Porcine Left Ventricular Myocardium Research Articles

Continuous Viscoelasticity Measurement of Cell Spheroids via Microfluidic Electrical Aspiration.

Measurement of viscoelastic characteristics of cells cultured in three-dimensional (3D) is critical to study many biological processes including tissue and organ growth. In this article, we present a unique electrical aspiration method to measure the viscoelastic properties of cell spheroids. A microfluidic sensor was created to demonstrate this method. Unlike the traditional optical aspiration method, the aspiration of the cell spheroids is monitored via monitoring the dynamic electrical resistance change of a symmetrical microfluidic aspiration channel. We first used the microfluidic device to measure the viscoelastic properties of two types of biological tissues derived from calfskin and porcine left ventricular myocardium. The equilibrium elastic modulus and creep time constants were measured to be 148.1 ± 24.1 kPa and 76.7 ± 3.5 s and 64.5 ± 7.7 kPa and 31.4 ± 2.7 s respectively, which matched well with reported data. The test validated the principle of the electrical aspiration method. Next, we applied the method for measuring cell spheroids made of human mesenchymal stem cells at different culture stages. The equilibrium elastic modulus and apparent viscosity decreased with increasing culture time. Compared to optical aspiration methods, this microfluidic electrical aspiration method has no reliance on transparent materials and image processing, which thus allows simple setup, fast data acquisition and analysis. The use of a symmetric aspiration channel and the linear half-space model enable measurements of a large number of viscoelastic properties via a single measurement with higher accuracy. This method will enable high throughput, continuous viscoelastic measurement of cell spheroids as well as other 3D cell culture models in flow conditions without the need for any optical measurements.

Evaluation of Porcine Psoas Major as a Scaffold Material for Engineered Heart Tissues.

Decellularized porcine myocardium is commonly used as scaffolding for engineered heart tissues (EHTs). However, structural and mechanical heterogeneity in the myocardium complicate production of mechanically consistent tissues. In this study, we evaluate the porcine psoas major muscle (tenderloin) as an alternative scaffold material. Head-to-head comparison of decellularized tenderloin and ventricular scaffolds showed only minor differences in mean biomechanical characteristics, but tenderloin scaffolds were less variable and less dependent on the region of origin than ventricular samples. The active contractile behavior of EHTs made by seeding tenderloin versus ventricular scaffolds with human-induced pluripotent stem cell-derived cardiomyocytes was also comparable, with only minor differences observed. Collectively, the data reveal that the behavior of EHTs produced from decellularized porcine psoas muscle is almost identical to those made from porcine left ventricular myocardium, with the advantages of being more homogeneous, biomechanically consistent, and readily obtainable.

Temperature dependence of speed of sound and attenuation of porcine left ventricular myocardium

The temperature dependence of soft tissue acoustic properties is relevant for monitoring tissue hyperthermia. In the current work, the propagation speed and attenuation of healthy porcine left ventricular myocardium (N=5) was investigated in a frequency range relevant for clinical diagnostic imaging, i.e. 2.5–13.0MHz. Each tissue sample was held in a water bath at a temperature T=25°C, heated to 45°C, and allowed to cool back down to 25°C. Due to initial tissue swelling, the data for decreasing temperatures was considered more reliable. In this case, the slope of the phase velocity versus temperature relation was measured to be 1.10±0.04m/s/°C, and the slope of the attenuation was −0.11±0.04dB/cm/°C at 10MHz, or −0.0041±0.0015dB/cm/MHz1.4336/°C as a function of frequency.

Porous decellularized extracellular matrix microcarriers for tissue-specific cell expansion and delivery

