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Cardiovascular research (2023). doi:10.1093/cvr/cvad037, Published by Oxford University Press, Oxford

To investigate the pathogenic mechanisms of calcified aortic valve disease (CAVD), it is necessary to develop a new three-dimensional model that contains valvular interstitial cells (VIC) and valvular endothelial cells (VEC). For this purpose, ovine aortic valves were processed to isolate VIC and VEC that were dissolved in an alginate/gelatin hydrogel. A 3D-bioprinter (3D-Bioplotter® Developer Series, EnvisionTec, Gladbeck, Germany) was used to print cell-laden tissue constructs containing VIC and VEC which were cultured for up to 21 days. The 3D-architecture, the composition of the culture medium, and the hydrogels were modified, and cell viability was assessed. The composition of the culture medium directly affected the cell viability of the multicellular tissue constructs. Co-culture of VIC and VEC with a mixture of 70% valvular interstitial cell and 30% valvular endothelial cell medium components reached the cell viability best tested with about 60% more living cells compared to pure valvular interstitial cell medium (p = 0.02). The tissue constructs retained comparable cell viability after 21 days (p = 0.90) with different 3D-architectures, including a “sandwich” and a “tube” design. Good long-term cell viability was confirmed even for thick multilayer multicellular tissue constructs. The 3D-bioprinting of multicellular tissue constructs with VEC and VIC is a successful new technique to design tissue constructs that mimic the structure of the native aortic valve for research applications of aortic valve pathologies.

OBJECTIVES Hypercholesterolaemia and obesity are risk factors for the development of calcified aortic valve disease and common comorbidities in respective patients. Peroxisome proliferator-activated receptor gamma activation has been shown to reduce the progression of native aortic valve sclerosis, while its effect on bioprosthetic valve degeneration is yet unknown. This project aims to analyse the impact of pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, on the degeneration of biological aortic valve conduits in an implantation model in obese and hypercholesterolaemic rats. METHODS Cryopreserved allogenic rat aortic valve conduits (n = 40) were infrarenally implanted into Wistar rats on high-fat (34.6%) diet. One cohort was treated with pioglitazone (75 mg/kg chow; n = 20, group PIO) and compared to untreated rats (n = 20, group control). After 4 or 12 weeks, conduits were explanted and analysed by (immuno-)histology and real-time polymerase chain reaction. RESULTS A significantly decreased intima hyperplasia occurred in group PIO compared to control after 4 (P = 0.014) and 12 weeks (P = 0.045). Calcification of the intima was significantly decreased in PIO versus control at 12 weeks (P = 0.0001). No significant inter-group differences were shown for media calcification after 4 and 12 weeks. Echocardiographically, significantly lower regurgitation through the implanted aortic valve conduit was observed in PIO compared to control after 4 (P = 0.018) and 12 weeks (P = 0.0004). Inflammatory activity was comparable between both groups. CONCLUSIONS Systemic peroxisome proliferator-activated receptor gamma activation decreases intima hyperplasia and subsequent intima calcification of cryopreserved allografts in obese, hypercholesterolaemic recipients. Additionally, it seems to inhibit functional impairment of the implanted aortic valve. Further preclinical studies are required to determine the long-term impact of peroxisome proliferator-activated receptor gamma agonists on graft durability.

The FASEB journal 36(11), e22591 (2022). doi:10.1096/fj.202200727RR, Published by Wiley, Hoboken, NJ

