On-chip magnetophoretic capture in a model of malaria-infected red blood cells.

The search for new rapid diagnostic tests for malaria is a priority for developing an efficient strategy to fight this endemic disease, which affects more than 3 billion people worldwide. In this study, we characterize systematically an easy-to-operate lab-on-chip, designed for the magnetophoretic capture of malaria-infected red blood cells (RBCs). The method relies on the positive magnetic susceptibility of infected RBCs with respect to blood plasma. A matrix of nickel posts fabricated in a silicon chip placed face down is aimed at attracting infected cells, while healthy cells sediment on a glass slide under the action of gravity. Using a model of infected RBCs, that is, erythrocytes with methemoglobin, we obtained a capture efficiency of about 70% after 10 min in static conditions. By proper agitation, the capture efficiency reached 85% after just 5 min. Sample preparation requires only a 1:10 volume dilution of whole blood, previously treated with heparin, in a phosphate-buffered solution. Nonspecific attraction of untreated RBCs was not observed in the same time interval.

In-vitro study of a porcine quadricuspid aortic valve.

BACKGROUND AND AIM OF THE STUDY: Quadricuspid aortic valve (QAV) is an extremely rare congenital defect in which the valve features an additional fourth cusp. It is often associated with an alteration in valve functionality such as valve regurgitation, stenosis and coronary disease. These associated pathologies entail surgical correction in about 50% of patients at a mean age of 50 years. METHODS: A swine QAV was studied in a pulsatile mock loop in the laboratory. Rest (70 bpm) and exercise (100 bpm) conditions were simulated, and opening and closing kinematics were evaluated from a high-speed video. Short- and long-axis echocardiograms were recorded. The pressure drop across the valve, valve competence and effective orifice area were evaluated and compared to data from healthy samples tested in the same experimental apparatus. RESULTS: Hemodynamic quantities were physiologic-like, despite the QAV showing an altered kinematics (longer closing and opening times compared to healthy samples) and an asynchronous closing phase (the extra cusps reached the closed configuration at the end of systole systematically earlier with respect to the other three cusps). Echocardiographic data showed an increased coaptation height between the left and right coronary cusps, and a mismatch between the lunule of the extra cusp and the non-coronary cusp. CONCLUSION: The altered kinematics, together with incorrect coaptation, can alter the biomechanics of the structure, inducing an anomalous distribution of local stress which could lead to structural failure over time.

Design and functional testing of a multichamber perfusion platform for three-dimensional scaffolds.

Perfusion culture systems are widely used in tissue engineering applications for enhancing cell culture viability in the core of three-dimensional scaffolds. In this work, we present a multichamber confined-flow perfusion system, designed to provide a straightforward platform for three-dimensional dynamic cell cultures. The device comprises 6 culture chambers allowing independent and simultaneous experiments in controlled conditions. Each chamber consists of three parts: a housing, a deformable scaffold-holder cartridge, and a 7 mL reservoir, which couples water-tightly with the housing compressing the cartridge. Short-term dynamic cell seeding experiments were carried out with MC3T3-E1 cells seeded into polycaprolactone porous scaffolds. Preliminary results revealed that the application of flow perfusion through the scaffold favored the penetration of the cells to its interior, producing a more homogeneous distribution of cells with respect to dropwise or injection seeding methods. The culture chamber layout was conceived with the aim of simplifying the user operations under laminar flow hood and minimizing the risks for contamination during handling and operation. Furthermore, a compact size, a small number of components, and the use of bayonet couplings ensured a simple, fast, and sterility-promoting assembling. Finally, preliminary in vitro tests proved the efficacy of the system in enhancing cell seeding efficiency, opening the way for further studies addressing long-term scaffold colonization.

Validation of long-term primary neuronal cultures and network activity through the integration of reversibly bonded microbioreactors and MEA substrates.

In vitro recording of neuronal electrical activity is a widely used technique to understand brain functions and to study the effect of drugs on the central nervous system. The integration of microfluidic devices with microelectrode arrays (MEAs) enables the recording of networks activity in a controlled microenvironment. In this work, an integrated microfluidic system for neuronal cultures was developed, reversibly coupling a PDMS microfluidic device with a commercial flat MEA through magnetic forces. Neurons from mouse embryos were cultured in a 100 µm channel and their activity was followed up to 18 days in vitro. The maturation of the networks and their morphological and functional characteristics were comparable with those of networks cultured in macro-environments and described in literature. In this work, we successfully demonstrated the ability of long-term culturing of primary neuronal cells in a reversible bonded microfluidic device (based on magnetism) that will be fundamental for neuropharmacological studies.

