Strategies to optimize engineered tissue towards native human aortic valves

unclear to what extent mechanical loading affects the collagen fibril morphology. To determine if local stresses affect the collagen fibril morphology (i.e. fibril diameter, its distribution, and fibril density), these parameters were investigated with transmission electron microscopy in adult human aortic valve leaflets. The mechanical behavior of human aortic valves was implemented in a computational model to predict the stress distribution in the valve leaflet during the diastolic phase of the cardiac cycle. The results showed that large tissue stress was associated with larger average fibril diameter, lower fibril density and wider fibril size distribution compared with low stress locations in the leaflets. These findings provide insight in the effect of mechanical loading on the collagen ultrastructure, and are valuable to optimize mechanical conditioning protocols for heart valve tissue engineering. The second objective of this thesis was to improve the mechanical properties of engineered tissues towards values of healthy native human aortic valves, which was considered an objective bench-mark for tissue engineering. Two strategies were adopted in the tissue engineering protocol to achieve this. The first approach involved modification of the scaffold design to provide sufficient mechanical support to engineered tissues. The currently used scaffold material is a mesh of rapid degrading polyglycolic acid coated with poly-4-hydroxybutyrate (PGA-P4HB). This material degrades within weeks, and does not provide mechanical integrity after implantation. As some tissue engineering applications do require a prolonged period of mechanical support by the scaffold, the feasibility of a slow degrading polymer scaffold of electrospun poly-"-caprolactone (PCL) was evaluated for cardiovascular tissue engineering, and compared with the PGA-P4HB scaffold. After optimization of the electrospun PCL scaffold, proper cell ingrowth and extracellular matrix biosynthesis were observed, while retaining elastic properties and mechanical integrity. PCL scaffolds appeared a promising alternative to PGA-P4HB scaffolds, specifically for tissue engineered blood vessels and the wall of an engineered heart valve, where prolonged mechanical support of the scaffold may be desired. As a second approach, the growing engineered tissues were biochemically stimulated to enhance tissue formation and strengthen the tissue. After an evaluation of biochemical factors known to promote protein synthesis, hypoxia and insulin were chosen for the experiments. A physiologically relevant oxygen tension, being lower than currently used in tissue engineering approaches, and insulin supplements were applied to the growing heart valve tissues to enhance their strength. Both insulin and hypoxia were associated with enhanced matrix production and improved mechanical properties, however, a synergistic effect was not observed. Although the amount of collagen and cross-links in the engineered tissues were still lower than native adult human aortic valves, tissues cultured under hypoxic conditions reached native human aortic valve values of tissue strength and stiffness after four weeks of culture, and were up to twice the values of the normoxic controls. These results strongly indicate that oxygen tension is a key parameter to achieve native-based bench-mark values of tissue strength in engineered heart valves. Engineered tissues, based on rapid degrading scaffolds, of such strength have not been achieved up to now. These findings bring the potential use for systemic applications a step closer, and can be considered an important improvement in heart valve tissue engineering. Heart valve replacement is a common treatment of end-stage valvular diseases to restore functionality of the valve. Although conventional valve replacements by mechanical or biological prosthesis offer prosperous function, they are associated with risks that limit their success. An important shortcoming of all prosthetic valves is their inability to grow, adapt and repair, which is particularly relevant for treatment of pediatric and adolescent patients. The lack of these features in current prosthesis drives the multidisciplinary approach of tissue engineering as a promising technique to create living heart valve substitutes. The concept of tissue engineering is based on seeding autologous cells onto a carrier of biodegradable material (the scaffold). This construct of cells and scaffold is stimulated to grow and develop in a mimicked physiological environment. Implantations of tissue engineered valves have been performed successfully at the pulmonary position in animal models. However, engineered valves did not possess sufficient mechanical integrity for implantation at the aortic position. Therefore, a major challenge in tissue engineering is to create tissue structures that resemble properties of native tissues to ensure durable functioning and in-vivo survival. For future human applications, one of the most important questions is: "How good is good enough for in vivo survival of tissue engineered heart valves?". The first objective of this work was to define qualitative and quantitative bench-marks for tissue engineered heart valves to determine when these valves qualify for implantation in patients. In this thesis these bench-marks were based on mechanical and structural characteristics of healthy human adult aortic valve leaflets. In native aortic valves, the collagen fiber architecture is the most prominent matrix component responsible for sustaining the load under high pressure conditions. Therefore, knowledge about the function of collagen in relation with the mechanical behavior of native heart valve tissue was an important research focus in the process to define bench-marks for tissue engineering. The relation between mechanical properties and collagen organization was investigated on a global and local scale in human adult aortic valve leaflets. Mechanical properties obtained by tensile tests of the leaflets were correlated to the amount of collagen and cross-links. Collagen cross-links, but not the collagen amount, appeared highly correlated to tissue stiffness in human heart valve leaflets. With these findings, the relevance of collagen cross-links for the mechanical integrity of engineered tissues should be given particular attention. Furthermore, in heart valve tissue, it remaine

Percutaneous Radiofrequency Ablation of Osteoid Osteomas with Use of Real-Time Needle Guidance for Accurate Needle Placement: A Pilot Study