Serum levels of cytokines interleukin 1 beta ( IL-1β) and interleukin 33 (IL-33) are highly abnormal in heart failure and remain elevated after mechanical circulatory support (MCS). However, local cytokine signaling induction remains elusive. Left (LV) and right ventricular (RV) myocardial tissue specimens of end-stage heart failure (HF) patients without (n = 24) and with MCS (n = 39, 594 ± 57 days) were analyzed for cytokine mRNA expression level of IL-1B, interleukin 1 receptor 1/2 (IL-1R1/2), interleukin 1 receptor-like 1 (IL-1RL1), IL-33 and interleukin-1 receptor accessory protein (IL-1RaP). MCS patients showed significantly elevated IL-1B expression levels (LV: 2.0 fold, p = 0.0058, RV: 3.3 fold, p &lt, 0.0001). Moreover, IL-1R1, IL-1RaP and IL-33 expression levels strongly correlated with each other. IL-1RL1 and IL-1R2 expression levels were significantly higher in RV myocardial tissue (RV/LV ratio IL-1R2 HF: 4.400 ± 1.359, MCS: 4.657 ± 0.655, IL-1RL1 HF: 3.697 ± 0.876, MCS: 4.529 ± 0.5839). In addition, IL1-RaP and IL-33 RV expression levels were significantly elevated in MCS. Furthermore, IL-33 expression correlates with C-reactive protein (CRP) plasma levels in HF, but not in MCS patients. Increased expression of IL-1B and altered correlation patterns of IL-1 receptors indicate enhanced IL-1β signaling in MCS patients. Correlation of IL-1 receptor expression with IL-33 may hint towards a link between both pathways. Moreover, diverging expression in LV and RV suggests specific regulation of local cytokine signaling.

Calcific aortic valve disease (CAVD) is a frequent cardiac pathology in the aging society. Although valvular interstitial cells (VICs) seem to play a crucial role, mechanisms of CAVD are not fully understood. Development of tissue-engineered cellular models by 3D-bioprinting may help to further investigate underlying mechanisms of CAVD. VIC were isolated from ovine aortic valves and cultured in Dulbecco’s modified Eagle’s Medium (DMEM). VIC of passages six to ten were dissolved in a hydrogel consisting of 2% alginate and 8% gelatin with a concentration of 2 × 106 VIC ml−1. Cell-free and VIC-laden hydrogels were printed with an extrusion-based 3D-bioprinter (3D-Bioplotter® Developer Series, EnvisionTec, Gladbeck, Germany), cross-linked and incubated for up to 28 d. Accuracy and durability of scaffolds was examined by microscopy and cell viability was tested by cell counting kit-8 assay and live/dead staining. 3D-bioprinting of scaffolds was most accurate with a printing pressure of P < 400 hPa, nozzle speed of v < 20 mm s−1, hydrogel temperature of T H = 37 °C and platform temperature of T P = 5 °C in a 90° parallel line as well as in a honeycomb pattern. Dissolving the hydrogel components in DMEM increased VIC viability on day 21 by 2.5-fold compared to regular 0.5% saline-based hydrogels (p < 0.01). Examination at day 7 revealed dividing and proliferating cells. After 21 d the entire printed scaffolds were filled with proliferating cells. Live/dead cell viability/cytotoxicity staining confirmed beneficial effects of DMEM-based cell-laden VIC hydrogel scaffolds even 28 d after printing. By using low pressure printing methods, we were able to successfully culture cell-laden 3D-bioprinted VIC scaffolds for up to 28 d. Using DMEM-based hydrogels can significantly improve the long-term cell viability and overcome printing-related cell damage. Therefore, future applications 3D-bioprinting of VIC might enable the development of novel tissue engineered cellular 3D-models to examine mechanisms involved in initiation and progression of CAVD.

Aims Donor heart shortage leads to increasing use of left ventricular assist device (LVAD) as bridge‐to‐transplant or destination therapy. Prolonged LVAD support is associated with aortic valve insufficiency, representing a relevant clinical problem in LVAD patients. Nevertheless, the impact of LVAD support on inflammation, remodelling, and chondro‐osteogenic differentiation of the aortic valve is still not clearly understood. The aim of the study is to evaluate the impact of LVAD support on structural and molecular alterations of the aortic valve. Methods and results During heart transplantation, aortic valves of 63 heart failure patients without (n = 22) and with LVAD support (n = 41) were collected and used for analysis. Data on clinical course as well as echocardiographic data were analysed. Calcification and markers of remodelling, chondro‐osteogenic differentiation, and inflammation were evaluated by computed tomography, by mRNA analysis and by histology and immunohistochemistry. Expression of inflammation markers of the LVAD group was analysed with regard to levels of C‐reactive protein and driveline infections. Calcium accumulation and mRNA expression of determined markers were correlated with duration of LVAD support. Data were also analysed relating to aortic valve opening and aortic valve insufficiency. There was no difference in the frequency of cardiovascular risk factors or comorbidities between the patient groups. Expression of matrix metalloproteinase‐9 (P = 0.007), alpha‐smooth muscle actin (P = 0.045), and osteopontin (P = 0.003) were up‐regulated in aortic valves of LVAD patients. Histological appearance of the aortic valve was similar in patients with or without LVAD, and computed tomography‐based analysis not yet revealed significant difference in tissue calcification. Expression of interferon gamma (P = 0.004), interleukin‐1 beta (P

