MyoLoop: Design, development and validation of a standalone bioreactor for pathophysiological electromechanical in vitro cardiac studies.

Mechanical load is one of the main determinants of cardiac structure and function. Mechanical load is studied in vitro using cardiac preparations together with loading protocols (e.g., auxotonic, isometric). However, such studies are often limited by reductionist models and poorly simulated mechanical load profiles. This hinders the physiological relevance of findings. Living myocardial slices have been used to study load in vitro. Living myocardial slices (LMS) are 300-µm-thick intact organotypic preparations obtained from explanted animal or human hearts. They have preserved cellular populations and the functional, structural, metabolic and molecular profile of the tissue from which they are prepared. Using a three-element Windkessel (3EWK) model we previously showed that LMSs can be cultured while performing cardiac work loops with different preload and afterload. Under such conditions, LMSs remodel as a function of the mechanical load applied to them (physiological load, pressure or volume overload). These studies were conducted in commercially available length actuators that had to be extensively modified for culture experiments. In this paper, we demonstrate the design, development and validation of a novel device, MyoLoop. MyoLoop is a bioreactor that can pace, thermoregulate, acquire and process data, and chronically load LMSs and other cardiac tissues in vitro. In MyoLoop, load is parametrised using a 3EWK model, which can be used to recreate physiological and pathological work loops and the remodelling response to these. We believe MyoLoop is the next frontier in basic cardiovascular research enabling reductionist but physiologically relevant in vitro mechanical studies.

Development of a Flow Evolution Network Model for the Stress-Strain Behavior of Poly(L-lactide).

Computational modeling is critical to medical device development and has grown in its utility for predicting device performance. Additionally, there is an increasing trend to use absorbable polymers for the manufacturing of medical devices. However, computational modeling of absorbable devices is hampered by a lack of appropriate constitutive models that capture their viscoelasticity and postyield behavior. The objective of this study was to develop a constitutive model that incorporated viscoplasticity for a common medical absorbable polymer. Microtensile bars of poly(L-lactide) (PLLA) were studied experimentally to evaluate their monotonic, cyclic, unloading, and relaxation behavior as well as rate dependencies under physiological conditions. The data were then fit to a viscoplastic flow evolution network (FEN) constitutive model. PLLA exhibited rate-dependent stress-strain behavior with significant postyield softening and stress relaxation. The FEN model was able to capture these relevant mechanical behaviors well with high accuracy. In addition, the suitability of the FEN model for predicting the stress-strain behavior of PLLA medical devices was investigated using finite element (FE) simulations of nonstandard geometries. The nonstandard geometries chosen were representative of generic PLLA cardiovascular stent subunits. These finite element simulations demonstrated that modeling PLLA using the FEN constitutive relationship accurately reproduced the specimen's force-displacement curve, and therefore, is a suitable relationship to use when simulating stress distribution in PLLA medical devices. This study demonstrates the utility of an advanced constitutive model that incorporates viscoplasticity for simulating PLLA mechanical behavior.

Artery buckling stimulates cell proliferation and NF-κB signaling.

Tortuous carotid arteries are often seen in aged populations and are associated with atherosclerosis, but the underlying mechanisms to explain this preference are unclear. Artery buckling has been suggested as one potential mechanism for the development of tortuous arteries. The objective of this study, accordingly, was to determine the effect of buckling on cell proliferation and associated NF-κB activation in arteries. We developed a technique to generate buckling in porcine carotid arteries using long artery segments in organ culture without changing the pressure, flow rate, and axial stretch ratio. Using this technique, we examined the effect of buckling on arterial wall remodeling in 4-day organ culture under normal and hypertensive pressures. Cell proliferation, NF-κB p65, IκB-α, ERK1/2, and caspase-3 were detected using immunohistochemistry staining and immunoblot analysis. Our results showed that cell proliferation was elevated 5.8-fold in the buckling group under hypertensive pressure (n = 7, P < 0.01) with higher levels of NF-κB nuclear translocation and IκB-α degradation (P < 0.05 for both). Greater numbers of proliferating cells were observed on the inner curve side of the buckled arteries compared with the outer curve side (P < 0.01). NF-κB colocalized with proliferative nuclei. Computational simulations using a fluid-structure interaction model showed reduced wall stress on the inner side of buckled arteries and elevated wall stress on the outer side. We conclude that arterial buckling promotes site-specific wall remodeling with increased cell proliferation and NF-κB activation. These findings shed light on the biomechanical and molecular mechanisms of the pathogenesis of atherosclerosis in tortuous arteries.

The effect of static and dynamic loading on degradation of PLLA stent fibers.

