Dimosthenis Mavrilas

An approach to the optimization of preparation of bioprosthetic heart valves

The stress and strain states of the valve leaflets during fixation with glutaraldehyde affect their final mechanical parameters. Comparative studies of the stress-strain relationships of aortic valve leaflet strips from fresh, statically and dynamically fixed porcine and human valves were made. Static pressures of 5 mmHg, 16 mmHg, and 95 mmHg result in stress-strain relationships which are in a region between that of fresh porcine and fresh human leaflet strips in the circumferential direction, while they are far from that of fresh porcine tissue (larger strains) in the radial direction. Leaflet strips, fixed under dynamic loading between zero and a predefined maximum load, set at an early post-transition state, give parameters not significantly different from those of human valves.

Physicochemical and microscopical study of calcific deposits from natural and bioprosthetic heart valves. Comparison and implications for mineralization mechanism.

Natural and bioprosthetic heart valves suffer from calcification, despite their differences in etiology and tissue material. The mechanism of developing calcific deposits in valve tissue is still not elucidated. The calcific deposits developed on human natural and bioprosthetic heart valves have been investigated and compared by physicochemical studies and microscopy investigations and the results were correlated with possible mechanisms of mineral crystal growth. Deposits from 16 surgically excised calcified valves (seven natural aortic and nine bioprosthetic porcine aortic valves) were examined by chemical analysis, FTIR, XRD, and SEM-EDS. The Ca/P molar ratio of the deposits from bioprosthetic valves (1.52±0.06) was significantly lower compared to that of the natural valves (1.83±0.03) (p=0.05, 1-way ANOVA). SEM-EDS examination of the two types of valve deposits revealed the coexistence of large (>20 μm) and medium (5–20 μm) plate-like crystals as well as microcrystalline (<5 μm) calcium phosphate mineral formations. The results confirmed the hypothesis that the mineral salt of calcified valves is a mixture of calcium phosphate phases such as dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP) and hydroxyapatite (HAP). DCPD and OCP are suggested to be precursor phases transformed to HAP by hydrolysis. The lower value of the Ca/P molar ratio found in the bioprostheses, in comparison with that corresponding in natural valves, was ascribed to the higher content in these deposits in precursor phases DCPD and OCP which were subsequently transformed into HAP. On the basis of chemical composition of the deposits and their morphology it is suggested that crystal growth proceeds in both types of valves by the same mechanism (hydrolysis of precursor phases to HAP) in spite of their differences in etiology, material, and possible initiation pathways.

Model experimental system for investigation of heart valve calcification in vitro

A model system was developed for the in vitro quantitative investigation of the calcification process occurring in heart valves. The process of heart valve calcification consists of the formation of calcium phosphates at the heart valve-biological fluid interface. Calcium phosphate deposits may consist of more than one calcium phosphate mineral phase, differing with respect to their physical and chemical properties. The kinetics of the formation of hydroxyapatite, the model inorganic compound for the calcified deposits, was precisely monitored in a reactor containing supersaturated calcium phosphate solutions in which the heart valves were immersed after being treated with glutaraldehyde and mounted on special racks. The precipitation process, accompanied with proton release in the solution, was monitored by a pair of glass-saturated calomel electrodes. Upon initiation of the formation of calcium phosphate deposits, the supersaturation in the working solution was reestablished through the addition of titrant solutions made with the appropriate concentration to compensate for the ions precipitated. With this methodology, not only the rates were measured very precisely but also the nucleation capability of the various substrates could be evaluated. Moreover, it was possible to identify the formation of intermediate calcium phosphate phases formed during the calcification process. Valves previously treated with glutaraldehyde were shown to nucleate octacalcium phosphate, which at lower supersaturations converted to the thermodynamically more stable hydroxyapatite. The rates measured were found to depend on the solution supersaturation, while the apparent order of the precipitation process was found to be 1.

