Customized 3D printed bioreactors for decellularization-High efficiency and quality on a budget.

Decellularization (DC) of biomaterials with bioreactors is widely used to produce scaffolds for tissue engineering. This study uses 3D printing to develop efficient but low-cost DC bioreactors. Two bioreactors were developed to decellularize pericardial patches and vascular grafts. Flow profiles and pressure distribution inside the bioreactors were optimized by steady-state computational fluid dynamics (CFD) analysis. Printing materials were evaluated by cytotoxicity assessment. Following evaluation, all parts of the bioreactors were 3D printed in a commercial fused deposition modeling printer. Samples of bovine pericardia and porcine aortae were decellularized using established protocols. An immersion and agitation setup was used as a control. With histological assessment, DNA quantification and biomechanical testing treatment effects were evaluated. CFD analysis of the pericardial bioreactor revealed even flow and pressure distribution in between all pericardia. The CFD analysis of the vessel bioreactor showed increased intraluminal flow rate and pressure compared to the vessel's outside. Cytotoxicity assessment of the used printing material revealed no adverse effect on the tissue. Complete DC was achieved for all samples using the 3D printed bioreactors while DAPI staining revealed residual cells in aortic vessels of the control group. Histological analysis showed no structural changes in the decellularized samples. Additionally, biomechanical properties exhibited no significant change compared to native samples. This study presents a novel approach to manufacturing highly efficient and low budget 3D printed bioreactors for the DC of biomaterials. When compared to standard protocols, the bioreactors offer a cost effective, fast, and reproducible approach, which vastly improves the DC results.

Generation of microbubbles in extracorporeal life support and assessment of new elimination strategies.

Occurrence of microbubbles (MB) is a major problem during venoarterial extracorporeal life support (ECLS) with partially severe clinical complications. The aim of this study was to establish an in vitro ECLS setup for the generation and detection of MB. Furthermore, we assessed different MB elimination strategies. Patient and ECLS circuit were simulated using reservoirs, a centrifugal pump, a membrane oxygenator, and an occluder (modified roller pump). The system was primed with a glycerin solution of 44%. Three different revolution speeds (2500, 3000, and 3400 rpm) were applied. For MB generation, the inflow line of the pump was either statically or dynamically (15 rpm) occluded. A bubble counter was used for MB detection. The effectiveness of the oxygenator and dynamic bubble traps (DBTs) was evaluated in regard to MB elimination capacities. MB generation was highly dependent on negative pressure at the inflow line. Increasing revolution speeds and restriction of the inflow led to increased MB activity. The significant difference between inflow and outflow MB volume identified the centrifugal pump as a main source. We could show that the oxygenator's ability to withhold larger MB is limited. The application of one or multiple DBTs leads to a significant reduction in MB count and overall gas volume. The application of DBT can significantly reduce the overall gas volume, especially at high flow rates. Moreover, large MB can effectively be broken down for faster absorption. In general, the incidence of MBs is significantly dependent on pump speed and restriction of the inflow. The centrifugal pump was identified as a major source of MB generation.

Is Transcatheter Aortic Valve Implantation of Living Tissue-Engineered Valves Feasible? An In Vitro Evaluation Utilizing a Decellularized and Reseeded Biohybrid Valve.