Background: We aimed to examine the anti-calcification and anti-inflammatory effects of pioglitazone as a PPAR-gamma agonist on bioprosthetic-valve-bearing aortic grafts in a rat model of diabetes mellitus (DM). Methods: DM was induced in male Wistar rats by high-fat diet with an intraperitoneal streptozotocin (STZ) injection. The experimental group received additional pioglitazone, and controls received normal chow without STZ (n = 20 each group). Cryopreserved aortic donor grafts including the aortic valve were analyzed after 4 weeks and 12 weeks in vivo for analysis of calcific bioprosthetic degeneration. Results: DM led to a significant media proliferation at 4 weeks. The additional administration of pioglitazone significantly increased circulating adiponectin levels and significantly reduced media thickness at 4 and 12 weeks, respectively (p = 0.0002 and p = 0.0107, respectively). Graft media calcification was highly significantly inhibited by pioglitazone after 12 weeks (p = 0.0079). Gene-expression analysis revealed a significant reduction in relevant chondro-osteogenic markers osteopontin and RUNX-2 by pioglitazone at 4 weeks. Conclusions: Under diabetic conditions, pioglitazone leads to elevated circulating levels of adiponectin and to an inhibition of bioprosthetic graft degeneration, including lower expression of chondro-osteogenic genes, decreased media proliferation, and inhibited graft calcification in a small-animal model of DM.

Type 2 diabetes mellitus (T2D) is one of the prominent risk factors for the development and progression of calcific aortic valve disease. Nevertheless, little is known about molecular mechanisms of how T2D affects aortic valve (AV) remodeling. In this study, the influence of hyperinsulinemia and hyperglycemia on degenerative processes in valvular tissue is analyzed in intact AV exposed to an either static or dynamic 3D environment, respectively. The complex native dynamic environment of AV is simulated using a software-governed bioreactor system with controlled pulsatile flow. Dynamic cultivation resulted in significantly stronger fibrosis in AV tissue compared to static cultivation, while hyperinsulinemia and hyperglycemia had no impact on fibrosis. The expression of key differentiation markers and proteoglycans were altered by diabetic conditions in an environment-dependent manner. Furthermore, hyperinsulinemia and hyperglycemia affect insulin-signaling pathways. Western blot analysis showed increased phosphorylation level of protein kinase B (AKT) after acute insulin stimulation, which was lost in AV under hyperinsulinemia, indicating acquired insulin resistance of the AV tissue in response to elevated insulin levels. These data underline a complex interplay of diabetic conditions on one hand and biomechanical 3D environment on the other hand that possesses an impact on AV tissue remodeling.

Calcific aortic valve disease is the most common valvular heart disease in industrialized countries. Pulsatile pressure, sheer and bending stress promote initiation and progression of aortic valve degeneration. The aim of this work is to establish an ex vivo model to study the therein involved processes. Ovine aortic roots bearing aortic valve leaflets were cultivated in an elaborated bioreactor system with pulsatile flow, physiological temperature, and controlled pressure and pH values. Standard and pro-degenerative treatment were studied regarding the impact on morphology, calcification, and gene expression. In particular, differentiation, matrix remodeling, and degeneration were also compared to a static cultivation model. Bioreactor cultivation led to shrinking and thickening of the valve leaflets compared to native leaflets while gross morphology and the presence of valvular interstitial cells were preserved. Degenerative conditions induced considerable leaflet calcification. In comparison to static cultivation, collagen gene expression was stable under bioreactor cultivation, whereas expression of hypoxia-related markers was increased. Osteopontin gene expression was differentially altered compared to protein expression, indicating an enhanced protein turnover. The present ex vivo model is an adequate and effective system to analyze aortic valve degeneration under controlled physiological conditions without the need of additional growth factors.