DYNAMIC MECHANICAL PROPERTIES OF ARTERIAL AND VENOUS GRAFTS USED IN CORONARY BYPASS SURGERY

In this work we studied the frequency dependence of the dynamic mechanical characteristic s of saphenous vein (SV) and internal mammary artery (IMA) grafts. Rectangular longitudinal strips from 14 patients were tested under cyclic uniaxial tensile loading in the frequency range of 0.1–20 Hz, at 37°C in wet conditions. The dynamic mechanical parameters (the storage modulus ES and the hysteresis ratio h (loading/loop area)) together with the collagen phase modulus EH were computed as a function of frequency. The results showed that in all graft types ES and EH varied with frequency in the range 0.5–10 Hz, presenting a maximum in the neighboring of 1 Hz. The hysteresis ratio h was increased in the frequency range 1–20 Hz. It seems from the results that the physical resonance frequency of the components of the tissue responsible for their elastic behavior may lay in the range around 1 Hz, while that for the viscous behavior in the range of 20 Hz or more. Early clinical outcomes of both grafting were studied in parallel. In a one-year postoperative period the follow-up (clinical examination, electrocardiography, echocardiography and stress test) did not reveal any sign of graft occlusion or severe stenosis except one perioperative infraction but without any correlation to the graft quality.

Aldosterone receptor blockade inhibits degenerative processes in the early stage of calcific aortic stenosis

Calcific aortic valve disease is associated with increased morbidity and mortality, especially in the elderly. To date, pharmacological therapies have not proven as effective as surgical intervention. Here, we used a hyperlipidemic rabbit model to investigate the potential effects of selective aldosterone inhibition on the early stages of aortic valve calcification, a pharmacological strategy that has not yet been tested. Forty New Zealand male rabbits fed a standard diet for 4 weeks were separated into three groups: (1) control (n=10), fed a standard diet; (2) vehicle (n=15), fed a hyperlipidemic diet (cholesterol 1%) plus vehicle; and (3) eplerenone (n=15), fed a hyperlipidemic diet plus 100 mg/kg/d eplerenone (last 4 weeks). After 8 weeks, animals were sacrificed and prepared aortic valve sections were examined with Von Kossa silver stain and by immunostaining for mineralocorticoid receptor, macrophages and angiotensin-converting enzyme. The presence of calcium deposits was confirmed by scanning electron microscopy. Eplerenone increased aldosterone levels but did not affect blood pressure, cholesterol or potassium levels. Hyperlipidemia induced macrophage accumulation and angiotensin-converting enzyme expression, as well as calcium deposition in the leaflets. All markers were decreased by eplerenone treatment. Immunohistochemistry for mineralocorticoid (aldosterone) receptors revealed similar expression in the leaflets of both control and hyperlipidemic groups. Collectively, these results indicate that aldosterone receptors are present in rabbit aortic valve leaflets and their selective blockade with eplerenone inhibits formation of the sclerotic lesions induced by a high fat diet.

Structural and biomechanical alterations in rabbit thoracic aortas are associated with the progression of atherosclerosis

BackgroundAtherosclerosis is a diffuse and highly variable disease of arteries that alters the mechanical properties of the vessel wall through highly variable changes in its cellular composition and histological structure. We have analyzed the effects of acute atherosclerotic changes on the mechanical properties of the descending thoracic aorta of rabbits fed a 4% cholesterol diet.MethodsTwo groups of eight male New Zealand White rabbits were randomly selected and fed for 8 weeks either an atherogenic diet (4% cholesterol plus regular rabbit chow), or regular chow. Animals were sacrificed after 8 weeks, and the descending thoracic aortas were excised for pressure-diameter tests and histological evaluation to examine the relationship between aortic elastic properties and atherosclerotic lesions.ResultsAll rabbits fed the high-cholesterol diet developed either intermediate or advanced atherosclerotic lesions, particularly American Heart Association-type III and IV, which were fatty and contained abundant lipid-filled foam cells (RAM 11-positive cells) and fewer SMCs with solid-like actin staining (HHF-35-positive cells). In contrast, rabbits fed a normal diet had no visible atherosclerotic changes. The atherosclerotic lesions correlated with a statistically significant decrease in mean vessel wall stiffness in the cholesterol-fed rabbits (51.52 ± 8.76 kPa) compared to the control animals (68.98 ± 11.98 kPa), especially in rabbits with more progressive disease.ConclusionsNotably, stiffness appears to decrease with the progression of atherosclerosis after the 8-week period.