Event Abstract Back to Event Porous decellularized extracellular matrix microcarriers for tissue-specific cell expansion and delivery Claire C. Yu1, Anna A. Kornmuller2 and Lauren Flynn3 1 Queen's University, Chemical Engineering, Canada 2 Western University, Chemical and Biochemical Engineering, Canada 3 Western University, Chemical and Biochemical Engineering & Anatomy and Cell Biology, Canada Introduction: The use of decellularized tissues in regenerative medicine is a promising approach due to the preservation of complex constituents and architecture of the native extracellular matrix (ECM). The tissue-specific ECM is crucial in regulating cellular behaviour and harnessing this innate function may be advantageous in strategies to direct cell proliferation, differentiation, and migration[1]. In this work, decellularized adipose tissue, dermis, or myocardium was used to fabricate non-chemically crosslinked porous microcarriers as cell expansion and injectable cell delivery vehicles for tissue-specific soft connective tissue regeneration. Materials and Methods: Decellularized human adipose tissue (DAT), porcine dermis (DDT), and porcine left ventricular myocardium (DLV) were prepared following established protocols (REB# CHEM-002-07)[2]-[4]. The tissue was enzymatically digested with α-amylase and homogenized in 0.2 M acetic acid to generate an ECM suspension. The microcarriers were fabricated by electrospraying the suspension (15, 25, and 35 mg/mL) into liquid nitrogen, followed by lyophilisation[2]. The size distribution was assessed, and the ECM composition and microarchitecture were characterized using IHC (collagen I, collagen IV, laminin, and fibronectin) and SEM. To assess the potential of the ECM-derived microcarriers as a tissue-specific cell culture and delivery platform, human adipose-derived stem cells (ASCs) were seeded onto the DAT microcarriers and dynamically cultured within a CELLSPIN system. Cell attachment, proliferation, and infiltration were monitored using Live/Dead® staining, Guava ViaCount Assay, and DAPI staining over 1 month in comparison to commercial Cultispher-S microcarriers, and the ASC immunophenotype was confirmed by flow cytometry. Viability of the ASCs seeded on the DAT microcarriers was also assessed following injection through a needle and after cryo-preservation. Results and Discussion: The DAT, DDT, and DLV microcarriers were structurally robust in culture over 1 month without the need for chemical crosslinking. SEM revealed a porous spherical morphology. IHC confirmed the preservation of collagen I and IV, fibronectin, and laminin in the DDT microcarriers, while only collagen I and IV were detected in the DAT and DLV microcarriers. Culture studies revealed enhanced ASC attachment and proliferation on the DAT microcarriers as compared to Cultispher-S microcarriers, with cell infiltration observed at 3 and 4 weeks. The injectability and ability to freeze the cell-seeded microcarriers may be advantageous in the translation of the technology as a minimally-invasive cell delivery platform for soft connective tissue regeneration. Conclusion: ECM-derived microcarriers hold promise as a clinically translatable strategy to regenerate a wide range of soft tissues and may be applied to advance current knowledge on how the tissue-specific ECM microenvironment influences cell behaviour. Funding for this study was provided by the Natural Sciences and Engineering Research Council (NSERC) of Canada and the Canadian Institutes of Health Research (CIHR). The authors would like to thank Dr. B.G. Amsden for access to his high-voltage DC apparatus and Dr. J.F. Watkins and Mrs. K. Martin for their clinical collaborations.

Comparison of Lesion Sizes Produced by Cryoablation and Open Irrigation Radiofrequency Ablation Catheters

The relative lesion sizes created by large electrode cryoablation catheter and irrigated radiofrequency (RF) ablation are not known. The purpose of this study was to directly compare lesion sizes created by cryoablation and irrigated RF under controlled conditions. Ablation lesions were created in freshly harvested porcine left ventricular myocardium in a blood-filled tissue bath using an 8-mm-tip cryoablation catheter and a 3.5-mm-tip open-irrigated RF ablation catheter. Lesions were created under all permutations of the following conditions: electrode orientation vertical (perpendicular) or horizontal (parallel) to the tissue, electrode contact pressure at 6 or 20 g, and blood flow at 0.2 or 0.4 m/s over the electrode-tissue interface. The largest lesion volumes created with cryoablation were 961 +/- 103 mm(3), compared with the largest lesions volumes created with RF of 680 +/- 48 mm(3) (P < 0.001). The 3-way interactions among electrode orientation, contact pressure, and superfusate blood velocity accounted for the variation in lesion volumes for both catheters (both r(2)= 0.97, both P < 0.0001). The greater contact pressure increased lesion size for both cryoablation and RF. For cryoablation, lesion sizes were increased by the horizontal orientation and by the lower blood flow velocity. For open-irrigated RF, lesion sizes were significantly reduced by the horizontal orientation, however. Depending on conditions of electrode orientation, contact pressure, and blood velocity, either 8-mm-tip cryoablation or open-irrigated RF may produce the larger lesion volumes. Open-irrigated RF lesion sizes are reduced in the horizontal catheter orientation.

