Transapical heart valve implantation is a common minimally invasive procedure for valve replacement in high-risk patients. However, the use of synthetic prostheses for this procedure is limited due to reduced long-term durability. Tissue engineered stented heart valves are promising as prospective transcatheter heart valve prostheses. Therefore, the aim of this study was to determine the mechanical integrity of tissue engineered stented heart valves after crimping procedure. Stented non-degradable polyurethane heart valves (sPUHV) were successively seeded with fibroblasts and endothelial cells derived from human saphenous vein segments. Dynamic seeding procedure was performed by using a special 3D-rotating bioreactor. Colonized stented heart valves were exposed to mechanical loadings by crimping in a conventional heart valve crimper and were analyzed by micro-computerized tomography (µ-CT), scanning electron microscopy (SEM), immunohistochemistry (IHC) and immunocytochemistry (ICC). µ-CT showed no damage of the sPUHV after crimping. A stable cellular coating with intact cell surfaces of crimped heart valves was demonstrated by SEM analysis. ICC and IHC observations also revealed the mechanical integrity of endothelial and fibroblast layers after crimping technique. It could be demonstrated that the crimping procedure does not affect the integrity of sPUHV structure and the cellular coating. This offers a potential use for tissue engineered stented heart valves for transapical heart valve replacement.
Ischemic heart disease is a major cause of mortality worldwide. Myocardial tissue engineering aims to create transplantable units of myocardium for the treatment of myocardial necrosis caused by ischemic heart disease - bioreactors are used to condition these bioartificial tissues before application.Our group developed a multimodal bioreactor consisting of a linear drive motor for pulsatile flow generation (500 ml/min) and an external pacemaker for electrical stimulation (10 mA, 3 V at 60 Hz) using LinMot-Talk Software to synchronize these modes of stimulation. Polyurethane scaffolds were seeded with 0.750 × 106 mesenchymal stem cells from umbilical cord tissue per cm2 and stimulated in our system for 72 h, then evaluated.After conditioning histology showed that the patches consisted of a cell multilayer surviving stimulation without major damage by the multimodal stimulation, scanning electron microscopy showed a confluent cell layer with no cell-cell interspaces visible. No cell viability issues could be identified via Syto9-Propidium Iodide staining.This bioreactor allows mechanical stimulation via pulsatile flow and electrical stimulation through a pacemaker. Our stem cell-polyurethane constructs displayed survival after conditioning. This system shows feasibility in preliminary tests.
After myocardial infarction, the implantation of stem cell seeded scaffolds on the ischemic zone represents a promising strategy for restoration of heart function. However, mechanical integrity and functionality of tissue engineered constructs need to be determined prior to implantation. Therefore, in this study a novel pulsatile bioreactor mimicking the myocardial contraction was developed to analyze the behavior of mesenchymal stem cells derived from umbilical cord tissue (UCMSC) colonized on titanium-coated polytetrafluorethylene scaffolds to friction stress. The design of the bioreactor enables a simple handling and defined mechanical forces on three seeded scaffolds at physiological conditions. The compact system made of acrylic glass, Teflon®, silicone, and stainless steel allows the comparison of different media, cells and scaffolds. The bioreactor can be gas sterilized and actuated in a standard incubator. Macroscopic observations and pressure-measurements showed a uniformly sinusoidal pulsation, indicating that the bioreactor performed well. Preliminary experiments to determine the adherence rate and morphology of UCMSC after mechanical loadings showed an almost confluent cellular coating without damage on the cell surface. In summary, the bioreactor is an adequate tool for the mechanical stress of seeded scaffolds and offers dynamic stimuli for pre-conditioning of cardiac tissue engineered constructs in vitro.
Aims: Multiple efforts were made to develop small-diameter tissue engineered vascular grafts by using different bioreactor systems. However, there is still an extensive need for a compact all-in-one system that allows multiple and simultaneous processing. The aim of this project was to develop a new device to fulfill the major requirements of an ideal bioreactor.