Simulation of functional tricuspid regurgitation using an isolated porcine heart model.

BACKGROUND AND AIM OF THE STUDY: The results of tricuspid annuloplasty to treat functional tricuspid regurgitation (FTR) are sometimes suboptimal, and alternative techniques are needed. In the absence of reliable FTR models, and in an effort to minimize the need for animal experiments, a reproducible bench-model was developed of FTR, that allowed the simulation of the anatomic features of the condition. METHODS: A fresh porcine heart was mounted on a rigid support that was placed into a basin filled with saline; a closed circuit was then created with a centrifugal pump, equipped with connection tubes. The inflow tube of the pump conveyed saline from the basin to the pump; the outflow cannula was inserted through the pulmonary artery, across the pulmonary valve, into the right ventricle. The pump was activated to pressurize the right ventricle, thus inducing tricuspid valve regurgitation (TVR). The regurgitant flow through the valve was quantified using a flow-meter. Radiopaque markers were sutured to the head of each papillary muscle and to the tricuspid annulus, in order to trace the geometric changes of the tricuspid valve at increasing pump rates, using fluoroscopy. The efficacy of the bench-model was validated with 10 hearts. RESULTS: The TVR was increased proportionally with the right ventricular pressure (RVP) (TVR = 0.089xRVP - 1.515; R2 = 0.89). The increase in TVR was associated with increases in the annular-to-papillary muscles distance (APML) (TVR = 0.059xAPML - 2.94; R2 = 0.96), of the inter-papillary muscles distance (PMD) (TVR = 0.058xPMD - 8.58; R2 = 0.94), and of the triscuspid annular dilatation (TAD) (TVR = 0.05xTAD - 1.85; R2 = 0.89). Of these parameters, APML was the strongest predictor of TVR. CONCLUSION: The porcine heart bench model represents a reproducible system to simulate the physiopathology of FTR, and has the potential to serve as a complementary method for the evaluation of new 'in vitro' technologies and therapies for FTR.

Trends in biomedical engineering: focus on Regenerative Medicine.

Regenerative medicine is a critical frontier in biomedical and clinical research. The major progresses in the last few years were driven by a strong clinical need which could benefit from regenerative medicine outcomes for the treatment of a large number of conditions including birth defects, degenerative and neoplastic diseases, and traumatic injuries. Regenerative medicine applies the principles of engineering and life sciences to enhance the comprehension of the fundamental biological mechanisms underlying the structure-function relationships in physiologic and pathologic tissues and to accomplish alternative strategies for developing in vitro biological substitutes which are able to restore, maintain, or improve tissue, and organ function. This paper reviews selected approaches currently being investigated at Politecnico di Milano in the field of regenerative medicine. Specific tissue-oriented topics are divided in three sections according to each developmental stage: in vitro study, pre-clinical study, and clinical application. In vitro studies investigate the basic phenomena related to gene delivery, stem cell behavior, tissue regeneration, and to explore dynamic culture potentiality in different applications: cardiac and skeletal muscle, cartilage, hematopoietic system, peripheral nerve, and gene delivery. Specific fields of regenerative medicine, i.e., bone, blood vessels, and ligaments engineering have already reached the preclinical stage providing promising insights for further research towards clinical applications. The translation of the results obtained during in vitro and preclinical steps into clinical organ replacement is a very challenging issue, which can offer a valid alternative to fight morbidity, organ shortage, and ethical-social problems associated with allotransplantation as shown in the clinical case reported in this review.

Fluid dynamics characterization and thrombogenicity assessment of a levitating centrifugal pump with different impeller designs.