Transcatheter aortic valve implantation (TAVI) is a fast-growing, exciting field of invasive therapy. During the last years many innovations significantly improved this technique. However, the prostheses are still associated with drawbacks. The aim of this study was to create cell-seeded biohybrid aortic valves (BAVs) as an ideal implant by combination of assets of biological and artificial materials. Furthermore, the influence of TAVI procedure on tissue-engineered BAV was investigated. BAV (n=6) were designed with decellularized homograft cusps and polyurethane walls. They were seeded with fibroblasts and endothelial cells isolated from saphenous veins. Consecutively, BAV were conditioned under low pulsatile flow (500 mL/min) for 5 days in a specialized bioreactor. After conditioning, TAVI-simulation was performed. The procedure was concluded with re-perfusion of the BAV for 2 days at an increased pulsatile flow (1100 mL/min). Functionality was assessed by video-documentation. Samples were taken after each processing step and evaluated by scanning electron microscopy (SEM), immunohistochemical staining (IHC), and Live/Dead-assays. The designed BAV were fully functioning and displayed physiologic behavior. After cell seeding, static cultivation and first conditioning, confluent cell layers were observed in SEM. Additionally, IHC indicated the presence of endothelial cells and fibroblasts. A significant construction of extracellular matrix was detected after the conditioning phase. However, a large number of lethal cells were observed after crimping by Live/Dead staining. Analysis revealed that the cells while still being present directly after crimping were removed in subsequent perfusion. Extensive regions of damaged cell-layers were detected by SEM-analysis substantiating these findings. Furthermore, increased ICAM expression was detected after re-perfusion as manifestation of inflammatory reaction. The approach to generate biohybrid valves is promising. However, damages inflicted during the crimping process seem not to be immediately detectable. Due to severe impacts on seeded cells, the strategy of living TE valves for TAVI should be reconsidered.

New perspectives in patient education for cardiac surgery using 3D-printing and virtual reality.

Background: Preoperative anxiety in cardiac surgery can lead to prolonged hospital stays and negative postoperative outcomes. An improved patient education using 3D models may reduce preoperative anxiety and risks associated with it. Methods: Patient education was performed with standardized paper-based methods (n = 34), 3D-printed models (n = 34) or virtual reality models (n = 31). Anxiety and procedural understanding were evaluated using questionnaires prior to and after the patient education. Additionally, time spent for the education and overall quality were evaluated among further basic characteristics (age, gender, medical expertise, previous non-cardiac surgery and previously informed patients). Included surgeries were coronary artery bypass graft, surgical aortic valve replacement and thoracic aortic aneurysm surgery. Results: A significant reduction in anxiety measured by Visual Analog Scale was achieved after patient education with virtual reality models (5.00 to 4.32, Δ-0.68, p < 0.001). Procedural knowledge significantly increased for every group after the patient education while the visualization and satisfaction were best rated for patient education with virtual reality. Patients rated the quality of the patient education using both visualization methods individually [3D and virtual reality (VR) models] higher compared to the control group of conventional paper-sheets (control paper-sheets: 86.32 ± 11.89%, 3D: 94.12 ± 9.25%, p < 0.0095, VR: 92.90 ± 11.01%, p < 0.0412). Conclusion: Routine patient education with additional 3D models can significantly improve the patients' satisfaction and reduce subjective preoperative anxiety effectively.

Use of a special bioreactor for the cultivation of a new flexible polyurethane scaffold for aortic valve tissue engineering.

BACKGROUND: Tissue engineering represents a promising new method for treating heart valve diseases. The aim of this study was evaluate the importance of conditioning procedures of tissue engineered polyurethane heart valve prostheses by the comparison of static and dynamic cultivation methods. METHODS: Human vascular endothelial cells (ECs) and fibroblasts (FBs) were obtained from saphenous vein segments. Polyurethane scaffolds (n = 10) were primarily seeded with FBs and subsequently with ECs, followed by different cultivation methods of cell layers (A: static, B: dynamic). Group A was statically cultivated for 6 days. Group B was exposed to low flow conditions (t1=3 days at 750 ml/min, t2=2 days at 1100 ml/min) in a newly developed conditioning bioreactor. Samples were taken after static and dynamic cultivation and were analyzed by scanning electron microscopy (SEM), immunohistochemistry (IHC), and real time polymerase chain reaction (RT-PCR). RESULTS: SEM results showed a high density of adherent cells on the surface valves from both groups. However, better cell distribution and cell behavior was detected in Group B. IHC staining against CD31 and TE-7 revealed a positive reaction in both groups. Higher expression of extracellular matrix (ICAM, Collagen IV) was observed in Group B. RT- PCR demonstrated a higher expression of inflammatory Cytokines in Group B. CONCLUSION: While conventional cultivation method can be used for the development of tissue engineered heart valves. Better results can be obtained by performing a conditioning step that may improve the tolerance of cells to shear stress. The novel pulsatile bioreactor offers an adequate tool for in vitro improvement of mechanical properties of tissue engineered cardiovascular prostheses.