Determinants of Lesion Sizes and Tissue Temperatures During Catheter Cryoablation

Factors which influence lesion size from catheter-based cryoablation have not been well described. This study describes factors which influence lesion size during catheter cryoablation. Cryoablation was delivered to porcine left ventricular myocardium in a saline bath using 4- or 8-mm electrode catheters. Ablation was delivered with the electrodes either vertical or horizontal to the tissue and both with and without superfusate flow over the electrode. The effect of electrode contact pressure was tested. Lesion dimensions were measured. All experiments were duplicated to measure tissue temperatures at 1-, 2-, 3-, and 5-mm deep to the ablation electrode. The 8-mm electrode produced lower tissue temperatures and larger lesion volumes when compared with the 4-mm electrode (all P < 0.05). Superfusate flow slowed the rate of tissue cooling, markedly warmed tissue temperatures, and reduced lesion volume when compared with no flow conditions. By linear regression modeling, lesion sizes and tissue temperatures were related to the presence of superfusate flow, electrode orientation, contact pressure and electrode size, or catheter refrigerant flow rate (r2 for models = 0.90-0.96, all P < 0.001). Electrode temperature predicted lesion size or tissue temperatures only when analyzed independent of electrode size or refrigerant flow rate. Lesion sizes and tissue temperatures during catheter cryoablation are related to convective warming, electrode orientation, electrode contact pressure, and any of the following: electrode size, catheter refrigerant flow rate or electrode temperature. However, electrode temperature may be a poor predictor of lesion size and tissue temperature for a given catheter size.

Tissue Temperatures and Lesion Size During Irrigated Tip Catheter Radiofrequency Ablation: An In Vitro Comparison of Temperature‐Controlled Irrigated Tip Ablation, Power‐Controlled Irrigated Tip Ablation, and Standard Temperature‐Controlled Ablation

The limited success rate of radiofrequency catheter ablation in patients with ventricular tachycardias related to structural heart disease may be increased by enlarging the lesion size. Irrigated tip catheter ablation is a new method for enlarging the size of the lesion. It was introduced in the power-controlled mode with high power and high infusion rate, and is associated with an increased risk of crater formation, which is related to high tissue temperatures. The present study explored the tissue temperatures during temperature-controlled irrigated tip ablation, comparing it with standard temperature-controlled ablation and power-controlled irrigated tip ablation. In vitro strips of porcine left ventricular myocardium were ablated. Temperature-controlled irrigated tip ablation at target temperatures 60 degrees C, 70 degrees C, and 80 degrees C with infusion of 1 mL saline/min were compared with standard temperature-controlled ablation at 70 degrees C and power-controlled irrigated tip ablation at 40 W, and infusion of 20 mL/min. Lesion size and tissue temperatures were significantly higher during all modes of irrigated tip ablation compared with standard temperature-controlled ablation (P < 0.05). Lesion volume correlated positively with tissue temperature (r = 0.87). The maximum recorded tissue temperature was always 1 mm from the ablation electrode and was 67 +/- 4 degrees C for standard ablation and 93 +/- 6 degrees C, 99 +/- 6 degrees C, and 115 +/- 13 degrees C for temperature-controlled irrigated tip ablation at 60 degrees C, 70 degrees C, and 80 degrees C, respectively, and 112 +/- 12 degrees C for power-controlled irrigated tip ablation, which for irrigated tip ablation was significantly higher than tip temperature (P < 0.0001). Crater formation only occurred at tissue temperatures > 100 degrees C. We conclude that irrigated tip catheter ablation increases lesion size and tissue temperatures compared with standard ablation in the temperature-controlled mode at the same or higher target temperatures and in the power-controlled mode. Furthermore, tissue temperature and delivered power are the best indicators of lesion volume during temperature-controlled ablation.

Lesion dimensions during temperature-controlled radiofrequency catheter ablation of left ventricular porcine myocardium: impact of ablation site, electrode size, and convective cooling.

