Search

Background: Platelet activation at the early stage of COVID-19 is poorly described. The need for antiplatelet therapy in patients with COVID-19 remains controversial. We characterized the platelet activation profile in hospitalized patients at the early stage of COVID-19 using the modified prothrombinase Platelet Activation State (PAS) assay. Methods: Sixteen patients admitted to the emergency department of the IRCCS San Raffaele Scientific Institute (Milano, Italy) between February 8 and April 2021 were enrolled. All patients presented with respiratory symptoms and tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Platelet activation was measured via the PAS assay within 24 hours from patients' hospital admission. Data were compared with those measured in n=24 healthy subjects (controls). Results: Platelet activation was significantly higher in COVID-19 patients with respect to controls (PAS = 0.63 [0.58-0.98]% vs. 0.46 [0.40-0.65]%, respectively; p=0.03). Of note, highest PAS values were measured in the two patients with the worst clinical outcome, i.e., death because of respiratory failure (PAS = 2.09% and 1.20%, respectively). No differences in standard coagulation parameters were noted between these two patients and those who were later discharged home. Conclusions: This study provides evidences of significant platelet activation state at the early stage of COVID-19 and suggests that the patient-specific platelet activation profile is a reliable clinical marker to stratify COVID-19 patients at high risk of poor clinical outcome who might potentially benefit from antiplatelet therapy.

OBJECTIVES To evaluate the competing pro-haemorrhagic contribution of acquired von Willebrand (vW) disease and antithrombotic therapy in patients implanted with continuous-flow left ventricular assist devices (LVADs). METHODS We compared the extent of vW factor (vWf) degradation [vWf antigen (vWf:Ag)] and a decrease of functional activity of large vWf multimers [vWf collagen binding (vWf:CB)] in LVAD patients who did and did not suffer from bleeding. Data were measured pre-implant, at short-term (t1: 12 months) follow-up. The occurrence of primary bleeding events, as well as bleeding recurrence, was correlated with patient-specific vWf profile and antithrombotic regimen. Indeed, patients were discharged on warfarin (international normalized ratio: 2–2.5) and aspirin, with the latter withhold after a first bleeding episode. RESULTS Fifty-three patients were enrolled. The median follow-up was 324 (226–468) days. We recorded 25 primary bleeding events (47% of patients). All primary events occurred in patients on warfarin and aspirin. Both vWf:Ag and vWf:CB decreased significantly post-implant (P = 0.0003 and P < 0.0001), and patients showing pathological vWf:CB/vWf:Ag ratio ( CONCLUSIONS Aspirin contributes significantly to haemorrhagic events in the background of acquired vW disease; its discontinuation significantly reduces bleeding recurrence. Clinical trial registration https://clinicaltrials.gov/ct2/show/NCT03255928; ClinicalTrials.gov Identifier: NCT03255928.

BACKGROUND No clinical standardized methods exist to identify the early stage of the development of pump thrombosis in the setting of HVAD (Medtronic Inc., USA) implantation. We aimed at developing a clinically relevant tool to evaluate HVAD operation during long-term support and at identifying a new reliable marker for the early diagnosis of pump thrombosis reflecting altered patient-pump physiological interplay. METHODS We developed a novel algorithm based on time-frequency analysis of the HVAD log files allowing the detection of the intrinsic circadian rhythmicity of the pump power consumption. With this tool, we retrospectively evaluated (1) post-operative restoration of circadian rhythm (n = 14 patients), (2) long-term stability of circadian rhythmicity in patients with no reported adverse events (n = 12), and (3) alteration of circadian fluctuations in patients who suffered from pump thrombosis (n = 19). RESULTS We demonstrate (1) progressive development of circadian rhythm following post-operative recovery (93% of the patients, 23 ± 15 days after implantation), (2) long-term stability of circadian rhythmicity in patients with no thrombotic complications (92% of the patients; 962 (445–1447) days of support), and (3) severe instability and loss of circadian fluctuations before the thrombotic event (89% of the patients, 12 ± 6 days ahead of the clinical manifestation of overt pump thrombosis). Furthermore, we provide the first clinical evidence of recovery of circadian rhythmicity following non-surgical resolution of pump thrombosis. CONCLUSIONS Time-frequency analysis of the HVAD log files provides a new tool for the early diagnosis of pump thrombosis. Loss of circadian rhythmicity might trigger medical evaluation, improving the results of medical management of pump thrombosis, and decreasing the need for pump exchange.