Supplemental Digital Content is Available in the Text., Aortic valve replacement for severe stenosis is a standard procedure in cardiovascular medicine. However, the use of biological prostheses has limitations especially in young patients because of calcifying degeneration, resulting in implant failure. Pioglitazone, a peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonist, was shown to decrease the degeneration of native aortic valves. In this study, we aim to examine the impact of pioglitazone on inflammation and calcification of aortic valve conduits (AoC) in a rat model. Cryopreserved AoC (n = 40) were infrarenally implanted into Wistar rats treated with pioglitazone (75 mg/kg chow; n = 20, PIO) or untreated (n = 20, controls). After 4 or 12 weeks, AoC were explanted and analyzed by histology, immunohistology, and polymerase chain reaction. Pioglitazone significantly decreased the expression of inflammatory markers and reduced the macrophage-mediated inflammation in PIO compared with controls after 4 (P = 0.03) and 12 weeks (P = 0.012). Chondrogenic transformation was significantly decreased in PIO after 12 weeks (P = 0.001). Calcification of the intima and media was significantly reduced after 12 weeks in PIO versus controls (intima: P = 0.008; media: P = 0.025). Moreover, echocardiography revealed significantly better functional outcome of the AoC in PIO after 12 weeks compared with control. Interestingly, significantly increased intima hyperplasia could be observed in PIO compared with controls after 12 weeks (P = 0.017). Systemic PPAR-gamma activation prevents inflammation as well as intima and media calcification in AoC and seems to inhibit functional impairment of the implanted aortic valve. To further elucidate the therapeutic role of PPAR-gamma regulation for graft durability, translational studies and long-term follow-up data should be striven for.

Reaktive Sauerstoffspezies (ROS) erlangen in der derzeitigen Diskussion um die Genese der kardiovaskularen Degeneration eine zunehmende Betrachtung als Schlusselfaktor. Verschiedene Modelle versuchen, uber nutritive oder enzymatische Ansatze deren kausale Rolle zu klaren, gehen jedoch mit systemischen Effekten und nichtabschatzbaren Interaktionsmustern einher. In dieser aktuellen Studie wurde das Potenzial einer fotodynamischen Reaktion (PDR), in einem fokal begrenzten Gebiet oxidativen Stress zu induzieren, analysiert. Dabei sollten die initiierten Prozesse und Ergebnisse mit dem klassischen Bild einer atherosklerotischen Gefasdegeneration verglichen werden. Die infrarenale Aorta von Ratten wurde einer PDR aus Bengalrot und einem Niedrigenergie-Laser ausgesetzt. Es wurden Exposition(ROS)-Gruppen und Kontrollgruppen (n = 5) mit 6 verschiedenen Follow-up-Zeitpunkten etabliert. Die Proben wurden sowohl hinsichtlich der morphologischen Veranderungen als auch der Zellkomposition und der Dynamik einer Zellmigration analysiert. Im Gegensatz zu den Kontrollgruppen wiesen die ROS-Tiere initial einen Thrombus auf, der spatestens zum Zeitpunkt t = 8 Tage nicht mehr nachweisbar war. Nach einer Phase der morphologischen Latenz mit signifikant erhohter Matrix-Metalloprotease(MMP)-Aktivitat zeigten sich in dem ROS-exponierten Areal eine signifikante Verdickung der Media (p

Optimized biocompatibility is crucial for the durability of cardiovascular implants. Previously, a combined coating with fibronectin (FN) and stromal cell-derived factor 1α (SDF1α) has been shown to accelerate the in vivo cellularization of synthetic vascular grafts and to reduce the calcification of biological pulmonary root grafts. In this study, we evaluate the effect of side-specific luminal SDF1α coating and adventitial FN coating on the in vivo cellularization and degeneration of decellularized rat aortic implants. Aortic arch vascular donor grafts were detergent-decellularized. The luminal graft surface was coated with SDF1α, while the adventitial surface was coated with FN. SDF1α-coated and uncoated grafts were infrarenally implanted (n = 20) in rats and followed up for up to eight weeks. Cellular intima population was accelerated by luminal SDF1α coating at two weeks (92.4 ± 2.95% versus 61.1 ± 6.51% in controls, p α coating inhibited neo-intimal hyperplasia, resulting in a significantly decreased intima-to-media ratio after eight weeks (0.62 ± 0.15 versus 1.35 ± 0.26 in controls, p α group as compared to the control group (area of calcification in proximal arch region 1092 ± 517 μm2 versus 11 814 ± 1883 μm2, p α promotes early autologous intima recellularization in vivo and attenuates neo-intima hyperplasia as well as calcification of decellularized vascular grafts.

