovine amniotic fluid-derived MSCs were screened for numeric chromosomal aberrations and successfully differentiated into several mesodermal phenotypes. Myofibroblastic ovine AFC lineages were then successfully used for the in vitro fabrication of small- and large-diameter tissue-engineered vascular grafts (n = 10) and cardiovascular patches (n = 34), laying the foundation for the use of this relevant pre-clinical in vivo assessment model for future amniotic fluid cell-based therapeutic applications., (Copyright © 2013 John Wiley & Sons, Ltd.)

Aims: To evaluate the short-, mid- and long-term safety, efficacy and vascular physiology of Axetis silicon dioxide (SiO2, abrading the micropores) inert-coated stent implantation in a randomised preclinical setting., Methods and Results: Coronary arteries of domestic pigs were randomised to receive either Axetis or BMS (same design) stents with one-, three- and six-month follow-up (FUP), controlled by coronary angiography, optical coherence tomography (OCT), intravascular ultrasound (IVUS) and histology (n=32). The time-dependent vasomotor reaction of coronary arteries to stenting was measured using modified myography (n=12). Complete endothelialisation of the Axetis stent was confirmed by OCT, IVUS and histology at one-month FUP. Histopathology revealed continuous healing of the vessel wall with a gradual reduction of inflammation and fibrin score during the six-month FUP in both stent types. Significantly smaller neointimal area and %area stenosis were measured in Axetis stents compared with BMS at each FUP time point. Vascular reactivity measurements showed significantly better endothelium-dependent vasodilation of stented arteries with Axetis implantation., Conclusions: Implantation of the Axetis SiO2-coated stent resulted in a significantly better safety, efficacy and vessel physiology profile compared with BMS of the same design with a continuous decrease in vessel inflammation during the six-month FUP.

Background: Decellularized tissue-engineered heart valves (TEHVs) are under investigation as alternative for current heart valve prostheses with the potential to rapidly repopulate with cells within the body. Ideally, these valves are stented for transapical or minimally invasive delivery. It is unclear if and how the matrix of these valves remodels under in vivo hemodynamic loading conditions and in the presence of a stent. Here, we study the evolution of collagen orientation and tissue maturation in the wall of stented decellularized TEHVs with time after implantation., Methods and Results: In a previous study, stented TEHVs based on rapidly degrading scaffolds were cultured in bioreactors, decellularized, and transapically implanted as pulmonary valve replacement in sheep. In the present study, collagen (re)orientation in the initially isotropic valvular wall was assessed using a fluorescent collagen probe combined with confocal imaging and image analysis of explanted tissue at 8, 16, and 24 weeks following implantation. Collagen tortuosity or waviness in the explants, as a measure of matrix maturity, was quantified using a Gabor wavelet method and compared with tortuosity in native sheep vascular wall tissue. Results indicate that on the luminal side of the valvular wall, fibers became aligned in circumferential direction, while tortuosity increased with implantation time, showing striking similarities with the native collagen structure after 24 weeks. On the outside of the wall, where the engineered tissue touches the stent, collagen fibers in the vicinity of the struts aligned along the struts, whereas collagen fibers in between struts were randomly oriented. Immunohistochemistry was performed to evaluate the presence of elastin and collagen type I and III. After 8 weeks, collagen types I and III were mostly present at the luminal side of the wall, whereas at 16 and 24 weeks, a homogenous distribution of collagen I and III was observed throughout the wall. Elastin was mostly expressed at the luminal side after 24 weeks. Biochemical assays showed that the amount of DNA (as a measure of cell number) increased significantly after 8 and 24 weeks, glycosaminoglycans increased significantly after 8, 16, and 24 weeks, and hydroxyproline, as a measure of collagen amount, increased significantly after 24 weeks compared to the controls., Conclusions: The collagen matrix in the wall of decellularized TEHVs shows clear structural remodeling and maturation with time. While collagen orientation rapidly remodels toward a native anisotropic architecture on the luminal side of the engineered valvular wall, it is dominated and guided by stent geometry on the outer side of the wall. Collagen tortuosity was increased with implantation time and was accompanied by an increase in elastin, especially on the luminal side of the vessel.

The vascular and the nervous system are responsible for oxygen, nutrient, and information transfer and thereby constitute highly important communication systems in higher organisms. These functional similarities are reflected at the anatomical, cellular, and molecular levels, where common developmental principles and mutual crosstalks have evolved to coordinate their action. This resemblance of the two systems at different levels of complexity has been termed the "neurovascular link." Most of the evidence demonstrating neurovascular interactions derives from studies outside the CNS and from the CNS tissue of the retina. However, little is known about the specific properties of the neurovascular link in the brain. Here, we focus on regulatory effects of molecules involved in the neurovascular link on angiogenesis in the periphery and in the brain and distinguish between general and CNS-specific cues for angiogenesis. Moreover, we discuss the emerging molecular interactions of these angiogenic cues with the VEGF-VEGFR-Delta-like ligand 4 (Dll4)-Jagged-Notch pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aims: Regulatory T cells (Treg) exert anti-inflammatory and atheroprotective effects in experimental atherosclerosis. Treg can be induced against specific antigens using immunization strategies associated with clonal restriction. No data exist on Treg in combination with clonal restriction of T cells in patients with acute coronary syndromes (ACS)., Methods and Results: Among T cell subsets characterized by flow cytometry, Treg (CD4(+) CD25(+) CD127(low)) were twice as frequent in coronary thrombi compared with peripheral blood. Treg prevailed among T cell subsets identified in coronary thrombi. To evaluate clonal restriction, genomic DNA was extracted from coronary thrombi and peripheral blood in order to evaluate T cell receptor (TCR) β chain diversity by means of Multi-N-plex PCR using a primer specific for all TCR β V gene segments and another primer specific for TCR β J gene segments. T cell receptor diversity was reduced in thrombi compared with peripheral blood (intra-individual comparisons in 16 patients) with 8 gene rearrangements in the TCR common in at least 6 out of 16 analysed coronary thrombi. Compared with age-matched healthy controls (n = 16), TCR diversity was also reduced in peripheral blood of patients with ACS; these findings were independent of peripheral T cell numbers., Conclusion: We provide novel evidence for a perturbed T cell compartment characterized by clonal restriction in peripheral blood and coronary thrombi from patients with ACS. Our findings warrant further studies on Treg as novel therapeutic targets aimed at enhancing this anti-inflammatory component of adaptive immunity in human atherothrombosis., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Cardiology.)

During development and in various diseases of the CNS, new blood vessel formation starts with endothelial tip cell selection and vascular sprout migration, followed by the establishment of functional, perfused blood vessels. Here we describe a method that allows the assessment of these distinct angiogenic steps together with antibody-based protein detection in the postnatal mouse brain. Intravascular and perivascular markers such as Evans blue (EB), isolectin B4 (IB4) or laminin (LN) are used alongside simultaneous immunofluorescence on the same sections. By using confocal laser-scanning microscopy and stereological methods for analysis, detailed quantification of the 3D postnatal brain vasculature for perfused and nonperfused vessels (e.g., vascular volume fraction, vessel length and number, number of branch points and perfusion status of the newly formed vessels) and characterization of sprouting activity (e.g., endothelial tip cell density, filopodia number) can be obtained. The entire protocol, from mouse perfusion to vessel analysis, takes ∼10 d.