Supraphysiological shear stress and surface-contact are recognized as driving mechanisms of platelet activation (PA) in blood contacting devices (BCDs). However, the competing role of these mechanisms in triggering thrombogenic events is poorly understood. Here, we characterized the dynamics of PA in response to the combined effect of shear stress and material exposure. Human platelets were stimulated with different levels of shear stress (500, 750, 1000 dynes/cm2) over a range of exposure times (10, 20, and 30 min) within capillary tubes made of various polymeric materials. Polyethylene (PE), polytetrafluoroethylene (PTFE), ethylene tetrafluoroethylene (ETFE), and polyether ether ketone (PEEK), used for BCDs fabrication, were investigated as compared to glass and thromboresistant Sigma™-coated glass. PA was quantified using the Platelet Activity State assay. Our results indicate that mechanical stimulation and polymer surface-contact both significantly contribute to PA. Notably, the contribution of the mechanical stimulus ranges between +36% and +43%, while that associated with polymer surface-contact ranges from +48% to +59%, depending on the exposure time. In more detail, our results indicate that: (i) PA increases with increasing shear stress magnitude; (ii) PA has a non-linear, time-dependent relationship to exposure time; (iii) PA is largely influenced by biomaterials, with PE and PEEK having respectively the lowest and highest prothrombotic potential; (iv) the effects of polymer surface-contact and shear stress are not correlated and can be studied separately. Our results suggest the importance of incorporating the evaluation of platelet activation driven by the combined effect of shear stress and polymer surface-contact for the comprehensive assessment, and eventually minimization, of BCDs thrombogenic potential.

Background: Metabolomic profiling has important diagnostic and prognostic value in heart failure (HF). We investigated whether left ventricular assist device (LVAD) support has an impact on the metabolomic profile of chronic HF patients and if specific metabolic patterns are associated with the development of adverse events.Methods: We applied untargeted metabolomics to detect and analyze molecules such as amino acids, sugars, fatty acids and other metabolites in plasma samples collected from thirty-three patients implanted with a continuous-flow LVAD. Data were analyzed at baseline, i.e., before implantation of the LVAD, and at long-term follow-up. Results: Our results reveal significant changes in the metabolomic profile after LVAD implant compared to baseline. In detail, we observed a pre-implant reduction in amino acid metabolism (aminoacyl-tRNA biosynthesis) and increased galactose metabolism, which reversed over the course of support [median follow-up 187 days (63–334 days)]. These changes were associated with improved patient functional capacity driven by LVAD therapy, according to NYHA functional classification of HF (NYHA class I-II: pre-implant=0% of the patients; post-implant =97% of the patients; P

We evaluated the real-time diagnostic capability of a new tool enhancing early diagnosis of pump thrombosis (PT) of the HeartWare left ventricular assist device via time-frequency analysis (TFA) of the log files. We analyzed 173 log files, including 24 (14%) associated with a clinical diagnosis of PT and 149 (86%) controls. The 30-day log file records were discretized into consecutive windows of a 24-h duration, which were iteratively acquired and processed via TFA. This way, we simulated longitudinal acquisition of pump parameters and provided real-time analysis of consecutive data, thus resembling the clinical scenario. Sensitivity and specificity of the tool were 79% and 84%, respectively. Sensitivity against PT events with progressive “build-up” thrombus increased up to 95%, and early signs of a forthcoming PT were identified 10±8 days prior to its clinical manifestation. This study demonstrates high reliability and the potential for effective clinical translation of this prognostic tool.