It is important to increase lesion size to improve the success rate for radiofrequency ablation of ischemic ventricular tachycardia. This study of radiofrequency ablation, with adjustment of power to approach a preset target temperature, ie, temperature-controlled ablation, explores the effect of catheter-tip length, ablation site, and convective cooling on lesion dimensions. In vitro strips of porcine left ventricular myocardium during different levels of convective cooling and in vivo pig hearts at 2 or 3 left ventricular sites were ablated with 2- to 12-mm-tip catheters. We found increased lesion volume for increased catheter-tip length </=8 mm in vitro (P<0.05) and 6 mm in vivo (P<0. 0001), but no further increase was found for longer tips. For the 4- to 10-mm catheter tips, we found smaller lesion volume in low-flow areas (apex) than in high-flow areas (free wall and septum) (P<0.05). Increasing convective cooling of the catheter tip in vitro increased lesion volume (P<0.0005) for the 4- and 8-mm tips but not for the 12-mm tip as the generator reached maximum output. In contrast to power-controlled ablation, we found a negative correlation between tip temperature reached and lesion volume for applications in which maximum generator output was not achieved (P<0. 0001), whereas delivered power and lesion volume correlated positively (P<0.0001). Lesion size differs in different left ventricular target sites, which is probably related to convective cooling, as illustrated in vitro. Longer electrode tips increase lesion size for tip lengths </=6 to 8 mm. For temperature-controlled ablation, the tip temperature achieved is a poor predictor of lesion size.

Temperature-controlled radiofrequency ablation of cardiac tissue: an in vitro study of the impact of electrode orientation, electrode tissue contact pressure and external convective cooling.

A variety of basic factors such as electrode tip pressure, flow around the electrode and electrode orientation influence lesion size during radiofrequency ablation, but importantly is dependent on the chosen mode of ablation. However, only little information is available for the frequently used temperature-controlled mode. The purpose of the present experimental study was to evaluate the impact during temperature-controlled radiofrequency ablation of three basic factors regarding electrode-tissue contact and convective cooling on lesion size. In vitro strips of porcine left ventricular myocardium were ablated in a tissue bath. Temperature-controlled ablation at 80 degrees C for 60 s was performed using a 7F 4 mm tip electrode in either perpendicular or parallel contact with the endocardium at a pressure of 10 or 20 g. Increased flow around the electrode was induced by circulating the saline in the tissue bath at a flow-velocity of 0.1 m/s. Lesion volume was determined by cutting lesions in 1 mm thick slices, staining with nitroblue tetrazolium and planimetering. A total of 107 lesions was created. Lesion size was significantly larger for perpendicular electrode orientation compared to parallel for both pressure-settings and both levels of flow around the electrode (p < 0.05). Increased flow around the electrode enlarged lesion size (p < 0.005). Electrode-tissue contact pressure had no significant impact on lesion size. During temperature-controlled radiofrequency ablation increased external cooling of the electrode tip due to either flow of the surrounding liquid or poor electrode tissue contact, as exemplified by perpendicular versus parallel electrode orientation, increases lesion size significantly. This is in contrast to the impact of these factors during power-controlled ablation due to the lack of increased power-delivery in the latter situation.

Triiodothyronine reverses depressed contractile performance after excessive catecholamine stimulation

Background. Conflicting results have been reported regarding the acute effects of triiodothyronine (T 3) on myocardial contractile performance. The present study analyzes the role of T 3 in reversing the depressant effect of excessive catecholamine stimulation in isolated porcine left ventricular myocardium. Methods. Thirty-six left ventricular trabeculae (0.4 × 6.0 mm) obtained from 6 pigs were used for measurements of isometric force development, isotonic shortening, and intracellular calcium in three experimental series (measurement conditions: 37°C; optimal length; supramaximal electrical stimulation, 1 Hz; calcium measurement, fura-2 ratio method; frequency, 225 Hz). In series 1, isometric force development was measured before and after a 60-minute incubation with 10 −7 mol/L epinephrine in preparations with (n = 6) and without (n = 6) preceding fura-2 loading for calcium measurements. In series 2, the acute effects of a 30-minute administration of T 3 (10 −9 mol/L) on isometric force and intracellular calcium were analyzed (n = 6). In series 3, after simultaneous fura-2 loading and a 6-hour 10 −7 mol/L epinephrine exposure the effects of T 3 (10 −9 mol/L, 30 minutes) on force development, shortening, and intracellular calcium transient were analyzed. Results. Long-term and high-dose epinephrine exposure induced a severe contractile depression with a significant reduction of isometric force development ( p < 0.05) and increased diastolic ( p < 0.001) and systolic calcium ( p < 0.001). In normal porcine myocardium T 3 had no effect on the extent of isometric force generation but accelerated the time course of force development ( p < 0.05) and increased the calcium transient ( p < 0.001). After induction of myocardial depression by epinephrine exposure T 3 accelerated the intracellular calcium transients and reduced diastolic calcium. Triiodothyronine increased the shortening amplitude and the force amplitude ( p < 0.01). Conclusions. Triiodothyronine reverses depressed contractile performance after preceding high-dose epinephrine exposure in isolated porcine myocardium. Increased force amplitudes and unaltered or even reduced intracellular calcium transients argue in favor of a resensitization of the contractile apparatus for calcium by T 3. The study supports a potential role for T 3 treatment in depressed myocardium after previous excessive catecholamine exposure (eg, brain death, catecholamine treatment, ischemia).

