Free emboli formation in the wake of bi-leaflet mechanical heart valves and the effects of implantation techniques

The high incidence of thromboembolic complications of mechanical heart valves (MHV), primarily due to platelet activation by contact with foreign surfaces and by non-physiological flow patterns past the valve, still limits their success as permanent implants. The latter include elevated shear and turbulent stresses and shed vortices formed in the wake of the valve's leaflets during the deceleration phase, potentially entrapping activated and aggregated platelets. It is hypothesized that these flow patterns induce the formation of free emboli which are the source of cerebrovascular microemboli associated with MHV. Implicit to this hypothesis is that free emboli formation will be affected by the implantation technique employed and the valve orientation, as those will alter the flow characteristics past the valve and the interaction of the platelets with the flow. In this study, numerical simulations of turbulent pulsatile flow past a St. Jude Medical bi-leaflet MHV were conducted. Platelet shear histories were calculated along pertinent turbulent platelet trajectories, and the effect of a misaligned valve on platelet activation was quantified and compared to that of an aligned valve. It demonstrated that the combination of a tilted valve and subannularly sutured pledgets had an explicit detrimental effect on platelet activation, with the following entrapment of the platelets within the shed vortices of the wake leading to a significant increase of the thromboembolic potential of the valve. This numerical model depicted a viable course for free emboli formation, and indicated how the implantation technique may enhance the risk of cardioembolism.     

新型机械心脏瓣膜的研究

目前临床使用的各种机械心脏瓣膜的主要问题是血栓栓塞和与抗凝治疗有关的出血,其缺陷在于瓣膜开启时,碟片和支架将瓣膜的整个血流通道分隔成三至四个较小的血流通道。在这种受阻隔的血流通宫,形成容易诱发血栓的高剪应力区、紊流和滞流区。我们研制的两种机械心脏瓣膜在瓣膜开启时,没有任何支架和碟片分隔瓣膜的血流通道,使血流与天然心脏瓣膜中的相类似,可减少对血液的危害,从而可减少换瓣病人对抗凝治疗的依赖程度。     

Vortex shedding as a precursor of turbulent electrical activity in cardiac muscle.

In cardiac tissue, during partial blockade of the membrane sodium channels, or at high frequencies of excitation, inexcitable obstacles with sharp edges may destabilize the propagation of electrical excitation waves, causing the formation of self-sustained vortices and turbulent cardiac electrical activity. The formation of such vortices, which visually resembles vortex shedding in hydrodynamic turbulent flows, was observed in sheep epicardial tissue using voltage-sensitive dyes in combination with video-imaging techniques. Vortex shedding is a potential mechanism leading to the spontaneous initiation of uncontrolled high-frequency excitation of the heart.     

Hyaluronate-alginate gel as a novel biomaterial: mechanical properties and formation mechanism

With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated to combine the gel-forming properties of alginate with the healing properties of hyaluronate. Gels were prepared by diffusion of calcium into alginate-hyaluronate mixtures, with an alginate content of 20 mg/mL. The hyaluronate source was shown to have significant effect on the aspect and the properties of the gels. The gels have viscoelastic behaviour and the transient measurements carried out in creep mode could be interpreted through a Kelvin-Voigt generalised model: experimental data led to the steady state hardness and a characteristic viscosity of the gel. Gels prepared from Na rooster comb hyaluronate with weight ratio up to 0.50 have satisfactory mechanical properties, and fully stable gels are obtained after a few days; on the contrary, use of lower molecular weight hyaluronate led to loose gels for hyaluronate contents over 0.25. Gel formation was investigated by measurements of the exchange fluxes between the calcium chloride solution and the forming gel, which allowed thorough investigations of the occuring diffusion phenomena of water, calcium ion and hyaluronate. Strong interactions of water with hyaluronate reduce significantly the rate of weight loss from the gel beads and allows higher water content in steady-state gels. Calcium content in the gel samples could be correlated to the actual alginate concentration, whatever the nature and the weight ratio of hyaluronate.     