The performance of blood-processing devices largely depends on the associated fluid dynamics, which hence represents a key aspect in their design and optimization. To this aim, two approaches are currently adopted: computational fluid-dynamics, which yields highly resolved three-dimensional data but relies on simplifying assumptions, and in vitro experiments, which typically involve the direct video-acquisition of the flow field and provide 2D data only. We propose a novel method that exploits space- and time-resolved magnetic resonance imaging (4D-flow) to quantify the complex 3D flow field in blood-processing devices and to overcome these limitations. We tested our method on a real device that integrates an oxygenator and a heat exchanger. A dedicated mock loop was implemented, and novel 4D-flow sequences with sub-millimetric spatial resolution and region-dependent velocity encodings were defined. Automated in house software was developed to quantify the complex 3D flow field within the different regions of the device: region-dependent flow rates, pressure drops, paths of the working fluid and wall shear stresses were computed. Our analysis highlighted the effects of fine geometrical features of the device on the local fluid-dynamics, which would be unlikely observed by current in vitro approaches. Also, the effects of non-idealities on the flow field distribution were captured, thanks to the absence of the simplifying assumptions that typically characterize numerical models. To the best of our knowledge, our approach is the first of its kind and could be extended to the analysis of a broad range of clinically relevant devices.

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.

As key cellular elements of hemostasis, platelets represent a primary target for thrombosis and bleeding management. Currently, therapeutic manipulations of platelet function (antithrombotic drugs) and count (platelet transfusion) are performed with limited or no real-time monitoring of the desired outcome at the point-of-care. To address the need, we have designed and fabricated an easy-to-use, accurate, and portable impedance aggregometer called &ldquo, MICELI&rdquo, (MICrofluidic, ELectrical, Impedance). It improves on current platelet aggregation technology by decreasing footprint, assay complexity, and time to obtain results. The current study aimed to optimize the MICELI protocol, validate sensitivity to aggregation agonists and key blood parameters, i.e., platelet count and hematocrit, and verify the MICELI operational performance as compared to commercial impedance aggregometry. We demonstrated that the MICELI aggregometer could detect platelet aggregation in 250 &mu, L of whole blood or platelet-rich plasma, stimulated by ADP, TRAP-6, collagen, epinephrine, and calcium ionophore. Using hirudin as blood anticoagulant allowed higher aggregation values. Aggregation values obtained by the MICELI strongly correlated with platelet count and were not affected by hematocrit. The operational performance comparison of the MICELI and the Multiplate&reg, Analyzer demonstrated strong correlation and similar interdonor distribution of aggregation values obtained between these devices. With the proven reliability of the data obtained by the MICELI aggregometer, it can be further translated into a point-of-care diagnostic device aimed at monitoring platelet function in order to guide pharmacological hemostasis management and platelet transfusions.

The paper discusses a Smoothed Particle Hydrodynamics (SPH) model for the analysis of the multiphase flow occurring in an experimental microfluidic device for conformal coating of pancreatic islets with a biocompatible and permeable polymer. The proposed numerical model, based on a weakly-compressible SPH approach, accurately mimics the encapsulation process while assuring phase conservation, thus overcoming potential limitations of grid-based models. The proposed SPH model is a triphasic multi-phase model that allows one: (i) to reproduce the physics of islet conformal coating, including the effects of surface tension at the interface of the involved fluids and of the islet diameter; and (ii) to evaluate how modulation of process parameters influences the fluid dynamics within the microfluidic device and the resulting coating characteristics. This model can represent a valuable, time- and cost-effective tool for the definition of optimized encapsulation conditions through in silico screening of novel combinations of conformal coating parameters, including polymeric coating blends, size range of insulin-secreting cell clusters, utilized chemical reagents, device geometry and scale.