Objectives: We compared the accuracy of NOGA endocardial mapping for delineating transmural and non-transmural infarction to the results of cardiac magnetic resonance imaging (cMRI) with late gadolinium enhancement (LE) for guiding intramyocardial reparative substance delivery using data from experimental myocardial infarction studies., Methods: Sixty domestic pigs underwent diagnostic NOGA endocardial mapping and cMRI-LE 60 days after induction of closed-chest reperfused myocardial infarction. The infarct size was determined by LE of cMRI and by delineation of the infarct core on the unipolar voltage polar map. The sizes of the transmural and non-transmural infarctions were calculated from the cMRI transmurality map using signal intensity (SI) cut-offs of>75% and>25% and from NOGA bipolar maps using bipolar voltage cut-off values of <0.8 mV and <1.9 mV. Linear regression analysis and Bland-Altman plots were used to determine correlations and systematic differences between the two images. The overlapping ratios of the transmural and non-transmural infarcted areas were calculated., Results: Infarct size as determined by 2D NOGA unipolar voltage polar mapping correlated with the 3D cMRI-LE findings (r = 0.504, p<0.001) with a mean difference of 2.82% in the left ventricular (LV) surface between the two images. Polar maps of transmural cMRI and bipolar maps of NOGA showed significant association for determining of the extent of transmural infarction (r = 0.727, p<0.001, overlap ratio of 81.6±11.1%) and non-transmural infarction (r = 0.555, p<0.001, overlap ratio of 70.6±18.5%). NOGA overestimated the transmural scar size (6.81% of the LV surface) but slightly underestimated the size of the non-transmural infarction (-3.04% of the LV surface)., Conclusions: By combining unipolar and bipolar voltage maps, NOGA endocardial mapping is useful for accurate delineation of the targeted zone for intramyocardial therapy and is comparable to cMRI-LE. This may be useful in patients with contraindications for cMRI who require targeted intramyocardial regenerative therapy.

Background: Chronic venous insufficiency (CVI) represents a major global health problem with increasing prevalence and morbidity. CVI is due to an incompetence of the venous valves, which causes venous reflux and distal venous hypertension. Several studies have focused on the replacement of diseased venous valves using xeno- and allogenic transplants, so far with moderate success due to immunologic and thromboembolic complications. Autologous cell-derived tissue-engineered venous valves (TEVVs) based on fully biodegradable scaffolds could overcome these limitations by providing non-immunogenic, non-thrombogenic constructs with remodeling and growth potential., Methods: Tri- and bicuspid venous valves (n=27) based on polyglycolic acid-poly-4-hydroxybutyrate composite scaffolds, integrated into self-expandable nitinol stents, were engineered from autologous ovine bone-marrow-derived mesenchymal stem cells (BM-MSCs) and endothelialized. After in vitro conditioning in a (flow) pulse duplicator system, the TEVVs were crimped (n=18) and experimentally delivered (n=7). The effects of crimping on the tissue-engineered constructs were investigated using histology, immunohistochemistry, scanning electron microscopy, grating interferometry (GI), and planar fluorescence reflectance imaging., Results: The generated TEVVs showed layered tissue formation with increasing collagen and glycosaminoglycan levels dependent on the duration of in vitro conditioning. After crimping no effects were found on the MSC level in scanning electron microscopy analysis, GI, histology, and extracellular matrix analysis. However, substantial endothelial cell loss was detected after the crimping procedure, which could be reduced by increasing the static conditioning phase., Conclusions: Autologous living small-diameter TEVVs can be successfully fabricated from ovine BM-MSCs using a (flow) pulse duplicator conditioning approach. These constructs hold the potential to overcome the limitations of currently used non-autologous replacement materials and may open new therapeutic concepts for the treatment of CVI in the future.

Objectives: This study sought to evaluate long-term in vivo functionality, host cell repopulation, and remodeling of "off-the-shelf" tissue engineered transcatheter homologous heart valves., Background: Transcatheter valve implantation has emerged as a valid alternative to conventional surgery, in particular for elderly high-risk patients. However, currently used bioprosthetic transcatheter valves are prone to progressive dysfunctional degeneration, limiting their use in younger patients. To overcome these limitations, the concept of tissue engineered heart valves with self-repair capacity has been introduced as next-generation technology., Methods: In vivo functionality, host cell repopulation, and matrix remodeling of homologous transcatheter tissue-engineered heart valves (TEHVs) was evaluated up to 24 weeks as pulmonary valve replacements (transapical access) in sheep (n = 12). As a control, tissue composition and structure were analyzed in identical not implanted TEHVs (n = 5)., Results: Transcatheter implantation was successful in all animals. Valve functionality was excellent displaying sufficient leaflet motion and coaptation with only minor paravalvular leakage in some animals. Mild central regurgitation was detected after 8 weeks, increasing to moderate after 24 weeks, correlating to a compromised leaflet coaptation. Mean and peak transvalvular pressure gradients were 4.4 ± 1.6 mm Hg and 9.7 ± 3.0 mm Hg, respectively. Significant matrix remodeling was observed in the entire valve and corresponded with the rate of host cell repopulation., Conclusions: For the first time, the feasibility and long-term functionality of transcatheter-based homologous off-the-shelf tissue engineered heart valves are demonstrated in a relevant pre-clinical model. Such engineered heart valves may represent an interesting alternative to current prostheses because of their rapid cellular repopulation, tissue remodeling, and therewith self-repair capacity. The concept of homologous off-the-shelf tissue engineered heart valves may therefore substantially simplify previous tissue engineering concepts toward clinical translation., (Copyright © 2014 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Ischemic Heart Disease (IHD) still represents the "Number One Killer" worldwide accounting for the death of numerous patients. However the capacity for self-regeneration of the adult heart is very limited and the loss of cardiomyocytes in the infarcted heart leads to continuous adverse cardiac-remodeling which often leads to heart-failure (HF). The concept of regenerative medicine comprising cell-based therapies, bio-engineering technologies and hybrid solutions has been proposed as a promising next-generation approach to address IHD and HF. Numerous strategies are under investigation evaluating the potential of regenerative medicine on the failing myocardium including classical cell-therapy concepts, three-dimensional culture techniques and tissue-engineering approaches. While most of these regenerative strategies have shown great potential in experimental studies, the translation into a clinical setting has either been limited or too rapid leaving many key questions unanswered. This review summarizes the current state-of-the-art, important challenges and future research directions as to regenerative approaches addressing IHD and resulting HF., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Following the coordinated efforts of five established scientific organizations, this report describes the "novel cellular therapy" activity (i.e., cellular treatments excluding hematopoietic stem cells [HSC] for the reconstitution of hematopoiesis) in Europe for the year 2011. Two hundred forty-six teams from 35 countries responded to the cellular therapy survey, 126 teams from 24 countries provided data on 1759 patients using a dedicated survey and 120 teams reported no activity. Indications were musculoskeletal/rheumatological disorders (46%; 99% autologous), cardiovascular disorders (22%; 100% autologous), hematology/oncology, predominantly including the prevention or treatment of graft-versus-host disease (18%; 2% autologous), neurological disorders (2%; 83% autologous), gastrointestinal (1%; 68% autologous), and other indications (12%; 77% autologous). Autologous cells were used predominantly for musculoskeletal/rheumatological (58%) and cardiovascular (27%) disorders, whereas allogeneic cells were used mainly for hematology/oncology (84%). The reported cell types were mesenchymal stem/stromal cells (56%), HSC (23%), chondrocytes (12%), dermal fibroblasts (3%), keratinocytes (2%), and others (4%). In 40% of the grafts, cells were delivered following ex vivo expansion, whereas cells were transduced or sorted, respectively, in 3% and 10% of the reported cases. Cells were delivered intraorgan (42%), intravenously (26%), on a membrane or gel (16%), or using 3D scaffolds (16%). Compared to last year, the number of teams participating in the dedicated survey doubled and, for the first time, all European Group for Blood and Marrow Transplantation teams reporting information on cellular therapies completed the extended questionnaire. The data are compared with those collected since 2008 to identify trends in the field. This year's edition specifically focuses on cardiac cell therapy.

