Bacterial adherence to graft tissues in static and flow conditions.

BACKGROUND: Various conduits and stent-mounted valves are used as pulmonary valve graft tissues for right ventricular outflow tract reconstruction with good hemodynamic results. Valve replacement carries an increased risk of infective endocarditis (IE). Recent observations have increased awareness of the risk of IE after transcatheter implantation of a stent-mounted bovine jugular vein valve. This study focused on the susceptibility of graft tissue surfaces to bacterial adherence as a potential risk factor for subsequent IE. METHODS: Adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus sanguinis to bovine pericardium (BP) patch, bovine jugular vein (BJV), and cryopreserved homograft (CH) tissues was quantified under static and shear stress conditions. Microscopic analysis and histology were performed to evaluate bacterial adhesion to matrix components. RESULTS: In general, similar bacteria numbers were recovered from CH and BJV tissue surfaces for all strains, especially in flow conditions. Static bacterial adhesion to the CH wall was lower for S sanguinis adhesion (P < .05 vs BP patch). Adhesion to the BJV wall, CH wall, and leaflet was decreased for S epidermidis in static conditions (P < .05 vs BP patch). Bacterial adhesion under shear stress indicated similar bacterial adhesion to all tissues, except for lower adhesion to the BJV wall after S sanguinis incubation. Microscopic analysis showed the importance of matrix component exposure for bacterial adherence to CH. CONCLUSIONS: Our data provide evidence that the surface composition of BJV and CH tissues themselves, bacterial surface proteins, and shear forces per se are not the prime determinants of bacterial adherence.

Ultrasound for In Vitro, Noninvasive Real-Time Monitoring and Evaluation of Tissue-Engineered Heart Valves.

Tissue-engineered heart valves are developed in bioreactors where biochemical and mechanical stimuli are provided for extracellular matrix formation. During this phase, the monitoring possibilities are limited by the need to maintain the sterility and integrity of the valve. Therefore, noninvasive and nondestructive techniques are required. As such, optical imaging is commonly used to verify valve's functionality in vitro. It provides important information (i.e., leaflet symmetry, geometric orifice area, and closing and opening times), which is, however, usually limited to a singular view along the central axis from the outflow side. In this study, we propose ultrasound as a monitoring method that, in contrast to established optical imaging, can assess the valve from different planes, scanning the whole three-dimensional geometry. We show the potential benefits associated with the application of ultrasound to bioreactors, in advancing heart valve tissue engineering from design to fabrication and in vitro maturation. Specifically, we demonstrate that additional information, otherwise unavailable, can be gained to evaluate the valve's functionality (e.g., coaptation length, and effective cusp height and shape). Furthermore, we show that Doppler techniques provide qualitative visualization and quantitative evaluation of the flow through the valve, in real time and throughout the whole in vitro fabrication phase.

Effect of Intensified Decellularization of Equine Carotid Arteries on Scaffold Biomechanics and Cytotoxicity.

Decellularized equine carotid arteries (dEAC) are suggested to represent an alternative for alloplastic vascular grafts in haemodialysis patients to achieve vascular access. Recently it was shown that intensified detergent treatment completely removed cellular components from dEAC and thereby significantly reduced matrix immunogenicity. However, detergents may also affect matrix composition and stability and render scaffolds cytotoxic. Therefore, intensively decellularized carotids (int-dEAC) were now evaluated for their biomechanical characteristics (suture retention strength, burst pressure and circumferential compliance at arterial and venous systolic and diastolic pressure), matrix components (collagen and glycosaminoglycan content) and indirect and direct cytotoxicity (WST-8 assay and endothelial cell seeding) and compared with native (n-EAC) and conventionally decellularized carotids (con-dEAC). Both decellularization protocols comparably reduced matrix compliance (venous pressure compliance: 32.2 and 27.4% of n-EAC; p < 0.01 and arterial pressure compliance: 26.8 and 23.7% of n-EAC, p < 0.01) but had no effect on suture retention strength and burst pressure. Matrix characterization revealed unchanged collagen contents but a 39.0% (con-dEAC) and 26.4% (int-dEAC, p < 0.01) reduction of glycosaminoglycans, respectively. Cytotoxicity was not observed in either dEAC matrix which was also displayed by an intact endothelial lining after seeding. Thus, even intensified decellularization generates matrix scaffolds highly suitable for vascular tissue engineering purposes, e.g., the generation of haemodialysis shunts.

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 patient's anatomy and pathological situation by producing a patient-specific rapid prototyped mold.

Pulsatile perfusion bioreactor system for durability testing and compliance estimation of tissue engineered vascular grafts.

The aim of the study was to design, construct, and test a bioreactor for the conditioning of tissue-engineered vascular grafts under physiological pressure, flow, and environmental conditions and up to supra-physiological pulse frequencies (5 Hz) as the first step towards durability testing. The system also allows the calculation of the compliance of vascular grafts as an indicator of tissue development. The system relies on the combination of a pulse-free pump and a linear magnetic actuator applying pressure pulses with controllable profile and frequency. The compliance estimation is based on the accurate measurement of the vessel's diameter by means of an optical micrometre. Software-based interface enables the control of a magnetic actuator and data acquisition to monitor the conditions of the system. Porcine carotid arteries were tested in the bioreactor for up to 4 weeks at different pulse frequencies. The tissue was analysed by means of histology and immunohistochemistry. Physiological pressures (~80 and 120 mmHg for diastolic and systolic phase, respectively) were generated in the system at frequencies between 1 and 5 Hz. The environmental conditions within the bioreactor were monitored and online determination of the compliance of the arteries was achieved under sterile conditions. Conditioning of the grafts resulted in the abundant production of ECM proteins. In conclusion, we developed a bioreactor for the conditioning of tissue engineered vascular grafts under controlled pressure conditions. The system is suitable to perform durability tests at supra-physiological pulse rates and physiological pressure levels under continuous monitoring of environmental variables (pH, pO2, pCO2, and temperature) and compliance.