In the setting of HeartWare left ventricular assist device (HVAD, Medtronic) implantation, pre-pump blood flow obstruction has been described due to intraventricular thrombus formation occluding the inflow cannula. This phenomenon often evolves in suboptimal pump performance, and requires prompt management to prevent its progression. However, to date, effective strategies and tools for the diagnosis and management of this complication are poorly described. We report a case of HVAD inflow cannula obstruction that drove later in-pump thrombosis and, eventually, complete cannula occlusion, and discuss gap of knowledge and limitations of currently available diagnostic and therapeutic tools in this scenario. Furthermore, we reinforce the value of time-frequency analysis of the HVAD log files to early identify abnormal pump operation associated with inflow cannula obstruction despite unremarkable trends of pump parameters.

We designed an experimental setup to characterize the thrombogenic potential associated with blood recirculating devices (BRDs) used in extracorporeal circulation (ECC). Our methodology relies on in vitro flow loop platelet recirculation experiments combined with the modified-prothrombinase platelet activity state (PAS) assay to quantify the bulk thrombin production rate of circulated platelets, which correlates to the platelet activation (PA) level. The method was applied to a commercial neonatal hollow fiber membrane oxygenator. In analogous hemodynamic environment, we compared the PA level resulting from multiple passes of platelets within devices provided with phosphorylcholine (PC)-coated and noncoated (NC) fibers to account for flow-related mechanical factors (i.e., fluid-induced shear stress) together with surface contact activation phenomena. We report for the first time that PAS assay is not significantly sensitive to the effect of material coating under clinically pertinent flow conditions (500 mL/min), while providing straightforward information on shear-mediated PA dynamics in ECC devices. Being that the latter is intimately dependent on local flow dynamics, according to our results, the rate of thrombin production as measured by the PAS assay is a valuable biochemical marker of the selective contribution of PA in BRDs induced by device design features. Thus, we recommend the use of PAS assay as a means of evaluating the effect of modification of specific device geometrical features and/or different design solutions for developing ECC devices providing flow conditions with reduced thrombogenic impact.

Glutaraldehyde-fixed pericardium of animal origin is the elective material for the fabrication of bio-prosthetic valves for surgical replacement of insufficient/stenotic cardiac valves. However, the pericardial tissue employed to this aim undergoes severe calcification due to chronic inflammation resulting from a non-complete immunological compatibility of the animal-derived pericardial tissue resulting from failure to remove animal-derived xeno-antigens. In the mid/long-term, this leads to structural deterioration, mechanical failure, and prosthesis leaflets rupture, with consequent need for re-intervention. In the search for novel procedures to maximize biological compatibility of the pericardial tissue into immunocompetent background, we have recently devised a procedure to decellularize the human pericardium as an alternative to fixation with aldehydes. In the present contribution, we used this procedure to derive sheets of decellularized pig pericardium. The decellularized tissue was first tested for the presence of 1,3 α-galactose (αGal), one of the main xenoantigens involved in prosthetic valve rejection, as well as for mechanical tensile behavior and distensibility, and finally seeded with pig- and human-derived aortic valve interstitial cells. We demonstrate that the decellularization procedure removed the αGAL antigen, maintained the mechanical characteristics of the native pig pericardium, and ensured an efficient surface colonization of the tissue by animal- and human-derived aortic valve interstitial cells. This establishes, for the first time, the feasibility of fixative-free pericardial tissue seeding with valve competent cells for derivation of tissue engineered heart valve leaflets. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 345–356, 2016.

In patients with left ventricular assist device support and aspirin allergy, the choice of effective antiplatelet strategy remains a challenge. We compared the antithrombotic effect of clopidogrel vs ticagrelor in an LVAD patient with aspirin allergy by using a modified protocol of the thrombin generation test, accounting selectively for the platelet contribution on thrombin generation. Our results demonstrate enhanced antithrombotic efficacy offered by ticagrelor. Consistent with experimental results, the patient has passed more than 300 days without thromboembolic complications. This study provides additional mechanistic rationale supporting clinical evidence and opens the perspective to identify individual poor responsiveness to drugs by specifically evaluating drug-mediated platelet function.