Objectives: While transcatheter aortic valve implantation (TAVI) has rapidly evolved for the treatment of aortic valve disease, the currently used bioprostheses are prone to continuous calcific degeneration. Thus, autologous, cell-based, living, tissue-engineered heart valves (TEHVs) with regeneration potential have been suggested to overcome these limitations. We investigate the technical feasibility of combining the concept of TEHV with transapical implantation technology using a state-of-the-art transcatheter delivery system facilitating the exact anatomical position in the systemic circulation., Methods: Trileaflet TEHVs fabricated from biodegradable synthetic scaffolds were sewn onto self-expanding Nitinol stents seeded with autologous marrow stromal cells, crimped and transapically delivered into the orthotopic aortic valve position of adult sheep (n = 4) using the JenaValve transapical TAVI System (JenaValve, Munich, Germany). Delivery, positioning and functionality were assessed by angiography and echocardiography before the TEHV underwent post-mortem gross examination. For three-dimensional reconstruction of the stent position of the anatomically oriented system, a computed tomography analysis was performed post-mortem., Results: Anatomically oriented, transapical delivery of marrow stromal cell-based TEHV into the orthotopic aortic valve position was successful in all animals (n = 4), with a duration from cell harvest to TEHV implantation of 101 ± 6 min. Fluoroscopy and echocardiography displayed sufficient positioning, thereby entirely excluding the native leaflets. There were no signs of coronary obstruction. All TEHV tolerated the loading pressure of the systemic circulation and no acute ruptures occurred. Animals displayed intact and mobile leaflets with an adequate functionality. The mean transvalvular gradient was 7.8 ± 0.9 mmHg, and the mean effective orifice area was 1.73 ± 0.02 cm(2). Paravalvular leakage was present in two animals, and central aortic regurgitation due to a single-leaflet prolapse was detected in two, which was primarily related to the leaflet design. No stent dislocation, migration or affection of the mitral valve was observed., Conclusions: For the first time, we demonstrate the technical feasibility of a transapical TEHV delivery into the aortic valve position using a commercially available and clinically applied transapical implantation system that allows for exact anatomical positioning. Our data indicate that the combination of TEHV and a state-of-the-art transapical delivery system is feasible, representing an important step towards translational, transcatheter-based TEHV concepts.

Valvular heart disease represents a leading cause of mortality worldwide. Transcatheter heart valve replacement techniques have been recently introduced into the clinical routine expanding the treatment options for affected patients. However, despite this technical progress toward minimally invasive, transcatheter strategies, the available heart valve prostheses for these techniques are bioprosthetic and associated with progressive degeneration. To overcome such limitations, the concept of heart valve tissue engineering has been repeatedly suggested for future therapy concepts. Ideally, a clinically relevant heart valve tissue engineering concept would combine minimally invasive strategies for both, living autologous valve generation as well as valve implantation. Therefore, merging transcatheter techniques with living tissue engineered heart valves into a trascatheter tissue engineered heart valve concept could significantly improve current treatment options for patients suffering from valvular heart disease. This report provides an overview on transcatheter tissue engineered heart valves and summarizes available pre-clinical data.

The pathogenesis of atherosclerosis involves dysfunctions of vascular endothelial cells and smooth muscle cells as well as blood borne inflammatory cells such as monocyte-derived macrophages. In vitro experiments towards a better understanding of these dysfunctions are typically performed in two-dimensional cell culture systems. However, these models lack both the three-dimensional structure and the physiological pulsatile flow conditions of native arteries. We here describe the development and initial characterization of a tissue engineered artery equivalent, which is composed of human primary endothelial and smooth muscle cells and is exposed to flow in vitro. Histological analyses showed formation of a dense tissue composed of a tight monolayer of endothelial cells supported by a basement membrane and multiple smooth muscle cell layers. Both low (LDL) and high density lipoproteins (HDL) perfused through the artery equivalent were recovered both within endothelial cells and in the sub-endothelial intima. After activation of the endothelium with either tumour necrosis factor alpha (TNFα) or LDL, monocytes circulated through the model were found to adhere to the activated endothelium and to transmigrate into the intima. In conclusion, the described tissue engineered human artery equivalent model represents a significant step towards a relevant in vitro platform for the systematic assessment of pathogenic processes in atherosclerosis independently of any systemic factors.