3D-printed heart models for hands-on training in pediatric cardiology - the future of modern learning and teaching?

Background: This project aims to develop a new concept in training pediatric cardiologists to meet the requirements of interventional cardiac catheterizations today in terms of complexity and importance. This newly developed hands-on training program is supposed to enable the acquisition of certain skills which are necessary when investigating and treating patients in a catheter laboratory. Methods: Based on anonymous CT-scans of pediatric patients' digital 3D heart models with or without cardiac defects were developed and printed three-dimensionally in a flexible material visible under X-ray. Hands-on training courses were offered using models of a healthy heart and the most common congenital heart defects (CHD). An evaluation was performed by quantifying fluoroscopy times (FL-time) and a questionnaire. Results: The acceptance of theoretical and practical contents within the hands-on training was very positive. It was demonstrated that it is possible to master various steps of a diagnostic procedure and an intervention as well as to practice and repeat them independently which significantly reduced FL-time. The participants stated that the hands-on training led to more confidence in interventions on real patients. Conclusion: With the development of a training module using 3D-printed heart models, basic and advanced training in the field of diagnostic cardiac examinations as well as interventional therapies of CHD is possible. The learning effect for both, practical skills and theoretical understanding, was significant which underlines the importance of integrating such hands-on trainings on 3D heart models in education and practical training.

Combining 3D-Printing and Electrospinning to Manufacture Biomimetic Heart Valve Leaflets.

Electrospinning has become a widely used technique in cardiovascular tissue engineering as it offers the possibility to create (micro-)fibrous scaffolds with adjustable properties. The aim of this study was to create multilayered scaffolds mimicking the architectural fiber characteristics of human heart valve leaflets using conductive 3D-printed collectors. Models of aortic valve cusps were created using commercial computer-aided design (CAD) software. Conductive polylactic acid was used to fabricate 3D-printed leaflet templates. These cusp negatives were integrated into a specifically designed, rotating electrospinning mandrel. Three layers of polyurethane were spun onto the collector, mimicking the fiber orientation of human heart valves. Surface and fiber structure was assessed with a scanning electron microscope (SEM). The application of fluorescent dye additionally permitted the microscopic visualization of the multilayered fiber structure. Tensile testing was performed to assess the biomechanical properties of the scaffolds. 3D-printing of essential parts for the electrospinning rig was possible in a short time for a low budget. The aortic valve cusps created following this protocol were three-layered, with a fiber diameter of 4.1 ± 1.6 µm. SEM imaging revealed an even distribution of fibers. Fluorescence microscopy revealed individual layers with differently aligned fibers, with each layer precisely reaching the desired fiber configuration. The produced scaffolds showed high tensile strength, especially along the direction of alignment. The printing files for the different collectors are available as Supplemental File 1, Supplemental File 2, Supplemental File 3, Supplemental File 4, and Supplemental File 5. With a highly specialized setup and workflow protocol, it is possible to mimic tissues with complex fiber structures over multiple layers. Spinning directly on 3D-printed collectors creates considerable flexibility in manufacturing 3D shapes at low production costs.

Bridging patients in cardiogenic shock with a paracorporeal pulsatile biventricular assist device to heart transplantation-a single-centre experience.