To correlate the dynamics of platelet activation with the development of thromboembolic events in patients with continuous-flow left ventricular assist device (cf-LVAD).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.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.

Electrical stimulation (ES) of cells has been shown to induce a variety of responses, such as cytoskeleton rearrangements, migration, proliferation, and differentiation. In this study, we have investigated whether monophasic and biphasic pulsed ES could exert any effect on the proliferation and differentiation of human cardiac progenitor cells (hCPCs) isolated from human heart fragments. Cells were cultured under continuous exposure to monophasic or biphasic ES with fixed cycles for 1 or 3 days. Results indicate that neither stimulation protocol affected cell viability, while the cell shape became more elongated and reoriented more perpendicular to the electric field direction. Moreover, the biphasic ES clearly induced the upregulation of early cardiac transcription factors, MEF2D, GATA-4, and Nkx2.5, as well as the de novo expression of the late cardiac sarcomeric proteins, troponin T, cardiac alpha actinin, and SERCA 2a. Both treatments increased the expression of connexin 43 and its relocation to the cell membrane, but biphasic ES was faster and more effective. Finally, when hCPCs were exposed to both monophasic and biphasic ES, they expressed de novo the mRNA of the voltage-dependent calcium channel Cav 3.1(α1G) subunit, which is peculiar of the developing heart. Taken together, these results show that ES alone is able to set the conditions for early differentiation of adult hCPCs toward a cardiac phenotype.

A combined physical–chemical protocol for whole full-thickness bladder decellularization is proposed, based on organ cyclic distention through repeated infusion/withdrawal of the decellularization agents through the urethra. The dynamic decellularization was intended to enhance cell removal efficiency, facilitating the delivery of detergents within the inner layers of the tissue and the removal of cell debris. The use of mild chemical detergents (hypotonic solution and non-ionic detergent) was employed to limit adverse effects upon matrix 3D ultrastructure. Inspection of the presence of residual DNA and RNA was carried out on decellularized matrices to verify effective cell removal. Histological investigation was focused on assessing the retention of adequate structural and functional components that regulate the biomechanical behaviour of the acellular tissue. Biomechanical properties were evaluated through uniaxial tensile loading tests of tissue strips and through ex vivo filling cystometry to evaluate the whole-organ mechanical response to a physiological-like loading state. According to our results, a dynamic decellularization protocol of 17 h duration with a 5 ml/min detergent infusion flow rate revealed higher DNA removal efficiency than standard static decellularization, resulting in residual DNA content

Introduction Mechanical circulatory support devices (MCSDs) are emerging as a valuable therapeutic option for the management of end-stage heart failure. However, although recipients are routinely administered with anti-thrombotic (AT) drugs, thrombosis persists as a severe post-implant complication. Conventional clinical assays and coagulation markers demonstrate partial ability in preventing the onset of thrombosis. Through years, different laboratory techniques have been proposed as potential tools for the evaluation of platelets' hemostatic response in MCSD recipients. Most rely on platelet aggregation tests; they are performed in static or low shear conditions, neglecting the prominent contribution of MCSD shear-induced mechanical load in enhancing platelet activation (PA). On the other hand, those tests able to account for shear-induced PA have limited possibility of effective clinical translation. Aims and Methods Advances on this side have been addressed by microfluidic technology. Microfluidic devices have been developed for AT drug monitoring under flow, able to replicate physiological and/or constant shear flow conditions in vitro. In this paper, we present a newly developed microfluidic platform able to expose platelets to MCSD-specific dynamic shear stress patterns. We performed in vitro tests circulating human platelets in the microfluidic platform and quantifying the dynamics of PA by means of the Platelet Activity State (PAS) assay. Results Our results prove the feasibility of using microfluidics for the diagnosis of MCSD-related thrombotic risk. This study paves the way for the development of a miniaturized point-of-care device for monitoring AT drug regimen. Such a system may have significant impact on limiting the incidence of thrombosis in MCSD recipients.

