Sabine Koch

Fibrin-polylactide-based tissue-engineered vascular graft in the arterial circulation

There is a clear clinical requirement for the design and development of living, functional, small-calibre arterial grafts. Here, we investigate the potential use of a small diameter, tissue-engineered artery in a pre-clinical study in the carotid artery position of sheep. Small-calibre ( approximately 5 mm) vascular composite grafts were molded using a fibrin scaffold supported by a poly(L/D)lactide 96/4 (P(L/D)LA 96/4) mesh, and seeded with autologous arterial-derived cells prior to 28 days of dynamic conditioning. Conditioned grafts were subsequently implanted for up to 6 months as interposed carotid artery grafts in the same animals from which the cells were harvested. Explanted grafts (n = 6) were patent in each of the study groups (1 month, 3 months, 6 months), with a significant stenosis in one explant (3 months). There was a complete absence of thrombus formation on the luminal surface of grafts, with no evidence for aneurysm formation or calcification after 6 months in vivo. Histological analyses revealed remodeling of the fibrin scaffold with mature autologous proteins, and excellent cell distribution within the graft wall. Positive vWf and eNOS staining, in addition to scanning electron microscopy, revealed a confluent monolayer of endothelial cells lining the luminal surface of the grafts. The present study demonstrates the successful production and mid-term application of an autologous, fibrin-based small-calibre vascular graft in the arterial circulation, and highlights the potential for the creation of autologous implantable arterial grafts in a number of settings.

Tissue-Engineered Small-Caliber Vascular Graft Based on a Novel Biodegradable Composite Fibrin-Polylactide Scaffold

Small-caliber vascular grafts (< or =5 mm) constructed from synthetic materials for coronary bypass or peripheral vascular repair below the knee have poor patency rates, while autologous vessels may not be available for harvesting. The present study aimed to create a completely autologous small-caliber vascular graft by utilizing a bioabsorbable, macroporous poly(L/D)lactide 96/4 [P(L/D)LA 96/4] mesh as a support scaffold system combined with an autologous fibrin cell carrier material. A novel molding device was used to integrate a P(L/D)LA 96/4 mesh in the wall of a fibrin-based vascular graft, which was seeded with arterial smooth muscle cells (SMCs)/fibroblasts and subsequently lined with endothelial cells. The mold was connected to a bioreactor circuit for dynamic mechanical conditioning of the graft over a 21-day period. Graft cell phenotype, proliferation, extracellular matrix (ECM) content, and mechanical strength were analyzed. alpha-SMA-positive SMCs and fibroblasts deposited ECM proteins into the graft wall, with a significant increase in both cell number and collagen content over 21 days. A luminal endothelial cell lining was evidenced by vWf staining, while the grafts exhibited supraphysiological burst pressure (>460 mmHg) after dynamic cultivation. The results of our study demonstrated the successful production of an autologous, biodegradable small-caliber vascular graft in vitro, with remodeling capabilities and supraphysiological mechanical properties after 21 days in culture. The approach may be suitable for a variety of clinical applications, including coronary artery and peripheral artery bypass procedures.

Development of a composite degradable/nondegradable tissue-engineered vascular graft.

The present study aimed to determine the feasibility of constructing a reinforced autologous vascular graft by combining the advantages of fibrin gel as an autologous cell carrier material with the inherent mechanical strength of an integrated mesh structure. It was hypothesized that the mesh and dynamic culture conditions could be combined to generate mechanically stable and implantable vascular grafts within a shorter cultivation period than traditional methods. A two-step moulding technique was developed to integrate a polyvinylidene fluoride (PVDF) mesh (pore size: 1-2 mm) in the wall of a fibrin-based vascular graft (I.D. 5 mm) seeded with carotid myofibroblasts. The graft was cultured under increasing physiological flow conditions for 2 weeks. Histology, burst strength, and suture retention strength were evaluated. Cell growth and tissue development was excellent within the fibrin gel matrix surrounding the PVDF fibers, and tissue structure demonstrated remarkable similarity to native tissue. The grafts were successfully subjected to physiological flow rates and pressure gradients from the outset, and mechanical properties were enhanced by the mesh structure. Mean suture retention strength of the graft tissue was 6.3 N and the burst strength was 236 mm Hg. Using the vascular composite graft technique, the production of tissue engineered, small-caliber vascular grafts with good mechanical properties within a conditioning period of 14 days is feasible.

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.

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-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.

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.