Julia Frese

Multiple-Step Injection Molding for Fibrin-Based Tissue-Engineered Heart Valves.

Heart valves are elaborate and highly heterogeneous structures of the circulatory system. Despite the well accepted relationship between the structural and mechanical anisotropy and the optimal function of the valves, most approaches to create tissue-engineered heart valves (TEHVs) do not try to mimic this complexity and rely on one homogenous combination of cells and materials for the whole construct. The aim of this study was to establish an easy and versatile method to introduce spatial diversity into a heart valve fibrin scaffold. We developed a multiple-step injection molding process that enables the fabrication of TEHVs with heterogeneous composition (cell/scaffold material) of wall and leaflets without the need of gluing or suturing components together, with the leaflets firmly connected to the wall. The integrity of the valves and their functionality was proved by either opening/closing cycles in a bioreactor (proof of principle without cells) or with continuous stimulation over 2 weeks. We demonstrated the potential of the method by the two-step molding of the wall and the leaflets containing different cell lines. Immunohistology after stimulation confirmed tissue formation and demonstrated the localization of the different cell types. Furthermore, we showed the proof of principle fabrication of valves using different materials for wall (fibrin) and leaflets (hybrid gel of fibrin/elastin-like recombinamer) and with layered leaflets. The method is easy to implement, does not require special facilities, and can be reproduced in any tissue-engineering lab. While it has been demonstrated here with fibrin, it can easily be extended to other hydrogels.

FMN-coated fluorescent USPIO for cell labeling and non-invasive MR imaging in tissue engineering

Non-invasive magnetic resonance imaging (MRI) is gaining significant attention in the field of tissue engineering, since it can provide valuable information on in vitro production parameters and in vivo performance. It can e.g. be used to monitor the morphology, location and function of the regenerated tissue, the integrity, remodeling and resorption of the scaffold, and the fate of the implanted cells. Since cells are not visible using conventional MR techniques, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are routinely employed to label and monitor the cells embedded in tissue-engineered implants. We here set out to optimize cell labeling procedures with regard to labeling efficiency, biocompatibility and in vitro validation during bioreactor cultivation, using flavin mononucleotide (FMN)-coated fluorescent USPIO (FLUSPIO). Efficient FLUSPIO uptake is demonstrated in three different cell lines, applying relatively short incubation times and low labeling concentrations. FLUSPIO-labeled cells were successfully employed to visualize collagen scaffolds and tissue-engineered vascular grafts. Besides promoting safe and efficient cell uptake, an exquisite property of the non-polymeric FMN-coating is that it renders the USPIO fluorescent, providing a means for in vitro, in vivo and ex vivo validation via fluorescence microscopy and fluorescence reflectance imaging (FRI). FLUSPIO cell labeling is consequently considered to be a suitable tool for theranostic tissue engineering purposes.

TexMi: development of tissue-engineered textile-reinforced mitral valve prosthesis.

Mitral valve regurgitation together with aortic stenosis is the most common valvular heart disease in Europe and North America. Mechanical and biological prostheses available for mitral valve replacement have significant limitations such as the need of a long-term anticoagulation therapy and failure by calcifications. Both types are unable to remodel, self-repair, and adapt to the changing hemodynamic conditions. Moreover, they are mostly designed for the aortic position and do not reproduce the native annular-ventricular continuity, resulting in suboptimal hemodynamics, limited durability, and gradually decreasing ventricular pumping efficiency. A tissue-engineered heart valve specifically designed for the mitral position has the potential to overcome the limitations of the commercially available substitutes. For this purpose, we developed the TexMi, a living textile-reinforced mitral valve, which recapitulates the key elements of the native one: annulus, asymmetric leaflets (anterior and posterior), and chordae tendineae to maintain the native annular-ventricular continuity. The tissue-engineered valve is based on a composite scaffold consisting of the fibrin gel as a cell carrier and a textile tubular structure with the twofold task of defining the gross three-dimensional (3D) geometry of the valve and conferring mechanical stability. The TexMi valves were molded with ovine umbilical vein cells and stimulated under dynamic conditions for 21 days in a custom-made bioreactor. Histological and immunohistological stainings showed remarkable tissue development with abundant aligned collagen fibers and elastin deposition. No cell-mediated tissue contraction occurred. This study presents the proof-of-principle for the realization of a tissue-engineered mitral valve with a simple and reliable injection molding process readily adaptable to the patients anatomy and pathological situation by producing a patient-specific rapid prototyped mold.

Nondestructive monitoring of tissue-engineered constructs

Abstract Tissue engineering as a multidisciplinary field enables the development of living substitutes to replace, maintain, or restore diseased tissue and organs. Since the term was introduced in medicine in 1987, tissue engineering strategies have experienced significant progress. However, up to now, only a few substitutes were able to overcome the gap from bench to bedside and have been successfully approved for clinical use. Substantial donor variability makes it difficult to predict the quality of tissue-engineered constructs. It is essential to collect sufficient data to ensure that poor or immature constructs are not implanted into patients. The fulfillment of certain quality requirements, such as mechanical and structural properties, is crucial for a successful implantation. There is a clear need for new nondestructive and real-time online monitoring and evaluation methods for tissue-engineered constructs, which are applicable on the biomaterial, tissue, cellular, and subcellular levels. This paper reviews current established nondestructive techniques for implant monitoring including biochemical methods and noninvasive imaging.

Non-destructive analysis of extracellular matrix development in cardiovascular tissue-engineered constructs.