Understanding how polymers such as PLLA degrade in vivo will enhance biodegradable stent design. This study examined the effect of static and dynamic loads on PLLA stent fibers in vitro. The stent fibers (generously provided by TissueGen, Inc.) were loaded axially with 0 N, 0.5 N, 1 N, or 0.125-0.25 N (dynamic group, 1 Hz) and degraded in PBS at 45 °C for an equivalent degradation time of 15 months. Degradation was quantified through changes in tensile mechanical properties. The mechanical behavior was characterized using the Knowles strain energy function and a degradation model. A nonsignificant increase in fiber stiffness was observed between 0 and 6 months followed by fiber softening thereafter. A marker of fiber softening, ß, increased between 9 and 15 months in all groups. At 15 months, the ß values in the dynamic group were significantly higher compared to the other groups. In addition, the model indicated that the degradation rate constant was smaller in the 1-N (0.257) and dynamic (0.283) groups compared to the 0.5-N (0.516) and 0-N (0.406) groups. While the shear modulus fluctuated throughout degradation, no significant differences were observed. Our results indicate that an increase in static load increased the degradation of mechanical properties and that the application of dynamic load further accelerated this degradation.

Extracellular vesicle transfer of lncRNA H19 splice variants to cardiac cells.

The delivery of therapeutic long non-coding RNAs (lncRNA) to the heart by extracellular vesicles (EVs) is promising for heart repair. H19, a lncRNA acting as a major regulator of gene expression within the cardiovascular system, is alternatively spliced, but the loading of its different splice variants into EVs and their subsequent uptake by recipient cardiac cells remain elusive. Here, we dissected the cellular expression of H19 splice variants and their loading into EVs secreted by Wharton-Jelly mesenchymal stromal/stem cells (WJ-MSCs). We demonstrated that overexpression of the mouse H19 gene in WJ-MSCs induces the expression of H19 splice variants at different levels. Interestingly, EVs isolated from the H19-transfected WJ-MSCs (EV-H19) showed similar expression levels for all tested splice variant sets. In vitro, we further demonstrated that EV-H19 was taken up by cardiomyocytes, fibroblasts, and endothelial cells (ECs). Finally, analysis of EV tropism in living rat myocardial slices indicated that EVs were internalized mostly by cardiomyocytes and ECs. Collectively, our results indicated that EVs can be loaded with different lncRNA splice variants and successfully internalized by cardiac cells.

Alterations of pulse pressure stimulate arterial wall matrix remodeling.

The effect of pulse pressure on arterial wall remodeling has not been clearly defined. The objective of this study was to evaluate matrix remodeling in arteries under nonpulsatile and hyperpulsatile pressure as compared with arteries under normal pulsatile pressure. Porcine carotid arteries were cultured for 3 and 7 days under normal, nonpulsatile, and hyperpulsatile pressures with the same mean pressure and flow rate using an ex vivo organ culture model. Fenestrae in the internal elastic lamina, collagen, fibronectin, and gap junction protein connexin 43 were examined in these arteries using confocal microscopy, immunoblotting, and immunohistochemistry. Our results showed that after 7 days, the mean fenestrae size and the area fraction of fenestrae decreased significantly in nonpulsatile arteries (51% and 45%, respectively) and hyperpulsatile arteries (45% and 54%, respectively) when compared with normal pulsatile arteries. Fibronectin decreased (29.9%) in nonpulsatile arteries after 3 days but showed no change after 7 days, while collagen I levels increased significantly (106%) in hyperpulsatile arteries after 7 days. The expression of connexin 43 increased by 35.3% in hyperpulsatile arteries after 7 days but showed no difference in nonpulsatile arteries. In conclusion, our results demonstrated, for the first time, that an increase or a decrease in pulse pressure from its normal physiologic level stimulates structural changes in the arterial wall matrix. However, hyperpulsatile pressure has a more pronounced effect than the diminished pulse pressure. This effect helps to explain the correlation between increasing wall stiffness and increasing pulse pressure in vivo.

The effects of isolation on chondrocyte gene expression.

Tissue engineering of articular cartilage usually requires the isolation and culture of chondrocytes. Previous studies have suggested that enzymatic isolation may alter the metabolic activity and growth rate of chondrocytes. This study examined the effects of 4 common isolation protocols on chondrocyte gene expression, morphology, and total cell yield immediately following the digest (t = 0) and after 2 culture periods (24 h and 1 week). Cartilage explants were digested using 1 of 4 protocols: (1) 6-h collagenase digest, (2) 22-h collagenase digest, (3) 45-min trypsin digest followed by a 3-h collagenase digest, or (4) 1.5-h pronase digest followed by a 3-h collagenase digest. Gene expression levels for glyceraldehyde-3-phosphate dehydrogenase, type I collagen, type II collagen, aggrecan, superficial zone protein, matrix metalloproteinase- 1, and tissue inhibitor of metalloproteinase-1 were measured at t = 0 h, 24 h, and 1 week using quantitative reverse transcriptase-polymerase chain reaction. In this study, cell yield was greatest for the 22-h collagenase and pronase-collagenase digests. However, the data indicate that a 6-h collagenase digest has the fewest gene expression changes compared to native cells. For tissue engineering, data from this study suggest that when cell yield is critical, a 22-h collagenase digest is preferable, but when obtaining cells closest to native chondrocytes is more desired, the 6-h collagenase digest is more beneficial.