Biomedical materials 14(3), 035014 (2019). doi:10.1088/1748-605X/ab0791, Published by Inst. of Physics, London

Type 2 diabetes is a known risk factor for cardiovascular diseases and is associated with an increased risk to develop aortic heart valve degeneration. Nevertheless, molecular mechanisms leading to the pathogenesis of valve degeneration in the context of diabetes are still not clear. Hence, we hypothesized that classical key factors of type 2 diabetes, hyperinsulinemia and hyperglycemia, may affect signaling, metabolism and degenerative processes of valvular interstitial cells (VIC), the main cell type of heart valves. Therefore, VIC were derived from sheep and were treated with hyperinsulinemia, hyperglycemia and the combination of both. The presence of insulin receptors was shown and insulin led to increased proliferation of the cells, whereas hyperglycemia alone showed no effect. Disturbed insulin response was shown by impaired insulin signaling, i.e. by decreased phosphorylation of Akt/GSK-3α/β pathway. Analysis of glucose transporter expression revealed absence of glucose transporter 4 with glucose transporter 1 being the predominantly expressed transporter. Glucose uptake was not impaired by disturbed insulin response, but was increased by hyperinsulinemia and was decreased by hyperglycemia. Analyses of glycolysis and mitochondrial respiration revealed that VIC react with increased activity to hyperinsulinemia or hyperglycemia, but not to the combination of both. VIC do not show morphological changes and do not acquire an osteogenic phenotype by hyperinsulinemia or hyperglycemia. However, the treatment leads to increased collagen type 1 and decreased α-smooth muscle actin expression. This work implicates a possible role of diabetes in early phases of the degeneration of aortic heart valves.

Degenerative aortic valve disease in combination with diabetes is an increasing burden worldwide. There is growing evidence that particularly small leucine-rich proteoglycans are involved in the development of degenerative aortic valve disease. Nevertheless, the role of these molecules in this disease in the course of diabetes has not been elucidated in detail and previous studies remain controversial. Therefore, the aim of this study is to broaden the knowledge about small leucine-rich proteoglycans in degenerative aortic valve disease and the influence of diabetes and hyperglycaemia on aortic valves and valvular interstitial cells is examined. Analyses were performed using reverse-transcription polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, (immuno)histology and colorimetric assays. We could show that biglycan, but not decorin and lumican, is upregulated in degenerated human aortic valve cusps. Subgroup analysis reveals that upregulation of biglycan is stage-dependent. In vivo, loss of biglycan leads to stage-dependent calcification and also to migratory effects on interstitial cells within the extracellular matrix. In late stages of degenerative aortic valve disease, diabetes increases the expression of biglycan in aortic valves. In vitro, the combinations of hyperglycaemic with pro-degenerative conditions lead to an upregulation of biglycan. In conclusion, biglycan represents a potential link between degenerative aortic valve disease and diabetes.