In the field of tissue engineering, there is an increasing demand for non-destructive methods to quantify the synthesis of extracellular matrix (ECM) components such as collagens, elastin or sulphated glycosaminoglycans (sGAGs) in vitro as a quality control before clinical use. In this study, procollagen I carboxyterminal peptide (PICP), procollagen III aminoterminal peptide (PIIINP), tropoelastin and sGAGs are investigated for their potential use as non-destructive markers in culture medium of statically cultivated cell-seeded fibrin gels. Measurement of PICP as marker for type I collagen synthesis, and PIIINP as marker of type III collagen turnover, correlated well with the hydroxyproline content of the fibrin gels, with a Pearson correlation coefficient of 0.98 and 0.97, respectively. The measurement of tropoelastin as marker of elastin synthesis correlated with the amount of elastin retained in fibrin gels with a Pearson correlation coefficient of 0.99. sGAGs were retained in fibrin gels, but were not detectable in culture medium at any time of measurement. In conclusion, this study demonstrates the potential of PICP and tropoelastin as non-destructive culture medium markers for collagen and elastin synthesis. To our knowledge, this is the first study in cardiovascular tissue engineering investigating the whole of here proposed biomarkers of ECM synthesis to monitor the maturation process of developing tissue non-invasively, but for comprehensive assessment of ECM development, these biomarkers need to be investigated in further studies, employing dynamic cultivation conditions and more complex tissue constructs.

Non-invasive Imaging of Tissue-Engineered Vascular Endothelium with Iron Oxide Nanoparticles

J. Frese, L. Hrdlicka, M. E. Mertens, L. Rongen, S. Koch, P. Schuster, V.N. Gesché, T. Lammers, P. Mela, F. Kiessling, S. Jockenhoevel Department of Tissue Engineering & Textile Implants, Institute of Applied Medical Engineering, RWTH Aachen University, Aachen, Germany, frese@hia.rwth-aachen.de Department of Experimental Molecular Imaging, RWTH-Aachen University, Aachen, Germany, Department of Tissue Engineering & Textile Implants, Institut für Textiltechnik, RWTH Aachen University, Aachen Germany

Generation and imaging of patient customized implants

Personalized medicine is the development of individual solutions and therapies tailored to the specific disease pattern of a patient. To enable patient customized medical solutions 40 partners of the Aachen Research Cluster “innovation medical technology in.nrw” are investigating a new generation of biomedical devices and systems. The subproject Patim addresses non-invasive monitoring techniques to observe dynamic changes in tissue engineered cardiovascular implants.

Bioreactor Development for the Study of Angiogenesis within Tissue Engineered Constructs

Tissue Engineering is an interdisciplinary field that combines cellular and molecular biology as well as material and mechanical engineering in order to replace damaged or diseased organs and tissues. The development of complex tissue engineered constructs is limited to a tissue thickness of less than 2mm. To overcome the limitations of diffusion, an internal capillary network is necessary to enable gas exchange, nutrient delivery and waste removal.

The Use of Fibrin as an Autologous Scaffold Material for Cardiovascular Tissue Engineering Applications: From In Vitro to In Vivo Evaluation

Introduction: Tissue engineering approaches are being investigated to construct living, autologous implantable cardiovascular structures, which have a post-implantation capacity for growth and remodeling. Our group is focusing on the development of implantable autologous heart valves and vascular grafts, by combining biodegradable scaffolds with an autologous fibrin cell carrier material. The current study reports the pre-clinical application of these fibrin-based structures in a large animal model. Method: For the construction of heart valves and vascular grafts, an autologous mixed cell population was expanded from ovine carotid artery. Heart valves and vascular grafts were cast in customized moulds by combining a cell/fibrinogen suspension (10×106 cells/ml) with a thrombin/CaCl2 solution to initiate polymerization and cell encapsulation around a supporting mesh. The constructs were subsequently conditioned for 3–4 weeks in vitro in a bioreactor system (pulsatile perfusion). Heart valve conduits were then implanted in the pulmonary trunk, while vascular grafts were interposed in the carotid artery. Tissue structure and remodeling were examined in all constructs after 3 months in vivo. Results: All tissue constructs exhibited sufficient mechanical properties for implantation periods of at least 3 months. Histological staining demonstrated excellent tissue development within the constructs. Remodeling of the constructs occurred post-implantation, with the deposition of extracellular matrix proteins, such as type I collagen, and resorption of the initial fibrin scaffold components. Scanning electron microscopy demonstrated a confluent layer of endothelial cells on the blood-contacting surfaces of the implants, while transmission electron microscopy demonstrated viable cells and mature collagen bundles throughout the tissue. Discussion: The use of fibrin as a cell carrier material in cardiovascular tissue engineering applications results in mechanically stable, autologous structures that undergo remarkable tissue development in vivo. Conclusion: Fibrin is a promising autologous scaffold material for the development of implantable structures for the replacement of diseased heart valves and blood vessels.

Novel Dynamic Bioreactor System for Heart Valve Cultivation under Echocardiographic Control

Dynamic bioreactor systems are widely used in heart valve tissue engineering as the means to apply defined mechanical stimuli on seeded myofibroblasts, thus promoting the development of sturdy yet elastic autologous tissue. Allowing for the delicate character of newly seeded cells as for the constant change of their mechanical properties over cultivation time, it is desirable to have a highly flexible dynamic bioreactor system adjustable to the changing demands. For this task a pulsatile bioreactor has been designed and built. This bioreactor renders possible the application of highly configurable pressure profiles on a heart valve and hence the simulation of the changing physiological environment of a growing heart valve. It consists of a pressure chamber where a PC-controlled linear actuator applies pulsatile pressure on the valve through a silicone membrane, and a reservoir chamber where several monitoring devices can be attached. Through the use of pressure sensors direct feedback to the actuator is possible and the development of the heart valve can be closely supervised. The system has been successfully tested on newly acquired porcine aortic and pulmonary valves.