Cardiovascular tissue engineering has emerged as a promising approach to overcome limitations of conventional heart valve substitutes regarding lack of growth, repair, and remodeling capability by mimicking a native heart valve. The present study has demonstrated that long-term conditioning of decellularized and re-seeded aortic homografts in a low-flow pulsatile bioreactor results in an improved quality of tissue engineering constructs. Cryopreserved and thawed homografts were decellularized by a detergent mixture. Decellularized homografts were primarily seeded with fibroblasts (FB) followed by colonization with endothelial cells (EC), both isolated from human saphenous vein segments. Re-seeded homografts were exposed to low-flow conditions (750-1 100 mL/min) for a time period of 12 d. Topographical examination was performed by scanning electron microscopy (SEM). Cell layer thickness, composition of extracellular matrix (ECM) and inflammatory response was investigated by immunohistochemistry (IHC). SEM analysis of re-seeded homografts showed a confluent and intact cellular coverage before and after conditioning. IHC demonstrated a distinct thickening of cellular layer. Cell specific staining demonstrated a confluent EC lining with a multilayer of FB underneath. The expression of ECM components, cytoskeletal and gap junctional proteins increased by conditioning. Inflammatory proteins were expressed in a low level. The novel pulsatile bioreactor provides a strong tool for conditioning of re-seeded decellularized homografts. Moreover, conditioning results in an increased quality of ECM in regard to connectivity, stability and cell communication, creating native-like heart valve prostheses.
Umbilical cord tissue comprises an attractive new source for mesenchymal stem cells. Umbilical cord tissue-derived mesenchymal stem cells (UCMSC) exhibit self-renewal, multipotency and immunological naivity, and they can be obtained without medical intervention. The transfer of UCMSC to the ischemic region of the heart may have a favorable impact on tissue regeneration. Benefit from typical cell delivery by injection to the infarcted area is often limited due to poor cell retention and survival. Another route of administration is to use populated scaffolds implanted into the infarcted zone. In this paper, the seeding efficiency of UCMSC on uncoated and titanium-coated expanded polytetrafluoroethylene (ePTFE) scaffolds with different surface structures was determined. Dualmesh® (DM) offers a corduroy-like surface in contrast to the comparatively planar surface of cardiovascular patch (CVP). The investigation of adherence, viability and proliferation of UCMSC demonstrates that titanium-coated scaffolds are superior to uncoated scaffolds, independent of the surface structure. Microscopic images reveal spherical UCMSC seeded on uncoated scaffolds. In contrast, UCMSC on titanium-coated scaffolds display their characteristic spindle-shaped morphology and a homogeneous coverage of CVP. In summary, titanium coating of clinically approved CVP enhances the retention of UCMSC and thus offers a potential cell delivery system for the repair of the damaged myocardium.
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.
Objectives: CD133pos cells are currently evaluated for use in cardiac cell therapy.We hypothesized that they exert their beneficial effects in a paracrine manner and investigated this in a cell culture ischaemia model.Furthermore, we checked whether purified CD133pos cells perform better than non-fractionated mononuclear cells (MNC).Methods: CD133pos cells were isolated from bone marrow MNC and conditioned medium was prepared from CD133pos and non-fractionated MNC.HL-1 cardiomyocytes were subjected to simulated ischaemia in the respective conditioned media or in control medium.After treatment, total remaining cells, apoptotic cells and nuclear shrinking were quantified using an automated imaging system.Furthermore, metabolic activity and phosphorylation of kinases Akt, Erk1/2, GSK3b and transcription factor Stat3 were investigated.Results: After simulated ischaemia, the rate of detached dead cells was lowest in CD133pos conditioned medium (26 ± 6%) and highest in control medium (36 ± 6%).In CD133pos conditioned medium, the fraction of nonapoptotic cells was most enhanced and nuclear shrinking as a consequence of apoptosis was reduced.Cell viability was also highest in CD133pos conditioned medium (109.4 ± 8.8% in relation to control).In both conditioned media, phosphorylation of Akt, Erk1/2, and GSK3b was lower than in control medium.Stat3 phosphorylation was sustained on the level of control.Conclusions: Factors released from purified CD133pos bone marrow cells exhibit more pronounced protective effects on HL-1 cardiomyocytes under simulated ischaemia than from non-fractionated MNC.These effects are not associated with the phosphorylation of cell survival promoting kinases Akt, Erk1/2, GSK3b and transcription factor Stat3.Although the molecular mechanism of cardioprotection by CD133pos cells requires further investigation, our results reinforce the advantage of enriching CD133pos cells for cardiac cell therapy.