We designed an experimental setup to characterize the thrombogenic potential associated with blood recirculating devices (BRDs) used in extracorporeal circulation (ECC). Our methodology relies on in vitro flow loop platelet recirculation experiments combined with the modified-prothrombinase platelet activity state (PAS) assay to quantify the bulk thrombin production rate of circulated platelets, which correlates to the platelet activation (PA) level. The method was applied to a commercial neonatal hollow fiber membrane oxygenator. In analogous hemodynamic environment, we compared the PA level resulting from multiple passes of platelets within devices provided with phosphorylcholine (PC)-coated and noncoated (NC) fibers to account for flow-related mechanical factors (i.e., fluid-induced shear stress) together with surface contact activation phenomena. We report for the first time that PAS assay is not significantly sensitive to the effect of material coating under clinically pertinent flow conditions (500 mL/min), while providing straightforward information on shear-mediated PA dynamics in ECC devices. Being that the latter is intimately dependent on local flow dynamics, according to our results, the rate of thrombin production as measured by the PAS assay is a valuable biochemical marker of the selective contribution of PA in BRDs induced by device design features. Thus, we recommend the use of PAS assay as a means of evaluating the effect of modification of specific device geometrical features and/or different design solutions for developing ECC devices providing flow conditions with reduced thrombogenic impact.

Left ventricular assist devices (LVADs) have emerged as vital life-saving therapeutic systems for patients with advanced and end-stage heart failure (HF). Despite their efficacy, VAD systems remain limited by post-implantation thrombotic complications. Shear-mediated platelet activation is the major driver of such complications in these devices. Nowadays few platelet function assays are routinely utilized in assessing the degree of platelet activation in VAD implanted patients. No assays exist that specifically target shear-mediated platelet activation. The platelet activity state (PAS) is a novel assay that has been well validated in vitro, measuring thrombin release as a surrogate for shear-mediated platelet activation. To date limited data exist as to the utility of this assay in the clinical setting. In the present study we evaluated eight LVAD patients' platelet activation level using the PAS assay. Simultaneous measurements of conventional prothrombotic and hemolysis markers, - i.e. fibrinogen and lactate dehydrogenase (LDH) - were also performed. Trends as to alteration from baseline were studied. We observed that the PAS assay allowed detection of an abnormal level of platelet activation in one patient in our series who suffered from an overt thrombosis. Interestingly in the same patient no signal of major abnormality in fibrinogen or LDH was detected. Further for 7/8 patients who were free of thrombosis, no significant level of platelet activation was detected via PAS assay, while elevation in fibrinogen and LDH were observed. As such, from our observational series it appears that the PAS assay is a sensitive and specific indicator of shear-mediated platelet activation. Further patients' experience will help elucidate the role of this promising assay in the management of LVAD implanted patients.