Lack of evidence of M-cells in porcine left ventricular myocardium.

The aim of this study was to analyze whether cells with long action potential duration, fast Vmax, and spike-and-dome configuration (M-cells) are present in porcine left ventricular myocardium. Transmembrane action potentials (n = 505) of the left ventricle were recorded with conventional glass microelectrodes in an epicardial-endocardial direction at 2000 ms basic cycle length in 14 pigs. In 3 pigs, potentials were obtained at 1000, 2000, and 5000 ms cycle length before and after superfusion with quinidine HCl 1 microgram/ml. In addition, transmembrane potentials (n = 52) were recorded in 4 dogs at 2000 ms cycle length to verify the ability of our protocol to detect M-cells. In pigs, action potential duration at 90% repolarization was shorter (ANOVA, P < 0.001) and Vmax slower (P < 0.001) in the epicardium than in the other transmural sites, but there were no regional differences in resting membrane potential or in action potential amplitude. Potentials with particularly long phase 3 or with spike-and-dome configuration were not observed. All myocardial sites displayed rate dependence of action potential duration (P = 0.02) which was transmurally homogeneous and persisted after quinidine exposure. The drug did not induce afterdepolarizations. In dogs, potentials with spike-and-dome configuration, long duration, and fast Vmax, like those described in M-cells, were detected in deep epicardial and midmyocardial areas. The porcine left ventricular myocardium shows transmural differences in action potential duration and Vmax, but, unlike dogs, it lacks M-cells.

Myocardial characteristics of thyroxine stimulated hypertrophy a structural and functional study

The effects of thyroxine-stimulated hypertrophy (TSH) were studied in the porcine left ventricular myocardium. Hypertrophy was produced in six adult pigs by administration of triiodothyronine (1 mg/kg; i.v.) for eight days. Six pigs served as controls. The degree of hypertrophy, determined by left ventricular-to-body weight ratio, was 47%. With hypertrophy there was a significant increase in heart rate, blood pressure and myocardial blood flows. Minimal coronary resistance measured during adenosine infusion was lower in the TSH group compared with the control group. Anatomic studies revealed a balanced proliferative response of mitochondria, myofibrils and the t-tubular system during TSH. Analysis of the microvasculature indicated that the capillary and arteriolar beds both experienced growth which paralleled myocyte growth during TSH. These results suggest that thyroxine administration promotes angiogenesis in the microvascular bed which provides a partial anatomic rationale for the lowered minimal coronary resistance.

Evidence for increased lipid peroxidation in the non-ischaemic portion of the heart with coronary occlusion.

Lipid peroxidation products, malondialdehyde (MDA), conjugated double bonds (CDB), and fluorescent end products (RF), were measured in porcine left ventricular myocardium. Myocardial samples were taken 15-20 min after left anterior descending coronary artery (LAD) ligation from ischaemic and non-ischaemic regions. Products of lipid peroxidation were also measured in the left ventricular myocardium of sham operated pigs. Non-ischaemic regions of the hearts with LAD occlusion showed a significantly higher content of lipid peroxidation products than intact hearts of sham operated pigs. The results suggest that enhancement of lipid peroxidation reflecting increased free radical generation may contribute to myocardial damage in the non-ischaemic, apparently "healthy" portion of the heart with acute coronary occlusion.