Technical guidelines nowadays recommend and regulate the use Computational Fluid Dynamics (CFD) to assess the performance of medical devices. CFD coupled to blood damage models has emerged as a powerful tool to evaluate the hemocompatibility of blood recirculating devices. The present study is aimed at evaluating the hydrodynamic performance and the thrombogenic potential of two prototypes of magnetically levitating centrifugal pumps. The two devices differ in the impeller configuration - 6-blades vs. 12-blades - and have been designed to be used in Cardiopulmonary Bypass (CPB) circuits during open heart surgery and in Extracorporeal Membrane Oxygenation (ECMO) to support patients with severe cardiac or respiratory failure. The pumps have been modelled using Direct Numerical Simulation coupled to Lagrangian analysis to predict platelet activation due to abnormal shear stress histories. Numerical results have been compared with experimental data in terms of head generation for different working points. Results show that the 6-blades pump has i) smaller stagnation areas, ii) lower stress levels and iii) higher strain rate, resulting in a lower thrombogenic potential, whereas the 12-blade impeller guarantees a more stable performance at high flow rates, suggesting its preferential use for more demanding applications, such as CPB.

Coronary artery mechanics induces human saphenous vein remodelling via recruitment of adventitial myofibroblast-like cells mediated by Thrombospondin-1.

Rationale: Despite the preferred application of arterial conduits, the greater saphenous vein (SV) remains indispensable for coronary bypass grafting (CABG), especially in multi-vessel coronary artery disease (CAD). The objective of the present work was to address the role of mechanical forces in the activation of maladaptive vein bypass remodeling, a process determining progressive occlusion and recurrence of ischemic heart disease. Methods: We employed a custom bioreactor to mimic the coronary shear and wall mechanics in human SV vascular conduits and reproduce experimentally the biomechanical conditions of coronary grafting and analyzed vein remodeling process by histology, histochemistry and immunofluorescence. We also subjected vein-derived cells to cyclic uniaxial mechanical stimulation in culture, followed by phenotypic and molecular characterization using RNA and proteomic methods. We finally validated our results in vitro and using a model of SV carotid interposition in pigs. Results: Exposure to pulsatile flow determined a remodeling process of the vascular wall involving reduction in media thickness. Smooth muscle cells (SMCs) underwent conversion from contractile to synthetic phenotype. A time-dependent increase in proliferating cells expressing mesenchymal (CD44) and early SMC (SM22α) markers, apparently recruited from the SV adventitia, was observed especially in CABG-stimulated vessels. Mechanically stimulated SMCs underwent transition from contractile to synthetic phenotype. MALDI-TOF-based secretome analysis revealed a consistent release of Thrombospondin-1 (TSP-1), a matricellular protein involved in TGF-ß-dependent signaling. TSP-1 had a direct chemotactic effect on SV adventitia resident progenitors (SVPs); this effects was inhibited by blocking TSP-1 receptor CD47. The involvement of TSP-1 in adventitial progenitor cells differentiation and graft intima hyperplasia was finally contextualized in the TGF-ß-dependent pathway, and validated in a saphenous vein into carotid interposition pig model. Conclusions: Our results provide the evidence of a matricellular mechanism involved in the human vein arterialization process controlled by alterations in tissue mechanics, and open the way to novel potential strategies to block VGD progression based on targeting cell mechanosensing-related effectors.

A new pulsatile volumetric device with biomorphic valves for the in vitro study of the cardiovascular system.

A pulsatile mock loop system was designed and tested. This prototype represents a versatile, adjustable, and controllable experimental apparatus for in vitro studies of devices meant to interface with the human circulatory system. The pumping system consisted of a ventricular chamber featuring two biomorphic silicone valves as the inlet and outlet valves. The chamber volume is forced by a piston pump moved by a computer-controlled, low-inertia motor. Fluid dynamic tests with the device were performed to simulate physiological conditions in terms of cardiac output (mean flow of 5 and 6 L/min, with beat rates from 60 to 80 bpm), of rheological properties of the processed fluid, and of systemic circulation impedance. The pulsating actuator performed a good replication of the physiological ventricular behavior and was able to guarantee easy control of the waveform parameters. Experimental pressure and flow tracings reliably simulated the physiological profiles, and no hemolytic subatmospheric pressures were revealed. The performance of the prototype valves was also studied in terms of dynamic and static backflow, effective orifice area, and pressure loss, resulting in their applicability for this device. Mechanical reliability was also tested over 8 h. The device proved to be a reliable lab apparatus for in vitro tests; the pumping system also represents a first step toward a possible future application of pulsating perfusion in the clinic arena, such as in short-term cardiac assist and pulsatile cardiopulmonary bypass.