In Vitro Evaluation for Potential Calcification of Biomaterials Used for Staple Line Reinforcement in Lung Surgery

Bovine pericardium (BPC) and polytetrafluoroethylene (PTFE) have been widely used to reinforce staple lines in lung resection. Since limited Information regarding the calcification of these biomaterials is available, we undertook an In vitro study to evaluate their calcification potential. Commercially available BPC and PTFE biomaterials were evaluated and compared with custom-prepared BPC tissue. In vitro calcification was performed via submersion in supersaturated solution In a double-walled glass reactor at 37.0°C ± 0.1°C, pH 7.4 ± 0.1, mimicking most ion concentrations of human blood plasma. In processing of calcification, the pH decrease of the solution simulated the addition of consumed H+, Ca2+, and PO43– ions from titrant solutions, the concentrations of which were based on the stolchiometry of octacalcium phosphate. The molar ion addition with time was recorded, and the initial slope of the curve was computed for each experiment. The rate of calcification developed (molar calcium phosphate ion addition rate per time and total surface area) (R) was computed after that with respect to the relative supersaturation (σ) used in each experiment. R for custom-prepared BPC tissues was found to be in the range of 0.19 ± 0.08 to 0.52 ± 0.19 (n = 17) in σ range of 0.72 to 1.42. Commercial BPC was found to be 0.016 to 0.052 (n = 4), and PTFE was 0.005 to 0.05 (n = 8) in the same σ range. Both clinically applied biomaterials, BPC and PTFE, seemed to be calcified with rates of at least one order of magnitude lower than the custom-prepared BPC tissue. This data suggested that BPC and PTFE biomaterials showed a similar, relatively very low tendency for calcification compared with custom-prepared BPC tissue. Although further studies are necessary, staple line reinforcement by these two biomaterials should be considered safe from the calcification point of view.

Development of a new combined test setup for accelerated dynamic pH-controlled in vitro calcification of porcine heart valves.

Fifty years after their first implantation, bioprosthetic heart valves still suffer from tissue rupture and calcification. Since new bioprostheses exhibit a lower risk of calcification, fast and reliable in vitro methods need to be evaluated for testing the application of new anti-calcification techniques. This report describes a modification of the well-known in vitro dynamic calcification test method (Glasmacher et al, Leibniz University Hannover (LUH)), combined with the pH-controlled, constant solution supersaturation (CSS) method (University of Patras (UP)). The CSS method is based on monitoring the pH of the solution and the addition of calcium and phosphate ion solutions through the implementation of two syringe pumps. The pH and the activities of all ions in the solutions are thus kept constant, resulting in higher calcification rates compared to conventional in vitro methods in which solution supersaturation is allowed to decrease without any further control. To verify this hypothesis, five glutaraldehyde preserved porcine aortic valves were tested. Three of the valves were tested according to a free-drift methodology: the valves were immersed in a supersaturated calcification solution, with an initial total calcium times total phosphate product of (CaxP)=10.5 (mmol/L)2 renewed weekly. Two valves were tested by the new pH-controlled loop system, implementing the CSS methodology. All valves were tested for a 4-week period, loaded at 300 cycles per minute, resulting in a total of 12 million cycles at the end of the testing period. The degree of calcification was determined weekly by means of μx-ray, and by conventional, clinical and micro-computer tomography (CT, μCT). The results showed that the valves mineralizing at constant solution supersaturation in vitro yielded higher rates of calcification compared to the valves tested at conditions of decreasing solution supersaturation without any control, indicating the development of a new, accelerated, controllable in vitro calcification method.