Reactive oxygen species (ROS) play an important role in the process of cardiovascular degeneration. We evaluated the potential of a controlled, local induction of ROS-release by application of rose bengal (RB) and photo energy to induce atherosclerosis-like focal vascular degeneration in vivo. After injection of RB, rats fed with a pro-degenerative diet underwent focal irradiation of the abdominal aorta by a green laser (ROS group), while the controls received irradiation without RB. Aortic tissue was analyzed by histology and immunohistochemistry at 0, 2, 4, 8, 28 and 56 days (n = 5). The intimal surface topography was analyzed by scanning electron microscopy. In the ROS group, an initial thrombus formation had disappeared by day 8. Similarly, ROS-derived products displayed the highest concentrations at day 0. Relative matrix metalloproteinase (MMP) activity achieved a maximum after 8 days (ROS group vs. control group: 1.60 ± 0.11 vs. 0.98 ± 0.01; p < 0.001). After 28 days, no significant differences in any aspect were found between the ROS group and the controls. However, after 56 days, the aortic tissue of ROS animals exhibited relative media-pronounced thickening (ROS vs. control: 2.15 ± 0.19 vs. 0.87 ± 0.10; p < 0.001) with focal calcification and reduced expression of alpha smooth muscle actin (aSMA). The ROS-releasing application of RB and photo energy allowed for the induction of vascular degeneration in a rodent model. This protocol may be used for the focal induction of vascular disease without systemic side effects and can thereby elucidate the role of ROS in the multifactorial processes of vessel degeneration and atherogenesis.

The native myocardium perfectly meets the demands of a healthy organism during a whole lifetime. But in case of congenital or acquired heart diseases, the native myocardium has to be surgically replaced by an appropriate substitute – the cardiac patch. The used cardiac patches should therefore feature long-term durability, should preferably be of autologous material to reduce immunogenicity eventually occurring in case of xenogenic substitutes, or should – in case of synthetic materials – behave like its autologous counterpart. In terms of pediatric surgery, patches should also be adaptable with the patient׳s growth to minimize the need of follow-on operations.

Although tissue-engineering approaches have led to significant progress in the quest of finding a viable substitute for dysfunctional myocardium, the vascularization of such bioartificial constructs still remains a major challenge. Hence, there is a need for model systems that allow us to study and better understand cardiac and vascular biology to overcome current limitations. Therefore, in this study, in toto decellularized rat hearts with a patent vessel system were processed into standardized coronary artery tissue flaps adherent to the ascending aorta. Protein diffusivity analysis and blood perfusion of the coronary arteries showed proper sealing of the de-endothelialized vessels. Retrograde aortic perfusion allowed for selective seeding of the coronary artery system, while surface seeding of the tissue flaps allowed for additional controlled coculture with cardiac cells. The coronary artery tissue-flap model offers a patent and perfusable coronary vascular architecture with a preserved cardiac extracellular matrix, therefore mimicking nature's input to the highest possible degree. This offers the possibility to study re-endothelialization and endothelial function of different donor cell types and their interaction with cardiac cells in a standardized biologically derived cardiac in vitro model, while establishing a platform that could be used for in vitro drug testing and stem cell differentiation studies.

Decellularization is a promising option to diminish immune and inflammatory response against donor grafts. In order to accelerate the autologous in vivo recellularization of aortic conduits for an enhanced biocompatibility, we tested fibronectin surface coating in a standardized rat implantation model. Detergent-decellularized rat aortic conduits (n = 36) were surface-coated with covalently Alexa488-labeled fibronectin (50 μg/ml, 24 h) and implanted into the systemic circulation of Wistar rats for up to 8 weeks (group FN; n = 18). Uncoated implants served as controls (group C; n = 18). Fibronectin-bound fluorescence on both surfaces of the aortic conduits was persistent for at least 8 weeks. Cellular repopulation was examined by histology and immunofluorescence (n = 24). Luminal endothelialization was significantly accelerated in group FN (p = 0.006 after 8 weeks), however, local myofibroblast hyperplasia with significantly increased ratio of intima-to-media thickness occurred (p = 0.0002 after 8 weeks). Originating from the adventitial surface, alpha-smooth muscle actin and desmin positive cell invasion into the media of fibronectin-coated conduits was significantly increased as compared to group C (p < 0.0001). In these medial areas, in situ zymography revealed enhanced matrix metalloproteinase activity. In both groups, inflammatory cell markers (CD3 and CD68) and signs of thrombosis proved negative. With regard to several markers of cell adhesion, inflammation and calcification, quantitative real-time PCR (n = 12) revealed no significant inter-group differences. Fibronectin surface coating of decellularized cardiovascular implants proved feasible and persistent for at least 8 weeks in the systemic circulation. Biofunctional protein coating accelerated the autologous in vivo endothelialization and induced a significantly increased medial recellularization. Therefore, this strategy may contribute to the improvement of current clinically applied bioprostheses.