An Overview of Mechanical Properties and Material Modeling of Polylactide (PLA) for Medical Applications.

This article provides an overview of the connection between the microstructural state and the mechanical response of various bioresorbable polylactide (PLA) devices for medical applications. PLLA is currently the most commonly used material for bioresorbable stents and sutures, and its use is increasing in many other medical applications. The non-linear mechanical response of PLLA, due in part to its low glass transition temperature (T g ≈ 60 °C), is highly sensitive to the molecular weight and molecular orientation field, the degree of crystallinity, and the physical aging time. These microstructural parameters can be tailored for specific applications using different resin formulations and processing conditions. The stress-strain, deformation, and degradation response of a bioresorbable medical device is also strongly dependent on the time history of applied loads and boundary conditions. All of these factors can be incorporated into a suitable constitutive model that captures the multiple physics that are involved in the device response. Currently developed constitutive models already provide powerful computations simulation tools, and more progress in this area is expected to occur in the coming years.

Smooth muscle cell contraction increases the critical buckling pressure of arteries.

Recent in vitro experiments demonstrated that arteries under increased internal pressure or decreased axial stretch may buckle into the tortuous pattern that is commonly observed in aging or diseased arteries in vivo. It suggests that buckling is a possible mechanism for the development of artery tortuosity. Vascular tone has significant effects on arterial mechanical properties but its effect on artery buckling is unknown. The objective of this study was to determine the effects of smooth muscle cell contraction on the critical buckling pressure of arteries. Porcine common carotid arteries were perfused in an ex vivo organ culture system overnight under physiological flow and pressure. The perfusion pressure was adjusted to determine the critical buckling pressure of these arteries at in vivo and reduced axial stretch ratios (1.5 and 1.3) at baseline and after smooth muscle contraction and relaxation stimulated by norepinephrine and sodium nitroprusside, respectively. Our results demonstrated that the critical buckling pressure was significantly higher when the smooth muscle was contracted compared with relaxed condition (97.3mmHg vs 72.9mmHg at axial stretch ratio of 1.3 and 93.7mmHg vs 58.6mmHg at 1.5, p<0.05). These results indicate that arterial smooth muscle cell contraction increased artery stability.

Alterations in Pulse Pressure Affect Artery Function.

Pulse pressure changes in response to cardiovascular diseases and interventions, but its effect on vascular wall structure and function is poorly understood. We examined the effect of increased or decreased pulse pressure on artery function, cellular function, and extracellular matrix remodeling. Porcine carotid arteries were cultured under non-pulsatile (100 mmHg), pulsatile (70-130 mmHg), or hyper-pulsatile pressure (50-150 mmHg) for 1 to 3 days. Vasomotor response, wall permeability, cell proliferation, apoptosis, extracellular matrix remodeling, and proteins involved in atherogenesis were examined. Our results showed that hyper-pulsatile pressure decreased the artery response to sodium nitroprusside, basal tone, and wall permeability after three days. Non-pulsatile pressure increased cell proliferation. Neither hyper-pulsatile nor non-pulsatile pressure caused a change in the extracellular matrix or in the expression of matrix metalloproteinase-2 (MMP-2), MMP-9, caveolin-1, or α-actin. Hyper-pulsatile pressure increased monocyte chemotactic protein-1 gene expression. Taken together, these changes indicate that pulse pressure has a limited effect on the artery immediately after its application. Specifically an increase in pulse pressure alters the artery tone and wall permeability while a decrease in pulse pressure alters cell proliferation. Overall these results provide insight into how the artery initially responds to changes in pulse pressure.

Effects of Axial Stretch on Cell Proliferation and Intimal Thickness in Arteries in Organ Culture.

Intimal hyperplasia (IH) remains the major cause of intermediate and long-term failure of vascular grafts and endovascular interventions. Arteries are subjected to a significant longitudinal stress in addition to the shear stress and tensile stress from the blood flow. The aim of this study was to determine the effect of axial stretch on cell proliferation and IH in arteries. Porcine carotid arteries, intact or endothelial cell (EC) denudated, were maintained ex vivo at different stretch ratios (1.3, 1.5, and 1.8) and flow rates (16 or 160 mL/min) while remaining at physiologic pressure for 7 days. The viability of the arteries was verified with norepinephrine, carbachol, and sodium nitroprusside stimulations, and the cell proliferation was detected using bromodeoxyuridine labeling and immunostaining. Our results showed that the axial stretch ratio did not significantly affect intimal thickness and cell proliferation in normal arteries. However, axial stretch increased the neointimal thickness in EC denudated arteries cultured under low flow conditions. The cell proliferation increased significantly in the intima and inner half of the media of the EC denudated arteries under normal or elevated axial stretch in comparison to intact arteries at the same stretch ratio. These results demonstrated that axial stretch with EC denudation and low flow increases neointimal formation and cell proliferation in the arteries.