Heart valve tissue engineering based on decellularized xenogenic or allogenic starter matrices has shown promising first clinical results. However, the availability of healthy homologous donor valves is limited and xenogenic materials are associated with infectious and immunologic risks. To address such limitations, biodegradable synthetic materials have been successfully used for the creation of living autologous tissue-engineered heart valves (TEHVs) in vitro. Since these classical tissue engineering technologies necessitate substantial infrastructure and logistics, we recently introduced decellularized TEHVs (dTEHVs), based on biodegradable synthetic materials and vascular-derived cells, and successfully created a potential off-the-shelf starter matrix for guided tissue regeneration. Here, we investigate the host repopulation capacity of such dTEHVs in a non-human primate model with up to 8 weeks follow-up. After minimally invasive delivery into the orthotopic pulmonary position, dTEHVs revealed mobile and thin leaflets after 8 weeks of follow-up. Furthermore, mild-moderate valvular insufficiency and relative leaflet shortening were detected. However, in comparison to the decellularized human native heart valve control - representing currently used homografts - dTEHVs showed remarkable rapid cellular repopulation. Given this substantial in situ remodeling capacity, these results suggest that human cell-derived bioengineered decellularized materials represent a promising and clinically relevant starter matrix for heart valve tissue engineering. These biomaterials may ultimately overcome the limitations of currently used valve replacements by providing homologous, non-immunogenic, off-the-shelf replacement constructs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Surgical revascularization is the most appropriate therapy for patients with significant left main coronary-artery disease (LMD). An incidence of perioperative stroke remains an issue when compared to the early outcomes to percutaneous coronary intervention (PCI). This study evaluates the safety and impact of standardized "clampless" OPCAB techniques, composed of either complete in situ grafting or "clampless" device enabled techniques for stroke reduction in patients undergoing surgical revascularization for LMD., Methods: Between 1999 and 2009, 1031 patients with LMD underwent myocardial-revascularization at our institution. Of these, 507 patients underwent "clampless" OPCAB and 524 patients underwent conventional on-pump CABG (ONCABG). Data-collection was performed prospectively and a propensity-adjusted regression-analysis was applied to balance patient characteristics. LMD was defined as a stenosis >50% and endpoints were mortality, stroke, a cardiac-composite (including death, stroke and myocardial-infarction); a non-cardiac composite and complete-revascularization., Results: In OPCAB patients, the cardiac composite (3.0% vs. 7.8%; propensity-adjusted (PA)OR=0.27; CI95% 0.12-0.65; p=0.003) as well as the occurrence of stroke (0.4% vs. 2.9%; PAOR=0.04; CI95% 0.003-0.48; p=0.012) were significantly lower while the mortality-rate was well comparable between groups (1.8% vs. 2.5%; PAOR=0.44; CI95% 0.11-1.71; p=0.24). The non-cardiac composite was also significantly decreased after OPCAB (8.9% vs. 19.7%; PAOR=0.55; CI95% 0.34-0.89; p=0.014) and complete revascularization was achieved for similar proportions in both groups (95.1% vs. 93.7%; p=0.35)., Conclusions: This study shows the superiority of OPCAB for patients with LMD with regards to risk-adjusted outcomes other than mortality. A "clampless OPCAB strategy", effectively reduces stroke yielding similar early outcomes as PCI., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Cardiac stem cell therapy has been proposed as a therapy option to treat the diseased myocardium. However, the low retention rate of transplanted single-cell suspensions remains a major issue of current therapy strategies. Therefore, the concept of scaffold-free cellular self-assembly into three-dimensional microtissues (3D-MTs) prior to transplantation may be beneficial to enhance retention and survival. We compared clinically relevant, human stem cell sources for their ability to generate 3D-MTs with particular regards to formation characteristics, proliferation-activity, viability and extracellular-matrix production. Single-cell suspensions of human bone marrow- and adipose tissue-derived mesenchymal stem cells (hBMMSCs and hATMSCs), Isl1(+) cardiac progenitors derived from human embryonic stem cells (hESC-Isl1(+) cells), and undifferentiated human induced pluripotent cells (hiPSCs) were characterized before to generate 3D-MTs using a hanging-drop culture. Besides the principal feasibility of cell-specific 3D-MT formation, a detailed head-to-head comparison between cell sources was performed using histology, immunocyto- and histo-chemistry as well as flow cytometry. Round-oval shaped and uniform 3D-MTs could be successfully generated from all cell types starting with a loose formation within the first 24 h that fully stabilized after 3 days and resulting in a mean 3D-MT diameter of 194.56 ± 18.01 μm (hBMMSCs), 194.56 ± 16.30 μm (hATMSCs), 159.73 ± 19.20 μm (hESC-Isl1(+) cells) and 120.95 ± 7.97 μm (hiPSCs). While all 3D-MTs showed a homogenous cell distribution, hiPSC-derived 3D-MTs displayed a compact cell formation primarily located at the outer margin. hESC-Isl1(+) and hiPSC-derived 3D-MTs maintained their proliferation-activity which was rather limited in the MSC-based 3D-MTs. All four 3D-MT types revealed a comparable viability in excess of 70% and showed a cell-specific expression profile being comparable to their single-cell counterparts. Extracellular matrix (ECM) production during 3D-MT formation was observed for all cell-specific 3D-MTs, with hiPSC-derived 3D-MTs being the fastest one. Interestingly, ECM distribution was homogenous for hATMSC- and hiPSC-based 3D-MTs, while it appeared to be primarily concentrated within in the center of hESC-Isl1(+) and hBMMSC-based 3D-MTs. The results of this head-to-head comparative study indicated that 3D-MTs can be successfully generated from hESC-derived Isl1(+) cells, hiPSCs and MSC lines upon hanging drop culture. Cell-specific 3D-MTs displayed sufficient viability and instant ECM formation. The concept of 3D-MT in vitro generation prior to cell transplantation may represent a promising delivery format for future strategies to enhance cellular engraftment and survival., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Transcatheter heart valve replacement is an attractive and promising technique for congenital as well as acquired heart valve disease. In this procedure, the replacement valve is mounted in a stent that is expanded at the aimed valve position and fixated by clamping. However, for this technique to be appropriate for pediatric patients, the material properties of the host tissue need to be determined to design stents that can be optimized for this particular application. In this study we performed equibiaxial tensile tests on four adult ovine pulmonary artery walls and compared the outcomes with one pediatric pulmonary artery. Results show that the pediatric pulmonary artery was significantly thinner (1.06 ± 0.36 mm (mean ± SD)) than ovine tissue (2.85 ± 0.40 mm), considerably stiffer for strain values that exceed the physiological conditions (beyond 50% strain in the circumferential and 60% in the longitudinal direction), more anisotropic (with a significant difference in stiffness between the longitudinal and circumferential directions beyond 60% strain) and presented stronger non-linear stress-strain behavior at equivalent strains (beyond 26% strain) compared to ovine tissue. These discrepancies suggest that stents validated and optimized using the ovine pre-clinical model might not perform satisfactorily in pediatric patients. The material parameters derived from this study may be used to develop stent designs for both applications using computational models., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Tissue engineering is aimed at the fabrication of autologous cardiovascular implants, for example, heart valves or vascular grafts. To date, the mechanical characterization of tissue-engineered vascular grafts (TEVGs) has focused mainly on the material's strength and not on the deformation behavior. A total of 31 samples obtained from 3 mature grafts (out of the cells of a single donor) were tested in uniaxial stress and uniaxial strain configurations to characterize their stiffness under uniaxial and biaxial stress states, respectively. Corresponding measurements were carried out on samples of an ovine artery. A physiological stiffness parameter was defined for data analysis and the uniaxial and multiaxial response compared, also in terms of anisotropy. The tension-strain curve of uniaxial stress tests is highly nonlinear, whereas the results show a more gradual deformation response of the material under a uniaxial strain configuration, which better represents the physiological state of biaxial stress. Stiffness parameters and anisotropy factors are significantly influenced by the selection of the testing configuration. Tangent stiffness of a TEVG at physiological loading conditions is significantly (p<0.05) higher for uniaxial stress as compared to uniaxial strain. The same is observed for the ovine tissue. The anisotropy of the scaffold is shown to partially transfer to the mature TEVG. The results of this study show that for a TEVG characterization, a physiological biaxial testing configuration should be preferred to the commonly used uniaxial stress.