A new method for evaluation of cavitation near mechanical heart valves

Evaluation of cavitation in vivo is often based on recordings of high-pass filtered random high-frequency pressure fluctuations. We hypothesized that cavitation signal components are more appropriately assessed by a new method for extraction of random signal components of the pressure signals. We investigated three different valve types and found a high correlation between the two methods (r2: 0.8806-0.9887). The new method showed that the cavitation signal could be extracted without a priori knowledge needed for setting the high-pass filter cut off frequency, nor did it introduce bandwidth limitation of the cavitation signal.     

Integrin shedding as a mechanism of cellular adaptation during cardiac growth

Integrin-mediated cell-extracellular matrix (ECM) interactions are essential for multiple cellular processes; however, little is known regarding integrin turnover during these events. Recent studies have demonstrated shedding of cell surface molecules and suggested this as a potential mechanism for integrin turnover. Confocal microscopy of mouse hearts under different physiological conditions demonstrated the presence of beta(1)-integrin-immunoreactive material in the interstitium. Culture media from neonatal rat cardiac myocytes and fibroblasts contained a 55-kDa fragment of beta(1)-integrin. Attachment to ECM components, response to phorbol 12-myristate 13-acetate stimulation, and matrix metalloproteinase inhibition assays demonstrated that fibroblasts responded differently to the fragment compared with myocytes. The beta(1)-integrin fragment stimulated myocyte attachment to collagen and the fragment itself bound a variety of ECM proteins. These studies indicate that as myocytes and fibroblasts change size and shape, cellular contacts with the ECM are altered, resulting in the liberation of a beta(1)-integrin fragment from the cell surface. Integrin shedding may represent a novel mechanism of rapidly modifying cell-ECM contacts during various cellular processes.     

Platelet activation of mechanical versus bioprosthetic heart valves during systole

Thrombus formation is a major concern for recipients of mechanical heart valves (MHVs), which requires them to take anticoagulant drugs for the rest of their lives. Bioprosthetic heart valves (BHVs) do not require life-long anticoagulant therapy but deteriorate after 10–15 years. The thrombus formation is initiated by the platelet activation which is thought to be mainly generated in MHVs by the flow through the hinge and the leakage flow during the diastole. However, our results show that the activation in the bulk flow during the systole phase might play an essential role as well. This is based on our results obtained by comparing the thrombogenic performance of a MHV and a BHV (as control) in terms of shear induced platelet activation under exactly the same conditions. Three different mathematical activation models including linear level of activation, damage accumulation, and Soares model are tested to quantify the platelet activation during systole using the previous simulations of the flow through MHV and BHV in a straight aorta under the same physiologic flow conditions. Results indicate that the platelet activation in the MHV at the beginning of the systole phase is slightly less than the BHV. However, at the end of the systole phase the platelet activation by the bulk flow for the MHV is several folds (1.41, 5.12, and 2.81 for linear level of activation, damage accumulation, and Soares model, respectively) higher than the BHV for all tested platelet activation models.     

The Jeanne Manery-Fisher Memorial Lecture 1991. Maturation of reticulocytes: formation of exosomes as a mechanism for shedding membrane proteins.

The transferrin receptor is a member of a group of reticulocyte surface proteins that disappear from the membranes of reticulocytes as the cells mature to the erythrocyte stage. The selective loss of membrane proteins appears to be preceded by the formation of multivesicular bodies (MVBs). At the reticulocyte stage, many species of mammalian red cells including man, and one nucleated avian species (chicken), contain these intracellular structures in both natural and induced anemias. Also characteristic of blood containing reticulocytes is the presence of circulating vesicles (exosomes), which contain proteins and lipids characteristic of the plasma membrane. These exosomes appear to arise from the contents of the MVBs, after the fusion of MVBs with the plasma membrane. The proteins in the exosomes are those frequently lost during red cell maturation (e.g., transferrin receptor). The major transmembrane proteins (such as the anion transporter) are fully retained into the mature red cell, indicating a highly selective mechanism of recognition of a specific group of proteins. The exosomes are largely devoid of soluble proteins and proteins associated with lysozomes or mitochondria. A speculative model is proposed which addresses the questions of the maturation-induced structural changes in a class of membrane proteins, their recognition and selective loss involving exosome formation, and the release of exosomes to the circulation.     

Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury

There has been considerable controversy regarding the role of oxygen free radicals as important mediators of cell damage in reperfused myocardium. This controversy regards whether superoxide and hydroxyl free radicals are generated on reperfusion and if these radicals actually cause impaired contractile function. In this study, EPR studies using the spin trap 5,5-dimethyl-1-pyroline-n-oxide (DMPO) demonstrate the formation of .OH and R. free radicals in the reperfused heart. EPR signals of DMPO-OH, aN = aH = 14.9 G, and DMPO-R aN = 15.8 G aH = 22.8 G are observed, with peak concentrations during the first minute of reperfusion. It is demonstrated that these radicals are derived from .O2- since reperfusion in the presence of enzymatically active recombinant human superoxide dismutase markedly reduced the formation of these signals while inactive recombinant human superoxide dismutase had no effect. On reperfusion with perfusate pretreated to remove adventitial iron, the concentration of the DMPO-OH signal was increased 2-fold and a 4-fold decrease in the DMPO-R signal was observed demonstrating that iron-mediated Fenton chemistry occurs. Hearts reperfused with recombinant human superoxide dismutase exhibited improved contractile function in parallel with the marked reduction in measured free radicals. In order to determine if the reperfusion free radical burst results in impaired contractile function, simultaneous measurements of free radical generation and contractile function were performed. A direct relationship between free radical generation and subsequent impaired contractile function was observed. These studies suggest that superoxide derived .OH and R. free radicals are generated in the reperfused heart via Fenton chemistry. These radicals appear to be key mediators of myocardial reperfusion injury.     

Platelet gel supernatant as a potential tool to repopulate acellular heart valves

Objective

The aim of this study was to repopulate decellularized heart valve matrices with ovine mesenchymal stem cells (oMSCs) by the use of platelet gel (PG) supernatant, a storage vehicle for growth factors.

Methods

oMSCs were exposed to different concentrations of PG‐released supernatant and cell proliferation was evaluated using the MTS assay. oMSC motility and invasiveness were assayed using a Boyden chamber. A quantitative sandwich enzyme immunoassay was used to examine amounts of bFGF and TGF‐β1 in the PG supernatant. Repopulation of acellular heart valve matrices was stimulated by seeding matrices with oMSCs supplemented with the PG supernatant.

Results

The most significant increase in proliferation induced by PG supernatant appeared at 1 × 10⁵ plts/ml concentration. Higher concentrations evoked reduction of the stimulatory process. oMSC motility was most significantly stimulated at 1 × 10⁶ plts/ml. Stimulating invasiveness of oMSCs needed the much higher concentration of 2 × 10⁶ plts/ml. Immunoassays revealed that sheep PG supernatant contains 184.8 pg/ml bFGF and 60.5 ng/ml TGF‐β1. Moreover, repopulation of acellular heart valve matrices was significantly enhanced by PG supernatant addition and resulted in upregulation of the myofibroblast marker alpha‐smooth muscle actin.

Conclusions

Growth factors released from platelets had the potential to induce cell repopulation in a heart valve tissue engineering procedure, through stimulation of mesenchymal stem‐cell migration and invasion.     