Inhibition of Atherosclerosis Progression, Intimal Hyperplasia, and Oxidative Stress by Simvastatin and Ivabradine May Reduce Thoracic Aorta’s Stiffness in Hypercholesterolemic Rabbits

Aims: This study aims to evaluate atherosclerosis, oxidative stress, and arterial stiffness attenuation by simvastatin and ivabradine in hyperlipidemic rabbits. Methods and Results: Forty rabbits were randomly divided into 4 groups: atherogenic diet (group C), atherogenic diet plus simvastatin (group S), atherogenic diet plus ivabradine (group I), and atherogenic diet plus simvastatin and ivabradine (group S + I). After 9 weeks, rabbits were euthanized and descending aortas excised for mechanical testing. Atherogenic diet induced the development of significant atherosclerotic lesions in group C animals but in none of groups S, I, and S + I. RAM-11 and HHF-35–positive cells were significantly reduced in groups S, I, and S + I compared with group C (P < .001). A significant neointimal hyperplasia and intima–media ratio reduction was demonstrated in groups S (P = .015 and P < .001), I (P = .021 and P < .001), and S + I (P = .019 and P < .001) compared with group C. Protein nitrotyrosine levels were significantly decreased in group S compared with group C (P = .009), and reactive oxygen species levels were decreased in group I compared with group C (P = .011). Aortic stiffness was significantly reduced in groups S, I, and S + I compared with group C (P = .003, P = .011, and P = .029). Conclusion: Simvastatin and ivabradine significantly inhibited intimal hyperplasia and oxidative stress contributing to aortic stiffness reduction in hyperlipidemic rabbits.

A sterilization method for decellularized xenogeneic cardiovascular scaffolds

Decellularized xenogeneic scaffolds have shown promise to be employed as compatible and functional cardiovascular biomaterials. However, one of the main barriers to their clinical exploitation is the lack of appropriate sterilization procedures. This study investigated the efficiency of a two-step sterilization method, antibiotics/antimycotic (AA) cocktail and peracetic acid (PAA), on porcine and bovine decellularized pericardium. In order to assess the efficiency of the method, a sterilization assessment protocol was specifically designed, comprising: i) controlled contamination with a known amount of bacteria; ii) sterility test; iii) identification of contaminants through MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry and iv) quantification by the Most Probable Number (MPN) method. This sterilization assessment protocol proved to be a successful tool to monitor and optimize the proposed sterilization method. The treatment with AA + PAA method provided sterile scaffolds while preserving the structural integrity and biocompatibility of the decellularized porcine and bovine tissues. However, surface properties and cellular adhesion resulted slightly impaired on porcine pericardium. This work developed a sterilization method suitable for decellularized pericardial scaffolds that could be adopted for in vivo tissue engineering. Together with the proposed sterilization assessment protocol, this decontamination method will foster the clinical translation of decellularized xenogeneic substitutes. STATEMENT OF SIGNIFICANCE Clinical application of functional and compatible xenogeneic decellularized scaffolds has been delayed due to the lack of appropriate sterilization methodologies. In this study, it was investigated an effective sterilization method optimized for porcine and bovine decellularized pericardia, based on the use of antibiotics/antimycotics followed by peracetic acid treatment. This treatment effectively sterilizes both species scaffolds, proves to maintain tissue overall structure and components, preserves biocompatibility and biomechanical properties. Furthermore, it was also developed a sterilization assessment protocol used to monitor and validate the previous method, consisting in three main parts: i) controlled contamination; ii) sterility test, and iii) identification and quantification of contaminants. Both methodologies were optimized for the tissues in study but can be applied to other scaffolds and accelerate their clinical translation.