Stem cells have been repeatedly suggested for cardiac regeneration after myocardial infarction (MI). However, the low retention rate of single cell suspensions limits the efficacy of current therapy concepts so far. Taking advantage of three dimensional (3D) cellular self-assembly prior to transplantation may be beneficial to overcome these limitations. In this pilot study we investigate the principal feasibility of intramyocardial delivery of in-vitro generated stem cell-based 3D microtissues (3D-MTs) in a porcine model. 3D-MTs were generated from iron-oxide (MPIO) labeled human adipose-tissue derived mesenchymal stem cells (ATMSCs) using a modified hanging-drop method. Nine pigs (33 ± 2 kg) comprising seven healthy ones and two with chronic MI in the left ventricle (LV) anterior wall were included. The pigs underwent intramyocardial transplantation of 16 × 10(3) 3D-MTs (1250 cells/MT; accounting for 2 × 10(7) single ATMSCs) into the anterior wall of the healthy pigs (n = 7)/the MI border zone of the infarcted (n = 2) of the LV using a 3D NOGA electromechanical mapping guided, transcatheter based approach. Clinical follow-up (FU) was performed for up to five weeks and in-vivo cell-tracking was performed using serial magnet resonance imaging (MRI). Thereafter, the hearts were harvested and assessed by PCR and immunohistochemistry. Intramyocardial transplantation of human ATMSC based 3D-MTs was successful in eight animals (88.8%) while one pig (without MI) died during the electromechanical mapping due to sudden cardiac-arrest. During FU, no arrhythmogenic, embolic or neurological events occurred in the treated pigs. Serial MRI confirmed the intramyocardial presence of the 3D-MTs by detection of the intracellular iron-oxide MPIOs during FU. Intramyocardial retention of 3D-MTs was confirmed by PCR analysis and was further verified on histology and immunohistochemical analysis. The 3D-MTs appeared to be viable, integrated and showed an intact micro architecture. We demonstrate the principal feasibility and safety of intramyocardial transplantation of in-vitro generated stem cell-based 3D-MTs. Multimodal cell-tracking strategies comprising advanced imaging and in-vitro tools allow for in-vivo monitoring and post-mortem analysis of transplanted 3D-MTs. The concept of 3D cellular self-assembly represents a promising application format as a next generation technology for cell-based myocardial regeneration., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Although stem-cell therapies have been suggested for cardiac-regeneration after myocardial-infarction (MI), key-questions regarding the in-vivo cell-fate remain unknown. While most available animal-models require immunosuppressive-therapy when applying human cells, the fetal-sheep being pre-immune until day 75 of gestation has been proposed for the in-vivo tracking of human cells after intra-peritoneal transplantation. We introduce a novel intra-uterine myocardial-infarction model to track human mesenchymal stem cells after direct intra-myocardial transplantation into the pre-immune fetal-sheep. Thirteen fetal-sheep (gestation age: 70-75 days) were included. Ten animals either received an intra-uterine induction of MI only (n = 4) or MI+intra-myocardial injection (IMI;n = 6) using micron-sized, iron-oxide (MPIO) labeled human mesenchymal stem cells either derived from the adipose-tissue (ATMSCs;n = 3) or the bone-marrow (BMMSCs;n = 3). Three animals received an intra-peritoneal injection (IPI;n = 3; ATMSCs;n = 2/BMMSCs;n = 1). All procedures were performed successfully and follow-up was 7-9 days. To assess human cell-fate, multimodal cell-tracking was performed via MRI and/or Micro-CT, Flow-Cytometry, PCR and immunohistochemistry. After IMI, MRI displayed an estimated amount of 1×10(5)-5×10(5) human cells within ventricular-wall corresponding to the injection-sites which was further confirmed on Micro-CT. PCR and IHC verified intra-myocardial presence via detection of human-specific β-2-microglobulin, MHC-1, ALU-Sequence and anti-FITC targeting the fluorochrome-labeled part of the MPIOs. The cells appeared viable, integrated and were found in clusters or in the interstitial-spaces. Flow-Cytometry confirmed intra-myocardial presence, and showed further distribution within the spleen, lungs, kidneys and brain. Following IPI, MRI indicated the cells within the intra-peritoneal-cavity involving the liver and kidneys. Flow-Cytometry detected the cells within spleen, lungs, kidneys, thymus, bone-marrow and intra-peritoneal lavage, but not within the heart. For the first time we demonstrate the feasibility of intra-uterine, intra-myocardial stem-cell transplantation into the pre-immune fetal-sheep after MI. Utilizing cell-tracking strategies comprising advanced imaging-technologies and in-vitro tracking-tools, this novel model may serve as a unique platform to assess human cell-fate after intra-myocardial transplantation without the necessity of immunosuppressive-therapy.

Living autologous tissue engineered vascular-grafts (TEVGs) with growth-capacity may overcome the limitations of contemporary artificial-prostheses. However, the multi-step in vitro production of TEVGs requires extensive ex vivo cell-manipulations with unknown effects on functionality and quality of TEVGs due to an accelerated biological age of the cells. Here, the impact of biological cell-age and tissue-remodeling capacity of TEVGs in relation to their clinical long-term functionality are investigated. TEVGs were implanted as pulmonary-artery (PA) replacements in juvenile sheep and followed for up to 240 weeks (∼4.5years). Telomere length and telomerase activity were compared amongst TEVGs and adjacent native tissue. Telomerase-activity of in vitro expanded autologous vascular-cells prior to seeding was <5% as compared to a leukemic cell line, indicating biological-aging associated with decreasing telomere-length with each cellular-doubling. Up to 100 weeks, the cells in the TEVGs had consistently shorter telomeres compared to the native counterpart, whereas no significant differences were detectable at 240 weeks. Computed tomography (CT) analysis demonstrated physiological wall-pressures, shear-stresses, and flow-pattern comparable to the native PA. There were no signs of degeneration detectable and continuous native-analogous growth was confirmed by vessel-volumetry. TEVGs exhibit a higher biological age compared to their native counterparts. However, despite of this tissue engineering technology related accelerated biological-aging, growth-capacity and long-term functionality was not compromised. To the contrary, extensive in-vivo remodeling processes with substantial endogenous cellular turnover appears to result in "TEVG rejuvenation" and excellent clinical performance. As these large-animal results can be extrapolated to approximately 20 human years, this study suggests long-term clinical-safety of cardiovascular in vitro tissue engineering and may contribute to safety-criteria as to first-in-man clinical-trials., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background and Aim of the Study: Living tissue-engineered heart valves (TEHVs) based on rapidly degrading scaffolds and autologous cells might overcome the limitations of today's valve substitutes. Following minimally invasive trans-apical implantation into an ovine model, TEHVs showed adequate in-vivo functionality, but a thickening of the leaflets was observed. In order to evaluate the impact of the substantial tissue deformations of TEHVs associated with the crimping procedure during minimally invasive delivery, trans-apical and conventional implantation technologies were compared in an ovine model., Methods: Trileaflet heart valves (n=11) based on PGA/P4HB-scaffolds, integrated into self-expandable stents, were engineered from autologous ovine vascular-derived cells. After in-vitro culture, the TEHVs were either implanted surgically (n=5), replacing the native pulmonary valve, or delivered trans-apically (n=6) into the orthotopic pulmonary valve position. In-vivo functionality was assessed by echocardiography and by angiography for up to eight weeks. The tissue compositions of the explanted TEHVs and corresponding control valves were analyzed., Results: TEHV implantations were successful in all cases. Independent of the implantation method, the explants demonstrated a comparable layered tissue formation with thickening and deposited fibrous layers. Active remodeling of these layers was evident in the explants, as indicated by vascularization of the walls, invasion of the host cells, and the formation of a luminal endothelial layer on the TEHV leaflets., Conclusion: This direct comparison of trans-apical and conventional surgical implantation techniques showed that crimping had no adverse effect on the integrity or functional outcome of TEHVs. This suggests that a thickening of TEHVs in vivo is neither caused by nor enhanced by the crimping procedure, but represents a functional tissue remodeling process.