OBJECTIVES: We evaluated the outcome of patients in cardiogenic shock receiving a paracorporeal pulsatile biventricular assist device as a bridge to transplantation. METHODS: We performed a retrospective single-centre analysis of all patients who received a Berlin Heart Excor® at our institution between 2004 and 2019. RESULTS: A total of 97 patients (90 adults, 7 paediatric) were analysed. Eighty-four patients were in Interagency Registry for Mechanically Assisted Circulatory Support level 1 (80 adults, 4 paediatric). Diagnoses were dilated cardiomyopathy (n = 41), ischaemic cardiomyopathy (n = 17) or myocardial infarction (n = 4), myocarditis (n = 15), restrictive cardiomyopathy (n = 2), graft failure after heart transplant (n = 7), postcardiotomy heart failure (n = 5), postpartum cardiomyopathy (n = 3), congenital heart disease (n = 1), valvular cardiomyopathy (n = 1) and toxic cardiomyopathy (n = 1). All patients were in biventricular heart failure and had secondary organ dysfunction. The mean duration of support was 63 days (0-487 days). There was a significant decrease in creatinine values after assist device implantation (from 1.83 ± 0.79 to 1.12 ± 0.67 mg/dl, P = 0.001) as well as a decrease in bilirubin values (from 3.94 ± 4.58 to 2.65 ± 3.61 mg/dl, P = 0.084). Cerebral stroke occurred in 16 patients, bleeding in 15 and infection in 13 patients. Forty-eight patients died on support, while 49 patients could be successfully bridged to transplantation. Thirty-day survival and 1-year survival were 70.1% and 41.2%, respectively. CONCLUSIONS: A pulsatile biventricular assist device is a reasonable therapeutic option in cardiogenic shock, when immediate high cardiac output is necessary to rescue the already impaired kidney and liver function of the patient.

Mapping of bovine pericardium to enable a standardized acquirement of material for medical implants.

OBJECTIVES: Bovine pericardium - native, fixed as well as decellularized - is one of the most common implant materials in modern cardiovascular surgery. Although used in everyday procedures, there are no recommendations in regard to which part of the pericardium to prefer. It was the aim of this study, to identify areas of the pericardium with consistent properties and high durability. METHODS: Fresh bovine pericardia were collected from a local slaughterhouse. The native pericardia were analyzed at 140 spots in regard to thickness and fiber orientation. Based on these results, five promising areas were selected for further evaluation. The pericardia were decellularized with detergents (0.5% sodiumdesoxycholate/0.5% sodiumdodecylsulfate) and subsequently incubated in DNAse. The two investigation groups native und DC consisted of 20 samples each. The efficiency of the decellularization was evaluated by DNA quantification, as well as DAPI and H&E staining. Biomechanical properties were determined using uniaxial tensile tests. To evaluate the microstructure, scanning electron microscopy, Picrosirius Red- and Movat's Pentachrome staining were utilized. To assess the long-term durability, patches were tested in a high-cycle system for a duration equaling the stress of three months in-vivo. Commercially available, fixed pericardium patches served as control group. RESULTS: Only a limited part of the pericardium showed a homogenous and usable thickness. The decellularization removed all cell nuclei, proven by negative DAPI and H&E staining, and also significantly reduced the DNA amount by 84%. The mechanical testing revealed that two investigated areas had an inconsistent tensile strength. Microscopical observations showed that the integrity of the extracellular matrix did not suffer by the decellularization procedure. During the long-term testing, most of the pericardia slowly lost tautness, though none of them got measurably damaged. Especially one area showed no decline of tensile strength after durability testing at all. Decellularized patches and fixed patches achieved comparable results in mechanical testing and microscopical evaluation after the durability testing. CONCLUSION: We could clearly document significant, location-based differences within single pericardia. Only one area showed consistent properties and a high durability. We highly recommend taking this into account for future implant material selections.

Design and 3D printing of variant pediatric heart models for training based on a single patient scan.

BACKGROUND: 3D printed models of pediatric hearts with congenital heart disease have been proven helpful in simulation training of diagnostic and interventional catheterization. However, anatomically accurate 3D printed models are traditionally based on real scans of clinical patients requiring specific imaging techniques, i.e., CT or MRI. In small children both imaging technologies are rare as minimization of radiation and sedation is key. 3D sonography does not (yet) allow adequate imaging of the entire heart for 3D printing. Therefore, an alternative solution to create variant 3D printed heart models for teaching and hands-on training has been established. METHODS: In this study different methods utilizing image processing and computer aided design software have been established to overcome this shortage and to allow unlimited variations of 3D heart models based on single patient scans. Patient-specific models based on a CT or MRI image stack were digitally modified to alter the original shape and structure of the heart. Thereby, 3D hearts showing various pathologies were created. Training models were adapted to training level and aims of hands-on workshops, particularly for interventional cardiology. RESULTS: By changing the shape and structure of the original anatomy, various training models were created of which four examples are presented in this paper: 1. Design of perimembranous and muscular ventricular septal defect on a heart model with patent ductus arteriosus, 2. Series of heart models with atrial septal defect showing the long-term hemodynamic effect of the congenital heart defect on the right atrial and ventricular wall, 3. Implementation of simplified heart valves and addition of the myocardium to a right heart model with pulmonary valve stenosis, 4. Integration of a constructed 3D model of the aortic valve into a pulsatile left heart model with coarctation of the aorta. All presented models have been successfully utilized and evaluated in teaching or hands-on training courses. CONCLUSIONS: It has been demonstrated that non-patient-specific anatomical variants can be created by modifying existing patient-specific 3D heart models. This way, a range of pathologies can be modeled based on a single CT or MRI dataset. Benefits of designed 3D models for education and training purposes have been successfully applied in pediatric cardiology but can potentially be transferred to simulation training in other medical fields as well.

Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training.

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training physicians' wire-skills as well as improving planning of interventional procedures. The aim of this study was to develop a manufacturing process of patient-specific 3D-printed models for cardiovascular interventions. To create a 3D-printed elastic phantom, different 3D-printing materials were compared to porcine biological tissues (i.e., aortic tissue) in terms of mechanical characteristics. A fitting material was selected based on comparative tensile tests and specific material thicknesses were defined. Anonymized contrast-enhanced CT-datasets were collected retrospectively. Patient-specific volumetric models were extracted from these datasets and subsequently 3D-printed. A pulsatile flow loop was constructed to simulate the intraluminal blood flow during interventions. Models' suitability for clinical imaging was assessed by x-ray imaging, CT, 4D-MRI and (Doppler) ultrasonography. Contrast medium was used to enhance visibility in x-ray-based imaging. Different catheterization techniques were applied to evaluate the 3D-printed phantoms in physicians' training as well as for pre-interventional therapy planning. Printed models showed a high printing resolution (~30 µm) and mechanical properties of the chosen material were comparable to physiological biomechanics. Physical and digital models showed high anatomical accuracy when compared to the underlying radiological dataset. Printed models were suitable for ultrasonic imaging as well as standard x-rays. Doppler ultrasonography and 4D-MRI displayed flow patterns and landmark characteristics (i.e., turbulence, wall shear stress) matching native data. In a catheter-based laboratory setting, patient-specific phantoms were easy to catheterize. Therapy planning and training of interventional procedures on challenging anatomies (e.g., congenital heart disease (CHD)) was possible. Flexible patient-specific cardiovascular phantoms were 3D-printed, and the application of common clinical imaging techniques was possible. This new process is ideal as a training tool for catheter-based (electrophysiological) interventions and can be used in patient-specific therapy planning.

3-dimensional printing for the diagnosis of left ventricular outflow tract obstruction after mitral valve replacement.

The objective of this study was to evaluate the use of the generation of 3D models and 3D prints of complex cases for physicians at the example of an intricate left ventricular outflow tract obstruction (LVOTO). LVOTO is a known complication of mitral valve surgery. A 38-year-old female patient with increasing dyspnoea after mitral valve replacement was referred to our centre. Echocardiography showed a strut of the bioprosthetic heart valve protruding into the left ventricular outflow tract. However, the diagnosis of a LVOTO was difficult based on echocardiography alone. Therefore, we fabricated a physical model of the left ventricular outflow tract, the mitral valve, the aortic valve and the left ventricle. With this physical model in hand, we were able to visualize the LVOTO and to discuss potential therapeutic options. Moreover, we were able to plan the subsequent redo surgery in detail using the model. This case shows the benefit of 3D printing technologies for surgeons and patients, not only for analysis, but also during the decision-making and pre-operative planning process.

Customized stent-grafts for endovascular aneurysm repair with challenging necks: A numerical proof of concept.