Prothrombotic activity of cytokine-activated endothelial cells and shear-activated platelets in the setting of ventricular assist device support.

BACKGROUND: We systematically analyzed the synergistic effect of: (i) cytokine-mediated inflammatory activation of endothelial cells (ECs) with and (ii) shear-mediated platelet activation (SMPA) as a potential contributory mechanism to intraventricular thrombus formation in the setting of left ventricular assist device (LVAD) support. METHODS: Intact and shear-activated human platelets were exposed to non-activated and cytokine-activated ECs. To modulate the level of LVAD-related shear activation, platelets were exposed to shear stress patterns of varying magnitude (30, 50, and 70 dynes/cm2, 10 minutes) via a hemodynamic shearing device. ECs were activated via exposure to inflammatory tumor necrosis factor-α (TNF-α 10 and 100 ng/ml, 24 hours), consistent with inflammatory activation recorded in patients on LVAD circulatory support. RESULTS: Adhesivity of shear-activated platelets to ECs was significantly higher than that of intact/unactivated platelets, regardless of the initial activation level (70 dynes/cm2 shear-activated platelets vs intact platelets: +80%, p < 0.001). Importantly, inflammatory activation of ECs amplified platelet prothrombinase activity progressively with increasing shear stress magnitude and TNF-α concentration: thrombin generation of 70 dynes/cm2 shear-activated platelets was 2.6-fold higher after exposure and adhesion to 100 ng/ml TNF-α‒activated ECs (p < 0.0001). CONCLUSIONS: We demonstrated synergistic effect of SMPA and cytokine-mediated EC inflammatory activation to enhance EC‒platelet adhesion and platelet prothrombotic function. These mechanisms may contribute to intraventricular thrombosis in the setting of mechanical circulatory support.

Microfluidic flow-based platforms for induction and analysis of dynamic shear-mediated platelet activation-Initial validation versus the standardized hemodynamic shearing device.

A microfluidic flow-based platform (µFP), able to stimulate platelets via exposure of shear stress patterns pertinent to cardiovascular devices and prostheses, was compared to the Hemodynamic Shearing Device (HSD)-a state-of-the-art bench-top system for exposure of platelets to defined levels and patterns of shear. Platelets were exposed to time-varying shear stress patterns in the two systems; in detail, platelets were recirculated in the µFP or stimulated in the HSD to replicate comparable exposure time. Shear-mediated platelet activation was evaluated via (i) the platelet activity state assay, allowing the measurement of platelet-mediated thrombin generation and associated prothrombotic tendencies, (ii) scanning electron microscopy to evaluate morphological changes of sheared platelets, and (iii) flow cytometry for the determination of platelet phosphatidylserine exposure as a marker of shear activation. The results revealed good matching and comparability between the two systems, with similar trends of platelet activation, formation of microaggregates, and analogous trends of activation marker exposure for both the HSD and microfluidic-stimulated samples. These findings support future translation of the microfluidic platform as a Point-of-Care facsimile system for the diagnosis of thrombotic risk in patients implanted with cardiovascular devices.

Routine clinical anti-platelet agents have limited efficacy in modulating hypershear-mediated platelet activation associated with mechanical circulatory support.

INTRODUCTION: Continuous flow ventricular assist devices (cfVADs) continue to be limited by thrombotic complications associated with disruptive flow patterns and supraphysiologic shear stresses. Patients are prescribed complex antiplatelet therapies, which do not fully prevent recurrent thromboembolic events. This is partially due to limited data on antiplatelet efficacy under cfVAD-associated shear conditions. MATERIALS AND METHODS: We investigated the efficacy of antiplatelet drugs directly acting on three pathways: (1) cyclooxygenase (aspirin), (2) phosphodiesterase (dipyridamole, pentoxifylline, cilostazol), and (3) glycoprotein IIb-IIIa (eptifibatide). Gel-filtered platelets treated with these drugs were exposed for 10min to either constant shear stresses (30dyne/cm2 and 70dyne/cm2) or dynamic shear stress profiles extracted from simulated platelet trajectories through a cfVAD (Micromed DeBakey). Platelet activation state (PAS) was measured using a modified prothrombinase-based assay, with drug efficacy quantified based on PAS reduction compared to untreated controls. RESULTS AND CONCLUSIONS: Significant PAS reduction was observed for all drugs after exposure to 30dyne/cm2 constant shear stress, and all drugs but dipyridamole after exposure to the 30th percentile shear stress waveform of the cfVAD. However, only cilostazol was significantly effective after 70dyne/cm2 constant shear stress exposure, though no significant reduction was observed upon exposure to median shear stress conditions in the cfVAD. These results, coupled with the persistence of reported clinical thrombotic complication, suggest the need for the development of new classes of drugs that are especially designed to mitigate thrombosis in cfVAD patients, while reducing or eliminating the risk of bleeding.