Objectives: This study sought to investigate the combination of transcatheter aortic valve implantation and a novel concept of stem cell-based, tissue-engineered heart valves (TEHV) comprising minimally invasive techniques for both cell harvest and valve delivery., Background: TAVI represents an emerging technology for the treatment of aortic valve disease. The used bioprostheses are inherently prone to calcific degeneration and recent evidence suggests even accelerated degeneration resulting from structural damage due to the crimping procedures. An autologous, living heart valve prosthesis with regeneration and repair capacities would overcome such limitations., Methods: Within a 1-step intervention, trileaflet TEHV, generated from biodegradable synthetic scaffolds, were integrated into self-expanding nitinol stents, seeded with autologous bone marrow mononuclear cells, crimped and transapically delivered into adult sheep (n = 12). Planned follow-up was 4 h (Group A, n = 4), 48 h (Group B, n = 5) or 1 and 2 weeks (Group C, n = 3). TEHV functionality was assessed by fluoroscopy, echocardiography, and computed tomography. Post-mortem analysis was performed using histology, extracellular matrix analysis, and electron microscopy., Results: Transapical implantation of TEHV was successful in all animals (n = 12). Follow-up was complete in all animals of Group A, three-fifths of Group B, and two-thirds of Group C (1 week, n = 1; 2 weeks, n = 1). Fluoroscopy and echocardiography displayed TEHV functionality demonstrating adequate leaflet mobility and coaptation. TEHV showed intact leaflet structures with well-defined cusps without signs of thrombus formation or structural damage. Histology and extracellular matrix displayed a high cellularity indicative for an early cellular remodeling and in-growth after 2 weeks., Conclusions: We demonstrate the principal feasibility of a transcatheter, stem cell-based TEHV implantation into the aortic valve position within a 1-step intervention. Its long-term functionality proven, a stem cell-based TEHV approach may represent a next-generation heart valve concept., (Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Decellularized xenogenic or allogenic heart valves have been used as starter matrix for tissue-engineering of valve replacements with (pre-)clinical promising results. However, xenografts are associated with the risk of immunogenic reactions or disease transmission and availability of homografts is limited. Alternatively, biodegradable synthetic materials have been used to successfully create tissue-engineered heart valves (TEHV). However, such TEHV are associated with substantial technological and logistical complexity and have not yet entered clinical use. Here, decellularized TEHV, based on biodegradable synthetic materials and homologous cells, are introduced as an alternative starter matrix for guided tissue regeneration. Decellularization of TEHV did not alter the collagen structure or tissue strength and favored valve performance when compared to their cell-populated counterparts. Storage of the decellularized TEHV up to 18 months did not alter valve tissue properties. Reseeding the decellularized valves with mesenchymal stem cells was demonstrated feasible with minimal damage to the reseeded valve when trans-apical valve delivery was simulated. In conclusion, decellularization of in-vitro grown TEHV provides largely available off-the-shelf homologous scaffolds suitable for reseeding with autologous cells and trans-apical valve delivery., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Prenatal heart valve interventions aiming at the early and systematic correction of congenital cardiac malformations represent a promising treatment option in maternal-fetal care. However, definite fetal valve replacements require growing implants adaptive to fetal and postnatal development. The presented study investigates the fetal implantation of prenatally engineered living autologous cell-based heart valves. Autologous amniotic fluid cells (AFCs) were isolated from pregnant sheep between 122 and 128 days of gestation via transuterine sonographic sampling. Stented trileaflet heart valves were fabricated from biodegradable PGA-P4HB composite matrices (n = 9) and seeded with AFCs in vitro. Within the same intervention, tissue engineered heart valves (TEHVs) and unseeded controls were implanted orthotopically into the pulmonary position using an in-utero closed-heart hybrid approach. The transapical valve deployments were successful in all animals with acute survival of 77.8% of fetuses. TEHV in-vivo functionality was assessed using echocardiography as well as angiography. Fetuses were harvested up to 1 week after implantation representing a birth-relevant gestational age. TEHVs showed in vivo functionality with intact valvular integrity and absence of thrombus formation. The presented approach may serve as an experimental basis for future human prenatal cardiac interventions using fully biodegradable autologous cell-based living materials., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Severely decreased ejection-fraction is an established risk-factor for worse outcome after cardiac surgery. We compare outcomes of off-pump coronary artery bypass grafting (OPCAB) and on-pump CABG (ONCABG) in patients with severely compromised EF. From 2004 to 2009, 478 patients with a decreased EF ≤35% underwent myocardial-revascularization. Patients received either OPCAB (n = 256) or ONCABG (n = 222). Propensity score (PS), including 50 preoperative risk-factors, was used to balance characteristics between groups. PS adjusted logistic regression analysis was performed to assess mortality and major adverse cardiac and cerebrovascular events (MACCE). A composite endpoint for major non-cardiac complications such as respiratory failure, renal failure, rethoracotomy was applied. Complete revascularization (CR) was assumed when the number of distal anastomoses was larger than that of diseased vessels. There was no difference for mortality (2.3 vs. 4.1%; PS-adjusted odds ratio (PS-OR) = 1.05; p = 0.93) and MACCE (13.7 vs. 17.6%; PS-OR = 1.22; p = 0.50) including myocardial-infarction (1.4 vs. 4.9%; PS-OR = 0.39; p = 0.26), low cardiac output (2.3 vs. 4.7%; PS-OR = 0.75; p = 0.72) and stroke (2.3 vs. 2.7%; PS-OR = 0.69; p = 0.66). OPCAB patients presented with a trend to less frequent occurrence of the non-cardiac composite (12.1 vs. 22.1%; PS-OR = 0.54; p = 0.059) including renal dysfunction (PAOR = 0.77; 95% CI 0.31-1.9; p = 0.57), bleeding (PAOR = 0.42; 95% CI 0.14-1.20; p = 0.10) and respiratory failure (PAOR = 0.39; 95% CI 0.05-3.29; p = 0.39). The rate of complete revascularization was similar (92.2 vs. 92.8%; PS-OR = 0.75; p = 0.50). OPCAB in patients with severely decreased EF is safe and feasible. It may even benefit these patients in regard to non-cardiac complications and does not come at cost of less complete revascularization.

Current implantation formats to deliver bone marrow-derived mesenchymal stem cells (MSCs) to the site of myocardial injury resulted only in limited cell retention and integration. As an alternative concept to single cell transplantation, we investigated the fate of cell tracker-labeled syngenic rat MSC microtissue implants, injected into the scar area in a chronic rat myocardial infarction model. Analysis of the explants after 2 and 7 days revealed substantial amounts of the cell tracker within the infarct region. However, the signal was associated with the extracellular matrix rather than with viable implanted cells. Following these results, we systematically evaluated the behavior of MSCs derived from mouse, rat, and human origin in the microtissue format in vitro. We found that MSC-composed microtissues of all three species displayed highly elevated levels of apoptotic activity and cell death. This effect could be attenuated by initiating osteogenic differentiation during the tissue formation process. We conclude that MSCs used for tissue regeneration undergo apoptosis in their new environment unless they get appropriate signals for differentiation that permit sustained survival. These findings may explain the limited cellular regeneration potential in current MSC-based clinical trials and may change therapeutic strategies away from pure, unmodulated cell delivery concepts.