Purpose: To evaluate the efficacy of aspirin (ASA) in inhibiting ex vivo thrombin generation in patients with durable Left Ventricular Assist Device (LVAD) and to compare the thrombin generation profile of patients administered low (100mg/day) vs high (300mg/day) ASA dose. Methods: The Thrombin Generation Test was performed on a cohort patients implanted with LVAD who did not suffer any thrombotic event (n= 14). Thrombin generation was triggered by 0.5 pmol/L Tissue Factor in normal platelet poor plasma with the addition of the patients’ purified platelets, to account for the role played by platelets in mediating thrombin generation and to exclude any influence of anticoagulation therapy (Warfarin) on the test. Patients were on different ASA dose, according to the pump model: a) HeartMate II (Thoratec Corp., USA): 100mg/day (n= 3, 21%); b) HeartMate III (St. Jude Medical Inc., USA): 100mg/day (n= 4, 29%); c) Heartware HVAD (Heartware Corp., USA): 300mg/day (n= 7, 50%). In addition, one patient with HVAD who was not on ASA because of allergy was also profiled. Results were compared with those obtained from ASA-free volunteers (controls, n= 14). Results: Following a median time of LVAD support of 210 (IQR: 138-477) days, platelet-mediated thrombin generation was comparable in ASA-treated LVAD recipients and in controls (Fig. 1A,B). Notably, the patient not on ASA (491 days of support) showed higher thrombin generation as compared to the rest of the population (Fig. 1A,B). Comparison of low vs high ASA dose revealed no differences in terms of platelet-mediated thrombin generation (Fig. 1C). Conclusion: We report that ASA inhibits ex vivo platelet-mediated thrombin generation in LVAD recipients and may eventually mitigate the risk of thrombotic complications. The thrombin generation profile of ASA-treated LVAD patients was similar to controls. Moreover, we suggest that high dose ASA does not add any significant effect in blunting platelet activation and the associated thrombin generation pattern.

We developed a numerical model, based on multi-physics computational fluid dynamics (CFD) simulations, to assist the design process of a plastic hollow-fiber bundle blood heat exchanger (BHE) integrated within the INSPIRE TM , a blood oxygenator (OXY) for cardiopulmonary by-pass procedures, recently released by Sorin Group Italia. In a comparative study, we analyzed five different geometrical design solutions of the BHE module. Quantitative geometrical-dependent parameters providing a comprehensive evaluation of both the hemo- and thermo-dynamics performance of the device were extracted to identify the best-performing prototypical solution. A convenient design configuration was identified, characterized by (i) a uniform blood flow pattern within the fiber bundle, preventing blood flow shunting and the onset of stagnation/recirculation areas and/or high velocity pathways, (ii) an enhanced blood heating efficiency, and (iii) a reduced blood pressure drop. The selected design configuration was then prototyped and tested to experimentally characterize the device performance. Experimental results confirmed numerical predictions, proving the effectiveness of CFD modeling as a reliable tool for in silico identification of suitable working conditions of blood handling medical devices. Notably, the numerical approach limited the need for extensive prototyping, thus reducing the corresponding machinery costs and time-to-market.

Computational fluid dynamics (CFD) models have become very effective tools for predicting the flow field within the carotid bifurcation, and for understanding the relationship between local hemodynamics, and the initiation and progression of vascular wall pathologies. As prescribing proper boundary conditions can affect the solutions of the equations governing blood flow, in this study, we investigated the influence to assumptions regarding the outflow boundary conditions in an image-based CFD model of human carotid bifurcation. Four simulations were conducted with identical geometry, inlet flow rate, and fluid parameters. In the first case, a physiological time-varying flow rate partition at branches along the cardiac cycle was obtained by coupling the 3D model of the carotid bifurcation at outlets with a lumped-parameter model of the downstream vascular network. Results from the coupled model were compared with those obtained by imposing three fixed flow rate divisions (50/50, 60/40, and 70/30) between the two branches of the isolated 3D model of the carotid bifurcation. Three hemodynamic wall parameters were considered as indicators of vascular wall dysfunction. Our findings underscore that the overall effect of the assumptions done in order to simulate blood flow within the carotid bifurcation is mainly in the hot-spot modulation of the hemodynamic descriptors of atherosusceptible areas, rather than in their distribution. In particular, the more physiological, time-varying flow rate division deriving from the coupled simulation has the effect of damping wall shear stress (WSS) oscillations (differences among the coupled and the three fixed flow partition models are up to 37.3% for the oscillating shear index). In conclusion, we recommend to adopt more realistic constraints, for example, by coupling models at different scales, as in this study, when the objective is the outcome prediction of alternate therapeutic interventions for individual patients, or to test hypotheses related to the role of local fluid dynamics and other biomechanical factors in vascular diseases.