Endovascular aortic repair (EVAR) is a challenging intervention whose long-term success strongly depends on the appropriate stent-graft (SG) selection and sizing. Most off-the-shelf SGs are straight and cylindrical. Especially in challenging vessel morphologies, the morphology of off-the-shelf SGs is not able to meet the patient-specific demands. Advanced manufacturing technologies facilitate the development of highly customized SGs. Customized SGs that have the same morphology as the luminal vessel surface could considerably improve the quality of the EVAR outcome with reduced likelihoods of EVAR related complications such as endoleaks type I and SG migration. In this contribution, we use an in silico EVAR methodology that approximates the deployed state of the elastically deformable SG in a hyperelastic, anisotropic vessel. The in silico EVAR results of off-the-shelf SGs and customized SGs are compared qualitatively and quantitatively in terms of mechanical and geometrical parameters such as stent stresses, contact tractions, SG fixation forces and the SG-vessel attachment. In a numerical proof of concept, eight different vessel morphologies, such as a conical vessel, a barrel shaped vessel and a curved vessel, are used to demonstrate the added value of customized SGs compared to off-the-shelf SGs. The numerical investigation has shown large benefits of the highly customized SGs compared to off-the-shelf SGs with respect to a better SG-vessel attachment and a considerable increase in SG fixation forces of up to 50% which indicate decreased likelihoods of EVAR related complications. Hence, this numerical proof of concept motivates further research and development of highly customized SGs for the use in challenging vessel morphologies.

Successful decellularization of thick-walled tissue: Highlighting pitfalls and the need for a multifactorial approach.

INTRODUCTION:: Decellularization of thick tissue is challenging and varying. Therefore, we tried to establish a multifactorial approach for reliable aortic wall decellularization. METHODS:: Porcine aortic walls were decellularized according to different procedures. Decellularization was performed for 24 (G1), 48 (G2), and 72 h (G3) with a solution of 0.5% desoxycholate and 0.5% dodecyl sulfate. The procedure was characterized using intermittent washing steps, the inclusion of sonication as well as DNase and α-galactosidase treatment. The decellularization efficiency was measured by the evaluation of 4',6-diamidino-2-phenylindole and hematoxylin and eosin staining and quantitative DNA assays. Pentachrome and picrosirius red staining, scanning electron microscopy as well as glycosaminoglycan assays were performed to evaluate the effect of the procedure on the extracellular matrix. RESULTS:: 4',6-Diamidino-2-phenylindole and hematoxylin and eosin staining revealed a large amount of remaining nuclei in all groups. However, consecutive DNase treatment had a significant effect. While the remaining DNA was detected in some samples of G1 and G2, samples of G3 were fully decellularized. Glycosaminoglycan content was significantly reduced to 50% after 24 h (G1) but remained constant for G2 and G3. Picrosirius red staining revealed an intact and stable collagen network without any visible defects. Pentachrome staining substantiated these results. Nonetheless, the fiber network remains intact, which could be confirmed by reflection electron microscopy analysis. CONCLUSION:: In this study, we developed a procedure that grants successful decellularization of porcine aortic wall while maintaining the fibrous microstructure. We highlighted the significant effect of DNase and α-galactosidase treatment. In addition, we could show the need for a multifactorial treatment and comprehensive evaluation protocols for thick tissue decellularization.

Tuning Tissue Ingrowth into Proangiogenic Hydrogels via Dual Modality Degradation.

The potential to control the rate of replacement of a biodegradable implant by a tissue would be advantageous. Here, we demonstrate that tissue invasion can be tuned through the novel approach of overlaying an enzymatically degradable hydrogel with an increasingly hydrolytically degradable environment. Poly(ethylene glycol) (PEG) hydrogels were formed from varying proportions of PEG-vinyl sulfone and PEG-acrylate (PEG-AC) monomers via a Michael-type addition reaction with a dithiol-containing matrix-metalloproteinase-susceptible peptide cross-linker. Swelling studies showed that PEG hydrogels with similar initial stiffnesses degraded more rapidly as the PEG-AC content increased. The replacement of subcutaneously implanted PEG hydrogels was also found to be proportional to their PEG-AC content. In addition, it would in many instances be desirable that these materials have the ability to stimulate their neovascularization. These hydrogels contained covalently bound heparin, and it was shown that a formulation of the hydrogel that allowed tissue replacement to occur over 1 month could trap and release growth factors and increase neovascularization by 50% over that time.