Objective: The purpose of this study was to assess the technical feasibility of a fetal trans-apical stent delivery into the pulmonary artery using a novel hybrid-intervention technique as a possible route for prenatal minimally invasive heart-valve-implantation approaches., Methods: Pregnant Pre-Alp sheep between 122 and 128 days' gestation (n = 3) underwent a midline laparotomy. The fetus was left in utero or partially externalized and its chest was opened via a left-sided minithoracotomy. The fetal heart was cannulated and a guide wire was introduced through the ductus arteriosus into the aorta. A 14-French delivery system was then mounted onto the guide wire and advanced to the landing zone in the pulmonary artery, where the stent was deployed. The position of the stent was confirmed using echocardiography, angiography as well as computed tomography., Results: The trans-apical implantation was successful in all animals. However, at necropsy in one animal, the stent was found to partly occlude one of the pulmonary valvular leaflets. Bleeding at the antero-apical incision occurred in all animals but could be managed without fetal demise. No fetal cardiopulmonary bypass was performed. In all animals, contrast angiography displayed normal perfusion of the pulmonary vasculature as well as the ductus arteriosus., Conclusions: Our study demonstrates the principal technical feasibility of a prenatal stent delivery into the pulmonary artery using a novel trans-apical hybrid-intervention technique. This approach demonstrates the first step towards possible future minimally invasive prenatal heart-valve-implantation procedures.

Objectives: We have previously demonstrated the functionality and growth of autologous, living, tissue-engineered vascular grafts (TEVGs) in long-term animal studies. These grafts showed substantial in vivo tissue remodeling and approximation to native arterial wall characteristics. Based on this, in vitro and in vivo matrix metalloproteinase (MMP) activity of TEVGs is investigated as a key marker of matrix remodeling., Methods: TEVGs fabricated from biodegradable scaffolds (polyglycolic-acid/poly-4-hydroxybutyrate, PGA/P4HB) seeded with autologous vascular cells were cultured in static and dynamic in vitro conditions. Thereafter, TEVGs were implanted as pulmonary artery replacements in lambs and followed up for 2 years. Gelatin gel zymography to detect MMP-2 and -9 was performed and collagen content quantified (n=5). Latent (pro) and active MMP-2 and -9 were detected., Results: Comparable levels of active MMP-9 and pro-MMP-2 were detected in static and dynamic culture. Higher levels of active MMP-2 were detected in dynamic cultures. Expression of MMP-2 and -9 was minimal in native grafts but was increased in implanted TEVG. Pro-MMP-9 was expressed 20 weeks post implantation and persisted up to 80 weeks post implantation. Collagen content in vitro was increased in dynamically conditioned TEVG as compared with static constructs and was increased in vivo compared with the corresponding native pulmonary artery., Conclusions: MMPs are up-regulated in vitro by dynamic culture conditions and could contribute to increased matrix remodeling, native analogous tissue formation and functional growth of TEVGs in vivo. Monitoring of MMP activity, for example, by molecular imaging techniques, may enable the non-invasive assessment of functional tissue quality in future clinical tissue-engineering applications.

Interventional closure of intracardiac wall defects using occluder devices has evolved as a highly attractive treatment option. However, incomplete and delayed healing reactions often result in a major risk of residual defects, thromboembolism, or device fractures. Biodegradable living tissue engineered occluder membranes (TEOMs) could provide autologous thromboresistant implants with growth and remodeling capacities. PGA-P4HB composite matrices were seeded with human umbilical cord-derived cells or vascular-derived control cells and exposed to static (n = 19) or dynamic (n = 13) conditioning. Harvested TEOMs were integrated into occluder frameworks, exposed to crimping and delivered into pre-formed defects of juvenile porcine hearts. Dynamically conditioned TEOM constructs showed higher collagen formation in histology than static constructs with significantly higher stiffness moduli in uniaxial tensile testing. Grating interferometry revealed substantial but inhomogeneous cone-like degradation of the composite matrices in dynamic conditioning. The crimping and delivery procedures resulted in no significant changes in macroscopy, histo-morphology, cellular viability, DNA or hydroxyproline content, and scanning electron microscopy findings. Here, we present the in vitro fabrication, crimping and experimental delivery of living human umbilical cord-cell derived TEOMs based on composite matrices as a potential future autologous therapy of intracardiac wall defects., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aims: Collagen degradation in atherosclerotic plaques with thin fibrous caps renders them more prone to rupture. Fibroblast activation protein (FAP) plays a role in arthritis and tumour formation through its collagenase activity. However, the significance of FAP in thin-cap human fibroatheromata remains unknown., Methods and Results: We detected enhanced FAP expression in type IV-V human aortic atheromata (n = 12), compared with type II-III lesions (n = 9; P < 0.01) and healthy aortae (n = 8; P < 0.01) by immunostaining and western blot analyses. Fibroblast activation protein was also increased in thin-cap (<65 µm) vs. thick-cap (≥ 65 µm) human coronary fibroatheromata (n = 12; P < 0.01). Fibroblast activation protein was expressed by human aortic smooth muscle cells (HASMC) as shown by colocalization on immunofluorescent aortic plaque stainings (n = 10; P < 0.01) and by flow cytometry in cell culture. Although macrophages did not express FAP, macrophage burden in human aortic plaques correlated with FAP expression (n = 12; R(2)= 0.763; P < 0.05). Enzyme-linked immunosorbent assays showed a time- and dose-dependent up-regulation of FAP in response to human tumour necrosis factor α (TNFα) in HASMC (n = 6; P < 0.01). Moreover, supernatants from peripheral blood-derived macrophages induced FAP expression in cultured HASMC (n = 6; P < 0.01), an effect abolished by blocking TNFα (n = 6; P < 0.01). Fibroblast activation protein associated with collagen-poor regions in human coronary fibrous caps and digested type I collagen and gelatin in vitro (n = 6; P < 0.01). Zymography revealed that FAP-mediated collagenase activity was neutralized by an antibody directed against the FAP catalytic domain both in HASMC (n = 6; P < 0.01) and in fibrous caps of atherosclerotic plaques (n = 10; P < 0.01)., Conclusion: Fibroblast activation protein expression in HASMC is induced by macrophage-derived TNFα. Fibroblast activation protein associates with thin-cap human coronary fibroatheromata and contributes to type I collagen breakdown in fibrous caps.

Aims: 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., Methods and Results: 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., Conclusion: 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.

Background: A severely impaired left ventricular ejection fraction (EF) (30%) increases the risk of surgical myocardial revascularization. We evaluated the safety and feasibility of off-pump coronary artery bypass (OPCAB) surgery in patients with a severely decreased EF., Methods: We compared 79 patients with an EF ≤30% (group A) with 863 patients with an EF >30% (group B) who underwent myocardial revascularization between 2003 and 2008. The relationship between EF and outcome after OPCAB was assessed by univariate and logistic regression analyses. A composite end point was constructed from 30-day mortality, renal failure, length of stay in the intensive care unit (ICU) >2 days, neurologic complications, and use of an intra-aortic balloon pump (IABP). Additionally, the completeness of revascularization was assessed., Results: The mortality rates for groups A and B were comparable (1.3% and 2.0%, respectively; P = .55), and the 2 groups did not differ with regard to serious postoperative complications, such as stroke (2.5% versus 1.4% for groups A and B, respectively; P = .42), peripheral neurologic complications (2.5% versus 0.7%, P = .14), renal failure (0% versus 1.1%, P = 1.00), use of an IABP (1.3% versus 0.8%, P = .50), ICU length of stay >2 days (17.7% versus 19.6%, P = .77). Similarly, groups A and B did not differ with regard to ventilation time (11.2 ± 12.7 hours versus 12.4 ± 15.5 hours, P = .82), indicating similar postoperative courses for the 2 groups of patients. In contrast, the composite end point occurred significantly more frequently in group A (43.0% versus 29.7%, P = .02), a result driven by the increased rate of rethoracotomy for bleeding in that group (11.4% versus 2.9%, P = .001). The 2 groups were similar with respect to the total number of grafts used per patient (3.82 ± 0.89 versus 3.63 ± 1.01, P = .10) and the completeness of revascularization (94% versus 93%, P = .49)., Conclusion: A standardized OPCAB approach is safe for patients with a severely decreased EF, and its use does not come at the cost of less complete revascularization.