A comprehensive computational study modelling the operation of a rotating hollow-fiber bioreactor for artificial liver (BAL) was performed to explore the interactions between the oxygenated culture medium and the cultured hepatocytes. Computational fluid dynamics investigations were carried out using two-dimensional (2D) and 3D time-dependent numerical simulations, integrating calculations of diffusion, convection, and multiphase fluid dynamics. The analysis was aimed at determining the rotational speed value of the chamber to ensure homogenous distribution of the floating microcarrier-attached aggregated cells (microCAACs) and avoid their sedimentation and excessive packing, analyzing oxygen (O(2)) delivery and cellular O(2) consumption as an index of cellular metabolic activity, and analyzing the fluid-induced mechanical stress experienced by cells. According to our results, homogeneous distribution of cells is reached at a rotational speed of 30 rpm; spreading of cellular concentration at around the initial value of 12% was limited (median = 11.97%, 5th percentile = 10.94%, 95th percentile = 13.2%), resulting in uniform suspension of microCAACs, which did not appear to be excessively packed. Mixing within the rotating fluid caused a maximum fluid-induced stress value of 0.05 Pa, which was neither endangering for liver-specific functions of cultured cells, nor causing disruption of the floating aggregates. Moreover, an inlet medium flow rate of 200 mL/m with a partial pressure of oxygen (pO(2)) value of 160 mmHg was found to guarantee an adequate O(2) supply for the hepatocytes (2.7 x 10(8) hepatocytes are simulated); under such conditions, the minimum pO(2) value (23 mmHg) is above the critical threshold value, causing the onset of cellular hypoxia (10 mmHg). We proved that numerical simulation of transport phenomena is a valuable tool for the computer-aided design of BALs, helping overcome the unsolved issues in optimizing the cell-environment conditioning procedure in rotating BALs.

Tissue engineering (TE) is the application of principles and methods of engineering and life sciences towards the fundamental understanding of structure–function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain or improve tissue function. One key component to TE is using three-dimensional porous scaffolds to guide cells during the regeneration process. These scaffolds are intended to provide cells with an environment that promotes cell attachment, proliferation, and differentiation. After sufficient tissue regeneration using in vitro culturing methods, the scaffold/tissue structure is implanted into the patient, where the scaffold will degrade away, thereby leaving only regenerated tissue; on a different approach, non-cellularised scaffolds are inserted into the patient to elicit in vivo cell recruitment, growth and tissue regeneration. Tissue-engineered scaffolds need to meet both the biological goals of tissue formation and the stresses and loading conditions present in the human body. For this reason, any design approach must ensure that the mechanical properties of the resulting scaffold structure are compatible and optimally match the requirements from the environment, that, respectively, are the cell adhesion transmembrane protein, the cytoskeleton structure, the cell population. The need to design scaffold structures, the need for precision control during their fabrication and for determining the metrological indices and the need to characterise their structural behaviour at different scales have lead to numerous experimental and computational challenges. In particular, there is a need for modelling and test tissue at multiple scales to gain insight into issues such as drug delivery, drug interaction, gene expression and cellular–environment interactions. The analysis of the tissue constructs at different scales includes a macro-scale model where the macro-scale tissue construct is characterised, a multi-cellular model where a sufficiently large multi-cellular representative element volume is selected to represent a microstructure of the tissue construct and a single cell model wherein the microstructures of the cell like the nucleus and the cytoplasm have been incorporated. A multi-scale approach is already being applied to bridge nano- and micro-scales as well as micro- and macro-scales within various research areas in TE. In this chapter, a review of the experimental and modelling techniques used for the evaluation, at different scales, of the mechanical and morphological properties of bioartificial scaffolds and matrices, such as compression testing, nanoindentation, AFM technique, Dynamical Mechanical Analysis (DMA), micro-CT, micro-MR, Asymptotic Homogenisation Theory, Finite Element Analysis (FEA), Rule-of- Mixtures, is proposed.