The cardiotomy reservoir - a preliminary evaluation of a new cell source for cardiovascular tissue engineering.

OBJECTIVES: Cell sources for cardiovascular tissue engineering (TE) are scant. However, the need for an ideal TE cardiovascular implant persists. We investigated the cardiotomy reservoir (CR) as a potential cell source that is more accessible and less ethically problematic. METHODS: CR (n = 10) were removed from the bypass system after surgery. Isolation was performed using different isolation methods: blood samples were taken from the cardiopulmonary bypass and centrifuged at low density. The venous filter screen was cut out and placed into petri dishes for cultivation. The spongelike filter was removed, washed and treated in the same way as the blood samples. After cultivation, cell lines of fibroblasts (FB) and endothelial cells (EC) were obtained for analysis. The cells were seeded on polyurethane patches and analyzed via scanning electron microscopy (SEM), Life/Dead assay and immunohistochemistry. RESULTS: No correlation between age, time of surgery and quality of cells was observed. The successful extraction of FB and was proven by positive staining results for TE-7, CD31 and vWF. Cell morphology, cytoskeleton staining and quantification of proliferation using WST-1 assay resembled the cells of the control group in all ways. The topography of a confluent and vital cell layer after cell seeding was displayed by SEM analysis, Life/Dead Assay and immunohistochemistry. The establishment of an extracellular matrix (ECM) was proven by positive staining for collagen IV, laminin, fibronectin and elastin. CONCLUSIONS: Viable FB and EC cell lines were extracted from the CR after surgery. Easy access and high availability make this cell source destined for widespread application in cardiovascular tissue engineering.

Noninvasive analysis of synthetic and decellularized scaffolds for heart valve tissue engineering.

Microcomputed tomography (µ-CT) is a nondestructive, high-resolution, three-dimensional method of analyzing objects. The aim of this study was to evaluate the feasibility of using µ-CT as a noninvasive method of evaluation for tissue-engineering applications. The polyurethane aortic heart valve scaffold was produced using a spraying technique. Cryopreserved/thawed homograft and biological heart valve were decellularized using a detergent mixture. Human endothelial cells and fibroblasts were derived from saphenous vein segments and were verified by immunocytochemistry. Heart valves were initially seeded with fibroblasts followed by colonization with endothelial cells. Scaffolds were scanned by a µ-CT scanner before and after decellularization as well as after cell seeding. Successful colonization was additionally determined by scanning electron microscopy (SEM) and immunohistochemistry (IHC). Microcomputed tomography accurately visualized the complex geometry of heart valves. Moreover, an increase in the total volume and wall thickness as well as a decrease in total surface was demonstrated after seeding. A confluent cell distribution on the heart valves after seeding was confirmed by SEM and IHC. We conclude that µ-CT is a new promising noninvasive method for qualitative and quantitative analysis of tissue-engineering processes.

In vitro comparison of novel polyurethane aortic valves and homografts after seeding and conditioning.

The aim of the study was to compare the behavior of seeded cells on synthetic and natural aortic valve scaffolds during a low-flow conditioning period. Polyurethane (group A) and aortic homograft valves (group B) were consecutively seeded with human fibroblasts (FB), and endothelial cells (EC) using a rotating seeding device. Each seeding procedure was followed by an exposure to low pulsatile flow in a dynamic bioreactor for 5 days. For further analysis, samples were taken before and after conditioning. Scanning electron microscopy showed confluent cell layers in both groups. Immunohistochemical analysis showed the presence of EC and FB before and after conditioning as well as the establishment of an extracellular matrix (ECM) during conditioning. A higher expression of ECM was observed on the scaffolds' inner surface. Real-time polymerase chain reaction showed higher inflammatory response during the conditioning of homografts. Endothelialization caused a decrease in inflammatory gene expression. The efficient colonization, the establishment of an ECM, and the comparable inflammatory cell reaction to the scaffolds in both groups proved the biocompatibility of the synthetic scaffold. The newly developed bioreactor permits conditioning and cell adaption to shear stress. Therefore, polyurethane valve scaffolds may offer a new option for aortic valve replacement.