In the last decade, cardiovascular tissue engineering has made great progress developing new strategies for regenerative medicine applications. However, while tissue engineered heart valves are already entering the clinical routine, tissue engineered myocardial substitutes are still restrained to experimental approaches. In contrast to the heart valves, tissue engineered myocardium cannot be repopulated in vivo because of its biological complexity, requiring elaborate cultivation conditions ex vivo. Although new promising approaches-like the whole-heart decellularization concept-have entered the myocardial tissue engineering field, bioreactor technology needed for the generation of functional myocardial tissue still lags behind in the sense of user-friendly, flexible and low cost systems. Here, we present a novel customizable modular bioreactor system that can be used for whole-heart cultivation. Out of a commercially obtainable original equipment manufacturer platform we constructed a modular bioreactor system specifically aimed at the cultivation of decellularized whole-hearts through perfusion and controlled 3D biomechanical stimulation with a simple but highly flexible operation platform based on LabVIEW. The modular setup not only allows a wide range of variance regarding medium conditioning under controlled 3D myocardial stretching but can also easily be upgraded for e.g. electrophysiological monitoring or stimulation, allowing for a tailor-made low-cost myocardial bioreactor system.

Hulsmann J, Grun K, El Amouri S, Barth M, Hornung K, Holzfus C, Lichtenberg A, Akhyari P. Transplantation material bovine pericardium: biomechanical and immunogenic characteristics after decellularization vs. glutaraldehyde-fixing. Xenotransplantation 2012; 19: 286–297. © 2012 John Wiley & Sons A/S. Abstract: Background: Today, bovine pericardium (BP) is extensively investigated as a biomaterial for the generation of various bioimplants. But despite the commercial distribution, and the development of methods either to remove (decellularization) or to mask (chemical cross-linking, for example by glutaraldehyde [GA] treatment) the xenogeneic antigen epitopes, yet questions around the immunogenic reactivity of BP remain. The aim of this study is the comparison of crucial tissue characteristics, that is, biomechanical properties, the presence of αGal epitopes, and residual DNA in acellular vs. GA-fixed BP. Methods: Bovine pericardium was either cross-linked with 0.6% GA or decellularized according to two common protocols using either sodium dodecyl sulfate (SDS) and desoxycholic acid (DCA) or trypsin and ethylenediaminetetraacetic acid (EDTA). The resulting extracellular matrix was prone to one-dimensional tensile testing. The tissue content for αGal was evaluated by immunoblotting, and residual DNA was determined by a commercial assay. Untreated BP served as control. Results: In contrast to previous reports, we found a pronounced decrease in the elastic modulus (E-Modulus) for common GA treatment and overall smaller values for the elastic moduli after decellularization (P

Cell–matrix interactions in a three-dimensional (3D) extracellular matrix (ECM) are of fundamental importance in living tissue, and their in vitro reconstruction in bioartificial structures represents a core target of contemporary tissue engineering concepts. For a detailed analysis of cell–matrix interaction under highly controlled conditions, we developed a novel ECM evaluation culture device (EECD) that allows for a precisely defined surface-seeding of 3D ECM scaffolds, irrespective of their natural geometry. The effectiveness of EECD was evaluated in the context of heart valve tissue engineering. Detergent decellularized pulmonary cusps were mounted in EECD and seeded with endothelial cells (ECs) to study EC adhesion, morphology and function on a 3D ECM after 3, 24, 48 and 96 h. Standard EC monolayers served as controls. Exclusive top-surface-seeding of 3D ECM by viable ECs was demonstrated by laser scanning microscopy (LSM), resulting in a confluent re-endothelialization of the ECM after 96 h. Cell viability and protein expression, as demonstrated by MTS assay and western blot analysis (endothelial nitric oxide synthase, von Willebrand factor), were preserved at maintained levels over time. In conclusion, EECD proves as a highly effective system for a controlled repopulation and in vitro analysis of cell–ECM interactions in 3D ECM. Copyright © 2012 John Wiley & Sons, Ltd.