Contains fulltext : 97211.pdf (publisher's version ) (Open Access)PURPOSE: To evaluate the accuracy and technical success of positioning a radiofrequency ablation (RFA) electrode in osteoid osteomas by use of a new real-time needle guidance technology combining cone-beam computed tomography (CT) and fluoroscopy. MATERIALS AND METHODS: Percutaneous RFA of osteoid osteomas was performed in five patients (median age 18 years), under general anesthesia, with the use of cone-beam CT and fluoroscopic guidance for electrode positioning. The outcome parameters were technical success, meaning correct needle placement in the nidus; accuracy defined as the deviation (in mm) from the center of the nidus; and clinical outcome at follow-up. RESULTS: In all five cases, positioning was possible within 3 mm of the determined target location (median nidus size 6.8 mm; range 5-10.2 mm). All procedures were technically successful. All patients were free of pain at clinical follow-up. No complications were observed. CONCLUSION: Real-time fluoroscopy needle guidance based on cone-beam CT is a useful tool to accurately position radiofrequency needles for minimally invasive treatment of osteoid osteomas

Poly(3-hydroxyoctanoate), a promising new material for cardiac tissue engineering

Cardiac tissue engineering (CTE) is currently a prime focus of research due to an enormous clinical need. In this work, a novel functional material, Poly(3-hydroxyoctanoate), P(3HO), a medium chain length polyhydroxyalkanoate (PHA), produced using bacterial fermentation, was studied as a new potential material for CTE. Engineered constructs with improved mechanical properties, crucial for supporting the organ during new tissue regeneration, and enhanced surface topography, to allow efficient cell adhesion and proliferation, were fabricated. Our results showed that the mechanical properties of the final patches were close to that of cardiac muscle. Biocompatibility of the P(3HO) neat patches, assessed using Neonatal ventricular rat myocytes (NVRM), showed that the polymer was as good as collagen in terms of cell viability, proliferation and adhesion. Enhanced cell adhesion and proliferation properties were observed when porous and fibrous structures were incorporated to the patches. Also, no deleterious effect was observed on the adults cardiomyocytes’ contraction when cardiomyocytes were seeded on the P(3HO) patches. Hence, P(3HO) based multifunctional cardiac patches are promising constructs for efficient CTE. This work will provide a positive impact on the development of P(3HO) and other PHAs as a novel new family of biodegradable functional materials with huge potential in a range of different biomedical applications, particularly CTE, leading to further interest and exploitation of these materials

Optimization of Suture-Free Laser-Assisted Vessel Repair by Solder-Doped Electrospun Poly(ε-caprolactone) Scaffold

Poor welding strength constitutes an obstacle in the clinical employment of laser-assisted vascular repair (LAVR) and anastomosis. We therefore investigated the feasibility of using electrospun poly(ε-caprolactone) (PCL) scaffold as reinforcement material in LAVR of medium-sized vessels. In vitro solder-doped scaffold LAVR (ssLAVR) was performed on porcine carotid arteries or abdominal aortas using a 670-nm diode laser, a solder composed of 50% bovine serum albumin and 0.5% methylene blue, and electrospun PCL scaffolds. The correlation between leaking point pressures (LPPs) and arterial diameter, the extent of thermal damage, structural and mechanical alterations of the scaffold following ssLAVR, and the weak point were investigated. A strong negative correlation existed between LPP and vessel diameter, albeit LPP (484 ± 111 mmHg) remained well above pathophysiological pressures. Histological analysis revealed that thermal damage extended into the medial layer with a well-preserved internal elastic lamina and endothelial cells. Laser irradiation of PCL fibers and coagulation of solder material resulted in a strong and stiff scaffold. The weak point of the ssLAVR modality was predominantly characterized by cohesive failure. In conclusion, ssLAVR produced supraphysiological LPPs and limited tissue damage. Despite heat-induced structural/mechanical alterations of the scaffold, PCL is a suitable polymer for weld reinforcement in medium-sized vessel ssLAVR

Effect of Strain Magnitude on the Tissue Properties of Engineered Cardiovascular Constructs

Mechanical loading is a powerful regulator of tissue properties in engineered cardiovascular tissues. To ultimately regulate the biochemical processes, it is essential to quantify the effect of mechanical loading on the properties of engineered cardiovascular constructs. In this study the Flexercell FX-4000T (Flexcell Int. Corp., USA) straining system was modified to simultaneously apply various strain magnitudes to individual samples during one experiment. In addition, porous polyglycolic acid (PGA) scaffolds, coated with poly-4-hydroxybutyrate (P4HB), were partially embedded in a silicone layer to allow long-term uniaxial cyclic mechanical straining of cardiovascular engineered constructs. The constructs were subjected to two different strain magnitudes and showed differences in biochemical properties, mechanical properties and organization of the microstructure compared to the unstrained constructs. The results suggest that when the tissues are exposed to prolonged mechanical stimulation, the production of collagen with a higher fraction of crosslinks is induced. However, straining with a large strain magnitude resulted in a negative effect on the mechanical properties of the tissue. In addition, dynamic straining induced a different alignment of cells and collagen in the superficial layers compared to the deeper layers of the construct. The presented model system can be used to systematically optimize culture protocols for engineered cardiovascular tissues