Versican is differentially regulated in the adventitial and medial layers of human vein grafts.

Changes in extracellular matrix proteins may contribute significantly to the adaptation of vein grafts to the arterial circulation. We examined the production and distribution of versican and hyaluronan in intact human vein rings cultured ex vivo, veins perfused ex vivo, and cultured venous adventitial and smooth muscle cells. Immunohistochemistry revealed higher levels of versican in the intima/media compared to the adventitia, and no differences in hyaluronan. In the vasa vasorum, versican and hyaluronan associated with CD34+ progenitor cells. Culturing the vein rings for 14 days revealed increased versican immunostaining of 30-40% in all layers, with no changes in hyaluronan. Changes in versican accumulation appear to result from increased synthesis in the intima/media and decreased degradation in the adventitia as versican transcripts were increased in the intima/media, but unchanged in the adventitia, and versikine (the ADAMTS-mediated cleavage product of versican) was increased in the intima/media, but decreased in the adventitia. In perfused human veins, versican was specifically increased in the intima/media in the presence of venous pressure, but not with arterial pressure. Unexpectedly, cultured adventitial cells express and accumulate more versican and hyaluronan than smooth muscle cells. These data demonstrate a differential regulation of versican and hyaluronan in human venous adventitia vs. intima/media and suggest distinct functions for these extracellular matrix macromolecules in these venous wall compartments during the adaptive response of vein grafts to the arterial circulation.

Platelet activation is a preoperative risk factor for the development of thromboembolic complications in patients with continuous-flow left ventricular assist device.

AIMS: To correlate the dynamics of platelet activation with the development of thromboembolic events in patients with continuous-flow left ventricular assist device (cf-LVAD). METHODS AND RESULTS: The platelet activity state (PAS) assay was utilized to evaluate platelet activation in 68 cf-LVAD patients implanted with the HeartMate II (n = 15, 22%), HeartMate 3 (n = 15, 22%), or HeartWare HVAD (n = 38, 56%). PAS was measured preoperatively, early post-implant, and at long-term follow-up (1, 3, 6, 12, 18, and 24 months post-implant). PAS was also measured at the occurrence of adverse events in patients who developed thrombotic complications. Data on patient demographics, medical history, antithrombotic therapy, and coagulation parameters were also analysed. Over a median follow-up of 602 (234-942) days, PAS values did not increase over time in the overall population (P = 0.15). However, PAS measured at event was 15-fold higher in the six patients (9%) who suffered pump thrombosis (n = 2) or ischaemic stroke (n = 4) vs. the rest of the population [6.67% (5.59%-11.98%) vs. 0.45% (0.33%-0.75%); P = 0.012], despite comparable coagulation profile. Pre-implant PAS values were 4.5-fold higher in these patients [1.90% (1.24%-3.17%) vs. 0.42% (0.32%-0.72%); P = 0.006]. Neither preoperative variables nor the type of the pump or the antiplatelet strategy were associated with a higher risk of complications. CONCLUSIONS: Thrombotic events are associated with altered PAS values. Moreover, baseline elevated PAS values in patients who developed thrombotic events suggest patient-specific tendency to post-implant thromboembolic complications. Prospectively, systematic monitoring of PAS might guide the development of refined patient-tailored antithrombotic strategies and the technological improvement of LVAD design.

Mastering Mitral Leaflets Coaptation After Valve Repair with Adjustable Mitral Annuloplasty Ring: Proof of Concept in Mock Loop Study.