With the introduction of heart valve prostheses cardiac valvular disease has become much more accessible to therapeutic options. However, currently available prostheses display significant limitations, such as limited long-term durability (biological prostheses) and a long-term necessity for anticoagulation therapy. Hence, alternative prosthesis types have been extensively explored in recent years particularly aiming at the development of vital and regenerative prostheses by means of tissue engineering. In the scientific field, different competing concepts have been introduced, including biological or synthetic scaffolds which can be further enhanced by cellular or extracellular components to promote further in vivo development of the prosthesis after implantation. Nowadays, decellularized donor heart valves are among the most advanced prosthesis types experiencing growing clinical attention and widespread use.

Acetylcholine (ACh) has always been regarded as a classical neurotransmitter that binds to nicotinic or muscarinic receptors and mediates signal transmission. The traditional view, that ACh acts solely as a neurotransmitter, has to be revised based on numerous findings demonstrating the existence of a non-neuronal cholinergic system. It is noteworthy that murine and human embryonic stem cells also synthesize ACh and express the enzyme acetylcholinesterase and muscarinic ACh receptors. Here, we investigated the possible role of ACh and AChRs in the regulation of embryonic stem cells. First, the expression of alpha3, alpha4, alpha7 and beta2 nicotinic receptor subunits in embryonic stem cells was investigated by RT-PCR. Second, in vitro studies have been conducted to assess the effects of ACh and its agonists on calcium dynamics, cell survival and proliferation. ACh and nicotine, but not muscarine could induce the mobilization of the intracellular Ca(2+). Interestingly, ACh increased the viability, but decreased the proliferation of embryonic stem cells. Our data provide evidence that ACh might exert its effect on stem cells by binding to specific receptors and modulating cell death and proliferation.

Postnatal development of mammalian cardiomyocytes in the working myocardium is characterized by a near-complete translocation of both kinds of adhering junctions (AJs), i.e. desmosomes and fasciae adhaerentes (FAs), to the polar intercalated disk (ID) regions where they cluster, fuse and molecularly amalgamate to extended hybrid intercellular junction structures, the area composita (composite junction; AC). Using immunofluorescence and immunoelectron microscopy we now report that the AJ structures of the conduction system, in particular those of the Purkinje fiber cells of cows and sheep are fundamentally different. Here the numerous AJs remain in lateral connections with other conductive cells. Desmosomal or desmosome-like junctions can still be distinguished from FA junctions, and a third type of AJs can be identified which shows colocalization of desmosomal and FA proteins, i.e. an AC character. These results, together with demonstrations of other cell type cytoskeletal markers such as alpha-cardiac actin and desmin, support the concept that conductive cells are derived from embryonal cardiomyocytes and are arrested at an early stage of differentiation. We also show that the conductive cells have extended plasma membrane regions characterized by an exceptionally high proportion of junctions with desmosomal character and proteins, amounting to 50% and more, resulting in the highest desmosome protein packing so far described in non-epithelial cells. The relevance of these junctions for the formation, maintenance and functions of the conductive system is discussed, together with the conclusion that the desmosome-rich regions of conductive cells are among the most vulnerable sites for functional disorders caused by desmosomal protein mutations.

Immunocytochemical, electron-, and immunoelectron-microscopical studies have revealed that, in addition to the four major "textbook categories" of cell-cell junctions (gap junctions, tight junctions, adherens junctions, and desmosomes), a broad range of other junctions exists, such as the tiny puncta adhaerentia minima, the taproot junctions (manubria adhaerentia), the plakophilin-2-containing adherens junctions of mesenchymal or mesenchymally derived cell types including malignantly transformed cells, the composite junctions (areae compositae) of the mature mammalian myocardium, the cortex adhaerens of the eye lens, the interdesmosomal "sandwich" or "stud" junctions in the subapical layers of stratified epithelia and the tumors derived therefrom, and the complexus adhaerentes of the endothelial and virgultar cells of the lymph node sinus. On the basis of their sizes and shapes, other morphological criteria, and their specific molecular ensembles, these junctions and the genes that encode them cannot be subsumed under one of the major categories mentioned above but represent special structures in their own right, appear to serve special functions, and can give rise to specific pathological disorders.

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology. Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).