A feasibility study of a multimodal stimulation bioreactor for the conditioning of stem cell seeded cardiac patches via electrical impulses and pulsatile perfusion.

BACKGROUND/OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

In vitro biological and mechanical evaluation of various scaffold materials for myocardial tissue engineering.

A cardiac patch is a construct devised in regenerative medicine to replace necrotic heart tissue after myocardial infarctions. The cardiac patch consists of a scaffold seeded with stem cells. To identify the best scaffold for cardiac patch construction we compared polyurethane, Collagen Cell Carriers, ePTFE, and ePTFE SSP1-RGD regarding their receptiveness to seeding with mesenchymal stem cells isolated from umbilical cord tissue. Seeding was tested at an array of cell seeding densities. The bioartificial patches were cultured for up to 35 days and evaluated by scanning electron microscopy, microscopy of histological stains, fluorescence microscopy, and mitochondrial assays. Polyurethane was the only biomaterial which resulted in an organized multilayer (seeding density: 0.750 × 10(6) cells/cm(2)). Cultured over 35 days at this seeding density the mitochondrial activity of the cells on polyurethane patches continually increased. There was no decrease in the E Modulus of polyurethane once seeded with cells. Seeding of CCC could only be realized at a low seeding density and both ePTFE and ePTFE SSP1-RGD were found to be unreceptive to seeding. Of the tested scaffolds polyurethane thus crystallized as the most appropriate for seeding with mesenchymal stem cells in the framework of myocardial 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.

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.

A Pulsatile Bioreactor for Conditioning of Tissue-Engineered Cardiovascular Constructs under Endoscopic Visualization.

Heart valve disease (HVD) is a globally increasing problem and accounts for thousands of deaths yearly. Currently end-stage HVD can only be treated by total valve replacement, however with major drawbacks. To overcome the limitations of conventional substitutes, a new clinical approach based on cell colonization of artificially manufactured heart valves has been developed. Even though this attempt seems promising, a confluent and stable cell layer has not yet been achieved due to the high stresses present in this area of the human heart. This study describes a bioreactor with a new approach to cell conditioning of tissue engineered heart valves. The bioreactor provides a low pulsatile flow that grants the correct opening and closing of the valve without high shear stresses. The flow rate can be regulated allowing a steady and sensitive conditioning process. Furthermore, the correct functioning of the valve can be monitored by endoscope surveillance in real-time. The tubeless and modular design allows an accurate, simple and faultless assembly of the reactor in a laminar flow chamber. It can be concluded that the bioreactor provides a strong tool for dynamic pre-conditioning and monitoring of colonized heart valve prostheses physiologically exposed to shear stress.

Mesenchymal stem cells from umbilical cord tissue as potential therapeutics for cardiomyodegenerative diseases - a review.

Heart failure is one of the leading causes of death worldwide. End stage disease often requires heart transplantation, which is hampered by donor organ shortage. Tissue engineering represents a promising alternative approach for cardiac repair. For the generation of artificial heart muscle tissue several cell types, scaffold materials and bioreactor designs are under investigation. In this review, the use of mesenchymal stem cells derived from human umbilical cord tissue (UCMSC) for cardiac tissue engineering will be discussed.

A novel pulsatile bioreactor for mechanical stimulation of tissue engineered cardiac constructs.

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.

Umbilical cord tissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypic and functional properties.

Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells that can differentiate into cell types of mesenchymal origin. Because of their immune properties and differentiation, potential MSCs are discussed for the use in tissue regeneration and tolerance induction in transplant medicine. This cell type can easily be obtained from the umbilical cord tissue (UCMSC) without medical intervention. Standard culture conditions include fetal bovine serum (FBS) which may not be approved for clinical settings. Here, we analyzed the phenotypic and functional properties of UCMSC under xeno-free (XF, containing GMP-certified human serum) and serum-free (SF) culture conditions in comparison with standard UCMSC cultures. Phenotypically, UCMSC showed no differences in the expression of mesenchymal markers or differentiation capacity. Functionally, XF and SF-cultured UCMSC have comparable adipogenic, osteogenic, and endothelial differentiation potential. Interestingly, the UCMSC-mediated suppression of T cell proliferation in an allogeneic mixed lymphocyte reaction (MLR) is more effective in XF and SF media than in standard FBS-containing cultures. Regarding the mechanism of action of MLR suppression, transwell experiments revealed that in neither UCMSC culture a direct cell-cell contact is necessary for inhibiting T cell proliferation, and that the major effector molecule is prostaglandin E2 (PGE2). Taken together, GMP-compliant growth media qualify for long-term cultures of UCMSC which is important for a future clinical study design in regenerative and transplant medicine.

Comparative analysis of adherence, viability, proliferation and morphology of umbilical cord tissue-derived mesenchymal stem cells seeded on different titanium-coated expanded polytetrafluoroethylene scaffolds.

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.