This investigation sought to determine the feasibility of a novel mitral ring designed to reshape mitral annulus on beating heart, after surgery. The mitral ring is intended to improve mitral leaflets coaptation to correct residual and recurrent mitral regurgitations. It could also provide progressive correction of mitral regurgitation. The device was tested in ex vivo beating heart model. The novel mitral ring is selectively deformable in P1, P2, and P3 segments using a dedicated angioplasty-type balloon. The deformation should increase leaflets coaptation, reducing distance between the two leaflets. It was implanted using standard surgical techniques. The mock loop is based on passive beating heart. Mitral valve (MV) functioning was evaluated in terms of leaflets coaptation height at P2 level using epicardial echocardiography. The test has been completed on eight swine hearts. Ring size was 30 mm. The balloons were inserted in the connecting line. Each segment of the posterior annulus was independently activated over three progressive positions. Balloon inflation pressures were between 15 and 21 bar. Maximum coaptation height increase was 7 mm. Mean pressure gradient across the MV was 1.7 ± 0.3 mm Hg after complete activation of the device. The device allowed significant increase in coaptation height at P2 level after adjustments at P1, P2, and P3. Results were consistent and reproducible. This feasibility study demonstrates the possibility to reshape the mitral annulus on beating heart to precisely increase MV leaflets coaptation height.

Microfludic platforms for the evaluation of anti-platelet agent efficacy under hyper-shear conditions associated with ventricular assist devices.

Thrombus formation is a major adverse event affecting patients implanted with ventricular assist devices (VADs). Despite anti-thrombotic drug administration, thrombotic events remain frequent within the first year post-implantation. Platelet activation (PA) is an essential process underling thrombotic adverse events in VAD systems. Indeed, abnormal shear forces, correlating with specific flow trajectories of VADs, are strong agonists mediating PA. To date, the ability to determine efficacy of anti-platelet (AP) agents under shear stress conditions is limited. Here, we present a novel microfluidic platform designed to replicate shear stress patterns of a clinical VAD, and use it to compare the efficacy of two AP agents in vitro. Gel-filtered platelets were incubated with i) acetylsalicylic acid (ASA) and ii) ticagrelor, at two different concentrations (ASA: 125 and 250 µM; ticagrelor: 250 and 500 nM) and were circulated in the VAD-emulating microfluidic platform using a peristaltic pump. GFP was collected after 4 and 52 repetitions of exposure to the VAD shear pattern and tested for shear-mediated PA. ASA significantly inhibited PA only at 2-fold higher concentration (250 µM) than therapeutic dose (125 µM). The effect of ticagrelor was not dependent on drug concentration, and did not show significant inhibition with respect to untreated control. This study demonstrates the potential use of microfluidic platforms as means of testing platelet responsiveness and AP drug efficacy under complex and realistic VAD-like shear stress conditions.

In vitro and in silico approaches to quantify the effects of the Mitraclip® system on mitral valve function.

Mitraclip® implantation is widely used as a valid alternative to conventional open-chest surgery in high-risk patients with severe mitral valve (MV) regurgitation. Although effective in reducing mitral regurgitation (MR) in the majority of cases, the clip implantation produces a double-orifice area that can result in altered MV biomechanics, particularly in term of hemodynamics and mechanical stress distribution on the leaflets. In this scenario, we combined the consistency of in vitro experimental platforms with the versatility of numerical simulations to investigate clip impact on MV functioning. The fluid dynamic determinants of the procedure were experimentally investigated under different working conditions (from 40bpm to 100bpm of simulated heart rate) on six swine hearts; subsequently, fluid dynamic data served as realistic boundary conditions in a computational framework able to quantitatively assess the post-procedural MV biomechanics. The finite element model of a human mitral valve featuring an isolated posterior leaflet prolapse was reconstructed from cardiac magnetic resonance. A complete as well as a marginal, sub-optimal grasping of the leaflets were finally simulated. The clipping procedure resulted in a properly coapting valve from the geometrical perspective in all the simulated configurations. Symmetrical complete grasping resulted in symmetrical distribution of the mechanical stress, while uncomplete asymmetrical grasping resulted in higher stress distribution, particularly on the prolapsing leaflet. This work pinpointed that the mechanical stress distribution following the clipping procedure is dependent on the cardiac hemodynamics and has a correlation with the proper execution of the grasping procedure, requiring accurate evaluation prior to clip delivery.

High Frequency Components of Hemodynamic Shear Stress Profiles are a Major Determinant of Shear-Mediated Platelet Activation in Therapeutic Blood Recirculating Devices.