Using the principal of tissue engineering, several groups have demonstrated the feasibility of creating heart valves, blood vessels, and myocardial structures using autologous cells and biodegradable scaffold materials. In the current cardiovascular clinical scenario, the main medical need for a tissue engineering solution is in the field of pediatric applications treating congenital heart disease. In these young patients, the introduction of autologous viable and growing replacement structures, such as tissue engineered heart valves and vessels, would substantially reduce today's severe therapeutic limitations, which are mainly due to the need for repeat reoperations to adapt the current artificial prostheses to somatic growth. Based on high resolution imaging techniques, an increasing number of defects are diagnosed already prior to birth around week 20. For interventions, cells should be obtained already during pregnancy to provide tissue engineered implants either at birth or even prenatally. In our recent studies human fetal mesenchymal stem cells were isolated from routinely sampled prenatal amniotic fluid or chorionic villus specimens and expanded in vitro. Fresh and cryopreserved samples were used. After phenotyping and genotyping, cells were seeded onto synthetic biodegradable scaffolds and conditioned in a bioreactor. Leaflets were endothelialized with either amniotic fluid- or umbilical cord blood-derived endothelial progenitor cells and conditioned. 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. Genotyping confirmed their fetal origin, and fresh versus cryopreserved cells showed comparable myofibroblast-like phenotypes. 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 morphologically cellular distribution throughout the polymer and smooth surfaces. Mechanical profiles approximated those of native heart valves. These in vitro studies demonstrated the principal feasibility of using various human cell types isolated from fetal sources for cardiovascular tissue engineering. Umbilical cord blood-, amniotic fluid- and chorionic villi-derived cells have shown promising potential for the clinical realization of this congenital tissue engineering approach. Based on these results, future research must aim at further investigation as well as preclinical evaluation of prenatally harvested stem- or progenitor cells with regard to their potential for clinical use., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Tissue-engineered heart valves (TEHV) are being explored as an alternative to conventional heart valve prostheses. Using the classic tissue engineering paradigm, a stented tri-leaflet valve is fabricated. Subsequently, the construct is implanted into the pulmonary position in a sheep. Follow-up by means of computed tomography, magnetic resonance imaging, and echocardiography was used to assess tissue formation. After 4 weeks, the scaffold of the TEHV has degraded and new tissue is formed. However, small areas without tissue formation were present at macroscopic inspection. This phenomenon was only visible on computed tomographic images. Therefore, computed tomography appears a promising technique for in vivo follow-up of tissue formation in tissue-engineered heart valves., (Copyright © 2011 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Objective: Diabetic patients often present with diffuse coronary disease than nondiabetic patients posing a greater surgical challenge during off-pump revascularization. In this study, the safety, feasibility, and completeness of revascularization for this subset of patients was assessed., Methods: From 2002 to 2008, 1015 diabetic patients underwent myocardial revascularization. Patients received either off-pump coronary artery bypass (OPCAB; n = 540; 53%) or coronary artery bypass grafting (CABG; n = 475; 47%). Data collection was performed prospectively and data analysis was done by propensity-score (PS)-adjusted regression analysis. Primary endpoints were mortality, major adverse cardiac and cerebrovascular events (MACCEs), and a composite endpoint including major noncardiac adverse events (MNCAEs) such as respiratory failure, renal failure, and rethoracotomy for bleeding was applied. An index of complete revascularization (ICOR) was defined to assess complete revascularization by dividing the total number of distal anastomoses by the number of diseased vessels. Complete revascularization was assumed when ICOR was >1., Results: OPCAB patients had a significantly lower mortality-rate (1.1% vs 3.8%; propensity-adjusted odds ratio (PAOR) = 0.11; p = 0.018) and displayed less frequent MACCE (8.3% vs 17.9%; PAOR = 0.66; p = 0.07) including myocardial infarction (1.3% vs 3.2%; PAOR = 0.33; p = 0.06) and stroke (0.7% vs 2.3%; PAOR = 0.28; p = 0.13). Similarly, a significantly lower occurrence of the noncardiac composite endpoint (MNCAE) (PAOR = 0.46; confidence interval (CI) 95% 0.35-0.91; p < 0.001) was detected. In particular, lesser respiratory failure (0.9% vs 4.3%; PAOR = 0.24; p = 0.63) and pleural effusions (3.3% vs 7.5%; PAOR = 0.45; p = 0.04) occurred, so that fast extubation (≤ 12 h postoperative) was more frequently possible (58.3% vs 34.2%; PAOR = 1.64; p = 0.007). The number of arterial grafts was significantly higher among OPCAB patients (1.54 ± 0.89 vs 1.33 ± 0.81; p = 0.006) due to a more frequent use of the right-internal mammary artery (35.6% vs 22.9%; p < 0.001). ICOR was significantly higher among CABG patients (1.24 ± 0.34 vs 1.30 ± 0.28; p = 0.001). However, for similar proportions in both groups, an ICOR > 1 was achieved clearly indicating complete revascularization (94.3% vs 93.7%; p = 0.24)., Conclusions: OPCAB offers a lower mortality and superior postoperative outcomes in diabetic patients with multivessel disease. Arterial grafts are used more frequently that may contribute to better long-term outcomes and the OPCAB approach does not come at the cost of less complete revascularization., (Copyright © 2010 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.)

We present a 68-year-old female who suffered extensive complications after severe myocardial infarction (MI) in the circumflex (CX) territory. At 24 hours after the initial event, the patient presented with a covered right ventricular free wall rupture (FWR) which was followed by a rupture of the left posterior wall ten days later. We report here on a rare case of delayed two-step biventricular FWR after severe MI in the CX territory., (© Georg Thieme Verlag KG Stuttgart · New York.)

Tissue engineering aims at the creation of living neo-tissues identical or close to their native human counterparts. As basis of this approach, temporary biodegradable supporter matrices are fabricated in the shape of a desired construct, which promote tissue strength and provide functionality until sufficient neo-tissue is formed. Besides fully synthetic polymer-based scaffolds, decellularized biological tissue of xenogenic or homogenic origin can be used. In a second step, these scaffolds are seeded with autologous cells attaching to the scaffold microstructure. In order to promote neo-tissue formation and maturation, the seeded scaffolds are exposed to different forms of stimulation. In cardiovascular tissue engineering, this "conditioning" can be achieved via culture media and biomimetic in vitro exposure, e.g., using flow bioreactors. This aims at adequate cellular differentiation, proliferation, and extracellular matrix production to form a living tissue called the construct. These living autologous constructs, such as heart valves or vascular grafts, are created in vitro, comprising a viable interstitium with repair and remodeling capabilities already prior to implantation. In situ further in vivo remodeling is intended to recapitulate physiological vascular architecture and function. The remodeling mechanisms were shown to be dominated by monocytic infiltration and chemotactic host-cell attraction leading into a multifaceted inflammatory process and neo-tissue formation. Key molecules of these processes can be integrated into the scaffold matrix to direct cell and tissue fate in vivo.