Secondary flow as a mechanism for the formation of biofilm streamers

In most environments, such as natural aquatic systems, bacteria are found predominantly in self-organized sessile communities known as biofilms. In the presence of a significant flow, mature multispecies biofilms often develop into long filamentous structures called streamers, which can greatly influence ecosystem processes by increasing transient storage and cycling of nutrients. However, the interplay between hydrodynamic stresses and streamer formation is still unclear. Here, we show that suspended thread-like biofilms steadily develop in zigzag microchannels with different radii of curvature. Numerical simulations of a low-Reynolds-number flow around these corners indicate the presence of a secondary vortical motion whose intensity is related to the bending angle of the turn. We demonstrate that the formation of streamers is directly proportional to the intensity of the secondary flow around the corners. In addition, we show that a model of an elastic filament in a two-dimensional corner flow is able to explain how the streamers can cross fluid streamlines and connect corners located at the opposite sides of the channel.     

Characterization of a simplified ice-free cryopreservation method for heart valves

The aim of the present study was to characterize the hemocompatibility of ice-free cryopreserved heart valves in anticipation of future human trials. Porcine pulmonary heart valves were infiltrated with either an 83 % cryoprotectant solution followed by rapid cooling and storage at ?80 °C or with 10 % DMSO and control rate freezing to ?80 °C and storage in vapor phase nitrogen as conventional frozen controls. Cryopreserved leaflets were compared with fresh, decellularized and glutaraldehyde-fixed control valve leaflets using a battery of coagulation protein assays after exposure to human blood. Von Willebrand Factor staining indicated that most of the endothelium was lost during valve processing prior to cryopreservation. Hemocompatibility, employing thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, beta-thromboglobulin and terminal complement complex SC5b-9, was preserved compared with both fresh and frozen leaflets. Hemocompatibility differences were observed for cryopreserved leaflets versus both decellularized and glutaraldehyde fixed controls. In conclusion, the hemocompatibility results support the use of ice-free cryopreservation as a simplified preservation method because no statistically significant differences in hemocompatibility were observed between the two cryopreservation methods and fresh untreated controls.     

Telocytes in human heart valves

Valve interstitial cells (VICs) are responsible for maintaining the structural integrity and dynamic behaviour of the valve. Telocytes (TCs), a peculiar type of interstitial cells, have been recently identified by Popescu's group in epicardium, myocardium and endocardium (visit www.telocytes.com ). The presence of TCs has been identified in atria, ventricles and many other tissues and organ, but not yet in heart valves. We used transmission electron microscopy and immunofluorescence methods (double labelling for CD34 and c‐kit, or vimentin, or PDGF Receptor‐β) to provide evidence for the existence of TCs in human heart valves, including mitral valve, tricuspid valve and aortic valve. TCs are found in both apex and base of heart valves, with a similar density of 27–28 cells/mm² in mitral valve, tricuspid valve and aortic valve. Since TCs are known for the participation in regeneration or repair biological processes, it remains to be determined how TCs contributes to the valve attempts to re‐establish normal structure and function following injury, especially a complex junction was found between TCs and a putative stem (progenitor) cell.     

Observation and quantification of gas bubble formation on a mechanical heart valve

Clinical studies using transcranial Doppler ultrasonography in patients with mechanical heart valves (MHV) have detected gaseous emboli. The relationship of gaseous emboli release and cavitation on MHV has been a subject of debate in the literature. To study the influence of cavitation and gas content on the formation and growth of stable gas bubbles, a mock circulatory loop, which employed a Medtronic-Hall pyrolytic carbon disk valve in the mitral position, was used. A high-speed video camera allowed observation of cavitation and gas bubble release on the inflow valve surfaces as a function of cavitation intensity and carbon dioxide (CO2) concentration, while an ultrasonic monitoring system scanned the aortic outflow tract to quantify gas bubble production by calculating the gray scale levels of the images. In the absence of cavitation, no stable gas bubbles were formed. When gas bubbles were formed, they were first seen a few milliseconds after and in the vicinity of cavitation collapse. The volume of the gas bubbles detected in the aortic track increased with both increased CO2 and increased cavitation intensity. No correlation was observed between O2 concentration and bubble volume. We conclude that cavitation is an essential precursor to stable gas bubble formation, and CO2, the most soluble blood gas, is the major component of stable gas bubbles.