We systematically analyzed the relative contributions of frequency component elements of hemodynamic shear stress waveforms encountered in cardiovascular blood recirculating devices as to overall platelet activation over time. We demonstrated that high frequency oscillations are the major determinants for priming, triggering and yielding activated "prothrombotic behavior" for stimulated platelets, even if the imparted shear stress has low magnitude and brief exposure time. Conversely, the low frequency components of the stress signal, with limited oscillations over time, did not induce significant activation, despite being of high magnitude and/or exposure time. In vitro data were compared with numerical predictions computed according to a recently proposed numerical model of shear-mediated platelet activation. The numerical model effectively resolved the correlation between platelet activation and the various frequency components examined. However, numerical predictions exhibited a different activation trend compared to experimental results for different time points of a stress activation sequence. With this study we provide a more fundamental understanding for the mechanobiological responsiveness of circulating platelets to the hemodynamic environment of cardiovascular devices, and the importance of these environments in mediating life-threatening thromboembolic complications associated with shear-mediated platelet activation. Experimental data will guide further optimization of the thromboresistance of cardiovascular implantable therapeutic devices.

Modelling of Lesions Associated with Functional Mitral Regurgitation in an Ex Vivo Platform.

Functional mitral regurgitation (FMR) is a complex pathology involving valvular and subvalvular structures reconfiguration, and its treatment is considered challenging. There is a lack of experimental models allowing for reliable preclinical FMR treatments' evaluation in a realistic setting. A novel approach to simulate FMR was developed and incorporated into an ex vivo passive beating heart platform. FMR was obtained by dilating the mitral annulus (MA) mainly in the antero-posterior direction and displacing the papillary muscles (PMs) apically and laterally by ad hoc designed and 3D printed dilation and displacing devices. It caused hemodynamic and valve morphology alterations. Isolated MA dilation (MAD) led to significantly increased antero-posterior distance (A-P) and decreased coaptation height (CH), tenting area (TA) and systolic leaflets angulation, resembling clinically recognized type I of mitral regurgitation with normal leaflet motion. Whereas concomitant MAD with PM displacement caused an increase in A-P, TA, CH. This geometrical configuration replicated typical determinants of type IIIb lesion with restricted leaflet motion. The proposed methods provided a realistic and repeatable ex vivo FMR model featuring two lesions clinically associated with the pathology. It bears a promise to be successfully utilized in preclinical studies, clinical training and medical education.

A novel system for the treatment of aortic annular dilation: an ex vivo investigation.

OBJECTIVES: The main reason for aortic repair failures is recurrent annular dilatation. The fibrous portion of left ventricular outflow tract dilates. A novel device was designed to tackle this problem. METHODS: The device consists of an internal ring applied at the aortic annulus plus an external flexible band at the level of the aortic root. The internal ring has a semi-rigid portion (40%, placed at ventriculo-arterial junction) and a flexible portion to allow it to conform along the curves of the non-coronary/right coronary leaflet and right coronary/left coronary leaflet commissures. The external band acts as a reinforcement to the internal ring. A pulsatile mock loop capable of housing porcine aortic valve was used. Working conditions were 60 bpm of heart rate, 75 of stroke volumes and 120-80 mmHg of simulated pressure. Mean gradient, effective orifice area, annular diameter, coaptation height and length were recorded on 11 aortic root units (ARUs). High-speed video and standard echocardiographic images were also recorded. All data were acquired in the following conditions: (i) basal (untreated ARU); (ii) pathological condition (left coronary/non-coronary triangle was dilated by suturing an aortic patch); and (iii) ARU treated with the device. RESULTS: Gradients and effective orifice area were respectively 0.9 ± 0.64 mmHg and 3.1 ± 0.7cm2 (pathological) and 3.7 ± 1.1 mmHg and 1.5 ± 0.2cm2 (treated, P < 0.05). Left coronary/non-coronary diameter decreased from 2.4 ± 0.2 cm (pathological) to 2.0 ± 0.2 (treated, P < 0.05). Coaptation length and height were fully restored to basal values following treatment. Visual inspection showed proper dynamics of the leaflet, confirmed by high-speed video and echocardiography. CONCLUSIONS: The device allowed for restoring physiologic-like coaptation in the experimental model, without inducing clinically relevant worsening of the haemodynamics of the treated ARU.